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BACKGROUND: The reconstructive ladder culminates in reconstruction with autologous and transplanted free tissue transfers. While these procedures play a significant role in the repair and reconstruction of major wounds, they present with donor site morbidity, increased length of procedures, risk of failure, and immunosuppression with transplanted tissue. Alternates and improvements are thus still needed. Recelluarization of decellularized tissue can fulfill this role: it maintain the architecture and structural proteins of host tissue, and provide a robust platform for regeneration of complex tissue. Where perfused skin and muscles are complicated by broad interconnected capillary beds hindering ex-vivo perfusion and regeneration of tissue, we hypothesize that the decellularized kidney, with its well-encapsulated structure and vascular circuit, would make it the ideal scaffold for regeneration of tissue for use for complex wound coverage.
METHODS: Bilateral kidneys with their associated renal arteries and veins were harvested from Adult Male Sprague Dawley Rats. The kidneys were decellularized and prepared for recellularization by sequential arterial perfusion with 1X PBS, 1% Sodium Dodecyl Sulfate, .1% Triton X-100, Deionized H20, 1X PBS, and 10% DMEM with FBS. Human Foreskin Fibroblasts (HFF1) passages 6-10 were injected into the substance of the tissue. The tissue was placed into a bioreactor, in a 5% CO2 incubator. Human Placental Pericytes (HPLP) passages 6-10 were arterially infused, followed by Human Umbilical Vein Endothelial Cells (HUVEC) passages 6-10 the subsequent day. Tissue was maintained in media, with arterial perfusion initiated on the second day, media was changed every two days for an additional 4 days.
RESULTS: Hemotoxylin and Eosin (H&E) histology demonstrated successful decellularization of renal tissue. H&E and Immunofluorescence for HPLP, HFF1, HUVEC, and Ki67- a marker of cellular proliferation, demonstrated successful incorporation and proliferation of seeded cells into the 4 decellularized scaffolds. Fibroblasts were seen throughout the renal matrix, endothelial cells and pericytes were seen lining the endothelium.
CONCLUSIONS: The decellularized tissue contained fibroblasts as the stromal component with pericytes and endothelial cells lining the vasculature. We thus have fabricated a recellularized vascularized fibrovascular flap from an acellular renal scaffold. This is a significant step towards creation of a tissue engineered free flap with neither donor site morbidity nor immunosuppression, while reducing surgical length and complexity.