Project 1: Preservation Solutions Used for Attenuating Ischemia-Reperfusion Injury in Vascularized Composite Allotransplantation

In our laboratory, we have established an animal model of VCA. A heterotopic rat hindlimb, composed of skin, muscle, bone, nerve and vessels, has been successfully transplanted as a perfused flap in both syngeneic and allogeneic rats with survival of the animals when ischemia is minimal. When ischemia time is prolonged in allogeneic animals, we have observed a significant reduction in survival time.  We have observed viability of the perfused flap in a syngeneic model while in the allogeneic model, there was eventual rejection and necrosis of the flap. The molecular events and tissue damage caused by IRI as well as vascular integrity following reperfusion in skin, muscle, nerve, vessels and bone (each of the components of the limb) were studied. We compared different preservation solutions with static cold storage (SCS) and found that heparinized saline resulted in higher markers of ischemia and decreased vascular integrity while perfadex appears to be protective. We have now expanded this work to focus on an Ex vivo preclinical model.

Project 3: Dynamic Re-endothelialization of Decellularized Composite Soft Tissue Allografts

To date, VCA perfusion decellularization has been investigated in vitro and in vivo with one significant limitation being sub-optimal graft vascularization. This study proposes a customized approach to decellularization and recellularization to engineer a diversity of soft tissues flaps. The four tissue flaps under study: 1) radial forearm fasciocutaneous, 2) latissimus dorsi muscle, 3) tensor fascia lata and 4) omentum were selected as representative of flaps currently in clinical practice. The first objective of this study is to optimize the decellularization protocol for four soft tissue flaps in porcine model and the second objective is for recellularization of soft tissue scaffolds using mixed cells types and dynamic re-endothelialization We hypothesize that dynamic re-endothelialization in vitro under pulsatile flow will promote recellularization of the vasculature without thrombogenesis or intimal hyperplasia while supporting tissue viability. Biochemical assays for collagen, laminin, and elastin will be performed. Cell attachment to the scaffold will be assessed qualitatively using modalities such as scanning electron microscopy. The microcirculatory network following decellularization will be examined qualitatively by immunohistochemistry for CD31 and smooth muscle actin (a smooth muscle cell marker) to determine removal of intimal and medial components of vascular tissue. Meanwhile, the microcirculatory channels in ECM will be imaged with angiographic techniques using Microfil contrast dye under microtomography to evaluate patency of the decellularized vascular conduits.