Dynamically crosslinked thermoresponsive granular hydrogels

Abstract

Granular hydrogels are a promising biomaterial for a wide range of biomedical applications, including tissue regeneration, drug/cell delivery, and 3D printing. These granular hydrogels are created by assembling microgels through the jamming process. However, current methods for interconnecting the microgels often limit their use due to the reliance on postprocessing for crosslinking through photoinitiated reactions or enzymatic catalysis. To address this limitation, we incorporated a thiol-functionalized thermo-responsive polymer into oxidized hyaluronic acid microgel assemblies. The rapid exchange rate of thiol-aldehyde dynamic covalent bonds allows the microgel assembly to be shear-thinning and self-healing, with the phase transition behavior of the thermo-responsive polymer serving as secondary crosslinking to stabilize the granular hydrogels network at body temperature. This two-stage crosslinking system provides excellent injectability and shape stability, while maintaining mechanical integrity. In addition, the aldehyde groups of the microgels act as covalent binding sites for sustained drug release. These granular hydrogels can be used as scaffolds for cell delivery and encapsulation, and can be 3D printed without the need for post-printing processing to maintain mechanical stability. Overall, our work introduces thermo-responsive granular hydrogels with promising potential for various biomedical applications.

Keywords: 3D printing; cell delivery; drug delivery; dynamic biomaterials; granular hydrogels; regenerative medicine.

FIGURE 1

Injectable and thermo-responsive granular hydrogels consisting of microgel assemblies formulated by synthetic polymers (PNAM) and hyaluronic acid-based microgels. (A) Thermo-responsive and thiolated polymers (PNAM) were blended with oxidized microgels to form shear-thinning and self-healing microgel assemblies by dynamic covalent crosslinking. Due to the hydrophobic interaction of PNAM at 37°C, the microgel assemblies became mechanically stable. (B) Methacylated and aldehyde modified hyaluronic acid (HA-ALD-MA) was used to fabricate oxidized HA microgels (HA-ALD-MG) by UV crosslinking and fragmentation. The thiol groups of PNAM can react with the aldehyde groups of HA-ALD-MGs to form hemithiolacetal. After heating, due to hydrophobic interaction of PNAM, the polymer aggregation facilitated the formation of disulfide bonds, which contributed to the structural mechanical stability of microgel assemblies.

FIGURE 2

The fabrication of microgel and granular hydrogels consisting of microgel assemblies. (A) The gelation kinetic of methacrylated and aldehyde-modified hyaluronic acid (HA-ALD-MA) by UV photocrosslinking. 4% of HA-ALD-MA reached a plateau of storage modulus at 3000 Pa within 2 min of 20 mW/cm² UV light. (B) aldehyde-modified microgels (HA-ALD-MGs) were produced by the fragmentation of methacrylated and aldehyde-modified hyaluronic acid bulk hydrogel with gradient sizes of needles from 18G to 27G. (C) The confocal image of the microgel assembly formed by PNAM (Red) and HA-ALD-MGs (Green). The PNAM phase occupied approximately 12% of the total volume, and the microgel sizes were around 50 μm, measured by ImageJ and fluorescent images. (D) The shear rate sweep analysis of the microgel assembly indicated its good injectability. (E) 1% and 500% of strain were applied to the microgel assembly to test its self-healing ability. Two pieces of the microgel assemblies can bind together in a few minutes.

FIGURE 3

Postinjection stability of the microgel assemblies was attributed to the phase transition ability and thiol groups of PNAM. (A) The temperature sweep analysis of PNAM, PNA, and PAM showed the phase transition behavior of PNAM and PNA by their enhancing storage modulus along the increment of temperature from 25 to 40°C. (B) The thiol concentrations of 0.5% PNAM, PNA, and PAM polymers were measured by Ellman’s assay (n = 3, mean ± SD). (C) The temperature sweep analysis of the PNAM, PNA, and PAM microgel assemblies. (D) Change in the transparency of the PNAM microgel assemblies from 25 to 37°C. (E) The strain sweeps of the PNAM, PNA, and PAM microgel assemblies at 25 and 37°C. (F) The yield stress of the PNAM, PNA, and PAM microgel assemblies at 37°C were obtained from the strain sweeps. (n = 4, mean ± SD). (G) The maximum compressive stress and compressive modulus of the PNAM, PNA, and PAM microgel assemblies before and after being incubated in PBS at 37°C for 48 h (n = 3, mean ± SD). Statistics analysis was performed using one-way or two-way ANOVA with *p < .05, **p < .01, ***p < .001, and ****p < .0001.

FIGURE 4

Injectable granular hydrogels consisting of microgel assembly for controlled drug release. (A) DOX was used as a model drug and covalently loaded to aldehyde-modified microgels (HA-ALD-MGs) by Schiff base reaction. 2.5% DOX-loaded HA-ALD-MGs, 2.5% HA-ALD-MGs, and 2.5% PNAM were mixed to form the DOX-loaded microgel assembly. (B) The release profile demonstrated that the DOX-loaded microgel assemblies are pH and redox responsive because of the formation of imine and disulfide bonds. (C) Live/dead assays of MCF-7 cells co-cultured with the microgel assembly (−DOX), DOX-loaded microgel assembly (+DOX; Control: TCPS, Calcein: live cells, EthD-1: dead cells). (D) Alarma blue assays of the MCF-7 cells cultured with the extracted solutions of −DOX and +DOX. All viability values were normalized to the control (Control: TCPS; n = 3, mean ± SD). (E) Live/dead assays were quantified by flow cytometry (n = 3, mean ± SD). Statistics analysis was performed using two-way ANOVA with *p < .05, **p < .01, ***p < .001, and ****p < .0001.

FIGURE 5

The applications of microgel assemblies in 3D printing and cell encapsulation. (A) 3D printing of the microgel assemblies extruded by a tapered extruder with 0.41 and 1 mm diameter. The snake line structures were used for assessing uniformity, and the printed grids showed that the microgel assemblies have better printability with a 1 mm tip (scale bar: 1 cm). (B) The printed microgel assembly was further reinforced by incubation at 37°C overnight. The printed cylinder was compressive and elastic. (C) The modification of cell adhesive peptide (RGD) to PNAM improved cell adhesion and metabolic activities of 3T3 fibroblasts seeded on the microgel assemblies (n = 3, mean ± SD). Cell morphology of the 3T3 fibroblasts seeded on the RGD-modified microgel assemblies (blue: nucleus, purple: actin). (D) Live/dead assays of 3T3 fibroblasts encapsulated in the microgel assemblies on days 1 and 7 (n = 4, mean ± SD). The results showed good cell viability on both days. (Calcein: live cells, EthD-1: dead cells) Statistics analysis was performed using one-way or two-way ANOVA with *p < .05, **p < .01, ***p < .001, and ****p < .0001.