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1 Transforming Growth Factor-ß 1 Accelerates Resorption of a Calcium Carbonate Biomaterial in Periodontal Defects Ki-Tae Koo The Graduate School Yonsei University Department of Dental Science

2 Transforming Growth Factor-ß 1 Accelerates Resorption of a Calcium Carbonate Biomaterial in Periodontal Defects A Dissertation Submitted to the Department of Dental Science and the Graduate School of Yonsei University in partial fulfillment of the requirements for the degree of Doctor of Philosophy of Dental Science Ki-Tae Koo December 2006

3 This certifies that the dissertation of Ki-Tae Koo is approved. Thesis Supervisor: Chong-Kwan Kim Jung-Kiu Chai Seong-Ho Choi Jeong-Hye Kim Hong-Seok Moon The Graduate School Yonsei University December 2006

4 감사의글 본논문이완성되기까지부족한저를항상격려해주시고사랑과관심으로이끌어주신김종관교수님께깊은감사를드립니다. 그리고, 많은격려와세심한지도편달을해주신채중규교수님, 조규성교수님, 최성호교수님, 김정혜교수님, 문홍석교수님, 김창성교수님께진심으로감사드립니다. 또한, 본연구내내많은도움을아끼지않은치주과교실원여러분께도고마움을전합니다. 이해와격려를베풀어주신양가부모님께감사드리고마지막으로사랑하는나의아내영선과아들영모에게진정으로사랑과고마움의마음을담아전합니다. 모든분께진심으로감사드립니다. 2006년 12월저자씀

5 Table of Contents Abstracts(English) ⅲ Ⅰ. Introduction 1 Ⅱ. Materials and Methods 4 A.Animals 4 B. Sugical Protocol 4 C. Experimental Protocols 5 D. Wound Management 6 E. Postsurgery Protocol 6 F. Histological Procedures 7 G.Histological Evaluation 8 H.Statistical Analysis 9 Ⅲ. Results 10 A. Clinical Observations 10 B. Radiographic Observations 10 C. Histological Observations 10 D. Histometric Analysis 11 Ⅳ. Discussion 16 Ⅴ. Conclusion 19 References 21 Figure Legends 23 Figures 24 Abstract(Korean) 30 i

6 List of Figures and Tables Table 1. Comparison of various parameters between the experimental groups Table 2. Effect of rhtgf- ß 1 on biodegradation after adjustment for wound and bone area. Table 3. Figure 1. Figure 2. Effect of rhtgf- ß 1 on bone formation after adjustment for wound and carrier area. Clinical application of calcium carbonate carrier before and after applying an eptfe-barrier for guided tissue regeneration at 4 weeks postsurgery Photomicrographs showing the critical-size, supraalveolar periodontal defects at 4 weeks postsurgery Figure 3. Carrier density by wound area. 26 Figure 4. Residual carrier area by wound area. 27 Figure 5. Bone height by wound area. 28 Figure 6. Bone area by wound area. 29 ii

7 Abstract Transforming Growth Factor-ß 1 Accelerates Resorption of a Calcium Carbonate Biomaterial in Periodontal Defects Background: In a previous study, recombinant human TGF-ß 1 (rhtgf-ß 1 ) in a calcium carbonate carrier was implanted into critical-size, supraalveolar periodontal defects under conditions for guided tissue regeneration (GTR) to study whether rhtgf-ß 1 would enhance or accelerate periodontal regeneration. The results showed minimal benefits of rhtgf-ß 1 and a clear account for this could not be offered. One potential cause may be that the rhtgf-ß 1 formulation was biologically inactive. Several growth or differentiation factors have been suggested to accelerate degradation of biomaterials used as carriers. The objective of this study was to evaluate possible activity of rhtgf-ß 1 on biodegradation of the calcium carbonate carrier. Methods: rhtgf-ß 1 in a putty-formulated particulate calcium carbonate carrier was implanted into critical-size, supraalveolar periodontal defects under conditions for GTR in five Beagle dogs. Contralateral defects received the calcium carbonate carrier combined with GTR without rhtgf-ß 1 (control). The animals were euthanized at week 4 week postsurgery when block-biopsies of the defect sites were collected for histologic and histometric analysis. Radiographs were obtained at defect creation, week 2 and week 4. iii

8 Results: No statistically significant differences were observed in new bone formation (bone height and area) among the treatments. However, total residual carrier was significantly reduced in sites receiving rhtgf-ß 1 compared to control (p=0.04). Similarly, carrier density was considerably reduced in sites receiving rhtgf-ß 1 compared to control, the difference being borderline statistically significant (p=0.06). Conclusions: Within the limitations of the study, it may be concluded that rhtgf-ß 1 accelerates biodegradation of a particulate calcium carbonate biomaterial indicating a biologic activity of the rhtgf-ß 1 formulation apparently not encompassing enhanced or accelerated periodontal regeneration. KEY WORDS Transforming growth factor- ß 1, calcium carbonate carrier, biodegradation, periodontal regeneration. iv

9 Transforming Growth Factor-ß 1 Accelerates Resorption of a Calcium Carbonate Biomaterial in Periodontal Defects Ki-Tae Koo, D.D.S., M.S.D. Department of Dental Science, Graduate School, Yonsei University (Directed by Prof. Chong-Kwan Kim, D.D.S., M.S.D., PhD.) I. Introduction Transforming growth factor-ß (TGF-ß) is a homodimeric peptide with multifunctional actions controlling growth, differentiation, and function of a broad range of target cells. 1 Tissue-specific and developmentally dependent expression strongly suggests a significant role in specific morphogenetic and histogenetic events. Thus far, five distinct TGF-ßs with 65-80% homology have been identified. 1 Currently thought to consist of at least 26 different proteins, TGF-ß 1 supports wound healing by augmenting angiogenesis and fibroblast collagen formation. 2,3 In addition, TGF-ß 1 is thought to be involved in regulating cell proliferation and differentiation and the production of extracellular matrix. 4 Also, a role of TGF-ß 1 in recruiting and stimulating osteoprogenitor cells to proliferate, providing a pool of early osteoblasts has been suggested. 5 In perspective, TGF-ß 1 technologies appear attractive candidate therapies to support periodontal wound healing/regeneration

10 In a previous study, recombinant human TGF-ß 1 (rhtgf-ß 1 ) in a puttyformulated particulate calcium carbonate carrier was implanted into critical-size, supraalveolar periodontal defects under conditions for guided tissue regeneration * (GTR) to study whether rhtgf-ß 1 would enhance or accelerate periodontal wound healing/regeneration. 6 Control sites received the calcium carbonate carrier combined with GTR without rhtgf-ß 1. The histometric analysis could not discern significant benefits of the rhtgf-ß 1 formulation. Overall, sites receiving rhtgf-ß 1 and control treatments exhibited limited bone formation and regeneration of the periodontal attachment suggesting marginal, if any, effects of rhtgf-ß 1. Although the results failed to discern a significant benefit of rhtgf-ß 1, an obvious account of the results could not be offered. Could possibly the putty-formulated particulate calcium carbonate carrier or the GTR device rendered the growth factor ineffective or biologically inactive? Parallel studies evaluating rhtgf-ß 1 using the supraalveolar periodontal defect model without GTR showed similar limited effects ruling out an inhibitory effect of the GTR device. 7 Other studies suggest that biomaterials implanted into periodontal sites indeed may obstruct bone formation and periodontal regeneration. 8 Still other studies may be interpreted to suggest that growth or differentiation factors may accelerate biodegradation/biotransformation of a biomaterial used as a carrier. 9,10 * Genentech Inc., San Francisco, CA, USA - 2 -

11 A re-examination of the study by Wikesjö et al. 6 using additional parameters was thus deemed necessary to discern possible effects of rhtgf-ß 1 on biodegradation of the putty-formulated particulate calcium carbonate carrier

12 II. Materials and Methods A. Animals Five male Beagle dogs (age months, weight kg) were used. Animal selection, management, surgery protocol, and periodontal defect preparation followed routines approved by the local Institutional Animal Care and Use Committee. The animals were fed a soft-consistency laboratory diet supplemented with vitamins throughout the study. A soft diet was chosen to alleviate potential mechanical interference with wound healing during food intake. B. Surgical Protocol Surgical procedures were performed under sodium pentobarbital anesthesia (20-30 mg/kg, IV) preceded by acepromazine.(1 mg/kg, IM) Routine dental infiltration anesthesia was used at the surgical sites. During surgery, the animals received lactated Ringer's solution. ( ml, IV) Bilateral, critical-size, supraalveolar periodontal defects were created at the 3 rd and 4 th mandibular premolar teeth in each animal. 11 Briefly, following sulcular incisions and elevation of buccal and lingual mucoperiosteal flaps, the alveolar bone was resected around the circumference of the teeth using chisels and water-cooled rotating burs. Nembutal Sodium Solution, Abbott Laboratories, North Chicago, IL, USA PromAce, Aveco Co Inc., Fort Dodge, IA, USA Lactated Ringer's Inj., USP, Abbott Laboratories - 4 -

13 The exposed root surfaces were instrumented with curettes, chisels, and watercooled rotating diamonds to remove the cementum. The resulting clinical defect approximated 5 mm from the cemento-enamel junction (CEJ) to the reduced alveolar crest. The 1 st and 2 nd mandibular premolar teeth were extracted and the crown of the 1 st molar amputated level with the reduced alveolar crest. The maxillary 1 st, 2 nd, and 3 rd premolar teeth were surgically extracted and the maxillary 4 th premolars reduced in height and exposed pulpal tissues were sealed with Cavit in order to alleviate potential trauma from the maxillary teeth to the experimental mandibular sites. C. Experimental Protocols Using a split-mouth design, contralateral, supraalveolar periodontal defects were implanted with rhtgf-ß 1 in a carrier or carrier alone (control). Experimental treatments were alternated between left and right jaw quadrants in consecutive animals. Both treatments were combined with GTR. The carrier comprised medical grade, natural, porous, particulate calcium carbonate and medical grade hydroxyethyl starch providing putty-like handling characteristics; 0.5% gelatin and 20 µm sodium acetate solution was mixed with hydroxyethyl starch to form a visco-elastic gel to contain the calcium carbonate particles in a manageable mass. Cavit, ESPE, Seefeld/Oberbayern, Germany Biocoral 1000, Inoteb, Saint-Gonnery, France - 5 -

14 For each defect scheduled to receive rhtgf-ß 1, 0.25 ml buffer containing 20 µg rhtgf-ß 1 was added to approximately 0.7 g calcium carbonate particles, the hydroxyethyl starch gel was then added to produce a homogenous putty-like mass. Final implant volume/defect approximated 0.8 ml. rhtgf-ß 1 and control constructs were prepared under aseptic conditions. D. Wound Management Defects receiving rhtgf-ß 1 or control treatments had the putty-like material shaped around the premolar teeth to the contour of the resected alveolar bone. The teeth were then fitted with an expanded polytetrafluoroethylene (eptfe) barrier # secured with an eptfe suture ** at the CEJ (Figure 1). Periostea were fenestrated at the base of the mucoperiosteal flaps and the flaps were advanced, adapted, and sutured using horizontal mattress sutures approximately 2 mm coronal to the CEJ. E. Postsurgery Protocol A long-acting opioid (0.015 mg/kg IM, BID, 2 days) was used for immediate pain control. # GORE-TEX Regenerative Material Transgingival Configuration, W.L. Gore & Associates Inc., Flagstaff, AZ, USA ** GORE-TEX Suture CV5, W.L. Gore & Associates Inc. Buprenex Injectable, buprenorphine HCl, Reckitt & Colman Pharmaceuticals Inc., Richmond, VA, USA - 6 -

15 A broad-spectrum antibiotic (2.5 mg/kg IM, BID, 2 weeks) was used for infection control Plaque control was maintained by twice daily topical application of chlorhexidine. (40 ml of a 2% solution) Observations of experimental sites with regards to gingival health, flap adaption, edema, and purulence were made daily. The eptfe devices were not removed. Gingival sutures were removed at day 10. Photographs were obtained at defect creation, suture removal, and at week 2 and 4. Radiographs were obtained at defect induction, and at week 2 and 4. Thiopental sodium anesthesia (20-25 mg/kg, IV) was used for suture removal and radiographic registrations. F. Histological Procedures The animals were euthanized at week 4 postsurgery using an intravenous injection of concentrated thiopental sodium. Tissue blocks including teeth, bone, and soft tissues were removed. The blocks were fixed in 10% buffered formalin for 3-5 days, decalcified in 5% formic acid for 8-10 weeks, trimmed, dehydrated, and embedded in butyl methacrylate-paraffin. Serial sections (7 µm) were cut in a buccal-lingual plane throughout the mesial-distal extension of the teeth. Baytril Brand of Enrofloxacin, Mobley Corporation, Shawnee, KS, USA Chlorhexidine Gluconate 20%, ICI Pharmaceutical Group, Wilmington, DE, USA Pentothal, Abbott Laboratories, North Chicago, IL Every 14th section was stainedwith Ladewig s connective tissue stain modified by - 7 -

16 Mallory allowing for observations at 100-µm intervals. G. Histological Evaluation One experienced, calibrated, masked examiner (KTK) performed the histometric analysis using a PC-based image analysis system with a custom application for the supraalveolar periodontal defect model. 11 The most central stained section for each root of the 3 rd and 4 th premolars, identified by the size of the root canal, was used for the analysis. 12 The following parameters were recorded for buccal and the lingual tooth surfaces for each section: Defect Height: distance between apical extension of the root planing and the CEJ. Device Height: distance between apical extension of the root planing and most coronal aspect of the eptfe device. Defect Area: area under the eptfe device circumscribed by the planed root, the width of the alveolar bone at apical extension of the root planing, and the device. Bone Regeneration (height): distance between apical extension of the root planing and the coronal extension of new alveolar bone formed along the planed root. Bone Regeneration (area): area represented by new alveolar bone formed along the planed root. Image-Pro Plus, Media Cybernetics, Silver Spring, MD, USA - 8 -

17 Total Residual Carrier: combined area of residual calcium carbonate carrier particles within the defect site. Carrier Density: ratio residual calcium carbonate carrier particles to bone within regenerated bone. H. Statistical Analysis Data was collected at tooth level and this was taken into consideration in the analysis. Standard errors of the mean were adjusted for the correlation of the observations within animals. Generalized estimating equations were used to assess the impact of different factors on carrier resorption. Measurements at tooth level were used and estimates were adjusted for the clustering of observations into animals using a robust variance estimator. Wald tests were used for multiple comparisons and the level of significance was set at 5%. A stratified analysis comparing residual carrier and new bone between the experimental groups was carried out using the median (5.1 mm 2 ) of the wound area as cut off point

18 III. Results A. Clinical Observations With the exception for one control site exhibiting gingival inflammation, the surgical sites exhibited healthy gingival conditions (Figure 1). There was no specific clinical characteristic differentiating rhtgf-ß 1 sites from the control. Two animals demonstrated limited exposure of the eptfe device. B. Radiographic Observations The radiographic appearance of the rhtgf-ß 1 and control sites was similar reflecting the particulate nature of the calcium carbonate biomaterial. Radiopacity compatible with the biomaterial was observed in all animals suggesting that significant amounts remained at week 4 postsurgery. C. Histological Observations All defect sites were available for analysis with the exception for one root in a control site that was lost in the histotechnical preparation. Generally, the barrier device was located near the CEJ and the epithelium arrested at the CEJ. Three animals exhibited an inflammatory infiltrate, partially or completely occupying the defect site, localized to the buccal and/or lingual aspect of the mesial and/or distal root of the premolar teeth in sites receiving rhtgf-ß 1. These animals also exhibited sites without an inflammatory infiltrate. Similarly, two control animals exhibited a bilateral

19 inflammatory infiltrate occupying the defect site. Defects in remaining animals did not exhibit an inflammatory infiltrate. Bone regeneration appeared limited to the apical aspect of the defect sites without notable differences between rhtgf-ß 1 and control sites (Figure 2). However, one animal exhibited considerably greater bone formation for both the rhtgf-ß 1 and control site. New cementum formation and regeneration of a functionally oriented periodontal ligament was limited, if at all appreciable, and thus not included in the histometric analysis. Similarly, root resorption appeared limited. Ankylosis was not observed. D. Histometric Analysis The rhtgf-ß 1 and control groups did not differ significantly with regards to defect characteristics (defect height, device height and wound area; Table 1). There were also no statistically significant differences in bone formation (height and area) among the treatments. On the other hand, total residual carrier was significantly smaller in sites that received rhtgf-ß 1 compared to that in the control (p=0.04). Similarly, carrier density was considerably smaller in the rhtgf-ß 1 group; this difference however did not reach statistical significance (p=0.06). This observation may indicate that rhtgf-ß 1 increased the resorption rate of the putty-formulated particulate calcium carbonate carrier, but this effect did not influence bone formation or regeneration of the periodontal attachment

20 Table 1. Comparison between experimental groups (mean ± SE) rhtgf-ß 1 Control p-value Defect height (mm) 4.1 ± ± Device height (mm) 4.1 ± ± Wound area (mm 2 ) 4.8 ± ± Bone height (mm) 2.1 ± ± Bone area (mm 2 ) 3.2 ± ± Residual carrier (mm 2 ) 0.9 ± ± Carrier density (%) 10.9 ± ±

21 A stratified analysis for wound area showed that carrier density and residual carrier area were significantly smaller for the rhtgf-ß 1 group in smaller wound areas (p<0.05) (Figures 3 and 4). No significant differences were observed for these parameters in larger wound areas (p>0.05). No significant differences were observed between experimental groups regarding new bone height and area irrespective of wound area (Figures 5 and 6). In the multivariable model, residual carrier area was significantly smaller for the rhtgf-ß 1 group, and this difference remained significant even after adjusting for wound and bone area (Table 2). On the other hand, no significant differences were observed in carrier density between experimental groups after adjusting for wound and bone area. Despite its positive effect on the resorption rate of the carrier, new bone height and area were not statistically different between groups, after adjusting for wound and carrier area (Table 3)

22 Table 2. Effect of rhtgf-ß 1 on biodegradation after adjustment for wound and bone area. ß ± SE p-value Residual carrier rhtgf-ß ± Wound area 0.24 ± Bone area ± Carrier density rhtgf-ß ± Wound area 1.79 ± Bone area ±

23 Table 3. Effect of rhtgf-ß 1 on bone formation after adjustment for wound and carrier area. ß ± SE p-value Bone height rhtgf-ß ± Wound area 0.27 ± Carrier area 0.05 ± Bone area rhtgf-ß ± Wound area 0.50 ± Carrier area ±

24 IV. Discussion The objective of this study was to evaluate the effect of rhtgf-ß 1 on biodegradation of a putty-formulated particulate calcium carbonate biomaterial used as a carrier for rhtgf-ß 1 in a well-characterized periodontal defect model. As previously observed, the rhtgf-ß 1 construct, alone or combined with GTR, showed no significant effects on periodontal wound healing/regeneration 6,7 potentially indicating the rhtgf-ß 1 formulation being biologically inactive. This study, on the other hand, also evaluating biodegradation of the calcium carbonate biomaterial, indicates that the rhtgf-ß 1 construct was biologically active accelerating degradation of the calcium carbonate biomaterial while not affecting periodontal wound healing/regeneration. Bone regeneration (height and area) was limited without significant differences between the rhtgf-ß 1 and control groups. The reason for this may be attributed to obscure effects of rhtgf-ß 1 combined with GTR or obstruction to bone formation by the calcium carbonate biomaterial obturating the defect site to migration and proliferation of a regenerate from the periodontal ligament. In previous studies, we also observed limited bone formation due to apparent obstruction of the wound space inflicted by biomaterials. 8,14 It is difficult to accept the limited effect of rhtgf-ß 1 on bone formation in the periodontal model; several studies have confirmed an osteoconductive potential of rhtgf-ß 1. 5,15,16 Potentially GTR may compromise osteoconductive properties of rhtgf-ß 1 or the 4-week healing interval may have been too short to reveal discernable effects of rhtgf-ß 1 ; the cause may only be speculated upon. Nevertheless,

25 the amount of residual particulate carrier biomaterial was significantly smaller in the rhtgf-ß 1 group compared to control in smaller wound areas. A stratified analysis dichotomizing the defect sites into smaller and larger wound areas showed that while bone formation (height and area) was not influenced by wound area, carrier density and residual carrier were significantly smaller for sites implanted with rhtgf-ß 1 while no difference was found in larger wound areas. This observation implies that rhtgf-ß 1 increased the degradation rate of the biomaterial. This is in agreement with previous studies where differentiation factors such as bone morphogenetic proteins have been shown to accelerate degradation of the biomaterials used as carrier technologies. 9,10 Additional statistical analysis was performed to determine if any other factors affected the outcome. When the two treatment groups were compared adjusting for wound and bone area, residual carrier area was significantly smaller for the rhtgf-ß 1 group while carrier density was not affected even after adjusting for wound and bone area. New bone height and area were again not affected for both groups even after adjusting for wound and carrier area. Histometric analysis of new cementum formation, regeneration of a functionally oriented periodontal attachment, root resorption, ankylosis was not included. The histological observations revealed limited, if any, new cementum formation. Root resorption and ankylosis were rare. These observations are in synchrony with previous studies evaluating GTR technologies and using a 4-week healing interval

26 Tissue maturation in this defect model apparently commands longer observation intervals that regeneration of the periodontal attachment is clearly distinguishable at least using incandescent and polarized light microscopy. 18 Within the limitations of the study, it may be concluded that rhtgf-ß 1 accelerates biodegradation of the particulate calcium carbonate biomaterial indicating a biologic activity of the rhtgf-ß 1 formulation apparently not encompassing enhanced or accelerated periodontal regeneration. This observation corroborates a previous study where recombinant human bone morphogenetic protein-2 accelerated biodegradation of a biomaterial used as a carrier in the supraalveolar periodontal defect model also without affecting regeneration of the periodontal attachment

27 V.Conclusion In a previous study, recombinant human TGF-ß 1 (rhtgf-ß 1 ) in a putty-formulated particulate calcium carbonate carrier was implanted into critical-size, supraalveolar periodontal defects under conditions for guided tissue regeneration (GTR) to study whether rhtgf-ß 1 * would enhance or accelerate periodontal wound healing/regeneration. 6 Control sites received the calcium carbonate carrier combined with GTR without rhtgf-ß 1. The histometric analysis could not discern significant benefits of the rhtgf-ß 1 formulation. Overall, sites receiving rhtgf-ß 1 and control treatments exhibited limited bone formation and regeneration of the periodontal attachment suggesting marginal, if any, effects of rhtgf-ß 1. Although the results failed to discern a significant benefit of rhtgf-ß 1, an obvious account of the results could not be offered. The present study re-evaluated the previous study utilizing the same parameters to examine if this finding holds true and additional parameters were added to discern any possible effects of rhtgf-ß 1 on biodegradation of the puttyformulated particulate calcium carbonate carrier. The following conclustions were made. 1. Bone regeneration (height and area) was limited without significant differences between the rhtgf-ß 1 and control groups. 2. The amount of residual particulate carrier biomaterial was significantly smaller in the rhtgf-ß 1 group compared to control in smaller wound areas. 3. Histological observations revealed limited, if any, new cementum formation

28 Root resorption and ankylosis were rare findings. 4. It may be concluded that rhtgf-ß 1 accelerates biodegradation of the particulate calcium carbonate biomaterial indicating a biologic activity of the rhtgf-ß 1 formulation apparently not encompassing enhanced or acclerated periodontal regeneration

29 References 1. Roberts AB. TGFß: activity and efficacy in animal models of wound healing. Wound Repair Regen 1995; 3: Hauschka PV, Mavrakos AE, Iafrati MD, Doleman SE, Klagsbrun M. Growth factors in bone matrix: Isolation of multiple types by affinity chromatography on heparin sepharose. J Biol Chem 1986; 261: Centrella M, McCarthy TL, Canalis E. Transforming growth factor ß is a bifunctional regulator of replication and collagen synthesis in osteoblast-enriched cell cultures from fetal rat bone. J Biol Chem 1987; 262: Hughes FJ, Turner W, Belibasakis G, Martuscelli G. Effects of growth factors and cytokines on osteoblast differentiation. Periodontol ; 41: Centrella M, Horowitz MC, Wozney JM, McCarthy TL. Transforming growth factor-beta gene family members and bone. Endocr Rev 1994; 15: Wikesjö UME, Razi SS, Sigurdsson TJ, Tatakis DN, Lee MB, Ongpipattanakul B, Nguyen T, Hardwick R. Periodontal repair in dogs: Effect of recombinant human transforming growth factor-beta 1 on guided tissue regeneration. J Clin Periodontol 1998; 25: Trombelli L, Lee MB, Promsudthi A, Guglielmoni PG, Wikesjö UME. Periodontal repair in dogs: Histologic observations of guided tissue regeneration with a prostaglandin E 1 analog/methacrylate composite. J Clin Periodontol 1999; 26: Sigurdsson TJ, Nygaard L, Tatakis DN, Fu E, Turek TJ, Jin L, Wozney JM, Wikesjö UME. Periodontal repair in dogs: Evaluation of rhbmp-2 carriers. Int J Periodontics Restorative Dent 1996; 16: Wikesjö UME, Sorensen RG, Kinoshita A, Wozney JM. rhbmp-2/α-bsm induces significant vertical alveolar ridge augmentation and dental implant osseointegration. Clin Implant Dent Relat Res 2002; 4:

30 10. Wikesjö UME, Kean CJC, Zimmerman GJ. Periodontal repair in dogs: Supraalveolar defect models for evaluation of safety and efficacy of periodontal reconstructive therapy. J Periodontol 1994; 65: Koo K-T, Polimeni G, Albandar JM, Wikesjö UME. Periodontal repair in dogs: Examiner reproducibility in the supraalveolar periodontal defect model. J Clin Periodontol 2004a; 31: Koo K-T, Polimeni G, Albandar JM, Wikesjö UME. Periodontal repair in dogs: Analysis of histometric assessments in the supraalveolar periodontal defect model. J Periodontol 2004b; 75: Tatakis DN, Wikesjö UME, Razi SS, Sigurdsson TJ, Lee MB, Nguyen T, Ongpipattanakul B, Hardwick R. Periodontal repair in dogs: Effect of transforming growth factor-ß 1 on alveolar bone and cementum regeneration. J Clin Periodontol 2000; 27: Koo K-T, Polimeni G, Qahash M, Kim CK, Wikesjö UME. Periodontal repair in dogs: Guided tissue regeneration enhances bone formation in sites implanted with a coral-derived calcium carbonate biomaterial. J Clin Periodontol 2005; 32: Robey PG, Young MF, Flanders KC, Roche NS, Kondaiah P, Reddi AH, Termine JD, Sporn MB, Roberts AB. Osteoblasts synthesize and respond to transforming growth factor-type beta (TGF-beta) in vitro. J Cell Biol 1987; 105: Janssens K, te Dijke P, Janssens S, van Hul W. Transforming growth factor-beta 1 to the bone. Endocr Rev 2005; 26: Haney JM, Nilvéus RE, McMillan PJ, Wikesjö UME. Periodontal repair in dogs: Expanded polytetrafluoroethylene barrier membranes support wound stabilization and enhance bone regeneration. J Periodontol 1993; 64: Polimeni G, Xiropaidis AV, Wikesjö UME. Biology and principles of periodontal wound healing/regeneration. Periodontol ; 41:

31 Figure Legends Figure 1. Critical-size, supraalveolar periodontal defect implanted with rhtgf-ß 1 in a putty-formulated particulate calcium carbonate carrier before and after application of an eptfe-barrier for GTR and at 4 weeks postsurgery (left). Figure 2. Photomicrographs showing the critical-size, supraalveolar periodontal defects at 4 weeks postsurgery. The left photomicrograph shows a defect site implanted with rhtgf-ß 1 in the calcium carbonate carrier under conditions for GTR and the right photomicrographs shows a control defect without rhtgf-ß 1. The green arrows delineate the base of the approximately 5-mm defects; the eptfe-barriers adapted to the teeth at the CEJ. Figure 3. Carrier density by wound area (* p<0.05). Note significantly smaller carrier density for the rhtgf-ß 1 group in the smaller wound areas. Figure 4. Residual carrier area by wound area (* p<0.05). Note significantly smaller carrier area for the rhtgf-ß 1 group in the smaller wound areas. Figure 5. Bone height by wound area. No significant differences were observed between the two groups regarding new bone height irrespective of the wound areas. Figure 6. Bone area by wound area. No significant differences were observed between the two groups regarding new bone area irrespective of the wound areas

32 Figures Figure 1. Critical-size, supraalveolar periodontal defect implanted with rhtgf-ß 1 in a putty-formulated particulate calcium carbonate carrier before and after application of an eptfe-barrier for GTR and at 4 weeks postsurgery (left)

33 Figure 2. Photomicrographs showing the critical-size, supraalveolar periodontal defects at 4 weeks postsurgery. The left photomicrograph shows a defect site implanted with rhtgf-ß 1 in the calcium carbonate carrier under conditions for GTR and the right photomicrographs shows a control defect without rhtgf-ß 1. The green arrows delineate the base of the approximately 5-mm defects; the eptfe-barriers adapted to the teeth at the CEJ

34 Figure 3. Carrier density by wound area (* p<0.05). Note significantly smaller carrier density for the rhtgf-ß 1 group in the smaller wound areas

35 Figure 4. Residual carrier area by wound area (* p<0.05). Note significantly smaller carrier area for the rhtgf-ß 1 group in the smaller wound areas

36 Figure 5. Bone height by wound area. No significant differences were observed between the two groups regarding new bone height irrespective of the wound areas

37 Figure 6. Bone area by wound area. No significant differences were observed between the two groups regarding new bone area irrespective of the wound areas

38 국문요약 변환성장유도단백질인 Tranforming growth factor-ß 1 은다양한종류의세포들의성장, 분화, 및세포기질의생성에관여한다고알려져왔다. 또, 골모세포들의초기생성에도 Tranforming growth factor-ß 1 단백질이활발하게관여한다고보고된바있어 TGF- ß 1 을사용한치주조직치유나재생실험들이많이진행되고있다. 예전의실험에서, Tranforming growth factor- ß 1 을 calcium carbonate 운반체를이용하여상치조결손부에적용한후차폐막으로덮고골형성에어떤영향을미치는지실험하였다. 대조군 (control group) 에서는 TGF-ß 1 을적용하지않은채 Biocoral 운반체와차폐막만사용하였다. 결과를살펴보면두실험군간에새롭게생성된골의양이나수직적인높이에있어서유의성있는차이가없었다. 이는 TGF- ß 1 이본상치조결손부에서치주조직재생에부가적인영향이없는것으로해석될수있으나몇가지의문점을남기게되었다. 본연구에서는이러한의문점중 TGF-ß 1 이운반체로서사용된 calcium carbonate (Biocoral) 이라는재료에미치는영향에대하여조사하기위해새로운조직계측학적기준들을첨가하여실험을재실시하였다. 새롭게생성된골의양 (area) 과수직적인높이 (height) 는두실험군간에유의성있는차이가없었고, 새로생성된백악질은극히제한적이었다. 잔존하는운반체 (calcium carbonate) 의양을비교해보았을때, rhtfg-ß 1 군에서운반체의양이현저히감소됨을관찰할수있었다. 결론적으로 rhtfg-ß 1 이 calcium carbonate 운반체를일련의생물학적인반응을통하여흡수시킬수있다는사실이입증되었고, 이를더넓은의미로해석한다면치주조직재생에사용되는여러성장인자들이운반체로사용되는생물학적물질들에영향을미칠수있다는결론을내릴수있겠다. 핵심되는말변환성장유도단백질 (Transforming growth factor Beta-1); 운반체 (calcium carbonate carrier); 생물학적흡수 (biodegradation)