In Vitro Cytotoxicity And Osteogenic Effect Of Mg-3Mn Alloy As Maxillofacial Bone Repairing Material

Objective:To study the effect of Mg-3Mn alloy maxillofacial bone repair material with high manganese content on Bone Marrow Mesenchymal Stem Cells(BMSCs)and Mouse embryo osteoblast precursor cells(MC3T3-E1)activity and its osteogenic differentiation.Methods:1.Cell culture experiment:1)Culture of BMSCs : taking 3-4 weeks old female SD rats to obtain primary BMSCs;BMSCs from SD rats were isolated and cultured to the third generation by whole bone marrow adherence method,and the phenotype of BMSCs was identified by flow cytometry.2)Culture of MC3T3-E1: MC3T3-E1 was purchased from Be Na Culture Collection and was passaged for 3 times for subsequent experiments.2.Effect of bone repair materials on cell activity in vitro:1)direct contact method: BMSCs and MC3T3-E1 were inoculated on the surface of Mg-3Mn alloy maxillofacial bone repair material for 1 and 7 days,respectively,and the adhesion and morphological changes of cells on the surface were observed by Scanningelectronmicroscopy(SEM),and the surface corrosion morphology of the material after soaking in DMEM medium for 7 days.2)Indirect contact method: prepare the leaching solution of pure magnesium and Mg-3Mn alloy maxillofacial bone repair materials,construct the microenvironment of cell proliferation and osteogenesis,and use ICP-OES to detect the concentration of metal ions in the leaching solution;BMSCs and MC3T3-E1 1,3,5 and 7 days were cultured in complete medium containing different concentrations of extracts,and CCK-8 kit was used to detect cell activity.The living dead cells of BMSCs and MC3T3-E1 were stained,and the effects of ion concentration in the extract on the activities of BMSCs and MC3T3-E1 were analyzed.3.In vitro experiment to explore the role of bone repair materials in inducing osteogenic differentiation of cells: According to the experimental results of cell activity in vitro,BMSCs and MC3T3-E114 days were cultured in osteogenic induction medium containing 25% extract,and the mRNA expression levels of ALP,OCN,Col-I and Runx2 were detected by q RT-PCR.BMSCs and MC3T3-E121 days were cultured,and the formation of mineralized nodules was detected by alizarin red staining.Results:1.Flow cytometry results: The third-generation SD rat BMSCs isolated and cultured by the whole bone marrow adherence method highly expressed the nonhematopoietic cell surface markers CD44 and CD90,and low expression of hematopoietic stem cell surface markers CD45 and CD11 b,confirming the culture cells are BMSCs.2.SEM results:1)Cell morphology: SEM results showed that BMSCs and MC3T3-E1 showed good cell adhesion on the surface of pure magnesium and Mg-3Mn alloy maxillofacial repair materials.Among them,BMSCs and MC3T3-E1 adhesion morphology on Mg-3Mn alloy maxillofacial bone repair materials was better than that of pure magnesium alloy.2)Corrosion morphology: pure magnesium and Mg-3Mn alloy maxillofacial bone repair materials were soaked in DMEM medium for 7 days,pure magnesium corrosion was more serious,the surface formed a loose and porous film structure;the corrosion morphology of Mg-3Mn alloy maxillofacial bone repair materials showed typical pitting and galvanic corrosion,and the corrosion pits expanded with the immersion time,and finally formed cracks.3.ICP-OES results: Pure magnesium releases metal Mg ions after soaking in complete medium,Mg-3Mn alloy maxillofacial bone repair materials release metal Mg and Mn ions after soaking in complete medium,and metal Mg ions released by Mg-3Mn alloy maxillofacial bone repair materials are significantly lower than that of pure magnesium,indicating that Mg-3Mn alloy maxillofacial bone repair materials have good biocorrosion resistance.4.CCK-8 results: The results of indirect contact test showed that Mg-3Mn alloy maxillofacial bone repair material had no cytotoxicity to BMSCs and MC3T3-E1.Among them,BMSCs and MC3T3-E1 showed higher cell activity in the extract of25% Mg-3Mn alloy maxillofacial repair materials,and the OD value of BMSCs was lower than that of the negative control group after 7 days,but the difference was not statistically significant(P > 0.05).After 7 days of MC3T3-E1 culture,the OD value was significantly lower than that of the negative control group.5.Live&dead viability assay: Both pure magnesium and Mg-3Mn alloy maxillofacial bone repair materials are beneficial to the proliferation of BMSCs and MC3T3-E1 cells.Among them,there was no significant difference among the three groups on the 5th day of cell culture.6.compared with pure magnesium,Mg-3Mn alloy maxillofacial bone repair material is beneficial to the relative expression of mRNA of ALP,Col-Ⅰ,OCN and Runx2(P< 0.01,there is significant difference),indicating that Mg-3Mn alloy maxillofacial bone repair material has good osteogenic induction.7.Alizarin red staining results: the alizarin red staining area of Mg-3Mn alloy maxillofacial bone repair material is higher than that of pure magnesium,indicating that the mineralization degree of Mg-3Mn alloy maxillofacial bone repair material is better than that of pure magnesium,which is beneficial to the growth of new bone tissue.Conclusion:1.Mg-3Mn alloy maxillofacial bone repair material has better biocorrosion resistance than pure Mg.2.Mg-3Mn alloy maxillofacial bone repair material is beneficial to the adhesion and proliferation of BMSCs and MC3T3-E1.3.Mg-3Mn alloy maxillofacial bone repair material significantly promoted the expression of mRNA of ALP,Col-I,OCN and Runx2 and induced osteogenic differentiation of BMSCs and MC3T3-E1.