Speaker
Description
Introduction
For 3D bioprinted structures to function effectively as tissues, it is essential to promote the proliferation of encapsulated cells. At the same time, it is important to be able to print with high structural fidelity to the blueprints designed to perform biological functions. Different techniques are currently used to meet each of these requirements. Cell proliferation is supported by a porous scaffold that facilitates the diffusion of nutrients and oxygen and provides sufficient space for growth. Conversely, structural fidelity to blueprints can be improved by incorporating nanofibers into bioinks to enhance shear-thinning properties. However, it is still difficult to achieve both improvements simultaneously. In this study, we propose a novel strategy to achieve enhancements in both cell proliferation and structural fidelity to blueprints using a single bioink component.
Our strategy involves immobilizing gelation-inhibiting molecules on nanofibers and incorporating them into the bioink. For gelation, we used a phenol-modified polymer that can be crosslinked by a horseradish peroxidase (HRP)-catalyzed reaction in the presence of hydrogen peroxide (H2O2). To inhibit gelation around the nanofibers, we immobilized catalase on silk fibroin nanofibers (SFNFs), which are known as cytocompatible material, to decompose H2O2 around the nanofibers. This is referred to as SFNF-catalase.
Method
Catalase was immobilized on SFNF and silk fibroin fiber (SFF) using carbodiimide chemistry. To observe crosslinking inhibition around catalase-immobilized SFF (SFF-catalase), phenol-modified hyaluronic acid (HA-Ph) (0.5% w/v) and HRP (5 U/mL) were mixed with SFF or SFF-catalase, and hydrogel films were prepared with H2O2 supplied from the air. The formation of dityrosine bonds through the crosslinking of phenol moieties was observed using a fluorescence microscope (ex: 320 nm, em: 404 nm). Cell-laden square structures were printed from a mixture of SFNF or SFNF-catalase (0.1 mg/mL), HA-Ph (0.5% w/v), HRP (5 U/mL), and HepG2 human liver cancer cells (1.4×106 cells/mL) with H2O2 supplied from the air and cultured for 7 days. The proliferation rate of the enclosed cells was evaluated by Calcein AM/PI staining.
Result and discussion
Fluorescence mapping of dityrosine formed via HRP-catalyzed crosslinking showed that the fluorescence intensity was lower around SFF-catalase than farther from the fibers. In contrast, no difference was observed when catalase-free SFF was used. This result demonstrates that the catalase immobilized on SFFs inhibited crosslinking of phenol groups around the fibers. As shown in the attached figure, independent of the immobilization of catalase, SFNFs contributed to achieving a structure close to the blueprint. Interestingly, the cells in the printed constructs containing SFNF-catalase showed greater proliferation compared to those in the constructs containing catalase-free SFNF. This result also supports the above-mentioned result, indicating the inhibition of gelation around SFNF-catalases.
In summary, our SFNF-catalase bioink formulation enables both enhanced cell proliferation and high structural fidelity through a biocompatible strategy, addressing a key limitation of current bioprinting technologies.
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