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Fabrication of Cellular Tissue Constructs from Autogenous Tissue
Karina A. Hernandez, D.O., Alyssa J. Reiffel, MD, Rachel Campbell, MD, Alejandro Pino, BS, Jeremiah Joyce, BA, Kadria N. Derrick, MD, Alice Harper, BA, Jason A. Spector, MD, FACS.
Weill Cornell Medical College, New York, NY, USA.
BACKGROUND: Although bioartificial tissue scaffolds created using decellularization techniques may obviate patient donor site morbidity associated with reconstructive surgery, these acellular, avascular tissues are dependent on neovascularization for their survival, making them vulnerable to degradation, infection and failure especially in suboptimal wounds. In previous work we have employed perfusion-based decellularization techniques to create a pre-vascularized acellular scaffold. We now seek to expand on this concept and develop a bioengineered matrix with a dominant vascular pedicle that would serve as a platform for cellular re-seeding and ultimately direct microsurgical anastomosis.
METHODS: Fasciocutaneous flaps supplied by the superficial inferior epigastric artery (SIEA) were harvested from Sprague Dawley rats. The femoral vessels were cannulated and a decellularization protocol was initiated via sequential perfusion with 4% sodium deoxycholate for 12 hours and DNAse I for 12 hours. Following decellularization, using a skin biopsy punch 6mm discs were obtained and allowed to equilibrate in standard culture medium for 48 hours. These acellular scaffolds were then topically seeded with 2x105 red fluorescent protein-expressing Human Dermal Fibroblasts (RFP-HDFn) with media changes performed daily. At 1, 3, 7 and 14 days the scaffolds were fixed and processed for histology. Unseeded scaffolds served as the control.
RESULTS: Perfusion of our scaffold via the femoral artery confirmed patency of the macro and microvasculature. Histological analysis with H&E staining of unseeded scaffolds confirmed successful decellularization and removal of all cellular material along with the stratum corneum layer of the epidermis. In contrast, a carpet of cells was observed along the surface of seeded scaffolds. These were confirmed as HDFn via fluorescent microscopy as evidenced by RFP expression. These cells were also seen lining the decellularized hair follicles. Invasion was evident as early as 3 days post-seeding. Further confirmation was obtained via fibroblast specific protein-1 (fsp-1) staining, which revealed dark stained cell bodies along the surface of our seeded scaffolds, which were absent on the unseeded controls.
CONCLUSIONS: We have established a successful method for perfusion-based decellularization of a fasciocutaneous flap with preservation of the inherent micro and macrovasculature. These data demonstrate that biological scaffolds derived from fasciocutaneous tissues can support the adhesion and proliferation of vital dermal components, taking us one step closer in the development of an off-the-shelf replacement tissue construct.
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