Genetically-achieved disturbances to the expression levels of TNFSF11 receptors modulate the effects of zoledronic acid on growing mouse skeletons.

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.

Bone resorption: an actor of dental and periodontal development?

Dental and periodontal tissue development is a complex process involving various cell-types. A finely orchestrated network of communications between these cells is implicated. During early development, communications between cells from the oral epithelium and the underlying mesenchyme govern the dental morphogenesis with successive bud, cap and bell stages. Later, interactions between epithelial and mesenchymal cells occur during dental root elongation. Root elongation and tooth eruption require resorption of surrounding alveolar bone to occur. For years, it was postulated that signaling molecules secreted by dental and periodontal cells control bone resorbing osteoclast precursor recruitment and differentiation. Reverse signaling originating from bone cells (osteoclasts and osteoblasts) toward dental cells was not suspected. Dental defects reported in osteopetrosis were associated with mechanical stress secondary to defective bone resorption. In the last decade, consequences of bone resorption over-activation on dental and periodontal tissue formation have been analyzed with transgenic animals (RANK (Tg) and Opg (-∕-) mice). Results suggest the existence of signals originating from osteoclasts toward dental and periodontal cells. Meanwhile, experiments consisting in transitory inhibition of bone resorption during root elongation, achieved with bone resorption inhibitors having different mechanisms of action (bisphosphonates and RANKL blocking antibodies), have evidenced dental and periodontal defects that support the presence of signals originating bone cells toward dental cells. The aim of the present manuscript is to present the data we have collected in the last years that support the hypothesis of a role of bone resorption in dental and periodontal development.

Role of RANKL (TNFSF11)-dependent osteopetrosis in the dental phenotype of Msx2 null mutant mice.

The MSX2 homeoprotein is implicated in all aspects of craniofacial skeletal development. During postnatal growth, MSX2 is expressed in all cells involved in mineralized tissue formation and plays a role in their differentiation and function. Msx2 null (Msx2 (-/-)) mice display complex craniofacial skeleton abnormalities with bone and tooth defects. A moderate form osteopetrotic phenotype is observed, along with decreased expression of RANKL (TNFSF11), the main osteoclast-differentiating factor. In order to elucidate the role of such an osteopetrosis in the Msx2 (-/-) mouse dental phenotype, a bone resorption rescue was performed by mating Msx2 (-/-) mice with a transgenic mouse line overexpressing Rank (Tnfrsf11a). Msx2 (-/-) Rank(Tg) mice had significant improvement in the molar phenotype, while incisor epithelium defects were exacerbated in the enamel area, with formation of massive osteolytic tumors. Although compensation for RANKL loss of function could have potential as a therapy for osteopetrosis, but in Msx2 (-/-) mice, this approach via RANK overexpression in monocyte-derived lineages, amplified latent epithelial tumor development in the peculiar continuously growing incisor.