Microenvironmental cues for vascular tissue engineering

Richard John Galas, Purdue University

Abstract

Therapeutic strategies aim to regulate vasculature either by encouraging vessel growth for tissue engineering or inhibiting vascularization around a tumor. We examined three microenvironmental cues for vascular tissue: soluble vascular endothelial growth factor (VEGF), VEGF covalently bound to a surface, and encapsulation in a degradable hydrogel. Mesenchymal stem cells (MSCs) were cultured at confluence for up to three weeks in either basal medium or medium containing VEGF. In both cases, expression of typical endothelial genes and proteins were elevated compared to proliferating MSCs. Both treatments resulted in cells capable of internalizing acetylated low density lipoproteins. These results indicate that soluble VEGF does not enhance endothelial characteristics in differentiating MSCs. Interestingly, VEGF did enhance the expression of the arterial protein Ephrin-B1. Thus, exogenous VEGF may prove to be a valuable tool for specification of arterial fate. We developed a method to control the surface density of VEGF that is covalently attached to tissue culture polystyrene (TCPS) and investigated the effect of VEGF surface density on cellular response. Endothelial cells cultured on surfaces of covalently-bound VEGF did not proliferate in response to cues from the surface. Interestingly, in the presence of soluble VEGF, low surface densities (0.04 ng VEGF/cm2) enhanced endothelial proliferation whereas high surface densities (5.9 ng VEGF/cm2) inhibited proliferation. Surfaces modified with high surface densities (5.9 ng VEGF/cm2) also acted in synergy with an inhibitor of VEGF receptors to further suppress endothelial cell proliferation. VEGF surfaces alone exhibited no effect on the endothelial differentiation of mesenchymal stem cells (MSCs). However, the VEGF surfaces acted in synergy with an inhibitor of VEGF receptors to decrease the ability of differentiated cells to form vascular networks. Together, these results suggest that surface density of bound VEGF affects promotion or inhibition of angiogenic responses. We developed degradable hydrogels for use in tissue engineering and drug delivery applications sensitive to reducing environments. Hydrogels of recombinant resilin-based proteins were formed using the amine-reactive crosslinker, 3,3´-Dithiobis(sulfosuccinimidylpropionate). Hydrogel formation occurred rapidly (within 131 ± 95 s) at 37°C, which facilitated homogenous cell encapsulation. The crosslinking was cytocompatible, and endothelial cells were cultured within the hydrogel for up to six days. Hydrogels were softer than previously reported for resilin-based hydrogels and had a storage modulus ranging from 71 ± 29 to 262 ± 66 Pa and a compressive modulus of 21.1 ± 6.2 kPa, making them suitable scaffolds for brain, adipose, and cochlear tissue replacements. Additionally, the hydrogels were degraded via reduction with physiologically-relevant concentrations of L-glutathione. These results thus demonstrate the potential for these hydrogels to be used in drug delivery systems targeting the reductive environments around tumors.

Degree

Ph.D.

Advisors

Liu, Purdue University.

Subject Area

Cellular biology|Biomedical engineering|Chemical engineering

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