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4 감사의글 본논문이완성되기까지부족한저를항상격려해주시고지도하여 주신최성호교수님께존경과깊은감사를드립니다. 또한본연구에많은도움을주었던이중석선생님을비롯한모든 치주과교실원여러분께무한한고마움을전합니다. 오늘이있기까지변함없는믿음과사랑으로이해해주시며 물심양면으로후원해주신부모님과가족들께진심으로감사의마음을 전합니다 년 6 월 저자씀 iv

5 Table of Contents Abstract (English) iii I. Introduction 1 II. Materials and Methods 5 1. Animals 5 2. Materials 5 3. Experimental design 6 4. Surgical protocol 7 5. Histologic processing 8 6. Histologic and histometric analysis 9 III. Results Clinical observations Histologic observation Histometric analysis 11 IV. Discussion 13 V. Conclusion 18 Conflict of Interest 19 References 20 Legends 26 Table 29 Figures 30 Abstract (Korean) 34 i

6 List of Figures Figure 1. A, Sugrically created, standardized dehiscence defects at the buccal side of the installed implants. B, Each defects was treated with graft materials and barrier membranes, or left untreated (control) 30 Figure 2. Illustration of the A, experimental defect and B, parameters for histometric analysis 30 Figure 3. Representative photomicrographs from A, control, B, biphasic calcium phosphate, and C, cyanoacrylate-combined calcium phosphate sites. 31 Figure 4. Representative photomicrographs from the A, barrier membrane only, B, biphasic calcium phosphate plus barrier membrane, and C, cyanoacrylate-combined calcium phosphate plus barrier membrane. 31 Figure 5. Higher-magnification photomicrograph of the outlined areas in Fig 4. B, D, polarized views of A,C respectively 32 Figure 6. Higher-magnification photomicrograph of the 2 top outlined areas in Fig 4B. 33 List of Table Table 1. Results of histometric analysis 29 ii

7 Abstract Guided Bone Regeneration Using Cyanoacrylate-Combined Calcium Phosphate in a Dehiscence Defect: A Histologic Study in Dogs Seung-Hee Ko, D.D.S., M.S.D. Department of Dental Science Graduate School, Yonsei University (Directed by Prof. Seong-Ho Choi, D.D.S., M.S.D., PhD.) Purpose: This study evaluated the effects of cyanoacrylate-combined calcium phosphate (CCP) as a candidate for a barrier membrane substitute in guided bone regeneration and the space maintenance capability of CCP placed in a dehiscence defect model. Materials and Methods: Six standardized dehiscence defects (5 3 mm; height width) around the dental implants were created on unilateral edentulous ridges in 5 dogs, where each defect was treated with sham surgery, biphasic calcium phosphate (BCP), CCP, barrier membrane (MEM), iii

8 BCP+MEM, and CCP+MEM. The animals were sacrificed after an 8-week healing interval for histologic and histometric analyses. Results: BCP and CCP sites showed increased bone formation compared to the control sites, although incomplete defect resolution occurred; bone regeneration heights (area) averaged 3.52±0.69mm (4.94±2.59mm 2 ), 3.51±0.16mm (4.10±1.99mm 2 ), and 1.53 ± 0.42mm (1.01±0.74mm 2 ) for BCP, CCP, and control sites, respectively. All the MEM sites showed more bone formation compared with the sites that received the same biomaterials without a MEM, and BCP+MEM and CCP+MEM sites showed extensive bone formation within the defect and on the top of the implant; bone regeneration heights (area) averaged 3.96±2.86mm (12.46±11.61mm 2 ), 5.45±0.25mm (11.63±1.97mm 2 ), and 2.62 ± 0.27mm (3.43±0.98mm 2 ) for BCP+MEM, CCP+MEM, and MEM sites, respectively. Conclusions: CCP can be a good scaffold for supporting a MEM as opposed to acting as a substitued for the MEM in guided bone regeneration. Key Words: cyanoacrylate, calcium phosphate, guided bone regeneration, dental implant, dehiscence defect iv

9 Guided Bone Regeneration Using Cyanoacrylate-Combined Calcium Phosphate in a Dehiscence Defect: A Histologic Study in Dogs Seung-Hee Ko, D.D.S., M.S.D. Department of Dental Science Graduate School, Yonsei University (Directed by Prof. Seong-Ho Choi, D.D.S., M.S.D., PhD.) Ⅰ. Introduction In clinical dentistry, various types of bony defect can occur either around or beside a dental implant in an edentulous alveolar ridge. In cases where a tooth has been extracted because of severe periodontal destruction owing to periodontitis or endodontic problems, the alveolar bone would be resorbed horizontally or vertically. These situations may result in exposure of an implant surface and can hamper the ideal placement of a dental implant in extreme cases (Esposito M et al., 2009; Aghaloo TL et al., 2007). Therefore, 1

10 such ridge defects should be carefully evaluated and classified and, if indicated, corrected. Seibert classified ridge defects into three categories: horizontal, vertical, and combined (Seibert JS., 1983). There have been numerous treatment proposals regarding these 3 classification. Implant placement at a horizontally resorbed ridge results in a dehiscence defect, most common type of defect, especially in the anterior area (Buser D et al., 1996; Hammerle CH et al., 1998; Jovanovic SA et al., 1992). This has been further tested in cases without periodontal destruction at the time of tooth extraction, where dimensional changes can result in a dehiscence defect around the dental implants (Araujo MG et al., 2006; Nevins M et al., 2006). However, many clinical and experimental studies have found successful dehiscence-defect resolution using guided bone regeneration (GBR) around the dental implant ( Hammerle CH et al., 1998; Jensen SS et al., 2009; Chiapasco M et al., 2009). GBR is a therapeutic technique that uses a barrier membrane to provide a protected space into which neighboring bone tissues can grow (Schenk RK et al., 1994). Occlusiveness had been regarded as the most important requirement of the barrier membrane for GBR (Karring T et al., 1993; Nyman S et al., 1987; Dahlin C et al., 1989). However, previous studies have found that bone can regenerate within the space protected by a non-occlusive membrane (Lundgren 2

11 A et al., 1998; Proussaefs et al., 2003; Wikesjo UM et al., 2003). Wikesjö et al. also reported comparable results in groups using occlusive and non-occlusive membranes and concluded that blood-clot stability might be a critical determinant of successful tissue regeneration (Wikesjo UM et al., 2003; Wikesjo UM et al., 1991). Barrier membranes used in the GBR technique not only occlude the space from other tissues but also provide stability for grafted biomaterials and blood clots. However, the barrier membranes are expensive and difficult to handle. In addition to the disadvantages mentioned earlier, a recent study has found a high rate of complications associated with barrier membranes, including the exposure of the membrane, during the postoperative period (Chiaspasco M et al., 2009). Overall, these drawbacks have created an interest in finding alternative methods for the GBR technique in bony defects. Many recent studies have focused on developing a substitute for the membrane in order to decrease the associated complications and the healing time (Thoma DS, Dard MM et al., 2011; Thoma DS, Subramani K et al., 2011; Jung RE et al., 2011; Park KJ et al., 2005). One candidate for such a substitute is cyanoacrylate glue. Cyanoacrylate is a tissue-adhesive material that promotes the rapid adhesion of soft and hard tissues (Ahn DK et al., 1997; Singer AJ et al 2004). Although inflammatory reactions occurred from tissue 3

12 toxicity in the early periods of its development, it was found that increasing the number of lateral chains in its molecular structure could decrease the associated toxicity (Pelissier P et al., 2001). This ultimately led to N-butyl-2- cyanoacrylate and octyl-cyanoacrylate being used for clinical use with minimal complications (Singer AJ et al., 2004; Inal S et al., 2006). Other studies have described the application of cyanoacrylate in block bone grafts and GBR procedures (Park KJ et al 2005; Bhumbra RS et al., 1998). Cyanoacrylate-combined calcium phosphate (CCP) is one of these substitutes; cyanoacrylate-based grafts can bind to and stabilize the biomaterials within the defect ( Park KJ et al., 2005; Chang YY et al., 2011). The purpose of this study was to evaluate the effects of CCP as a candidate barrier-membrane substitute in GBR. The investigators hypothesized that cyanoacryalte-based techniques could simplify the manipulation of the graft particles and to provide the space required for bone formation. The specific aims of the study were: 1) to compare the effects of CCP with or without barrier membrane in GBR, and 2) to characterize the space maintenance capability of CCP placed in a dehiscence defect. 4

13 Ⅱ. Materials and Methods 1. ANIMALS Five male mongrel dogs, approximately 12 to 15 months old and weighing approximately 30 kg, were chosen for this experiment. The animal selection, management, preparation, and the surgical protocols were performed according to routine procedures approved by the Institutional Animal Care and Use Committee, Yonsei Medical Center, Seoul, Korea (09-067). The animals had ad libitum access to water and a pelleted laboratory diet, with the exception of 2 weeks immediately after surgery, when they were fed a diet of canned soft dog food (Prescription Diet Canine i/d, Hill s Pet Nutrition, Topeka, KS, USA). 2. MATERIALS Dental Implants. Implants with dimensions of mm (diameter length) and whose surfaces had been sand-blasted with large-grit and etched with acid (Implantium, Dentium, Seoul, Korea) were used in this study. 5

14 Cyanoacrylate-Combined Calcium Phosphate. CCP (Ceragem Biosys, Seoul, Korea) was prepared by mixing two pastes, one containing monocalcium phosphate 0.23 g (particle size, 50 to 100 μm), dicalcium phosphate 0.3 g (particle size, 10 to 20 μm), and 2-octyl-cyanoacrylate 0.1 g, and the other containing β-tricalcium phosphate (β-tcp) 0.22g (particle size, 10 to 50 μm) and glycerin 0.5g. Biphasic Calcium Phosphate (BCP). The biphasic calcium phosphate (BCP) used in this study was a commercially provided biomaterial by GenOss (Osteon; Dentium, Suwon, Korea), which had a pore size of 300 to 500 μm. Hydroxyapatite (HA) was coated with β-tcp at an HA:β-TCP ratio of 70:30. Barrier membrane. A nonabsorbable, expanded polytetrafluoroethylene membrane (Gore-Tex Regenerative membrane, W L Gore and Associates, Flagstaff, AZ) was used to cover some of the defects in this study. 3. EXPERIMENTAL DESIGN Experimental defects were divided into 6 groups according to the materials applied to the dehiscence defect around the implant: defects filled with BCP (BCP group) or CCP (CCP group), defects covered with a barrier membrane 6

15 alone (MEM group) or with BCP (BCP+MEM group) or CCP (CCP+MEM group), and a negative control group, in which the defect was left without any graft material (ie, sham operated). 4. SURGICAL PROTOCOL All surgical procedures, including tooth extraction and subsequent experiments, were performed under general anesthesia. All animals were anesthetized using an intravenous injection of atropine (0.04 mg/kg; Kwangmyung Pharmaceutical, Seoul, Korea) and an intramuscular injection of a combination of xylazine (Rompun, Bayer Korea, Seoul, Korea) and ketamine (Ketara, Yuhan, Seoul, Korea). General anesthesia was maintained with inhalation anesthesia (Gerolan, Choongwae Pharmaceutical, Seoul, Korea). The mandibular premolars and first molar were extracted bilaterally. After an 8-week healing period, animals were prepared for the experiments. A crestal incision was made and a full-thickness flap was elevated. The edentulous ridges used were flattened using a ridge-contouring bur with a rotary engine, and 6 unilateral sites were identified for the placement of dental implants. Contralateral sites received other treatments will be reported elsewhere. Surgical preparations for implant installation were performed 7

16 according to the manufacturer s instructions. After implant preparation, dehiscence defects of a standardized size (5 3 mm, height width) were made in the buccal side of all implants (Fig 1, 2). A dental implant was placed at each of the 6 sites, and each of the associated defects was immediately treated with 1 graft materials (i.e, BCP or CCP), with a barrier membrane alone (MEM), with a barrier membrane together with either BCP (BCP+MEM) or CCP (CCP+MEM), or left untreated (control), as outlined earlier (Fig 1). The flaps were repositioned and sutured with resorbable suture materials (Monosyn 4/0, B. Braun Aesculap Academy, Tuttlingen, Germany). The sutures were removed after 7 days. The animals were sacrificed by an overdose of ketamine at 8 weeks after surgery. 5. HISTOLOGIC PROCESSING Block sections, including the segments with implants, were preserved and fixed in 10% neutral-buffered formalin. The specimens were dehydrated in ethanol, embedded in methacrylate, and sectioned in the buccolingual plane using a diamond saw. The central section from each implant site was reduced to a final thickness of 50 μm by microgrinding and polishing with a cuttingand-grinding device (Exakt, Apparatebau, Norderstedt, Germany). The sections were stained with Goldner s trichrome stain. 8

17 6. HISTOLOGIC AND HISTOMETRIC ANALYSIS One experienced examiner blind to the experimental conditions of each of the defect samples performed the histologic and histometric analysis using incandescent and polarized-light microscopy (BX51 Microscope, Olympus Research System, Tokyo, Japan) and a personal computer-based imageanalysis system (Image-Pro Plus, Media Cybernetics, Silver Spring, MD). The following measurements were made within the defect area at a magnification of 40 (Fig 2): Bone regeneration height: distance from the base of the defect (center of the length of implant) to the most coronal extension of the newly formed bone along the implant surface. Bone regeneration area: extent of newly formed bone within the defect area. Bone-to-implant contact: proportion of the newly formed bone in contact with the implant surface in the defect site (the most coronal 5-mm area of the implant). 9

18 Ⅲ. Results 1. CLINICAL OBSERVATIONS All experimental and control sites healed uneventfully, with the exception of 2 sites in which wound dehiscence occurred and the barrier membranes were exposed. These sites were excluded from the study analysis. 2. HISTOLOGIC OBSERVATIONS Newly formed bone was observed within all defect sites, but to various extents depending upon the experimental condition (Fig 3, 4). Sites that received sham surgery only (control group) exhibited slight formation of new bone originating from the base of the defect; this bone was in a mature state (Fig 3A). In the BCP- and CCP-treated sites, extensive bone formation was observed in an immature woven bone state along the implant surface. BCP sites exhibited new bone formation around the residual biomaterials at the lower area of the defect. However, residual biomaterials in the upper area of the defect were encapsulated by connective tissue without bone formation (Fig 3B). The volume of newly formed bone was lower at CCP sites than at BCP sites, with no residual biomaterials (Fig 3C). 10

19 All membrane-applied sites showed more bone formation compared with the sites receiving the same biomaterials without a barrier membrane (Fig 3, 4). Newly formed, mature bone with a marrow space was found in sites where only the membrane was applied (MEM group); however, in these cases the exposed implant surface in the defect was partially covered with the newly formed bone. In sites treated with BCP+MEM or CCP+MEM, there was an extensive bone formation of bone within the defect and on the top of the implant. The BCP+MEM sites exhibited the latgest amount of newly formed bone, with residual biomaterials located all over the defect. However, this bone was in an immature woven bone state and could be distinguished from the native bone under the defect (Fig 4B, 5A, B). In some areas, the residual biomaterials were encapsulated by loose connective tissue, which encapsulated the scattered debris from the biomaterials (Fig 6), whereas the outer surface of this area was filled with newly formed bone. In contrast, lamellar structures of newly formed bone and no residual biomaterials were observed in CCP+MEM specimens (Fig 4 C). 3. HISTOMETRIC ANALYSIS The results of histometric analysis are presinted in Table 1. The regenerated bone height was 1.53 ± 0.42 (mean ± standard deviation), 3.52 ± 0.69, 3.51 ± 11

20 0.16, 2.62 ± 0.27, 3.96 ± 2.86, and 5.45 ± 0.25 mm in the control, BCP, CCP, MEM, BCP+MEM, and CCP+MEM groups, respectively. The corresponding values for bone regeneration area were 1.01 ± 0.74, 4.94 ± 2.59, 4.10 ± 1.99, 3.43 ± 0.98, ± 11.61, and ± 1.97 mm 2, whereas those for bone to implant contact were 27.54% ± 13.58%, 35.30% ± 5.75%, 49.21% ± 11.74%, 21.31% ± 21.69%, 47.77% ± 29.55%, and 80.66% ± 12.37%, respectively. 12

21 Ⅳ. Discussion The purpose of this study was to determine the effects of CCP as a suitable candidate substitute for barrier membranes in GBR and to characterize the space maintenance capability of CCP placed in a dehiscence defect. The investigators hypothesized that cyanoacrylate-based techniques could simplify the manipulation of the graft particles and provide the space required for bone formation. The BCP and CCP sites showed increased bone formation compared with the control sites, although incomplete defect resolution was observed. However, all membrane-applied sites showed more bone formation compared with the sites receiving the same biomaterials without a barrier membrane. Cyanoacrylate can be expected to exert a stabilizing effect on graft biomaterials and blood clots because of its adhesive properties as a tissue-glue (de Oliveira Neto PJ et al., 2010; Saska S et al., 2009). A previous study applied fibrin sealant as a tissue adhesive for GBR instead of employing membrane protection. However, Carmagnola et al. described the separation of graft particles from the native tissue and the implant surface by a well-defined connective tissue capsule at sites receiving bone substitutes with fibrin sealant 13

22 and concluded that the fibrin sealant may jeopardize bone regeneration. However, results from previous studies do not concur with the present study findings such that newly formed bone and the bone-to-implant contact increased at sites receiving biomaterials combined with cyanoacrylate (CCP and CCP + MEM groups). These opposing results may be attributable to 2 major differences between these studies. Carmagnola et al used a horizontal defect of the entire edentulous ridge, whereas in the present study the investigators used the dehiscence defect model that limited the site to a single implant ( Carmagnola D et al., 2000; Carmagnola D et al. 2002). Furthermore, the model in the present study had lateral walls beside the defect (Fig 2), which could provide stability for the CCP and be a source of regeneration. Although the instantly created defect model could be different from a clinical situation, this defect model was used in this study to compare the treatments in a standardized model encompassing the same dimensions. Moreover, HA, exhibiting a slow biodegradation rate, was used as the bone substitute in the previous study, whereas various calcium phosphates with fast biodegradation rates were used in the present study (Mordenfeld A et al., 2010). The calcium phosphates would allow the newly formed bone to replace the space for the calcium phosphate during the early healing period ( Koo KT et al., 2007). The 6 treatment conditions in this study can be divided into 2 categories 14

23 according to the use of a barrier membrane: control, BCP, and CCP versus MEM, BCP + MEM, and CCP + MEM. All sites that received a barrier membrane exhibited increased bone regeneration compared with those that received the same biomaterial only without a barrier membrane. In particular, the BCP + MEM and CCP + MEM sites displayed newly formed bone, which exceeded the size of the defect site and grew over the top of the implant (Fig 4). However, 2 of 5 animals exhibited wound dehiscence and membrane exposure, which can result in extensive loss of soft tissues and the failure of regeneration (Kim YK et al., 2009). The rates of these complications exceeded those reported previously in humans, where 20% of cases exhibited membrane exposure at sites that received a nonresorbable membrane (Chiapasco M et al., 2009). Moreover, because multiple teeth were extracted from individual animals in the present study, the bite forces that were exerted during food intake could have caused the relatively increased rate of wound dehiscence compared with the previous studies. However, no complications occurred at sites that received BCP or CCP only, but 60% of the defect height was resolved with newly formed bone, where the remaining exposed surface of the implant was in direct contact with the connective tissue (Fig 3). Although more bone formed at CCP sites than at control sites, this was considerably less than at sites that received CCP and 15

24 membrane protection. This showed that the cyanoacrylate-induced stability of the graft biomaterials was not as effective as expected in cases with membrane protection. However, the increase of linear new bone formation in sites that received CCP may be caused by the increased adhesiveness to the implant surface and the increased stability of graft biomaterials afforded by cyanoacrylate. One of the critical factors for successful GBR is space maintenance (Bartold PM et al., 2000). However, within the sites that received the membrane only (MEM group) in the present study, the membrane collapsed and the space was compressed against the implant surface (Fig 4A). Therefore, bone substitutes were used in conjunction with a barrier membrane in particular situations to provide space during the healing period. In this study, BCP was used as a positive control for evaluating the space-maintaining capability of CCP. To clarify, BCP is a combination of HA and β-tcp and has been widely applied in clinical and research fields because of its capacity for osteoconductivity and osteoinductivity (Jang JW et al., 2011; Lee JH et al., 2008). The results of the study showed that the residual biomaterials occupied a large portion of newly formed bone area in the BCP sites, and all CCP sites showed no residual biomaterials. This could be due to the HA composition of BCP and the difference of particle size between BCP and CCP. CCP was completely 16

25 resorbed within the defect, whereas some BCP particles were encapsulated by the connective tissue, and small fragments of BCP were scattered within this area (Figs 4B, 6). However, newly formed bone increased similarly in height and area at the sites receiving BCP and CCP. Although all CCP particles were resorbed at 8 weeks after the graft procedure, increased bone formation could occur within the space maintained by biomaterials at the early healing phase. The sites that received CCP with the protection of a barrier membrane (CCP + MEM), the augmented and newly formed bone areas, were comparable to the BCP + MEM sites (Fig 4). However, the CCP + MEM sites showed remodeled mature bone in the augmented area, whereas the BCP + MEM sites showed the bony resorption and formation process around the residual biomaterials (Fig 5). The disintegration of BCP particles can result in a temporary high concentration of calcium ions in the adjacent area, and this can exert short-term adverse effects on the cellular events in some localized areas during the regeneration process (Fig 6) (Orrenius S et al., 1992). Therefore, further long-term studies are needed to compare the space-maintaining capabilities of BCP and CCP because the BCP sites appeared to be actively remodeling at 8 weeks after surgery. 17

26 Ⅴ. Conclusion Within the limitations of this study, bone graft alone with CCP and BCP could increase new bone formation within the small dehiscence defect, which was limited to single implant. However, the CCP sites showed less new bone formation compared with the membrane-protected sites. Therefore, CCP could be used as a scaffold within the membrane-protected space as opposed to acting as a substitute for the barrier membrane during a GBR procedure. 18

27 Conflict of Interest The authors declare no conflicts of interest. 19

28 References Aghaloo TL, Moy PK: Which hard tissue augmentation techniques are the most successful in furnishing bony support for implant placement? Int J Oral Maxillofac Implants 22 special supplement:49, 2007 Ahn DK, Sims CD, Randolph MA, et al: Craniofacial skeletal fixation using biodegradable plates and cyanoacrylate glue. Plast Reconstr Surg 99:1508, 1997 Araujo MG, Sukekava F, Wennstrom JL, et al: Tissue modeling following implant placement in fresh extraction sockets. Clin Oral Implants Res 17:615, 2006 Bartold PM, McCulloch CA, Narayanan AS, et al: Tissue engineering: a new paradigm for periodontal regeneration based on molecular and cell biology. Periodontol :253, 2000 Bhumbra RS, Berman AB, Walker PS, et al: Enhanced bone regeneration and formation around implants using guided bone regeneration. J Biomed Mater Res 43:162, 1998 Buser D, Dula K, Hirt HP, et al: Lateral ridge augmentation using autografts and barrier membranes: a clinical study with 40 partially edentulous patients. J Oral Maxillofac Surg 54:420, 1996 Carmagnola D, Berglundh T, Araujo M, et al: Bone healing around implants placed in a jaw defect augmented with Bio-Oss. An experimental study in dogs. J Clin Periodontol 27:799,

29 Carmagnola D, Berglundh T, Lindhe J: The effect of a fibrin glue on the integration of Bio-Oss with bone tissue. A experimental study in labrador dogs. J Clin Periodontol 29:377, 2002 Chang YY, Dissanayake S, Yun JH, et al: The biological effect of cyanoacrylatecombined calcium phosphate in rabbit calvarial defects. J Periodontal Implant Sci 41:123, 2011 Chiapasco M, Zaniboni M: Clinical outcomes of GBR procedures to correct periimplant dehiscences and fenestrations: a systematic review. Clin Oral Implants Res 20 Suppl 4:113, 2009 Dahlin C, Sennerby L, Lekholm U, et al: Generation of new bone around titanium implants using a membrane technique: an experimental study in rabbits. Int J Oral Maxillofac Implants 4:19, 1989 de Oliveira Neto PJ, Cricchio G, Hawthorne AC, et al: Tomographic, Histological, and Immunohistochemical Evidences on the Use of N-Butyl-2-Cyanoacrilate for Onlay Graft Fixation in Rabbits. Clin Implant Dent Relat Res DOI: /j x, 2010 Esposito M, Grusovin MG, Felice P, et al: Interventions for replacing missing teeth: horizontal and vertical bone augmentation techniques for dental implant treatment. Cochrane Database Syst Rev:CD003607, 2009 Hammerle CH, Chiantella GC, Karring T, et al: The effect of a deproteinized bovine bone mineral on bone regeneration around titanium dental implants. Clin Oral Implants Res 9:151,

30 Inal S, Yilmaz N, Nisbet C, et al: Biochemical and histopathological findings of N- butyl-2-cyanoacrylate in oral surgery: an experimental study. Oral Surg Oral Med Oral Pathol Oral Radiol Endod 102:e14, 2006 Jang JW, Yun JH, Lee KI, et al: Osteoinductive activity of biphasic calcium phosphate with different rhbmp-2 doses in rats. Oral Surg Oral Med Oral Pathol Oral Radiol Endod DOI: /j.tripleo , 2011 Jensen SS, Terheyden H: Bone augmentation procedures in localized defects in the alveolar ridge: clinical results with different bone grafts and bone-substitute materials. Int J Oral Maxillofac Implants 24 Suppl:218, 2009 Jovanovic SA, Spiekermann H, Richter EJ: Bone regeneration around titanium dental implants in dehisced defect sites: a clinical study. Int J Oral Maxillofac Implants 7:233, 1992 Jung RE, Kokovic V, Jurisic M, et al: Guided bone regeneration with a synthetic biodegradable membrane: a comparative study in dogs. Clin Oral Implants Res 22:802, 2011 Karring T, Nyman S, Gottlow J, et al: Development of the biological concept of guided tissue regeneration--animal and human studies. Periodontol :26, 1993 Kim YK, Yun PY, Kim SG, et al: In vitro scanning electron microscopic comparison of inner surface of exposed and unexposed nonresorbable membranes. Oral Surg Oral Med Oral Pathol Oral Radiol Endod 107:e5,

31 Koo KT, Susin C, Wikesjo UM, et al: Transforming growth factor-beta1 accelerates resorption of a calcium carbonate biomaterial in periodontal defects. J Periodontol 78:723, 2007 Lee JH, Jung UW, Kim CS, et al: Histologic and clinical evaluation for maxillary sinus augmentation using macroporous biphasic calcium phosphate in human. Clin Oral Implants Res 19:767, 2008 Lundgren A, Lundgren D, Taylor A: Influence of barrier occlusiveness on guided bone augmentation. An experimental study in the rat. Clin Oral Implants Res 9:251, 1998 Mordenfeld A, Hallman M, Johansson CB, et al: Histological and histomorphometrical analyses of biopsies harvested 11 years after maxillary sinus floor augmentation with deproteinized bovine and autogenous bone. Clin Oral Implants Res 21:961, 2010 Nevins M, Camelo M, De Paoli S, et al: A study of the fate of the buccal wall of extraction sockets of teeth with prominent roots. Int J Periodontics Restorative Dent 26:19, 2006 Nyman S, Gottlow J, Lindhe J, et al: New attachment formation by guided tissue regeneration. J Periodontal Res 22:252, 1987 Orrenius S, Burkitt MJ, Kass GE, et al: Calcium ions and oxidative cell injury. Ann Neurol 32 Suppl:S33, 1992 Park KJ, Park JH, Lee SB, et al: Bioactive Cyanoacrylate-Based Filling Material for Bone Defects in Dental Applications. Key Eng Mater :933,

32 Pelissier P, Casoli V, Le Bail B, et al: Internal use of n-butyl 2-cyanoacrylate (Indermil) for wound closure: an experimental study. Plast Reconstr Surg 108:1661, 2001 Proussaefs P, Lozada J, Kleinman A, et al: The use of titanium mesh in conjunction with autogenous bone graft and inorganic bovine bone mineral (bio-oss) for localized alveolar ridge augmentation: a human study. Int J Periodontics Restorative Dent 23:185, 2003 Saska S, Hochuli-Vieira E, Minarelli-Gaspar AM, et al: Fixation of autogenous bone grafts with ethyl-cyanoacrylate glue or titanium screws in the calvaria of rabbits. Int J Oral Maxillofac Surg 38:180, 2009 Schenk RK, Buser D, Hardwick WR, et al: Healing pattern of bone regeneration in membrane-protected defects: a histologic study in the canine mandible. Int J Oral Maxillofac Implants 9:13, 1994 Seibert JS: Reconstruction of deformed, partially edentulous ridges, using full thickness onlay grafts. Part I. Technique and wound healing. Compend Contin Educ Dent 4:437, 1983 Singer AJ, Thode HC, Jr.: A review of the literature on octylcyanoacrylate tissue adhesive. Am J Surg 187:238, 2004 Thoma DS, Dard MM, Halg GA, et al: Evaluation of a biodegradable synthetic hydrogel used as a guided bone regeneration membrane: an experimental study in dogs. Clin Oral Implants Res DOI: /j x,

33 Thoma DS, Subramani K, Weber FE, et al: Biodegradation, soft and hard tissue integration of various polyethylene glycol hydrogels: a histomorphometric study in rabbits. Clin Oral Implants Res 22:1247, 2011 Wikesjo UM, Claffey N, Egelberg J: Periodontal repair in dogs. Effect of heparin treatment of the root surface. J Clin Periodontol 18:60, 1991 Wikesjo UM, Lim WH, Thomson RC, et al: Periodontal repair in dogs: gingival tissue occlusion, a critical requirement for GTR? J Clin Periodontol 30:655,

34 Legends FIGURE 1. A, Surgically created, standardized dehiscence defects at the buccal side of the installed implants. B, Each defect was treated with one or the other of the graft materials (ie, biphasic calcium phosphate or cyanoacrylate-combined calcium phosphate), with a barrier membrane alone, with a barrier membrane plus biphasic calcium phosphate or cyanoacrylatecombined calcium phosphate, or left untreated (control). FIGURE 2. Illustrations of the A, experimental defect and B, parameters for histometric analysis. Bone regeneration height (BH) is the distance from the base of the defect to the most coronal extension of the newly formed bone along the implant surface. The bone regeneration area (dark yellow) is depicted. FIGURE 3. Representative photomicrographs (original magnification 40; Goldner s trichrome stain) from A, control, B, biphasic calcium phosphate, and C, cyanoacrylate-combined calcium phosphate sites. A, The control site shows slight but mature bone formation in the defect area, whereas extensive newly formed bone in an immature state is observed in the B, bihasic calcium 26

35 phosphate and C, cyanoacrylate-combined calcium phosphate sites B, Especially in biphasic calcium phosphate site, the surgically created defect line (arrowhead) is distinguished between the mature native bone and the newly formed woven bone, and residual biomaterials (stars) are observed all around the defect area. Residual biomaterials in upper area of the defect are capsulated with connective tissues. FIGURE 4. Representative photomicrographs (original magnification 40; Goldner s trichrome stain) from A, barrier membrane only, B, biphasic calcium phosphate plus barrier membrane, and C, cyanoacrylate-combined calcium phosphate plus barrier membrane sites. A, The barrier membrane site shows partial defect resolution with mature newly formed bone. In contrast, the B, biphasic calcium phosphate plus barrier membrane and C, cyanoacrylate-combined calcium phosphate plus barrier membrane sites show the extensive bone formation within the defect and over the top of the implant. C, The cyanoacrylate-combined calcium phosphate plus barrier membrane site shows homogeneous bone formation without residual biomaterials, B, whereas residual biomaterials (stars) appear interspersed in the newly formed woven bone at the biphasic calcium phosphate plus barrier membrane site. 27

36 FIGURE 5. Higher-magnification photomicrographs (original magnification, x100) of the outlined areas in Fig 4. B, D, Polarized views of A, C, respectively. A, B, Biphasic calcium phosphate plus barrier membrane site shows a clearly distinguishable defect line (arrowheads) between the newly formed and the native bone, and B, lamellar structures are observed only in the native bone area in a polarized view. A, Some areas show no bone formation around the residual biomaterials (stars). D, The polarized view of the cyanoacrylate-combined calcium phosphate plus barrier membrane site shows lamellar structures in the native and newly formed bone areas. C, The base of the dehiscence defect is displayed (arrow) FIGURE 6. Higher-magnification photomicrographs (original magnification, x200) of the 2 top outlined areas in Fig 4B. A, Newly formed bone is observed contacting the residual biomaterials all around the defect area. B, In some areas, the residual biomaterials are encapsulated by loose connective tissue, within which debris from the biomaterials is scattered extensively 28

37 Table Table 1. RESULTS OF HISTOMETRIC ANALYSIS Control BCP CCP MEM BCP+MEM CCP+MEM BH (mm) 1.53 ± ± ± ± ± ± 0.25 BA (mm 2 ) 1.01 ± ± ± ± ± ± 1.97 BIC (%) ± ± ± ± ± ± Abbreviations: BA, regenerated bone area; BCP, biphasic calcium phosphate; BH, regenerated bone height; BIC, proportion of bone-to-implant contact; CCP, cyanoacrylate-combined calcium phosphate; MEM, barrier membrane. 29

38 Figures Figure 1 Figure 2 30

39 Figure 3 Figure 4 31

40 Figure 5 32

41 Figure 6 33

42 국문요약 임플란트주위열개형결손부에서시아노아크릴레이트 - 인산칼슘 골이식재를이용한골유도재생술에대한조직학적 / 조직계측학적연구 연세대학교대학원치의학과 ( 지도최성호교수 ) 고승희 이연구의목적은시아노아크릴레이트가결합된인산칼슘 (CCP) 을 성견의열개형결손부에적용했을때골재생에있어공간유지능력과차단막 대체제로서의효과을알아보기위함이다. 5 마리의성견에서편측으로표준화된여섯개의열개형결손부 (5 X 3 mm, 높이 X 너비 ) 를형성한후, 아무것도처치하지않은대조군을포함하여각각의결손부에인산칼슘 (BCP), CCP, 차단막 (MEM), BCP+MEM, 그리고 CCP+MEM 을처치하였다. 8 주간의치유기간후에실험동물을희생시켜조직학적그리고조직계측학적연구를시행하였다. 34

43 차단막을사용하지않은경우에, 전반적으로불완전재생이관찰되었지만 BCP 와 CCP 부위에서대조군보다증가된골재생을보여주었다. BCP, CCP 그리고대조군에서의평균적인골재생높이 ( 면적 ) 는각각 3.52±0.69mm (4.94±2.59mm 2 ), 3.51±0.16mm (4.10±1.99mm 2 ), and 1.53 ± 0.42mm (1.01±0.74mm 2 ) 이었다. 그리고차단막을사용한경우에는차단막을사용하지않은경우보다더많은골형성이관찰되었고 BCP 와 MEM 그리고 CCP 와 MEM 을적용한부위에서는임플란트의상부에서광범위한골형성이관찰되었다. BCP 와 MEM, CCP 와 MEM 그리고차단막만적용한부위에서의평균적인골재생높이 ( 면적 ) 는각각 3.96±2.86mm (12.46±11.61mm 2 ), 5.45±0.25mm (11.63±1.97mm 2 ), and 2.62 ± 0.27mm (3.43±0.98mm 2 ) 이었다. 결론적으로시아노아크릴레이트가결합된인산칼슘은골재생에있어 차단막대체제라기보다는차단막을지지하는좋은골격으로서의역할을 한다고볼수있겠다. 핵심되는말 : 시아노아크릴레이트, 인산칼슘, 골재생, 치과용임플란트, 열개형결손부. 35