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Introduction: Three dimensional (3D) bioprinting provides a wide avenue to design complex and customized constructs for tissue regeneration, disease modelling, and drug testing applications. Bioink formulations in 3D bioprinting usually lack the presence of micrometre-sized and interconnected pores, resulting in reduce cell viability and prevent biological communications with host tissues, which limits the therapeutic efficacy. Previously, we reported porous injectable hydrogels with microcapillary network (µCN) utilizing liquid-liquid phase separation (LLPS).[1] This study aims to design an LLPS bioink and porous 3D scaffold using the extrusion bioprinting technique for muscle tissue regeneration. This strategy enables to form fibrous microporous structures. In this presentation, the orientation of 3D structure, cell differentiation in 3D printed hydrogels, and cell survival in mouse muscle defect model will be presented.
Experiments: Gelatin methacrylate (GelMA) was synthesized by the reaction of gelatin with methacrylic anhydride. Ureidopyrimidinone (UPy) modified gelatin (GUPy) was synthesized by reaction of gelatin with UPy-isocyanate. To prepare bioink (Gel+), GelMA (10 wt%), GUPy (12 wt%) solutions, lithium phenyl (2,4,6-trimethylbenzoyl) phosphinate (LAP), and C2C12 cells were mixed thoroughly using pipette. While for bioink (Gel), GUPy replaced with gelatin. 3D printed µCN structure was engineered through photo crosslinking of GelMA matrix and dissolution of GUPy. 25-gauge (G) needle and 27G nozzle was used to print 3D structures. The stiffness of µCN bioink evaluated using a rheometer. µCN formation, cell adhesion, and differentiation in hydrogels were observed using confocal laser scanning microscopy (CLSM). Additionally, muscle defect models of mice were prepared by cutting the tibialis anterior (TA) muscles and DiI stained MSCs encapsulated in 3D scaffold were transplanted into the defects. Cell engraftment was observed at day 7 after the transplantation.
Results and discussion: Both Gel+ and Gel bioink enabled 3D printing of hydrogels with promising structure fidelity and stability. It was also confirmed that the µCN structures were formed uniformly in printed hydrogels. Moreover, bioink Gel+ exhibits excellent stiffness and less swelling which are necessary for the stability of 3D construct. C2C12 cells showed excellent adhesion, extension, migration, and proliferation in the 3D-printed Gel+ construct due to the presence of µCN, but these traits are not observed in the Gel construct. The CLSM observation revealed that the porous 3D structure not only improved material permeability, but also functioned as a void for cell infiltration which upholds our previous finding.[1] Furthermore, bioink Gel+ represented higher cell survival than that of Gel bioink. Therefore, these 3D printed porous tissues constructs may have versatile applications in the individualized therapy of tissue defects.
[1] Nishiguchi, A., Ito, S., Nagasaka, K., Komatsu, H., Uto, K. & Taguchi, T. Biomaterials 305, 122451 (2024).
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