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The effects of burn blister fluid on cultured keratinocytes.

OBJECTIVE
Previous studies have suggested that burn blister fluid (BBF) may be detrimental to the healing of the underlying wound bed. In this study, the effects of burn blister fluid on cultured keratinocyte proliferation and differentiation were examined and quantitated using various techniques.


METHODS
At three different concentrations (2%, 10%, 20% in 20% fetal bovine serum/complete culture medium), 19 BBFs were tested in triplicate using 12 populations of cultured keratinocytes. All BBFs were collected from partial thickness burns within 72 hours of injury,. BBF was added on day 4 of the keratinocyte culture. The effect on proliferation and viability was assessed using trypan blue dye exclusion. Multiparameter flow cytometric analysis was used to quantitate population kinetics and cell size distribution. Keratinocyte differentiation was determined using immunohistochemical staining of differentiation markers and quantitation of cornified envelope formation.


RESULTS
Relative to control fluid, the BBF caused a variable effect on proliferation, ranging from 67% inhibition to 103% stimulation with an overall 4% inhibition. The range of keratinocyte viability was narrower, with a similar overall 4% reduction. Using flow cytometry to analyze RNA/DNA content and cell size, the BBF caused a subtle shift in keratinocyte population kinetics and cell size distribution toward larger, less rapidly dividing cells. The BBF had no significant effect on expression of the differentiation markers, filaggrin and involucrin. Finally, the BBF did not alter terminal differentiation as it did not influence formation of cornified envelopes (BBF = 9.1 +/- 4.8%, control = 9.9 +/- 6.6%).


CONCLUSION
Previous biochemical analysis has shown that BBF consists primarily of human serum filtrate with locally produced acute reactants. Our study suggests that BBF is biologically similar to serum and does not significantly alter keratinocyte proliferation or differentiation in vitro.