Fartasch, M., M. Ponec, and M. Rosdy. The Development of A Structurally Competent Epidermal Barrier in Air-Exposed Keratinocyte Cultures: A Time-Course Study. J. Invest. Dermatol. 1996. 107(4): 656. Abstract #3. [Reprinted with permission from Blackwell Science, Inc.].

Recently we could show that an air-exposed (A/E) keratinocyte culture reconstituted in chemically defined medium formed a structurally and biochemically normal permeability barrier. To characterize the critical events associated with the development of the barrier, the A/E cultures were harvested on day 7, 10, 13 and 16 of air-exposure. At each time point, the cultures were split for immunohistochemical (differentiation markers: involucrin, filaggrin, loricrin; proliferation markers, PCNA, KI-67), and ultrastructural (OsO4 and ruthenium tetroxide (RuO4) post-staining) studies. Quantification of the layers of the viable parts of the epidermis (vE) was performed after a standardized procedure. At time points 7, 10, and 13 days a disturbed differentiation with spongiosis of the basal cell layer was evident. With increasing thickness of the vE (7-11-12 layers) the intercellular edema decreased, and stratum granulosum (SG) layers and stratum corneum (SC) increased (SG: 1-3-3 layers, SC: 12-12-21). Further evaluation of SG revealed rounded keratohyalin granules (KH) instead of normal stellate KH. With RuO4-staining an impaired structural organization and distribution of SC intercellular lipids and intracorneocytic lipid droplets were seen, resulting in barrier immaturity. At time point 16 days, normalization of differentiation and proliferation occurred with the formation of regular epidermal lipid layers. The study shows that a barrier maturation process seems to be functioning in A/E cultures, comparable to the ontogeny of the epidermal barrier in mammalian skin.