Determining a hydrogel scaffold milieu for stem cell differentiation into endothelial lineage

Abstract

Event Abstract Back to Event Determining a hydrogel scaffold milieu for stem cell differentiation into endothelial lineage Kendra Clark1 and Amol Janorkar1 1 University of Mississippi Medical Center, Biomedical Materials Science, School of Dentistry, United States Introduction: It is known that cells, scaffolds, signaling molecules, and blood supply are key factors in regenerative medicine and the future of periodontal tissue engineering[1]. Unfortunately, clinical applications of tissue engineering are limited by the lack of adequate blood supply[2]. Endothelial cells are a vital component of the capillaries that provide adequate blood supply and excretion of wastes in tissues. Hence, induction of neo-angiogenesis utilizing endothelial cells in hydrogel scaffolds has been a promising tissue engineering strategy. Studies have shown human adipose-derived stem cells (hASCs)’s ability to differentiate into various cell types depending on their microenvironment consisting of growth factor cocktail, scaffold formulation, and other culture conditions[3]. The aim of this study was to determine a hydrogel scaffold milieu to aid the growth-factor stimulated differentiation of hASCs into endothelial lineage. Methods: Elastin-like polypeptide (ELP) expression and purification was performed as describe elsewhere[4],[5]. hASCs isolated from elective liposuction aspirates under an IRB-approved protocol were seeded in hydrogels (40,000 cells/hydrogel) prepared by varying concentrations of either collagen (2 and 6mg/mL) or collagen-ELP (1:3 mass ratio). Additionally, some hydrogels were cross-linked utilizing ethyl(dimethylaminopropyl) carbodiimide (EDC) and N-Hydroxysuccinimide (NHS). Cells were cultured in DMEM/F12 with 10% FBS to acclimate for 24h (called ‘day 0’) followed by addition of endothelial differentiation medium (EGM-2-MV) for 7, 14 or 21 days. Successively, assays for endothelial differentiation (acLDL), viability (Live/Dead), total protein content, and total DNA content were performed using manufacturers’ protocols. Statistical evaluation was performed with ANOVA and Games-Howell post hoc test. Results: The extracellular matrix proteins elastin and collagen play an essential role in rendering elasticity and mechanical strength to blood vessels[6]. With our previous studies showing successful differentiation of 3T3-L1 adipocytes[7] and 3T3-E1 osteoblasts[5] in collagen and ELP containing hydrogels, we hypothesized that hASCs would proliferate and differentiate toward endothelial lineage when encapsulated in collagen-ELP composite hydrogels. Though differences in cell viability were observed among the various hydrogel conditions, viability remained high (>70%) in most hydrogels except the crosslinked hydrogels (Fig. 1a). Figure 1b,c show total protein and DNA content did not differ across hydrogel conditions on Day 0 indicating uniform seeding across hydrogels. There was a significant increase in total protein and DNA content in 2mg/mL collagen and collagen-ELP (2:6 mg/mL) hydrogels. In contrast, either no differences or decreases were seen in 6mg/mL collagen and cross-linked hydrogels. All hydrogels contained cells of endothelial lineage that fluoresced green due to uptake of fluorescently labeled acetylated low-density lipoprotein (acLDL) (Fig. 2). Conclusion: Our data shows hASCs remained viable and differentiated into endothelial cell lineage in hydrogel scaffolds via the addition of specific growth factors. The endothelial cells showed the most proliferation in 2mg/mL collagen and collagen-ELP (2:6 mg/mL) hydrogel scaffolds compared to 6mg/mL collagen and cross-linked hydrogel scaffolds. These results suggest collagen and collagen-ELP hydrogels support endothelial cell differentiation, proliferation and survival, which are prerequisites for neo-angiogenesis. Further studies will elucidate hydrogel mechanical properties and co-culturing strategies aiming to an enhanced endothelial differentiation using hASCs. Supported by NIH/NIDCR R03DE024257