Comparison of cultured and uncultured keratinocytes seeded into a collagen-GAG matrix for skin replacements

A well-characterised collagen-glycosaminoglycan (CG) matrix functions as an extracellular matrix analogue (ECMA) of dermis on full-thickness wounds. T he epidermis can be reconstituted by seeding autologous uncultured keratino cytes into the matrix prior to grafting. We hypothesised that seeding the C G matrix with keratinocytes cultured to sub-confluence may provide the ECMA with more proliferating keratinocytes than with uncultured keratinocytes. Autologous cells were isolated from split-thickness skill grafts and cultur ed to sub-confluence. ECMAs were seeded by centrifuging cultured (n = 8) or uncultured (n = 8) autologous keratinocytes into a CG matrix at a density of 100 000 cells/cm(2), then applied onto full-thickness wounds on Yorkshir e pigs. Gross and histologic observations were made up to 21 days post-graf ting. At 14 days, a fully differentiated epidermis was present on all graft sites, but the epidermis of the cultured-cell-seeded matrices was thicker, 180 (19) mu m, than the uncultured-cell-seeded matrices, 110 (18) mu m. Th e epidermis of cultured-cell-seeded matrices was acanthotic, containing 14 (4) cell layers, as compared to uncultured-cell-seeded matrices, 9 (1) cell layers. The number of subepithelial keratinocyte cysts/cm cross-section pr esent in the neodermis was also greater in cultured-, 1.35 (0.37), than in uncultured-cell-seeded matrices, 0.47 (0.35). Epidermal confluence on day 1 4 was 96 (3)% on cultured-cell-seeded grafts and 50 (17)% on uncultured-cel l-seeded grafts. These results are consistent with the hypothesis that the process of in vitro cell cultivation increases the proportion of dividing c ells in preference to differentiated cells. This technology may be useful i n reconstruction of specialised bilayer tissues with minimal donor sites.