Three methods to isolate osteoblasts: stem cell induction, cell line induction and primary isolation / 中国组织工程研究

ABSTRACT

BACKGROUND: Osteoblasts have become a kind of important seed cells in bone tissue engineering. However, it is difficult to harvest osteoblasts, and the purity and calcification ability of osteoblasts isolated by different methods are inconsistent. OBJECTIVE: To compare the purity and calcification ability of osteoblasts induced from mouse bone marrow mesenchymal stem cells, MC3T3 cell lines, and cultured primarily from the neonatal mouse cranium. METHODS: Mouse bone marrow mesenchymal stem cells were isolated by differential adhesion method, and after passaing, passage 3 cells were cultured in osteogenic induction medium for 21 days. MC3T3 cell lines were cultured in osteogenic induction media 1 and 2 for 21 days. Osteoblasts were cultured primarily from neonatal mouse cranium by type Ⅰ coll agenase digestion method. Calcium nodules of osteoblasts obtained by three methods were observed by Alizarin red staining to detect osteogenic activity of cells. RESULTS AND CONCLUSION: (1) There were average 16.3 calcium nodules per low-power field after osteogenic induction of bone marrow mesenchymal stem cells. (2) There were sparsely distributed calcium nodules in MC3T3 cells after induction with osteogenic induction medium 1, accounting for 1.7 calcium nodules per low-power field, while there were dense calcium nodules in MC3T3 cells after induction with osteogenic induction medium 2, accounting for 44.6 calcium nodules per low-power field. There was a significant difference in the calcium nodule formation ability between the two groups (P < 0.01). (3) After primary culture, there was only 0.6 calcium nodule per low-power field. (4) Except for the insignificant difference between osteogenic induction medium 1 and primary culture groups, there were significant differences in pair-wise comparison of any other two groups. Except the insignificant difference between group I of MC3T3 inducing conditional media and primary culture osteoblasts, there were significant differences in the osteogenic ability between groups (P < 0.01). In conclusion, it is a better method to culture MC3T3 cells in osteogenic induction medium 2 containing dexamethasone, because many uncontrol able factors are involved in the isolation and culture of bone marrow mesenchymal stem cells.