The Influence Of Autophagy On The Osteogenic Differentiation In Osteoporotic Bone Marrow Mesenchymal Stem Cell

Primary osteoporosis(OP)is an age-related metabolic bone disease,whose morbidity showes a trend of increasing year by year in this aging society.The osteoporotic fracture(OF)is the most serious complication,which easily leads to lose self-care ability of daily living in senile patients due to pain and dysfunction.Because of its high disability and mortality rate,OP has became a severe public health problem.The osteoporotic vertebral compression fracture(OVCF)is the most common issue in all the OF suffering.The main pathogenesis of OP is defective osteogenic capability caused by aging.Meanwhile,autophagy is an important physiological mechanism of cells to maintain self-stabilization.Recent studies has demonstrated that autophagy plays a critical regulatory role of self-renewal and self-differentiation in mesenchymal stem cells.However,whether autophagy could improve the osteogenic capability in osteoporotic bone marrow mesenchymal stem cells(BMSCs)remains unclear.In this study,we took the BMSCs from osteoporotic patients as the research object,to investigate the influence of autophagy on osteogenic capability in BMSCs from the following four aspects,to provide a new idea for OP treatment.Section OneSenile Phenotypes Comparation in Human BMSCsObjective To compare the senile phenotype of human vertebral BMSCs from senile OP and young non-OP patients,respectively.Methods Ten patients with vertebrae resection were included in this study,of which 5 patients were diagnosed as primary senile OP;and the other 5 patients were young,who had no medical history about spinal disease.After signing informed consent,the patients’ injured vertebrae were cut,and the BMSCs were conventionally isolated and cultured as previous report.The primary passage cells were used in the study of this section.The senile phenotype was detected by SA-β-gal staining,the protein expressions of P53 and P21 were detected by western blot,and telomerase activity was detected by ELISA.Results The rate of SA-β-gal positive cells in OP group were significantly increased(P ﹤ 0.05),and the expressions of senescenceassociated proteins,P53 and P21 were also significantly increased in OP group(P﹤0.05),furthermore,the ELISA test showed that the telomerase activity in BMSCs from OP group obviously decreased(P ﹤ 0.05).Conclusion The BMSCs from senile OP patients have more obviously senile phenotypes.Section Two Autophagic Level and Ostegenic Capability Comparation in Human BMSCsObjective To compare the autophagic level and osteogenic capability of human vertebral BMSCs from senile OP and young non-OP patients,respectively.Methods The primary passage of BMSCs were used in the study of section two.Western blot was used to detect the expression of autophagy associated proteins,LC3 and P62;the adenovirus containing LC3-GFP fusion protein was used to transfect BMSCs,and autophagy was evaluated by analyzing the number of green fluorescent puncta of autophagosomes under laser confocal microscopy;after osteoinduced differentiation culture,alkaline phosphatase staining was used to detect the early stage of osteoblast differentiation and alizarin red staining was for the the late stage of osteoblast differentiation;fluorogenic quantitative PCR(q-PCR)was used to detect the osteogenic gene,collagen type I(COL I),osteocalcin(OCN),osteopontin(OPN)and Runt-related transcription factor 2(RUNX2).Results Western blot analysis showed that the protein expression ratio of LC3 II/I significantly decreased(P ﹤ 0.05),on the contrary,the protein expression of P62 significantly increased(P﹤0.05).In addition,after adenovirus with LC3-GFP transfection,the laser confocal microscopy observation showed a significant decline of green LC3 puncta that represented the number of autophagosome in BMSCs from OP group(P﹤0.05).Furthermore,after osteogenic induction,the optical microscope observation showed that the positive degree of alkaline phosphatase and alizarin red staining were obviuosly weaker than those in young control group.The q-PCR results further indicated that the osteogenic gene,COL I,OCN,OPN and RUNX2 all significantly decreased in BMSCs from OP group,whose difference was statistically significant(P﹤0.05).Conclusion There are low autophagic level and osteogenic capability in the osteoporotic BMSCs.Section ThreeAutophagy Promotes Osteogenic Differentiation in Osteoporotic BMSCs in VitroObjectvie To investigate the influence of autophagy on the osteogenic differentiation in osteoporotic BMSCs in vitro.Methods The primary BMSCs were pretreated by autophagy inhibitor,3-methyladenine(3-MA)and agonist,rapamycin(RAP)2 h prior the subsequent processing.The changes of autophagic level were detected by western blot and laser confocal microscopy observation.Furthermore,after osteoinduced differentiation culture,alkaline phosphatase and alizarin red staining were used to compare the effect of osteoblast differentiation;and fluorogenic quantitative PCR(qPCR)was used to detect the osteogenic gene,collagen type I(COL I),osteocalcin(OCN),osteopontin(OPN)and Runt-related transcription factor 2(RUNX2).Results The results of western blot and laser confocal microscopy both indicated that 3-MA treatment significantly decreased the autophgic level,on the contrary,RAP treatment significantly activated autophagy in osteoporotic BMSCs(P﹤0.05).After the autophagy regulation,there was an obvious changes on osteoblast differentiation in BMSCs.With the suppression of autophagy,3-MA treatment significantly decreased the positive degree of alkaline phosphatase and alizarin red staining(P﹤0.05),whose results from RAP group just suggested contrary.Furthermore,The qPCR results indicated that the osteogenic gene,COL I,OCN,OPN and RUNX2 all significantly decreased in BMSCs from RAP group,whose difference was statistically significant(P﹤0.05).Conclusion Autophagy plays an important role of regulating osteogenic differentiation,which is able to significantly promote the osteogenic capability.Section FourAutophagy Promotes Osteogenic Differentiation in Osteoporotic BMSCs in VivoObjective To investigate the influence of autophagy on osteogenic differentiation in osteoporotic BMSCs in nude mice.Methods The BMSCs pretreated with osteogenesis induction medium in the presence of 3-MA or RAP were implanted into nude mice to compare the osteogenic capability in vivo.At 8 weeks after implantation,the mice were terminated and scaffolds were harvested for HE and Masson staining.In addition,micro-computed tomography(micro-CT)was used to test the new bone formation.The volume of interest(VOI)was defined as the relative changes in bone volume density(BV/TV%),which is the percentage of bone volume(BV)to the total tissue volume(TV).Results Eight weeks after implantation,bony masses formed at the injection site were removed and analyzed.The size of new bone was significantly larger in rapmaycin group than that in control group(P﹤0.05),however,3-MA treated hBMSCs exhibited lower osteogenetic ability,whose bone size was smaller than that in control group(P﹤0.05).These results were confirmed by micro-CT analysis that was the VOI value was significantly increased in RAP group,while it was significantly decreased in 3-MA group,whose difference was statistically significant(P﹤0.05).HE and masson staining revealed that rapmaycin markedly induced bone-like tissue formation in vivo;while after 3-MA treatment,this autophagy-induced osteogenetic ability was significantly decreased.Conclusion Autophagy activation by pharmacological regulation could significantly promote the osteogenic differentiation of BMSCs in vivo,which provide a new idea for OP treatment.