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A novel methodology to generate tissue-engineered vascular grafts for skin flaps
Gurtej Singh, PhD, Kai-li Liang, BS, John Cordero, BS, Brody Wiles, BS, Gabriel Klein, MD, Marcia Simon, PhD, Miriam Rafailovich, PhD, Duc Bui, MD, Sami Khan, MD, Nicos Labropoulos, PhD, Alexander Dagum, MD.
Stony Brook University, Stony Brook, NY, USA.

BACKGROUND: Patient receiving reconstructive surgeries may require the use of an autologous vascular graft in order to provide an extra length to the artery or vein during anastomosis to the native vessels. Additionally, an autologous graft may also be necessary in turbocharging a vessel. Currently, the standard for these procedures is to use the patient’s own saphenous vein to serve as a graft. Unfortunately, this option is not always available and could result in complications related to its harvesting. Here, we propose a novel modular approach (provisional patent filed) to construct a tissue engineered vascular graft that results in uniform deposition of various biomaterials that could potentially alleviate problems associated with existing grafts.
METHODS: Rat tail collagen type I was used as a scaffold material to provide structural support to the vascular graft. For the design of the vascular conduit, a cylindrical Polydimethylsiloxane (PDMS) mold of 5mm in diameter was used. The developed lumen was then coated with fibronectin and matrigel, followed by seeding with 3 x 10^6 cells/mL human umbilical vein endothelial cells (HUVECs). Following incubation, the tissue architecture was characterized through immunostaining, histology and imaging using confocal microscopy.
RESULTS: Endothelial cells exhibited high viability (>90%) as measured via confocal microscopy using Hoechst 33342 and propidium iodide staining. Following 7 days of incubation, the necessary structural integrity of the endothelial monolayer lining the lumen was achieved. This was proven by the formation of a well-defined network of actin-filaments, stained with Alexa Fluor 488® phalloidin stain. The diffusional permeability of the lumen was also characterized through the use of fluorescent labeled molecules such as FITC labeled dextran of different molecular weights and Alexa Fluor® 594 conjugated with bovine serum albumin. No permeation of these molecules was observed beyond the monolayer of endothelial cells as compared to the lumen without cells where permeation was up to 250 microns. Current experiments are focused on characterizing the functionality and integrity of the graft under physiological flow conditions. The graft is connected to a perfusion pump to mimic flow conditions in the body and blood/media will be made to flow at different rates to determine the optimum operating conditions. Parameters including the burst strength, fatigue strength, maturation processes, vascular remodeling and angiogenesis are being evaluated.
CONCLUSIONS: We have successfully developed a novel method to generate a vascularized conduit while maintaining precise spatial control over the deposition of different biomaterials. The developed vascular graft has the potential to aid in flap based tissue reconstructions when combined with a tissue engineered construct.

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