Thermoplastic moulding of regenerated silk

Abstract

Early insights into the unique structure and properties of native silk suggested that β-sheet nanocrystallites in silk would degrade prior to melting when subjected to thermal processing. Since then, canonical approaches for fabricating silk-based materials typically involve solution-derived processing methods, which have inherent limitations with respect to silk protein solubility and stability in solution, and time and cost efficiency. Here we report a thermal processing method for the direct solid-state moulding of regenerated silk into bulk 'parts' or devices with tunable mechanical properties. At elevated temperature and pressure, regenerated amorphous silk nanomaterials with ultralow β-sheet content undergo thermal fusion via molecular rearrangement and self-assembly assisted by bound water to form a robust bulk material that retains biocompatibility, degradability and machinability. This technique reverses presumptions about the limitations of direct thermal processing of silk into a wide range of new material formats and composite materials with tailored properties and functionalities.

Figure 1 |. Process to generate silk-based bulk materials from native silk.

a, Schematic combining top-down and bottom-up approaches to transform natural silk fibers into silk-based bulk materials by thermal processing method. Step 1, chemical treatment to dissolve natural silk fibers and generate purified silk fibroin solution; step 2, freeze-drying and milling to generate amorphous silk nanomaterials; step 3, mechanical hot-pressing with high pressure for densification, fusion and structural transition of the amorphous silk nanomaterials to semi-crystalline bulk materials. b-e, Photographs of silk bar and rod (b), plate (c), tube with caps (d), screws (e) fabricated by the thermal processing method with/without post-machining (scale bars are 1 cm).

Figure 2 |. Comparison of degummed natural silk fiber and ASN.

a-c, Photograph (a) and SEM images (b, c) of degummed natural silk fibers. d-f, Photograph (d) and SEM images (e, f) of amorphous silk nanomaterials (ASN). g, 1D WAXS profiles of degummed natural silk fiber and ASN. h, Solid-state NMR spectra of degummed natural silk fiber and ASN. i, FTIR spectra of degummed natural silk fiber and ASN. j, Quantitative analysis of secondary structures present in degummed silk fiber and amorphous silk nanomaterials. Data are shown as mean±s.d.; n=3 independent experiments; the error bars represent the sample variation, measurement uncertainty, and the deconvolution uncertainty. Scale bars are 50 μm, 5 μm, 5 μm, and 500 nm for b, c, e, f, respectively.

Figure 3 |. Thermal processing of ASN.

a, Photographs and cross-sectional SEM images of the silk plates prepared at different temperatures: 25 °C (A1-A3), 65°C (B1-B3), 95°C (C1-C3), 125°C (D1-D3), 145°C (E1-E3), 175°C (F1-F3). b, FTIR spectra of ASN powder incubated at different temperatures for 15 mins (dashed line) and bulk silk plates prepared at 632 MPa and different temperatures (solid line). c, 1D WAXS profiles of the bulk silk plates prepared at 632 MPa and different temperatures. Inset shows a zoom-in plot for q values from 10 nm⁻¹ to 20 nm⁻¹. d, β-sheet content of the silk plates quantified from FTIR spectra. Data are shown as mean±s.d.; n=3 independent experiments. e, Degree of crystallinity of bulk silk plates estimated from 1D WAXS profiles. Data are shown as mean±s.d.; n=3 independent experiments. f, Schematic showing a proposed mechanism for the structural transition of ASN during thermal processing. The scales bars in (A2 to F2) and (A3 to F3) are 200 μm and 1 μm, respectively.

Figure 4 |. Physical properties of fabricated silk-based bulk materials.

a. Three-point bending curves for silk plates prepared at different temperatures. b. Flexural strength and modulus of the silk plates prepared at different temperatures. Data are shown as mean±s.d.; n=3 independent experiments. c. Compared with other natural and synthetic structural materials,^, the bulk silk materials developed in this work showed excellent mechanical properties. d. DSC curves of the silk plates prepared at 125 °C and 632 MPa. e-f. Photographs of patterned silk dime (e) and silk structures (f) prepared by thermal forming. Scale bars in are 1 cm.

Figure 5 |. In vitro and In vivo testing of functional silk-based medical devices.

a-c, In vitro degradation profiles of silk bone screws in PBS solution (a), protease XIV (5 U/mL) PBS solution (b), and α-chymotrypsin (40 U/mL) PBS solution (c). The silk bone screws were machined from bulk silk materials prepared at three different conditions (95 °C, 632 MPa; 125 °C, 632 MPa; 145 °C, 632 MPa) and the photograph of as-fabricated silk screws was shown as an inset in c. Data are shown as mean±s.d.; n=3 independent experiments. d-f, In vivo testing of silk bone screw (fabricated at 145°C, 632 MPa) after 4 weeks implantation in rats: μCT (d), Hematoxylin and eosin staining (e) and Masson trichrome staining (f) demonstrated active new bone formation (long arrow) observed at the screw (S) surface along with multinucleated, macrophage-like cells (short arrow) lining the screw surface. Data are shown as mean±s.d.; n=3 independent experiments. Scale bars are 1 mm, 50 μm, and 50 μm for d, e, f, respectively. g. Photographs of fabricated silk ear tubes with or without protease XIV doping (1 wt%). The silk ear tubes were machined from pure silk or silk-protease XIV bulk materials prepared at two different conditions (125 °C, 632 MPa; 145 °C, 632 MPa). h-i. In vitro degradation analysis of silk ear tubes with or without protease XIV doping: (h) photographs of silk ear tubes incubated in PBS at 37 °C for different times (5 min, 3 h, and 48 h) and (i) residual mass ratios of silk ear tubes after continuous incubation in PBS at 37 °C for 72 h. Red dashed lines in (h) indicates the observed degradation of silk ear tubes. * in (i) denotes statistically significant differences obtained from two-way ANOVA analysis, P < 0.0001.