European Cells & Materials (Jun 2025)
Enhancing human osteoblast to osteocyte-like cell differentiation using 25-hydroxyvitamin D3 and all-trans retinoic acid combination
Abstract
Background: The individual effects of the 25-hydroxyvitamin D3 (25(OH)D3) and all-trans retinoic acid (ATRA) on osteogenesis have been studied in two-dimensional (2D) in vitro models, but the combined effect of these vitamins remains largely unexplored. Therefore, to address this knowledge gap we evaluated the effect of different concentrations of 25(OH)D3 and ATRA, individually and in combination, on differentiation of primary human osteoblasts (hOBs) over a 21-day period. Methods: The growth of hOBs, alkaline phosphatase (ALP) activity (cell bound and secreted), and the expression of bone-related markers were comprehensively evaluated at both gene expression level using real-time polymerase chain reaction (RT-PCR) and the proteins secretion through multiplex immunoassays. Results: Both 25(OH)D3 and ATRA enhanced hOBs differentiation. However, the combination of 10−6 M 25(OH)D3 and 10−5 M ATRA demonstrated the most pronounced effect, characterized by reduction in cell growth and enhancement in differentiation. This was further validated by elevated relative expression of osteoblast and osteocyte markers at both mRNA level (collagen type I alpha 1 (COL1A1), podoplanin (E11), and dentin matrix acidic phosphoprotein 1 (DMP1), and at protein level (osteopontin (OPN), osteocalcin (OCN), sclerostin (SOST), and fibroblast growth factor 23 (FGF23)). Additionally, 25(OH)D3 and ATRA were found to regulate the expression of key receptors involved in osteoblasts differentiation, including retinoic acid receptor alpha (RARα), retinoid X receptor beta (RXRβ), and vitamin D receptor (VDR). Conclusions: This study demonstrates that a combination of 10−6 M 25(OH)D3 and 10−5 M ATRA effectively enhanced the differentiation of hOBs to osteocyte-like cells in vitro.
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