Preclinical evidence of potential craniofacial adverse effect of zoledronic acid in pediatric patients with bone malignancies

Bone 68 (2014) 146–152

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Original Full Length Article

Preclinical evidence of potential craniofacial adverse effect of zoledronic acid in pediatric patients with bone malignancies Frédéric Lézot a,b, Julie Chesneau a,b, Séverine Battaglia a,b, Régis Brion a,b, Beatriz Castaneda c, Jean-Christophe Farges d,e, Dominique Heymann a,b, Françoise Rédini a,b,⁎ a

INSERM, UMR-957, Nantes, F-44035, France Université de Nantes Nantes Atlantique Université, Faculté de Médecine, Laboratoire de physiopathologie de la résorption osseuse et thérapie des tumeurs osseuses primitives, Nantes F-44035, France c INSERM, UMR1138, Paris F-75006, France d IGFL, CNRS UMR-5242, ENS de Lyon, Lyon F-69364, France e Université de Lyon 1, Faculté d'odontologie, Equipe odontoblastes et régénération du tissu dentaire, Lyon F-69372, France b

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Article history: Received 29 April 2014 Revised 18 July 2014 Accepted 27 August 2014 Available online 2 September 2014 Edited by: Michael Amling Keywords: Zoledronic acid Tooth eruption Skull bone formation Osteosarcoma

a b s t r a c t High doses of zoledronic acid (ZOL), one of the most potent inhibitors of bone resorption, are currently evaluated in phase III clinical trials in Europe for the treatment of malignant pediatric primary bone tumors. The impact of such an intensive treatment on the craniofacial skeleton growth is a critical question in the context of patients with actively growing skeleton; in particular, in light of our previous studies evidencing that endochondral bone formation was transiently disturbed by high doses of ZOL. Two protocols adapted from pediatric treatments were developed for newborn mice (a total of 5 or 10 injections of ZOL 50 μg/kg every two days). Their impact on skull bones and teeth growth was analyzed by X-rays, microCT and histology up to 3 months after the last injection. ZOL administrations induced a transient delay of skull bone growth and an irreversible delay in incisor, first molar eruption and root elongation. Other teeth were affected, but most were erupted by 3 months. Root histogenesis was severely impacted for all molars and massive odontogenic tumor-like structures were observed in all mandibular incisors. High doses of ZOL irreversibly disturbed teeth eruption and elongation, and delayed skull bone formation. These preclinical observations are essential for the follow-up of onco-pediatric patients treated with ZOL. © 2014 Elsevier Inc. All rights reserved.

Introduction Bisphosphonates (BPs) are synthetic, non-hydrolysable analogs of the naturally occurring pyrophosphate, sharing a common P-x-P structure in which the central atom of oxygen in pyrophosphate has been replaced by a carbon atom in BPs, allowing the addition of two side chains. BPs are broadly divided into nitrogen- and non-nitrogen containing families [1]. Among the nitrogen-containing BPs (N-BPs), zoledronic acid (ZOL) is one of the most potent inhibitors of bone resorption, characterized by a heterocyclic substituent. BPs have high affinity for calcium and therefore target bone mineral, where they appear to be internalized selectively by bone-resorbing osteoclasts inhibiting their activity and function via the inhibition of protein prenylation [2]. BPs

Abbreviations: BP, bisphosphonate; ZOL, zoledronic acid. ⁎ Corresponding author at: INSERM UMR957, UNAM, Université de Nantes, EA 3822, Faculté de Médecine, 1 rue Gaston Veil, 44 035 Nantes Cedex 1, France. Fax: +33 240 412 860. E-mail address: [email protected] (F. Rédini).

http://dx.doi.org/10.1016/j.bone.2014.08.018 8756-3282/© 2014 Elsevier Inc. All rights reserved.

are extensively used in clinical practice for the treatment of high bone degradation associated diseases, mainly osteoporosis, Paget's disease, and cancer related bone diseases such as multiple myeloma or bone metastasis from prostate and breast carcinoma [3,4]. They have also been shown to benefit children with skeletal pediatric disorders such as osteogenesis imperfecta, juvenile rheumatoid arthritis or juvenile idiopathic osteoporosis [5–8]. More recently, high doses of ZOL have been proposed as adjuvant therapy for young patients (median age: 15–18 years) with malignant primary bone tumors in Europe and, in the clinical phase III OS2006 trial in France (osteosarcoma) and the EWING2008 (Ewing sarcoma) in Germany. Its association with conventional therapy is also discussed for the future EWING2012 (European protocol for French and English patients with Ewing's sarcoma) and EURAMOS (European consortium which will join the French OS2006 protocol in 2013) protocols. The rationale for the therapeutic use of ZOL in bone tumors is linked to the existence of a stimulatory feedback loop (called the ‘vicious cycle’) between tumor cell proliferation and bone resorption during tumor development in bone [9]. Thus, ZOL may have complementary actions on bone tumors: an indirect anti-

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tumor effect by inhibiting osteoclastogenesis and a direct action on tumor cells. Several preclinical data confirm this hypothesis in osteosarcoma models [10,11] and in Ewing's sarcoma [12,13]. Other preclinical studies and clinical observations have demonstrated that BPs, in particular alendronate and ZOL, delay or inhibit tooth eruption causing several dental abnormalities [14–17] and may, as in the juvenile Paget's disease of bone, exert an inhibitory effect on bone mineralization [18]. Such inhibition of mineralization has been observed for various BPs using calvaria osteoblast culture [19]. According to the fact that cumulative doses of ZOL used in pediatric cancer protocols are 5 times higher (50 μg/kg every 4 weeks, 10 times: 500 μg/kg/year) than those given for other pediatric bone disorders (same dose, once/6 months: 100 μg/kg/year), we propose to investigate the impact of high doses of ZOL on the peculiar craniofacial skeleton growth, knowing that such doses induce transitory arrest of axial and appendicular skeleton long bone growth as demonstrated in our previous works [20]. Craniofacial skeleton growth is a complex process requiring a harmonious and timely controlled growth of the diverse skeleton entities (bones, teeth…) and fusions of some of them, for instance flat bones of skull. Any defect in bone fusions timing and any ruptures of the harmonious growth of the different skeleton elements are responsible of craniofacial pathologies [21]. Skull bones and teeth are representative elements of the craniofacial skeleton whose growth processes have been widely documented [21,22]. Therefore, the present experimental study concerning the impact of high doses of ZOL on craniofacial development is focused on skull membranous bone and teeth growth. Materials and methods Animals and drug administration Pregnant C57BL/6J mice (14 days of gestation) were purchased at Janvier's breeding (Le Genest Saint Isle, France). Mice were housed under pathogen-free conditions at the Experimental Therapy Unit (Faculty of Medicine, Nantes, France) in accordance with the institutional European guidelines (EU directive 2010/63/EU) and of the French Ethical Committee (CEEA-PdL-06) under the supervision of authorized investigators. Newborn mice were used for experiments. After weaning, mice were routinely fed by liquid diet. Two protocols were carried out independently (Fig. 1A) according to pharmacokinetics data of zoledronic acid already published [23,24]. The first protocol was developed in order to approximate the time schedule of the clinical protocol administered in onco-pediatric patients: 10 perfusions (4 preoperative and 6 postoperative) of 50 μg/kg zometa® (Novartis Pharmaceuticals Corporation) at 4 week intervals that approximately correspond to 2 days in mouse life. The second one is a short-term protocol developed to determine potential reversible skeleton defects induced by ZOL injections. Protocol 1—long-term treatment At birth, C57BL/6J newborn mice (from naive mothers) were randomized into two groups that received ten subcutaneous injections of ZOL (50 μg/kg in PBS kindly provided as the disodium hydrate by Pharma Novartis AG, Basel, Switzerland) or PBS alone (controls) every 2 days beginning at day 1 after birth. Animals were sacrificed at day 21 (end of treatment). Protocol 2—short-term treatment At birth, C57BL/6J newborn mice were randomized into two groups for five subcutaneous injections of ZOL (50 μg/kg in PBS) or PBS alone (controls) every 2 days beginning at day 1. Animals were sacrificed on day 11 (end of treatment), on day 21 (to compare with protocol 1) or 1 (day 39) and 3 (day 99) months after the end of treatment. Heads and tibias were analyzed by micro-CT, micro-radiography and histology.

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Body mass The animals were weighed every two days all along each treatment and once a week after the end of treatment until the latest endpoint. Micro-radiography Tibias and heads were collected at day 11, 21, 39 and 99 after birth, and were analyzed with the radiography apparatus Faxitron (Edimex, Angers, France). Micro-CT analysis Analyses of bone microarchitecture were performed using a Skyscan 1076 in vivo micro-CT scanner (Skyscan, Kontich, Belgium). Tests were performed after sacrifice on tibias and heads for each treatment group. All tibias and heads were scanned using the same parameters (pixel size 18 μm, 50 kV, 0.5-mm Al filter, 10 min of scanning). The reconstruction was analyzed using NRecon and CTan software (Skyscan). For long bones, the volumes of interest (VOI) were determined as 15% and 50% of the trabecular bone (tibias). Thresholds used for VOI were 55 and 255. The specific bone volume was quantified as the relative bone volume/ total volume measured for each VOI. 3D visualizations of tibias and heads were realized using ANT software (Skyscan) at sacrifice. Histology Tibias and heads were collected from euthanized mice from control and ZOL treated groups and were fixed in 4% buffered paraformaldehyde. Tibias and heads were decalcified in 4.13% EDTA/0.2% paraformaldehyde pH 7.4 over 4 days in KOS sw10 (Milestone, Sorisole, Italy). The specimens were dehydrated and embedded in paraffin. Then 3-μm-thick sagittal sections stained with Masson's trichrome (three-color staining protocol which stains muscle fibers in red, collagen and bone in green, cytoplasm in light red, and nuclei in dark brown) were observed using a DMRXA microscope (Leica, Nussloch, Germany). Tartrate resistant acid phosphatase (TRACP) staining was performed on tibia and head sections to identify multinucleated osteoclast-like cells after 90 minute incubation in a 1 mg/mL of Naphthol AS-TR phosphate, 60 mmol/L N, Ndimethylformamide, 100 mmol/L sodium tartrate, and 1 mg/mL Fast red TR salt solution (all from Sigma Chemical Co., St Louis, MO, USA) and counterstained with hematoxylin. Flow cytometry Spleen's cells were collected and washed in cold PBS. Three hundred thousand cells were stained with the following FITC conjugated rat antibodies: anti-mouse CD45R/B220 (clone RA3-6B2), anti-mouse CD11b (clone M1/70) and anti-mouse CD3 (clone 17A2) all from BD Biosciences Pharmingen (Le Pont de Claix, France). Staining was assessed using a FC500 cytometer and CXP analysis software (Beckman Coulter, Villepinte, France). FITC labeled rat anti IgG2a antibodies were used as negative controls. Statistical analyses For micro-CT analysis, a one-way ANOVA test followed by a Dunnett post-test was performed to evidence statistical significant differences in VOI between groups. For TRAP positive cells, in order to obtain numerical values representative of the osteoclast number, the surface covered by TRAP positive cells was measured for each section and normalized in relation to the total volume (TrapV/TV) using Image-J software. A Student t-test was performed to evidenced statistical significant differences.

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Fig. 1. Chronograph of the two protocols used and their impacts on tibia micro-architecture. A: Chronograph of long term (ZOL10: 10 injections of 50 μg/kg ZOL at 2 day intervals, beginning at day 1 after birth) and short term (ZOL5: 5 injections of 50 μg/kg ZOL) treatments presenting times of injection and sacrifice. B: Micro-radiographies of the tibias of young mice (day 21) treated or not with ZOL. Tibia sizes of ZOL treated animals were reduced comparatively to controls (CT). Tibias of ZOL10 treated mice evidenced an elevated mineral density in both the primary spongiosa and the bone marrow cavity. Tibias of ZOL5 treated mice presented an intermediary density with however remnant dense horizontal streaks corresponding to injection periods (arrow-heads). C–D: Three-dimensional representation of bone micro-architecture analyzed by micro-CT at day 21 (C) and 11 (D). The specific bone volume can be calculated and compared between controls and ZOL treated mice, on the 15 or 50% of tibia length. Presented values correspond to measures performed on two representative animals from each group of six mice. In tibias of ZOL5 treated mice, remnant dense horizontal streaks (arrow-heads) were visible at day 21 while a huge global density was present at day 11. *:p b 0.05; ***:p b 0.001.

Results Zoledronic acid impact on newborn's growth Newborn mice were treated with zoledronic acid (50 μg/kg) a total of 10 times (ZOL 10) from day 1 after birth to day 19, every 2 days (Fig. 1A). Pups were further sacrificed two days after the last ZOL injection (day 21). A short term protocol with only 5 injections (ZOL 5) was also developed: the animals were sacrificed two days after the last ZOL injection (day 11) or at day 21 to compare with long term protocol. Two others time points have been added to this protocol: two mice were sacrificed 1 and 3 months (respectively 39 and 99 days) after the end

of the treatment (Fig. 1A). The weight of ZOL treated and untreated pups has been monitored for the all duration of the treatment (Supplementary Fig. 1). During the first 21 days, no significant body mass differences have been observed between the treated and un-treated groups (supplementary Fig. 1). However, from day 21 (after weaning) a significant decrease of the total body mass gain was observed in short term treated animals (Supplementary Fig. 1B). One may notice that mice were fed with a liquid diet after weaning to deal with potential consequences of dental defects on mouse intake. Concerning the size, treated animals remained smaller than their untreated littermates at all ages (data not shown). Micro-radiographic and micro-CT analyses of tibias evidenced a growth delay of long bone elongation at the end of

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treatments (day 21, Figs. 1B and C) as it has been previously reported [20]. This phenomenon was due to a huge bone formation observed at the growth plate that can be quantified as a significant augmentation of the specific bone volume (Figs. 1C–D: *: p b 0.05; ***: p b 0.001). Indeed, the specific bone volume calculated in the 50% of the total length was around 20% and 60% respectively for short term- and long termtreated pups versus less than 5% in controls (4.7 and 4.8%; Fig. 1C). The specific bone volume (in the 15% of the total length) represents around 8% in the control bones, versus about 9% and 70% respectively in ZOL short term- and long term-treated pups (Fig. 1C). Interestingly, the fact that specific bone volumes were lower at 21 days in the short term- versus long term-treated animals can be explained by the difference of time period following the last injection and the opportunity of recovery. This was supported by the observation at day 11 of elevated specific bone volumes in short term-treated animals versus controls (Fig. 1D) and by comparative analyses of tibia longitudinal sections at day 21 (Supplementary Fig. 2). Indeed, an enlargement of the growth plate hypertrophic zone, characteristic of osteopetrosis, was observed for long term-treated mice whereas the size of this zone remained unchanged in short term-treated animals (Supplementary Fig. 2A). Surprisingly, a significant increase of the number of TRAP positive cells was observed at the end of treatments whatever the bone considered:

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long bone or mandible (Supplementary Figs. 2B and 3). However, this number was unchanged two weeks after the last injection in the case of short-term treatment (Supplementary Fig. 2B). As ZOL induced an osteopetrosis-like phenotype at the end of treatments, we wondered whether these treatments might interfere with medullar hematopoiesis, inducing potential extra-medullar hematopoiesis in the spleen. T-lymphocytes, B-lymphocytes and monocytes populations of the spleen were thus quantitatively compared by flow cytometry analysis between ZOL treated (5 and 10 injections) and untreated mice at day 21. No difference was observed between treated and untreated animals (Supplementary Fig. 4). Zoledronic acid impact on craniofacial skeleton growth The impact of ZOL treatments on craniofacial skeleton at day 21 has been evaluated micro-radiography (Fig. 2A) and micro-CT (Fig. 2B) analyses, demonstrating a blockage of all teeth eruption and root elongation, independently of the treatment period (arrow-heads in Fig. 2). An enlargement of the craniofacial sutures was also observed by micro-CT (stars in Fig. 2B) in the ZOL treated group. This may be associated to perturbations of osteoblast functions as revealed by the whole cranium alizarin red and alcian blue double staining (Fig. 2C). The

Fig. 2. Effects of early zoledronic acid treatments (5 and 10 injections every two days beginning at day 1) on general tooth eruption and suture formation analyzed at day 21 by microradiography (A), micro-CT (B) and alcian blue/alizarin red double staining (C). Both treatments induced an arrest of all teeth eruption (arrow-heads and higher magnification) and an important delay of calvaria bone growth with an enlargement of the sutures (stars in B). Moreover, the skull of treated mice presented a domed morphology (dotted-line) associated with a perturbed suture mineralization process evidenced by alizarin red staining (stars in C).

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suture enlargement was observed whatever the protocol used, however it seemed to be more important for the long-term protocol. The presence of a domed skull (doted-line in Fig. 2C), characteristic of osteopetrotic animals, was associated to the suture enlargement. In order to establish whether the observed craniofacial defects could be reversible or not, animals were sacrificed one and three months after the last ZOL injection in the short-term protocol. Short-term protocol was chosen taking into account that the irreversible defects observed with this protocol would be also present and eventually reinforced in longer treatment. Micro-CT analyses evidenced that the suture mineralization defects and the dome morphology of the skull were gradually normalized by three months after the end of ZOL treatment as represented by doted lines (Fig. 3A: 1 month and 3B: 3 months). In contrast, the dental phenotype was not restored. Micro-CT analyses performed one and three months after the end of ZOL treatment evidenced that incisors and first molars were definitively included (arrow-heads in Figs. 3A–B) and that second and third molar eruptions were highly delayed (Figs. 3A–B). Moreover, deformation of the mandibular bone was observed in the basal region (arrows in Figs. 3A–B). Micro-CT sections in the frontal plane at the second molar level (Figs. 3C–D) evidenced the formation in the lower incisor area of an abnormal mineralized structure associated with mandibular bone perforations (arrows in Figs. 3C–D). Histological frontal sections of treated mouse mandible in the different molar planes showed that eruption of all molars and root elongation were severely delayed one month after the last ZOL injection (Figs. 4D–F vs A–C) and confirmed that first molars were still included three months after the last injection (supplementary Fig. 5D vs A). Root hypercementosis (stars in Figs. 4D and H, and Supplementary Figs. 5D and G) and partial ankylosis (arrows in

Figs. 4D,E,H and I and supplementary Figs. 5D–E) were observed at one and three months. TRAP histo-enzymology enabled to see the presence of root resorption pits (arrow-heads in Fig. 4J and Supplementary Figs. 5F–G) and a global increase of the TRAP expressing cells (red staining) around molars (Figs. 4H–J versus G). Under the second and third molars, in place of the incisor, an odotongenic tumor-like structure was observed (T in Figs. 4E,F,I and J, and Supplementary Figs. 5E–H). Enamel and dentin matrices (stained in brown and green respectively in Fig. 4F and Supplementary Fig. 5F) were present in this structure showing its mixed epithelial and mesenchymal origins. An important TRAP staining was observed around the structures (Figs. 4I–J and arrow-heads in Supplementary Figs. 5G–H) with perforations of the mandibular bone in the lingual region at one month (Circles in Figs. 4F and J) and a total destruction of this bone area at three months (ovoids in Supplementary Figs. 5F–H). Discussion The present study was devoted to the analysis of high doses zoledronic acid (ZOL) impact on craniofacial skeleton growth. High doses of ZOL have been proposed as adjuvant therapy for the treatment of malignant primary bone tumors (osteosarcoma and Ewing sarcoma; 4, 13) that mainly affect children and young adults [25,26]. The principal objectives of high doses zoledronic acid protocol were not only to protect bone from the tumor-induced osteolysis but also to target tumor cells [27], ZOL being a powerful inhibitor of the mevalonate pathway [28]. Our previous studies have evidenced that high doses of ZOL in mouse, similar to the human pediatric treatment, induced a transient arrest of the axial and appendicular skeleton growth of long bones [20]. These

Fig. 3. Micro-CT analyses of the potential reversibility of the craniofacial phenotype of ZOL treated animals, one (A and C) and three (B and D) months after the last ZOL injection. A graded amelioration of the skull phenotype was observed (dotted lines for the domed skull) with a less important suture enlargement at one month (star in A) and no visible suture defect at three months (B). The tooth eruption was definitively blocked for incisors and first molars (arrow-heads) whereas other molars were erupted at three months. Micro-CT sections in the frontal plane through the second molars confirmed these molar eruptions and enable to visualize the presence of an abnormal mineralized structure in place of the incisor. This structure appeared to increase in size from one to three months and was associated with perforation and finally destruction of the mandibular bone (arrows).

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Fig. 4. Histological analyses of the dental phenotype of animals treated with ZOL5 one month after the last ZOL injection (A–F: Masson staining; G–J: TRAP histo-enzymology). Frontal sections in the planes of the different molars enabled to see eruption delay of all teeth in ZOL treated mice (D–F) versus untreated controls (A–C). The presence of a hypercementosis (stars in D and H), an ankylosis (arrows in D, E, H and I) and some root resorptions (arrow-heads in J) were also observed. An odontogenic tumor-like structure was present in place of the incisor (T in E, F, I and J) surrounded by an important number of TRAP positive cells (I–J) with holes in the mandible basal bone (circles in F and J). M1: first molar; M2: second molar; M3: third molar; I: incisor; B: bone; T: odontogenic tumor-like structure; CT: control; ZOL5: mouse receiving 5 injections of zoledronic acid (50 μg/kg) at 2 day intervals beginning at day 1 after birth.

observations were further supported by clinical data [20], but currently not sufficient to restrict the use of high doses of ZOL to adult patients. However, the impact of elevated cumulative doses of ZOL on craniofacial skeleton growth has never been fully documented and is necessary in the context of onco-pediatric treatment. Available data are related to

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the consequence of osteopetrosis induced by low cumulative doses of ZOL on tooth eruption and tooth growth in rat [16], and on tooth eruption in children with osteogenesis imperfecta [17]. The inhibition of tooth eruption and the consequent dental abnormalities were also reported for high cumulative doses of other bisphosphonates such as alendronate [14] or pamidronate [15] known to be less efficient than ZOL. Moreover, the potential reversibility of bisphosphonate induced dental defects has never been analyzed. The present longitudinal study of ZOL effect in mice up to three months after the end of treatment enables us to answer the question of the reversibility of the dentoalveolar phenotype induced by high doses of ZOL injection during growth. Moreover, this study also considered the reversibility of ZOL impact on the formation of other elements of the craniofacial skeleton, more precisely skull bones. Presented results obtained in mice evidenced that only part of the craniofacial skeleton phenotype induced by high doses of ZOL was reversed three months after the treatment. The craniofacial suture enlargement which represents the likely consequence of osteoblast differentiation and mineralization defect [29,30], and the domed form of the skull were gradually normalized. Moreover, the second and third molar eruptions were finally effective. However, incisors and first molars were definitively included and important dental defects (ankylosis, hypercementosis and root resorptions) were observed in the different molars. The development of an odontoblastic tumor-like structure was observed after ZOL treatment in the distal area of the mouse mandible continuously growing incisor. Such tumor development is a characteristic feature of osteopetrosis in mouse [31] with a limited relevance for human. Indeed, it appeared to be restricted to continuously growing teeth, and only rare cases of odontoma formation have been described in osteopetrotic patients [32]. However, it is known from clinicians that included tooth (mainly the third molar) may convert into odontoma [33]. So a continuous and rigorous odontologic follow-up of onco-pediatric patients receiving ZOL injections appears to be necessary to ensure all teeth eruption and to rapidly take in charge dental defects potentially induced by the treatment. A better knowledge of the origin of the dental defects induced by ZOL treatment may help the clinicians to answer this question. It is too restrictive to consider that the dental defects are only the consequences of osteopetrosis. Indeed, a direct effect of ZOL on dental cell differentiation and functions has to be considered as it was previously reported for bisphosphonates from first generation [34]. In addition, an important increase of TRAP positive cell number is observed on the bone surface of ZOL treated mice (this study; [35]) as previously reported in pamidronate treated rats [15] and in alendronate treated patients [36]. These cells are unable to resorb bone but may secrete signaling molecules, for instance Semaphorin 4D [37] and Ephrin [38] that could affect dental cell differentiation and functions [39,40]. Interestingly, an effect of osteoclasts on dental cells was already reported in a transgenic mouse model of expansile familial osteolysis [41]. Finally, further studies will be necessary to decipher in depth the mechanism by which ZOL induces irreversible dental defect in mouse. To conclude, high dose injections of ZOL in a mouse model reproducing onco-pediatric protocol induce craniofacial skeleton defects, some of which being irreversible after the end of the treatment, such as dental flaws. Such observation requires caution, and a strict preventive odontologic follow-up is therefore necessary when children are enrolled in therapeutic trials with high doses of bisphosphonate, as in the French OS2006 and German EWING2008 protocols. Supplementary data to this article can be found online at http://dx. doi.org/10.1016/j.bone.2014.08.018. Acknowledgments The authors wish to thank Y. Allain, G. Hamery, and P. Monmousseau from the Therapeutic Experimental Unit (Nantes, France) for their technical assistance and Dr B. Ory for critical reading of the manuscript.

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The project has received the financial support of the French Association for Cancer Research (ARC, Project # ECL2010R00778 for the small animal radiography device Faxitron, Edimex). This work was supported by a grant from Novartis Pharma (Grant 2006/051/Novartis RueilMalmaison, France).

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