Effects of the Mechanical Properties of Collagen Gel on the In Vitro Formation of Microvessel Networks by Endothelial Cells

Abstract

Vascularization by endothelial cells (ECs) is an essential element in tissue-engineering of organoids. Morphogenesis of these cells is regulated not only by the biochemical properties of the extracellular matrix (ECM) but also by its mechanical properties. Here, we investigated the effect of substrate mechanical properties on the formation of capillary-like networks by ECs; in particular, we examined the three-dimensional (3D) configurations of the resulting networks. Bovine pulmonary microvascular ECs (BPMECs) were cultured on a series of collagen gels of different stiffness but the same collagen concentration. Imaging techniques revealed that cells cultured in rigid and flexible gels formed 3D networks via different processes; cells formed dense, thin networks in the flexible gel, whereas thicker and deeper networks were formed in the rigid gel. Cross-sections of the networks revealed that those formed within the rigid gel had large lumens composed of multiple cells, whereas those formed within the flexible gel had small, intracellular vacuoles. The expression of vinculin, a focal adhesion protein, appeared to change with the mechanical properties of collagen gel. Our results indicate that the mechanical properties of adhesion substrates play an important role in regulating 3D network formation.

Adhesion, proliferation, and gene expression profile of human umbilical vein endothelial cells cultured on bilayered polyelectrolyte coatings composed of glycosaminoglycans

“…17,18,21,[48][49][50] This invariably results in a relatively thick multilayered coating that tends to be soft and gel-like due to the natural physiological role of glycosaminoglycans in absorbing and retaining water, 51 as in the case of synovial fluid 52 and vitreous humor. 53 Generally, most mammalian cells, including endothelial cells, prefer a stiff rigid surface for attachment, 54,55 thus a soft gel-like substrate is most often nonconducive for cellular adhesion and subsequent growth. Indeed, apoptosis ͑programed cell death͒ has been reported to manifest in cells cultured on soft nonrigid substrates.…”

Increased stiffness of the rat liver precedes matrix deposition: implications for fibrosis

“…It is now understood that matrix stiffness (and the mechanical tension that results from cellular adhesion to stiff substrates) is instrumental in determining the phenotype of many cell types in culture (1,3,9,13,16,19,20,35,38,42,45; for review, see Ref. 11).…”

Towards in vitro vascularisation of collagen-GAG scaffolds

“…The rising PECAM and VCAM and falling α smooth muscle actin (SMA) for MSCs in EC medium suggest differentiation towards the endothelial phenotype, however no increases were seen for TIE-2, vWF and VEGFR2 suggesting this process did not follow through to completion. GP Duffy et al Pre-vascularisation of collagen-GAG scaffold et al, 2007;Yamamura et al, 2007). It is likely that this factor is the main vasculogenic component in our EC medium, and it has been found to itself induce VEGF expression in ECs (Seghezzi et al, 1998); if the same is true in MSCs, or the MSCs are directly stimulated towards an EC phenotype first by the bFGF, this may be why additional VEGF did not promote further vascular structure formation by MSCs.…”