Tussah and B. mori silk (Haian County So HO SILK-MAKING Co., Ltd.), anhydrous sodium carbonate (Na₂CO₃, China National Pharmaceutical Group Corporation), protease XIV (active unit: 3.5 U/mg, Sigma-Aldrich), glutaraldehyde (50%, Sinopharmaceutical Group), anhydrous ethanol (Sinopharmaceutical Group), mouse embryonic fibroblasts (NIH-3T3, Beina Chuanglian Biotechnology Co., Ltd.), DMEM high-sugar medium (Gibco), fetal bovine serum (Procell), amino benzylpenicillin (Sigma), 0.25% trypsin digestion solution (Sigma), dimethyl sulfoxide (DMSO, Sigma), paraformaldehyde (Sigma), and CCK-8 kit (Shanghai Biyuntian Biotechnology Co., Ltd.) were used in this study.

As shown in Figure 1A, the tussah silk was degummed three times in boiling 0.5 wt% Na₂CO₃ solution for 30 min with a bath ratio of 1:50 (w/v) and rinsed thoroughly with deionized water to remove sericin. The degummed silk was cut into short fibers with about 5 mm length. These fibers were physically sheared in a high-speed shearing machine (Joyoung, Shandong, China) at 32,000 r/min for 10 min (10TSF), 20 min (20TSF), and 30 min (30TSF) to obtain TSF nanofiber solution. The solution was poured onto the Petri dish (90 mm diameter). After drying, the TSF nanofiber mats were prepared. The B. mori silk fibroin (30BSF) nanofiber mats were also prepared using the same process as a control in the biocompatibility experiment.

The 30 TSF nanofiber mats (n = 3 per group and time point) were incubated at 37°C in 0.1 U/mL protease XIV in phosphoric acid buffer solution at a ratio of 1:25. The solution was changed every 3 days, and the degraded samples were collected at designated time points. After drying, the samples were observed by SEM, and the mass remaining (MR) after degradation was calculated using the following equation: MR ( % ) = W b W a × 100 % .

In the formula, MR represents mass remaining, W_b represents the sample weight before degradation, and W_a represents the sample weight after degradation.

The morphology of TSF nanofiber mats was observed by scanning electron microscopy (SEM) (8,100, Hitachi, Tokyo, Japan) after sputter-coated with gold for 90s. The Fourier transform infrared (FTIR) spectra of TSF nanofiber mats were obtained using an ATR-FTIR spectrometer (NicoleT iS5, Thermal Nicolet Company, United States) in the spectral region of 400–4,000 cm⁻¹. The XRD spectra of TSF nanofiber mats were performed with an X-ray diffractive analyzer (40 kV, 40 mA) (Panalytical, Netherlands). The tensile properties of TSF nanofiber mats (50 mm × 10 mm) were measured using an Instron5967 universal material testing machine (Boston, United States) at 25 ± 0.5°C and 60 ± 5% relative humidity. The experiment was conducted at a cross-head speed of 10 mm/min with a gauge length of 30 mm and pretension of 0.2cN. At least five measurements for each sample were performed. The JY-PHA type contact angle tester was used to test water contact angle. The TSF nanofiber mats were randomly selected and cut into 5 cm × 5 cm, and then pasted on the contact angle tester platform. The high-precision CCD camera shot the image and sent it to the computer to display the high-definition image, and the computer automatically processed the relevant data.

The 30TSF nanofiber mats and 30BSF nanofiber mats with a diameter of 6 mm were prepared and sterilized at high temperature and high pressure. The mouse embryonic fibroblasts (NIH-3T3) were seeded at a density of 5×10⁴ cells (10 μl) on the mats. The cell was grown on the culture plate as a control. The cell culture medium was changed every 48 h. At the designated time points (1, 3, and 7 days; n = 3 for each time point), the culture medium was sucked out, and 250 μl cell counting kit-8 (CCK-8) solution was added to each well and incubated in a cell incubator for 2 h. After that, 100 ml supernatant was removed from each well and added to a 96-well plate and transferred to a microplate to detect the absorbance value (OD) at 450 nm. The cell morphology on nanofiber mats was observed by SEM at 3 day and 7 day.

One-way analysis of variance (ANOVA) was used for comparison between groups. The t-test was used for comparison between the two groups. The data obtained are expressed as mean ± standard deviation. SPSS20.0 software (IBM SPSS Statistics, US) was used to analyze statistically significant difference defined as p < 0.05 (*), p < 0.01 (**), and p < 0.001 (***).

Figure 1 shows the process to isolate tussah silk fibroin (TSF) nanofibers. First, Na₂CO₃ was used to extract TSF while removing sericin and impurities. Then, the degummed tussah silk was split into nanofibers by physical shearing. The degummed tussah silk is about 30 μm, presenting a ribbon-like shape with nanofibrils stacked neatly and tightly (Shimanovich et al., 2018). The micro-sized tussah silk consists of a bundle of nanofibrils with the diameter of ∼15 nm. These nanofibrils can be separated by the physical or chemical method due to their weak adhesion (Zhang et al., 2018). To isolate these TSF nanofibers, the degummed silk was treated by physical shearing to break the weak interfaces between nanofibrils.

Figure 2 shows the morphological change of degummed tussah silk with physical shearing. The increased microfiber and nanofiber were isolated from tussah silk with the increase of physical shearing time. Obviously, the diameter of isolated nanofibers was closely related to the shearing time. The fiber split and nanofiber exfoliation occurred after 2–8 min physical shearing (Figures 2A–C). With the increase of shearing time to 10 min, the intact tussah silk disappeared. The diameter of the isolated nanofiber decreased from 196.5 ± 65.1 nm to 152.2 ± 63.7 nm and 139.7 ± 52.9 nm with the increase of physical shearing time from 10 to 20 min and 30 min, respectively (Figure 3A). Of note, the diameter decrease is significant when the physical shearing time is increased from 10 to 20 min, but it is hard to further reduce the fiber diameter significantly through prolonging treatment time. The resulting TSF nanofibers can be stably and uniformly dispersed in aqueous solution for at least one month due to the electrostatic repulsion between anionically charged TSF nanofibers (Figures 3B–D) (Zheng et al., 2018). The TSF nanofiber solution after 10-, 20-, and 30-min physical shearing was cast to form TSF nanofiber mats for further study, including secondary structure, mechanical property, in vitro degradation, and biocompatibility.

The FTIR spectroscopic analysis was carried out for TSF nanofiber mats to determine their secondary structure, as shown in Figure 4. All the TSF nanofiber mats showed prominent amide I, II, III, IV, and V peaks at 1629 cm⁻¹, 1,517 cm⁻¹, 1,222 cm⁻¹, 965 cm⁻¹, and 700 cm⁻¹, respectively, attributed to the β-sheet conformation (Yang et al., 2018b; Fang et al., 2017), which revealed that the physical shearing had no effect on their secondary structure. To further determine the secondary structure of TSF nanofiber mats, the XRD analysis was also conducted, as shown in Figure 5. The XRD patterns of TSF nanofiber mats showed diffraction peaks around 16.8°(0.53 nm), 20.3°(0.44 nm), and 24.1°(0.37 nm) assigned to β-sheet structure, confirming the similar result to the FTIR analysis (Fu et al., 2011). It is consistent with previous studies that the physical shearing breaks only the weak interfaces between nanofibers in silk but does not destroy the strong β-sheet crystal structure in nanofibers (Okahisa et al., 2019; Wang et al., 2020a; Narita et al., 2020). So, the secondary structure of silk fibroin can be well retained by this moderate approach.

The mechanical properties of the biomaterial are important for its application in biomedicine. Figure 6 shows the stress–strain curve, breaking stress and breaking strain, and modulus of TSF nanofiber mats. The stress–strain curves of TSF nanofiber mats had a yield point followed by mild strain hardening, which was reminiscent of B. mori silk (Fu et al., 2011). The breaking stress was found to be 26.31 ± 2.54 MPa, 54.98 ± 3.79 MPa, and 72.68 ± 10.77 MPa for TSF nanofiber mats with 10-, 20-, and 30-min physical shearing, respectively. These values were better than the previous results from electrospinning BSF mats and electrospinning TSF nanofiber mats (Li et al., 2017; Shao et al., 2016; Wang et al., 2017). For example, the random and aligned electrospun TSF nanofibers showed much lower breaking stress of 1.7 and 2.7 MPa (Shao et al., 2016). The breaking stress in the range of approximately 72 MPa represented the strongest silk nanofiber mats to date, possibly due to the preservation of hierarchical structure and the dense packing of TSF nanofibers as observed by SEM (Figures 2, 7). Although the breaking strain (the highest value is 8.2%) was low, the TSF nanofiber mats were flexible which was likely due to the nanofiber nature and nanofibrous structure (Wang et al., 2020b). Young’s modulus of TSF nanofiber mats increased with the increase of shearing time, and the highest value exceeded 3 GPa, which was close to that of silk film (Zhang et al., 2015).

To further understand the failure behavior of TSF nanofiber mats, we conducted SEM imaging of the cross sections after tensile fracture, as shown in Figure 7. The fracture surfaces of TSF nanofiber mats were very rough due to their nonwoven nanofiber structure. The interface was not flat, and the individual nanofiber protrusion could be identified, likely originating from the weak bound interfaces of nanofibers. The morphological feature of cross sections clearly confirms the fracture mechanism of nanofiber fracture and nanofiber pull-out. The nanofiber fracture contributes to the high breaking stress of TSF nanofiber mats, and the nanofiber pull-out responds for its flexibility. The TSF mats derived from 10-min physical shearing was composed of thicker nanofibers (Figure 7A). After fracture, the pull-out of the layered structure was identified. The layer pull-out significantly influenced the stress and strain at break, as shown in Figure 6. The result was completely different in the TSF nanofiber mats composed of thin nanofibers (Figures 7B,C) (Benítez et al., 2013). The pull-out of the nanofiber instead of the layer appeared, thus significantly improving the breaking stress and strain. Taken together, the observed deformation behavior of TSF nanofiber mats included layer pull-out, nanofiber pull-out, and nanofiber fracture, which gave rise to the robust performance of TSF nanofiber mats.

The measurement results of contact angle are shown in Figure 8 and Table 1. It is known that the smaller contact angle represents higher hydrophilicity. The contact angles of the three kinds of mats were all within 90, which were close to that of the electrospun TSF nanofiber mats (Shao et al., 2017; Wang et al., 2017), indicating the excellent hydrophilicity of TSF nanofiber mats. The finer nanofiber had a larger specific surface area and more contact area with water on the mat surface, which endowed the nanofiber mat with more excellent hydrophilicity. However, with the increase of shearing time, the contact angle of the TSF nanofiber mats gradually increased. This was because of the decreased nanofiber diameter, which resulted in smoother surface and compact fiber structure that prevented water entering into the mats. The hydrophilic material can promote the adhesion and proliferation of cells on the surface through the absorption of nutrients (He et al., 2010; Bhattacharjee et al., 2015). Therefore, the superior hydrophilicity made the TSF nanofiber mats an excellent biomaterial.

To evaluate the in vitro degradability, the TSF nanofiber mat derived from 30-min physical shearing was immersed in protease XIV, which is known to effectively degrade silk fibroin (Horan et al., 2005; Taddei et al., 2006). As shown in Figure 9A, the TSF nanofiber mat lost about 11% of its original weight after 15 days. SEM imaging showed that transverse and longitudinal cracking and nanoscale fragments degraded from the TSF nanofiber mat. Similar results of degraded silk fragments were reported in previous studies (Numata et al., 2010; Zhou et al., 2010; Umuhoza et al., 2020). Compared with B. mori silk, tussah silk showed enhanced stability with slow degradation due to its high crystal content (Zhao et al., 2011; Sahu et al., 2015). Therefore, the TSF nanofiber mat featured slow degradation behavior, which could provide durable support or protect cell adhesion or tissue growth (Perrone et al., 2014).

The proliferation activity of NIH-3T3 cells on the BSF and TSF nanofiber mats was quantitatively analyzed by the CCK-8. The cultured plate was used as a control. It could be seen from Figure 10 that the cell number increased on both BSF and TSF nanofiber mats with the increase of culture time, suggesting their good biocompatibility. More cell adhesion on 1 day and more proliferated cell number on 3 and 7 day were observed on TSF nanofiber mats than those on BSF nanofiber mats. Previous studies had reported that the biocompatibility of TSF was superior to BSF, such as enhanced cell proliferation and tissue regeneration (Zhang et al., 2020). The inherent RGD sequence in TSF material was a well-known cell-adhesion site and contributed significantly to the superior biocompatibility of TSF material (Wang et al., 2019).

The morphology of NIH-3T3 cells grown on nanofiber mats was observed using a scanning electron microscope, as shown in Figure 11. The BSF and TSF nanofiber mats all support cell adhesion and proliferation. After the 3-day culture, more NIH-3T3 cells and their clusters were observed on TSF nanofiber mats than those on BSF nanofiber mats. With the increase of culture time to 7 days, the number of cells on the surface of the nanofiber mats significantly increased, and the cell sheet formed on both nanofiber mats. The SEM results further confirmed the good biocompatibility of TSF nanofiber mats, which would be a promising biomaterial.