Another approach attempts to provide an engineered tissue with a preformed structural template of the desired vasculature. One series of studies performed by Donald E. Ingber and coworkers has defined how the two-dimensional geometry of the ECM substrate influences EC growth and differentiation. Thus, EC were cultured on self-assembled monolayers generated by stamping microscopic patterns of alkanethiols on gold-coated substrata, resulting in micro-patterns of alkanethiols that were further modified by the addition of various ECM molecules such as fibronectin. This approach allows for the generation of ECM substrata in the forms of islands, stripes, or geometric patterns of various dimensions, on which EC can be cultured and examined for their adhesive, growth, and differentiation responses. A main finding was that substrate geometry and area dictates whether an EC would adhere at all, proliferate, or undergo apoptosis. Thus, on single ECM islands of 10 mm or less, cells could not adhere; those ranging in sizes between 10 to 25 mm supported attachment but also promoted apoptosis, and on ECM islands of 50 mm or more, cells proliferated. Of particular relevance to the engineering of angiogenic scaffolds, it was further shown that EC cultured on 10-|mm-wide fibronectin stripes formed vascular-like cords complete with contiguous lumens, but on 30-|mm-thick stripes, the cells proliferated and did not form lumens.
Some investigators have proposed the development of three-dimensional templates for the generation of the microvasculature. One such template is described as a micro-bioreactor or angiochip. The device has as its basic design a solid-state silicon capsule or wafer containing thousands of microscopic chambers measuring 1 mm or less, which can be loaded with nanoliter volumes of bioactive agents. After filling, chambers are sealed with a thin gold layer to retain their contents until a later time. After implantation in a host, the contents of individual chambers on such chips could be selectively released by electro-dissolving the gold layers limiting the chambers. For some applications, the device may be surface-tooled and treated with an appropriate ECM scaffold to render them amenable to EC seeding before implantation. Potential uses for non-EC-seeded variety of implants would be for the sustained delivery of angio-genic or angiostatic drugs, targeted to expand or reduce the endogenous microvasculature. Angiochips preseeded with EC may be useful in promoting the outgrowth of the exogenous EC to form a new microvascular network in the host tissue, which also might anastamose with the microvascular network of the host.
Another potential approach to creating microvascular conduits was discovered using a mouse transgenic model with a targeted overexpression of chemoattractant protein-1 in the myocardium. The cardiac muscle was found to contain thin channels presumably formed by the enzymatic boring of macrophages, via their release of metalloelastase, through the tissue interstitium. To apply this phenomenon toward the rational engineering of the microvasculature will require directing the formation of channels to generate a desired microvascular configuration. Some possible approaches may include imprinting a matrix scaffold with a chemotactic gradient and seeding one end of a scaffold with macrophages. The cells may respond by boring channels through the ECM in a directed fashion, in response to the gradient. A variant of this approach would be to drive the cell-boring activities actively, using an external stimulus such as a magnetic field to spatially direct the migration of the macrophages; perhaps iron-conjugated beads may be affixed to the cells to confer their response to the magnetic field. Subsequently, the macrophage-generated channels would have to be populated with the appropriate complement of vascular cells before its use as a tissue implant.
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