저작자표시 - 비영리 - 변경금지 2.0 대한민국 이용자는아래의조건을따르는경우에한하여자유롭게 이저작물을복제, 배포, 전송, 전시, 공연및방송할수있습니다. 다음과같은조건을따라야합니다 : 저작자표시. 귀하는원저작자를표시하여야합니다. 비영리. 귀하는이저작물을영리목적으로이용할

저작자표시 - 비영리 - 변경금지 2.0 대한민국 이용자는아래의조건을따르는경우에한하여자유롭게 이저작물을복제, 배포, 전송, 전시, 공연및방송할수있습니다. 다음과같은조건을따라야합니다 : 저작자표시. 귀하는원저작자를표시하여야합니다. 비영리. 귀하는이저작물을영리목적으로이용할수없습니다. 변경금지. 귀하는이저작물을개작, 변형또는가공할수없습니다. 귀하는, 이저작물의재이용이나배포의경우, 이저작물에적용된이용허락조건을명확하게나타내어야합니다. 저작권자로부터별도의허가를받으면이러한조건들은적용되지않습니다. 저작권법에따른이용자의권리는위의내용에의하여영향을받지않습니다. 이것은이용허락규약 (Legal Code) 을이해하기쉽게요약한것입니다. Disclaimer

[UCI]I804:11046-000000515053 Dehydrothermally cross-linked collagen membrane with a bone graft improves bone regeneration in rat calvarial defect model Yin-Zhe An Department of Dentistry The Graduate School, Yonsei University

Dehydrothermally cross-linked collagen membrane with a bone graft improves bone regeneration in rat calvarial defect model Directed by Professor Seong-Ho Choi The Doctoral Dissertion submitted to the Department of Dentistry, the Graduate school of Yonsei University in partial fulfillment of the requirements for the degree of Ph.D. in Dental Science Yin-Zhe An December 2017

감사의글 우선박사학위과정을마치고논문을완성할수있도록아낌없는조언과격려해주신모든분들께깊은감사의마음을전하고싶습니다. 2014 년 9 월부터연세대학교치주과연구원으로일하면서정말많은것을배우며성장할수있었습니다. 특히, 최성호교수님의도움으로대학원을시작할수있었고, 이렇게졸업할수있게되었습니다. 교수님께진심으로감사드립니다. 감사의마음을어떻게글로다표현할지모르겠습니다. 교수님의가르침을본받아참된연구자의길을계속걷겠습니다. 또한바쁘신와중에도심사를맡아주시고연구방향을제시해주신정의원교수님, 이중석교수님, 한상선교수님, 김영택교수님께도감사의마음을전합니다. 학위과정동안언제나가슴따뜻한조언을해주시고늘마음써주시고아낌없는조언과격려를해주신허영구원장님께가슴깊이감사드립니다. 파이펫을잡을줄도모르는저를데리고기초부터가르쳐주시고진심어린조언을해주신장향란교수님께진심으로감사드립니다. 그외에도연구에많은도움을주셨던실험실선생님들께감사드립니다. 마지막으로지금까지키워주시고항상곁에서응원해주고지지해주신부모님께고개숙여진심으로감사드립니다. 공부하면서어렵고힘든일이많았지만좋은맺음을할수있게도와주신모든분들께감사드리며, 박사과정이끝이아닌앞으로새로시작하는마음으로치의학분야에도움이되는훌륭한치과의사가되도록노력하겠습니다. 2017 년 12 월 안은철올림

TABLE OF CONTENTS List of figures ⅲ List of tables ⅳ Abstract ν I. Introduction 1 II. Materials and Methods 5 2.1. Experimental DHT collagen membrane 5 2.2. BG material 5 2.3. Experimental animals 6 2.4. Study design 6 2.5. Surgical procedure 7 2.6. Micro-computed tomography analysis 8 2.7. Histological and histomorphometric analysis 9 2.8. Immunohistochemical analysis 10 2.9. Statistical analysis 11 III. Results 12 3.1. Clinical observations 12 3.2. Micro-CT findings 12 3.3. Histological findings 14 3.4. Histomorphometric analysis 16 3.5. Immunohistochemical findings 17 Ⅳ. Discussion 18 Ⅴ. Conclusion 24 i

References 25 Figures 31 Tables.. 35 Abstract (in Korean) 37 ii

LIST OF FIGURES Figure 1. Three-dimensional reconstruction images obtained at 2 and 8 weeks after surgery 31 Figure 2. Histologic transversal sections obtained at 2 weeks after surgery (hematoxylin and eosin staining) 32 Figure 3. Histologic transversal sections obtained at 8 weeks after surgery (hematoxylin and eosin staining) 33 Figure 4. Expression patterns of proliferating cell nuclear antigen (PCNA) in tissue sections detected by immunohistochemistry 34 iii

LIST OF TABLES Table 1. Micro-computed tomography measurements of the rat calvarial defect at 2 and 8 weeks after surgery 35 Table 2. Histomorphometric measurements at 2 and 8 weeks after surgery 36 iv

ABSTRACT Dehydrothermally cross-linked collagen membrane with a bone graft improves bone regeneration in rat calvarial defect model Yin-Zhe An Department of Dentistry The Graduate School, Yonsei University (Directed by Professor Seong-Ho Choi, D.D.S., M.S.D., PhD.) Purpose The aim of this study was to evaluate bone regeneration efficacy of dehydrothermally (DHT) cross-linked collagen membrane with or without a bone graft (BG) material in a critical-sized rat model. Materials and Methods An 8-mm-diameter defect was created in the calvaria of 40 rats, which were randomized into four groups: (1) control, (2) DHT, (3) BG, and (4) DHT+BG. Evaluations were made at 2 and 8 weeks after surgery using micro-computed tomographic (micro-ct), histological, and histomorphometric analysis. v

Results Micro-CT analysis showed an increase in the new bone volume (NBV) in the BG and DHT+BG groups at 2 weeks after surgery, representing a significant difference (P<0.05). At 8 weeks after surgery, the NBV increased in all four groups. However, larger NBVs were observed in the BG and DHT+BG groups, and a significant difference was no longer observed between the two groups. Histologic analysis demonstrated that the graft materials sustained the center of the defect in the BG and DHT+BG groups, which was shown in histomorphometric analysis as well. Conclusions These results suggest that DHT membrane is a safe biomaterial with adequate tissue integration; moreover, the best effects were achieved when DHT was used in conjunction with BG materials. Key Words: allograft, bone regeneration, collagen, cross-linking, dehydrothermal vi

Dehydrothermally cross-linked collagen membrane with a bone graft improves bone regeneration in rat calvarial defect model Yin-Zhe An Department of Dentistry The Graduate School, Yonsei University (Directed by Professor Seong-Ho Choi, D.D.S., M.S.D., PhD.) Ⅰ. INTRODUCTION The presence of sufficient bone volume is a prerequisite for the predictable osseointegration of a dental implant. However, alveolar ridge resorption over time, combined with the tooth loss and the presence of anatomical structures such as the maxillary sinus, nasal cavity, and inferior alveolar nerve all limit the amount of available bone for implant placement (Greenstein et al., 2008; Laino et al., 2014). Therefore, a bone graft (BG) is often required for the bone regeneration of osseous defects prior to or simultaneous with implant placement. 1

Guided bone regeneration (GBR) is a well-established and widely used technique that promotes new bone formation using a barrier membrane to exclude epithelial and connective tissue proliferation within the defect (Zubery et al., 2007; Bornstein et al., 2007). The barrier membranes utilized in GBR procedure should meet the following requirements: biocompatibility, cell occlusion, host tissue integration, easy manageability, space-maintaining ability during the early stage of healing, and comfort for the patient (Rothamel et al., 2014). Various non-degradable and degradable membranes have been developed according to these requirements, and several non-degradable membranes, such as titanium mesh and expanded polytetrafluoroethylene, showed successful outcomes in clinical and animal studies (Lundgren et al., 1988; Von Arx et al., 1988; Van Steenberghe et al., 2003; Hammerle et al., 2003). Although these membranes are currently considered gold-standard materials, they have a fatal disadvantage since they are susceptible to exposure, resulting in an increased risk of infection, often requiring a second surgery. To avoid a second surgery and to overcome the disadvantages of available membranes, several degradable materials such as collagen, polyglycolide, and polylactic acid have been identified and developed for the use in GBR (Bornstein et al., 2007; Rothamel et al., 2014; Bottino et al., 2012; Bunyaratavej et al., 2001). However, degradable membranes made from polymers may not be suitable for this purpose because of the presence of non- 2

enzymatic cleavage, leading to acid production with subsequent adverse events (Bottino et al., 2012). Since collagen is the main component of periodontal tissues, collagen-based materials are representative degradable membranes with many advantageous properties, including minimal inflammation rates, low immunogenicity and cytotoxicity, hemostasis, and the ease of manipulation during surgical procedures (Bunyaratavej et al., 2001). Collagen can be extracted industrially from the bovine and porcine dermis and tendons, and it has multiple applications in periodontal and implant surgeries. Successful GBR, using a non-cross-linked collagen membrane, has been reported (Zubery et al., 2007; Bornstein et al., 2007; Tal et al., 2008). However, the key disadvantage of such membranes is that they can be quickly resorbed and do not maintain the underlying secluded space sufficiently long time to allow the coagulum to appropriately mature and achieve selective repopulation (Tatakis et al., 1999). To prolong the resorption rate, many physical and chemical cross-linking techniques have been applied, such as ultraviolet radiation, dehydrothermal (DHT), glutaraldehyde, and diphenyl-phosphorylation-azide treatment (Bunyaratavej et al., 2001; Chen et al., 1997; Park et al., 2015). However, cross-linking using glutaraldehyde has been reported to induce a cytotoxic effect accompanied by the failure to integrate with the host tissue. Moreover, previous studies reported that collagen membranes cross-linked 3

using different methods show substantial differences in terms of biodegradation, biocompatibility, and angiogenesis (Rothamel et al., 2014; Park et al., 2015). Several investigations have been performed in a rat calvarial defect model to evaluate the effects of various materials on new bone formation. This model is established by forming a bone defect with the diameter of 8 mm in the center of the parietal bone of rat calvarium using a trephine bur. This defect is regarded as a critical-sized defect, as it does not spontaneously heal during the lifetime of the animal (Teng et al., 2008). This model is also relevant for periodontal research, as the physiological remodeling of calvaria is similar to that of the human mandibular bone (Spicer et al., 2012). However,animal study limitations are that they become the valid predictors of human response to the exposure and treatment only if there is substantial improvement in the scientific methods and systematic review of the animal study-related literature as it involves. It is essential for collagen membranes utilized in GBR to maintain the physical integrity long enough to allow bone regeneration. However, there are few available membranes that can completely satisfy all requirements. Therefore, the purpose of the present study was to evaluate bone regeneration efficacy in a critical-sized rat calvarial defect model using DHT cross-linked collagen membrane with or without BG material. 4

Ⅱ. MATERIALS & METHODS 2.1. Experimental DHT collagen membrane The DHT collagen membrane was obtained from the native porcine dermis with additional physical cross-linking and was mainly composed of type I collagen. The experimental membrane was processed by mechanical cleaning, chemical treatment, lyophilization, and compression, followed by the crosslinking using DHT in a vacuum at 110 C and sterilization. Various DHT membrane properties were investigated, including morphology, enzyme resistance, and mechanical properties. The surface of the DHT membrane had macro micro interconnective porous structure to permit cellular invasion. 2.2. BG material Freeze-dried bone allograft (FDBA) was used as the graft material in this study. The allograft (Regenoss, Cellumed, Seoul, Korea), a particle-type graft material, was composed of a cortical and cancellous powder with a weight ratio of 80/20. The sizes of the particle and pore were 0.2-1.0 mm and 250 µm, respectively. 5

2.3. Experimental animals A total of 40 albino rats of the Wistar strain (male, 11±0.5 weeks old) weighing between 350 g and 370 g were used in the present study. All rats were housed individually in standard cages under specific pathogen-free conditions and fed a standard laboratory diet and water. The animals were allowed to acclimate to the new environment for 7 days before performing the surgery. The animal selection, management, preparation and surgical protocol were evaluated and approved by the institutional Animal Care and Use Committee, Yonsei Medical Center, Seoul, Korea (approval number 2014-0332). 2.4. Study design A critical-sized defect of 8 mm in diameter was created in the parietal bone of each rat calvarium. A total of 40 defects were formed. Ten rats were allocated to each of the following groups, which were split into two subgroups for the examination at 2 and 8 weeks after surgery: (1) sham surgery control group, in which the bone discs were removed and the surgical defects were not filled with any membrane or material; (2) DHT membrane group, in which the surgical defects were covered by DHT collagen membranes; (3) BG group, in which the surgical defects were filled with FDBA, and (4) DHT+BG group, in 6

which the surgical defects were filled with FDBA and covered with DHT collagen membranes. 2.5. Surgical procedure Initially, the animals were sedated in a chamber with 4% isoflurane (Ifran, Hana Pharm, Kyonggi-Do, Korea) in 100% O 2 and then anesthetized by intraperitoneal injection of 15 mg/kg zoletile (Zoletil 50, Virbac, Carros, France) and 10 mg/kg rompun (Bayer, Ansan, Gyeonggi-do, Korea). Under local anesthesia with 2% lidocaine hydrochloride containing 1:100,000 epinephrine, after disinfection with povidone iodine (Povidin, Firson, Cheonan, Chungcheongnam-do, Korea), a middle skin incision was made on the skull, and a full-thickness flap was reflected. Under copious saline irrigation, a standardized, round transosseous defect of 8 mm in diameter was created in the center of the calvaria with a trephine bur. After removal of the trephined calvarial disc, four groups of ten animals each received the DHT collagen membrane, BG, BG with collagen membrane, or sham-surgery control procedure. For the groups with the collagen membrane, it was cut to a size of 10 10 mm squares to cover the outer surface of the bony defect, and then placed over the defect. For the groups involving BG, a sufficient amount of graft material was applied to completely fill the defect by applying a gentle pressure using a surgical instrument. After obtaining adequate hemostasis, the 7

periosteum and skin were repositioned and sutured. Post-operatively, to minimize post-operative pain and prevent infection, all rats were injected subcutaneously with antibiotics (10 mg/day enfloxacin, once daily for 5 days) and analgesics (1 mg/kg meloxicam, once daily for 5 days). After healing periods of 2 and 8 weeks, five rats in each group were euthanized in a CO 2 chamber. Subsequently, block sections of the rat calvaria were harvested and fixed in a 10% neutral buffered formalin solution. 2.6. Micro-computed tomography (Micro-CT) analysis All samples were scanned using high-resolution micro-ct system (SkyScan 1173, Kontich, Belgium) at a pixel size of 14.91 µm. Prior to scanning the samples, calibration was performed using water, air, and synthetic bone samples. The digital images were obtained under a source voltage of 130 kv and a current of 60 µa. The scanned images were then reconstructed using CT-analyzer software (Ondemand 3D, Cybermed Inc., version 1.0, Seoul, Korea). The regions of interest of each sample were determined in three dimensional (3D) images for the analysis of total augmented volume (TV), new bone volume (NBV), bone volume fraction (BVF: calculated as the NBV divided by TV), and bone mineral density (BMD). 8

2.7. Histological and histomorphometric analysis After obtaining micro-ct scans, block sections of the experimental sites were fixed in a 10% neutral-buffered formalin solution for 10 days. The fixed specimens were decalcified in 5% formic acid for 14 days and embedded in paraffin. Serial sections of 5 µm were cut through the central portion of each experimental site. Only the central sections were chosen and stained with hematoxylin-eosin for histological and histomorphometric analysis. An experienced researcher, blinded to the specific experimental conditions, performed the microscopic examination and histomorphometric analysis. Digital images of histologic slides were obtained using a binocular microscope (Leica DM LB, Leica Microsystems, Wetzlar, Germany) coupled with a color camera (Leica DC300F, Leica Microsystems, Wetzlar, Germany), and saved as digital files. Histometric measurements in the defects were made using automated image analysis software (Image-Pro Plus, Media Cybernetics, Silver Spring, MD, USA) at 12.5 magnification. The following parameters were measured: (1) total augmented area (TA), including newly formed bone, connective tissue, and remaining membrane, and grafted materials within the defect; (2) new bone area (NB), representing the area of newly formed bone within the defect; (3) remaining membrane area (RMA), representing the area of residual membrane within the defect; and (4) residual materials (RM), grafted bone materials within the defect. 9

2.8. Immunohistochemical analysis Activity of cell proliferation in new bone area was identified using immunohistochemical analysis with anti-proliferating cell nuclear antigen (PCNA) monoclonal antibody. Endogenous peroxidase activity was inactivated with 1% hydrogen peroxide solution for 30 min. The sections were blocked with PBS containing 5% bovine serum albumin at room temperature for 10 min and reacted with the primary antibody (1:100, ab29, Abcam, Cambridge, UK) at room temperature for 1 h. After washing in tris-buffered saline, the sections were reacted with secondary antibody (Vectastain ABC kit, Vector laboratories, Burlingame, CA, USA) at room temperature for 30 min. Then, the bound antibodies were visualized with avidin-biotin DAB system (Dako, Glostrup, Denmak), counter-stained with hematoxylin. The PCNApositive cells were observed under optical microscopy (Olympus, Tokyo, Japan). 10

2.9. Statistical analysis The statistical analysis was performed using a commercially available software program (SPSS 20.0, SPSS Inc., Chicago, IL, USA). The bone defect of each rat was regarded as a statistical unit. Data obtained in each group are expressed as mean values and standard deviations. Kruskal-Wallis one-way analysis of variance based on ranks and the post-hoc Mann Whitney U test were used to assess the differences among groups at each time point, and Wilcoxon signed rank test was used to compare the data obtained in the same group between two healing periods. A P-value less than 0.05 was considered statistically significant. 11

Ⅲ. RESULTS 3.1. Clinical observations The post-operative soft tissue healing was uneventful, with no complications (including the membrane and graft material exposure) or other inflammatory reactions observed in any of the rats. At 2 and 8 weeks post-surgery, surgical sites showed the evidence of incision and sutures. After harvesting of the surgical sites, the bone defect areas were clearly surrounded by periosteum and dura mater under visual inspection. 3.2. Micro-CT findings The results of micro-ct measurements are summarized in Table 1 and Figure 1. At 2 weeks after surgery, there was no statistically significant difference between the control and DHT groups in terms of TV, NBV, BVF, and BMD. In contrast to this, TV was significantly increased in both groups receiving BG compared with that in the control group (BG, P=0.031; DHT+BG, P=0.002), while that of the DHT+BG group was significantly greater than those of the DHT and BG groups (P=0.001 and P=0.016, respectively). Both BG and DHT+BG groups showed significantly higher NBV (all, P=0.008), BVF (vs. control: P=0.009 and 0.008, respectively; vs. DHT group: P=0.008 for both), 12

and BMD (vs. control: P=0.008 and 0.002, respectively; vs. DHT group: P=0.008 for both). Moreover, the NBV and BVF values were significantly higher in the DHT+BG group than in the BG group (P=0.008 and P=0.016, respectively). At 8 weeks after surgery, all values were shown to be increased, and no statistically significant difference was observed between the control group and DHT group in the four variables. In contrast, the BG and DHT+BG groups showed significantly higher TV values compared with that in the control group (P=0.008 and P<0.001, respectively). Additionally, the TV value in the DHT+BG group was also significantly higher than those in the DHT and BG groups (P=0.009 and P=0.008, respectively). Similar to the results obtained at 2 weeks, both BG and DHT+BG groups showed significantly higher NBV values (vs. control: P=0.008 and 0.008, respectively; vs. DHT group: P=0.003 and P<0.001, respectively), BVF (vs. control: P=0.002 and P<0.001, respectively; vs. DHT: P=0.003 and P<0.001, respectively), and BMD (vs. control: P=0.004 and P<0.001, respectively; vs. DHT group: P=0.003 and P<0.001, respectively) values. However, no statistically significant difference was observed in NBV, BVF, and BMD values between the BG and DHT+BG groups. 13

Comparisons within the same experimental groups showed only statistically significant differences in NBV and BVF values in the BG and DHT+BG groups with time (P=0.043 and P=0.043, respectively). 3.3. Histological findings At 2 weeks of healing, the early phase of newly formed bone was observed at the margin of the defect in all four groups. In the control and DHT groups, the center of the defect was flattened and mainly occupied by the connective tissue or the grafted collagen membrane. In contrast, in the two groups receiving BG, the center of the defect was sustained by the bone materials (Figure 2a, c, e, and g). Under high magnification, a normal post-operative inflammatory response was observed in all four groups. Mononuclear cell infiltration rate in the connective tissue was shown to be pronounced, which was followed by the infiltration of macrophages and lymphocytes. In all four groups, osteoblast and osteoclasts were found to be in a close contact with the newly formed bone of the margin of defect. In this period, in the DHT and DHT+BG groups, the membrane was shown to integrate with the surrounding connective tissue and its resorption started. However, the appearance of the membrane body could be easily distinguished from the connective tissue, and the formation of vascular endothelial cells could be observed beneath the 14

membrane. In the BG and DHT+BG groups, multinucleated giant cells were observed around the grafted bone materials (Figure 2b, d, f, and h). At 8 weeks, the woven bone formed in the early phase was shown to be matured, and most of the newly formed bone was apparently regenerated through a centripetal extension from the defect margin in all four groups. In the control group, the center of the defect was depressed and loose connective tissue was shown to be transformed into a well-arranged bundle of fibers. In the DHT group, the collagen membrane body was almost completely resorbed and replaced by newly formed connective tissue, and sparser distribution of inflammatory cells could be observed, accompanied by a reduction in their numbers in comparison with those detected at 2 weeks. In a few specimens belonging to the control and DHT groups, bony islands were found in the defect area. In the BG and DHT+BG groups, the defect was well maintained by the allograft materials. Although the number of grafted bone particles was reduced in comparison with those detected at 2 weeks after the surgery, the particles were not completely replaced by the newly formed bone. Bone regeneration was slightly more activated both through the centripetal extension from the margin of the defect and by formation on the superficial layer of the grafted bone particles (Figure 3). 15

3.4. Histomorphometric analysis The results of histomorphometric analysis are summarized in Table 2. At 2 weeks of healing, no significant differences in TA and NB were observed between the control and DHT groups. In contrast, these values were significantly higher in the BG and DHT+BG groups compared with those determined in the control group (all P<0.001) and DHT group (P=0.003 and P<0.001, respectively), while a significant difference was obtained between two BG groups (P=0.009). At 8 weeks after surgery, no statistically significant differences were observed in TA and NB values between the control and DHT groups. However, the TA (P=0.008 and P<0.001) and NB (P=0.001 and P<0.001) values in the BG and DHT+BG groups, respectively, were significantly higher compared with those determined in the control group. The TA values in two BG groups were shown to be significantly different (P=0.009). The NB values were significantly higher in the BG (both P<0.001) and DHT+BG (P=0.001 and 0.003) groups, compared with those in the control and DHT groups, respectively. TA values were significantly higher in both BG and DHT+BG groups compared with that of determined in the DHT group (P=0.004 and P=0.009, respectively). In the BG and DHT+BG groups, significant difference in the NB values were detected between 2- and 8-week healing periods (P=0.043 and P=0.043, 16

respectively). The amount of graft materials, including the bone material and membrane, decreased in all three experimental groups between 2 and 8 weeks after surgery. 3.5.Immunohistochemical findings Proliferating cells were highlighted using PCNA antibody by immunohistochemistry both in control and experimental group. We found that control group was mainly showed negative patterns for PCNA expression (Fig. 4a-b). By contrast, positive patterns of PCNA expression were frequently detected in the each experimental group (Fig. 4c-d). 17

Ⅳ. DISCUSSION Biologically, an optimal barrier membrane must induce hemostasis, be integrated by the host tissues, maintain chemotaxis for periodontal ligament fibroblasts, and possess low cytotoxicity. Moreover, the membrane should be able to exclude unwanted cells to protect the wound area and prevent infection (Zubery et al., 2007; Bornstein et al., 2007; Bunyaratavej et al., 2001). Therefore, a barrier membrane must be able to maintain its structural integrity during the early healing period. From the clinical point of view, easy manageability and cost-effectiveness are also of great concern when developing a new material. Therefore, many studies focused on these two aspects to develop barrier membranes. Porcine skin-derived collagen membranes are widely used in GBR because their 3D structure is similar to that of the native extracellular matrix and moreover, DHT cross-linking technique in porcine collagen membrane may promote mechanical properties (Li et al., 2013). In the present study, a porcine skin- derived type I collagen membrane was evaluated using a rat calvarial defect model. The DHT collagen membrane has a positive effect on new bone formation, confirming the results of previous studies (Park et al., 2015; Chung et al., 2014). Additionally, the DHT membrane displayed excellent integration into the surrounding connective tissue during the early healing period with minimal 18

immune reactions, as histological analysis demonstrated. Specifically, the histomorphometric analysis showed that the new bone was formed in all four groups at 2 and 8 weeks after surgery. However, no statistically significant differences were observed between the control and DHT groups. In contrast to this, significantly greater NB values were detected in the BG and DHT+BG groups, compared with those in the control and DHT groups, and the differences in NB were significant between the BG and DHT+BG groups as well. Micro-CT analysis yielded similar results, and the bone of better quality was shown to be produced in the groups containing BG materials. Notably, these results demonstrated that the highest level of new bone formation occurred when the DHT collagen membrane was used in combination with BG materials. Therefore, the DHT membrane shows may be a suitable barrier membrane for GBR. Good biocompatibility and tissue integration are crucial elements for the reduction of the inflammatory response and membrane exposure (Bunyaratavej et al., 2001). These properties are closely related to the cross-linking agents used, which can induce foreign body reactions. The cross-linking of collagen was shown to be associated with decreased tissue integration and angiogenesis, and a slower resorption rate (Rothamel et al., 2014). Tai et al. reported that a cross-linked collagen membrane was more resistant than a non-cross-linked collagen membrane, with more adverse 19

events and lower rate of new bone regeneration in a human study. However, certain cross-linked techniques were shown to be suitable despite some membrane exposure (Zubery et al., 2007). Although the early membrane exposure to the oral environment was reported to cause disintegration, the capacity for resistance to proteolysis of a cross-linked membrane was found to be much stronger than that of a non-cross-linked membrane (Sela et al., 2009). Unlike materials reported in previous studies, the DHT cross-linked collagen membrane did not induce soft tissue dehiscence at any surgical cites. Furthermore, histological evidence suggested that the DHT membrane was well-integrated with the surrounding connective tissues at the early healing stage (i.e., at 2 weeks after surgery), and had the ability to form vascular endothelial cells, which play an important role during the early new bone formation. These results indicate that the DHT cross-linking technique is a suitable method for achieving a balance between membrane stability and functional remodeling (Hu et al., 2013). Various graft materials have been developed and used to obtain favorable outcomes in the periodontal and implant surgeries, including autogenous bones, allografts, xenografts, and bone substitutes (Laino et al., 2015). FDBA may represent a good substitute for autogenous bone. In the present study, FDBA was used to fill the bone defect, which showed a positive effect on the new bone formation: both BG and DHT+BG groups exhibited a significant 20

increase in the NBV compared with those in the control and DHT groups at 8 weeks after surgery. In a previous study, various BG materials in conjunction with a titanium membrane were evaluated in a rabbit calvarial defect model at 8 and 16 weeks post-surgery, demonstrating that the most of newly formed bone was observed even at a relatively early stage (at 8 weeks), while a considerable amount of new bone was formed in the FDBA group at 16 weeks (Kim et al., 2006), supporting the results of this study. Here, newly formed bone was observed to be in a close contact with the FDBA particles. FDBA particles may play an osteoconductive role and serve as the core of new bone deposition; this is supported by the results obtained by Froum et al. and Kolerman et al. Moreover, Piatelli et al. observed the presence of osteoclasts actively resorbing the bone on the outer surface of particles located far from the mother bone (Froum et al., 2006; Kolerman et al., 2008; Piattelli et al., 1996). Collectively, these results show that the particles are resorbed, allowing the deposition of the new bone. The aim of this study was to evaluate new bone formation using DHT crosslinked collagen membrane with or without bone materials. Some studies reported unfavorable mechanical strength and inadequate barrier function as the major disadvantages of the use of collagen membrane (Hurzeler et al., 1997; Owens et al., 2001). Appropriate space was shown to be obtained when the bone defect morphology is superior, and if the defect cannot be sustained, 21

the membrane itself will inevitably become depressed (Luepke et al., 1997). Here, the histological evidence revealed that the central portion was depressed or flattened by surrounding connective tissue in the DHT membrane group. Therefore, the results of the present study show that when performing GBR with a collagen membrane, supporting the space with the use of graft material is essential for bone regeneration. Previous studies reported that a cross-linked collagen membrane promotes new bone formation in bone defects. Park et al. histologically and histomorphometrically evaluated the efficiency of a 1-ethyl-3-(3- dimethylaminopropyl) carbodiimide cross-linked collagen membrane in a rabbit calvarial defect model. Chung et al. clinically and radiographically assessed the bone regeneration capacity of two collagen membranes in human periodontal defects. Although histological analysis and histomorphometric analysis are considered the gold standard in evaluating bone regeneration, a significant correlation between micro-ct and histological analysis has been reported in previous studies (Baek et al., 2015; Nooh et al., 2016). In the present study, both histological and histomorphometric analysis were performed to ascertain the effectiveness of the DHT cross-linked collagen membrane for GBR, and the results were further corroborated by the results of micro-ct analysis and 3D reconstruction. Micro-CT and a reconstruction program have the advantage of quantifying the parameters accurately, which 22

can serve as guidelines while applying GBR in the large animal experiments or human studies (Laino et al., 2015). 23

Ⅴ. CONCLUSION According to the results of micro-ct analysis, TV values in the regions of interest increased in a time-dependent manner in all groups. Significant differences in the NBV, BVF, and BMD values, which are the key indices of bone regeneration, were observed between the BG and DHT+BG groups and the control group, together with significant difference observed between the BG and DHT+BG groups. Based on these results, DHT cross-linked collagen membrane represents a safe biomaterial, showing good potential to serve as a barrier membrane in the GBR procedure. Given the short evaluation period and the limitations of this study, further investigations are necessary to confirm the effectiveness of the DHT membrane for the GBR in clinical practice. 24

REFERENCES Greenstein, G., Cavallaro, J., Romanos, G., & Tarnow, D. (2008) Clinical recommendations for avoiding and managing surgical complications associated with implant dentistry: a review. Journal of Periodontology 79: 1317-1329. Laino, L., Iezzi, G., Piattelli, A., Lo Muzio, L., & Cicciu, M. (2014) Vertical ridge augmentation of the atrophic posterior mandible with sandwich technique: bone block from the chin area versus corticocancellous bone block allograft--clinical and histological prospective randomized controlled study. BioMed Research International 2014, 982104. Zubery, Y., Goldlust, A., Alves, A., & Nir, E. (2007) Ossification of a novel cross-linked porcine collagen barrier in guided bone regeneration in dogs. Journal of Periodontology 78: 112-121. Bornstein, M.M., Bosshardt, D., & Buser, D. (2007) Effect of two different bioabsorbable collagen membranes on guided bone regeneration: a comparative histomorphometric study in the dog mandible. Journal of Periodontology 78: 1943-1953. Rothamel, D., Benner, M., Fienitz, T., Happe, A., Kreppel, M., Nickenig, H.J., & Zoller, J.E. (2014) Biodegradation pattern and tissue integration of native and cross-linked porcine collagen soft tissue augmentation matrices - an experimental study in the rat. Head & Face Medicine 10: 10. 25

Lundgren, A., Lundgren, D., & Taylor, A. (1998) Influence of barrier occlusiveness on guided bone augmentation. An experimental study in the rat. Clinical Oral Implants Research 9: 251-260. Von Arx, T., Wallkamm, B., & Hardt, N. (1998) Localized ridge augmentation using a micro titanium mesh: a report on 27 implants followed from 1 to 3 years after functional loading. Clinical Oral Implants Research 9: 123-130. Van Steenberghe, D., Johansson, C., Quirynen, M., Molly, L., Albrektsson, T., & Naert, I. (2003) Bone augmentation by means of a stiff occlusive titanium barrier. Clinical Oral Implants Research 14: 63-71. Hammerle, C.H., & Jung, R.E. (2003) Bone augmentation by means of barrier membranes. Periodontology 2000 33: 36-53. Bottino, M.C., Thomas, V., Schmidt, G., Vohra, Y.K., Chu, T.M., Kowolik, M.J., & Janowski, G.M. (2012) Recent advances in the development of GTR/GBR membranes for periodontal regeneration--a materials perspective. Dental Materials 28: 703-721. Bunyaratavej, P., & Wang, H.-L. (2001) Collagen membranes: a review. Journal of Periodontology 72: 215-229. 26

Tal, H., Kozlovsky, A., Artzi, Z., Nemcovsky, C.E., & Moses, O. (2008) Cross-linked and non-cross-linked collagen barrier membranes disintegrate following surgical exposure to the oral environment: a histological study in the cat. Clinical Oral Implants Research 19: 760-766. Tatakis, D.N., Promsudthi, A., & Wikesjö, U.M. (1999) Devices for periodontal regeneration. Periodontology 2000 19: 59-73. Chen, Y.T., Wang, H.L., Lopatin, D.E., O'Neal, R., & MacNeil, R.L. (1997) Bacterial adherence to guided tissue regeneration barrier membranes exposed to the oral environment. Journal of Periodontology 68: 172-179. Park, J.Y., Jung, I.H., Kim, Y.K., Lim, H.C., Lee, J.S., Jung, U.W., & Choi, S.H. (2015) Guided bone regeneration using 1-ethyl-3-(3- dimethylaminopropyl) carbodiimide (EDC)-cross-linked type-i collagen membrane with biphasic calcium phosphate at rabbit calvarial defects. Biomaterials Research 19: 15. Teng, S.H., Lee, E.J., Wang, P., Shin, D.S., & Kim, H.E. (2008) Three-layered membranes of collagen/hydroxyapatite and chitosan for guided bone regeneration. Journal of Biomedical Materials Research Part B Applied Biomaterials 87: 132-138. Spicer, P.P., Kretlow, J.D., Young, S., Jansen, J.A., Kasper, F.K., & Mikos, A.G. (2012) Evaluation of bone regeneration using the rat critical size calvarial defect. Nature Protocols 7: 1918-1929. 27

Li, J., Ren, N., Qiu, J., Jiang, H., Zhao, H., Wang, G., Boughton, R.I., Wang, Y., & Liu, H. (2013) Carbodiimide crosslinked collagen from porcine dermal matrix for high-strength tissue engineering scaffold. International Journal of Biological Macromolecules 61: 69-74. Chung, Y.M., Lee, J.Y., & Jeong, S.N. (2014) Comparative study of two collagen membranes for guided tissue regeneration therapy in periodontal intrabony defects: a randomized clinical trial. Journal of Periodontal & Implant Science 44: 194-200. Tal, H., Kozlovsky, A., Artzi, Z., Nemcovsky, C.E., & Moses, O. (2008) Long term bio degradation of cross linked and non cross linked collagen barriers in human guided bone regeneration. Clinical Oral Implants Research 19: 295-302. Sela, M.N., Babitski, E., Steinberg, D., Kohavi, D., & Rosen, G. (2009) Degradation of collagen-guided tissue regeneration membranes by proteolytic enzymes of Porphyromonas gingivalis and its inhibition by antibacterial agents. Clinical Oral Implants Research 20: 496-502. Hu, Y., Liu, L., Dan, W., Dan, N., Gu, Z., Yu, X. (2013) Synergistic effect of carbodiimide and dyhydrothermal crosslinking on acellular dermal matrix. International Journal of Biological Macromolecules 55: 221-230. 28

Laino, L., Troiano, G., Giannatempo, G., Graziani, U., Ciavarella, D., Dioguardi, M., Lo Muzio, L., Lauritano, F., & Cicciu, M. (2015) Sinus Lift Augmentation by Using Calcium Sulphate. A Retrospective 12 Months Radiographic Evaluation Over 25 Treated Italian Patients. The Open Dentistry Journal 9: 414-419. Kim, Y., Kown, Y., Park, J., Chung, J., Lim, H., Jue, S., Cho, M., & Herr, Y. (2006) Porous Titanium membranes combined with various graft materials induce exophytic bone formation in rabbit calvaria. Key Engineering Materials 309-311: 427-432. Froum, S.J., Wallace, S.S., Elian, N., Cho, S.C., & Tarnow, D.P. (2006) Comparison of mineralized cancellous bone allograft (Puros) and anorganic bovine bone matrix (Bio-Oss) for sinus augmentation: histomorphometry at 26 to 32 weeks after grafting. The International Journal of Periodontics and Restorative Dentistry 26: 543-551. Kolerman, R., Tal, H., & Moses, O. (2008) Histomorphometric analysis of newly formed bone after maxillary sinus floor augmentation using ground cortical bone allograft and internal collagen membrane. Journal of Periodontoogy 79: 2104-2111. Piattelli, A., Scarano, A., Corigliano, M., & Piattelli, M. (1996) Comparison of bone regeneration with the use of mineralized and demineralized freeze-dried bone allografts: a histological and histochemical study in man. Biomaterials 17: 1127-1131. 29

Hurzeler, M.B., Quinones, C.R., & Schupbach, P. (1997) Guided bone regeneration around dental implants in the atrophic alveolar ridge using a bioresorbable barrier. An experimental study in the monkey. Clinical Oral Implants Research 8: 323-331. Owens, K.W., & Yukna, R.A. (2001) Collagen membrane resorption in dogs: a comparative study. Implant Dentistry 10: 49-58. Luepke, P.G., Mellonig, J.T., & Brunsvold, M.A. (1997) A clinical evaluation of a bioresorbable barrier with and without decalcified freeze-dried bone allograft in the treatment of molar furcations. Journal of Clinical Periodontology 24: 440-446. Baek, W.S., Yoon, S.R., Lim, H.C., Lee, J.S., Choi, S.H., & Jung, U.W. (2015) Bone formation around rhbmp-2-coated implants in rabbit sinuses with or without absorbable collagen sponge grafting. Journal of Periodontal & Implant Science 45: 238-246. Nooh, N., Ramalingam, S., Al-Kindi, M., Al-Rasheed, A., Al-Hamdan, K.S., & Al-Hezaimi, K. (2016) Real-Time Assessment of Guided Bone Regeneration in Standardized Calvarial Defects in Rats Using Bio- Oss With and Without Collagen Membrane: An In Vivo Microcomputed Tomographic and Histologic Experiment. The International Journal of Periodontics and Restorative Dentistry 36: s139-149. 30

FIGURES Figure 1. Three-dimensional reconstruction images obtained at 2 and 8 weeks after surgery. (a, e) Control group, (b, f) DHT membrane group, (c, g) BG group, (d, h) DHT+BG group. Gray: mother bone; green: new bone; purple: bone graft material. 31

Figure 2. Histologic transversal sections obtained at 2 weeks after surgery (hematoxylin and eosin staining). (a, b) Control group, (c, d) DHT membrane group, (e, f) BG group, (g, h) DHT+BG group. The boxed areas in the left panels (40 magnification) are magnified in the corresponding panels on the right (200 magnification). Arrowhead: defect margin. MB: mother bone; NB: new bone; CM: collagen membrane; RM: residual material; black arrow: vascular endothelial cells. 32

Figure 3. Histologic transversal sections obtained at 8 weeks after surgery (hematoxylin and eosin staining). (a, b) Control group, (c, d) DHT membrane group, (e, f) BG group, (g, h) DHT+BG group. The boxed areas in the left panels (40 magnification) are magnified in the corresponding panels on the right (200 magnification). Arrowhead: defect margin. CT: connective tissue; NB (black arrow): new bone; CM: collagen membrane; RM: residual material. 33

Figure 4. Expression patterns of proliferating cell nuclear antigen (PCNA) in tissue sections detected by immunohistochemistry. Example of a negative (a, b) and positive (c, d) patterns of PCNA expression in tissue sections. The boxed areas in the left panels (200 magnification) are magnified in the corresponding panels on the right (1000 magnification). 34

TABLES Table 1. Micro-CT measurements of the rat calvarial defect at 2 and 8 weeks after surgery. Group TV (mm 3 ) NBV (mm 3 ) BVF (%) BMD (mg ml -1 ) 2 weeks Control 75.17 ± 3.43 1.43 ± 0.67 1.90 ± 1.28 0.29 ± 0.09 DHT 87.26 ± 9.18 1.05 ± 0.36 1.20 ± 0.37 0.36 ± 0.14 BG 97.58 ± 13.93 a 10.48 ± 2.48 ab 10.74 ± 1.76 ab 1.92 ± 0.48 ab DHT +BG 122.35 ± 13.05 abc 16.41 ± 1.99 abc 13.42 ± 0.86 abc 2.58 ± 0.49 ab 8 weeks Control 85.76 ± 8.12 2.18 ± 1.04 2.52 ± 1.12 0.50 ± 0.13 DHT 98.78 ± 6.76 3.21 ± 1.10 3.04 ± 1.19 0.68 ± 0.39 BG 110.02 ± 6.06 a 21.38 ± 6.38 abd 19.38 ± 5.48 abd 3.28 ± 0.99 ab DHT +BG 137.40 ± 3.66 abc 26.77 ± 2.11 abd 19.52 ± 1.99 abd 4.25 ± 0.48 ab TV: total tissue volume NBV: new bone volume BVF: bone volume fraction BMD: bone mineral density a Statistically significant difference from the control group. b Statistically significant difference from the DHT group. c Statistically significant difference from the BG group. d Statistically significant difference from the same experimental group at 2 weeks. 35

Table 2. Histomorphometric measurements at 2 and 8 weeks after surgery. Group TA NB Residual Materials Remaining membrane 2 weeks Control 2.71 ± 0.36 0.11 ± 0.08 - - DHT 4.85 ± 0.83 0.20 ± 0.06-1.63 ± 0.44 BG 7.04 ± 1.09 ab 0.62 ± 0.07 ab 3.30 ± 0.69 - DHT+BG 8.46 ± 0.93 abc 0.72 ± 0.07 ab 2.79 ± 0.53 2.16 ± 0.27 8 weeks Control 4.15 ± 0.73 0.24 ± 0.13 - - DHT 8.09 ± 0.63 0.39 ± 0.08-0.27 ± 0.18 BG 9.91 ± 0.66 abc 1.35 ± 0.13 abd 1.64 ± 0.88 - DHT+BG 14.45 ± 0.57 abc 1.52 ± 0.38 abd 1.52 ± 0.45 0.03 ± 0.02 TA: total augmented area NB: new bone area a Statistically significant difference from the control group. b Statistically significant difference from the DHT group. c Statistically significant difference from the BG group. d Statistically significant difference from the same experimental group at 2 weeks. 36

국문요약 백서두개골결손부에서 dehydrothermal 가교화기법으로제작한콜라겐차폐막과골이식재를이용한골재생효과 < 지도교수최성호 > 연세대학교대학원치의학과 안은철 본연구의목적은백서두개골모델에서 dehydrothermal (DHT) 가교화기법으로제작한콜라겐차폐막과골이식재 (BG) 를이식하여골재생효과를평가하는것이다. 40 마리의백서두개골에직경이 8mm 임계크기의결손부를형성하였고다음과같이무작위로 4 개그룹으로분류하였다. 대조군은결손부형성후아무런재료도이식하지않았다. 나머지 3 군은실험군으로 DHT 군은결손부형성후콜라겐차폐막을이식, BG 군은결손부형성후골이식재를이식, DHT+BG 군은결손부형성후콜라겐차폐막과골이식재를이식하였다. 2 주, 8 주후에 37

40 마리의백서를모두희생하여조직을적출하였고마이크로전산화단층촬영, 조직학적및조직계측학적분석을시행하였다. 2 주후의방사선학적분석결과대조군과비교시 BG 및 DHT+BG 군에서신생골이많이형성된것을관찰할수있었고통계학적유의한차이를보였다 (P<0.05). 8 주후에 4 개의그룹에서신생골은모두증가하였고 BG 및 DHT+BG 군에서 2 주군의결과처럼신생골이많이형성된것을관찰할수있었지만통계학적유의한차이는보이지않았다. 조직학적분석결과골이식재가결손부를받쳐주는것을관찰할수있었고조직계측학적분석결과방사선학적분석이랑같은양상을보였다. 이러한결과는 DHT 가교화기법으로제작한콜라겐차폐막이조직융합을유도하는안전한생체재료라는것을알수있다. 또한본연구에서는콜라겐차폐막과골이식재를같이이식하였을때최상의효과를얻을수있었다. 핵심되는말 : 이종골 ; 골재생 ; 콜라겐 ; 가교화 ; dehydrothermal 38