ORIGINAL ARTICLE Platelet gel: applications in dental regenerative surgery

Franco Forni, Massimo Marzagalli, Patrizia Tesei, Alessandra Grassi

Hospital Dentistry Service, Foundation I.R.C.C.S, San Matteo Hospital, Pavia, Italy

Background. Platelet growth factors obtained from platelet-rich plasma (PRP) are used in prosthetic and periodontal regenerative therapy in dentistry. Increased microvascular proliferation in the first 20 days is followed by enhanced osteoblast activity and immature osteoid formation within 3-6 weeks, improving the quality and quantity of newly formed bone tissue. The aim of this study was to evaluate, through three-dimensional X-ray monitoring, the amount of bone obtained after bone regeneration treatment with platelet gel from autologous PRP. Materials and methods. Patients eligible for regenerative treatment of atrophic alveolar bone of the maxilla or mandible were studied. The patients' autologous whole blood was collected at the Department of Immunology and Transfusion of San Matteo Hospital for the preparation of platelet gel. The bone at the treated sites was analysed prior to and 4 months after the treatment using the three-dimensional X-ray system Galileos. Results. Over a period of 6 years, 133 patients were treated: 304 implants were inserted and there were five cases of failure. The regenerated bone area consisted of histologically immature osteoid tissue composed of thin trabeculae of vital bone and nuclei of osteocytes, associated with fibro-connective tissue. Discussion. This co-operative trial between the Transfusion Centre, for standardised production and validation of the platelet gel, and the Dental Surgery room for its application showed that the technique appears effective and safe. Although difficulties were encountered because of the small sample size and the inability to carry out long-term histological controls, the use of small amounts of PRP (5-10%) combined with autologous bone (15 -20%) and alloplastic material appears to reduce the need for bone grafting. Keywords: PRP, growth factors, alloplastic bone, pre-prosthetic surgery, cone beam computer tomography (CBTC).

Introduction
Platelet-rich plasma (PRP), platelet gel and multiple platelet growth factors have been studied for many years to evaluate their biological effects and clinical applications in tissue regeneration in various therapeutic fields1 such as maxillofacial surgery2, wound healing3 and orthopaedic surgery4,5. In dentistry, PRP preparations are useful for all types of pre-prosthetic surgery and periodontal regeneration (guided tissue regeneration - GTR)6 requiring the application of autologous bone grafts or alloplastic material. PRP works as an autologous source of growth factors essential for bone regeneration5,7-9. There are many studies on the effectiveness of PRP in periodontal therapy and in the healing of
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soft and hard tissues. Some interesting data have been provided by clinical trials and systematic reviews which evaluated the combined use of PRP with therapeutic bioactive agents in guided tissue regeneration for the treatment of periodontal vertical bone defects10,11. The use of PRP increases the vascularity in the first 20 days, with an increase of osteoblast and immature osteoid tissue formation within 3-6 weeks, improving the quality and quantity of newly formed bone tissue12,13. Histomorphological observations showed that 54% of patients treated with PRP achieved 100% bone regeneration within 24 weeks, compared with 38% of the control group. This implies that the acceleration of bone growth with PRP may be clinically useful to reduce alveolar

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defects in the initial stages of the healing processes13. The use of PRP seems to minimise the amount of autologous bone necessary during regenerative surgery, thus decreasing the need for extra-oral tissue13. In addition, PRP promotes the regeneration of alveolar bone and other periodontal tissues, including root cementum and the periodontal ligament without any epithelial invasion, which represents the characteristic feature of an inadequate healing process and periodontal disease13. Unfortunately, studies in clinical dentistry with PRP, such as jaw bone grafting from the iliac crest with extraction, healing of extraction sites and lifting of the maxillary sinus do not provide statistically significant data supporting the use of PRP to promote bone healing because the sample sizes are too small. However, PRP appears to have significant positive effects on the healing of hard and soft tissues combined with graft material and to improve management10,14-21, despite the fact that its clinical efficacy is unpredictable, probably due to inefficient delivery of growth factors and inconsistent PRP derivatives7-8. The aim of this study was to evaluate, through three-dimensional X-ray monitoring, the amount of bone obtained after bone regeneration treatment with PRP.

Materials and methods

Preparation of the platelet gel The platelet gel was prepared in the Transfusion Centre of the IRCCS Policlinico San Matteo Foundation (Pavia, Italy). The PRP used in this research was obtained with two different techniques. The patients were selected according to previously described criteria22, and the platelet gel was derived from a pre-donation of an autologous whole blood unit. PRP was obtained by classical whole blood centrifugation and separation of 350 mL pre-donated blood. Platelet gels were prepared by a manual method, mixing PRP aliquots, obtained from standard platelet concentrate, with plasma from the same blood unit activated by re-calcification with calcium gluconate or calcium chloride. After bacteriological sampling the aliquots were frozen immediately and stored at 80 C until required22. Because the amount of blood collected in a standard blood bag is greater than the small amount of required platelet

gel aliquots, the Regen PRP system (RegenKit, RegenLab, Le Mont-sur-Lausanne, Switzerland) was subsequently adopted, with an 8 mL sample of whole blood collected in a tube. This extraction process is similar to the classical technique (initially used), but reproduced at a smaller scale. It ensures the same results and advantages obtained with the initial method, without introducing any variables. After collecting blood, the tube is centrifuged at 1,500 x g for 8 minutes according to manufacturer's recommendations, resulting in separation into three layers: red blood cells, the buffy-coat and the plasma supernatant. About 2 mL of plasma supernatant are removed with a syringe, leaving an amount of plasma equal to the desired final volume of the material (usually 2 mL or less) above the buffy-coat. The volume extracted is transferred under a laminar flow hood into a Vacutainer blood test tube (without anticoagulants) and either 0.5 mL of calcium chloride or calcium gluconate are added. The solution is then left to rest horizontally for about 10 minutes, in order for a fibrin clot to form in which the coagulation cascade, which allows the activation of platelets, is triggered. The other tube is gently agitated to mix the buffy-coat with the remaining supernatant. The contents are removed with a long-needled syringe and transferred into a sterile Petri dish; the calciumstimulated, activated plasma is added gradually. After gentle mixing, a buffy-coat-derived red-coloured platelet gel is obtained (red because of the presence of erythrocytes in the buffy-coat), containing growth factors released from the alpha granules in the activated platelets. Platelet gel obtained from 350 mL of blood appears white, because it is composed of platelets and plasma, without the buffy-coat. At this point, a sample is taken for bacteriological cultures to determine the sterility of the product and the product is frozen at 80 C for safe storage for up to 1 year22. The whole procedure of platelet gel preparation is performed under a laminar flow hood with sterile handling techniques. The GALILEOS radiographic system GALILEOS is a three-dimensional (3D) X-ray system, consisting of hardware and software components23. The equipment provides images with a 15x15x15 cm3 volume, consisting of one billion voxels, with a resolution of 0.3 mm. This resolution

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Platelet gel: applications in dental regenerative surgery

enables reproduction of all bones of the face and the analysis of their smallest anatomical details in three dimensions. In addition, partial volumes, chosen as desired, can be reconstructed at a later time, with a maximum resolution of 0.15 mm using data already recorded, without requiring additional X-rays to be taken. The radiological scanning takes place in 14 seconds, using a pulsed technique so that the actual time of exposure to radiation is 2-6 seconds. This guarantees a reduced dose of radiation and provides many types of representations: 3D volumes, panoramic and cephalometric (CEFH) views, cross-sections and details of all layers of the radiation sections (axial, sagittal, coronal). Through ConeBeam technology GALILEOS exploits interactions between hardware and software and between the 3D volume reconstruction and the 3D diagnostics. During scanning, data from 200 individual shots using a cone-shaped beam are recorded, giving a 3-D representation of the patient's skull. The GALAXIS software of this X-ray system can produce a 3D volumetric reconstruction of the maxillofacial area to be treated. The software initially provides the traditional X-ray images and the CEFH, and then guides the operator, thanks to its intuitive "examination window", to every desired view in axial, sagittal, coronal planes in real time. Using almost exclusively the left-hand button on the mouse the bone volume can be viewed, measurements can be made, and new views can be created or modified. Furthermore the images from the classical scanning slices (axial, coronal, and sagittal) can be displayed and rotated. Alternatively the software can compare multiple views such as the panoramic, lateral, anterior-posterior or posterioranterior cephalometric views and orthoradial slices which are used more frequently by dentists. John B. Ludlow and colleagues 23 measured the radiation dose emitted by GALILEOS and compared the actual radiation dose with the dose emitted by other cone beam computed tomography (CBTC) devices and by conventional computed axial tomography scans. The study showed that GALILEOS offers good image quality while subjecting the patient to the lowest amount of radiation (28 Sv, 21 mAs, 85 kV) compared with other devices.

Clinical methods The patients considered in this study underwent removal of a unit of autologous whole blood to obtain the platelet gel at the Transfusion Centre of San Matteo Hospital. The patients had been selected to undergo regeneration procedures of the alveolar bone crest of atrophic upper jaw, with or without a sinus lift, or lower jaw in order to prepare the area for insertion of an implant to which a fixed prosthetic construction was then to be attached. The amount of bone present at the treated sites was measured with the Galileos X-ray system 4 months after regenerative surgery with application of platelet gel, in order to assess the amount of bone present for implant insertion in patients treated in two stages, or the peri-implant bone levels present in patients treated in one stage. The software of the GALAXIS X-ray system enables a 3D volumetric reconstruction of the maxillofacial area treated. The software first provides the standard views and the CEFH and then, in real-time, guides the user, thanks to its intuitive "examination window", to every desired view in the axial, sagittal and coronal planes within the bone volume. The bone graft is prepared during surgery. The platelet gel, transported still frozen from the Transfusion Centre, is cut into small pieces with thin-tipped scissors and is mixed with alloplastic bone (antigen-deprived bovine bone; BIO-OSS), with a grain size varying between 0.25 and 1 mm, and with autologous bone, taken during surgery from the oral cavity (maxillary tuberosity, retromolar zone or mandibular symphysis) and with fluid taken from the Petri dish which contained the PRP24. The protocol involves the use of different percentages of material: 5-10% of platelet gel, 15-20% of autologous bone and 70-80% of alloplastic bone (BIO-OSS). This method is suitable for use in various types of interventions. The regeneration of atrophic alveolar bone crest of the upper or lower jaw bone includes flap raising, a large sinus lift when necessary, or the creation of microretentions in the cortex. The bone particles, consisting of platelet gel, autologous bone and the plastic BIO-OSS, are then positioned to induce vertical and horizontal augmentation of the alveolar ridge. An absorbable membrane (BIOGIDE) is stretched over the bone and the flap is sutured to avoid

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tension displacement. In all cases we proceeded to the immediate placement of implants only if the right conditions existed (at least 4 mm of bone at baseline to guarantee primary stability). The subsequent activation of the implant must be gradual: indeed, whenever an implant is inserted into bone grafts or into regenerated structures, it is crucial to proceed first with a temporary prosthesis, for 4-5 weeks, with resins in order to stimulate and complete the process of osteogenesis and then proceed with the placement of the definitive prosthesis.

cm, examined at the Department of Human Pathology at San Matteo Hospital. This examination revealed the presence of immature osteoid tissue composed of thin trabeculae of viable bone, in which osteocyte nuclei could be seen, associated with some fibroconnective tissue.
Table I - Patients undergoing regenerative therapy with PRP.
N. of patients Total Treated with 1 graft Treated with 2 grafts Regenerative therapy Regenerative therapy and prosthetic implant rehabilitation Failure 133 129 4 25 108 5

Results
This research was based on 133 patients undergoing bone regeneration treatment with PRP; 25 of these patients continued their prosthetic implant treatment in private dental practices. The 108 patients who continued their prosthetic implant management at San Matteo Hospital were examined periodically (Table I). These controls were performed monthly during therapy, every 3 months from the end of rehabilitation for the first year and then every 6 months. In the 6 years of the trial 112 grafts were performed and underwent rehabilitation and 304 implants were inserted. In the cases in which the primary stability was sufficient, the implants were inserted in one session immediately after the bone regeneration with platelet gel; otherwise the implants were inserted 4 months after the bone regeneration treatment. In 79 cases the grafts were used with immediate implants, whereas in 31 cases the implant insertion was delayed: two grafts were lost because of infection at the regenerated site and the onset of sinusitis (Tables II-III). The percentage of failures in the 108 patients monitored over time is low. Overall, there were five failures, of which three occurred in regenerated sites with immediate implants, and only two in patients with deferred implant insertion (Table II). The three-dimensional X-ray reconstruction of the treated sites showed a zone that stood out from the surrounding area of mature bone because of its different radiographic opacity, being sharper, brighter and more compact than the usual grey colour of mature bone. Using the panoramic view provided by the GALAXIS software, this area was measured precisely, to the tenth of a millimetre, in the orthogonal projection. Histomorphometric evaluation of the newly formed bone was performed on samples of 0.3x0.2

Table II - Failures.
Failure of the graft Perforation of the maxillary sinus membrane Exposure of the graft and the membrane Infection Incomplete regeneration site Sinusitis Failure of the graft with implants 1 0 0 0 2

0 0 1 0 1

Table III - Cases treated.

N. of grafts Total Not controlled Controlled (CT) Maxilla and sinus lift Mandibolar Grafts with delayed implants Grafts with immediate implants Failures 137 25 112 Total 106 (controlled 84) Total 31 (controlled 28) 31 79 5 N. of implants 304 0 304 227 77 87 217 8

Discussion
Considering the results obtained in the 6 year trial carried out by the Department of Dentistry at San Matteo Hospital in Pavia, it was possible to "design" a specific protocol for the regeneration

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Platelet gel: applications in dental regenerative surgery

technique. Initially, the graft consisted of the three components present in equal amounts; subsequently the percentage of PRP (20%) was decreased while the percentage of the alloplastic and autologous bone (40%) was increased in order to avoid the formation of fibrous tissue and to decrease bone reabsorption from graft. The best results that led to the establishment of an effective protocol were obtained by increasing the amount of alloplastic bone to 70-80%, the autologous bone to 15-20% and by further reducing the percentage of platelet gel to 5-10%. Given the minimal amount of platelet gel necessary for application to small sites it was possible to modify the technique used for platelet gel production by markedly decreasing the autologous blood collected - from 350 mL to 8 mL - in a standardised, certified collection device. This minimises the discomfort experienced by the patient and avoids any kind of interference from underlying conditions which could create a large variety of patient-related clinical problems, such as hypotension or vasovagal reactions. Various techniques have been developed to extract PRP, starting from the Marx's original and modified procedures, which are not feasible outside a modern operating theatre, to newer extraction techniques. The technique described by Sacchi and Bellanda's group, which is feasible in a dental practice, involves withdrawal of 60 mL of blood, preparation of the gel in 30 minutes and immediate clinical use to avoid bacterial contamination25. The greatest differences in the preparation are related to the velocity, duration and strength of the centrifugation cycles, the different amounts of blood required and the choices of anticoagulant used25. Correctly prepared PRP must be based on a "double centrifugation technique", which must be sterile and suitable for separating platelets from red blood cells and obtaining an adequate concentrations of platelets. Not all currently available commercial PRP devices fulfil all these characteristics and some probably do not concentrate active platelets in sufficient numbers to promote healing8. Furthermore, investigations have shown that current methods of PRP preparation result in premature release of the relevant bone stimulatory factors 7 . This may explain the variability of the clinical efficacy of PRP7,8.

The technique for PRP production at San Matteo Hospital in Pavia meets all the centrifugation requirements necessary to obtain a platelet-rich gel that can be used as an autologous source of growth factors. The technique also meets stringent safety criteria, such as those applied to blood products. Bacteriological sampling and subsequent sealed packaging, freezing and maintenance at 80 C ensures sterility and stable conditions of storage. All platelet gel units are treated as blood components and tracked with the same database system to provide complete traceability of the product from donation to use in the patient. The main difficulties encountered during this study were related to the small populations common in clinical dental trials15. In fact, it was difficult to implement long-term controls of the treated patients because many patients only come to the hospital for regenerative surgery and then complete the prosthetic implant work at private dental practices. Furthermore none of the patients treated with PRP and implants positioned in one session agreed to undergo subsequent further surgery to allow a histological evaluation of the newly formed bone. To overcome these problems samples of regenerated tissue were taken during surgery for the implant positioning from patients treated in two stages. Of course, postponing the insertion of the implant screws from 4 to 6 months after bone regeneration prevented us from having a histological analysis 1 year after surgery.

Conclusions
The use of PRP in oral surgery seems to decrease the need for autologous bone grafts collected from extra-oral sites, avoiding cutaneous incisions and reducing the extent of mucosal incisions. The technique for producing PRP that we used minimised the discomfort of the patient, since only 8 mL of blood were withdrawn, while ensuring the formation of a sterile gel rich in growth factors. The use of low percentages of this PRP (5-10%), together with 15-20% of autologous bone and the remainder consisting of alloplastic material, appears to produce a quantity of very immature, hard osteoid tissue already within 4 months after the regenerative surgery, thereby providing a sufficient amount of viable bone to ensure primary stability for placement of the implant.

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The Authors declare no conflicts of interest.

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Arrived: 17 January 2012 - Revision accepted: 29 March 2012 Correspondence: Alessandra Grassi P.le Franco Quarleri n. 2 27058 Voghera, Pavia, Italy e-mail: alessandra.7479@libero.it

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