Title:

Novel 3D Printing Process for Improved Mechanical Properties of Tissue Engineering Scaffolds

3D bioprinting is a relatively new area of tissue engineering that allows for complex tissue-like structures to be constructed in a layer-by-layer process, providing precise placement of cells and extracellular matrix (ECM). The application of 3D bioprinting could assist in the shortage of donor tissues for transplantation but finding a bio-ink that prints at a high resolution and is both biocompatible and has good mechanical properties remains a challenge. The proposed bio-ink was composed of 5% gelatin, 5% methacrylated gelatin (GelMA), and 1% alginate, crosslinked through an enzymatic reaction by microbial transglutaminase (mTG), photopolymerization of methacrylate groups by UV radiation and ionic crosslinking by calcium ions (Ca2+), respectively. Rheology data showed substantially higher stiffness and ultimate strength of the hydrogels crosslinked with all three mechanisms (UV+mTG+Ca2+) than the other sample groups (UV only and UV+mTG). 5x10^6 cells (human dermal fibroblasts (hDFs))/mL were mixed into the bio-ink and printed using the BioAssemblyBot 400 at 27 ℃ and 10-15 psi in a 3D grid. LDH cytotoxicity results showed a gradual decrease in cytotoxicity over 7 days in all groups. Confocal imaging with live/dead staining showed that a majority of cells were viable in all groups, demonstrating excellent biocompatibility of the bioink and crosslinking processes. The UV+mTG+Ca2+ group resulted in the lowest cell spreading due to the increased stiffness of the hydrogel. However, delayed Ca2+ crosslinking showed potential for an increase in cell proliferation and cell spreading compared to immediate Ca2+ crosslinking. These results showed the feasibility of using the novel bioink and crosslinking processes to generate 3D printed tissue-like structures with improved mechanical stability.

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