Mineralization of three‐dimensional osteoblast cultures is enhanced by the interaction of 1α,25‐dihydroxyvitamin D3 and BMP2 via two specific vitamin D receptors

1α,25‐Dihydroxyvitamin D3 [1α,25(OH)2D3] and bone morphogenetic protein‐2 (BMP2) are both used to stimulate osteoblastic differentiation. 1α,25(OH)2D3 regulates osteoblasts through classical steroid hormone receptor mechanisms and through rapid responses that are mediated by two receptors, the traditional vitamin D receptor (VDR) and protein disulphide isomerase family A member 3 (Pdia3). The interaction between 1α,25(OH)2D3 and BMP2, especially in three‐dimensional (3D) culture, and the roles of the two vitamin D receptors in this interaction are not well understood. We treated wild‐type (WT), Pdia3‐silenced (Sh‐Pdia3) and VDR‐silenced (Sh‐VDR) pre‐osteoblastic MC3T3‐E1 cells with either 1α,25(OH)2D3, or BMP2, or with 1α,25(OH)2D3 and BMP2 together, and measured osteoblast marker expression in 2D culture and mineralization in a 3D poly(ε‐caprolactone)–collagen scaffold model. Quantitative PCR showed that silencing Pdia3 or VDR had a differential effect on baseline expression of osteoblast markers. 1α,25(OH)2D3 + BMP2 caused a synergistic increase in osteoblast marker expression in WT cells, while silencing either Pdia3 or VDR attenuated this effect. 1α,25(OH)2D3 + BMP2 also caused a synergistic increase in Dlx5 in both silenced cell lines. Micro‐computed tomography (μCT) showed that the mineralized volume of untreated Sh‐Pdia3 and Sh‐VDR 3D cultures was greater than that of WT. 1α,25(OH)2D3 reduced mineral in WT and Sh‐VDR cultures; BMP2 increased mineralization; and 1α,25(OH)2D3 + BMP2 caused a synergistic increase, but only in WT cultures. SEM showed that mineralized matrix morphology in 3D cultures differed for silenced cells compared to WT cells. These data indicate a synergistic crosstalk between 1α,25(OH)2D3 and BMP2 toward osteogenesis and mineral deposition, involving both VDR and Pdia3. Copyright © 2013 John Wiley & Sons, Ltd.