Study of Cell-derived Extracellular Matrix for Angiogenesis and Novel Growth Factor Delivery System

다국어 초록 (Multilingual Abstract)

Extracellular matrix (ECM) is a highly organized network of proteins and other macromolecules that plays a critical role in cell adhesion, migration, and differentiation. In this study, we hypothesize that ECM derived from in-vitro-cultured cells possesses unique surface texture, topography, and mechanical property, and consequently carries some distinct cues for vascular morphogenesis of human umbilical vein endothelial cells (ECs). Cell-derived matrix (CDM) was obtained by culturing fibroblasts, preosteoblasts, and chondrocytes, respectively, on coverslips and then by decellularizing them using detergents and enzymes. These matrices were named fibroblast-derived matrix (FDM), preosteoblast-derived matrix (PDM), and chondrocyte-derived matrix (CHDM). Immunofluorescence of each CDM shows that some of the matrix components are fibronectin (FN), type I collagen, and laminin. Atomic force microscopy analysis presented that average fiber diameter ranged from 2 to 7 µm and FDM holds much larger fibers. The matrix elasticity measurements revealed that average Young’s modulus of CHDM (17.7 – 4.2 kPa) was much greater than that of PDM (10.5 – 1.1 kPa) or FDM (5.7 – 0.5 kPa). During 5-day culture, EC morphologies were dramatically changed on PDM and FDM, but those on CHDM and gelatin were rather stable, regardless of time lapse. Cell migration assay discovered quicker repopulation of the scratched areas on PDM and FDM than on gelatin and CHDM. A capillary-like structure (CLS) assembly was also notable only in the PDM and FDM, as compared with CHDM, gelatin, or FN that were very poor in CLS formation. Quantitative analysis of mean CLS branch points and branch lengths demonstrated much better angiogenic activity of ECs on PDM and FDM. Interestingly, CLS formation was closely associated with matrix remodeling by ECs and the matrix clearance on PDM with time was sharply contrasted with that on CHDM that majority of the matrix FN was reserved. It was notable that membrane type 1-matrix metalloprotease was deeply involved in the process of matrix remodeling. This study indicates that specific matrix microenvironments are very critical for vascular morphogenesis of ECs, and thus, provide a nice platform for angiogenesis study as well as vascular tissue engineering.

다국어 초록 (Multilingual Abstract)

Vascular endothelial growth factor (VEGF) is one of the most important signaling cues during angiogenesis. Since many delivery systems of VEGF have been reported, the presentation of VEGF using a more physiologically relevant extracellular matrix (ECM), however, has yet to be thoroughly examined. In this study, we propose that fibroblast-derived extracellular matrix (FDM) is a novel platform for angiogenic growth factor delivery and that FDM-mediated VEGF delivery can result in an advanced angiogenic response. The FDMs, activated by EDC/NHS chemistry, were loaded with varying amounts of heparin. Different doses of VEGF were subsequently immobilized onto the heparin-grafted FDM (hep-FDM); 19.6±0.6, 39.2±3.2, and 54.8 ±8.9 ng of VEGF were tethered using 100, 300, and 500 ng of initial VEGF, respectively. VEGF-tethered FDM was found chemoattractive and VEGF dose-dependent in triggering human umbilical vein endothelial cells (ECs) migration in vitro. When hep-FDM-bound VEGF (H-F/V) was encapsulated into alginate capsules (A/H-F/V) and subjected to release test for 28 days, it exhibited a significantly reduced burst release at early time point compared to that of A/V. The cell proliferation results indicated a substantially extended temporal effect of A/H-F/V on EC proliferation compared to those treated with soluble VEGF. For a further study, A/H-F/V was transplanted subcutaneously into ICR mice for up to 4 weeks to assess its in vivo effect on angiogenesis; VEGF delivered by hep-FDM was more competitive in promoting blood vessel ingrowth and maturation compared to other groups. Taken together, this study successfully engineered an FDM-mediated VEGF delivery system, documented its capacity to convey VEGF in a sustained manner, and demonstrated the positive effects of angiogenic activity in vivo as well as in vitro.