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Background: Although autologous tissue transfer has been established as a reliable approach to the reconstruction of complex defects, there are associated consequences including donor site pain, functional loss, paresthesias, dysthesthia, and scarring. The ability to synthesize vascularized constructs for the management of these complex wounds would represent a quantum leap in the field of tissue engineering. In previous work we synthesized and performed an in vivo microvascular anastomosis of a collagen construct containing an unseeded internal longitudinal microchannel with inlet and outlet. Here we fabricate and microsurgically anastomose collagen constructs containing an internal endothelialized microchannel.
Methods: Pluronic F127 microfibers were embedded in neutralized type I collagen, then sacrificed leaving a central “loop” microchannel, 1.5 mm in diameter. Constructs contained an inlet and outlet and were reinforced with polyglactone mesh for tensile strength at the anastomotic site. Microchannels were seeded with 5 x106 cells/mL human umbilical vein endothelial cells (HUVEC) and constructs placed in static culture for 7 days with daily media changes. Seeded and unseeded constructs were microsurgically anastomosed to the femoral artery and vein of nude rats. Following completion of anastomoses, patency was evaluated via venous strip tests and in vivo microdoppler assessment. Unseeded constructs were perfused for 2.5 and 5 hours. Seeded constructs were perfused for up to 24 hours. Following perfusion, all constructs were fixed in 10% formalin, embedded, stained and analyzed.
Results: Polyglactone mesh provided the necessary tensile strength, allowing microchannel-containing constructs to be successfully anastomosed to the femoral artery and vein of nude rats. In vivo gross inspection and H&E staining of seeded and unseeded constructs following harvest revealed intact microchannels capable of withstanding physiologic perfusion pressures. Patency was confirmed via venous strip tests and auscultation of continuous pulsatile blood flow via microdoppler. Post-perfusion analysis of unseeded constructs demonstrated microchannels with host inflammatory cells adherent along the walls. Post-harvest histology following perfusion of seeded channels revealed HUVEC largely attached to the endoluminal surface of the microchannel, taking on an elongated appearance, despite the relatively high perfusion pressure upon arterial unclamping. Immunohistochemical analysis of seeded microchannels demonstrated CD31 expressing endothelial cells.
Conclusions: We have successfully created custom vascularized biodegradable, biocompatible constructs that support microchannel endothelialization and microsurgical anastomosis in vivo. Constructs with their own inherent vascular network can be directly anastomosed to host vasculature providing immediate perfusion, thus increasing the survival of cellular constituents within the scaffold as well as the rate of incorporation into the host. This represents a major advance in tissue engineering and opens the door to the creation and application of larger, more complex surgically relevant constructs.