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Optimizing Endothelial Cell Adhesion and Invasion in Tissue Engineering with Naturally-Derived, Biodegradable Hybrid Hydrogel Scaffolds
Alyssa J. Reiffel, MD1, Justin L. Perez, BS1, Allie M. Sohn, MD2, Edo Israely, BA1, Natalia Jimenez, BS1, Nikola Lekic, MS3, Jason A. Spector, MD, FACS1.
1Weill Cornell Medical College, New York, NY, USA, 2Albert Einstein College of Medicine, New York, NY, USA, 3Georgetown University School of Medicine, Washington, DC, DC, USA.

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 penetration, 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 4% w/v, alginate 4% + collagen type I 1% w/v in 5:1 and 10:1 w/w ratios, alginate 4% + chitosan 4% w/v in 1:1 and 2:1 w/w ratios, and alginate+collagen+chitosan in a 10:2:5 w/w ratio. Additional alginate+collagen 10:1 scaffolds were fabricated to contain an internal biodegradable polyglactone mesh to structurally reinforce the scaffold and allow for suture fixation to a recipient site. All scaffolds were Arg-Gly-Asp (RGD)-modified to enhance cellular adhesion. Scaffolds were seeded with 3.5x105 human umbilical vein endothelial cells (HUVECs) and maintained under standard cell culture conditions. After 14 days, HUVECS were labeled with DiI-Ac-LDL (an LDL tagged with a red flurophore that is endocytosed by HUVECs), and the scaffolds were fixed and mounted. Cell density and invasion into scaffolds were evaluated using 3-dimensional confocal fluorescent imaging.
RESULTS: HUVECs were maximally confluent on alginate+collagen (5:1 and 10:1) hybrid scaffolds when compared with pure alginate scaffolds (163.6±27.6 and 221±19.8 v. 5.9±0.9 cells/HPF, p<0.001). Cellular adhesion to alginate+chitosan 1:1 scaffolds was greater than to pure alginate scaffolds (9.8±1.4 v. 5.9±0.9, p=0.02) but less than collagen-containing scaffolds (p<1.0x10-5). Cellular adhesion to alginate+chitosan 2:1 scaffolds was greater than to pure alginate scaffolds (151.6±21.4 v. 5.9±0.9, p<1.0x10-7), unchanged from alginate+collagen 5:1 scaffolds, and less than to alginate+collagen 10:1 scaffolds (p=0.02). Tubulization (representing HUVEC activation and early vessel formation) was seen in chitosan-containing scaffolds. While three-dimensional imaging revealed a small degree of cellular invasion into chitosan-containing scaffolds, cells traversed the greatest distance through collagen-containing hydrogels. HUVEC adhesion to mesh-reinforced alginate+collagen 10:1 hybrid scaffolds (433.9±22.5) was greater to non-mesh containing scaffolds (p<1.0x10-7).
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. Not only did the incorporation of mesh reinforcement fail to diminish cellular adhesion, cellular adhesion was actually increased in its presence. These findings provide important insights into the optimization of endothelial cell adhesion and invasion by appropriate substrate selection and should serve to guide tissue-replacement scaffold production in the future.
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