Cobalt Chloride Promotes Osteogenesis of Rat Bone Marrow Mesenchymal Stem Cells In Vitro and In Vivo

Abstract

This work aimed to investigate the effect of low-concentration cobalt chloride on the in vitro and in vivo proliferation of rat bone mesenchymal stem cells, the expression of hypoxia-inducing fraction-1 alpha and the related osteogenic genes. The bone mesenchymal stem cells were isolated from bone marrow of rats, and the surface biomarkers were identified by immunofluorescence staining and flow cytometry. The differentiation of bone mesenchymal stem cells was measured by alizarin red and alkaline phosphatase staining. The bone mesenchymal stem cells were treated with cobalt chloride at different concentrations and cell viability was assessed by cell counting kit 8. Cell migration was detected by Transwell and wound healing assay. The expression of collagen type 1, bone morphogenetic protein-2, runt-related transcription factor 2, alkaline phosphatase and hypoxia-inducing fraction-1 alpha were measured by quantitative real time polymerase chain reaction assay. The large segmental defect model was made to mimic the in vivo bone defect. The bone mesenchymal stem cells-collagen sponge scaffold was adopted for in vivo treatment and histological analysis of femur structure was performed to assess the effects of cobalt chloride-treated bone mesenchymal stem cells in promoting bone formation. X-ray and micro computed tomography were performed to observe bone structure. The cluster of differentiation 90 and cluster of differentiation 44 were highly expressed and cluster of differentiation 45 expression was relatively low in isolated bone mesenchymal stem cells. The cobalt chloride at 300 and 400 μmol/l could effectively suppress cell proliferation, compared with the control group. The low dose of cobalt chloride (50 μmol/l and 100 μmol/l) could promote cell proliferation. The cobalt chloride at 100 μmol/l could increase the positive staining of alkaline phosphatase and alizarin red. The expression of collagen type 1, bone morphogenetic protein-2, runt-related transcription factor 2, alkaline phosphatase and hypoxiainducing fraction-1 alpha were statistically up-regulated by cobalt chloride treatment. The cobalt chloridebone mesenchymal stem cells-collagen scaffold effectively accelerated the bone regeneration, increased new bone deposition and bone morphogenetic protein-2 expression in femur tissues. Appropriate concentration of cobalt chloride can promote the proliferation of bone mesenchymal stem cells and up-regulate the expression of hypoxia-inducing fraction-1 alpha and osteogenic genes in vitro, simultaneously promoted bone regeneration in vivo.