Microfibre-Functionalised Silk Hydrogels

Abstract

Silk hydrogels have shown potential for tissue engineering applications, but several gaps and challenges, such as a restricted ability to form hydrogels with tuned mechanics and structural features, still limit their utilisation. Here, Bombyx mori and Antheraea mylitta (Tasar) silk microfibres were embedded within self-assembling B. mori silk hydrogels to modify the bulk hydrogel mechanical properties. This approach is particularly attractive because it creates structured silk hydrogels. First, B. mori and Tasar microfibres were prepared with lengths between 250 and 500 μm. Secondary structure analyses showed high beta-sheet contents of 61% and 63% for B. mori and Tasar microfibres, respectively. Mixing either microfibre type, at either 2% or 10% (w/v) concentrations, into 3% (w/v) silk solutions during the solution-gel transition increased the initial stiffness of the resulting silk hydrogels, with the 10% (w/v) addition giving a greater increase. Microfibre addition also altered hydrogel stress relaxation, with the fastest stress relaxation observed with a rank order of 2% (w/v) > 10% (w/v) > unmodified hydrogels for either fibre type, although B. mori fibres showed a greater effect. The resulting data sets are interesting because they suggest that the presence of microfibres provided potential 'flow points' within these hydrogels. Assessment of the biological responses by monitoring cell attachment onto these two-dimensional hydrogel substrates revealed greater numbers of human induced pluripotent stem cell-derived mesenchymal stem cells (iPSC-MSCs) attached to the hydrogels containing 10% (w/v) B. mori microfibres as well as 2% (w/v) and 10% (w/v) Tasar microfibres at 24 h after seeding. Cytoskeleton staining revealed a more elongated and stretched morphology for the cells growing on hydrogels containing Tasar microfibres. Overall, these findings illustrate that hydrogel stiffness, stress relaxation and the iPSC-MSC responses towards silk hydrogels can be tuned using microfibres.

Keywords: fibre; gel; iPSC; mechanics; silk fibroin; stem cells; tissue engineering.

Figure 1

Manufacture of Bombyx mori and Antheraea mylitta (Tasar) silk microfibres and microfibre-functionalised B. mori hydrogels.

Figure 2

Morphology of silk microfibres. Bombyx mori and Antheraea mylitta (Tasar) silk microfibre characterisation using (A) size measurements (analyses of light microscopy images) and (B) surface morphological assessment of silk microfibres with scanning electron microscopy (scale bar, 200 µm; zoom, 50 µm).

Figure 3

Silk hydrogel composites containing 2% and 10% (w/v) Bombyx mori and Antheraea mylitta (Tasar) silk microfibres. The hydrogel morphology was assessed using scanning electron microscopy (SEM) (scale bar, 400 µm; zoom, 100 µm). Silk hydrogels without microfibres were used as controls. Silk microfibres are indicated by arrows.

Figure 4

Secondary structure of silk samples. (A) Fourier transform infrared (FTIR) spectra and peak assignment. FTIR spectra of Bombyx mori and Antheraea mylitta (Tasar) silk microfibres, silk hydrogels, an air-dried film (negative control) and a 70% ethanol-treated silk film (positive control). Dashed lines indicate the β-sheet peak (1621 cm⁻¹) and the α-helix peak (1640 cm⁻¹). (B) Quantitative secondary structure analyses of the respective samples. Secondary structures and analysis are as detailed previously [30].

Figure 5

Impact of the microfibre amount and type on silk hydrogel mechanical properties. (A) Stiffness, (B) stress relaxation time profiles and (C) normalised stress relaxation time for 3% (w/v) self-assembled silk hydrogels (control) with and without microfibres. Data are presented as mean ± SD; n = 3 independent experiments; * p ≤ 0.05, ** p ≤ 0.01.

Figure 6

Biological response of human stem cells (iPSC-MSCs) towards two-dimensional silk hydrogel culture substrates. (A) Cell metabolic activity at 24 h after seeding on various silk hydrogels or on tissue culture-treated polystyrene (control). (B) Quantification of iPSC-MSC cell attachment. Control: iPSC-MSCs seeded on tissue culture-treated plastic were used as control. Data are presented as mean ± SD; n = 3 independent biological experiments. (C) Impact of substrate mechanics on iPSC-MSC cytoskeletal organisation. The images show F-actin cytoskeleton staining. All analyses were performed with reference to cell area, aspect ratio, roundness and circularity. Scale bars = 20 and 40 μm. Quantification of the morphological characteristics of iPSC-MSCs (106 cells in n = 21 images from three pooled experiments). One-way analysis of variance (ANOVA), followed by Tukey’s multiple comparison test, was used to compare the silk hydrogel composites with tissue culture-treated plastic; * p ≤ 0.05, ** p ≤ 0.01 *** p ≤ 0.005 and **** p ≤ 0.0001.