Controlling homogeneous 3D network of gelatin-carboxymethyl cellulose-chitosan self-crosslinked hydrogel with digital biomixing for multilayered extrusion 3D bioprinting

The controlled combination and balance of self-crosslinking bioprintable hydrogel components and extrusion-based three-dimensional (3D) bioprinting technologies draw high interest in developing precisely tailored complex scaffolds for biomedical and tissue engineering applications. This study developed two 3D bioprintable, gelatin-carboxymethyl cellulose-chitosan structurally balanced physically and chemically crosslinked hydrogels by homogeneous mixing of their components using a mechanically driven Biowork Pen®. Terpolymeric hydrogels were fabricated with chitosan and carboxymethyl cellulose with (C) or without (OC) oxidation by varying the concentration of gelatin. The hydrogels crosslinked physically (G-C) and chemically (G-OC) were tested to analyze their better 3D printability, mechanical, and self-healing properties in complex 3D bioprinting applications. G-C demonstrated moderate extrusion ability and 3D printability, while G-OC can print complex, self-standing, and intricate scaffolds. The biomixing Biowork Pen® significantly enhanced the mechanical properties of both G-C and G-OC by imparting the structural balance due to homogeneity in the mixing of the highly viscous gel components. The swelling and degradation rate of the G-OC hydrogel decreased by almost half due to increased chemical crosslinking density. Further, G-OC gel successfully fabricated a multilayered, large complex structure, 120 layers with a 2.9 cm height and 1.2 cm base diameter, using balanced gelatin concentration with advanced Biowork Pen®. In vitro cell culture (NIH3T3 cells) showed the wound-healing and biocompatible nature of the hydrogel with high cell proliferation and effective in vitro wound healing. Our results are paving a simple way for designing a biocompatible, biodegradable, and balanced structure of bioink components, resulting in post-printing stability by introducing balanced mechanical biomixing.