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Coculture assays allow the investigation of the role of endothelial cell and mural cell interactions in small vessel development and function. Different setups for coculture can be used to assay questions of interest. We include here methods for direct coculture, indirect coculture, and coculture in a three-dimensional extracellular matrix scaffold for studies of either a simple and direct association between the two cell types, the exchange of soluble molecules, or the interaction within a biomimetic tissue microenvironment.

Small intestinal submucosa grafts for vascular regeneration have produced variable patency (0-100%) that has been concurrent with variability in fabrication techniques. We hypothesized that 1) preservation (P) or removal (R) of the stratum compactum layer of the intestine and 2) a dehydrated (D) or hydrated (H) state of the graft, affect early patency and tissue regeneration. We combined both parameters through a 2(2) factorial experimental design into four groups (PD, RD, PH, RH), and compared them in an in vivo early response predictive model (swine, ID 4.5 mm, 7d, n = 4). Patency, thrombogenicity, vascularization, fibroblast infiltration, macrophage polarization profile, endothelialization, and biaxial mechanics were assessed. PD grafts remained patent (4/4) but had scarce vascularization and fibroblast infiltration. RD and RH had extensive vascularization and fibroblast infiltration, however, RD had sustained patency (4/4) and the highest number of regeneration-associated phenotype macrophages (M2), whereas RH had lower patency (3/4) and less M2 macrophages. PH had a modest cellular infiltration, but the lowest patency (2/4) and a dominant adverse macrophage phenotype. Elasticity of R grafts evolved toward that of native carotids (particularly RD), while P grafts kept their initial stiffness. We concluded that fabrication parameters drastically affected early patency and regeneration, with RD providing the best results.

In small intestinal submucosa (SIS) scaffolds for functional tissue engineering, the impact of scaffold fabrication parameters on cellular response and tissue regeneration may relate to the mechanotransductory properties of the final arrangement of collagen fibres. We previously proved that two fabrication parameters, (a) preservation (P) or removal (R) of a dense collagen layer present in SIS, and (b) SIS in a final dehydrated (D) or hydrated (H) state, have an effect on the micromechanical environment of SIS. In a continuation of our studies, we herein hypothesized that these fabrication parameters also modulate early mechanotransduction in cells populating the scaffold. Mechanotransduction was investigated by seeding human umbilical vein endothelial cells (HUVECs) on scaffolds, exposing them to pulsatile shear stress (12 ± 4 dyne/cm ) for 1 h (n = 5) in a cone-and-plate shear system, and evaluating the expression of the mechanosensitive genes Pecam1 and Enos by immunofluorescence and qPCR. Expression of mechanosensitive genes was highest in PD grafts, followed by PH and RH grafts. The RD group had similar expression to that of unsheared control cells, suggesting that the RD combination potentially reduced mechanotransduction of shear to cells. We concluded that the two fabrication parameters studied, which modify SIS micromechanics, also potentially modulated the early shear-induced expression of mechanosensitive genes in seeded HUVECs. Our findings suggest that fabrication parameters influence the outcome of SIS as a therapeutic scaffold. Copyright © 2015 John Wiley & Sons, Ltd.

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