Isolation, culture and induced differentiation of rabbit mesenchymal stem cells into osteoblasts

Abstract

Mesenchymal stem cells (MSCs) may be easily isolated from the bone marrow, and possess multi-lineage differentiation potential and various therapeutic applications. The differentiation of MSCs into osteoblasts is a complex process that is regulated by multiple internal and external factors. In the present study, the differentiation of MSCs isolated from rabbit bone marrow into osteoblasts using different osteoblast inductive media in the presence of dexamethasone, bone morphogenetic protein 2 (BMP-2), 1,25-dihydroxyvitamin D3, transforming growth factor β (TGFβ), platelet lysate and cyclooxygenase 2 (COX2), respectively. Alkaline phosphatase (ALP) activity, mineralization, collagen type (Ct) I and osteocalcin activities, and the mRNA and protein expression levels of vascular endothelial growth factor (VEGF), BMP-2 and Ct II were measured during the differentiation process in MSCs treated with different inducers. Rabbit MSCs were successfully isolated and were observed to be predominantly circular in shape after culture for 24 h. Following subculture for 5 days, the cells demonstrated a spindle shape. ALP, Ct I and osteocalcin activities were higher in cells cultured in dexamethasone, BMP-2 and TGFβ compared with the activities in control cells. Following differentiation, the dexamethasone, BMP-2 and TGFβ groups demonstrated significantly enhanced mineralization of MSCs detected by Alizarin Red S staining. The mRNA and protein expression levels of VEGF, BMP-2 and Ct II were significantly increased in the same groups compared with the levels in the control group. In conclusion, rabbit MSCs were successfully isolated from bone marrow and differentiated into osteoblasts indicated by raised ALP, Ct I and osteocalcin activities, mineralization and expression of osteogenesis-inducing genes and proteins. The present study revealed that dexamethasone, BMP-2 and TGFβ have a positive effect on cell differentiation.

Dorsomorphin attenuates Jagged1‐induced mineralization in human dental pulp cells

“…Dexamethasone supplementation up‐regulated BMP2 expression in Jagged1‐treated condition similar to those cultured in osteogenic induction medium. Correspondingly, previous report demonstrated that dexamethasone induced BMP2 mRNA and protein expression in mesenchymal stem cells (Liu et al, 2018a, 2018b). Dexamethasone interacted with BMP2 then regulated osteogenic differentiation in human MSC (Jager et al, 2008).…”

“…SB431542 treatment (as low as 0.1 μmol L −1 ) significantly increased mineralization in human gingival mesenchymal stem cells and the addition of SB431542 at 1 μmol L −1 markedly increased osteogenic differentiation both in vitro and in vivo (Shi et al, 2019). It has also been shown that the optimal dose for SB505124 in osteogenic induction of bone marrow derived from Sh3bp2 KI/KI mice was 100–250 nmol L −1 (Liu et al, 2018a, 2018b). Further, SB505124 (10–1000 nmol L −1 ) inhibited chorion membrane extracts‐induced calcium deposition under osteogenic induction of MG‐63 cells (Go et al, 2017).…”

Bone Regeneration Using Adipose-Derived Stem Cells in Injectable Thermo-Gelling Hydrogel Scaffold Containing Platelet-Rich Plasma and Biphasic Calcium Phosphate

“…Being expressed at the early to middle stages of osteo-differentiation, ALP and COL were up-regulated during the first 2 weeks of cell culture without further increase of mRNA expression after day 21 [ 59 ]. On the other hand, the late expression of OCN could be confirmed from the continued increase of mRNA expression up to 28 days [ 60 ]. The scaffold-dependent analysis on gene expression further endorses the effect of PRP/BCP in controlling the expression of various osteogenic markers.…”

Expression Pattern of CircRNA in Naringin-induced Fate Dicision of BMSCs

“…At the same time, when osteoblasts are cultured in vitro, they will cause the deposition of calcium salts, thereby forming calci ed nodules. The more calcium salts are deposited, the better the osteogenic differentiation [24].…”