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20(S)-hydroxycholesterol and simvastatin synergistically enhance osteogenic differentiation of marrow stromal cells and bone regeneration by initiation of Raf/MEK/ERK signaling

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Abstract

Previous studies have demonstrated the significant roles of simvastatin (SVA) and oxysterols in the osteogenesis process. In this study, we evaluate the effect of a combination of SVA and 20(S)-hydroxycholesterol (20(S)OHC) on the cell viability and osteogenic differentiation of bone marrow stromal cells (BMSCs). After treatment with a control vehicle, SVA (0.025, 0.10, 0.25 or 1.0 μM), 20(S)OHC (5 μM), or a combination of both (0.25 μM SVA + 5 μM 20(S)OHC), the proliferation, apoptosis, ALP activity, mineralization, osteogenesis-related gene expression and Raf/MEK/ERK signaling activity in BMSCs were measured. Our results showed that high concentrations of SVA (0.25 and 1.0 μM) enhanced osteogenesis-related genes expression while attenuating cell viability. The addition of 5 μM 20(S)OHC induced significantly higher proliferative activity, which neutralized the inhibitory effect of SVA on the viability of BMSCs. Moreover, compared to supplementation with only one of the additives, combined supplementation with both SVA and 20(S)OHC induced significantly enhanced ALP activity, calcium sedimentation, osteogenesis-related genes (ALP, OCN and BMP-2) expression and Raf/MEK/ERK signaling activity in BMSCs; these enhancements were attenuated by treatment with the inhibitor U0126, indicating a significant role of Raf/MEK/ERK signaling in mediating the synergistically enhanced osteogenic differentiation of BMSCs by combined SVA and 20(S)OHC treatment. Additionally, histological examination confirmed a synergistic effect of SVA and 20(S)OHC on enhancing bone regeneration in a rabbit calvarial defect model. This newly developed SVA/20(S)OHC formulation may be used as an osteoinductive drug to enhance bone healing.

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Acknowledgements

This work was supported by the National Nature Science Foundation of China under Grant (81170998); and the Science and Technology Plan Projects funds of Taishan City under Grant (201634611).

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Authors and Affiliations

  1. Center of Oral Implantology, Stomatological Hospital, Southern Medical University, 366 South Jiangnan Road, Guangzhou, 510280, Guangdong, China

    Yinghe Huang, Mingdeng Rong, Weizhen Liu & Lei Zhou

  2. The Department of Stomatology, Taishan People’s Hospital, Affiliated to Guangdong Medical University, Taishan, Guangdong, China

    Yinghe Huang

  3. The Department of Stomatology, Jieyang Affiliated Hospital, SunYat-sen University, Jieyang, Guangdong, China

    Yao Lin & Junbing He

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Huang, Y., Lin, Y., Rong, M. et al. 20(S)-hydroxycholesterol and simvastatin synergistically enhance osteogenic differentiation of marrow stromal cells and bone regeneration by initiation of Raf/MEK/ERK signaling. J Mater Sci: Mater Med 30, 87 (2019). https://doi.org/10.1007/s10856-019-6284-0

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