Optimizing Endothelial Cell Adhesion and Invasion with Naturally-Derived, Biodegradable Hybrid Hydrogel Scaffolds

BACKGROUND:  Cellular ingrowth and neovascularization of acellular scaffolds represent the rate-limiting steps of permanent scaffold integration.  While a number of tissue-regeneration matrices are available both commercially and experimentally, the optimal material composition of such products has yet to be defined.  Therefore, in an effort to determine the material specifications that allow for maximal cell adhesion and invasion, we evaluated human umbilical vein endothelial cell (HUVEC) adherence and invasion into naturally-derived, biodegradable hybrid hydrogel scaffolds in an in vitro wound healing model.

METHODS:  Hydrogel scaffolds were fabricated to consist of the following compositions: alginate, alginate+collagen type I in 5:1 and 10:1 ratios, alginage+chitosan in 1:1 and 2:1 ratios, and alginate+collagen+chitosan in a 10:2:5 ratio.  All scaffolds were Arg-Gly-Asp (RGD)-modified.  Scaffolds were seeded with 3.5x10⁵ HUVECs and maintained under standard cell culture conditions.  After 15 days, HUVECS were fluorescently labeled with DiI-Ac-LDL and DAPI.  Cell density and invasion into scaffolds were evaluated using 3-dimensional confocal fluorescent imaging.  All experiments were performed in triplicate.  Data is given as mean ± SEM.

RESULTS:  HUVECs were maximally confluent on alginate/collagen (5:1, 10:1) hybrid scaffolds (Figure) compared with pure alginate scaffolds (164 ± 28 and 222 ± 20 v. 5.0 ± 0.9 cells/HPF, p<2.0x10^-6).  Alginate+chitosan (2:1, 1:1) hybrid scaffolds also supported greater HUVEC adherence and growth than alginate alone (152 ± 21 cells/HPF, p<1.0x10^-7 and 10 ± 1.4 cells/HPF, p=0.022, respectively).  HUVECS adhered to alginate+collagen+chitosan scaffolds in greater numbers than to alginate-alone scaffolds (88 ± 15, p < 4.0x10^-6), but to a lesser degree than alginate+collagen 5:1 and 10:1 scaffolds (p=0.02 and p<5.0x10^-6, respectively).  Filopodial extension (representing HUVEC activation and early vessel formation) was seen in chitosan-containing scaffolds.  While three-dimensional confocal imaging revealed a small degree of cellular invasion into chitosan-containing scaffolds, collagen-containing scaffolds demonstrated a greater number and depth of invasive cell fronts.

CONCLUSIONS: Even at low concentrations, the addition of collagen type I to alginate matrices results in a substantial increase in HUVEC adherence and invasion.   Such hybrid hydrogels harness both the low-cost and availability of alginate and the cellular appeal of collagen.  These findings provide important insights into the optimization of endothelial cell adhesion and invasion by appropriate substrate selection and should serve to guide and enhance the fabrication of tissue-replacement scaffolds in the future.