Mechanism of tissue-engineered bone recruiting endogenous mesenchymal stem cells towards bone regeneration

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 higher than those in the DBM 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. Key words: Tissue engineering; Mesenchymal stem cells; Stromal cell-derived factor-1