Dynamic behavior of type IV collagen at cell-biomaterial interface

Abstract

The initial molecular events that take place at biomaterials interface mimic to a certain extent the natural interaction of cells with the extracellular matrix (ECM). In this thesis we describe the fate of adsorbed type IV collagen (Col IV) - the main structural component of the basement membrane (BM) - as an important target in vascular tissue engineering. We studied the adsorption kinetic of Col IV on different model surfaces varying in wettability, chemistry and charge, and followed how it alters the molecular organization of the adsorbed protein layer. We strived to learn how it will affect the subsequent cellular interaction. AFM studies revealed specific substratum– dependent adsorption pattern of Col IV ranging from single molecular deposition to fine meshwork formation at high coating concentrations, which is characteristic for hydrophilic and NH2 functionalized substrates. Conversely, the formation of a complex networks consisting of molecular aggregates were found on hydrophobic and COOH modified surfaces. Complex structures were found also when a family of model substrates with tailored density of OH, CH3 and NH2 functions were used. Human umbilical endothelial cells (HUVEC) and fibroblasts were employed to study the biological response on these substrata. We found that fibroblast not only interact with adsorbed Col IV but also tend to reorganize it in fibril like pattern, which is strongly dependent on the materials surface properties. Following the trend of adsorption NH2>CH3>COOH>OH the reorganization pattern of Col IV improve with lowering the amount of protein. However, the cells interact better with hydrophilic and NH2 surfaces, thus acting independently on the amount of adsorbed Col IV. This trend was confirmed by the quantitative measurements of cell adhesion and spreading, as well as, the expression of p-FAK, α1 and α2 integrins – all reflecting the proper functioning of cell adhesion machinery. This is the first study that addresses the relationship between microscopic observation for remodeling of surface associated Col IV and it´s dynamic behavior in nano scale. We found that cells remodel Col IV in two ways: by mechanical reorganization and via proteolytic degradation. We identify the role of FN in the reorganization process and the involvement of MMP2 and MMP9 in the pericellular degradation activity of both HUVEC and fibroblasts. The later was further quantified via FITC labeled Col IV release and zymography. We found that in hydrophobic environment the degradation activity can override the Col IV organization process, which corroborates with the altered cell morphology, abrogated cell adhesion machinery and altered capability of HUVEC to form capillary-like structures. Taken together these results support our view that the ability of cells to remodel surface associated proteins affects strongly the biological performance of a biomaterial. They also show that the appropriate chemical functionalization (NH2, OH), combined with Col IV pre-adsorption, comprises a prospective biomimetic modification that might provide insights for the improved endothelization of cardiovascular implants.