A cytocompatible inkjet bioprinting approach that enables the use of a variety of bioinks to produce hydrogels with a wide range of characteristics is developed. Stabilization of bioinks is caused by horseradish peroxidase (HRP)-catalyzed cross-linking consuming hydrogen peroxide (H O ). 3D cell-laden hydrogels are fabricated by the sequential dropping of a bioink containing polymer(s) cross-linkable through the enzymatic reaction and H O onto droplets of another bioink containing the polymer, HRP, and cells. The ≈95% viability of enclosed mouse fibroblasts and subsequent elongation of the cells in a bioprinted hydrogel consisting of gelatin and hyaluronic acid derivatives suggest the high cytocompatibility of the developed printing approach. The existence of numerous polymers, including derivatives of polysaccharides, proteins, and synthetic polymers, cross-linkable through the HRP-catalyzed reaction, means the current approach shows great promise for biofabrication of functional and structurally complex tissues.

We report an extrusion-based bioprinting approach, in which stabilization of extruded bioink is achieved through horseradish peroxidase (HRP)-catalyzed cross-linking consuming hydrogen peroxide (H2O2) supplied from HRP and glucose. The bioinks containing living cells, HRP, glucose, alginate possessing phenolic hydroxyl (Ph) groups, and cellulose nanofiber were extruded to fabricate 3D hydrogel constructs. Lattice- and human nose-shaped 3D constructs were successfully printed and showed good stability in cell culture medium for over a week. Mouse 10T1/2 fibroblasts enclosed in the printed constructs remained viable after 7 days of culture. It was also able to switch a non-cell-adhesive surface of the printed construct to cell-adhesive surface for culturing cells on it through a subsequent cross-linking of gelatin possessing Ph moieties. These results demonstrate the possibility of utilizing the presented cross-linking method for 3D bioprinting.

Three-dimensional (3D) bioprinting technology is now one of the best ways to generate new biomaterial for potential biomedical applications. Significant progress in this field since two decades ago has pointed the way toward use of natural biopolymers such as polysaccharides. Generally, these biopolymers such as alginate possess specific reactive groups such as carboxylate able to be chemically or enzymatically functionalized to generate very interesting hydrogel structures with biomedical applications in cell generation. This present review gives an overview of the main natural anionic polysaccharides and focuses on the description of the 3D bioprinting concept with the recent development of bioprinting processes using alginate as polysaccharide.

Hydrogels were obtained from aqueous solution containing polymer(s) possessing phenolic hydroxyl moieties through horseradish peroxidase (HRP)-catalyzed reaction without direct addition of H 2 O 2 . In this hydrogelation process, H 2 O 2 was generated from HRP and glucose contained in the aqueous solution, that is, HRP functioned not only as a catalyst, but also as a source of H 2 O 2 . The gelation time and mechanical properties of the resultant hydrogel could be altered by changing the concentrations of HRP and glucose. Cytocompatibility of the hydrogelation process was confirmed from cell studies using mouse 10T1/2 fibroblast cells.

A simple fabrication method for cell micropatterns on hydrogel substrates was developed using an inkjet printing system that induced hydrogel micropatterns. The hydrogel micropatterns were created from inks resulting in cell-adhesive and non-cell-adhesive printed regions by horseradish peroxidase-catalyzed reaction onto non-cell-adhesive and cell-adhesive hydrogel substrates, respectively, to obtain the cell micropatterns. Cell-adhesive and non-cell-adhesive regions were obtained from gelatin and alginate derivatives, respectively. The cells on the cell-adhesive regions were successfully aligned, resulting in recognizable patterns. Furthermore, the proposed system permits the patterning of multiple cell types by switching the non-cell-adhesive region to the cell-adhesive region in the presence of growing cells. Also, we could fabricate disc- and filament-shaped small tissues by degrading the non-cell-adhesive substrates having dot- and line-shaped cell-adhesive micropatterns using alginate-lyase. These results indicate that our system is useful for fabrication of tailor-made cell patterns and microtissues with the shape defined by the micropattern, and will be conducive to a diverse range of biological applications.