Bioengineering Vascularized Composite Allograft Flaps For Large Soft Tissue Defect Repair
Michael S. Xu, MD1, Tom Waddell, MD, PhD, FRCSC1,2, Siba Haykal, MD, PhD, FRCSC, FACS1,3.
1Latner Thoracic Surgery Research Laboratories, University Health Network, Toronto, ON, Canada, 2Division of Thoracic Surgery, University of Toronto, Toronto, ON, Canada, 3Division of Plastic and Reconstructive Surgery, University of Toronto, Toronto, ON, Canada.
PURPOSE: Large volume soft tissue defects impose substantial effects on patient quality of life, cause loss of function, and can result in permanent disability. Engineered tissues using decellularization/recellularization methods can circumvent issues of donor site morbidity or immunosuppression associated with conventional autologous free flap reconstruction or vascularized composite allotransplantation (VCA). We describe the decellularization and recellularization of vascularized soft tissue flaps (omentum, tensor fascia lata, radial forearm, and latissimus dorsi muscle) in a porcine model using an ex vivo perfusion bioreactor.
METHODS: Soft tissue flap scaffolds based on their main vascular pedicle were surgically procured from Yorkshire pigs (~35-40kg), cannulated, then placed in an ex vivo perfusion bioreactor. Flaps were immediately flushed with heparinized saline (15 U/mL) then perfused at 2mL/min in a perfusion bioreactor with a sequence of 0.05% sodium dodecyl sulfate (SDS), DNase (0.1 mg/mL), phosphate-buffered saline, followed by sterilization with 0.1% peracetic acid/4% ethanol. Recellularization was performed using 2 x 107 human umbilical vein endothelial cells (HUVEC) suspended in EGM2 growth media (Lonza) introduced into the scaffold arterial inlet by manual syringe seeding. Scaffolds were statically cultured for 4 hours to allow cell attachment and then perfused with EGM2 for 1 day. Biochemical analysis for DNA and glycosaminoglycan (GAG) content was performed to quantitatively assess decellularized scaffolds. Decellularized and recellularized scaffolds were also assessed histologically by hematoxylin & eosin staining.
RESULTS: Four vascularized soft tissue flaps in a porcine model were procured and perfusion decellularized in a bioreactor. The omentum, tensor fascia lata, and radial forearm flaps were decellularized with 0.05% SDS for two, three, and five days, respectively and demonstrated absent nuclear staining on H&E. However, latissimus dorsi decellularization is incomplete even after 14 days (Fig. 1). DNA content in the acellular scaffolds was significantly lowered in the omentum, tensor fascia lata, and radial forearm flaps although no significant change in GAG content was observed (Fig. 2). Preliminary experiments to recellularize the omentum and tensor fascia lata with HUVECs demonstrated evidence of cell attachment at the vascular lumen surface after one day of culture (Fig. 3).
CONCLUSION: Acellular omentum, tensor fascia lata, and radial forearm flap scaffolds can be successfully decellularized with low concentration SDS, although latissimus dorsi decellularization is heterogenous and requires further optimization. Scaffold recellularization with HUVEC cells is also feasible with evidence of cell attachment onto the acellular omentum and tensor fascia lata. Future work will expand recellularization conditions for each porcine flap model and characterize the vascular networks in recellularized flaps using microtomography. We will also examine recellularization using endothelial cells co-cultured with human mesenchymal stem cells.
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