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Optimizing Vascular Invasion into Hydrogel Scaffolds Using Bioactive Sphingolipids
Andrew I. Abadeer, M.Eng1,2, Brandon Gold, BA1, Julia Jin, B.S.1, Xue Dong, B.A.1, Peyton Delgorio, B.S.1, Omer Kaymakcalan, M.D.1, Sarah Karinja, B.A.1, Jaime Bernstein, B.S.1, Ross Weinreb, M.S.1, Jason Spector, M.D.1.
1Weill Cornell Medical College, New York, NY, USA, 2Columbia University College of Physicians and Surgeons, New York, NY, USA.

PURPOSE: The promise of an ideal “off-the-shelf” regenerative template has long been predicated upon the availability of an adequate inherent vasculature. Due to this, current dermal regenerative templates such as Integra™ are clinically limited in scope secondary to their dependence on excellent host wound bed vascularity. This reliance in turn restricts their applicability in any but the most optimal wound beds, all but prohibiting their use in wound beds with exposed bone, tendon, or hardware. Recently, sphingosine-1-phosphate (S1P), a bioactive sphingolipid stored in platelets and found in micromolar quantities in the plasma and nearly absent in the interstitium, has gained widespread attention for its effects on endothelial motility and vascular maturation. In fact, it is thought that a reversal of the physiologic gradient causing S1P to be present in the interstitium, such as in wound healing, is responsible for inducing reparative angiogenesis. It is further hypothesized that gradients of S1P are a major signaling component in activating homeostatically nascent angiogenic mechanisms by promoting endothelial sprouting, motility, and stability. Herein we build upon our novel scaffolds by incorporating a bioactive sphingolipid into a collagen matrix in order to select for and amplify endothelial invasion to create a rapidly integrated, vascularized, tissue engineered construct with potentially profound implications for application in poorly vascularized wound beds.
METHODS: Raft cultures were created by seeding 100 ul of 3 mg/ml type I collagen onto 8um pore rafts. GFP labeled HUVECs were topically seeded and cultured for 7 days. The media in contact with the cells was devoid of S1P and a gradient in the collagen established by filling the bottom well in 1uM S1P. Gradients of VEGF, FGF, and concentrations of fetal bovine serum were also tested in the absence of S1P. Rafts were cross-sectioned at 100 um using a vibratome and sections stained with toluidine blue. Sections were quantified for number of sprouts per square micron, average length of sprouts, number of cells per structure, and number of junctions. Confocal reflectance z-stacks were taken to confirm the presence of lumens within the sprouts.
RESULTS: Robust and complex invasion was seen as early as day 3 in cultures containing S1P. S1P rafts demonstrated multicellular, luminal, complex sprouts that anastomosed to adjacent sprouts as early as day 14. At seven days of culture, these constructs exhibited an average sprout density of 37±8 sprouts/mm2 with an average depth of invasion of 129±58 um. Origin endoluminal diameters were on the order of 10-20 um. Rafts without S1P exhibited confluent superficial growth but no invasion into the collagen matrix regardless of VEGF, FGF, or serum gradient.
CONCLUSION: With current dermal replacement products currently limited by an inadequate induction of endothelial invasion in poorly vascularized wound beds, we have herein demonstrated the remarkable ability of sphingosine-1-phosphate, a bioactive sphingolipid, to induce robust and complex endothelial invasion in a collagen template. Future work will focus on the incorporation of this sphingolipid into our novel hydrogel microsphere drug delivery platform towards a powerful new dermal regenerative template capable of inducing endothelial invasion.


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