The Role Of MT1-MMP In Pre-vascularization Of The MSC Cell Sheet

This study aimed to explore the interaction between endothelial cells (ECs) and bone marrow-derived mesenchymal stem cells (BMSCs) cell sheet in the process of the formation of blood vessels when the BMSCs cell sheet and EC were co-cultured in vitro. Method:(1) The human bone marrow-derived mesenchymal stem cells (hBMSCs) were cultured continuously for 14 days in H-DMEM medium in six-well plates at a high-density to form MSCs cell sheet (MSCs group); (2) The green fluorescent GFP-labeled human umbilical vein endothelial cells(HUVECs) were seeded on hBMSCs cell sheet, the EC medium were used to culture the pre-vascularized tissue engineering cell sheet for 7 days (co-cultured normal group); At the same time, HUVECs were seeded on a tissue culture plate as a control(HUVECs group); (3) HUVECs at the density of 5x104 were seeded on MSCs cell sheet and were cultured in the EC medium added a broad-spectrum inhibitor GM6001 for 7 days (co-cultured inhibitor group);(4) A fluorescent microscope was used to observe the HUVECs cell migration on the hMSCs sheet at designated time points-1,3,7days, and immunofiuorescence staining for Col I, FN, MT1-MMP and TIMP-2 were finished at the 7th days of co-culturing. At the same time, the pre-vascularized cell sheets were digested and separated endothelial cells and stem cells by using flow cytometry, and then the genes of MT1-MMP, MMP-2 and the surface markers CD31 from endothelial cells, TIMP-2, Col I, FN from BMSCs were detected by RT-PCR. Results:(1) For MSCs sheets, stem cells gradually lost the typical morphology after continuously cultured, and grew into a layer of milky white film-like substance attached at the bottom of the six-well plates at 14 days. The film presented a slight edge contraction and could be peeled off. Immunofiuorescence staining for Col I and FN: Col I arranged without rules, FN showed reticulate arrangement. And the amount of secretion of Col I is higher than that of FN by analysis of Image J software; but MT1-MMP and TIMP-2 were unable to be detected in MSCs sheet;(2)When HUVECs were seeded onto the MSCs sheet, cell morphology changed and cell vacuoles appear after co-cultured for 3 d; lumen structure formed after co-cultured for 7d. It showed that Col I became orderly arrangement, and mean fluorescence intensity(MFI) of Col I and FN declined after seeding HUVECs, but the amount of Col I was still higher than that of FN; the expression of MT1-MMP, MMP-2, CD31, TIMP-2 were upregulated compare to HUVECs group and MSCs sheet group; (3) When GM6001 was added into the culture medium, it showed an irregular arrangement for HUVECs in the first days and there was no significant lumen formation after 7th days; Immunofluorescence for Col I showed that the Col I arranged in disorder, MFI of Col I and FN increase, and the expression of MT1-MMP, MMP-2, CD31 genes were downregulated compared to co-cultured normal group, but the expression of TIMP-2 had no significant effect. Conclusion:the co-cultured of hBMSCs cell sheet and HUVECs can pre-fabricate vascularized networks in vitro, and in this process, MT1-MMP play a key role to ECM remodeling and the formation of blood vessels.