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Ex Vivo Perfusion Decellularization Of Rat Hindlimb For Vascular Composite Allotransplantation
Aisha Adil, HBSc1,2, Golnaz Karoubi, PhD1,3, Siba Haykal, MD, PhD, FRCSC, FACS1,4.
1Latner Thoracic Surgery Research Laboratories, University Health Network, Toronto, ON, Canada, 2Institute of Medical Science, Temerty Faculty of Medicine, University of Toronto, Toronto, ON, Canada, 3Department of Laboratory Medicine and Pathobiology, University of Toronto, Toronto, ON, Canada, 4Division of Plastic & Reconstructive Surgery, University Health Network, University of Toronto, Toronto, ON, Canada.

PURPOSE: Vascular composite allotransplantation (VCA) is a promising reconstructive surgical avenue for treating severe musculoskeletal and tissue loss in patients with limb trauma. However, due to high allograft immunogenicity and required long-term immunosuppression which poses increased risk of infections, malignancies, and toxicity, VCA's clinical implications are limited. Engineering acellular composite tissue allografts can help circumvent the need for immunosuppression and significantly advance VCA strategies.
METHODS: Rat hindlimbs were procured from cadaveric male Lewis rats whereby the common femoral artery was cannulated. A customized perfusion-based, single-pass, closed-system bioreactor was constructed to apply detergent perfusion at 0.1 mL/minute via. the cannulated artery using 0.25% sodium dodecyl sulfate (SDS) concentration. Endpoint of decellularization was determined by visual examination for systemic white, translucent appearance and by histological analyses. All tissue compartments including the skin, femoral vessels, nerves, muscle, and femur were individually assessed histologically for preservation of tissue architecture and absence of cellular content. For recellularization, human umbilical vein endothelial cells (HUVECs) and L6 rat myoblasts were selected.
RESULTS: Gross morphology showed systemic white, translucent appearance of decellularized hindlimbs in 0.25% SDS condition relative to native (Figure 1A). Histologically, 0.25% SDS concentration preserved tissue architecture across all tissue compartments. Notably, it retained up to the innermost nerve tissue, the endoneurium, and retained similar morphology relative to native tissues. Cellular content was absent across all decellularized tissues (Figure 1D). Additionally, the construction of a perfusable, single-pass, and closed-system bioreactor circuit was suitable for decellularization (Figure 1B). For recellularization, HUVECs and rat myoblasts will be used (Figure 1C).
CONCLUSION: Perfusion decellularization was successfully implemented and 0.25% SDS perfusion retained all tissue compartments and respective tissue architectures of the rat hindlimb. The successful bioreactor design can serve as a foundational model for sterilization and recellularization. Next steps include further characterization via. biochemical analyses for DNA and extracellular matrix content. This decellularized hindlimb model will be recellularized using HUVECs and rat myoblasts cells.


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