Insights into Osteocytic Signaling in Bone Regeneration
Osteocytes, previously believed to be passive cells, are key players in bone modeling and remodeling by orchestrating osteoblast and osteoclast differentiation and function. Embedded inside the bone matrix, elaborate osteocyte networks sense a wide spectrum of external stimuli and react by expressing cytokines and other signaling mediators such as WNTs, sclerostin, and receptor activator of NF-κB (RANK) ligand (RANKL). Bone modeling and remodeling is strictly regulated by osteocytic signaling. Notwithstanding, the exact mechanisms of osteocyte signaling in bone regeneration remain elusive. Bone regeneration predominantly occurs following trauma to the bone. At the trauma site, osteocytes undergo apoptosis and trigger RANKL production in neighboring bystander osteocytes. The consequent osteocytic expression of RANKL, the key factor of osteoclastogenesis, is responsible for early resorption, i.e., the removal of necrotic bone. There is conflicting evidence regarding the exact role of osteocytic signaling in the consequent bone formation. The sclerostin pathway appears to be of importance. However, it remains to be determined how the signaling of apoptotic osteocytes at the trauma site influence sclerostin expression and, consequently, bone formation. Based on previous work, we hypothesize that suppressed osteocytic apoptosis and/or RANKL expression leads to less bone resorption and, through the suppression of the subsequent cascade, also less bone formation.
Here we investigate these two main hypotheses using murine Cre/loxP conditional knockout models. We create Casp3fl/fl;Dmp1-Cre osteocyte specific caspace-3 knockout and Tnfsf11fl/fl;Dmp1-Cre osteocyte specific RANKL knockout mice. We then perform four preclinical studies in total, with Dmp1-Cre mice serving as controls to the conditional knockout mice. First, we characterize the dental and periodontal phenotypes of Casp3fl/fl;Dmp1-Cre and Tnfsf11fl/fl;Dmp1-Cre mice. We then study the resorption of the alveolar bone lamella following tooth extraction using a mandibular first molar extraction model. We use a calvarial defect model to assess the healing of a standardized critical size bone defect. Finally, we use a live bone allograft transplantation model to evaluate early graft resorption and consequent overall graft consolidation. In all four preclinical studies, we use micro-computed tomography supported by advanced bioimaging algorithms for tissue segmentation, as well as histology and histomorphometry to assess and visualize bone regeneration in the experimental conditional knockout models. Taken together, we comprehensively assess the importance of osteocytic signaling in bone regeneration with a focus on the signaling of apoptotic osteocytes as well as osteocytic RANKL expression. Our ultimate goal is to elucidate the mechanisms of osteocytic signaling and leverage the knowledge gained to possibly optimize bone regeneration in a wide range of clinical indications.
Methods and Skills
Preclinal studies; Cre/loxP systems; clinical studies
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