Interactions of angiogenic microvessels with the extracellular matrix

摘要

Angiogenesis, the formation of new blood vessels from existing ones, is critical to development, repair, tumorigenesis, and artificial construct design. It is regulated by soluble factors, cell-matrix interactions, and mechanical loading. Sprouting endothelial cells degrade their basement membrane and surrounding extracellular matrix (ECM) by matrix metalloprotease (MMP) activity, migrate along ECM components, and deposit a new basement membrane to form a patent capillary network. It is recognized that survival and integration of tissue engineered grafts depend on their vascularity. A better understanding of the angiogenic process and its interactions with the ECM is critical from a basic science perspective and in the fabrication of vascularized grafts. The angiogenic process by virtue of the associated invasion and growth of new vasculature can influence the local mechanical properties. The objective of this dissertation research was to study the effects of angiogenesis on mechanical properties of 3D in vitro vascularized constructs and relate the alterations to changes in gene expression and proteolytic activity at similar culture times, and to characterize the role of different mechanical boundary conditions on the morphology of microvascular networks. The dynamic mechanical properties of cell-free collagen constructs were evaluated at different collagen concentrations, strain magnitudes, and strain rates. The mechanical properties were shown to only alter minimally over 7 days in culture. Thus, any change in the mechanical properties of vascularized constructs was attributed to angiogenesis.;An increase in MMP gene expression and proteolysis paralleled the decrease in normalized dynamic stiffness of vascularized collagen constructs on the 6th day of culture. This was followed by a rise in dynamic stiffness, accompanied by a reduction in proteolysis in spite of elevated expression of MMP mRNA. The alignment of angiogenic sprouts from microvessels in a 3D in vitro model under different boundary conditions was also examined. Significant microvessel orientation was observed along the direction of anchorage and stretch, recapitulating in vivo behavior of vasculature in soft tissues such as muscles, ligaments and tendons. Free floating constructs lacked any preferred orientation. The results of this work provide fundamental insights into the process of angiogenesis, the interaction of angiogenic microvessels with the ECM, and the influence of mechanical conditioning on angiogenic microvessels, and will be beneficial in engineering of functional constructs with defined orientation of vasculature.