Mechanism of tissue-engineered bone recruiting endogenous mesenchymal stem cells towards bone regeneration / 中华创伤杂志

ABSTRACT

Objective To investigate the mechanism of implanted tissue-engineered bone (TEB)recruiting endogenous mesenchymal stem cells (BMSCs) towards bone regeneration after traumatic bone defect.Methods In vivo experiments:2 mm of diaphysis and periosteum were removed from the middle of the femoral shaft in 8 week old FVB/N mice to form a large segment of bone defect.Demineralized bone matrix (DBM) and TEB were implanted into the defect area and fixated.All mice were randomly divided into DBM group (n =18) and TEB group (n =18).The results were observed 24 hours after implantation:(1) flow cytometry was used to evaluate the number of mobilized host BMSCs into the blood;(2) non-invasive bioluminescent imaging was used to observe the ability of two groups in recruiting mouse bone marrow derived mesenchymal stem cells (mBMSCs) in peripheral blood to the defect area;(3) ELISA was used to evaluate the stromal cell-derived factor 1 (SDF-1) content in peripheral blood of two groups.In vitro experiments:(1) transwell assay was conducted to evaluate the ability of SDF-1 (100 ng/ml) in promoting the migration of human bone marrow derived mesenchymal stem cells (hBMSCs).SDF-1/C-X-C motif chemokine receptor-4 (CXCR4) pathway was blocked by the selective CXCR4 antagonist Plerixafor (AMD3100).The experimental groups were divided into control group,SDF-1 group,and SDF-1 + AMD3100 group.(2) The co-culture system of human umbilical vein endothelial cells (hUVECs) and hBMSCs was established,and cells were stimulated by SDF-1.The experimental groups were divided into hBMSCs group,hBMSCs + hUVECs group,and hBMSCs + hUVECs (AMD3100 pretreatment) group.Transwell assays were used to compare the migration of hBMSCs in each group.ELISA was used to detect the concentration of hepatocyte growth factor (HGF) in the co-culture supernatant.(3) In vitro cultured hUVECs were stimulated by SDF-1 and SDF-1/CXCR4 pathway was antagonized by AMD3100.The experimental groups were divided into control group,SDF-1 group,and SDF-1 + AMD3100 group.Quantitative real-time polymerase chain reaction (qRT PCR) was used to evaluate the expression of HGF in each group.Results In vivo experiments:24 h after transplantation,the number of BMSCs and SDF-1 concentration in the TEB group were significantly highcr than those in the DBM group (P ＜ 0.05).The number of recruited mBMSCs into the circulation in the TEB group was larger than that in the DBM group (P＜ 0.01).In vitro experiments:(1) compared with the control group and the SDF-1 + AMD3100 group,the SDF-1 group significantly enhanced the migration ability of hBMSCs in Transwell migration experiments (P ＜ 0.01);(2) compared with the hBMSCs group and the hBMSCs + hUVECs (AMD3100 pretreatment) group,the number of migrated cells and HGF concentration in the hBMSCs + hUVEC group significantly increased (P ＜ 0.01),but there were no significant differences between the hBMSCs group and the hBMSCs + hUVECs (AMD3100 Pretreatment) group (P ＞0.05);(3) qRT-PCR showed that the expression of HGF was significantly increased in the SDF-1 group compared with the control group (P ＜ 0.05).After antagonizing SDF-1/CXCR4,HGF expression in the SDF-1 + AMD3100 group was significantly lower than that in the SDF-1 group.Conclusions TEB transplantation in traumatic bone defect can significantly increase the concentration of chemokine SDF-1 in vivo and effectively promote the mobilization of endogenous MSCs and recruitment of circulating MSCs.SDF-1 not only directly promotes the migration of hBMSCs through SDF-1/CXCR4 pathway,but also up-regulates the expression and secretion of HGF in vascular cells to further amplify the chemotactic effect of SDF-1 on hBMSCs.