Development of a skin model based on insoluble fibrillar collagen

A biocompatible, 3-dimensional, noncontracting, crosslinked collagen matrix was adapted to promote differentiation of epidermal keratinocytes. To produce the matrix, a 3% wt/wt dispersion of insoluble bovine collagen containing 5 mg polylysine/g collagen in 0.001 N HCl was blended, lyophilized, and crosslinked using a dehydrothermal technique. Matrices 4 cm2 and 3 mm thick were seeded with human dermal fibroblasts (1 x 10(5)/cm2). After 5 days in culture, the matrices were seeded with human epidermal keratinocytes (1 x 10(5)/cm2). The cultures were grown submerged for 1 week and raised to the liquid/air interface for 3 weeks to promote epidermal differentiation. Based on morphology and immunological staining with antibodies for human involucrin, keratin 1 (k1), filaggrin, and loricrin, the state of differentiation of the epidermal layer was nearly equivalent to that seen with cultures grown on contracted collagen lattices produced according to the methodology described in the literature and similar to the pattern produced in normal neonatal foreskin. These results demonstrate the usefulness of an in vitro skin model employing a crosslinked collagen matrix that permits the incorporation of additional covalently linked bioactive molecules during matrix formation.