| Research objectives:To study the effect of Schizandrin A on the proliferation and osteogenic differentiation of BMSCs,to prepare DBM scaffolds loaded with Schizandrin A in a sterile environment,and to study its related biological activities,so as to lay a foundation for the clinical application of new scaffolds.Research Methods:1.Primary BMSCs were isolated and cultured from bilateral femur and tibia of Wistar rats by the whole bone marrow adherent screening method.The primary BMSCs were successively passed to the second generation for osteogenesis induction and adipogenesis induction and subsequent experiments.2.BMSCs were cultured in different concentrations(0.01μg/ m L,0.1μg/ m L,1μg/ m L,10μg/ m L)of Schizandrin A,and the effect of Schizandrin A on the proliferation of BMSCs was detected by CCK-8 method.After BMSCs induction,ALP staining,ALP activity detection,alizarin red staining and real-time PCR quantitative detection were performed to detect the transcription expression of Runx-2,BMP-2,OPN and ALP osteogenic related genes in BMSCs after osteogenesis induction,so as to comprehensively evaluate the optimal drug concentration for BMSCs proliferation and osteogenic differentiation.3.DBM scaffolds were prepared by defatting,decalcification and deproteinization of bovine cancellous bone in vitro.DBM scaffolds were treated with different concentrations of 10μg/ m L,20μg/ m L,30μg/ m L,40μg/ m L and 50μg/ m L of Schizandrin A solution,and the scaffolds loaded with Schizandrin A were prepared by freeze-drying method.4.DBM scaffolds loaded with Schizandrin A were co-cultured with BMSCs,and the effect of drug-loaded scaffolds on the proliferation activity of BMSCs was detected by CCK-8 method;After selecting the optimal drug loading concentration,the surface morphology of the scaffold was observed under a scanning electron microscope,and the porosity,swelling rate and degradation rate of the scaffold were measured.5.BMSCs with good growth conditions were inoculated on sterile DBM scaffolds and DBM scaffolds loaded with Schizandrin A.The growth of BMSCs in the scaffolds was observed under scanning electron microscope.BMSCs cells were inoculated onto DBM scaffolds before and after drug loading.After 12 h,the cells were treated with DAPI solution,and the adhesion of BMSCs to DBM and SCH drug loading scaffolds was observed under laser confocal microscope.Results:1.Long fusiform BMSCs were successfully isolated and cultured from bilateral femur and tibia of Wistar rats.After osteogenesis induction,calcium nodules and alkaline phosphatase expression were observed,and lipid droplets were observed after adipogenesis induction.2.In CCK-8 cell proliferation detection,compared with blank group and 10μg/m L concentration group,0.01μg/ m L,0.1μg/ m L and 1μg/ m L concentration groups promoted the proliferation of BMSCs(P<0.05),and 0.1μg/ m L and 1μg/ m L concentration groups had more significant effects.Compared with blank group,both0.1μg/ m L and 1μg/ m L groups promoted osteogenic differentiation of BMSCs.Compared with 0.1μg/ m L group,1μg/ m L group significantly increased extracellular calcium salt deposition,deeper ALP staining,higher ALP activity expression level(P<0.05),and higher transcription levels of Runx-2,BMP-2,OPN and ALP osteogenic related genes(P<0.05).3.DBM scaffolds have loose and porous spongy structures.The pore sizes in the scaffolds are different,and most of the shapes are irregular round or oval,and the internal pores communicate with each other.Compared with blank group and other concentration SCH-DBM groups,the proliferation of BMSCs was significantly promoted in 20μg/ m L SCH-DBM scaffold group(P<0.05).The porosity,swelling rate and degradation rate of the scaffold were decreased after modified with 20μg/ m L Schizandrin A(P<0.05).4.Good growth of cells was observed on both DBM and SCH-DBM scaffolds.After DAPI staining,the nuclei were observed under a confocal laser microscope,showing a round blue stain structure,and scattered in the scaffold of each group showed a three-dimensional structure.Research Conclusions:1.Long fusiform BMSCs were successfully extracted from Wistar rats and identified by osteogenesis induction and adipogenesis induction,which were consistent with the characteristics of BMSCs.2.The concentration of 0.1μg/ m L and 1μg/ m L Schizandrin A can promote the proliferation and osteogenic differentiation of BMSCs,and the effect of 1μg/ m L concentration is more obvious.3.Both the DBM scaffold and the DBM scaffold coated with Schizandrin A had good cytocompatibility.The DBM scaffold modified with 20μg/ m L Schizandrin A could promote the proliferation activity of BMSCs... |
PDF Full Text Request