Abstract
Background: We have previously developed a prevascularized platform by seeding endothelial cells on the mesenchymal stem cell sheet and the technology provides a promising vascularization potential for wide applications in tissue engineering. However, the mechanism behind the interactions between stem cell sheet-derived matrix and endothelial cells (ECs) are still unknown. The purpose of this study is to investigate the role of MMPs in angiogenesis process in vitro.Methods: Human bone marrow-derived mesenchymal stem cells (hBMSCs) were cultured continually to form a thin cell sheet, and then green fluorescent protein (GFP)-labeled human umbilical vein endothelial cells (HUVECs) were seeded on the cell sheet to form a prevascularized system. Fibronectin, collagen I and MMPs immunofluorescence staining were used to observe their interaction changes. After that, HUVECs and hBMSCs were sorted by flow cytometry and RT-PCR was performed to quantify related gene expression of ECs and stem cells. In order to further explore the possible mechanism, the broad-spectrum MMPs inhibitor GM6001 was added into the medium to observe the effects of vascular network formation. Results: The immunofluorescence staining results showed that HUVECs secreted MMP-2 and membrane-type matrix metalloproteinase-1 (MT1-MMP) in the early stage of angiogenesis. MMPs degraded and remodeled the ECM to create space for vascular formation. HUVECs also promoted hBMSCs to secrete tissue inhibitors of metalloproteinase-2 (TIMP-2), which can stabilize blood vessels by avoiding excessive vascularization or vascular degradation in the later stage. Thus, HUVECs/hBMSCs co-culture group were beneficial for angiogenesis than the other two control groups. With the addition of the broad-spectrum matrix metalloproteinase inhibitor GM6001, RT-PCR results showed that the formation of EC lumen was blocked, while the mean fluorescence density of fibronectin and collagen I were increased.Conclusions: our study suggests that the MMP-2, MT1-MMP and TIMP-2 can synergistically regulate the angiogenesis. In addition, HUVECs and hBMSCs interact with each other in the process of vascular formation, and jointly regulate and promote the construction of lumen. These results also indicate a promising potential using the hBMSCs sheet as a platform for broad applications in engineering vascularized tissues.
Enhancing chondrogenic potential via mesenchymal stem cell sheet multilayering
