Ultrastructural features of composite skin cultures grafted onto athymic mice.

Skin substitutes composed of cultured keratinocytes with or without a dermal substrate are now being used in the treatment of burns and other cutaneous wounds. Composite skin cultures (Graftskin, LSE), consisting of epidermal keratinocytes seeded on a fibroblast-containing collagen matrix and maintained at the air-liquid interface, develop a well differentiated epidermis in vitro with many of the morphological and biochemical features of intact skin. Basement membrane-associated antigens, developing hemidesmosomes and short segments of lamina densa are present at the dermal-epidermal junction in vitro, although the LSE lacks a continuous basement membrane. As epidermal differentiation proceeds, the culture develops a stratum corneum composed of electron-dense corneocytes surrounded by extracellular lipid. However, the intercorneocyte lipid lamellae do not exhibit the repeating pattern of broad and narrow electron lucent bands observed in electron micrographs of the stratum corneum of intact skin. In this study, LSE were grafted onto full thickness wounds in athymic mice. Animals were killed 6, 15, 30 and 60 d after surgery for examination by light and electron microscopy to identify any ultrastructural changes which occurred in the culture in response to the host environment. The grafted LSE integrated well into the host tissue and remained intact throughout the 60 d study period. At the dermal-epidermal junction, a continuous basement membrane with a well defined lamina densa was established by 15 d after surgery. An extensive network of anchoring fibrils was present by 30 d after surgery. Collagen fibrils within the dermal matrix condensed by 6 d after surgery and began organising into loosely packed bundles by 15 d after surgery.(ABSTRACT TRUNCATED AT 250 WORDS)

Development of a stratum corneum and barrier function in an organotypic skin culture.

The stratum corneum of human skin is responsible for maintaining the epidermal permeability barrier. We have developed a bilayered skin culture (SC) which forms a corneum 35 +/- 1 cell layers thick 21 days after being raised to the air-liquid (A/L) interface. By the 7th day after raising to the A/L interface the corneocytes were irregularly shaped and had cross-sectional areas (CSA) of > or = 300 microns 2. By the 21st day the corneocytes had assumed polygonal shapes and had a CSA (100-250 microns 2) similar to that of human foreskin. The total lipid (TL) content of the corneum averaged 5-7% of the lyophilized weight. Ceramide content increased from 20% of TL at day 7 of A/L interface culture to 30% at day 21. Triglycerides decreased from 43% to 17% of TL during the same period. Free fatty acids comprised 5.5% of TL at day 21 of A/L interface culture. The intercorneocyte spaces contained stacks of lipid lamellae. However, the stacks lacked the Landmann unit repeat. Abnormal lamellar structures were observed in both the intra- and extracorneocyte spaces. Transepidermal water loss (TEWL) was > 4 mg/cm2 per h throughout the culture period. Lipid supplementation of the culture medium and culturing in a low humidity environment improved barrier function by 50%. However, the effects were not additive. The SC developed a near-normal corneum, but did not achieve barrier competence, due at least partially to abnormalities in lipid composition and organization.(ABSTRACT TRUNCATED AT 250 WORDS)