| ?Background? The treatment of large segmental bone defect caused by severe trauma and so on,has always been a difficult problem in orthopaedic clinic.With the rapid development of tissue engineering,tissue engineered bone provides a feasible method for the treatment of large segmental bone defects.So far,however,tissue engineered bone has not been widely used in repairing large bone defects in clinic.The main obstacle is that the tissue engineered bone implanted in vivo lacks the support of blood vessels and nerves,which leads to poor osteogenesis of tissue engineered bone.Our previous animal experiments showed that the implantation of sensory nerve bundles into tissue engineered bone could significantly promote the osteogenesis of tissue engineered bone.However,the cytological mechanism of sensory nerve promoting tissue engineered bone formation in vitro remains unclear.?Objectives? 1.To explore the cytological mechanism of sensory nerve promoting osteogenesis of tissue engineered bone in vitro;2.To investigate the effects of dorsal root ganglion(DRG)on the proliferation and differentiation of bone marrow mesenchymal stem cells(BMSCs)in coculture system;3.To explore the intrinsic mechanism of the effect of DRG on BMSCs in coculture system.?Methods? 1.BMSCs were extracted by bone marrow flushing and adherent culture,and BMSCs were identified by cell morphology,multidirectional differentiation potential and specific CD molecule.2.Construction of Transwell coculture system.Transwell coculture system was constructed by using dorsal root ganglion(DRG)of neonatal SD rats and BMSCs of green fluorescent rats.The experimental group contained DRG and BMSCs in a Transwell coculture system(referred to as DRG+BMSCs group).DRG neurons were located in Transwell chamber and BMSCs were normally cultured in 6-well plates.The control group only contained BMSCs(referred to as BMSCs group).3.The proliferation of BMSCs was detected by CCK8 test,and the ability of osteogenic,adipogenic and chondrogenic differentiation of BMSCs was evaluated by Alizarin red,oil red O and alicin blue staining.4.The self-renewal ability of BMSCs was detected by cloning formation test(CFU)and the expression of BMSCs stemness genes were detected by RT-PCR to determine whether the stemness of BMSCs was enhanced or not.5.The expressionof autophagy-related protein LC3 was detected by Western blot and immunofluorescence staining,and the formation and number of autophagosomes were observed by transmission electron microscopy to explore the changes of autophagy.6.The activation of AMPK/mTOR and AKT/mTOR signaling pathways in BMSCs were detected by Western blot to determine the signal pathways mediating autophagy in this experiment.7.Compound C,an inhibitor of AMPK,was used to block the AMPK/mTOR pathway.The autophagy-related protein LC3 was detected by immunofluorescence and Western blot.The formation of autophagosomes was observed by transmission electron microscopy.The changes of autophagy intensity and stemness gene expression in coculture group before and after adding Compound C were compared to clarify the role of AMPK/mTOR signaling pathway in the enhancement of autophagy and upregulation of stemness gene expression in BMSCs.8.The effect of substance P(SP)produced by DRG was blocked by neurokinin-1 receptor(NKIR)inhibitor aprepitant on BMSCs.The changes of autophagy-related protein LC3 in coculture group were detected by Western blot and the changes of stemness genes were detected by RT-PCR to identify the cytokines of DRG acting on BMSCs in coculture system.?Results? 1.Characterization of BMSCs.Morphological observation showed that the cell morphology of P3 generation was typical spindle or spindle-shaped with green fluorescence;Alizarin red staining,oil red O staining and alicin blue staining were all positive;the marker molecules of hematopoietic cell CD34(2.3±0.37%,n=5)and CD45(2.4±0.19%,n=5)were low expression,and medullary cell marker CD11b/c(1.4±0.13%,n=5)was also low expression,while one of the markers of BMSCs CD90(99.6±0.24%,n=5)was high expression.Therefore,the cultured cells are bone marrow mesenchymal stem cells with high purity.2.Results of experiments of proliferation and differentiation of BMSCs in coculture system.The CCK8 results showed that on the 2nd,4th,6th,8th and 10 th day of the experiment,the absorbance value of CCK8 in coculture group was larger than that in control group(P<0.05),which meant the proliferation ability of BMSCs in coculture group was stronger than that in control group,and the difference of proliferation ability between the two groups was most significant on the 8th day of coculture.CCK8 experiment showed that coculture with DRG could promote the proliferation of BMSCs.The result of Alizarin red showed that the calcium nodules formed by coculture group after 14 days of osteogenic induction were larger and more than those in control group.The result of Oil red O staining showed that lipid droplets formed in coculture group after 24 days of adipogenic induction were fuller and more well-stacked than those in control group.The results of Alicin blue staining showed that the cartilage matrix formed by coculture group after 20 days of chondrogenic induction was larger and thicker than those in control group.Therefore,the ability of osteogenic,adipogenic and chondrogenic differentiation of BMSCs in coculture group was stronger than that of control group,which meant the multi-directional differentiation potential of BMSCs was maintained or enhanced after coculture with DRG compared with control group.The coculture of DRG and BMSCs not only promotes the proliferation of BMSCs,but also maintains or enhances the multidirectional differentiation potential of BMSCs,suggesting that the stemness of BMSCs might be maintained or enhanced.3.Results of CFU test and RT-PCR test of stemness genes.(1)Results of CFU test showed that the clone formation rate of BMSCs in coculture group was significantly higher than that in control group(P < 0.05),that is,the clone formation ability of BMSCs in coculture group was stronger than that in control group.(2)Results of RT-PCR test showed that the gene expression of Sox 2,Nanog and Oct 4 of BMSCs in coculture group was higher than that in control group(P < 0.05).The coculture with DRG could enhance the stemness of BMSCs.4.Results of BMSCs autophagy test.(1)Laser confocal microscopy showed that the fluorescence intensity of autophagy-related protein LC3 of BMSCs in coculture group was stronger than that in control group.Image J fluorescence intensity semi-quantitative analysis showed that the average OD value of autophagy-related protein LC3 of BMSCs in coculture group was larger than that in control group(P < 0.05);(2)Semi-quantitative analysis of Western blot showed that the LC3-II/LC3-I ratio of BMSCs in coculture group was higher than that in control group(P < 0.05);(3)Transmission electron microscopy showed that the number of autophagosome of BMSCs in coculture group was significantly larger than that in control group.The autophagy intensity of BMSCs in coculture group was higher than that in control group.5.Results of Western blot test of AMPK/mTOR pathway and AKT/mTOR pathway.Western blot results showed that the expression of P-AMPK was upregulated and P-mTOR was downregulated in AMPK/mTOR signaling pathway of BMSCs in coculture group compared with the control group,while the expression of P-AKT in the AKT/mTOR signaling pathway remained unchanged.Therefore,the enhancement of autophagy of BMSCs during coculture was accompanied by activation of AMPK/mTOR signaling pathway,while AKT/mTOR signaling pathway was not significantly changed.6.Results of AMPK blocking experiment.Compound C inhibited the activation of AMPK and blocked the AMPK/mTOR signaling pathway.(1)Results of Western blot showed that the expression of autophagy-related proteins LC3-II,LC3-I and LC3-II/ LC3-I ratio in DRG+BMSCs+Compound C group were lower than those in DRG+BMSCs group(P < 0.05),and higher than those in control group(P < 0.05).(2)Results of RT-PCR test showed that the expression of stemness genes,Sox2,Nanog and Oct4,in DRG+BMSCs+Compound C group was lower than that in DRG+BMSCs group(P < 0.05),and higher than that in control group(P < 0.05).(3)Transmission electron microscopy showed that the number of autophagosome in DRG+BMSCs+Compound C group was significantly less than that in DRG+BMSCs group and more than that in control group.Compound C,a specific inhibitor of AMPK,can significantly reduce the autophagy level and the expression of stemness genes in BMSCs in coculture group.The activation of AMPK/mTOR signaling pathway mediates the enhancement of autophagy level in DRG+BMSCs coculture system.At the same time,the expression level of stemness genes in DRG+BMSCs+Compound C group was significantly lower than that in DRG+BMSCs coculture group,which further demonstrated that autophagy enhancement mediated the enhancement of cell stemness in coculture group.7.Result of NK1 R inhibition experiment.The result of Western blot showed that the ratio of LC3-II/ LC3-I in DRG+BMSCs+aprepitant group was significantly lower than that in DRG+BMSCs group(P < 0.05),but still higher than that in BMSCs group(P < 0.05).(2)Result of RT-PCR test showed that the expression level of stemness genes in DRG+BMSCs+aprepitant group was significantly lower than that in DRG+BMSCs group(P < 0.05),but higher than that in BMSCs group(P < 0.05).DRG-derived SP plays an important role in the increase of autophagy level and the upregulation of stemness gene expression in BMSCs.?Conclusion? In the Transwell coculture system composed of DRG and BMSCs,DRG could enhance the autophagy level of BMSCs by activating AMPK/mTOR pathway,thereby maintaining or enhancing the stemness of BMSCs.In this process,the sensory neuropeptide SP produced by DRG plays an important role.This study is of great significance in revealing the mechanism of sensory nerve promoting tissue engineered bone osteogenesis. |
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