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HRP plus glucose-mediated bioprinting
is desirable to use multiple materials that give a the rapid hydrogelation when it was contacted
function suitable for each part and multiple cross- with the ink containing HRP. Furthermore, there
linking systems suitable for each material. Many was an approach that uses air containing H O
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attempts have been made to develop cross-linking instead of an aqueous H O solution to control
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methods which can exert powerful influences its concentration at the ppm level . All of these
[28]
on printability, mechanical properties, and cell approaches are the direct supply of H O , which
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compatibility of hydrogels. Even today, it is might cause inhomogeneity of the resultant
desired to develop a novel bioprinting system that hydrogel network as well .
[31]
can be achieved by a biocompatible cross-linking Recently, we have developed a way to supply
process without using toxic substances because it H O indirectly to this enzymatic reaction in the
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will further enlarge the potential of bioprinting in presence of reducing sugars, such as glucose,
the fields of tissue engineering and regenerative galactose, and mannose [32,33] . In this system, the
medicine. The existing cross-linking pathways redox reactions between thiol groups in HRP
in bioprinting include physical and chemical and formed disulfide bond gradually generate
cross-linking that rely on external stimuli, such H O by consuming reducing sugar under aerobic
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as temperature, ions, or light [22,23] . More recently, conditions. It was confirmed that living cells can
enzymatic reactions have newly attracted attention be enclosed/cultured inside or on the surface
as mild and cell-friendly cross-linking methods for of resultant hydrogel with high cell viability
3D bioprinting . Specifically, bioinks containing and proliferation. Herein, we utilized glucose-
[24]
horseradish peroxidase (HRP) that has a function mediated enzymatic reaction for extrusion-based
to catalyze the conjugation of phenol and aniline bioprinting as a comixable cross-linker with living
derivatives by consuming hydrogen peroxide cells to expand its potential application. Our
(H O ) have been used for printing cell-laden bioink contains living cells, HRP, a supporting
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microparticles , 3D hydrogel constructs [26-28] , material, polymer possessing phenolic hydroxyl
[25]
or patterned hydrogels for cell immobilization (Ph) groups (Polymer-Ph), and reducing sugar
[29]
using inkjet or extrusion-based bioprinting. (Figure 1). Alginate and glucose were chosen as
Despite the advantage of a wide range of material a representative polymer chain and reducing sugar.
choices, a major consideration in this reaction In a preliminary study of the application of the
system is a way of supplying H O . To print a 3D cross-linking system, we realized that a drawback of
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hydrogel construct with living cells, the exposure the system was a non-instantaneous hydrogelation
time or the concentration of H O should be at of deposited ink. A possible approach to suppress a
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non-cytotoxic level and harmless to the activity of dispersion of the deposited ink was an enhancement
the enzyme itself . In the previous studies, H O of the viscosity of ink. To enhance the viscosity of
[30]
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was mixed into an aqueous bath solution [25,26] , a ink and the shape fidelity of printed constructs until
hydrogel substrate , or another ink to achieve the stabilization through the enzyme-mediated
[29]
[27]

Figure 1. Schematic of extrusion-based bioprinting through glucose-mediated enzymatic hydrogelation.

44 International Journal of Bioprinting (2020)–Volume 6, Issue 1