Comparative Study Of The Effects Of Boneceramic And Bio-Oss On The Osteogenic Differentiation Of Dog Bone Marrow Mesenchymal Stem Cells In Vitro

Objective Autologous bone transplantation has always been the gold standard for the treatment of bone defects,but due to various complications,it is difficult to promote clinically,so people are paying more and more attention to "bone graft substitutes".A suitable bone graft substitute must not only have good biocompatibility and bone differentiation properties,but also have a suitable degradation rate.This study mainly compares the effects of Bone Ceramic,a new pure synthetic bone graft material and Bio-Oss bone meal on the proliferation,adhesion and osteogenic differentiation of dog bone marrow mesenchymal stem cells(DBMSCs).Methods After the beagle was anesthetized by 3% pentobarbital sodium intravenously,the posterior iliac ridge was selected as the puncture point,bone marrow was extracted,and the primary cells were cultured by density gradient centrifugation.When the cells reached 80～90% confluence,0.25% pancreatin was added The digested cells were subcultured,and the DBMSCs isolated in vitro were identified for osteogenic,adipogenic,and chondrogenic differentiation.Take two kinds of bone meal granules,Bio-Oss and Bone Ceramic,respectively,add DMEM medium with 10% FBS and soak for 48 hours to obtain the extract with a concentration of 0.1g/ml.DBMSCs were seeded in a 96-well plate at a density of 4×104 cells/ml.After the cells were cultured for 24 hours,the original culture medium was replaced with the extract,and the control medium was not replaced.After culturing for 1,3,and 5 days,CCK8 working solution was added,placed in a constant temperature incubator for 2 hours,and then the absorbance of each well was measured at a wavelength of 450 nm.Put Bio-Oss and Bone Ceramic particles into a 15 ml centrifuge tube,add DBMSCs with a density of1×106/m L,culture for 3 days,aspirate the culture medium,wash with PBS,add 2.5%glutaraldehyde,fix for 12 h,PBS rinse,use 60%,70%,80%,90% and 100% absolute ethanol to dehydrate the cells in a gradient,critical point drying,after completion of gold plating 90 s,the gold-plated particles under a scanning electron microscope Observed.In a sterile environment,after the Bio-Oss and Bone Ceramic particles are co-cultured with the cells for 1 day,the growth medium(GM)is replaced with the osteogenic medium(OM),and the medium is changed every 2 days.The groups are as follows: Bio-Oss implanted cells(OM): group A,Bone Ceramic implanted cells(OM):group B,pure DBMSCs(OM): group C,pure DBMSCs(GM): group D.The levels of alkaline phosphatase ALP in the cell culture supernatant were measured after the 4th,7th,and 10 th days of culture.According to the grouping method of ALP quantitative detection,total cell RNA was extracted 7 and 14 days later,and RT-PCR was performed to quantitatively detect the relative expression levels of OPN,OCN,RUNX-2 and ALP genes.Results The cells isolated and cultured from dog bone marrow conformed to the characteristics of mesenchymal stem cells.CCK-8 results showed that the stock solutions of Bio-Oss and Bone Ceramic had no obvious cytotoxic effect on DBMSCs.SEM showed that DBMSCs distributed more widely on the surface of Bone Ceramic than that of Bio-Oss.ALP quantitative detection showed that DBMSCs on the Bone Ceramic surface were more conducive to its osteogenic differentiation than Bio-Oss,and it also increased the expression of OPN,OCN,RUNX-2 and ALP genes.Conclusion In vitro studies show that both Bio-Oss and Bone Ceramic have good biocompatibility.Compared with Bio-Oss bone meal,Bone Ceramic is more conducive to the osteogenic differentiation of DBMSCs.