Synthesis, Characterization, and Digital Light Processing of a Hydrolytically Degradable Hyaluronic Acid Hydrogel

Abstract

Numerous chemical modifications of hyaluronic acid (HA) have been explored for the formation of degradable hydrogels that are suitable for a variety of biomedical applications, including biofabrication and drug delivery. Thiol-ene step-growth chemistry is of particular interest due to its lower oxygen sensitivity and ability to precisely tune mechanical properties. Here, we utilize an aqueous esterification route via reaction with carbic anhydride to synthesize norbornene-modified HA (NorHA_CA) that is amenable to thiol-ene crosslinking to form hydrolytically unstable networks. NorHA_CA is first synthesized with varying degrees of modification (∼15-100%) by adjusting the ratio of reactive carbic anhydride to HA. Thereafter, NorHA_CA is reacted with dithiol crosslinker in the presence of visible light and photoinitiator to form hydrogels within tens of seconds. Unlike conventional NorHA, NorHA_CA hydrogels are highly susceptible to hydrolytic degradation through enhanced ester hydrolysis. Both the mechanical properties and the degradation timescales of NorHA_CA hydrogels are tuned via macromer concentration and/or the degree of modification. Moreover, the degradation behavior of NorHA_CA hydrogels is validated through a statistical-co-kinetic model of ester hydrolysis. The rapid degradation of NorHA_CA hydrogels can be adjusted by incorporating small amounts of slowly degrading NorHA macromer into the network. Further, NorHA_CA hydrogels are implemented as digital light processing (DLP) resins to fabricate hydrolytically degradable scaffolds with complex, macroporous structures that can incorporate cell-adhesive sites for cell attachment and proliferation after fabrication. Additionally, DLP bioprinting of NorHA_CA hydrogels to form cell-laden constructs with high viability is demonstrated, making them useful for applications in tissue engineering and regenerative medicine.

Figure 1.

Synthesis of NorHA_CA macromer and hydrogels. (a) Reaction scheme for modification of sodium hyaluronate (HA) with cis-5-norbornene-endo-2,3-dicarboxylic anhydride (CA) to form norbornene-modified HA (NorHA_CA) (b) The degree of modification of HA with norbornene is tuned by changing the molar ration of CA to HA repeat units. Data are reported as mean ± SD; n ≥ 3; **p < 0.01; ****p < 0.0001 (c) Schematic representation of network formation by visible light induced thiol-ene step growth reaction between NorHA_CA and dithiothreitol (DTT) in the presence of photoinitiator (lithium phenyl-2,4,6-trimethylbenzoylphosphinate, LAP).

Figure 2.

Rheological characterization of NorHA_CA hydrogels. Storage modulus, G′ (closed symbol) and loss modulus, G″ (open symbol) as a function of time for NorHA_CA formulations with varying macromer concentrations (1, 3, 5 wt.%) and degrees of modification: (a) 15% mod., (b) 40% mod., (c) 100% mod. Shaded box indicates the time period between 120 and 420 seconds for which visible light is introduced. Note that 1 and 3 wt.% of the 100% mod. are not testable past reaching the plateau, as samples likely separate from the plates. (d) G′, (e) G″, and (f) time to gel point (t_g) for various NorHA_CA hydrogel formulations investigated. Statistical comparisons of all groups within each degree of modification shown on graphs. Data are reported as mean ± SD; n ≥ 3; ns = not significant; *p < 0.05; **p < 0.01; ***p < 0.001; ****p < 0.0001.

Figure 3.

Characterization of NorHA_CA hydrogel degradation. Cumulative uronic acid released, compressive modulus (E_C), and mass swelling ratio (Q_m) over time (t) for varying NorHA_CA macromer concentrations (1, 3, 5 wt.%) and degrees of modification: (a) 15% mod., (b) 40% mod., and (c) 100% mod. Data are reported as mean ± SD; n ≥ 3.

Figure 4.

Modeling the degradation behavior of NorHA_CA hydrogels. a) Schematic overview of the modeling approach employed to characterize NorHA_CA hydrogel degradation. Model fit (dotted line, k = 0.350 day⁻¹) for the (b) compressive modulus (E_C) and (c) mass swelling ratio (Q_m) of a select NorHA_CA hydrogel (5 wt.%, 40% mod.) over time, with comparisons to experimental data (symbols). Shaded box indicates time point for complete degradation of the hydrogel. Data are reported as mean ± SD; n ≥ 3. Hydrogel schematics created using BioRender.

Figure 5.

Tuning hydrogel degradation behavior by combining degradable and non-degradable macromers. (a) Schematic illustration of networks composed of NorHA_CA alone, NorHA alone, and NorHA_CA/NorHA mixtures (4.75:0.25, 4.50:0.50, 4.00:1.00). (b) Cumulative uronic acid released, (c) compressive modulus (E_C), and (d) mass swelling ratio (Q_m) over time (t) for hydrogels formed with NorHA_CA, NorHA, and NorHA_CA/NorHA mixtures. The total macromer concentration was kept fixed at 5 wt.%. Data are reported as mean ± SD; n ≥ 3.

Figure 6.

3D printing and patterning of NorHA_CA hydrogels. (a) Schematic illustration of digital light processing (DLP)-based fabrication of NorHA_CA hydrogels (5 wt.%, 40% mod.) and post-print curing to improve mechanical properties. (b) Photographs of pyramid, femoral condyle, and 3D gyroid (in air). Scale bar: 5 mm. (c) Fluorescence images of 3D building blocks, wheel, and knotted pattern printed with NorHA_CA resin. Scale bar: 2 mm. (d) Comparison of uronic acid release and compressive modulus (E_C) over time (t) for casted and 3D printed NorHA_CA hydrogels. (e) Schematic illustration of maskless photo-patterning of NorHA_CA hydrogels (5 wt.%, 40% mod.) with fluorescent thiol (GCDDD-Fluor) on the DLP-printer. (f) Fluorescence maximum projection image of 2D patterned CU buffalo, checkerboard, triangle on NorHA_CA bulk hydrogel and printed macroporous lattice (shown in red), respectively. Scale bar: 5 mm.

Figure 7.

Cell interactions with NorHA_CA hydrogels. (a) Representative fluorescence micrographs of bovine bone marrow derived mesenchymal stromal cells (bMSCs) seeded on NorHA_CA hydrogels (5 wt.%, 40% mod.) over time (1,3,7 days). F-actin (magenta) and nuclei (cyan). Scale bar: 200 μm. (b) Quantification of the number of cells per unit area over time. Data are reported as mean ± SD; n ≥ 3; ****p < 0.0001 (c) Schematic illustration of digital light processing (DLP)-based fabrication of NorHA_CA hydrogels with cell adhesive peptides (GCGYGRGDSPG, 2 mM) and post-seeded with cells. (d) Representative maximum projection image of bMSCs seeded on a NorHA_CA macroporous lattice at day 3. F-actin (magenta) and nuclei (cyan). Scale bars: 1 mm and 500 μm. Dotted line indicates lattice boundary. (e) Representative fluorescence micrographs of bMSCs encapsulated in NorHA_CA (5 wt.%, 40% mod.) bulk hydrogels over time (1,3,7 days). Live (calcein AM, green), Dead (ethidium homodimer-1, red). Scale bar: 200 μm. (f) Percentage of viable (live) cells over time in NorHA_CA hydrogels. Data are reported as mean ± SD; n ≥ 3; *p < 0.05 (g) Schematic representation of DLP-based 3D printing of NorHA_CA hydrogels (5 wt.%, 40% mod.) with bMSCs. (h) Representative maximum projection image of bMSCs encapsulated in NorHA_CA macroporous lattice at day 1. Live (calcein AM, green), Dead (ethidium homodimer-1, red). Scale bars: 1 mm and 500 μm. Dotted line indicates lattice boundary.

Figure 8.

Fabrication of macroporous constructs from degradable NorHA_CA, non-degradable NorHA, and NorHA_CA/NorHA mixtures. (a) (top) Fluorescence images of 3D printed macroporous discs. Scale bar: 1 mm. (bottom) Representative maximum projection images of the pores within the discs. Scale bar: 200 μm. (b) Cumulative uronic acid release and compressive modulus (E_C) over time (t) for 3D printed macroporous NorHA_CA, NorHA, and NorHA_CA/NorHA discs. The total macromer concentration was kept fixed at 5 wt.% and the NorHA_CA/NorHA mixture consists of 4.5 wt.% NorHA_CA and 0.5 wt.% NorHA. (c) Fluorescence images of macroporous NorHA discs (magenta) filled with NorHA_CA (cyan) over the course of degradation. Scale bar: 1 mm. (d) Cumulative uronic acid and encapsulated bovine serum albumin (BSA) release from filled NorHA_CA hydrogels over time (t).