Ultrastructural features of composite skin cultures grafted onto athymic mice
Nolte, C.J.; Oleson, M.A.; Hansbrough, J.F.; Morgan, J.; Greenleaf, G.; Wilkins, L.
Journal of Anatomy 185: 325-333
1994
ISSN/ISBN: 0021-8782 PMID: 7961139 Document Number: 426528
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 intercomeocyte 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. Tightly packed bundles of collagen fibrils with a circular cross-section were observed at 30 d after surgery. Landmann unit repeats were identified in the intercomeocyte lipid lamellae at 30 d after surgery. The ultrastructural analysis of the grafted LSE demonstrates that the culture, highly differentiated in vitro, not only persisted after grafting, but responded to the biochemical features of the in vivo environment, rapidly developing additional morphological features of intact skin which may be critical to the establishment of a stable and durable skin replacement.