An in vitro Experimental Insight into the Osteoblast Responses to Vitamin D3 and Its Metabolites

25-hydroxyvitamin D3 (25[OH]VD3) has recently been found to be an active hormone. Its biological actions are also demonstrated in various cell types. However, the precise influences of vitamin D3 (VD3) and its metabolites (25[OH]VD3, 1α,25-dihydroxyvitamin D3 [1α,25-(OH)2VD3]) on the osteoblast differentiation remain largely unknown. In this work, we investigated the effects of VD3 and its metabolites in different concentrations on the early and later osteoblast differentiation and biomineralization.

We first used quantitative real-time polymerase chain reaction (RT-qPCR) to evaluate the responsiveness of osteoblasts to VD3, 25(OH)VD3 or 1α,25-(OH)2VD3. We also evaluated the proliferation, differentiation and biomineralization of osteoblast at different time points via cell counting kit-8 assay and the analysis of osteogenic markers.

The experimental results confirmed that osteoblasts could be responsive to 25(OH)VD3 and 1α,25-(OH)2VD3 but could not directly metabolize VD3 and 25(OH)VD3. Only 200 nmol/L VD3 significantly promoted osteoblast proliferation, while 25(OH)VD3 and 1α,25-(OH)2VD3 did not show obvious actions. Moreover, the early osteogenic markers were increased by 25(OH)VD3 and 1α,25-(OH)2VD3 in a dose-dependent manner. More importantly, only 25(OH)VD3 had accelerated the gene and protein expressions of osteocalcin and the biomineralization level of osteoblasts.

Our findings provide reliable evidence that 25(OH)VD3 at 100-200 nmol/L can induce the early and later osteoblast differentiation and biomineralization for clinical bone tissue engineering.