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Mechanically robust and personalized silk fibroin-magnesium composite scaffolds with water-responsive shape-memory for irregular bone regeneration – Nature Communications

We have complied with all relevant ethical regulations declared in the manuscript, and disclosed the name(s) of the board and institution in the methods part. All procedures were approved by the animal care and use committee of Beijing Jishuitan Hospital regarding the Guiding Principles for the care and use of experimental animals (Number: 2022-12-03).

Materials

Bombyx mori cocoons were procured from Favorsun Medical Technology (Shanghai) Co., Ltd., China. Lithium bromide (LiBr, 99%), EGDE, Epoxy value ≥ 0.7, N,N,N’,N’-Tetramethylethylenediamine (TEMED, 99%), dialysis tubes (8000–14,000 MWCO) and dialysis tubes (3500 MWCO) were obtained from Sigma-Aldrich, United States. Polyethylene glycol (PEG, Mn ~ 100,000 g mol-1) and sodium bicarbonate (NaHCO3, 99.8%) were purchased from Macklin Chemical Reagent Co., Ltd., China. MgO particles (50 nm, 99%) were obtained from Aladdin, Shanghai. Simulated body fluid (SBF, pH = 7.4) and phosphate buffered saline (PBS, pH = 7.4) were purchased from Beijing Solarbio Science & Technology Co., Ltd.

Preparation of silk fibroin solution

The SF solution was extracted from Bombyx mori cocoons. Briefly, silk cocoons (10 g) were degummed in a boiling NaHCO3 solution (2 L, 5 g/L) for 1 h, and sericin was removed with water. The degummed silk fibres were oven-dried at 45 °C for 24 h. Then, the dry degummed silk fibres (8 g) were dissolved in LiBr solution (100 mL, 9.3 M) for 3 h at 40 °C. The SF solution was subsequently dialysed (MWCO 8000−14,000) against deionized water for 3 days; and the deionized water was changed 3 times a day to remove ions and impurities. The purified SF solution (~20 mg/mL) was acquired. Finally, the highly concentrated SF solution (50−60 mg/mL) was obtained by concentration in 15% PEG solutions using dialysis membrane (3500 MWCO).

Fabrication of silk fibroin/magnesium composite scaffolds

The silk fibroin/magnesium composite scaffolds were synthesised through cryogelation. The process could be found in Supplementary Fig. 1. Briefly, SF solutions (60 mg/ml), EGDE (2 mmol/g) and TEMED (0.25 v/v%) were added in a centrifuge tube. Next, MgO nanoparticles were added to deionised water to prepare a 90 mg/mL suspension. MgO suspension was added to the above SF mixture solution according to the ratio of the solute mass of the SF solution to the mass of the MgO nanoparticles at 10:0 (SF), 10:1 (SF-1nMgO) and 10:3 (SF-3nMgO), respectively. The above solution was uniformly mixed, and the cryogel reaction was performed at −10 °C for 24 h. After thawing in a water bath (room temperature) for 12 h, SF/MgO composite scaffolds were obtained. The residual crosslinkers and catalysts in SF/MgO scaffolds were removed by rinsing with ultrapure water. The SF/MgO scaffolds were frozen at −20 °C for 12 h, followed by freeze-drying at -50 °C for 24 h. The dry scaffolds were then sterilised using cobalt-60 (25 kGy) radiation for 2 h.

Characterisation of silk fibroin/magnesium composite scaffolds

The pore size and micromorphology of SF/MgO scaffolds were observed by scanning electron microscopy with an energy-dispersive spectrometer (SEM-EDS, S-4800, Hitachi, Japan) at an accelerating voltage of 10 kV. The cryogeled SF/MgO composite scaffolds were first frozen at −20 °C for 12 h, and vacuum-dried at −50 °C for 24 h. The dry SF/MgO scaffolds were sputter-coated with gold for the SEM test.

The wide-angle X-ray diffraction (XRD) patterns of the SF/MgO scaffolds were conducted using an XRD (D8 Advance, Bruker, Germany) and a Cu Ka radiation source (1.54 Å). The SF/MgO scaffolds were scanned from 2θ = 10° to 60°. The surface chemical and conformation analysis of the SF/MgO scaffolds were measured by attenuated total reflection Fourier-transform infra-red spectroscopy (ATR-FTIR, Thermo Scientific Nicolet iS20) over a wavenumber range of 400 to 4000 cm−1. 32 scans with a resolution of 4 cm-1 were accumulated for each spectrum at 25 °C. The conformation contents of the SF/MgO scaffolds in the amide I region (1595–1705 cm−1) was analyzed by Fourier self-deconvolution (FSD) using PeakFit software (version 4.12)42. The 1−2 mm thick slices of freeze-dried scaffolds were compacted for the XRD and FTIR tests.

Degradation behaviour of silk fibroin/magnesium composite scaffolds

The dry scaffolds were prepared using 1 ml of homogeneous reaction solution (SF, SF-1nMgO, SF-3nMgO) by cryogel reaction and used as samples for degradation experiments. The SF, SF-1nMgO and SF-3nMgO scaffolds were incubated at 37 °C in a PBS solution that contained 2 U/mL protease (Type XIV from Streptomyces griseus (3.5 U/mg)). The enzyme solutions were changed every 2 days to maintain the enzyme activity. The scaffolds were removed from the enzyme solution and rinsed with deionised water at indicated time (3, 7, 14 and 28 days), freeze-dried and weighed. Weight and morphology were recorded. The weight loss (%) of the scaffolds was calculated as follows:

$${{{{{rm{Weight; loss}}}}}}(%)=({{{{{{rm{W}}}}}}}_{0}-{{{{{{rm{W}}}}}}}_{{{{{{rm{t}}}}}}})/{{{{{{rm{W}}}}}}}_{0} times 100%,$$

(1)

where Wt and W0 are the weights of the remaining and initial scaffolds at different time points, respectively.

In vitro Mg2+ release behaviour of silk fibroin/magnesium composite scaffolds

The immersion test was performed to measure the accumulative release profile of magnesium ion (Mg2+) from the SF/MgO composite scaffolds. The samples were prepared using 1 ml of homogeneous reaction solution (SF-1nMgO, SF-3nMgO) using a cryogel reaction. The two samples (SF-1nMgO and SF-3nMgO) were incubated individually in 2 mL of SBF (pH = 7.4) at 37 °C. The Mg2+ concentrations in the extract liquids of the samples were measured by inductively coupled plasma-optical emission spectrometry (ICP-OES; Thermo Fisher Scientific, USA) at 3, 7, 14 and 28 days, respectively. The pH change of the extract liquids was measured using a pH metre (Mettler, Switzerland) and recorded.

Zeta potential measurement

The zeta potential of the SF solution, SF-1nMgO solution (SF: MgO, 10:1, w/w) and SF-3nMgO solution (SF: MgO, 10:3, w/w) solution was measured at 25 °C (Malvern Zetasizer Nano ZS90) according to ISO-13099-2. The sample concentration was diluted to one-half of the initial concentration for testing. Three measurements were obtained for each group.

Mechanical properties of silk fibroin/magnesium composite scaffolds

The mechanical retention during degradation and cyclic compression properties of the SF and SF/MgO scaffolds were measured using a dynamic mechanical analyser (DMA) Q800 (TA Instruments, Waters Ltd. USA). The compression testing samples were cut to 10 mm × 10 mm (diameter × height). The mechanical retention of samples was incubated at 37 °C in SBF solution and removed at indicated time points (0, 14 and 28 days). On day 0, all samples were adequately hydrated in PBS for 12 h before testing. The compression test was carried out at a strain rate of 30%/min at room temperature. Young’s modulus of samples was calculated from the initial linear strain range of the curve obtained from the stress-strain. Five cycles compression testing of the SF-1nMgO scaffolds was measured at a strain rate of 30%/min with a maximum strain of 30%. Fatigue-resistant ability testing of the SF-1nMgO scaffolds was conducted between −2% and −22% strain at a strain rate of −100%/min for 1000 cycles.

Shape-memory effect and personalised customisation of silk fibroin/magnesium composite scaffolds

The SF/MgO scaffolds were fully hydrated in PBS solution and subsequently subjected to bending, compression and torsion deformation, followed by exposure to room-temperature water to evaluate their ability to restore their original shape. Multiple scaffolds were completely compressed together and subsequently exposed to room-temperature water and blood to record the rate and extent of recovery of the scaffolds to their original shape in different media environments. In addition, the compressed wet SF-1nMgO scaffolds were placed at a weight of 50 g. The scaffolds were then incubated in room-temperature water and the images were recorded. Furthermore, the potential of SF-1nMgO scaffolds to be trimmed during clinical surgery for personalised implantation was explored. The bulk SF-1nMgO scaffolds were prepared and then roughly and precisely trimmed according to the shape of the bone defect. Finally, the SF-1nMgO scaffolds were wetted and compressed to achieve the implantation of small pore-size irregular bone defects.

In vitro cell biocompatibility and migration of silk fibroin/magnesium composite scaffolds

Mouse embryo osteoblast precursor (MC3T3-E1) cells (PUMC000012, Cell Resource Center, IBMS, CAMS/PUMC) were utilised for evaluating cell viability, proliferation and migration. The extract liquid of the SF, SF-1nMgO and SF-3nMgO scaffolds was prepared according to ISO 10993-12. First, the scaffolds were sterilised using cobalt-60 (25 kGy) radiation for 2 h. Then, the sterilised scaffolds with a fixed volume to medium volume ratio (1.25 cm2 ml−1) were immersed in Dulbecco’s modified Eagle’s medium (DMEM, high glucose, Invitrogen, USA) supplemented with 10% (v/v) foetal bovine serum (FBS, Gibco, USA) and 1% (v/v) penicillin/streptomycin (Gibco, USA) (30 ml in total) for 24 h at 37 °C in 5% CO2 chamber. For live/dead staining, MC3T3-E1 cells were incubated with the extraction in a 24-well culture plate with a density of 2 × 104 cells/well in an incubator under 37 °C and 5% CO2 for 3 days. The viability of the cells was assessed using a live/dead viability/cytotoxicity kit (Thermo Fisher, USA). The fluorescence photograph was obtained using confocal laser scanning microscopy (CLSM, Leica SP8, Germany). To determine cell proliferation, a Cell Counting Kit-8 (CCK-8, Dojindo, Japan) assay was conducted after being incubated for 1, 3, 5 and 7 days according to the manufacturer’s instructions. The optical density at 450 nm (OD450) was measured using a spectrophotometer (ThermoFisher, USA). The spreading morphology of the cells cultured with the extraction was determined using CLSM. After being fixed with 4% paraformaldehyde, the cells were treated with 0.2% Triton X-100 (Sigma, USA) in PBS. After rinsing, the cells were stained with phalloidin-FITC (1:200 dilution, Solarbio, China) for 30 min and the mounting medium (with DAPI) (Solarbio, China) was used to cover the slides for CLSM observation. A scratch assay was performed to investigate the effect of scaffolds on the migration of MC3T3-E1 cells. When the fusion rate of the MC3T3-E1 cells reached 90%, a cell scratch was prepared using the tip of a 200 μl pipette. After rinsing with PBS, the scaffold extracts were added to the well and cultured at 37 °C. The photographs were obtained at 12 and 24 h using a light microscope. Cell migration images were analysed using ImageJ software. The cell migration ratio was calculated using the following equation:

$${{{{{rm{Cell}}}}}}; {{{{{rm{migration}}}}}}; {{{{{rm{ratio}}}}}}(%)=({{{{{{rm{L}}}}}}}_{0}-{{{{{{rm{L}}}}}}}_{{{{{{rm{t}}}}}}})/{{{{{{rm{L}}}}}}}_{0} times 100%,$$

(2)

where L0 and Lt are the scratched widths before and after the addition of the extract, respectively.

In vitro osteogenic differentiation study of silk fibroin/magnesium composite scaffolds

Bone mesenchymal stem cells (CP-R131, Procell Life Science & Technology Co,. Ltd) were utilised to evaluate the osteogenic differentiation. The bone mesenchymal stem cells (BMSCs) were cultured in a fresh osteogenic induction medium (ascorbic acid 50 μg/mL, dexamethasone 100 nM, β-glycerol phosphate 10 mM). The culture plate was incubated in a 5% CO2 chamber at 37 °C. The extract liquid of the SF, SF-1nMgO and SF-3nMgO scaffolds was prepared as mentioned above. Alkaline phosphatase (ALP), ALP activity, Alizarin Red S (ARS) staining, quantitative polymerase chain reaction (qPCR), and western blot analysis were conducted to assess the osteogenic differentiation of the BMSCs. ALP activity was detected with p-nitrophenyl phosphate (pNPP) (Beyotime, China) at 3, 7 and 14 days after the addition of scaffold extracts to the BMSCs. ALP activity was quantified by absorbance measurements at 405 nm. The mineralisation of the BMSCs was identified by ARS solution (Solarbio, China) according to the manufacturer’s protocol after 14 and 21 days. Images were photographed using an optical microscope (Nikon, Japan). The amount of calcium deposition was further investigated quantificationally.

The gene expression of osteogenic differentiation markers runt-related transcription factor 2 (Runx2), osteocalcin (OCN), osteopontin (OPN) and collagen I (COL I) were evaluated through qPCR after the cells were cultured for 7 and 14 days. The total RNA of the cells was extracted using TRIzol reagent (15596026CN, Invitrogen, USA) and reverse-transcribed to complementary DNA (cDNA) using a PrimeScript RT kit (Takara, Tokyo, Japan). SYBR Green Master Mix (Roche Applied Science, Germany) was used to perform real-time PCR. The primer pairs used are shown in Supplementary Table 1. The relative mRNA expression levels of genes were normalised to the GAPDH using CT values. For western blot analysis, after 14 days of culture, the total proteins from BMSCs were lysed with Radio Immuno Precipitation Assay (RIPA) and the concentration of protein was measured using the BCA protein assay kit (Thermo Fisher Scientific, USA). After that, the proteins were boiled and separated by SDS-PAGE followed by transformation to PVDF membranes. Membranes were incubated with primary antibodies of Runx2 (1:1000, Affinity, China), OCN (1:1000, Affinity, China), OPN (1:1000, Affinity, China) and COL I (1:500, Affinity, China) overnight at 4 °C, followed by the incubation of HRP‑conjugated secondary antibodies. The proteins were visualised with an enhanced chemiluminescence (ECL)-chemiluminescent kit (Thermo Fisher Scientific, USA), and the images were acquired using Scion image software.

Subcutaneous implantation of silk fibroin/magnesium composite scaffolds

Twelve 6-week-old male Sprague-Dawley rats weighing 220−230 g were used for in vivo subcutaneous implantation. All procedures were approved by the animal care and use committee of Beijing Jishuitan Hospital regarding the Guiding Principles for the care and use of experimental animals (Number: 2022-12-03). The scaffolds with a cylindrical shape (1 cm diameter, 2 mm height) were sterilised using cobalt-60 (25 kGy) radiation for 2 h. All the rats were anaesthetised with 0.5% pentobarbital sodium. Two small midline incisions were made on the dorsum of each rat, and the scaffolds were introduced into bilateral subcutaneous pockets created by blunt dissection. After 7, 14 and 28 days, the rats were sacrificed, and the implanted scaffolds with the surrounding tissue were removed. The samples were fixed and processed for histology studies.

Rat skull defect model of silk fibroin/magnesium composite scaffolds

Twenty-four 6-week-old male Sprague-Dawley rats weighing 220−230 g were used to assess the skull regeneration ability of the scaffolds. All procedures of the animal experiments were approved by the Beijing Jishuitan Hospital Animal Care and Use Committee (Number: 2022-12-03). Blank group (defects only), control group (defects received SF scaffolds) and experimental group (defects received SF/1MgO scaffolds). After anaesthesia and routine preparation, two critical-size defects with a diameter of 4 mm were created in each rat. The scaffolds with a cylindrical shape (4 mm diameter, 1 mm height) were sterilised using cobalt-60 (25 kGy) radiation for 2 h. The scaffolds were implanted and the defective tissues were supported. The physical examination was monitored daily throughout the experimental period after the operation. At 4 and 8 weeks post-surgery, the rats were sacrificed by injecting an overdose of sodium pentobarbital, and the implanted scaffolds with the surrounding tissue were removed. The samples were fixed and processed for further studies.

Micro-CT evaluation

All samples were scanned by a micro-CT machine (SCANCO Medical, MicroCT 100, Switzerland) to determine the 3D structure of the bone and the growth of newly grown bone tissue. The scanning parameters were 1 mm aluminium, 9 μm resolution, 70 kV voltage and 120 μA current. After scanning, 3D reconstruction of the bone was realised using a CT analyser, and the percentages of bone volume to total bone volume (BV/TV) and the local volumatric BMD were determined.

Histology, immunohistochemistry and immunofluorescence staining

For in vivo histocompatibility, the sliced sections of subcutaneous implantation were subjected to H&E staining and Masson’s trichrome staining to assess the tissue integration and FBR, including cell infiltration, the number of FBGC and collagenous fibrotic capsules formation of the scaffolds. For ectopic bone formation, the sliced sections of subcutaneous implantation were incubated with anti-OCN (1:100, Affinity, China) and anti-CD31 (1:200, Affinity, China) primary antibodies, followed by treatment with HRP-conjugated or Alexa Fluor 594-labelled secondary antibodies according to a standard protocol. For histological analysis of skull reconstruction, H&E and Masson’s trichrome staining were performed. To further evaluate bone formation and angiogenesis, the OCN (1:100, Affinity, China), bone morphogenetic protein type 2 (BMP-2) (1:100, Affinity, China), Runx2 (1:100, Affinity, China), VEGF (1:100, Affinity, China) and CD31 antibodies (1:200, Affinity, China) were detected via immunofluorescence or immunohistochemistry staining. Images from stained sections were obtained using a digital slide scanner (3DHISTECH, Hungary), and image analysis was performed using IPP 6.0 to quantify the expression.

Statistical analysis

At least three times each experiment was repeated independently with similar results and the results could be reproduced according to this method. Statistical analyses were carried out using SPSS version 20.0. The results are expressed as mean ± SD (standard deviation). The two-tailed unpaired Student’s t test was used to compare two groups, and a one-way ANOVA with Tukey’s multiple comparisons was used to compare more than two groups. Statistical differences were shown with three significance levels. ns: P  >0.05, *P < 0.05, **P < 0.01, and ***P < 0.001.

Reporting summary

Further information on research design is available in the Nature Portfolio Reporting Summary linked to this article.