Three-dimensional (3D) cultures are known to promote cell differentiation. Previously, we investigated the differentiation of rat dermal fibroblasts to α-smooth muscle actin (α-SMA)-positive myofibroblasts through transforming growth factor (TGF)-β production using a 3D culture model. Here, we investigated the phenotypic change from dermal mesenchymal cells (mostly fibroblasts) to osteoblast-like cells, being inspired by the roles of smooth muscle cells or fibroblasts during vascular calcification. Spindle-shaped cells that grew in heterologous populations out of dermal explants from 2-day-old Wistar rats were cultured within a collagen matrix. α-SMA and alkaline phosphatase (ALP) messenger RNA (mRNA) levels initially increased, followed by a rise in Runx2 and osteocalcin (OCN) mRNA levels without calcification. Calcium deposits were produced in the presence of a high concentration of inorganic phosphate (2.1 mM) or β-glycerophosphate (βGP, 10 mM) after 2 weeks of culture, and both were sensitive to an inhibitor of type III phosphate transporters. An ALP inhibitor decreased only βGP-induced calcification. Inhibition of TGF-β type-I receptors attenuated ALP mRNA levels and βGP-induced calcification, suggesting that endogenous TGF-β stimulates ALP activity and then βGP breakdown. An increase in the number of cells embedded in the collagen gel enhanced the mRNA levels of Runx2 and OCN, but not of ALP. Collectively, several factors are likely to promote the differentiation of dermal mesenchymal cells into osteoblast-like cells and ectopic calcification in a 3D collagen matrix, implying the utility of these cells as a potential autologous cell source for tissue engineering.
Actins ; Alkaline Phosphatase ; Animals ; Calcium ; Cell Differentiation ; Collagen ; Dermis ; Fibroblasts ; Myocytes, Smooth Muscle ; Myofibroblasts ; Osteocalcin ; Phosphate Transport Proteins ; Rats* ; Rats, Wistar ; RNA, Messenger ; Tissue Engineering ; Transforming Growth Factors ; Vascular Calcification