*Purpose: Given therapeutic limitations in limb salvage, we sought to demonstrate the efficacy of combining local mesenchymal stem cells (MSCs) infusion with fresh whole blood in an ex vivo porcine vascularized composite allotransplantation (VCA) model utilizing normothermic machine perfusion (NMP) for 24 hours. Local intravascular infusion of MSCs during NMP should facilitate the attachment of MSCs to the VCA vasculature and lead to enhanced tissue repair as well as aid in the prevention of additional cellular injury; reducing inflammatory responses during the ex vivo preservation phase.

*Methods: A porcine semitendinosus myocutaneous free flap was used to model VCA. Seven Yorkshire swine were randomly divided into an experimental group (n=3) or control group (n=4). Each VCA flap contained skin, subcutaneous tissue, fascia, muscle, and a vascular pedicle (terminal branch of deep femoral/profunda artery and vein). 5 Units of autologous whole blood was collected from the donor animal post tissue procurement. In an antegrade fashion, the harvested flap was flushed with 20 ml ± 20 ml heparinized saline. The flaps underwent 3-hour cold ischemia at 4°C, preserved with University of Wisconsin solution administered via a 1-hour drip infusion. The free flap was connected to NMP with whole blood and additive supplements (trace elements, multi-vitamin, nutrients, antibiotics, antioxidants, and vasodilator) ± MSCs. Flow rate was set at 5-10 ml/min for 24 hours. The flaps were monitored for various parameters in both blood perfusate and tissue biopsy within serial time points. Finally, the tracing of fluorescence-labelled MSCs in the VCA flap was assessed by an in vivo imaging system (IVIS).

*Results: A slower progression of VCA necrosis after 24-hour NMP was observed in MSC-treated group compared with the control group. All MSC-treated VCA flaps showed fluorescent intensity by the IVIS throughout muscle, fat, and skin. Lab results varied greatly given variables between flaps and groups. Further analysis on cellular injury and inflammatory markers, histology, and comparison between groups at different time points is warranted.

*Conclusions: Our preliminary data suggest that the local infusion of MSCs to VCA during 24-hour NMP lead to a reduction in ischemic tissue injury and degeneration. The induction of MSC is feasible and attaches to the VCA vasculature. This is demonstrated by the IVIS which show fluorescent illumination minimally throughout flap and largely throughout skin. It is therefore important for this information to continue forward with further trials on local stem cell therapy in ex-vivo VCA preservation. This study shows great promise for improving outcomes on delayed limb salvage surgeries in combat trauma by reducing ischemia reperfusion injury and incidence of graft rejection.

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