A fundamental limitation to tissue engineering vital organs is the need to create a vascularised support structure for these large 3-D organs. Hence the objectives of this proposal are (1) to devise a modular approach to the fabrication of tissue engineering constructs that are scaleable, with a uniform cell distribution, can accommodate multiple cell types and in which the porosity is created after cell incorporation and (2) To use this modular approach to create a vascularised construct by seeding the construct, containing smooth muscle cells (SMC), with endothelial cells (EC). The prototype to be developed and studied here is based on the porous structure that is created when a column or tube is packed, randomly, with solid objects (here, short cylindrical rods). Cells (eg., SMC) are encapsulated in solid gelatin rods (200 microm diameter, aspect ratio 5 to 10) on to which endothelial cells (eg., HUVEC) can adhere. The gelatin rods are randomly packed into a larger tube and then coated with HUVEC. The interstitial gaps among the rods form interconnected channels, which become lined by the endothelial cells. The resulting endothelial cell lining is expected to enable whole blood to percolate around the rods and through the interstitial channels. We expect good nutrient and oxygen supply (and waste removal) from the encapsulated cells, all of which should be <100 microm away from a 'pseudo-capillary' in this system. In this exploratory research (R21) proposal our focus is on demonstrating the feasibility of the concept and in defining its potential and its limits.
Our specific aims are: (1) Prepare a modular construct using gelatin cylinders encapsulating smooth muscle cells (SMC) or SMC spheroids (2) Seed the construct with endothelial cells before or after assembly of the modules into the construct, in the presence and absence of the SMC. (3) Assess the thrombogenicity of the endothelialised construct in vitro and ex vivo. (4) Assess the phenotype of the two cell types in the context of the construct, when used separately or together. The emphasis is on proof-of-principle studies to support our novel approach to (a) creating porous constructs and (b) generating vascularised structures. However, we note that the endothelialised construct may be subject to unique biomechanical and biochemical cues as a consequence of the un-natural architecture - e.g., there are no arterioles and venules in the construct. This may lead to a unique co-culture model for exploring the cellular interactions of vascular biology.
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