Enzymatically crosslinked silk-hyaluronic acid hydrogels

Abstract

In this study, silk fibroin and hyaluronic acid (HA) were enzymatically crosslinked to form biocompatible composite hydrogels with tunable mechanical properties similar to that of native tissues. The formation of di-tyrosine crosslinks between silk fibroin proteins via horseradish peroxidase has resulted in a highly elastic hydrogel but exhibits time-dependent stiffening related to silk self-assembly and crystallization. Utilizing the same method of crosslinking, tyramine-substituted HA forms hydrophilic and bioactive hydrogels that tend to have limited mechanics and degrade rapidly. To address the limitations of these singular component scaffolds, HA was covalently crosslinked with silk, forming a composite hydrogel that exhibited both mechanical integrity and hydrophilicity. The composite hydrogels were assessed using unconfined compression and infrared spectroscopy to reveal of the physical properties over time in relation to polymer concentration. In addition, the hydrogels were characterized by enzymatic degradation and for cytotoxicity. Results showed that increasing HA concentration, decreased gelation time, increased degradation rate, and reduced changes that were observed over time in mechanics, water retention, and crystallization. These hydrogel composites provide a biologically relevant system with controllable temporal stiffening and elasticity, thus offering enhanced tunable scaffolds for short or long term applications in tissue engineering.

Keywords: Biomaterials; Enzymatic crosslinking; Hydrogel blends; Polymer composites; Temporal stiffening.

Figure 1. Hydrogel Gelation

(a) A schematic representing the single step covalent crosslinking between tyrosine residues on silk and tyramine side chains on HA creating a composite hydrogel. (b) Images showing gelation of silk-HA hydrogels during a vial inversion test. (c) Gelation times, as determined by the vial inversion test, show hydrogels consisting of more than 1% HA decreased gelation time (n=4, ***p≤0.001). (d) In addition, the increasing of HA concentration affected crosslinking kinetics by reducing the lag period seen most prominently in 0% hydrogels and decreasing time at which crosslinking was complete (n=7).

Figure 2. Rheological Properties

(a) Representative time sweeps, (b) strain sweeps, and (c) final shear storage moduli are shown. HA only hydrogels reached a final modulus much quicker (a), had a lower maximum strain (b), and had a much lower final storage modulus (c) as compared to composite and silk only hydrogels. (n=3, *p≤0.05, ***p≤0.001).

Figure 3. Unconfined Compression

(a) The compressive moduli of the hydrogels over time, expressed on a log scale, are dependent on HA concentration where increasing concentration reduces the amount the modulus increases after 1 month. (b) Average stress-strain curves show that stress and hysteresis increase for all samples after 4 weeks, except for HA only hydrogels, with the extent of hysteresis reduced by increasing HA concentration. (n=5, +p≤0.05 compared to 0%, *p≤0.05, ***p≤0.001. Statistical analysis was performed after log transformation).

Figure 4. FTIR Absorbance Spectra

(a) The average FTIR absorbance spectrum in the amide I region is shown for hydrogels over time. Hydrogels with lower HA concentration exhibit a peak shift from ~1640cm⁻¹ to ~1620cm⁻¹ at 3 weeks. Additionally, the peak at ~1620 cm⁻¹, which is representative of ß-sheet formation, becomes larger and wider as HA concentration decreases. (b) The ratio of ß-sheet to random coil conformation was calculated by dividing the average of peak absorbance at 1620–1625 and 1640–1650cm⁻¹ showing that increasing HA concentration reduced the ratio over time. (n=5, *p≤0.05 and ***p≤0.001 compared to week 0).

Figure 5. Percent Water

The percent water, based on mass, was calculated for hydrogels over time. Initially, all hydrogels, with the exception of HA only hydrogels, contained ~97% water. At weeks1, 2, 3, and 4, all composite hydrogels had higher percentages of water as compared to 0% hydrogels, showing that the addition of HA reduces the amount of water loss over time. (n=5, **p≤0.01, ***p≤0.001 compared to 0% HA).

Figure 6. In vitro Degradation

Degradation was determined by calculating the fraction of initial mass after soaking in an enzymatic cocktail consisting of 1 U/mL and 0.001 U/mL of hyalruonidase and protease. A direct relation between degradation rate and HA concentration was seen when increasing HA concentration above 5% significantly increased the degradation rate at day 8 (n=4, **p≤0.01, ***p≤0.001).

Figure 7. 2D hMSC Response

(a) Bright-field images of 2D hMSCs are shown at day 0 and 7. Hydrogels containing silk fibroin enhanced cell spreading whereas HA only hydrogels inhibited spreading (scale bar=100 μm). (b) The fold change of hMSC DNA content as compared to day 0 was calculated for days 3, 5, and 7. Silk only and silk-HA (10% HA) hydrogels promoted cellular growth similar to that of TCP whereas HA only hydrogels showed inhibited growth (n=6, * p≤0.05, **p≤0.01, ***p≤0.001).