ALVELaC CLINICaL EVIDENCES

TM
EDITION NO. 01

CONTENTS
1. 2.

Introduction Summary of Clinical Trial How about bone density ?

European Symposium on Calcied Tissues, Barcelona, Spain 2008 poster European Symposium on Calcied Tissues, Barcelona, Spain 2009 poster Micro CT analysis of human bone

01 02-03 04 05 06 07 08 09 11-14 15

3.

4. 5. 6.

A positive control comparison trial with xenograft

National Oral and Maxillofacial Scientic Conference, Kochi, India, poster presentation

Case Studies Conclusion

IntRoDUction
After tooth extraction, the extraction socket heals by forming a blood clot which leads to the formation of new bone within 3-4 months. Although bone deposition in the socket will continue for several months, it will not reach the crestal level of the neighbouring teeth (Fig. 1). The resulting alveolar bone loss may lead to problems such as poor aesthetic appearance and diculty in placement of implants or prostheses for partially or totally toothless patients (Fig. 2). Various techniques have been proposed to limit alveolar bone loss such as atraumatic extraction, immediate post-extraction removable prosthesis, immediate placement of dental implant and immediate bone-lling of extraction socket. Guided bone regeneration (GBR) and guided tissue regeneration (GTR) has been gaining attention in recent years for repair of bone defects and had been used in patients after tooth extraction with the aim of preserving alveolar bone. However, none of the currently available solutions work on the principle of providing a strong mechanical support that is also bio-inert. The need for such a design gave birth to the development of AlvelacTM, a strong, yet porous synthetic polymer-based bio-scaold which has degradation rates matching that of natural bone healing and bone remodelling process. The bio-scaold is made of PLGA (Poly lactic coglycolic acid) material and acts as a mechanical support to hold the blood clot at the crest level (Fig. 03). To analyze the ecacy of the bio-scaold for alveolar socket preservation, a multi site clinical trial was initiated and bio-scaold used in post-extraction sockets of patients analyzed over 12 weeks with dental OPG.

Fig. 1: Natural bone healing process after extraction

a. Bone loss with gum collapse b. Knife-edge ridge (need bone graft to put implants) c. Thin jaw bone caused exposed implant thread

b c

Fig. 2: Poor aesthetic appearance and diculty in placement of implant

Fig. 3: Bone healing process enhanced with application of AlvelacTM

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SUMMaRY oF Clinical TRial ResUlt

RaDiographic assessment of bone regeneration in alveolar sockets With PLGA bio-scaffolD (BioscaffTM AlvelacTM) after teeth eXtraction Clinical trial.gov registration no: NCT00836797 Objective: To assess the eectiveness of ridge height preservation with AlvelacTM after tooth extraction.

Trial Site: A multi-site clinical study at the Oral and Maxillofacial Surgery Department, Faculty of Dentistry, National University Hospital, Singapore, in collaboration with the Saveetha University, Chennai, India. Procedure: OPGs were taken at 1st week, 8th week and 12th week. Bone height dierences were measured between the 1st week and 12th week to determine height gained, lost or neutral.

Sample Size: Final report: seventeen cases and sixteen controls with seven dropouts. Results: Out of these seventeen cases (Blue bars in Fig. 1), nine cases (53%) show height gain, three cases (18%) shows insignicant height loss (height preservation) and another ve cases (29%) shows minimum height loss compared to the controls. All the controls (Red bars in Fig. 1) show height loss after 12 weeks.

Fig. 1: Graphical presentation of seventeen cases and sixteen controls.

Result Analysis: Statistical analysis by SPSS (statistical analysis software) showed that, the mean dierence of bone gain/loss (+/) between two group is 1.69mm which is signicant based on the Mann-Whitney U test conducted (p < 0.0005,). The medians of bone loss were 0.06mm (range -1.01 to 1.81) for cases with scaold and -1.80mm(range -3.09 to -0.18) for controls without scaold. This conrms the clinical trial hypotheses of bone height preservation with the scaold in the alveolar socket. Out of the nine cases that showed bone height gain, we analyzed one patient (random pick) to see how the height gain was achieved.

This summary clinical report is not for circulation as it has been submitted for international publication
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Fig. 2a: Extraction done

Fig. 2b: Socket size measured

Fig. 2c: Bio-scaold placed

Fig. 2d: Closed with simple suture

This case showed a bone gain of 0.84mm after 12 weeks. It was an upper right 1st premolar and an atraumatic extraction was done with forceps. After extraction, socket size was measured with a perio probe and bio-scaold size was selected. Bio-scaold was placed buccopalatally at the alveolar crest level and secured in place with two interrupted sutures. This case showed very good healing and after 12 weeks, it showed 0.84mm of bone gain. It demonstrates that proper bio-scaold placement can help in achieving bone height gain. One explanation for the height gain could be due to the bio-scaold being placed higher than the alveolar crest level measured at 1st week. This could well be the case to explain those cases where there were height loss, i.e. bio-scaold was placed lower than crest level at 1st week. Conclusions: Bio-scaold successfully preserved height after the tooth extraction. The result shows statistical signicance in preserving alveolar bone height with the placement of a rigid bio-scaold. The bio-scaold material exhibited excellent biocompatibility with no signicant reactions at the placement site. The PLGA bio-scaold, AlvelacTM, demonstrated a high degree of osteoconductive capabilities favouring the regeneration of bone tissue. The placement of the bio-scaold higher in the socket could explain the height gain of the nine cases. Overall 71% shows good height preservation or better results.

This summary clinical report is not for circulation as it has been submitted for international publication
3

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HoW AboUt Bone DensitY ?

Though the clinical trial successfully concluded the ecacy of bio-scaold in preserving the alveolar bone socket height, however, the density of the bone could not be assessed through the x-ray analysis and a dierent method of analysis was required. To achieve that goal, bone was harvested from one of the bio-scaold preserved site and underwent a conventional histopathological examination. To be more assured, a micro CT analysis method was further adopted to show the micro structure of the bone in a 3D model to show the quality and density of the analyzed bone. This was a new testing method of assessing the bone density and we had to be assured of its eectiveness. So a joint collaboration was initiated with Xradia Inc, Concord, CA, USA and the nished project was presented at 3 dierent international Symposiums European Symposium on Calcified Tissues, Barcelona, Spain 2008. European Tissue Engineering Symposium, Poland, 2008 European Symposium on Calcified Tissues, Barcelona Spain 2009.

The project clearly showed the evidence of good bone density and structural quality in a 3D model through the eective use of micro CT. Below are the posters presented at the Symposiums -

BIO-SCAFFOLD YIELDS A GOOD SOCKET BONE DENSITY

Bone sample was harvested from a bio-scaold preserved site and assessed through micro CT scan and was compared against the normal histopathological slides. Micro CT successfully conrmed the evidence of good bone density and proved its eectiveness and power of detailing in a 3D model.

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EURopean SYMposiUM on CalciFieD TissUes, BaRcelona, Spain 2008

RAPID 3D CHARACTERIZATION OF CARTILAGE, BONE QUALITY & SCAFFOLD TO SUBMICRON RESOLUTION, WITH A NOVEL MICROCT
S H Lau1, M. Chandrasekaran2, V. Fan3 , M. Islam3 , S. Candell1, T. Case1, L. Chitra2, T. Fong1, H. Chang1, W. Broderick1 Inc, Concord, CA, USA International Pte. Ltd, Singapore; 3 Faculty of Dentistry, National University of Singapore
2Bio-scaffold 1Xradia

Email Contact: shlau@xradia.com

Abstract
The current study is focused on developing a new rapid non invasive 3D imaging technique for bone quality, bioscaffold, cartilage and its related drug efficacy evaluation. Bone quality evaluation is critical in patients suspected with osteoporosis or those treated with synthetic scaffolds for restoration of bone. Similarly success of synthetic scaffolds depends much on the micro architecture of the scaffold. These evaluation plus those involving cartilage thickness in osteoarthritis are mainly carried out with conventional histology, which requires experienced personnel and time consuming sample preparation techniques. In addition, histology studies can take up to a few weeks, results are often operator dependant, and are only available as individual 2D slices. While in recent years there are several publications on the use of MicroCT for such evaluation, the biggest deficiency of conventional microCT is the lack of contrast and resolution to detect fine microstructures on bones and low Z ( low contrast) bioscaffold materials and soft tissue. It is also not possible to image cartilage without contrast enhancing agents. In the current work we have used a novel microCT system for rapid virtual histology in 3D for bone quality, cartilage and scaffold microchannel evaluation to submicron pixel resolution, without contrast agents. Examples using human and murine bones will be illustrated, including a clinical study involving a human cancellous bone after bioscaffold implant and its comparison with conventional histology

Results & Discussion

Human Cancellous Cancellous Bone Bone Quality Quality Evaluation Evaluation Human
Remains of PLGA scaffold in trabeculae

Characterizing Cartilage Cartilage Thickness Thickness Characterizing without Contrast Contrast Agent Agent in in Murine Murine Model Model without
Fig 5 compares CT images of a rat knee joint ( a & c) with the cra corresponding ck MRI (b) and conventional histology slides (d)

5a

Fig. 2 MicroCT 3D image of cancellous bone sample extracted for analyzing the quality for implant placement @ 0.7 m detector resolution PLGA Scaffold

Osteoclasts
R

Methods
Four different samples are used in this study to test the use of the novel MicroCT as a possible means for rapid 3D imaging technique for bone quality and bioscaffold evaluation. These includes: 1. Human Cancellous Bone extracted from a dental alveolar socket after placement of bioscaffold for 4 months in the socket 2. Rat knee joint for cartilage imaging in Osteoarthritis 3. Resorption Pit Assays for Osteoporosis 4. Bioscaffold microchannel and microporosity evaluation after fabrication and post processing.

C Fig 3a microCT slice of bone sample @ 0.7m S resolution, compared with conventional histology (3b)

Evaluation of the bone sample shows trabecular bone structure with a cortical layer similar to Type III bone structure suited for implant placement. The Fig 3b shows the connective tissues, the cancellous bone and the cortical layers in the histology which can be also clearly seen in the CT slice (Fig 3a) where the cancellous bone is seen as a dull grey region while the cortical boundaries as white regions. The osteoclasts spread on the cancellous regions (dots seen on the dull grayish surface) is also seen. The connective tissues are seen as dark grey regions identical to porosity while scaffold remains are seen within the connective tissue regions. While the conventional histology required extensive sample preparation techniques along with staining to identify these (up to a few weeks), the microCT was able to provide the data rapidly (couple of hours) and with very little sample 1 m preparation.

f = after The patient had a placement of bioscaffold (fig 4) in the socket Extraction for about 4 months within which sufficient bone volume was achieved in the socket. Patient opted for an implant placement and a bone piece was harvested from the proposed implant site to ensure bone quality and absence of any inflammation. The bone sample was 500 analyzed both using conventional histology and microCT which is m shown in the Figures 2 & 3

OD R N

Fig 6 shows CT slices of a rat tibia demonstrating high contrast imaging of b cartilage-bone interface (a & b) 6a 1 m and the quantitative evaluation of cartilage layer 100 thickness m without the aid of contrast c d agents (c & d ) Evaluation of cartilage degeneration in osteoarthritis using murine model is possible with a microCT (only with contrast agents), with a MRI or through conventional histology. The novel high contrast CT can image cartilage structure and its bone interface quickly without contrast agent (Fig 5 & 6) [2]. Fig 5 compares the novel CT imaging at low and high resolution and the equivalent images from MRI and conventional histology. MRI are typically very low resolution, while conventional histology generally takes 2 to 3 weeks to prepare.
.

Characterizing Bone Bone Resorption Resorption pits pits in in Characterizing Osteoporosis Assays Assays Osteoporosis

Rat knee joints were harvested, fixed in paraffin and imaged at different Resolutions (Fig 5 to 7). Comparison with histology is shown. In case of bioscaffold, the fabricated samples were analyzed using xray microCT to characterize the micro porosities and macro channels present. The slices were taken both in the vertical and horizontal directions to evaluate the distribution of pores and pore sizes while the 3 dimensional images were constructed from the slices to analyze the macro channels in the scaffold. Conventional imaging technique can only produce 2 D images which will be giving information on one plane alone (Fig 4).

Characterizing 3D 3D Microchannels Microchannels in in Characterizing Bioscaffolds Bioscaffolds

7a b

With detector pixel resolution to < 1 micron range, microscopic bone resorption pits may be detected in drug assays for osteoporosis in murine models ( Fig 7).

Fig 1: Apparatus: schematic of the novel microCT with unique high resolution and high contrast optics

The apparatus used for this study (Fig 1) is based on the Xradias MicroXCT[1], which is capable of submicron detector pixel resolution. Unlike conventional MicroCTs which uses point projection technique where resolution is limited by source spot size and its sample-source distance- resolution of the novel microCT is not dependant on these parameters. Relatively large biological materials of several mm diameter may be imaged to 1 micron resolution. With PhaseEnhancedTM optics, significant increase in contrast is realized, making it possible to image inherently low contrast samples such as cartilage and biomaterials without contrast agents.

c 300 100 m m CT images of the Fig 4 a b & c 3D and 2D Bioscaffold surface and the CT slice image. It can be clearly seen from the images, that microCT combines the advantage of conventional optical microscope, scanning electron microscope and that of a confocal microscope, without the associated sample preparation requirements. Fig 4a shows the 3 D structure of the scaffold with macro channel and micro porosity on the sample surface. The fig 4b clearly shows the macro channel size and configuration while also reflecting the microporosity along the sides of the channel. Fig 4c shows an exploded view of the slice depicting the microporosity distribution critical for the scaffold. The ideal architecture desired for a scaffold with about 70-80 % space (pores and channels) and microarchitecture in terms of size distribution is clearly seen from the CT

From the above figures and discussion, it is evident that the novel microCT is an effective tool for characterizing any solids with very low attenuation factors including human cancellous bones, murine cartilage and bones and resorbable bio-scaffolds non-invasively. As microCT technique also requires little or no sample preparation thereby minimizing the time required while preserving the integrity of sample and information required. It is therefore envisioned that this technique could supplement conventional histology for these assays in preclinical and possibly clinical applications.
[1] S H Lau, et al., : Virtual Non Invasive 3D Imaging of Biomaterials and Soft Tissue with a Novel High Contrast CT, with Resolution from mm to sub 30 nm, Symposium on Adv Functional Biomaterials, Proceedings of ICMAT, Singapore 2007 [2] S H Lau, et al., : Rapid Virtual Histology in 3D for Cartilage, Soft tissue, Bones, Scaffolds and Cells with a novel micro and nano CT without contrast agents, Paper # 10, 6TH Combined Meeting, Orthopedic Research Society 2007

Conclusion

References

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EURopean SYMposiUM on CalciFieD TissUes, BaRcelona Spain 2009

RAPID TECHNIQUE FOR BONE QUALITY & OSTEOCYTES LACUNAE EVALUATION USING A NOVEL MICROCT WITH HISTOLOGY RESOLUTION
M. Chandrasekaran1, S H Lau2, R.Gunaseelan3 , V. Prabhu3 1Bio-scaffold International Pte. Ltd, Singapore; 2Xradia Inc, Concord, CA, USA 3 Oral and Maxillofacial Implant Dentistry Surgery, Rajan Dental Institute, Chennai, India
Email Contact: shlau@xradia.com

Abstract

Results & Discussion

It can be seen from the above pictures that Micro CT is able to clearly identify the osteocytes and the compact bone and even to the extent of remnants of PLGA scaffold with similar resolution if not better than the conventional histopathology. In addition to it, the Micro CT is able to distinguish between th type of bone and stages of healing when different grafting 5a b materials were used. In addition to this, the power of 3 D Micro CT is its ability to render 3 D image with clear indication of bone density distribution and soft tissue presence across the three dimension which is not possible wit conventional histology. c d

The current study is focused on developing a rapid technique to Bone Quality Evaluation: BioOss vs Bioscaffold compare and evaluate bone quality and osteocyte lacunae distribution in human and murine bones in high resolution comparable to histology. We will present results in regenerated human maxillary jaw bone sample comparing a novel resorbable bioscaffolds and those obtained from xenograft sources in dental implants. Preliminary results of the 3D distribution of osteocyte lacunae distribution and its relationship to its canal network will also be shown using the cortical bone of a lactating mouse. Conventional histology studies can take up to a few weeks, results are often operator dependant, and are only available as individual 2D slices. While in recent years there are a number of publications on the use of MicroCT for bone quality evaluation, contrast and resolution is still vastly inadequate compared to histology. It is also not possible to characterize the distribution of osteocyte lacunae satisfactory in laboratory based micro or nanoCTs.
In the current work we have used a novel microCT system for rapid virtual histology in 3D for bone quality, and osteocyte ultrastructural evaluation at submicron resolution and without contrast agents. Results are comparable to some existing studies using micro and nanotomography in synchrotron radiation.

cra ck

Fig 2 Images on the top rows are CT slices of BioOss implant showing a lack of new bone growth while images in the bottom row shows new bone regeneration through evidence of osteocytes, which the lighter regions shows compacted mineralized bones

Osteocyte lacunae and canal network: cortical bone, lactating mouse

Methods
In the current work, the bone quality obtained with two different types of dental bone grafting materials were evaluated using novel Micro CTBone quality of two different types of dental scaffold materials in a mandibular socket using novel MicroCT. The socket after tooth extraction was grafted with

1 m

6a

300 m

100 m

c
100 m

Fig 3 3D render image comparing BioOss (left) and Bioscaffold (right). There is greater presence of soft tissues in the bone sample grafted with bioscaffold a. Novel Resorbable Bioscaffold (PLGA) manufactured by Bioscaffold It can be clearly seen that the Micro CT is able to clearly International (Singapore) distinguish between the bone extracted from the two b. & BioOss, a xenograft most commonly used material in dental bone different sockets. The socket with the bioscaffold had grafting natural bone with the osteocytes while the bone sample from the socket which had Bio Oss shows lack of The patient had placement of bioscaffold and Bio Oss filling in two osteocytes. In addition sample grafted with bioscaffold mandibular first incisor sockets respectively following OD a tooth R N shows greater presence of soft tissues. The correlation extraction. After 3 months, the bone from the respective f = incisor between the conventional histology and novel micro CT sockets were harvested to assess the quality of bone for planning the was demonstrated in the earlier paper by the authors on dental implant. The bone sample was analyzed using micro CT which comparison of histology of bone extracted and the is shown in the Figures 2 & 3. correcponding Micro CT. The picture below provides a typical example of correlation between histology and 500 m Micro CT
Fig 1: Apparatus: schematic of the novel microCT with unique high resolution and high contrast optics

Fig 6 microCT slice of lactating bone sample @ 0.5 m c d

Fig 7 3D rendered image of lactating mouse bone showing osteocyte lacunae and canal network microCT slice of lactating bone sample @ 0.5 m

Human Bone Quality Evaluation: microCT vs Histology

Remains of PLGA scaffold in trabeculae

The above image shows the Micro CT of cortical bone of a lactatng mouse. It clearly shows the canal network and absence of osteocytes. The 3 D rendered image shows compact bone (cortical) at the edges while the central region shows the canal network.

XXXX
XXX 7a b
R C S

The apparatus used for this study (Fig 1) is based on the Xradias MicroXCT[1], which is capable of submicron detector pixel resolution. Unlike conventional MicroCTs which uses point projection technique where resolution is limited by source spot size and its sample-source distance- resolution of the novel microCT is not dependant on these parameters. Relatively large biological materials of several mm diameter may be imaged to <1 micron resolution. With PhaseEnhancedTM optics, significant increase in contrast is realized, making it possible to image inherently low contrast samples such as cartilage and biomaterials without contrast agents.

1 m

Conclusion
From the above figures and discussion, it is evident that the novel microCT is an effective and noninvasive tool for bones quality and osteocyte lacumae-canular matrix evaluation. With this technique the relative effectiveness of the different scaffold materials for bone regeneration can be established quickly. With submicron resolution where osteocytes lacunae can visualized clearly could help us understand how these cells respond to strain and regulate bone remodeling. This is turn will lead to new bone therapeutics to prevent bone loss resulting in fracture. , Acknowledgement
Authors wish to thank the following individuals for their support in this study: Dr Victor Fan and Dr Mohd Nazrul Faculty of Dentistry, National University of Singapore for providing the histology Prof Lynda Bonewald, School of Dentistry, Dept. of Oral Biology, Univ. of Missouri at Kansas City Missouri, USA for providing the lactating mouse bone and valuable insight. Tiffany Fong, Application lab, Xradia Inc, Concord, CA, USA

Fig. 4 MicroCT 3D image of Bioscaffold regenerated bone sample @ 1 m resolution

For ultrastructural evaluation of Osteocytes lacunae and its canal network using the microCT, a piece of cortical bone about 500 microns in diameter was prepared from a lactating mouse. The sample was scanned with the microCT and preliminary results are shown.

Fig 5 microCT slice of bone sample @ 1 m resolution, compared with conventional histology.

References
[1] S H Lau, G. Wang, M.Chandrasekeran, V. Fan, M. Nazrul , H.Chang, T. Fong, J. Gelb, M. Feser, W. Yun, Multiscale 3D Bioimaging: from cell, tissue to whole organism , Proceedings SPIE Scanning Microscopy 2009

[2] S H Lau, et al., : Rapid Virtual Histology in 3D for Cartilage, Soft tissue, Bones, Scaffolds and Cells with a novel micro and nano CT without contrast agents, Paper # 10, 6TH Combined Meeting, Orthopedic Research Society 2007

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MicRo CT analYsis oF hUMan bone

With the conrmation of micro CT usefulness to show bone quality and density, it was used to analyze one case where the bone was harvested during the implant placement after 5 months of scaold socket preservation (Fig. 01 & Fig. 02). The case is presented below with conventional histopathology (Fig. 03) and novel micro CT analysis (Fig. 04) showing a very healthy, natural and normal bone through bio-scaold socket preservation. Histopathological Report: L.S sections taken from trephine column of bone removed from the tooth socket grafted with PLGA (Poly lactic Co Glycolic Acid) 6 months back show part cortico- cancellous bone and many interconnecting trabeculae of new bone formed in association with small pieces of PLGA materials which undergoing varying stages of degradation and being replaced by new bone (woven bone) associated with previous cancellous bone. There is also demonstrable conversion of woven bone in to lamellar bone/mature bone with the trabeculae of woven bone. Bony section shows almost no or few inammatory cells within the marrow spaces. No sign of bony resorption have taken place but rather layers of new bone seen to be forming in some places around the degrading PLGA materials. There is however a foci of PLGA associated with inammatory inltrate. Micro CT Report: The upper top left slide (Fig 04: 4a) is taken from the top part of the speciman sent and shows good and compact bony structure. The upper top right slide (Fig 04: 4b) was taken from a side of the speciman and also shows very compact bony that resembles a normal bone. The lower left slide (Fig 04: 4c) was taken from the opposite side of the speciman and also shows the same result. The lower right picture (Fig 04: 4d) is presenting the 3D view of the speciman itself which shows a cancellus good bony pattern that can be compared with any healthy good quality bone. After going through all the dierent level micro CT pictures, we can condently say that the speciman scan shows good cancellus bony structures of spongy nature that resembles a normal healthy bone. At closer examination we also can identify the spongy characteristics of the bone with its bone marrow space and the havertian system. So overall the bony speciman proves to have the same consistency and structure as that of a healthy normal bone.

Fig. 01: Bio-scaold preserved site after Fig. 02: Bone piece 5 months Cortical bone Scaold residue Connective tissue

Fig. 03: Histopathological slide 4a 4b

4c

4d

Fig. 04: Micro CT picture

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A positiVe contRol coMpaRison tRial With XenoGRaFt

After conrming the bone quality and density, a positive control comparison with other socket preservation procedure with conventional bone grafting materials was arranged. Xenograft granules are the most popular bone grafting product that is used widely in dentistry. In spite of the necessity for a surgical procedure for xenografting, it is being used as a socket preservation option. The ecacy of bio-scaold over xenograft in terms of bone growth and regeneration capability was compared over the period of 3 month. A clinical case study is being conducted by Dr R. Gunaseelan of Rajan Dental Institute, Chennai, India involving 10 patients with bilateral extraction in the same patient. 3 patients have already completed the trial and another 7 will be done soon. One completed case was presented as a poster at the National Oral and Maxillofacial Scientic Conference held at Kochi, India, in November 2009. It was showed and concluded from the analysis of this case that compared to xenograft, synthetic bio-scaold is able to assist the natural healing process better and provides a better bone quality while preserving the ridge after extractions in the anterior segment. Materials and method: Patients who have requirement of removing two teeth in the same ridge (mandible/maxilla) were selected and in one socket PLGA bio-scaold was used while the other socket was packed with a xenograft widely used for grafting applications which is used as an alternative in socket preservation. The xenograft was lled into the second socket and was closed with a collagen membrane. The OPG radiograph was taken immediately after extraction of the teeth with the bio-scaold and the xenograft placed in the other socket. In order to get a better picture of healing, a bone sample was extracted from both the socket and was examined using a novel micro CT technique which gives a 3D picture of the bone. Result: Bone sample retrieved from socket grafted with xenograft showes a lack of new bone growth while scaold preserved site demonstrates new bone regeneration through evidence of osteocytes. Sample extracted from the socket preserved using bio-scaold shows compact mineralized bone. The socket with the bio-scaold had natural bone with the osteocytes while the bone sample from the socket which had xenograft shows lack of osteocytes.
1a 1b

(1a) Upper left picture showing packed xenograft granules after 3 months. (1b) Upper right picture showing resorbing bio-scaold after 3 months. (1c) Lower left picture is showing granule surface with very few osteocytes.

1c

1d

(1d) Lower right picture showing scaold surface with osteocytic activity.

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National ORal anD MaxilloFacial ScientiFic ConFeRence, Kochi, InDia, PosteR PResentation
BIO-SCAFFOLD SHOWED BETTER BONE REGENERATION THAN CONVENTIONAL XENOGRAFT

Evaluation Of Bone Quality In Dental Socket Using Two Different Approaches For Ridge Preservation Using A Novel Micro CT
RAJAN DENTAL INSTITUTE, Chennai, India - R.Gunaseelan, V.Prabhu, B. Praveen BIO-SCAFFOLD INTERNATIONAL PTE. LTD, Singapore - Margam Chandrasekaran, Md Nazrul Islam XRADIA INC, CONCORD, United States - S.H. Lau Presented at the National OMS conference 2009, Kochi, India. www. rajandental.com www.bio-scaffold.com www.xradia.com

Abstract
The current study is focused on comparing the healing and bone quality after using two different approaches to ridge preservation in the mandibular arch. The study also highlights the recent development of rapid technique to compare bone quality in regenerated human bone using resorbable bioscaffolds and those obtained from using HA/TCP or xenograft and allograft sources in dental alveolar sockets. Conventional methods use analysis of healing using a OPG which gives very little information on the bone quality. Despite developments such as Dental CT, which pose concerns on radiation exposure of patient it is very difficult to assess the bone quality and most of the implant procedures are done based on assessment of the surgeon. Implant stability is largely dependent on the bone quality and currently most common method of assessment is using histopathological assessment. Conventional histology studies can take up to a few weeks and the results are often operator dependant. Moreover, the results are only available as individual 2D slices and so many slices are required from the bone sample before making a decision on the quality of bone. In the current work, we have used a novel Micro CT with histology resolution and superior contrast as a rapid means to evaluate bone quality in 2D and 3D between bone samples extracted from patients who used a synthetic resorbable bioscaffold against conventional grafting materials.

For ultrastructural evaluation of Osteocytes lacunae and its canal network using the Micro CT, a piece of cortical bone about 500 microns in diameter was prepared . The sample was scanned with the Micro CT and the results are shown below

It can be seen from the above pictures that Micro CT is able to clearly identify the osteocytes and the compact bone and even to the extent of remnants of PLGA scaffold with similar resolution if not better than the conventional histopathology. In addition to it, the Micro CT is able to distinguish between the type of bone and stages of healing when different grafting materials were used. In addition to this, the power of 3 D Micro CT is its ability to render 3 D image with clear indication of bone density distribution and soft tissue presence across the three dimension which is not possible wit conventional histology.

Results & Discussion

Bone Quality Evaluation: Xenograft vs Bioscaffold

OPG of Posterior tooth socket treated with Xenograft/ Bio-scaffold

Fig. 6a & b 1st and 3rd month OPG of socket 35/36 (Bio-scaffold/Xenograft)

The OPG shows the distinguished healing difference between xenograft used 36 and bio-scaffold used 35 in a posterior lower jaw segment. The 7 day post extraction OPG shows does not show a significant difference
Fig. 2 Images on the top rows are CT slices of bone sample retrieved from socket grafted with Xenograft showing a lack of new bone growth while images in the bottom row shows new bone regeneration through evidence of osteocytes, which the lighter regions shows compacted mineralized bones in the sample extracted from the socket preserved using Bioscaffold

between these two sockets but 3 month follow up OPG clearly shows a good radio opacity at the root socket site of 36 where the root socket site of 35 still shows radiolucency proving the healing and bone growth difference between the two site after 3 month. This radiographic comparison re enforce the ability of synthetic bio-scaffold to allow a more natural healing than xenografts. In order to verify the observation, image analysis was done to check the average grey value distribution and the standard deviation. Figure 7 shows the typical grey value for the sockets after 7 days and Figure 8 shows the grey value distribution after 3 months
Histogram of Grey Value Distribution
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Introduction
After tooth extraction, the extraction socket heals by forming a blood clot which leads to the formation of new bone within 3-4 months. Although bone deposition in the socket will continue for several months, it will not reach the crestal level of the neighbouring teeth. The resulting alveolar bone loss may lead to problems such as poor aesthetic appearance and difficulty in placement of implants or prostheses for partially or totally toothless patients. Various techniques have been proposed to limit alveolar bone loss such as atraumatic extraction, immediate postextraction removable prosthesis, immediate placement of dental implant and immediate bone-filling of extraction socket. Guided bone regeneration (GBR) and guided tissue regeneration (GTR) has been gaining attention in recent years for repair of bone defects and had been used in patients after tooth extraction with the aim of preserving alveolar bone. In the current work, two different methods of ridge preservation techniques is compared for the healing and quality of bone regenerated after grafting.

Histogram of Grey value distribution

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Fig. 3 3D render image comparing Xenograft (left) and Bioscaffold (right). There is greater presence of soft tissues in the bone sample grafted with bioscaffold

Fig. 7a & b Area histogram analysis of socket 35 & 36 after 7 days

Histogram of Grey value distribution
300 250 200 150 100 50 0

It can be clearly seen that the Micro CT is able to clearly distinguish between the bone extracted from the two different sockets. The socket with the bioscaffold had natural bone with the osteocytes while the bone sample from the socket which had Xenograft shows lack of osteocytes. In addition sample grafted with bioscaffold shows greater presence of soft tissues. The correlation between the conventional histology and novel Micro CT was demonstrated in the earlier paper by the authors on comparison of histology of bone extracted and the correcponding Micro CT. The picture below provides a typical example of correlation between histology and Micro CT
500 400 300 200 100 0

Histogram of Grey value distribution Mean 34 Std Dev. 9.29156

1 15 29 43 57 71 85 99 113 127 141 155 169 183 197 211 225 239 253

1 17 33 49 65 81 97 113 129 145 161 177 193 209 225 241

a)

Methods
In the current study, patients who were having multiple extractions in the same jaw were selected and after the tooth extraction, the adjacent sockets were treated with two different solutions. One of the socket was grafted with commercially available xenograft and covered with a membrane while BioscaffTM AlvelacTM ( a synthetic scaffold made of polylactic-co-glycolic acid) was placed and was closed with a simple interrupted suture. The OPG of the patient was taken immediately after the procedure and was subsequently followed up after 3 months. Figure 1 and 2 shows a typical OPG taken on the patient immediately after placement of scaffold/grafting the socket size and after 3 months.

Fig. 8a & b Area histogram analysis of socket 35 & 36 after 3 months

From the above histograms we can clearly see that the mean value is more or less same initially but there is significant difference after 3 months. However, the standard deviation increases for the grey values for socket 35 compared to socket 36 which does not show significant difference in standard deviation compared to initial value. Moreover, the socket 36 has a normal distribution of grey values while the socket 35 has initially positive skew in distribution but the 3rd month OPG grey shows a negative skew and increase in width of the histogram. The standard deviation of distribution also increases which possibly indicates new bone at site.

Human Bone Quality Evaluation: Micro CT vs Histology

Remains ofbPLGA scaffold in trabeculae

Conclusion
From the above figures and discussion, it is evident that the synthetic Bioscaffold is able to better assist the natural healing process and provides a
Fig. 4 Micro CT 3D image of Bioscaffold regenerated bone sample @ 1 m resolution

better bone quality while preserving the ridge after extractions in the anterior segment. The posterior segment indicates new bone growth but needs further analysis to yield conclusive evidence. Micro CT provides a good insight in to the bone healing and the bone quality in 3 dimension compared to conventional histopathology.

a)

b)

Fig. 1a & b 1st and 3rd month OPG of 31/41 (Xenograft/ Bio-scaffold)

The above picture shows a typical healing of the anterior socket with Xenograft and Bioscaffold respectively. The conventional xenograft was placed in the extraction socket 31 and Bioscaffold was placed in socket 41. The xenograft was radio opaque to certain extent and the granules occupy the space to a certain extent. So clear indication of healing could not be obtained from the OPG. In order to get a better picture of healing a bone sample was extracted from both the sockets and were examined using a novel micro CT Technique which gives a 3 dimensional picture of the bone.
Fig. 5 Micro CT slice of bone sample @ 1 m resolution, compared with conventional histology

References
[1] S H Lau, G. Wang, M.Chandrasekeran, V. Fan, M. Nazrul , H.Chang, T. Fong, J. Gelb, M. Feser, W. Yun, Multiscale 3D Bioimaging: from cell, tissue to whole organism , Proceedings SPIE Scanning Microscopy 2009

[2] S H Lau, et al., : Rapid Virtual Histology in 3D for Cartilage, Soft tissue, Bones, Scaffolds and Cells with a novel micro and nano CT without contrast agents, Paper # 10, 6TH Combined Meeting, Orthopedic Research Society 2007. (3). M. Chandrasekeran, S. Lau, R. Gunaseelan, V. Prabhu, Rapid technique to evaluate human bone quality using novel Micro CT with histology reolution, Presented at the 36th European Symposium on Calcified Tissues, 23-25 May,2009 Vienna, Austria

1 15 29 43 57 71 85 99 113 127 141 155 169 183 197 211 225 239 253

b)

1 14 27 40 53 66 79 92 105 118 131 144 157 170 183 196 209 222 235 248

a)

Mean 34 Std Dev. 7.32246

b)

Mean 35 Std Dev 7.76309

Mean 49 Std Dev. 8.26817

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CASE STUDIES
To gather more clinical evidences, case studies are continuously conducted. Dentists who are using bio-scaold for their patients were welcome to share their cases as a form of case study. Presented below are some of the case studies with bio-scaold socket preservation.

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CASE STUDY 01

Courtesy of NUH (Clinical Trial Case)

A female patient aged 34 came for an extraction of the right lower first molar due to endodontic treatment failure. Extraction was done with forceps. After the extraction, bio-scaffolds were placed horizontally in the extraction site engaging the buccal and lingual walls. Subsequently, the buccal and lingual flaps were sutured using vicryl suture. X-ray was taken at one week follow up and sutures were removed. Healing was good with no complains and patient was advised for the next follow up in two months. Second and third month follow ups showed normal tissue contour at the extracted site with no scarring effect or tissue defect (Fig. 01). The patient came back after five months for implant placement at the healed site. At fifth month, the same site showed good bony contour with well maintained height and width with no bony defects (Fig. 02 and Fig. 03).

Fig. 01 Good soft tissue healing.

Fig. 02 Good bony socket width after 5 months.

Fig. 03 Good alveolar height preservation.

To assess the bone quality of the healed socket site, a sample of bone was trephine out from the healed socket area for histopathological examination and micro CT (Fig. 04). The histopathological slides were made in the histopathology laboratory, Faculty of Dentistry, NUH, Singapore and the report was given by A/P Yeo Jin Fei, Head of the OMS Department, NUH, Singapore (Fig. 05). The histopathological report shows cortico-cancellous bone with many interconnecting trabeculae of new bone formed in association with small fragments of PLGA bio-scaffold materials while undergoing varying stages of degradation and being replaced by new bone associated with previous cancellous bone. Section shows almost no or few inflammatory cells within the marrow space. The micro CT of the trephine bone (Fig. 06) shows normal healthy bony structure similar to that of the normal bone (Fig. 07).
Cortical Bone Scaffold residue Connective tissue

Fig. 04 Extracted bone piece (ruler in mm).

Fig. 05 Histopathological picture of the trephined bone.

Fig. 06 Micro CT of trephine bone.

After the bone piece was trephine out, an implant (Fig. 08) was placed at the same prepared site with good initial stability.

Fig. 07 Micro CT of normal bone.

Fig. 08 Implant.

Conclusion: Use of bio-scaffolds in the socket after extraction helps to promote good healing. The bio-scaffolds also helped to preserved the height and width of the alveolar ridge after 5 months with very little residue of bio-scaffold material at the site. The quality of bone was good and healthy at the post socket site after 5 months.

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CASE STUDY 02

Courtesy of Dr Hui Chee Wah, Singapore

A male patient of age 38 had a fractured 11 which was root canal treated and restored with a post crown. The tooth was fractured transversely and subgingivally. Fractured post crown and remaining tooth was beyond salvage (Fig. 01). There was an existing apical lesion from the failed root canal treatment and could be seen in the OPG. Patient oral hygiene was bad with lots of plaques and calculus. An OPG was taken before extraction clearly showing the apical lesion (Fig. 02). Extraction was done and the coronal portion was removed first. The root was extracted atraumatically with periotomes (Fig. 03).

Fig. 01 Picture before extraction showing bad oral hygiene.

Fig. 02 OPG showing root canal treated 11 with apical lesion.

Fig. 03 Dislodged crown was taken out first.

Socket was cleaned of all infected debris (Fig. 04) and bio-scaffold was placed horizontally at the crestal level (Fig. 05). Healing was good after a week and suture was removed. There was no complicacy or complains from the patient. The patient came back after 10 month for a follow up visit as he was planning to have implant for his missing 11. While the patients oral hygiene continues to be bad, it was however clear that the extracted socket showed minimal height loss as seen in the picture (Fig. 06). Width loss was observed to be insignificant and is likely to be due to the patients early apical infections and bad periodontal condition.

Fig. 04 Socket cleaned of all infected debris.

Fig. 05 Scaffold placed horizontally at crest level.

Fig. 06 10 month follow up shows well tissue contour. Socket height is preserved.

Due to financial situation, patient is still waiting for a suitable time for his implant restoration. Conclusion: Bio-scaffolding of this extracted 11 preserved the height of the socket and also prevented severe collapse of width of the socket despite having an apical infection and periodontal conditions.

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CASE STUDY 03

Courtesy of Dr Hui Chee Wah, Singapore

A female patient of age 59yrs came to the clinic with periodontically involved two upper central incisor and the teeth were beyond salvation (Fig. 01). An OPG was taken before extraction clearly showing the condition of the teeth and surrounding bone which was not so good (Fig. 02). The bone height is already far below the cervical margin and only half or so socket was remaining. So to preserve the remaining socket bone, socket preservation was done with AlvelacTM. An atraumatic extraction was done with periotomes and 21 was removed first followed by 11 (Fig. 03 & Fig. 04).

Fig. 01 Before extraction showing two upper central incisors.

Fig. 02 OPG showing periodontically involved 11 and 21 with severe bone resorption.

Fig. 03 Atraumatic extraction of 21 done.

Sockets were cleaned of all infected debris and bio-scaffolds were placed horizontally at the crestal level (Fig. 05). Two 3.54.0mm bio-scaffolds were used which resembles the width of the socket at crest level (Fig. 06).

Fig. 04 Atraumatic extraction of 11 and 21.

Fig. 05 Bio-scaffold placed horizontally at crest level.

Fig. 06 Bio-scaffold placed at crest level which is far below the cervical margin.

Healing was good after a week and suture was removed. There was no complicacy or complain from the patient. The patient came back after seven months for a follow up visit (Fig. 07) as she was planning to have implant for her missing 11 and 21. At seventh month re-entry, the alveolar ridge height is almost at the same level as the time of extraction though there was a bit of height loss at the 21 site (Fig. 08). The thickness of the ridge was compromised at the time of extraction and now it is just suitable for a 411mm implant (Fig. 09).

Fig. 07 Seven month follow up shows well tissue contour.

Fig. 08 Socket height is preserved at almost the same level as after extraction.

Fig. 09 Two 411mm Osstem implant is placed in site.

As the thickness of the ridge was compromised, there was some labial exposure of implant threads and needed bone grafting. Xenograft was used for grafting and was covered with a resorbable membrane (Fig. 10). Mucoperiosteal flap was replaced and sutured with 3.0 vicryl. An immediate denture was given to restore the aesthetic look of the patient (Fig. 11).

Fig. 10 Labial defect was grafted with xenograft.

Fig. 11 An immediate denture is given to the patient for aesthetic purpose.

Conclusion: Bio-scaffolding of extracted 11 and 21 preserved the height of the socket and also prevented severe collapse of width of the socket despite having a very bad periodontal condition and bone resorption.
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CASE STUDY 04

Courtesy of Dr Hui Chee Wah, Singapore

A female patient of age 70 had a fractured 45 which was grossly carious. The tooth fractured vertically and was beyond salvage, thus extraction were performed (Fig. 01). Other then the fracture, there was an existing apical lesion seen in the OPG (Fig. 02). The patients oral hygiene was moderate. OPG was taken before and after extraction (Fig. 03).

Fig. 01 Picture of tooth socket after extraction.

Fig. 02 OPG showing fractured 45 with apical lesion.

Fig. 03 OPG post extraction.

Extraction was done and the tooth was extracted atraumatically with periotomes. Socket was healthy with normal bleeding and blood clot. Socket was cleaned of all infected debris (Fig. 04) and bio-scaffold was placed vertically at the crestal level (Fig. 05). Healing was good after a week and suture was removed. After 3 weeks, bio-scaffold was visible with good soft tissue healing (Fig. 06). There was no complicacy or complain from the patient.

Fig. 04 Socket cleaned of all infected debris.

Fig. 05 Scaffold placed vertically at crest level.

Fig. 06 Scaffold visible with good soft tissue healing around it after 3 weeks.

The patient came back after four months for a follow up visit (Fig. 07) as she wanted to have implant for her missing 45. Implant was placed at the end of the fourth month with good initial stability (Fig. 08).

Fig. 07 OPG showing good bony healing with height of the alveolar ridge well maintained.

Fig. 08 Implant placement after 4 month.

Fig. 09 5 months follow up during the restoration procedure.

Restoration was done at fifth month (Fig. 09) and extracted socket did not show any height loss as seen in the picture (Fig. 10). The soft tissue profile looks normal without any marked deformation and depression.

Fig. 10 5 month follow up shows implant placement.

Conclusion: Bio-scaffolding of this extracted 45 preserved the height of the socket, prevented severe collapse of width of the socket and preserved the soft tissue aesthetic nicely for the implant restoration.

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ConclUsion
Amongst the various osteoconductive materials that are widely used, biopolymers occupy a principal position due to their relatively faster degradation and metabolisation rates compared with other synthetic substitutes. The most commonly used material includes PLA and PGA where degradation time and strength are critical. The PLGA bio-scaold, AlvelacTM, developed by Bio-Scaold International has a typical strength of 6 MPa and a degradation time of 2-6 months. The material is biocompatible to the surrounding tissue and does not induce any inammatory reaction or damage to the local tissue. Osteogenesis and brogenesis were seen in the scaold preserved sites. Through a well design multi site clinical trial, the ecacy and eectiveness of the bio-scaold was proven with a statistical signicance. Collaboration with Rajan Dental Institute, Chennai, India, Xradia Inc, CA, USA, has further strengthened evidences of bone quality and density after socket preservation with bio-scaold and established the fact that AlvelacTM is a better choice for bone regeneration in alveolar socket after extraction.

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BSI-M-CLI-001-V1.0