The formation of a stably perfused microvasculature continues to be a major challenge in tissue engineering. Previous work has suggested the importance of a sufficiently large transmural pressure in maintaining vascular stability and perfusion. Here we show that a system of empty channels that provides a drainage function analogous to that of lymphatic microvasculature in vivo can stabilize vascular adhesion and maintain perfusion rate in dense, hydraulically resistive fibrin scaffolds in vitro. In the absence of drainage, endothelial delamination increased as scaffold density increased from 6 mg/mL to 30 mg/mL and scaffold hydraulic conductivity decreased by a factor of twenty. Single drainage channels exerted only localized vascular stabilization, the extent of which depended on the distance between vessel and drainage as well as scaffold density. Computational modeling of these experiments yielded an estimate of 0.40–1.36 cm H2O for the minimum transmural pressure required for vascular stability. We further designed and constructed fibrin patches (0.8 by 0.9 cm2) that were perfused by a parallel array of vessels and drained by an orthogonal array of drainage channels; only with the drainage did the vessels display long-term stability and perfusion. This work underscores the importance of drainage in vascularization, especially when a dense, hydraulically resistive scaffold is used.
The low stiffness of reconstituted collagen hydrogels has limited their use as scaffolds for engineering implantable tissues. Although chemical crosslinking has been used to stiffen collagen and protect it against enzymatic degradation in vivo, it remains unclear how crosslinking alters the vascularization of collagen hydrogels. In this study, we examine how the crosslinking agents genipin and 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide (EDC) alter vascular stability and function in microfluidic type I collagen gels in vitro. Under moderate perfusion (~10 dyn/cm2 shear stress), tubes of blood endothelial cells exhibited indistinguishable stability and barrier function in untreated and crosslinked scaffolds. Surprisingly, under low perfusion (~5 dyn/cm2 shear stress) or nearly zero transmural pressure, microvessels in crosslinked scaffolds remained stable, while those in untreated gels rapidly delaminated and became poorly perfused. Similarly, tubes of lymphatic endothelial cells under intermittent flow were more stable in crosslinked gels than in untreated ones. These effects correlated well with the degree of mechanical stiffening, as predicted by analysis of fracture energies at the cell-scaffold interface. This work demonstrates that crosslinking of collagen scaffolds does not hinder normal endothelial cell physiology; instead, crosslinked scaffolds promote vascular stability. Thus, routine crosslinking of scaffolds may assist in vascularization of engineered tissues.
The low stiffness of reconstituted collagen hydrogels has limited their use as scaffolds for engineering implantable tissues. Although chemical crosslinking has been used to stiffen collagen and protect it against enzymatic degradation in vivo, it remains unclear how crosslinking alters the vascularization of collagen hydrogels. In this study, we examine how the crosslinking agents genipin and 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide (EDC) alter vascular stability and function in microfluidic type I collagen gels in vitro. Under moderate perfusion (~10 dyn/cm 2 shear stress), tubes of blood endothelial cells exhibited indistinguishable stability and barrier function in untreated and crosslinked scaffolds. Surprisingly, under low perfusion (~5 dyn/cm 2 shear stress) or nearly zero transmural pressure, microvessels in crosslinked scaffolds remained stable, while those in untreated gels rapidly delaminated and became poorly perfused. Similarly, tubes of lymphatic endothelial cells under intermittent flow were more stable in crosslinked gels than in untreated ones. These effects correlated well with the degree of mechanical stiffening, as predicted by analysis of fracture energies at the cell-scaffold interface. This work demonstrates that crosslinking of collagen scaffolds does not hinder normal endothelial cell physiology; instead, crosslinked scaffolds promote vascular stability. Thus, routine crosslinking of scaffolds may assist in vascularization of engineered tissues.
The zona pellucida (ZP) is a thick glycoprotein shell surrounding the mammalian egg cell (oocyte) that regulates spermatozoa access during fertilization and protects the zygote during early embryonic development [1]. Hardening of the zona pellucida over the cell fertilization cycle is a well-recognized phenomenon and has been investigated using contact methods to measure shear and bending elasticity from indentation and micropipette aspiration [2, 3]. However, the area elasticity of the ZP, which provides resistance to cell swelling under variable osmotic environments, has not yet been reported. A recently devised theoretical model [4] suggests that the ZP area expansion modulus may be determined through non-contact hypo-osmotic loading of the oocyte. If successful, this method may be suited for implementation by practicing fertility health professionals during routine manipulation.
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