Abstract:

Objective：To examine the effect of CaCl2, a sodium alginate crosslinker, to stimulate cells for a short time period on human adipose-derived mesenchymal stem cells (hASCs) proliferation and osteogenic differentiation ability, and to determine the appropriate concentration of CaCl2 for post three-dimensional biological experiments. Methods: hASCs stimulated with or without CaCl2 at various concentrations were seeded and cultured in control medium and osteogenic medium, respectively. The cell counting kit-8 (CCK8) was used to estimate the cell proliferation level of each group. After 7 days of osteogenic induction, alkaline phosphatase (ALP) staining and activity assays were performed using an ALP kit. After 14 days of osteogenic induction, alizarin red staining and quantitative detection were used to determine the calcium mineral density. The results were analyzed using analysis of variance (ANOVA) and Student-Newman-Keuls (SNK) tests for pairwise comparisons implemented in the SPSS 17.0 software. Results: The CCK-8 assays showed that the differences between the control groups and experimental groups were not statistically significant, so different concentrations of CaCl2 had no significant effect on hASCs proliferation. The ALP staining and activity assays showed that ALP activity first increased and then decreased as the CaCl2 concentration increased. Furthermore, the differences between all the groups were statistically significant (P<0.05), except the difference between the 50 mmol/L CaCl2 group and the 100 mmol/L CaCl2 group, and between the osteogenetic medium(OM) group and the 200 mmol/L CaCl2 group. Alizarin red staining and quantitative detection showed that the differences between all pairwise combinations of the groups were statistically significant (P<0.05). As the CaCl2 concentration increased, the calcium deposition increased, initially in the form of a scattered sheet and eventually a laminated sheet. Conclusion: Stimulation by a high concentration of CaCl2 over a short time period can enhance hASCs osteogenic differentiation ability, but has no effect on hASCs proliferation.

Key words: Adipose tissue, Mesenchymal stromal cells, Calcium chloride, Cell proliferation, Cell differentiation