(580f) 3D Printing of in Situ-Gelling Dynamic Crosslinked Hydrogels Using Varying Mixing Strategies

Introduction: Hydrogels provide a favorable 3D environment for cells to grow, proliferate and differentiate, mimicking the function of the extracellular matrix ofsoft tissues. However, in the context of 3D bioprinting, relatively few options exist in terms of the types of hydrogels that can be printed to form functional tissue constructs. Current approaches for printing hydrogels are limited by the use of templates that must subsequently be removed [1], the weak mechanics of the printed features [2], and/or a need to work at non-physiological temperature/pH to enable the required fast gelation [3]. Recently, we have developed in situ-gelling hydrogels based on poly(oligoethylene glycol methacrylate) (POEGMA) or [2-(methacryloyloxy)ethyl]dimethyl-(3-sulfopropyl)ammonium hydroxide (PDMAPS) based on dynamic hydrazone chemistry that proceeds without any UV crosslinking, templating, or additional catalysts, providing an excellent platform for directly incorporating cells during printing. Both POEGMA and PDMAPS hydrogels have significant biological benefits such as low inflammation [4], low non-specific adsorption [5] and thermo-reversible cell adhesion suitable for tissue scaffolds [6]. However, the mixing of in situ-gelling hydrogels presents a challenge to ensure sufficient crosslinking during the 3D printing process without clogging the printing needle given that crosslinking is occurring simultaneous to crosslinking [7]. Herein, we demonstrate the direct use of dynamic in situ-gelling synthetic hydrogels for the design of a modular synthetic bioink platform in extrusion bioprinting applications using three distinct mixing modalities that should be applicable to any in situ-gelling polymer-based bioink: diffusion in a support bath, pre-mixed shear thinning hydrogels, and coaxial needle printing.

Materials and Methods: A 3D printer has been customized to ensure direct mixing between hydrazide and aldehyde or ketone-functionalized polymers to allow for the formation of in situ-gelling synthetic POEGMA or PDMAPS hydrogels. Specifically, the printer consists of a four-axis stage that controls the printing speed and design of the construct via defined G-codes, a control system that allows input from four step motors, and a customized 3D printed clamp system that can hold up to two syringes. Three mixing strategies have been tested (Figure 1A): 1) embedded printing using the FRESH (freeform reversible embedding of suspended hydrogels [8]) method by printing hydrazide-functionalized POEGMA polymer into a support bath containing aldehyde-functionalized POEGMA bath; 2) direct free-form printing using a pre-mixed bioink formulation based on ketone and hydrazide-functionalized PDMAPS polymers; and 3) a coaxial printing approach in which hydrazide (core needle) and aldehyde (shell)-functionalized POEGMA polymers are co-delivered to allow for circumferential gelation upon extrusion. Each mixing strategy was evaluated based on the degree of polymer mixing (using fluorescent probes), scaffold stability and 3D resolution, cell viability, and cell adhesion (using NIH/3T3 fibroblasts and human umbilical vein endothelial cells as model cells); for direct-printed gels, co-printing of HepG2 and NIH/3T3 cells was assessed. In vivo subcutaneous implants were also performed in BALB/c mice to assess the local tissue responses and stability of each type of printed scaffold.

Results: The custom extrusion bioprinting platform is capable of fabricating high-resolution three-dimensional prints of dynamic POEGMA hydrogels using three different mixing strategies. For the embedded printing approach, the bioink composed of hydrazide-functionalized POEGMA (including collagen to promote cell adhesion) was printed directly in the gelatin support bath containing aldehyde-functionalized POEGMA. Depending on the polymer concentration (ranging from 3 to 10 wt%), the resulting gel can be made to be homogeneous (low concentration) or to resemble a more core-shell structure (high concentration) within the printed features; the latter re-arranges to a more uniform structure after three days due to the dynamic nature of hydrazone chemistry (Figure 1B). Both 3T3 cells and HUVECs can be successfully co-printed and have high cell viability and good adhesion over 14 days of culture (Figure 1B). For the pre-mixing approach, ketone/hydrazide-functionalized PDMAPS precursor polymers were pre-mixed to form a gel and printed by taking advantage of the dynamic nature of both zwitterion fusion interactions as well as hydrazone crosslinks to enable printing via a shear-thinning strategy. Pre-mixing the low viscosity precursor polymers allows for easy mixing with cells, well-defined structures upon gelation, and high cell viability (>90%) following co-printing with HepG2 hepatocytes (Figure 1C); in addition, by co-printing HepG2 cells with fibroblasts, albumin secretion increased 4-fold in a small-scale liver model. Finally, the coaxial printing approach was optimized using computational fluid dynamics modelling to determine the best inner needle gauge (gauges 20 to 26), inner needle length difference (5-20 mm) and flow rate of hydrazide/aldehyde functional POEGMA polymers to achieve uniform printed structures. Increasing the length of the inner needle length difference (i.e. the mixing zone) significantly improved the quality of mixing (Figure 1D) and permitted the fabrication of 3D cellularized constructs (including co-cultures) with high maintained cell viabilities. In vivo implantation of each of the printed scaffolds indicated that high dimensional and mechanical stability of the prints could be maintained over several weeks, significantly longer than many prints based on conventional alginate-calcium bioinks.

Significance: Unlocking the potential of 3D bioprinting depends in part on developing more diverse bioinks that can provide more options in terms of stability, cell-scaffold interactions, and biological responses. Herein, we demonstrate the successful 3D printing of highly cell compatible in situ-gelling bioinks based on hydrazide and aldehyde/ketone-functionalized POEGMA polymers that facilitate good print fidelity, high maintained cell viability, potential for co-printing multiple cell types, and long-term stability in the in vivo environment. Access to such bioinks is essential to address different bioink needs in diverse bioprinting applications.

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