Endothelialization and Microsurgical Anastomosis of Constructs Containing Sacrificial Polymer-Derived Vascular Channels: Advancement toward the Creation of Surgically Relevant Tissue Replacements
Peter W. Henderson, MD MBA1, Allie M. Sohn, BS1, Aleid Koppius, BA1, Alyssa J. Reiffel, MD1, Alice Harper, BA1, Lawrence J. Bonassar, PhD2, Margaret W. Frey, PhD2, Jason A. Spector, MD FACS1.
1Weill Cornell Medical College, New York, NY, USA, 2Cornell University, Ithaca, NY, USA.
BACKGROUND: The development of artificial tissue replacements is an inevitability that will revolutionize reconstructive surgery in the next 30 years, eventually replacing the autologous free tissue transfers that have revolutionized the field of over the past 30 years. Attempts to fabricate bioengineered tissues that are surgically relevant, however, have been encumbered by an inability to fabricate constructs that contain a microchannel network that is in continuity with inflow and outflow macrochannels that are amenable to microsurgical anastomosis and manipulation. Our laboratory has developed an innovative technique to overcome this obstacle that utilizes sacrificial polymers that are first embedded within a polymer construct of opposite solubility, and then subsequently dissolved, leaving in place an internal channel network. Having accomplished this, we next sought to endothelialize the channels and demonstrate the feasibility of in vivo studies.
METHODS: The constructs were fabricated by injecting polar-soluble 4% Ca2+-crosslinked alginate mixed with 1% collagen into custom-designed molds containing fibers of the non-polar-soluble polymer polylactic acid (PLA). PLA was then sacrificed by flushing the construct with the non-polar solvent chloroform, leaving a patent network of channels that were in continuity with inflow and outflow macrochannels. The entire network was subsequently covalently modified with RGD peptide in order to increase endothelial cell adhesiveness. Both macrochannels were cannulated, injected with media containing 500μl fluorescently-labeled human umbilical vein endothelial cells (HUVECs; 105 cells/ml), and clamped. The construct was rotated for 4h to optimize circumferential cell deposition within the channels. In situ fluorescent imaging was performed 5 days later to detect the presence of the fluorescently-labelled HUVECs. Finally, microsurgical anastomosis of a rat vessel to the inflow macrochannel was attempted ex vivo to demonstrate the feasibility of subsequent in vivo studies.
RESULTS: Fluorescent imaging 5 days after HUVEC infusion confirmed the presence of a dense endothelial coating of the internal network. Using 8-0 nylon on spatulated ophthalmic needles, a representative rat aorta was successfully anastomosed ex vivo to the alginate block. Subsequent successful perfusion with contrast confirmed both the integrity of the anastomosis and the patency of the endothelialized channels within the alginate.
CONCLUSIONS: This series of experiments has demonstrated the capability of this novel technique to produce surgically relevant, complex bioengineered constructs that contain an endothelialized network that are amenable to microsurgical manipulation and anastomosis. This important series of innovations has led to a construct that is ready for first of its kind in vivo testing, and thereby represents a significant advancement towards the ultimate development of surgically-relevant engineered replacement tissues.
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