Periodontitis is based on a complex inflammatory over-response combined with possible genetic predisposition factors. The RANKL/RANK/OPG signaling pathway is implicated in bone resorption through its key function in osteoclast differentiation and activation, as well as in the inflammatory response. This central element of osteo-immunology has been suggested to be perturbed in several diseases, including periodontitis, as it is a predisposing factor for this disease. The aim of the present study was to validate this hypothesis using a transgenic mouse line, which over-expresses RANK (RTg) and develops a periodontitis-like phenotype at five months of age. RTg mice exhibited severe alveolar bone loss, an increased number of TRAP positive cells, and disorganization of periodontal ligaments. This phenotype was more pronounced in females. We also observed dental root resorption lacunas. Hyperplasia of the gingival epithelium, including Malassez epithelial rests, was visible as early as 25 days, preceding any other symptoms. These results demonstrate that perturbations of the RANKL/RANK/OPG system constitute a core element of periodontitis, and more globally, osteo-immune diseases.

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.

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 primary retention of molars observed in clinic corresponds to a still-unexplained absence of molar eruption despite the presence of an eruption pathway, resembling the experimental transient inhibition of RANKL signaling in mice. The aim of the present study was to confront the hypothesis according to which the primary retention of molars is associated with transitory perturbations to RANKL signaling during growth as part of a wider craniofacial skeleton pattern. The experimental strategy was based on combining a clinical study and an animal study corresponding to the characterization of the craniofacial phenotypes of patients with primary retention of molars and analyses in mice of the consequences of transient inhibition of RANKL signaling on molar eruption and craniofacial growth. The clinical study validated the existence of a particular craniofacial phenotype in patients with primary retention of molars: a retromandibular skeletal class II typology with reduced mandibular dimensions which manifests itself at the dental level by a class II/2 with palatoversion of the upper incisors and anterior overbite. The animal study demonstrated that transient invalidation of RANKL signaling had an impact on the molar eruption process, the severity of which was dependent on the period of inhibition and was associated with a reduction in two craniofacial morphometric parameters: total skull length and craniofacial vault length. In conclusion, primary retention of molars may be proposed as part of the craniofacial skeleton phenotype associated with a transitory alteration in RANKL signaling during growth.

La formation du squelette cranio-facial et plus particulièrement le développement alvéolo-dentaire suivent un programme génétique finement régulé et contrôlé. En effet, une combinatoire spatiale et temporelle de signaux moléculaires détermine la typologie et la position intraet inter-maxillaires de chaque unité alvéolo-dentaire. La croissance volumétrique ainsi que les déplacements des germes dentaires dépendent d’un processus de modelage adaptatif et coordonné de l’os alvéolaire périphérique supporté par des voies de signalisation entre les cellules de l’épithélium dentaire, du sac folliculaire et de l’alvéole osseuse. Ces voies de signalisation impliquent des facteurs de transcription, des proto-oncogènes et des facteurs diffusibles dits « de croissance ». Des altérations transitoires de ces signalisations, au cours des fenêtres de temps correspondant à la formation des différents types dentaires, pourraient expliquer les atteintes localisées et spécifiques de l’éruption d’un type dentaire comme cela peut être observé pour les deuxièmes molaires. Des travaux en cours montrent que les perturbations de la résorption osseuse en relation avec des atteintes de la signalisation RANKL/RANK/OPG ont des répercussions sur l’éruption dentaire et sur la morphogenèse des racines avec une relation claire entre la fenêtre temporelle de survenue des perturbations et le type de dent atteint. The craniofacial skeleton formation and more specifically the alveolar bone development follow a deeply and timely regulated and controlled genetic program. Indeed, spatial and temporal combination of molecular signals determines the type and position of each dental-alveolar unit in the maxillary. The volumetric growth and movements of tooth germs depend on an adaptive and coordinated alveolar bone modeling process which is supported by signaling pathways between cells of the dental epithelium, the follicle and the alveolar bone. These signaling pathways involve transcription factors, proto-oncogenes and diffusible factors called “growth factors”. Any alterations of these signals during windows of time corresponding to the development of the different dental types could explain specific and localized alterations such as observed for second molars. Ongoing studies show that disturbances of bone resorption related to damages of the RANKL/ RANK/OPG signaling pathway affect tooth eruption and the root morphogenesis with a clear relationship between the temporal window of the disturbance and the affected tooth type.