Proangiogenic Activity of Endometrial Epithelial and Stromal Cells in Response to Estradiol in Gelatin Hydrogels

Biomaterial vascularization remains a major focus in the field of tissue engineering. Biomaterial culture of endometrial cells is described as a platform to inform the design of proangiogenic biomaterials. The endometrium undergoes rapid growth and shedding of dense vascular networks during each menstrual cycle mediated via estradiol and progesterone in vivo. Cocultures of endometrial epithelial and stromal cells encapsulated within a methacrylamide-functionalized gelatin hydrogel are employed. It is reported that proangiogenic gene expression profiles and vascular endothelial growth factor production are hormone dependent in endometrial epithelial cells, but that hormone signals have no effect on human telomerase reverse transcriptase (hTERT)-immortalized endometrial stromal cells. This study subsequently examines whether the magnitude of epithelial cell response is sufficient to induce changes in human umbilical vein endothelial cell network formation. Incorporation of endometrial stromal cells improves vessel formation, but co-culture with endometrial epithelial cells leads to a decrease in vascular formation, suggesting the need for stratified cocultures of endometrial epithelial and stromal cells with endothelial cells. Given the transience of hormonal signals within 3D biomaterials, the inclusion of sex hormone binding globulin (SHBG) to alter the bioavailability of estradiol within the hydrogel is reported, demonstrating a strategy to reduce diffusive losses via SHBG-mediated estradiol sequestration

The induction of pro-angiogenic processes within a collagen scaffold via exogenous estradiol and endometrial epithelial cells

Nutrient transport remains a major limitation in the design of biomaterials. One approach to overcome this constraint is to incorporate features to induce angiogenesis-mediated microvasculature formation. Angiogenesis requires a temporal presentation of both pro- and anti-angiogenic factors to achieve stable vasculature, leading to increasingly complex biomaterial design scheme. The endometrium, the lining of the uterus and site of embryo implantation, exemplifies a non-pathological model of rapid growth, shedding, and re-growth of dense vascular networks regulated by the dynamic actions of estradiol and progesterone. In this study, we examined the individual and combined response of endometrial epithelial cells and human umbilical vein endothelial cells to exogenous estradiol within a three-dimensional collagen scaffold. While endothelial cells did not respond to exogenous estradiol, estradiol directly stimulated endometrial epithelial cell transduction pathways and resulted in dose-dependent increases in endogenous VEGF production. Co-culture experiments using conditioned media demonstrated estradiol stimulation of endometrial epithelial cells can induce functional changes in endothelial cells within the collagen biomaterial. We also report the effect of direct endometrial epithelial and endothelial co-culture as well as covalent immobilization of estradiol within the collagen biomaterial. These efforts establish the suitability of an endometrial-inspired model for promoting pro-angiogenic events within regenerative medicine applications. These results also suggest the potential for developing biomaterial-based models of the endometrium

Strategies to balance covalent and non-covalent biomolecule attachment within collagen-GAG biomaterials

Strategies to integrate instructive biomolecular signals into a biomaterial are becoming increasingly complex and bioinspired. While a large majority of reports still use repeated treatments with soluble factors, this approach can be prohibitively costly and difficult to translate in vivo for applications where spatial control over signal presentation is necessary. Recent efforts have explored the use of covalent immobilization of biomolecules to the biomaterial, via both bulk (ubiquitous) as well as spatially-selective light-based crosslinking, as a means to both enhance stability and bioactivity. However, little is known about how processing conditions during immobilization impact the degree of unintended non-covalent interactions, or fouling, that takes place between the biomaterial and the biomolecule of interest. Here we demonstrate the impact of processing conditions for bulk carbodiimide (EDC) and photolithography-based benzophenone (BP) crosslinking on specific attachment vs. fouling of a model protein (concanavalin A, ConA) within collagen- glycosaminoglycan (CG) scaffolds. Collagen source significantly impacts the selectivity of biomolecule immobilization. EDC crosslinking intensity and ligand concentration significantly impacted selective immobilization. For benzophenone photoimmobilization we observed that increased UV exposure time leads to increased ConA immobilization. Immobilization efficiency for both EDC and BP strategies was maximal at physiological pH. Increasing ligand concentration during immobilization process led to enhanced immobilization for EDC chemistry, no impact on BP immobilization, but significant increases in non-specific fouling. Given recent efforts to covalently immobilize biomolecules to a biomaterial surface to enhance bioactivity, improved understanding of the impact of crosslinking conditions on selective attachment versus non-specific fouling will inform the design of instructive biomaterials for applications across tissue engineering. This journal is © the Partner Organisations 2014

Photopatterning of vascular endothelial growth factor within collagen-glycosaminoglycan scaffolds can induce a spatially confined response in human umbilical vein endothelial cells

Biomolecular signals within the native extracellular matrix are complex, with bioactive factors found in both soluble and sequestered states. In the design of biomaterials for tissue engineering applications it is increasingly clear that new approaches are required to locally tailor the biomolecular environment surrounding cells within the matrix. One area of particular focus is strategies to improve the speed or quality of vascular ingrowth and remodeling. While the addition of soluble vascular endothelial growth factor (VEGF) has been shown to improve vascular response, strategies to immobilize such signals within a biomaterial offer the opportunity to optimize efficiency and to explore spatially defined patterning of such signals. Here we describe the use of benzophenone (BP) photolithography to decorate three-dimensional collagen-glycosaminoglycan (CG) scaffolds with VEGF in a spatially defined manner. In this effort we demonstrate functional patterning of a known agonist of vascular remodeling and directly observe phenotypic effects induced by this immobilized cue. VEGF was successfully patterned in both stripes and square motifs across the scaffold with high specificity (on:off pattern signal). The depth of patterning was determined to extend up to 500 μm into the scaffold microstructure. Notably, photopatterned VEGF retained native functionality as it was shown to induce morphological changes in human umbilical vein cells indicative of early vasculogenesis. Immobilized VEGF led to greater cell infiltration into the scaffold and the formation of immature vascular network structures. Ultimately, these results suggest that BP-mediated photolithography is a facile method to spatially control the presentation of instructive biological cues to cells within CG scaffolds