短时CaCl2刺激对人脂肪干细胞增殖与成骨向分化的影响

目的:观察CaCl2 溶液作为海藻酸钠/明胶交联剂短时作用于人脂肪间充质干细胞( human adipose-de-rived mesenchymal stem cells,hASCs)时,对其增殖和成骨向分化的影响,从而为后期三维生物打印实验选择合适浓度的CaCl2 溶液打下研究基础. 方法:取P3代hASCs,实验组分别用50、100、200、500 mmol/L的CaCl2 溶液对细胞处理5 min,对照组细胞不处理. P3代hASCs处理后第1、3、5、7天用CCK-8(cell counting kit-8)法检测细胞增殖情况. P3代hASCs成骨诱导7d后,采用碱性磷酸酶染色法观察细胞成骨向分化情况,并用碱性磷酸酶活性定量法测定各实验组与对照组碱性磷酸酶活性表达的差异. P3代hASCs成骨诱导14天后,用茜素红染色法观察细胞矿化结节形成情况进行定量分析. 采用SPSS 17. 0软件,运用单因素方差分析对结果进行分析,两两比较采用SNK法.结果:不同浓度CaCl2 溶液处理组细胞的增殖水平在第1、3、5、7天的差异均无统计学意义;成骨诱导7天后,随着CaCl2 溶液浓度的升高,碱性磷酸酶活性先升高后降低,除50 mmol/L与100 mmol/L CaCl2 溶液处理组之间,以及成骨诱导培养基组与200 mmol/L CaCl2 溶液处理组之间差异无统计学意义外,其余组间差异均有统计学意义( P<0. 05);成骨诱导14天后,随着CaCl2 溶液浓度的升高,矿化结节由散在片状逐步变为层叠片状,矿化结节定量结果显示各实验组之间以及与各对照组间的差异均有统计学意义(P<0. 05). 结论:短时高浓度钙离子刺激对hASCs增殖无影响,但对hASCs的成骨向分化有促进作用,且随着钙离子浓度增加,促进作用加强,因此可以选用高浓度CaCl2 溶液作为三维生物打印用材料的交联剂.

Short-term effect of CaCl2 on human adipose-derived mesenchymal stem cells proliferation and osteogenic differentiation

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 os-teogenic differentiation ability, and to determine the appropriate concentration of CaCl2 for post three-di-mensional biological experiments. Methods:hASCs stimulated with or without CaCl2 at various concen-trations 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 os-teogenic 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 soft-ware. Results:The CCK-8 assays showed that the differences between the control groups and experimen-tal 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/LCaCl2 group. Alizarin red staining and quantitative detection showed that the differences between all pair-wise combinations of the groups were statistically significant (P<0. 05). As the CaCl2 concentration in-creased, the calcium deposition increased, initially in the form of a scattered sheet and eventually a lami-nated sheet. Conclusion:Stimulation by a high concentration of CaCl2 over a short time period can en-hance hASCs osteogenic differentiation ability, but has no effect on hASCs proliferation.