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

Shinji Sakai,Ryohei Harada,Takashi Kotani

doi:10.3390/biom11121908

Abstract

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.

Highlights

This study aimed to report a freeform bioprinting process involving gelation mediated by the enzymatic cascade reaction of choline oxidase (COD) and horseradish peroxidase (HRP) (Figure 1)
We studied the effect of the 3D printing process, involving extrusion from a microneedle and COD/HRP-mediated ink gelation in a support bath, on cells
Demonstrated that risk of of damaging cells due to the unwanted generation of by the oxidase remaining in damaging cells due to the unwanted generation of H2 O2 by the oxidase remaining in thethe printed hydrogel constructs cancan be be reduced byby using

Summary

Introduction

Three-dimensional (3D) printing, known as additive manufacturing or rapid prototyping, has been driving innovations in various fields, such as manufacturing, education, and medicine. Bioprinting is a subcategory of 3D printing and is defined as the technology for the fabrication of biological tissues and organs. It allows the fabrication of cell-laden constructs with spatially complex structures that are otherwise difficult to fabricate using conventional biofabrication techniques [1,2]. The ink that forms cell-laden hydrogels in the manufacturing process has gained much popularity [3]. A hydrogel is composed of a network of cross-linked hydrophilic polymer chains in an aqueous solution

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