Freeform 3D Bioprinting Involving Ink Gelation by Cascade Reaction of Oxidase and Peroxidase: A Feasibility Study Using Hyaluronic Acid-Based Ink

Freeform bioprinting, realized by extruding ink-containing cells into supporting materials to provide physical support during printing, has fostered significant advances toward the fabrication of cell-laden soft hydrogel constructs with desired spatial control. For further advancement of freeform bioprinting, we aimed to propose a method in which the ink embedded in supporting materials gelate through a cytocompatible and rapid cascade reaction between oxidase and peroxidase. To demonstrate the feasibility of the proposed method, we extruded ink containing choline, horseradish peroxidase (HRP), and a hyaluronic acid derivative, cross-linkable by HRP-catalyzed reaction, into a supporting material containing choline oxidase and successfully obtained three-dimensional hyaluronic acid-based hydrogel constructs with good shape fidelity to blueprints. Cytocompatibility of the bioprinting method was confirmed by the comparable growth of mouse fibroblast cells, released from the printed hydrogels through degradation on cell culture dishes, with those not exposed to the printing process, and considering more than 85% viability of the enclosed cells during 10 days of culture. Owing to the presence of derivatives of the various biocompatible polymers that are cross-linkable through HRP-mediated cross-linking, our results demonstrate that the novel 3D bioprinting method has great potential in tissue engineering applications.