Keratinocytes undergo a complex process of differentiation to form the stratified stratum corneum layer of the skin. In most biomimetic skin models, a 3D hydrogel fabricated out of collagen type I is used to mimic human skin. However, native skin also contains keratin, which makes up 90% of the epidermis and is produced by the keratinocytes present. We hypothesized that the addition of keratin (KTN) in our collagen hydrogel may aid in the process of keratinocyte differentiation compared to a pure collagen hydrogel. Keratinocytes were seeded on top of a 100% collagen or 50/50 C/KTN hydrogel cultured in either calcium-free (Ca-free) or calcium+ (Ca+) media. Our study demonstrates that the addition of keratin and calcium in the media increased lysosomal activity by measuring the glucocerebrosidase (GBA) activity and lysosomal distribution length, an indication of greater keratinocyte differentiation. We also found that the presence of KTN in the hydrogel also increased the expression of involucrin, a differentiation marker, compared to a pure collagen hydrogel. We demonstrate that a combination (i.e., containing both collagen and kerateine or “C/KTN”) hydrogel was able to increase keratinocyte differentiation compared to a pure collagen hydrogel, and the addition of calcium further increased the differentiation of keratinocytes. This multi-protein hydrogel shows promise in future models or treatments to increase keratinocyte differentiation into the stratum corneum.
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