Optimization of Vasculogenesis within Naturally-Derived, Biodegradable Hybrid Hydrogel Scaffolds

INTRODUCTION:  Cellular ingrowth and neovascularization of acellular tissue-regeneration scaffolds represent the rate-limiting steps of permanent integration.  In previous work, we have demonstrated that naturally-derived, biodegradable hybrid hydrogel scaffolds fabricated from a 10:1 w/w combination of alginate and type I collagen allow for maximal human umbilical vein endothelial cell (HUVEC) adherence and invasion compared with other substrate combinations in an in vitro wound healing model.  We next sought to stimulate endothelial tubule formation within these scaffolds as the next step towards the creation of pre-formed vascularized tissue.

METHODS:  Hybrid hydrogel scaffolds were fabricated and Arg-Gly-Asp (RGD)-modified as before.  Scaffolds were seeded with 3.0x10⁵ HUVECs and maintained under standard cell culture conditions.  After 3 days, cell culture media was supplemented with basic fibroblast growth factor (bFGF) at varying physiologic concentrations (1ng/mL, 5ng/mL, 10ng/mL, and 20ng/mL).  A second set of scaffolds was seeded with 3.0x10⁴, 6.0x10⁴, 1.5x10⁵, or 3.0x10⁵ human aortic smooth muscle cells (HASMCs).  After 3 days of HASMC culture, scaffolds were seeded with 3.0x10⁵ HUVECs.  Following 2 weeks of HUVEC culture, all scaffolds were labeled with DiI-Ac-LDL (an LDL tagged with a red fluorophore that is endocytosed by HUVECs) and DAPI, and the scaffolds were fixed, mounted and imaged using 3-dimensional confocal fluorescent microscopy.

RESULTS:  HUVECs were maximally confluent on hybrid scaffolds when cultured with media supplemented with bFGF 20ng/mL (372.5±37.4) compared with 0ng/mL (250.7±18.2, p=0.005), 1ng/mL (179.0±17.4, p<1.0x10^-2), 5ng/mL (198.5±17.2, p<1.0x10^-3), and 10ng/mL (273.3±22.9, p=0.030).  However, the addition of growth factor stimulated neither an increase in HUVEC invasion nor tubule formation within the scaffold substrate.  In contrast, a "dose-dependent" increase in tubule content and network-like organization was observed as the co-culture ratio of HASMCs to HUVECS from increased from 1:10 to 1:1 (Figures).

CONCLUSIONS: Even at high concentrations, the presence of the mitogenic factor bFGF alone is insufficient to stimulate HUVECs to form tubules within hydrogel scaffolds.  Instead, the complex chemical and possibly contextual cues offered by vascular smooth muscle cells are required to instruct endothelial cells to assemble into vasculogenic configurations.  Although further work is necessary to engineer vascular networks according to pre-determined (rather than random) patterns, these findings provide important insights into the instruction of endothelial tubule and network formation and bring us one step closer to the fabrication of pre-vascularized artificial tissue.