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Vascular Smooth Muscle Cells in the Presence of Fibronectin Functionalized Collagen Scaffold Increases the Size of Endothelial Cell-based Vascular Aggregates
Kaiti Duan1, Daniel Sasson1, Biraja Dash1, Henry Hsia, MD1,2
1Section of Plastic Surgery, Department of Surgery Yale School of Medicine, Yale University, New Haven, USA, 2Department of Biomedical Engineering, Yale University, New Haven, USA

Purpose:: Fibronectin-functionalized collagen scaffolds can promote induced-pluripotent-stem-cell-derived-vascular smooth muscle cell (iPSC-VSMC) to enhance their pro-angiogenic paracrine profiles. However, the influence of fibronectin collagen on human umbilical vein endothelial cells(HUVEC),a key component of angiogenesis, is still unknown. In this study, our objectives was to evaluate the integrity of iPSC-VSMC:HUVEC combination vascular formation in the setting of fibronectin-functionalized collagen scaffolds.
Methods: Fibronectin was added to type I collagen-based scaffolds. HUVECs were incubated within the scaffold for 7 days, and after the first 24 hours Echistatin, an integrin inhibitor of Alpha-v Beta-3, was added to scaffolds. The resultant scaffolds were evaluated for cellular viability via AlamarBlue assay. Also, we combined iPSC-VSMC and HUVEC at a ratio of 1:4 and embedded them into fibronectin-functionalized collagen for 7 days. Confocal microscope was used to count total number of endothelial vascular aggregates, and then categorized based on sizes, greater or less than 50um. Result:: Fibronectin-functionalized collagen scaffolds improved IPSC-VSMCs viability(p-value=0.001), but not HUVECs. However, the addition of Echistatin, an inhibitor of fibronectin, to fibronectin scaffolds resulted in significant decrease in HUVEC viability (p-value=0.0001). The total number of vascular aggregates was significantly higher in fibronectin scaffolds than in control and Echistatin scaffolds(P value=0.03). There was no significance between the number of large or small vascular aggregates amount the 3 groups. iPSC-VSMC:HUVEC combination scaffolds that were functionalized fibronectin scaffolds demonstrated increased number of vascular aggregates when compared to control and Echistatin scaffolds (P value=0.003 and 0.002, respectively). The number of large vascular aggregates was increased in the fibronectin scaffolds containing iPSC-VSMC and HUVEC combination when compared control and Echistatin scaffolds(P value=0.0001, both). The number of large vascular aggregates was significantly decreased in the scaffolds treated with Echistatin(P value=0.0001).(Figure1) Conclusion: Fibronectin plays a key role in maintaining the HUVECís viability, since the addition of Echistatin dramatically decreased the HUVECís viability. This suggested fibronectin to have an agonistic effect on HUVEC via interaction of Alpha-v Beta-3 integrin expressed on the cells. The higher number of large vascular aggregates in iPSC-VSMC and HUVEC combination in fibronectin scaffolds, suggested that fibronectin was promoting iPSC-VSMC interaction with HUVEC to promote formation larger vascular aggregates. These findings resulted in a better understanding of potentially an underlying mechanism of how to optimize iPSC-VSMC and HUVEC interaction to improve vascular formations by specifically targeting the Alpha-v Beta-3 integrin, which may translate to more effective cell therapy-based wound healing regimens.


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