The development of crosslinked keratin-alginate sponges as novel biomaterials

Abstract

As one of the prospective alternative biomaterials that has recently been explored, keratin offered several advantages over other natural protein-based biomaterials such as its natural abundance, biocompatibility, presence of cell binding motifs that could improve cellular attachment and possibility to obtain autologous material that could minimize immunoresponse. Nonetheless, their further application in the biomedical field has been limited by their fragile and brittle characteristics as well as their poor mechanical properties. In this study, keratin extracted from human hair was crosslinked with alginate, a relatively bioinert material, using 1-Ethyl-3-dimethylaminopropyl Carbodiimide (EDC) as a crosslinking agent in order to improve its mechanical properties. Variations of crosslinking agent concentration and keratin-alginate ratio were studied in order to determine the effects of these differences on the physical and mechanical properties, as well as cell compliance. Successful crosslinking was confirmed with free amine groups determination as well as analysis of IR spectra, where increasing crosslinking degree (up to 83%) was achieved with higher crosslinking agent concentration. Higher crosslinking degree and higher alginate content were confirmed to increase the strength and modulus of the resulting material (from tensile, compression and flexural tests). Additionally, increasing the alginate content would increase the water uptake capacity by up to 6 times its original weight while increasing the crosslinking degree would initially increase the water uptake capacity to a certain point before reducing it. The crosslinked sponges were also shown to exhibit lower water vapor transmission rate, a characteristic that is desirable for wound dressing applications, compared to commercially available wound dressing Kaltostat®. Moreover, sponges with the highest keratin content were also revealed to be degraded by proteinase K by up to 75% of their original weight. Our results also revealed that matrices with higher keratin content enhanced the proliferation of both L929 murine fibroblasts and human dermal fibroblasts (HDF) in both 2D and 3D environment compared to matrices with higher alginate content. The resulting matrices with higher keratin content were also shown to support cell viability as well as extracellular matrice proteins, cytokines, and growth factor production with even distribution of cells inside the matrices. Matrices with higher keratin content were also revealed to upregulate production of tissue factor by HDF, suggesting it would be beneficial for hemostasis application. Based on these findings, crosslinked keratin-alginate matrices were shown to be a promising tunable materials for cell carriers or wound dressing purposes.