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Purpose: The decellularization of Vascularized Composite Allotransplantation (VCA) represents a first step in circumventing the need for immunosuppression by depleting donor cells from VCAs. Here we present a model of decellularization of VCAs in rats. Methods: Rat hind limbs based upon the common femoral artery were harvested and perfused through the vascular pedicle at 1ml/min through with 1% sodium dodecyl sulfate (SDS) (group 1) or 0.2% SDS (group 2) or were immersed in SDS 1% (group 3), followed by perfusion (group 1 and 2) or immersion (group 3) in 1% Triton X-100. The degree of decellularization was assessed quantitatively by determining DNA content and qualitatively by histology in all groups at the same locations. The group yielding best decellularization outcome was selected for further assessment for the preservation of ECM content and vasculature and was transplanted orthotopically to assess the ability to reperfuse these decellularized limbs in vivo. RESULTS - Gross examination and DNA quantification of less than 50 ng/mg tissue demonstrated successful decellularization only in group 2 while preserving tissue matrix architecture, including vessels. The arterial network was found to be perfusable and intact during in vivo orthotopic transplantation of the decellularized limb. No outflow was observed, however, after 115 minutes of arterial reperfusion. Conclusions: We describe a successful decellularization protocol for rat hindlimb VCAs by perfusion ex-vivo of 1% SDS. During its in vivo transplantation, decellularization alone allows for a perfusable arterial network and with further optimization, we aim to achieve venous perfusion as well.

Purpose: The current gold standard in organ storage is static cold storage (SCS) at 4°C in a preservation solution (UW, HTK). While kidneys can be stored for 24 hours in SCS, Vascularized Composite Allografts (VCA) have been transplanted up to 12 hours in this condition. We aimed to determine the limits of VCA viability with SCS. We used a partial heterotopic hindlimb transplantation model on rodent to assess VCA survival at various durations of SCS. Methods: Right partial hind limbs in Lewis rats were procured and a 24G Angiocath inserted in the femoral artery. Limbs were flushed with 2ml of heparin saline (100UI/ml) at room temperature and then 5ml of ice cold HTK solution. The limbs were then immersed in HTK in a sterile bag and stored at 4°C for a period of 12(group 1), 18(group 2), 24(group 3), 48 (group 4) or 0 hours (fresh controls Group 5), before microsurgical transfer to the opposite groin. Graft weight was assessed before and after cold storage. At post-operative day (POD) 21, VCAs were harvested for histological analysis. Results: In all groups, there was no significant difference of weight gain in SCS groups after extended SCS times. In group 1 (n=4), 3 rats survived until POD21; clinical evolution showed a limited epidermolysis (