25-Hydroxyvitamin D₃ induces osteogenic differentiation of human mesenchymal stem cells

25-Hydroxyvitamin D₃ [25(OH)D₃] has recently been found to be an active hormone. Its biological actions are demonstrated in various cell types. 25(OH)D₃ deficiency results in failure in bone formation and skeletal deformation. Here, we investigated the effect of 25(OH)D₃ on osteogenic differentiation of human mesenchymal stem cells (hMSCs). We also studied the effect of 1α,25-dihydroxyvitamin D₃ [1α,25-(OH)₂D₃], a metabolite of 25(OH)D₃. One of the vitamin D responsive genes, 25(OH)D₃-24-hydroxylase (cytochrome P450 family 24 subfamily A member 1) mRNA expression is up-regulated by 25(OH)D₃ at 250-500 nM and by 1α,25-(OH)₂D₃ at 1-10 nM. 25(OH)D₃ and 1α,25-(OH)₂D₃ at a time-dependent manner alter cell morphology towards osteoblast-associated characteristics. The osteogenic markers, alkaline phosphatase, secreted phosphoprotein 1 (osteopontin), and bone gamma-carboxyglutamate protein (osteocalcin) are increased by 25(OH)D₃ and 1α,25-(OH)₂D₃ in a dose-dependent manner. Finally, mineralisation is significantly increased by 25(OH)D₃ but not by 1α,25-(OH)₂D₃. Moreover, we found that hMSCs express very low level of 25(OH)D₃-1α-hydroxylase (cytochrome P450 family 27 subfamily B member 1), and there is no detectable 1α,25-(OH)₂D₃ product. Taken together, our findings provide evidence that 25(OH)D₃ at 250-500 nM can induce osteogenic differentiation and that 25(OH)D₃ has great potential for cell-based bone tissue engineering.