142 Epidermal equivalents on nonwoven collagen scaffolds

Abstract

The use of natural polymers such as collagen for tissue engineering has many advantages providing cells with an environment of natural topography and rich of bioactive cues. Nevertheless, as compared to synthetic polymers, electrospinning of such materials is still a methodological challenge. Two nonwoven scaffolds were fabricated by collagen dispersion in hexafluoroisopropanol (HFIP) (microfibrous) and in acetic acid (nanofibrous) and stabilized by genipin. The helical structure of collagen was retained in acetic acid, but not in HFIP. Epidermal equivalents were prepared using human primary keratinocytes on both types of materials. There were up to seven layers of live cells with well-defined granular layer in the stratified epithelium obtained on nanofibrous scaffolds. The stratum corneum (SC) was well pronounced and desquamation was evident. The patterns of involucrin expression were similar, but not identical to the normal epidermis: it demarcated all suprabasal layers, not the uppermost ones as it was shown for filaggrin. The basement membrane (BM) component laminin 5 was deposited on the surface of the fibers, but a compact BM structure was not formed. CK5 expression was not limited to the basal layer, but observed in all living layers of the equivalent. The stratified epithelium formed on the microfiber materials also had a well-distinguishable granular layer and SC. But SC was thinner as compared to nanofiber samples. The expression pattern of involucrin was much closer to normal than on nanofiber scaffold while filaggrin and CK5 showed localization similar to nanofiber scaffolds. The laminin 5 positivity was insignificant. Thus, we have managed to obtain nonwoven collagen matrices of two different types (nanofibrous and microfibrous) which both support the formation of stratified epithelium. Of note, the use of nanofibrous material yields epidermal equivalents with more prominent basal layer and synthesis of BM components, while equivalents on microfibrous material gave more “normal” patterns of involucrin and filaggrin expression.