Swine Fasciocutaneous Flap Decellularization: Protocol Optimization For Microvascular Structure Preservation
Corentin B. Taveau, MD1,2, Aylin Acun, PhD1, Alexandre G. Lellouch, MD, MSc1,2, Ruben Oganesyan, MD, FACS, FAAP1, Alec R. Andrews1, Philipp Tratnig-Frankl, MD1, Laurent Lantieri, MD2, Mark A. Randolph, MAS1, Basak Uygun, PhD1, Curtis L. Cetrulo, Jr., MD, FACS, FAAP1.
1Massachusetts General Hospital, Boston, MA, USA, 2Service de Chirurgie Plastique, Hôpital Européen Georges Pompidou, Assistance Publique-Hôpitaux de Paris (APHP), Paris, France.
PURPOSE: Vascularized Composite Allotransplantation (VCA) emerged as a valuable option in treating critical tissue defects, at the cost of life long immunosuppressive drug regimen. To avoid the risk of such therapy and overcome organ compatibility, decellularization/recellularization is an attractive approach. Decellularization of fasciocutaneous flaps has been achieved previously, however the recellularization of small vessels failed. Our hypothesis was that failure in recellularization was due to aggressive decellularization.
METHODS: Saphenous fasciocutaneous flaps were procured from swine in surgical conditions, under general anesthesia. After pedicle ligation, the VCAs were immediately flushed with 30mL of 100 U/mL heparinized saline. Before beginning decellularization perfusion, vasculature integrity was assessed by flap angiography. Pressure-controlled perfusion decellularization was then started with a sequence of phosphate-buffered saline (PBS), 0.1% sodium dodecyl for 5 days, deionized water, 1% Triton X-100, deionized water and finally PBS again. The target pressure was 70-100mmHg. After perfusion was complete, ethyl-cyanoacrylate glue was used on the subcutaneous side of the flaps to fix leakage. Flaps were assessed clinically, with angiography and histology for overall extracellular matrix and microvasculature integrity and complete removal of cellular materials.
RESULTS: Twelve flaps were used to optimize this protocol. Perfusion flowrate was 0.2-1mL/min to maintain the target pressure. Outflow was preserved throughout perfusion decellularization. Clinically, the flaps lost all coloration and appeared morphologically and biomechanically intact (image 1). Flap angiography showed preservation of microvasculature and venous return (image 2). Histology showed no remaining cell nuclei and a preserved extracellular matrix (ECM) structure (image 3).
CONCLUSION: Fasciocutaneous flap decellularization protocol optimization through use of milder detergent solutions and lower perfusion flow rates enabled better preservation of the microvasculature and the venous outflow which is crucial for successful recellularization. Further analysis of structural and compositional preservation of the ECM structure will confirm the recellularization potential of decellularized fasciocutaneous skin flaps.
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