Iej 10 2009

doi:10.1111/j.1365-2591.2009.01577.
REVIEW
Apexication: the beginning of its end
G. T.-J. Huang
Department of Endodontics, Prosthodontics and Operative Dentistry, College of Dental Surgery, University of Maryland, Baltimore, MD, USA
Abstract
Huang G.T.-J. Apexication: the beginning of its end. International Endodontic Journal, 42, 855866, 2009.
Apexication is a procedure for treating and preserving immature permanent teeth that have lost pulp vitality. It contrasts apexogenesis in terms of its outcome in that apical maturation and normal root thickness cannot be obtained. Apexication has been a routine practice for such teeth for many decades, and despite a literature replete with discussion, including recent articial barrier methods with mineral trioxide aggregate, ultimately there has been no major breakthrough to improve this treatment. Recently, two new clinical concepts have emerged. One involves a revitalization approach to achieve tissue generation and regeneration. In this method, new living tissue is expected to
form in the cleaned canal space, allowing continued root development in terms of both length and thickness. The other is the active pursuit of pulp/dentine regeneration via tissue engineering technology to implant or re-grow pulps. Although the technology is still at its infancy, it has the potential to benet immature pulpless teeth by allowing continued growth and maturation. With this understanding, it may be predicted that apexication will become less needed in years to come. This study will overview the recent concept of pulp revitalization in the treatment of immature teeth with nonvital pulps and the emerging research on pulp tissue engineering and regeneration. Keywords: apexication, calcication, pulp/dentine tissue regeneration, stem cells.
Received 15 July 2008; accepted 26 February 2009
Introduction
Apexication is a procedure to promote the formation of an apical barrier to close the open apex of an immature tooth with a nonvital pulp such that the lling materials can be contained within the root canal space (Rafter 2005). The capacity of materials such as calcium hydroxide [Ca(OH)2] to induce the formation of this calcic barrier at the apex made apexication possible and allowed the preservation of many
Correspondence: George T.-J. Huang, DDS, MSD, DSc, Department of Endodontics, Prosthodontics and Operative Dentistry, College of Dental Surgery, Dental School, University of Maryland, 650 West Baltimore St, Baltimore, 21201 MD, USA (Tel.: +410 706 7680; fax: +410 706 3028; e-mail: ghuang@umaryland.edu).
compromised, immature teeth with nonvital pulps by endodontic and restorative means. Clinically, when the pulpal diagnosis of an immature tooth is nonvital, apexication is undertaken to close the root-end, but with an understanding that there will be no more development of the root in terms of apical maturation and thickening of its dentine walls. The clinical decision as to whether to perform apexogenesis or apexication for immature teeth appears to be clear cut with the teeth deemed to contain vital pulp tissue being subject to apexogenesis and teeth deemed to have nonvital pulp tissue receiving apexication. However, certain clinical observations reported recently have broken this clear-cut guideline by showing that apexogenesis may occur in teeth which have nonvital pulps (Iwaya et al. 2001, Banchs & Trope 2004, Chueh & Huang 2006). Moreover, it is
2009 International Endodontic Journal
International Endodontic Journal, 42, 855866, 2009
855
Apexication, end in sight Huang
likely that many clinicians had been treating some cases by an apexogenesis approach despite apparent pulp necrosis, but never reporting the outcome. A new protocol has been suggested in which a haemorrhage is induced to ll the canal with blood clot as a scaffold to allow generation of live tissues in the canal space and continued root formation (length and wall thickness) (Banchs & Trope 2004, Thibodeau & Trope 2007, Thibodeau et al. 2007). Instead of using Ca(OH)2 as the intracanal medicament between visits to disinfect and to induce apical barrier formation, an antibiotic paste is used for the purpose of disinfection only (Iwaya et al. 2001, Banchs & Trope 2004). This new protocol of treatment coincides with the recent concept of regenerative medicine which promotes the research and practice of tissue regeneration (National Institutes of Health 2006). On another front, pulp/dentine tissue may be regenerated using tissue engineering technologies. Attempts to regenerate pulp tissue have been considered impossible until recently and major developments in two basic research, namely tissue engineering and stem cell biology. Investigations on dental pulp tissue engineering began in the late 1990s (Mooney et al. 1996, Bohl et al. 1998, Buurma et al. 1999). The isolation and characterization of dental pulp stem cells (DPSCs) (Gronthos et al. 2000), stem cells from exfoliated deciduous teeth (SHED; Miura et al. 2003) and stem cells from apical papilla (SCAP) (Sonoyama et al. 2006) has capitalized the possibility for pulp/ dentine regeneration (Huang et al. 2006, 2008, Murray et al. 2007a, Cordeiro et al. 2008, Prescott et al. 2008). Because of the wide-open apex of the immature tooth, vascularization via apical ingrowth of blood vessels into an engineered construct containing stem cells may facilitate a successful regeneration of pulp/dentine within the canal space (Huang et al. 2008). This study will overview the shifting concept of treating immature teeth using revitalization rather than apexication and the current status of pulp tissue engineering and regeneration. The review will analyse the fate of apexication as a rst-line treatment for immature teeth with nonvital pulps and how this is affected by the shifting paradigm of the management and the coming era of pulp/dentine tissue regeneration. Again, apexication does not allow generation or regeneration of vital tissues in the canal space whereas the revitalization or tissue regeneration approaches provide a new chance for those affected teeth to regain biological tissue recovery and growth.
From this point of view, it seems inevitable that in the interest of patients, apexication may become a lessdesirable and less needed clinical treatment in the foreseeable future.
Apexication
Immature teeth undergoing apexication are usually disinfected with irrigants including NaOCl, chlorhexidine, EDTA and iodinepotassium iodide (Rafter 2005). The canal is then lled with Ca(OH)2 paste for the purpose of further disinfection and induction of an apical calcic barrier. Ca(OH)2 is antimicrobial because of its release of hydroxyl ions which can cause damage to the bacterial cellular components. The best example is the demonstration of its effect on lipopolysaccharide (LPS). Ca(OH)2 chemically alters LPS which affects its various biological properties (Safavi & Nichols 1993, 1994, Barthel et al. 1997, Nelson-Filho et al. 2002, Jiang et al. 2003). Filling the root canal is undertaken normally when the apical calcic barrier is formed. Without the barrier, there is nothing against which the traditional guttapercha lling material can be condensed. Besides the fact that Ca(OH)2 functions as a potent disinfectant, early evidence has suggested osteo-inductive properties (Mitchell & Shankwalker 1958), although it has been difcult to demonstrate this effect in vitro (Raquel Assed Bezerra da et al. 2008). It was considered that the high pH may be a contributing factor for the induction of hard tissue formation (Javelet et al. 1985). The time required for apical barrier formation in apexication using Ca(OH)2 may be considerable, often as long as 20 months and other conditions such as age and presence of symptoms or periradicular radiolucencies may affect the time needed to form an apical barrier. Refreshing the Ca(OH)2 paste usually takes place every 3 months (Rafter 2005). A number of shortcomings can be summarized for Ca(OH)2 apexication: (i) long time-span of the entire treatment; (ii) multiple visits with heavy demands on patients and carers and inevitable clinical costs; (iii) increased risk of tooth fracture using Ca(OH)2 as a long-term root canal dressing (Cvek 1992, Andreasen et al. 2002). These drawbacks led to the use of mineral trioxide aggregate (MTA) to ll the apical end without the need for calcic barrier formation. In comparison to Ca(OH)2, some data suggest that MTA appears to be more predictable with consistent hard-tissue formation based on in vivo studies in dogs (Shabahang et al. 1999). Using MTA for apexication may shorten the treatment period with
856
Huang Apexication, end in sight
more favourable results and improved patient compliance (Maroto et al. 2003, El-Meligy & Avery 2006, Pace et al. 2007). Many authors and clinicians propose a one-visit apexication protocol with MTA, which presents a major advantages over traditional Ca(OH)2 methods (Witherspoon & Ham 2001, Steinig et al. 2003). This expedient cleaning and shaping of the root canal system followed by its apical seal with MTA makes the rapid placement of a bonded restoration within the root canal possible, which may prevent potential fractures of immature teeth. While advances with MTA and bonded restorations go some way towards a better outcome, ultimately no apexication method can produce the outcome that apexogenesis can achieve, i.e. apical maturation with increased thickness of the root. As noted above, clinical experience on the outcome of apexied teeth with thin and weak roots after successful treatment is that they are highly susceptible to fracture (Cvek 1992, Katebzadeh et al. 1998). Therefore, alternative approaches that allow the increase of root thickness and/or length should be pursued.
A paradigm shift in the management of immature teeth

Although the standardized clinical approach for apexogenesis or apexication has been widely practiced, some clinicians inevitably modify their treatment procedures based on their clinical judgement. Some reported their cases using alternative approaches, with three appearing to capture great interest from the endodontic community. The rst, reported by Iwaya et al. (2001) presented an immature mandibular premolar with a sinus tract and periradicular radiolucency. During canal preparation, they did not instrument to full working length because the patient felt discomfort on the insertion of instruments. The canal was mainly irrigated with NaOCl and hydrogen peroxide and further disinfected with antibiotic agents (metronidazole and ciprooxacin). Thirty-ve months after the completion of these procedures, they observed complete maturation of the root apex with thickened root structure. The tooth also responded positively to electronic pulp testing. After observing the success of this alternative approach, the same idea was applied to treatment of a mandibular premolar having a similar condition but with more extensive periradicular bone loss. During careful follow-up to 2 years after the treatment, complete maturation of the root was observed with a positive response to cold testing
(Banchs & Trope 2004). Chueh & Huang (2006) later reported four mandibular premolars in a similar clinical condition that were treated between 1988 and 2000, all again demonstrating healing and apical maturation. These reports raised a great response and encouraged further reports (Thibodeau & Trope 2007, Hargreaves et al. 2008, Jung et al. 2008). A more conservative approach and a shifting paradigm for the treatment of nonvital immature teeth has thus been proposed (Huang 2008). Furthermore, the Regenerative Endodontics Committee of the American Association of Endodontists has initiated a pilot study by encouraging endodontists to submit their cases to a data base (http://www.aae.org/members/revascularizationsurvey. htm). The study is designed to determine the incidence and predictors of healing of apical periodontitis in cases considered to have nonvital pulps when treated by nonconventional, biologically based revitalization methods. Currently, the success rate of this type of approach is only available from an animal study model (Thibodeau et al. 2007) and a pilot clinical study in humans (Shah et al. 2008). In the animal model, it was found that after disinfection of the root canals, 43.9% of the cases had thickened canal walls, 54.9% had apical closure and 64.6% had no radiographic evidence of periapical radiolucency or showed improvement/ healing of previous periapical radiolucencies (Thibodeau et al. 2007). The clinical pilot study involving teeth in 14 patients demonstrated 93% resolution of periradicular radiolucencies, thickening of lateral dentinal walls in 57%, and increased root length in 71%. None of the cases presented with pain, reinfection or radiographic enlargement of pre-existing periapical lesions (Shah et al. 2008). However, due the preliminary nature of the study, the clinical success rates should be interpreted with caution (Messer 2008). Regarding the use of Ca(OH)2 versus antimicrobial paste, it was suggested that the former may not be suitable if there is remaining vital pulp tissue in the canal. The direct contact of Ca(OH)2 paste with the tissue will induce the formation of a layer of calcic tissue which may occlude the pulp space, therefore preventing pulp tissue from regeneration (Huang 2008). Another concern is that Ca(OH)2 may damage the Hertwigs epithelial root sheath (HERS) and thereby destroy its ability to induce the nearby undifferentiated cells to become ododontoblasts (Banchs & Trope 2004). The effectiveness of a triple-antibiotic regimen to disinfect root canal space was rst tested and veried by Sato et al. (1996) and the clinical use of the mixture has shown success in terms of clinical outcome (Sato
857
et al. 1996, Banchs & Trope 2004, Jung et al. 2008). Whether the three antibiotics originally described (i.e. metronidazole, minocycline and ciprooxacin) must be used for this purpose or if other choices may serve this purpose requires further investigation. These clinical case reports demonstrate that despite the formation of periapical abscesses with extensive periradicular bone resorption as the result of root canal infection in immature teeth, conservative treatment may allow roots to increase in length and thickness or even reach mature form. One explanation is that the clinical diagnosis of pulp status is inaccurate and that some of those teeth must have contained vital tissues in the apical pulp space despite negative pulp testing and periapical lucencies. It is also acknowledged that there is a lack of scientic studies on the diagnosis of pulpal pathology in permanent teeth with open apices (Camp 2008). It has been considered that, to have continued root development, HERS and the recently identied tissue, apical papilla, must be functional (Huang et al. 2008). On the other hand, if the pulp, HERS and apical papilla are completely lost, the root may still gain some level of thickness by the ingrowth of cementum from the periapical areas onto the internal root canal dentine walls. Additionally, this cementum ingrowth is accompanied by periodontal ligament (PDL) and bone tissue (Kling et al. 1986, Andreasen et al. 1995a,b).
stem cells, SCAP that have been recently described to be more robust stem cells than DPSCs (Sonoyama et al. 2006). The SCAP may survive the infection and retain the capacity to give rise to new odontoblasts inuenced by HERS, allowing new root dentine to form and root maturation to proceed to completion. It was speculated that the surviving DPSCs in the remaining vital pulp may rebuild the lost pulp tissue in the canal and differentiate into replacement odontoblasts to substitute for the damaged primary odontoblasts (Sonoyama et al. 2008). Under this circumstance, one may anticipate the newly formed odontoblasts from SCAP to produce root dentine that leads to the apical extension of the root. Additionally, the existing primary odontoblasts that survived in the residual pulp tissue and perhaps some new replacement odontoblasts may continue to lay down dentine on the dentinal walls, causing the root to increase its thickness (Fig. 1). Whilst this explanation is conjecture and requires further basic and clinical investigation, some data on the recovery of pulp tissue after tooth replantation appear to support this speculation (Kling et al. 1986, Ritter et al. 2004).
Pulp space lled with periodontal tissues

In cases where the entire pulp, apical papilla tissues and the HERS are lost, current understanding is that self-regeneration of pulp and new dentine formation is unlikely to occur. There is abundant evidence in the literature demonstrating that when the pulp tissue of
The outcome of guided generation and regeneration approach

The use of the term revascularization was adapted by Iwaya et al. (2001) to describe the clinical healing of periapical abscesses and continued root formation in immature teeth with nonvital pulps. Other authors adapted the term without questioning until Huang & Lin (2008) considered that revascularization did not encompass the actual healing and repair process that takes place in these clinical cases (Huang & Lin 2008). The term revitalization used by earlier studies attempting to revive tissues in the pulp space would perhaps describe the phenomenon more accurately (Nevins et al. 1976).
Apical papilla Epithelial diaphragm Bone
Cementum PDL Dentin Pulp
Odontoblasts
Pulp space lled with regenerated pulp

The ideal situation is that there is surviving pulp and apical papilla tissue after root canal disinfection. Continued root formation to its maturity and an increased thickness of root dentine may then be anticipated. The dental papilla at the apex contains
?
Figure 1 Hypothetical pulp regeneration from the remaining recovered pulp. The question mark indicates that the regeneration of pulp into the empty pulp space is uncertain at present.
858
(a)
c (b)
Bone
Bone Cementum Cementum
Figure 2 Ingrowth of periodontal tissue
into pulp space. (a) Radiographs showing an immature tooth 11 (FDI notation) with an open apex which was re-implanted and healed. At recalls, the ingrowth of bone tissue with the PDL space and lamina dura is evident [adapted from Kling et al. (1986) with permission). (b) Illustration depicting the ingrown tissues of bone, PDL and cementum into the canal space.
PDL Cementum PDL Dentin PDL Bone PDL
immature teeth with wide-open apices undergoes complete necrosis but in a sterile environment, other tissues are capable of lling the canal space. As shown in the radiographic images (Fig. 2), the replanted avulsed immature tooth lost pulp vitality but the pulp
space became occupied by the ingrowth of alveolar bone from the periapex (Kling et al. 1986). There is a space separating the ingrown bone and the canal dentinal walls. If one traces this space, it is apparent that it is continuous with the PDL space on the external
859
root surfaces. Lamina dura also appears to have been established in the ingrown bone occupying the pulp space. The contents of the pulp space were described by Holan (1998) as tube-like mineralization and following histological examination it was interpreted that secondary dentine and the pulp tissue existed in the canal space. In fact, this secondary dentine was actually cementum and the pulp tissue was PDL. Careful examination of the characteristics of the ectopic cementum and PDL in the canal space should be the basis of further research. There also seems to have been some degree of vertical and horizontal extension of the root over time (Fig. 2). Since the pulp tissue has been entirely lost, it has not been possible to deposit new dentine, and the newly acquired calcied tissue has to come from a tissue source where the cellular components are capable of proliferating and producing new tissues. Cementum has the capacity to fulll this purpose. Histologically, the hard tissues, bone, cementum and dentine can usually be distinguished unambiguously merely for their anatomical location. However, when ectopic formation of these tissues occurs, discerning them without specic markers may be difcult. Nonetheless, the ingrown hard tissues within the pulp space have been veried by histological examination, revealing the deposition of cementum onto the dentine surface in the canal, extending from the outside surface of the apex (Nevins et al. 1977, Lieberman & Trowbridge 1983). The apical extension of roots resulting from the apposition of cementum is a normal physiological process. The apposition of calcied cemental tissue on the internal canal wall also increases the thickness of the root. A distinct feature of cementum is its connection with the PDL by Sharpeys bres, which can also be observed in the ingrown tissues in the pulp space. The ingrowth of periodontal tissue may reach all the way to the coronal pulp chamber (Nevins et al. 1977, 1978, Ellis et al. 1985, Hitchcock et al. 1985). Similar results were observed in a dog as a study model (Thibodeau et al. 2007). When the pulp space is lled with periodontal tissues, the situation is totally different from normal because the pulp space is no longer part of the root canal system, but part of periapical tissues. If the tooth becomes reinfected causing destruction of the periodontal tissue in the canal space, the understanding of a root canal infection to this type of infection cannot be applied, but perhaps more appropriately that of a periapical tissue pathosis. It is known that periapical tissue loss will recover if the source of infection from the
root canal space is eliminated though the establishment of a biolm by the invading microbes may complicate management (de Paz 2007). From this perspective, disinfection does not have to involve with the aggressive entrance into the canal space, but rather dealing mainly with the source of infection in the crown. Currently, there is no case report showing the management and the outcome of infected canal space that has been lled with periodontal tissues.
Severe disorganized calcication of the pulp space

Whether the pulp space is lled with regenerated pulp or periodontal tissues, long-term radiographic observations demonstrate that the pulp space becomes severely narrowed or lled with radio-opaque mineralized tissue over time. Histologically, the mineralizing tissues are either bone-like or dentine-like (Robertson et al. 1997). The hard tissues may begin as calcic particles that have been observed to originate or are closely associated with blood vessels and perineurium sheaths (Pashley et al. 2002). Interestingly, these are also the locations where pulp stem cells are believed to exist (Shi & Gronthos 2003). Whether these stem cells are activated by the low-grade inammation to undergo osteogenic differentiation is unclear at present. Over time, these particles merge into larger calcic masses and obliterate the pulp space (Fig. 3). Although this calcifying phenomenon within the pulp has been welldocumented, the mechanisms underlying this process are still elusive. Prolonged inammation causes calcication in many parts of the body, e.g. calcifying tendonitis (Uhthoff 1996). Arthritic joints tend to build osteophytes as a result of the expanding bone tissue over the damaged cartilaginous tissues (van der Kraan & van den Berg 2007). Another phenomenon named heterotropic ossication is characterized by the formation of mineralized inclusions within the soft tissues (McCarthy & Sundaram 2005), e.g. muscles of patients who suffered from severe trauma to their extremities including soldiers injured by bomb explosions (Owens et al. 2006). It has been speculated that the causes of such phenomena include systemic factors and/or local inammatory conditions. Stem cells in the muscle have been investigated for their potential contributory role in this disease. Deciencies in osteopontin may lead to vascular calcication (Giachelli 2005). There has been an ongoing debate on the relative benets of calcied material or gutta-percha lled canals. From a physiological point of view, calcic
860
(a)
(b)
(c)
Figure 3 Common feature of pulp undergoing calcic metamorphosis. (a) Pulp tissue from a tooth which had been previously restored with old llings and a clinical diagnosis of normal pulp (arrows indicate mineral deposits that appear to have been associated with vascular structures) (b, c) Pulp tissues from teeth diagnosed with irreversible pulpitis. Arrows indicate heavy mineral deposits.
metamorphosis is a degenerative disease. Moreover, from a technical perspective, calcied canals pose a challenge if they need treatment. Most of the literature does not support endodontic intervention in the case of mineralized obliteration unless periradicular pathoses is detected or the involved tooth becomes symptomatic (Robertson et al. 1997, Gopikrishna et al. 2004). Surgical intervention may be the only option to contain the infection from the periradicular tissues if calcied canals are not accessible for nonsurgical root canal treatment.
Progress on pulp/dentine tissue engineering and regeneration

The potential of pulp tissue to regenerate lost dentine is well-known. Direct pulp capping therapy to induce dentinal bridge formation is practiced on the basis of this understanding. The use of various cement-based materials such as Ca(OH)2 and MTA is believed to promote such activity. Long-term success using MTA for direct pulp capping has been reported recently (Bogen et al. 2008). The application of recombinant growth factors to the injured site to enhance the regeneration of dentine has also been investigated (Rutherford & Gu 2000). Cell-based therapy using isolated pulp cells or DPSCs, with genetic manipulation to express bone morphogenic proteins, to augment the generation of new dentine bridge formation is an additional area of exploration (Rutherford 2001, Iohara et al. 2004). When dealing with the initial phases of dentine destruction where there is minimal damage, applying a
complicated biotechnological approach appears impractical. When the tooth is further damaged, regeneration of dentine becomes difcult as it needs a healthy pulp which may be compromised by the disease. Ideally, the regenerated dentine should not replace the pulp space. Two types of pulp regeneration can be considered based on the clinical situations: (i) partial pulp regeneration and (ii) de novo synthesis of pulp. It has been observed that pulpal infection and inammation is compartmentalized until the entire pulp tissue undergoes necrosis (Seltzer et al. 1963, Trowbridge 2002). Before the end stage, the remaining pulp tissue may be recoverable and help regenerate the lost portion. To enhance the regeneration, engineered pulp tissues may be inserted into the pulp space to facilitate the entire recovery of pulp tissue and the generation of new dentine. When the entire pulp tissue is lost, de novo synthesis of pulp must take place to regenerate the tissue.
Early efforts on pulp regeneration

Regenerating pulp tissue has been a long quest. Ostby (1961) studied the tissue re-organization in the canal space lled with blood clot. It was observed that the tissue formed in the canal was not pulp but granulation or brous tissues and in some cases the ingrowth of cementum and bone occurred. Similar ndings were observed by Myers & Fountain (1974) in a primate study using blood clot as a scaffold. The average generation of soft connective tissue into the canal was only 0.11.0 mm, although the authors mentioned
861
that teeth with open apices had a few more millimetres of ingrowth than those with mature apicies (Myers & Fountain 1974). It appears that in a natural situation, regeneration of pulp cannot occur following total loss of pulp tissue. Pulp cells have been isolated for various studies for many decades and they have been shown to have the capacities to differentiate into mineral forming odontoblast-like cells in vitro (Tsukamoto et al. 1992, About et al. 2000, Couble et al. 2000). However, it was not until it was demonstrated the formation of ectopic dentine/pulp-like complex in vivo by isolated pulp cells that the isolation of odontoblast progenitor cells or pulp stem cells was truly conrmed (Gronthos et al. 2000). These cells were termed postnatal DPSCs.
Pulp tissue engineering

Before the isolation of DPSCs, pulp regeneration was tested using modern tissue engineering concepts by growing pulp cells onto synthetic polymer scaffolds of polyglycolic acid (PGA) and in vitro and in vivo analyses performed (Mooney et al. 1996, Bohl et al. 1998, Buurma et al. 1999). These approaches are basically a proof-of-principle to test whether cultured pulp cells can grow well and produce matrix on PGA, and whether the engineered pulp can be vascularized using in vivo study models. This approach reected the emphasis on providing a three-dimensional structure for cells to attach to which simulates the in vivo environment. Using a tooth slice model, generation of well-vascularized pulp-like tissue has been reported (Cordeiro et al. 2008, Prescott et al. 2008).
Issues in cell-based pulp tissue engineering

The following questions must be considered when attempting to engineer and regenerate pulp tissue: (i) vascularization: can the angiogenesis from the limited apical blood supply extend to the coronal end if the entire pulp is to be regenerated? (ii) New odontoblast formation: can the new odontoblasts form against the existing dentinal wall that has been chemically disinfected during the root canal procedures? (iii) New dentine formation: can the newly differentiated odontoblasts produce new dentine and how much would they produce? (iv) Cell source: autologous cells are still the best cell source to avoid potential immune rejection. However, where can one nd the cells needed for pulp regeneration in the clinical setting? These points will now be discussed in turn.
Vascularization While vascularization is a universal issue for an engineered tissue, it is of special concern for pulp tissue engineering because of the lack of a collateral source of blood supply. It was considered that the use of angiogenic inducing factors such as vascular endothelial growth factor (VEGF) could enhance and accelerate pulp angiogenesis. Alternatively, the insertion of engineered pulp tissue may have to be separated into multiple steps to allow progressive vascularization (Huang et al. 2008). The choice of scaffold and the source of angiogenic factors have become integrated issues. Articial synthetic scaffolds such as co-polymer of d,l-lactide and glycolide can be fabricated with impregnated growth factors such as VEGF and/or platelet-derived growth factor (Sheridan et al. 2000, Richardson et al. 2001, Peters et al. 2002, Kanematsu et al. 2004, Stiver et al. 2004, Sun et al. 2005). The size of apical opening would affect the ingrowth of blood vessels into the engineered pulp tissue. It is assumed that the larger the opening, the more likely that angiogenesis can occur. Immature teeth with open apices are therefore the best candidates for pulp tissue regeneration. It is a misconception to adapt the concept of engineering/regenerating bone for pulp tissue. Certain scaffolds that have osteo-inductive or conductive properties and are suitable for bone regeneration, such as hydroxyapatite and tricalcium phosphate have been proposed as scaffolds for pulp regeneration. The misconception is based on the fact that dentine production has many aspects similar to bone formation. However, it is important to recognize the key differences. An obvious one is the anatomic characteristics. Bone mass contains compact or trabecular bone and marrow, whereas dentine and pulp in a tooth have a rigid anatomic location. When regenerating pulp and dentine, the dentine should be located peripherally to the pulp, not within it. Therefore, the scaffold that carries the cells to regenerate pulp and dentine should not induce dentine formation randomly within the regenerated pulp. New odontoblast formation To address the question whether new odontoblasts can form on the existing dentine walls, in vitro experiments have shown that by seeding DPSCs onto the existing dentine, some cells transformed into odontoblast-like cells with a cellular process extending into dentinal tubules (Huang et al. 2006). A tooth slice model has been utililzed and seeded SHED onto synthetic scaffolds
862
of poly-l-lactic acid cast in the pulp chamber of the thin tooth slice. They observed odontoblast-like cells arising from the stem cells and localized against the existing dentine surface in their in vivo study model (No r 2006, Cordeiro et al. 2008). From these observations, it appears that stem cells seeded in the scaffold will be attracted to the dentinal wall, differentiate into odontoblast-like cells and extend their cellular processes into the dentinal tubules. The mechanism behind this phenomenon has been speculated to be the released growth factors such as TGF-b by the dentine, which attracts and induces the differentiation of odontoblasts (Huang et al. 2006). Chemical disinfection of the root canal space may damage these embedded growth factors. Further investigation is needed to seek for ways to avoid this potential damage, and positively promote odontoblast-like colonization. New dentine formation The next question is whether these newly formed odontoblast-like cells will make new dentine. In an in vivo study model, DPSCs were seeded onto dentine and the construct implanted into the subcutaneous space of immunocompromised mice. Deposition of reparative dentine-like structures by odontoblast-like cells was observed (Batouli et al. 2003). This nding suggests the possibility of forming additional new dentine on existing dentine if new odontoblasts can emerge. Huang G.T.-J., Shea L.D., Shi S. & Tuan R.S. (upubl. data) also demonstrated that new dentine-like or osteodentine structure can deposit onto the existing dentine throughout the entire canal wall in an in vivo pulp engineering/regeneration study model. Cell source With respect to the cell source, there are several potential sources to obtain autologous cells for pulp/ dentine tissue regeneration: DPSC, SCAP and SHED. Immature third molars are one of the best sources for DPSCs and SCAP. The latter have been shown to be more potent dental stem cells than DPSCs in terms of their level of immaturity and potentiality. They give rise to odontoblast-like cells and make ectopic dentine in in vivo study models (Sonoyama et al. 2006) . SHED also produce ectopic dentine in vivo (Miura et al. 2003). The problem is the availability of this source. Banking personal teeth for future use appears to be a direction that must be explored and established to ensure this availability. Allogenic cells are an alternative and convenient source. The nding of the immunosuppressive capacity of mesenchymal stem cells to avoid
immuno-rejection provides a great possibility that allogenic stem cells may be a good source (Pierdomenico et al. 2005, Chen et al. 2006). However, in vivo studies to verify the long-term survival of transplanted allogenic dental stem cells are lacking.
Prospects
The above analysis points out the potential future fate of apexication procedures. Such procedures may no longer be the preferred rst option to treat immature permanent teeth with nonvital pulps. Induced generation and regeneration of vital tissues in the pulp space can thicken the root structure leading to a stronger tooth with a potentially reduced fracture risk. The progress of pulp/dentine regeneration so far has been promising and is likely to work in the not so distant future. There is some concern caused by the uncertainty as to how pulp regeneration would affect the future of endodontic practice (Murray et al. 2007b) . One may anticipate that to feasibly deliver stem cell-based endodontic therapy for pulp/dentine regeneration in endodontic practice, an uncomplicated clinical protocol would need to be established. If not, technology transfer to the commercial sector would be difcult (Rutherford 2007).
Acknowledgements
This work was supported in part by an Endodontic Research Grant from the American Association of Endodontists Foundation (G.T.-J.H.).
Reference
About I, Bottero MJ, de Denato P, Camps J, Franquin JC, Mitsiadis TA (2000) Human dentin production in vitro. Experimental Cell Research 258, 3341. Andreasen JO, Borum MK, Jacobsen HL, Andreasen FM (1995a) Replantation of 400 avulsed permanent incisors. 1. Diagnosis of healing complications. Endodontics and Dental Traumatology 11, 518. Andreasen JO, Borum MK, Jacobsen HL, Andreasen FM (1995b) Replantation of 400 avulsed permanent incisors. 2. Factors related to pulpal healing. Endodontics and Dental Traumatology 11, 5968. Andreasen JO, Farik B, Munksgaard EC (2002) Long-term calcium hydroxide as a root canal dressing may increase risk of root fracture. Dental Traumatology 18, 1347. Banchs F, Trope M (2004) Revascularization of immature permanent teeth with apical periodontitis: new treatment protocol? Journal of Endodontics 30, 196200.
863
Barthel CR, Levin LG, Reisner HM, Trope M (1997) TNF-alpha release in monocytes after exposure to calcium hydroxide treated Escherichia coli LPS. International Endodontic Journal 30, 1559. Batouli S, Miura M, Brahim J et al. (2003) Comparison of stem-cell-mediated osteogenesis and dentinogenesis. Journal of Dental Research 82, 97681. Bogen G, Kim JS, Bakland LK (2008) Direct pulp capping with mineral trioxide aggregate: an observational study. Journal of the American Dental Association 139, 30515. Bohl KS, Shon J, Rutherford B, Mooney DJ (1998) Role of synthetic extracellular matrix in development of engineered dental pulp. Journal of Biomaterials Science. Polymer Edition 9, 74964. Buurma B, Gu K, Rutherford RB (1999) Transplantation of human pulpal and gingival broblasts attached to synthetic scaffolds. European Journal of Oral Sciences 107, 2829. Camp JH (2008) Diagnosis dilemmas in vital pulp therapy: treatment for the toothache is changing, especially in young, immature teeth. Journal of Endodontics 34, S612. Chen XI, Armstrong MA, Li G (2006) Mesenchymal stem cells in immunoregulation. Immunology and Cell Biology 84, 413 21. Chueh L-H, Huang GTJ (2006) Immature teeth with periradicular periodontitis or abscess undergoing apexogenesis: a paradigm shift. Journal of Endodontics 32, 120513. Cordeiro MM, Dong Z, Kaneko T et al. (2008) Dental pulp tissue engineering with stem cells from exfoliated deciduous teeth. Journal of Endodontics 34, 9629. Couble ML, Farges JC, Bleicher F, Perrat-Mabillon B, Boudeulle M, Magloire H (2000) Odontoblast differentiation of human dental pulp cells in explant cultures. Calcied Tissue International 66, 12938. Cvek M (1992) Prognosis of luxated non-vital maxillary incisors treated with calcium hydroxide and lled with gutta-percha. A retrospective clinical study. Endodontics and Dental Traumatology 8, 4555. Ellis E III, Cox CF, Hitchcock R, Baker J (1985) Vital apicoectomy of the teeth: a 14 week histopathological study in Macaca mulatta. Journal of Oral Pathology 14, 718 32. El-Meligy OA, Avery DR (2006) Comparison of apexication with mineral trioxide aggregate and calcium hydroxide. Pediatric Dentistry 28, 24853. Giachelli CM (2005) Inducers and inhibitors of biomineralization: lessons from pathological calcication. Orthodontics and Craniofacial Research 8, 22931. Gopikrishna V, Parameswaran A, Kandaswamy D (2004) Criteria for management of calcic metamorphosis: review with a case report. Indian Journal of Dental Research 15, 54 7. Gronthos S, Mankani M, Brahim J, Robey PG, Shi S (2000) Postnatal human dental pulp stem cells (DPSCs) in vitro and in vivo. Proceedings of the National Academy of Sciences of the United States of America 97, 1362530.
Hargreaves KM, Geisler T, Henry M, Wang Y (2008) Regeneration potential of the young permanent tooth: what does the future hold? Journal of Endodontics 34, S516. Hitchcock R, Ellis E III, Cox CF (1985) Intentional vital root transection: a 52-week histopathologic study in Macaca mulatta. Oral Surgery, Oral Medicine, Oral Pathology 60, 214. Holan G (1998) Tube-like mineralization in the dental pulp of traumatized primary incisors. Dental Traumatology 14, 279 84. Huang GTJ (2008) A paradigm shift in endodontic management of immature teeth: conservation of stem cells for regeneration. Journal of Dentistry 36, 37986. Huang GTJ, Lin LM (2008) Letter to the editor: comments on the use of the term revascularization. Journal of Endodontics 34, 511. Huang GTJ, Sonoyama W, Chen J, Park S (2006) In vitro characterization of human dental pulp cells: various isolation methods and culturing environments. Cell and Tissue Research 324, 22536. Huang GTJ, Sonoyama W, Liu Y, Liu H, Wang S, Shi S (2008) The hidden treasure in apical papilla: the potential role in pulp/dentin regeneration and bioroot engineering. Journal of Endodontics 34, 64551. Iohara K, Nakashima M, Ito M, Ishikawa M, Nakasima A, Akamine A (2004) Dentin regeneration by dental pulp stem cell therapy with recombinant human bone morphogenetic protein 2. Journal of Dental Research 83, 5905. Iwaya SI, Ikawa M, Kubota M (2001) Revascularization of an immature permanent tooth with apical periodontitis and sinus tract. Dental Traumatology 17, 1857. Javelet J, Torabinejad M, Bakland LK (1985) Comparison of two pH levels for the induction of apical barriers in immature teeth of monkeys. Journal of Endodontics 11, 3758. Jiang J, Zuo J, Chen SH, Holliday LS (2003) Calcium hydroxide reduces lipopolysaccharide-stimulated osteoclast formation. Oral Surgery, Oral Medicine, Oral Pathology, Oral Radiology and Endodontics 95, 34854. Jung I-Y, Lee S-J, Hargreaves KM (2008) Biologically based treatment of immature permanent teeth with pulpal necrosis: a case series. Journal of Endodontics 34, 87687. Kanematsu A, Yamamoto S, Ozeki M et al. (2004) Collagenous matrices as release carriers of exogenous growth factors. Biomaterials 25, 451320. Katebzadeh N, Dalton BC, Trope M (1998) Strengthening immature teeth during and after apexication. Journal of Endodontics 24, 2569. Kling M, Cvek M, Mejare I (1986) Rate and predictability of pulp revascularization in therapeutically reimplanted permanent incisors. Endodontics and Dental Traumatology 2, 839. van der Kraan PM, van den Berg WB (2007) Osteophytes: relevance and biology. Osteoarthritis and Cartilage 15, 237 44.
864
Lieberman J, Trowbridge H (1983) Apical closure of nonvital permanent incisor teeth where no treatment was performed: case report. Journal of Endodontics 9, 25760. Maroto M, Barberia E, Planells P, Vera V (2003) Treatment of a non-vital immature incisor with mineral trioxide aggregate (MTA). Dental Traumatology 19, 1659. McCarthy E, Sundaram M (2005) Heterotopic ossication: a review. Skeletal Radiology 34, 60919. Messer HH (2008) To the editor. Journal of Endodontics 34, 1157. Mitchell DF, Shankwalker GB (1958) Osteogenic potential of calcium hydroxide and other materials in soft tissue and bone wounds. Journal of Dental Research 37, 115763. Miura M, Gronthos S, Zhao M et al. (2003) SHED: stem cells from human exfoliated deciduous teeth. Proceedings of the National Academy of Sciences of the United States of America 100, 580712. Mooney DJ, Powell C, Piana J, Rutherford B (1996) Engineering dental pulp-like tissue in vitro. Biotechnology Progress 12, 8658. Murray PE, Garcia-Godoy F, Hargreaves KM (2007a) Regenerative endodontics: a review of current status and a call for action. Journal of Endodontics 33, 37790. Murray PE, Garcia-Godoy F, Hargreaves KM (2007b) Reply. Journal of Endodontics 33, 1277. Myers WC, Fountain SB (1974) Dental pulp regeneration aided by blood and blood substitutes after experimentally induced periapical infection. Oral Surgery Oral Medicine, Oral Pathology 37, 44150. National Institutes of Health (2006) Regenerative Medicine. National Institutes of Health Fact Sheet. Available at: http:// www.nih.gov/about/researchresultsforthepublic/Regen.pdf. Nelson-Filho P, Leonardo MR, Silva LA, Assed S (2002) Radiographic evaluation of the effect of endotoxin (LPS) plus calcium hydroxide on apical and periapical tissues of dogs. Journal of Endodontics 28, 6946. Nevins AJ, Finkelstein F, Borden BG, Laporta R (1976) Revitalization of pulpless open apex teeth in rhesus monkeys, using collagen-calcium phosphate gel. Journal of Endodontics 2, 15965. Nevins A, Wrobel W, Valachovic R, Finkelstein F (1977) Hard tissue induction into pulpless open-apex teeth using collagen-calcium phosphate gel. Journal of Endodontics 3, 4313. Nevins A, Finkelstein F, Laporta R, Borden BG (1978) Induction of hard tissue into pulpless open-apex teeth using collagen-calcium phosphate gel. Journal of Endodontics 4, 7681. No r JE (2006) Tooth regeneration in operative dentistry. Operative Dentistry 31, 63342. Ostby BN (1961) The role of the blood clot in endodontic therapy. An experimental histologic study. Acta Odontologica Scandinavica 19, 32453. Owens BD, Wenke JC, Svoboda SJ, White DW (2006) Extremity trauma research in the United States Army.
Journal of the American Academy of Orthopaedic Surgeons 14, S3740. Pace R, Giuliani V, Pini Prato L, Baccetti T, Pagavino G (2007) Apical plug technique using mineral trioxide aggregate: results from a case series. International Endodontic Journal 40, 47884. Pashley DH, Walton RE, Slavkin HC (2002) Histology and physiology of the dental pulp, Chapter 2. In: Ingle JI, Bakland LK, eds. Endodontics, 5th edn. Hamilton, ON, Canada: BC Decker Inc, pp. 2538. de Paz LC (2007) Redening the persistent infection in root canals: possible role of biolm communities. Journal of Endodontics 33, 65262. Peters MC, Polverini PJ, Mooney DJ (2002) Engineering vascular networks in porous polymer matrices. Journal of Biomedical Materials Research 60, 66878. Pierdomenico L, Bonsi L, Calvitti M et al. (2005) Multipotent mesenchymal stem cells with immunosuppressive activity can be easily isolated from dental pulp. Transplantation 80, 83642. Prescott RS, Alsanea R, Fayad MI et al. (2008) In vivo generation of dental pulp-like tissue by using dental pulp stem cells, a collagen scaffold, and dentin matrix protein 1 after subcutaneous transplantation in mice. Journal of Endodontics 34, 4216. Rafter M (2005) Apexication: a review. Dental Traumatology 21, 18. rio Roberto L, da LA a Assed da Raquel Assed Bezerra S, MA Bezerra S, Larissa Moreira Spinola de C, Adalberto Luiz R, de Paulo Tambasco O (2008) Effects of the association between a calcium hydroxide paste and 0.4% chlorhexidine on the development of the osteogenic phenotype in vitro. Journal of Endodontics 34, 14859. Richardson TP, Peters MC, Ennett AB, Mooney DJ (2001) Polymeric system for dual growth factor delivery. Nature Biotechnology 19, 102934. Ritter AL, Ritter AV, Murrah V, Sigurdsson A, Trope M (2004) Pulp revascularization of replanted immature dog teeth after treatment with minocycline and doxycycline assessed by laser Doppler owmetry, radiography, and histology. Dental Traumatology 20, 7584. Robertson A, Lundgren T, Andreasen JO, Dietz W, Hoyer I, Noren JG (1997) Pulp calcications in traumatized primary incisors. A morphological and inductive analysis study. European Journal of Oral Sciences 105, 196206. Rutherford RB (2001) BMP-7 gene transfer to inamed ferret dental pulps. European Journal of Oral Sciences 109, 4224. Rutherford B (2007) To the editor. Journal of Endodontics 33, 1277. Rutherford RB, Gu K (2000) Treatment of inamed ferret dental pulps with recombinant bone morphogenetic protein7. European Journal of Oral Sciences 108, 2026. Safavi KE, Nichols FC (1993) Effect of calcium hydroxide on bacterial lipopolysaccharide. Journal of Endodontics 19, 768.
865
Safavi KE, Nichols FC (1994) Alteration of biological properties of bacterial lipopolysaccharide by calcium hydroxide treatment. Journal of Endodontics 20, 1279. Sato I, Ando-Kurihara N, Kota K, Iwaku M, Hoshino E (1996) Sterilization of infected root-canal dentine by topical application of a mixture of ciprooxacin, metronidazole and minocycline in situ. International Endodontic Journal 29, 118 24. Seltzer S, Bender IB, Ziontz M (1963) The dynamics of pulp inammation: correlations between diagnostic data and actual histologic ndings in the pulp. Oral Surgery, Oral Medicine, Oral Pathology 16, 96977. Shabahang S, Torabinejad M, Boyne PP, Abedi H, McMillan P (1999) A comparative study of root-end induction using osteogenic protein-1, calcium hydroxide, and mineral trioxide aggregate in dogs. Journal of Endodontics 25, 15. Shah N, Logani A, Bhaskar U, Aggarwal V (2008) Efcacy of revascularization to induce apexication/apexogensis in infected, nonvital, immature teeth: a pilot clinical study. Journal of Endodontics 34, 91925; discussion 1157. Sheridan MH, Shea LD, Peters MC, Mooney DJ (2000) Bioabsorbable polymer scaffolds for tissue engineering capable of sustained growth factor delivery. J Control Release 64, 91102. Shi S, Gronthos S (2003) Perivascular niche of postnatal mesenchymal stem cells in human bone marrow and dental pulp. Journal of Bone and Mineral Research 18, 696 704. Sonoyama W, Liu Y, Fang D et al. (2006) Mesenchymal stem cell-mediated functional tooth regeneration in swine. PLoS ONE 1, e79. Sonoyama W, Liu Y, Yamaza T et al. (2008) Characterization of the apical papilla and its residing stem cells from human
immature permanent teeth: a pilot study. Journal of Endodontics 34, 16671. Steinig TH, Regan JD, Gutmann JL (2003) The use and predictable placement of mineral trioxide aggregate in onevisit apexication cases. Australian Endodontic Journal 29, 3442. Stiver SI, Tan X, Brown LF, Hedley-Whyte ET, Dvorak HF (2004) VEGF-A angiogenesis induces a stable neovasculature in adult murine brain. Journal of Neuropathology and Experimental Neurology 63, 84155. Sun Q, Chen RR, Shen Y, Mooney DJ, Rajagopalan S, Grossman PM (2005) Sustained vascular endothelial growth factor delivery enhances angiogenesis and perfusion in ischemic hind limb. Pharmaceutical Research 22, 11106. Thibodeau B, Trope M (2007) Pulp revascularization of a necrotic infected immature permanent tooth: case report and review of the literature. Pediatric Dentistry 29, 4750. Thibodeau B, Teixeira F, Yamauchi M, Caplan DJ, Trope M (2007) Pulp revascularization of immature dog teeth with apical periodontitis. Journal of Endodontics 33, 6809. Trowbridge H (2002) Histology of pulpal inammation. Chapter 10. In: Hargreaves KM, Goodis HE, eds. Seltzer and Benders Dental Pulp. Carol Stream, IL, USA: Quintessence Publishing Co., Inc, pp. 22745. Tsukamoto Y, Fukutani S, Shin-Ike T et al. (1992) Mineralized nodule formation by cultures of human dental pulp-derived broblasts. Archives of Oral Biology 37, 104555. Uhthoff HK (1996) Calcifying tendinitis. Annales Chirurgiae et Gynaecologiae 85, 1115. Witherspoon DE, Ham K (2001) One-visit apexication: technique for inducing root-end barrier formation in apical closures. Practical Procedures and Aesthetic Dentistry 13, 45560; quiz 462.
866
doi:10.1111/j.1365-2591.2009.01581.x
Polymerization stress, ow and dentine bond strength of two resin-based root canal sealers
S. F. C. Souza1,2, A. C. Bombana3, C. Francci2, F. Gonc alves2, C. Castellan2 & R. R. Braga2

1 School of Dentistry, Federal University of Maranha o, Sa o Luiz, MA; Departments of 2Dental Materials and 3Restorative Dentistry University of Sa o Paulo, Sa o Paulo, SP, Brazil
Abstract
Souza SFC, Bombana AC, Francci C, Gonc alves F, Castellan C, Braga RR. Polymerization stress, ow and
dentine bond strength of two resin-based root canal sealers. International Endodontic Journal, 42, 867873, 2009.
Aim To compare two resin-based root canal sealers (AH Plus and dual cure Epiphany) in terms of ow, polymerization stress and bond strength to dentine. Methodology Flow was evaluated by measuring the diameter of uncured discs of sealer (0.5 mL) after 7 min compression (20N) between two glass plates (n = 5). Polymerization stress was monitored for 60 min in 1-mm thick discs bonded to two glass rods ( = 5 mm) attached to a universal testing machine (n = 3). Bond strength was analyzed through micropush-out test (n = 10) and failure mode was examined with scanning electron microscope (100 and 2500). Data
were statistically analyzed using the Students t-test (a = 0.05). Results Polymerization stress was 0.32 0.07 MPa for Epiphany self-cure, 0.65 0.08 MPa for Epiphany light-cure and zero for AH Plus (P < 0.05). Flow data and bond strength values were 30.9 1.1, 28.6 0.7 mm and 6.3 5.3, 17.8 7.5 MPa for Epiphany and AH Plus, respectively (P < 0.001). Failure mode was predominantly cohesive in the sealer for both materials. Conclusions Epiphany had higher ow and polymerization stress and lower bond strength values to dentine than AH Plus. In view of these ndings it can be implied that AH Plus would provide a better seal. Keywords: apical gap, ow, micropush-out, polymerization stress, root canal sealer.
Received 3 November 2008; accepted 5 March 2009
Introduction
Complete lling of the root canal system with biocompatible and dimensionally stable lling materials is an important factor in achieving endodontic success (Sjo gren et al. 1990). Gutta-percha in combination with sealers of different chemical compositions has been widely used in clinical practice. However, lling completely the root canals system remains a challenge despite the large number of techniques and materials available (Schwartz 2006). Adhesive bonding and resin cements developed for endodontic use have emerged as
tima Carvalho Souza, Correspondence: Dra Soraia de Fa Faculdade de Odontologia, Universidade Federal do Maranha o (UFMA), Av. dos Portugueses s/n, Bacanga, Sa o Luis, MA 65085-580, Brazil (Tel.: +55 98 21098575; e-mail: sosocarvalho@usp.br).
a possibility to improve root canal lling (Weis et al. 2004). In 2004, a new adhesive root lling material, Epiphany Root Filling System, was patented by Pentron Clinical Technologies (Wallingford, CT, USA). This system contains a polyester-based thermoplastic root canal core material (Resilon; Resilon Research LLC, Madison, CT, USA), a dual-cure methacrylate-based sealer and a self-etching primer. This material can promote hybridization with the dentine substrate and a chemical bond with Resilon, improving resistance to bacterial leakage (Shipper et al. 2004, 2005) and root fracture (Teixeira et al. 2004a) due to a potential resin monoblock formation (Teixeira et al. 2004b). Nevertheless, an ultrastructural evaluation revealed a weak link between Resilon and dentine (Tay et al. 2005a). Methacrylate-based sealers shrink during polymerization (Ferracane 2005), generating stress within the material and at the tooth-restoration interface that can
867
Physicomechanical properties of endodontic sealers Souza et al.
lead to gap formation (Carvalho et al. 1996, Braga et al. 2002, De Munck et al. 2005). The magnitude of stress is inuenced by several factors, such as composition and volume of the material and cavity conguration factor (factor-C) (Davidson & de Gee 1984, Davidson et al. 1984, Davidson & Feilzer 1997). In composite restorations, the use of low viscosity materials has been associated with a reduced incidence of marginal gaps at the tooth/restoration interface (Uno & Asmussen 1991, Peutzfeldt & Asmussen 2004) and better adaptation to cavity walls (Ferdianakis 1998, Fruits et al. 2002). On the other hand, viscosity is directly related to degree of conversion (Lovell et al. 1999, Sideridou et al. 2002) which, in turn, is a determinant factor on polymerization stress development (Braga & Ferracane 2002, Stansbury et al. 2005). The high C-factor situation represented by the lling of root canals may originate high polymerization stresses (Goracci et al. 2004), exceeding bond strength to root dentine and causing debonding of the interface for stress relief (Tay et al. 2005b). Furthermore, resin sealer photoactivation for immediate coronal sealing hinders the resin viscous ow and increases stress build-up (Ferracane 2005), resulting in inappropriate bond strength or gap formation between sealer and root dentine (Nagas et al. 2007). The aim of this study was to compare an epoxy- and a methacrylate-based root canal sealer in terms of several characteristics involved in apical gap formation. The null hypothesis was that AH Plus (Maillefer, Dentsply polis, RJ, Brazil) or Epiphany Ind. e Com. Ltda., Petro (Pentron Clinical Technologies, Wallingford, CT, USA) would show no difference terms of ow, polymerization stress and dentine bond strength.
Polymerization stress
Polymerization stress was determined using an established method (Condon & Ferracane 2000, Witzel et al. 2007, Gonc alves et al. 2008). One end of two glass rods (B 5 mm 13 or 28 mm height) was sand-blasted with alumina (150250 lm), silanated (RelyX Ceramic primer S; 3M ESPE, St Paul, MN, USA) and coated with a layer of unlled resin (Adper Scotchbond Multipurpose, bottle 3; 3M ESPE), which was exposed to the light source with 300 mW cm)2 for 40 s. The nontreated ends were attached to the opposite xtures of a universal testing machine (Model 5565; Instron, Canton, MA, USA), and the distance between the treated surfaces was adjusted to 1.0 mm. The 28-mm rod was connected to a crosshead/load cell, whilst the 13-mm rod was connected to a stainless steel xture containing a slot that allowed, when necessary the distal end of the light-curing guide to contact the rod opposite to the treated surface which was highly polished. Resin sealer (19.6 mm3) was inserted between the treated glass surfaces and formed into a cylinder and excess was removed. An extensometer (Model 2630101; Instron) was attached to the rods in order to monitor specimen height. The approximation of the glass rods due to composite shrinkage was registered by the extensometer and caused the crosshead to move in the opposite direction to restore the initial distance, with 0.01 lm accuracy. Therefore, the values registered by the load cell corresponded to the force necessary to maintain the initial height of the specimen in opposition to the contraction force exerted by the resin sealer (Fig. 1). Three specimens were tested in each experimental condition at 37 C, and force development was monitored for 60 min, starting 3 min after mixing. Experimental conditions were AH Plus, Epiphany self-cure (SC) and Epiphany light-cure (LC). Epiphany-LC was photoactivated (VIP Ju nior; BISCO, Schaumburg, IL, USA) 17 min after mixing with 475 mW cm)2 for 51 s (24 J cm)2), following manufacturers instructions. Maximum nominal stress (r, in MPa) was calculated by dividing the maximum contraction force [F (N)] by the cross-sectional area of the rods (A) as follows: r F N Amm2
Materials and methods Flow

According to ADA 57 Specication (American National Standard/American Dental Association, 2000), 0.5 mL of sealers was mixed and placed using a graduated syringe, on a glass plate (40 40 5 mm). After 180 5 s another glass plate was placed on top of the sealer, followed by load application of 20 N. Then, 10 min after mixing, the load was removed and maximum and minimum diameters of compressed discs were measured with a digital caliper with a 0.01 mm resolution (Mitutoyo MTI Corporation, Tokyo, Japan). Results were recorded only if both diameters were uniform and were within 1.0 mm. Flow was calculated by averaging ve specimens.
Micropush-out bond strengths

Twenty mandibular single-rooted human premolar teeth with straight root canals, anatomically similar
868
Souza et al. Physicomechanical properties of endodontic sealers
1 2 5 3 4
Figure 1 Schematic representation of the experimental set-up used for polymerization stress determination: (1) xture conectect to the load cell; (2) long glass rod; (3) short glass rod; (4) stainless steel xture with a slot to allow for the positioning of the light guide in contact with the glass rod; (5) extensometer.
dimensions, fully developed apices and patency foramen were collected after patients informed consent had been obtained under a protocol reviewed and approved by the Ethical Research Committee of Sa o Paulo University (protocol number, 177/05). Teeth were cleaned and the working length of each root was established with a size 15 K le (Dentsply Maillefer Ballaigues, Switzerland) 1.0 mm short of the apical foramen. Canals were prepared with a crown-down technique up to size 50 and irrigated with 0.5% NaOCl after every change of instrument. Five millilitres of 17% EDTA was used as nal rinse to remove canal wall smear layer. EDTA solution was neutralized with 0.5% NaOCl and then the canal was rinsed with saline solution (15 mL) and dried with paper points. Prepared root canals were randomly (http://www. random.org) divided into two experimental groups (n = 10): AH Plus (Dentsply Ind. e Com. Ltda.) and Epiphany-SC (Pentron Clinical Technologies). Three disc slices of one-millimetre thick (0.1 mm) were obtained after transverse sectioning (Isomet 1000 Precision Saw; Buehler Ltd., Lake Bluff, IL, USA) the apical 5.0 mm of each root under water cooling. The thickness of each root slice was measured by means of
a digital caliper (Mitutoyo MTI Corporation, Tokyo, Japan). The diameters of each apical and cervical slice were photographed by a digital camera (Q-Color 5; Olympus America Inc., Center Valley, PA, USA) attached to a stereomicroscope (SZ61; Olympus America Inc., Miami, FL, USA) and was measured using Image J software (http://rsb.info.nih.gov/ij/; National Institute of Health) under 25 magnication. Specimens with noncircular shape were discarded to avoid nonuniform stress distributions during testing, resulting in approximately 25 slices per group. Endodontic sealers were mixed according to manufacturers instructions and used to ll the entire root canal space. Prior to lling with Epiphany sealer, root canal dentine was etched for 30 s with Epiphany primer. Specimens were stored for 72 h at 37 C and 100% relative humidity. For the micropush-out test, a compressive load was applied to the specimen via a cylindrical stainless steel punch attached to a universal testing machine (Kratos metros, Embu, SP, Brazil). For each specimen, a Dinamo punch tip 0.2 mm smaller than its apical diameter was selected and positioned such that it touched only the sealer and did not stress the surrounding root canal walls. The apical aspect of the each specimen was positioned facing the punch tip. Loading was performed at a crosshead speed of 0.5 mm min)1 until the sealer was dislodged from the root slice. Tensile bond strength of each slice was calculated as the force (N) of failure divided by the bonded cross-sectional surface area and expressed in MPa (Patierno et al. 1996).
Failure mode analysis

For scanning electron microscope (SEM) observation (100 and 2500, LEO Stereoscan 440, Electron Microscopy Ltd., Cambridge, UK) micropush-out specimens were cut longitudinally and root segments were covered with platinum (Coating System MED 020; BAL-TEC AG, Balzers, Liechtenstein). To estimate the percentage of free substrate the interface area was divided into eight segments. This approach, suggested by Fowler et al. (1992), was used to classify failure mode as: (75%); cohesive within sealer (25%) adhesive-cohesive (>25% to <75%).
Statistical analysis
Data from bond strength to dentine, ow and polymerization stress were analyzed using the Students t-test. For the bond strength test each tooth derived one single value. The level of signicance was xed at 5%.
869
Results
Table 1 summarizes average and SD of the micropushout test and ow of both sealers. Epiphany presented signicantly high ow than AH Plus (P < 0.001). A signicant difference was detected between polymerization stress for Epiphany-SC (0.32 0.07 MPa) and Epiphany-LC (0.65 0.08 MPa) as shown in Fig. 2 (P < 0.05). Epiphany-SC started to generate stress 20 min after mixing. Epiphany-LC was photoactivated after 17 min from the beginning of the test, when an abrupt increase on polymerization stress curve occurred. AH Plus revealed zero polymerization stress values during 60 min, and for this reason was excluded from statistical analysis. For the micropush-out test Epiphany-SC had lower values when compared with AH Plus (P < 0.001). Failure mode distribution is shown in Fig. 3: 79.2% cohesive within sealer and 20.8% adhesive for AH Plus, 78.3% cohesive within sealer and 21.7% adhesivecohesive for Epiphany-SC.
Figure 3 Failure mode distribution for experimental groups
(%).
Discussion
Apical gap formation is inuenced by local factors such as substrate morphology (Wu et al. 1998, Ferrari et al.
Table 1 Mean values and standard deviations of bond
strength to dentine and ow for AH Plus and Epiphany sealers

Groups AH Plus Epiphany Micropush-out (MPa) 17.8 (7.5)a 6.3 (5.3)b Flow (mm) 28.6 (0.7)b 30.9 (1.1)a
Different letters on the same column show statistically significant differences (P < 0.001).
Figure 2 Polymerization stress (MPa) as a function of time (s)
of Epiphany self-cure (SC) and light-cure (LC).
2000, Mjo r et al. 2001), C-factor (Goracci et al. 2004, Tay et al. 2005b), and also material-related factors such as physical properties of sealers (i.e. ow, polymerization contraction) (Bergmans et al. 2005, Braga et al. 2005) and bond strength to dentine (Tagger et al. 2002, Bouillaguet et al. 2003). This study assessed the possible relationship between ow, polymerization stress and bond strength of AH Plus and Epiphany sealers with apical gap formation. The fact that no stress development was observed for AH Plus up to 60 min after mixing agrees with the manufacturer information that states a setting time of 8 h at 37 C. However, running the polymerization stress test for such long periods is impractical. Notwithstanding, this information is interesting for comparative purposes with the other sealer evaluated. For Epiphany, polymerization stress tests were performed for both curing modes: self-cured, relying only on the peroxideamine reaction and dual-cured. Epiphany was tested in SC mode because clinically the light from photoactivation does not reach the middle or apical root regions (Hiraishi et al. 2005). The increased polymerization time in SC mode allows materials to ow in a pre-gel state, which could provide stress relief at the dentine/ resin interface (Braga et al. 2002, Braga & Ferracane 2004), and be advantageous for this material. However, polymerization stress when light-curing was used (Epiphany-LC) doubled when compared with Epiphany-SC (Fig. 2; P < 0.05). This nding is related to an increase in polymerization rate caused by light activation. Nagas et al. (2007) suggested that a decreased polymerization time can adversely affect Epiphany bond strength to dentine. In fact, one could speculate that an
870
(a)
(b)
(c)
(d)
Figure 4 Representative scanning electron microscope (SEM) micrographs of failure mode for AH Plus (a and b) and Epiphany (c and d): (a) sealer cohesive failure showing dentine surface recovered by a thick organic matrix layer with different sizes llers; (b) adhesive failure showing clean dentine surface only with small llers and dentinal tubules with organic matrix tags; (c) sealer cohesive failure indicating dentine surface recovered by an organic matrix layer with granular small llers, and major llers with a thin plaque format, and also some empty spaces; (d) cohesive and adhesive failure demonstrating dentine surface covered by Epiphany primer and some sealers fragments with llers closing total or partially dentinal tubules (pointer).
increased polymerization rate conferred by light activation can restrict the chances for polymerization stress release during the pre-gel state (Tay et al. 2005b). In theory, total bond strength is the sum of the strengths of resin tags, hybrid layer and surface adhesion (Pashley et al. 1995). The low viscosity and hydrophilic nature of resin-based sealers in association with pressure caused by condensation technique allowed the sealer to inltrate into dentinal tubules, forming long tags and secondary branchings (Bergmans et al. 2005, Tay et al. 2005a) In this study, both resin sealers differed in ow (P < 0,001; Table 1), and both of them exceeded specication 57 of American National Standard/American Dental Association (2000). Despite that, Tay et al. (2005a) showed in SEM and Transmission Electron Microscope (TEM) the loss of integrity at dentine/Epiphany sealer and gutta-percha/AH Plus sealer interfaces. These gaps, presumably created by polymerization contraction forces (Tay et al. 2005b), suggest that hybrid layer and long tags do not guarantee the absence of gaps (Bergmans et al. 2005). Bond strength between endodontic cements and dentine may be an important property to provide a seal (Tagger et al. 2002). Micropush-out values for Epiphany were lower than for AH Plus (P < 0.001; Table 1). Epiphany polymerization stress may have
contributed to its lower bond strength value. The amount of stress associated with shrinkage may result in separation of resin-based sealer and dentinal walls, and consequently, bond strength values of this interface would decrease (Hiraishi et al. 2005). In this study, bond strength results for Epiphany sealer are comparable with other experiments that showed values between 0.32 and 3.73 MPa (Gesi et al. 2005, Ungor et al. 2006, Fisher et al. 2007, Sly et al. 2007, Kaya et al. 2008, Lawson et al. 2008, Lee et al. 2008) though towards the high end range. Although lling the root canal only with the sealer does not accurately represent the clinical situation, this experimental model was chosen because it represents a worst case scenario, as polymerization stress development is directly related to the volume of shrinking material (Tay et al. 2005b). Moreover, by not using gutta-percha and resilon cones, it can be assured that the tested interface is comprised of sealer and dentine only. Epiphany-LC was not included in the micropush-out test because the study was designed to simulate the clinical conditions found at the apical third of the root canal, where the effect of light-curing is likely to be zero. It is reasonable to speculate that, when used in SC mode, the sealer does not totally polymerize. The incomplete polymerization can impair cement mechanical proper-
871
ties and chemical stability (Braga et al. 2002). In fact, failure mode analysis revealed a high incidence of sealer cohesive failure for Epiphany (Figs 3 and 4). The integrity loss on dentine/Epiphany interface can be explained by comparing its bond strength to dentine with stress generated during the polymerization contraction. Apparently, shrinkage stress was high enough to surpass bond strength (Bouillaguet et al. 2003, Tay et al. 2005a). The apparently negligible polymerization stress values determined in the mechanical test (Fig. 2) might be of a much higher magnitude in the root canal, where geometric shape and material connement are obstacles for stress release. According to Tay et al. (2005b), C-factor of adhesive bonding root lling materials in root canals is highly unfavourable, challenging the concept of total bonding in root canals.
Conclusion
The null hypothesis was rejected for the three variables analyzed. Epiphany had higher ow, lower bond strength to dentine and also developed higher polymerization stress than AH Plus. Within the limitations of this laboratory study and in view of the results it can be speculated that, clinically, a better interfacial sealing could be expected with AH Plus. The higher bond strength to dentine obtained with AH Plus can be partially explained by its lower polymerization stress. Moreover, its higher viscosity compared with Epiphany did not seem to impair its bond strength.
Acknowledgements
This study was partially supported by CAPES (Coordenac a o de Aperfeic oamento de Pessoal de N vel Superior) Institutional Qualication Program (PQI no.: via Rodri0090/034). The authors are grateful to Fla gues for providing the polymerization stress test diagram.
References
American National Standard/American Dental Association, Council on Scientic Affairs (2000) Specication No 57 for Endodontic Sealing Material. Standards Committee on Dental Products. Chicago, IL: ANSI/ADA. Bergmans L, Moisiadis P, De Munck J, Van Meerbeek B, Lambrechts P (2005) Effect of polymerization shrinkage on the sealing capacity of resin llers for endodontic use. The Journal of Adhesive Dentistry 7, 3219. Bouillaguet S, Troesch S, Wataha JC, Krejci I, Meyer JM, Pashley DH (2003) Microtensile bond strength between
adhesive cements and root canal dentin. Dental Materials 19, 199205. Braga RR, Ferracane JL (2002) Contraction stress related to degree of conversion and reaction kinetics. Journal of Dental Research 81, 1148. Braga RR, Ferracane JL (2004) Alternatives in polymerization contraction stress management. Critical Reviews in Oral Biology and Medicine 15, 17684. Braga RR, Cesar PF, Gonzaga CC (2002) Mechanical properties of resin cements with different activation modes. Journal of Oral Rehabilitation 29, 25762. Braga RR, Ballester RY, Ferracane JL (2005) Factors involved in the development of polymerization shrinkage stress in resin-composites: a systematic review. Dental Materials 21, 96270. Carvalho RM, Pereira JC, Yoshiyama M, Pashley DH (1996) A review of polymerization contraction: the inuence of stress developmentversusstressrelief. OperativeDentistry 21,1724. Condon JR, Ferracane JL (2000) Assessing the effect of composite formulation on polymerization stress. Journal of American Dental Association 131, 497503. Davidson CL, de Gee AJ (1984) Relaxation of polymerization contraction stresses by ow in dental composites. Journal of Dental Research 63, 1468. Davidson CL, Feilzer AJ (1997) Polymerization shrinkage and polymerization shrinkage stress in polymer-based restoratives. Journal of Dentistry 25, 43540. Davidson CL, de Gee AJ, Feilzer A (1984) The competition between the composite-dentin bond strength and the polymerization contraction stress. Journal of Dental Research 63, 13969. De Munck J, Van Landuyt K, Peumans M et al. (2005) A critical review of the durability of adhesion to tooth tissue: methods and results. Journal of Dental Research 84, 11832. Ferdianakis K (1998) Microleakage reduction from newer esthetic restorative materials in permanent molars. The Journal of Clinical Pediatric Dentistry 22, 2219. Ferracane JL (2005) Developing a more complete understanding of stresses produced in dental composites during polymerization. Dental Materials 21, 3642. Ferrari M, Mannocci F, Vichi A, Cagidiaco MC, Mjor IA (2000) Bonding to root canal: structural characteristics of the substrate. American Journal of Dentistry 13, 25560. Fisher MA, Berzins DW, Bahcall JK (2007) An in vitro comparison of bond strength of various obturation materials to root canal dentin using a push-out test design. Journal of Endodontics 33, 8568. Fowler CS, Swartz ML, Moore BK, Rhodes BF (1992) Inuence of selected variables on adhesion testing. Dental Materials 8, 2659. Fruits TJ, VanBrunt CL, Khajotia SS, Duncanson MG Jr (2002) Effect of cyclical lateral forces on microleakage in cervical resin composite restorations. Quintessence International 33, 20512.
872
Gesi A, Raffaelli O, Goracci C, Pashley DH, Tay FR, Ferrari M (2005) Interfacial strength of Resilon and gutta-percha to intraradicular dentin. Journal of Endodontics 31, 80913. Gonc alves F, Pfeifer CS, Meira JB, Ballester RY, Lima RG, Braga RR (2008) Polymerization stress of resin composites as a function of system compliance. Dental Materials 24, 64552. Goracci C, Tavares AU, Fabianelli A et al. (2004) The adhesion between ber posts and root canal walls: comparison between microtensile and push-out bond strength measurements. European Journal of Oral Sciences 112, 35361. Hiraishi N, Papacchini F, Loushine RJ et al. (2005) Shear bond strength of Resilon to a methacrylate-based root canal sealer. International Endodontic Journal 38, 75363. Kaya BU, Kec eci AD, Orhan H, Belli S (2008) Micropush-out bond strengths of gutta-percha versus thermoplastic synthetic polymer-based systems: an ex-vivo study. International Endodontic Journal 41, 2118. Lawson MS, Loushine B, Mai S et al. (2008) Resistance of a 4META-containing, methacrylate-based sealer to dislocation in root canals. Journal of Endodontics 34, 8337. Lee BS, Lai EH, Liao KH, Lee CY, Hsieh KH, Lin CP (2008) A novel polyurethane-based root canal-obturation material and urethane-acrylate-based root canal sealer-part 2: evaluation of push-out bond strengths. Journal of Endodontics 34, 5948. Lovell LG, Stansbury JW, Syrpes DC, Bowman CN (1999) Effects of composition and reactivity on the reaction kinetics on dimethacrylate copolymerizations. Macromolecules 32, 391321. Mjo r IA, Smith MR, Ferrari M, Mannocci F (2001) The structure of dentine in the apical region of human teeth. International Endodontic Journal 34, 34653. Nagas E, Cehreli ZC, Durmaz V, Vallittu PK, Lassila LV (2007) Regional push-out bond strength and coronal microleakage of Resilon after different light-curing methods. Journal of Endodontics 33, 14648. Pashley DH, Ciucchi B, Sano H, Carvalho RM, Russell CM (1995) Bond strength versus dentine structure: a modelling approach. Archives of Oral Biology 40, 110918. Patierno JM, Rueggeberg FA, Anderson RW, Weller RN, Pashley DH (1996) Push-out strength and SEM evaluation of resin composite bonded to internal cervical dentin. Endodontic Dental Traumatolology 12, 22736. Peutzfeldt A, Asmussen E (2004) Determinants of in vitro gap formation of resin composites. Journal of Dentistry 32, 109 15. Schwartz RS (2006) Adhesive dentistry and endodontics. Part 2: bonding in the root canal system-the promise and the problems: a review. Journal of Endodontics 32, 112534. Shipper G, Orstavik D, Teixeira FB, Trope M (2004) An evaluation of microbial leakage in roots lled with a thermoplastic synthetic polymer-based root canal lling material (Resilon). Journal of Endodontics 30, 3427. Shipper G, Teixeira FB, Arnold RR, Trope M (2005) Periapical inammation after coronal microbial inoculation of dog
roots lled with gutta-percha or resilon. Journal of Endodontics 31, 916. Sideridou I, Tserki V, Papanastasiou G (2002) Effect of chemical structure on degree of conversion in light-cured dimethacrylate-based dental resins. Biomaterials 23, 1819 29. Sjo gren U, Hagglund B, Sundqvist G, Wing K (1990) Factors affecting the long-term results of endodontic treatment. Journal of Endodontics 16, 498504. Sly MM, Moore BK, Platt JA, Brown CE (2007) Push-out bond strength of a new endodontic obturation system (Resilon/ Epiphany). Journal of Endodontics 33, 1602. Stansbury JW, Trujillo-Lemon M, Lu H, Ding X, Lin Y, Ge J (2005) Conversion-dependent shrinkage stress and strain in dental resins and composites. Dental Materials 21, 5667. Tagger M, Tagger E, Tjan AH, Bakland LK (2002) Measurement of adhesion of endodontic sealers to dentin. Journal of Endodontics 28, 3514. Tay FR, Loushine RJ, Weller RN et al. (2005a) Ultrastructural evaluation of the apical seal in roots lled with a polycaprolactone-based root canal lling material. Journal of Endodontics 31, 5149. Tay FR, Loushine RJ, Lambrechts P, Weller RN, Pashley DH (2005b) Geometric factors affecting dentin bonding in root canals: a theoretical modeling approach. Journal of Endodontics 31, 5849. Teixeira FB, Teixeira EC, Thompson JY, Trope M (2004a) Fracture resistance of roots endodontically treated with a new resin lling material. Journal of American Dental Association 135, 64652. Teixeira FB, Teixeira EC, Thompson J, Leinfelder KF, Trope M (2004b) Dentinal bonding reaches the root canal system. Journal of Esthetic Restorative Dentistry 16, 34854. Ungor M, Onay EO, Orucoglu H (2006) Push-out bond strengths: the Epiphany-Resilon endodontic obturation system compared with different pairings of Epiphany, Resilon, AH Plus and gutta-percha. International Endodontic Journal 39, 6437. Uno S, Asmussen E (1991) Marginal adaptation of a restorative resin polymerized at reduced rate. Scandinavian Journal Dental Research 99, 4404. Weis MV, Parashos P, Messer HH (2004) Effect of obturation technique on sealer cement thickness and dentinal tubule penetration. International Endodontic Journal 37, 65363. Witzel MF, Ballester RY, Meira JB, Lima RG, Braga RR (2007) Composite shrinkage stress as a function of specimen dimensions and compliance of the testing system. Dental Materials 23, 20410. Wu MK, de Gee AJ, Wesselink PR (1998) Effect of tubule orientation in the cavity wall on the seal of dental lling materials: an in vitro study. International Endodontic Journal 31, 32632.
873
doi:10.1111/j.1365-2591.2009.01582.x
Evaluation of the cost-effectiveness of root canal treatment using conventional approaches versus replacement with an implant
M. W. Pennington1, C. R. Vernazza2, P. Shackley1,3, N. T. Armstrong1, J. M. Whitworth2 & J. G. Steele2

1 Institute of Health and Society; 2School of Dental Sciences, Newcastle University; and 3Shefeld Vascular Institute, University of Shefeld, Shefeld, UK
Abstract
Pennington MW, Vernazza CR, Shackley P, Armstrong NT, Whitworth JM, Steele JG. Evaluation of
the cost-effectiveness of root canal treatment using conventional approaches versus replacement with an implant. International Endodontic Journal, 42, 874883, 2009.
Aim To evaluate the cost-effectiveness of root canal treatment for a maxillary incisor tooth with a pulp infection, in comparison with extraction and replacement with a bridge, denture or implant supported restoration. Methodology A Markov model was built to simulate the lifetime path of restorations placed on the maxillary incisor following the initial treatment decision. It was assumed that the goal of treatment was the preservation of a xed platform support for a crown without involving the adjacent teeth. Consequently, the model estimates the lifetime costs and the total longevity of tooth and implant supported crowns at the maxillary incisor site. The model considers the initial treatment decisions, and the various subsequent
treatment decisions that might be taken if initial restorations fail. Results Root canal treatment extended the life of the tooth at an additional cost of 58 per year of tooth life. Provision of orthograde re-treatment, if the root canal treatment fails returns further extension of the expected life of the tooth at a cost of 1215 per year. Surgical re-treatment is not cost-effective; it is cheaper, per year, to extend the life of the crown by replacement with a single implant restoration if orthograde endodontic treatment fails. Conclusion Modelling the available clinical and cost data indicates that, root canal treatment is highly costeffective as a rst line intervention. Orthograde re-treatment is also cost-effective, if a root treatment subsequently fails, but surgical re-treatment is not. Implants may have a role as a third line intervention if re-treatment fails. Keywords: cost-effectiveness, decision implant, Markov, root canal treatment. analysis,
Received 16 September 2008; accepted 17 March 2009
Introduction
Clinical decisions could be consistent and straightforward, if they were informed by unequivocal evidence, supported by clear and accepted guidelines, and if the recommended actions were universally acceptable to
Correspondence: Mark W. Pennington, MSc, PhD, Research Associate, Institute of Health and Society, Newcastle University, 21 Claremont Place, Newcastle upon Tyne NE2 4AA, UK (Tel.: +44 191 222 3544; fax: +44 191 222 6043; e-mail: mark.pennington@ncl.ac.uk).
patients and care providers. But few areas of practice are so clear-cut. Patients are not always equipped with the information they need to make rational decisions on their short and long-term care, and healthcare agencies might equally be ill-equipped to advise on best actions for the short and long term. As a consequence, patients may submit to the paternalistic decisionmaking of a healthcare professional (Kaba & Sooriakumaran 2007) whose priorities may be expected to be objective, consistent and based on the same values as their own. But observations from medicine and dentistry suggest that the decisions of healthcare
874
Pennington et al. C-E of root canal treatment
professionals themselves may be highly variable, even in the case of relatively simple interventions (Dome jean-Orliaguet et al. 2004, Lanning et al. 2005, van der Sanden et al. 2005, Calnan et al. 2007, Tickle et al. 2007), and inuenced by a number of personal, educational and economic considerations (McColl et al. 1999, Brennan & Spencer 2006). The picture is complicated further in the case of complex interventions, and interventions that may not be the nal solution within the lifetime of the patient. Here, the decision-making process may be limited to a consideration of the next step, and informed by short-term success rates, assessment of immediate costs, or of the willingness of the patient to pay for that individual step. Rarely is the decisionmaking process informed by a detailed understanding of the relative lifespan of alternative interventions or the ongoing costs, both nancial and otherwise (White et al. 2006, Balevi 2008), which may ow from a particular treatment decision. Restorative dental treatments are an example of such an intervention, and if patients faced with treatment decisions, or healthcare providers stewarding nite resources are to make properly informed decisions, they must be presented with information on cost and outcome which they understand and which accounts for the long-term. The uncertainties inherent in modelling the costs of combinations of interventions over a lifetime require a fundamentally different approach to the use of evidence to that, with which most clinicians are comfortable. Decision analytic modelling provides a rational framework for decision making based on expected costs and outcomes (Raiffa 1968). Many decision analytic models are based on Markov modelling, a mathematical means of investigating stochastic or random events over time (Sonnenberg & Beck 1993). Such modelling lends itself well to the study of long-term medical conditions, dening a clear starting point or condition, and identifying a number of states into which the individual may or may not move at dened points in the future. The probability of remaining in the starting condition or moving to an alternative state is informed by best outcome and survival data, and the costs of initial and future interventions estimated from professional sources. Markov models are increasingly used in evaluating the long-term cost effectiveness of clinical interventions from the chemoprevention of prostatic cancer to the management of heart failure (Chan et al. 2008, Svatek et al. 2008, Takao et al. 2008).
By contrast, the economic models applied to dentistry have generally been quite simple decision trees (Mileman & van den Hout 2003) or Markov models (Edwards et al. 1999) extrapolating over a xed number of years or the assumed lifetime of a specied intervention (for example, a dental restoration), rather than over the lifetime of the patient. Whilst previous publications have investigated the costs of dental treatments over a xed time span (Bra gger et al. 2005), as far as the authors are aware, this report represents the rst attempt to provide a denitive examination of the cost effectiveness of common dental interventions and look at all realistic options that ow from this over the lifetime of a patient. The starting point of the Markov model is a common clinical scenario; a damaged and irreversibly pulpitic maxillary central incisor tooth, where initial treatment options include root canal treatment and restoration, or extraction and prosthetic replacement. The model explores the long-term consequences and cost effectiveness of initial and subsequent decisions for individuals at different ages. The question at the heart of this investigation is whether root canal treatment and restoration of a damaged maxillary central incisor is a legitimate and cost-effective intervention over the lifetime of an adult patient, and in comparison with the alternatives of extraction followed by either a conventional or an implantsupported restoration.
Methods Building the model

For this study, a Markov model was built with TreeAge decision analysis software (TreeAge Software Inc., Williamstown, MA, USA, http://www.treeage.com/ index.htm). The starting point was a damaged, irreversibly pulpitic maxillary central incisor in an otherwise healthy adult male of varying age. The loss of coronal tooth tissue was dened as sufcient to require restoration with a post-retained crown. Assuming that the patient requests some treatment to ll the space, and from this starting position, the patient could occupy any of the six health states listed below at any given point in time, until the end of their life: Tooth extracted with resin bonded bridge (RBB) in situ Tooth extracted with a conventional bridge (xed dental prosthesis, FDP) in situ
875
C-E of root canal treatment Pennington et al.
Tooth extracted with removable partial denture (RPD) in situ Tooth root canal treated (RoCT) with a postretained crown in situ (there may be repair or replacement of any of the parts of the restoration or root lling within this state) Tooth extracted with an implant-supported single crown (ISC) in situ (again this could be a rst, second or subsequent restoration) An implant in situ prior to abutment connection (the transitional state during osseointegration assuming there is no immediate loading) Death of the patient. The model calculated the probability of the incidence of all signicant mechanical and biological complications that might arise in each of these states, over each 6 month period of the patients life, based on existing evidence (see Outcome data later). A repair event or no event occurring meant that the simulated patient remained in the same restoration state, whereas complete failure resulted in transition to a different state (e.g. the event of root fracture would require extraction and replacement of the tooth with a prosthesis of some description). The analysis was simplied by modelling the selection of a bridge or denture prosthesis as a random parameter based on likely distributions in the UK population rather than a treatment choice. The simulation terminated when the patient reached 100 years of age or died (using age-related mortality probabilities govt. actuaries dept., life tables 20022004, http:// www.gad.gov.uk/). The number of possible pathways through these various states in a lifetime is clearly massive. The initial treatment decision and then the
potential subsequent treatments necessitated by failure of a restoration are captured in the ten major strategies outlined in Fig. 1. Whilst these cannot capture every single possibility, they were considered the most likely 10 pathways by consensus of two senior clinical academics in Restorative Dentistry (JGS and JMW). Strategy 1 illustrates a decision to extract the irreversibly pulpitic tooth and to replace it with a conventional removable or xed prosthesis, not an implant. The remaining nine strategies involved either retaining the tooth by root canal treatment, removing it and placing an implant or a combination of these. In comparing each of the 10 major strategies, the costs and expected outcomes of both the initial treatment strategy (rst intervention) and supplementary interventions (second to fourth intervention) are predicted. Estimations of cost and treatment longevity are central to the model. To examine fully the costeffectiveness of three initial options (bridge/denture, implant, orthograde endodontics) the costs which might follow them are required. Clearly a RoCT is less expensive than an implant at the point of delivery but will the implant save money in the long term? To do this, it was necessary to model at least the second and third interventions and their costs and outcomes. It is not known what the patient might or should choose when the restoration fails, so all of the reasonable subsequent choices if that happened were considered and evaluated as different strategies. The strategy of placing an implant initially was also evaluated. One of these will be the most cost-effective. It was necessary to look at all of the likely second and third interventions if implants were to be given a fair comparison against RoCT.
Strategy 1 Extraction 2 One RoCT 3 RoCT then re-treatment 4 RoCT then surgery 5 RoCT then Implant 6 RoCT/Implant/2nd Implant
1st Intervention Bridge/denture
2nd Intervention
3rd Intervention 4th Intervention
Orthograde RoCT Bridge/denture Orthograde RoCT Orthograde RoCT Bridge/denture Orthograde RoCT Surgical RoCT Orthograde RoCT First implant Orthograde RoCT First implant Bridge/denture Bridge/denture 2nd implant Bridge/denture Bridge/denture Bridge/denture
7 RoCT/re-treatment/Implant Orthograde RoCT Orthograde RoCT First implant 8 RoCT/Surgery/Implant 9 Implant 10 Implant then 2nd implant Orthograde RoCT Surgical RoCT First implant First implant Bridge/denture Second implant Bridge/denture First implant
Figure 1 Sequence of interventions in
the ten treatment strategies.
876
Cost-effectiveness analysis: data sources

Outcome data In order to function, the model was parameterized with information on expected treatment longevity/ failure rates, and likely maintenance needs of different treatment options. Extensive Searching of MEDLINE, EMBASE, DARE and Cochrane Library databases (from inception to June 2006) was undertaken for all papers with terms including failure, fracture, success, treatment, re-treatment, replacement, complications, survival, (meta)analysis and terms describing the tooth state such as root canal, endodont#, #apical. This was supplemented by systematically checking the references of all papers retrieved for further relevant studies. Meta-analyses were utilized, where available, otherwise parameters were chosen based on the size, quality, age and selection criteria of the study. In the very rare instances where no appropriate data were available, the expert opinions of two senior clinical academics in Restorative Dentistry (JGS and JMW) were sought to dene the likely limits of parameters. Three meta-analyses were retreived on the survival of ISCs. The meta-analysis of Branemark implants (Lindh et al. 1998) was selected to parameterize implant survival as it differentiates between implant loss after loading and failure to osseo-integrate. A metaanalysis of prospective studies (Berglundh et al. 2002) provided data to parameterize complications in the implant states. However, the exclusion criteria limited the paper to a small number of studies. Hence, the analysis was judged less satisfactory than those reported by Lindh et al. (1998). The FDP state was parameterized using the most recent and largest metaanalysis (Tan et al. 2004). There are fewer reports on the survival and complication rates for RBBs and no meta-analyses were retrieved. The available data on RPDs is minimal. These states were parameterized from published individual trial or longitudinal studies where available. The heterogeneity of success criteria in reports on RoCT has deed meta-analysis to date (Creugers et al. 1993). Creugers analysis selected only three papers of which one (Mentink et al. 1993) was by far the largest, hence this report was prioritized when parameterizing the post-supported crown states. Rates of failure of root canal after re-treatment were taken from a 10-year Swedish study (Sjo gren et al. 1990) whilst rates of treatment failure following surgical endodontics were based on an evaluation of apical surgery (Buhler 1988).
Costs For the purposes of this model, typical staff time and resource use for each procedure was estimated based on a UK National Health Service (NHS) secondary care setting. Staff costs were taken from published reference costs (Curtis 2006), and costs are in UK 2006 pounds. The base case analysis for this study assumes that all implant procedures were carried out by a senior specialist (consultant) dentist. All of the conventional dental procedures were costed at more junior specialist staff (Specialist Registrar or Senior House Ofcer) rates reecting the more routine nature of such interventions. Staff costs were based on mid-band salaries and included overheads, training costs and administrative support. Costs and outcomes are discounted at 3.5% according to NICE guidelines for economic analyses. Mortality is parameterized using data for UK males (20022004 Government actuaries department). It is important to note that the costs used are based on standard data and represent the costs to the NHS, not the price that may be paid, for example in private practice where there are a range of additional considerations, such as prot margins and variations in overhead costs.
Cost-effectiveness analysis: assumptions

In order to develop an economic model such as this, a number of assumptions need to be made. Where possible these are supported by published evidence. The following assumptions were made for this model: That the patient retains most of the dentition over his/her lifetime (Kelly et al. 2000) That the longevity of the restoration is proportional to the lifetime benet of the restoration to the patient ISCs and crowned and root treated teeth provide the same Oral Health Quality of Life (OHQoL) Apical surgery is undertaken alongside orthograde re-treatment to enhance success rates, and not as a response to a distinct clinical indication such as a cyst RBBs, RPDs and conventional FDPs provide the same OHQoL, inferior to that of the ISC or crowned tooth. This assumption infers that the retention of a tooth unit in the maxillary anterior region in the form of the original tooth or an implant is preferable to loss of a xed platform (natural or articial) for restoration. Whilst it is acknowledged that this is not universally the case, this was considered a reasonable working rule, which was necessary to allow the model to compare endodontic strategies with implant strategies
877
A constant hazard rate is assumed for mechanical and biological complications following an intervention The same hazard rate applies to an event, such as tooth fracture, in the post-supported crown states regardless of whether a surgical or nonsurgical endodontic re-treatment had occurred. The exception to this was the rate of root canal treatment failure for which there was available data (see above) Probability of implant loss and peri-implantitis are independent. These were modelled independently on the basis of data reported in the literature (Berglundh et al. 2002) Results are presented for UK males only on the assumption that dental costs and benets are independent of gender. As life expectancy rather than gender dictates costs, results for females would be similar to those for a slightly younger cohort of males with the same life expectancy The literature consists predominantly of follow-up of patients treated in dental hospitals, or in specialist clinics in the case of implants. This may not accurately reect outcomes achieved in primary-care settings, but robust data in these environments are generally lacking. However, sensitivity analysis allowed the cost variables related to hospital staff costs to be varied (see below).
analysis of the model to be undertaken for each of these parameters. This re-running of the model with different starting parameters illustrates the impact that the inevitable inaccuracies might have on the overall model. The overall costs of each strategy are clearly a product of the estimated dental procedure costs. Dental costs are considerably lower in eastern European countries but average wages and hence patient budgets are also likely to be lower. However, varying the costs of dental wages or implant components will inuence the relative cost-effectiveness of each treatment strategy. The relative effect of decreasing component costs or increasing dental salaries is likely to be similar implant costs will fall relative to alternative restorative procedures and implant strategies will be more costeffective. We simulated three different potential cost environments to illustrate the impact of higher and lower wage costs and the impact of lower implant component costs.
Results
Table 1 shows both the expected total lifetime costs and the expected longevity of the root canal treated tooth and/or implant supported crowns for a male aged 35, 55 and 75 years, without ination. The values have been discounted to take account for change in perceived value with time, using standard measures recommended by NICE (http://www.nice.org.uk/ media/F13/6E/ITEM3FINALTAMethodsGuidePostConsultationForBoardCover.pdf) and this partly accounts for the relatively low monetary values in all strategies. Crown longevity is the sum of the total lifetimes of root canal treated tooth and/or implant supported crowns at that site prior to failure and replacement with a bridge or denture. It is assumed that if no endodontic or implant treatment is provided there will still be a need over the lifetime to ll the space, with a cost consequence [statistically, unlled anterior spaces are very rare in the UK (Kelly et al. 2000)]. The model predicts superior survival of the ISC over a conventional root canal treated tooth with a postcrown based on published evidence. After 20 years around 25% of root canal treated and re-treated teeth are predicted to have been lost, whereas 10% of rst implants have failed, necessitating a further implant or replacement with a bridge or denture. Despite improved longevity, the implant based strategies still require more interim interventions if a two stage procedure is assumed.
Cost effectiveness: ratio calculation

The outcome measure used in the cost-effectiveness analysis is the total longevity of a xed platform supported crown, both root canal treated and postcrowned natural tooth, and implant supported crowns. After reviewing the costs and longevity for all ten strategies and ranking them by cost, strategies that were clearly less cost effective (those that were dominated or extendedly dominated, see results) were removed and the rest retained for the calculation of an incremental cost-effectiveness ratio (ICER). This widely used index of cost-effectiveness (Drummond et al. 2005) is the additional nancial cost divided by the additional effectiveness (in this case the prolonged longevity of the crown) of that strategy over the next cheapest alternative.
Cost-effectiveness analysis: sensitivity analysis

The key parameters (such as costs and survival) are all estimates and, by denition, likely to be imprecise. To allow for this, plausible ranges for key parameters (such as survival of restorations) were estimated by the academic dental authors, allowing one-way sensitivity
878
Table 1 Base case results cost and total crown longevity for each strategy
Strategy 1 (Extraction) 2 (One RoCT) 3 (RoCT then re-treatment) 4 (RoCT then Surgery) 7 (RoCT/re-treatment/Implant) 8 (RoCT/Surgery/Implant) 5 (RoCT then Implant) 6 (RoCT/Implant/2nd Implant) 9 (Implant) 10 (Implant then 2nd Implant)
Male age 35 Cost () Longevity 731 805 828 847 1071 1079 1113 1140 1623 1717 0 15.81 17.29 17.51 21.58 21.59 21.47 21.85 20.12 21.73
Male age 55 Cost () Longevity 649 717 730 746 916 924 967 983 1570 1642 0 12.62 13.56 13.66 15.78 15.78 15.73 15.88 14.96 15.83
Male age 75 Cost () Longevity 540 597 601 611 694 701 736 741 1487 1527 0 7.1 7.41 7.43 8 8 7.99 8.02 7.74 8.01
1800 1600 1400 1200 1000
800 600 400 200 0 35

Implant/2nd implant RoCT/Implant/2nd implant RoCT/Surg/Implant RoCT/Surg One RoCT Implant RoCT/Implant RoCT/re-treat/Implant RoCT/re-treat Extraction
45
55
65
75
85
95
Age
Figure 2 Cumulative costs of each strategy (male age 35 years).
Figure 2 shows the cost accumulation (discounted) for each strategy over 65 years for a male aged 35 years. The signicantly greater initial outlay on placing an implant is evident but slightly mitigated by lower ongoing costs, illustrated by the rather shallow curve. The ongoing costs of strategies ve (RoCT/ Implant) and six (RoCT/Two implants) show the steepest gradient, due to a combination of relatively high failure rates of the rst treatment (RoCT), and the high cost of the second treatment (implant).
reviewed. When this was done, some strategies were clearly less cost-effective because they have poorer longevity but still cost more than others. They are said to be dominated. Strategies ve (RoCT/Implant), nine (One Implant) and 10 (Two Implants) were dominated for patients at all ages analysed (35, 45, 55, 65, 75, 85) and have been excluded. The remaining strategies are each more effective than less expensive alternatives, but some are signicantly more expensive than a comparator but only marginally more effective. It would not make sense to choose such a strategy if, by paying only a little more, we could get a much bigger increase in effectiveness, hence these strategies are excluded (they are said to be extendedly dominated). Both strategies involving a surgical endodontic re-treatment (strategies four and eight) fell in to this category at each age analysed. Whilst surgical endodontic re-treatment has a higher reported success rate than nonsurgical re-treatment in some studies, this has generally followed endodontic re-treatment. The overall increase in longevity, relative to the increased cost, is small. Additional crown years (longevity) can actually be achieved at a lower cost per year with implants. The results of the cost-effectiveness analysis are shown in Table 2.1 Strategy 1 (No Treatment) is the least effective and the cheapest, and so this is the comparator for calculating the ICER for strategy two
Cost-effectiveness analysis
The 10 strategies model both the initial intervention and the possible subsequent interventions required to maintain a tooth or prosthesis at that site for the patients lifetime. To establish cost effectiveness these are ranked in order of cost and their longevity
1 The costs generated by the model are the expected future costs discounted to the present and not the actual costs faced by a patient if he/she was to receive each of the interventions in the strategy. We would expect many patients to die with an intact root treated tooth, only a proportion will go onto to receive subsequent interventions and the model presents the average costs given the likelihood of failure of restorations undertaken.
879
Table 2 Incremental cost-effectiveness ratios (ICERs) for non-
dominated strategies over the age range 3585

ICERs for males aged 3585 () Strategy 2 3 7 6 35 45 55 65 75 85
(One RoCT) 5 5 5 6 8 ED (RoCT then re-treatment) 15 15 14 13 11 12 (RoCT/re-treatment/Implant) 57 67 84 111 158 241 (RoCT/Implant/2nd Implant) 252 383 654 1272 2813 6916
ED-extendedly dominated.
(One RoCT). The comparator for each subsequent strategy is the next best alternative after excluding dominated and extendedly dominated options. All the cost-effective strategies involve initial root treatment. Strategy 2 is expected to cost 58 more per year of longevity of the root treated tooth than replacement with a bridge or denture. The table reveals that patients who would choose othograde re-treatment should the root canal treatment fail (strategy three) can expect to extend the longevity of the root treated tooth at a cost per year of additional life of 1115 over and above the expected cost if a bridge or denture is tted on failure of the root treated tooth. Patients who would choose an implant rather than a bridge or denture should the re-treatment fail (strategy 2) can expect to extend the longevity of xed platform supported crown at a likely additional cost of 57241 per year.
higher or lower wage rates on overall costs is marked, the impact on ICERs is small. Unsurprisingly, lowering both wage rates and component costs only for implant procedures leads to a signicant reduction in the costs of implant based strategies, but they are still more expensive than conventional treatment. Only when component costs are radically reduced to 10% of the current costs does an implant strategy (strategy ve, RoCT/Implant) extendedly dominate an endodontic strategy (strategy three, two RoCTs), in this case for younger males below the age of 37 years.
Discussion
It is unrealistic to expect most dental restorations to last for life (Richardson et al. 1999). Although data may be scarce, one systematic review estimated that 50% of all routine dental restorations may be anticipated to last between 10 and 20 years (Downer et al. 1999), whilst life-expectancy for women is now currently 80 years or more (http://www.statistics.gov.uk/cci/nugget.asp?id= 168). As our urban populations continue to age and expectations of dental function and aesthetics continue to rise, patients, dentists and health planners need to recognize that the next intervention may not be the last, particularly in younger patients. Decisions made at a xed point in time may set individuals on a pathway with long-term ramications. The example considered in this study was a compromised, irreversibly pulpitic maxillary central incisor, with the starting expectation that very few would opt for no treatment at the point of presentation. The immediate choice facing the theoretical patient is whether to preserve the tooth by root canal treatment and a post-retained crown, or whether to have the tooth extracted and replaced with a prosthesis, including the possibility of a single implant. This decision may be inuenced by patient and practitioner-based factors, including perceptions of success, the special interests of the practitioner, and the attitudes and nancial considerations of the patient (Brennan & Spencer 2006, White et al. 2006). Debates on the merits of individual treatment decisions are not new and have been recognized clearly at the endodontic/implant interface, where strong arguments have been made on both sides that certain options are more likely to succeed or to be more economic at the point of delivery (Felton 2005, Trope 2005). But debates on survival and immediate costs cannot always account for the lifetime implications, including maintenance and repair, and costs of replacement after outright failure. A decision analytic
Sensitivity analysis
When each of the key parameters was altered over the limits of likely variation and the models re-run, the impact on the overall cost-effectiveness of each strategy was small, and no changes in the overall rankings were observed. General diffusion of implant technology is likely to lead to lower potential component costs and also more efcient provision by general dentists. The impact of halving all of the implant component costs, and re-costing implant procedures at lower professional rates (50/hour instead of 87/hour) was examined. The impact of a higher wage setting (such as the US) was simulated by costing all procedures using the UK consultant rate (87/hour) for dentists and by increasing labwork costs by 50%. The impact of a lower wage setting was examined by reducing all wage costs (dentists, assistants and hygienists) to 30% of the UK estimates and by reducing dental laboratory costs by 50%. Costs and ICERs for each scenario for nondominated strategies are presented for a 55-year-old male in Table 3. It can be seen that whilst the absolute effect of
880
Table 3 Impact of varying wages and implant component costs on cost-effectiveness (55 years old)
Base case Strategy 1 2 3 7 6 (Extraction) (One RoCT) (RoCT then re -treatment) (RoC T/re-treatment/Implant) (RoCT/Implant/2nd Implant) Cost () 649 717 730 916 983 ICER () Cheaper implants Cost () 649 717 730 822 848 ICER () Higher wages Cost () 993 1088 1103 1242 1286 ICER () Lower wages Cost () 281 315 321 451 501 ICER ()
5 14 84 654
5 14 41 254
8 16 63 437
3 7 59 486
ICER, incremental cost-effectiveness ratio.
framework combines expected costs and expected benets in a manner that aids decision making. In the absence of data on patient utility, it was assumed that benets are proportional to the longevity of a root canal treated tooth or implant; the presentation of ICERs guides the decision according to the value placed on those benets by the decision maker. For the clinician, the patient, the commissioner or the policy maker the model reported here gives a reasonably strong guide to the general courses of action that are likely to be the most cost effective in this relatively common scenario. It suggests that root treatment in the rst instance is a cost effective strategy, and that the lifetime costs are relatively low, even compared with extraction and replacement with a denture or bridge. Where root treatment fails, in general terms, orthograde re-root treatment is still a reasonably cost effective approach. The lifetime costs are a little higher, but still not a great deal higher, than extraction and bridge or denture placement. Following endodontic re-treatment with surgery was not cost effective in a typical presentation, though this does not rule-out the clinical need for surgery in the event of lesions requiring a biopsy, or the diagnosis of a lesion unlikely to heal by orthograde endodontic means. Implant placement is expensive, and is cost effective in this scenario only after endodontic treatment has failed twice. It is not costeffective as an initial option. Of course these calculations do not take into account the value that an individual patient may place on any given treatment. Markov modelling presents a valuable tool for examining such complex lifetime events. Central to the model is a body of survival and outcome data, which informs the probability of a patient remaining in a given health state or moving to a new health state at dened points in time. It allows extrapolation of the clinical data to estimate the expected costs and outcomes over the patients lifetime. The ICERs combine costs and outcome data in a manner which facilitates rational decision making at the level of the
individual, the insurer or the state. It would be easy to misinterpret these ndings as some sort of clinical guidance they are explicitly not that. The model deals in probabilities spread across the generality of patients. Technical or patient issues will tip the balance in favour of one or other approach to treatment for individual patients. However, an understanding of costs and cost effectiveness may help the clinician to advise their patients about the long term costs of any given course of action, or to help insurers or health planners to decide on the basic treatment strategies that give the best value for money. For example, based on this evidence, a reasonable starting point for an insurer may be to provide high quality endodontic treatment, and perhaps to put a premium on high endodontic standards, in the rst instance rather than funding implant provision as a rst line treatment. The substantial body of evidence that dened the current model is available in the on-line Appendix S1. The literature was unable to provide the very best quality of evidence on all of the interventions considered, so the model was informed by the best available evidence. It is likely that survival of restorations will vary widely according to patient characteristics and the skill of the dentist. The evidence for survival of implants and root treatments was meagre, though of reasonable quality. The weakest evidence related to the survival of partial dentures and bridges. This problem is of course not restricted to Markov modelling, and impacts on any attempt to conduct dental care on a base of evidence. Long-term, prospective clinical trials with large sample sizes and clearly dened outcome criteria are desperately needed (Torabinejad et al. 2007). The costs incorporated within the current model were specic to the state funded healthcare system currently operating in the UK. Clearly salary and labwork costs vary signicantly in different countries and the impact on overall strategy costs is large. However, it is the relative costs between strategies rather than the actual values that are important. The
881
relative impact of changing wage costs is surprisingly small. ICERs are changed, but not by an order of magnitude, and overall ranking of strategies remains the same. Hence recommendations based on the calculated ICERs are less susceptible to care costs in different settings. The sensitivity analysis, which demonstrated the stability of the strategy rankings to changes in event probabilities and costs, suggests the ndings are robust.
Conclusions
Root canal treatment is an appropriate and costeffective intervention to extend the life of a maxillary incisor tooth with a diseased pulp. Orthograde re-treatment is also cost-effective, but unless clinically indicated the benets of additional apical surgery do not justify the additional cost. Increased longevity of the crown can be achieved at a lower cost per year with an implant. At current costs the role of implants is limited to a third line intervention if re-treatment fails.
Acknowledgements
The authors are grateful for the advice of Pelham Barton on the appropriate analysis of sequential treatment decisions, and the critical review and comments from Rob Anderson. This work was undertaken by Mark Pennington at Newcastle University without external nancial support.
References
Balevi B (2008) The management of incipient or suspicious occlusal caries: a decision tree analysis. Community Dentistry & Oral Epidemiology 36, 392400. Berglundh T, Persson L, Klinge B (2002) A systematic review of the incidence of biological and technical complications in implant dentistry reported in prospective longitudinal studies of at least 5 years. Journal of Clinical Periodontology 29(Suppl. 3), 197212. Bra gger U, Krenander P, Lang NP (2005) Economic aspects of single-tooth replacement. Clinical Oral Implants Research 16, 33541. Brennan DS, Spencer AJ (2006) Dentist preferences for patients: dimensions and associations with provider, practice, and service characteristics. International Journal of Behavioral Medicine 13, 6978. Buhler H (1988) Evaluation of root-resected teeth. Results after 10 years. Journal of Periodontology 59, 80510. Calnan M, Payne S, Kemple T, Rossdale M, Ingram J (2007) A qualitative study exploring variations in GPs out-of-hours
referrals to hospital. British Journal of General Practice 57, 70613. Chan DC, Heidenreich PA, Weinstein MC, Fonarow GC (2008) Heart failure disease management programs: a costeffectiveness analysis. American Heart Journal 155, 3328. Creugers NH, Mentink AG, Ka yser AF (1993) An analysis of durability data on post and core restorations. Journal of Dentistry 21, 2814. Curtis LNA. (2006) Unit Costs of Health and Social Care. [WWW document]. http://www.pssru.ac.uk/ URL| [accessed on 26 September 2008]. jean-Orliaguet S, Tubert-Jeannin S, Riordan PJ, Espelid I, Dome Tveit AB (2004) French dentists restorative treatment decisions. Oral Health & Preventive Dentistry 2, 12531. Downer MC, Azli NA, Bedi R, Moles DR, Setchell DJ (1999) How long do routine dental restorations last? A systematic review. British Dental Journal 187, 4329. Drummond M, Sculpher M, Torrance G, OBrien B, Stoddart G (2005) Methods for the Economic Evaluation of Health Care Programmes, 3rd edn. Oxford: Oxford University Press. Edwards MJ, Brickley MR, Goodey RD, Shepherd JP (1999) The cost, effectiveness and cost effectiveness of removal and retention of asymptomatic, disease free third molars. British Dental Journal 187, 3804. Felton DA (2005) Implant or root canal therapy: a prosthodontists view. Journal of Esthetic & Restorative Dentistry: Ofcial Publication of the American Academy of Esthetic Dentistry 17, 1979. Kaba R, Sooriakumaran P (2007) The evolution of the doctorpatient relationship. International Journal Of Surgery 5, 57 65. Kelly M, Steele J, Nuttall N et al. (2000) Adult Dental Health Survey: Oral Health in the United Kingdom in 1998. London: TSO. Lanning SK, Pelok SD, Williams BC et al. (2005) Variation in periodontal diagnosis and treatment planning among clinical instructors. Journal of Dental Education 69, 325 37. Lindh T, Gunne J, Tillberg A, Molin M (1998) A meta-analysis of implants in partial edentulism. Clinical Oral Implants Research 9, 8090. McColl E, Smith M, Whitworth J, Seccombe G, Steele J (1999) Barriers to improving endodontic care: the views of NHS practitioners. British Dental Journal 186, 5648. Mentink AG, Meeuwissen R, Ka yser AF, Mulder J (1993) Survival rate and failure characteristics of the all metal post and core restoration. Journal of Oral Rehabilitation 20, 455 61. Mileman PA, van den Hout WB (2003) Preferences for oral health states: effect on prescribing periapical radiographs. Dentomaxillofacial Radiology 32, 4017. Raiffa H (1968) Decision Analysis: introductory lecture on choices under uncertainty. Reading, MA: Addison-Wesley. Richardson R, Treasure E, Sheldon T (1999) On the evidence. Dental restoration. Health Service Journal 109, 289.
882
van der Sanden WJM, Mettes DG, Plasschaert AJM, Grol RPTM, Mulder J, Verdonschot EH (2005) Effectiveness of clinical practice guideline implementation on lower third molar management in improving clinical decision-making: a randomized controlled trial. European Journal of Oral Sciences 113, 34954. Sjo gren U, Ha gglund B, Sundqvist G, Wing K (1990) Factors affecting the long-term results of endodontic treatment. Journal of Endodontics 16, 498504. Sonnenberg FA, Beck JR (1993) Markov models in medical decision making: a practical guide. Medical Decision Making 13, 32238. Svatek RS, Lee JJ, Roehrborn CG, Lippman SM, Lotan Y (2008) Cost-effectiveness of prostate cancer chemoprevention: a quality of life-years analysis. Cancer 112, 105865. Takao H, Nojo T, Ohtomo K (2008) Treatment of ruptured intracranial aneurysms: a decision analysis. British Journal of Radiology 81, 299303. Tan K, Pjetursson BE, Lang NP, Chan ES (2004) A systematic review of the survival and complication rates of xed partial dentures (FPDs) after an observation period of at least 5 years. Clinical Oral Implants Research 15, 65466. Tickle M, Threlfall AG, Pilkington L, Milsom KM, Duggal MS, Blinkhorn AS (2007) Approaches taken to the treatment of young children with carious primary teeth: a national crosssectional survey of general dental practitioners and paediatric specialists in England. British Dental Journal 203, 1023.
Torabinejad M, Anderson P, Bader J et al. (2007) Outcomes of root canal treatment and restoration, implant-supported single crowns, xed partial dentures, and extraction without replacement: a systematic review. Journal of Prosthetic Dentistry 98, 285311. Trope M (2005) Implant or root canal therapy: an endodontists view. Journal of Esthetic & Restorative Dentistry: Ofcial Publication of the American Academy of Esthetic Dentistry 17, 13940. White SN, Miklus VG, Potter KS, Cho J, Ngan AYW (2006) Endodontics and implants, a catalog of therapeutic contrasts. The Journal of Evidencebased Dental Practice 6, 1019.
Supporting Information Additional Supporting Information may be found in the online version of this article: Appendix S1. A detailed description of the model including all of the data sources used to parameterise it. Please note: Wiley-Blackwell are not responsible for the content or functionality of any supporting materials supplied by the authors. Any queries (other than missing material) should be directed to the corresponding author for the article.
883
doi:10.1111/j.1365-2591.2009.01583.x
Long-term sealing ability of Resilon apical root-end llings
M. A. A. De Bruyne & R. J. G. De Moor

Department of Operative Dentistry and Endodontology, Dental School, Ghent University, Ghent University Hospital, Gent, Belgium
Abstract
De Bruyne MAA, De Moor RJG. Long-term sealing ability
of Resilon apical root-end llings. International Endodontic Journal, 42, 884892, 2009.
Aim To evaluate ex vivo the long-term sealing ability of the SE Resilon Epiphany system as an apical root-end lling material. Methodology A total of 60 standardized horizontal bovine root sections were divided into three groups lled with either gutta-percha with AH 26, tooth-coloured mineral trioxide aggregate (MTA) or Resilon pellets with Epiphany SE, and submitted to capillary ow porometry at 48 h, 1 and 6 months to assess the minimum, mean ow and maximum pore diameters. Results of the different materials and results by material and time were analysed statistically using nonparametric tests; the level of signicance was set at 0.05. Results Resilon had smaller pore diameters than gutta-percha and MTA at 48 h and smaller mean ow
and maximum pore diameters than gutta-percha and MTA at 1 month. At 6 months Resilon had larger minimum pore diameters than gutta-percha. Although not always statistically signicant, the minimum, mean ow and maximum pore diameters of guttapercha and MTA diminished with time. This was not the case for Resilon, where the same parameters increased. Conclusions All materials leaked at all times. Resilon performed better than gutta-percha and MTA in the short-term, but the seal of MTA and gutta-percha improved over time whereas the seal of Resilon deteriorated. It is critical to evaluate the performance of materials in the long-term contrary to most studies which are short-term. Keywords: capillary ow porometry, leakage, Resilon, root-end lling, seal. Epiphany,
Received 14 October 2008; accepted 17 March 2009
Introduction
When orthograde root canal treatment is associated with post-treatment disease, surgical endodontics may be indicated. The procedure involves surgical debridement of pathological periradicular tissue, apical rootend resection, root-end cavity preparation and the placement of a root-end lling in an attempt to seal the root canal (Gutmann & Harrison 1994). The root-end lling should ideally produce a uid-tight seal that
Correspondence: Dr M. A. A. De Bruyne, Department of Operative Dentistry and Endodontology, Dental School, Ghent University, Ghent University Hospital, De Pintelaan 185 P8, 9000 Gent, Belgium (Tel.: +32/9/332 58 35; fax: +32/9/332 38 51; e-mail: mieke.debruyne@UGent.be).
prevents residual irritants and oral contaminants from exiting the root canal system and entering the periradicular tissues (Arens et al. 1998). An ideal root-end lling material would adhere and adapt to the walls of the root-end preparation, prevent leakage of micro-organisms and their toxins into the periradicular tissues, be biocompatible, be insoluble in tissue uids and dimensionally stable and remain unaffected by the presence of moisture (Arens et al. 1998). It is generally accepted that the most uid-tight apical seal possible is required for successful periapical healing (Hirsch et al. 1979). If the seal is not uid-tight, microleakage may occur. Leakage of various root-end lling materials has been investigated widely, mainly using dye penetration methods. However, there are certain disadvantages in using the linear measurement
884
De Bruyne & De Moor Resilon root-end llings
of dye penetration, including the destruction of the specimen, which makes further evaluation of samples impossible, and the lack of reproducible and comparable results (Schuurs et al. 1993, Wu & Wesselink 1993). The reported pattern of leakage in endodontics differs according to the various techniques adopted (Wu et al. 2003). The uid transport method was rst reported by Greenhill & Pashley (1981) and adapted by Wu et al. (1993). This method investigates through-and-through voids and the result when using this technique indicates the diameter of the void. The dye penetration method investigates through-and-through as well as cul-de-sac voids and the result when using this technique indicates the length of the void rather than the diameter (Wu et al. 2003). Capillary ow porometry which was rst introduced in dentistry in 2005 (De Bruyne et al. 2005) is also used to evaluate through-and-through voids. This technique is used in membrane and lter media testing to measure through pores (Jena & Gupta 2002), as does the uid transport method. In contrast to the uid transport method, which gives an indication on the diameter of the void, CFP provides exact information on the diameter of the minimum, mean ow and maximum pore diameter at its most constricted part. The method has been approved by the American Society of Testing and Materials (1999) and was adapted successfully in collaboration with VITO (Flemish Institute for Technological Research, Mol, Belgium) to evaluate through pores in lled root canals or root sections (De Bruyne et al. 2005). The method also provides information on pore distribution. A variety of substances have been proposed as rootend lling materials including amalgam, gutta-percha, zinc oxideeugenol cements, dentine bonding agents, glassionomer cements, mineral trioxide aggregate (MTA) and other restorative materials (Gutmann & Harrison 1994). MTA shows excellent biocompatibility (De Bruyne & De Moor 2004) and, in spite of the limited clinical research, is considered by many clinicians as a standard during apical surgery (Nicholson et al. 1991, Asrari & Lobner 2003, Pistorius et al. 2003, Sousa et al. 2004). After the introduction of grey MTA a tooth-coloured or white MTA was introduced (Matt et al. 2004, Tselnik et al. 2004). Gutta-percha has been used frequently as a root-end lling material in the past and often the lling material is exposed apically when no root-end lling is placed. The Epiphany endodontic obturation system (Pentron, Wallingford, CT, USA) consists of Resilon obturation material available in
points and pellets, and a dual-cure, hydrophilic resin sealer. The Resilon points or pellets can be processed in the same way as gutta-percha. Recently, a self-etch (SE) version of this sealer was introduced. Resilon material is a formulation of polymers of polyester with llers and radiopacifers in a soft resin matrix. The pellets are used with a delivery system (Obtura-Spartan, Fenton, MO, USA). The manufacturer claims that after curing the combination of obturation material and sealer will create a monoblock in the canal that effectively resists leakage. After periradicular surgery, the surface of the root-end lling is exposed to the periapical environment. Because of this exposure, decomposition of the material may occur and the seal of the lling may degrade. In order to obtain information on the performance of root-end lling materials on the long-term, the seal of root-end lling materials should be tested at different intervals after lling (Wu et al. 1998, De Bruyne et al. 2006). The purpose of this study was to evaluate the sealing ability of the SE Resilon-Epiphany system as a root-end lling material and to compare it with warm guttapercha and white MTA in standard bovine root sections at 48 h and after 1 and 6 months.
Materials and methods Preparation and lling of root sections

Roots of freshly extracted bovine incisors with an external diameter of approximately 7 mm were selected and prepared into standardized sections 3 mm high. The central pulp lumen was drilled to 2.5 mm in diameter. For this purpose, the sections which were veried to have a natural internal diameter smaller than 2.5 mm were xed in a clamp. A bur of 2.5 mm in diameter which was secured in a xed position was passed once through the lumen. Sixty of these sections were divided into three different groups and each group was lled according to the following scheme: Group 1: warm gutta-percha (Obtura II, ObturaSpartan) and AH 26 (Dentsply De Trey, Konstanz, Germany) (gutta-percha). Group 2: Pro-Root MTA Tooth-Colored Formula (Dentsply Tulsa, Tulsa, OK, USA) (MTA). Group 3: Resilon pellets (Pentron) (Obtura II delivery system; Obtura-Spartan) and Epiphany SE (Pentron) (Resilon). The root sections were rinsed with physiological saline solution, dried with paper points and air spray
885
Resilon root-end llings De Bruyne & De Moor
and placed on a glass plate on top of a strip of polyester. All materials were mixed and handled according to the manufacturers instructions and the root sections were lled. The lling materials were condensed with a plugger (RCPS 12P; Hu Friedy, Chicago, IL, USA) and excess material was removed. The root sections were kept for 24 h at a temperature of 37 C and 95100% relative humidity and then immersed in demineralized water for 24 h before measurement. After the rst capillary ow measurement at 48 h the root sections were removed from the capillary ow porometer and stored in demineralized water at a temperature of 37 C. They remained under these conditions except during the follow-up measurements that were undertaken at 1 and 6 months.
liquid permeability. The pore diameter (D) is derived from the following equation: D = 4 c cos h/p (c = surface tension of the wetting liquid, h = contact angle of the wetting liquid, p = differential pressure required to displace the wetting liquid from the pore) (Jena & Gupta 2003). All measurements were performed at VITO (Vlaamse Instelling voor Technologisch Onderzoek or Flemish Institute for Technological Research).
Results were analysed statistically using nonparametric tests. Comparisons were made between the leakage results of the different materials at 48 h, 1 and 6 months using KruskalWallis tests; two by two analyses were performed by MannWhitney U-tests with Bonferroni correction. Comparisons between the leakage results of each material at the specied time intervals were completed using Friedman tests and two by two comparisons were carried out by Wilcoxon Signed Ranks tests with Bonferroni correction. The level of signicance was set at 0.05.
Measurement of capillary ow
Capillary ow porometry (CFP-1200-A; PMI, Ithaca, NY, USA) provides fully automated through pore analysis. A wetting liquid (Galwick: 15.9 Dynes cm)1, PMI) was used to ll the pores of the sample. Because the wetting liquids liquid/solid surface free energy is less than the solid/gas surface free energy, lling of the pores is spontaneous, but removal of the liquid from the pores is not. In order to remove the wetting liquid from pores and permit gas ow, pressure must be applied to the sample. The fully wetted sections were xed in the sample chamber after which the sample chamber was sealed. Air was then allowed to ow into the chamber behind the sample. When the pressure reaches a point, it overcomes the capillary action of the uid within the largest pore (maximum pore), and the samples bubble point pressure is identied. After determination of the bubble point pressure, the pressure is increased and the ow is measured until all pores are empty, and the sample is considered dry. At this time the smallest or minimum pore has been identied. The mean ow pore is described as follows: half of the ow through a dry sample is through pores having a diameter greater than the mean ow pore diameter. The other half of the ow is through pores having a diameter smaller than the mean ow pore diameter. Pressure in CFP ranges from 0 to 200 psi or 1.4 MPa and the pore size range that can be measured lies between 0.035 and 500 lm. The ow meters detect the presence of pores by sensing the increase in ow rate due to emptying of pores. Differential pressures and ow rates through wet and dry samples are measured. Application of differential pressure on excess liquid on the sample causes liquid displacement. Measurement of the volume of displaced liquid allows computation of
Results
Measurements were obtained for each sample at each point in time, conrming the presence of through pores regardless of which root-end lling material was being tested. Exact values for minimum, mean ow and maximum pore diameters of each sample were obtained. The results of the study are summarized in Tables 1 3. For reasons of completeness the range and median of minimum, mean ow and maximum pore diameters of gutta-percha and MTA as reported in De Bruyne et al. (2006) are repeated in Tables 13.
Leakage results at 48 h, 1 and 6 months

From the KruskalWallis tests and the MannWhitney U-tests with Bonferroni correction the following results were obtained. At 48 h signicant differences between the minimum (P < 0.001), mean ow (P < 0.001) and maximum (P < 0.001) pore diameters could be demonstrated. No signicant differences between gutta-percha and MTA could be demonstrated but there were signicant differences between gutta-percha and Resilon and between MTA and Resilon for minimum, mean ow and maximum pore diameters. At 48 h Resilon showed
886
Table 1 Range and median of minimum, mean ow and maximum pore diameters by root-end lling material at 48 h (lm)
Minimum pore diameter (lm) Group 1 2 3 Filling material GP + AH 26 MTA Resilon Range 0.0750.355 0.0700.258 0.0820.201 Median 0.1995 0.2210 0.1165 Mean ow pore diameter (lm) Range 0.1410.395 0.1830.925 0.1000.272 Median 0.2630 0.2760 0.1465 Maximum pore diameter (lm) Range 0.1771.714 0.1931.304 0.1270.433 Median 0.4375 0.4440 0.2140
MTA, mineral trioxide aggregate.
Table 2 Range and median of minimum, mean ow and maximum pore diameters by root-end lling material at 1 month (lm)
Table 3 Range and median of minimum, mean ow and maximum pore diameters by root-end lling material at 6 months (lm)
smaller pore diameters than gutta-percha and MTA. The range and median of minimum, mean ow and maximum pore diameters at 48 h are shown in Table 1. At 1 month there was no signicant difference between the minimum pore diameters of the different materials, but signicant differences between the mean ow (P < 0.001) and maximum (P < 0.001) pore diameters could be demonstrated. Concerning the mean ow and maximum pore diameters, no signicant differences between gutta-percha and MTA could be demonstrated, but there were signicant differences between gutta-percha and Resilon and between MTA and Resilon. At 1 month Resilon showed smaller mean ow and maximum pore diameters than gutta-percha and MTA. The range and median of minimum, mean ow and maximum pore diameters at 1 month are shown in Table 2. At 6 months a signicant difference between the minimum pore diameters could be demonstrated
(P < 0.05), but there were no signicant differences between the mean ow and maximum pore diameters of the different materials. Concerning the minimum pore diameters, there was a signicant difference between gutta-percha and Resilon. No signicant differences between gutta-percha and MTA and between MTA and Resilon could be demonstrated. At 6 months Resilon showed larger minimum pore diameters than gutta-percha. The range and median of minimum, mean ow and maximum pore diameters at 6 months are shown in Table 3.
Leakage results by material

From the Friedman tests the following results were obtained. Concerning the minimum pore diameters there were signicant differences between the different points in time for gutta-percha and MTA, but not for Resilon. Results of the two by two comparisons are summarized in Table 4. Statistically signicant
887
Root-end lling material GP + AH 26 MTA Minimum pore diameter Mean ow pore diameter Maximum pore diameter Minimum pore diameter Mean ow pore diameter Maximum pore diameter Minimum pore diameter Mean ow pore diameter Maximum pore diameter
Two by two comparisons Friedmans test *(P *(P *(P *(P *(P < < < < < 0.05) 0.001) 0.001) 0.01) 0.005) 48 h vs. 48 h vs. 1 month vs. 1 month 6 months 6 months > > > > >
Table 4 Summary of signicant differ-
> > > >
ences (marked by an asterisk) between minimum, mean ow or maximum pore diameters at 48 h, 1 and 6 months and for two by two comparisons by material (> means the pore diameter is larger at the former than at the latter measurement)
Resilon
decreases in size were found between 48 h and 6 months for gutta-percha and MTA and between 1 and 6 months for MTA. Concerning the mean ow pore diameters there were signicant differences between the different points in time for gutta-percha and MTA but not for Resilon. Results of the two by two comparisons are summarized in Table 4. Statistically signicant decreases in size were found for gutta-percha and MTA between 48 h and 6 months and between 1 and 6 months. Concerning the maximum pore diameters there were signicant differences between the different points in time for gutta-percha but not for MTA and Resilon. Results of the two by two comparisons are summarized in Table 4. Statistically signicant decreases in size were found for gutta-percha between 48 h and 6 months and between 1 and 6 months.
Discussion
Capillary ow porometry generates highly reproducible and accurate data (Gupta & Jena 1999). Therefore, because of its nondestructive nature and following a previous study (De Bruyne et al. 2006) CFP was chosen as the evaluation method for the present study. It provides, as the rst and only method in leakage research, exact data on pore diameters which can be compared statistically and gives an indication whether bacteria or their metabolites will be able to pass through the sample. This is in contrast to other methods, which only compare materials without giving any information on the size of pores. As such, the method can overcome the problem of limited reproducibility and comparability of conventional methods for evaluating leakage (Wu & Wesselink 1993). CFP uses a wetting liquid with a low surface tension such
that pores as small as 0.035 lm can be measured, which assures the detection of gaps of about 2 lm which were already observed between the root dentine and the Resilon primer. These gaps might be too small to be detected by, for example, bacterial penetration models (De-Deus et al. 2007). The relatively high pressures used during CFP may be a concern. It needs to be emphasized, however, that during the present study and during all previous studies none of the llings were dislodged. Results from a pilot study also showed that no statistically signicant differences were evaluated between measurements when samples were measured multiple times immediately after each other (De Bruyne 2006). Apart from this the results from push-out tests revealed that micropush-out bond strengths of all materials tested were higher than the pressures used in the present study (Yan et al. 2006, Sly et al. 2007, Ureyen et al. 2008). This implies that the lling materials used in the present study will not be damaged during CFP. As the purpose of the study was to compare root-end lling materials, standardized root sections were essential. Because human teeth are too small to be used to prepare standardized samples that are easy to handle, x and evaluate in a reliable way, bovine teeth were used. As bovine teeth are easy to obtain and as the sections are large enough to adjust the central pulp lumen to the exact diameter, standardization is straightforward. Consequently cavities of equal size could be lled with different materials and compared under the same conditions, although these differ from the clinical situation. From the study of Nakamichi et al. (1983) it appeared that no statistically signicant difference was found in adhesion of various materials to human or bovine dentine. Because of the larger diameter and same height, the C-factor in the present samples will be lower
888
than in human teeth which results in less inuence from contraction forces (Tay et al. 2005a). As the manufacturer claims that after curing the combination of obturation material and sealer will create a monoblock that effectively resists leakage, it seemed that Resilon would be a perfect root-end lling material. It can be applied easily in the same way as gutta-percha, and the material sets fast, which is an advantage during surgery. Because of the similarity to gutta-percha and because of the fact that MTA is often considered as a standard of care, both these materials were selected as controls. Similar to the results of previous studies performed with CFP on root-end llings (De Bruyne et al. 2005), measurements were obtained for each sample at each time interval. The average length of bacteria varies between 0.2 and more than 10 lm, the width between 0.2 and 1.5 lm (Hobot 2002); and their metabolites are even smaller. Apart from this, one has to keep in mind the fact that bacteria are not rigid structures but can alter their outline. Therefore, in general the maximum pore diameter and the size of bacteria and their metabolites will be indicative of the possible leakage along the root-end lling materials. The minimum and mean ow pore diameters are relevant in terms of pore size distribution. Looking at the results, this means that some bacteria and denitely their metabolites will be able to pass along root end llings. At 48 h the minimum, mean ow and maximum pore diameters were smaller for Resilon than for guttapercha and MTA. At 1 month this was not the case for the minimum pore diameter, but remained so for the mean ow and maximum pore diameters. At 6 months the difference for the mean ow pore and maximum pore diameters had disappeared, whereas at this time Resilon had larger minimum pore diameters than gutta-percha. Looking at the tables, although not always statistically signicant, it appears that the minimum, mean ow and maximum pore diameters of gutta-percha and MTA diminished in the course of time, which was not the case for Resilon. For Resilon there was an increase. As the maximum pore diameter will determine the eventual seal of the material, this diameter is of major importance. Until now improvement of the seal was seen over time for all materials tested by CFP (De Bruyne et al. 2006); Resilon seems to act differently. In a short-term study by Maltezos et al. (2006), which also tested Resilon as a root-end lling material, the bacterial leakage analysis (4-week observation) showed that Super-EBA leaked signicantly more than Resilon and that there was no
difference between Resilon and white Pro Root MTA. This is contrary to the present study where after 1 month Resilon still performed better than MTA. In contrast to the above, most other studies evaluated root llings and not root-end llings. In a recent study which evaluated short-term coronal leakage, Epiphany SE sealer and Resilon as a root lling was compared with gutta-percha with AH 26 or AH plus sealer using dye leakage and performed better (Bodrumlu & Tunga 2007a). Shipper et al. (2005) evaluated the prevention of apical periodontitis in an in vivo dog model and concluded that the Resilon Monoblock System was associated with less apical periodontitis than guttapercha with AH 26, maybe because of its superior resistance to coronal microleakage. In other studies on root llings, Resilon performed better, equal or worse than gutta-percha (Shipper et al. 2004, Aptekar & Ginnan 2006, Biggs et al. 2006, Bodrumlu & Tunga 2006, 2007b, von Fraunhofer et al. 2006, Onay et al. 2006, Pitout et al. 2006, Sagsen et al. 2006, Shemesh et al. 2006, 2007, Stratton et al. 2006, Tunga & Bodrumlu 2006, Almeida et al. 2007, Baumgartner et al. 2007, De-Deus et al. 2007, Ishimura et al. 2007, Paque & Sirtes 2007, Raina et al. 2007, Silveira et al. 2007, Verissimo et al. 2007, Pasqualini et al. 2008), sometimes depending on the sealer used in combination with gutta-percha or the leakage assessment method. Interesting in the context of root-end llings though is the fact that Resilon had signicantly less leakage than gutta-percha with Grossmans cement in moist canals in a (short-term) study (Zmener et al. 2008). In surgical circumstances, which often are not ideal, this might be a major benet. On the other hand, the biodegradation of Resilon (polycaprolactone) as mentioned by Tay et al. (2005b), but contradicted by Trope (2006) might also be of relevance. Different from most studies which are short-term, Paque & Sirtes (2007) performed a long-term study using a uid transportation model in which they showed that initially there was no difference in leakage between gutta-percha with AH Plus sealer and Resilon/ Epiphany but after 16 months gutta-percha retained its seal whereas Resilon/Epiphany lost its sealing capacity. The results of the present study conrmed these longterm results. Different factors might contribute to this loss of seal over time. Although the conguration factor (C-factor) of the specimens in the present study was not as high as in root canals (Tay et al. 2005a) one or more bonded areas might pull off or debond in the course of time. De Munck et al. (2005), in their review on the durability of adhesion to tooth tissue, reported that
889
after about 3 months all adhesives exhibited mechanical and morphological evidence of degradation, which probably will also be true for Epiphany SE. Colour discharge from the Resilon pellets, according to the manufacturer only food grade dye leaching out into the tooth, was seen in 11 samples at 1 month and in 13 samples at 6 months in the present study. This might also have contributed to the increased leakage (Shemesh et al. 2006). As it is not common during surgery, in the present study no EDTA was used to remove the smear layer as suggested by the manufacturer. Removing the smear layer might positively inuence the results. This evolution of the seal over time of Resilon is in contrast to the improvement of seal over time of guttapercha and MTA, which has already been discussed extensively in a former study (De Bruyne et al. 2006). Changes in these root-end llings occurred probably at the interface with the root dentine as the absence of voids within the materials was conrmed earlier (De Bruyne et al. 2005). Dimensional changes during time (Orstavik et al. 2001) and further hydration of MTA powder (Wu et al. 1998) are factors which might have contributed to the improvement of their seal.
Conclusion
Irrespective of the root-end lling material, each sample leaked along the lling material at all times. Resilon was unable to provide a uid-tight seal. Whereas in the present study Resilon performed better than guttapercha and MTA in the short-term, the seal of these materials improved over time whereas the seal of Resilon deteriorated. As not all materials evolve the same way, it is important to evaluate on the long-term basis contrary to most studies which are short-term.
References
Almeida JF, Gomes BP, Ferraz CC, Souza-Filho FJ, Zaia AA (2007) Filling of articial lateral canals and microleakage and ow of ve endodontic sealers. International Endodontic Journal 40, 6929. American Society of Testing and Materials (1999) Standard Test Method for Pore Size Characteristics of Membrane Filters Using Automated Liquid Porosimeter. ASTM Designation: E1294-89. West Conshohocken, PA, USA, pp. 12. Aptekar A, Ginnan K (2006) Comparative analysis of microleakage and seal for 2 obturation materials: Resilon/ Epiphany and gutta-percha. Journal of the Canadian Dental Association 72, 245.
Arens DE, Torabinejad M, Chivian N, Rubinstein R (1998) Practical Lessons in Endodontic Surgery. Carol Stream, IL, USA: Quintessence Publishing Co, Inc., pp. 1213. Asrari M, Lobner D (2003) In vitro neurotoxic evaluation of root-end-lling materials. Journal of Endodontics 29, 7436. Baumgartner G, Zehnder M, Paque F (2007) Enterococcus faecalis type strain leakage through root canals lled with Gutta-Percha/AH plus or Resilon/Epiphany. Journal of Endodontics 33, 457. Biggs SG, Knowles KI, Ibarrola JL, Pashley DH (2006) An in vitro assessment of the sealing ability of resilon/epiphany using uid ltration. Journal of Endodontics 32, 75961. Bodrumlu E, Tunga U (2006) Apical leakage of Resilon obturation material. Journal of Contemporary Dental Practice 7, 4552. Bodrumlu E, Tunga U (2007a) Coronal sealing ability of a new root canal lling material. Journal of the Canadian Dental Association 73, 623. Bodrumlu E, Tunga U (2007b) The apical sealing ability of a new root canal lling material. American Journal of Dentistry 20, 2958. De Bruyne MAA (2006) Ultrasonic root-end preparation and sealing ability of conventionally-setting glass ionomer cements in surgical endodontics, PhD Thesis. Gent, Belgium: Ghent University. De Bruyne MA, De Moor RJ (2004) The use of glass ionomer cements in both conventional and surgical endodontics. International Endodontic Journal 37, 91104. De Bruyne MA, De Bruyne RJ, Rosiers L, De Moor RJ (2005) Longitudinal study on microleakage of three root-end lling materials by the uid transport method and by capillary ow porometry. International Endodontic Journal 38, 12936. De Bruyne MA, De Bruyne RJ, De Moor RJ (2006) Long-term assessment of the seal provided by root-end lling materials in large cavities through capillary ow porometry. International Endodontic Journal 39, 493501. De Munck J, Van Landuyt K, Peumans M et al. (2005) A critical review of the durability of adhesion to tooth tissue: methods and results. Journal of Dental Research 84, 11832. De-Deus G, Audi C, Murad C, Fidel S, Fidel RA (2007) Sealing ability of oval-shaped canals lled using the System B heat source with either gutta-percha or Resilon: an ex vivo study using a polymicrobial leakage model. Oral Surgery, Oral Medicine, Oral Pathology, Oral Radiology and Endodontology 104, e1149. von Fraunhofer JA, Kurtzman GM, Norby CE (2006) Resinbased sealing of root canals in endodontic therapy. General Dentistry 54, 2436. Greenhill JD, Pashley DH (1981) The effects of desensitizing agents on the hydraulic conductance of human dentin in vitro. Journal of Dental Research 60, 68698. Gupta V, Jena AK (1999) Substitution of alcohol in porometers for bubble point determination. Advances in Filtration and Separation Technology 13b, 83344.
890
Gutmann JL, Harrison JW (1994). Surgical Endodontics. St. Louis, MO: Ishiaku EuroAmerica, Inc., pp. 20377. Hirsch JM, Ahlstrom U, Henrikson PA, Heyden G, Peterson LE (1979) Periapical surgery. International Journal of Oral Surgery 8, 17385. Hobot JA (2002) Molecular Medical Microbiology. London, UK: Academic Press, p. 7. Ishimura H, Yoshioka T, Suda H (2007) Sealing ability of new adhesive root canal lling materials measured by new dye penetration method. Dental Materials Journal 26, 2905. Jena A, Gupta K (2002) Characterization of pore structure of lter media. Fluid/Particle Separation Journal 14, 22741. Jena A, Gupta K (2003) Measuring pore characteristics without mercury. Ceramic Industry 153, 338. Maltezos C, Glickman GN, Ezzo P, He J (2006) Comparison of the sealing of Resilon, Pro Root MTA, and Super-EBA as root-end lling materials: a bacterial leakage study. Journal of Endodontics 32, 3247. Matt GD, Thorpe JR, Strother JM, McClanahan SB (2004) Comparative study of white and gray mineral trioxide aggregate (MTA) simulating a one- or two-step apical barrier technique. Journal of Endodontics 30, 8769. Nakamichi I, Iwaku M, Fusayama T (1983) Bovine teeth as possible substitutes in the adhesion test. Journal of Dental Research 62, 107681. Nicholson JW, Braybrook JH, Wasson EA (1991) The biocompatibility of glass-poly (alkenoate) (GlassIonomer) cements: a review. Journal of Biomaterials Science. Polymer Editon 2, 27785. Onay EO, Ungor M, Orucoglu H (2006) An in vitro evaluation of the apical sealing ability of a new resin-based root canal obturation system. Journal of Endodontics 32, 9768. Orstavik D, Nordahl I, Tibballs JE (2001) Dimensional change following setting of root canal sealer materials. Dental Materials 17, 5129. Paque F, Sirtes G (2007) Apical sealing ability of Resilon/ Epiphany versus gutta-percha/AH Plus: immediate and 16-months leakage. International Endodontic Journal 40, 7229. Pasqualini D, Scotti N, Mollo L et al. (2008) Microbial Leakage of Gutta-percha and ResilonTM root canal lling material: a comparative study using a new homogeneous assay for sequence detection. Journal of Biomaterials Applications 22, 33752. Pistorius A, Willershausen B, Briseno MB (2003) Effect of apical root-end lling materials on gingival broblasts. International Endodontic Journal 36, 6105. Pitout E, Oberholzer TG, Blignaut E, Molepo J (2006) Coronal leakage of teeth root-lled with gutta-percha or Resilon root canal lling material. Journal of Endodontics 32, 87981. Raina R, Loushine RJ, Weller RN, Tay FR, Pashley DH (2007) Evaluation of the quality of the apical seal in Resilon/ Epiphany and Gutta-Percha/AH Plus-lled root canals by using a uid ltration approach. Journal of Endodontics 33, 9447.
Sagsen B, Er O, Kahraman Y, Orucoglu H (2006) Evaluation of microleakage of roots lled with different techniques with a computerized uid ltration technique. Journal of Endodontics 32, 116870. Schuurs AH, Wu MK, Wesselink PR, Duivenvoorden HJ (1993) Endodontic leakage studies reconsidered. Part II. Statistical aspects. International Endodontic Journal 26, 44 52. Shemesh H, Wu MK, Wesselink PR (2006) Leakage along apical root llings with and without smear layer using two different leakage models: a two-month longitudinal ex vivo study. International Endodontic Journal 39, 968 76. Shemesh H, van den BM, Wu MK, Wesselink PR (2007) Glucose penetration and uid transport through coronal root structure and lled root canals. International Endodontic Journal 40, 86672. Shipper G, Orstavik D, Teixeira FB, Trope M (2004) An evaluation of microbial leakage in roots lled with a thermoplastic synthetic polymer-based root canal lling material (Resilon). Journal of Endodontics 30, 3427. Shipper G, Teixeira FB, Arnold RR, Trope M (2005) Periapical inammation after coronal microbial inoculation of dog roots lled with gutta-percha or resilon. Journal of Endodontics 31, 916. Silveira FF, Soares JA, Nunes E, Mordente VL (2007) Negative inuence of continuous wave technique on apical sealing of the root canal system with Resilon. Journal of Oral Science 49, 1218. Sly MM, Moore BK, Platt JA, Brown CE (2007) Push-out bond strength of a new endodontic obturation system (Resilon/ Epiphany). Journal of Endodontics 33, 1602. Sousa CJ, Loyola AM, Versiani MA, Bif JC, Oliveira RP, Pascon EA (2004) A comparative histological evaluation of the biocompatibility of materials used in apical surgery. International Endodontic Journal 37, 73848. Stratton RK, Apicella MJ, Mines P (2006) A uid ltration comparison of gutta-percha versus Resilon, a new soft resin endodontic obturation system. Journal of Endodontics 32, 6425. Tay FR, Loushine RJ, Lambrechts P, Weller RN, Pashley DH (2005a) Geometric factors affecting dentin bonding in root canals: a theoretical modeling approach. Journal of Endodontics 31, 5849. Tay FR, Loushine RJ, Weller RN et al. (2005b) Ultrastructural evaluation of the apical seal in roots lled with a polycaprolactone-based root canal lling material. Journal of Endodontics 31, 5149. Trope M (2006) Resilon will biodegrade from lipases released by bacteria or by bacterial or salivary enzymes. Journal of Endodontics 32, 856. Tselnik M, Baumgartner JC, Marshall JG (2004) Bacterial leakage with mineral trioxide aggregate or a resin-modied glass ionomer used as a coronal barrier. Journal of Endodontics 30, 7824.
891
Tunga U, Bodrumlu E (2006) Assessment of the sealing ability of a new root canal obturation material. Journal of Endodontics 32, 8768. Ureyen KB, Kececi AD, Orhan H, Belli S (2008) Micropush-out bond strengths of gutta-percha versus thermoplastic synthetic polymer-based systems an ex vivo study. International Endodontic Journal 41, 2118. Verissimo DM, do Vale MS, Monteiro AJ (2007) Comparison of apical leakage between canals lled with gutta-percha/AHPlus and the Resilon/Epiphany System, when submitted to two lling techniques. Journal of Endodontics 33, 2914. Wu MK, Wesselink PR (1993) Endodontic leakage studies reconsidered. Part I. Methodology, application and relevance. International Endodontic Journal 26, 3743. Wu MK, De Gee AJ, Wesselink PR, Moorer WR (1993) Fluid transport and bacterial penetration along root canal llings. International Endodontic Journal 26, 2038.
Wu MK, Kontakiotis EG, Wesselink PR (1998) Long-term seal provided by some root-end lling materials. Journal of Endodontics 24, 55760. Wu MK, Van Der Sluis LW, Ardila CN, Wesselink PR (2003) Fluid movement along the coronal two-thirds of root llings placed by three different gutta-percha techniques. International Endodontic Journal 36, 53340. Yan P, Peng B, Fan B, Fan M, Bian Z (2006) The effects of sodium hypochlorite (5.25%), Chlorhexidine (2%), and Glyde File Prep on the bond strength of MTA-dentin. Journal of Endodontics 32, 5860. Zmener O, Pameijer CH, Serrano SA, Vidueira M, Macchi RL (2008) Signicance of moist root canal dentin with the use of methacrylate-based endodontic sealers: an in vitro coronal dye leakage study. Journal of Endodontics 34, 769.
892
doi:10.1111/j.1365-2591.2009.01590.x
Sealing ability, water sorption, solubility and toothbrushing abrasion resistance of temporary lling materials
C. M. Pieper1, C. H. Zanchi1, S. A. Rodrigues-Junior1, R. R. Moraes2, L. S. Pontes1 & M. Bueno1

1 Department of Restorative Dentistry, School of Dentistry, Federal University of Pelotas, Pelotas, Brazil; and 2Department of Restorative Dentistry, Dental Materials Area, Piracicaba Dental School, State University of Campinas, Campinas, Brazil
Abstract
Pieper CM, Zanchi CH, Rodrigues-Junior SA, Moraes RR, Pontes LS, Bueno M. Sealing ability, water sorption,
solubility and toothbrushing abrasion resistance of temporary lling materials. International Endodontic Journal, 42, 893899, 2009.
Aim To evaluate marginal seal, water sorption, solubility and loss of mass after brushing of several temporary lling materials. Methodology For marginal seal, Class I cavities, including endodontic access preparations, were made in human molar teeth and restored using one or other of several temporary lling materials (n = 10): zinc oxide/ calcium sulphate-based cement (Cavit, 3M,ESPE, St. Paul, MN, USA), zinc oxide/eugenol cement (IRM, Dentsply Caulk, Milford, DE, USA), glass ionomer cement (Vidrion R, SSWhite, Rio de Janeiro, RJ, Brazil) or a mica, dimethacrylate-based lling (Bioplic, Biodina Londrina, PR, Brazil). Dye penetration was assessed after thermocycling and immersion in 0.5% basic fuchsine solution. For water sorption, solubility and loss of mass analyses, disc-shaped specimens were made. Water sorption and solubility were evaluated by mass alteration
after storage in distilled water for 7 days (n = 7). Loss of mass was calculated based on the difference of mass after abrasion with a toothbrush (n = 5), and surfaces were analysed by SEM. Data of water sorption, solubility and loss of mass were submitted to anova and Tukeys test, and marginal sealing data to KruskalWallis test (P < 0.05). Results Statistically signicant differences were observed for marginal sealing (P < 0.0001), water sorption (P < 0.01), solubility (P < 0.01) and loss of mass (P < 0.05). Bioplic had the best marginal seal. Cavit had the greatest water sorption and solubility. Vidrion R and Bioplic had the lowest solubility. Loss of mass after brushing was higher for Cavit, followed by Bioplic, IRM and Vidrion R. Cavit and Vidrion R were worn aggressively by brushing. Conclusions The resin-based temporary lling Bioplic produced the best marginal seal, and was associated with the lowest water sorption, solubility and loss of mass. Keywords: loss of mass, marginal sealing, temporary lling,toothbrushingabrasion, watersorption/solubility.
Received 13 October 2008; accepted 26 March 2009
Introduction
The outcome of root canal treatment depends, amongst other factors, upon the sealing capacity of temporary restorations that prevents bacterial inltration and
Correspondence: Sinval Adalberto Rodrigues-Junior, Rua Gonc alves Chaves 457, Centro, 96015-560, Pelotas, RS, Brazil (Tel.: +55 53 3222-6690; e-mail: rodriguesjr2002@ yahoo.com.br).
recontamination of the root canal system (Torabinejad et al. 1990, Ray & Trope 1995, Hommez et al. 2002). Besides avoiding bacterial percolation, temporary llings may help to protect weakened coronal tooth tissue from fractures when they have adhesive properties (Soares & Goldberg 2002). Conversely, llings that expand during or after setting, due to hygroscopic expansion, may cause cusp deection or fractures (Laustsen et al. 2005). Characteristically, restorative materials undergo degradation in contact with water, such as leaching of
893
Sealing of temporary llings Pieper et al.
components that may weaken their structure (Ferracane 2006). In addition, the oral environment is inhospitable for restorative materials, with extremes of thermal and mechanical challenges. The mechanical action of toothbrushing might also abrade the materials (Moraes et al. 2008). Several temporary lling materials with different microstructures, compositions and setting mechanisms are available commercially. Cavit (3M; ESPE, St. Paul, MN, USA) is a premanipulated eugenol-free material that sets in contact with moisture, but has given conicting marginal sealing results (Naoum & Chanmica, Londrina, PR, Brazil) dler 2002). Bioplic (Biodina is a resin-based material that sets upon light-curing, characteristically presenting volumetric shrinkage during polymerization. This contraction, however, is usually followed by expansion due to water sorption (Deveaux et al. 1992), although whether this hygroscopic expansion is sufcient to adequately seal the cavity is still unknown. Conventional glassionomer cements (GIC) are considered suitable materials for restorations for several reasons: they form a hard material upon setting, present relatively little or no exothermic reaction or shrinkage during setting, have no free monomer in the set matrix, and adhere to tooth structure (Culbertson 2001). Based on its adhesion potential, it could be expected that the marginal sealing produced by GICs is good. Naoum & Chandler (2002) have concluded that GIC is a satisfactory endodontic temporary lling, even in the long-term. IRM (Dentsply Caulk, Milford, DE, USA), a zinc oxide-eugenol (ZOE) based cement, has been associated with antibacterial activity (Naoum & Chandler 2002). Together with Cavit, IRM has been the most used temporary lling in endodontics (Koagel et al. 2008), even though its sealing capability has generated conicting results (Mayer & Eickholz 1997, Naoum & Chandler 2002, Zmener et al. 2004, Koagel et al. 2008).
Discrepancies between studies still raise concerns about the capacity of temporary lling materials with different compositions to avoid bacterial percolation that could lead to post-treatment disease. As these materials have different setting mechanisms, different reactions with moisture and variable dimensional stability, there is a potential for them to produce different marginal sealing abilities. In addition, few studies have evaluated the in vitro performance of temporary llings. Therefore, the aim of this study was to evaluate the marginal sealing ability, water sorption, solubility and toothbrushing abrasion resistance of different lling materials used as temporary restoration in root lled teeth.
Material and method Temporary lling materials

Four temporary lling materials with different constituents and setting mechanisms were evaluated: a ZOEbased cement (IRM; Dentsply Caulk, Milford, DE, USA), a eugenol-free ZO cement (Cavit; 3M ESPE, St. Paul, MN, USA), a GIC (Vidrion R; SS White, Rio de Janeiro, RJ, Brazil), and a resin-based cement (Bioplic; Biodinamica, Londrina, PR, Brazil). Table 1 presents the composition of all materials.
Marginal sealing
Forty unrestored, caries-free human rst and second molar teeth were selected under approval of the institutional Ethics Committee of School of Dentistry/Federal University of Pelotas (UFPel), Brazil (protocol no. 16/ 04). All teeth were examined at 10 magnication, and those with microcracks were excluded. The teeth were stored in 0.2% thymol solution for 7 days, after which the periodontal ligament was removed with a razor blade
Material Vidrion R
Composition Powder: aluminium silicate glass Liquid: copolymers of polyacrylic, itaconic and tartaric acids Zinc oxide, calcium sulphate, zinc sulphate Silicium dioxide, dimethacrylates, inorganic ller Powder: Zinc oxide, polymethyl methacrylate Liquid: Eugenol
Manufacturer SS White
Batch no. 6040306
Table 1 Temporary lling materials
Cavit Bioplic IRM
3M ESPE mica Biodina Dentsply Caulk
215000 632/05 679307
894
Pieper et al. Sealing of temporary llings
and the teeth cleaned at low-speed with a water-pumice slurry. They were then stored in saline at 5 C. Class I endodontic access cavities with standardized outline were prepared using a handpiece under watercooling. The coronal access to the pulp chamber started with a cylindrical diamond bur no. 1014 (KG Sorensen, Barueri, SP, Brazil) in enamel, and carbide burs no. 245 (SS White) in dentine. The burs were changed after 10 preparations. The pulp cavity and the root canals were rinsed with 1% NaOCl solution in order to remove debris. Root canals were dried through aspiration and using cotton pellets, and their entrance was lled with gutta-percha. To standardize the cavity depth, a periodontal probe was used to assure the existence of at least 4 mm between the cavity outline and the entrance of the root canals (Cruz et al. 2002). Since unrestored, caries-free molar teeth were used, the dentine surfaces after cavity preparation were sound. The teeth were randomly assigned into four groups, dened by the temporary restorative llings (Table 1). All materials were manipulated according to the manufacturers specications. IRM was prepared in a 6-g mL)1 powder/liquid ratio, and inserted and adapted to the cavity walls with a dental spatula. Vidrion R was manipulated and inserted with a Centrix syringe. For Cavit, the cavity was left slightly moist, the material inserted with a dental spatula and allowed to set in contact with a moist cotton pellet. Bioplic was inserted into the cavity, carved and light-cured for 40 s with a quartztungstenhalogen light-curing unit (Ultralux; Dabi Atlante, Ribeira o Preto, SP, Brazil irradiance >400 mW cm)2). The root apices were sealed with self-cured epoxy resin (Durepox; Alba Qu mica Ind. e Com. Ltda., Sa o Paulo, SP, Brazil) and teeth were covered with two coats of nail polish, except the restorations and a 1-mm area surrounding them. After storage in saline for 7 days, at 37 C, the teeth were submitted to 500 thermal cycles between 5 5 and 55 5 C, with 30 s dwell time and 3 s interval time. The teeth were then immersed in 0.5% basic fuchsine solution for 24 h, at room temperature, and washed for 24 h in running tap water. Sectioning was performed bucco-lingually to the long axis of the tooth using a diamond disc. Two previously calibrated examiners analysed both sections using a stereomicroscope, at 40 magnication, recording the highest penetration score. Dye penetration was determined based on the following scores: 0 no visible dye penetration at the tooth/lling interface; 1 dye penetration limited to the dentineenamel junction; 2 dye penetration up to half of the pulp chamber; 3
dye penetration over half of the pulp chamber. Data were submitted to nonparametric KruskalWallis test (P < 0.05).
Water sorption and solubility

Disc-shaped specimens (n = 7), 6 mm in diameter (D) and 1 mm in height (h) were prepared for each material. The GIC specimens were prepared and allowed to set in the mould with polyester strips for 2 days, in order to avoid dehydration of the material. All specimens were stored in a desiccator at 37 C with silica gel, and were weighed daily to verify mass stabilization (dry mass, m1), which was represented by mass variations lower than 0.1 mg in any 24 h interval. Thereafter, the specimens were stored in distilled water at 37 C for 7 days to obtain the mass after saturation with water (m2). The specimens were then placed in the desiccator again, at 37 C, and reweighed again until a constant dry mass (m3) was obtained. Weighing was performed using an analytical balance with 0.1 mg accuracy (AG 200; Gehaka, Sa o Paulo, SP, Brazil). The volume (V) of each specimen was calculated based on the following equation: V = pR2h, where R is the specimen radius. Water sorption and solubility, given in lg mm)3, were calculated as follows: WS = m2 ) m3/V; SL = m1 ) m3/ V. Data were submitted to One-Way Analysis of Variance and Tukeys test (P < 0.05).
Toothbrushing abrasion and loss of mass

Five disc-shaped specimens were prepared for each material following the same procedures previously described. The specimens were ultrasonically cleaned (MaxiClean 750; Unique, Indaiatuba, SP, Brazil) in distilled water for 10 min and dry-stored at 37 C for stabilization of specimen mass. The pre-brushing mass (m1) was obtained by weighing the specimens every 24 h until a constant mass was achieved. The abrasion test was carried out in a multi-station brushing device. Each sample was brushed in a different station, using a soft nylon-bristled toothbrush with a brush-head load of 200 g. During the brushing cycle, the specimens were completely immersed in slurry of dentifrice (Colgate Total, Sa o Bernardo do Campo, SP, Brazil) and distilled water (1 : 2 wt ratio). In total, 5000 strokes (forward and reverse movement) were performed with a frequency of 4 Hz at 37 C. After testing, the specimens were cleaned with a air/ water spray for 1 min and in a ultrasonic bath for
895
10 min. They were then dry-stored at 37 C to constant mass (m2). Mass loss, expressed in mg, was calculated by the difference between m2 and m1. Data were submitted to One-Way Analysis of Variance and Tukeys test (P < 0.05). Representative specimens for each material before and after brushing were goldsputter coated (Denton Vacuum Desk II; Denton Vacuum, Moorestown, NJ, USA) for observation with scanning electron microscopy (SEM). Imaging of the surfaces was performed in secondary electron mode (JSM-5600LV; Jeol Inc., Peabody, MA, USA) at accelerating voltage of 15 kV.
Figure 2 Results for water sorption and solubility. Distinct letters indicate statistical differences amongst materials (P < 0.05).
Results Marginal sealing

Results are shown in Fig. 1. The KruskalWallis test revealed statistically signicant differences between groups (P < 0.0001). Bioplic produced the best marginal seal (all specimens with score 0), followed by Cavit. Vidrion R presented intermediate results, whilst IRM resulted in the poorest marginal seal (9 out of 10 specimens presenting score 3).
lower solubility was observed for Vidrion R and Bioplic compared with the other materials (P < 0.05).
Toothbrushing abrasion and loss of mass

Results of loss of mass after toothbrushing are shown in Fig. 3.One specimen of Vidrion R fractured during the brushing cycling and was replaced. Signicant differences were observed between materials (P < 0.05). Loss of mass after brushing was signicantly higher for Cavit (P < 0.05). Bioplic had intermediate loss of mass values, similar to IRM and to Vidrion R, which had the lowest loss of mass of all groups. SEM micrographs of the control and brushed surfaces are shown in Fig 4. Before abrasion, a relatively smooth surface was observed for all groups, especially for Bioplic and IRM. After toothbrushing, all materials had characteristic worn surfaces, with Cavit and Vidrion R showing an aggressive wear pattern characterized by extensive loss of substance for Cavit, and deep grooved scratches
Water sorption and solubility

Results are shown in Fig. 2. Signicant differences occurred between materials for water sorption (P < 0.01) and solubility (P < 0.01). Both parameters were signicantly higher for Cavit. IRM and Vidrion R presented similar intermediate values for water sorption, whilst Bioplic had the lowest values. Signicantly
Figure 1 Marginal leakage observed for the different tempo-
rary lling materials. Distinct letters indicate statistical differences amongst materials (P < 0.05).
Figure 3 Mass loss (mg) of the temporary lling materials after toothbrushing abrasion. Distinct letters indicate statistical differences amongst materials (P < 0.05).
896
BP
IR
CA
VD
Before
After
Figure 4 Representative SEM micrographs of the temporary lling materials before and after toothbrushing abrasion. A relatively smooth surface was observed for all groups before abrasion, especially for BP and IR. After toothbrushing, all materials presented characteristics of worn surfaces, with CA and VD showing an aggressive pattern of wear, characterized by extensive loss of substance for CA, and deep grooved scratches for VD. BP showed the least altered surface after abrasion.
for Vidrion R. Bioplic had the least altered surface after abrasion.
Discussion
The sealing ability of temporary llings can be evaluated in several ways (Cruz et al. 2002, Naoum ia et al. & Chandler 2002, Balto et al. 2005, Saua 2006). According to Raskin et al. (2001), lack of standardization of the test methods compromises comparisons and, therefore, the reliability of marginal sealing results. Methodological aspects of the test used in the study, namely basic fuchsine as leakage tracer, the thermocycling protocol and the assessment of dye penetration through sections of the specimen, have been reported as the most frequent choices in marginal sealing evaluations (Raskin et al. 2001). In this sense, the test protocol employed allows comparisons with similar studies, besides being a rapid way to determine the sealing ability of the materials used. Conventional GICs adhere to tooth tissue as a result of a chelation reaction with calcium (Culbertson 2001). Therefore, one could expect dye penetration in enamel to be lower than in dentine, since the former has more calcium available. However, 9 out of 10 specimens of Vidrion R group had dye penetration up to the enamel dentine junction, which might reect the effect of the thermocycling on the interaction between GIC and enamel and their different coefcients of thermal expansion. The bond strength of conventional GIC to tooth tissue is difcult to evaluate, due to the extremely
brittle nature of the cement, which leads to cohesive failure within the material (Mount 1991). Thus, one could hypothesize that the tracer percolated through fracture lines within the cement, close to the tooth/ restoration interface. Bioplic, a dimethacrylate-based temporary lling, prevented dye penetration in all the specimens. This material has the advantage of not requiring etching of the dental surface or application of an intermediate bonding material, thus eliminating additional clinical steps. According to the manufacturers information, Bioplic tends to expand in contact with moisture, improving its adaptation to the cavity walls. The lightcuring characteristic of Bioplic seems to be an important factor on its sealing ability, as the contact with the wet environment occurs after polymerization. In a previous study, Jenkins et al. (2006) observed considerably higher marginal sealing ability for a resin-based light-cured material in comparison with conventional self-curing cements and other temporary llings. Moreover, the translucency of Bioplic allows the passage of the curing light through the material, requiring a single light activation step, even with layers thicker than 2 mm. The eugenol-free ZO cement Cavit sets in contact with moisture, and has produced conicting sealing ia results (Uranga et al. 1999, Jenkins et al. 2006, Saua et al. 2006). The hygroscopic properties result in expansion of the material, potentially sealing the ia et al. tooth/lling interface (Cruz et al. 2002, Saua 2006), and might explain the absence of interfacial dye penetration in 70% of the samples. The presence of dye, though, was observed in the material itself, conrming
897
previous ndings (Cruz et al. 2002) and indicating the possibility of recontamination of the canal by bacterial inltration through the material itself. The sealing ability of the eugenol-based ZOE cement (IRM) was poor, conrming previous reports (Deveaux et al. 1999, Balto et al. 2005). Extensive degradation was observed, with the presence of dye within the body of the material (Zmener et al. 2004). Studies have pointed out that stress, such as the one imposed by thermocycling, promotes a signicant degradation of IRM (Gilles et al. 1975), whilst others indicate that variations in volume resulting from contraction of the material and the inhomogeneous mixing process could partially explain the poor sealing results with this lling (Deveaux et al. 1999). In addition, it has been reported that ZOE-based cements may impair the polymerization of resin composites, and should be avoided when nal restorations of such materials are to be made (Naoum & Chandler 2002). In contrast, temporary llings such as Bioplic and Vidrion R are compatible with resin-based materials, and theoretically do not need to be completely removed to execute the nal restoration. Water sorption and solubility were calculated by weight differences of specimens, and were used as a measure of the degradation of the llings (Carvalho Ju nior et al. 2003, Ferracane 2006) (Fig. 2). Carvalho Ju nior et al. (2003), also determined the sealing ability of temporary llings, and recommend that water sorption and solubility should be minimal. Usually, the absorption of water precedes events such as volumetric changes, swelling and softening of the materials (Ferracane 2006), which may compromise their microstructure and, as a consequence, the seal produced by the restoration. Water uptake is a key factor in the setting mechanism of Cavit. The expansion caused by the water diffusion is responsible for the sealing of the tooth/ restoration interface, but also allows the swelling of components from the spaces occupied by water (Ferracane 2006), explaining the high solubility observed for this material (Fig. 2). The intermediate sorption results observed with IRM and Vidrion R reect the cement nature of these materials, which characteristically absorb water. IRM had greater solubility than Vidrion R, conrming the previously reported disintegration this cement undergoes in contact with moisture. This process was explained by Wilson & Batchelor (1970) as eugenol loss of the cement matrix by aqueous leaching, resulting in microstructural degradation and reduction of mechanical strength. It is important to highlight that Vidrion R specimens were dehydrated in order to reach
the rst dry mass. Although this procedure does not mimic the in vivo situation, it is inherent to the test and might have caused appreciable structural modications in the GIC that might have inuenced the results. Resin-based materials have different patterns of water uptake, depending upon the chemical structure of the resin (Sideridou et al. 2007), which involves the hydrophilic nature of the monomers and differences between the solubility parameter of the monomers and the solvent (Ferracane 2006). In addition, the crosslink density of the polymer network is also important, since it dictates the presence and the amount of pendant molecules that could be swelled following water uptake (Ferracane 2006). In this sense, lightcured materials, such as Bioplic, justify their low water sorption and solubility by being able to set prior to contact with moisture. Brushing simulation was used in the present study to test the surface wear and degradation of the llings under cyclic mechanical challenge (Moraes et al. 2008). The eugenol-free ZO cement underwent considerable disintegration after brushing, as shown by the substantial loss of mass and the rough surface pattern observed (Figs 3 and 4). SEM images of the GIC Vidrion (Fig. 4) also depicted aspects of aggressive wear in the surface of the cement. Nevertheless, Vidrion had the lowest mass loss, indicating that the brushing action might affect only the surface of the material. SEM images of Bioplic and IRM (Fig. 4) revealed smoother surfaces after brushing, which indicate a more homogeneous wear pattern.
Conclusions
The resin-based light-cured temporary lling material Bioplic produced the best marginal sealing and was associated with the lowest water sorption, solubility and loss of mass in comparison with all other materials.
References
Balto H, Al-Nahan S, Al-Mansour K, Al-Otaibi M, Siddiqu Y (2005) Microbial leakage of Cavit, IRM, and Temp Bond in post-prepared root canals using two methods of guttapercha removal: An in vitro study. The Journal of Contemporary Dental Practice 6, 18. Carvalho Ju nior JR, Guimara es LFL, Correr Sobrinho L, cora J, Sousa Neto MD (2003) Evaluation of solubility, Pe desintegration, and dimensional alterations of a glass ionomer root canal sealer. Brazilian Dental Journal 14, 1148.
898
Cruz EV, Shigetani Y, Ishikawa K, Kota K, Iwaku M, Goodis HE (2002) A laboratory study of coronal microleakage using four temporary restorative materials. International Endodontic Journal 35, 31520. Culbertson BM (2001) Glass-ionomer dental restoratives (2001). Progress in Polymer Science 26, 577604. Deveaux E, Hilderbert P, Neut C, Boniface B, Romond C (1992) Bacterial microleakage of Cavit, IRM, and TERM. Oral Surgery, Oral Medicine, Oral Pathology 74, 63443. Deveaux E, Hildelbert P, Neut C, Romond C (1999) Bacterial microleakage of Cavit, IRM, TERM, and Fermit: A 21-day in vitro study. Journal of Endodontics 25, 6539. Ferracane JL (2006) Hygroscopic and hydrolytic effects in dental polymer networks. Dental Materials 22, 21122. Gilles G, Huget EF, Stone RC (1975) Dimensional stability of temporary restoratives. Oral Surgery Oral Medicine Oral Pathology 40, 796800. Hommez GM, Coppens CR, De Moor RJ (2002) Periapical health related to the quality of coronal restorations and root llings. International Endodontic Journal 35, 6809. Jenkins S, Kulild J, Williams K, Lyons W, Lee C (2006) Sealing ability of three materials in the orice of root canal systems obturated with gutta-percha. Journal of Endodontics 32, 2257. Koagel SO, Mines P, Apicella M, Sweet M (2008) In vitro study to compare the coronal microleakage of Tempit UltraF, Tempit, IRM, and Cavit by using the uid transport model. Journal of Endodontics 34, 4424. Laustsen MH, Munksgaard EC, Reit C, Bjrndal L (2005) A temporary lling material may cause cusp deection, infractions and fractures in endodontically treated teeth. International Endodontic Journal 38, 6537. Mayer T, Eickholz P (1997) Microleakage of temporary restorations after thermocycling and mechanical loading. Journal of Endodontics 23, 3202. Moraes RR, Ribeiro DS, Klumb MM, Brandt WC, CorrerSobrinho L, Bueno M (2008) In vitro toothbrushing abrasion of dental composites: packable, microhybrid,
nanohybrid and microlled materials. Brazilian Oral Research 22, 1128. Mount GJ (1991) Adhesion of glass-ionomer cement in the clinical environment. Operative Dentistry 16, 1418. Naoum HJ, Chandler NP (2002) Temporization of endodontics. International Endodontic Journal 35, 96478. jou J (2001) Raskin A, DHoore W, Gonthier S, Degrange M, De Reliability of in vitro microleakage tests: a literature review. Journal of Adhesive Dentistry 3, 295308. Ray HA, Trope M (1995) Periapical status of endodontically treated teeth in relation to the technical quality of the root lling and the coronal restoration. International Endodontic Journal 28, 812. ia TS, Brenda PF, Gomes BPFA et al. (2006) Microleakage Saua evaluation of intraorice sealing materials in endodontically treated teeth. Oral Surgery, Oral Medicine, Oral Pathology, Oral Radiology, and Endodontology 102, 2426. Sideridou ID, Karabela MM, Bikiaris DN (2007) Aging studies of light cured dimethacrylate-based dental resins and a resin composite in water or ethanol/water. Dental Materials 23, 11429. Soares IJ, Goldberg F (2002) Materais para restaurac o es rias em endodontia. In: Soares IJ, Goldberg F, eds. proviso cnica e Fundamentos, 1st edn. Porto Alegre, Endodontia: Te Brazil: Artmed, pp. 21729. Torabinejad M, Ung B, Kettering JD (1990) In vitro bacterial penetration of coronally unsealed endodontically treated teeth. Journal of Endodontics 16, 5669. Uranga A, Blum JY, Esber S, Parahy E, Prado C (1999) A comparative study of four coronal obturation materials in endodontic treatment. Journal of Endodontics 25, 178 80. Wilson AD, Batchelor RF (1970) Zinc oxide-eugenol cements: II. Study of erosion and disintegration. Journal of Dental Research 49, 5938. Zmener O, Banegas G, Pameijer CH (2004) Coronal microleakage of three temporary restorative materials: An in vitro study. Journal of Endodontics 30, 5824.
899
doi:10.1111/j.1365-2591.2009.01593.x
A comparative study of image quality and radiation exposure for dental radiographs produced using a charge-coupled device and a phosphor plate system
S. L. Farrier, N. A. Drage, R. G. Newcombe, S. J. Hayes & P. M. H. Dummer

School of Dentistry, Cardiff University, Wales, UK
Abstract
Farrier SL, Drage NA, Newcombe RG, Hayes SJ, Dummer PMH. A comparative study of image quality and
radiation exposure for dental radiographs produced using a charge-coupled device and a phosphor plate system. International Endodontic Journal, 42, 900907, 2009.
Aim To investigate the quality of periapical radiographic images produced by two digital dental radiography systems, a charge-coupled device (CCD) and a photostimulable phosphor (PSP) image plate system, and to examine the overall radiation exposure when using these systems in a clinical setting. Methodology Patients were randomly allocated to both systems and the resultant radiographs rated for quality. The expected radiation exposure for an investigation was calculated.
Results Overall, 98 images were acquired using the CCD system and 108 with the PSP system. The PSP system produced signicantly higher quality (P < 0.001) periapical images compared with the CCD system. The CCD system required signicantly more (P < 0.001) repeat exposures to obtain a diagnostic image than the PSP system but at a lower expected radiation exposure. Conclusions The image quality was superior using the phosphor plate system. Although more repeat radiographs were required using the CCD system, the images were produced with a lower expected radiation exposure. Keywords: dental digital radiography, radiation doses.
Received 3 February 2009; accepted 1 April 2009
Introduction
Digital radiography is increasingly being used in clinical practice. Two common systems employed use either a charge-coupled device based sensor (CCD) or a photostimulable phosphor (PSP) imaging plate system. The literature is replete with studies, conducted ex vivo, comparing the quality of image between CCD and PSP systems for diagnosis of a specic pathological condition, either naturally occurring or mechanically formed (Lim et al. 1996, Borg & Grondahl 1996a, Borg et al. 2000, Boscolo et al. 2001, de Almeida et al. 2003). Subsequently, various advantages and disad-
Correspondence: Nicholas Drage, Department of Dental Radiology, University Dental Hospital, Cardiff, CF14 4XY, UK (Tel.: 029 20766483; fax: 029 20743605; e-mail: nicholas. drage@cardiffandvale.wales.nhs.uk).
vantages of both CCD and PSP systems have been suggested but results tend to show both systems comparable in terms of image quality, with neither signicantly superior (Wenzel & Borg 1995, Kang et al. 1996, Velders et al. 1996, Borg et al. 1997, 1998, Cederberg et al. 1998, Versteeg et al. 1998, Syriopoulos et al. 2000). However, within the clinical environment there are many variables that may inuence the quality of the image obtained. Few studies have examined the effectiveness of either system for diagnostic purposes in vivo (Morner-Svalling et al. 2003) . It is well documented that the optimum individual exposure using the CCD system requires a lower radiation exposure than the PSP systems (van der Stelt 2005), but this does not take into account any repeat exposures that may be necessary. The aims of the study were therefore to investigate whether there were any differences in image quality and radiation exposure
900
Farrier et al. Subjective quality assessment of digital intraoral radiographs
between a CCD and a PSP digital system for periapical radiography.
Material and methods

The study was approved by the Cardiff & Vale NHS Trust Research and Development Committee (reference number 04-DH-3089), and South East Wales Local Research Ethics Committee. Adult patients, referred to the Radiology Department at the University Dental Hospital, Cardiff, UK and requiring periapical radiographs of at least one individual tooth, were recruited into the study. Written, informed consent was obtained from each patient, by the principal investigator (SF), prior to the radiograph being exposed. Two digital radiography systems were compared: the Sidexis CCD system (Sirona Dental Systems GmbH, Bensheim, Germany), and the Vistascan PSP system (Du rr Dental GmbH, Bissingen, Germany). The resolution for Sidexis CCD system is measured at <10lp mm)1. For the Vistascan the high resolution setting was chosen which corresponds to a measured resolution of 8 lp mm)1 (horizontal) and 10 lp mm)1 (vertical) The sensor sizes used were 31 mm 41 mm and 22 mm 35 mm for the Vistascan PSP system. For the Sidexis CCD system, the universal sensor which measured 25.4 mm 36.8 mm 6.6 mm (11 mm over cable insert) and the full size sensor which measured 29.9 mm 40.1 mm 6.8 mm (11.2 mm over the cable insert) were used. The active area of the Sidexis CCD system is 26 mm 34 mm for the full size sensor and 20 mm 30 mm for the universal sensor.
of 120 (60 per system) gives a power of 80% to detect this difference. The intention was to increase the total sample size to 240 (120 per system) in order to detect a difference of the order of 15% with a power of 80%. It was planned to use each system uniformly across six areas of the dentition, namely incisors and canines, premolars and molars, in both the maxillary and mandibular arches. Thus, 40 radiographs were to be used in each of the six regions, 20 allocated to each system according to a predetermined concealed randomization scheme. The chief investigator was blind to the system allocation until it was disclosed in the clinical setting. Some patients had requests for more than one tooth to be radiographed. If this was the case, the same digital system was used for all exposures, but only one radiograph formed part of this particular study. This was chosen by the principal investigator, before meeting the patient and taken rst.
Radiological process
Each radiograph was taken by the principal investigator using the paralleling technique, using an appropriate sensor holder and beam aiming device. The manufacturers instructions regarding exposure factors were followed for all examinations (Table 1). If the resultant image was deemed nondiagnostic by the principal investigator, a repeat exposure was carried out immediately using the same system. In the situation that a patient could not tolerate the sensor and holder, or a repeated intraoral digital image was again assessed undiagnostic, a conventional lm based intraoral radiograph or an extraoral radiograph was used to obtain the necessary information. These additional images were not evaluated in the study.
Sample size
The primary outcome was identied as image quality assessment rated on a 3-point scale as described later. Assuming 70% excellent, 20% satisfactory and 10% unsatisfactory are the quality assessment scores for one system, and 50% excellent, 20% satisfactory and 30% unsatisfactory are the scores for the other, a sample size
Table 1 Exposure factors for the digital
Evaluation of images
The principal investigator evaluated all images immediately after the exposure on a Fujitsu Siemens computer monitor (Hansol Electronics Inc., Jinchon-Kun
Time of exposure (seconds)
systems
Region Maxilla Incisors and canines Premolars Molars Incisors and canines Premolars Molars mA 7 7 7 7 7 7 kV 60 60 60 60 60 60
CCD, Sidexis 0.05 0.06 0.06 0.05 0.06 0.08
PSP, Vistascan 0.12 0.16 0.25 0.12 0.16 0.25
Mandible
CCD, charge-coupled device; PSP, photostimulable storage phosphor.
901
Subjective quality assessment of digital intraoral radiographs Farrier et al.
Table 2 Three-point scale for assessment of radiograph quality

Rating 1 2 3 Quality Excellent Diagnostically acceptable Unacceptable Basis No errors of patient preparation, exposure, positioning, processing or handling Some errors of patient preparation, exposure, positioning, processing or handling, but which do not detract from the diagnostic utility of the radiograph Errors of patient preparation, exposure, positioning, processing or handling, which render the radiograph diagnostically unacceptable
Choongbuk, Korea). Each image was assessed in a systematic fashion, within its own software and enhanced if necessary, and assigned a Quality Score (13), based on National Radiological Protection Board (NRPB 2001) guidelines (Table 2). To assess inter- and intra-observer variability, the set of images was reviewed and any with particularly memorable features were excluded. Then 60 images were selected using a stratied random sampling scheme. Three observers, the principal investigator, an experienced endodontic specialist and a maxillofacial radiologist assigned these a Quality Score (13), using the same guidelines and viewing conditions. The specialist endodontist and radiologist were also allowed to enhance the images if necessary. Images were selected equally from both systems, and from all areas of the mouth. Should images chosen be associated with a repeat exposure, this second image was also graded in the same manner. Each observer analysed the images in the same order and no knowledge of the previous quality score was available.
Inter- and intra-observer agreement was assessed using quadratic weighted kappa for the 3-point scale (Fleiss & Cohen 1973). For the binary decision as to whether repeat radiography should be performed, Scotts pi was used (Scott 1955, Newcombe 1996), with CIs calculated by the method of Donner & Eliasziw (1992). Proportions of radiographs rated as excellent, acceptable and unacceptable were compared to NRPB targets using upper and lower tail probabilities based on summation of trinomial probabilities generalizing P-values (Newcombe & Farrier 2008).
Results Sample
In total, 209 patients were included in the study; 108 subjects were male and 101 female, with ages ranging from 17 to 90 years. Table 3 shows the number of radiographs from each of the six areas of the mouth allocated to each system. Ideally 240 images should have been obtained. However, it was not possible to collect the planned numbers of mandibular incisors and premolars in the time available for the study. In total, 206 images from 206 different patients were assessed as three patients could not tolerate the digital sensor.
Radiation exposure
The observed probability of requiring a repeat radiograph and the standard exposure times given for the different regions for the two systems were considered. From this both the overall average exposure time and radiation exposure (surface entrance doses) were calculated, with condence intervals.
Quality of the original periapical radiographs

Table 3 shows the quality scores assigned to the 206 images obtained by the principal investigator. A 1 df trend component chi-square indicates a highly signicant preference for the PSP system (v2 = 26.3, P < 0.001), with a very large difference commensurate with what was initially assumed in the power calculation. Clinically the most relevant dichotomization was obtained by combining categories 1 and 2, (excellent and clinically acceptable), in contrast to category 3 (unacceptable, undiagnostic). The proportion of images judged excellent or acceptable was 94% for PSP and 78% for CCD. The estimated difference in the proportions unacceptable is 17% with 95% CI from 8% to 27%.
Chi-squared tests were used to compare radiographic quality scores between the two systems. A 1 degree of freedom v2 was used for the binary variable indicating whether a repeat was required, and a 1 df trend component is reported for analyses relating to the 3-point ordinal radiographic quality score. Condence intervals (CIs) for differences between proportions were calculated using method 10 of Newcombe (Newcombe 1998), and CIs for weighted means of proportions analogously.
902
Table 3 Quality scores for the two radiography systems. These show the numbers of radiographs taken for each area of the dentition, and the quality scores of the original periapical radiographs as assessed by the principal investigator
Number of radiographs 20 20 20 7 13 18 98 20 20 20 10 18 20 108 Quality score Excellent 6 7 8 3 6 6 36 11 18 14 7 11 16 77 (30%) (35%) (40%) (43%) (46%) (33%) (37%) (55%) (90%) (70%) (70%) (61%) (80%) (71%) Acceptable 13 11 6 4 2 4 40 7 2 5 3 6 2 25 (65%) (55%) (30%) (57%) (15%) (22%) (41%) (35%) (10%) (25%) (30%) (33%) (10%) (23%) Unacceptable 1 2 6 0 5 8 22 2 0 1 0 1 2 6 (50%) (10%) (30%) (38%) (44%) (22%) (10%) (5%) (6%) (10%) (6%)
Radiography system CCD
Area of dentition Maxillary incisors Maxillary premolars Maxillary molars Mandibular incisors Mandibular premolars Mandibular molars Total Maxillary incisors Maxillary premolars Maxillary molars Mandibular incisors Mandibular premolars Mandibular molars Total
PSP
CCD, charge-coupled device; PSP, photostimulable phosphor.
Repeat required for clinical purposes

Repeat exposures were actually performed for 27 (27%) of 98 radiographs using the CCD system and 8 (7%) of 108 radiographs using the PSP system. This was because the clinician in charge of the patients care deemed some of the images unacceptable when SF deemed them acceptable and vice versa. Nevertheless, this 20% difference (95% CI 10% to 30%, v2 = 14.8, P < 0.001) was closely in line with the results shown in Table 3.
Quality scores in relation to area of dentition

Table 3 shows the quality scores of the original periapical radiograph as graded by the principal investigator with regard to the area of the dentition. There were marked differences (>20%) between the two systems in favour of the PSP system, for all areas of the dentition. The greatest differences were for maxillary premolars and molars, and mandibular molars. The observed overall difference in repeat rates between the CCD and PSP systems needed slight adjustment to allow for the imbalance in numbers of radiographs taken in each area of the dentition. In the maxillary arch the required samples of 20 incisors, 20 premolars and 20 molars were studied. However, in the mandibular arch, this was not the case and fewer subjects used the CCD system than the PSP system. Therefore, a balanced scorecard, consisting of equal numbers of radiographs in all areas of the dentition was used to provide a more accurate representation. The projected proportion requiring a repeat periapical radiograph for the CCD system is 27% and for the PSP system 6.8%, a difference of 20.2% (95% CI 10 30%, v2 = 14.8, P < 0.001). It is therefore reasonable to say that the difference between the two systems is not substantially related to tooth type.
Quality of the repeat periapical radiographs

Analyses for the quality scores for the repeat radiographs and whether a further repeat exposure was required were restricted to 34 patients (Table 4). The results of this showed a similar pattern to the original radiographs but statistical signicance was not reached due to the very small sample size.
Table 4 Quality scores of repeat radiographs

Radiography system Quality score Excellent Acceptable Unacceptable Total CCD 5 (20%) 14 (54%) 7 (27%) 26 PSP 5 (63%) 2 (25%) 1 (13%) 8
Inter- and intra-observer variability

Weighted kappa for the 3 point scale of quality of the original images ranged from 0.460.72 for inter-
CCD, charge-coupled device; PSP, photostimulable phosphor.
903
observer agreement and was 0.98 for intra-observer agreement (SF), with broadly similar results for the repeat radiographs. For the binary outcome of whether a repeat radiograph was judged necessary, Scotts pi ranged from 0.60 (95% CI 0.300.80) to 0.87 (95% CI 0.590.96) for inter-observer agreement and was 1.0 (95% CI 0.761.0) for intra-observer agreement.
Performance targets
The 3 point quality scores for each system were compared to the minimum target for radiographic quality (70% excellent, 10% unsatisfactory) suggested by the National Radiological Protection Board (NRPB 2001). It is clear that the results obtained for the CCD system fell short of these targets. Conversely, the results obtained for the PSP system, with 71% excellent and only 6% unsatisfactory, were ostensibly slightly better than the ultimate target performance for quality. For the PSP system, the probability of observing performance as good as, or better than, the observed proportions of 71% excellent and 6% unacceptable, assuming that in the underlying population the method yields exactly 70% excellent and 10% unacceptable quality of images, may be calculated as a summation of multinomial probabilities. For these results, an upper tail probability U = 0.058 was obtained, which represents the probability that results as good as or better than those observed would arise if the true population proportions were 70% excellent, 20% acceptable and 10% unacceptable. A corresponding lower tail probability of L = 0.64 was obtained, representing the probability of results no better than those observed. Both U and L use a mid-P accumulation of tail probabilities (Lancaster 1949). A high value of L with a low value of U indicates that the observed results surpass the standard, whereas a high U and a low L indicate results that fall short of the standard. For the CCD system, U > 0.99 and L < 0.0001 which indicated strongly that the performance of this system fell short of the target.
5.98 mGy/s. Using this value an entrance doses for the examinations was derived. Based on a balanced scorecard and disregarding repeat exposures, the mean time of exposure of the CCD system was 0.06 s. The expected additional exposure due to the 27.6% risk of requiring a repeat exposure, based on all areas of the dentition is 0.0171 s, with a 95% condence interval of 0.01310.0232 s. Taking account of the original periapical radiograph and a single repeat if required, the average time for the CCD system was 0.0771 s with a 95% CI from 0.0731 to 0.0832. This translated into an expected 0.46 mGy radiation entrance dose, with a 95% CI of 0.44 to 0.50 mGy. Similarly, for the PSP system the mean exposure time averaged across the dentition was 0.1767 s. The expected additional exposure due to the 7.4% risk of requiring a repeat was 0.0122 s, with a 95% CI from 0.0077 to 0.0274. Thus, taking into account the original periapical radiograph and a single repeat if required, the average exposure time with the PSP system was 0.1889 s, with a 95% CI from 0.1844 to 0.2041. This translated into a 1.13 mGy expected radiation surface dose, with a 95% CI of 1.10 1.22 mGy. Therefore, the difference in average exposure time between the CCD and PSP systems based on the balanced scorecard and taking into account the original radiograph and repeat radiograph if required, was estimated to be 0.18890.0771 = 0.1118 s, in favour of the CCD system. The 95% condence interval for this difference is from 0.10420.1275 s. This translated into a 0.67 mGy difference in expected radiation surface dose with a condence interval of 0.620.76 mGy.
Discussion
In this study comparing CCD and PSP systems in the clinical environment, patients were allocated randomly to a digital system and stratication methods used so that each area of the dentition was uniformly examined. The principal investigator, a qualied dentist, was responsible for exposing and assessing all the radiographs and was blind to the system allocation until it was disclosed in the clinical setting to ensure a more fair comparison. The subjective image quality rating score as described by the NRPB was chosen since it is based on the diagnostic potential of the image produced (NRPB 2001). This rating system is recommended when
Radiation exposure
The physical characteristics of the X-ray machine used (Siemens Heliodent DS, Bensheim, Germany) were measured by the radiation physicist as part of the annual radiation safety survey. The 60 kV machine gave a dose rate at the end of the spacer cone of
904
conducting audits based on the quality of radiographs and is thus familiar and clinically relevant. Since this is an established grading system used in the UK and by its very nature is subjective no calibration of the investigators was performed. Each investigator was familiar with the grading scheme before the study commenced and deemed whether the radiographs were diagnostically acceptable independently. Various studies have been carried out which suggest that digital equipment may improve the quality of the image, especially if the contrast and density are not optimum (Wenzel 1993, Gotfredsen et al. 1996, Yoshiura et al. 1999, Svanaes et al. 2000, Li 2004). Therefore, manipulation of the digital images was allowed by all observers within the appropriate system software, since this made the study more reminiscent of the clinical environment. The same monitor was used to view both types of image so that confounding screen factors were not introduced. Each image was analysed within its own software and it is possible that this could lead to observer bias. An alternative approach would have been to export the digital images to one common viewing environment, as was done by Berkhout et al. (2004), this would have made the observers blind to the identity of the imaging systems. However, a more realistic assessment was desired by enabling the observers to manipulate the image within their own software so as to simulate a true clinical situation. The active areas of the CCD sensor and PSP imaging plates are also different and thus masking the borders would also have been required for all bias to be eliminated. This would have resulted in some of the image from the PSP systems being absent from view, since it has a larger active pixel area and thus some vital radiographic data could be prevented from being viewed and this would have had a direct impact on the relevant quality score. The overall quality of the PSP system was found to be signicantly better than the quality of the images produced with the CCD system. This agrees with Borg & Grondahl (1996b) and Boscolo et al. (2001), but is the opposite to that reported by de Almeida et al. (2003), however, these studies were ex vivo and involved imaging dried specimens with soft tissue equivalents where control of radiographic positioning is more consistent and reproducible. In addition, these studies did not use the NRPB system for grading image quality. There was a 20% difference between the two systems in favour of the PSP digital system as to whether a repeat image was required (95% CI 1030%, P < 0.001). This is potentially a very important benet
since such a difference could have a marked inuence on the patient dose received, the ease at which the process is carried out by both the patient and clinician and the time taken for a successful radiological examination. Suggested explanations for the improved quality of the PSP images and the less frequent need for a repeat exposure can be sensibly combined. The CCD sensor is far more bulky and stiff than the PSP imaging plates and has a cable attached; it is also associated with a larger sensor holder and beam aiming device. The CCD sensor was found to be more difcult to position than the PSP imaging plates. Patients susceptible to gagging also found the CCD more difcult to tolerate than the PSP sensor. The active pixel area of the two sensors also differs, which may be a contributing factor to the image quality. The larger active image size of the PSP imaging plates enables more information to be captured and a greater probability that all the relevant information required is actually recorded. Difculties in positioning CCD sensors have been reported before (Wenzel & Moystad 2001, Berkhout et al. 2002). In two surveys of dental practitioners, there were signicant problems with the positioning of CCD sensors with an increase in the number of CCD images taken compared with PSP systems (Wenzel & Moystad 2001, Berkhout et al. 2002). In addition, when compared to conventional lm it has been shown that there is an increase in the number of unsatisfactory images with the CCD systems (Berkhout et al. 2003). The balanced scorecard allowed for the imbalance in numbers throughout the areas of the dentition and between the two digital systems. This gives a projected difference of 20.2% between the CCD and PSP systems in favour of the PSP system regarding whether or not a repeat radiograph is required. The CCD system did not reach the suggested NRPB quality targets. The PSP system performed slightly better than the ultimate targets. These performance targets do not take into consideration the radiation dose received by patients in order to eventually obtain the correct diagnostic information. The mean exposure time and radiation surface dose for the PSP is greater than that for the CCD system by a factor of 2.45. Therefore, despite the CCD system requiring more repeat exposures, the radiation received by the patient is less. In a questionnaire based survey comparing CCD and PSP systems, CCD systems showed a larger dose reduction in comparison to PSP imaging plates (Berkhout et al. 2004). However, the authors
905
also raised concerns that the radiation reduction may be less than originally perceived as more CCD exposures were carried out than PSP exposures. In the context of the present study, it might have been possible to reduce the mean exposure time without affecting the quality for the PSP system, since the sensor has a very wide exposure latitude; this is an area for further research. In this study, if conventional radiographs or extraoral radiographs were required because the patient could not tolerate the sensor or there had been two failures using the digital sensor the additional radiation exposure was not included in the calculations. Another study reported that the dose reduction as a result of shorter exposure times exceeded the increase in doses as a result of the greater number of radiographs with both digital systems (Berkhout et al. 2003). However, with the CCD sensors the dose reduction per exposure was almost cancelled out by the increase in the number of radiographs taken. These results are very different to the ndings of the present study.
Conclusion
The PSP Vistascan system produced signicantly higher quality intraoral periapical images compared with the CCD Sidexis system. The CCD system did not reach the set performance targets of 70% excellent and 10% unsatisfactory. There was also a signicantly higher repeat rate using the CCD system compared to the PSP system. The mean exposure time and radiation exposure for the PSP system is greater than for the CCD system.
Acknowledgement
The authors are grateful to Paul Beere and his staff in the dental radiology department for their help in this research project.
References
de Almeida SM, de Oliveira AE, Ferreira RI, Boscolo FN (2003) Image quality in digital radiographic systems. Brazilian Dental Journal 14, 13641. Berkhout WE, Sanderink GC, Van der Stelt PF (2002) A comparison of digital and lm radiography in Dutch dental practices assessed by questionnaire. Dentomaxillofacial Radiology 31, 939. Berkhout WE, Sanderink GC, Van der Stelt PF (2003) Does digital radiography increase the number of intraoral radio-
graphs? A questionnaire study of Dutch dental practices. Dentomaxillofacial Radiology 32, 1247. Berkhout WE, Beuger DA, Sanderink GC, van der Stelt PF (2004) The dynamic range of digital radiographic systems: dose reduction or risk of overexposure? Dentomaxillofacial Radiology 33, 15. Borg E, Grondahl HG (1996a) Endodontic measurements in digital radiographs acquired by phostimulable storage phosphor system. Endodontic Dental Traumatology 12, 204. Borg E, Grondahl HG (1996b) On the dynamic range of different X-ray photon detectors in intra-oral radiography. A comparison of image quality in lm, charge-coupled device and storage phosphor systems. Dentomaxillofacial Radiology 25, 828. Borg E, Grondahl K, Grondahl HG (1997) Marginal bone level buccal to mandibular molars in digital radiographs from charge-coupled device and storage phosphor systems. An in vitro study. Journal of Clinical Periodontology 24, 30612. Borg E, Kallqvist A, Grondahl K, Grondahl HG (1998) Film and digital radiography for detection of simulated root resorption cavities. Oral Surgery Oral Medicine Oral Pathology Oral Radiology & Endodontics 86, 1104. Borg E, Attaelmanan A, Grondahl HG (2000) Subjective image quality of solid-state and photostimulable phosphor systems for digital intra-oral radiography. Dentomaxillofacial Radiology 29, 705. Boscolo FN, Oliveira AE, Almeida SM, Haiter CF, Haiter Neto F (2001) Clinical study of the sensitivity and dynamic range of three digital systems, E-speed lm and digitized lm. Brazilian Dental Journal 12, 1915. Cederberg RA, Tidwell E, Frederiksen NL, Benson BW (1998) Endodontic working length assessment. Comparison of storage phosphor digital imaging and radiographic lm. Oral Surgery Oral Medicine Oral Pathology Oral Radiology & Endodontics 85, 3258. Donner A, Eliasziw M (1992) A goodness-of-t approach to inference procedures for the kappa statistic: condence interval construction, signicance-testing and sample size estimation. Statistics in Medicine 11, 15119. Fleiss JL, Cohen J (1973) The equivalence of weighted kappa and the intraclass correlation as measures of reliability. Educational and Psychological Measurement 33, 6139. Gotfredsen E, Wenzel A, Grondahl HG (1996) Observers use of image enhancement in assessing caries in radiographs taken by four intra-oral digital systems. Dentomaxillofacial Radiology 25, 348. Kang BC, Farman AG, Scarfe WC, Goldsmith LJ (1996) Observer differentiation of proximal enamel mechanical defects versus natural proximal dental caries with computed dental radiography. Oral Surgery Oral Medicine Oral Pathology Oral Radiology & Endodontics 82, 45965. Lancaster HO (1949) The combination of probabilities arising from data in discrete distributions. Biometrika 36, 37082.
906
Li G (2004) Comparative investigation of subjective image quality of digital intraoral radiographs processed with 3 image-processing algorithms. Oral Surgery Oral Medicine Oral Pathology Oral Radiology & Endodontics 97, 7627. Lim KF, Loh EE, Hong YH (1996) Intra-oral computed radiography an in vitro evaluation. Journal of Dentistry 24, 35964. Morner-Svalling AC, Tronje G, Andersson LG, Welander U (2003) Comparison of the diagnostic potential of direct digital and conventional intraoral radiography in the evaluation of peri-implant conditions. Clinical Oral Implants Research 14, 7149. Newcombe RG (1996) The relationship between chi-square statistics from matched and unmatched analyses. Journal of Clinical Epidemiology 49, 1325. Newcombe RG (1998) Interval estimation for the difference between independent proportions: comparison of eleven methods. Statistics in Medicine 17, 87390. Newcombe RG, Farrier SL (2008) A generalisation of the tailbased P-value to characterise the conformity of trinomial proportions to prescribed norms. Statistical Methods in Medical Research 17, 60919. NRPB (2001) Guidance Notes for Dental Practitioners on the Safe Use of X-Ray Equipment. Department of Health, London. Scott WA (1955) Reliability of content analysis: the case of nominal scale coding. Public Opinion Quarterly 19, 3215. van der Stelt PF (2005) Filmless imaging: the uses of digital radiography in dental practice. Journal of the American Dental Association 136, 137987.
Svanaes DB, Moystad A, Larheim TA (2000) Approximal caries depth assessment with storage phosphor versus lm radiography. Evaluation of the caries-specic Oslo enhancement procedure. Caries Research 34, 44853. Syriopoulos K, Sanderink GC, Velders XL, van der Stelt PF (2000) Radiographic detection of approximal caries: a comparison of dental lms and digital imaging systems. Dentomaxillofacial Radiology 29, 3128. Velders XL, Sanderink GC, van der Stelt PF (1996) Dose reduction of two digital sensor systems measuring le lengths. Oral Surgery Oral Medicine Oral Pathology Oral Radiology & Endodontics 81, 60712. Versteeg CH, Sanderink GC, van Ginkel FC, van der Stelt PF (1998) An evaluation of periapical radiography with a charge-coupled device. Dentomaxillofacial Radiology 27, 97 101. Wenzel A (1993) Computer-aided image manipulation of intraoral radiographs to enhance diagnosis in dental practice: a review. International Dental Journal 43, 99108. Wenzel A, Borg E (1995) Accuracy of caries diagnosis in digital images from charge-coupled device and storage phosphor systems: an in vitro study. Dentomaxillofacial Radiology 24, 2504. Wenzel A, Moystad A (2001) Experience of Norwegian general dental practitioners with solid state and storage phosphor detectors. Dentomaxillofacial Radiology 30, 2038. Yoshiura K, Kawazu T, Chikui T et al. (1999) Assessment of image quality in dental radiography, part 1: phantom validity. Oral Surgery Oral Medicine Oral Pathology Oral Radiology & Endodontics 87, 11522.
907
doi:10.1111/j.1365-2591.2009.01594.x
Microora in teeth associated with apical periodontitis: a methodological observational study comparing two protocols and three microscopy techniques
N. Richardson, N. J. Mordan, J. A. P. Figueiredo, Y-L. Ng & K. Gulabivala

Units of Endodontology & Microscopy, Divisions of Restorative Dental Science and Biomaterials & Tissue Engineering, UCL Eastman Dental Institute, University College London, London, UK
Abstract
Richardson N, Mordan NJ, Figueiredo JAP, Ng Y-L, Gulabivala K. Microora in teeth associated with apical
periodontitis: a methodological observational study comparing two protocols and three microscopy techniques. International Endodontic Journal, 42, 908921, 2009.
Aim The aim of this study was to compare two protocols to examine bacterial colonization in teeth associated with chronic apical periodontitis with acute episodes (ap), using light microscopy (LM), transmission electron microscopy (TEM) and scanning electron microscopy (SEM). Methodology Nine root samples (seven teeth) were processed using either Eastman Dental Institute (EDI) (n = 4 teeth/4 roots) or Zurich (n = 3 teeth/5 roots) protocols. The roots were sectioned longitudinally; one root portion was viewed with SEM, descriptively dividing its length into apical, middle and coronal; semi-thin and ultra-thin transverse sections were viewed under LM and TEM from each third of the other root portion. Each root was therefore examined using all microscopy techniques. Observations of bacterial presence, description and distribution within the root canal lumen and root dentine were systematically recorded using pre-determined criteria.
Results The Zurich technique gave a more predictable division of the root, but the surface was slightly smeared and demineralization was incomplete. The Eastman Dental Institute (EDI) approach appeared to provide better ultrastructural detail. Bacteria were detected in eight of the nine roots. Bacterial biolms were commonly seen adhering to the root canal surface, containing various cellular morphotypes: rods, cocci, laments and spirochaetes. Bacteria were more evident apically than coronally, associated with the canal wall but were more commonly evident coronally than apically within the dentinal tubules. Polymorphs (PMNs) were found in all the root thirds, especially apically, often numerous and walling off the bacterial biolm from the remaining canal lumen. Conclusions Both protocols had merits and de-merits. The combination of microscopy techniques offered complementary views of intra-radicular bacterial colonization. The perception of connement of the host/ microbial interface at the apical foramen is not entirely correct; PMNs may be found even in the coronal third of root canals containing necrotic pulp tissue. Keywords: intra-radicular bacteria, microscopy, protocols, root canal.
Received 12 August 2008; accepted 25 March 2009
Introduction
The role of a polymicrobial infection of the root canal system in apical periodontitis is well established (Kakehashi et al. 1965, Sundqvist 1976, Fabricius et al. 1982, Tani-Ishii et al. 1994) but deep insights into the ecology and physiology of the bacterial
Correspondence: Professor Kishor Gulabivala, Unit of Endodontology, UCL Eastman Dental Institute, 256 Grays Inn Road, London WC1X 8LD, UK (Tel.: 020 7915 1033; fax: 020 7915 2371; e-mail: k.gulabivala@eastman.ucl.ac.uk).
908
Richardson et al. In situ microscopy of endodontic microora
colonization remain elusive. Much of the current knowledge of intra-radicular infection stems from in vivo and ex vivo culture studies of sampled bacteria; such approaches tend to bias the revealed micro-ora (Akpata 1976, Kumar et al. 2002). The picture of bacterial diversity is inuenced by many factors, including growth conditions, sub-culture strategy and the nature of bacterial identication (Rolph et al. 2001, Kumar et al. 2002, Munson et al. 2002, Gulabivala 2004). The number of detected and identied taxa per tooth has increased from 1 to 12 cultured varieties up to 20 phylotypes using culture-independent techniques, with estimates of actual numbers up to 90 (Rolph et al. 2001, Munson et al. 2002). Whilst, the known diversity of the microora has increased with improved culture techniques and culture-independent techniques, direct microscopy suggests, as indeed it did even in the time of Miller (1894) that a proportion of the ora still remains uncultured. Furthermore, the process of sampling disturbs insights about the intimate and intricate relationships between bacteria and their abiotic environment (Nair 1987). Microscopically, bacterial strains are evident as cocci, rods, lamentous or spiral morphotypes and have been shown in a landmark paper to exist mainly in a biolm lining the root canal wall in the root apex (Nair 1987). This paper provided the rst real insight into the morphological distribution of the root canal ora in the root apex and its association with the host response. Study of the excellent photo-micrographs provides visual evidence to support the predicted ecological and physical spatial relationships between bacteria (Sundqvist 1992). Different microscopy techniques possess different properties and propensities to reveal the inherent truth about the bacterial distribution and its structure. Light microscopy (LM) remains a useful base-line technique to provide an overall perspective but lacks resolution to reveal ner details. In contrast, transmission electron microscopy (TEM) possesses the high resolution to reveal ultra-structural details, losing something of the perspective as a trade-off. Hence, Nair used the approach he described as correlative LM and electron microscopy studies to decipher both aspects. Although not using the term biolm, he provided the rst real detailed description of the root canal biolm within root apices, as it related to the aetio-pathogenesis of apical periodontitis. The structure of the microora within the entire tooth, as it relates to approaches to treatment has been little studied.
The validity of the observations made by microscopy rests on the assumption that the processing stages have accurately preserved the anatomical structures and that the imaging system possesses the means and resolution to highlight the relevant features. Knowledge of imaging principles is essential but empirical studies are also necessary to reveal the true in situ potential of microscopy techniques. Distortion of tissues and translocation of structural components are possible but need to be minimized or else recognized as artefacts. Detection of such artefacts may not be straightforward but is an important element in the critical appraisal of ndings. To this end, the nature of sample xation and processing may also inuence results. The aim of this methodological observational study was to compare different tooth processing protocols and microscopy (LM, SEM and TEM) techniques to examine bacterial colonization within the coronal, middle and apical thirds of roots associated with apical periodontitis.
Materials and methods Sample collection and storage

The material for this study consisted of extracted human teeth with radiographically evident periapical lesions (and associated acute episodes) and an absence of periodontal disease or previous pulpal therapy. The teeth were carefully extracted by General Dental Practitioners with minimal pumping motion (Kapalas et al. 2001, 2002) and immersed into tubes containing 3% glutaraldehyde (Agar Scientic, Stanstead, UK) in 0.1 mol L)1 sodium cacodylate (Agar Scientic) after de-coronation with a sterile diamond bur. The sample teeth were stored at 4 C to provide a total xation period of 1 week. Informed consent had been obtained from the patients prior to inclusion in the study pool; seven teeth meeting the above criteria were selected for the study.
Processing for microscopy

Two methods of sample processing were used: (i) the EDI protocol (Vrahopoulos 1989), which involved demineralization after embedding; and (ii) the Zurich protocol (Nair 1987), which involved demineralization before embedding. The seven selected teeth were randomly assigned to the two processing groups; EDI protocol (n = 4 teeth/4 roots with apical periodontitis) and Nair protocol (n = 3 teeth/5 roots/4 roots with
909
In situ microscopy of endodontic microora Richardson et al.
apical periodontitis). An overview of the key stages in the two processing protocols is shown in Fig. 1.
LM and TEM examination, whilst the other half was used for SEM examination. Processing for SEM The root halves allocated for SEM examination were dehydrated in a graded series of alcohol (20%, 50%, 70%, 90% and 3 100% for 10 min each), placed in hexamethyldisilazane (HMDS) (TAAB Laboratories Ltd, Reading, UK) for 5 min, then removed and left on lter paper for 23 h for the HMDS to evaporate. The samples were attached to aluminium SEM stubs (Agar Scientic) using carbon conducting cement (Neubauer Chemikalien, Munster, Germany) and sputter-coated with gold/palladium in a Polaron E5000 Sputter Coater (Quorum Technologies Ltd, Newhaven, UK).
Tooth collection
EDI protocol
Longitudinal splitting of the roots The roots were grooved longitudinally using an ultrane diamond disc (Metrodent, Hudderseld, UK) along the narrowest surface of the root in a fume cupboard (Labcaire, Clevedon, UK). The root was then rmly pressed into unset lab putty (Optosil and Xantopren; Heraeus Kulzer, Hanau, Germany) and allowed to set. Splitting of the root was completed using an osteotome, exposing the pulp canal space in both sections. The section containing more hard tissue was used for the
EDI protocol
Fixed in 3% glutaraldehyde in 0.1% sodium cacodylate
Zurich protocol
Tooth split longitudinally with an osteotome
Placed in EDTA
Dehydrated
4 months later, teeth cut longitudinally with razor blade
Embedded in resin
SEM processing
Dehydrated
Cut into 1 mm slices & placed in EDTA
SEM processing
Embedded in resin
Coronal
Re-dehydrated & re-embedded
1 mm
Middle
Cut into 4 sections
Apical
Ultra-thin sectioning for TEM
Semi-thin sectioning for LM
Ultra-thin sectioning for TEM
Semi-thin sectioning for LM
Figure 1 Flow chart showing the succession of stages for each processing protocol.
910
The specimens were viewed in a Cambridge Stereoscan 90B (LEO Electron Microscopy Ltd, Cambridge, UK) operating at 15 kV and digital images were captured using i-scan 2000 software (ISS Group, Manchester, UK). Processing for LM and TEM The root halves allocated for LM and TEM were dehydrated in a graded series of alcohol (20%, 50%, 70% and 3 90% for 10 min each) and inltrated with LR White resin (The London Resin Company, London, UK). This was performed in stages as follows: initial immersion in LR White resin and 90% alcohol (ratio of 1 : 1) for 2 h at 4 C; immersion in pure fresh LR White for 30 min at 4 C; immersion in fresh LR White overnight (1012 h) at 4 C and the following morning, for 1 h, at 4 C. The sections were embedded in tinfoil containers (Buyrite UK Ltd, Aldershot, UK) containing 20 mL of LR White and 30 lL LR White accelerator. Air was excluded from the setting process using paralm (Agar Scientic) over the exposed resin mix, which was polymerized for 1 h in the freezer, then overnight at 4 C and then removed to warm up to room temperature. The embedded roots were sliced transversely using a high-speed diamond saw (Exact, Aberdeen, UK) into 1mm thick sections. The slices were decalcied in 0.15 mol L)1 EDTA in specimen tubes for 38 weeks at room temperature on a tissue rotator at 2 rpm (TAAB, Rotator type N; Agar Scientic). The EDTA solution was changed every 23 days until the dentine could be easily cut with a single edge carbon steel razor blade (Agar Scientic). The slices were dehydrated again and re-embedded in LR White resin as described above. Sectioning for LM Semi-thin sections of 0.5 and 1 lm were cut with a Diatome (Diatome AG, Biel, Switzerland) diamond knife on an ultramicrotome (Reichert UltracutE; Cambridge Instruments, Cambridge, UK). These were stained with toluidine blue and used to check sample orientation before proceeding with LM and TEM. Slides were viewed on an Olympus BX50 optical microscope (Olympus, Southall, UK). Sectioning for TEM Ultra-thin sections (90100 nm) were cut using the same technique, and collected on either carbon-formvar coated copper 200 mesh grids (Agar Scientic) or gold 400 mesh grids (Agar Scientic). The sections were then stained on the grid with 0.4% (w/v) uranyl
acetate in absolute alcohol for 5 min, followed by 5 min in Reynolds (1963) lead citrate. Sections were examined on a TEM (100CXII; JEOL, Welwyn Garden City, UK) operating at 80 kV and images were recorded onto Kodak 4 EM lm (TAAB Laboratories Ltd).
Zurich protocol
Demineralization The roots were placed in 0.15 mol L)1 EDTA and 0.5% glutaraldehyde (Agar Scientic) in specimen tubes on a tissue rotator at 2 rpm. Initially, the EDTA solution was replaced every 23 days over 3 months, and then changed everyday for the remaining 1 month. Progress was checked by carefully inserting a single-edged carbon steel razor blade (Agar Scientic) into the dentine, taking care not to penetrate to the root canal. Longitudinal cutting of the roots After approximately 4 months in EDTA, the roots were demineralized sufciently to allow, gentle, controlled, longitudinal cutting of the roots. At this point, one-half of each root was randomly designated for SEM and the other half for LM and TEM. The root associated with the periapical lesion was used from each tooth, except for tooth R6, a molar, from which all three roots were used for comparison, although only two were radiographically associated with periapical lesions (R6 a, b Table 1). The root halves designated for SEM were dehydrated to 100% ethanol, immersed in HMDS and allowed to dry as for the EDI protocol, handling with greater care because of the demineralization. Those samples due for TEM examination were dehydrated to 90% ethanol and embedded in LR White resin in the same manner as the initial part of the EDI protocol without the necessity for demineralization and re-embedding.
Selection of elds of view for both protocols

Scanning electron microscopy The entire root half was rst examined under low magnication. Then, starting coronally, the root was examined horizontally millimeter by millimeter, using the lbar on the image as a guide. At each millimeter level, the site of examination was magnied to 5000. This horizontal scanning was repeated at the next adjacent apical level until the entire root canal had been traversed. Observations were made on this basis and representative photographic images were recorded
911
Table 1 Summary of viewable elds for each protocol (EDI/Zurich) and the presence/absence of bacteria by microscopy technique,
tooth, root and segment

Periapical lesion visible on radiograph 4 4 4 4 4 4 4 4 4 4 4 4 4 4 4 4 4 4 4 4 4 4 4 4 SEM Dentinal tubules o 4 o 4 4 o LM Dentinal tubules o o o o 4 4 4 4 4 4 4 4 4 4 4 4 4 TEM Dentinal tubules o o o o 4 4 4 4 4 4 4 4 4
Protocol EDI protocol
Root no. R1
Root portion Coronal Middle Apical Coronal Middle Apical Coronal Middle Apical Coronal Middle Apical Coronal Middle Apical Coronal Middle Apical Coronal Middle Apical Coronal Middle Apical Coronal Middle Apical
Lumen o 4 4 o 4 4 4 4 4 4 4 4 4 4 4 o 4 4 4
Lumen o o 4 o 4 o 4 4 4 4 4 4 4 4 4 4 4 4 4 4 4 4 4
Lumen o o 4 o o 4 4 4 4 4 4 4 4 4 4 4 4
R2
R3
R4
Zurich protocol
R5
R6A
R6B
R6C
R7
4, bacteria detected; , Bacteria not detected; , insufcient demineralization; o, canal not visible. R6A, root; from tooth 6; root A.
or when bacterial colonization patterns worthy of note were discerned. Light microscopy For the EDI protocol, 1-lm sections were cut from the most coronal, middle and most apical slices of the root. The Zurich protocol involved cutting the whole embedded root into four equal corono-apical portions and then 1-lm thick sections were cut from the coronal, apical sections and from either of the two middle sections (Fig. 1). Stained sections were examined to verify presence of the canal in the section; upon conrmation 57 sections of either 0.5 or 1 lm were cut and examined at 200, 400 and 1000 (oil immersion) magnications. Representative photographs were taken at both low and high magnication for maintaining perspective and obtaining the highest resolution.
Transmission electron microscopy The LM ndings informed the further sectioning for TEM for both protocols. Two sections were cut and examined from the same sites as for the LM sections for each third of the root. The sections were initially examined at the lowest magnication for perspective before zooming in at higher magnications. Photographic images were recorded at a number of magnications to illustrate ndings.
Comparison between EDI and Zurich protocols

The EDI and Zurich protocols were subjectively compared using the following measures: 1 Ease of processing. This was judged by the ability to split or section the root in a controlled manner to view the root canal and its contents, as well as the time taken for complete processing of the roots;
912
2 Accuracy of ndings. Note was made of actual or apparent artefacts, distortion or evident bacterial translocation.
D
Analysis of ndings
Observational data were collected as systematically as possible to build a coherent picture of the intraradicular infection, in particular highlighting any common, surprising or unusual ndings. An attempt was made to record presence or absence and density of bacteria as objectively as possible to enable comparison. Simple descriptive statistics were used to analyse the ndings.
CC
Results Comparison of processing protocols

The principal difference between the processing for the two protocols was the length of time to progress from unxed sample to SEM/TEM examination. The Zurich protocol was several weeks longer than the EDI protocol because of longer decalcication times. However, once demineralization was complete, the Zurich protocol allowed more controlled and accurate bisecting of the root, than the less predictable root splitting required for the EDI protocol. Table 1 summarizes the viewable elds for each protocol (EDI/Zurich) and the presence/absence of bacteria by microscopy technique, tooth, root and segment.
1 mm
Figure 2 R5 (Zurich protocol) SEM low magnication LS root
showing dentine (D), the root canal and cellular material (CC) (lbar represents 1 mm).
as supercial cells and debris (Fig. 3) but within the canal this was less easy to identify. Bacterial cell morphology (rods, cocci and laments) was easily distinguished with SEM (Fig. 4), but only at the sample surface, and the presence of a thick extracellular matrix masked underlying bacteria. It was, however, possible to discern the relative thickness of
Comparison of techniques by SEM

From each root, one-half was prepared for SEM, four from the EDI protocol and ve from the Zurich protocol. The tooth structure and root canal contents observed in samples processed using the two protocols were similar (Fig. 2) although it was noted that in some of the Zurich samples the dentine surface had a smeared appearance (Fig. 3). As a result of the more accurate dividing of the root with the Zurich protocol, there were more root portions, 14 of 15, in which the root canal was visible as opposed to 10 of 12 with the EDI protocol. In both of the EDI samples without a visible canal, this occurred in the important apical portion. Translocation of root canal contents as a result of processing was sometimes observed with both protocols. On the cut (Zurich Fig. 3) or fractured (EDI Fig. 6) dentine surface, this could be clearly discerned
60 m
Figure 3 R5 (Zurich protocol) SEM showing the smeared
dentine and some translocated RBCs (arrows) (l bar represents 60 lm).
913
the bacterial layer in some instances where a fortuitous cut through the thickness of the biolm revealed the inner topography (Fig. 5). The appearance of the bacterial biolm within the canal seemed similar for both protocols and the relationship between the bacterial biolm and the canal anatomy was clear (Fig. 5). The SEM examination detected bacteria within the canal in seven of nine roots, and 16 of 27 root portions. In only three roots were bacteria observed in the dentine tubules, two from the EDI protocol (Fig. 6) and one from the Zurich protocol, although the slight smearing of the dentine made examination more difcult.
Comparison of techniques by LM
Light microscopy provided the best overall perspective of the root canal, enabling larger areas to be observed at low magnication (Fig. 7). There was little difference between the two protocols in terms of the type of information gained from the samples, providing details of the structure and distribution of bacterial biolms and cells, and also an indication of the bacterial morphology, although care should always be taken interpreting cross-sections of cells. It was evident from the LM observation of all three portions from root R1 that this was, in fact, a vital pulp. However, a difference between the protocols was noted, a consequence of the splitting of the roots with
60 m
Figure 5 R5 (Zurich protocol) SEM apical section through dentine (D) and biolm (B) within the canal lumen (L) (l bar represents 60 lm).
the EDI protocol, in which the whole canal was within the SEM portion and therefore no canal could be found in the LM samples. In all the Zurich samples, the lumen was present in the LM sections whereas in two of the roots processed by the EDI method there was no visible lumen in two of the three portions. The dentine tubules were easily visible in all the LM sections and, in 12 of 23 portions, were observed to contain bacteria, even
30 m
Figure 4 R4 (EDI protocol) SEM middle section showing
10 m
Figure 6 R4 (EDI protocol) SEM apical section showing bacteria (arrows) within the dentine tubules (l bar represents 10 lm).
bacteria morphotypes, laments (F) and cocci (arrows) (l bar represents 30 lm).
914
thick over the bacterial biolm (Fig. 9). This was observed in two teeth and was most prominent in the apical segments but less so in the middle and coronal segments. In one root, a second bacterial biolm (although less dense) could be observed on the luminal aspect of the PMN layer (Fig. 9), thus a layer of PMNs was sandwiched between two bacterial biolms.
B
Comparison of technique by TEM

As the same samples were used for both the semi-thick LM and ultra-thin TEM sections, the reported absence of a root canal in both was due to inadequate demineralization. However, in ve of the 15 root portions processed by the Zurich technique, although the dentine was demineralized sufciently for the LM sectioning, it was insufcient for the ultra-thin sectioning and therefore these were not viewed by TEM. In four of the ve cases, this was a middle portion of the root (Table 1). In most cases, TEM provided similar information to LM except that TEM conferred the considerable advantage over the other techniques in the detail of visual information available on the cells and bacteria. The TEM of the biolm in Figs 10 and 11 showed the close arrangement and morphology of the cells, including spirochaetes. Furthermore, the PMNs in this Zurich processed sample (Fig. 11) appeared to be leached of
100 m
Figure 7 R5 (Zurich protocol) light microscopy (LM) apical section showing the overall view. The canal wall has a thick biolm (B) with the luminal part containing some poorly visible amorphous substance (l bar represents 100 lm).
when the bacterial lm was sparse (Fig. 8). In 10 of these, bacteria were found in the LM sections where SEM had not found them, and in one, the opposite occurred. In some samples, it was observed that polymorphs (PMNs) and some RBCs formed a layer several cells
30 mm 10 m
Figure 8 R4 (EDI protocol) LM middle section showing Figure 9 R5 (Zurich protocol) LM apical section showing
bacteria (arrows) within the dentine tubules (l bar represents 10 lm).
bacterial biolm (B) adherent to the canal surface and walled in by PMNs and RBCs (I) beyond which there is a further biolm (l bar represents 30 lm).
915
cytoplasmic contents, whereas in EDI samples the immune cells appeared healthy (Fig. 12). When LM detected bacteria in the dentinal tubules, this was conrmed by TEM, except for those samples that were not sufciently demineralized. In many of these samples, there was an apparent attachment of some bacteria to the collagen (Fig. 13) that must have been present before demineralization and may indicate exposed or available collagen epitopes within the canal.
Summary of observations
The Zurich technique allowed examination of the root canal in most SEM samples, all LM sections but only half of the TEM sections. In contrast, for the EDI technique, most of the canals were visible in the SEM, but only three-quarters could be used for LM and TEM. Generally, the correlation between LM and TEM was good but SEM provided rather different information. When bacteria were detected in the canal using LM or TEM, their presence was not always found in the SEM samples, although this may reect the use of different halves of the root canal for each type of technique.
Figure 10 R5 (Zurich protocol) transmission electron microscopy (TEM) apical section showing bacterial biolm (B) extending from the canal surface with palisading of the bacterial cells. The initial attachment to the canal dentine wall (D) appears to be due to lamentous morphotypes with coccal forms further out towards the canal lumen (TEM 5000).
Figure 12 R2 (EDI protocol) TEM coronal section a healthy inammatory cell, probably a lymphocyte, within the canal lumen (TEM 6700).
Figure 11 R5 (Zurich protocol) TEM apical section showing the layer of PMNs () and RBCs (*) covering the bolm. Note the loss of cellular contents from the PMNs (TEM 2700).
Figure 13 R4 (EDI protocol) TEM middle section showing the apparent attachment of a bacterium to the collagen bres (C) (TEM 40 000).
916
Bacteria were detected in eight of the nine roots examined, including root R6C, apparently not associated with a periapical lesion, but bacteria were not found in root R1, which although positive for presence of periapical lesion, was found to be a vital pulp. The pooled data from all microscopy techniques (Table 1) showed that in the bacteria-positive teeth, bacteria were detected in the canal lumens in all the root segments except 3 (R3 coronal and apical; R6c apical). In contrast, they were less frequently detected in the dentinal tubules, especially in the apical portions, and detection was more accurate by LM and TEM techniques. The pattern of bacterial distribution, both in the canal lumen and on the canal walls, varied enormously both from root to root and within each root. Continuous biolms were only evident in teeth with grossly carious exposures and continuous communication with the oral environment. The structure, thickness and morphotypic composition varied considerably. The bacterial biolm was mainly evident on the canal wall with interspersed bacterial aggregates in what seemed to be residual necrotic pulp tissue. Sometimes bacterial cells seemed to be present in the canal lumen in apparent isolation (perhaps planktonic forms). In teeth with intact pulp chambers, the canal lumen appeared empty but was lled with some amorphous material (Fig. 7). In some teeth, the relative abundance of detectable bacteria was greater coronally, usually associated with carious crowns. Rarely did the middle portion have the greater abundance but, in teeth with intact pulp chambers, the relative abundance of bacteria was greater in the apical segments. Each tooth seemed to have its own variation of infection pattern. The patterns of colonization of the dentinal tubules appeared to follow relatively more predictable but nevertheless variable behaviour. Dentinal tubules usually appeared to be colonized as a continuation of the canal wall infection, although the diversity of morphotypes were more restricted. Other instances showed variable colonization of adjacent tubules sometimes with highly dense colonization of the tubules in the predentine with reducing density of colonization further away from the canal lumen into the dentine. The observation regarding the presence and close association of PMNs and sometimes RBCs with bacterial aggregates and lms was not consistent. It was more prominent in some teeth than others and, where present, was always observed in the apical portions and frequently in the middle and coronal portions. In one tooth, a second bacterial biolm, although less
dense, was observed on the luminal aspect of the PMN layer, implying that this was not a result of tooth preparation.
Discussion
The prime purpose of this study was to evaluate the utility of different microscopy techniques and protocols to gain visual insights into the presence, distribution and structure of bacterial colonization in teeth associated with apical periodontitis, regardless of the clinical condition of the tooth; the intention was to use a wide selection of tooth conditions meeting selection criteria to evaluate the breadth of morphotypic bacterial diversity. Many studies on the microora of infected roots have used teeth with gross caries, presumably because of their easier availability (Nair 1987, Baumgartner & Falkler 1991, Sen et al. 1995). An additional clinical parameter in this study was the acute presentation of the selected teeth. A subsidiary but important aim was to compare two tooth processing protocols. The importance of this aspect is that the validity of microscopy observations rest on accurate preservation of the in situ anatomical structures and relationships. Distortion of tissues or translocation of structural components may obscure the truth, and they correctly need to be recognized as artefacts. The problem for the observer is to be able to distinguish real from artefact without a positive control. To be able to do so requires a good appreciation of what is to be expected based on understanding of biology, familiarity with the technical aspects of the procedures and critical interpretation. Purely morphological studies are able to give morphological insight but cannot enable dissection of the relationships and roles of bacterial species and their interaction with host cells. Such insights may only be obtained in the future through in situ labelling studies, which require the preservation or exposure of target cell surface, structural or chemical elements (Lam et al. 2000, Tan et al. 2000); the preserving protocols therefore become important. The purpose of this study was not to explore the effect of protocols on such cell surface targets but to evaluate the effect of such protocols on normal structural viewing, in the rst instance. Studies on in situ hybridization will be reported separately. Another key factor is that each microscopy technique requires an independent section; the same section may not be viewed by all techniques. Absolute comparison between microscopy techniques is therefore impossible and relative comparison reliant on viewing adjacent sections that are thin enough to
917
represent, more-or-less, the same structures. This was more easily possible for LM/TEM views than for SEM, because, of necessity, the opposite halves were viewed and these could theoretically have different bacterial colonization, particularly in teeth with intact pulp chambers. Some key features of difference between the protocols bear discussion. Previous microscopy studies have split their test teeth using a technique similar to the EDI protocol (Lin & Langeland 1981, Molven et al. 1991, Sen et al. 1995) but none commented on its inherent problem of unpredictability; a feature mostly reduced by practicing on spare rather than sample teeth. Furthermore, the lack of comment may reect that the studies could select from both halves, whereas in the present study, the portion with the larger canal component was reserved for LM and TEM, theoretically compromising that used for SEM. Numerous approaches have been used to split teeth and alternative methods have been reported (Rapp 1985) but without tested consensus. The Zurich protocol was favoured for its more predictable cutting of the demineralized root compared with the splitting of mineralized tissues in the EDI protocol. The predictable cuts in the Zurich protocol were, however, associated with an apparently smeared appearance of the dentine, in contrast to the rougher fractured surface produced by the EDI protocol (Fig. 3 vs. Fig. 6); the signicance of this feature is unknown although it may affect an appraisal of dentine tubule content. It should be added that such a feature was absent in the published material from the Zurich laboratory and could be a feature of adaptation in another laboratory. A further putative advantage of the Zurich protocol is that the natural morphological relationships and conjunction of the structures would be less likely to be disturbed. In contrast, in the EDI protocol, the various washings of the pre-split and open canal surface prior to sputter-coating could potentially result in translocation of loose structures such as planktonic bacteria or pulp debris (Nair 1987, and personal communication) (Fig. 3). Translocated debris and bacteria are sometimes evident in publications using the SEM (Sen et al. 1995). In general though, the SEM views did not seem much distorted or different between the protocols in this study. Furthermore, the enmeshed and matted appearance of the bacterial biolm was conrmed by the different microscopy techniques and appeared to suggest that at least this feature of the bacterial colonization remained preserved regardless of the protocol used.
The careful and slow approach used by Nair (1987) to demineralize the test specimens is laudable and is most likely to yield accurate images representing the truth, nevertheless, within the time constraints imposed in this study, the slower process resulted in several middle root segments remaining un-demineralized and therefore un-viewable (Table 1). The counterargument against the Zurich protocol was that the more aggressive demineralization, albeit slower, and associated long xation periods, may damage surface antigens (Hobot & Newman 1991) and probe targets for in situ hybridization (Binder 1992). It would be intuitively expected that each microscopy technique with its own unique characteristics would yield different perspectives on the objects under scrutiny; each hopefully yielding unique accuracy in some way so that they together complement ndings to build a more accurate overview. The ndings of this study conrm these expectations and potential, whilst at the same time highlighting the advantages and disadvantages of each microscopy technique. The SEM, with its propensity for revealing surface topography was generally useful for deciphering detail over the entire canal surface, whilst retaining contextual perspective at lower magnications; this also enabled the proportion of the surface colonized to be estimated. The technique was also useful for describing cell morphotypes but by the same token, surface coverage with cells or extra-cellular matrices precluded revelatory insight into biolm structure and relationships. The LM provided an excellent overview of the collective bacterial colonization and its variation from site to site within the selected section, particularly on the canal wall. Its main limitation is the level of magnication and resolution necessary to determine inter-cellular and cellular-abiotic relationships. Furthermore, morphotypic differentiation was relatively gross and lacked discriminatory detail. Transmission electron microscopy was the most discriminating technique for providing ne detail of the microora and its relationship to adjacent structures, as well as cell-to-cell contacts. Furthermore, the internal cellular morphology was also most clearly seen by TEM. It is evident that correlative studies using LM and TEM provide the best conjunction, as reported by Nair (1987). Furthermore, the combination with SEM provides further insights but the processing required is different from that for LM and TEM and may be more prone to distortion of surface detail.
918
The pictures of bacterial quantity and density were broadly comparable between the microscopy techniques (Table 1), conrming the utility of using adjacent serial sections for LM and TEM. However, differences were sometimes apparent, both between sections, and by microscopy technique, the latter mainly because of SEM sections, which would by denition have viewed geographically different locations from those viewed by LM or TEM. Bacteria were not detected in one root apparently associated with a periapical lesion, otherwise, a variety of morphotypes were found in all canal segments consisting of cocci, rods, laments, spiral forms and yeasts. The existence of a periapical lesion associated with an inamed but vital pulp is not a novel nding. The present study found that of all the roots which could be examined fully, only one had fewer bacteria apically than coronally. In the other roots (including those with intact pulp chambers), there was a transition from the coronal segment to the apical segment, of greater relative bacterial abundance apically. This contrasts with other work (Shovelton 1964), where the sample was also made up of both open and closed pulp systems. There was a lack of consistency in the middle root segments, where some roots had fewer bacteria than either the coronal or apical segments and others where the bacterial abundance formed a continuous transition from coronal to apical. The distribution could potentially be explained by abundance of nutritive sources coronally and apically. A carious exposure may allow seepage of salivary components from the coronal aspect, forming a diffusion gradient towards the apex. Once the bacteria are established apically, the stimulation of inammation apically may then play a part in deriving nutrition from the inammatory serum exudate (Khot et al. 2004). The relative scarcity of bacteria in the middle segment could be explained by its farthest location from opposing sources of nutrition (coronal or apical); it being the lowest point on two opposing gradients. The evidence of dividing bacterial cells in the middle segments suggests the presence of sufcient nutrients in this part of the canal at some point. In some species, such as staphylococci, divided cells may remain joined for some time after division. The patterns of bacterial distribution in the canal lumen and on canal walls varied. Some teeth had discontinuous biolms together with variable density and layers of cells, whilst others had thick continuous, dense biolm layers. The structure, thickness and morphotypic composition also varied considerably but
the species diversity of the ora may only be speculated upon without in situ hybridization. Some niches in the root canal seemed apparently more suited to biolm growth than others, although the main bacterial colonization seemed to be on the canal walls; that within the lumen, in the middle and coronal segments, seemed more scattered. It is not known whether these bacteria, apparently oating without attachment represent planktonic phenotypes or are biolm phenotypes attached to a surface of degrading tissue that is invisible in the chosen microscopy technique. Each tooth seemed to have its own variation of infection, corroborating the ndings of various culture and culture-independent studies (Sundqvist 1976, Rolph et al. 2001, Munson et al. 2002). The impression in some teeth was that, indeed this was a nutrient-depleted environment but in others, the canal system appeared to be nutrient-rich with active bacterial growth and propagation. It is possible that acute apical symptoms may be due to such rapid and proliferative bacterial growth rather than because of specic species. Associations between species and acute symptoms although often made, have not proved fruitful, because the presence of the same species can be conrmed in asymptomatic teeth. The answers may lie in strain variation. Yeast cells were detected in 3/7 (43%) teeth in this study, a value that ts within the range previously reported: by microscopy, 840% (Molven et al. 1991, Sen et al. 1995); by culture, 555% (Slack 1975, Egan et al. 2002); and by molecular detection, 21% (Baumgartner et al. 2000). Yeasts have been implicated in failed cases, raising the suggestion that reduction of bacteria during treatment may allow yeasts to overgrow and predominate in the low-nutrient environment (Sundqvist 1992). Bacterial invasion of dentinal tubules was predominantly seen in the coronal and middle root segments; in contrast Sen et al. (1995) reported dentinal tubule invasion in the middle and apical root segments. The presence of bacteria on inter-tubular dentine casts some doubt on the SEM ndings. The ndings in the apical root segments are consistent with reports of fewer dentinal tubules in this region (Mjo r et al. 2001). Dentinal colonization was heaviest in the pre-dentine and mainly conned towards the canal lumen end of tubules than the cementum; in agreement with some (Shovelton 1964, Nair 1987) but contradicting others (Peters et al. 2001). The nding of apparent bacterial association or attachment to dentine collagen (Fig. 13) would appear to be in situ conrmation of the suggestion previously made by Love & Jenkinson (2002).
919
Another interesting nding in the present study was the presence of PMNs in all thirds of the roots with necrotic pulps; the nding was particularly surprising in the coronal segments but would be consistent with pus exudation into the canal. Nair (1987) had previously reported PMNs amongst bacteria in the apical sections of roots associated with apical periodontitis, but these were described as isolated wandering cells. Their presence was explained by virtue of chemotactic signals from intra-canal bacteria. The extensive presence of PMNs in the root canals of teeth associated with apical periodontitis (with acute episodes), apparently strategically attempting to wall off the bacterial biolm adherent to the canal wall was unexpected and unique in the endodontic published literature. The observation which was consistent between LM/TEM techniques and different teeth and roots, alters the perception of the root canal ecology in such acute cases. First, it implies the presence of sufcient moisture or a water-saturated medium through which they can propel themselves to such distances into the canal. Second, there should be sufcient nutrients to allow them to migrate and survive in such locations (technically beyond the viable part of the body), bearing in mind their short life-span [34 days (Taussig 1984)]. Third, it changes the perception of the host/microbial interface as being conned to the apical foramen. Clearly, at least one branch of this host/microbial interaction is capable of extending into the length of the necrotic, infected canal associated with symptoms. Given the short life span of PMNs, the growth of a bacterial biolm on the luminal aspect of the layer of PMNs suggests a very dynamic ecological niche in such a tooth. The relative equality between the protocols, at least in terms of quality of viewable sections, opens the doors towards use of microscopy with immuno-labelling to further dissect the root canal ecology and the dynamics of infection. The PMNs would appear to play an important role in apical periodontitis and perhaps apical healing.
infection but all exhibited bacterial biolms on canal walls; 8/9 roots showed bacteria. Bacteria in the canal lumen were often associated with other structures but sometimes appeared free-oating. In general, bacteria appeared more abundant apically than coronally but dentinal tubule colonization was more common in coronal and middle thirds. PMNs were often found walling off bacterial biolm along the entire length of the root canal wall, although they were in higher numbers apically. The ndings provide interesting insights into the nature of host/microbial interaction and the ecology of infected root canals.
Acknowledgement
The authors are grateful to Dr PNR Nair for his generous advice on the adoption of the Zurich protocol, as well as on the ner points of microscopy.
References
Akpata ES (1976) Effect of endodontic procedures on the population of viable microorganisms in the infected root canal. Journal of Endodontics 2, 36973. Baumgartner CJ, Falkler WA (1991) Bacteria in the apical 5 mm of the infected root canals. Journal of Endodontics 17, 3803. Baumgartner JC, Watts CM, Xia T (2000) Occurrence of Candida albicans in infections of endodontic origin. Journal of Endodontics 26, 6958. Binder M (1992) In situ hybridization at the electron microscope level. In: Wilkinson DG, ed. In Situ Hybridization A Practical Approach. Oxford: Oxford University Press, 10520. Egan MW, Spratt DA, Ng Y-L, Lam JM, Moles DR, Gulabivala K (2002) Prevalence of yeasts in saliva and root canals of teeth associated with apical periodontitis. International Endodontic Journal 35, 3219. n G, Holm G, Mo Fabricius L, Dahle ller AJR (1982) Inuence of combination of oral bacteria on periapical tissues of monkeys. Scandinavian Journal of Dental Research 90, 2006. Gulabivala K (2004) Species Richness of Gram-Positive Coccoid Morphotypes Isolated from Untreated and Treated Root Canals of Teeth Associated with Periapical Disease. PhD Thesis. London: University of London. Hobot JA, Newman GR (1991) Strategies for improving the cytochemical and immunocytochemical sensitivity of ultrastructurally well-preserved, resin embedded biological tissue for light and electron microscopy. Scanning Microscopy Supplement 5, S2740. Kakehashi S, Stanley HR, Fitzgerald W (1965) The effects of surgical exposures of dental pulps in germ-free and conventional laboratory rats. Oral Surgery, Oral Medicine, and Oral Pathology 20, 3409.
Conclusions
The Zurich protocol was more predictable than the EDI protocol in creating longitudinal sections and possibly bacterial detection by microscopy but the quality of observed sections seemed equivalent. Each microscopy technique provided a unique perspective and together allowed complementary synthesis of the presence and morphological distribution of bacteria within roots. Each tooth presented a unique pattern of bacterial
920
Kapalas A, Spratt DA, Ng Y-L, Gulabivala K (2001) An in vitro investigation of the bulk ow of uid through apical foramina during simulated tooth extraction: a potential confounder in microbiological studies? International Endodontic Journal 34, 335. Kapalas A, Spratt DA, Ng Y-L, Gulabivala K (2002) Investigation of bulk ow of bacterial suspension through apical foramina during simulated tooth extraction. International Endodontic Journal 35, 83. Khot A, Spratt DA, Ng Y-L, Gulabivala K (2004) Utilisation of relevant nutritional resources by root canal isolates. International Endodontic Journal 37, 3456 (abstract). Kumar T, Spratt DA, Ng Y-L, Gulabivala K (2002) A preliminary evaluation of a new method for sampling the intra-radicular bacterial ora. International Endodontic Journal 35, 85. Lam JM, Gulabivala K, Barrett W, Speight P, Smallwood E, Spratt DA (2000) The identication of bacteria in dental histological specimens using molecular methods. International Endodontic Journal 33, 73. Lin L, Langeland K (1981) Light and electron microscopic study of teeth with carious pulp exposures. Oral Surgery, Oral Medicine,and Oral Pathology 51, 292316. Love RM, Jenkinson HF (2002) Invasion of dentinal tubules by oral bacteria. Critical Reviews in Oral Biology and Medicine 13, 17183. Miller WD (1894) An introduction to the study of the bacteriopathology of the dental pulp. Dental Cosmos 36, 50528. Mjo r IA, Smith MR, Ferrari M, Mannocci F (2001) The structure of dentine in the apical region of human teeth. International Endodontic Journal 34, 34653. Molven O, Olsen I, Kerekes K (1991) SEM of bacteria in the apical 5 mm of infected root canals in permanent teeth with periapical lesions. Endodontics and Dental Traumatology 7, 2269. Munson M, Pitt-Ford T, Chong B, Weightman A, Wade WG (2002) Molecular and cultural analysis of the microora associated with endodontic infections. Journal of Dental Research 81, 7616. Nair PN (1987) Light and electron microscopic studies of root canal ora and periapical lesions. Journal of Endodontics 13, 2939.
Peters LB, Wesselink PR, Buijs JF, van Winkelhoff AJ (2001) Viable bacteria in root dentinal tubules of teeth with apical periodontitis. Journal of Endodontics 27, 7681. Rapp R (1985) A procedure for splitting human teeth to obtain intact pulp tissue, enamel and dentin. Stain Technology 60, 3943. Reynolds ES (1963) The use of lead citrate at high pH as an electron-opaque stain in electron microscopy. Journal of Cell Biology 17, 20812. Rolph HJ, Lennon A, Riggio MP et al. (2001) Molecular identication of microorganisms from endodontic infections. Journal of Clinical Microbiology 39, 32829. Sen BH, Piskin NB, Demirci T (1995) Observation of bacteria and fungi in infected root canals and dentinal tubules by SEM. Endodontics and Dental Traumatology 11, 69. Shovelton DS (1964) The presence and distribution of microorganisms within non-vital teeth. British Dental Journal 117, 1017. Slack G (1975) The resistance to antibiotics of microorganisms isolated from root canals. British Dental Journal 18, 4934. Sundqvist G (1976) Bacteriologic Studies of Necrotic Dental , Sweden: University of Umea . Pulps. PhD Dissertation. Umea Sundqvist G (1992) Associations between microbial species in dental root canal infections. Oral Microbiology and Immunology 7, 25762. Tan L, Spratt DA, Lam JM, Speight P, Gulabivala K (2000) The effect of tissue-xation conditions on the amplication of 16S rRNA genes from parafn-embedded teeth using polymerase chain reaction (PCR). International Endodontic Journal 33, 1667. Tani-Ishii N, Wang CY, Tanner A, Stashenko P (1994) Changes in root canal microbiota during the development of rat periapical lesions. Oral Microbiology and Immunology 9, 12935. Taussig MJ (1984) Chapter 1 inammation. In: Taussig MJ, ed. Processes in Pathology and Microbiology, 2nd edn. Oxford, England: Blackwell Scientic Publications, pp. 15. Vrahopoulos TP (1989) Ultrastructure of the Periodontal Lesion in a Case of Papillon-Lefevre Syndrome (PLS). PhD Thesis. London: University of London.
921
doi:10.1111/j.1365-2591.2009.01595.x
An in vivo experimental model to assess furcal lesions as a result of perforation
M. J. B. Silva1, M. V. Caliari3, A. P. R. Sobrinho2, L. Q. Vieira1 & R. M. E. Arantes3

ncias Biolo gicas, Universidade Federal de Minas Gerais, Belo Departamento de Bioqu mica e Imunologia, Instituto de Cie Horizonte, MG, Brazil; 2Departamento de Odontologia Restaurativa, Faculdade de Odontologia, Universidade Federal de Minas ncias Biolo gicas, Universidade Federal de Gerais, Belo Horizonte, MG, Brazil; and 3Departmento de Patologia Geral, Instituto de Cie Minas Gerais, Belo Horizonte, MG, Brazil
1
Abstract
Silva MJB, Caliari MV, Sobrinho APR, Vieira LQ, Arantes RME. An in vivo experimental model to assess furcal lesions as
a result of perforation. International Endodontic Journal, 42, 922929, 2009.
Aim To design and validate a rat molar model of furcal perforation to allow investigation of the biological phenomena that follow and to explore its potential for evaluating repair materials under standardized conditions. Methodology Eighteen male Wistar rats were used. Surgical aseptic procedures were carried out in order to open the pulp chamber of a rst molar tooth. A cavity was prepared on the oor of the pulp chamber using a round bur that created a communication between the furcation and the periodontal tissues. Six animals for each time point were sacriced on days 14, 21 and 28 to assay morphological changes at the furcation region of molars. Maxillary bone was processed,
removed and sectioned. Cellular inltration, collagen deposition and bone resorption were assessed by histological analysis. Cellularity in the lesion area was determined by morphometric analysis. Data were analysed using parametric Students t-test. Results A furcal perforation model was standardized in which both radiological outcome and periodontal tissue reactions could be assessed through evaluation of cellularity, osteoclast activity and collagen deposition. The morphometric analysis revealed a greater number of cells 21 day post-surgery when compared with 14 days. Conclusion This animal model was suitable for radiological and histological evaluation of the processes that accompany surgical furcal perforation. Keywords: animal models, biomaterials, furcal perforations.
Received 3 September 2008; accepted 14 April 2009
Introduction
Accidental root canal or furcal perforation complicates the treatment and compromises the outcome of root canal treatment (Seltzer & Bender 1990, Walton & Torabinejad 2002). The prognosis of treatment of
Correspondence: Rosa Maria Esteves Arantes, MD, PhD, rio de Neuro-imunopatologia Experimental, DepartaLaborato ncias Biolo gicas, mento de Patologia Geral, Instituto de Cie nio Carlos, Universidade Federal de Minas Gerais, Av. Anto 6627, 31270-901, Belo Horizonte, MG, Brazil (Tel.: +5531 3409 2896/2884; fax: +5531 3409 2879; e-mail: rosa@icb.ufmg.br).
perforations is dependent on site, size, setting time of the repair material, and the efcacy of the material in terms of to seal (Walton & Torabinejad 2002). All of these factors are related to the ability to prevent or eliminate bacterial inltration (Daoudi & Saunders 2002). The chronic inammatory reaction after accidental furcal perforation has been the object of several studies (Bhaskar & Rappaport 1971, Meister et al. 1979, Beavers et al. 1986, Seltzer & Bender 1990, Balla et al. 1991, Arens & Torabinejad 1996, Wu et al. 2005, Vajrabhaya et al. 2006, Al-Daafas & Al-Nazhan 2007,). This inammatory reaction provokes alveolar bone damage around the perforation site where bone is progressively substituted by granulation tissue. The
922
Silva et al. Model of furcal perforation
lack of bone tissue leads to loss of periodontal attachment that can affect tooth stability (Arens & Torabinejad 1996, Wu et al. 2005, Vajrabhaya et al. 2006, Al-Daafas & Al-Nazhan 2007). Accidental furcal perforation has stimulated evaluation of the immunological responses that occur in the periodontal tissues and also the materials to seal the defects. It is believed that an ideal sealing material should seal the communication between the periodontium and pulp chamber and, at the same time, be biocompatible so as to induce bone and cement deposition (Hartwell & & Holland 2004). However, so England 1993, Bernabe far no ideal sealing material has been available. To evaluate the prognosis and the best treatment choice different animal models have been used. Small rodents have many advantages as experimental models: (i) age and genetic rodent background can be well dened; (ii) better cost benets; (iii) mouse and rats may be kept in controlled environments easily; (iv) there is a wide variety of gene knockout models. The rat bears much resemblance to man with respect to periodontal anatomy, development and composition of dental plaque, histopathology of periodontal lesions, and basic immunobiology (Klausen 1991). In this context, the characterization of alternative rodent models for dental research is important. Therefore, this study aimed at describing a new model to evaluate the outcome of furcal perforation including histopathological aspects using rats as the experimental model.
Agribrands do Brasil Ltda, Paul nia, SP, Brazil). The absence of ick reex to hindpaw interdigital skin stretch was documented before the beginning of surgical procedures.
Surgical procedures
All experimental procedures were carefully performed under aseptic condition to avoid contamination. The disinfection of the surgical eld was accomplished as proposed by Mo ller (1966). Access to the pulp chamber in the rat molar tooth was prepared via the occlusal surface using a number 33 carbide bur (KG Sorensen, Barueri, SP, Brazil) coupled to a controlled rotation handpiece (Driller, Sa o Paulo, SP, Brazil) under air cooling. Once the pulp was exposed, the roof of the chamber was removed with an Endo Z bur (Dentsply Maillefer, Catanduva, SP, Brazil). Coronal pulp tissue was removed using an endodontic probe until the level of root canal orices. Haemorrhage was controlled by irrigating the chamber with saline solution and by applying pressure with sterilized cotton balls. As soon as bleeding was controlled, the chamber was irrigated again with saline solution and dried with cotton pellets. Subsequently, a perforation was created in the centre of the pulp chamber oor towards the alveolar bone using a number carbide drill (KG Sorensen, Barueri, SP, Brazil). To standardize the depth of the perforation, a cursor was glued to the drill 1 mm from its tip; the width was limited to the bur diameter (0.5 mm). A layer of gutta percha was inserted on the pulp chamber oor, avoiding contact between the sealing material and the perforation site. Condensation of gutta-percha was accomplished with the endodontic plugger and the excess was removed and contoured using a Hollemback 3S (SS White, Rio de Janeiro, RJ, Brazil). The tooth was restored with silver amalgam that was burnished using Dycal instrument (SS White); nally, the waste materials was removed using sterilized cotton balls. Prior to sacrice the integrity of the amalgam was checked under an endodontic microscopy (Alliance microscopia, Sao Paulo, SP, Brazil).
Material and methods Animals

Wistar male rats weighing 240260 g were obtained from CEBIO (Centro de Bioterismo da UFMG, Belo Horizonte, MG, Brazil) and kept in a conventional animal house with barriers and controlled light cycle. The experimental protocol was approved by the institutional animal ethical committee (protocol number 097/04, CETEA UFMG). Six rats were used for each time point: 14, 21 and 28 days after the surgical procedure. Three rats were used as controls.
Anaesthesia
All experimental procedures were carried out under general anaesthesia. Rats were injected intra-muscularly with 100 mg kg)1 of ketamine hydrochloride (Dopalen, Division Vetbrands Animal Health, Jacare , SP, Brazil) and 10 mg kg)1 of Xilazine (Anasedan,
MTA sealing procedure: testing the model application

To evaluate if it would be feasible to seal the experimental perforation and to analyse the surrounding periodontium tissues, six animals had their perforations ngelus, Londrina, PR, Brazil). lled with grey MTA (A This material was manipulated according to the
923
Model of furcal perforation Silva et al.
manufacturers recommendation and inserted using a probe. Animals were killed on day 21 post-surgery. A video animation was designed using the blender software (Blender Foundation, Amsterdam, the Netherlands) to represent the entire surgical procedure (see Video Clip S1 of Supporting Information).
Radiographic procedures
Prior to the sacrice, the animals were anaesthetized and killed by decapitation on days 14, 21 and 28 after the procedure. The hemi-maxilla was removed and radiographs were taken using a dental X-ray unit (Bioatlant, Ribeira o Preto, SP, Brazil). The optimal exposure parameters were determined as 0.12 s (70 kVp, 10 mA). Ekta-speed plus X-ray lm (Eastman Kodak Co, Rochester NY, USA) was placed and xed on a wooden surface to avoid movement. Then, the buccal side of the maxillary bone was placed so that side faced the machine. To take the image, the X-ray cone was set perpendicular to the X-ray lm and 5 cm from it.
the same animal. The images were obtained at 40 magnication through a JVC TK-1270-RGB adapted to a microscope and analysed using KS300 software built into a Kontron Elektronick/Carl Zeiss image analyzer (Caliari 1997). An automatic macro recorder assembler (an algorithm of the KS300 software) was elaborated for capture, image processing and segmentation, denition of morphometrical conditions and counts of all the cells detected in each image. Image processing techniques were applied. Segmentation permitted the separation of these nuclei from cytoplasm and other structures in the section, such as blood vessels and extracellular space. Consequently, a binary image was created containing just the nuclei and other spaces (Pacheco et al. 2008). The nucleus from the cellular types usually found in the furca area and newly recruited leukocytes were then counted. It was imperative that only connective tissue, excluding bone and root dentine were visualized. This approach limited the acquisition of more than three images. The measurements were made by an observer who was unaware of the nature of the tissue sample. Three nonoperated rats were used as reference of normal periodontal cellularity.
Histological preparation
The hemi-maxillas were immersed to 10% buffered formalin for 72 h at room temperature. Demineralization was performed for 30 days in EDTA solution (10% and pH 7.2) (Merck; Darmstadt; Germany). After demineralization, the samples were washed in running tap water for 24 h. Restorations were removed from the access cavities and the tissues were routinely processed for parafn embedding. The hemi-maxillas were embedded with the buccal side of molars facing the base of the parafn block. Consecutive (buccallingual direction) sections of 5 lm encompassing the perforation of the furcation area and both mesial and distal roots were stained with Haematoxylin/Eosin (H&E) and Gomoris Trichome and used to assess inammatory inltration, periodontal ligament organization and bone resorption.
Statistical analysis of mean values of cellular numbers obtained by morphometry was carried out using parametric Students t-test. Statistical signicance was set at P < 0.05.
Results Clinical aspects

After anaesthesia and prior to sacrice the animals were submitted to clinical evaluation. Animals tolerated the surgical procedure well and did not have signs of distress, weight loss or oral swelling. Colour and texture of the gingival regions were not altered.
Radiographic assessments Morphometric analysis

The inammatory cells inltrating the tissue adjacent to the perforation area was quantied. Images taken from the furcal areas were used to assay quantitatively the longitudinal inammatory cell inltration that occurred around the perforation, as shown in Fig. 3. Quantication of the inammatory cells in the furca area was conducted from three images selected from Figure 1a represents the nonoperated furcation and its adjacent tissue. Radiographs were performed to allow visualization of bone rebsorption. Radiographs were effective in revealing the three molar teeth as well as their associated alveolar bone and periodontal ligament space (Fig. 1b). Radiographic analyses demonstrated the position of the perforation (Fig. 1c) and, in some animals, interdental crestal destruction. In perforation
924
(a)
(b)
(c)
(d)
(e)
Figure 1 Histological aspect of the rst left maxillary molar and radiograph aspects of tooth and periodontium tissue. (a) Panoramic normal histological aspect of the molar, (T, pulp tissue; PF, pulp chamber oor; PL, periodontal ligament; R, root; B, interradicular bone), H&E, 40. (b) Radiological aspects of nonoperated group. White arrows mark the three molars. (c) Operated molar at day 14 post-surgery. White arrow marks the amalgam restoration. Note the gutta-percha material (arrow head). Open white arrow marks periodontal bone loss. (d) and (e) Operated and MTA-treated molar at day 21 post surgery. The white arrow represents MTA material sealing the furcal perforation. Note the MTA periodontium extrusion (open white arrow) and alveolar bone loss (arrow head).
sites sealed by MTA, radiographs demonstrated that it extended into the periodontal tissues (Fig. 1d,e).
Histopathology
Figure 2 shows the furcation area from operated animals on days 14, 21 and 28 post-surgery. On day 14, the periodontal ligament presented signicant alterations in its architecture, showing intense cellular inltration and edema, especially close to the perforation area (Fig. 2a). The following aspects were observed: mononuclear and polymorphonuclear cells prevailed in the underlying inammatory inltrate; edema interposition between tissue elements; intense vascular neoformation (Fig. 2b, inset); cement and root dentine reabsorption (Fig. 2f); intense collagen and connective tissue deposition and osteoclastic activity (Fig. 2f inset). On day 21 post-surgery, the samples showed chronic mononuclear inammatory inltrate around the perforation area. Collagen deposition was observed in the surrounding tissue apart from the perforation area (Fig. 2c). Fig. 2e represents samples from animals sacriced 28 days after surgery. It shows a discrete chronic inammatory inltrate (Fig. 2e) and evidence of periodontal reorganization. In one sample, small necrotic areas surrounded by polymorphonuclear cells and debris were observed close to the deep bone layer, indicating that in some cases the surgical procedure resulted in bone microabscesses (data not show).
Figure 2d represents samples from rats that were killed on day 21 after surgery in which perforations were sealed by MTA. It demonstrates the presence of debris inside the perforation sites which could be MTA residue. The adjacent connective tissue presented a prevalence of mononuclear cells in the inltrate, despite the presence of polymorphonuclear cells in some areas.
Morphometric analysis
As an example of quantitative analysis made possible by this model, we quantied the inammatory cells inltrating the tissue adjacent to the perforation area. Images taken from furcal area were used to assay quantitatively the longitudinal inammatory cell inltration that occurred around the perforation, as shown in Fig. 3. It was observed that cell numbers were much higher in furcal areas of operated animals than in animals that were not submitted to surgery.
Discussion
It is fundamental to the treatment of a perforation that the site does not become infected, and that it is sealed immediately. Prognosis of treatment depends on the perforation site, size of damage, adjacent periodontal condition and type of sealing material (Walton & Torabinejad 2002). The sealing ability of the material and its possible extrusion into the periodontal area should also be considered. Furthermore, good visibility
925
(a)
(b)
(c)
(d)
(e)
(f)
Figure 2 Histological aspects of the rat furcal lesion. (a) Day 14 post-surgery, perforation area showing granulation tissue (arrow).
(b) Higher magnication of (a) inammatory cells at the periphery of the perforation and disorganization of periodontal ligament (inset, arrow), the perforation site (arrow) and MTA fragments (arrow heads). (c) and (e) day 21 and 28, respectively showing the evolution of lesion towards repair (d) MTA-treated perforation at day 21, residues of MTA are present (arrows heads), notice the granulation tissue at the border of the perforation (arrows). (f) Intense vascular neorformation and deposition of collagen in the periodontal tissue, cement reabsorption (arrows), and osteoclastic activity (inset, arrow). C, cement; Co, collagen deposition T, pulp tissue; R, root; Bo, inter root bone D, dentine; P, perforation; Pl, periodontal ligament. (a), (c), (d) and (e) H&E, 100; (b), H&E, 200; (f) Gomoris trichome, 400 and insets, 400.
926
(a)
(b)
(c)
(d) 300
250
Non-operated
200 Cell numbers
14 days 21 days 28 days
150
100
50
0 Time after surgery
Figure 3 (a), (b) and (c) show, respectively, the intensity of periodontal tissue cellularity on days 14, 21 and 28 post-surgery. Also notice the presence of vascular congestion (small black arrows) and polimorfonuclear cells (encircled) on day 21 (H&E, 400); (d) Morphometry of furcal lesion periodontal tissue. The graphic shows the average of cell numbers counted in two elds per animal sample at 400 magnication, at each time point. Data are presented as mean values of counted cells in each animal (n = 3). Statistically signicant difference (P < 0.05) between data from the non-operated group and the operated group 14 days postsurgery. Statistically signicant difference (P < 0.05) between data from the operated groups on days 14 and 21 post-surgery.
of the perforation will help to facilitate the repair procedure (Daoudi & Saunders 2002). Hence, a model that simulates as much as possible the human clinical situation is crucial to the systematic assessment of the many biological aspects involved in this procedural accident. In this study, all efforts were undertaken to reproduce the clinical conditions in an experimental model. Thus, the design of the stainless steel clamp was modied to increase its ability to completely grasp the tooth (Sampaio 1967). All surgical procedures were performed under aseptic conditions, since a rubber dam could isolate the tooth in a similar fashion to the practice in humans. It should be recognized that the inammatory response in rodents is different from that found in humans, however, the rat was selected as a model because the periodontal anatomy, the development of plaque and histopathology of the periodontal lesion in this animal is similar to that found in humans (Klausen
1991, Genco et al. 1998). Other advantages include knowledge of animal age, genetic background, and the ethical and economical issues that contraindicate the choice of large animals for research (Beavers et al. 1986, Balla et al.1991, Arens & Torabinejad 1996, Bramante et al. 2004). In addition, the rat model provides good access to the tooth and allows visibility of the surgical eld. As manipulation of genetic and pharmacological parameters is possible in the rat, the establishment of this model will allow further studies on furcal perforation. Histopathological results demonstrated the effects of the furcal lesion on the periodontal tissues. An increased number of polymorphonuclear cells at 14 day after surgery could be observed, counterbalanced by prevailing mononuclear cells on days 21 and 28. At 28 days, an increase of collagen deposition and an exuberance of granulation tissue were observed. Furthermore, morphometric assessments were in agreement with outcomes observed by previous workers (Balla et al. 1991,
927
Sousa et al. 2004) and showed an increase in inammatory cell numbers around furcal perforations, which decreased with time. Other histological parameters such as periodontal ligament thickness, bone resorption area, collagen deposition could be accessed easily. Furthermore, despite its limitations this model was useful in accomplishing qualitative and quantitative (by morphometry) assessments. It is well-known that failure of perforation repairs may be the result of the absence of a seal (Saunders & Saunders 1994). Therefore, in this study, perforations were sealed with MTA and histopatological analysis was performed. This is an example of the application of this model to analyse the biocompatibility aspects of sealing materials. The preliminary results presented here indicate that the presence of MTA may interfere positively with lesion progression. The assessment was taken on day 21 post-surgery, but further and systematic investigations are necessary. The radiographic technique proposed revealed the anatomical structures of the normal periodontal and also demonstrated the adaptation of the lling materials, the perforation position and, in some cases, the destruction of interdental crest of bone. Alveolar bone mineral density was previously documented in mandibular radiographs and the lm used was tested in rats previously (Xiong et al. 2007, Mahl & Fontanella 2008). Although morphometrical evaluation of alveolar bone loss was not documented, correlation between bone resorption and histological parameters has been described previously (Waterman et al.1998, Teixeira et al. 2000, Xiong et al. 2007).
` was supported by grants from Fundac a o de Amparo a Pesquisa do Estado de Minas Gerais FAPEMIG (CBB APQ-1323-3.13/07 and CNPq (grant number 350567/1995-6 and 571093/2008-6). The authors nia Aparecida do are indebted to the technicians Va Nascimento Silva for histopatological processing.
References
Al-Daafas A, Al-Nazhan S (2007) Histological evaluation of contaminated furcal perforation in dogs teeth repaired by MTA with or without internal matrix. Oral Surgery Oral Medicine Oral Pathology Oral Radiology and Endodontology 103, e929. Arens DE, Torabinejad M (1996) Repair of furcal perforations with mineral trioxide aggregate: two case reports. Oral Surgery Oral Medicine Oral Pathology Oral Radiology and Endodontology 82, 848. Balla R, LoMonaco CJ, Skribner J, Lin LM (1991) Histological study of furcation perforations treated with tricalcium phosphate, hydroxylapatite, amalgam, and Life. Journal of Endodontics 17, 2348. Beavers RA, Bergenholtz G, Cox CF (1986) Periodontal wound healing following intentional root perforations in permanent teeth of Macaca mulatta. International Endodontic Journal 19, 3644. PFE, Holland R (2004) Cirurgia paraendodo ntica: Bernabe -la com embasamento cient como pratica co. In: Estrela C, ncia Endodo ntica, 3rd edn. Sao Paulo, Brazil: Artes Cie Medicas, pp. 657797. Bhaskar SN, Rappaport HM (1971) Histologic evaluation of endodontic procedures in dogs. Oral Surgery Oral Medicine Oral Pathology 31, 52635. Bramante CM, Berbert A, Bernardineli N, Gomes de Moraes I, Garcia RB (2004) Acidentes e complicac o es no tratamento ntico: soluc nicas, 2nd edn. Sao Paulo, Brazil: endodo o es cl Santos. pios Ba sicos de Morfometria Digital: Caliari MV (1997) Princ KS300 para iniciantes, Editora UFMG edn. Belo Horizonte, Minas Gerais, Brazil: UFMG. Daoudi MF, Saunders WP (2002) In vitro evaluation of furcal perforation repair using mineral trioxide aggregate or resin modied glass lonomer cement with and without the use of the operating microscope. Journal of Endodontics 28, 5125. Genco CA, Van DT, Amar S (1998) Animal models for Porphyromonas gingivalis-mediated periodontal disease. Trends in Microbiology 6, 4449. Hartwell GR, England MC (1993) Healing of furcation perforations in primate teeth after repair with decalcied freeze-dried bone: a longitudinal study. Journal of Endodontics 19, 35761. Klausen B (1991) Microbiological and immunological aspects of experimental periodontal disease in rats: a review article. Journal of Periodontology 62, 5973.
Conclusion
This experimental animal model for furcal perforation is not totally comparable to human models of furcal lesions. However, under carefully standardized conditions, this model can be used to simulate the inammatory process induced iatrogenically in humans during root canal treatment as an effective tool to study several aspects of tissue reaction to this specic kind of injury. Therefore, it is suitable for other studies on the triggering and control of the inammatory reaction, as well as on the search for suitable sealing materials.
Acknowledgements
R.M.E. Arantes and L.Q. Vieira are supported by research fellowships from Conselho Nacional de Desen gico (CNPq). This work volvimento Cientico e Tecnolo
928
Mahl CR, Fontanella V (2008) Optimal parameters for lateral oblique radiographs of rat mandibles. Dentomaxillofacial Radiology 37, 2247. Meister F, Lommel TJ, Gerstein H, Davies EE (1979) Endodontic perforations which resulted in alveolar bone loss. Report of ve cases. Oral Surgery Oral Medicine Oral Pathology 47, 46370. Mo ller AJR (1966) Microbiological examination of root canals and periapical tissues of human teeth. Odontol Tidskr 20, 745. Pacheco CMF, Queiroz-Junior CM, Maltos KLM et al. (2008) Crucial role of peripheral kappa-opioid receptors in a model of periodontal disease in rats. Journal of Periodontal Research 43, 7306. Sampaio P (1967) Placement of a rubber dam on rat molars. Journal of Dental Research 46, 1102. Saunders WP, Saunders EM (1994) Coronal leakage as a cause of failure in root-canal therapy: a review. Endodontics & Dental Traumatology 10, 1058. Seltzer RE, Bender IB (1990) The Dental Pulp, 3rd edn. Philadelphia: JB Lippincott. Sousa CJ, Loyola AM, Versiani MA, Bif JC, Oliveira RP, Pascon EA (2004) A comparative histological evaluation of the biocompatibility of materials used in apical surgery. International Endodontic Journal 37, 73848. Teixeira FB, Gomes BP, Ferraz CC, Souza-Filho SC, Zaia AA (2000) Radiographic analysis of the development of periapical lesions in normal rats, sialoadenectomized rats and sialoadenectomized-immunosuppressed rats. Dental Traumatology 16, 1547. Vajrabhaya LO, Korsuwannawong S, Jantarat J, Korre S (2006) Biocompatibility of furcal perforation repair material using cell culture technique: Ketac Molar versus ProRoot
MTA. Oral Surgery Oral Medicine Oral Pathology Oral Radiology and Endodontology 102, e4850. Walton RE, Torabinejad M (2002) Principles and Practice of Endodontics, 3rd edn. Oxford, UK: W B Saunders Co. Waterman PA, Torabinejad M, McMillan PJ, Kettering JD (1998) Development of periradicular lesions in immunosuppressed rats. Oral Surgery Oral Medicine Oral Pathology Oral Radiology and Endodontology 85, 7205. Wu MK, van der Sluis LW, Wesselink PR (2005) The risk of furcal perforation in mandibular molars using Gates-Glidden drills with anticurvature pressure. Oral Surgery Oral Medicine Oral Pathology Oral Radiology and Endodontology 99, 37882. Xiong H, Peng B, Wei L, Zhang X, Wang L (2007) Effect of an estrogen-decient state and alendronate therapy on bone loss resulting from experimental periapical lesions in rats. Journal of Endodontics 33, 13048.
Supporting Information Additional Supporting Information may be found in the online version of this article: Video Clip S1. A video animation was designed using the blender software (Blender Foundation, Amsterdam, the Netherlands) to represent the entire surgical procedure. The video clip is in avi format. Please note: Wiley-Blackwell are not responsible for the content or functionality of any supporting materials supplied by the authors. Any queries (other than missing material) should be directed to the corresponding author for the article.
929
doi:10.1111/j.1365-2591.2009.01597.x
The morphology of the apical foramen in posterior teeth in a North Indian population
S. Arora & S. Tewari

Department of Operative Dentistry and Endodontics, Government Dental College, Post Graduate Institute of Medical Sciences, Rohtak, Haryana, India
Abstract
Arora S, Tewari S. The morphology of the apical foramen in
posterior teeth in a North Indian population. International Endodontic Journal, 42, 930939, 2009.
Aim To determine the position and shape of the apical foramina in posterior teeth derived from an Indian population. Methodology A total of 800 freshly extracted maxillary and mandibular premolar and molar teeth from a native Haryana population were collected. Apices of teeth were stained with methylene blue and then examined stereomicroscopically (40). The following observations were made: number of apical foramina; size and shape of the minor apical foramen; accessory foramina frequency, and deviation of the minor apical foramina (frequency and distance) from the apex. Results The mean maximum and minimum diameter of the minor apical foramina ranged from 0.158 to 0.323 mm. The most common minor apical foramen shape was oval (81%). Frequency of accessory
foramina was between 2% and 41% for the various tooth types. The frequency of deviation of the minor apical foramina from the anatomic apex varied from 43% to 83% and the distance of deviation in all the teeth was between 0.052 and 2.921 mm. Conclusions The incidence of oval canals was higher in this Indian population compared to other populations. In 92% and 96% of teeth the difference between the maximum and the minimum diameter of all foramina was less than or equal to 0.20 and 0.25 mm, respectively. Therefore, four to ve instrument sizes larger than the rst binding le would have been necessary to shape the minor apical foramen of more than 95% of the teeth included in this study to make them round. Keywords: anatomical apex, computer-aided stereomicroscope, major apical foramina, minor apical foramina.
Received 14 October 2008; accepted 16 April 2009
Introduction
The main objective of root canal treatment is thorough mechanical and chemical cleansing of the pulp cavity and its complete lling with an inert material (Kuttler 1958). From the early work of Hess & Zurcher (1925) to the most recent studies demonstrating anatomic complexities of the root canal system, it has been
Correspondence: Dr Sanjay Tewari, Principal and Professor & Head of Department of Operative Dentistry and Endodontics, Government Dental College, Post Graduate Institute of Medical Sciences, Rohtak, Haryana, 124001 India (Tel.: +91 12622 13737; fax: +91 1262213737; e-mail: tewarisanjayrohtak@ yahoo.co.in).
established that a root with a tapering canal and a single foramen is the exception rather than the rule. Root canal morphology especially in the apical third is a critically important factor during conventional root canal treatment and surgical endodontics. The apical constriction, when present, is the narrowest part of the root canal, and preparation to this point should result in a small wound and optimal healing conditions (Ricucci & Langeland 1998). The dimension of the apical constriction has been a focus of debate. The horizontal dimension of the root canal system is not only more complicated than the vertical dimension (root canal length or working length) but also more difcult to investigate. These measurements could provide clues for the size of master apical le
930
Arora & Tewari Morphology of apical foramina
during root canal preparation (Ricucci 1998, Jou et al. 2004) and can have an impact on the selection of the best instrumentation technique (Cheung et al. 2007). The clinical philosophy that apical sizes should be kept as small as possible (Buchanan 2000), rather than as large as required, disregards the existing scientic facts. Studies have reported better debridement and reduced bacterial load with larger apical preparation (Ram 1977, Chow 1983, Dalton et al. 1998, Siqueira et al. 1999, Shuping et al. 2000). Dental manufacturers and some individuals are suggesting apical instrumentation with rotary instruments of sizes 20, 25 and 30 (Spangberg 2001). However this gives the erroneous impression that apical diameters of canals are small in size; even though the dimensions of the apical foramina and the apical canal region are reported to be larger (Mors et al. 1994, Gani & Visvisian 1999, Wu et al. 2000, Marroquin et al. 2004). Wide variations in the dimensions of the apical constriction have been reported. Cheung et al. (2007) reported the mean value for the longest and the shortest diameter of the apical constriction of mesial and distal canals of C-shaped mandibular second molars to be 0.26 : 0.15 mm and 0.36 : 0.22 mm, respectively. Marroquin et al. (2004) reported mean narrow (0.20 mm) and wide (0.26 mm) diameters of minor apical foramina in mandibular molars, 0.18 0.25 mm in the mesio-buccal and distobuccal roots and 0.220.29 mm in the palatal root of the maxillary molars. Mors et al. (1994) reported a mean diameter of 0.26 and 0.39 mm for the mesial and distal canals, respectively, in mandibular molars (without dening the exact site of measurement). Previous studies have frequently demonstrated that the apical foramen is not always located at the tip of the root. The frequency of deviation of the major foramen from the anatomical apex ranged from 46% to 92% and the mean distance between them ranged from 0.2 to 1.38 mm (Kuttler 1955, Green 1956, 1960, Palmer et al. 1971, Burch & Hulen 1972, Pineda & Kuttler -Subat et al. 1992, Mors et al. 1994, 1972, Blaskovic Marroquin et al. 2004, Cheung et al. 2007). Furthermore, these studies have shown that frequency of deviation varies in different races. If the foramen deviates in the buccal/lingual plane, it is difcult to locate its position using radiographs alone (Schaeffer et al. 2005). ElAyouti et al. (2001, 2002) also reported that a seemingly accurate working length ending radiographically 02 mm short of the radiographic apex can result in overestimation of working length in 51% of the root canals. Therefore, misinterpretation of
dental radiographs may lead to an incorrect determination of working length and to subsequent complications of over-instrumentation and overlling of the root canal (Seltzer et al. 1971). The cosmopolitan nature of urban populations means that endodontists treat an increasing number of patients of different and mixed racial origin. It is, therefore, important to be aware of the frequency of racially determined anatomic variations. A number of studies have shown different trends in morphology of roots and canals amongst the different races (Caliskan et al. 1995, Gulabivala et al. 2001, 2002, Ng et al. 2001, Wasti et al. 2001, Sert & Bayirli 2004, Al-Qudah & Awawdeh 2006). Additionally, no such morphometric study has yet been conducted in Indian populations. In view of conicting ndings regarding the apical zone and scarce reports on teeth of Indian origin, the objective of this study was to determine further the number, shape, diameter of the apical foramina as well as the incidence of their deviation from the anatomical apex, the distance between them and the frequency of accessory foramina in an Indian population.
Materials and methods

A total of 800 freshly extracted human permanent maxillary and mandibular posterior teeth, with completely formed apices, obtained from a North Indian (Haryana) population were included. Teeth were collected from a general district hospital attended by local population. Ethnicity of the population was further veried from the outpatient records. Teeth were extracted because of periodontal or pulpal disease. Following extraction teeth were washed under tap water, and stored in 5% sodium hypochlorite (Aroma Agencies, Mumbai, Maharashtra, India) and used within 6 months of extraction. The teeth were identied as maxillary or mandibular rst and second premolars, or rst and second molars. One hundred teeth, with intact crowns (for clear identication), in each group were selected according to strict inclusion and exclusion criteria as described by Marroquin et al. (2004). Primary teeth and roots with fractures, resorption, or underdevelopment (40magnication) or that had received any previous endodontic treatment were discarded (Marroquin et al. 2004). Soft tissues around and in the foramen area were removed with a size 6 K le (Dentsply Maillefer, Ballaigues, Switzerland) at 40magnication. The roots were then placed in methylene blue (Macsen Laboratories, Udaipur, Rajasthan, India), washed under
931
Morphology of apical foramina Arora & Tewari
running water for 10 min, and dried with pressurized air before examination. A computer-aided stereomicroscope with 40magnication (Stereo zoom microscope RSM-9; Radical Scientic Equipments Pvt. Ltd., Ambala Cantt, Haryana, India) and VideoTesT-Size 5.0 measurement software (VideoTesT, St Petersburg, Russia) was used. Measurement accuracy was assured through calibration between a micro scale with 0.1 mm markings and the software. The measuring dialogue menu was set in millimetres and adjusted to three decimal places. On the external root surface, the opening of the root canal was called the apical foramen (AF) and its outermost diameter was termed the major apical foramen (Fig. 1). The minor apical foramen (apical constriction) was considered to be the region of the apical foramen with the smallest diameter. From the minor apical foramen the canal widens as it approaches the major apical foramen (Fig. 1). In clinical practice, the minor apical foramen is a more consistent anatomical feature (Ponce & Vilar Fernandez 2003) and is the preferred landmark for the apical end-point for root canal treatment. The anatomic apex was dened as the most apical root structure (Fig. 1), and was traced by marking with red ink. These three anatomical entities could, theoretically, coincide in one. A foramen was categorized as accessory when its diameter was narrower than 0.10 mm (Marroquin et al. 2004). After initial conrmation of tooth type, the root morphology of the apical area was examined under 40magnication. Teeth were oriented until the major apical foramen was located in the middle of and parallel to the objective lens. The image of the minor apical foramen was then captured by regulating the focus.
Here, minor foramen was that part of the apical foramen with the smallest planar dimension, as observed by focusing below the major apical foramen (Cheung et al. 2007). If a root had more than one apical foramen, then each foramen was focused separately parallel to objective lens by changing the orientation of tooth and individual photographs were captured. The following observations were then made: 1. size of minor apical foramen, 2. shape of minor apical foramen, 3. accessory foramina frequency (if found), and 4. deviation of the minor apical foramen (in mm) from the apex.
Diameters of the minor apical foramen

The widest and narrowest diameters of each minor apical foramen were measured using the length measuring mode of the software and dened as the maximum and the minimum diameters, respectively (Fig. 2).
Shape of the minor apical foramen

A minor apical foramen with a difference greater than or equal to 0.02 mm between its maximum and minimum diameters was considered to have an oval shape. This criterion was established according to Marroquin et al. (2004) in consideration of the ISO tolerances for root canal instruments. The shape of the minor apical foramen was accordingly determined to
Figure 1 Diagrammatic representations of the morphological
features investigated.
Figure 2 Two minor apical foramina and one accessory foramen (top most) at apex, measurement of maximum and minimum diameters of each foramen has been made.
932
Figure 3 Method for measuring distances between the minor apical foramen and the anatomic apex. Each foramen was separately focused parallel to the objective lens and measurements were made on different photographs. AA denotes anatomic apex, traced by marking with red ink.
have either a round, oval, or irregular (triangular, kidney, or irregular) form.
Frequency and distance of deviation of minor apical foramen from anatomical apex
If the minor apical foramen was not located at the anatomical root apex, but at a more cervical position on the long axis of the root, a straight line parallel to the root from the most apical point of the foramen to a tangent line at the most apical point of the anatomical apex was used to determine the distance between the minor apical foramen and anatomical apex (Marroquin et al. 2004) (Fig. 3). The statistical data were arranged by means, maximum, minimum and SD.
deviation are reported separately for each root with one, two, three, four, and ve foramina. In tables S15 S16 minimum and maximum diameters of accessory foramina have been reported.
Number of apical foramina

Mandibular teeth The number of apical foramina in all posterior teeth is described in Table 1. In the mandibular posterior teeth the incidence of a single apical foramen ranged from 64% to 81% except in the mesial root of mandibular rst molars (33%). As many as ve apical foramina were observed in 1% teeth in mandibular rst and second premolars and mesial roots of rst molars. The greatest variation from a single apical foramina was observed in the mesial roots of mandibular rst molars with an incidence of two, three, four, ve foramina in 46%, 16%, 4% and 1%, respectively. Maxillary teeth In maxillary teeth the greatest variations from a single apical foramina were observed in both rst and second premolars followed by the mesiobuccal root of rst molars. The incidence of two foramina was 57%, 38% and 37% and more than two foramina in 17%, 21% and 9% of teeth, respectively. In maxillary teeth ve apical
Results
A total of 2004 foramina were investigated. The distance between the minor apical foramen and anatomical apex; frequency of accessory foramina; number, shape and diameter of apical foramina in each root of maxillary and mandibular premolars and rst and second premolars are shown in Tables 15. In tables S1S14 (supporting information) the mean values for minimum and maximum diameters and distance of
933
Table 1 Number of apical foramina by tooth type

Single Two Three Four Five Mandibular teeth Mandibular rst premolar Mandibular second premolar Mandibular rst molar Mesial root Distal root Mandibular second molar Mesial root Distal root Maxillary teeth Maxillary rst premolar Maxillary second premolar Maxillary rst molar Mesiobuccal root Distobuccal root Palatal root Maxillary second molar Mesiobuccal root Distobuccal root Palatal root
70 74 33 75 64 81 25 41 54 91 78 61 80 88
24 16 46 22 33 18 57 38 37 8 20 31 13 11
3 6 16 2 3 X 13 16 6 X 2 5 X 1
2 3 4 1 X 1 4 2 3 X X 3 X X
1 1 1 X X X X 3 X X X X X X
(Table 2). The highest frequency of accessory foramina was in maxillary second premolars (41%). Values as low as 2%, for palatal roots of maxillary second molars were also obtained.
Deviation of minor apical foramina from the anatomical apex

Deviation of the minor apical foramina from the anatomical apex was seen in all teeth but there was no conclusive pattern of variation (Table 3). Frequency of deviation of minor apical foramina (Table 3) from the apex varied from 43% in maxillary rst premolars to 83% in mandibular rst premolars. The highest mean value of the distance between the minor apical foramen and the anatomical apex was observed in the mesiobuccal root of maxillary rst molars (0.996 mm). The lowest value of 0.632 mm was observed for the distobuccal root of maxillary second molars (Table 3).
Table 2 Frequency of accessory foramina by tooth type

0 Mandibular teeth Mandibular rst premolar Mandibular second premolar Mandibular rst molar Mesial root Distal root Mandibular second molar Mesial root Distal root Maxillary teeth Maxillary rst premolar Maxillary second premolar Maxillary rst molar Mesiobuccal root Distobuccal root Palatal root Maxillary second molar Mesiobuccal root Distobuccal root Palatal root 1 2 3 4 5 Total
Diameters of minor apical foramina

The mean maximum diameter (Table 4) of the minor apical foramina ranged from 0.230 mm (distobuccal root of maxillary second molars) to 0.323 mm (distal root of mandibular second molars). The mean minimum diameter (Table 5) of the minor apical foramina ranged from 0.158 mm (mandibular second premolars) to 0.227 mm (distal root of mandibular second molars).
91 91 86 96 95 94 87 72 92 95 93 86 96 98
7 6 13 4 5 6 8 12 5 5 5 7 4 2
2 0 1 X X X 1 3 2 X 2 5 X X
X 1 X X X X 3 3 1 X X X X X
X 2 X X X X X 1 X X X 2 X X
X X X X X X 1 2 X X X X X X
11 17 15 4 5 6 24 41 12 5 9 25 4 2
Discussion
A digital stereomicroscope with integrated software was used to provide accurate measurement of a large number of teeth. Teeth were oriented until the apical foramen was located in the middle of and parallel to the objective lens to allow measurement of the true dimensions of the minor apical foramen irrespective of the curvature that a canal follows inside the root. If the apical foramen was located at a more cervical position on the long axis of the root, a straight line parallel to the root from the most apical point of the foramen to a tangent line at the most apical point of the anatomical apex was drawn for each foramina. This determined the true distance not the vertical distance between the apical foramen and anatomical apex. However this distance can not be measured in vivo because of limitations of radiographs in identifying accurately the foramina on buccal/lingual root surfaces
foramina were found in maxillary second premolars only, with an incidence of 3% (Fig. 4). Distobuccal roots of rst and second molars showed simpler anatomy having a maximum two apical foramina only, and no main apical foramina in 1% teeth.
Frequency of accessory foramen

There was a higher frequency of accessory foramina in maxillary premolars followed by mandibular premolars
934
Table 3 Distance (in mm) and %age deviation of minor apical foramina from the apex by tooth type
Minimum Maximum Average SD Mandibular teeth First premolar Second premolar First molar: mesial root First molar: distal root Second molar: mesial root Second molar: distal root Maxillary teeth First premolar Second premolar First molar: mesiobuccal root First molar: distobuccal root First molar: palatal root Second molar: mesiobuccal root Second molar: distobuccal root Second molar: palatal root
%Deviation
0.112 0.052 0.154 0.167 0.127 0.065 0.18 0.122 0.176 0.225 0.26 0.276 0.106 0.122
2.186 2.892 2.808 1.617 2.202 2.031 2.03 2.523 2.921 2.224 2.455 2.527 1.433 0.873
0.796 0.781 0.834 0.817 0.78 0.809 0.78 0.984 0.996 0.824 0.924 0.992 0.632 0.719
0.415 0.463 0.496 0.297 0.465 0.322 0.391 0.407 0.676 0.441 0.466 0.580 0.319 0.313
83 78 50 68 46 44 43 49 59 54 68 59 49 61
Table 4 Maximum diameter (in mm) of minor apical foramina by tooth type
Mandibular teeth First premolar Second premolar First molar: mesial root First molar: distal root Second molar: mesial root Second molar: distal root Maxillary teeth First premolar Second premolar First molar: mesiobuccal root First molar: distobuccal root First molar: palatal root Second molar: mesiobuccal root Second molar: distobuccal root Second molar: palatal root
Minimum
Maximum
Average
SD
0.1 0.104 0.102 0.116 0.112 0.122 0.106 0.103 0.1 0.103 0.108 0.105 0.103 0.117
1.169 1.45 1.265 0.585 0.793 1.168 0.553 1.394 0.835 0.524 0.794 0.509 0.43 0.794
0.256 0.241 0.261 0.300 0.303 0.323 0.24 0.254 0.263 0.257 0.320 0.244 0.230 0.309
0.122 0.144 0.153 0.104 0.143 0.142 0.092 0.139 0.125 0.082 0.146 0.098 0.078 0.12
(Schaeffer et al. 2005), as well as the inability of the electronic apex locators in locating the canal terminus with 100% accuracy (Wrbas et al. 2007, ElAyouti et al. 2009). The frequency of deviation of the minor apical foramen from the apex (4383%), distances of the minor apical foramina from the apex (0.052 2.921 mm) and mean distance of minor apical foramina from anatomic apex reported in the present study (0.6320.996 mm) compare with the literature (Kuttler 1955, Green 1956, 1960, Palmer et al. 1971, Pineda & Kuttler 1972, Vertucci 1984, Blask -Subat et al. 1992, Mors et al.1994, Marroquin ovic et al. 2004). The minor differences observed between various studies may be explained by the different measuring methods, by the different apical foramen denitions used and by difference in reference points to measure the distances. These results demonstrate the
complexity of the apical zone in this Indian population and the similarity with other parts of the world. Thus, they suggest that the endodontic principles and practices being followed in the other parts of the world can also be applied to this Indian population. The unpredictable nature of the position of the apical constriction with respect to the radiographic apex further strengthens the need of using apex locators rather than relying on radiographs for canal length determination. These ndings also support the current practice of cutting 3 mm of the root apex (Kim & Kratchman 2006) during surgical procedures to ensure the removal of most of the unprepared and unlled canals. Unlike most of the previous studies mesial and mesiobuccal roots of mandibular and maxillary molars respectively had a higher frequency of single foramen. This may be interpreted that there was a high tendency for Type II (two canals merging into one at apex) canals
935
Minimum Mandibular teeth First premolar Second premolar First molar: mesial root First molar: distal root Second molar: mesial root Second molar: distal root Maxillary teeth First premolar Second premolar First molar: mesiobuccal root First molar: distobuccal root First molar: palatal root Second molar: mesiobuccal root Second molar: distobuccal root Second molar: palatal root
Maximum
Average
SD
Table 5 Minimum diameter (in mm) of
minor apical foramina by tooth type
0.064 0.06 0.065 0.074 0.061 0.101 0.059 0.032 0.045 0.043 0.073 0.045 0.052 0.065
0.512 0.439 0.495 0.544 0.654 0.581 0.493 0.406 0.428 0.398 0.714 0.422 0.387 0.506
0.173 0.158 0.178 0.222 0.198 0.227 0.171 0.169 0.174 0.183 0.226 0.168 0.171 0.218
0.074 0.065 0.078 0.084 0.098 0.083 0.064 0.068 0.068 0.069 0.108 0.07 0.07 0.087
Figure 4 Photomicrograph of maxillary second premolar
(40), with ve minor apical foramina and one accessory foramen, identied by regulating the focus.
in mesial/mesiobuccal roots in this north Indian population. This anatomy is best treated by preparing and lling the straighter canal (generally palatal/ lingual) to the apex and the other (buccal) canal to point of juncture. If both canals are enlarged to the apex, an hour glass preparation results, which leaves voids in apical third during lling (Vertucci 2005). The presence of two root canals with two apical foramina in the palatal root of maxillary molars is uncommon. However, the present study revealed that approximately 20% of palatal roots of maxillary rst molars and 11% of maxillary second molars had two minor apical foramina of similar dimensions. About 2% and 1% palatal roots of maxillary rst and second molars, respectively, had three foramina. This nding
may indicate the presence of two or more root canals or one root canal with an apical ramication in the palatal roots of maxillary molars. Up to ve apical foramina were observed in 0.75% samples in mandibular and maxillary premolars and mesial and mesiobuccal roots of rst molars similar to the study of Gutierrez & Aguayo (1995). There was a high frequency of accessory foramina in both maxillary and mandibular premolars amongst all tooth type; supporting the ndings of Mors et al. (1994) that maxillary and mandibular premolars have the most complicated apical morphologic make up with respect to main foramina and accessory foramina. A high frequency of accessory foramina as well as multiple foramina suggests the extensive branching of the root canal or the presence of multiple canals at the apex and thus relates to high incidence of post-treatment apical periodontitis (Green et al. 1997, Barthel et al. 2004) due to non negotiation of extra canals by orthograde instrumentation alone and further suggests scope of surgical endodontics for management of such teeth. The mean maximum diameter of the minor apical foramina ranged from 0.23 to 0.32 mm and the mean minimum diameter was in the range 0.158 0.227 mm. These values were in accordance with Marroquin et al. (2004) (0.200.29 mm), Cheung et al. (2007) (0.32 mm) and Wu et al. (2000) (0.13 0.46 mm) but lower than those reported by Mors et al. (0.4180.977 mm) and by Gani & Visvisian (0.3320.594 mm). These results support that instrument sizes 10 or 15 often do not have contact at the minor apical foramen but rather encounter resistance elsewhere because of root canal irregularities or
936
curvature. So, in this Indian population the rst le that will truly bind at the apex, i.e. initial working width at working length would correspond to at least a size 20 le. The most common shape of the minor apical foramen was oval. The frequencies were between 79% and 89% for various roots. This prevalence of oval canals were higher than Marroquin et al. (2004) and Wu et al. (2000) and lower than Gani & Visvisian (1999). Other forms of apical foramina such as triangular, kidney, or irregular forms were observed in 0.5% of the roots (Fig. 5). One of the major point of interest when planning this investigation was its clinical signicance when shaping and cleaning the root canal of this Indian population. The determination of the rst le that binds in the apical part of the root canal does not allow a reliable prediction of the appropriate nal instrument size required for complete apical enlargement. The nal instrument size must be large enough to touch all walls. Because most canals are oval in their cross sectional shape, the goal should be to make the nal apical instrument size correspond to the largest diameter of the oval to make these canals round. The difference between the wide and narrow diameters of the apical constriction region in mandibular premolars in this population, was less than or equal to 0.15 mm in 86% teeth therefore enlarging up to three instrument sizes larger than the rst binding le in apical constriction region (FAB) will shape the minor apical foramen area round only in 86% teeth. This difference between the wide and narrow diameters in these premolars was less than or equal to 0.20 mm in 91%
and less than or equal to 0.25 mm in 97% teeth so shaping up to 4 instrument sizes larger than FAB will shape apical area in 91% and ve instrument size larger than FAB will shape the apical area in 97% teeth to a round outline. Similarly, for the rest of the teeth it was found that up to three instrument sizes larger than FAB would shape the apical constriction round in only 84% of mandibular molars, 88% of maxillary premolars and 87% of maxillary molars. While four instrument sizes larger than FAB will shape apical constriction round in 89% of mandibular molars, 93% of maxillary premolars and 94% of maxillary molars, and ve instrument sizes larger than the FAB will shape apical constriction round in 94% of mandibular molars, 97% of maxillary premolars and 96% of maxillary molars. These ndings suggest that in the absence of any predictable method to measure accurate working width, enlargement of apical third 45 sizes larger than rst le to bind at the apex may ensure complete involvement of the largest diameter of an oval canal in more than 95% teeth and will produce a round shape of apical preparation for proper lling with a round gutta percha cone.
Conclusions
The incidence of oval canals was higher in this Indian population (81%) compared to other populations and occurred in 7988% of roots depending on tooth type. In 92% and 96% of teeth the difference between the maximum and the minimum diameter of all foramina was less than or equal to 0.20 and 0.25 mm, respectively, therefore four to ve instrument sizes larger than the rst binding le would have been necessary to shape the minor apical foramen of more than 95% of the teeth included in this study to make them round.
References
Al-Qudah AA, Awawdeh LA (2006) Root canal morphology of mandibular incisors in a Jordanian population. International Endodontic Journal 39, 8737. Barthel CR, Zimmer S, Trope M (2004) Relationship of radiologic and histologic signs of inammation in human root-lled teeth. Journal of Endodontics 30, 759. -Subat V, Maricic B, Sutalo J (1992) Asymmetry of Blaskovic the root canal foramen. International Endodontic Journal 25, 15864. Buchanan LS (2000) The standardized-taper root canal preparation Part 1. Concepts for variably tapered shaping instruments. International Endodontic Journal 33, 51629.
Figure 5 Minor apical foramina of various irregular shapes (irregular, kidney shaped, triangular).
937
Burch JG, Hulen S (1972) The relationship of the apical foramen to the anatomic apex of the tooth root. Oral Surgery, Oral Medicine and Oral Pathology 34, 2628. Caliskan MK, Pehlivan Y, Sepetcioglu F, Turkun M, Tuncer SS (1995) Root canal morphology of human permanent teeth in a Turkish population. Journal of Endodontics 21, 2004. Cheung GSP, Yang J, Fan B (2007) Morphometric study of the apical anatomy of C-shaped root canal systems in mandibular second molars. International Endodontic Journal 40, 23946. Chow T (1983) Mechanical effectiveness of root canal irrigation. Journal of Endodontics 9, 4759. Dalton BC, Orstavik D, Phillips C, Pettiette M, Trope M (1998) Bacterial reduction with nickeltitanium rotary instrumentation. Journal of Endodontics 24, 7637. ElAyouti A, Weiger R, Lo st C (2001) Frequency of overinstrumentation with an acceptable radiographic working length. Journal of Endodontics 27, 4952. ElAyouti A, Weiger R, Lo st C (2002) The ability of root ZX apex locator to reduce the frequency of overestimated radiographic working length. Journal of Endodontics 28, 1169. ElAyouti A, Dima E, Ohmer J, Sperl K, von Ohle C, Lo st C (2009) Consistency of apex locator function. Journal of Endodontics 35, 17981. Gani O, Visvisian C (1999) Apical canal diameter in the rst upper molar at various ages. Journal of Endodontics 25, 689 91. Green D (1956) A stereomicroscopic study of the root apices of 400 maxillary and mandibular teeth. Oral Surgery, Oral Medicine and Oral Pathology 9, 122432. Green D (1960) Stereomicroscopic study of 700 root apices of maxillary and mandibular posterior teeth. Oral Surgery, Oral Medicine and Oral Pathology 13, 72833. Green TL, Walton RE, Taylor JK, Merrell P (1997) Radiographic and histologic periapical ndings of root canal treated teeth in cadaver. Oral Surgery, Oral Medicine, Oral Pathology, Oral Radiology, and Endodontics 83, 70711. Gulabivala K, Aung TH, Alavi A, Ng YL (2001) Root and canal morphology of Burmese mandibular molars. International Endodontic Journal 34, 35970. Gulabivala K, Opasanon A, Ng YL, Alavi A (2002) Root canal morphology of Thai mandibular molars. International Endodontic Journal 35, 5662. Gutierrez JH, Aguayo P (1995) Apical foraminal openings in human teeth. Number and location. Oral Surgery, Oral Medicine, Oral Pathology, Oral Radiology, and Endodontics 79, 76977. Hess W, Zurcher E (1925) The Anatomy of Root Canals of the Teeth of the Permanent and Deciduous Dentitions. New York: William Wood & Co. Jou YT, Karabucak B, Levin J, Liu D (2004) Endodontic working width: current concepts and techniques. Dental Clinics of North America 48, 32335.
Kim S, Kratchman S (2006) Modern endodontic surgery concepts and practice: a review. Journal of Endodontics 32, 60123. Kuttler Y (1955) Microscopic investigation of root apexes. Journal of the American Dental Association 50, 54452. Kuttler Y (1958) A Precision and Biologic root canal lling technic. Journal of the American Dental Association 56, 38 50. Marroquin BB, El-Sayed MA, Wilershausen-Zo nnchen B (2004) Morphology of the physiological foramen. I. Maxillary and mandibular molars. Journal of Endodontics 30, 3218. Mors A, Sylaras SN, Georgopoulou M, Kernani M, Prountzos F (1994) Study of the apices of human permanent teeth with the use of a scanning electron microscope. Oral Surgery, Oral Medicine and Oral Pathology 77, 1726. Ng YL, Aung TH, Alavi A, Gulabivala K (2001) Root and canal morphology of Burmese maxillary molars. International Endodontic Journal 34, 62030. Palmer MJ, Weine FS, Healey H (1971) Position of the apical foramen in relation to endodontic therapy. Journal of the Canadian Dental Association 37, 3058. Pineda F, Kuttler Y (1972) Mesiodistal and buccolingual roentgenographic investigation of 7,275 root canals. Oral Surgery, Oral Medicine and Oral Pathology 33, 10110. Ponce EH, Vilar Fernandez JA (2003) The cemento-dentinocanal junction, the apical foramen, and the apical constriction: evaluation by optical microscopy. Journal of Endodontics 29, 2149. Ram Z (1977) Effectiveness of root canal irrigation. Oral Surgery, Oral Medicine and Oral Pathology 44, 30612. Ricucci D (1998) Apical limit of root canal instrumentation and obturation. Part 1. Literature review. International Endodontic Journal 31, 38493. Ricucci D, Langeland K (1998) Apical limit of root canal instrumentation and obturation. Part 2. A histological study. International Endodontic Journal 31, 394409. Schaeffer M, White R, Walton R (2005) Determining the optimal obturation length: a meta-analysis of the literature. Journal of Endodontics 31, 2714. Seltzer S, Soltanoff W, Sinai I (1971) Biologic aspects of endodontics. III. Periapical tissue reactions to root canal instrumentation. Oral Surgery, Oral Medicine and Oral Pathology 33, 10110. Sert S, Bayirli GS (2004) Evaluation of the root canal congurations of the mandibular and maxillary permanent teeth by gender in the Turkish population. Journal of Endodontics 30, 3918. Shuping G, Orstavik D, Sigurdsson A, Trope M (2000) Reduction of intracanal bacteria using nickel-titanium rotary instrumentation and various medications. Journal of Endodontics 26, 7515. Siqueira J, Lima K, Magalhaes F, Lopes H, de Uzeda M (1999) Mechanical reduction of the bacterial population in the root canal by three instrumentation techniques. Journal of Endodontics 25, 3325.
938
Spangberg L (2001) The wonderful world of rotary root canal preparation. Oral Surgery, Oral Medicine, Oral Pathology, Oral Radiology and Endodontics 92, 479. Vertucci FJ (1984) Root canal anatomy of the human permanent teeth. Oral Surgery, Oral Medicine and Oral Pathology 58, 58999. Vertucci FJ (2005) Root canal morphology and its relationship to endodontic procedures. Endodontic Topics 10, 329. Wasti F, Shearer AC, Wilson NH (2001) Root canal systems of the mandibular and maxillary rst permanent molar teeth of south Asian Pakistanis. International Endodontic Journal 34, 2636. Wrbas KT, Ziegler AA, Altenburger MJ, Schirrmeister JF (2007) In vivo comparison of working length determination with two electronic apex locators. International Endodontic Journal 40, 1338. Wu M-K, Roris A, Barkis D, Wesselink PR (2000) Prevalence and extent of long oval canals in the apical third. Oral Surgery, Oral Medicine, Oral Pathology, Oral Radiology and Endodontics 89, 73943.
Table S3. Mandibular First Molar Mesial Root. Table S4. Mandibular First Molar Distal Root. Table S5. Mandibular Second Molar Mesial Root. Table S6. Mandibular Second Molar Distal Root. Table S7. Maxillary First Premolar. Table S8. Maxillary Second Premolar. Table S9. Maxillary First Molar (Mesiobuccal Root). Table S10. Maxillary First Molar (Distobuccal Root). Table S11. Maxillary First Molar (Palatal Root). Table S12. Maxillary Second Molar (Mesiobuccal Root). Table S13. Maxillary Second Molar ( Distobuccal Root). Table S14. Maxillary Second Molar (Palatal Root). Table S15. Maximum diameter (in mm) of accessory foramina by tooth type. Table S16. Minimum diameter (in mm) of accessory foramina by tooth type. Please note: Wiley-Blackwell are not responsible for the content or functionality of any supporting materials supplied by the authors. Any queries (other than missing material) should be directed to the corresponding author for the article.
Supporting Information Additional Supporting Information may be found in the online version of this article: Table S1. Mandibular First Premolar. Table S2. Mandibular Second Premolar.
939
doi:10.1111/j.1365-2591.2009.01598.x
Carbon dioxide laser irradiation stimulates mineralization in rat dental pulp cells
Y. Yasuda1, E. Ohtomo1, T. Tsukuba2, K. Okamoto2 & T. Saito1

1 Division of Clinical Cariology and Endodontology, Department of Oral Rehabilitation, School of Dentistry, Health Sciences University of Hokkaido, Hokkaido, Japan; and 2Division of Oral Pathopharmacology, Department of Medical and Dental Sciences, Nagasaki University Graduate School of Biomedical Sciences, Nagasaki, Japan
Abstract
Yasuda Y, Ohtomo E, Tsukuba T, Okamoto K, Saito T.
Carbon dioxide laser irradiation stimulates mineralization in rat dental pulp cells. International Endodontic Journal, 42, 940946, 2009.
Aim To examine the effect of carbon dioxide laser irradiation on mineralization in dental pulp cells. Methodology Rat dental pulp cells were irradiated with a carbon dioxide laser at 2 W output power for 20, 40 and 60 s, and were cultured in ascorbic acid and b-glycerophosphate containing media. Cell viability was examined 24 h after laser irradiation by a modied MTT assay. Alizarin Red S staining was performed 10 days after laser irradiation. The amounts of secreted collagen from the cells after irradiation were quantied following Sirius Red staining. The expression levels of collagen type I and HSP47, collagen-binding stress protein, were analysed by real-time PCR. HSP47 protein expression was examined by Western blotting. Statistical analysis was performed using one-way analysis of variance (anova) followed by the Tukeys multiple comparison test.
Results The cell viability was not affected by laser irradiation at 2 W for up to 40 s. However, it was signicantly decreased by 20% at 60 s (P < 0.05). The amount of mineralization after 10 days of irradiation at 2 W for 40 s was signicantly increased in comparison to the other conditions (P < 0.05). The extracellular collagen production was signicantly increased by 73% on day 2 and 38% on day 4 after laser irradiation (P < 0.05). Although collagen type I gene expression was not changed by laser irradiation, HSP47 gene and protein expression was induced within 12 and 24 h, respectively. Conclusions These results suggested that carbon dioxide laser irradiation stimulated mineralization in dental pulp cells. The laser irradiation also increased HSP47 expression but not collagen gene expression. Keywords: carbon dioxide laser, collagen, dental pulp cells, heat shock protein 47, mineralization.
Received 28 December 2007; accepted 16 April 2009
Introduction
Reparative dentine forms in the dental pulp in response to various external stimuli such as caries and abrasion (Kamal et al. 1997, Lee et al. 2006). Direct pulp capping with calcium hydroxide has been advocated
Correspondence: Yoshiyuki Yasuda, DDS, PhD, Division of Clinical Cariology and Endodontology, Department of Oral Rehabilitation, School of Dentistry, Health Sciences University of Hokkaido, 1757 Kanazawa, Ishikari-Tobetsu, Hokkaido 061-0293, Japan (Tel.: +81 133 23 2841; fax: +81 133 23 1423, e-mail: yasuda@hoku-iryo-u.ac.jp).
to accelerate reparative dentine formation on the exposed pulp surface. However, calcium hydroxide is highly alkaline and causes an inammatory response. In addition, it has not always been clinically highly efcacious in the uniform formation of reparative dentine (Scarano et al. 2003). Recently, the use of mineral trioxide aggregate (MTA) in direct pulp capping has been reported (Aeinehchi et al. 2003, Chacko & Kurikose 2006). MTA is superior to calcium hydroxide for pulp capping of mechanically exposed human teeth; however, a variety of histological responses were still observed (Caicedo et al. 2006). Furthermore, no data from long-term clinical results
940
Yasuda et al. Effect of laser irradiation
are yet available. In an ideal situation, the exposed pulp surface should be covered promptly with reparative dentine and the dental pulp should not demonstrate an inammatory response. The application of lasers has expanded into various elds and has also been frequently used in clinical dentistry (Pearson & Schuckert 2003, Parker 2007). In cultured cells, irradiation by low power laser, such as a diode laser, has been reported to accelerate cell differentiation and mineralization in calvarial and dental pulp cells (Ozawa et al. 1998, Ohbayashi et al. 1999, Ueda & Shimizu 2003). On the other hand, Moritz et al. (1998a,b) reported that the utility of a high power laser, such as a carbon dioxide laser, is useful on exposed pulp surfaces in direct pulp capping experiments. Furthermore, Melcer et al. (1987) observed that a neo-dentine bridge was formed in the pulp tissue after carbon dioxide laser irradiation on teeth, suggesting that this laser is effective in mineralization. However, the mechanism by which carbon dioxide laser irradiation stimulates mineralization in direct pulp capping treatment is not fully elucidated. Laser irradiation may affect the collagen production in dental pulp cells, because the collagenous network plays an important role in mineralization (Linde 1989). Heat shock proteins (HSPs) are induced by stress from heat and chemical stimuli (Noda et al. 2002). HSPs have been known to suppress the aggregation of denatured protein (Guzhova & Margulis 2006). They are also constitutively expressed in normal cells, and are associated with important functions such as protein synthesis and intracellular transport (Eisenberg & Greene 2007). In particular, HSP47 is a collagenspecic molecular chaperone. It specically binds to collagen and plays an essential role in collagen production (Masuda et al. 1994, Koide et al. 2002). The purpose of this study was to examine the effect of a carbon dioxide laser on mineralization in rat dental pulp cells. Moreover, the amount of extracellular secreted collagen and the HSP47 expression levels were examined to clarify the stimulatory effects of carbon dioxide laser irradiation on mineralization of dental pulp cells.
out under the control of the Universitys Guidelines for Animal Experimentation. The dental pulp cells were isolated from incisors of Wistar rats (female, 5-weekold) as described previously (Yokose et al. 2000). The cells were cultured in Dulbeccos modied eagle medium (DMEM; Sigma, St Louis, MI, USA) supplemented with 10% foetal bovine serum (Sigma), 10 000 U mL)1 penicillin (Invitrogen, Grand Island, NY, USA), and 10 mg mL)1 streptomycin (Invitrogen) at 37 C in a humidied atmosphere of 5% carbon dioxide.
Laser irradiation
A carbon dioxide laser apparatus (Bel Luxar LX-20SP, Takara, Kyoto, Japan) with a wavelength of 10.6 lm and a power output of 2.0 W (A4 mode, 10 pps, average power output of 0.3 W) was used. Rat dental pulp cells (5 104 cells per well) were seeded out in 24-well plates and cultured for 24 h, and then serumstarved for 24 h. After withdrawal of medium, the cells were irradiated at 2 W output power for 20, 40 and 60 s using the scanning method as applied in clinical laser irradiation. The tip was moved gradually at a constant rate, avoiding concentrating laser light on one site, and the whole area was irradiated. The laser beam was delivered by a ceramic tip (0.8 mm diameter) with the distance from the tip of the bre to the cell layer being 2 cm (irradiation diameter approximately 2 mm). The total energy of irradiation time of 40 s was 382.2 J cm)2. Irradiated or nonirradiated (control) cells were cultured in DMEM containing 50 lg mol L)1 ascorbic acid (AA, Sigma) and 10 m mL)1 b-glycerophosphate (b-GP, Sigma) for 10 days.
Cell viability assay

Rat dental pulp cells were cultured in DMEM containing 50 lg mL)1 AA and 10 mmol L)1 b-GP for 24 h after irradiation or nonirradiation (control). Cell viability was determined by a modied MTT assay (WST-8 assay: Dojindo, Kumamoto, Japan), and data are presented as a percentage of viability values seen under control culture conditions. The assay is based on the cleavage of tetrazolium salt WST-8 to formazan by cellular mitochondrial dehydrogenase. The amount of the dye generated by activity of dehydrogenase is directly proportional to the number of living cells. For the WST-8 assay, a 10-lL quantity of WST-8 dye solution was added directly to 100 lL of culture medium per well. The absorbance of the dye was
Materials and methods Cells and cell culture conditions

All animal protocols were approved by the Institutional Animal Care and Use Committee of the Health Sciences University of Hokkaido, and experiments were carried
941
Effect of laser irradiation Yasuda et al.
measured at 450 nm using a Model 680 microplate reader (Bio-Rad, Hercules, CA, USA).
Quantitative Alizarin Red S staining

Rat dental pulp cells were cultured as before in 50 lg mL)1 AA and 10 mmol L)1 b-GP-containing media for 10 days. Cells were xed in 70% ice-cold ethanol for 1 h and rinsed with distilled water. Cells were stained with 40 mmol L)1 Alizarin Red S (Sigma), pH 4.2, for 10 min with gentle agitation. Alizarin Red S staining is specic for calcium deposition. Cells were rinsed thrice with distilled water and then rinsed with PBS for 15 min. Dye was extracted from xed cells by treatment with 500 lL 10% cetylpyridinium chloride (Nakarai Tesque., Kyoto, Japan) for 20 min with gentle agitation. The absorbance of the extracted dye was measured at 570 nm using a Model 680 microplate reader. The amount of Alizarin Red S was determined according to an Arizarin Red S standard curve.
Quantitative analysis of extracellular secreted collagen

The amount of extracellular secreted collagen was measured on day 210 using the method described by Ohbayashi et al. (1999). Conditioned media (100 lL) were dispensed into wells of 96 well plates, and plates were incubated at 37 C for 24 h until dry. After rinsing with distilled water, 0.2% Sirius Red (Sigma) in saturated picric acid (w/v) was placed in each well for 30 min. The plates were washed with 0.5% NaOH. The eluted stain was then drawn up and down several times in a pipette and placed into a second plate. Absorbance was read at 540 nm in a Model 680 microplate reader, and the amount of extracellular secreted collagen was estimated from a standard curve.
Real-time PCR
The mRNA expression of collagen type I, HSP47 and glyceraldehyde 3-phosphate dehydrogenase (GAPDH) was determined by real-time PCR with rat-specic primers. Total RNA was extracted using RNeasy (Qiagen Inc., Chatworth, CA, USA) and was digested with DNase I (Sigma), according to the manufacturers instructions. Single-strand cDNA was synthesized with SuperScript II reverse transcriptase (Invitrogen) and random primers. Real-time PCR was performed on a volume of 15 lL containing 1.5 lL (50 ng) of cDNA and 13.5 lL of master mix containing 7.5 lL of mix
(SYBR Green PCR Master Mix, Invitrogen), 0.75 lL of each primer (10 pmol L)1), and 4.5 lL of diethyl pyrocarbonate-treated water using an ABI PRISM 7500 Sequence Detection System Thermal Cycler (Applied Biosystems, Foster City, CA, USA). The sequences of the rat-specic primers were as follows: collagen type I, forward 5-TTGACCCTAACCAAG GATGC-3, reverse 5-CACCCCTTCTGCGTTGTATT-3; HSP47, forward 5-GTGCGCTCCCTCAGTAACTC-3, reverse 5-CCACATCCTTGGTGACCTCT-3; Control primers specic for GAPDH were: forward 5-TCCACC ACCCTGTTGCTGTA-3, reverse 5-ACCACAGTCCAT GCCATCAC-3. The program was set at 50 C for 2 min and 95 C for 10 min followed by 40 cycles of denaturation at 95 C for 15 s and annealing at 60 C for 60 s. SYBR green uorescence was monitored after each elongation period. The threshold was set above the nontemplate control background and within the linear phase of target gene amplication to calculate the cycle number at which the transcript was detected (denoted CT). Samples were amplied in triplicate, averages were calculated, and differences in CT data were evaluated by Sequence Detection Software V1.3. (Applied Biosystems). For each primers set, validation experiments demonstrated that the efciencies of target and reference gene amplication were approximately equal; the absolute value of the slope of log input amount versus CT was <0.1. For data analysis, we used the comparative CT method (DDCT method) with the following formula: DCT = CT (Target))CT (GAPDH). The comparative DDCT calculation involved nding the difference between DCT of irradiated cells and the mean value of the DCT from the control cells. Fold increase in the expression of specic mRNA in irradiated cells compared to control cells was calculated as 2(DDCT). The data are expressed as relative quantity (RQ) and differences are shown in the gures as the expression ratio of the normalized target gene according to the software results.
Western blotting
For investigating the expression of HSP47 protein in dental pulp cells, immunoblot analysis was performed. The extracts were prepared from irradiated or nonirradiated cells using lysis buffer [100 mmol L)1 Tris HCl (pH 7.2) containing 150 mmol L)1 NaCl, 0.1 mmol L)1 DTT/EDTA, 0.1% Triton X-100]. The protein concentrations were determined using protein assay kit (Bio-Rad). Protein (20 lg) was loaded onto
942
Alizarin red S (mg/mL)
10% SDSPAGE gel. After electrophoresis, the SDS PAGE separated proteins were transferred to nitrocellulose membrane at 60 V for 2 h. The membrane was blocked with 10% bovine serum albumin in TBST [10 mmol L)1 TrisHCl (pH8.0), 150 mmol L)1 NaCl, 0.05% Tween 20] for 30 min, and incubated with a 1 : 1000 dilution of polyclonal rabbit IgG against human HSP47 (Stressgen, Ann Arbor, MI, USA) in TBST for 1 h. Then, the membrane was incubated with a 1 : 2000 dilution of goat anti-rabbit IgG conjugated with horseradish peroxidase (Sigma) for 1 h. Horseradish peroxidase activity was detected using the ECL system (Amersham Biosciences, Piscataway, NJ, USA).
(a) 100 80 60 40 20 0 Control (c) 0.5 0.4 0.3 0.2 0.1 0 Control
(b) Control
Cell viability (% of control)
2W 20 s
2W 40 s
2W 60 s
2 W, 40 s
Statistical analysis was performed with data obtained from three independent experiments. The data are expressed as mean SD and analysed using one-way analysis of variance (anova) followed by the Tukeys multiple comparison test. Statistical signicance was accepted at P < 0.05.
2W 20 s
2W 40 s
2W 60 s
Figure 1 Effect of laser irradiation on cell viability and miner-
Results
Rat dental pulp cells were irradiated with a carbon dioxide laser at 2 W output power for 20, 40, and 60 s. Thereafter, the cell viability was measured 24 h after irradiation (Fig. 1a). There was no difference in the cell viability between the control and the cells which were irradiated 20 or 40 s. However, it was signicantly decreased by 20% in the cells which were irradiated for 60 s in comparison to the controls (P < 0.05). Next, the effect of laser irradiation was examined on the mineralization in dental pulp cells. The cells which were irradiated for 40 s had a clearly increased number and total area of calcied nodules stained by Alizarin Red S (Fig. 1b). In addition, when the mineralization was determined quantitatively on day 10, the cells with 40-s irradiation had signicantly increased the degree of mineralization in comparison to the other conditions (P < 0.05). However, no signicant differences were observed between the controls and the cells with 20- or 60-s irradiation (P > 0.05; Fig. 1c). Next, the culture media were collected every 2 days up to 10 days and the amount of extracellular secreted collagen was determined quantitatively after Sirius Red staining. The amount of secreted collagen signicantly increased after laser irradiation in comparison to the controls 73% and 38% on day 2 and 4, respectively
alization of dental pulp cells. (a) Cell viability after carbon dioxide laser irradiation at 2 W for 20, 40 and 60 s was analysed by a modied MTT (3-(4,5-Dimethylthiazol-2-yl)-2,5diphenyltetrazolium bromide) assay. (b) After laser irradiation at 2 W for 20, 40 and 60 s, rat dental pulp cells were cultured in DMEM containing AA and b-GP for 10 days. Representative photographs of Alizarin Red S staining are shown (Original magnication 200 ). (c) Quantication of Alizarin Red S staining. Bar represents mean SD (n = 3). The data were analysed using one-way anova: *P < 0.05 versus control.
(P < 0.05; Fig. 2). However, there was no difference in comparison to the controls after day 6. To clarify the mechanism of increased collagen secretion after irradiation, the effect of laser irradiation on the expression of collagen type I and HSP47 was examined by real-time PCR method. There was no signicant difference in the expression of the collagen type I gene between the irradiated cells and the controls at any time point (P > 0.05; Fig. 3a). Interestingly, the expression of the HSP47 gene in the irradiated cells was signicantly increased compared to the controls by 54%, 57% and 24% at 12, 24 and 48 h, respectively (P < 0.05). In addition, Western blot analysis showed that HSP47 protein with a molecular weight of 47 kDa was increased in the cells 24 h after irradiation compared to that in control cells (Fig. 3b).
Discussion
A carbon dioxide laser has a photothermal effect that is applied when making incisions in soft tissue and obtaining haemostasis. It also has a photochemical
943
40 Secreted collagen (g/well)
# * *
Control Irradiation
30
20
10
6 Day
10
Figure 2 The amount of collagen secreted into the culture
media of dental pulp cells. The dental pulp cells were irradiated at 2 W for 40 s and cultured in DMEM supplemented with AA and b-GP for 10 days. Conditioned media from control and irradiated cells were collected every 2 days. The amount of collagen (lg per well) was measured by the Sirius Red staining method. Bar represents mean SD (n = 3). The data were analysed using one-way anova. Signicantly different from the control at each time point: *P < 0.05. Signicantly different from the day 2: #P < 0.05.
effect used in alleviating pain (Posten et al. 2005). Melcer et al. (1987) reported that neo-dentine bridge formation was observed in pulp tissue after carbon dioxide laser irradiation on teeth of dogs and monkeys. This nding indicates that carbon dioxide laser may be
useful for the induction of mineralization. The current study showed a decrease of cell viability for irradiation at 2 W for 60 s using a carbon dioxide laser. In other words, if the irradiation time is long, then the energy density is increased even with a low power laser and injuries to cells occur. Therefore, the effect of carbon dioxide laser on mineralization, collagen secretion and HSP47 expression was examined under conditions (2 W, 40 s) that did not injure the cells. Collagen, which constitutes almost 90% of the dentine matrix protein, is synthesized by the odontoblasts and secreted into predentine, where collagen molecules are arranged into bres. These bres form the collagenous network in which the mineral crystals are deposited (Linde 1989). Furthermore, it has been reported that collagen type I time-dependently stimulates the expression of osteopontin and alkaline phosphatase (ALP), whilst also inducing the differentiation of bone marrow cells into osteoblasts (Mizuno & Kuboki 2001). The DGEA (Asp-Gly-Glu-Ala) domain of type I collagen binds with integrin on the cellular membrane. Differentiation is thought to be promoted through its binding with integrin (Mizuno et al. 2000). In this study, carbon dioxide laser irradiation signicantly increased in the secretion of collagen into culture media on day 2 and 4. This nding suggests that increased collagen in culture media acted on the integrin of the cells, and mineralization was thus
Relative quality (collagen type I/GAPDH)
(a) 2.5
Control Irradiation
# #
Relative quantity (HSP47/GAPDH)
3.0 2.5 2.0 1.5 1.0 0.5 0 0 12
*# *#
2.0 1.5 1.0 0.5 0 0 12
# #
*# #
24 (h)
(b)
48
72
24 (h)
48
72
24 h + Irradiation HSP47
Figure 3 The expression of collagen type I and HSP47 in control and irradiated cells. (a) The dental pulp cells were irradiated at
2 W for 40 s and cultured in DMEM supplemented with AA and b-GP. The mRNA expression of collagen type I and HSP47 was analysed at the indicated time points by real-time PCR. Bar represents mean SD (n = 3). The data were analysed using one-way anova. Signicantly different from the control at each time point: *P < 0.05. Signicantly different from time 0: #P < 0.05. (b) The HSP47 protein expression was examined by Western blotting.
944
stimulated. Most recently, Lee et al. (2008) reported that heat stress at 42 C for 30 min signicantly elevated ALP activity on days 7 and 14 in rat pulp cells compared to control groups, revealing the possibility that heat stress generated by laser elevated ALP activity, thereby stimulating mineralization. HSP47 knockout mice cannot produce collagen with the correct triple helix. Therefore, they die by 11.5 days post-coitus due to apoptosis in various tissues and vascular ruptures because they cannot form collagen bres and basement membranes (Nagai et al. 2000). In addition, HSP47 expression is induced by thermal stimuli, and its constitutive expression is closely coupled with the amount of the collagen matrix. For example, an increase in the HSP47 expression has been reported in pulmonary brosis in which there is increased production of collagen (Razzaque et al. 1998). Therefore, HSP47 expression, which has a close relationship with collagen production, was examined. The results clearly showed that the HSP47 gene was induced by laser irradiation within 12 h and HSP47 protein was induced within 24 h. The observation that the expression level of HSP47 was correlated with the amount of collagen secretion is consistent with the ndings of a previous study (Razzaque et al. 1998). Although collagen gene expression was not altered by carbon dioxide laser irradiation, extracellular collagen secretion did increase. Regarding this discrepancy, increased HSP47 production by laser irradiation have led to efcient assembly of procollagen molecules prior to their secretion, thereby & promoting extracellular collagen secretion (Lamande Bateman 1999). To date, the studies of the mechanism of mineralization induction by laser have been conducted using a low power laser. Irradiation by a low power laser on osteoblasts resulted in increased expression of ALP and osteocalcin (Ozawa et al. 1998, Ohbayashi et al. 1999, Ueda & Shimizu 2003). It has been reported that these increases are one cause of mineralization induction. Hamajima et al. (2003) indicated that the gene expression of a bone-inducing factor called osteoglycin increased by twofold within 2 h when MC3T3-E1 osteoblast-like cells were irradiated by a low power laser. These ndings indicate that the mechanism of mineralization induction might differ according to the cells, type of laser, and irradiation conditions.
dental pulp cells. Furthermore, laser irradiation enhanced HSP47 gene and protein expressions but not type I collagen gene expression. Further study will be needed to elucidate the role of HSP47 on laser-induced mineralization in dental pulp cells.
Acknowledgements
This work was supported by Grant-in-Aid for Scientic Research 18659563, and Grant-in-Aid for Young Scientists (B) 18791407 and 20791390 from the Japan Society for the Promotion of Science, and by a Grant from the Research Center, Health Sciences University of Hokkaido. The authors give special thanks to Toru Kawamorita (Division of Clinical Cariology and Endodontology, Department of Oral Rehabilitation, School of Dentistry, Health Sciences University of Hokkaido) for his technical assistance in the molecular biology portion of this study.
References
Aeinehchi M, Eslami B, Ghanbariha M, Saffar AS (2003) Mineral trioxide aggregate (MTA) and calcium hydroxide as pulp-capping agents in human teeth: a preliminary report. International Endodontic Journal 36, 22531. Caicedo R, Abbott PV, Alongi DJ, Alarcon MY (2006) Clinical, radiographic and histological analysis of the effects of mineral trioxide aggregate used in direct pulp capping and pulpotomies of primary teeth. Australian Dental Journal 51, 297305. Chacko V, Kurikose S (2006) Human pulpal response to mineral trioxide aggregate (MTA): a histologic study. Journal of Clinical Pediatric Dentistry 30, 2039. Eisenberg E, Greene LE (2007) Multiple roles of auxilin and hsc70 in clathrin-mediated endocytosis. Trafc 8, 6406. Guzhova I, Margulis B (2006) Hsp70 chaperone as a survival factor in cell pathology. International Review of Cytology 254, 10149. Hamajima S, Hiratsuka K, Kiyama-Kishikawa M et al. (2003) Effect of low-level laser irradiation on osteoglycin gene expression in osteoblasts. Lasers in Medical Science 18, 7882. Kamal AM, Okiji T, Kawashima N, Suda H (1997) Defense responses of dentin/pulp complex to experimentally induced caries in rat molars: an immunohistochemical study on kinetics of pulpal Ia antigen-expressing cells and macrophages. Journal of Endodontics 23, 11520. Koide T, Takahara Y, Asada S, Nagata K (2002) Xaa-Arg-Gly triplets in the collagen triple helix are dominant binding sites for the molecular chaperone HSP47. Journal of Biological Chemistry 277, 617882. SR, Bateman JF (1999) Procollagen folding and Lamande assembly: the role of endoplasmic reticulum enzymes and
Conclusion
Carbon dioxide laser irradiation stimulated collagen production and calcied nodules formation on rat
945
molecular chaperones. Seminars in Cell & Developmental Biology 10, 45564. Lee YL, Liu J, Clarkson BH, Lin CP, Godovikova V, Ritchie HH (2006) Dentin-pulp complex responses to carious lesions. Caries Research 40, 25664. Lee MW, Muramatsu T, Uekusa T, Lee JH, Shimono M (2008) Heat stress induces alkaline phosphatase activity and heat shock protein 25 expression in cultured pulp cells. International Endodontic Journal 41, 15862. Linde A (1989) Dentin matrix proteins: composition and possible functions in calcication. Anatomical Record 224, 15466. Masuda H, Fukumoto M, Hirayoshi K, Nagata K (1994) Coexpression of the collagen-binding stress protein HSP47 gene and the a 1(I) and a 1(III) collagen genes in carbon tetrachloride-induced rat liver brosis. Journal of Clinical Investigation 94, 24818. Melcer J, Chaumette MT, Melcer F (1987) Dental pulp exposed to the CO2 laser beam. Lasers in Surgery and Medicine 7, 34752. Mizuno M, Kuboki Y (2001) Osteoblast-related gene expression of bone marrow cells during the osteoblastic differentiation induced by type I collagen. Journal of Biochemistry 129, 1338. Mizuno M, Fujisawa R, Kuboki Y (2000) Type I collageninduced osteoblastic differentiation of bone-marrow cells mediated by collagen-a2b1 integrin interaction. Journal of Cellular Physiology 184, 20713. Moritz A, Schoop U, Goharkhay K, Sperr W (1998a) The CO2 laser as an aid in direct pulp capping. Journal of Endodontics 24, 24851. Moritz A, Schoop U, Goharkhay K, Sperr W (1998b) Advantages of a pulsed CO2 laser in direct pulp capping: a long-term in vivo study. Lasers in Surgery and Medicine 22, 28893. Nagai N, Hosokawa M, Itohara S et al. (2000) Embryonic lethality of molecular chaperone hsp47 knockout mice is
associated with defects in collagen biosynthesis. Journal of Cell Biology 150, 1499506. Noda M, Wataha JC, Kaga M, Lockwood PE, Volkmann KR, Sano H (2002) Components of dentinal adhesives modulate heat shock protein 72 expression in heat-stressed THP-1 human monocytes at sublethal concentrations. Journal of Dental Research 81, 2659. Ohbayashi E, Matsushima K, Hosoya S, Abiko Y, Yamazaki M (1999) Stimulatory effect of laser irradiation on calcied nodule formation in human dental pulp broblasts. Journal of Endodontics 25, 303. Ozawa Y, Shimizu N, Kariya G, Abiko Y (1998) Low-energy laser irradiation stimulates bone nodule formation at early stages of cell culture in rat calvarial cells. Bone 22, 34754. Parker S (2007) Low-level laser use in dentistry. British Dental Journal 202, 1318. Pearson GJ, Schuckert KH (2003) The role of lasers in dentistry: present and future. Dental Update 30, 704. Posten W, Wrone DA, Dover JS, Arndt KA, Silapunt S, Alam M (2005) Low-level laser therapy for wound healing: mechanism and efcacy. Dermatologic Surgery 31, 33440. Razzaque MS, Hossain MA, Kohno S, Taguchi T (1998) Bleomycin-induced pulmonary brosis in rat is associated with increased expression of collagen-binding heat shock protein (HSP) 47. Virchows Archiv 432, 45560. Scarano A, Manzon L, Di Giorgio R, Orsini G, Tripodi D, Piattelli A (2003) Direct capping with four different materials in humans: histological analysis of odontoblast activity. Journal of Endodontics 29, 72934. Ueda Y, Shimizu N (2003) Effects of pulse frequency of lowlevel laser therapy (LLLT) on bone nodule formation in rat calvarial cells. Journal of Clinical Laser Medicine and Surgery 21, 2717. Yokose S, Kadokura H, Tajima Y et al. (2000) Establishment and characterization of a culture system for enzymatically released rat dental pulp cells. Calcied Tissue International 66, 13944.
946
doi:10.1111/j.1365-2591.2009.01602.x
Torsional behaviour of rotary NiTi ProTaper Universal instruments after multiple clinical use
E. P. Vieira1, R. K. L. Nakagawa1, V. T. L. Buono2 & M. G. A. Bahia1

1 2
Department of Restoration Dentistry, Faculty of Dentistry, Federal University of Minas Gerais, Belo Horizonte-MG, Brazil; and Department of Metallurgical and Materials Engineering, Engineering School, Federal University of Minas Gerais, Belo HorizonteMG, Brazil
Abstract
Vieira EP, Nakagawa RKL, Buono VTL, Bahia MGA.
Torsional behaviour of rotary NiTi ProTaper Universal instruments after multiple clinical use. International Endodontic Journal, 42, 947953, 2009.
Aim To assess the inuence of multiple clinical uses on the torsional behaviour of ProTaper Universal rotary NiTi instruments. Methodology Root canal treatments were performed on patients using the ProTaper Universal rotary system to prepare canals. Ten sets of instruments were used by an experienced endodontist, each set being used in ve molar teeth. After clinical use, S1, S2, F1 and F2 instruments were analysed for damage by optical and scanning electron microscopy. The used sets, along with a control group of 10 sets of new instruments, were then torsion tested based on the ISO 3630-1 specication. Data obtained were subjected to a one-way analysis of variance (anova) with a = 0.05.
Results The use of the ProTaper Universal rotary instruments by an experienced endodontist allowed for the cleaning and shaping of the root canal system of ve molar teeth without fracture. The maximum torque for instruments S2, F1 and F2, and the angular deection at fracture for instruments S2 and F1 were signicantly lower following clinical use. The largest decrease in maximum torque was 18.6% (P = 0.014) for S2 instruments. The same maximum percent decrease was found for angular deection at fracture for F1 instruments (P = 0.009). Conclusions Torsional resistance and angular deection of used instruments, as compared to that of new instruments, were reduced following clinical use. Keywords: clinical use, endodontic instruments, nickeltitanium, ProTaper Universal, torsional resistance.
Received 21 January 2009; accepted 28 April 2009
Introduction
Reasons for the fracture of rotary NiTi instruments include variations in canal anatomy, such as merging, curving, re-curving, dilacerating or dividing canals (Ruddle 2002). In addition, other factors can affect the fracture resistance of endodontic instruments, such as size, taper, alloy composition, manufacturing methods, exibility and rigidity, instrument shape and direction
Correspondence: Vicente T. L. Buono, Professor, Department of Metallurgical and Materials Engineering, Federal University of Minas Gerais, Rua Esp rito Santo 35 room 206, 30160-030, Belo Horizonte, MG, Brazil (Tel.: +55 31 3409 1859; fax: +55 31 3409 1815; e-mail: vbuono@demet.ufmg.br).
of rotation (Hilt et al. 2000). The cross-sectional prole also has a signicant inuence on the mechanical behaviour of NiTi instruments (Scha fer et al. 2003, Melo et al. 2008). The factors affecting the performance include the depth of the ute, the area of the inner core, the radial land and the peripheral ground surface (Gambarini 2005, Xu & Zheng 2006). The fatigue life of a rotary endodontic instrument is related to the degree to which it is exed when placed in a curved root canal, with greater exures leading to a shorter fatigue life expectation (Pruett et al. 1997, Melo et al. 2002, Bahia & Buono 2005). Torsional failure occurs when the tip or another part of the instrument is locked in the canal, whilst the shaft continues to rotate. If the elastic limit of the metal is
947
Torsional behaviour of clinically used ProTaper Universal Vieira et al.
exceeded, the instrument undergoes plastic deformation, which can be followed by fracture if the load is high enough (Blum et al. 1999, Gambarini 2000). Peters et al. (2003) established that torque is correlated not only with apically exerted force, but also with preoperative canal volume. Hence, the preparation of narrow and constricted canals can subject rotary NiTi instruments to higher torsional loads and high-apically directed forces. The problem of fracture by torsional overload has been dealt with by determining the maximum torque at separation for each type of instrument and then using low-torque endodontic motors, which can be programmed in such a way as to avoid the application of torque values higher than that of each instrument can support without failing. Nevertheless, this approach does not take into account the fact that fatigue loads developed during curved root canal shaping may decrease the torsional resistance of endodontic instruments. This effect was studied by various authors (Yared et al. 2003, Ullmann & Peters 2005, Bahia et al. 2006), who reported a reduction in the maximum torque to failure for all instruments evaluated. The reuse of rotary instruments of NiTi is a constant concern. The cumulative effects of multiple clinical uses on the incidence of fatigue, deformation and instrument separation have been analysed (Yared et al. 2000, Gambarini 2001, Fife et al. 2004, Bahia & Buono 2005), with the conclusion that their clinical reuse progressively reduced their resistance to fatigue. During canal preparation, especially in curved root canals in molar teeth, these instruments are submitted to a high degree of cyclic deformation that may consume a considerable amount of their fatigue life (Bahia & Buono 2005). In a recent study, Vieira et al. (2008) observed that the exural fatigue resistance of ProTaper instruments, used clinically by an experienced endodontist for the cleaning and shaping of ve molars, was reduced up to 52% when compared with that of new instruments. The present work was undertaken to assess the inuence of multiple clinical uses on the torsional behaviour of ProTaper Universal rotary NiTi instruments.
Material and methods

Twenty sets of ProTaper Universal instruments (Dentsply Maillefer, Ballaigues, Switzerland), type S1, S2, F1 and F2, totalling 88 les, were analysed. They were divided into two groups: (i) control group (CG), with 10
sets of new instruments tested in torsion until fracture to establish the mean values of maximum torque and angular deection at fracture for each type of instrument and (ii) experimental group (EG), with 10 sets of instruments, each set used clinically by an endodontist with experience using the ProTaper Universal system in ve molar teeth to shape between 15 and 20 root canals. The instruments of the EG were tested subsequently in torsion until fracture. The SX and F3 instruments used in the clinical procedures were not included in the study, since these instruments work only in the straight portion of the canals (SX) or in preparation of straight canals (F3). Direct and angled radiographs of each tooth were obtained using a paralleling technique to evaluate anatomy, as well as to determine the canal radius and angle of curvature, as dened by Pruett et al. (1997), and its approximate length. The measurement of these parameters was performed by projecting the radiographic images using a prole projector (Mitutoyo, Tokyo, Japan) at 10 magnication. The canal radius of curvature was measured along the outer canal wall. After the orices were located and the canal explored with sizes 10 and 15 stainless steel K-les (Dentsply Maillefer), cleaning and shaping of the canals were completed in accordance with a crowndown technique recommended by Ruddle (2005). Once a glide path had been created, the ProTaper Universal shaping instruments were used like a brush to laterally and selectively cut dentine on the outstroke. The preparation was nished using the ProTaper Universal nishing instruments F1 and F2 in a nonbrushing manner. The clinical protocol was followed with recapitulations until the working length, established at 0.5 mm of the canal patency length, could be reached by at least an F2 instrument, at which point shaping was considered complete. A 5.25% sodium hypochlorite solution was used for irrigation and Rc-prep (Premier Dental Products, Norristown, PA, USA) was used as a lubricant. The rotational speed was 300 rpm, applied by an endodontic electric motor (Endo Plus, VK Driller, Sa o Paulo, SP, Brazil), operating at a torque of 5 Ncm together with a hand piece of 16 : 1 reduction (W&H 975, Dentalwerk, Bu rmoos, Austria). After use in each patient, the instruments were washed, ultrasonically cleaned for 5 min in ethanol and steam autoclave sterilized. The S1, S2, F1 and F2 instruments of the EG were observed by optical microscopy (Mitutoyo TM, Tokyo, Japan), at 30 magnication, to determine the presence of distortion,
948
Vieira et al. Torsional behaviour of clinically used ProTaper Universal
unwinding defects and macroscopic deformation. Before torsion testing, three sets of instruments were randomly selected and examined by scanning electron microscopy (SEM) (Jeol JSM 6360, Tokyo, Japan) to assess their surface characteristics. Torsion testing was based on ISO 3630-1 specica gica, Belo tion, and using a torsion machine (Analo Horizonte, MG, Brazil) was described in Bahia et al. (2006). The rotation speed was set clockwise to 2 rpm. The end of the shaft was clamped into a chuck connected to a reversible geared motor. Three millimetres of the instruments tip was clamped in another chuck with brass jaws to prevent sliding. Continuous recording of torque and angular deection, as well as measurements of the maximum torque and angular deection to failure, was provided by a specically designed computer program. To determine the statistical signicance of differences in the measured parameters amongst different groups, data obtained were subjected to a one-way analysis of variance (anova). Signicance was determined at the 95% condence level.
(a)
(b)
Results
During the clinical part of the study, none of the instruments fractured or deformed permanently. The mean values (and standard deviations) of radius and angle of curvature characterizing the geometry of the root canals of the 50 molars instrumented with the 10 sets of les (ve molars for each set) were 4.0 mm (1.7 mm) and 33.1 (11.1), respectively. The results of the torsion tests are summarized in Fig. 1, which shows mean values of the maximum torque and angular deection at fracture of new instruments (CG) and of those previously used in the clinical practice (EG). As is common, torsional resistance increased as the diameter of the instruments increased, with the mean values of maximum torque appearing statistically different when instruments in the CG were compared one to another: S1S2, S2F1 and F1F2. A similar tendency was observed for angular deection at fracture in the CG, and statistically signicant differences were found when comparing instruments S1 with S2, and F1 with F2, but not when comparing S2 with F2 instruments. The mean values in Fig. 1 indicated that multiple clinical uses caused a reduction in maximum torque and angular deection at fracture of ProTaper Universal instruments. Comparison between the values of maximum torque, measured for the same type of
Figure 1 Mean values of maximum torque (a) and angular
deection at fracture (b) of ProTaper Universal instruments from the control and experimental groups. Error bars represent the standard deviations.
instruments from the Control and EGs, showed that this tendency was signicant for instruments S2 (P = 0.014), F1 (P = 0.007) and F2 (P = 0.006), but not for S1 (P = 0.475). When a similar analysis was performed for angular deection at fracture, statistically signicant reduction in this parameter was found for S2 (P = 0.003) and F1 (P = 0.009) instruments, but not for S1 (P = 0.546) and F2 (P = 0.097). After canal shaping, all instruments examined by SEM had microcracks and widening of machine grooves, as well as wear and blunting of the cutting edges. These surface characteristics were qualitatively similar in all three sets of randomly selected instruments of the EG. The SEM images shown in Fig. 2 illustrate typical microcracks found in used S2 instruments. The majority of the cracks were transverse to
949
(a)
(a)
Figure 2 SEM images of the surface of an S2 ProTaper
mara et al. 2009, Kim et al. 2009). The et al. 2008, Ca results for the CG depicted in Fig. 1 are thus in agreement with the general observation that the maximum torque of endodontic instruments increases as instrument diameter becomes larger. On the other hand, measurements of angular deection at fracture showed that this parameter does not correlate with instrument diameter in the same way (Gambarini 2001, Bahia et al. 2006). The results shown in Fig. 1 for new instruments conrm this observation. In straight root canals, rotary endodontic instruments operate by cutting and removing organic tissue and debris, experiencing mostly frictional forces, which run in opposition to their torsional motion. However, when the instrument rotates inside a curved root canal, it is bent and thus submitted to tensilecompressive strain cycles in the region of the canal curvature, in addition to the torsional restraints. The strain levels attained by endodontic instruments during this cyclic loading depend on the root canal and instrument geometries, being concentrated at the portion of the instrument positioned in the maximum curvature region of the root canal (Bahia & Buono 2005, Cheung & Darvell 2007). These cyclic forms of stress cause exural fatigue, involving crack nucleation and growth. The value of the tensile strain amplitude, eT, on the surface of an instrument of diameter D inserted into a canal of radius of curvature R can be estimated by the expression: eT D 2R D 1
Universal instrument used for the cleaning and shaping of ve molars showing (a) cracks transversal to the cutting edge and (b) longitudinal cracks.
the cutting edge (Fig. 2a), but longitudinal cracks, parallel to the long axis of the instrument, were also observed (Fig. 2b).
which is valid when the canal radius is measured at the outer canal wall (Bahia & Buono 2005), as was done in the present study. Alternatively, when R is measured at the canal central axis, this expression becomes (Cheung & Darvell 2007): eT D 2R 2
Discussion
The torsional behaviour of rotary NiTi endodontic instruments is affected by a variety of factors, such as size, taper, design, alloy chemical composition and thermomechanical processes applied during manufacturing (Kuhn & Jordan 2002, Bahia et al. 2005, Miyai et al. 2006). Nevertheless, there is a strong relationship between the maximum torque an instrument can withstand and its diameter (Peters & Barbakow 2002, Bahia & Buono 2005). It has also been suggested that the cross-sectional shape of instruments affects the stress distribution pattern as well as their torsional properties (Turpin et al. 2000, Berutti et al. 2003, Melo
If the maximum amplitude is assumed to occur at 3 mm from the instrument tip, the region of the instrument subject to the maximum tensile strain amplitude is D3. Table 1 shows the values of D3 measured for ProTaper Universal instruments by mara et al. (2009) and the corresponding estimated Ca values of eT, calculated using equation 1 for the average radius of curvature, 4.0 mm, of the root canals instrumented in the present study. Cyclic exural straining by the amounts shown in Table 1 would certainly cause damage to the
950
Table 1 Diameter of the ProTaper instruments at 3 mm from their tip, D3, and corresponding maximum tensile amplitudes, eT, estimated for the average radius of curvature of 4.0 mm
Instrument S1 S2 F1 F2
a
D3 (mm)a 0.29 0.35 0.42 0.50
eT (%) 3.8 4.6 5.5 6.7
mara et al. (2009). Ca
instruments. The microcracks exemplied in Fig. 2 constitute evidence of this damage. The presence of longitudinal cracks, that is, cracks parallel to the long axis of the le, has previously been described (Peng et al. 2005, Tripi et al. 2006, Vieira et al. 2008), and is thought to reect the direction of the stress on the surface of the instrument under torsional load. Similar cracking patterns have been observed on other rotary NiTi endodontic instruments subjected to cyclic torsional straining (Bahia et al. 2008). During this type of cyclic deformation, planes with a maximum shear stress are either perpendicular or parallel to the longitudinal axis, whilst the normal stress component on the slip plane is zero. Microscopic investigations have shown that microcracks nucleate in a slip band under cyclic torsion and then grow further in a direction perpendicular to the main stress. In a cylindrical bar, this direction makes an angle of 45 with the axis of the bar. Consequently, cracks in a round axle under cyclic torsion grow in the form of a spiral around its surface (Schijve 2001). The longitudinal appearance of the cracks observed in endodontic instruments is because of the fact that the instruments have helical shapes and that the cracks, being rather small in size, require large magnications to be observed (Bahia et al. 2008). When the torsional resistance of similar instruments belonging to CG and EG was compared, a tendency for this property to decrease with the clinical use in ve molars was observed for all instruments analysed (Fig. 1). This tendency was statistically signicant for S2, F1 and F2 instruments. Previous studies (Yared et al. 2003, Ullmann & Peters 2005, Bahia et al. 2006) reported that simulated clinical use lowered the mean values of maximum torque when compared with that of new instruments. Regarding the behaviour of angular deection at fracture, Yared et al. (2003) and Ullmann & Peters (2005) found no statistically signicant changes in this parameter between new instruments and those submitted to simulated clinical use. In the present study, angular
deection at fracture tended to decrease for the used instruments (Fig. 1b) and statistically signicant decreases were found for S2 and F1 instruments. This result conrms previous ndings on ProFile instruments submitted to simulated clinical use (Bahia et al. 2006). However, it is important to mention that angular deection at fracture has little clinical significance, because at a typical rotational speed of 300 rpm, one complete revolution of a tip-locked instrument will occur in one-fth of a second. Thus, differences in this parameter will not be perceived in clinical practice. The reduction in maximum torque measured in the present study were, on average, 6%, 19%, 12% and 13% for S1, S2, F1 and F2 ProTaper Universal instruments, respectively. These results conrmed the role played by exural fatigue in the torsional resistance of these instruments. However, in a previous work (Vieira et al. 2008) considerably higher values were found for the reduction of exural fatigue life of ProTaper instruments clinically employed for the cleaning and shaping of ve molars: 33%, 52%, 45% and 44% for S1, S2, F1 and F2 instruments, respectively. Taken together, these results indicated that the cumulative effects of multiple clinical uses on rotary NiTi endodontic instruments have a stronger inuence on exural fatigue behaviour than on their torsional resistance. Although exural fatigue appears to have a cumulative effect on rotary endodontic instruments, causing weakening over time, clinical studies have failed to demonstrate the extent of the cumulative effects of multiple clinical uses on the fatigue resistance of these instruments. For instance, Fife et al. (2004) did not observe statistically signicant differences when the remaining fatigue life of ProTaper instruments used in two and four molars were compared, whilst Vieira et al. (2008) obtained a similar result after shaping of ve and eight molars. Moreover, simulated clinical use of ProFile instruments up to one of two and three-fourth of their fatigue life (Bahia et al. 2006) and of ProTaper instruments up to 30%, 60% and 90% of their fatigue life (Ullmann & Peters 2005) did not significantly alter their torsional resistance when the prestrained instruments were compared. These results were interpreted as indicating that crack nucleation occurs early during exural fatigue of NiTi rotary instruments, low-crack growth occupying a large fraction of their low-cycle fatigue life (Bahia & Buono 2005).
951
Conclusions
Torsional resistance of used instruments was reduced by average amounts varying from 6% to 19%, when compared with that of new instruments. Structural fatigue took place during the clinical use of the instruments and, in addition to the usual transversal cracks generate by exural fatigue, longitudinal cracks were also observed on the surface of the used instruments. Comparisons with data on ProTaper instruments indicate that the cumulative effects of multiple clinical uses on rotary NiTi endodontic instruments have a stronger inuence on exural fatigue behaviour than on their torsional resistance.
Acknowledgements
This work was partially supported by Fundac a o de ` Pesquisa do Estado de Minas Gerais Amparo a FAPEMIG, Belo Horizonte, MG, Brazil and Conselho gico Nacional de Desenvolvimento Cient co e Tecnolo CNPq, Bras lia, DF, Brazil.
References
Bahia MGA, Buono VTL (2005) Decrease in the fatigue resistance of nickeltitanium rotary instruments after clinical use in curved root canals. Oral Surgery, Oral Medicine, Oral Pathology, Oral Radiology and Endodontology 100, 24955. Bahia MGA, Martins RC, Gonzalez BM, Buono VTL (2005) Physical and mechanical characterization and the inuence of cyclic loading on the behaviour of nickel-titanium wires employed in the manufacture of rotary endodontic instruments. International Endodontic Journal 38, 795801. Bahia MGA, Melo MCC, Buono VTL (2006) Inuence of simulated clinical use on the torsional behavior of nickel titanium rotary endodontic instruments. Oral Surgery, Oral Medicine, Oral Pathology, Oral Radiology and Endodontology 101, 67580. Bahia MGA, Melo MCC, Buono VTL (2008) Inuence of cyclic torsional loading on the failure of rotary nickel-titanium instruments. International Endodontic Journal 41, 88391. Berutti E, Chiandussi G, Gaviglio I, Ibba A (2003) Comparative analysis of torsional and bending stresses in two mathematical models of nickel-titanium rotary instruments: ProTaper versus ProFile. Journal of Endodontics 29, 159. Blum JY, Machtou P, Micallef JP (1999) Location of contact areas on rotary Prole instruments in relationship to the forces developed during mechanical preparation on extracted teeth. International Endodontic Journal 32, 10814. mara AS, Martins RC, Viana ACD, Leonardo RT, Buono VTL, Ca Bahia MGA (2009) Flexibility and torsional strength of ProTaper and ProTaper Universal rotary instruments
assessed by mechanical tests. Journal of Endodontics 35, 1136. Cheung GSP, Darvell BW (2007) Fatigue testing of a NiTi rotary instrument. Part 1: strain-life relationship. International Endodontic Journal 40, 62632. Fife D, Gambarini G, Britto LR (2004) Cyclic fatigue testing of ProTaper NiTi rotary instruments after clinical use. Oral Surgery, Oral Medicine, Oral Pathology, Oral Radiology and Endodontics 97, 2516. Gambarini G (2000) Rationale for the use of low-torque endodontic motors in root canal instrumentation. Endodontics Dental Traumatology 16, 95100. Gambarini G (2001) Cyclic fatigue of ProFile rotary instruments after prolonged clinical use. International Endodontic Journal 34, 3869. Gambarini G (2005) The K3 rotary nickel titanium instrument system. Endodontic Topics 10, 17982. Hilt BR, Cunningham CJ, Shen C, Richards N (2000) Torsional properties of stainless steel and nickeltitanium les after multiple autoclave sterilizations. Journal of Endodontics 26, 7680. Kim TO, Cheung GSP, Lee JM, Kim BM, Hur B, Kim HC (2009) Stress distribution of three NiTi rotary les under bending and torsional conditions using a mathematic analysis. International Endodontic Journal 42, 1421. Kuhn G, Jordan L (2002) Fatigue and mechanical properties of nickel-titanium endodontics instruments. Journal of Endodontics 28, 71620. Melo MMC, Bahia MGA, Buono VTL (2002) Fatigue resistance of engine-driven rotary nickeltitanium endodontic instruments. Journal of Endodontics 28, 7659. Melo MCC, Pereira ESJ, Viana ACD, Fonseca AMA, Buono VTL, Bahia MGA (2008) Dimensional characterization and mechanical behaviour of K3 rotary instruments. International Endodontic Journal 41, 32938. Miyai K, Ebihara A, Hayashi y, Doi H, Suda h, Yoneyama T (2006) Inuence of phase transformation on the torsional and bending properties of nickel-titanium rotary endodontic instruments. International Endodontic Journal 39, 11926. Peng B, Shen Y, Cheung GSP, Xia TJ (2005) Defects in ProTaper S1 instruments after clinical use: longitudinal examination. International Endodontic Journal 38, 5507. Peters OA, Barbakow F (2002) Dynamic torque and apical forces of ProFile .04 rotary instruments during preparation of curved canals. International Endodontic Journal 35, 379 89. Peters OA, Peters CI, Schonenberger K, Barbakow F (2003) ProTaper rotary root canal preparation: assessment of torque and force in relation to canal anatomy. International Endodontic Journal 36, 939. Pruett JP, Clement DJ, Carnes DL (1997) Cyclic fatigue testing of nickeltitanium endodontic instruments. Journal of Endodontics 23, 7785. Ruddle CJ (2002) Broken instrument removal. The endodontic challenge. Dent Today 21, 702.
952
Ruddle CJ (2005) The ProTaper technique. Shaping the future of endodontics. In: Castellucci A, ed. Endodontics, vol II. Florence: IL Tridente, pp. 54863. Scha fer E, Dzepina A, Danesh G (2003) Bending properties of rotary nickel-titanium instruments. Oral Surgery Oral Medicine Oral Pathology, Oral Radiology and Endodontics 96, 75763. Schijve J (2001) Fatigue of Structures and Materials. 1st edn. Dordrecht, The Netherlands: Kluwer Academic Publishers. Tripi TR, Bonaccorso A, Condorelli GG (2006) Cyclic fatigue of different nickel-titanium endodontic rotary instruments. Oral Surgery Oral Medicine Oral Pathology, Oral Radiology and Endodontics 102, e10614. Turpin YL, Chagneau F, Vulcain JM (2000) Impact of two theoretical cross-sections on torsional and bending stress of nickel-titanium root canal instrument models. Journal of Endodontics 26, 4147.
Ullmann CJ, Peters OA (2005) Effect of cyclic fatigue on static fracture loads in ProTaper nickeltitanium rotary instruments. Journal of Endodontics 31, 1836. Vieira EP, Franc a EC, Martins RC, Buono VTL, Bahia MGA (2008) Inuence of multiple clinical use on fatigue resistance of ProTaper rotary nickeltitanium instruments. International Endodontic Journal 41, 16372. Xu X, Zheng Y (2006) Comparative study of torsional and bending properties for six models of nickeltitanium root canal instruments with different cross-sections. Journal of Endodontics 32, 3725. Yared GM, Bou Dagher FE, Machtou P (2000) Cyclic fatigue of Prole rotary instruments after clinical use. International Endodontic Journal 33, 2047. Yared G, Kulkarni GK, Ghossayn F (2003) An in vitro study of the torsional properties of new and used K3 instruments. International Endodontic Journal 36, 7649.
953
doi:10.1111/j.1365-2591.2009.01628.x
Erratum
Somma F, Leoni D, Plotino G, Grande NM, Plasschaert A. Root canal morphology of the mesiobuccal root of maxillary rst molars: a micro-computed tomographic analysis. International Endodontic Journal 42, 16574. 2009. The above paper did not include the following acknowledgements:
Acknowledgements
The authors express their gratitude to Drs. Rossella Bedini and Drs. Raffaella Pecci and to ISS Italian Superior Health Institute for the micro CT scan images and reconstructions. The publisher apologizes for this error.
954

Iej 10 2009

Uploaded by

Document Information

Original Description:

Original Title

Copyright

Available Formats

Share this document

Share or Embed Document

Sharing Options

Did you find this document useful?

Is this content inappropriate?

Copyright:

Available Formats

Iej 10 2009

Uploaded by

Copyright:

Available Formats

doi:10.1111/j.1365-2591.2009.01577.

Apexication: the beginning of its end

2009 International Endodontic Journal

International Endodontic Journal, 42, 855866, 2009

Apexication, end in sight Huang

International Endodontic Journal, 42, 855866, 2009

2009 International Endodontic Journal

Huang Apexication, end in sight

A paradigm shift in the management of immature teeth

2009 International Endodontic Journal

International Endodontic Journal, 42, 855866, 2009

Apexication, end in sight Huang

Pulp space lled with periodontal tissues

The outcome of guided generation and regeneration approach

Apical papilla Epithelial diaphragm Bone

Cementum PDL Dentin Pulp

Pulp space lled with regenerated pulp

International Endodontic Journal, 42, 855866, 2009

2009 International Endodontic Journal

Huang Apexication, end in sight

Bone Cementum Cementum

Figure 2 Ingrowth of periodontal tissue

PDL Cementum PDL Dentin PDL Bone PDL

2009 International Endodontic Journal

International Endodontic Journal, 42, 855866, 2009

Apexication, end in sight Huang

Severe disorganized calcication of the pulp space

International Endodontic Journal, 42, 855866, 2009

2009 International Endodontic Journal

Huang Apexication, end in sight

Progress on pulp/dentine tissue engineering and regeneration

Early efforts on pulp regeneration

2009 International Endodontic Journal

International Endodontic Journal, 42, 855866, 2009

Apexication, end in sight Huang

Pulp tissue engineering

Issues in cell-based pulp tissue engineering

International Endodontic Journal, 42, 855866, 2009

2009 International Endodontic Journal

Huang Apexication, end in sight

2009 International Endodontic Journal

International Endodontic Journal, 42, 855866, 2009

Apexication, end in sight Huang

International Endodontic Journal, 42, 855866, 2009

2009 International Endodontic Journal

Huang Apexication, end in sight

2009 International Endodontic Journal

International Endodontic Journal, 42, 855866, 2009

Apexication, end in sight Huang

International Endodontic Journal, 42, 855866, 2009

2009 International Endodontic Journal

S. F. C. Souza1,2, A. C. Bombana3, C. Francci2, F. Gonc alves2, C. Castellan2 & R. R. Braga2

2009 International Endodontic Journal

International Endodontic Journal, 42, 867873, 2009

Physicomechanical properties of endodontic sealers Souza et al.

Materials and methods Flow

Micropush-out bond strengths

International Endodontic Journal, 42, 867873, 2009

2009 International Endodontic Journal

Souza et al. Physicomechanical properties of endodontic sealers

Failure mode analysis

2009 International Endodontic Journal