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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

[UCI]I804:11046-000000517063[UCI]I804:11046-000000517063





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

감사의 글

1991년 초등학교 1학년 입학을 시작으로 박사 논문을 마무리하는 지금까지

23년간 대장정의 학업을 끝마치는 데 정말 많은 분들의 도움과 격려가 있었습니

다. 가장 먼저, 통합치의학과에서 수련을 시작할 때부터 존경해 온 김기덕 지도

교수님의 가르침에 진심으로 감사드립니다. 진료와 연구로 바쁘신 와중에도 저

의 박사 논문을 위해 혼신의 힘을 쏟아 주신 박원서 교수님, 통합치의학과를 이

끌어 주시느라 온 힘을 다하고 계신 정복영 교수님께 감사드립니다. 직접 조직

사진을 보며 지도해 주시고 부족했던 논문임에도 아낌없이 조언을 주셨던 김현

실 교수님, 제가 생각지 못했던 부분을 정확하고 꼼꼼하고 날카롭게 검토해 주

셨던 이중석 교수님께 감사드립니다. 위 네 분의 심사위원 교수님들께 진심으로

감사드립니다. 못난 제자가 지치고 힘들어 하소연을 할 때 항상 곁에서 응원과

칭찬을 아끼지 않으셨던 방난심 교수님께도 말로 전할 수 없는 감사를 드립니다.

연구에 대한 열정과 탁월한 능력으로 제 논문을 함께 쓴 것이나 다름없는

박경미 연구원, 연구를 위해 언제나 무슨 일이든 흔쾌히 도와준 김서율 연구원,

최이슬 연구원께 감사드립니다. 박사 기간 내내 구체적이고 세심한 조언을 해주

셨던 임지영 선생님, 힘든 일이 있을 때 마다 공감해주시고 격려해 주셨던 이강

희 선생님께 감사합니다. 영어를 잘하는 탓에 고생한 양수진 선생님 감사합니다.

병원에서 늘 저의 마음의 안식처가 되어 주는 변혜덕 선생님 감사합니다.

자주 보지도 못하면서 어쩌다가 만날 때면 바쁘고 힘들다고 늘 투정만 부리

는 딸의 박사 졸업을 묵묵히 기다려 주신 엄마, 아빠에게 사랑과 감사를 전합니

다. 연락도 자주 드리지 못한 못난 며느리임에도 볼 때 마다 제 걱정을 먼저 해

주시는 어머님, 아버님 그리고 형님께도 감사드립니다. 형제 자매가 없는 저에게

모든 것을 포기하고 싶은 순간 항상 위로가 되고 용기를 주는 가족과 같은 친구

들, 고맙고 사랑한다! 누구보다도 같은 길을 가는 치과의사로서, 통합치의학과

의국 동문으로서, 평생을 함께할 인생의 동반자로서 논문을 시작할 때부터 끝나

는 순간까지 제 곁에서 모든 것을 감수하고 응원해주며 인내해 준 사랑하는 남

편 김병노, 이 모든 사랑하는 가족들과 통합치의학과 식구들에게 제 논문을 바

칩니다.

2018년 6월

정 지 은

i

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

ii

5. Conclusion ························································································ 37

6. References ························································································ 38

Abstract (in Korean) ················································································ 42

iii

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


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

iv

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

v

Abstract





Jieun Cheong


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

vi

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

vii

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

1





Jieun Cheong


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

2

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 BP-

associated 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.

3

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 disease12-14 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.

4

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).

5

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

6

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.

7

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.

8

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/cm2) of each group were assessed for trabecular bone morphometry.

9


morphometry of the tibia.


morphometry of the extraction sites.

10

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).

11

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% H2O2 for 0.5 hour at room temperature.

The sections were then incubated with normal 1% bovine serum for 1 hour to block non-

specific 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).

12

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.

13

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 non-

ligation 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.

14

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).

15

Table 1. Micro-CT analysis of trabecular bone in the tibia.

Mean and standard deviation (SD) of all parameters. BV/TV, Bone volume/tissue volume

(%); BS/BV, bone surface/bone volume (mm2/mm3); BS/TV, bone surface/tissue volume

(mm2/mm3); Tb.N, trabecular numbers (1/mm); Tb.Th, trabecular thickness (mm); Tb.Sp,

trabecular separation (mm); BMD, bone mineral density (g/cm2)

* Significant differences compared CONT group and ZA group (P value < 0.05)

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*

16

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.

17

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)

18

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 (mm2/mm3); BS/TV, bone surface/tissue volume

(mm2/mm3); 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.


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.


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

19

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).

20

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).

21

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).

22

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))

23

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))

24

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% 0.000*

ZA 20 21 17 15/22 68.1%

25

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).

26

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).

27

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).

28

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).

29

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).

30

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)

31

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.

32

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

33

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%

34

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.

35

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 pseudo-

epitheliomatous-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

36

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.

37

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.

38

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42

국문 요약

Zoledronic acid를 투여한 폐경기 골다공증 백서에서

치주병변에 의한 발치 후 발생하는

MRONJ (Medication related osteonecrosis of the jaw) 모델 개발

연세대학교 대학원 치의학과

정 지 은

지도 교수 : 김 기 덕

비스포스포네이트 (Bisphosphonate, BP)는 골다공증 치료에 널리 쓰이는 대표적인

골대사 약물이다. BP는 파골세포의 활성화를 방해하여 골흡수를 막는다. 이러한 BP의 가

장 큰 부작용은 악골 괴사인데, 최근에는 항흡수성 약물과 항혈관성 약물을 포함하여

Medication-related osteonecrosis of jaw (MRONJ)로 정의하고 있다. 하지만 MRONJ의

발생기전은 명확히 밝혀지지 않았으며 적절한 치료 방법 개발을 위한 동물 모델 개발도

정확히 이루어지지 않은 상태이다.

본 연구의 목적은 BP의 일종인 zoledronic acid가 투여된 골다공증이 유발된 쥐에

43

서 치주 병변이 유발된 치아를 발치하고 MORNJ의 발생을 관찰함으로써 이후 연구에서

유용하게 사용될 MRONJ 동물 모델의 개발하기 위함이다.

12주령의 골성숙 쥐 40마리를 모두 난소 절제술 하였다. 이후 8주의 기간을 주어

골다공증을 유발하였다. 난소절제술 8주 뒤 우측 상, 하악 제 1 대구치와 제 2 대구치에

4-0 black silk를 이용하여 치경부에 결찰 하였다. 결찰 4주 후, 대조군 20마리, 실험군

20마리로 나누어 대조군에서는 생리 식염수, 실험군에서는 zoledronic acid (ZA)를 4주

간 일주일에 한 번 정맥 투여하였다. 4주간 투여 후에는 결찰 했던 우측 상, 하악 제 1,

2 대구치를 발치 하였다.

경골의 방사선 사진 관찰에서 실험군은 대조군에 비해 ZA의 효과로 높은 골밀도를

보였으며 방사선 사진 정량분석에서도 유의한 차이로 높은 골밀도를 보였다. 발치와에서

방사선 사진 정량분석에서는 유의한 차이가 나타난 값은 없었으나 방사선 사진 관찰에

서 뚜렷한 치유 양상의 차이를 보였다. 대조군은 정상적인 치유가 일어난데 반해 실험군

은 발치와 치유가 지연되는 양상을 보였다.

경골의 조직 사진 관찰에서, 대조군에서는 얇은 성장판과 골소주의 소실이 관찰되었

으나 실험군에서는 두꺼운 성장판과 많은 골소주를 나타내었다. 발치와에서는 대조군에

44

서 정상적인 상피 치유와 골세포를 보인 반면 실험군에서 골괴사, 염증 세포 침윤, 상피

세포 치유 지연, 핵이 소실된 골세포 등의 MRONJ의 특징을 나타내는 조직학적 특징이

관찰되었다. 조직학적 기준에 따라 MRONJ의 특성을 보이는 병소 발생률 분석 결과 실

험군에서 대조군에 비해 통계적으로 유의한 차이로 높게 나타났다.

결론적으로 본 실험을 통하여 zoledronic acid의 정맥 투여로 전신적인 효과가 있

었으며 치주 병소로 인해 발치한 발치와의 치유가 지연된다는 것을 확인하였다.

핵심되는 말: 비스포스포네이트, 약물관련 악골괴사, zoledronic acid, 발치, 난소절제술

disclaimer - yonsei university · 2019-06-28 · the pathophysiology of mronj has not been...

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