The influence of thermal treatments on the secondary structure of silk fibroin scaffolds and their interaction with fibroblasts

Preparation of silk fibroin sponge (SFS)

Silk fibroin aqueous solutions were prepared from the degummed silk thread of Bombyx mori by dissolution in 9 M LiBr, followed by dialysis against pure water, and SFSs were fabricated as described previously (Tamada, 2005; Kameda, Hashimoto & Tamada, 2011). Briefly, 1% v/v of DMSO (the sponge formation reagent) was added to the fibroin aqueous solution (6% w/v) and mixed thoroughly. The mixture was then poured into a mold (thickness, two mm), and the solution was placed at −20 °C and kept at this temperature for 17 h using a thermoregulated bath (EYELA, Tokyo, Japan). The mold was then thawed to room temperature to yield a porous structure. The SFSs were then washed with pure water in order to remove DMSO and subsequently stored in water at 4 °C until needed. Some SFSs were freeze-dried and sputter-coated with gold for field emission scanning electron microscopy (FESEM) imaging using JSM-6700F (JEOL, Tokyo, Japan).

Heat treatments

Wet-heating treatments were performed by immersing the SFSs (5.0 × 3.3 cm) in ion-exchanged water at predetermined temperatures (37 °C, 50 °C, 60 °C, 80 °C and 95 °C) for 360 min. Wet-heating treatments at 95 °C were also carried out for various other durations (3, 5, 10, 20, 90 and 180 min). For autoclaving, SFSs were heated to 120 °C (at a pressure of 0.11 MPa for 20 min) in ion-exchanged water using an autoclave (TOMY, Tokyo, Japan). Dry-heating treatment involved oven-based heating (SANYO, Tokyo, Japan) at 180 °C for 30 min. Prior to dry-heating treatments, the SFSs were dried at 25 °C for at least 12 h using a dryer equipped with a vacuum pump (YAMATO, Tokyo, Japan). These conditions are summarized in Table 1. The abbreviations used in this paper are the following: NON, non-treated SFSs; AC, autoclaved SFSs; and DH, dry-heated SFSs.

¹³C CP/MAS NMR measurements

High-resolution solid-state ¹³C NMR spectra were recorded on a Bruker Avance 600 WB NMR spectrometer (Bruker, Karlsruhe, Germany) equipped with a magnetic field of 14.1 T. The spectrometer was operated at a ¹³C NMR frequency of 150.94 MHz. A 4.0 mm ϕ zirconia rotor was used as the sample tube, and magic angle spinning (MAS) was performed at a speed of 10.0 kHz. In cross-polarization (CP) experiments, a ¹H π/2 pulse length of 3.5 µs and a ¹H-¹³C CP contact of 70 kHz were employed. The repetition time was set at 3.0 s. High-power ¹H decoupling was employed by the SPINAL-64 method. Approximately 16,000 scans were accumulated for each spectrum to attain a reasonable S/N ratio. The chemical shifts of all spectra were calibrated through the adamantane CH₂ peak (29.5 ppm) relative to the tetramethylsilane (TMS) peak (0 ppm).

Thermogravimetry

Weight losses as a function of temperature were measured using a thermogravimetric (TG) apparatus (Rigaku, Tokyo, Japan). Non-treated SFSs were heated from room temperature to the given temperature at a rate of 3 °C/min using uniform air flow (200 mL/min). Weight loss as a function of time of non-treated SFSs at various temperatures were measured.

Mechanical properties

The mechanical properties of the SFSs were measured using an EZ test (Shimadzu Corp., Kyoto, Japan) with a 50 N load cell. SFSs in the wet state (thickness: around two mm) were cut into a dumbbell shape and stretched with a head speed of five mm/min. The force at break is generally evaluated as the tensile strength (N/m²) by converting it into the force per unit cross-sectional area. However, because the wet SFSs contain many voids including water, it is difficult to accurately determine the cross-section of only SFS. Therefore, using the samples with identical thickness and width, the observed values of force at break were directly used for comparison. Statistical analysis was performed to compare mechanical properties using ANOVA and the Tukey Post Hoc test (n = 4).

Cell culture

Cell adhesion and proliferation experiments on/in heat-treated SFSs were performed using the NIH/3T3 murine fibroblast cell line. NIH/3T3 cells were grown in Eagle’s medium (EMEM; Nissui, Japan) containing 10% FBS (Invitrogen, Life Technologies Corp., USA), 2 mM L-glutamine (Invitrogen, Life Technologies Corp., USA) and 0.1 mg/mL kanamycin (Invitrogen, Life Technologies Corp., USA) in a humidified incubator (5% CO2 and 95% air at 37 °C).

Wet heating or dry heating was carried out before the cells were introduced onto the sponge surface. Prior to cell seeding, six mm diameter circles were cut from ACs using a biopsy punch and placed in EMEM for 1 h at 37 °C. DHs were first immersed in PBS before excision of six mm circles. And then, PBS was replaced with EMEM. NIH/3T3 cells were then seeded at a density of 1 × 10⁵ cells/sponge. After 1, 24, 72, 120 and 168 h of incubation at 37 °C and 5% CO2, wells were washed with PBS to remove non-adherent cells, and 0.5% Triton X-100 in PBS was added to each well to permeabilize the cells. Lactate dehydrogenase (LDH) activity in each well was measured as described previously (Tamada & Ikada, 1994). The cell number at each time point was determined by comparison to a standard curve of cell number versus LDH activity and then averaged. The doubling times of the cells on the surfaces were calculated by exponential curve-fitting.

Cell migration

Cell migration experiments were performed using silk fibroin-coated glass to mimic the surface for sponges. Silk fibroin-coated glass was prepared using cover slips (15 mm in diameter, Thermo Fisher Scientific K.K., Japan) and by incubation in a fibroin aqueous solution that had been adjusted to 1% (w/v). After incubation for 30 min at room temperature, the fibroin solution was removed, and the fibroin surfaces were dried in an oven at 50 °C (for AC-mimicking surfaces) or at room temperature (for DH-mimicking surfaces) for at least 12 h. In order to induce the structural changes from random to β-sheet (Min et al., 2004), the AC-mimicking surfaces were treated with 80% MeOH solution for 30 min at room temperature and then dried at 50  °C for at least 12 h. Additionally, some AC-mimicking surfaces were autoclaved and treated with 70% EtOH solution for 1 h. The DH-mimicking surfaces were vacuum-dried at 25 °C for at least 12 h and then dry-heated in an oven at 180 °C for 30 min. The secondary structures of these mimicking surfaces were measured using FTIR spectroscopy (FT/IR-4100, JASCO, Tokyo, Japan) equipped with an ATR unit (PRO450-S, JASCO, Tokyo, Japan) to determine whether these surfaces did indeed mimic sponges. FTIR-ATR spectra of mimicking surfaces were recorded at a resolution of four cm⁻¹ between 500 and 4,000 cm⁻¹. The contact angles of the mimicking surfaces were measured using the sessile drop method. Prior to cell culture experiments, the AC-mimicking surfaces were autoclaved for 20 min at 120  °C and then immersed in 70% v/v ethanol. Quantitative evaluations of cell migration on the mimicking surfaces were performed using the MtrackJ software as described previously (Hashimoto et al., 2013). Cell motility on various surfaces was averaged between five individual cells.

Effects of heat treatments on the morphologies and secondary structures of SFSs

FESEM was conducted to examine the morphological difference between the porous structures of SFSs before and after heat treatments, as well as the difference between the three kinds of treatment (Fig. 1). Uniform porous structures were observed in all SFSs, indicating that heat treatment with or without water did not result in a morphological difference between SFSs.

To investigate the secondary structure of the protein molecules in the SFSs and in order to assess any structural changes resulting from the heat treatments, SFSs before and after the heat treatment were analyzed using ¹³C CP/MAS NMR. The focus of the analysis was on the C_α and C_β peaks of Ala and Ser residues because these peaks are sensitive to structural changes and are relatively stronger and better resolved. Peak assignments were made by comparing the chemical shifts observed in the spectra of the peptides and those of the other silk fibers (obtained from silkworms, spiders, etc.) in solution and the solid state. Figure 2A shows the C_α and C_β regions of the protein molecules in the ¹³C CP/MAS NMR spectra of NON, AC and DH. The NMR spectrum of DH was very similar to that of NON, and the AC spectrum differed markedly. An expansion of the Ala C_β peak area of the heated SFSs is shown in Fig. 2B. An independent peak for Ala C_β was used to determine the structure of the fibroin molecules because of a large chemical shift in the structure (Saitô, Tuzi & Naito, 1998). The peaks at 17.4 and 20.5 ppm in Fig. 2B represent chemical shifts attributable to Ala in silk I and silk II structures, respectively. The intensity of the peak at 17.4 ppm decreased with the increasing treatment temperatures in the wet-treated SFSs, although AC exhibited only a small shoulder. In contrast, the peaks at 20.5 ppm that were attributable to silk II increased with increasing wet-heating temperatures. No structural changes were observed in DH even after the SFSs were heated above 120 °C, as described above (DH and NON in Fig. 2A).

To compare the effects of heat treatments on the SFS structure in detail, the ratio of silk II to silk I (II/I) was determined from the peak intensities of Ala C_β and plotted (Fig. 3) (Kim et al., 2003; Zhou et al., 2004). Structural changes from silk I to silk II were observed in SFSs wet-heated at 95 °C, which reached a plateau at around 30 min (Fig. 3A). When SFSs were wet-heated at 37 °C, 50 ° C, 60 °C, 80 °C, 95 ° C and 121 °C (AC) for 360 min, the II/I ratios were 0.92, 0.93, 1.06, 1.25, 1.55 and 1.64 respectively, indicating that the heat treatments in the presence of water induced structural changes from silk I to silk II (Fig. 3B). The II/I ratios for all of the SFSs in this temperature titration plateaued within the 360 min treatment time. The transformation from silk I to silk II occurred above 60 °C, and the II/I ratio increased with increasing temperature, with the highest II/I ration occurring in the autoclaved SFSs. Fibroin films have been reported to exhibit structural changes from random coils to silk I (and subsequently to silk II), which occurs in water at > 70 °C (Magoshi et al., 1979). Moreover, this transformation in films occurred in water even below 60 °C, whereas here, no transformation was observed in the SFS in water below 60 °C. Autoclave sterilization (exposure to steam at 121 °C for 20 min) of SFS also induced structural changes, as evident from the marked differences between the spectra shown in Fig. 2, indicating that the conformational transition from silk I to silk II occurs during autoclave treatment. In contrast, the ratio of DH was 0.95, which is close to the ratio of 0.92 for NON, suggesting that no structural changes occurred during the dry-heating treatment.

Properties of heated SFSs

Thermogravimetry (TG) curves of SFSs are shown in Fig. 4A. Following dehydration-induced weight loss at temperatures up to 180 °C, additional weight loss was also observed above 200 °C because of thermal degradation. Next, sponge samples were rapidly heated (100 °C/min) to an elevated temperature (after heating to 100 °C for removing residual absorbed moisture), and their weight loss due to thermal degradation was measured over time (Fig. 4B). The weights of SFSs heated to 180.5 °C and 190.2 °C remained unchanged over 180 min.

Maximum forces at break for heat-treated SFSs were quite similar as shown in Fig. 5. This result was consistent with the fact that there was no significant difference in morphology due to heat treatment conditions as described in Fig. 1.

Interactions with cells

Figure 6 shows the proliferation of NIH/3T3 cells cultured on AC and DH over 120 h. The calculated doubling times (from 24 h to 120 h) were 26 h (AC) and 34 h (DH) and were significantly different (P < 0.005, n = 3). However, both AC and DH contained similar cell numbers 168 h after seeding. Since both sponges were autoclaved and both dry-heating methods used the same pore size (Fig. 1), it appears that different structures of fibroin molecules affected the doubling times of fibroblast at early stages in culture.

To evaluate cell functions apart from proliferation, cell motilities were also measured quantitatively using single-cell tracking with time-lapse image capture coupled to computer image analysis. Cell migration is known to heavily depend on the structure and properties of the extracellular matrix (ECM) (Li et al., 2004). Fibroin-coated glass was prepared in order to mimic AC and DH surfaces and was used because of the difficulty inherent in measuring cellular migration in the confines of the three-dimensional structure of the SFSs. Figure 7 shows the FTIR-ATR spectra of the AC- and DH-mimicking surfaces. To analyze the contents of each structure in the mimicking surfaces, the amide I band was deconvoluted into individual bands by Gaussian curve-fitting (Tretinnikov & Tamada, 2001; Hu, Kaplan & Cebe, 2006; (Dong, Huang & Caughey, 1990). Bands centered at around 1,610, 1,619, 1,625, 1,633, 1,642, 1,651, 1,659 and 1,680 cm⁻¹ were assigned to Tyr side chain/aggregated strand (SC), aggregated β-strand/ β-sheet (B), β-sheet (B), β-sheet (B), random coil/extended chain (R), α-helix (A) and turn (T), respectively. A band centered at 1,700 cm⁻¹ and assigned to β-sheet (B) was detected in AC- mimicking surfaces. Contents of β-sheets, α-helices, random coils and turns obtained from amide I band measurements are shown in Table 2. Compared to the structure contents of DH-treated mimicking surfaces, AC-mimicking surfaces clearly exhibited higher levels of β-sheet structure. These results demonstrate that secondary structures in AC-mimicking surfaces changed to β-sheet upon autoclave treatment, similarly to the structural changes in SFSs detected by NMR measurements. This evaluation indicated that AC-mimicking and DH-mimicking surfaces could indeed mimic autoclaved and dry-heated SFSs (AC and DH), although the absolute values of silk I or silk II contents differed slightly. The contact angle for uncoated glass was 37.7 ±  3.4°, which increased in fibroin-coated surfaces. The AC-mimicking surfaces had a contact angle of 49.1 ±  9.2° and were found to be more hydrophilic than the DH-mimicking surfaces (78.5 ±  0.4°). Figure 8 shows the cell migration on the mimicking surface. The movements of the fibroblasts 23 h after attachment to AC- and DH-mimicking surfaces were 747 and 588 µm, respectively. Thus, cell motility was slightly higher on AC-mimicking surfaces.