Influence of Microgel Fabrication Technique on Granular Hydrogel Properties

Abstract

Bulk hydrogels traditionally used for tissue engineering and drug delivery have numerous limitations, such as restricted injectability and a nanoscale porosity that reduces cell invasion and mass transport. An evolving approach to address these limitations is the fabrication of hydrogel microparticles (i.e., "microgels") that can be assembled into granular hydrogels. There are numerous methods to fabricate microgels; however, the influence of the fabrication technique on granular hydrogel properties is unexplored. Herein, we investigated the influence of three microgel fabrication techniques (microfluidic devices (MD), batch emulsions (BE), and mechanical fragmentation by extrusion (EF)) on the resulting granular hydrogel properties (e.g., mechanics, porosity, and injectability). Hyaluronic acid (HA) modified with various reactive groups (i.e., norbornenes (NorHA), pentenoates (HA-PA), and methacrylates (MeHA)) were used to form microgels with an average diameter of ∼100 μm. The MD method resulted in homogeneous spherical microgels, the BE method resulted in heterogeneous spherical microgels, and the EF method resulted in heterogeneous polygonal microgels. Across the various reactive groups, microgels fabricated with the MD and BE methods had lower functional group consumption when compared to microgels fabricated with the EF method. When microgels were jammed into granular hydrogels, the storage modulus (G') of EF granular hydrogels (∼1000-3000 Pa) was consistently an order of magnitude higher than G' for MD and BE granular hydrogels (∼50-200 Pa). Void space was comparable across all groups, although EF granular hydrogels exhibited an increased number of pores and decreased average pore size when compared to MD and BE granular hydrogels. Furthermore, granular hydrogel properties were tuned by varying the amount of cross-linker used during microgel fabrication. Lastly, granular hydrogels were injectable across formulations due to their general shear-thinning and self-healing properties. Taken together, this work thoroughly characterizes the influence of the microgel fabrication technique on granular hydrogel properties to inform the design of future systems for biomedical applications.

Keywords: granular hydrogels; hyaluronic acid; hydrogels; injectable; microfluidics; microgels.

Figure 1.

Chemically-modified hyaluronic acid (HA) structures, crosslinking, and bulk hydrogel formation. Chemical structures (top), schematics of crosslinking (middle), and representative rheology time sweeps (1 Hz, 0.5% strain; storage [G′, closed black circles] and loss [G″, open gray circles] moduli) during photocrosslinking (bottom, purple: UV light at 20 mW/cm²) of HA modified with a) norbornene (NorHA, (15 ± 2)% degree of modification), b) pentenoate (HA-PA, (22 ± 2)% degree of modification), or methacrylate (MeHA, (20 ± 1)% degree of modification) groups. NorHA and HA-PA undergo primarily thiol-ene radical addition photocrosslinking (shown as line linking the reactive groups in cartoon) in the presence of dithiothreitol (DTT) and photoinitiator (Irgacure 2959, I2959), whereas MeHA undergoes photocrosslinking through the formation of kinetic chains (shown as dotted lines) in the presence of I2959.

Figure 2.

Microgel fabrication and size characterization. a) Schematic representation of microgels fabricated using microfluidic devices (MD, blue), batch emulsions (BE, pink), and extrusion fragmentation (EF, yellow). b) Fluorescently labelled microgels in suspension (scale bar = 200 μm) and c) size quantification of NorHA (top), HA-PA (middle), and MeHA (bottom) microgels showing average size per batch (left) and size distribution of 50 microgels within a single batch (right). For the EF method, the microgel diameter was determined by quantifying the area of a fluorescently labelled microgel in suspension and calculating an effective diameter for the area. n ≥ 3, **p < 0.01, ns = not significant.

Figure 3.

Degree of consumption of crosslinking groups within microgels. a) Overview of quantification method showing a representative ¹H NMR spectrum of pure NorHA prior to microgel fabrication (left), microgel fabrication and processing steps to digest in hyaluronidase (center), and representative ¹H NMR spectra of the digested microgel showing unconsumed norbornene peaks (right). Quantified reactive group consumption during microgel fabrication using MD, BE, and EF techniques for b) NorHA, c) HA-PA, and d) MeHA. n ≥ 3, *p < 0.05, **p < 0.01, ***p < 0.001, ns = not significant.

Figure 4.

Granular hydrogel formation and rheological characterization. a) Schematic of jamming microgels in suspension using vacuum filtration to form granular hydrogels with a representative macroscopic image of NorHA EF granular hydrogels on a spatula (left, scale bar = 1 cm), and representative confocal images of fluorescently labelled NorHA granular hydrogels (right, scale bar = 100 μm). Quantified rheological behavior of b) NorHA, c) HA-PA, and d) MeHA granular hydrogels, showing (top) shear-yielding with increase in strain (0.5–500%, 1 Hz) and (bottom) quantified storage moduli (G’) of granular hydrogels at low strain (0.5%, 1 Hz). n ≥ 3, *p < 0.05, ***p < 0.001, ****p < 0.0001, ns = not significant.

Figure 5.

Porosity in NorHA granular hydrogels. a) Representative confocal image slices of NorHA granular hydrogels, showing microgels (black) and fluorescently-labelled pores (white). Scale bar = 100 μm. b) Quantified porosity measurements of NorHA microgels showing void space % (top), number of pores per (500 μm)² (middle), and distribution of pore sizes (bottom). Error bars in pore size distribution (bottom) show median and inner quartile ranges. n ≥ 3 (at least 20 images were analyzed per region of interest), *p < 0.05, ****p < 0.0001, ns = not significant.

Figure 6.

Tuning granular hydrogel properties through microgel crosslinking. NorHA granular hydrogels were formed from extrusion fragmented NorHA microgels fabricated with varied dithiothreitol (DTT) concentrations of 1, 3, and 5 mM. a) Fluorescently labelled microgels in suspension (scale bar = 200 μm). b) Size characterization of microgels showing average size per batch (left) and size distribution of 50 microgels within a single batch (right). c) Quantified ¹H NMR peak integration showing functional groups consumed during microgel fabrication. d) Shear-yielding behavior with increase in strain (0.5–500%, 1 Hz) (left) and storage moduli (G’) at low strain (0.5%, 1 Hz) (right) of granular hydrogels. e) Quantification of porosity showing void space % (left), number of pores per (500 μm)² (center), and distribution of pore sizes (right). n ≥ 3, *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001, ns = not significant.

Figure 7.

Injectability of NorHA granular hydrogels. a) Representative images of extruding NorHA granular hydrogels (MD: blue; BE: pink; EF: yellow) through an 18G needle. b) Representative rheological characterization of shear-thinning behavior through decreased viscosity with continuously increasing shear rates (0–50⁻¹ s) (left) and a rapid reduction and recover of G’ with low (unshaded, 0.5% strain, 1 Hz) and high (shaded, 500% strain, 1 Hz) strain cycles (right). c) Extrusion force measurements through an 18G (left) and 23G (right) needle at a flow rate of 10 mL/hr. A DTT concentration of 3 mM was used with EF fabrication, whereas a 5 mM concentration of DTT was used for MD and BE fabrication, to maintain similar degrees of consumption (~50%). n ≥ 3, *p < 0.05, ****p < 0.0001.