Expression of insulin-like growth factor I by cultured skin substitutes does not replace the physiologic requirement for insulin in vitro.
Clinical efficacy of cultured skin substitutes may be increased if their carbohydrate metabolism is optimized by understanding whether endogenous insulin-like growth factor I can substitute for exogenous insulin. Cultured skin substitutes were prepared and incubated at the air-liquid interface for 4 wk in media containing 0.5 or 5 microg per ml insulin, 10 or 50 ng per ml insulin-like growth factor I, or 0 insulin and 0 insulin-like growth factor I (negative control). In situ hybridization showed that the epidermal and dermal cultured skin substitute components express insulin-like growth factor I mRNA throughout the 28 d interval. Immunohistochemistry confirmed the expression of insulin-like growth factor I protein by the human keratinocytes and fibroblasts in cultured skin substitutes. Insulin-like growth factor I at 10 or 30 ng per ml could partially replace insulin in a clonal assay of keratinocyte growth. 3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT) assays showed significantly higher values in cultured skin substitutes incubated with insulin at incubation days 14 and 28 compared to negative control or the 10 ng per ml insulin-like growth factor I condition. Cultured skin substitutes incubated in 50 ng per ml insulin-like growth factor I had MTT values similar to the insulin-treated cultured skin substitutes at day 14, but were significantly lower by day 28. Light microscopy agreed with MTT data showing that cultured skin substitutes grown with insulin media had multiple layers of nucleated keratinocytes and stratum corneum at days 14 and 28. The negative control and 10 ng per ml insulin-like growth factor I exhibited poor cultured skin substitute epidermal morphology throughout the experiment. In contrast, the cultured skin substitutes in 50 ng per ml insulin-like growth factor I were similar to the insulin-treated cultured skin substitutes at day 14, but by day 28 had deteriorated to resemble the negative control. Bromodeoxyuridine incorporation at day 28 was significantly higher for 5 microg per ml insulin cultured skin substitutes versus all other treatment groups. These data suggest that medium containing 5 microg per ml insulin supports greater physiologic stability in cultured skin substitutes over time, and that expression of insulin- like growth factor I by keratinocytes and fibroblasts in cultured skin substitutes is not sufficient to fully replace the requirement for exogenous insulin in vitro.
Swope
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VB
Department of Surgery, University of Cincinnati College of Medicine, Cincinnati, Ohio, USA.
Supp
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Greenhalgh
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DG
Warden
G D
GD
Boyce
S T
ST
D001973:Bromodeoxyuridine / Q000493:pharmacokinetics; D002455:Cell Division / Q000187:drug effects; D002478:Cells, Cultured; D003470:Culture Media / Q000737:chemistry; D000078404:Epidermal Cells; D004817:Epidermis / Q000378:metabolism / Q000502:physiology; D006801:Humans; D007328:Insulin / Q000494:pharmacology*; D007334:Insulin-Like Growth Factor I / Q000378:metabolism* / Q000494:pharmacology; D015603:Keratinocytes / Q000166:cytology; D017768:Skin, Artificial
D003470:Culture Media; D007328:Insulin; D007334:Insulin-Like Growth Factor I; D001973:Bromodeoxyuridine