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

Abstract

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.

Keywords: ECM; Keratinocytes; Papillary; Reticular.

Fig. 1

Skin explants cultured upside down at an air–liquid interface for 14 day support keratinocyte proliferation and tissue infiltrations. Representative H&E stains of split-thickness skin shaved to a papillary and b the reticular dermal layer depth. c Schematic representation of the upside-down skin explant culture and keratinocyte seeding onto exposed dermis. d HE stained skin explants after 7 and 14 days in culture. BM is membrane and KL is keratinised layer. e Representative fluorescence micrographs of anti-CK14 stained explant sections after 14 days in culture with and without keratinocytes (red arrows). Epidermal keratinocytes (bottom green fluorescence) were not evaluated. Scale bars are 200 µm.

Fig. 2

Keratinocytes cultured on the papillary and reticular dermal surfaces at the liquid–air interface for 14 days remodelled dermal-ECM differently. Movat-Russell’s modified pentachrome stain showing: a Control sections cultured with no keratinocytes seeded and b magnification of the control surface; Papillary (c) and reticular (d) dermal sections seeded with keratinocytes. Representative micrographs of the papillary and reticular sections stained with col VII (e, f respectively), laminin (g, h respectively), and both Cx26 and 43 (j, k respectively). (i) Representative laminin stained reticular section after 21 days in culture. (l) Graphical representation of Cx26/43 and laminin immunostains of the KC seeded layer. Laminin in blood vessels and basement membrane (BM) was not factored in (n = 3 and error bars = SEM). (M) Representative control section with no antibody.