Back to 2014 Annual Meeting Abstracts

BACKGROUND:
Although bioartificial tissue scaffolds created using decellularization techniques may obviate patient donor site morbidity associated with reconstructive surgery, survival and durability of these acellular, avascular tissues are dependent on neovascularization and effective host cell invasion for their survival, making them vulnerable to degradation, infection and failure. We sought to develop a bioengineered matrix with a dominant vascular pedicle that would serve as a platform for decellularization, re-seeding and ultimately direct donor-recipient 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 24 hours and DNAse I for 3 hours. Following decellularization, the tissue was gently washed in PBS for 7 days, then transferred to standard culture medium for 72 hours. Following washing, 6mm discs were obtained and equilibrated in standard culture medium for 24 hours. These acellular scaffolds were topically seeded with either RFP-expressing Human Dermal Fibroblasts (HDFn) or a 2:1 ratio of HDFn and Human Aortic Smooth Muscle Cells (HASMC). Using a cellular concentration of 20x106 cells/mL, HDFn-only and HDFn:HASMC co-cultured cellular suspension were obtained, (HDFn-only cellular suspension: 6x105, 8x105 and 10x105 cells; HDFn:HASMC cellular suspension: 6x105:3x105, 8x105:4x105 and 10x105:5x105 cells). Culture media changes were performed daily. At 3 and 7 days, the scaffolds were fixed and processed for histology. Unseeded scaffolds served as the control.
RESULTS:
Histological analysis with H&E staining of unseeded scaffolds confirmed successful decellularization and removal of all cellular material. Cells were observed lining the surface of all the seeded scaffolds (single-cultured and co-cultured). They were confirmed as HDFn and HASMC via fluorescent microscopy as evidenced by RFP, DAPI, and Smooth Muscle Actin (SMA) expression. The cell lining appeared to be thicker with higher seeding concentrations. An increase in the degree of cellular invasion into the scaffold was seen in samples with higher seeding concentrations and in HDFn-HASMC co-cultured samples (in comparison to single-cultured HDfn-seeded samples).
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.