Primary Cilia Enhance Osteogenic Response of Jaw Mesenchymal Stem Cells to Hypoxia and Bisphosphonate

Abstract

Primary cilia play a significant role in mesenchymal stem cell (MSC) lineage commitment, skeletal development, and bone homeostasis. MSC responsiveness to metabolic stress is associated with radiation and drug-induced jaw osteonecrosis. Therefore, we hypothesize that orofacial MSCs (OFMSCs) osteogenic commitment in response to cellular stressors hypoxia and bisphosphonates is a survival response coupled to primary cilia biogenesis. Human OFMSCs were subjected to cellular stress using severe hypoxia, nitrogen-containing bisphosphonate (pamidronate) and low serum starvation. OFMSC primary cilia formation, as well as cell survival and proliferation, were detected using immunofluorescence, CellTitre-Glo, and WST-1 assays respectively. OFMSC differentiation was tested using Alizarin Red S staining. OFMSCs survival and osteogenic markers were assessed by western blotting relative to primary cilia number and associated acetylated tubulin levels. Baseline OFMSC proliferation was stable under short-term severe hypoxia and pamidronate treatments whether combined with or without serum starvation. Hypoxia and pamidronate decreased the number of OFMSCs positive for primary cilia that was consistent with increased HIF-1α and caspase 3 but decreased cyclin D1. Combined effects of hypoxia and pamidronate on OFMSCs significantly reduced ciliation but did not completely abrogate it. Combination of serum deprivation, hypoxia, and pamidronate promoted OFMSCs osteogenic differentiation that was consistent with upregulated HIF-1α levels. Partial rather than complete loss of OFMSC ciliation and enhanced osteogenic commitment represent adaptive survival response of OFMSCs to severe hypoxia and pamidronate-induced metabolic stress. Hypoxia and drug-induced OFMSC stress may be significant events governing the pathogenesis and clinical outcomes of jaw osteonecrosis.