Jammed Microgel Inks for 3D Printing Applications

Abstract 3D printing involves the development of inks that exhibit the requisite properties for both printing and the intended application. In bioprinting, these inks are often hydrogels with controlled rheological properties that can be stabilized after deposition. Here, an alternate approach is developed where the ink is composed exclusively of jammed microgels, which are designed to incorporate a range of properties through microgel design (e.g., composition, size) and through the mixing of microgels. The jammed microgel inks are shear‐thinning to permit flow and rapidly recover upon deposition, including on surfaces or when deposited in 3D within hydrogel supports, and can be further stabilized with secondary cross‐linking. This platform allows the use of microgels engineered from various materials (e.g., thiol‐ene cross‐linked hyaluronic acid (HA), photo‐cross‐linked poly(ethylene glycol), thermo‐sensitive agarose) and that incorporate cells, where the jamming process and printing do not decrease cell viability. The versatility of this particle‐based approach opens up numerous potential biomedical applications through the printing of a more diverse set of inks.

generated droplets were crosslinked by UV exposure (320 -390 nm, 15 mW cm -2 , ~30 s). For fabrication of agarose microgels, a solution of 1 wt% ultra low melting temperature agarose (Lonza) was flowed through devices, and the droplets were crosslinked by cooling outlet tubing and a collecting reservoir (4 ºC). To generate cell-containing microgels, NIH 3T3 fibroblasts were introduced into the 3 wt% NorHA solution (with 2 mM thiolated RGD added, GenScript) at a density of 10 million cells ml -1 and visible light (400 -500 nm, 200 W, 12 cmgap from the tubing, ~12 s) was introduced to crosslink the microgels. Microgels were suspended in PBS and centrifuged at 1,000 G for 5 min, and the oil layer on top was aspirated.
The rinsed microgels in pure PBS were jammed by vacuum-driven filtration (Steriflip,0.22 μm-pores, Millipore) to use as inks for 3D printing. Image J was used to quantify diameters and roundness of microgels. Support Hydrogel Synthesis and Formation: All chemicals were purchased from Sigma-Aldrich, unless indicated otherwise. AdNorHA [2] and CDHA [3] used throughout the experiments were synthesized, as described previously. Briefly, AdNorHA was obtained by functionalizing the tetrabutylammonium salt of HA (HA-TBA) with 1-adamantane acetic acid (Ad) and 5-norbornene-2-carboxylic acid (Nor) simultaneously via esterification through ditert-butyl dicarbonate (B OC 2O)/4-dimethylaminopyridine (DMAP). 1 H NMR was used to determine that ~10.8 % and ~30.5 % of HA repeat units were modified with Ad and Nor, respectively ( Figure S10a). For CDHA, HA-TBA was functionalized with aminated βcyclodextrin (β-CD, TCI America) via amidation through benzotriazole-1-yl-oxy-tris-(dimethylamino)-phosphonium hexafluorophosphate (BOP). 1 H NMR was used to confirm that ~19.8 % of HA repeat units were modified with β-CD ( Figure S10b). To form support hydrogels, AdNorHA and CDHA macromers (final polymer concentration: 3 wt%, Ad and CD ratio: 1:1) were separately dissolved in PBS containing 0.05 wt% Irgacure 2959 and 10 mM DTT. Next, the two macromer solutions were mixed thoroughly and centrifuged at >10,000 G to remove entrapped air. The self-assembled hydrogels were then loaded into custom PDMS molds to enable printing of the jammed microgel inks into the support material.
Cell Culture and Live Dead Staining: NIH 3T3 fibroblasts were expanded in growth medium (Minimum essential medium, 10% fetal bovine serum (FBS), 1% L-glutamine, and 1% penicillin-streptomycin). To assess post-printing cell viability, the printed cell-laden microgel structures were gently immersed in calcein-AM/ethidium homodimer solution for 60 min without post-crosslinking processes. Fluorescence microscopy (Olympus BX51) was used to image the stained cell-laden structures and Image J was used to quantify cell viability.
Pressure Measurement during the Extrusion of Inks: 3 experimental groups were loaded into 1 mL tuberculin syringes fitted with 6.35 mm-length 25 G needles. These groups were: jammed microgel ink, crosslinked polymer, and polymer solution before crosslinking.
Syringes were loaded onto an Instron fitted with a 100 N-load cell and plungers were depressed by the Instron at a constant rate of 1 mm s -1 . The resulting forces measured were converted to pressures based on the plunger diameter, and reported with respect to net material extruded at the constant extrusion rate.
Stability Analysis of 3D Printed Structures: 3D printed cuboid structures were immersed in cell culture media consisting of Minimum Essential Medium (MEM, Gibco) with 20 % fetal bovine serum and 1 % penicillin/streptomycin without or with post-crosslinking.