Vascularized Tissue-Engineered Constructs for In Vivo Microanastomosis

Abstract

INTRODUCTION: Reconstruction following extensive tissue loss from trauma, burns, oncologic resections, and diabetic soft tissue infections remains challenging. While feasible, autologous tissue transfer has inherent consequences including donor site pain, functional loss, paresthesias, dysthesthia, and scarring. The synthesis of vascularized constructs for the management of such 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 internal longitudinal microchannel with inlet and outlet. Here we fabricate and microsurgically anastomose endothelial cell-lined microchannel containing collagen constructs. METHODS: Pluronic F127 microfibers were embedded in neutralized type I collagen, then sacrificed leaving a central longitudinal or “loop” microchannel, 1.5 mm in diameter. Constructs contained an inlet and outlet and were reinforced with polyglactone mesh. Microchannels were seeded with 3 x105 human umbilical vein endothelial cells (HUVEC) and constructs placed in static culture for 7 days. Seeded and unseeded constructs were microsurgically anastomosed to the femoral artery and vein of nude rats. After perfusion, all constructs were fixed in 10% formalin, embedded, stained with hematoxylin and eosin (H &E), and imaged via light microscopy. 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. Examination of unseeded constructs post-perfusion demonstrated microchannels lined by host cells in time-dependent manner, with 5h demonstrating greater cellular deposition than 2.5h. Following microsurgical anastomosis of seeded constructs, HUVEC remained largely attached to the microchannel despite the relatively high perfusion pressure upon arterial unclamping. CONCLUSION: We have successfully created vascularized biodegradable, biocompatible constructs that support microchannel endothelialization and microsurgical anastomosis in vivo. Constructs containing their own inherent vascular network can be directly anastomosed to host vasculature. This will provide immediate perfusion, increasing the survival of cellular constituents within as well as permanent 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.