Novel female murine alveolar osteoblasts responsive to hormones and mechanical forces.

Abstract

There is a need for a comprehensive understanding of alveolar bone biology, given its unique characteristics compared to other skeletal sites. To this end, we established novel female murine alveolar osteoblastic cell lines and analyzed their responses to hormones and mechanical cues. Cell lines were obtained by conditionally immortalizing primary alveolar osteoblasts isolated from the jaws of wild-type female mice and screened for high expression of alkaline phosphatase. Three clones were selected for further characterization. All clones differentiate into mature osteoblasts and mineralize the matrix. Two clones, ObB5-2 and ObC4-2, were further analyzed for their responsiveness to hormones and mechanical forces. Both responded to parathyroid hormone (PTH), 1,25OH₂D₃, and mechanical stimulations. Continuous treatment with PTH or 1,25OH₂D₃, inhibited mineralization and significantly suppressed Dmp1 expression. Intermittent PTH (iPTH) also inhibited mineralization in both clones. In ObB5-2, iPTH suppressed Dmp1, Sost, and Opg expression at doses as low as 1 nM PTH. Next, we assessed whether these cells support osteoclastogenesis. Both clones promoted the formation of TRAP-positive osteoclasts when cultured with bone marrow macrophages. Lastly, cells were subjected to fluid flow shear stress and simulated microgravity prior to gene expression analysis. ObC4-2 was highly sensitive to mechanical loads whereas ObB5-2 responded more to simulated microgravity. In summary, these findings describe novel female murine alveolar osteoblastic cell lines that provide a valuable model for studying the cellular and molecular mechanisms of alveolar bone remodeling. Unlike long bone derived cells and primary osteoblasts, these cells did not increase mineralization upon intermittent PTH, highlighting site specific differences in osteoblast function.