INVESTIGATING THE MECHANISM OF INTRACELLULAR SIGNALING OF MAGNESIUM IN HUMAN BONE MARROW STROMAL CELLS (hBMSCs): POTENTIAL ROLE IN OSTEOGENESIS

Magnesium metal and its alloys have been investigated as promising biomaterials for internal bone fixation devices. Previous reports have shown that magnesium fixation devices enhance fracture healing in animal models while exhibiting biocompatibility and biodegradability. Moreover, mechanistic studies have indicated that mesenchymal stem cells (MSCs) display an osteogenic response to increased magnesium (Mg2+) concentrations in culture medium. We sought to study the signaling pathways underlying Mg-mediated osteogenesis in human bone marrow stromal cells (hBMSCs). We hypothesized that Mg2+ could be inducing the osteogenic response of hBMSCs in a calcium sensing receptor (CaSR) dependent manner. In the present study, hBMSCs were stimulated with basal medium (BM) supplemented with 10 mM MgSO4 (10-Mg), 4 mM CaCl2 (4-Ca) or 10 mM CaCl2 (10-Ca) at different time points. Western blot results showed a similar trend of CaSR phosphorylation in response to 10-Mg and 4-Ca media. Although this phosphorylation peaked at 15 minutes with both 10-Mg and 4-Ca media, it was not significantly higher than with BM alone. PKC-δ and Erk 1/2 are two downstream kinases of the CaSR. Both kinases have been reported to play a pivotal role in the differentiation of osteoprogenitors by regulating the expression of key osteogenic markers. We showed that hBMSCs exposure to 10-Mg medium resulted in significant phosphorylation of PKC-δ relative to BM. In contrast, Erk1/2 did not exhibit significant changes in its activity with 10-Mg medium. In addition, qPCR data showed upregulation of Cx43, ALPL and Col10A1 following hBMSCs treatment with 10-Mg medium. Similarly, a trend of upregulation of Runx2 mRNA was seen with 10-Mg medium; however, this upregulation was not statistically significant. VEGFA, a key angiogenic marker, was downregulated at the mRNA level by 10-Mg medium at a late time point (3 weeks). Chemical blocking of CaSR by its selective antagonist NPS2143 only potentiated ALPL expression, but did not have any effect on VEGFA or Col10A1. In conclusion, we propose a potential mechanism by which PKC-δ and Cx43 could mediate the osteogenic response of hBMSCs to increased Mg2+ concentrations. The suggested functional activity of PKC-δ might be regulated by a CaSR upstream signaling mechanism