Tensile Strain Promotes The Angiogenicosteogenic Effect Of BMSCs/VECs Coculture And Its Paracrine Mechanism Research

OBJECTIVES: Tensile strain and neovascularization both play an essential role in new bone formation.The purpose of present study was to evaluate the impact of cyclic tensile strain on the angiogenic-osteogenic effect of bone marrow stromal cells(BMSCs)/vascular endothelial cells(VECs)coculture system and to clarify its paracrine mechanism.METHODS: Primary BMSCs and VECs were isolated from Sprague-Dawley rats and cocultured at various ratio(1:2,1:4,4:1,2:1,1:1).Optimized cell ratio was selected based on best osteogenic effect.Cells were subjected to cyclic tensile strain at various magnitudes(0%,3%,6%,9%)using an FX5000 T mechanical loading system,and the optimized magnitude was selected on the basis of best ALP activity.The expression of paracrine factors was evaluated by q PCR and ELISA.Transwell coculture and conditioned medium(CM)coculture were employed to investigate the paracrine effect and its mechanism.The angiogenic activity was assessed by MTT assay?scratch wound assay?Transwell chemotactic migration and Matrigel angiogenesis,while the osteogenic activity was assessed by ALP activity?matrix mineralization and osteogenic gene expression.Tivozanib,an inhibitor of vascular endothelial growth factor(VEGF)receptor,and recombinant VEGF were used to verify the paracrine effect of VEGF induced by tensile strain.Statistical analyses were performed by SPSS 19.0 software.RESULTS:(1)BMSCs/VECs cocultured at 1:1 ratio exhibited best osteogenic effect,with increased ALP activity and matrix mineralization;(2)BMSCs/VECs exhibited highest ALP activity when loaded by 6% tensile strain,with Col-1 m RNA increased by 1.6-fold,OCN by 2.0-fold and OPN by 3.2-fold,respectively(P<0.05);(3)Tensile strain induced VEGF expression by BMSCs,with 1.3-fold increased m RNA and 2.5-fold increased VEGF proteins compared to non-loading control after 48 h loading(P<0.05);(4)CM from loaded BMSCs activated VECs' functions,with enhanced proliferation?migration and tubular formation,while inducing BMP-2 and IGF-1 secretion by VECs(P<0.05);(5)Activated VECs in turn enhanced BMSCs osteogenesis via paracrine factors,with more matrix mineralization?1.4-fold increased ALP activity?3.5-fold increased OCN m RNA and 4.4-fold increased OPN m RNA(P<0.05);(6)Tivozanib abrogated the paracrine effect of VEGF induced by tensile strain,with decreased VECs proliferative activity,less cell migration and tubular formation.Tivozanib also suppressed the secretion of paracrine factors by VECs,and then impaired BMSCs osteogenesis.However,addition of recombinant VEGF significantly promoted the angiogenic-osteogenic effect of BMSCs/VECs.CONCLUSIONS: 6% Cyclic tensile strain promotes the angiogenicosteogenic activity of BMSCs/VECs coculture system,and this effect is based on BMSC-source VEGF induced by tension.VEGF enhances VECs' activity,with increased BMP-2 and IGF-1 release,and then induces BMSCs osteogenesis via paracrine pathway.