Treatment of burns with skin substitutes.

More than 2 million persons sustain thermal injuries in the United States annually (Monafo and Crabtree, 1985) and more than 10,000 burn victims die (Collini and Kealey, 1989). The principal factors affecting mortality are the total area burned and the area of third degree (full thickness) burns (Tompkins et al., 1985) with wound sepsis being the leading cause of mortality. Early aggressive excision and immediate covering of the wounds improve survival (Herndon and Parks, 1986). Various biological and synthetic substrates have been employed to replace the injured skin. Most of these provide a permeability barrier which substitutes for the epidermal function of the lost skin. An ideal skin replacement should also provide a substitute for dermis, which provides both support and stability for the epidermal replacement and prevents wound contraction. The dermal and epidermal replacement should be firmly integrated by a complete basement membrane zone (BMZ).

Dermal mast cell granules bind interstitial procollagenase and collagenase.

In order to identify structures in human skin that bind collagenase, sections from frozen or paraffin-embedded skin were incubated with either procollagenase or activated collagenase. After washing, bound procollagenase or collagenase was detected by immunofluorescence microscopy. In normal skin, procollagenase bound only to isolated granular dermal cells that were identified as mast cells on the basis of staining with fluoresceinated avidin and pinacyanol erythrosinate. When mast cells were degranulated by exposure to the ionophore A23187, extracellular granules bound procollagenase. Of various pathologic conditions examined, the highest binding of procollagenase occurred in specimens of urticaria pigmentosa. Procollagenase bound to granular cells and to abundant granules scattered throughout the dermis. Binding could be abolished by pre-treatment of tissue sections with heparinase or by pre-incubation of procollagenase with soluble heparin, suggesting that heparin is the binding agent in the granules. Activated collagenase also bound to dermal mast cells but in addition bound strongly to the dermal collagen. Enzymatic activity of activated collagenase was not inhibited by heparin in concentrations up to 10 mg/ml. There is evidence that mast cell tryptase can contribute to procollagenase activation. This study further supports a role for mast cells in collagenolysis by demonstrating that heparin from mast cells binds procollagenase and possibly serves as a reservoir for procollagenase, which may then subsequently be activated.

Epithelial differentiation in the absence of extracellular matrix.

To investigate the regulation of epithelial differentiation, normal human epidermal keratinocytes were cultured floating on the surface of culture medium without attachment to a solid substrate. Keratinocytes spread out on the surface of the medium, proliferated and differentiated either into several flat lacy sheets 1 to 3 cells thick (on medium containing 0.15 mM calcium) or formed one single aggregate of cells from 5 to 15 cells in thickness on medium containing 1.15 mM calcium. The cell aggregates demonstrated a pattern of ordered epithelial differentiation. Levels of progressive differentiation resembling the structure of normal human epidermis were identified by light microscopy, immunohistochemistry, and electron microscopy. Differentiation proceeded from cells at the air side toward cells at the medium side with basal appearing cells on the air side and keratinocytes expressing filaggrin and involucrin on the side toward the medium. These results demonstrate that organized epithelial differentiation can occur in the absence of extracellular matrix. In contrast with other air-liquid interface cultures, epithelial differentiation in the absence of extracellular matrix progresses from air towards medium.

In vitro reconstitution of skin: fibroblasts facilitate keratinocyte growth and differentiation on acellular reticular dermis.

Extensive full-thickness burns require replacement of both epidermis and dermis. We have described a method in which allogeneic dermis from engrafted cryopreserved cadaver skin was combined with cultured autologous keratinocytes. In the present study we combined human keratinocytes and fibroblasts, and acellular human dermis in vitro and transplanted this "reconstituted skin" into athymic mice. Both human papillary dermis in which the basement membrane zone has been retained and human reticular dermis that has been repopulated with human dermal fibroblasts are good substrates for keratinocyte attachment, stratification, growth, and differentiation. Both of these dermal preparations can be lyophilized and stored at room temperature without losing their ability to support keratinocyte growth. In contrast, human papillary dermis that has been treated with trypsin lacks laminin and collagen type IV in the BMZ and supports keratinocyte attachment and differentiation less well.