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Zoledronate induces osteonecrosis of the jaw in sheep
Journal of Cranio-Maxillo-Facial Surgery xxx (2015) 1e6
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Zoledronate induces osteonecrosis of the jaw in sheep* Pit Jacob Voss a, *, Martin Stoddart b, Thomas Ziebart c, Stephan Zeiter b, Katja Nelson a, Gido Bittermann a, Rainer Schmelzeisen a, Philipp Poxleitner a, b a
Department of Oral and Maxillofacial Surgery, University Medical Center Freiburg, (Head: Prof. Dr. R. Schmelzeisen), Hugstetter Str. 55, 79106 Freiburg im Breisgau, Germany b AO Research Institute Davos, (Head: Prof. Dr. G. Richards), Clavadeler Str. 8, 7270 Davos Platz, Switzerland c Department of Oral and Maxillofacial Surgery, University Hospital Mainz, (Head: Prof. Dr. W. Wagner), Augustusplatz 2, 55131 Mainz, Germany
a r t i c l e i n f o
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Article history: Paper received 26 February 2015 Accepted 23 April 2015 Available online xxx
Introduction: The treatment of bisphosphonate-related osteonecrosis of the jaw has become routine in maxillofacial hospitals. However, the etiopathology has not yet been fully understood. The aim of this study was to develop a large animal model for medication-related osteonecrosis of the jaw (MRONJ). Material and methods: Eight Swiss mountain sheep were randomly assigned into two groups. Group I received 0.075 mg/kg zoledronate (ZOL) intravenously every third week for 16 weeks. After 16 weeks, extraction of the first and second lower left premolar was performed. Group II underwent surgery and no ZOL was administered. After surgery, Group I continued to receive ZOL infusions; after 16 weeks, all animals were euthanized. The jaw bones were investigated macroscopically, radiographically (computed tomography) and histologically. Results: Osteonecrosis of the jaw was observed at all extraction sites in all the animals receiving ZOL, and at none of the sites in animals without ZOL. All ZOL-treated animals spontaneously developed exposed bone lesions in the oral cavity at sites where no surgical intervention was performed. CT imaging shows persistent alveolar extraction sockets 16 weeks after surgery in all animals of the ZOL-group, and healed alveolar extraction sockets in non-ZOL-treated animals. Conclusion: Sheep treated with ZOL reproducibly demonstrated osteonecrosis of the jaw after tooth extraction, and spontaneous development of exposed bone in the oral cavity at sites where no manipulation was performed. This animal model can be used for further research in the fields of BP-ONJ etiopathology, oral implantology, bone and fracture healing and periodontology. © 2015 European Association for Cranio-Maxillo-Facial Surgery. Published by Elsevier Ltd. All rights reserved.
Keywords: Bisphosphonates Oral and maxillofacial surgery Oral pathology Osteonecrosis Wound healing
1. Introduction Bisphosphonates (BPs) effectively reduce skeletal-related events in patients with osteoporosis and bone metastases of malignant diseases, mainly by inhibiting bone turnover through interference with osteoclasts. Following the introduction of the third generation of BPs with high pharmacological potency, an association between
* This study was funded by the AO Foundation by way of the AOCMF R&D Research Commission (Grant No C-11-11-V). * Corresponding author. Tel.: þ49 761 270 47010; fax: þ49 761 270 48000. E-mail addresses: [email protected] (P.J. Voss), martin.stoddart@ aofoundation.org (M. Stoddart), [email protected] (T. Ziebart), [email protected] (S. Zeiter), [email protected] (K. Nelson), [email protected] (G. Bittermann), rainer. [email protected] (R. Schmelzeisen), philipp.poxleitner@ uniklinik-freiburg.de (P. Poxleitner).
treatment with nitrogen containing BPs and bone necrosis of the jaws became evident (Marx, 2003). At present, patients treated with zoledronate (ZOL), the most potent BP available, show the highest incidence of MRONJ (Hoff et al., 2011). Several groups reported good results for a surgical approach to cure MRONJ; complete resection of the altered bone, thorough rounding of bony edges and meticulous closure of the soft tissue are important measures for healing of the oral mucosa without leaving any exposed bone. However, bony reconstruction and dental rehabilitation of the resection areas still present clinical challenges (Carlson and Basile, 2009; Markose et al., 2009; Stockmann et al., 2010; Voss et al., 2012; Wilde et al., 2011; Williamson, 2010). Even though several theories have been proposed, until now, the exact pathogenesis of the condition is not understood (Ruggiero et al., 2014). In the clinical setting, MRONJ mostly arises in patients with periodontal disease or after tooth extraction without primary wound closure (Hoff et al., 2011). One theory is that bone turnover
http://dx.doi.org/10.1016/j.jcms.2015.04.020 1010-5182/© 2015 European Association for Cranio-Maxillo-Facial Surgery. Published by Elsevier Ltd. All rights reserved.
Please cite this article in press as: Voss PJ, et al., Zoledronate induces osteonecrosis of the jaw in sheep, Journal of Cranio-Maxillo-Facial Surgery (2015), http://dx.doi.org/10.1016/j.jcms.2015.04.020
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in the jaws is increased due to local factors such as bacterial oral flora and masticatory forces, presumably resulting in high concentrations of BPs in the alveolar bone and leading to an oversuppression of bone turnover in the ‘stressed’ areas. The reduced capability of the tissue to respond to local factors is followed by necrosis (Reid, 2009). Other possible factors are the suppression of angiogenesis under the influence of BPs and the activation of inert BPs that are bound to the bone through the decrease of local pHvalues (Otto et al., 2010). During the last few years, a plethora of case series and in vitro studies concerning the pathology, occurrence, treatment and prevention has been published. A large number of rodent models have been described and used (Sharma et al., 2013); however, a canine model (Allen and Burr, 2008; Burr and Allen, 2009) in which the amount of necrotic bone could only be evaluated histologically, and a minipig model showing MRONJ lesions (Pautke et al., 2012), are the only large animal models published to date (Allen, 2009). Burr and Allen performed several studies in healthy, nonovariectomized beagle dogs, in which the animals were treated with different dosages of the BPs alendronate and ZOL (Allen and Burr, 2008). Conventional histology (basic fuchsin staining) showed islands of necrotic bone within the alveolar bone but no evidence of intraoral exposure. Furthermore, using dynamic histomorphometry they demonstrated that BPs significantly suppressed (75%) the level of intracortical remodeling in the alveolar bone. One of these canine studies included a surgical intervention, which showed compromised healing of the extraction wounds using mCT and dynamic histomorphometry only (Allen et al., 2011). A high incidence of mucosal dehiscence observed in miniature pigs in previous investigations after oral surgery without ZOL administration is supposedly provoked by the habits and parafunctional masticatory load. Despite a softened diet, the animals exert excessive stress by heavy chewing patterns and by gnawing their pens, compromising successful healing after intraoral surgical treatment (Ruehe et al., 2011). Sheep are a convenient large animal model due to easy housing and handling, animal costs, and acceptance as a research animal. They have a long history in bone research and are frequently used in orthopedic research. Sheep show a similar weight, size, bone and joint structure and display both cortical and trabecular bone remodeling cycles comparable to humans. Sheep are subject to research in the maxillofacial area, including osteosynthesis and fracture healing and dental implants, and facilitate the ideal anatomical conditions to assess augmentation procedures such as sinus augmentations (Gutwald et al., 2010, 2011). The aim of this study was to establish a MRONJ model in sheep for further evaluation of the pathogenesis of this condition and the study of fracture healing, dental implantology and augmentation procedures. 2. Material and methods All procedures during the study were carried out in an AAALAC (Association for Assessment and Accreditation of Laboratory Animal Care) accredited facility according to the Swiss Laws of animal welfare and were approved by the local Animal Welfare Commission of the official veterinary authorities (Authorization Number 2012_10). Eight, skeletally mature, white alpine ewes were included in the study and randomly divided into two groups. Mean age at start of the study was 2.5 years. A pre-operative physical examination revealed no gross abnormalities in any of the sheep. Animals were housed in groups of four animals. Weights were taken weekly after surgery for 3 weeks and afterwards every 4 weeks. Animals were scored for animal welfare every week by an experienced animal care-taker or veterinarian.
ZOL was provided by the Department of Chemistry of the University of Southern California and dissolved in PBS at 0.05 mg/ml at pH 7. Every third week, each animal in the ZOL group received an infusion of 0.075 mg/kg bodyweight ZOL intravenously via a 16 G catheter in the jugular vein. All surgical procedures were performed under general anesthesia. General anesthesia was induced with 4 mg/kg ketamine IV and 0.2 mg/kg diazepam IV. Sheep were intubated and anesthesia was maintained with 2% isoflurane (Pharmacia and Upjohn AG, Dübendorf, Switzerland) and oxygen. Heart rate, oxygen saturation and body temperature were continuously monitored. The first and second lower left premolars were removed using dental levers. The extraction wound of the first premolar was closed with 3.0 monocryl suturing material; the second premolar alveolus was left open. Following surgery, sheep were housed in single stalls for 3 days and then group housed, fed a maintenance ration of hay and had ad libitum access to water for the whole duration of the study. ZOL injections were continued in group I every third week. Sixteen weeks after surgery animals were euthanized with an injection of 20 ml phenobarbital (Esconarcon 300 mg/ml). A CTscan was performed using SIEMENS Somatom Emotion 6 with a slice thickness of 0.63 mm. Mandible and maxilla were harvested, investigated and photographed and stored in 70% ethanol for further processing. For the 3D analysis, microcomputed tomography (mCT 40, Scanco Medical AG, Brüttisellen, Switzerland) was used. Imaging analysis was performed using Osirix open source software (Osirix v. 5.6, Pixmeo, Geneva, Switzerland). Two oral and maxillofacial surgery specialists performed macroscopic evaluation. The staging was adapted to the MRONJ stages described by Ruggiero et al. (2009). Stage 0 ¼ healed mucosa and no apparent necrotic bone at extraction site, 1 ¼ no clinical evidence of necrotic bone but unspecific findings at extraction site, 2 ¼ exposed/necrotic bone with no signs of infection at extraction site, 3 ¼ exposed/necrotic bone with signs of infection at site of extraction, 4 ¼ exposed and necrotic bone with pain, infection, and one or more of the following: exposed and necrotic bone extending beyond the region of alveolar bone (ie, inferior border and ramus in the mandible, maxillary sinus and zygoma in the maxilla) resulting in pathologic fracture, extraoral fistula, oral antral/oral nasal communication, or osteolysis extending to the inferior border of the mandible or the sinus floor. DICOM images from the CT scan were transferred and multiplanar reconstructions were assessed. The reconstructed images were assessed in a window with settings appropriate for osseous structures and evaluated by an experienced radiologist in a blinded manner. The evaluation used a score previously described by Pautke et al. (2012): 0 ¼ healed bone, 1 ¼ osteolysis or nonregeneration without cortical erosion, 2 ¼ osteolysis with cortical erosion. Also the thickness of periosteal reactions was measured. After harvesting the extraction sites of the mandibles, the specimens were processed in 70% ethanol and embedded in poly(methyl methacrylate) (PMMA) resin. They were then cut in a buccolingual direction with an EXAKT cutting machine to a thickness of 100 mm and then prepared with an EXAKT grinding machine to 15 mm (EXAKT, Cutting-Grinding System, Exakt-Apparatbau, Norderstedt, Germany). The sections were thereafter stained in pararosaniline-azure-II and inspected using light microscopy. After a gross examination, regions of interest were defined and the amount of new bone and old bone quantified. Descriptive statistics of all variables were determined, including means and standard deviations. For the calculation of differences between the groups we used a mixed linear model that took the use of dependent variables into account. For multiple testing Scheffe's method was adapted for the correction. Analysis was performed
Please cite this article in press as: Voss PJ, et al., Zoledronate induces osteonecrosis of the jaw in sheep, Journal of Cranio-Maxillo-Facial Surgery (2015), http://dx.doi.org/10.1016/j.jcms.2015.04.020
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with SPSS 22 and STATA 13.1. Results were considered significant at p < 0.05. This study is in compliance with the ARRIVE (Animal Research: Reporting of In Vivo Experiments) guidelines. 3. Results ZOL administration was successfully performed in all animals of the ZOL group and dental surgery was performed in all animals. The recovery time after surgery was uneventful. No significant weight loss occurred between the groups. All ZOL-treated animals suffered from impaired wound healing and exposed bone at all extraction sites during the 16-week postoperative period (Fig. 1A). The exposed bone was present for more than 8 weeks and thus meets the MRONJ definition criteria (Ruggiero et al., 2014). According to the MRONJ staging system, all of the ZOL-treated animals were stage 3. In addition, all ZOL-treated animals showed exposed bone in the oral cavity at sites where no surgical manipulation was performed (Fig. 1B, C, D). In the control group, normal wound healing occurred within the first two weeks (Fig. 2A, B). The MRONJ stage was 0 in all animals (Fig. 4). CT analysis of the animals immediately after euthanasia revealed typical signs of MRONJ in all ZOL-treated animals. Osteolysis, sequestration and periosteal reaction were especially seen around the extraction sites and the sites of spontaneous exposed bone. The alveolar defect was not replaced by cancellous bone formation in the ZOL group; in the control group, complete cortical regeneration was seen in all animals (Fig. 3A, B). Normal bone healing was observed in the control group (Fig. 3C, D). The CT evaluation score revealed a mean score of 2 in the ZOL-treated animals versus 0 in the controls (Fig. 4). No osteolysis or osseous defects were detected in the control group, one animal showed eruption of a fragmented root with no signs of inflammation or infection. The periosteal reaction at the buccal and lingual aspect of the mandible was between 1.92 ± 0.33 and 1.60 ± 0.94 mm in all ZOL-treated animals. The mild and dense periosteal reaction of 0.23 ± 0.34 and 0.18 ± 0.76 mm in the control group was interpreted as a normal sign of new bone formation (Fig. 5A). Two experienced examiners analyzed all histological sections in a blinded manner. Typical signs of MRONJ bone necrosis, such as non-vital bone with rough margins and empty lacunae and no osteocytes were detected in all extraction sites of the ZOL group. Signs of bone remodeling were absent in these animals. No signs of
Fig. 2. (A and B) Clinical aspect in the control group. No exposed bone was visible in any of the animals. Mucosa covered the defect and no signs of infection or inflammation were noticed.
osteolysis were seen in the extraction sites of the control group. ZOL-treated animals showed significantly less new bone formation compared with the control group (0.21 ± 0.18% vs. 2.37 ± 1.41%) (Fig. 5B). The ratio of new bone to old bone in the ZOL-treated animals was significantly decreased 0.009 ± 0.009 compared with the control group 0.070 ± 0.04. 4. Discussion Bisphosphonates are effective drugs used widely in the treatment of bony metastases of malignant diseases, osteoporosis and Paget's disease (Hatoum et al., 2008; Sambrook and Cooper, 2006). Despite a generally low level of side effects, the treatment of MRONJ has become a clinical routine in oral and maxillofacial hospitals as a result of the large numbers of patients receiving BPs. The current definition includes BP-treatment history, exposed bone in the oral cavity for a period longer than 8 weeks and no radiation to the jaws (Ruggiero et al., 2014). Animal models are important to understand the underlying pathological mechanism and to develop and establish an evidence-based treatment option (Ruggiero and Drew, 2007).
Fig. 1. (A) Representing the clinical aspect of the unhealed extraction socket in an animal from the ZOL group. Exposed dark-colored bone can be noted beside pus draining from the anterior part as sign of infection. (B, C and D) Clinical image of the spontaneous exposed bone at parts of the mandible and maxilla where no surgical manipulation was performed.
Please cite this article in press as: Voss PJ, et al., Zoledronate induces osteonecrosis of the jaw in sheep, Journal of Cranio-Maxillo-Facial Surgery (2015), http://dx.doi.org/10.1016/j.jcms.2015.04.020
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Fig. 3. (A and B) Histological and microcomputed tomography section of the unhealed extraction socket in a ZOL-treated animal. Note the periosteal reaction around the buccal and lingual aspect of the mandible. (C and D) Transverse section of the former extraction socket from an animal of the control group. The socket is filled with cancellous bone formation and the mucosa is closed. New bone formation can be noticed around the mandible as a sign of ongoing bone remodeling.
The ideal animal model mimics the clinical situation of the MRONJ lesion in the patient and is consistent with the criteria used in clinics. Various animal models of experimental MRONJ have been described. The most commonly used animal species are rodents, however, the use of dogs and minipigs as large animal models also have been published (Sharma et al., 2013). Especially in rodents, meeting the clinically relevant diagnostic criteria is difficult. While Kobayashi et al. (2010) failed to establish MRONJ in C57BL/6J mice with ZOL only, Bi et al. (2010) reported MRONJ-like lesions in the same model treated with ZOL and dexamethasone. Several authors reported MRONJ-like lesions in Sprague-Dawley and Wistar rats. Guevarra et al. (2013) demonstrated vascular changes in mandibular alveolar bone after tooth extraction in rats receiving ZOL. Barba-Recreo et al. (2014) showed recently, that the highest cumulative doses of ZOL had the highest incidence of osteonecrosis in these animals, with lower numbers of osteoclasts. While Tsurushima et al. (2013) promoted MRONJlike lesions in the mandible and femur of Wistar rats after injecting Aggregatibacter actinomycetemcomitans into the bone marrow, Berti-Couto et al. (2014) identified diabetes mellitus as a
risk factor for developing MRONJ after tooth extraction in these animals. Even though animal studies using rodents are less expensive, the animals show different bone morphology, and are usually too small for testing clinical materials. Because intracortical turnover seems to play a major role in the manifestation of MRONJ, the generalized absence of normal intracortical remodeling in rodents limits their usefulness as a model for MRONJ (Jowsey, 1966; Kubek et al., 2010). €ttingen minipigs have been described as Beagle dogs and Go large animal models for MRONJ. Allen et al. (2011) described compromised osseous healing of extraction sites in beagle dogs treated with ZOL, but only one of six dogs developed exposed bone. In a minipig model, extraction of six teeth led to impaired wound healing and exposed bone in at least three of the extraction sites in all five animals (Pautke et al., 2012). The most common trigger factor for inducing MRONJ is dentoalveolar surgery. However, according to the literature, in 30e40% of cases no local surgical trauma was noted; even though sometimes prosthetic trauma or periodontitis are present, spontaneous
Please cite this article in press as: Voss PJ, et al., Zoledronate induces osteonecrosis of the jaw in sheep, Journal of Cranio-Maxillo-Facial Surgery (2015), http://dx.doi.org/10.1016/j.jcms.2015.04.020
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Fig. 4. (A) The macroscopic score of the MRONJ staging system adapted by Ruggiero et al. (2009) was used to score the mandibles: Stage 0 ¼ healed mucosa and no apparent necrotic bone at extraction site, 1 ¼ no clinical evidence of necrotic bone but unspecific findings at extraction site, 2 ¼ exposed/necrotic bone with no signs of infection at extraction site, 3 ¼ exposed/necrotic bone with signs of infection at site of extraction, 4 ¼ exposed and necrotic bone with pain, infection, and one or more of the following: exposed and necrotic bone extending beyond the region of alveolar bone (ie, inferior border and ramus in the mandible, maxillary sinus and zygoma in the maxilla) resulting in pathologic fracture, extraoral fistula, oral antral/oral nasal communication, or osteolysis extending to the inferior border of the mandible or the sinus floor. (B) The computed tomographic score of the mandible adapted by Pautke et al. (2012): 0: healed bone, 1: osteolysis or non-regeneration without cortical erosion, 2: osteolysis with cortical erosion.
evolution seems to be frequent (Vescovi et al., 2011). Despite one mouse model that recently showed spontaneous osteonecrosis of the jaw after antiresorptive treatment, no other animal study displayed this phenomenon (de Molon et al., 2014). In this sheep model MRONJ was observed, not only at all the dental extraction sites, but also spontaneous exposed bone in the oral cavity developed where no manipulation was performed. This is similar to the situation in many patients. One possible factor could be the dosage of 0.075 mg/kg body weight, which is about 50% higher than doses given for oncological reasons in human patients. We decided to elevate the ZOL doses to have a more reliable development of necrosis, especially given the fact that the animals received only five ZOL infusions every third week before surgery. Repeating of the dose every third week instead of a monthly dose, as it is carried out in oncological patients, reflects the higher bone turnover of sheep (Corlett et al., 1990). Regarding the literature, high cumulative doses of bisphosphonates are a risk factor for the development of MRONJ, and the use of high single doses is more likely to cause transient side effects such as hypophosphatemia, hypocalcemia, hypomagnesemia, renal failure and acute phase symptoms (Body et al., 1999;
Hoff et al., 2008). None of our animals showed any of these transient side effects. In most of the other large animal studies, ZOL dosages vary. While weekly infusions of 0.05 mg ZOL/kg body weight for 6 weeks led to inconsistent development of BP-ONJ in minipigs, (Pautke et al., 2012), monthly infusions of 0.1 mg/kg body weight for 4 months in beagle dogs did not lead to the development of MRONJ lesions (Huja et al., 2011). In another study on beagle dogs, 0.06 mg/ kg every second week was used for a 3 month period. ZOL led to a severe suppression of intracortical remodeling and exposed bone in one out of six animals (Allen et al., 2011). These findings are heterogeneous and make comparison of dosing schemes between different animal species difficult. The right timing and dosage for each species needs to be determined for each species individually. Further research is needed to define the No and Lowest Observed Effect Concentration (NOEC, LOEC) and dose responsiveness for ZOL in sheep. In our study, spontaneous open bone lesions mostly arose in the interdental spaces where bits of hay had accumulated. This supports the theory, that periodontal infections trigger MRONJ, which is also suggested by Aghaloo et al. (2011) who induced ONJ using a
Fig. 5. (A) Mean periosteal reaction in mm at the oral and lingual aspect of the mandible at the extraction socket (B) Ratio of new bone to old bone on the region of interest in the extraction socket of the mandible.
Please cite this article in press as: Voss PJ, et al., Zoledronate induces osteonecrosis of the jaw in sheep, Journal of Cranio-Maxillo-Facial Surgery (2015), http://dx.doi.org/10.1016/j.jcms.2015.04.020
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periodontitis model with wire ligatures and 66 mg/kg of ZOL in rats. In the histological analysis of our animals, we found a significantly higher fraction of old bone and a smaller fraction of new bone in the periodontal region of ZOL-treated sheep compared with the control group. Following a widespread theory, the reduced boneremodeling rate does not allow adequate reaction of the bony tissue to inflammatory and physical stimuli. Given the fact that low concentrations of ZOL rapidly and directly affect the oral fibroblasts and epithelial cells (Scheper et al, 2009), it is not unlikely that the oral mucosal tissues disconnect from the bone through shearing forces which occur during chewing cycles. This exposed bone will react as surgically denudated bone. Further studies concerning the periodontal cause of MRONJ in sheep need to confirm this theory. 5. Conclusion ZOL administrations consistently lead to MRONJ after tooth extractions in sheep. In interdental spaces the direct effect of low ZOL concentrations on mucosal tissues in interaction with local irritation and shearing forces during chewing cycles and the reduced bone remodeling in the alveolar process may lead to spontaneously exposed bone. This large animal model is feasible for further evaluation of bone and fracture healing, augmentation procedures and oral implantology and periodontitis models for the pathogenesis of MRONJ. Acknowledgements This study was funded by the AO Foundation by way of the AOCMF R&D Research Commission (Grant No C-11-11-V). The study was conducted in an AAALAC accredited facility and followed the ARRIVE guidelines. We would also thank Dr. Vincent Stadelmann for his help with the CT. We thank Luca Werner for his help with preparing and analyzing the histological samples. The authors declare no conflict of interests. References Aghaloo TL, Kang B, Sung EC, Shoff M, Ronconi M, Gotcher JE, et al: Periodontal disease and bisphosphonates induce osteonecrosis of the jaws in the rat. J Bone Miner Res 26(8): 1871e1882. http://dx.doi.org/10.1002/jbmr.379, 2011 Aug Allen MR: Bisphosphonates and osteonecrosis of the jaw: moving from the bedside to the bench. Cells, tissues, organs 189: 289e294, 2009 Allen MR, Burr DB: Mandible matrix necrosis in beagle dogs after 3 years of daily oral bisphosphonate treatment. J Oral Maxillofac Surg 66: 987e994, 2008 Allen MR, Kubek DJ, Burr DB, Ruggiero SL, Chu TM: Compromised osseous healing of dental extraction sites in zoledronic acid-treated dogs. Osteoporos Int 22: 693e702, 2011 Barba-Recreo P, Del Castillo Pardo de Vera JL, Garcia-Arranz M, Yebenes L, Burgueno M: Zoledronic acid-related osteonecrosis of the jaws. Experimental model with dental extractions in rats. J Craniomaxillofac Surg 42: 744e750, 2014 Berti-Couto SA, Vasconcelos AC, Iglesias JE, Figueiredo MA, Salum FG, Cherubini K: Diabetes mellitus and corticotherapy as risk factors for alendronate-related osteonecrosis of the jaws: a study in Wistar rats. Head Neck 36: 84e93, 2014 Bi Y, Gao Y, Ehirchiou D, Cao C, Kikuiri T, Le A, et al: Bisphosphonates cause osteonecrosis of the jaw-like disease in mice. Am J Pathol 177: 280e290, 2010 Body JJ, Lortholary A, Romieu G, Vigneron AM, Ford J: A dose-finding study of zoledronate in hypercalcemic cancer patients. J Bone Miner Res 14: 1557e1561, 1999 Burr DB, Allen MR: Mandibular necrosis in beagle dogs treated with bisphosphonates. Orthod Craniofac Res 12: 221e228, 2009 Carlson ER, Basile JD: The role of surgical resection in the management of bisphosphonate-related osteonecrosis of the jaws. J Oral Maxillofac Surg 67: 85e95, 2009 Corlett SC, Couch M, Care AD, Sykes AR: Measurement of plasma osteocalcin in sheep: assessment of circadian variation, the effects of age and nutritional status and the response to perturbation of the adrenocortical axis. Exp Physiol 75: 515e527, 1990 de Molon RS, Cheong S, Bezouglaia O, Dry SM, Pirih F, Cirelli JA, et al: Spontaneous osteonecrosis of the jaws in the maxilla of mice on antiresorptive treatment: a novel ONJ mouse model. Bone 68: 11e19, 2014
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Oral Dis 19: 747e754, 2013 Stockmann P, Vairaktaris E, Wehrhan F, Seiss M, Schwarz S, Spriewald B, et al: Osteotomy and primary wound closure in bisphosphonate-associated osteonecrosis of the jaw: a prospective clinical study with 12 months follow-up. Support Care Cancer 18: 449e460, 2010 Tsurushima H, Kokuryo S, Sakaguchi O, Tanaka J, Tominaga K: Bacterial promotion of bisphosphonate-induced osteonecrosis in Wistar rats. Int J Oral Maxillofac Surg 42: 1481e1487, 2013 Vescovi P, Campisi G, Fusco V, Mergoni G, Manfredi M, Merigo E, et al: Surgerytriggered and non surgery-triggered Bisphosphonate-related Osteonecrosis of the Jaws (BRONJ): a retrospective analysis of 567 cases in an Italian multicenter study. Oral Oncol 47: 191e194, 2011 Voss PJ, Joshi Oshero J, Kovalova-Muller A, Veigel Merino EA, Sauerbier S, AlJamali J, et al: Surgical treatment of bisphosphonate-associated osteonecrosis of the jaw: technical report and follow up of 21 patients. J Craniomaxillofac Surg 40: 719e725, 2012 Wilde F, Heufelder M, Winter K, Hendricks J, Frerich B, Schramm A, et al: The role of surgical therapy in the management of intravenous bisphosphonates-related osteonecrosis of the jaw. Oral Surg Oral Med Oral Pathol Oral radiol Endod 111: 153e163, 2011 Williamson RA: Surgical management of bisphosphonate induced osteonecrosis of the jaws. Int J Oral Maxillofac Surg 39: 251e255, 2010
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Zoledronate induces osteonecrosis of the jaw in sheep* Pit Jacob Voss a, *, Martin Stoddart b, Thomas Ziebart c, Stephan Zeiter b, Katja Nelson a, Gido Bittermann a, Rainer Schmelzeisen a, Philipp Poxleitner a, b a
Department of Oral and Maxillofacial Surgery, University Medical Center Freiburg, (Head: Prof. Dr. R. Schmelzeisen), Hugstetter Str. 55, 79106 Freiburg im Breisgau, Germany b AO Research Institute Davos, (Head: Prof. Dr. G. Richards), Clavadeler Str. 8, 7270 Davos Platz, Switzerland c Department of Oral and Maxillofacial Surgery, University Hospital Mainz, (Head: Prof. Dr. W. Wagner), Augustusplatz 2, 55131 Mainz, Germany
a r t i c l e i n f o
a b s t r a c t
Article history: Paper received 26 February 2015 Accepted 23 April 2015 Available online xxx
Introduction: The treatment of bisphosphonate-related osteonecrosis of the jaw has become routine in maxillofacial hospitals. However, the etiopathology has not yet been fully understood. The aim of this study was to develop a large animal model for medication-related osteonecrosis of the jaw (MRONJ). Material and methods: Eight Swiss mountain sheep were randomly assigned into two groups. Group I received 0.075 mg/kg zoledronate (ZOL) intravenously every third week for 16 weeks. After 16 weeks, extraction of the first and second lower left premolar was performed. Group II underwent surgery and no ZOL was administered. After surgery, Group I continued to receive ZOL infusions; after 16 weeks, all animals were euthanized. The jaw bones were investigated macroscopically, radiographically (computed tomography) and histologically. Results: Osteonecrosis of the jaw was observed at all extraction sites in all the animals receiving ZOL, and at none of the sites in animals without ZOL. All ZOL-treated animals spontaneously developed exposed bone lesions in the oral cavity at sites where no surgical intervention was performed. CT imaging shows persistent alveolar extraction sockets 16 weeks after surgery in all animals of the ZOL-group, and healed alveolar extraction sockets in non-ZOL-treated animals. Conclusion: Sheep treated with ZOL reproducibly demonstrated osteonecrosis of the jaw after tooth extraction, and spontaneous development of exposed bone in the oral cavity at sites where no manipulation was performed. This animal model can be used for further research in the fields of BP-ONJ etiopathology, oral implantology, bone and fracture healing and periodontology. © 2015 European Association for Cranio-Maxillo-Facial Surgery. Published by Elsevier Ltd. All rights reserved.
Keywords: Bisphosphonates Oral and maxillofacial surgery Oral pathology Osteonecrosis Wound healing
1. Introduction Bisphosphonates (BPs) effectively reduce skeletal-related events in patients with osteoporosis and bone metastases of malignant diseases, mainly by inhibiting bone turnover through interference with osteoclasts. Following the introduction of the third generation of BPs with high pharmacological potency, an association between
* This study was funded by the AO Foundation by way of the AOCMF R&D Research Commission (Grant No C-11-11-V). * Corresponding author. Tel.: þ49 761 270 47010; fax: þ49 761 270 48000. E-mail addresses: [email protected] (P.J. Voss), martin.stoddart@ aofoundation.org (M. Stoddart), [email protected] (T. Ziebart), [email protected] (S. Zeiter), [email protected] (K. Nelson), [email protected] (G. Bittermann), rainer. [email protected] (R. Schmelzeisen), philipp.poxleitner@ uniklinik-freiburg.de (P. Poxleitner).
treatment with nitrogen containing BPs and bone necrosis of the jaws became evident (Marx, 2003). At present, patients treated with zoledronate (ZOL), the most potent BP available, show the highest incidence of MRONJ (Hoff et al., 2011). Several groups reported good results for a surgical approach to cure MRONJ; complete resection of the altered bone, thorough rounding of bony edges and meticulous closure of the soft tissue are important measures for healing of the oral mucosa without leaving any exposed bone. However, bony reconstruction and dental rehabilitation of the resection areas still present clinical challenges (Carlson and Basile, 2009; Markose et al., 2009; Stockmann et al., 2010; Voss et al., 2012; Wilde et al., 2011; Williamson, 2010). Even though several theories have been proposed, until now, the exact pathogenesis of the condition is not understood (Ruggiero et al., 2014). In the clinical setting, MRONJ mostly arises in patients with periodontal disease or after tooth extraction without primary wound closure (Hoff et al., 2011). One theory is that bone turnover
http://dx.doi.org/10.1016/j.jcms.2015.04.020 1010-5182/© 2015 European Association for Cranio-Maxillo-Facial Surgery. Published by Elsevier Ltd. All rights reserved.
Please cite this article in press as: Voss PJ, et al., Zoledronate induces osteonecrosis of the jaw in sheep, Journal of Cranio-Maxillo-Facial Surgery (2015), http://dx.doi.org/10.1016/j.jcms.2015.04.020
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in the jaws is increased due to local factors such as bacterial oral flora and masticatory forces, presumably resulting in high concentrations of BPs in the alveolar bone and leading to an oversuppression of bone turnover in the ‘stressed’ areas. The reduced capability of the tissue to respond to local factors is followed by necrosis (Reid, 2009). Other possible factors are the suppression of angiogenesis under the influence of BPs and the activation of inert BPs that are bound to the bone through the decrease of local pHvalues (Otto et al., 2010). During the last few years, a plethora of case series and in vitro studies concerning the pathology, occurrence, treatment and prevention has been published. A large number of rodent models have been described and used (Sharma et al., 2013); however, a canine model (Allen and Burr, 2008; Burr and Allen, 2009) in which the amount of necrotic bone could only be evaluated histologically, and a minipig model showing MRONJ lesions (Pautke et al., 2012), are the only large animal models published to date (Allen, 2009). Burr and Allen performed several studies in healthy, nonovariectomized beagle dogs, in which the animals were treated with different dosages of the BPs alendronate and ZOL (Allen and Burr, 2008). Conventional histology (basic fuchsin staining) showed islands of necrotic bone within the alveolar bone but no evidence of intraoral exposure. Furthermore, using dynamic histomorphometry they demonstrated that BPs significantly suppressed (75%) the level of intracortical remodeling in the alveolar bone. One of these canine studies included a surgical intervention, which showed compromised healing of the extraction wounds using mCT and dynamic histomorphometry only (Allen et al., 2011). A high incidence of mucosal dehiscence observed in miniature pigs in previous investigations after oral surgery without ZOL administration is supposedly provoked by the habits and parafunctional masticatory load. Despite a softened diet, the animals exert excessive stress by heavy chewing patterns and by gnawing their pens, compromising successful healing after intraoral surgical treatment (Ruehe et al., 2011). Sheep are a convenient large animal model due to easy housing and handling, animal costs, and acceptance as a research animal. They have a long history in bone research and are frequently used in orthopedic research. Sheep show a similar weight, size, bone and joint structure and display both cortical and trabecular bone remodeling cycles comparable to humans. Sheep are subject to research in the maxillofacial area, including osteosynthesis and fracture healing and dental implants, and facilitate the ideal anatomical conditions to assess augmentation procedures such as sinus augmentations (Gutwald et al., 2010, 2011). The aim of this study was to establish a MRONJ model in sheep for further evaluation of the pathogenesis of this condition and the study of fracture healing, dental implantology and augmentation procedures. 2. Material and methods All procedures during the study were carried out in an AAALAC (Association for Assessment and Accreditation of Laboratory Animal Care) accredited facility according to the Swiss Laws of animal welfare and were approved by the local Animal Welfare Commission of the official veterinary authorities (Authorization Number 2012_10). Eight, skeletally mature, white alpine ewes were included in the study and randomly divided into two groups. Mean age at start of the study was 2.5 years. A pre-operative physical examination revealed no gross abnormalities in any of the sheep. Animals were housed in groups of four animals. Weights were taken weekly after surgery for 3 weeks and afterwards every 4 weeks. Animals were scored for animal welfare every week by an experienced animal care-taker or veterinarian.
ZOL was provided by the Department of Chemistry of the University of Southern California and dissolved in PBS at 0.05 mg/ml at pH 7. Every third week, each animal in the ZOL group received an infusion of 0.075 mg/kg bodyweight ZOL intravenously via a 16 G catheter in the jugular vein. All surgical procedures were performed under general anesthesia. General anesthesia was induced with 4 mg/kg ketamine IV and 0.2 mg/kg diazepam IV. Sheep were intubated and anesthesia was maintained with 2% isoflurane (Pharmacia and Upjohn AG, Dübendorf, Switzerland) and oxygen. Heart rate, oxygen saturation and body temperature were continuously monitored. The first and second lower left premolars were removed using dental levers. The extraction wound of the first premolar was closed with 3.0 monocryl suturing material; the second premolar alveolus was left open. Following surgery, sheep were housed in single stalls for 3 days and then group housed, fed a maintenance ration of hay and had ad libitum access to water for the whole duration of the study. ZOL injections were continued in group I every third week. Sixteen weeks after surgery animals were euthanized with an injection of 20 ml phenobarbital (Esconarcon 300 mg/ml). A CTscan was performed using SIEMENS Somatom Emotion 6 with a slice thickness of 0.63 mm. Mandible and maxilla were harvested, investigated and photographed and stored in 70% ethanol for further processing. For the 3D analysis, microcomputed tomography (mCT 40, Scanco Medical AG, Brüttisellen, Switzerland) was used. Imaging analysis was performed using Osirix open source software (Osirix v. 5.6, Pixmeo, Geneva, Switzerland). Two oral and maxillofacial surgery specialists performed macroscopic evaluation. The staging was adapted to the MRONJ stages described by Ruggiero et al. (2009). Stage 0 ¼ healed mucosa and no apparent necrotic bone at extraction site, 1 ¼ no clinical evidence of necrotic bone but unspecific findings at extraction site, 2 ¼ exposed/necrotic bone with no signs of infection at extraction site, 3 ¼ exposed/necrotic bone with signs of infection at site of extraction, 4 ¼ exposed and necrotic bone with pain, infection, and one or more of the following: exposed and necrotic bone extending beyond the region of alveolar bone (ie, inferior border and ramus in the mandible, maxillary sinus and zygoma in the maxilla) resulting in pathologic fracture, extraoral fistula, oral antral/oral nasal communication, or osteolysis extending to the inferior border of the mandible or the sinus floor. DICOM images from the CT scan were transferred and multiplanar reconstructions were assessed. The reconstructed images were assessed in a window with settings appropriate for osseous structures and evaluated by an experienced radiologist in a blinded manner. The evaluation used a score previously described by Pautke et al. (2012): 0 ¼ healed bone, 1 ¼ osteolysis or nonregeneration without cortical erosion, 2 ¼ osteolysis with cortical erosion. Also the thickness of periosteal reactions was measured. After harvesting the extraction sites of the mandibles, the specimens were processed in 70% ethanol and embedded in poly(methyl methacrylate) (PMMA) resin. They were then cut in a buccolingual direction with an EXAKT cutting machine to a thickness of 100 mm and then prepared with an EXAKT grinding machine to 15 mm (EXAKT, Cutting-Grinding System, Exakt-Apparatbau, Norderstedt, Germany). The sections were thereafter stained in pararosaniline-azure-II and inspected using light microscopy. After a gross examination, regions of interest were defined and the amount of new bone and old bone quantified. Descriptive statistics of all variables were determined, including means and standard deviations. For the calculation of differences between the groups we used a mixed linear model that took the use of dependent variables into account. For multiple testing Scheffe's method was adapted for the correction. Analysis was performed
Please cite this article in press as: Voss PJ, et al., Zoledronate induces osteonecrosis of the jaw in sheep, Journal of Cranio-Maxillo-Facial Surgery (2015), http://dx.doi.org/10.1016/j.jcms.2015.04.020
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with SPSS 22 and STATA 13.1. Results were considered significant at p < 0.05. This study is in compliance with the ARRIVE (Animal Research: Reporting of In Vivo Experiments) guidelines. 3. Results ZOL administration was successfully performed in all animals of the ZOL group and dental surgery was performed in all animals. The recovery time after surgery was uneventful. No significant weight loss occurred between the groups. All ZOL-treated animals suffered from impaired wound healing and exposed bone at all extraction sites during the 16-week postoperative period (Fig. 1A). The exposed bone was present for more than 8 weeks and thus meets the MRONJ definition criteria (Ruggiero et al., 2014). According to the MRONJ staging system, all of the ZOL-treated animals were stage 3. In addition, all ZOL-treated animals showed exposed bone in the oral cavity at sites where no surgical manipulation was performed (Fig. 1B, C, D). In the control group, normal wound healing occurred within the first two weeks (Fig. 2A, B). The MRONJ stage was 0 in all animals (Fig. 4). CT analysis of the animals immediately after euthanasia revealed typical signs of MRONJ in all ZOL-treated animals. Osteolysis, sequestration and periosteal reaction were especially seen around the extraction sites and the sites of spontaneous exposed bone. The alveolar defect was not replaced by cancellous bone formation in the ZOL group; in the control group, complete cortical regeneration was seen in all animals (Fig. 3A, B). Normal bone healing was observed in the control group (Fig. 3C, D). The CT evaluation score revealed a mean score of 2 in the ZOL-treated animals versus 0 in the controls (Fig. 4). No osteolysis or osseous defects were detected in the control group, one animal showed eruption of a fragmented root with no signs of inflammation or infection. The periosteal reaction at the buccal and lingual aspect of the mandible was between 1.92 ± 0.33 and 1.60 ± 0.94 mm in all ZOL-treated animals. The mild and dense periosteal reaction of 0.23 ± 0.34 and 0.18 ± 0.76 mm in the control group was interpreted as a normal sign of new bone formation (Fig. 5A). Two experienced examiners analyzed all histological sections in a blinded manner. Typical signs of MRONJ bone necrosis, such as non-vital bone with rough margins and empty lacunae and no osteocytes were detected in all extraction sites of the ZOL group. Signs of bone remodeling were absent in these animals. No signs of
Fig. 2. (A and B) Clinical aspect in the control group. No exposed bone was visible in any of the animals. Mucosa covered the defect and no signs of infection or inflammation were noticed.
osteolysis were seen in the extraction sites of the control group. ZOL-treated animals showed significantly less new bone formation compared with the control group (0.21 ± 0.18% vs. 2.37 ± 1.41%) (Fig. 5B). The ratio of new bone to old bone in the ZOL-treated animals was significantly decreased 0.009 ± 0.009 compared with the control group 0.070 ± 0.04. 4. Discussion Bisphosphonates are effective drugs used widely in the treatment of bony metastases of malignant diseases, osteoporosis and Paget's disease (Hatoum et al., 2008; Sambrook and Cooper, 2006). Despite a generally low level of side effects, the treatment of MRONJ has become a clinical routine in oral and maxillofacial hospitals as a result of the large numbers of patients receiving BPs. The current definition includes BP-treatment history, exposed bone in the oral cavity for a period longer than 8 weeks and no radiation to the jaws (Ruggiero et al., 2014). Animal models are important to understand the underlying pathological mechanism and to develop and establish an evidence-based treatment option (Ruggiero and Drew, 2007).
Fig. 1. (A) Representing the clinical aspect of the unhealed extraction socket in an animal from the ZOL group. Exposed dark-colored bone can be noted beside pus draining from the anterior part as sign of infection. (B, C and D) Clinical image of the spontaneous exposed bone at parts of the mandible and maxilla where no surgical manipulation was performed.
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Fig. 3. (A and B) Histological and microcomputed tomography section of the unhealed extraction socket in a ZOL-treated animal. Note the periosteal reaction around the buccal and lingual aspect of the mandible. (C and D) Transverse section of the former extraction socket from an animal of the control group. The socket is filled with cancellous bone formation and the mucosa is closed. New bone formation can be noticed around the mandible as a sign of ongoing bone remodeling.
The ideal animal model mimics the clinical situation of the MRONJ lesion in the patient and is consistent with the criteria used in clinics. Various animal models of experimental MRONJ have been described. The most commonly used animal species are rodents, however, the use of dogs and minipigs as large animal models also have been published (Sharma et al., 2013). Especially in rodents, meeting the clinically relevant diagnostic criteria is difficult. While Kobayashi et al. (2010) failed to establish MRONJ in C57BL/6J mice with ZOL only, Bi et al. (2010) reported MRONJ-like lesions in the same model treated with ZOL and dexamethasone. Several authors reported MRONJ-like lesions in Sprague-Dawley and Wistar rats. Guevarra et al. (2013) demonstrated vascular changes in mandibular alveolar bone after tooth extraction in rats receiving ZOL. Barba-Recreo et al. (2014) showed recently, that the highest cumulative doses of ZOL had the highest incidence of osteonecrosis in these animals, with lower numbers of osteoclasts. While Tsurushima et al. (2013) promoted MRONJlike lesions in the mandible and femur of Wistar rats after injecting Aggregatibacter actinomycetemcomitans into the bone marrow, Berti-Couto et al. (2014) identified diabetes mellitus as a
risk factor for developing MRONJ after tooth extraction in these animals. Even though animal studies using rodents are less expensive, the animals show different bone morphology, and are usually too small for testing clinical materials. Because intracortical turnover seems to play a major role in the manifestation of MRONJ, the generalized absence of normal intracortical remodeling in rodents limits their usefulness as a model for MRONJ (Jowsey, 1966; Kubek et al., 2010). €ttingen minipigs have been described as Beagle dogs and Go large animal models for MRONJ. Allen et al. (2011) described compromised osseous healing of extraction sites in beagle dogs treated with ZOL, but only one of six dogs developed exposed bone. In a minipig model, extraction of six teeth led to impaired wound healing and exposed bone in at least three of the extraction sites in all five animals (Pautke et al., 2012). The most common trigger factor for inducing MRONJ is dentoalveolar surgery. However, according to the literature, in 30e40% of cases no local surgical trauma was noted; even though sometimes prosthetic trauma or periodontitis are present, spontaneous
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Fig. 4. (A) The macroscopic score of the MRONJ staging system adapted by Ruggiero et al. (2009) was used to score the mandibles: Stage 0 ¼ healed mucosa and no apparent necrotic bone at extraction site, 1 ¼ no clinical evidence of necrotic bone but unspecific findings at extraction site, 2 ¼ exposed/necrotic bone with no signs of infection at extraction site, 3 ¼ exposed/necrotic bone with signs of infection at site of extraction, 4 ¼ exposed and necrotic bone with pain, infection, and one or more of the following: exposed and necrotic bone extending beyond the region of alveolar bone (ie, inferior border and ramus in the mandible, maxillary sinus and zygoma in the maxilla) resulting in pathologic fracture, extraoral fistula, oral antral/oral nasal communication, or osteolysis extending to the inferior border of the mandible or the sinus floor. (B) The computed tomographic score of the mandible adapted by Pautke et al. (2012): 0: healed bone, 1: osteolysis or non-regeneration without cortical erosion, 2: osteolysis with cortical erosion.
evolution seems to be frequent (Vescovi et al., 2011). Despite one mouse model that recently showed spontaneous osteonecrosis of the jaw after antiresorptive treatment, no other animal study displayed this phenomenon (de Molon et al., 2014). In this sheep model MRONJ was observed, not only at all the dental extraction sites, but also spontaneous exposed bone in the oral cavity developed where no manipulation was performed. This is similar to the situation in many patients. One possible factor could be the dosage of 0.075 mg/kg body weight, which is about 50% higher than doses given for oncological reasons in human patients. We decided to elevate the ZOL doses to have a more reliable development of necrosis, especially given the fact that the animals received only five ZOL infusions every third week before surgery. Repeating of the dose every third week instead of a monthly dose, as it is carried out in oncological patients, reflects the higher bone turnover of sheep (Corlett et al., 1990). Regarding the literature, high cumulative doses of bisphosphonates are a risk factor for the development of MRONJ, and the use of high single doses is more likely to cause transient side effects such as hypophosphatemia, hypocalcemia, hypomagnesemia, renal failure and acute phase symptoms (Body et al., 1999;
Hoff et al., 2008). None of our animals showed any of these transient side effects. In most of the other large animal studies, ZOL dosages vary. While weekly infusions of 0.05 mg ZOL/kg body weight for 6 weeks led to inconsistent development of BP-ONJ in minipigs, (Pautke et al., 2012), monthly infusions of 0.1 mg/kg body weight for 4 months in beagle dogs did not lead to the development of MRONJ lesions (Huja et al., 2011). In another study on beagle dogs, 0.06 mg/ kg every second week was used for a 3 month period. ZOL led to a severe suppression of intracortical remodeling and exposed bone in one out of six animals (Allen et al., 2011). These findings are heterogeneous and make comparison of dosing schemes between different animal species difficult. The right timing and dosage for each species needs to be determined for each species individually. Further research is needed to define the No and Lowest Observed Effect Concentration (NOEC, LOEC) and dose responsiveness for ZOL in sheep. In our study, spontaneous open bone lesions mostly arose in the interdental spaces where bits of hay had accumulated. This supports the theory, that periodontal infections trigger MRONJ, which is also suggested by Aghaloo et al. (2011) who induced ONJ using a
Fig. 5. (A) Mean periosteal reaction in mm at the oral and lingual aspect of the mandible at the extraction socket (B) Ratio of new bone to old bone on the region of interest in the extraction socket of the mandible.
Please cite this article in press as: Voss PJ, et al., Zoledronate induces osteonecrosis of the jaw in sheep, Journal of Cranio-Maxillo-Facial Surgery (2015), http://dx.doi.org/10.1016/j.jcms.2015.04.020
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periodontitis model with wire ligatures and 66 mg/kg of ZOL in rats. In the histological analysis of our animals, we found a significantly higher fraction of old bone and a smaller fraction of new bone in the periodontal region of ZOL-treated sheep compared with the control group. Following a widespread theory, the reduced boneremodeling rate does not allow adequate reaction of the bony tissue to inflammatory and physical stimuli. Given the fact that low concentrations of ZOL rapidly and directly affect the oral fibroblasts and epithelial cells (Scheper et al, 2009), it is not unlikely that the oral mucosal tissues disconnect from the bone through shearing forces which occur during chewing cycles. This exposed bone will react as surgically denudated bone. Further studies concerning the periodontal cause of MRONJ in sheep need to confirm this theory. 5. Conclusion ZOL administrations consistently lead to MRONJ after tooth extractions in sheep. In interdental spaces the direct effect of low ZOL concentrations on mucosal tissues in interaction with local irritation and shearing forces during chewing cycles and the reduced bone remodeling in the alveolar process may lead to spontaneously exposed bone. This large animal model is feasible for further evaluation of bone and fracture healing, augmentation procedures and oral implantology and periodontitis models for the pathogenesis of MRONJ. Acknowledgements This study was funded by the AO Foundation by way of the AOCMF R&D Research Commission (Grant No C-11-11-V). The study was conducted in an AAALAC accredited facility and followed the ARRIVE guidelines. We would also thank Dr. Vincent Stadelmann for his help with the CT. We thank Luca Werner for his help with preparing and analyzing the histological samples. The authors declare no conflict of interests. References Aghaloo TL, Kang B, Sung EC, Shoff M, Ronconi M, Gotcher JE, et al: Periodontal disease and bisphosphonates induce osteonecrosis of the jaws in the rat. J Bone Miner Res 26(8): 1871e1882. http://dx.doi.org/10.1002/jbmr.379, 2011 Aug Allen MR: Bisphosphonates and osteonecrosis of the jaw: moving from the bedside to the bench. Cells, tissues, organs 189: 289e294, 2009 Allen MR, Burr DB: Mandible matrix necrosis in beagle dogs after 3 years of daily oral bisphosphonate treatment. J Oral Maxillofac Surg 66: 987e994, 2008 Allen MR, Kubek DJ, Burr DB, Ruggiero SL, Chu TM: Compromised osseous healing of dental extraction sites in zoledronic acid-treated dogs. Osteoporos Int 22: 693e702, 2011 Barba-Recreo P, Del Castillo Pardo de Vera JL, Garcia-Arranz M, Yebenes L, Burgueno M: Zoledronic acid-related osteonecrosis of the jaws. Experimental model with dental extractions in rats. J Craniomaxillofac Surg 42: 744e750, 2014 Berti-Couto SA, Vasconcelos AC, Iglesias JE, Figueiredo MA, Salum FG, Cherubini K: Diabetes mellitus and corticotherapy as risk factors for alendronate-related osteonecrosis of the jaws: a study in Wistar rats. Head Neck 36: 84e93, 2014 Bi Y, Gao Y, Ehirchiou D, Cao C, Kikuiri T, Le A, et al: Bisphosphonates cause osteonecrosis of the jaw-like disease in mice. Am J Pathol 177: 280e290, 2010 Body JJ, Lortholary A, Romieu G, Vigneron AM, Ford J: A dose-finding study of zoledronate in hypercalcemic cancer patients. J Bone Miner Res 14: 1557e1561, 1999 Burr DB, Allen MR: Mandibular necrosis in beagle dogs treated with bisphosphonates. Orthod Craniofac Res 12: 221e228, 2009 Carlson ER, Basile JD: The role of surgical resection in the management of bisphosphonate-related osteonecrosis of the jaws. J Oral Maxillofac Surg 67: 85e95, 2009 Corlett SC, Couch M, Care AD, Sykes AR: Measurement of plasma osteocalcin in sheep: assessment of circadian variation, the effects of age and nutritional status and the response to perturbation of the adrenocortical axis. Exp Physiol 75: 515e527, 1990 de Molon RS, Cheong S, Bezouglaia O, Dry SM, Pirih F, Cirelli JA, et al: Spontaneous osteonecrosis of the jaws in the maxilla of mice on antiresorptive treatment: a novel ONJ mouse model. Bone 68: 11e19, 2014
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Please cite this article in press as: Voss PJ, et al., Zoledronate induces osteonecrosis of the jaw in sheep, Journal of Cranio-Maxillo-Facial Surgery (2015), http://dx.doi.org/10.1016/j.jcms.2015.04.020