Regulation of Matrix Metalloproteinase in Vascularization of Endothelial Cells On Extracellular Matrix Secreted By Mesenchymal Stem Cell Sheet

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.

Human Mesenchymal Stem Cell Sheets Improve Uterine Incision Repair in a Rodent Hysterotomy Model

Objective The study aimed to assess the feasibility of creating and transplanting human umbilical cord mesenchymal stem cell sheets applied to a rat model of hysterotomy, and additionally to determine benefits of human umbilical cord mesenchymal stem cell sheet transplantation in reducing uterine fibrosis and scarring. Study Design Human umbilical cord mesenchymal stem cell sheets are generated by culturing human umbilical cord mesenchymal stem cells on thermo-responsive cell culture plates. The temperature-sensitive property of these culture dishes facilitates normal cell culture in a thin contiguous layer and allows for reliable recovery of intact stem cell sheets without use of destructive proteolytic enzymes.We developed a rat hysterotomy model using nude rats. The rat uterus has two distinct horns: one horn provided a control/untreated scarring site, while the second horn was the cell sheet transplantation site.On day 14 following surgery, complete uteri were harvested and subjected to histologic evaluations of all hysterotomy sites. Results The stem cell sheet culture process yielded human umbilical cord mesenchymal stem cell sheets with surface area of approximately 1 cm2.Mean myometrial thickness in the cell sheet-transplanted group was 274 µm compared with 191 µm in the control group (p = 0.02). Mean fibrotic surface area in the human umbilical cord mesenchymal stem cell sheet-transplanted group was 95,861 µm2 compared with 129,185 µm2 in the control group. Compared with control horn sites, cell sheet-transplanted horns exhibited significantly smaller fibrotic-to-normal myometrium ratios (0.18 vs. 0.27, respectively, p = 0.029). Mean number of fibroblasts in cell sheet-transplanted horns was significantly smaller than the control horns (483 vs. 716/mm2, respectively, p = 0.001). Conclusion Human umbilical cord mesenchymal stem cell sheet transplantation is feasible in a rat model of hysterotomy. Furthermore, use of stem cell sheets reduces fibroblast infiltration and uterine scar fibrotic tissue formation during hysterotomy healing, potentially mitigating risks of uterine scar formation. Key Points

Integration of mesenchymal stem cell sheet and bFGF-loaded fibrin gel in knitted PLGA scaffolds favorable for tendon repair

The integration of mesenchymal stem cell sheet and bFGF-loaded fibrin gel in knitted PLGA scaffolds synergistically promoted rat At regeneration