Construction of SF substrates with different conformations

B. mori silk cocoons were first degummed in boiled 0.02 mol·L⁻¹ Na₂CO₃ solution for 30 min and then washed with Milli-Q water. After drying, the extracted silk was dissolved in 9.3 mol·L⁻¹ LiBr solution at 60 °C for 4 h and then dialyzed with Milli-Q water.¹⁰ Insoluble residues were removed by centrifugation. Finally, the SF aqueous solution was diluted to 4 wt%.

To facilitate good handling and avoid morphological changes of SF materials during the experiments, we prepared silk substrates by casting 200 μL of SF solution onto titanium disks (15 mm diameter). To enhance the contact area and adhesion of SF film with titanium disk and avoid detachment of SF film during the whole experiments, we first acid etched the titanium disks with HCl/H₂SO₄ for 30 min at 60 °C, ultrasonically cleaned them in acetone, ethanol and water, and finally subjected them to argon plasma glow discharge (Radio frequency glow discharge machine, Harrick Scientific Corp., U.S.A.) for 5 min before the SF solution casting. The SF films were dried in a vacuum oven to avoid structural changes.³⁸ Finally, the films were annealed in a water vapor-filled vacuum chamber at 4 °C for 6 h (SFL group), 37 °C for 12 h (SFM group), or 90 °C for 24 h (SFH group) to achieve different β-sheet contents in the SF substrates. The thickness of the SF films made by this method was ~50 μm.^10,38

Fourier transform infrared spectroscopy (FTIR)

FTIR analysis of the silk films was performed using attenuated total reflectance infrared spectroscopy (UATR Two, PerkinElmer, the Netherlands). For calculation of the β-sheet content in the different SF substrates, the contribution of the different SF conformations to the amide I region (1 595–1 705 cm⁻¹) was determined by Fourier self-deconvolution using PerkinElmer software and subsequent curve fitting by OriginPro software according to a previously reported step-by-step method (n = 3).^15,51

Surface topography of the SF substrates observed via SEM

The surface topography of SF substrates dried in a vacuum oven was examined by SEM (Zeiss, Sigma-300, Germany) after being coated with a 10 nm chromium layer.

AFM

The surface topography and roughness of the SF substrates were measured using AFM (Bruker, multimode 8, U.S.A.) (n = 3, three random points per sample). The stiffness of the SF films was measured by using AFM cantilevers (SNL-10, Bruker, multimode 8, U.S.A.) with a nominal spring constant of 0.35 N·m⁻¹. Samples for stiffness measurement were first hydrated with PBS solution,³⁸ and the force vs. indentation curves were obtained in PBS on each SF substrate. Elastic modulus values were analyzed by NanoScope Analysis software.

Wettability

The wettability of different SF substrates was determined by detecting the static water contact angles of the surfaces with an optical tensiometer (Theta Lite Attension®, Biolin Scientific, Sweden) (n = 3).

Surface stability of the SF substrates

For detection of the surface stability in an aqueous environment, samples were immersed in 2 mL of PBS solution at 37 °C for 14 days. PBS was refreshed every 24 h. At specified time points, a mild ultrasonic treatment was applied to remove any loosened components from the material surfaces before removing the samples from PBS. The probe of the sonicator (UP50H, Hielscher, Germany) was located 1 cm right above the sample, and mild ultrasonic treatment lasted for 6 s at 20% amplitude with a pulse rate of 1 s on and 1 s off according to previous references.⁴¹ Then, the samples were gently rinsed with Milli-Q water and dried in a vacuum oven. The remaining mass of SF materials at specified time points was calculated using the following formula:

where W₀ represents the initial weight of the sample and W_t represents the weight of the sample at a specified time point (n = 3).

Detachment resistance of FN

FN (5 μg·mL⁻¹) from human plasma (F1056, Sigma, U.S.A.) was dissolved in PBS solution according to previous references.^19,22 The SF substrates were immersed in FN solution for 24 h. Then, the samples were treated without or with the same ultrasonic treatment used in the surface stability experiments before removing the samples from the solution. Subsequently, all samples were incubated with the corresponding primary and secondary antibodies (Supplementary Table 2) to fluorescently label FN. Images were captured by a fluorescence microscope (Axio Imager Microscope Z1, Zeiss, Germany), and fluorescence density was analyzed by ImageJ (n = 3, three random fields per sample).

For determination of the FN detachment ratio under external stimuli, FN concentrations in solutions were determined by a FN Human ELISA kit (BMS2028, Invitrogen, U.S.A.). The detachment ratio was calculated using the following formula:

where C₀ represents the initial concentration of FN added to the PBS solution, C₁ represents the concentration of FN remaining in the PBS solution after 24 h of immersion, and C₂ represents the concentration of FN detached under external stimuli (n = 3).

In addition, immersion of samples in 50 mmol·L⁻¹ Tris buffer pH 7.4 containing 1 mmol·L⁻¹ EDTA for 24 h at 37 °C⁵² was chosen as an alternative artificial external stimulus for reference.

Cell isolation and identification

BMSCs were isolated from the femurs of 3-week-old male rats (Charles River) with the approval of the Institutional Animal Care and Use Committee of Tongji Medical College (IACUC Number: 539). For each batch, primary cells derived from at least five rats were pooled together, and three batches of cells were made to verify the reproducibility of the experiments. Cells were cultured in growth medium consisting of α-MEM medium (Gibco, Invitrogen Corp., Paisley, Scotland) supplemented with 10% fetal bovine serum (FBS, Gibco, Invitrogen Corp., Paisley, Scotland) and 1% penicillin-streptomycin according to a standard protocol.^40,53 The pluripotency of BMSCs was identified with a flow cytometer (LSRFortessa, BD, U.S.A.). Cells were harvested and suspended to a concentration of 1 × 10⁶ cells per mL in ice-cold PBS, and CD90, CD44, and CD29 were used as positive markers, while CD31 was used as a negative marker (Supplementary Table 3). Cells were passaged to the 3rd generation at a confluency of 70%–80% before use.

Cell culture

Untreated SF substrates were sterilized by ultraviolet light for 30 min. For analysis of the detachment resistance of FN in a cell culture environment, the SF substrates were first immersed in serum-free (to avoid the interference of FN from serum) cell culture medium (α-MEM) containing 5 μg·mL⁻¹ FN for 2 h.^19,22 Then, BMSCs were seeded at a density of 2 × 10³ cells per cm² in serum-free medium and cultured for 24 h, while SF substrates immersed in the same medium without cells were used as controls.

For other cell assays, untreated SF substrates were first immersed in complete osteogenic medium (α-MEM, 10% FBS, 50 μg·mL⁻¹ ascorbic acid (A4544, Sigma, U.S.A.), 10 mmol·L⁻¹ β-glycerophosphate (G9422, Sigma, U.S.A.), 10⁻⁸ M dexamethasone (D4902, Sigma, U.S.A.), and 1% penicillin-streptomycin) for 2 h before cell seeding. Then, cells were seeded in this complete osteogenic medium. For the individual cell-based fluorescent measurements, a seeding density of 2 × 10³ cells per cm² was applied for the cell adhesion, spreading, and intracellular mechanotransduction experiments according to the literature.³² For the fluorescent staining of RUNX2 and YAP/TAZ at day 3, the SF substrates were treated with mitomycin C (10 μg·mL⁻¹, Sigma, U.S.A.) for 2 h after 12 h of cell seeding to inhibit proliferation, which can interfere with their individual cell-based fluorescence measurements.³² In addition, 1 × 10⁴ cells per cm² BMSCs were seeded to assess cell proliferation and osteogenic differentiation.

Immunofluorescence staining

At specific experimental time points, cells were fixed with 4% paraformaldehyde and permeated with 0.1% Triton X-100. Subsequently, the cells were blocked with 1% goat serum for 30 min, incubated with the corresponding primary and secondary antibodies (Supplementary Table 3) and incubated for 1 h. Samples were incubated with phalloidin for 30 min and DAPI for 10 min to label F-actin and mark cell nuclei, respectively.

CKB treatment could remove the proteins that were not associated with the cytoskeleton. CKB treatment was applied as described previously to distinguish the amount of cytoskeleton-associated vinculin-containing FAs from the total amount of vinculin-containing FAs.³⁶ Briefly, to observe the cytoskeleton-associated (i.e., CKB treatment-resistant) vinculin-containing FAs, cells were first treated twice with CKB (0.1% Triton X-100, 10 mmol·L⁻¹ PIPES, pH 6.8, 50 mmol·L⁻¹ NaCl, 3 mmol·L⁻¹ MgCl₂, 300 mmol·L⁻¹ sucrose) at 4 °C for 30 s, followed by a standard fixation step with 4% paraformaldehyde. Images of stained samples were captured by confocal fluorescence microscopy (Zeiss, LSM780, Germany).

Fluorescent image analysis

All images were analyzed by ImageJ (NIH, U.S.A.).

For collagen Ι quantification, the mean intensity of four random fields across three samples per group was measured.

FN detachment induced by cell contractility was measured using a method as previously described.²² In brief, the cellular masks were determined by the thresholding method from F-actin fluorescent images and used to delineate the cell outline. We calculated the ratio of the dark area underlying the cell outline in FN staining images to the total cell area. In addition, we calculated the ratio of FN intensity underlying the cell outline to the background (the other area beyond the cell outline) FN intensity. Four random fields across three samples per group were measured.

At least 30 individual cells across three samples per group were selected for all cell-based measurements. For measurement of the FA area per cell, the grayscale vinculin image was thresholded to produce a black and white image from which the pixels representing FAs were counted and summed, following a step-by-step quantitative FA analysis protocol as previously reported.⁵⁴

For measurement of cell area and perimeter, the cellular masks were determined by a thresholding method from F-actin fluorescent images, and then, they were used to calculate cell area and cell perimeter. The CSI was calculated using the formula as previously reported:²⁸

where a line and a circle have CSI values of 0 and 1, respectively.

F-actin anisotropy was measured by the ImageJ plug-in “FibrilTool” as previously reported,⁵⁵ where disordered F-actin (purely isotropic fibers) and perfectly ordered F-actin (parallel fibers) have anisotropy values of 0 and 1, respectively.

The nuclear/cytosolic (nuc/cyto) ratio of YAP/TAZ and RUNX2 was determined by measuring the intensity of a region of the nucleus and a region of equal size in the cytosol immediately adjacent to the nuclear region as previously described.³⁶

Cell spreading observed by SEM

Samples were fixed in 2% glutaraldehyde for 20 min and then immersed in 0.1 mol·L⁻¹ Na-cacodylate for 10 min. Then, the samples were dehydrated in a graded series of ethanol (5 min in 70%, 80%, 90%, 96%, and 10 min in 100% ethanol twice). The samples were then treated with 1 drop of tetramethylsilane, air-dried, coated with 10 nm chromium, and examined via SEM.

ALP and mineralization

After 7 days of cell culture, ALP activity in BMSCs was measured using the p-nitrophenyl phosphate (Sigma, U.S.A.) method as previously described.¹⁹ The ALP activity was normalized by DNA content per sample via a QuantiFluor dsDNA System kit (E2670, Promega Corporation, Madison, U.S.A.). For ALP staining, cells were fixed and stained with an ALP Staining Kit (AP100B-1, SBI, U.S.A.), followed by taking images via a stereomicroscope (MZ12, Leica, Germany). After 14 days, calcium contents were assayed using the ortho-cresolphthalein complexone (Sigma, U.S.A.) method as previously described.⁴⁸ For mineralization staining, cells were fixed and then stained with an Alizarin Red S staining kit (TMS-008-C, Merck Millipore, U.S.A.), followed by taking images via stereomicroscope.

Quantitative reverse transcription polymerase chain reaction (qRT-PCR)

The RNA of BMSCs on different silk substrates (n = 3) was extracted using the RNAprep Pure Micro Kit (DP420, Tiangen, China) according to the manufacturer’s instructions. The RNA was reverse transcribed to cDNA using HiScript III RT SuperMix for qPCR (R323-01, Vazyme, China). Subsequently, cDNA was added to ChamQ SYBR qPCR Master Mix (Q311-02/03, Vazyme, China) and complemented by a real-time PCR system (ABI 7300, Applied Biosystems, U.S.A.). The primers for the genes are listed in Supplementary Table 4. The mRNA levels of target genes were normalized to the level of GAPDH mRNA and calculated via the 2^-ΔΔCt method.

Inhibition of cytoskeleton organization

For analysis of the inhibition of cell spreading and cytoskeletal organization, Y27632 (50 μmol·L⁻¹, Selleck, U.S.A.) was supplemented daily with the cell culture medium, and the treatment lasted 4 h prior to cell harvesting as previously described²⁴ for fluorescent staining and qRT-PCR.

Cell proliferation

Cell proliferation on different material surfaces (n = 3) was assessed by a cell counting kit (CCK-8, Dojindo, Japan) according to the manufacturer’s instructions.

Statistical analysis

One-way ANOVA was used to determine statistical significance followed by post hoc analysis using the Tukey test. All statistical analyses were performed with GraphPad Prism and Origin software.