Rapid and effective healing of burn wounds with cultured analogs of human skin is the central objective of this proposal. Medical benefits from improved healing may include, but not be limited to; reduced requirements for split- thickness autograft, shorter hospitalization time; and reduced long-term morbidity after recovery. However, anatomic and physiologic deficiencies of all current models of cultured skin have restricted realization of these benefits. Major deficiencies result from culture conditions that do not generate an epidermal analog with fully functional barrier properties, from irregular pigmentation after healing, and from absence of a vascular plexus in the dermal analog which delays vascularization. The current model of cultured skin substitute (CSS) in this laboratory is a collagen- based sponge populated with cultured human keratinocytes and fibroblasts. An investigative cycle has been established to: A) design experiments to address clinical deficiencies of CSS, B) perform mechanistic studies in vitro to generate new prototypes of CSS; C) transplant experimental CSS to athymic mice to measure efficacy and tissue phenotypes, and, D) study the improved prototype of CSS for reduction of mortality and morbidity in critically injured burn patients.
Six specific aims will be pursued: 1 ) Regulation of cellular viability (DNA synthesis, mitochondrial metabolism) and phenotypes (epidermal barrier, basement membrane) by culture conditions (media, biophysical environment); 2) Identification of molecular mediators (cytokines, extracellular matrix) of wound healing processes (angiogenesis, matrix structure); 3) Regulation of melanocyte distribution (cell density) and pigment expression (melanin content) to restore normal skin color; 4) Stimulation of angiogenesis by addition of human dermal microvascular endothelial cells and morphogenesis of vascular analogs; 5) Regulation and automation of keratinocyte growth rates and metabolism (reduction of lactic acid and ammonia) in the Kerator bioreactor; and, 6) Treatment of extensive burns with cultured skin substitutes in the clinic by paired-site comparison to meshed, split-thickness skin autograft. Comparative parameters will include: a) quantitative wound closure with skin substitutes (rates of engraftment, ratio of healed area to biopsy area, frequency of regraftng); and, b) qualitative outcome (scarring, contraction, function and cosmesis). The investigators possess all of the required expertise in cell biology, skin biochemistry and biophysics, wound physiology, chemical engineering and clinical burn care to perform these studies successfully. Accomplishment of these objectives will contribute to reduced mortality and morbidity from burns, improved materials for plastic and reconstructive surgery, and development of other tissue and organ substitutes.

National Institute of Health (NIH)
National Institute of General Medical Sciences (NIGMS)
Research Project (R01)
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Surgery, Anesthesiology and Trauma Study Section (SAT)
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Ikeda, Richard A
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University of Cincinnati
Schools of Medicine
United States
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Boyce, Steven T; Simpson, Peggy S; Rieman, Mary T et al. (2017) Randomized, Paired-Site Comparison of Autologous Engineered Skin Substitutes and Split-Thickness Skin Graft for Closure of Extensive, Full-Thickness Burns. J Burn Care Res 38:61-70
Boyce, Steven T; Zimmerman, Rachel L; Supp, Dorothy M (2015) Tumorigenicity Testing in Athymic Mice of Cultured Human Melanocytes for Transplantation in Engineered Skin Substitutes. Cell Transplant 24:1423-9
Powell, Heather M; Armour, Alexis D; Boyce, Steven T (2011) Fluorescein diacetate for determination of cell viability in 3D fibroblast-collagen-GAG constructs. Methods Mol Biol 740:115-26
Kalyanaraman, Balaji; Boyce, Steven T (2009) Wound healing on athymic mice with engineered skin substitutes fabricated with keratinocytes harvested from an automated bioreactor. J Surg Res 152:296-302
Kalyanaraman, Balaji; Supp, Dorothy M; Boyce, Steven T (2008) Medium flow rate regulates viability and barrier function of engineered skin substitutes in perfusion culture. Tissue Eng Part A 14:583-93
Armour, Alexis D; Powell, Heather M; Boyce, Steven T (2008) Fluorescein diacetate for determination of cell viability in tissue-engineered skin. Tissue Eng Part C Methods 14:89-96
Supp, Dorothy M; Karpinski, Andrea C; Boyce, Steven T (2004) Vascular endothelial growth factor overexpression increases vascularization by murine but not human endothelial cells in cultured skin substitutes grafted to athymic mice. J Burn Care Rehabil 25:337-45
Supp, Dorothy M; Wilson-Landy, Kaila; Boyce, Steven T (2002) Human dermal microvascular endothelial cells form vascular analogs in cultured skin substitutes after grafting to athymic mice. FASEB J 16:797-804