Coculture Assays for Endothelial Cells-Mural Cells Interactions.

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.

Effects of Fabrication on Early Patency and Regeneration of Small Intestinal Submucosa Vascular Grafts.

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.

Effects of fabrication on the mechanics, microstructure and micromechanical environment of small intestinal submucosa scaffolds for vascular tissue engineering.

In small intestinal submucosa scaffolds for functional tissue engineering, the impact of scaffold fabrication parameters on success rate may be related to the mechanotransductory properties of the final microstructural organization of collagen fibers. We hypothesized that two fabrication parameters, 1) preservation (P) or removal (R) of a dense collagen layer present in SIS and 2) SIS in a final dehydrated (D) or hydrated (H) state, have an effect on scaffold void area, microstructural anisotropy (fiber alignment) and mechanical anisotropy (global mechanical compliance). We further integrated our experimental measurements in a constitutive model to explore final effects on the micromechanical environment inside the scaffold volume. Our results indicated that PH scaffolds might exhibit recurrent and large force fluctuations between layers (up to 195 pN), while fluctuations in RH scaffolds might be larger (up to 256 pN) but not as recurrent. In contrast, both PD and RD groups were estimated to produce scarcer and smaller fluctuations (not larger than 50 pN). We concluded that the hydration parameter strongly affects the micromechanics of SIS and that an adequate choice of fabrication parameters, assisted by the herein developed method, might leverage the use of SIS for functional tissue engineering applications, where forces at the cellular level are of concern in the guidance of new tissue formation.