Role of 1,25-Dihydroxyvitamin D3 on Osteogenic Differentiation and Mineralization of Chicken Mesenchymal Stem Cells

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.

Effects of l-Tryptophan and 1,25-Dihydroxycholecalciferol on Proliferation and Osteogenic Differentiation of Mesenchymal Stem Cells Isolated from the Compact Bones of Broilers and Layers

“…Therefore, further study could be conducted to investigate the effect of L-Trp on osteogenic differentiation during shorter time intervals in layer MSCs, which might show consistent results with broiler MSCs. The negative effect of 1,25OHD 3 on ALP activity and calcium deposition compared with the control in broiler MSCs was consistent with Chen et al, 23 but no significant negative effect of 1,25OHD 3 was found in layer MSCs, which illustrated the main role of 1,25OHD 3 in bone formation and remodeling was to improve MSC proliferation, but the effect of 1,25OHD 3 was also affected by bone matrix competency and the differentiation status from MSCs to osteoblasts. 35 The regulation of gene expression during osteogenic differentiation is complicated.…”

“…The reason might be the intricate regulation of the vitamin D system on the transcription of osteogenic genes involving many factors such as the origins of MSCs, treatment time, concentration, ambient condition, etc. 23 Interestingly, 1 μM NPS-2143 inhibited the gene expression of CaSR in both broiler and layer MSCs, but in most osteogenic genes, CaSR NAM (NPS-2143) supplementation upregulated their gene expression in broiler MSCs. The results were similar to Koori et al 38 who found that NPS-2143 addition was conducive to the expression of OCN and OPN and increasing doses of NPS-2143 showed more mineralization in cell line 1− 17 (human periodontal ligament stem cells).…”

“…To reveal the regulation mechanism of CaSR and VDR during chicken MSC proliferation and osteogenic differentiation, in addition to Ca 2+ and the 5 mM L-Trp, we included 1 nM 1,25OHD 3 and 1 μM NPS-2143 (CaSR antagonist). The selection of 1 nM 1,25OHD 3 was based on the results of Chen et al 23 who have reported that increasing the dose of 1,25OHD 3 had a negative effect on osteogenic differentiation of broiler MSCs. Therefore, 1 nM 1,25OHD 3 was a proper concentration to investigate the regulation mechanism.…”

“…Alizarin Red S (ARS) Staining. Monolayers of chicken MSCs differentiated by different treatments in 6-well plates for 7 days were used for mineralization determination by the ARS staining method as described by Gregory et al 30 and Chen et al 23 The cells were washed with phosphate-buffered saline (PBS) and fixed with 1 mL of 10% formalin (Fisher Scientific, Waltham, MA) per well for 15 min at room temperature. The monolayers were stained with 1 mL of 40 mM ARS solution at pH 4.1−4.3 (Sigma-Aldrich) for 45 min in the dark.…”

Effects of rearing systems on the eggshell quality, bone parameters and expression of genes related to bone remodeling in aged laying hens

“…However, the humerus in the CCS group showed less bone formation. Active vitamin D and estrogen are involved in osteogenesis and bone formation after binding with the target receptor (mainly VDR and ER, respectively) ( Hayashi et al, 2019 ; Chen et al, 2021 ). The deficiency of either of them accelerated bone loss and led to an OP phenotype ( Chokalingam et al, 2012 ; Yang et al, 2020 ).…”