| Bone Mesenchymal Stem Cells are a kind of stem cell, which are the common cell source of osteoblasts, osteocytes and chondrocytes. BMSCs have become an important stem cell with its good self- renewal and multi-directional differential potential, and have good ability to repair bone defects. However, the differentiation of BMSCs into osteoblasts is a very complicated process, which can be interacted and influenced by many factors. In recent years, studies confirm that DDR2(Disco idin Domain Receptor 2) as a specific receptors of type I collagen, plays an important role in regulation of osteoblast differentiation. However, the function of DDR2 on the osteogenic differentiation of BMSCs is rarely studied, and its functional mechanis m is also unclear. In this study, we isolated, cultured and identified the BMSCs derived from DDR2 gene knockout mice and explored the influence of DDR2 on the biological characteristics and osteogenic ability of BMSCs, to lay the theoretical foundation fo r the promotion of BMSCs ability on bone regeneration. Objectives and MethodsTo further investigate the osteogenesis of DDR2, we isolated, cultured and identified BMSCs derived from DDR2 knockout mice and normal wild C57 mice. We investigated the effect of DDR2 on growth circle, apoptosis, and multi-directional differentiation of BMSCs. And we studied the role of DDR2 on the process of osteogenesis. Furthermore, we demonstrated the DDR2 gene function in the bone repair ability of BMSCs after DDR2 gene deletion.We isolated and cultured BMSCs derived from DDR2 knockout mice and wild type mice by using the improved whole bone marrow adherent method. Then we used inverted microscope to observe the cell morphology. Next we also analyzed the phenotypes and apoptosis of BMSCs by flow cytometry. The activity of BMSCs proliferation was also detected with MTT assay. The osteogenic and adiopogenic differentiation experiment was desighed for multi-differentiation potential of BMSCs. Real time-PCR was used to analyse osteogenic marker genes expression after osteoinduction by day of 3, 7, 14, and 21. ALP staining, ALP assay and OCN detection were used to determine ALP enzymatic activity and OCN secretion. We established the mice skull bone defect model and then implanted the bone defects of the cells and HA/TCP combination immediately. Gross observation, Micro-CT, histological HE stain were used to observe the osteogenesis of two kinds of BMSCs. Results and ConclusionsThere are two kinds of cultured BMSCs showed a vorte x-like confluent population growth and behaved elongated spindle or rhomboid-shaped. The mesenchymal markers showed immunopositve in the cells, but they showed negative for the hematopoietic markers. The results of osteogenic or adipogenic induction expreiments displayed that after treated in induction media the cells could be differentiated into adipocytes and osteoblasts.DDR2 gene deletion could slightly inhibit the apoptosis of BMSCs, but had no effect on the differentiation and proliferation of BMSCs.DDR2 gene deletion significantly decreased the osteoblast differentiation ability of cells, but had a little influence on the ability of differentiation into adipocytes. DDR2 gene deletion could reduce the activity of alkaline phosphatase and the secretion o f OCN, and the expression of osteogenic related genes have obviously decreased. The animal study has also showed the same results. The bone repair ability of BMSCs derived from DDR2 knockout mice was worser than the BMSCs derived from the wild type mice.The results showed that DDR2 gene deletion could not change the phenotype and growth characteristics of BMSCs.However, DDR2 gene deletion had significantly decreased the osteoblast differentiation ability of cells. And it also could reduce the activity of alkaline phosphatase, the secretion of OCN, and the expression of osteogenic related genes. And the bone repair ability of BMSCs derived from DDR2 knockout mice was decreased which further proved by in vivo study. |
PDF Full Text Request