Fabrication of Keratin Intermediate Filament Proteins (KIFP) Cryogels for Wound Dressing Purposes

Abstract

Protein based biomaterials have had many successes over the year especially for its biomedical application due to its abundance in nature and safety. Keratin is one of the most abundant protein in human body and often studied in the field of biomaterials. It can be extracted from human hair and nails or even animal feather, wools, and hooves. Furthermore, it also has a good biocompatibility for fibroblast cells, supporting its adhesion and proliferation that is crucial in skin regeneration. Although, keratin scaffolds for wound dressing often lacks a good physical properties and stability in aqueous environment. Cryogelation method is a purely physical method to prepare and improve polymer or scaffolds physical characteristics without any addition of chemical cross-linker that could affect its biocompatibility. It consisted of 3 stages; freezing, storage, and thawing. It utilizes the solvent crystallization, leading to formation of unfrozen liquid microphase which is the keratin that is not frozen. Gelation process will begin in the unfrozen part which will cause the formation of microgel fraction that may achieve interconnected structure with each other. The use of cryogelation for keratin therefore hypothesized to improve mechanical properties of keratin scaffold without significantly affecting its biocompatibility towards human fibroblasts. Shindai’s method of extraction will be utilized in the extraction of KIF. KIF and thiol content in the extracted solution will then be measured using Bicinchoninic Acid Assay (BCA) and ellman's assay. Samples then diluted or concentrated to the desired concentration and then critical concentration (CCG) and gelation test will be done to found out the CCG and the amount of freeze thaw cycles needed to gel each KIF sample concentrations. Cryogel then fabricated according to the result of CCG and gelation assay. Cryogels then measured for its viscosity and rheology and the rest of the samples will be freezedried to form a porous scaffold. The final scaffold then analyzed using mechanical compression test and cyclic compression test, FTIR analysis, SEM imaging, water uptake test, 3D cell study using alamar blue assay, and cell growth pattern study using SYTOX green and hoechst. Statistical vii analysis was also done to assess the data significance. It is shown from the result that cryogelation method was able to improve the physicochemical properties of sponges without significantly affect the biocompatibility towards human dermal fibroblasts. Furthermore, it is also feasible to fabricate a wound dressing material out of KIF using optimized cryogelation method.