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Tissue electrophoresis for generation of porcine acellular dermal matrices




Duran, Celso, Jr., author
Orton, Christopher, advisor
Dasi, Lakshmi Prasad, committee member
James, Susan P., committee member

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Background: Acellular dermal matrices have several applications including treatment of burns, reconstructive surgery, and treatment of chronic ulcers. Xenogeneic acellular dermal matrices have the advantage of increased availability compared to matrices derived from human cadavers (i.e. allogeneic dermal matrices), however they have a higher potential for generating an inflammatory response in the recipient. One approach to creating an acellular dermal matrix is through chemical and detergent-based processes collectively known as decellularization. Concerns regarding the completeness of soluble protein and antigen removal associated with current detergent-based decellularization treatments have been raised. The aim of this study was to compare the efficacy of a standard detergent-based decellularization and a novel electrophoresis-based method at removing soluble protein and protein antigens. Hypothesis: I hypothesized that tissue electrophoresis would enhance the removal of soluble proteins and protein antigens from porcine dermis compared to a standard detergent-based decellularization protocol. Methods: Skin was harvested from 6 pig cadavers. A portion of skin from each pig was assigned to four treatment groups: 1. Epidermis removal without sodium dodecyl sulfate (SDS) (positive or untreated control) 2. Epidermis removal with 0.5% SDS (epidermis removal control) 3. Epidermis removal with 0.5% SDS and standard 0.5% SDS decellularization treatment with a 6 h passive diffusion washout period 4. Epidermis removal with 0.5% SDS and Tissue Electrophoresis-based decellularization (0.5% SDS, 2% agarose gel, and 0.5 Amp) for 6 h The completeness of soluble protein and antigen removal was evaluated by SDS-PAGE and immunoblot analysis, respectively. Rabbit anti-porcine and human IgG serums were the primary antibodies for immunoblot analysis. Results: Tissue electrophoresis decellularization increased removal of soluble proteins from porcine dermis when compared to standard passive detergent-based decellularization, based on SDS-PAGE analysis. Antigen removal, based on immunoblot analysis, was increased compared to untreated dermis, but was not significantly different between standard detergent-based and tissue electrophoresis-based decellularization treatments. Conclusion: Tissue electrophoresis enhances removal of soluble proteins from porcine dermis compared to standard detergent-based decellularization. This enhanced removal of soluble proteins may translate into reduced inflammatory response to xenogeneic acellular dermal matrices implanted into humans. Optimization of electrophoretic parameters may further increase the efficiency of tissue electrophoresis as a decellularization method.


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