The creation of partial or complete human epidermis represents a critical aspect and the major challenge of skin tissue engineering. This work was aimed at investigating the effect of nano- and micro-structured CHT membranes on human keratinocyte stratification and differentiation. To this end, nanoporous and microporous membranes of chitosan (CHT) were prepared by phase inversion technique tailoring the operational parameters in order to obtain nano- and micro-structured flat membranes with specific surface properties. Microporous structures with different mean pore diameters were created by adding and dissolving, in the polymeric solution, polyethylene glycol (PEG Mw 10,000 Da) as porogen, with a different CHT/PEG ratio. The developed membranes were characterized and assessed for epidermal construction by culturing human keratinocytes on them for up to 21 days. The overall results demonstrated that the membrane surface properties strongly affect the stratification and terminal differentiation of human keratinocytes. In particular, human keratinocytes adhered on nanoporous CHT membranes, developing the structure of the corneum epidermal top layer, characterized by low thickness and low cell proliferation. On the microporous CHT membrane, keratinocytes formed an epidermal basal lamina, with high proliferating cells that stratified and differentiated over time, migrating along the z axis and forming a multilayered epidermis. This strategy represents an attractive tissue engineering approach for the creation of specific human epidermal strata for testing the effects and toxicity of drugs, cosmetics and pollutants.
Keywords: cell-material interactions; chitosan; differentiation; epidermis; keratinocytes; membranes; microporous; nanoporous.