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Zoledronic acid (ZOL), a nitrogen bisphosphonate (N-BP), is currently used to treat and control pediatric osteolytic diseases. Variations in the intensity of the effects and side effects of N-BPs have been reported with no clear explanations regarding their origins. We wonder if such variations could be associated with different levels of RANKL signaling activity in growing bone during and after the treatment with N-BPs. To answer this question, ZOL was injected into neonate C57BL/6J mice with different genetically-determined RANKL signaling activity levels (Opg+/+\RankTg−, Opg+/+\RankTg+, Opg+/−\RankTg−, Opg+/−\RankTg+, Opg−/−\RankTg− and Opg−/−\RankTg+ mice) following a protocol (4 injections from post-natal day 1 to 7 at the dose of 50 μg/kg) that mimics those used in onco-pediatric patients. At the end of pediatric growth (1 and half months) and at an adult age (10 months), the bone morphometric and mineral parameters were measured using μCT in the tibia and skull for the different mice. A histologic analysis of the dental and periodontal tissues was also performed. At the end of pediatric growth, a delay in long bone and skull bone growth, a blockage of tooth eruption, some molar root alterations and a neoplasia-like structure associated with incisor development were found. Interestingly, the magnitude of these side effects was reduced by Opg deficiency (Opg−/−) but increased by Rank overexpression (RankTg). Analysis of the skeletal phenotype at ten months confirmed respectively the beneficial and harmful effects of Opg deficiency and Rank overexpression. These results validated the hypothesis that the RANKL signaling activity level in the bone microenvironment is implicated in the modulation of the response to ZOL. Further studies will be necessary to understand the underlying molecular mechanisms, which will help decipher the variability in the effects of N-BPs reported in the human population. Significant statements The present study establishes that in mice the RANKL signaling activity level is a major modulator of the effects and side-effects of bisphosphonates on the individual skeleton during growth. However, the modulatory actions are dependent on the ways in which this level of activity is increased. A decrease in OPG expression is beneficial to the skeletal phenotype observed at the end of growth, while RANK overexpression deteriorates it. Far removed from pediatric treatment, in adults, the skeletal phenotypes initially observed at the end of growth for the different levels of RANKL signaling activity were maintained, although significant improvement was associated only with reductions in OPG expression.

Introduction : Des observations récentes effectuées dans le service d’ODF de la Pitié-Salpêtrière à Paris montrent une augmentation des altérations de l’éruption des molaires permanentes non-familiales. Nos travaux récents au laboratoire montrent l’implication des ostéoclastes (OC) dans les processus d’éruption et de rétention dentaires avec implication de la voie de signalisation RANKL/RANK/OPG. Ces faits nous ont amenés à émettre l’hypothèse d’une étiologie environnementale à l’origine de ces défauts d’éruption qui correspondrait à la perturbation des voies de signalisation cellulaires autocrines/paracrines telles que la voie RANKL/RANK/OPG. Matériels et méthodes : Des souris C57BL/6 ont subi des injections d’anticorps anti- RANKL à intervalles réguliers au cours des neuf premiers jours après la naissance. Une comparaison phénotypique avec les souris transgéniques RANK a permis la caractérisation fonctionnelle de la voie RANK/RANKL. Le complexe dento-alvéolaire a été analysé par micro-CT pour la densité osseuse, et la coloration au trichrome de Masson pour les examens histologiques. Résultats : L’invalidation transitoire de RANKL a conduit à un arrêt du développement radiculaire des molaires et l’inhibition de l’éruption dentaire contrairement au phénotype des souris surexprimant RANK. Le recrutement et l’activité des ostéoclastes ont été fortement altérés. Discussion : Ces recherches présentent un intérêt clinique tant direct concernant la compréhension des pathologies de l’éruption qu’indirect pour l’établissement des protocoles de traitements orthodontiques pour les cas particuliers.

The purpose of the present study was to assess the early stages of development of mouse first molar roots in the osteopetrotic context of RANKL invalidation in order to demonstrate that the radicular phenotype observed resulted not only from defective osteoclasts, but also from loss of cell-to-cell communication among dental, periodontium and alveolar bone cells involving RANKL signaling. Two experimental models were used in this study: Rankl mutants with permanent RANKL invalidation, and C57BL/6J mice injected during the first postnatal week with a RANKL neutralizing antibody corresponding to a transient RANKL invalidation. The dento-alveolar complex was systematically analyzed using micro-CT, and histological and immunohistochemical approaches. These experiments showed that the root elongation alterations observed in the Rankl-/- mice were associated with reduced proliferation of the RANK-expressing HERS cells with a significant decrease in proliferating cell nuclear antigen (PCNA) expression and a significant increase in P21 expression. The phenotypic comparison of the adult first molar root at 35 days between permanent and transitory invalidations of RANKL made it possible to demonstrate that alterations in dental root development have at least two origins, one intrinsic and linked to proliferation/differentiation perturbations in dental-root-forming cells, the other extrinsic and corresponding to disturbances of bone cell differentiation/function.

The purpose of the present study was to assess the early stages of development of mouse first molar roots in the osteopetrotic context of RANKL invalidation in order to demonstrate that the radicular phenotype observed resulted not only from defective osteoclasts, but also from loss of cell-to-cell communication among dental, periodontium and alveolar bone cells involving RANKL signaling. Two experimental models were used in this study: Rankl mutants with permanent RANKL invalidation, and C57BL/6J mice injected during the first postnatal week with a RANKL neutralizing antibody corresponding to a transient RANKL invalidation. The dento-alveolar complex was systematically analyzed using micro-CT, and histological and immunohistochemical approaches. These experiments showed that the root elongation alterations observed in the Rankl-/- mice were associated with reduced proliferation of the RANK-expressing HERS cells with a significant decrease in proliferating cell nuclear antigen (PCNA) expression and a significant increase in P21 expression. The phenotypic comparison of the adult first molar root at 35 days between permanent and transitory invalidations of RANKL made it possible to demonstrate that alterations in dental root development have at least two origins, one intrinsic and linked to proliferation/differentiation perturbations in dental-root-forming cells, the other extrinsic and corresponding to disturbances of bone cell differentiation/function.

The muscle segment homeogenes Msx1 and Msx2 play a major role in tooth and bone formation. Periodontal osteoclast impairment also occurs in Msx2 null mutant mice, which is restored by overexpression of the receptor activator of NF-κB targeted in osteoclast lineage. Here, we investigated the role of Msx2 in dentinogenesis. Experiments were performed on Msx2(-/-) mice and the MDPC-23 odontoblastic cell line. After Msx2 gene silencing, real-time quantitative RT-PCR data showed significant overexpression of Runx2, Bglap, Dspp, and Alpl. Of three inhibitors of Wnt/β-catenin signaling (Dkk1, SostDc1, and Sost/Sclerostin), only Sost was expressed in postnatal teeth and overexpressed in Msx2(-/-) tooth samples. Initial crown dentin formation-primary dentinogenesis-occurred fairly normally in Msx2(-/-) teeth, albeit with distorted cusp patterns. Later stages of tooth development were characterized by a deviation from secondary toward tertiary dentinogenesis with osteodentin formation and impaired dentin deposition leading to limited root elongation. In Msx2(-/-)/receptor activator of NF-κB-transgenic double mutants, the dentin phenotype, notably in the roots, was rescued and sclerostin levels were normalized. These data suggest that Msx2 may act indirectly on dentinogenesis by controlling osteoclast activity and the signaling network related to eruption, supporting and further extending the concept that Msx2 controls formation of mineralized tissues by inhibition of the Wnt/β-catenin pathway; Sost in dentin and Dkk1 in bone, as previously demonstrated.

RANKL signalization is implicated in the morphogenesis of various organs, including the skeleton. Mice invalidated for Rankl present an osteopetrotic phenotype that was less severe than anticipated, depending on RANKL’s implication in morphogenesis. The hypothesis of an attenuated phenotype, as a result of compensation during gestation by RANKL of maternal origin, was thus brought into question. In order to answer this question, Rankl null mutant pups from null mutant parents were generated, and the phenotype analyzed. The results validated the presence of a more severe osteopetrotic phenotype in the second-generation null mutant with perinatal lethality. The experiments also confirmed that RANKL signalization plays a part in the morphogenesis of skeletal elements through its involvement in cell-to-cell communication, such as in control of osteoclast differentiation. To conclude, we have demonstrated that the phenotype associated with Rankl invalidation is attenuated through compensation by RANKL of maternal origin.

Recent observations performed in the orthodontic department of La Pitié-Salpêtrière hospital in Paris reported an increase of non-familial eruption defects of permanent molars. Our recent data have evidenced the involvement of osteoclasts (OC) in both the eruption and the dental retention processes through the RANKL/RANK/OPG signaling pathway. These facts are at the origin of the hypothesis of the existence of an environmental etiology for those eruption defects that would correspond to the perturbation of cellular autocrine/paracrine signaling pathways as the RANKL/ RANK/OPG.C57BL/6 mice were submitted to repeated injections with anti-RANKL neutralizing antibody during the nine days following birth. A phenotypic comparison with transgenic mice overexpressing RANK was performed for the functional characterization of the RANKL/RANK/OPG pathway. The dento-alveolar complex was analyzed using micro-CT for bone density and Masson's trichrome staining for histological examination.The RANKL transient invalidation of RANKL stopped the molar root development and tooth eruption contrary to transgenic mice overexpressing RANK. The recruitment and the OC activity were strongly impacted.This research is of direct clinical interest in understanding the pathology of eruption as indirect in establishing orthodontic treatment protocols for particular cases.

Mast cells are important in the initiation of ocular inflammation, but the consequences of mast cell degranulation on ocular pathology remain uncharacterized. We induced mast cell degranulation by local subconjunctival injection of compound 48/80. Initial degranulation of mast cells was observed in the choroid 15 minutes after the injection and increased up to 3 hours after injection. Clinical signs of anterior segment inflammation paralleled mast cell degranulation. With the use of optical coherence tomography, dilation of choroidal vessels and serous retinal detachments (SRDs) were observed and confirmed by histology. Subconjunctival injection of disodium cromoglycate significantly reduced the rate of SRDs, demonstrating the involvement of mast cell degranulation in posterior segment disorders. The infiltration of polymorphonuclear and macrophage cells was associated with increased ocular media concentrations of tumor necrosis factor-α, CXCL1, IL-6, IL-5, chemokine ligand 2, and IL-1β. Analysis of the amounts of vascular endothelial growth factor and IL-18 showed an opposite evolution of vascular endothelial growth factor compared with IL-18 concentrations, suggesting that they regulate each other's production. These findings suggest that the local degranulation of ocular mast cells provoked acute ocular inflammation, dilation, increased vascular permeability of choroidal vessels, and SRDs. The involvement of mast cells in retinal diseases should be further investigated. The pharmacologic inhibition of mast cell degranulation may be a potential target for intervention.

The muscle segment homeogenes Msx1 and Msx2 play a major role in tooth and bone formation. Periodontal osteoclast impairment also occurs in Msx2 null mutant mice, which is restored by overexpression of the receptor activator of NF-κB targeted in osteoclast lineage. Here, we investigated the role of Msx2 in dentinogenesis. Experiments were performed on Msx2(-/-) mice and the MDPC-23 odontoblastic cell line. After Msx2 gene silencing, real-time quantitative RT-PCR data showed significant overexpression of Runx2, Bglap, Dspp, and Alpl. Of three inhibitors of Wnt/β-catenin signaling (Dkk1, SostDc1, and Sost/Sclerostin), only Sost was expressed in postnatal teeth and overexpressed in Msx2(-/-) tooth samples. Initial crown dentin formation-primary dentinogenesis-occurred fairly normally in Msx2(-/-) teeth, albeit with distorted cusp patterns. Later stages of tooth development were characterized by a deviation from secondary toward tertiary dentinogenesis with osteodentin formation and impaired dentin deposition leading to limited root elongation. In Msx2(-/-)/receptor activator of NF-κB-transgenic double mutants, the dentin phenotype, notably in the roots, was rescued and sclerostin levels were normalized. These data suggest that Msx2 may act indirectly on dentinogenesis by controlling osteoclast activity and the signaling network related to eruption, supporting and further extending the concept that Msx2 controls formation of mineralized tissues by inhibition of the Wnt/β-catenin pathway; Sost in dentin and Dkk1 in bone, as previously demonstrated.

High doses of bone resorption inhibitors are currently under evaluation in pediatric oncology. Previous works have evidenced transient arrest in long bone and skull bone growth and tooth eruption blockage when mice were treated with zoledronic acid (ZOL). The question of potential similar effects with a RANKL-blocking antibody (IK22.5) was raised. Sensitivity disparities in these inhibitors between mouse strains and synergic effects of zoledronic acid and a RANKL-blocking antibody were subsidiary questions. In order to answer these questions, newborn C57BL/6J and CD1 mice were injected every two or three days (4 injections in total so 7 or 10 days of treatment length) with high doses of a RANKL-blocking antibody. The consequences on the tibia, craniofacial bones and teeth were analyzed by μCT and histology at the end of the treatment and one, two and three months later. The results obtained showed that RANKL-blocking antibody injections induced a transient arrest of tibia and skull bone growth and an irreversible blockage of tooth eruption in C57BL/6J mice. In CD1 mice, tooth eruption defects were also present but only at much higher doses. Similar mouse strain differences were obtained with zoledronic acid. Finally, a synergic effect of the two inhibitors was evidenced. In conclusion as previously observed for bisphosphonates (ZOL), a RANKL-blocking antibody induced a transient arrest in long bone and skull bone growth and a blockage of tooth eruption with however disparities between mouse strains with regard to this last effect. A synergic effect of both bone resorption inhibitors was also demonstrated.

Four approaches co-exist for who is willing to leverage the bone turnover in order to accelerate the orthodontic tooth movement (OTM) : biomodulation, physical assisted devices, biomechanics, and several surgical protocols. This litterature review describes the principles of action and clinical protocols available for each of these techniques.; En orthodontie, il existe 4 approches pour accélérer le Mouvement Dentaire Provoqué (MDP). Premièrement, l’approche biologique, dans laquelle les méthodes d’accélération reposent sur l’administration de biomodulateurs du métabolisme osseux. Ces recherches sont fondamentales in vitro, ou in vivo sur modèle animal, mais rarement cliniques. Deuxièmement, l’approche physique dans laquelle les méthodes d’accélération sont les forces cycliques, les courants électromagnétiques, la photobiomodulation par lampes LED ou laser basse intensité. Ces techniques disposent d’études cliniques. Troisièmement, l’approche Biomécanique, dans laquelle l’amélioration continue des arcs et des brackets a permis une avancée récente dans l’efficacité des pressions orthodontiques. Quatrièmement, l’approche chirurgicale, dont les effets sont plus prévisibles : corticotomies, corticision, distraction alvéolo-dentaire, distraction desmodontale et fibrotomie supra crestale. L’approche chirurgicale est le domaine de recherche le plus avancé dans les techniques d’accélération du déplacement dentaire.

Les inclusions palatines des canines maxillaires constituent un accident d'évolution dont les conséquences peuvent être néfastes pour les canines elles-mêmes et pour les dents adjacentes (déplacement, résorption, ankylose, ...). Leur mise en place peut nécessiter l'établissement d'un traitement orthodontique afin de rétablir la continuité d'arcade et rendre aux canines maxillaires leurs fonctions occlusales, fonctionnelles et esthétiques. Leur étiologie est aujourd'hui encore mal comprise et semble multifactorielle. Par ailleurs, de nombreuses études permettent de mieux saisir le mécanisme d'éruption dentaire, d'un point de vue cytologique et moléculaire. En effet, le sac folliculaire paraît jouer un rôle essentiel dans la conduite de l'éruption en étant le siège d'une cascade moléculaire. D'ailleurs, il semble également exister une implication du phénotype osseux. Ainsi, il se pourrait qu'un défaut de signalisation puisse contribuer aux inclusions palatines des canines maxillaires. Une meilleure connaissance de leur cause permettrait de mettre en place des thérapeutiques interceptives efficaces et une bonne caractérisation squelettique phénotypique pourrait s'avérer utile à une détection précoce de ces troubles d'éruption.