Tailoring the Printability of Photocrosslinkable Polypeptide and Polysaccharide-based Bioinks for Extrusion-based 3D Bioprinting

In extrusion-based bioprinting, a bioink with appropriate rheological properties is used to achieve 3D-printed constructs. A bioink is a printable hydrogel precursor that has properties similar to those of hydrogels, including biocompatibility, biofunctionality, and biodegradability. In particular, natural polymers such as polypeptides and polysaccharides are widely used and are suitable for hydrogel and bioink research. On the other hand, natural polymers without chemical modification lack the mechanical strength and stability to maintain 3D constructs. In bioink development, natural polymers are often functionalized with methacrylate anhydride to enable photopolymerization via free-radical polymerization. Photocrosslinking approachhas been the most used in bioink development due to its tunability, accessibility and cost-effectiveness. This provides the freedom to optimize the physical and chemical properties of the precursor by varying the degree of modification, polymer concentration, photoinitiator concentration and crosslinking time.

To the best of our knowledge, this is the first comprehensive study, which offered simple tools to improve printability in respect of the connection between polymer properties, chemical modifications, rheological behaviors, and 3D biofabrication. The most suitable bioink properties should harness shear-thinning, yield stress, and recovery behaviors. Although some bioinks may exhibit these properties, they are still unable to achieve the desired printing outcomes. The two-step crosslinking strategy is the main focus of this thesis, as it comprises physical crosslinking as the pre-crosslinking step and photocrosslinking as the post-crosslinking step. The initial crosslinking was employed to modulate the rheological properties of the hydrogel precursors for 3D printing. The secondary crosslinking provided stability to the 3D constructed via covalent crosslinking from photocrosslinking. A wide range of photocrosslinkable precursors were investigated and screened as biomaterial inks, including GelMA, ColMA, HAMA, AlgMA, GGMA, GelMAGA, HAGA and GGMAGA. In addition to the methacrylate group, gallic acid was used to improve the multifunctionality and flexibility such as stimuli-responsiveness and tissue adhesion. Different polymer sources and chemical functionalization require different pre-crosslinking techniques such as temperature, ionic crosslinking (CaCl2), catechol-metal complex (FeCl3), pH modulation and controlled photocrosslinking (low UV intensity).

These pre-crosslinked precursor formulations were formed through different chemistries to obtain printable precursors based on physical or covalent crosslinking. The optimization of the pre-crosslinking parameters (temperature, precursor concentration and amount of pre-crosslinker agents) influenced the printability of the hydrogel precursor in extrusion-based 3D bioprinting. For photocrosslinking, the properties of printed hydrogel, including gelation kinetics, crosslinking degree, mechanical strength, average mesh size and swelling behaviors, could be modulated by the degree of methacrylation, photoinitiator, UV light intensity and exposure time. The printability of the biomaterial inks was assessed by pre-screening via visual analysis, flow behavior, and structural integrity of 3D printed constructs. Printable precursors were defined as those that could form long, smooth, and coherent fibers and stack on top of each fiber. The printable precursors exhibited non-Newtonian fluid, sufficient yield stress, and recovery behaviors to retain their shape fidelity after the force was applied. During printing, rational printing parameters are the most important factors in achieving 3D constructs, such as printing temperature, nozzle size/type, pressure, speed, curing time, and the selection of CAD models. The results showed that the use of pre-crosslinkers (GGMA-CaCl2 and GelMAGA-FeCl3) offered the highest printing resolution, followed by thermal crosslinking, pH modulation and controlled photocrosslinking. For 3D printed constructs, GelMA60 at 16 °C, GGMA-CaCl2, GelMAGA-FeCl3 and HAGA20-HAMA15 inks were successfully printed in cylinders with high structural integrity and stability in the swelling studies. In summary, a two-step crosslinking technique in the hydrogel precursor not only enhanced the printability but also significantly improved the mechanical strength and stability of the final printed structure. This doable crosslinking strategy demonstrates the potential for producing robust and durable constructs through 3D printing technology in the field of hydrogel-based scaffolds. The proposed characterization in this study could be used as the key to screen the printable precursors in bioink development in the future.