In Vivo Evaluation of Three-Dimensional Printed, Keratin-Based Hydrogels in a Porcine Thermal Burn Model

Keratin is a natural material that can be derived from the cortex of human hair. Our group had previously presented a method for the printed, sequential production of three-dimensional (3D) keratin scaffolds. Using a riboflavin-sodium persulfate-hydroquinone (initiator-catalyst-inhibitor) photosensitive solution, we produced 3D keratin-based constructs through ultraviolet crosslinking in a lithography-based 3D printer. In this study, we have used this bioink to produce a keratin-based construct that is capable of delivering small molecules, providing an environment conducive to healing of dermal burn wounds in vivo, and maintaining stability in customized packaging. We characterized the effects of manufacturing steps, such as lyophilization and gamma irradiation sterilization on the properties of 3D printed keratin scaffolds prepared for in vivo testing. Keratin hydrogels are viable for the uptake and release of contracture-inhibiting Halofuginone, a collagen synthesis inhibitor that has been shown to decrease collagen synthesis in fibrosis cases. This small-molecule delivery provides a mechanism to reduce scarring of severe burn wounds in vitro. In vivo data show that the Halofuginone-laden printed keratin is noninferior to other similar approaches reported in literature. This is indicative that the use of 3D printed keratin is not inhibiting the healing processes, and the inclusion of Halofuginone induces a more organized dermal healing after a burn; in other words, this treatment is slower but improves healing. These studies are indicative of the potential of Halofuginone-laden keratin dressings in dermal wound healing. We aim to keep increasing the complexity of the 3D printed constructs toward the production of complex scaffolds for the treatment and topographical reconstruction of severe burn wounds to the face. Impact statement Keratin-based photosensitive bioink can be used to three-dimensionally (3D) print complex scaffolds for the topical treatment of dermal burn wounds. We have developed reproducible protocols that allow us to 3D print large volumes of keratin-based hydrogels, and we now have a better understanding of how 3D printed geometrical features, crosslinking properties, or mass are altered due to the manufacturing processes. The printed hydrogels improved healing parameters in vivo on a porcine thermal burn model, indicative of the potential of the scaffolds for the regeneration of complex dermal wounds. Overall, our approach elucidates on physiological and topographical 3D healing of burn wounds.