Effects Of Strontium And DBM-Sr On Proliferation And Differentiation Of Human Bone Marrow Derived Mesenchymal Stem Cells

Objective:To study the effects of strontium (Sr) on proliferation and differentiation of human bone marrow derived mesenchymal stem cells (hMSCs) in vitro and determine at which concentration strontium can promote the effect best. The decalcified bone matrix (DBM) with different concentration of Sr2+ was prepared and culturd with hMSCs to observe the proliferation and differentiation.Methods:1. The hMSCs were isolated and cultured in vitro, and cell surface markers were tested by flow cytometer. Cells at passage 4 were used for experiments and were randomly divided into 5 groups. The osteogenic agent was used alone in the control group (Group A) , and the combinations of strontium [ in different concentrations (3.75×10-3mmol/L,3.75×10-2mmol/L,3.75×10-1mmol/L and 3.75 mmol/L, group B~E, respectively) ] and the osteogenic agent was used in Groups B~E. The MTT colorimetric assay was employed to evaluate the proliferation, and the differentiation of hMSCs into osteoplasts was evaluated by testing the activities of alkaline phosphatase (ALP) , the content of osteocalcin (OCN) and the formation of bone nodule.2. The femoral heads were obtained from the surgery of hip replacement, and cut into small bone block with the size of 5×5×5 mm3, then prepared the DBM by means of modified Urist method. The solution of typeⅠcollagen with SrCl2 with different concentrations was mixed, and the final concentrations of Sr2+ in commixing solutions were 0 mmol/L (Group A'), 3.75×10-3 mmol/L (Group B'), 3.75×10-2 mmol/L (Group C'), 3.75×10-1 mmol/L (Group D'), and 3.75 mmol/L (Group E') respectively. The DBM was immersed in commixing solutions as mentioned above for 1h , then were processed by vacuum adsorption, and sterilized by epoxy ethane. The hMSCs at passage 4 were cocultured with DBM, and the proliferation rate, activity of ALP and the content of OCN were observed at regular intervals.Results:1.The proliferation and osteogenic differentiation rate of each group was significantly increased with the culture time (P <0.01). The results of MTT test showed the proliferation rate and survival rate of hMSCs of B~E groups were significantly higher than that of group A at 7 days(P <0.05). In ALP detection, the enzyme activity of groups B~E was incresed at 7 and 14 days (P<0.05,compared to group A). The OCN contents of groups B~E were higher than that of group A at 14 and 21 days(P <0.05). The quantitative analysis revealed that the amount and the area of calcium nodule of groups B~E were also higher than these of groupA at 21 days (P <0.05). More detailedly, in both MTT test at 7days and ALP detection at 14 day, the maximun values were presented in group D, there were also a statistics difference compared to groups A~C(P <0.05), but no significant difference compared to group E. In the quantitative analysis of calcium nodule and OCN content at 21 days, the maximun value was also presented in group D(P <0.05; compared to other groups).2.In DBM coculture experiments, the proliferation rate of each group was also significantly increased with the culture time (P <0.01). The results of MTT analysis showed an increased number of cells in each group at 1, 4, 7 days, but there was no statistical difference among the groups (P>0.05). In ALP analysis, the enzyme activity of each group was incresed at 7 and 14 days, and the increase of groups with Sr2+ was more significant than control group(P <0.01),but there was no statistical difference among the groups with Sr2+ (P>0.05). The OCN content was almost invariable higher in all groups at 7, 14 and 21 days. The supplemented experiment revealed that DBM without hMSCs contained OCN although its concentration was not permanant.Conclusions:1.Strontium could promote hMSCs to proliferate and differentiate into osteoblasts, the optimal strontium concentration was 3.75×10-1mmol/L; 2. The DBM containing strontium have a good biocompatibility, after cultured with hMSCs, hMSCs could proliferate and differentiate into osteoblasts, and the osteogenic differentiation rate of the groups containing strontium is better than the control group. Becouse of the difference of biological characteristics of DBM, the difference of the proliferation and osteogenic differentiation can not be confirmed among the groups.