The Investigation On Osteoblastic Differentiation Of Mesenchymal Stem Cells Induced By Strontium-containing Hydroxyapatite

Objective To investiagate the effect of strontium-containing hydroxyapatite(Sr-HA) bioceramics on osteobalstic differentiation of marrow mesenchymal stem cells (MSCs) and explore their mechanism by comparing the different action of Sr-HA to the proliferation and differentiation of MSCs.Methods MSCs were purified and proliferated by adherent method.Maceration extract was prepared by Sr-HA ,HA power mixed with 10%FBS L-DMEM,they were divided into 4 groups:Group A(maceration extract of 10% Sr-HA), GroupB(maceration extract of 5% Sr-HA), Group C(maceration extract of 1% Sr-HA), Group D(maceration extract of HA),the effect of each maceration extract to the proliferation and differentiation of MSCs was investigated and compared.The concentration of Sr and Ca ion in each maceration extract were measured with inductively coupled plasma-atomic emission spectrometry.MTT was used to assay cell proliferation.Osteoblasts were identified by immunocytochemical alkaline phosphatase(ALP) and alizarin red stained mineralized nodules; Osteoblastic differentiation of MSCs was compared by the activity of cell ALP which was measured by colorimetry and the expression of cell core binding factor a1(Cbfa1) gene which was measured by semi-quantitative reverse transcription polymerase chain reaction(Rt-PCR) in each group.Results MSCs were validated by flow cytometry.Sr ion concentration were range from 5.05ug/ml to 19.46ug/ml,and Ca ion concentration were range from 5.03ug/ml to 23.26ug/ml in maceration extract of 1-10% Sr-HA. At various time points of cell culture, MTT value showed that there were no significant difference in the absorbance of each maceration extract (p>0.05).It is found that ALP staining on day 14th, alizarin red stained mineralized nodules on day 21st are both positive in each group after induced culture. There was a significant increase in ALP activity and Cbfa1 mRNA expression of the Sr-HA group when compared with that of HA group.The activity of ALP in Group A,B,C was Significantly higher than in Group D on day 6th,9 th,12 th,15 th (p<0.01).It was found that Cbfa1 mRNA had expressed in Group A,B,C on day 6 th. Reverse transcript-PCR showed that the expression of cbfa1 mRNA in Group A,B,Cwere elevated significantly higher than in Group D on day 9 th,12 th(p<0.01).Conclusions The highly pure and active MSCs could be acquired by adherent method. Sr-HA had no significant effect on the proliferation of MSCs.There was a significant increase in osteoblastic differentiation of MSCs of 1-10%Sr-HA when compared with that of HA.It is evident that strontium had beneficial role on osteoblastic differentiation of MSCs for HA. We also found that strontium ion in the 1%-10%Sr-HA was an essential factor that could further enhance osteoblastic differentiation of MSCs.