Engineered small diameter vascular grafts by cell sheet engineering with human umbilical cord vein perivascular cells

摘要

Pericytes are perivascular cells that surround endothelial cells of capillaries, microvessels and larger vessels; arteries and veins. They have important roles in vascular development (vasculogenesis and angiogenesis), stability, maturation and remodeling, regulation of vessel diameter and blood How, blood pressure control, contractility and tone of vascular smooth muscle cells. The cell sheet engineering is based on the capability of cells to secrete and organize their own extracellular matrix (ECM). In this study our aim is to isolate human umbilical cord vein pericytes (CD146+ cells) then differentiate into smooth muscle cells and fibroblasts that will be used for generating tissue engineering vascular grafts and also reduce the time required to produce a tissue-engineered vascular graft. Human perivascular cells were isolated from umbilical cord vein and pericytes were purified by using MACs cell separation with CD146 microbeads. Perivascular cells were cultured in FGM-2, SMCGM-2 and EGM-2 medium for 21 days to investigate fibroblast, smooth muscle cell and endothelial cell differentiation. Cell differentiation was confirmed by immunofluorescence staining and flow cytometry analysis (fibrobtast markers; Tenascin-C and Collagen type Ⅰ, smooth muscle cell markers; Caldesmon and alpha smooth muscle actin, endothelial cell markers; VEGFR1, VEGFR2 and CD31). Next, a tissue-engineered vascular graft was fabricated by rolling the perivascular cells derived endothelial cell, smooth muscle cell and fibroblast sheets (Rapid cell sheet detachment from Poly(N-isopropylacrylamide)) around a mandrel. Different human ECM molecules such as collagen type Ⅱ and Ⅳ, fibrin, elastin, fibronectin and glycosaminoglycans were used among the vascular layers to increase the mechanical properties of the graft. The results indicated that perivascular cells could differentiate into fibroblast, smooth muscle, endothelial cells and formed tubular vascular graft with ECM proteins. As a conclusion, differentiated pericytes offer an alternative cell source for constructing tissue engineered vascular graft. The authors wish to thank The Scientific and Technological Research Council of Turkey (Project Number: 113S815) for their financial support.