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In this study, we characterised the effect that seeding keratinocytes on the papillary and reticular dermis had on the extracellular matrix and tissue integrity ex vivo. Human skin explants from consented patients (n = 6) undergoing routine surgery were cultured at a liquid-air interface, dermal-side up, and autologous keratinocytes seeded on the exposed papillary or reticular layer. After 7-21 days, histological and immunohistochemical evaluation of the morphology and extracellular matrix was performed. While the dermis remained robust in all explants cultures, keratinocytes seeded on the papillary layer showed less tissue infiltration and remodelling and formed clusters across the tissue. In contrast, keratinocytes seeded on the reticular layer infiltrated the tissue homogenously with an intact single-cell-layer surface coverage and structural changes characterised by increased deposition of ground substance, glycosaminoglycans, and collagen VII in 14 days. In addition, while the papillary section showed more new laminin deposition by 14 days than the reticular section, the latter expressed more connexin 43. These differences in re-epithelialisation and extracellular matrix characteristics suggest that wound depth and graft thickness may play a key role in wound healing and indicate that ECM characteristics should be factored in when designing biomaterials for wound applications and in the selection of recipient sites when using cells for grafting.

Ex vivo culture of keratinocytes on papillary and reticular dermal layers remodels skin explants differently: towards improved wound care