In Vitro Study Of SrO/nHA Bone Defect Repairing Materials With Nonhuman Primate Bone Marrow Derived Mesenchymal Stem Cells

Objective: In the previous work,the research team had conducted a series of experimental studies on the preparation process,characterization,porosity,and pore size of strontium oxide(Sr O)/nanohydroxyapatite(n HA)composites with different mass concentration ratios of Strontium(Sr)by spark plasma sintering(SPS).It was found that Sr O/n HA composites with a mass concentration ratio of 3% to 7% have good the properties of the degradation and sedimentation mineralization.According to the Biological Evaluation Standards for Biological Materials and Medical Devices(ISO 10993),biological properties(cell compatibility and osteogenic activity)of Sr O/n HA composites with different Sr concentrations were evaluated and screened out Sr O/n HA composites with the best Sr content in vitro.Methods: In this experiment,the 0% Sr O/n HA composites without Sr and the Sr O/n HA composites with 1%,3%,5% and 7% Sr were prepared by spark plasma sintering(SPS).Then,the experiments were divided into 6 groups,namely the control group without material,0%,1%,3%,5% and 7% Sr O/n HA groups.Based on these groups,the experiment to verify the cellular compatibility of Sr O/n HA composites was firstly divided into two parts.The experiment one: the 0%,1%,3%,5%,and 7% Sr O/n HA groups were used to directly adhere to the rhesus monkey bone marrow derived mesenchymal stem cells(RMBMSCs).The adherence rates of RMBMSCs were calculated 24 hours later,and after 1 day,4 days,and 7 days,the effects of five kinds of materials on the adhesion of RMBMSCs were observed by scanning electron microscopy(SEM)at different times.The experiment two: in order to avoid the effect of the surface properties of the materials on the adhesion quantity of RMBMSCs,the extracts of five materials were prepared.Then in combination with the blank control group,the effects of different materials on proliferation and apoptosis of RMBMSCs were analyzed by the WST-1 method and the flow cytometry at different times.Finally,on the based of the experimental results of the above two parts,a Sr O/n HA composite with the best Sr mass concentration ratio was selected.Then,combined with the blank control group and 0% Sr O/n HA group,RMBMSCs were seeded on the bottom surface of the 6-well plates and the surface of the materials respectively.The osteogenic differentiation and the mineralization of extracellular matrix of the three groups of RMBMSCs were compared using q RT-PCR and the alizarin red staining.Results:(1)With comprehensive experimental results on adhesion,proliferation and apoptosis of RMBMSCs,it was found that 3% Sr O/n HA composite had better cell activity in the experimental study.(2)The results of osteogenic differentiation of RMBMSCs showed that 3% Sr O/n HA composites containing Sr can upregulate the expression of osteoblast-related genes(Runx2,SP7,ALP,OCN,and OPN)and downregulate the expression of the osteoclast-associated genes(OPG,RNAKL,and OPG/RNAKL).This promoted the differentiation of RMBMSCs into osteoblasts.In addition,through the mineralization experiments of extracellular matrix,the results did not show a significant difference between the three groups,but the mineralization of the extracellular matrix occurred.Conclusion:(1)SrO/nHA composites had good the cell compatibility,and the osteogenic compatibility still needs further studies.(2)In the study of this subject,when the mass concentration ratio of Sr is 3%,the Sr O/n HA composite had the best cell compatibility and can promote the osteogenic differentiation of RMBMSCs.