Abstract
1,25-dihydroxyvitamin D3 (1,25OHD) has been suggested to play an important role in osteogenic differentiation and mineralization. However, limited data have been reported in avian species. In the present study, the direct role of 1,25OHD on osteogenic differentiation and mineralization in chicken mesenchymal stem cells (cMSCs) derived from day-old broiler bones was investigated. cMSCs were treated with control media (C), osteogenesis media (OM), OM with 1, 5, 10, and 50 nM 1,25OHD, respectively. The messenger RNA (mRNA) samples were obtained at 24 and 48 h and 3 and 7 days to examine mRNA expression of key osteogenic genes [runt related transcription factor 2 (RUNX2), bone morphogenetic protein 2 (BMP2), collagen type I alpha 2 chain (COL1A2), bone gamma-carboxyglutamate protein (BGLAP), secreted phosphoprotein 1 (SPP1), and alkaline phosphatase (ALP)]. Cells were stained at 7, 14, and 21 days using Von Kossa (mineralization), Alizarin Red (AR; mineralization), and Alkaline Phosphatase (early marker) staining methods. From the mRNA expression results, we found a time-dependent manner of 1,25OHD on osteoblast differentiation and mineralization. In general, it showed an inhibitory effect on differentiation and mineralization during the early stage (24 and 48 h), and a stimulatory effect during the late cell stage (3 and 7 days). The staining showed 1,25OHD had an inhibitory effect on ALP enzyme activities and mineralization in a dosage-dependent manner up to 14 days. However, at 21 days, there was no difference between the treatments. This study provides a novel understanding of the effects of 1,25OHD on osteogenic differentiation and mineralization of cMSCs depending on cell stage and maturity.
Highlights
Vitamin D3 requires two steps of hydroxylation to become 1,25-dihydroxyvitamin D3 (1,25OHD) to exert its biological functions (St-Arnaud, 2008)
In order to study the role of 1,25OHD in chicken osteogenic differentiation of chicken mesenchymal stem cells (cMSCs), the samples were obtained at 24 and 48 h and 3 and 7 days post-treatment for the assessment of key osteogenic gene expression (RUNX2: runt related transcription factor 2; BMP2: bone morphogenetic protein 2, COL1A2: collagen type I alpha 2 chain; BGLAP: bone gamma-carboxyglutamate protein; SPP1: secreted phosphoprotein 1; and ALP: alkaline phosphatase)
osteogenesis media (OM) induced messenger RNA (mRNA) expression of key osteogenic genes at all time points compared to control (C; p < 0.05), except for RUNX2 and BMP2 at 7 days (Figures 1–3), which confirmed that the experimental conditions were adequate to induce osteogenic differentiation in cMSCs
Summary
Vitamin D3 requires two steps of hydroxylation to become 1,25-dihydroxyvitamin D3 (1,25OHD) to exert its biological functions (St-Arnaud, 2008). 1,25OHD mostly showed stimulatory effects on osteogenic differentiation and mineralization (Prince et al, 2001; Chen et al, 2002; Jorgensen et al, 2004; Zhou et al, 2006; Tourkova et al, 2017; Li et al, 2018), but with a few exceptions (Viereck et al, 2002); In rat osteoblasts, the responses to 1,25OHD showed either inhibitory effects or no effects (Harrison et al, 1989; Kim and Chen, 1989); In mouse osteoblasts, the differentiation and mineralization of osteoblasts showed the most inconstant responses to 1,25OHD with either no effects, inhibition, or facilitation (van Driel and van Leeuwen, 2014; Chen et al, 2016; Kim et al, 2016; Xiong et al, 2017); Limited studies have been done in chicken osteoblasts, but the available data suggested, in general, that 1,25OHD showed an inhibitory effect on osteoblast differentiation and mineralization (Broess et al, 1995; Pande et al, 2015). This study filled a research gap regarding roles of 1,25OHD on chicken osteogenic differentiation and mineralization
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