Silk degumming time controls horseradish peroxidase-catalyzed hydrogel properties

Abstract

Hydrogels provide promising applications in tissue engineering and regenerative medicine, with silk fibroin (SF) offering biocompatibility, biodegradability and tunable mechanical properties. The molecular weight (MW) distribution of SF chains varies from ∼80 to 400 kDa depending on the extraction and purification process utilized to prepare the protein polymer. Here, we report a fundamental study on the effect of different silk degumming (extraction) time (DT) on biomaterial properties of enzymatically crosslinked hydrogels, including secondary structure, mechanical stiffness, in vitro degradation, swelling/contraction, optical transparency and cell behaviour. The results indicate that DT plays a crucial role in determining material properties of the hydrogel; decrease in DT increases β-sheet (crystal) formation and mechanical stiffness while decreasing degradation rate and optical transparency. The findings on the relationships between properties of silk hydrogels and DT should facilitate the more rational design of silk-based hydrogel biomaterials to match properties needed for diverse purpose in biomedical engineering.

Figure 1:

Representative ATR-FTIR spectra of hydrogels of different silk DT crosslinked in the presence of HRP/H₂O₂. D1 and D7 samples were incubated in 1X PBS (phosphate buffered saline) for 1 and 7 days, respectively, after hydrogelation. a, b, c) Time-dependent FTIR spectra for 1, 30 and 120 DT silk hydrogels, respectively. For D7, all samples showed strong β-sheet absorption band at 1620 cm⁻¹. However, at D1, low DT (1 DT) showed strong β-sheet absorption bands whereas higher DT silk (30 and 120 DT) showed weak absorption bands. d) Percentage β-sheet content in the hydrogels increased with time after incubation in PBS at 37°C. (data are presented at mean ± standard deviation; n=3, ***p < 0.001 by two-way ANOVA with Tukey’s post hoc test)

Figure 2:

Analysis of dityrosine bonds using LC-MS/MS analysis. (a) LC-MS/MS chromatograms obtained from analysis of silk hydrogels formed with 1 DT (black), 30 DT (red), and 120 DT (blue) samples, where the concentration of the silk solutions was fixed at 2.5% w/v. (b) Relative peak area of dityrosine in peak area in the hydrogels, as compared within the sample groups based on the peak areas (data are presented as mean ± standard deviation; n = 3, **p < 0.01 by one-way ANOVA with Tukey’s post hoc test).

Figure 3:

Compressive mechanical properties of silk hydrogels after 24h of hydrogel formation. (a) Representative compressive stress-strain curves for 1 DT (black), 30 DT (red), and 120 DT (blue) samples. (b) Energy loss calculated from the area between the loading and unloading in the stress-strain curve. (c) Compressive modulus of hydrogels. (data are presented as mean ± standard deviation; n = 3, *p<0.05, **p < 0.01 and ***p < 0.001 by one-way ANOVA with Tukey’s post hoc test).

Figure 4:

In vitro enzymatic degradation of silk hydrogels of different DT over 7 days of incubation in 1X PBS with 0.001 U/mL chymotrypsin. Data at D1 and D7 are presented as residual percentage mass remaining of D0. Data are presented as mean ± standard deviation; n = 3, **p < 0.01 and ***p < 0.001 by two-way ANOVA with Tukey’s post hoc test.

Figure 5:

Investigation into swelling (a) and deswelling (contraction) (b) of different DT SF hydrogels in ultrapure water and 1X PBS, respectively. The Table under each figure corresponds to percent swelling and deswelling by mass at different time intervals (1h, 2h, 24h) for the different MW SFs. Data are presented as mean ± standard deviation; n = 3, *p<0.05, **p < 0.01 and ***p < 0.001 by two-way ANOVA with Tukey test).

Figure 6:

(a) Digital images of silk hydrogels formed from different DT. Appearance of the bottom letters depicts the macroscopic transparency of each MW hydrogel after 24h. The hydrogels were prepared in a PDMS mold. (b) Percent transmittance of SF hydrogels at different wavelengths measured after 24h. (c) Percent transmittance of SF hydrogels at different wavelengths. Data are presented as mean ± standard deviation; n = 3.

Figure 7:. Cytocompatibility of the hydrogels.

(a) Fluorescent micrographs of Live/Dead stained L929 fibroblasts cultured on the SF hydrogels. Green: calcein (live), red: EthD-1(dead), scale bars: 200 μm. (b) Survival rates of the cells at day 1. (c) Fold changes in metabolic activity of the cells cultured on hydrogel surfaces compared to day 1. (n = 4, *p < 0.05, **p < 0.01 and ***p < 0.001).

Scheme 1:

Scheme depicting the effect of DT on the enzymatic crosslinking of SF hydrogels in the presence of HRP and H₂O₂. All MWs formed into hydrogels, while the content of chemical crosslinks and crystals varied, impacting the properties of the different hydrogels formed in these reactions.