Introduction: Injectable hydrogel systmes have received a great attention in the biomedical research fields due to the minimally invasiveness as well as the structural similarity to the natural extracellular matrix and multi-tunable properties. Up to date, various kinds of biomaterials have been utilized to create injectable hydrogel matrices for therapeutic implants and therapeutic vesicles. Keratin, derived from hair, has emerged as a fascinating biomaterial due to excellent biocompatibility, biodegradability, low immunogenecity, and favorable cellular interactions. While the natural polymer has been widely used for biomedical applications, poor solubility of keratin in aqueous solutions has been recognized as a critical limitation for use in a broad range of applications including development of injectable biomaterial systems. Herein, we developed keratin-based in situ crosslinkable hydrogels through the enzymatic reaction. We fisrt desinged a water soluble karatin by conjugating poly(ethylene glycol) (PEG) molecules tethered with tyrmine (TA), which can be crosslinked to form the hydrogel network. We next evaluated its physico-chemical and biological properteis, showing a great potential as bioactive injectable materials Materials and Methods: Human hair keratin was extracted by slight modified Shindai method. Keratin-PEG-TA (KPT), a water soluble keratin, was synthesized by conjugating amine-PEG-TA (PEG M.W. = 4,000) to keratin backbone via EDC/NHS chemistry with different PEG feed amount. The chemical structure of the KPT was confirmed using 1H NMR and TGA analysis. The physico-chemical properties of KPT hydrogels were examined, such as gelation time, rheological properties depending on HRP or H_2O_2 concentrations. In vitro cytocompatibility of hydrogels was also evaluated using human dermal fibroblasts cultured with KPT hydrogel extracts. Results and Discussion: The chemical structures of KPT were confirmed by ~1H-NMR and TGA analysis, demonstrating PEG and TA were sucessfully conjugated into keratin. We conjugated PEG as a linker to increase water solubility of keratin and confirmed that 5 wt% of KPT conjugate was quickly (-10 sec) and homogeneously dissolved in distilled water though the same concentration of intact keratin was not completely dissolved even with the vigorous vortexing for 30 min. Figure 1a illustrates the schemetic representation of polymer synthesis and hydrogel formation through HRP-mediated crosslinking reaction, forming a C-C bond or C-O bond between phenol moieties. We also evaluated the physico-chemical properties, such as gelation time (ranged from 2 to 100 sec) and mechanical strength (ranged from 100 to 2400 Pa) of the KPT hydrogels (Figure 1b) depending on concentrations of HRP and H_2O_2, showing the multi-tunable properties. Futhermore, we carried out in vitro cell study to confirm cytocompatibility of the KPT hydrogels, exhibiting excellent cytocompatibility. Conclusions: We have developed a keratin-based in situ cross-linkable hydrogels. It was demonstrated that water solubility of KPT was significantly increased by introducing PEG molecules between the polymer backbone and TA molecules, and the physico-chemical properties could be controlled easily by varying the concentrations of HRP and H_2O_2 with excellent cytocompatibility. Taken together, we believe that our Keratin-based hydrogels have a great potential for use as injectable materials for a broad range of biomedical applications, including either therapeutic implnats or therapeutic vesicles.
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