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[UCI]I804:11046-000000517063 Development of an animal model for medication related osteonecrosis of the jaw in ovariectomized rats treated with zoledronic acid before dental extractions caused by periodontal lesion Jieun Cheong The Graduate School Yonsei University Department of Dentistry

Development of an animal model for medication related osteonecrosis of the jaw in ovariectomized rats treated with zoledronic acid before dental extractions caused by periodontal lesion Directed by Professor Kee-Deog Kim, D.D.S., M.S.D., Ph.D. A Dissertation Thesis Submitted to the Department of Dentistry and the Graduate School of Yonsei University in partial fulfillment of the requirements for the degree of Doctor of Philosophy in Dental Science Jieun Cheong June 2018

감사의글 1991년초등학교 1학년입학을시작으로박사논문을마무리하는지금까지 23년간대장정의학업을끝마치는데정말많은분들의도움과격려가있었습니다. 가장먼저, 통합치의학과에서수련을시작할때부터존경해온김기덕지도교수님의가르침에진심으로감사드립니다. 진료와연구로바쁘신와중에도저의박사논문을위해혼신의힘을쏟아주신박원서교수님, 통합치의학과를이끌어주시느라온힘을다하고계신정복영교수님께감사드립니다. 직접조직사진을보며지도해주시고부족했던논문임에도아낌없이조언을주셨던김현실교수님, 제가생각지못했던부분을정확하고꼼꼼하고날카롭게검토해주셨던이중석교수님께감사드립니다. 위네분의심사위원교수님들께진심으로감사드립니다. 못난제자가지치고힘들어하소연을할때항상곁에서응원과칭찬을아끼지않으셨던방난심교수님께도말로전할수없는감사를드립니다. 연구에대한열정과탁월한능력으로제논문을함께쓴것이나다름없는박경미연구원, 연구를위해언제나무슨일이든흔쾌히도와준김서율연구원, 최이슬연구원께감사드립니다. 박사기간내내구체적이고세심한조언을해주셨던임지영선생님, 힘든일이있을때마다공감해주시고격려해주셨던이강희선생님께감사합니다. 영어를잘하는탓에고생한양수진선생님감사합니다. 병원에서늘저의마음의안식처가되어주는변혜덕선생님감사합니다.

자주보지도못하면서어쩌다가만날때면바쁘고힘들다고늘투정만부리는딸의박사졸업을묵묵히기다려주신엄마, 아빠에게사랑과감사를전합니다. 연락도자주드리지못한못난며느리임에도볼때마다제걱정을먼저해주시는어머님, 아버님그리고형님께도감사드립니다. 형제자매가없는저에게모든것을포기하고싶은순간항상위로가되고용기를주는가족과같은친구들, 고맙고사랑한다! 누구보다도같은길을가는치과의사로서, 통합치의학과의국동문으로서, 평생을함께할인생의동반자로서논문을시작할때부터끝나는순간까지제곁에서모든것을감수하고응원해주며인내해준사랑하는남편김병노, 이모든사랑하는가족들과통합치의학과식구들에게제논문을바칩니다. 2018 년 6 월 정지은

TABLE OF CONTENTS TABLE OF CONTENTS i LIST OF FIGURES iii LIST OF TABLES iv Abstract v 1. Introduction 1 2. Materials and Methods 4 2.1 Animals 4 2.2 Experimental design 5 2.3 Radiographic analysis: micro-computed tomography (micro-ct) 8 2.4 Microscopic analysis: Histologic analysis 10 2.5 Microscopic analysis: Immunohistochemistry 11 2.6 Statistical analysis 12 3. Results 13 3.1. Radiographic analysis: micro-computed tomography (micro-ct) 13 3.2. Microscopic analysis: Histologic analysis 19 3.3. Microscopic analysis: Immunohistochemistry 25 4. Discussion 32 i

5. Conclusion 37 6. References 38 Abstract (in Korean) 42 ii

List of Figures Figure 1. Experimental design 7 Figure 2. Clinical photo of tooth ligation for inducing periodontal lesion 7 Figure 3. The region of interest (ROI) in micro-ct analysis for trabecular bone morphometry of the tibia 9 Figure 4. The region of interest (ROI) in micro-ct analysis for trabecular bone morphometry of the extraction sites 9 Figure 5. In vivo micro-ct 3D reconstructed images of the mandible at the buccal side 14 Figure 6. Micro-CT images of the tibia 14 Figure 7. Micro-CT images of the extraction site in the axial view 17 Figure 8. Histologic analysis in the tibia of CONT group 20 Figure 9. Histologic analysis in the tibia of ZA group 21 Figure 10. Histologic analysis in the extraction site of CONT group 22 Figure 11. Histologic analysis in the extraction site of ZA group 23 Figure 12. Immunohistochemical staining for anti-cd68 and anti-cd86 in CONT group 26, 27 Figure 13. Immunohistochemical staining for anti-cd68 and anti-cd86 in ZA group 28, 29 Figure 14. Comparison of Anti-CD68 and anti-cd86 immunohistochemical staining between two groups 30, 31 iii

List of Tables Table 1. Micro-CT analysis of trabecular bone in the tibia 15 Table 2. Micro-CT analysis of trabecular bone in the extraction site 18 Table 3. Micro-CT analysis of trabecular bone in the extraction site of maxilla 18 Table 4. Micro-CT analysis of trabecular bone in the extraction site of mandible 18 Table 5. Comparison of the incidence of MRONJ lesions in the extraction sites 24 iv

Abstract Development of an animal model for medication related osteonecrosis of the jaw in ovariectomized rats treated with zoledronic acid before dental extractions caused by periodontal lesion Jieun Cheong Department of Dentistry The Graduate School, Yonsei University (Directed by Professor Kee-Deog Kim, D.D.S., M.S.D., Ph.D.) Bisphosphonate (BP) is a representative drug affecting bone metabolism that is widely used in treatment of osteoporosis. BPs prevent bone resorption by inhibiting the activation of osteoclasts. However, current studies have continuously reported critical side effects of such agents, mainly known as osteonecrosis of jaw (ONJ). ONJ has recently been defined as medication related osteonecrosis of the jaw including anti-resorptive or anti-angiogenic v

agents such as denosumab. But, the pathophysiology of MRONJ has not been completely elucidated and animal models constructed for the development of treatment methods in previous studies prove inadequate. The purpose of this study was to investigate the occurrence of medication related osteonecrosis of the jaw lesion after tooth extraction caused by periodontal lesion in ovariectomized female rats after administration zoledronic acid. 40 skeletal mature female Sprague-Dawley rats were randomly divided into 2 groups; zoledronic acid group (ZA group, n=20) and control group (CONT group, n=20). Bilateral ovariectomy were performed to all specimens at 12-week-old. 8 weeks after ovariectomy, the right maxillary and right mandibular first molar (M1) and second molar (M2) of all specimens were ligated with 4-0 silk. 4 weeks after tooth ligation, zoledronic acid (Zometa ready ) in ZA group and saline in CONT group were administered intravenously once a week during the next 4 weeks. 4 weeks after, ligated teeth (M1, M2) were extracted and all rats were sacrificed 8 weeks after tooth extraction. In micro-computed tomography analysis of the tibia, bone mineral density of ZA group was higher than that of CONT group. There was no significant difference in trabecular bone analysis in the extraction sites. In histologic analysis of the tibia, the proliferative zone of growth plate of ZA group was thick and there were many trabecular patterns in bone marrow were present because of the effect of zoledronic acid. The CONT group showed rare trabecular patterns and a vi

thin proliferative zone of growth plate. In the histologic analysis of the extraction site, the incidence of MRONJ lesion was significantly higher in the ZA group. Within the limitations of this study, zoledronic acid had systemic effects in ovariectomized rats and the healing of the extraction site were delayed because of the effect of zoledronic acid. Key words: Bisphosphonate, Medication-related osteonecrosis of the jaw, zoledronic acid, Tooth extraction, Ovariectomy vii

Development of an animal model for medication related osteonecrosis of the jaw in ovariectomized rats treated with zoledronic acid before dental extractions caused by periodontal lesion Jieun Cheong Department of Dentistry The Graduate School, Yonsei University (Directed by Professor Kee-Deog Kim, D.D.S., M.S.D., Ph.D.) 1. Introduction Bisphosphonate (BP) is a representative drug affecting bone metabolism that is widely used in treatment of osteoporosis. BPs prevent bone resorption by inhibiting the activation of osteoclasts. 1-3 BP agents are readily applied in clinical situations to treat bone disease in multiple myeloma and bone metastases associated with solid tumors by inhibiting bone resorption and angiogenesis. 4-6 1

The most drastic side effect of BP is osteonecrosis of jaw (ONJ). In 2003, Marx first reported on ONJ occurring after dental treatment in BP treated patients. 7 Many reports discussing the side effects associated with BP agents followed Marx s report, and BPassociated jaw necrosis was defined as bisphosphonate-related osteonecrosis of the jaw (BRONJ). 8 Recently, the term has been re-defined as medication related osteonecrosis of the jaw (MRONJ) including anti-resorptive or anti-angiogenic agents such as denosumab. 9,10 The incidence of MRONJ has been reported to be related to the duration, type and dosage of BP administration. In particular, high dose intravenous zoledronic acid (ZA) is showed to have a higher incidence of MRONJ than oral BP. 8,11 Dental causes considered as risk factors of MRONJ include surgical treatment exposing alveolar bone such as tooth extractions by periodontal or periapical disease, dental surgery such as periodontal flap surgery, or micro-endodontic surgery. The major manifestation of MRONJ is exposure of alveolar bone accompanied by pain, edema, paresthesia, suppuration and soft tissue ulceration. The most challenging part considering MRONJ is that it is hard to treat despite the presence of the best oral healthcare practice, including antibiotic therapy, parathyroid hormone therapy and surgical debridement/resection. Due to the fact that treatment of MRONJ is usually difficult and recurrence is frequent, efficient prevention of MRONJ in patients is fundamental. 2

The pathophysiology of MRONJ has not been completely elucidated and several hypotheses have been suggested in attempt to explain the mechanism of MRONJ. The majority of previous studies has used animal models that are remotely related to actual patients in clinic. In fact, most patients with MRONJ are female, old and are in menopausal status. Local inflammatory conditions such as periodontal or periapical disease 12-14 are present, and patients are usually in immunosuppressed conditions. Therefore, it is important to develop an animal model which can represent the patients situation in which both local risk factors and systemic factors exert influence to MRONJ, in order to perform an animal model experiment that can readily be applied to clinic. The purpose of this study was to investigate the occurrence of MRONJ lesion after tooth extraction caused by periodontal lesion in ovariectomized female rats with osteoporosis after administration zoledronic acid. This experiment can contribute to the process for developing the MRONJ animal model. 3

2. Materials and Methods 2.1 Animals Forty skeletal mature (12-week-old) female Sprague-Dawley rats (Orientbio Co., Ltd.; Seongnam-si, Gyenggi-do, Korea) were used in this study. The mean weight was 281 g at 12-week-old and 421 g just before sacrifice. Animals were housed at 2~3 rats per cage and were individually marked. Animals were housed under standard laboratory conditions (temperature 20 C ± 5 C, humidity 50% ± 10%, lighting cycle, 12 h light/12 h dark). All animals had ad libitum access to standard laboratory pellet diet and water. The animal selection, management, surgical protocol, and preparation followed the routine protocol approved by the Institutional Animal Care and Use Committee of Yonsei Medical Center, Seoul, Korea (IACUC No. 2016-0171). 4

2.2 Experimental design (Figure 1) Forty animals were randomly divided into 2 groups (n = 20 per group). Bilateral ovariectomy (OVX) on all animals was performed under general anesthesia by intraperitoneal injection of combined Zoletil (tiletamine and zolazepam, 50 mg/ml, 0.6 ml/kg body mass; Virbac lab. Carros, France) and Rompun (zylazine, 23.32 mg/ml, 0.4 ml/kg body mass; Bayer, Leverkusen, Germany) when the rats were 12 weeks old. After surgery, meloxicam (1 mg/kg, once a day for 5 days; Metacam, Boehringer Ingelheim, Rhein, Germany) and enfloxacin (10 mg/kg/day, once a day for 5 days, Baytril, Bayer, Germany) were subcutaneously administered. 8 weeks after, 4-0 black silk ligation was performed on the cervical portion of the right maxillary and right mandibular first molar (M1) and second molar (M2) under general anesthesia in all rats (Figure 2). 4 weeks after, the zoledronic acid group (ZA group) was administered zoledronic acid (Zometa ready, Novartis, Basel, Switzerland) and the control group (CONT group) was administered saline. 40 μg/kg body weight of zoledronic acid (ZA group) and saline (CONT group) was intravenously injected. All rats were injected once a week during the next 4 weeks. Tooth extraction was performed 4 weeks later under general anesthesia by intraperitoneal injection of combined Zoletil (tiletamine and zolazepam, 50 mg/ml, 0.6 ml/kg body mass; Virbac lab. Carros, France) and Rompun (zylazine, 23.32 mg/ml, 0.4 ml/kg body mass; Bayer, Leverkusen, Germany) in all animals. Local anesthesia was 5

administered with 2 % lidocaine (1: 80,000 epinephrine). The ligated right maxillary and mandibular M1 and M2 were extracted with a sharp dental explorer after sufficient mobility was achieved. Bleeding was controlled by gauze pressure and suture was not performed. After tooth extraction, meloxicam (1 mg/kg, once a day for 5 days; Metacam, Boehringer Ingelheim, Rhein, Germany) and enfloxacin (10 mg/kg/day, once a day for 5 days, Baytril, Bayer, Germany) were subcutaneously administered for prevention of post-surgical infection and pain control. All animals were sacrificed 8 weeks after tooth extraction. Specimens were sacrificed with perfusion after general anesthesia. After removing the skin, organs were extracted and fixed in 10% formalin. 6

Figure 1. Experimental design. ZA group is shown in the upper line and CONT group is shown in the lower line. OVX, bilateral ovariectomy; Lig, 4-0 black silk ligation of right maxilla and mandible M1, M2; ZA inj/saline inj, Zoledronic acid (Zometa ready ) injection/saline injection; Ext, tooth extraction of right maxilla and right mandible M1, M2; Sac, sacrifice. Figure 2. Clinical photo of tooth ligation for inducing periodontal lesion. 4-0 black silk ligation was performed on the cervical portion of the right maxillary and right mandibular M1 and M2 under general anesthesia in all rats. Figure shows ligated right mandibular M1 and M2. 7

2.3 Radiographic analysis: micro-computed tomography (micro-ct) To confirm bone loss induced by tooth ligation, in vivo micro-ct (NFR-Polaris- G90MVC, Nano Focus Ray, Kwangju, Korea) was performed under general anesthesia. In vivo micro-ct images were obtained at 65 kvp, 115 μa and 9 μm pixel size. In vivo micro- CT was performed immediately after ligation and 4 weeks after ligation. Ex vivo images of all specimens were obtained by micro-computed tomography (micro-ct) (Skyscan1173, Skyscan, Konitch, Belgium) at 100 kv, 100 μa and 13.9 μm pixel size in the tibia and 18.1 μm pixel size in the jaw. The images were reconstructed and analyzed via CTAn (Skyscan, Aartselaar, Belgium). Ex vivo micro-ct were taken for evaluation of the tibia and the extraction sites. To measure trabecular bone morphometry of the tibia, the region of interest (ROI) in the tibia included the growth plate and trabecular bone 3.5 mm upper to the growth plate (Figure 3). To measure the trabecular bone morphometry of the extraction site, the ROI was focused on inter-radicular bone with a 0.6 mm 0.6 mm-sized round shape in coronal view. The posterior border was the mesial root surface of the third molar, and the anterior border was the 4mm point anterior from the posterior border (Figure 4). Bone volume/tissue volume (BV/TV, %), bone surface/bone volume (BS/BV, 1/mm), bone surface/tissue volume (BS/TV, 1/mm), trabecular numbers (Tb.N, 1/mm), trabecular thickness (Tb.Th, mm), trabecular separation (Tb.Sp, mm) and bone mineral density (BMD, g/cm 2 ) of each group were assessed for trabecular bone morphometry. 8

Figure 3. The region of interest (ROI) in micro-ct analysis for trabecular bone morphometry of the tibia. Figure 4. The region of interest (ROI) in micro-ct analysis for trabecular bone morphometry of the extraction sites. 9

2.4 Microscopic analysis: Histologic analysis All specimens were decalcified with 10% EDTA and embedded in paraffin. 4 μm serial sections of coronal direction were obtained. Hematoxylin and eosin (H&E) staining were done according to the manufacturer s recommendations. The images were scanned by light microscope (Leica DM 2500, Leica Microsystem, German and Virtual microscope VS120, Olympus Corporation, Japan) and histological images were examined at 100 magnification using Caseviewer (3DHISTECH Ltd., Budapest, Hungary). 10

2.5 Microscopic analysis: Immunohistochemistry Three specimens of each group were randomly selected for anti-cd68 and anti-cd86 immunohistochemistry. Immunohistochemistry was performed only in the extraction site. Anti-CD68 antibody was used for detection of M1 macrophage and anti-cd86 antibody was used for detection of M1, M2 macrophage. Selected block sections embedded in paraffin were cut into 5 μm thickness and mounted on slides. The slides were then immersed in heat-induced sodium citrate 10 mm, ph 6.0 for 5minutes with a cooker and left in 3% H 2 O 2 for 0.5 hour at room temperature. The sections were then incubated with normal 1% bovine serum for 1 hour to block nonspecific binding of immunoglobulin, and then incubated with primary antibody at appropriate dilution (1:200) buffer for 1 hour at 37. The primary antibody used for incubation in this study were CD68 and CD86 (Dako, Dopenhage, Denmark). The sections were next incubated with secondary antibody for 0.5 hour at 37. Envision System Plus HRP (Dako ) (ready to use) was utilized for the secondary antibody incubation. Counterstaining was done with Hematoxylin. The images were scanned by light microscope (Leica DM 2500, Leica Microsystem, German and Virtual microscope VS120, Olympus Corporation, Japan) and histological images were examined at 100 magnification using Caseviewer (3DHISTECH Ltd., Budapest, Hungary). 11

2.6 Statistical analysis Statistical analysis was performed using a commercially available software program (IBM SPSS 23.0, IBM Corp., Armonk, NY, USA). Mann-Whitney U test and Chi-squared test were used to compare parameters between control group and experimental group. Statistical significance was set at P<0.05. 12

3. Results 3.1 Radiographic analysis: micro-computed tomography (micro-ct) 7 rats in CONT group and 9 rats in ZA group deceased during the experiment. As a result, 13 and 11 rats in CONT groups and ZA group were analyzed, respectively. Both ZA group and CONT groups showed alveolar bone resorption and furcation involvement in the ligated right maxillary and right mandibular M1 and M2 when compared to the left side without ligation. Figure 5 shows the 3D micro-ct reconstructed images of the left nonligation site and right periodontal lesion induced area. Figure 6 shows the difference in the tibia between two groups after sacrifice. In the CONT group, a thin and low-density growth plate was observed, while a thick high-density growth plate and high-density trabecular patterns were observed in ZA group. Also, regarding the cortical bone of the tibia, ZA group showed higher bone mineral density than the CONT group. Table 1 shows micro-ct analysis of trabecular bone in the tibia. ZA group showed higher value for BV/TV, BS/TV, Tb.N and BMD than CONT group with statistically significant difference (P<0.05). Mann-Whitney U test was used for comparing all parameters between CONT group and ZA group. 13

Figure 5. In vivo micro-ct 3D reconstructed images of the mandible at the buccal side. Images taken immediately after ligation, on the left mandible (a) and right mandible (c). 4 weeks after ligation, little change is found on the left (b) yet alveolar bone resorption and furcation involvement around M1, M2 area are observed on the right (d). (A, anterior; P, posterior) Figure 6. Micro-CT images of the tibia. Two-dimensional colored ex vivo micro-ct image of proximal part of tibia. Bone mineral density is expressed as a color scale. The white arrow indicates thin and low-density growth plate in CONT group (a). The growth plate of ZA group showed thicker than CONT group and shows more trabecular patterns (b). 14

Table 1. Micro-CT analysis of trabecular bone in the tibia. BV/TV BS/BV BS/TV Tb.Th Tb.N Tb.Sp BMD CONT 13.81±1.83 39.18±3.41 5.41±0.85 0.1±0.01 1.39±0.24 0.57±0.12 0.32±0.03 ZA 31.21±22.9 32.97±13.4 7.62±1.83 0.14±0.08 2.03±0.62 0.42±0.15 0.52±0.02 p value 0.028* 0.798 0.010* 0.574 0.010* 0.065 0.002* Mean and standard deviation (SD) of all parameters. BV/TV, Bone volume/tissue volume (%); BS/BV, bone surface/bone volume (mm 2 /mm 3 ); BS/TV, bone surface/tissue volume (mm 2 /mm 3 ); Tb.N, trabecular numbers (1/mm); Tb.Th, trabecular thickness (mm); Tb.Sp, trabecular separation (mm); BMD, bone mineral density (g/cm 2 ) * Significant differences compared CONT group and ZA group (P value < 0.05) 15

Figure 7 shows the healing status of the extraction site in the axial view. Different healing patterns were present between the ZA group and CONT group. CONT group showed almost complete healing of the extraction socket, whereas ZA group showed partial healing and delayed healing. Table 2 shows micro-ct analysis of trabecular bone in the extraction site. There was no significant difference between the two groups. Table 3, 4 shows micro-ct analysis of the extraction site in maxilla and mandible, respectively. There was a significant difference in value of Tb.Th in maxilla (P<0.05). However, there was no remarkable difference in Tb.Th mean value (Table 3). There was a significant difference in value of BV/TV in mandible (P<0.05) (Table 4). Mann-Whitney U test was used for comparing all parameters between the CONT group and ZA group. 16

Figure 7. Micro-CT images of the extraction site in the axial view. Bony healing status of the extraction site was observed. CONT group showed almost complete healing of the extraction socket (a, white dashed box), whereas ZA group showed delayed healing (b, white dashed box). (A, anterior; P, posterior; red arrow, M3 root; white asterisk, incisor root space) 17

Table 2. Micro-CT analysis of trabecular bone in the extraction site. BV/TV BS/BV BS/TV Tb.Th Tb.N Tb.Sp CONT 18.20±20.43 45.23±59.37 4.00±3.82 0.15±0.66 0.97±0.96 0.44±0.16 ZA 31.27±17.37 20.66±5.90 5.71±2.11 0.24±0.08 1.26±0.48 0.36±0.08 P value 0.541 0.584 0.548 0.841 0.310 0.679 Mean and standard deviation (SD) of all parameters. BV/TV, Bone volume/tissue volume (%); BS/BV, bone surface/bone volume (mm 2 /mm 3 ); BS/TV, bone surface/tissue volume (mm 2 /mm 3 ); Tb.N, trabecular numbers (1/mm); Tb.Th, trabecular thickness (mm); Tb.Sp, trabecular separation (mm) * Significant differences compared CONT group and ZA group (P value < 0.05) Table 3. Micro-CT analysis of trabecular bone in the extraction site of maxilla. BV/TV BS/BV BS/TV Tb.Th Tb.N Tb.Sp CONT 21.47±16.86 23.49±10.43 4.72±2.84 0.24±0.23 1.21±0.67 0.46±0.23 ZA 24.28±12.60 20.40±2.47 4.76±2.11 0.24±0.07 1.02±0.48 0.41±0.08 P value 0.875 0.313 1.000 0.042* 0.713 0.958 Table 4. Micro-CT analysis of trabecular bone in the extraction site of mandible. BV/TV BS/BV BS/TV Tb.Th Tb.N Tb.Sp CONT 35.55±22.12 64.74±80.84 2.93±4.57 0.13±0.07 0.71±1.13 0.49±0.20 ZA 12.89±23.53 21.03±8.40 6.16±2.41 0.24±0.10 1.40±0.52 0.33±0.10 P value 0.04* 0.055 0.099 0.055 0.075 0.165 18

3.2 Microscopic analysis: Histologic analysis Figure 8, 9 shows the histological comparison between the two groups at the proximal growth plate of tibia. The proliferative zone of growth plate of CONT group appeared thin and trabecular pattern was not distinct (Figure 8). ZA group showed a thick proliferative zone of growth plate and many trabecular patterns in bone marrow were visible (Figure 9). Normal bone healing status with completely healed epithelium in extraction site of CONT group was present. Normal osteocytes were observed in CONT group (Figure 10), whereas abnormal empty osteocytes in necrotic bone and inflammation infiltration were observed in extraction socket of ZA group (Figure 11). 19

Figure 8. Histologic analysis in the tibia of CONT group. Longitudinal cross-section image of the proximal tibia stained with H&E. 50 magnification. Scale bar: 400μm. Thin proliferative zone of growth plate (black double arrow) of CONT is shown (a) and the trabecular pattern is not clear (b). 20

Figure 9. Histologic analysis in the tibia of ZA group. Longitudinal cross-section image of the proximal tibia stained with H&E. 50 magnification. Scale bar: 400μm. Thicker proliferative zone of growth plate (black double arrow) of ZA group than that of CONT group (a). Many trabecular patterns (black arrowhead) in bone marrow are visible (b). 21

Figure 10. Histologic analysis in the extraction site of CONT group. Histologic findings in extraction site stained with H&E staining. Normally healed epithelium (black arrow) and normal bone healing state (black asterisk) are observed (a). ( 50 magnification. Scale bar: 400μm in (a)) The inside of the black dashed box of (a) is enlarged in (b). Normal bone healing (black asterisk) and normal osteocytes (black arrowhead) are observed (b). Yellow arrowhead shows reversal line that occurs during bone formation (b). ( 200 magnification. Scale bar: 100μm in (b)) 22

Figure 11. Histologic analysis in the extraction site of ZA group. Delayed healing of epithelium (black arrow) and necrotic bone under the lesion (black asterisk) are observed in (a). ( 100 magnification. Scale bar: 200μm in (a)) Extraction site of ZA group showed inflammation infiltration (red asterisk), necrotic bone (black asterisk) and empty osteocytes (black arrowhead) in necrotic bone (b). ( 200 magnification. Scale bar: 100μm in (b)) 23

Table 5 shows the incidence of MRONJ lesions based on histological criteria. MRONJ lesions were diagnosed based on following histological criteria in the experimental condition. 8,15 In the ZA group, 68.1% of all extraction sites showed a lesion, while in the CONT group, the incidence of MRONJ lesions was 7.7%. Compared with the two groups, the incidence of MRONJ lesions was significantly higher in the ZA group than in the CONT group (P<0.05). Chi-squared test was used for comparison between CONT group and ZA group. Table 5. Comparison of the incidence of MRONJ lesions in the extraction sites Exposed & necrotic bone Inflammatory cell infiltration Sequestrum Sites with all lesion /Total sites Incidence (%) Chi-squared test (P< 0.05) CONT 6 12 2 2/26 7.7% ZA 20 21 17 15/22 68.1% 0.000* 24

3.3 Microscopic analysis: Immunohistochemistry Figure 12 shows immunohistochemical staining for anti-cd68 and anti-cd86 in the CONT group, and Figure 13 shows immunohistochemical staining for Anti-CD68 and anti- CD86 in the ZA group. In ZA group, more CD68 positive cells were found within the trabecular bone and soft tissues than in CONT group. More CD86 positive cells were also found in the ZA group rather than in the CONT group. The difference between CONT group and ZA group was more pronounced in CD68 than in CD86 (Figure 14). 25

Figure 12. Immunohistochemical staining for anti-cd68 (a, b) in CONT group. 50 magnification. Scale bar: 200μm in (a) 200 magnification. Scale bar: 50μm in (b). 26

Figure 12. (continued) Immunohistochemical staining for anti-cd86 (c, d) in CONT group. 50 magnification. Scale bar: 200μm in (c). 200 magnification. Scale bar: 50μm in (d). 27

Figure 13. Immunohistochemical staining for anti-cd68 (a, b) in ZA group. 50 magnification. Scale bar: 200μm in (a). 200 magnification. Scale bar: 50μm in (b). 28

Figure 13. (continued) Immunohistochemical staining for anti-cd86 (c, d) in ZA group. 50 magnification. Scale bar: 200μm in (c). 200 magnification. Scale bar: 50μm in (d). 29

Figure 14. Comparison of anti-cd68 (a, b) immunohistochemical staining between two groups. 200 magnification. Scale bar: 50μm. CONT group in (a) and ZA group in (b) 30

Figure 14. (continued) Comparison of anti-cd86 (c, d) immunohistochemical staining between two groups. 200 magnification. Scale bar: 50μm. The difference between CONT group (c) and ZA group (d) is more pronounced in CD68 (a, b) than in CD86. 31

4. Discussion In this study, the occurrence of MRONJ lesion after tooth extraction caused by periodontal lesion in ovariectomized female rats treated with ZA was investigated. Zoledronic acid induced systemic effects in ovariectomized rats and thus the healing after tooth extraction caused by periodontal lesion was delayed in rats administered with zoledronic acid. Micro-CT analysis of the tibia showed a significant difference in the ZA group compared to CONT group, indicating that ZA is systemically effective. This was also evident in histologic analysis of the tibia of the ZA group, showing a thicker proliferative zone of the growth plate and more trabecular pattern compared to the CONT group. Although micro-ct analysis of trabecular bone in the extraction site showed no significant difference statistically, delayed healing of the extraction sites was observed in the ZA group in micro-ct images and histologic analysis. The incidence of MRONJ lesions was significantly higher in the ZA group than in CONT group. 12-week-old adult female rats with skeletal maturity were used and all specimens were waited for 8 more weeks after OVX before inducing periodontal lesion to mimic a situation similar to that of a postmenopausal women's general condition. The rat model with osteoporosis induced by ovariectomy (OVX) had been used in many MRONJ studies, and by those studies had been reported that 8 weeks after OVX were sufficient to induce osteoporosis. Bone loss occurs in estrogen deficient environment due to enhanced bone resorption and impaired osteoblast function. 16-18 32

Periodontal disease is one of the most common causes of tooth extraction. MRONJ in patients appear after tooth extraction that cannot be restored owing to periapical infection with advanced dental caries or periodontal disease. 19-21 Inducement of periodontal lesion was attempted by placing tooth ligation on the cervical portion for 8 weeks before tooth extraction. Duarte et al. reported bacterial species commonly observed in humans were found in biofilm around ligature 42 days after ligation in rats, and Liu et al. reported 28- day ligature could cause significant loss in the trabecular of alveolar bone in rats. 22,23 Therefore, tooth ligation was kept for 8 weeks and it was judging the duration to be long enough to cause periodontal lesion. In vivo 3D micro-ct reconstructed images (Figure 5) showed that the 8-week ligation induced periodontal lesion. Alveolar bone resorption and furcation involvement around M1, M2 area were observed in all specimens. However, there was no visible gingival inflammation or mobility of teeth 8 weeks after ligation, which was an unexpected outcome. The amount of alveolar bone resorption was also less than expected. This can be explained by rat s rapid metabolism of the rat and remarkable ability to regenerate. Many previous studies have attempted to overcome the limitations of tooth ligation used to induce periodontal disease for animal model in various ways. 24,25 It is necessary to include experimental data using additionally different methods of in causing periodontal disease. In this study, ZA was administered intravenously. Previous studies have shown that intravenous administration of ZA in patients increases the risk of MRONJ. The risk of MRONJ development for patients on intravenous BPs is variously estimated at between 1% 33

and 11%. 26,27 The incidence of MRONJ has varied across reports (66-95%) according to the dose and duration of ZA injection. 28-30 Patients receive ZA intravenously because intravenous ZA administration is absorbed better than oral BP and has advantages in patients who do not tolerate or adhere to oral BPs, including those with gastrointestinal pathology, polypharmacy, or the inability to remain upright. 31 However, several previous experiments of MRONJ in animal models had injected ZA via intraperitoneal injection or subcutaneous injection. 32 Due to the fact that rodents have relatively rapid bone metabolism compared to humans, intravenously injected ZA can be released from the body more quickly. For this reason, the effects of ZA may have been less pronounced at the extraction site. In this study, 40 μg/kg body weight of ZA was administered once a week for 4 weeks. Previous studies have shown that intraperitoneal injection of 66 μg/kg ZA 3 times per week is the most common method for inducing MRONJ. 15,33-35 Gasser et al. reported the minimum intravenous dose of zoledronic acid that fully blocks ovariectomy-induced bone loss in adult rats is 8 µg/kg for 4 weeks. 36 It was expected in this study that the effect would be stronger when administered intravenously compared to other methods of administration. Thus, the dose of ZA was reduced compared to that of intraperitoneal or subcutaneous injection. At the same time, the dose of ZA was considered to be sufficient to induce MRONJ lesion and was administered and confirmed in our pilot study. As above, the intravenous dose of ZA was determined by reference to previous studies and our pilot study. 34

In micro-ct images of the extraction site, the ZA group showed delayed healing (Figure 7), but trabecular bone morphometry showed no statistically significant difference between the two groups. This is because the effect of ZA is more pronounced in cortical bone, as observed in the micro-ct images of the tibia (Figure 6). Another reason is the incidence of MRONJ lesion in CONT group. Because some specimens showed MRONJ lesion in CONT group only histologically, the difference between two groups could not be recognized in micro-ct analysis. Diagnosis of MRONJ was done according to a histological criteria in the extraction site. This was because there were MRONJ lesions in some rat specimens that could not be clinically found and only observed in microscopic analysis. Though the definition of MRONJ is based on macroscopic appearance in the clinic, MRONJ lesions were diagnosed based on following histological criteria in the experimental condition (1) presence of ulcerative lesion with exposed and necrotic bone and/or osteolysis, (2) presence of pseudoepitheliomatous-like hyperplasia of the epithelium accompanied by inflammatory cell infiltration, and (3) presence of sequestrum and bacterial colonies. 8,15 The incidence of MRONJ lesions in the ZA group was significantly higher than that in the CONT group. On the other hand, the incidence of 7.7% in the CONT group was unexpected. The reasons for these results are suggested to be the general condition of rats and the rats root morphology. The rats used in this experiment can be regarded as immunosuppressed animals because they represent postmenopausal women with osteoporosis. Therefore, the extraction site of CONT group may also have shown 35

inflammatory findings. Immunohistochemistry showed more CD68 positive cells in ZA group than in CONT group. However, in the CONT group, CD68 positive cells were easily observed in the soft tissues. This is probably related to unexpected incidence of MRONJ lesions in the CONT group. The association of immune-related cells with MRONJ has been reported in recent studies. 37 Nevertheless, more studies are needed to show that MRONJ is dependent on immune modulation. Another reason is the morphological characteristics of rat molars. Rat molars have more divergent roots than those of humans, resulting in more chances to leave root rests. Root rests may affect healing of the extraction sites. Following this study, a comparison between the incidence of MRONJ lesion between ligated tooth and non-ligated tooth is necessary. To investigate whether the periodontal disease-induced extraction is a risk factor for MRONJ, ligated tooth and non-ligated tooth will be extracted simultaneously in one specimen and we will be analyzed to compare the differences in the extraction sites. The ligation period and dose of ZA should be further be adjusted by additional experiments. In the future, it is necessary to study the experimental design reflecting the withdrawal period before and after the extraction to simulate the patient's situation. 36

5. Conclusion In conclusion, zoledronic acid inflicted systemic effects on ovariectomized rats of zoledronic acid group. Micro-CT analysis on the rat tibia of the ZA group showed significant differences compared to the control group. Although the result of micro-ct analysis of trabecular bone in the extraction site showed no statistical significance between the two groups, histologic analysis and micro-ct images showed delayed healing in extraction site of the ZA group. Considering the immunohistochemistry analysis of the extraction socket, the ZA group showed a significantly enhanced number of macrophages compared to the CONT group. The incidence of MRONJ lesion in histologic analysis was significantly higher in ZA group than in CONT group. 37

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국문요약 Zoledronic acid 를투여한폐경기골다공증백서에서 치주병변에의한발치후발생하는 MRONJ (Medication related osteonecrosis of the jaw) 모델개발 연세대학교대학원치의학과 정지은 지도교수 : 김기덕 비스포스포네이트 (Bisphosphonate, BP) 는골다공증치료에널리쓰이는대표적인 골대사약물이다. BP 는파골세포의활성화를방해하여골흡수를막는다. 이러한 BP 의가 장큰부작용은악골괴사인데, 최근에는항흡수성약물과항혈관성약물을포함하여 Medication-related osteonecrosis of jaw (MRONJ) 로정의하고있다. 하지만 MRONJ 의 발생기전은명확히밝혀지지않았으며적절한치료방법개발을위한동물모델개발도 정확히이루어지지않은상태이다. 본연구의목적은 BP 의일종인 zoledronic acid 가투여된골다공증이유발된쥐에 42

서치주병변이유발된치아를발치하고 MORNJ 의발생을관찰함으로써이후연구에서 유용하게사용될 MRONJ 동물모델의개발하기위함이다. 12 주령의골성숙쥐 40 마리를모두난소절제술하였다. 이후 8 주의기간을주어 골다공증을유발하였다. 난소절제술 8 주뒤우측상, 하악제 1 대구치와제 2 대구치에 4-0 black silk 를이용하여치경부에결찰하였다. 결찰 4 주후, 대조군 20 마리, 실험군 20 마리로나누어대조군에서는생리식염수, 실험군에서는 zoledronic acid (ZA) 를 4 주 간일주일에한번정맥투여하였다. 4 주간투여후에는결찰했던우측상, 하악제 1, 2 대구치를발치하였다. 경골의방사선사진관찰에서실험군은대조군에비해 ZA 의효과로높은골밀도를 보였으며방사선사진정량분석에서도유의한차이로높은골밀도를보였다. 발치와에서 방사선사진정량분석에서는유의한차이가나타난값은없었으나방사선사진관찰에 서뚜렷한치유양상의차이를보였다. 대조군은정상적인치유가일어난데반해실험군 은발치와치유가지연되는양상을보였다. 경골의조직사진관찰에서, 대조군에서는얇은성장판과골소주의소실이관찰되었 으나실험군에서는두꺼운성장판과많은골소주를나타내었다. 발치와에서는대조군에 43

서정상적인상피치유와골세포를보인반면실험군에서골괴사, 염증세포침윤, 상피 세포치유지연, 핵이소실된골세포등의 MRONJ 의특징을나타내는조직학적특징이 관찰되었다. 조직학적기준에따라 MRONJ 의특성을보이는병소발생률분석결과실 험군에서대조군에비해통계적으로유의한차이로높게나타났다. 결론적으로본실험을통하여 zoledronic acid 의정맥투여로전신적인효과가있 었으며치주병소로인해발치한발치와의치유가지연된다는것을확인하였다. 핵심되는말 : 비스포스포네이트, 약물관련악골괴사, zoledronic acid, 발치, 난소절제술 44