Animal model for medication-related osteonecrosis of the jaw with precedent metabolic bone disease

    Animal model for Medication-related Osteonecrosis of the Jaw with Precedent Metabolic Bone Disease Jin-Woo Kim, Jacquiline Tatad, Erika Landayan, Sun-Jong Kim, Myung-Rae Kim PII: DOI: Reference:

S8756-3282(15)00323-3 doi: 10.1016/j.bone.2015.08.012 BON 10839

To appear in:

Bone

Received date: Accepted date:

21 June 2015 14 August 2015

Please cite this article as: Kim Jin-Woo, Tatad Jacquiline, Landayan Erika, Kim SunJong, Kim Myung-Rae, Animal model for Medication-related Osteonecrosis of the Jaw with Precedent Metabolic Bone Disease, Bone (2015), doi: 10.1016/j.bone.2015.08.012

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Animal model for Medication-related Osteonecrosis of the Jaw with

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Precedent Metabolic Bone Disease

Jin-Woo Kim, DDS, MSD, PhD

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Jacquiline Tatad, DDS, MSD Erika Landayan, DDS, MSD Sun-Jong Kim, DDS, MSD, PhD

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Myung-Rae Kim, DDS, MSD, PhD

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Graduate School of Clinical Implant Dentistry, Ewha Womans University, Seoul, Korea; Research Institute for Intractable Osteonecrosis of the Jaw, School of Medicine, Ewha

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Womans University, Seoul, Korea

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*JW Kim and J Tatad contributed equally to this study.

Corresponding authors Sun-Jong Kim, DDS, MSD, PhD Professor Department of Oral & Maxillofacial Surgery Ewha Womans University Medical Center Anyangcheon-ro 1071, Yangcheon-gu, Seoul, 158-710, Korea Tel: 82-2-2650-2720, Fax: 82-2-2650-2754 E-mail: [email protected]

ACCEPTED MANUSCRIPT Abstract

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Despite the fact that the medications used to treat abnormal bone conditions often induce

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osteonecrosis of the jaw (ONJ), previous attempts to establish an animal model for ONJ have shown insufficient consideration for this important prerequisite for the development of the disease. The

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purpose of this study was to establish an animal model with the most common metabolic bone disease, osteoporosis.

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Ninty-six rats were randomly divided into ovariectomy (Ov) group (n=48) and sham-operated group (n=48). Six weeks after Ov or sham surgery, rats in each group were subdivided into

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bisphosphonate group (n=36 each) and control group (n=12 each) and injected with zoledronic acid and normal saline, respectively, once a week. After additional 6 weeks, surgical intervention was

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performed, and the injections were continued for 8 more weeks. The animals were then sacrificed for

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further macroscopic, histological, histomorphometric, radiological, and bone biomarker investigations.

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As histologically determined, the Ov group (77.8%) showed higher ONJ prevalence compared to the sham group (47.2%; P<0.05). Micro-structural and histomorphometric assessments revealed that rats with ONJ (ONJ group) presented with deteriorated bone architectures with higher

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necrotic bone fraction and lower number of osteoclasts (P<0.05). Compared to the sham-operated ONJ group, the Ov ONJ group showed significantly lower values of Tb.N, Tb.Sp, Conn.D, N.Oc/T.Ar, and TRACP 5b and CTX/TRACP (P<0.05). The ovariectomized rat model in this study successfully mimicked human ONJ lesions with an underlying bone disease and showed different bone characteristics than that of the previous ONJ model. Based on the differences, further researches for investigating pathophysiology of ONJ, including various pharmacological responses for deteriorated bone environment, are required. Keywords: Medication-related osteonecrosis of the jaw; Osteoporosis; Ovariectomy; Bisphosphonates; Animal model

ACCEPTED MANUSCRIPT Introduction

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Antiresorptive medications have been widely used for the non-hormonal treatment of osteoporosis as

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well as for cancer metastasis and other various bone diseases. [1] However, a serious complication associated with these antiresorptive administrations, osteonecrosis of the jaws (ONJ), has been

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reported several years ago. [2] Although there have been many efforts to establish the pathophysiologic nature of this disease, its pathogenesis is still poorly understood. [3-5] As part of

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this effort, ONJ-like lesions have been induced in animals with the administration of anti-resorptive medications and surgical interventions, yet showed controversial results since experimental induction

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and manipulation of disease progression do not always reflect clinical reality. [6-8] Medication-related ONJ (MRONJ) has been observed in patients with a primary diagnosis of various

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bone diseases such as osteoporosis, bone metastatic cancer, multiple myeloma, and etc. These pre-

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existing abnormal bone conditions not only change skeletal microarchitecture and physiologic characteristics of hard tissues, but also the pharmacological responses to therapeutic medications like

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antiresorptive medications.

For mimicking a model for post-menopausal osteoporosis in humans, ovariectomized animal models have effectively been utilized. [9-11] Ovariectomy induced sex-hormone deficiency stimulated the

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accelerated loss of bone that occurs in women following menopause, in addition to increased rate of bone turnover with resorption exceeding formation and greater loss of cancellous bone than cortical bone with decreased intestinal calcium resorption. [10] Also, the model presented similar skeletal response to medications, which was different from those of normal bone conditions. [10, 12] Since MRONJ has been thought to be associated mainly with the oversuppression of bone remodeling based on the pharmacological effects of antiresorptives [3-5], reflection of altered bone physiology and responses to medications due to the underlying bone diseases are thought to be important. Thus, the authors hypothesized that previous efforts to develop MRONJ animal model have shown insufficient consideration for an important prerequisite, that is, different physiological and pharmacological tissue responses to due to precedent abnormalities of bone. Therefore, this study aimed to establish an MRONJ animal model with osteoporosis, a representative metabolic bone

ACCEPTED MANUSCRIPT disease, using clinical, radiological, and histological measures and compare with the conventional

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MRONJ animal model.

ACCEPTED MANUSCRIPT Materials and Methods

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Animal and Study design

from 273

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Female Sprague-Dawley (SD) rats (n = 96) at 16-weeks of age with mean weight of 308g (ranging to 347g) were purchased from the Institute of Ewha Medical Research (Seoul, Korea) and

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used in this study. The cages were placed in a room with filtered air at a temperature of 22 ± 2°C and 50% ± 10% relative humidity. A 12-h light/dark cycle was maintained. The animals were fed a

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normal rodent diet and water ad libitum. and acclimated for 1 week prior to the beginning of the investigation. This study was approved by and performed in accordance with the guidelines of the

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institutional animal research ethics committee.

After acclimation, the animals were randomly divided into ovariectomy (Ov) group (n = 48) and

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sham-operated (Sh) group (n = 48; Figure 1). For the animals subjected to ovariectomy, the bilateral

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ovaries were resected, while the ovaries of Sh rats were dissected and returned to their original positions. The rats of each group were further divided into bisphosphonate group (n = 36 each), that

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were injected subcutaneously once a week with zoledronic acid (100 ug/kg, Zometa, Novartis Pharma AG, Basel, Switzerland), and the control group (n = 12) that were injected with normal saline (0.9 % NaCl) once a week. [6, 13, 14] Weekly injections were administered till the day of euthanasia. Given

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that the pharmacodynamics of bisphosphonates have not been determined in rodents, the authors determined dosing according to the following factors[14]: (1) the oncologically relevant zoledronate doses in humans (67 μg/kg/4 weeks); (2) the relatively rapid bone metabolism of rodents; (3) the route of bisphosphonate administration; lower plasma concentrations when using the subcutaneous route compared to intravenous route; and (4) maximizing drug exposure during the relatively short experimental period.

Surgical Intervention: Tooth Extraction After 6 weeks of injection, tooth extraction was performed as a surgical intervention to induce ONJ. Animals were anesthetized with an intramuscular injection of ketamine (100 mg/kg ketamine, Yuhan Pharmacy, Seoul, Korea), xylazine (10 mg/kg Rompun, Bayer AG, Leverkusen, Germany), and

ACCEPTED MANUSCRIPT meloxicam (1.5 mg/kg Metacam, Boehringer Ingelheim, Germany). After syndesmotomy using a sharpened dental explorer, adequate luxation was performed until sufficient mobility of the tooth was

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achieved. In this manner, the lower left first to third molars were extracted using dental explorer in a

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consecutive order. Gauze pressure was applied to control bleeding, and the wounds were left open

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without any sutures. Weekly injections were continued for 8 more weeks, a timeframe chosen according to the pathogenic progression of MRONJ [15]. The animals were then sacrificed for further

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assessment.

Macroscopic and Radiographic Analysis

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Following euthanasia, photographs of each tissue section were taken for documentation. The entire dissected mandible underwent microarchitectural assessment using microcomputed tomography (μCT,

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SKyscan 1076, Aartselaar, Belgium). The acquisition settings were at a voxel size of 6 µm3 with an

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X-ray tube voltage of 70 kVp and the intensity current of 140 µA. An entire region of the mandible (1335 slices; each slice = 8 µm) was scanned for each sample. Data sets were reconstructed using a

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modified Feldkamp algorithm and segmented into 8 bit BMP images using adaptive local thresholding. Microarchitectural properties of the mandible specimens were evaluated within a

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conforming volume of interest (VOI), which was defined as the area surrounded by the outer wall of the extraction socket and the upper limit of the extraction socket as determined by lines passing through the bone crest from the first molar to third molar. Morphometric indices included tissue volume (TV; mm3), bone volume (BV; mm3), bone mineral density (BMD), trabecular number (Tb.N; 1/mm), trabecular thickness (Tb.Th; μm), and trabecular separation (Tb.Sp; μm).

Histomorphometric analysis In preparation for histologic assessment, the samples were fixated with 4% phosphatebuffered formalin for 2 weeks and decalcified in 10% EDTA (pH 7.2) at 4° C for 4 weeks prior to immersion in paraffin. Sectioning, deparaffinization, and rehydration and staining with hematoxylin and eosin were executed to determine the presence of ONJ, based on the following histological

ACCEPTED MANUSCRIPT criteria [14, 15]: (1) presence of ulcerative lesion with exposed and necrotic bone and/or osteolysis, (2) presence of pseudoepitheliomatous-like hyperplasia of the epithelium accompanied by

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inflammatory cell infiltration, and (3) presence of sequestrum and bacterial colonies.

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Image capture was carried out using an Eclipse 50i light microscope (Nikon, Tokyo, Japan),

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equipped with CCD camera (MicroPublisher 3.3 RTV cooled, QImaging, Bethesda, MD, USA), and Image Pro Capture Kit Platform (Media Cybernetics, Bethesda, MD, USA). In a blind-coded fashion, histomorphometric evaluation was performed in 4 to 6 regions of interest (ROI) located at the

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osteonecrotic lesion or prior extraction site utilizing Image Pro Plus 6.2 software (Media Cybernetics, Bethesda, MD, USA). Measurements included tissue area, (T.Ar, mm2), bone area (B.Ar, mm2), bone

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surface (B.Pm; mm), and number of osteoclasts (N.Oc, #). To normalize indices, B.Ar/T.Ar (%), N.Oc/B.Ar (#/mm2) and N.Oc/B.Pm (#/mm2), and empty lacunae (#/mm2) were calculated for further

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analysis. [16]

Serum chemistry and Statistical Analysis

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Blood samples were obtained at sacrifice by cardiac puncture and then allowed to coagulate for 20 min. Collected serum samples were separated by centrifugation (1600×g for 15 min at 4°C),

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aliquoted, and stored at -70°C until analysis. The concentrations of serum CTx (RatLaps EIA, IDS, Boldon, Tyne & Wear, UK) and serum TRACP 5b (RatTRAP, IDS, Boldon, Tyne & Wear) were determined in accordance with the manufacturer’s instructions. [14, 17] All marker measurements were performed blinded and in duplicates. The normal distribution and homogeneity of measurements were checked before further statistical analyses. According to the results of histological assessment, rats in the bisphosphonate group were additionally classified into the non-ONJ and ONJ subgroups. Bone morphometric, histomorphometric indices, and serum measurements were compared using analysis of variance with Bonferroni correction. A P value of less than 0.05 was considered statistically significant. Statistical analysis was carried out using SPSS 20.0 (IBM Corp., Armonk, NY, USA).

ACCEPTED MANUSCRIPT Results All the Ov control (OvC; n=12) and sham control (ShC; n=12) animals presented normal

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healing course. Of the 36 rats in the Ov bisphosphonates group (OvB), 28 rats (77.8%) were

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histologically classified into the ONJ subgroup (OvB-ONJ), and 8 rats were classified into the non-

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ONJ group (OvB-Non). In the Sh bisphosphonates group (ShB), 17 out of 36 rats (47.2%) were determined to have ONJ, indicating lower prevalence of ONJ occurrence compared to OvB (P<0.05). Three-dimensional μCT imaging demonstrated the presence of osteolytic lesions accompanied

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by extensive destruction and disorganization of trabecular patterns with cortical disruption and sequestrum formation in the ONJ group (Figure 2). In the control group, the extraction sockets were

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nearly filled with newly formed bone exhibiting normal trabecular patterns. On the other hand,

changes of trabecular patterns.

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bisphosphonate-injected non-ONJ group showed insufficient amount of bone fill with osteosclerotic

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Morphometric assessment by μCT revealed that both bisphosphonate-injected groups (ShB-

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Non and OvB-Non) resulted in improved bone architectural indices compared to each control (ShC and OvC; Figure 3, Supplement Table 1). Contrastively, both ONJ groups (ShB-ONJ and OvB-ONJ) presented deteriorated architectures with OvB-ONJ group showing lower values of Tb.N (2.21 ±

(P<0.05).

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0.90), Tb.Sp (39.16 ± 17.05), and Conn.D (72.11 ± 11.12) compared to those of ShB-ONJ group

Histologically, both control groups showed normal course of healing of extraction sockets with newly filled bone and complete mucosal coverage (Figure 4). On the other hand, ONJ group showed extensive ulcerative lesion accompanied by exposed and necrotic bone with sequestrum and bacterial colonies. Also, pseudoepitheliomatous-like hyperplasia of epithelium with inflammatory cell infiltration to the connective tissue was observed. The Non-ONJ group showed mucosal coverage over the extraction socket, but the decrease in bone formation and the presence of empty lacunae were found compared to the control group. In the histomorphometric analysis, bisphosphonate-injected groups (ShB-Non and OvB-Non) showed lower osteoclast number (N.Oc/B.Pm and N.Oc/T.Ar) than each control group (P<0.05; Figure 5; Supplement table). Both ONJ groups (ShB-ONJ and OvB-ONJ)

ACCEPTED MANUSCRIPT showed higher necrotic bone fractions, empty lacunae, and lower N.Oc/B.Pm and N.Oc/T.Ar compared to each control group (P<0.05). The OvB-ONJ group demonstrated significantly higher

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empty lacunae (4.47 ± 1.16) and lower N.Oc/T.Ar (2.57 ± 1.03) than those of the ShB-ONJ group

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(P<0.05).

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The serum bone biomarker measurements demonstrated that bisphosphonate injection induced decrease in TRACP 5b (P<0.05; Figure 5), but the decreased CTX values were not statistically significant. There were significant differences between the ShB-ONJ and OvB-ONJ

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groups for TRACP 5b and CTX/TRACP 5b, which is a reliable index for the ovariectomy model [18].

ACCEPTED MANUSCRIPT Discussion

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In this study, the ovariectomized rat model successfully mimicked human ONJ lesions with a

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precedent bone disease and showed different bone characteristics than that of the previous ONJ model. Clinical, histological, and radiographic features of ovariectomized animal with ONJ did not

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only confirm the results of previous studies [19-21], but also presented quantifiable evidences of similar characteristics with MRONJ in humans. To establish a MRONJ model with underlying

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metabolic disease, ovariectomized rat model, which has been widely used for its proven quantifiable effects in the bone physiology [22], was adopted. This model has shown the physiologic disparity of

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bone integrity compared to a healthy animal model [23-25]. Also, ovariectomized rats are known to effectively reflect the osteoporotic condition of the mandible[11].

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Several previous ONJ animal models were diverse in terms of design resulting in inconsistent

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clinical characteristics of MRONJ [7, 26-29]. Given that the establishment of an animal model is essential to form a standard in discovering the cellular mechanisms responsible for the disease process

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[7, 23], this study produced ONJ lesions in 80% of the bisphosphonate-treated OV animals, which were not only determined clinically but also with the histological and radiographic confirmations. Compared to the sham-operated ONJ rats, the ovariectomized ONJ rats showed significantly lower

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trabecular thickness, connectivity density, and higher trabecular separation, indicating severely deteriorated bone integrity. Also, higher prevalence of ONJ occurrence among bisphosphonateinjected groups demonstrated different pathogenesis and pharmacologic responses in the diseased bone. Based on the primary pharmacological effects of bisphosphonates on osteoclasts, ONJ has been thought to be associated mainly with the oversuppression of bone remodeling [3-5]. Histomorphometrically lower number of osteoclasts in the OvB-ONJ group compared to the ShBONJ group and higher prevalence of ONJ in OvB rats support this hypothesis. The CTX values decreased after bisphosphonates administrations, yet there was no significant difference between the OvB-ONJ and ShB-ONJ groups. On the other hand, TRACP 5b, which is known to reflect the number of osteoclast [14, 18], were significantly lower in the OvB-ONJ group compared to the ShB-ONJ

ACCEPTED MANUSCRIPT group. This disparity was greater in the index for ovariectomized rat, CTX/TRACP 5b.[18] These results are consistent with the idea that osteoclast-mediated bone remodeling would play a key role in

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ONJ pathogenesis.

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Histologically, osteoporosis is presented through an elevated number of osteoclasts resulting in an

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imbalance in bone remodeling. In this study, ovariectomized control rats showed similar features; lower bone volume fraction, lower trabecular integrity, higher osteoclast number per bone perimeter, that is, increase in bone turnover and osteoclastic resorption, were presented. In addition,

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bisphosphonate-injected rats (OvB-Non) showed improved overall bone integrity. However, the OvBONJ group presented a sudden decrease in the number of osteoclasts with greater empty lacunae and

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ONJ prevalence. These results may imply both/either vulnerability of deteriorated bone condition upon bisphosphonate administration and/or differed pharmacological responses of ONJ-inducing

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medications on bone tissue. Therefore, in future studies, further elucidation of the pathogenesis of

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MRONJ is required.

The group of ovariectomized rats was established as a model for osteoporosis that may play an

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important role in the drug development. The significant difference between the ovariectomized and healthy animal models, as presented, reiterate the importance of the incorporation of precedent

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metabolic disease in utilizing animal models for the prospective studies of MRONJ. Exclusion of this factor could produce inconsistent pharmacologic effects on the bone architecture with physiologic changes represented histologically in the tissues. Based on the results of this study, the importance of incorporating abnormal bone condition in animals to be utilized for well-controlled future studies is emphasized. Also, the data presented are sufficient to favor the use of this animal model to expound on the different risk factors and etiology, further leading to the discovery of effective treatment strategies for MRONJ.

ACCEPTED MANUSCRIPT Acknowledgments This study was supported by the Ewha Global Top5 Grant 2013 and of the Ewha Womans

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University, Seoul, Korea. The authors declare no potential conflicts of interest with respect to the

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authorship and/or publication of this article.

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ACCEPTED MANUSCRIPT Figure Legends

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Figure 1. Study Design

Figure 2. Representative Clinical and Microcomputed tomographic images. (A) OvB-Non rat showed normal OvB-ONJ rat showed ulcerative lesion with necrotic

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mucosal healing 8 weeks after surgical intervention. (B)

bone. Three-dimensional μCT imaging demonstrated healing with newly formed bone in the control group (C) while OvB-ONJ group (E) presented osteolytic lesions accompanied by extensive destruction and

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disorganization of trabecular patterns with cortical disruption and sequestrum. (D) OvB-Non group showed

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insufficient amount of bone fill with osteosclerotic changes of trabecular patterns.

Figure 3. Bone micro-architectural morphometric assessment by μCT

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Abbreviations; BV, bone volume; TV, total volume; Tb.Th, trabecular thickness; Tb.Sp, trabecular separation;

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BMD, bone mineral density; Conn.D, connectivity density Column and bar indicate mean and standard error, respectively. indicates P<0.05 between ShB-ONJ and OvB-ONJ; * indicates P<0.05 compared to each control

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**

Figure 4. Representative histologic images of each group. (A) Ovariectomized control rat showing complete

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mucosal healing and sufficient bony healing (B, C) OvB-Non rat showing minimal bony fill and inflammatory cell infiltration with mucosal healing (D, E) OvB-ONJ rat showing ulceration with necrotic bone with empty lacunae, and pseudoepitheliomatous hyperplasia with the presence of sequestrum

Figure 5. Histomorphometric and bone biomarkers assessments on the influence of ovariectomy and development of ONJ Abbreviations; CTX, C-terminal crosslinked telopeptide of type I collagen; TRACP 5b, Tartrate-resistant acid phosphatase isoform 5b Column and bar indicate mean and standard error, respectively. **

indicates P<0.05 between ShB-ONJ and OvB-ONJ; * indicates P<0.05 compared to each control

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 Previous attempts to establish an animal model for ONJ have shown insufficient consider

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ation for underlying abnormal bone conditions

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 The authors investigated animal model with the most common metabolic bone disease, ost eoporosis.

 The rat model successfully mimicked human ONJ with underlying osteoporosis, showing

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different bone characteristics than that of previous model.