SYNTHESIS AND TESTING OF LIVING SKIN EQUIVALENT

Project: Research project

Project Details

Description

Rapid burn wound closure improves patient survival. Multi-center studies
have shown that mortality rates decrease with increasing speed of
full-thickness burn wound closure. The purpose of this study is to demonstrate that replacement of injured
skin with a combination of prosthesis and cultured skin cells will be
faster, safer, less expensive and will decrease morbidity and mortality
compared to current techniques of dressing, debridement, and autografting.
The syngeneic Buffalo rat will be employed as the experimental animal. A
living skin equivalent will be made from a prosthetic dermis and Buffalo
rat keratinocytes grown in cell culture. Current cell culture techniques require several weeks to produce clinically
useful quantities of synthetic skin. Our experimental plan involves
manipulation of the culture medium using recognized methods that accelerate
the proliferative rate of keratinocytes. These manipulations include the
isolated addition of cholera toxin, 8-bromo c-AMP, and insulin to culture
medium. The reduction of medium calcium concentration will also be done to
accelerate cell growth. The proliferative rate of keratinocytes will be
measured by [3H]-thymidine pulse labeling, DNA determination, Lowry protein
assay, autoradiography, and scintillation counting. Progression of
differentiation will be assessed by Kreyberg staining, [3H]-leucine pulse
labeling, extraction of proteins from epidermal keratinocyte cultures,
determination of protein concentration (Lowry) assay, slab gel sodium
dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and
fluorography. Once the characteristics of keratinocytes grown at an accelerated rate are
established, a living skin substitute will be fabricated. This epidermis
will consist of rat keratinocytes. Biologic and synthetic dermal
substitutes will be tested. This skin equivalent will be grafted onto
full-thickness wounds created on the backs of adult Buffalo rats.
Adherence of the grafts will be determined, being the most important
characteristic of a skin substitute. The wounds will be observed for graft
take and healing. Biopsy and histologic examination of the skin substitute
will be performed at appropriate intervals. The ultimate goal of our efforts is to develop a living skin substitute
useful in treating the severely burned patient. This skin equivalent
produced using tissue culture techniques will provide rapid burn wound
closure.
StatusFinished
Effective start/end date12/1/845/31/88

Funding

  • National Institutes of Health
  • National Institutes of Health
  • National Institutes of Health

ASJC

  • Medicine(all)
  • Biochemistry, Genetics and Molecular Biology(all)

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