Speaker
Description
Introduction
To advance 3D bioprinting, it is essential to develop bioinks with appropriate rheological (e.g., flow behavior, yield stress) and gelation (e.g., kinetics, storage modulus) properties to enhance printability. Previous studies have incorporated additional components into bioinks, such as rheology modifiers (e.g., nanofibers, nanoparticles) and secondary crosslinking agents (e.g., radical generators, other polymers). However, these additional components can complicate printing systems and potentially compromise biocompatibility. Therefore, improving printability by developing bioinks without additional components is necessary.
Here, we developed a bioink consisting of horseradish peroxidase (HRP) and phenylboronic acid-functionalized hyaluronic acid (HA-nAPBA), and applied it to a printing method that utilizes exposure to air containing ppm-level H2O2 (Figure 1). This printing system utilizes two key features: (1) shear-responsive rheological behavior via dynamic boronic acid–diol bonds, and (2) gelation via H2O2-responsive boron–carbon (B–C) bond cleavage followed by HRP-mediated crosslinking. In this study, we first investigated how the type of n-aminophenylboronic acid (nAPBA; n = 2, 3, 4) grafted onto HA, as well as the concentrations of HRP and H2O2, affected the rheological and gelation properties. Then, the relationship between these parameters and printability was evaluated. Finally, mammalian cells were cultured within the fabricated constructs.
Methods
Each nAPBA was grafted onto the HA by amide coupling. The binding affinity of each nAPBA for diols was spectroscopically evaluated using fluorescent Alizarin Red S. Gelation was tested by mixing PBS (500 µL) containing HA-nAPBA (0.80 w/v%), HRP (1.25–125 U/mL), and H₂O₂ (1.25–10 mM). Printability was assessed by printing a 32 × 32 mm, 4-layer lattice under ppm-level H₂O₂ using a 3D printer (BioX, CELLINK, Gothenburg, Sweden) with a 27G conical nozzle. Cell-laden constructs were printed with a bioink with HepG2 cells (1.0 × 10⁶ cells/mL), HA-3APBA (1.5 w/v%), and HRP (50 U/mL), and cultured for 14 days.
Results and Discussion
The binding affinity between nAPBA and diols followed the ratio HA-2APBA: HA-3APBA: HA-4APBA = 1: 18.8: 7.2, and the viscosity of 1.0 w/v% solutions followed the same order. HA-2APBA solution did not form a hydrogel, while HA-3APBA and HA-4APBA solutions formed a hydrogel. Increasing the HRP concentration from 2.5 to 125 U/mL shortened the gelation time of HA-4APBA from 15 ± 3.3 s to 2.7 ± 0.7 s, and that of HA-3APBA from 119 ± 5.3 s to 12 ± 2.4 s. These gelation kinetics are probably explained by the combined effects of B-C bond cleavage and HRP-mediated reaction. Subsequently, HRP and H2O2 concentration-dependent printability of HA-3APBA ink was evaluated. Increased HRP and H2O2 concentrations improved printability, but also resulted in non-uniform line width and ink clogging. HepG2 cells in the printed constructs proliferated and formed aggregates until day 10, and cell-laden constructs were stable until day 14 (Figure 2).
Conclusion
This study demonstrated that bioinks composed of HA-nAPBA and HRP can improve printability without the need for additional components. In the future, printability may be further improved by combining polymers with phenylboronic acid derivatives that have higher binding affinity.
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