Abstract
Recent studies indicated that alendronate enhanced osteogenesis in osteoblasts and human bone marrow-derived stem cells. However, the time- and dose-dependent effects of Aln on ostegenic differentiation and cytotoxicity of hBMSCs remain undefined. In present study, we investigated the effective dose range and timing of hBMSCs. hBMSCs were treated with various Aln doses (1, 5 and 10 µM) according to the following groups: group A was treated with Aln during the first five days of bone medium, groups B, C and D were treated during the first, second, and final five days of osteo-induction medium and group E was treated throughout the entire experiment. The mineralization level and cytotoxicity were measured by quantified Alizarin Red S staining and MTT assay. In addition, the reversal effects of farnesyl pyrophosphate and geranylgeranyl pyrophosphate replenishment in group B were also investigated. The results showed that Aln treatment in groups A, B and E enhanced hBMSC mineralization in a dose-dependent manner, and the most pronounced effects were observed in groups B and E. The higher dose of Aln simultaneously enhanced mineralization and caused cytotoxicity in groups B, C and E. Replenishment of FPP or GGPP resulted in partial or complete reverse of the Aln-induced mineralization respectively. Furthermore, the addition of FPP or GGPP also eliminated the Aln-induced cytotoxicity. We demonstrated that hBMSCs are susceptible to 5 µM Aln during the initiation stage of osteogenic differentiation and that a 10 µM dose is cytotoxic.
Highlights
Bisphosphonates are clinically used to inhibit osteoclast activity and to treat osteoporosis
In group B, the mineralization levels were significantly elevated after Aln treatments in dose-dependent manner (Figure 2B). 1 mM and 5 mM Aln significantly elevated mineralization when compared to the control (Figure 2B, 1 and 5 mM, p,0.01), and 10 mM Aln treatment enhanced mineralization to a great degree than other doses at days 15 and 20 (Figure 2B, p,0.01)
The mineralization levels of Human bone marrow mesenchymal stem cells (hBMSCs) treated with Aln were not significantly different in group C (Figure 2E) and only weakly elevated in group D when compared to the control (Figure 2F)
Summary
Bisphosphonates are clinically used to inhibit osteoclast activity and to treat osteoporosis. Aln was found to enhance osteogenic activities, including cell proliferation and osteo-differentation. A wide range of effective osteo-inducing Aln doses has been reported for different cell types. 5 mM Aln sufficiently enhanced osteo-differentiation in human ADSCs [11]. The effective dose, treatment time and duration of Aln required for enhancing osteogenesis among different cell types were varied and those in mesenchymal stem cells have not been well characterized. In this study, we identified the appropriate treatment times and doses required for Aln-induced osteogenesis in human BMSCs. Simultaneously, the cell cytotoxic effects of Aln treatment were investigated
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