Compression and heat-assisted production of silk-based materials

Abstract

To produce silk-based materials using a novel compression and thermal based method.SOLUTION: The present disclosure provides methods including the steps of (i) providing silk fibroin material comprising substantially amorphous structure, and (ii) applying at least one of elevated temperature and elevated pressure to the silk fibroin material to form a silk fibroin article, wherein the applying induces fusion between at least a portion of the silk fibroin and structural change of fibroin in the silk fibroin material. In some embodiments, the present disclosure also provides silk fibroin articles made in accordance with the methods disclosed herein.SELECTED DRAWING: Figure 1

Description

[Technical Field]

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS This application is related to and claims priority from U.S. Provisional Patent Application No. 62 / 743,975, filed October 10, 2018, and is incorporated herein by reference in its entirety for all purposes.

[0002] Statement Regarding Federally Funded Research This invention was made with government support under grants AR068048 and DE016525 awarded by the National Institutes of Health, and grant FA9550-17-1-0333 awarded by the U.S. Air Force. The government has certain rights in this invention. [Background technology]

[0003] background Silk is generally defined as a protein polymer naturally produced by lepidopteran larvae, including silkworms, spiders, scorpions, and flies. Silkworm silk has been used commercially for centuries in textile production and is recognized as one of the most studied protein-based materials in history. Silkworm silk and spider silk are the most widely studied and widely used, and their biocompatibility, biodegradability, and exceptional mechanical properties make them excellent materials for biomedical applications such as drug delivery and tissue engineering. Silkworm silk has been used as a suture material for centuries, but in recent years, it has attracted significant attention as a biomaterial with diverse medical applications due to its tunable degradation rate in vivo and its ability to be fabricated into multiple types of materials, including fibers, films, gels, and foams. However, compared to the traditional polymer industry, the technologies developed for silk processing and the production of silk-based materials are very limited. Summary of the Invention [Means for solving the problem]

[0004] Abstract The compositions and methods described herein relate, inter alia, to the production of silk-based materials using novel compression- and heat-based methods. The silk-based materials herein include pure silk materials (e.g., silk films, silk monolith materials) and / or composite materials (e.g., silk-graphene composites, silk-enzyme composites). The processes described herein have been shown to produce a variety of silk materials with properties comparable to or even superior to those of materials produced by solution-based processes, such as transparent silk films, silk orthopedic devices, and functional silk patterns. Furthermore, in some embodiments, these processes can be used to produce functional silk formats not achievable by solution-based processes, such as silk materials with controllable degradability, by incorporating silk-degrading enzymes and conductive silk-graphene composites. In some embodiments, the manufacturing techniques described herein can be extended to other structural biopolymers, such as collagen and recombinant silk proteins.

[0005] In some embodiments, the present disclosure provides a method comprising: (i) providing a silk fibroin material comprising a substantially amorphous structure; and (ii) applying at least one of high temperature and high pressure to the silk fibroin material to form a silk fibroin article, wherein the applying step induces fusion between at least some of the silk fibroin and a conformational change of the fibroin in the silk fibroin material. In some embodiments, the silk fibroin material is transformed into a solid silk article that has undergone a conformational change of the silk, comprising at least some of the silk fibroin fused together.

[0006] In some embodiments, the present disclosure provides a method comprising the steps of: (i) selecting a high temperature and a high pressure to produce a desired silk fibroin article of a desired crystallinity and desired material properties; and (ii) applying the high temperature and high pressure to a silk fibroin material comprising a substantially amorphous structure to form a silk fibroin article, wherein the silk fibroin article has the desired crystallinity and desired material properties.

[0007] In some embodiments, the present disclosure also provides silk fibroin articles made according to the methods disclosed herein. In some embodiments, the silk fibroin article is or comprises a packaging material. In some embodiments, the packaging material is suitable for use in the manufacture of electronic devices, drug delivery systems, patterning, molding, and any combination thereof. In some embodiments, the silk fibroin article comprises semi-crystalline silk fibroin, and the glass transition temperature of the silk fibroin article is between about 40°C and 135°C. In some embodiments, the provided composition comprises both crystalline and amorphous silk fibroin. In some embodiments, the flexural strength of the silk fibroin article is at least 5 MPa, and the substantially overall density of the silk fibroin article is at least 1.20 g / cm. 3 In some embodiments, the silk fibroin article comprises about 10% (w / w) or more silk. In some embodiments, the silk fibroin article degrades by at least 1% by weight after being exposed to an aqueous environment at 37° C. for 30 days.

[0008] In various embodiments, the present disclosure encompasses the discovery that application of a specific combination of high temperature and / or high pressure can result in the crystallization of silk molecules in a silk fibroin material. In some embodiments, crystallization can include a transition from an amorphous state to a semi-crystalline or crystalline structure and / or a β-sheet structure. In some embodiments, applying high temperature and / or pressure can increase the amount of β-sheet in the silk fibroin article by at least 1% compared to the level of β-sheet in the silk fibroin material. In some embodiments, the applying step increases the amount of β-sheet in the silk fibroin article by at least 50% compared to the level of β-sheet in the silk fibroin material. As discussed herein, the present disclosure provides a series of novel methods for providing silk fibroin articles, including the application of at least one period of exposing the silk fibroin material to high temperature and at least one period of exposing the silk fibroin material to high pressure. In some embodiments, the high temperature and high pressure are applied simultaneously. In some embodiments, the high temperature is applied to the silk fibroin material after high pressure has been applied. In some embodiments, the applying step occurs at least partially within the mold. In some embodiments, the applying does not occur within the mold.

[0009] As discussed herein, the present disclosure provides a series of novel methods for providing silk fibroin articles, including applying at least one period of time to expose a silk fibroin material to an elevated temperature and at least one period of time to expose the silk fibroin material to an elevated pressure. In some embodiments, the elevated temperature and elevated pressure are applied simultaneously. In some embodiments, the elevated temperature is applied to the silk fibroin material after the elevated pressure has been applied. In some embodiments, the applying step is performed at least partially in a mold. In some embodiments, the applying step is not performed in a mold.

[0010] In some embodiments, the applying step is or includes heat pressing. In some embodiments, the heat pressing is performed at a pressure of at least 1 MPa. In some embodiments, the heat pressing is performed at a temperature between 25°C and 200°C.

[0011] In some embodiments, the silk fibroin article is substantially transparent. In some embodiments, the silk fibroin article is bioabsorbable. In some embodiments, the silk fibroin article has thermoformable properties and can be remolded into a desired shape at high temperature or pressure.

[0012] The present disclosure also encompasses compositions comprising one or more additives (e.g., 2, 3, 4, 5, 6, 7, 8, 9, 10, or more additives) in some embodiments. In some embodiments, the additives can enhance one or more properties (e.g., physical properties, mechanical properties, etc.) of the provided composition. In some embodiments, prior to the applying step, the silk fibroin material is mixed with at least one additive to form a composite silk fibroin article. In some embodiments, the additive is or includes at least one of small organic or inorganic molecules, organic polymers, inorganic polymers, biopolymers such as peptides and proteins; conductive materials, carbon-based materials, antibodies and antigen-binding fragments thereof; antigens; nucleic acids; nucleic acid analogs and derivatives; saccharides; immunogens; natural compounds and extracts from biological systems such as cells, bacteria, or tissues; synthetic materials; metallic materials; alloys; hydrophobic materials; hydrophilic materials; nanomaterials; and any combination thereof. In some embodiments, the organic polymer is or includes at least one enzyme, such as a protease. In some embodiments, the activity of the enzyme is stabilized by the silk under high temperature and pressure. In some embodiments, the protease is or includes one or more of protease XIV, proteinase K, α-chymotrypsin, collagenase, matrix metalloproteinase-1 (MMP-1), and MMP-2. In some embodiments, the conductive material is or includes an inorganic conductive material (e.g., silver, gold, iron oxide), an organic conductive material (e.g., graphene), a metal, an alloy, a semiconductor material, and / or a conjugated polymer. In some embodiments, the additive is mixed with the silk fibroin material at a ratio of between 0.001% and 95.0% by weight.

[0013] The provided methods and compositions (e.g., articles) can include one or more physical and / or mechanical properties not observed using conventional known methods. By way of example, in some embodiments, the silk fibroin article is substantially homogeneous. In some embodiments, the provided silk article is considered to be substantially homogeneous if the article exhibits a consistent structure throughout the majority of the article (e.g., at least 70%, 80%, 90%, 95% or more of the silk article exhibits a consistent structural pattern).

[0014] Furthermore, in certain embodiments, the provided methods enable the production of complex articles using multiple techniques that would result in significantly different articles (e.g., in terms of physical and / or mechanical properties) when applied using conventional methods. According to some embodiments, the provided methods can be used to produce any of a variety of silk fibroin articles. In some embodiments, the silk fibroin article can be or can include a film, fiber, mesh, needle, tube, plate, screw, rod, or any desired shape.

[0015] Furthermore, in certain embodiments, the provided methods enable the production of complex articles using multiple techniques that would result in significantly different articles (e.g., in terms of physical and / or mechanical properties) when applied using conventional methods. According to some embodiments, the provided methods can be used to produce any of a variety of silk fibroin articles. In some embodiments, the silk fibroin article can be or can include a film, fiber, mesh, needle, tube, plate, screw, rod, or any desired shape.

[0016] The provided methods and compositions enable the production of silk articles that exhibit one or more enhanced properties. In some embodiments, the silk fibroin articles may have enhanced thermal or electrical properties. In some embodiments, the silk fibroin articles are electrically conductive. According to various embodiments, the provided compositions may be suitable for patterning (e.g., on a surface of the composition).

[0017] In some embodiments, the provided compositions may be biocompatible and / or biodegradable. According to various embodiments, the provided methods and compositions allow for any of a variety of degradation profiles, which in turn allow for a wide variety of potential applications. For example, in some embodiments, the silk article degrades by at least 50% by weight after about 96 hours of exposure to an aqueous environment at 37°C. As a further example, in some embodiments, the silk fibroin article does not degrade by more than 10% after six months of exposure to an in vivo environment or conditions.

[0018] Any of a variety of forms of silk fibroin material can be used in accordance with the various provided methods and compositions. Generally, any silk fibroin material containing a significant amount of amorphous silk can be used in accordance with some embodiments. By way of non-limiting example, in some embodiments, the silk fibroin material can be or include particles, films, and / or fibers. In some embodiments, the particles can be or include at least one of microparticles and nanoparticles.

[0019] In some embodiments, the provided silk article may be made by a process that includes converting silk fibroin material into a solid silk article that has undergone a conformational change of the silk, with at least some silk fibroin having transitioned from an amorphous state to a semi-crystalline structure.

[0020] Citations herein to publications, patents, or patent applications are incorporated by reference in their entirety. Numbers used in this application, whether about / approximate or not, are intended to cover normal variations that would be understood by one of ordinary skill in the relevant art.

[0021] Other features, objects, and advantages of the present specification will become apparent in the detailed description which follows. It should be understood, however, that the detailed description, while indicating embodiments of the present invention, is given by way of illustration only and not of limitation. Various changes and modifications within the scope of the present invention will become apparent to those skilled in the art from the detailed description. [Brief explanation of the drawings]

[0022] [Figure 1] Panel (a) of Figure 1 shows natural degummed silk fibers. Panels (b) and (c) are SEM images of natural degummed silk fibers. Panel (d) shows amorphous silk nanomaterials. Panels (e) and (f) show SEM images of amorphous silk nanomaterials. Panel (g) shows X-ray diffraction (XRD) profiles of degummed silk fibers and amorphous silk nanomaterials. Panel (h) shows solid-state NMR spectra of degummed silk fibers and amorphous silk nanomaterials. Panel (i) shows FTIR spectra of degummed silk fibers and amorphous silk nanomaterials. Panel (j) shows a quantitative analysis of the secondary structures present in degummed silk fibers and amorphous silk nanomaterials.

[0023] [Figure 2] Panel (A) of Figure 2 shows exemplary thermogravimetric analysis (TGA) and differential thermogravimetric (DTG) profiles of freeze-dried silk powder and degummed silk fibers. Panel (B) shows exemplary differential scanning calorimetry (DSC) profiles of freeze-dried silk powder and degummed silk fibers. The heating rate for TGA, DTG, and DSC characterization was 10 °C / min.

[0024] [Figure 3]Figure 3 shows an exemplary DSC profile of freeze-dried silk powder with step heating. The heating rate is 10°C / min.

[0025] [Figure 4] Figure 4 shows an exemplary DSC profile of freeze-dried silk powder. The solid line represents standard DSC with a heating rate of 2 °C / min, and the dashed line represents temperature-modulated DSC (TMDSC) with a heating rate of 2 °C / min. TMDSC uses a temperature amplitude of 0.318 K and a period of 1 min.

[0026] [Figure 5] Figure 5 shows a schematic diagram of silk processing by heat and pressure. Process A (top row) shows that a silk monolith is obtained by heat pressing, which is then machined into a silk screw. Process B (middle row) shows that a silk screw is obtained by directly molding the silk with heat and pressure. Process C (top row) shows that a silk monolith is obtained by heat pressing, which is then molded into the shape of a screw.

[0027] [Figure 6] Panel (a) of Figure 6 shows a schematic diagram of the method for converting amorphous silk nanomaterials into bulk natural silk by combining top-down and bottom-up approaches. Step 1: Chemical treatment to dissolve natural silk fibers and create a silk fibroin solution; Step 2: Freeze-drying treatment to create amorphous silk nanomaterials; Step 3: Mechanical hot pressing at 632 MPa at various temperatures (which leads to densification and structural transition of the silk fibroin). Panel (b) shows a schematic diagram of the protein structure of silk fibroin.

[0028] [Figure 7] Panel (a) of Figure 7 shows a photograph of silk bars, pins, plates, rods, tubes, and screws. Panel (b) shows a large silk plate measuring 4 cm (length) x 2 cm (width). The scale bar is 1 cm.

[0029] [Figure 8] Panel (a) of Figure 8 shows photographs and cross-sectional SEM images of bulk silk plates fabricated at various temperatures. The scale bars in A2–F2 and A3–F3 are 200 μm and 1 μm, respectively. Panel (b) shows FTIR spectra of bulk silk plates fabricated at various temperatures. Panel (c) shows X-ray diffraction spectra of bulk silk plates fabricated at various temperatures. Panel (d) shows the β-sheet content of the bulk silk plates quantified from FTIR spectroscopy. Panel (e) shows the crystallinity of the bulk silk plates estimated from X-ray diffraction spectroscopy. Panel (f) shows a schematic diagram illustrating the proposed mechanism of structural transition of amorphous silk nanomaterials (ASNs) during thermal processing.

[0030] [Figure 9] Panel (a) of Figure 9 shows exemplary three-point bending curves of silk plates fabricated at various temperatures. Panel (b) shows the bending strength and modulus of silk plates fabricated at various temperatures. Panel (c) shows the mechanical properties of natural materials compared to synthetic structural materials.

[0031] [Figure 10] Figure 10 shows three-point bending tests of silk plates made from various raw materials (degumed silk powder (black), amorphous silk nanomaterial (gray)). The silk plates were fabricated at 145 °C and 632 MPa. The bending stress of the silk plate made from degummed silk powder (45.9 ± 9.1 MPa) is much lower than that of the one made from amorphous silk nanomaterial (147.1 ± 14.2 MPa).

[0032] [Figure 11]Panel (a) of Figure 11 shows the density of silk plates fabricated at various press temperatures. The density was calculated based on the measured mass, thickness, and dimensions of the silk plates. Panel (b) shows three-point bending tests of silk plates fabricated at 145 °C under different pressures (632 MPa and 125 MPa). The bending stress of silk plates fabricated at low pressures is lower than that of silk plates fabricated at high pressures.

[0033] [Figure 12] FIG. 12 shows an exemplary image of a directly formed silk screw.

[0034] [Figure 13] Panel (a) of Figure 13 shows the DSC curve of a silk plate pressed at 125 °C and 632 MPa for 15 minutes. Panel (b) shows a silk thread obtained by forming a cylindrical silk monolith under heat and pressure. Panel (c) is a photograph of a silk structure produced by thermoforming a silk plate pressed at 125 °C and 632 MPa for 15 minutes. Panel (d) shows an image of a patterned silk dime. Panel (e) shows an SEM image of the nanostructures in a nanostructure-patterned silk film pattern.

[0035] [Figure 14] Figure 14 shows photographs and SEM images of the machined silk screws (a, b, c) and ear tubes (d, e, f). The scale bar is 1 mm.

[0036] [Figure 15] Figure 15 shows the degradation profiles of silk screws fabricated at various temperatures (95 °C, 125 °C, 145 °C) in pure PBS (panel a), 5 U / mL protease in PBS (panel b), and 40 U / mL chymotrypsin in PBS (panel c). Corresponding SEM images of silk screws after 30 days of degradation were presented. Scale bars are 1 mm and 500 μm for the top and bottom images, respectively.

[0037] [Figure 16] Figure 16 shows the water absorption and swelling test (PBS, 37°C) of silk screws by machining silk bars made under different processing conditions (95°C and 632 MPa; 125°C and 632 MPa; 145°C and 632 MPa).

[0038] [Figure 17] FIG. 17 shows an exemplary degradation profile of a silk-protease XIV composite (panel (a)) and an image of the silk-protease XIV composite (panel (b)).

[0039] [Figure 18] FIG. 18 shows an exemplary conductivity test of silk graphene films.

[0040] [Figure 19] FIG. 19 shows SEM images of the silk fibroin material containing freeze-dried silk powder shown in panel (A) and the internal structure of a silk article pressed at 145°C and 632 MPa for 15 minutes in panel (B), with a more magnified SEM image shown in panel (C).

[0041] [Figure 20] FIG. 20 shows the solution NMR spectrum of the regenerated silk aqueous solution (6 wt %).

[0042] [Figure 21] FIG. 21 shows the 13C cross-polarization magic angle spinning (CP-MAS) spectra of native degummed silk and freeze-dried silk.

[0043] [Figure 22] FIG. 22 shows the 13C chemical shifts (ppm of TMS) of amino acid residues in B. mori gland liquid silk, regenerated B. mori silk solution, degummed B. mori silk, and freeze-dried B. mori silk.

[0044] [Figure 23] Figure 23 shows the deconvolution of the amide I peak from the FTIR spectrum (Panel A) and the diffraction pattern from XRD (Panel B). For the FTIR spectrum, spectral correction and decomposition were performed using a proprietary Matlab package. The spectrum was first smoothed with a five-point triangular smoothing method and then baseline-corrected using a cubic spline of the amide I band. Deconvolution was performed using the second derivative method, and the four primary peaks were assigned to various secondary structures: 1620 cm-1 (β-sheet), 1645-1655 cm-1 (random coil / helix), 1685 cm-1 (β-turn), and 1698 cm-1 (β-sheet). For the XRD, multiple Gaussian peaks were used to fit the one-dimensional intensity profile. The broad component at 1.48 cm-1 represents the amorphous component.

[0045] [Figure 24] Figure 24 shows an SEM image of the nanopattern-imprinted silk film.

[0046] [Figure 25] Figure 25 shows the standard DSC profiles of silk plates prepared at various temperatures. The samples were first heated from -50 to 200°C at a heating rate of 10°C / min under a dry nitrogen gas flow of 50 mL / min. After cooling to -50°C, the samples were heated back to 200°C at a heating rate of 10°C / min. The first heating profile is shown by the solid line, and the second heating profile is shown by the dashed line.

[0047] [Figure 26] Figure 26 shows exemplary temperature-modulated DSC (TMDSC) profiles of silk plates prepared at different temperatures (95°C, 125°C, and 145°C). The samples were heated from -50 to 200°C with a heating rate of 10°C / min, a modulation period of 60 seconds, and a temperature amplitude of 1.59°C. The total and reverse heat flow curves are shown in panels (A) and (B), respectively. The dashed tangent line indicates the step change that occurs at the glass transition temperature.

[0048] [Figure 27] FIG. 27 is a table showing a comparison of different methods for producing silk monoliths.

[0049] [Figure 28] Figure 28 shows the molecular weight distribution of silk fibroin in aqueous solution after different degumming times (10, 30, and 60 minutes). Sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) was performed using a 0.5 wt% silk solution. This shows that the average molecular weight of silk fibroin decreases with increasing degumming time. After a 30-minute degumming time, the average molecular weight of silk fibroin is approximately 260 kDa.

[0050] [Figure 29] FIG. 29 shows the complete protein sequence of B. mori silk fibroin.

[0051] [Figure 30] Figure 30 shows the in vitro degradation analysis of silk ear tubes with or without protease XIV doping: (a) Photographs of silk ear tubes incubated in PBS at 37°C for different times (5 min, 1 h, 3 h, 6 h, 24 h, 48 h, and 72 h). Silk ear tubes were machined from pure silk or silk protease XIV bulk material fabricated under two different conditions (125°C, 632 MPa; 145°C, 632 MPa). (b) Photographs of the fabricated silk ear tubes (left) and silk ear tubes after continuous incubation in PBS solution for 72 h (right). DETAILED DESCRIPTION OF THE INVENTION

[0052] Detailed Description definition

[0053] In this application, unless otherwise clear from the context, (i) the term "a" may be understood to mean "at least one"; (ii) the term "or" may be understood to mean "and / or"; (iii) the terms "comprising" and "including" may be understood to encompass the listed components or steps, whether presented by themselves or with one or more additional components or steps; (iv) the terms "about" and "approximately" are used as equivalents and may be understood to allow for standard variations that would be appreciated by one of ordinary skill in the art; and (v) when ranges are stated, the endpoints are included.

[0054] Approximately: As used herein, the term "approximately" or "about" as applied to one or more values of interest refers to a value similar to the stated reference value. In certain embodiments, the term "approximately" or "about" refers to a range of values that falls within 25%, 20%, 19%, 18%, 17%, 16%, 15%, 14%, 13%, 12%, 11%, 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, 1%, or less in either direction (greater or lesser) of the stated reference value, unless otherwise specified or apparent from the context (unless such number exceeds 100% of the possible values).

[0055] Biocompatible: As used herein, the term "biocompatible" refers to materials that do not cause significant harm to living tissues when placed in contact with such tissues, for example, in vivo. In certain embodiments, materials are "biocompatible" if they are not toxic to cells. In certain embodiments, a material is "biocompatible" if its addition to cells in vitro results in 20% or less cell death and / or if its administration in vivo does not induce significant inflammation or other adverse effects.

[0056] Biodegradable: As used herein, the term "biodegradable" refers to a material that, when introduced into cells, breaks down into components that the cells can reuse or process without significant toxic effects to the cells (e.g., by cellular mechanisms, such as by enzymatic degradation, hydrolysis, and / or a combination thereof). In certain embodiments, the components produced by the degradation of a biodegradable material are biocompatible and therefore do not induce significant inflammation and / or other adverse effects in vivo. In some embodiments, a biodegradable polymeric material breaks down into its constituent monomers. In some embodiments, the degradation of a biodegradable material (e.g., including a biodegradable polymeric material) involves hydrolysis of ester bonds. Alternatively, or in addition, in some embodiments, the degradation of a biodegradable material (e.g., including a biodegradable polymeric material) involves cleavage of urethane bonds. Examples of biodegradable polymers include polymers of hydroxy acids, such as, for example, lactic acid and glycolic acid, including, but not limited to, poly(hydroxyl acid), poly(lactic acid) (PLA), poly(glycolic acid) (PGA), poly(lactic-co-glycolic acid) (PLGA), and copolymers with PEG, polyanhydrides, poly(ortho)esters, polyesters, polyurethanes, poly(butyric acid), poly(valeric acid), poly(caprolactone), poly(hydroxyalkanoates), poly(lactide-co-caprolactone), blends and copolymers thereof. Many naturally occurring polymers Polymers are also biodegradable and include, for example, proteins such as albumin, collagen, gelatin, and prolamines, e.g., zein, and polysaccharides such as alginates, cellulose derivatives, and polyhydroxyalkanoates, e.g., polyhydroxybutyrate blends and copolymers thereof. One of ordinary skill in the art will understand or be able to determine that such polymers are biocompatible and / or biodegradable derivatives thereof (e.g., those related to the parent polymer with substantially identical structure, differing only in the substitution or addition of certain chemical groups, as known in the art).

[0057] Compaction: As used herein, the term "compaction" refers to the process by which a material gradually loses its porosity due to the influence of a load.

[0058] Composition: As used herein, may be used to refer to a separate physical entity containing one or more specified components. Generally, unless otherwise specified, a composition may be in any form, e.g., gas, gel, liquid, solid, etc. In some embodiments, a "composition" may refer to a combination of two or more entities for use in a single embodiment or as part of the same article. In all embodiments, the combination of entities need not result in a physical mixture; i.e., combination of each component of the composition as a separate co-entity is possible. However, many practitioners in the art may find it advantageous to create a composition in which two or more components are mixed in a pharmaceutically acceptable carrier, diluent, or excipient, allowing the combination of components to be administered simultaneously.

[0059] Fusion: As used herein, the term "fusion" refers to the process of combining two or more separate entities into a new whole.

[0060] Hydrophilic: As used herein, the terms "hydrophilic" and / or "polar" refer to a tendency to be miscible with or readily soluble in water.

[0061] Hydrophobic: As used herein, "hydrophobic" and / or "non-polar" refer to a tendency to repel water, not to bind water, or not to dissolve readily in water.

[0062] Improve, increase, or decrease: As used herein, or grammatical equivalents thereof, refers to a value relative to a baseline measurement, such as a measurement on a similar composition made according to previously known methods.

[0063] Macroparticle: As used herein, the term "macroparticle" refers to a particle having a diameter of at least 1 millimeter. In some embodiments, a macroparticle is a micelle in that it comprises an enclosed compartment separated from the bulk solution by a micellar membrane, and is generally composed of amphiphilic entities that surround a space or compartment (e.g., to define a lumen). In some embodiments, the micellar membrane is composed of at least one polymer, such as, for example, a biocompatible and / or biodegradable polymer. In some embodiments, a population of particles is considered a population of macroparticles if the average diameter of the population is equal to or exceeds 1 millimeter.

[0064] Microparticle: As used herein, the term "microparticle" refers to a particle having a diameter between 1 micrometer and 1 millimeter. In some embodiments, a microparticle is a micelle in that it comprises an enclosed compartment separated from the bulk solution by a micellar membrane, and is generally composed of amphiphilic entities that surround a space or compartment (e.g., to define a lumen). In some embodiments, the micellar membrane is composed of at least one polymer, such as, for example, a biocompatible and / or biodegradable polymer. In some embodiments, a population of particles is considered a population of microparticles if the average diameter of the population is between 1 micrometer and 1 millimeter.

[0065] Nanoparticle: As used herein, the term "nanoparticle" refers to a particle having a diameter of less than 1000 nanometers (nm). In some embodiments, the diameter of a nanoparticle is less than 300 nm as defined by the National Science Foundation. In some embodiments, the diameter of a nanoparticle is less than 100 nm as defined by the National Institutes of Health. In some embodiments, a nanoparticle is a micelle in that it comprises an enclosed compartment separated from the bulk solution by a micellar membrane, and is generally composed of amphiphilic entities surrounding a space or compartment (e.g., to define a lumen). In some embodiments, the micellar membrane is composed of at least one polymer, e.g., a biocompatible and / or biodegradable polymer. In some embodiments, a population of particles is considered a population of nanoparticles if the average diameter of the population is less than or equal to 1000 nm.

[0066] Physiological conditions: As used herein, the term has its art-recognized meaning as conditions for cells or organisms to survive and / or reproduce. In some embodiments, the term refers to the external or internal environmental conditions that may naturally occur in an organism or cell system. In some embodiments, physiological conditions are conditions present inside a human or non-human animal, particularly at and / or within a surgical site. Physiological conditions generally include, for example, a temperature range of 20-40°C, an atmospheric pressure of 1, a pH of 6-8, a glucose concentration of 1-20 mM, an oxygen concentration at atmospheric pressure, and gravity as encountered on Earth. In some embodiments, laboratory conditions are manipulated and / or maintained at physiological conditions. In some embodiments, physiological conditions are those found in an organism.

[0067] Pure: As used herein, a material, additive, and / or entity is "pure" if it is substantially free of other elements. For example, a preparation that contains greater than about 90% of a particular agent or entity is generally considered to be a pure preparation. In some embodiments, the agent or entity is at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% pure.

[0068] Reference: As used herein, describes a standard or control against which a comparison is made. For example, in some embodiments, a material, article, additive, entity, or other sample, sequence, or value of interest is compared to a reference or control material, article, additive, entity, or other sample, sequence, or value. In some embodiments, the reference or control is tested and / or determined substantially contemporaneously with the test or determination of interest. In some embodiments, the reference or control is a historical reference or control, optionally embodied in a tangible medium. Generally, as understood by those of skill in the art, a reference or control is determined or characterized under conditions or circumstances comparable to those being evaluated. One of skill in the art will understand when there is sufficient similarity to justify reliance on and / or comparison to a particular possible reference or control.

[0069] Solid Forms: As is known in the art, many chemicals (particularly many organic molecules and / or many small molecules) can exist in a variety of different solid forms, such as, for example, amorphous and / or crystalline forms (e.g., polymorphs, hydrates, solvates, etc.). In some embodiments, such entities may be utilized as a single such form (e.g., as a pure preparation of a single polymorph). In some embodiments, such entities may be utilized as a mixture of such forms.

[0070] Substantially: As used herein, the term "substantially" refers to the qualitative condition of exhibiting the entire or nearly entire extent or degree of a characteristic or property of interest. Those skilled in the art of biology will understand that biological and chemical phenomena rarely, if ever, go to completion and / or proceed perfectly, or achieve or avoid absolute results. As such, the term "substantially" is used herein to capture the potential lack of completeness inherent in many biological and chemical phenomena.

[0071] Compositions and Methods

[0072] This description encompasses, inter alia, the recognition that applying one or more periods of high temperature and high pressure in a particular manner results in previously unknown changes to the structure of the silk fibroin, for example, in a silk fibroin material. In some embodiments, the present disclosure encompasses the surprising discovery that silk fibroin articles can be made directly from amorphous silk fibroin material without the need for intermediate solubilization, which is widely used in the art. While not wishing to be bound by any particular theory, it is specifically contemplated that the novel structures enabled by the methods disclosed herein result, at least in part, from a combination of compression and fusion of the silk fibroin material.

[0073] As a natural protein-based biopolymer, silk in various material forms has promising features, including excellent mechanical properties, biocompatibility, and biodegradability. Therefore, silk is expected to be a promising candidate for biomedical applications, including drug delivery, tissue engineering, and regenerative medicine. 1-4 It has been utilized for decades as a material option for biomaterials and scaffolds in the field. Natural silk is a semi-crystalline biopolymer material, composed of nanocrystallites of β-sheets embedded in a disorganized, less crystalline continuous phase due to the amphiphilic nature of its protein chemistry. 5-7The strong hydrogen-bonding network of β-sheet nanocrystallites contributes significantly to the cellulose-like stability and excellent mechanical properties of silk, but this poses a challenge to the ability to thermally process silk-based materials without degradation. Indeed, there are limited reports on the thermal melting and reconstitution of silk materials, which requires ultrafast laser heating. 8、9 Furthermore, historically, starting from Pauling's studies on the fundamental structural features of silk fibroin, it was discovered that the antiparallel β-pleated sheets (antiparallel β-sheets) that form the crystalline phase of silk are highly stable due to the hydrogen bonding of well-aligned N-H-O bonds. 6、10 .

[0074] Previously known methods for silk protein processing include solution-based processing, which often results in material decomposition before melting. Over the past few decades, researchers have devoted considerable effort to developing techniques for processing silk fiber materials, focusing on extracting silk fibroin from the fibers and creating silk solutions. For example, silk fibers after removing the outer sericin (degummed silk fibers) can be dissolved in a high-salt LiBr / CaCl2 aqueous solution to produce an aqueous silk solution. Furthermore, degummed silk fibers are soluble in several organic solvents, such as formic acid, trifluoroacetic acid, and 1,1,1,3,3,3-hexafluoro-2-propanol (HFIP). These methods are aimed at downstream processing (solvent removal) to produce silk materials, including gels, foams, films, new fiber forms, and related materials. 16-18 The addition and removal of solvents associated with protein solubility limitations can lead to new and useful materials, but the solubility limitations also limit the material properties and incur significant costs for various processing steps.

[0075] Prior to the present invention, methods using aqueous silk solutions as starting materials were favored because they are more user-friendly and can be further used to fabricate diverse material formats, such as films, foams, sponges, hydrogels, tubes, and bulk materials. However, silk fibroin's tendency to self-assemble in aqueous solutions makes scale-up production and product quality control challenging. Furthermore, other than solution-based processing methods, established technologies for the engineering and production of silk-based structural materials with tunable physical properties are very limited. To overcome these limitations and better process silk materials, a simple and effective strategy for directly converting amorphous natural silk powder into high-performance structural materials with tunable mechanical properties is disclosed herein.

[0076] In some embodiments, the methods disclosed herein involve the production of amorphous silk nanomaterials (ASNs) generated from aqueous silk fibroin solutions. The ASNs can then be processed by hot pressing to fuse and densify the silk (e.g., into silk articles). The resulting bulk silk materials exhibit higher specific strengths than most natural structural materials and have been shown to be effective for the production of silk-based composites. Furthermore, engineered silk materials have been shown to possess thermoforming properties, allowing the material to be further deformed into desired shapes under appropriate conditions. Figure 6 shows a schematic diagram of a specific method provided that combines top-down and bottom-up approaches to convert natural silk fibers into bulk silk moieties. In some embodiments, the compositions and methods described herein demonstrate a heat- and pressure-based, time-efficient, and controllable method for converting silk fibroin directly into bulk structural materials from silk fibroin materials containing significant amounts of amorphous silk fibroin (e.g., in powder form). In some embodiments, the methods and compositions described herein may enable the application of more traditional processes and molding techniques to silk materials that have not previously been successfully used with silk. Furthermore, in some embodiments, the processing methods described herein avoid the need for solvent or aqueous approaches and provide a direct route for converting silk fibroin materials into moulds. According to various embodiments, the methods described herein provide for converting silk fibroin from amorphous material to semi-crystalline, high-performance structural materials through the controlled application of heat and pressure. In some embodiments, the provided processes induce a conformational transition of silk molecules from random coil to β-sheet. In some embodiments, the provided methods include processing natural silk fibres into amorphous silk material (e.g., powder) by degumming, silk fibroin solubilisation and lyophilisation to create a suitable preformed material; feeding the amorphous silk material into a pre-designed mould; and inducing conformational and structural changes in the silk by applying heat and pressure.Furthermore, this method can be processed with silk alone or with the addition of inorganic fillers or a second polymer to create composite devices.

[0077] The methods described herein, which utilize heat and pressure-assisted processing of amorphous silk precursor materials, allow for the preparation of various forms of silk materials, including plates, rods, screws, and tubes, with tunable mechanical and thermoformable properties while maintaining the material's favorable biocompatibility and degradability characteristics. In some embodiments, the methods described herein enable a more environmentally friendly and cost-effective method for converting natural fibers into silk monoliths compared to previously reported methods (Figure 27). This discovery significantly changes the landscape in terms of processing methods for silk-based materials, allowing traditional processing and shaping techniques that have not previously been successfully employed for silk to be applied to silk materials. The fabricated silk-based devices have potential for diverse biomedical applications, including orthopedic implants. Furthermore, the heat and pressure-assisted method can be extended to other protein-based materials (e.g., recombinant proteins) to produce protein-based monoliths.

[0078] In some examples, the methods described herein may involve selecting an elevated temperature and pressure to produce a desired silk fibroin article with a desired crystallinity and desired material properties, and then applying the elevated temperature and pressure to a silk fibroin material having a substantially amorphous structure. That is, the methods described herein can predictably select and apply temperatures and pressures to produce articles with desired crystallinity and material properties. This differs from other applications in which heat and / or pressure can be applied to a silk material without predetermining the desired outcome in terms of crystallinity and material properties.

[0079] The amount of plasticizer in the silk fibroin material can be adjusted to produce the desired crystallinity and material properties. This is particularly effective in low-temperature embodiments, where the amount of plasticizer is selected to produce the desired crystallinity and material properties. In some instances, the plasticizer is water.

[0080] silk material

[0081] Any of a variety of silk materials can be used according to various embodiments. In some embodiments, the silk material can be or include silk fibroin (e.g., degummed or substantially sericin-free silk fibroin). In some embodiments, the silk material can be or include silk powder (e.g., including a plurality of silk particles).

[0082] In some embodiments, the silk material may be or include silk particles (e.g., microparticles or nanoparticles). As used herein, the term "particle" includes spheres, rods, shells, prisms, and related structures. While any particle size suitable for any application is contemplated within the scope of this disclosure, in some embodiments, a single silk particle has a diameter between 1 nm and 1,000 μm (e.g., between 1 nm and 1 μm, between 1 μm and 1,000 μm, etc.). In some embodiments, the diameter of the silk particle may be greater than 1,000 μm.

[0083] Various methods for producing silk particles (e.g., nanoparticles and microparticles) are known in the art. For example, silk powder can be produced using a mill (e.g., a Retsch planetary ball mill). Ball mills generally consist of two or four sample cups arranged around a central axis, with each cup adjusted to rotate both centrally and locally. Each ceramic cup is filled with small ceramic spheres. Various sizes are available. For the milling operation described in this disclosure, balls with a diameter of 10 millimeters were used. As the cups rotate, the spheres crush the material in the cups into small, characteristic sizes. Both degummed and undegummed silk can be converted from finely ground material to powder form in a ball mill.

[0084] In other embodiments, alternative powder-forming techniques can be used (e.g., freeze-drying or flash-freezing and crushing). In other embodiments, alternative grinder grates with larger holes can be used, which can produce larger silk particle sizes.

[0085] In some embodiments, silk particles can be produced using the freeze-drying method described in U.S. Provisional Patent Application No. 61 / 719,146, filed October 26, 2012, the contents of which are incorporated herein by reference in their entirety. Specifically, silk foams can be produced by freeze-drying a silk solution. The foam can then be reduced to particles. For example, the silk solution can be cooled to a temperature at which the liquid carrier transforms into a plurality of solid crystals or particles, and at least a portion of the plurality of solid crystals or particles is removed to leave a porous silk material (e.g., silk foam). After cooling, the liquid carrier can be at least partially removed by sublimation, evaporation, and / or freeze-drying. In some embodiments, the liquid carrier can be removed under reduced pressure. After formation, the silk fibroin foam can be subjected to grinding, cutting, crushing, or any combination thereof to form silk particles. For example, the silk fibroin foam can be blended in a conventional blender or ground in a ball mill to form silk particles of a desired size.

[0086] In some embodiments, silk fibroin materials containing a significant amount of amorphous structure are produced from silk solutions and are composed of nanostructures (as shown in Figure 1). This may be referred to as nanosized silk powder (NSP) and may be part of a material called amorphous silk nanomaterial (ASN). As used herein, these terms are equivalent and may be used interchangeably.

[0087] Without wishing to be bound by any particular theory, in some embodiments, the present disclosure encompasses the recognition that the use of certain starting materials (e.g., silk fibroin materials containing a significant amount of amorphous structure) allows for the production of compositions that were previously unattainable. In some embodiments, the silk material is not made from solubilized silk. In some embodiments, the silk material may be freeze-dried.

[0088] Silk fibroin

[0089] According to various embodiments, any silk fibroin can be used in the provided methods. In some embodiments, the silk fibroin is selected from the group consisting of spider silk (e.g., from Nephila ciavipes), silkworm silk (e.g., from Bombyx mori), and recombinant silk (e.g., silk produced / engineered from bacterial cells, yeast cells, mammalian cells, transgenic animals, and / or transgenic plants). According to various embodiments, the silk used in the provided methods and compositions is degummed silk (i.e., silk fibroin from which at least a portion of the native sericin has been removed). Degummed silk can be produced by conventional methods known to those skilled in the art. For example, B. mori cocoons are boiled in an aqueous solution for a predetermined period of time. Generally, longer degumming times result in the production of silk fibroin with a lower molecular weight. In some embodiments, the silk cocoons are boiled for at least 60 minutes, at least 70 minutes, at least 80 minutes, at least 90 minutes, at least 100 minutes, at least 110 minutes, at least 120 minutes, or longer. Additionally or alternatively, in some embodiments, the silk cocoons can be heated or boiled at temperatures such as about 101.0°C, about 101.5°C, about 102.0°C, about 102.5°C, about 103.0°C, about 103.5°C, about 104.0°C, about 104.5°C, about 105.0°C, about 105.5°C, about 106.0°C, about 106.5°C, about 107.0°C, about 107.5°C, about 108.0°C, about 108.5°C, about 109.0°C, about 109.5°C, about 110.0°C, about 110.5°C, about 111.0°C, about 111.5°C, about 112.0°C, about 112.5°C, about 113.0°C, about 114.0°C, about 114.5°C, about 115.0°C, about 115.5°C, about 116.0°C, about 116.5°C, about 117.0°C, about 117.5°C, about 118.0°C, about 118.5°C, about 119.0°C, about 119.5°C, about 120.0°C, about 121.5°C, about 122.0°C, about 123.0°C, about 124.0°C, about 125.0°C, about 126.0°C, about 127.0°C, about 128.0°C, about 129.0°C, about 130.0°C, about 131.5 °C, about 111.0°C, about 111.5°C, about 112.0°C, about 112.5°C, about 113.0°C, 113.5°C, about 114.0°C, about 114.5°C, about 115.0°C, about 115.5°C, about 116.0°C, about 116.5°C, about 117.0°C, about 117.5°C, about 118.0°C, about 118.5°C, about 119.0°C, about 119.5°C, about 120.0°C, or higher.In some embodiments, such elevated temperatures can be achieved by performing at least a portion of the heating process (e.g., boiling) under pressure. For example, suitable pressures capable of producing the silk fibroin fragments described herein are generally between about 10 and 40 psi, e.g., about 11 psi, about 12 psi, about 13 psi, about 14 psi, about 15 psi, about 16 psi, about 17 psi, about 18 psi, about 19 psi, about 20 psi, about 21 psi, about 22 psi, about 23 psi, about 24 psi, about 25 psi, about 26 psi, about 27 psi, about 28 psi, about 29 psi, about 30 psi, about 31 psi, about 32 psi, about 33 psi, about 34 psi, about 35 psi, about 36 psi, about 37 psi, about 38 psi, about 39 psi, or about 40 psi.

[0090] In some embodiments, the aqueous solution used in the silk cocoon degumming process contains approximately 0.02M Na2CO3. The cocoons are rinsed, for example, with water to extract the sericin protein. The degummed silk can be dried and used to make silk powder. Alternatively, the extracted silk can be dissolved in an aqueous salt solution. Salts useful for this purpose include lithium bromide, lithium thiocyanate, calcium nitrate, or other chemicals capable of solubilizing silk. In some embodiments, the extracted silk can be dissolved in an approximately 8M-12M LiBr solution. The salt is then removed, for example, using dialysis.

[0091] In some embodiments, the silk fibroin is substantially depleted of its native sericin content (e.g., 5% (w / w) or less sericin remaining in the final extracted silk). In some embodiments, the silk fibroin is completely free of its native sericin content. As used herein, the term "completely free" (i.e., "consisting of") means that the substance cannot be detected or its presence cannot be confirmed within the detection range of the instrument or process being used. In some embodiments, the silk fibroin is essentially free of its native sericin content. As used herein, the term "essentially free" (or "essentially consisting of") means that only trace amounts of a substance can be detected, that it is present in an amount below detection, or that it is non-existent.

[0092] If necessary, the silk solution can be concentrated using, for example, dialysis against a hygroscopic polymer, such as PEG, polyethylene oxide, amylose, or sericin. In some embodiments, the PEG has a molecular weight of 8,000 to 10,000 g / mol and a concentration of about 10% to about 50% (w / v). A slide-a-lyzer dialysis cassette (Pierce, MW CO3500) can be used. However, any dialysis system can be used. Dialysis can be carried out for a time sufficient to achieve a final concentration of about 10% to about 30% in aqueous silk. In most cases, 2 to 12 hours of dialysis may be sufficient. See, for example, International Patent Application Publication No. WO 2005 / 012606, the contents of which are incorporated herein by reference in their entirety. Another method for producing a concentrated silk solution involves drying a dilute silk solution (e.g., by evaporation or lyophilization). A dilute solution can be partially dried to reduce its volume, thereby increasing the silk concentration. After the dilute solution is completely dried, the dried silk fibroin can be dissolved in a smaller amount of solvent compared to the dilute silk solution. In some embodiments, the silk fibroin solution can be filtered and / or centrifuged at an appropriate time, if necessary. For example, in some embodiments, the silk fibroin solution can be filtered and / or centrifuged after the heating or boiling step, if necessary. In some embodiments, the silk fibroin solution can be filtered and / or centrifuged after the dialysis step, if necessary. In some embodiments, the silk fibroin solution can be filtered and / or centrifuged after the concentration adjustment step, if necessary. In some embodiments, the silk fibroin solution can be filtered and / or centrifuged after the reconstitution step, if necessary. In any of these embodiments, one or more filtration and / or centrifugation steps can be performed to remove insoluble materials. In any of these embodiments, one or more filtration and / or centrifugation steps can be performed to selectively enrich silk fibroin fragments of a specific molecular weight.

[0093] In some embodiments, silk fibroin and / or silk fibroin articles may comprise a protein structure that substantially comprises β-turn and / or β-strand regions. Without wishing to be bound by theory, the β-sheet content of silk may affect the gel function and in vivo longevity of the composition. It should be understood that compositions (e.g., e-gels) that comprise non-β-sheet content may also be utilized. In some embodiments, the silk fibroin has a protein structure that comprises, for example, about 5% β-turn and β-strand regions, about 10% β-turn and β-strand regions, about 20% β-turn and β-strand regions, about 30% β-turn and β-strand regions, about 40% β-turn and β-strand regions, about 50% β-turn and β-strand regions, about 60% β-turn and β-strand regions, about 70% β-turn and β-strand regions, about 80% β-turn and β-strand regions, about 90% β-turn and β-strand regions, or about 100% β-turn and β-strand regions. In other aspects of these embodiments, the silk fibroin has a protein structure that includes, for example, at least 10% β-turn and β-strand regions, at least 20% β-turn and β-strand regions, at least 30% β-turn and β-strand regions, at least 40% β-turn and β-strand regions, at least 50% β-turn and β-strand regions, at least 60% β-turn and β-strand regions, at least 70% β-turn and β-strand regions, at least 80% β-turn and β-strand regions, at least 90% β-turn and β-strand regions, or at least 95% β-turn and β-strand regions.In still other aspects of these embodiments, the silk fibroin may have, for example, about 10% to about 30% β-turn and β-strand regions, about 20% to about 40% β-turn and β-strand regions, about 30% to about 50% β-turn and β-strand regions, about 40% to about 60% β-turn and β-strand regions, about 50% to about 70% β-turn and β-strand regions, about 60% to about 80% β-turn and β-strand regions, about 70% to about 90% β-turn and β-strand regions, about 80% to about 100% β-turn and The silk fibroin composition may have a protein structure comprising about 10% to about 40% β-turn and β-strand regions, about 30% to about 60% β-turn and β-strand regions, about 50% to about 80% β-turn and β-strand regions, about 70% to about 100% β-turn and β-strand regions, about 40% to about 80% β-turn and β-strand regions, about 50% to about 90% β-turn and β-strand regions, about 60% to about 100% β-turn and β-strand regions, or about 50% to about 100% β-turn and β-strand regions. In some embodiments, silk β-sheet contents of less than 10% to about 55% can be used in the silk fibroin compositions disclosed herein.

[0094] In some embodiments, the silk fibroin or silk fibroin article has a protein structure that is substantially free of α-helix and / or random coil regions. In aspects of these embodiments, the silk fibroin has a protein structure that includes, for example, about 5% α-helix and / or random coil regions, about 10% α-helix and / or random coil regions, about 15% α-helix and / or random coil regions, about 20% α-helix and / or random coil regions, about 25% α-helix and / or random coil regions, about 30% α-helix and / or random coil regions, about 35% α-helix and / or random coil regions, about 40% α-helix and / or random coil regions, about 45% α-helix and / or random coil regions, or about 50% α-helix and / or random coil regions. In other aspects of these embodiments, the silk fibroin has a protein structure that includes, for example, up to 5% α-helix and / or random coil regions, up to 10% α-helix and / or random coil regions, up to 15% α-helix and / or random coil regions, up to 20% α-helix and / or random coil regions, up to 25% α-helix and / or random coil regions, up to 30% α-helix and / or random coil regions, up to 35% α-helix and / or random coil regions, up to 40% α-helix and / or random coil regions, up to 45% α-helix and / or random coil regions, or up to 50% α-helix and / or random coil regions.In still other aspects of these embodiments, the silk fibroin has, for example, about 5% to about 10% α-helix and / or random coil regions, about 5% to about 15% α-helix and / or random coil regions, about 5% to about 20% α-helix and / or random coil regions, about 5% to about 25% α-helix and / or random coil regions, about 5% to about 30% α-helix and / or random coil regions, about 5% to about 40% α-helix and / or random coil regions, or .... coil regions, about 5% to about 50% α-helices and / or random coil regions, about 10% to about 20% α-helices and / or random coil regions, about 10% to about 30% α-helices and / or random coil regions, about 15% to about 25% α-helices and / or random coil regions, about 15% to about 30% α-helices and / or random coil regions, or about 15% to about 35% α-helices and / or random coil regions.

[0095] high temperature

[0096] As discussed herein, the provided methods and compositions include one or more exposures to elevated temperatures. As used herein, the term "elevated temperature" refers to a temperature higher than standard room temperature (i.e., higher than 25°C). In some embodiments, the provided methods or compositions include one exposure to elevated temperatures. In some embodiments, the provided methods or compositions include at least two (e.g., 3, 4, 5, 6, 7, 8, 9, 10 or more) exposures to elevated temperatures. In some embodiments where the composition method includes two or more elevated temperatures, at least two of the elevated temperatures are different from each other.

[0097] In some embodiments, the elevated temperature may be between 25° C. and 200° C. As specific exemplary ranges, in some embodiments, the elevated temperature may be between 25° C. and 150° C., between 25° C. and 100° C., between 25° C. and 95° C., between 25° C. and 50° C., between 50° C. and 200° C., between 50° C. and 150° C., between 50° C. and 100° C., between 25° C. and 100° C., between 125° C. and 200° C., or any other range between 125° C. and 175° C.

[0098] In some embodiments, the elevated temperature may be at least 25° C. By way of further example, in some embodiments, the elevated temperature may be at least 26° C., 27° C., 28° C., 29° C., 30° C., 35° C., 40° C., 45° C., 50° C., 55° C., 60° C., 65° C., 70° C., 75° C., 80° C., 85° C., 90° C., 95° C., or 100° C. In some embodiments, enhanced crystallization of the silk fibroin material is observed at temperatures of 95° C. or higher.

[0099] In some embodiments, the elevated temperature may be up to 125° C. By way of further example, in some embodiments, the elevated temperature may be up to 126° C., 127° C., 128° C., 129° C., 130° C., 135° C., 140° C., 145° C., 150° C., 155° C., 160° C., 165° C., 170° C., 175° C., 180° C., 185° C., 190° C., or 195° C.

[0100] The application of one or more elevated temperatures to the provided compositions or methods may be performed by any suitable method for application. By way of non-limiting example, in some embodiments, the application of one or more elevated temperatures may be performed by a heat press, via a heating device such as an oven, a heating stage, an exposed flame, or other mechanism.

[0101] The one or more applications of elevated temperature may occur for any of a variety of periods or over a period of time. For example, in some embodiments, the one or more applications of elevated temperature occur substantially immediately (e.g., by placing over a flame or in an oven). In some embodiments, the one or more applications of elevated temperature occur over a period of several seconds, minutes, or hours. In some embodiments, the one or more applications of elevated temperature occur over a period of between 1 second and 1 hour.

[0102] High pressure

[0103] As discussed herein, the provided methods and compositions include one or more exposures to high pressure. As used herein, the term "high pressure" refers to a pressure higher than standard atmospheric pressure (i.e., 1.013 bar). In some embodiments, the provided methods or compositions include one exposure to high pressure. In some embodiments, the provided methods or compositions include at least two (e.g., 3, 4, 5, 6, 7, 8, 9, 10 or more) exposures to high pressure. In some embodiments, where the composition method includes two or more high pressures, at least two of the high pressures are different from each other.

[0104] Any suitable method for one or more applications can be used to generate the high pressure applied to the provided compositions or provided methods. By way of non-limiting example, in some embodiments, the high pressure can include the use of a vacuum, a press (e.g., a heat press), and combinations thereof.

[0105] In some embodiments, the application of high pressure may be or may include uniaxial compression, hi some embodiments, the application of high pressure may be or may include multiaxial compression (e.g., biaxial compression).

[0106] While any suitable level of high pressure may be used for application, in some embodiments, high pressures between 1 MPa and 1 GPa are used. As specific exemplary ranges, in some embodiments, the high pressure may be between 10 MPa and 1 GPa, between 50 MPa and 1 GPa, between 100 MPa and 1 GPa, between 200 MPa and 1 GPa, between 300 MPa and 1 GPa, between 400 MPa and 1 GPa, or between 500 MPa and 1 GPa. In some embodiments, the high pressure may be and include at least 1 MPa (e.g., at least 2 MPa, 3 MPa, 4 MPa, 5 MPa, 6 MPa, 7 MPa, 8 MPa, 9 MPa, 10 MPa, 20 MPa, 30 MPa, 40 MPa, 50 MPa, 60 MPa, 70 MPa, 80 MPa, 90 MPa, 100 MPa, 150 MPa, 200 MPa, 250 MPa, 300 MPa, 350 MPa, 400 MPa, 450 MPa, 500 MPa, 550 MPa, 600 MPa, 650 MPa, 700 MPa, or 750 MPa).

[0107] Silk products

[0108] The provided methods and compositions enable the production of previously unattainable silk articles, as well as silk articles with enhanced properties. In some embodiments, the provided silk articles exhibit a substantially homogeneous structure (e.g., as shown in Figure 25, panel A). As used herein, "substantially homogeneous structure" means that silk fibroin molecules are distributed and / or organized in a consistent manner throughout substantially all parts or throughout the article. Furthermore, in some embodiments, the silk article may exhibit a significant amount of silk fibroin in a semi-crystalline structure (see, e.g., Figure 25, panels C and E). In some embodiments, production of silk articles according to the provided methods includes a structural transition of silk fibroin from a substantially amorphous state to a semi-crystalline state, as observed, for example, by X-ray diffraction.

[0109] In some embodiments, the silk article may exhibit a substantial amount of beta-sheet structure. For example, in some embodiments, the silk article may exhibit at least 10% or more (e.g., at least 20%, 30%, 40%) by weight of beta-sheet structure compared to the starting silk fibroin material. In some embodiments, the silk article may exhibit at least 50% or more (e.g., at least 60%, 70%, 80%, 90%, 95%) by weight of beta-sheet structure compared to the starting silk fibroin material.

[0110] In some embodiments, the crystallinity of a silk article can be controlled by the application of temperature and pressure. For example, in some embodiments, when amorphous silk is processed at a temperature ranging from about 25°C to 125°C, the silk article may contain about 10-15% β-sheet structure. In some embodiments, when amorphous silk is processed at a temperature ranging from about 125°C to 175°C, the silk article may contain, for example, about 20-35% β-sheet structure, or, for example, greater than 40% β-sheet structure.

[0111] In some embodiments, the provided methods and compositions allow for the production of homogeneous silk articles in which the round amorphous powder is packed together through bonding between adjacent raw silk powders, for example, at processing temperatures of about 25° C. to 95° C. In some embodiments, the provided methods and compositions allow for the production of homogeneous silk articles in which the silk molecules of the amorphous powder are heated above their glass transition temperature, gaining greater mobility and self-organizing into entangled nanoglobules, for example, at processing temperatures of about 125° C. to 175° C.

[0112] In some embodiments, the provided methods and compositions allow for the production of silk articles (e.g., thin films) that can be subjected to heat softening and molded into desired shapes, hi some embodiments, the provided methods and compositions allow for the production of machinable silk articles.

[0113] The provided methods and compositions enable the production of complex silk articles in ways not previously achievable (e.g., silk screws that can resist the torsional forces associated with in vivo use). By way of non-limiting example, in some embodiments, the provided methods and compositions may be used to produce silk articles such as films, fibers, meshes, needles, tubes, plates, screws, rods, and any combination thereof.

[0114] In some embodiments, the silk article may be suitable for one or more types of patterning. In some embodiments, the patterning may be or include macropatterning. In some embodiments, the patterning may be or include micropatterning (i.e., patterning with microscale features). In some embodiments, the patterning may be or include nanopatterning (i.e., patterning with nanoscale features). In some embodiments, the patterning may be or include etching, lithography-based patterning, carving, cutting, and any combination thereof.

[0115] In some embodiments, the silk article may be subjected to one or more types of processing (e.g., 2, 3, 4, 5, 6, 7, 8, 9, 10 or more). While any form of processing suitable for application is contemplated within the scope of this disclosure, in some embodiments, processing may be or include machining, rolling, drilling, milling, sanding, punching die cutting, extruding, chemical etching, coating, molding, turning, thread rolling, and any combination thereof.

[0116] Examples of properties or characteristics of silk goods

[0117] In some embodiments, the provided composition (e.g., silk article) may be substantially transparent. In some embodiments, the provided composition (e.g., silk article) may be translucent. In some embodiments, the provided composition (e.g., silk article) may be substantially opaque. As used herein, the term "transparent" refers to an object's tendency to transmit light (with or without scattering of said light). In some embodiments, a composition / article is said to be substantially transparent if it transmits 80% or more of light to which it is exposed in the visible range (400 nm to 800 nm). In some embodiments, a composition / article is said to be translucent if it transmits 50% to 80% of light to which it is exposed in the visible range (400 nm to 800 nm). In some embodiments, a composition / article is said to be substantially opaque if it transmits 50% or less of light to which it is exposed in the visible range (400 nm to 800 nm).

[0118] In some embodiments, provided compositions may be biocompatible and / or biodegradable. In some embodiments, provided compositions may exhibit one or more particular degradation profiles. As a specific example, in some embodiments, provided compositions may degrade at least 50% by weight after exposure to an aqueous environment at 37° C. for about 96 hours. In some embodiments, provided compositions may not degrade by more than 10% after exposure to an in vivo environment or conditions for one month.

[0119] In some embodiments, provided compositions may exhibit one or more desirable properties, including, but not limited to, electrical conductivity, enhanced machinability, and / or enhanced thermoformability.

[0120] additives

[0121] In some embodiments, provided methods or compositions include one or more additives (e.g., 2, 3, 4, 5, 6, 7, 8, 9, 10, or more additives). In some embodiments, at least one additive may be mixed with or otherwise associated with the silk fibroin material prior to the applying step (e.g., one or more exposures to elevated temperature and pressure). In some embodiments, at least one additive may be mixed with or otherwise associated with the silk fibroin material substantially simultaneously with the applying step. In some embodiments, at least one additive may be mixed with or otherwise associated with the silk fibroin material following the applying step.

[0122] The provided methods and compositions are suitable for the addition of any of a variety of additives. By way of non-limiting example, in some embodiments, the additive may be or include a small molecule, an organic polymer, an inorganic polymer, a conductive material, an inorganic material, a hydrophobic material, a hydrophilic material, a nanomaterial, and any combination thereof.

[0123] The processing of silk-based materials, including pure silk materials and silk-based composite materials, can be modified by adding one or more additives. In some embodiments, the function of the additive can be to adjust the processing conditions and product properties. In some embodiments, the additive can be selected from water; glycerol; sugars; biopolymers, such as peptides, proteins; antibodies and antigen-binding fragments; nucleic acids; immunogens; antigens; enzymes; synthetic polymers, such as poly(ethylene) glycol, polylactic acid, poly(lactic-co-glycolic acid), etc., although any additive suitable for the application is specifically contemplated within the scope of the present disclosure.

[0124] In some embodiments, e.g., in some embodiments intended for in vivo use, provided compositions may include one or more proteases. In some embodiments, the organic polymer is or includes at least one protease. In some embodiments, the protease is or includes one or more of proteinase XIV, proteinase K, α-chymotrypsin, collagenase, matrix metalloproteinase-1 (MMP-1), and MMP-2. In some embodiments, the protease may be useful for adjusting the degradation profile (e.g., in an in vivo environment) of a particular provided composition.

[0125] In some embodiments, the conductive material may be or include an organic conductive material and / or an inorganic conductive material (e.g., a metal), hi some embodiments, the conductive material may be or include at least one of a conductive polymer, graphene, silver, gold, aluminum, copper, platinum, steel, brass, bronze, and iron oxide.

[0126] According to various embodiments, one or more additives can be useful in amounts appropriate for any application. By way of non-limiting example, in some embodiments, the additives can be present in a provided composition in an amount between 0.001% and 95% by weight. In some embodiments, the one or more additives can be mixed with the silk fibroin material in an amount ranging from 0.001% to 95% by weight of the silk fibroin material. [Example]

[0127] Example 1 - Preparation of aqueous silk solution for the production of freeze-dried silk

[0128] The silk solution was prepared by dissolving degummed silk fibers (silk fibers after removing their outer sericin) in a high-salt LiBr / CaCl2 solution. Bombyx mori (B. mori) cocoons were cut into small pieces and boiled in 0.02 M Na2CO3 aqueous solution (Sigma-Aldrich, USA) for 30 minutes, then rinsed with distilled water to remove the Na2CO3 and sericin. The degummed silk was dried overnight at room temperature. Next, 20 grams of dried degummed silk was dissolved in 100 mL of 9.3 M LiBr solution at 60 °C for 3–4 hours. The solution was then dialyzed against distilled water for 3 days using a Slide-a-Lyzer dialysis cassette (MWCO 3,500, Pierce, USA). The water was changed five times during dialysis (1 h, 4 h, 8 h, 24 h, and 48 h). After dialysis, the solution was centrifuged at 13,000 rpm for 10 min to remove insoluble impurities. The concentration of the final silk solution was determined by drying a known amount of solution and measuring the mass of the residual solids (approximately 6 w / v%).

[0129] Example 2 - Characterization of amorphous silk materials

[0130] Natural silk fibers from the silkworm cocoon (Bombyx mori) contain silk fibroin, a naturally occurring protein with heavy chains of approximately 390 kDa and light chains of approximately 25 kDa. 5、19 The silk solution is obtained by breaking the hydrogen bond network of degummed natural silk fibers with a highly concentrated salt solution to form a regenerated silk solution. The fresh solution is then further freeze-dried to obtain amorphous silk material (Figure 1 Panels D, E, and F) in a solid state. This is more stable than the silk solution and can be stored at ambient conditions for years without significant structural changes.

[0131] In this example, fresh solubilized silk was freeze-dried and then further processed into particles, which were then shaped by heat and compression to form various final structures. To understand the properties of the final materials, the starting materials were characterized and compared to the material properties of natural silk.

[0132] Fresh solubilized silk can be converted back into a solid silk form by lyophilization to produce freeze-dried silk. Here, freeze-dried silk has been found to have a fibroin structure (predominantly random coil) very similar to that in solution. More importantly, dry, freeze-dried silk can be stored under ambient conditions for years without noticeable structural changes. Compared to raw silk cocoon fibers and degummed silk fibers, freeze-dried silk lacks a well-defined hierarchical structure composed of secondary structures (e.g., β-sheets). It is an amorphous material with a random coil structure. Due to these significant structural differences, the thermal and mechanical behavior of freeze-dried silk differs from that of raw silk cocoon fibers and degummed silk fibers.

[0133] Panel (A) of Figure 2 shows the thermal properties of degummed silk fiber and freeze-dried silk powder. Thermogravimetric analysis (TGA) revealed that the freeze-dried silk had a higher moisture content (5.0 wt%) than the degummed silk fiber (2.8 wt%).

[0134] Characterization by differential scanning calorimetry (DSC) further showed that the freeze-dried silk possessed a water-related glass transition near 65 °C and a glass transition of pure silk fibroin at 178 °C, which were not observed in the degummed silk fibers (Figure 2, panel B).

[0135] To understand the structural changes during heating, we performed a more detailed DSC study of freeze-dried silk, and the results are shown in Figure 3. During heating, the silk-water system undergoes dynamic conformational and compositional changes due to a combination of silk fibroin chain reorganization and the evaporation of bound water. Specifically, silk fibroin may first reorganize to form new conformations using bound water as a plasticizer, which was demonstrated in the low temperature range (35°C–80°C). A well-resolved glass transition of the silk-water system was demonstrated at 65°C. Further heating of the silk simultaneously led to the release of bound water molecules. Two types of bound water molecules were found in this system, including weakly bound and strongly bound water molecules. Weakly bound water molecules began to evaporate around 35°C with a maximum evaporation rate of 95°C, whereas strongly bound water molecules began to evaporate around 55°C with a maximum evaporation rate of 125°C. When the temperature reached 160°C, all bound water molecules evaporated. Continuous heating showed that the stable glass transition of pure silk fibroin was around 178 °C. The heating rate for TGA, DTG, and DSC characterization was 10 °C / min.

[0136] DSC measurements were performed with samples sealed in aluminum pans under a dry nitrogen gas flow rate of 50 mL / min. In standard DSC measurements, samples were heated from -50 °C to 200 °C at a heating rate of 10 °C / min. The water content of ASN is similar to that reported for solution-cast silk fibroin, which has a less ordered structure. 27、28 .

[0137] Thermal analysis showed that the water content of ASN was 5.0 ± 0.5 wt% (Figure 2, Panel A), significantly higher than that of degummed natural silk fibers (approximately 2.8 ± 0.3 wt%). Differential scanning calorimetry (DSC) showed that ASN had a water-related glass transition of 65 °C and a stable glass transition temperature of 178 °C (Figure 2, Panel B), which is in good agreement with previous studies on amorphous silk films (Hu, X., Kaplan, D. & Cebe, P. Determining Beta-Sheet Crystallinity in Fibrous Proteins by Thermal Analysis and Infrared Spectroscopy. Macromolecules 39, 6161-6170 (2006). Detailed step-scan profiles further revealed two types of water bound to the amorphous silk material: weakly bound water with a maximum evaporation rate of 95 °C and strongly bound water with a maximum evaporation rate of 125 °C (Figure 2, panel C). Characterization by differential scanning calorimetry (DSC) further demonstrated that the freeze-dried silk possessed a water-related glass transition near 71 °C (from TMDSC) and a glass transition of pure silk fibroin at 178 °C, which were not observed in the degummed silk fibers (Figure 4). In this example, the silk fibroin exhibited a well-resolved glass transition of the silk-water system at 71 °C. Upon continued heating, a stable glass transition of pure silk fibroin was observed near 178 °C. The heating rate was 2°C / min, the temperature amplitude was 0.318 K, and the period was 1 min.

[0138] X-ray diffraction (XRD), Fourier transform infrared spectroscopy (FTIR), and solid-state NMR spectroscopy showed that the silk fibroin in the amorphous silk material was mainly amorphous, with a low content of β-sheet structure (approximately 1.0%) (Figure 1, Panels G, H, I, and J).

[0139] Example 3 - Engineering silk monoliths using amorphous silk materials at various temperatures and pressures

[0140] Natural silk fibers have a highly stable molecular structure containing a strong hydrogen-bonding network under ambient and high-pressure conditions. 26 This limits the direct thermal processing of raw silk fibers. By restructuring natural silk into amorphous silk, the molecular structure of silk fibroin becomes less ordered. At high pressure and temperature, the free energy change of the material system (Gibbs free energy G = E + PV - TS) may provide new opportunities to tailor the phase or molecular structure of the material. Therefore, we decided to investigate how external pressure (or stress) and heat affect the assembly of silk proteins in the solid state.

[0141] Amorphous silk is obtained by freeze-drying and used as raw material for densification and thermal processing. A combination of molecular structure (XRD, FTIR) and morphological analysis (SEM) reveals that increasing the processing temperature during densification promotes the self-assembly of amorphous silk fibroin. Pressure-driven densification of raw ASN can promote intermolecular interactions, including hydrogen bonding and van der Waals interactions, which lead to the formation of new β-sheet structures.

[0142] Regenerated amorphous silk prepared from silk solutions of various concentrations (<7 wt%) was used as a raw material to fabricate silk materials by a heat / pressure process. In this example, regenerated amorphous silk powder prepared from a 1% silk solution was used as an exemplary starting material for the production of silk materials in various formats. This example further explored the effects of heat and pressure on silk structure and further demonstrated various processing routes for engineering amorphous silk materials into functional devices (e.g., silk screws) using heat and pressure (Figure 5). A collection of silk parts fabricated by various processing routes is shown in Figure 7.

[0143] The method used to engineer and induce structural changes in ASNs is the controlled application of pressure and heat. The applied pressure ranges from 1 MPa to over 1 GPa, and the temperature ranges from 0 °C to 200 °C. Because silk fibroin begins to decompose around 200 °C, the temperature is controlled below 200 °C. ASNs were filled into a predesigned mold and compressed into silk plates at 632 MPa and various temperatures. As the temperature increased, the silk plates changed appearance from opaque to transparent and then to a pale yellowish color. Scanning electron microscopy analysis showed significant differences in the internal structure of silk plates processed at low temperatures (25, 65, and 95 °C) and high temperatures (125, 145, and 175 °C). At low processing temperatures, features similar in size to raw silk powder still exist, despite the increased packing density and plastic deformation of raw silk powder with increasing temperature (Figure 8, panels A2–C2 and A3–C3). Silk globules of approximately 30 nm were observed at high processing temperatures (Figure 8, panels a: D3–F3), which resemble the spherical structure of natural silkworm and spider silk fibers.

[0144] Fourier transform infrared spectroscopy (FTIR) analysis showed that a small amount of β-sheet structure (approximately 10–15%) was formed at low processing temperatures (25 °C, 65 °C, 95 °C, and 125 °C), while a larger amount (>40%) of β-sheet structure was formed at higher processing temperatures (145 °C and 175 °C) (Figure 8, panels B and D). Furthermore, X-ray diffraction (XRD) characterization showed that the crystallinity of the densified silk plates increased slowly with increasing temperature in the temperature range of 25–95 °C. In contrast, when hot-pressed above 125 °C, the crystallinity increased significantly with temperature (Figure 8, panels C and E). At low temperatures (25, 65, and 95 °C), amorphous silk powders may pack together through bonds between adjacent particles, resulting in a low content of β-sheet structure; at higher temperatures (125, 145, and 175 °C), silk molecules may gain greater mobility above the glass transition temperature and self-assemble into structures characterized by entangled nanoglobules and moderate crystallinity (approximately 20–35%). Furthermore, when hot-pressed above 95 °C, XRD revealed a silk II structure (similar to natural silk fibers), indicating a structural transition (from silk I to silk II) during processing (Figure 8, Panel C). A simple mechanism for the structural transition of ASN during thermal processing has been proposed (Figure 8, Panel F).

[0145] In one example, the mechanical properties of thermally processed bulk silk materials were characterized by three-point bending tests. As the processing temperature increased (from 25 °C to 145 °C), the strength of the bulk silk material increased (Figure 9, Panels A and B). However, at 175 °C, the strength began to decrease. This can be attributed to high crystallinity and partial thermal decomposition. The maximum strength was 109 ± 10 MPa / g cm for the silk plate processed at 145 °C. -3 This exceeds the strength of some natural structural materials, such as CP nacre and wood (Figure 9, panel C). In contrast, densification of degummed silk powder with high β-sheet content and low crystallinity results in poor mechanical properties. This may indicate weaker fusion of the degummed silk powder (Figure 10).

[0146] In another example, the role of pressure in the transformation of amorphous to structured silk materials was investigated. At room temperature, pressure-driven densification of raw amorphous silk material leads to the formation of β-sheet structures (β-sheet content is approximately 10% compared to approximately 1% in amorphous silk material, Figure 8 panels B and D). When densification is performed at elevated temperatures (65–175 °C), the density of the densified silk material is approximately 1.35 g / cm at high pressure (632 MPa). 3 The densification temperature reaches a certain value (Figure 11, panel A). Densification under low external pressure exhibits low bending strength in three-point bending tests (Figure 11, panel B). A combination of molecular structure (XRD, FTIR) and morphological analysis (SEM) revealed that increasing the processing temperature during densification promotes the self-organization of amorphous silk fibroin, as indicated by the formation of significant β-sheet structure at high temperatures (>145 °C). The pressure- and heat-induced structural transition of amorphous silk likely helps fuse the silk fibroin and transform the amorphous silk nanomaterial into a robust bulk material.

[0147] Example 4 - Direct molding of amorphous silk nanomaterials into designed shapes

[0148] The amorphous silk material can be directly loaded into a pre-designed mold for molding, as shown in Process B in Figure 5. After molding, the silk material is deformed into the designed shape. In this case, the amorphous silk material was loaded into a screw-shaped mold, and then the material was molded into the shape at 120 °C and 800 MPa. The resulting silk screw is shown in Figure 12.

[0149] Example 5 - Thermoforming and Patterning

[0150] In another example, silk monoliths obtained by heat and pressure can be used for thermoforming and patterning. Standard DSC was performed on a silk plate obtained at 125 °C and 632 MPa (Figure 13, panel A). This material exhibited a glass transition temperature (Tg) of 97 °C, followed by an exothermic peak due to crystallization at around 137 °C. The silk plate contains a significant amount of amorphous structure (approximately 79% non-crystalline structure), which becomes flexible when heated above the Tg of the plate. This thermal softening property can be utilized to further mold pre-pressed silk material into desired shapes, such as threads, patterns, and other complex shapes (Figure 13, panels B–E). For example, to replicate the features of a dime, 200 mg of ASN was first pressed in a 13 mm mold set at 700 MPa to create a small pellet. The pellet was then placed on top of the dime and subjected to thermal compression (100 MPa, 140 °C) to imprint the pattern on the coin. Panel (D) of Figure 13 shows a photograph of the patterned silk coin. The transparency of the silk plate allows the molded pattern to be seen from both sides of the silk coin. Patterning of micro / nanostructures can also be achieved using this method. Figure 13, panel (E) shows an SEM image of the nanoscale patterned silk film.

[0151] Example 6 - Engineering silk monoliths used for machining into designed shapes

[0152] By applying different processing conditions, silk monoliths with tunable molecular structures can be fabricated. More specifically, silk monoliths with low β-sheet content can be prepared at 95 °C and 632 MPa, while silk monoliths with high β-sheet content can be prepared at 145 °C and 632 MPa. The silk monoliths can be further machined into designed shapes, as shown in Process A in Figure 14. In this example, silk screws and ear tubes were machined from the silk monoliths. Furthermore, water absorption tests were performed to investigate the swelling properties of silk screws with different molecular structures.

[0153] The swelling and degradability of these products can be controlled by manipulating the processing conditions. In one example, silk-based bone screws were fabricated by machining silk bars prepared under different processing conditions (95 °C and 632 MPa; 125 °C and 632 MPa; 145 °C and 632 MPa) to vary the internal molecular structure characteristics (β-sheet content and crystallinity). The degradation profile showed that processing at 145 °C provided the slowest degradation rate in enzyme solution over time, demonstrating tunable silk screw degradability (Figure 15). Furthermore, silk screws processed at 95 °C and 125 °C exhibited a weight loss of over 60% in a 5 U / mL protease XIV solution and were found to rapidly degrade within 30 days. Furthermore, water absorption in PBS at 37 °C showed that silk screws machined from silk bars prepared at 95 °C and 125 °C rapidly absorbed water, up to approximately 30 wt %, within 15 minutes (Figure 16, panel B). In comparison, silk screws machined from silk bars prepared at 145 °C showed much slower water absorption, up to about 20 wt %. These differences reflect the underlying control of crystalline content.

[0154] Furthermore, degradation studies showed that the degradability of silk screws was tunable, where highly crystalline silk screws provided low degradation rates in both PBS and enzyme solutions over a 30-day period ( Figure 15 ).

[0155] Example 7 - Preparation of composite materials, including silk-enzyme composites

[0156] The methods described in the previous examples can be extended to produce composite materials other than pure silk materials. Composite materials can include, among others, silk-small molecule systems, silk-polymer systems, silk-inorganic materials systems, silk-hydrophobic / hydrophilic materials systems, and / or silk-nanomaterial systems. In one example, silk-enzyme composites were fabricated by mixing dried silk powder with protease XIV powder, an enzyme that degrades silk. The ratios of protease XIV were varied (0.2 wt%, 1.0 wt%, and 5.0 wt%). The mixtures were then pressed into small pellets using a 6 mm mold set at 700 MPa. The pellets were then incubated in methanol for 48 hours to induce a beta-sheet structure in the silk, improving its mechanical properties and water resistance.

[0157] Degradation tests were conducted in PBS solution at 37 °C. The results showed that the degradation of silk materials could be controlled by varying the ratio of protease XIV in the composite. For example, at a protease XIV loading rate of 5 wt%, silk degraded 60 wt% after 4 days of incubation in PBS solution at 37 °C (Figure 17, panel (a)). At a protease XIV loading rate of 0.2 wt%, silk degraded approximately 30 wt%. Degradation was observed for silk screws containing 0.5% protease XIV machined from silk bars pressed at 145 °C and 632 MPa (Figure 16, panel (b)). It is noteworthy that during heat pressing (145 °C and 632 MPa), the enzyme activity, which would be lost in the absence of silk, was maintained in the silk / enzyme system. This suggests that silk can stabilize the enzyme at high temperature and pressure. These methods demonstrate the ability to fabricate silk-based bulk materials with controllable degradability.

[0158] The components added to the silk matrix / article can be organic or inorganic molecules. The components can be incorporated into the silk matrix by mixing the silk solution with the components and then freeze-drying the solution. The components can include, but are not limited to, micro / nano materials, such as metal micro / nano materials, graphene, graphene oxide, and / or hydroxyapatite.

[0159] Components may be added to create a fiber-reinforced silk matrix / article. Natural or synthetic fibers can be incorporated into the silk matrix using the described methods. Natural or synthetic fibers can include raw silk fibers, synthetic nylon fibers, and / or synthetic polymer fibers.

[0160] Example 8 - Fabrication of composite materials, including silk-graphene composites

[0161] In this example, silk-graphene composites were fabricated using freeze-dried silk material. Graphene is hydrophobic, making it difficult to directly fabricate silk-graphene composites using solution methods. However, using the method described in the previous example, silk-graphene composites were formed by mixing graphene and silk powder in the solid state. In one example, graphene was mixed with fine silk powder at a loading rate of 70.0 wt. %. The mixture was then pressed in a 6 mm mold set at 700 MPa to produce compressed silk-graphene films. The as-prepared silk-graphene films were highly conductive, demonstrating great potential in bioelectronics fabrication (Figure 18).

[0162] Example 9 - Investigation of structural differences between freeze-dried silk and solid silk articles

[0163] In this example, certain structural features of freeze-dried silk and solid silk articles subjected to the high temperatures and pressures described herein were investigated.

[0164] Figure 19, panel (A), shows an SEM image of freeze-dried silk powder. The internal structure of silk articles pressed at 145 °C and 632 MPa for 15 minutes is shown in Figure 19, panels (B) and (C). The loosely freeze-dried silk powder transformed into solid silk articles. A close-up of the internal structure of the silk article (Figure 19, panel C) revealed spherical features in the nanometer range. These features show similarities to the spherical structure of natural silkworm and spider silk fibers. The SEM images demonstrate that the freeze-dried raw silk material undergoes a fusion process, rather than a simple physical compression of the raw silk powder to form the pressed silk articles in Figure 19, panels (B) and (C). The molecular conformation of the silk partially changed from a random coil / amorphous state to a crystalline beta-sheet structure. The level of uniformity observed in the structure of these pressed silk articles was not achievable using previous methods and is therefore likely only possible using the method described herein.

[0165] Example 10 - Methods for converting silk into new materials

[0166] Materials and Chemicals. Sodium carbonate, lithium bromide, protease XIV, and chymotrypsin were purchased from Sigma-Aldrich (Sigma-Aldrich, St. Louis, MO) and used as received.

[0167] Regenerated silk fibroin preparation. Bombyx mori (B. mori) cocoons were cut into small pieces and boiled in 0.02 M Na2CO3 aqueous solution (Sigma-Aldrich, USA) for 30 min, then rinsed with distilled water to remove Na2CO3 and sericin. The degummed silk was dried overnight at room temperature. The dried silk was dissolved in 9.3 M LiBr solution at 60 °C for 3–4 h. The solution was then dialyzed in distilled water for 3 days using a Slide-A-Lyzer dialysis cassette (MWCO 3,500, Pierce, USA). The water was changed five times during dialysis (1 h, 4 h, 8 h, 24 h, and 48 h). After dialysis, the solution was centrifuged at 13,000 rpm for 10 min to remove insoluble impurities. The concentration of the final silk solution was determined by measuring the volume of the solution and the final dry weight (approximately 6 w / v%). The solution was diluted with distilled water and frozen in liquid nitrogen. The frozen silk was then freeze-dried at −80° C. until complete sublimation. The freeze-dried silk was ground into a fine powder and stored under ambient dry conditions to prevent rehydration of the freeze-dried solid until use in the following processing step.

[0168] Hot pressing of amorphous silk nanomaterials (ASN). ASN was packed into a pre-designed mold and subsequently hot pressed at 632 MPa and variable temperatures (25°C, 65°C, 95°C, 125°C, 145°C, and 175°C) for 15 min. After hot pressing, the samples were cooled to room temperature and used for characterization.

[0169] Thermal analysis. The thermal decomposition of silk samples was measured by thermogravimetric analysis (TGA) from 30 to 800 °C in N2 (99.99%) at a scan rate of 5 °C / min. For all measurements, samples were held under N2 in a furnace to reach a stable weight before heating. Differential scanning calorimetry (DSC) measurements were performed on a TA Instruments Q100 DSC (TA Instruments, New Castle, DE) with samples sealed in aluminum pans under a 50 mL / min dry nitrogen gas flow. Both standard DSC and temperature-modulated DSC (TMDSC) measurements were performed. For standard DSC measurements, samples were heated from -50 to 200 °C at a heating rate of 10 °C / min. For TMDSC measurements, samples were heated from -50 to 200 °C at 10 °C / min with a modulation period of 60 s and a temperature amplitude of 1.59 °C.

[0170] Scanning Electron Microscopy (SEM). The morphology of the scaffolds was characterized by scanning electron microscopy (SEM) (Ultra55 field emission scanning electron microscope, Carl Zeiss AG, Harvard University Center for Nanoscale Systems). SEM images were collected at a voltage of 5 kV, and the samples were sputter-coated with a thin layer of gold.

[0171] Fourier transform infrared spectroscopy (FTIR). Fourier transform infrared spectroscopy (FTIR) was performed on a JASCO FTIR6200 spectrometer (JASCO, Tokyo, Japan) equipped with a MIRacle attenuated total reflectance (ATR) Ge crystal cell in absorbance mode. For each measurement, the spectrum was analyzed using 32 scans and a 4.0 cm -1 The secondary structure of the protein was recorded at a resolution of 1000 MHz. The secondary structure of the protein was determined by the amide I region (1600-1700 cm). -1 The peaks were determined by performing peak deconvolution using the FTIR (FFT) method. The number and positions of the peaks were defined from the second derivative spectra and fixed during the deconvolution process. A Gaussian model was selected for the band shape and bandwidth, which were automatically adjusted by the software.

[0172] X-ray diffraction. X-ray diffraction was performed on a SAXSLAB small-angle / wide-angle X-ray scattering system (MIT Center for Materials Science and Engineering). The wavelength of the X-ray beam was 1.5409 Å and the fixed energy was 45 kV. The sample-to-detector distance was 109.1 mm, and an exposure time of 60 seconds was used in the experiment.

[0173] Three-point bending test. Three-point bending tests were performed on unnotched dry and wet specimens at loading rates of 0.2 mm / min and 2 mm / min, respectively, using an Instron 3366 testing machine (Instron, Norwood, USA) in bending mode at 25°C and 50% RH. The specimens were 12 mm long, 7 mm wide, and 1 mm thick.

[0174] In vitro hydration and swelling tests. Machined silk screws (n = 4 per condition) were placed in phosphate-buffered saline (PBS) at 37°C for various times (0 min, 15 min, 60 min, 3 h, 24 h, 48 h, 72 h, 9 days, 15 days, and 30 days) to observe changes in weight and diameter, indicating fluid uptake and swelling. Surface moisture was removed from the samples by wiping with Kimwipes (Kimberly-Clark, USA), and the wet weight (Ws) and head and screw diameters of the collected samples were measured. The hydration rate (%) was calculated.

[0175] Water absorption rate (%)=[(Ws-Wd) / Ws]×100(1)

[0176] In vitro enzymatic degradation test. Machined silk screws (n = 3 per condition) were incubated at 37 °C in 2 mL of 5 U / mL protease XIV and 40 U / mL chymotrypsin in PBS or PBS as a negative control. The solution was changed every 2–3 days. At designated time points (2, 5, 10, 20, and 30 days), the sample groups were rinsed with DI water, dried, and weighed. The remaining mass of each sample was collected, and SEM images of the samples were collected.

[0177] NMR spectrum of an aqueous solution of regenerated silk (6 wt%). Solution NMR experiments were performed using a Bruker 850 MHz laser equipped with a 5 mm cryogenic helium-cooled triple-resonance TCL CryoProbe. Spectroscopy was performed on an Avance III HD spectrometer. The Larmor frequencies for 1H, 13C, and 15N were 850.28 MHz, 213.82 MHz, and 86.17 MHz. A sealed capillary tube containing DO was used for NMR locking. NOESY was performed with a mixing time of 150 ms, a spectral width of 11 ppm, and 512 and 2048 complex points in both the t1 and t2 dimensions, 16 scans, and a 1-second relaxation delay. TOCSY was performed with a mixing time of 60 ms, a spectral width of 11 ppm, and 512 and 2048 complex points in both the t1 and t2 dimensions, 16 scans, and a 1-second relaxation delay. 1H-13C HSQC was collected with a spectral width of 165.8 ppm in t1, 11 ppm in the t2 dimension, 256 and 1024 complex points in t1 and t2, and 64 scans. 1H-15N HSQC was collected with a spectral width of 28.0 ppm in t1, 11 ppm in the t2 dimension, 512 and 1024 complex points in t1 and t2, and 32 scans. NMR data were processed and analyzed using MestReNova. Figure 20 shows the solution NMR spectrum of the regenerated silk aqueous solution (6 wt%).

[0178] Natural degummed silk and freeze-dried silk 13 C cross-polarization magic angle spinning (CP-MAS) spectrum. The spectrum was analyzed using double resonance ( 1 H / 13 C) mode. 1 Hπ 13 The CP condition for the C CP-MAS experiment was 2.25 μs. 1The experiment consisted of a H π / 2 pulse followed by a 1.0 ms ramp (3%) H spin-lock pulse at 70 kHz radio frequency (rf) field strength. Experiments were performed for all samples with a 25 kHz sweep width, a 3.0 s recycle delay, 8192 scans, and a 91 kHz two-pulse phase-modulated (TPPM) H decoupling level at a 20 kHz MAS speed. Figure 23 shows the results of the analysis of the native degummed silk and freeze-dried silk. 13 C shows the cross-polarization magic angle spinning (CP-MAS) spectrum.

[0179] Example 11 - In vitro degradation analysis of silk ear tubes with or without protease XIV doping

[0180] Using the methods described in the previous examples, ear tubes were fabricated from pure silk and silk-Protease XIV composite bulk materials prepared under two different conditions: a temperature of 125°C and a pressure of 632 MPa; and a temperature of 145°C and a pressure of 632 MPa.

[0181] The degradation test was carried out in PBS solution at 37° C. for various times: 5 minutes, 1 hour, 3 hours, 6 hours, 24 hours, 48 hours, and 72 hours. The results are shown in FIG.

[0182] REFERENCES The following list of references is identified in the above disclosure by the number immediately preceding the reference rather than by the paragraph number within parentheses.

[0183] 1. Altman, GH et al. Silk-based Biomaterials. Biomaterials 24, 401-416 (2003).

[0184] 2. Kundu, B., Rajkhowa, R., Kundu, S. C. & Wang, X. Silk fibroin biomaterials for tissue regenerations. Advanced Drug Delivery Reviews 65, 457-470 (2013).

[0185] 3. Koh, L.-D. et al. Structures, mechanical properties and applications of silk fibroin materials. Prog. Polym. Sci. 46, 86-110 (2015).

[0186] 4. Vepari, C. & Kaplan, D. L. Silk as a biomaterial. Prog. Polym. Sci. 32, 991-1007 (2007).

[0187] 5. Zhou, C. et al. Silk Fibroin: Structural Implications of a Remarkable Amino Acid Sequence. Proteins: Struct., Funct., Genet. 44, 119-122 (2001).

[0188] 6. Marsh, R. E., Corey, R. B. & Pauling, L. An Investigation of the Structure of Silk Fibroin. Biochim. Biophys. Acta 16, 1-34 (1955).

[0189] 7. Lefevre, T., Rousseau, M.-E. & Pezolet, M. Protein Secondary Structure and Orientation in Silk as Revealed by Raman Spectromicroscopy. Biophys. J. 92, 2885-2895 (2007).

[0190] 8. Cebe, P. et al. Beating the Heat - Fast Scanning Melts Silk Beta Sheet Crystals. Sci. Rep. 3, 741-7 (2013).

[0191] 9. Sidhu, M. S., Kumar, B. & Singh, K. P. The processing and heterostructuring of silk with light. Nat. Mater. 16, 938-945 (2017).

[0192] 10. Pauling, L. & Corey, R. B. Configurations of Polypeptide Chains with Favored Orientations Around Single Bonds: Two New Pleated Sheets. Proc. Natl. Acad. Sci. U.S.A. 37, 729-740 (1951).

[0193] 11. Cheng, G., Wang, X., Tao, S., Xia, J. & Xu, S. Differences in regenerated silk fibroin prepared with different solvent systems: From structures to conformational changes. Journal of Applied Polymer Science 132, n / a-n / a (2015).

[0194] 12. Um, I. C., Kweon, H., Park, Y. H. & Hudson, S. Structural characteristics and properties of the regenerated silk fibroin prepared from formic acid. Int. J. Biol. Macromol. 29, 91-97 (2001).

[0195] 13. Ha, S.-W., Tonelli, A. E. & Hudson, S. Structural studies of Bombyx mori silk fibroin during regeneration from solutions and wet fiber spinning. Biomacromolecules 6, 1722-1731 (2005).

[0196] 14. Trabbic, K. A. & Yager, P. Comparative structural characterization of naturally- and synthetically-spun fibers of Bombyx mori fibroin. Macromolecules 31, 462-471 (1998).

[0197] 15. Ha, S.-W., Park, Y. H. & Hudson, S. Dissolution of Bombyx mori silk fibroin in the calcium nitrate tetrahydrate-methanol system and aspects of wet spinning of fibroin solution. Biomacromolecules 4, 488-496 (2003).

[0198] 16. Rockwood, D. N. et al. Materials fabrication from Bombyx mori silk fibroin. Nat. Protoc. 6, 1612-1631 (2011).

[0199] 17. Perrone, G. S. et al. The use of silk-based devices for fracture fixation. Nat. Commun. 5, 1-9 (2014).

[0200] 18. Li, C. et al. Regenerated silk materials for functionalized silk orthopedic devices by mimicking natural processing. Biomaterials 110, 24-33 (2016).

[0201] 19. Yamaguchi, K. et al. Primary Structure of the Silk Fibroin Light Chain Determined by cDNA Sequencing and Peptide analysis. J. Mol. Biol. 210, 127-139 (1989).

[0202] 20. Wray, L. S. et al. Effect of processing on silk-based biomaterials: reproducibility and biocompatibility. J. Biomed. Mater. Res. Part B Appl. Biomater. 99, 89-101 (2011).

[0203] 21. Marelli, B. et al. Programming function into mechanical forms by directed assembly of silk bulk materials. Proc. Natl. Acad. Sci. USA 114, 451-456 (2017).

[0204] 22. Kluge, J. A., Kahn, B. T., Brown, J. E., Omenetto, F. G. & Kaplan, D. L. Optimizing molecular weight of lyophilized silk as a shelf-stable source material. ACS Biomater. Sci. Eng. 2, 595-605 (2016).

[0205] 23. Hu, X., Kaplan, D. & Cebe, P. Determining Beta-Sheet Crystallinity in Fibrous Proteins by Thermal Analysis and Infrared Spectroscopy. Macromolecules 39, 6161-6170 (2006).

[0206] 24. Lu, Q. et al. Silk Self-Assembly Mechanisms and Control From Thermodynamics to Kinetics. Biomacromolecules 13, 826-832 (2012).

[0207] 25. Koebley, S. R. et al. Silk Reconstitution Disrupts Fibroin Self-Assembly. Biomacromolecules 16, 2796-2804 (2015).

[0208] 26. Krywka, C., Krasnov, I., Figuli, R., Burghammer, M. & Mueller, M. Determination of silkworm silk fibroin compressibility using high hydrostatic pressure with in situ X-ray microdiffraction. Macromolecules 47, 7187-7193 (2014).

[0209] 27. Hu, X.; Kaplan, D.; Cebe, P. Thermochim. Acta 2007, 461 (1-2), 137-144.

[0210] 28. Hu, X.; Kaplan, D.; Cebe, P. Macromolecules 2008, 41, 3939-3948.

[0211] Equivalents and Scope. The recitation of a list of elements in any definition of a variable herein includes definition of that variable as any single element or combination (or subcombination) of the listed elements. The recitation of an embodiment herein includes that embodiment as any single embodiment or in combination with any other embodiment or portion thereof. Those skilled in the art will recognize, or be able to ascertain using no more than routine experimentation, many equivalents to the specific embodiments of the invention described herein. The scope of the invention is not intended to be limited to the above description, but is as set forth in the following claims. The present invention provides, for example, the following items. (Item 1) (i) providing a silk fibroin material comprising a substantially amorphous structure; (ii) applying at least one of elevated temperature and pressure to the silk fibroin material to form a silk fibroin article, wherein the applying step induces fusion between at least a portion of the silk fibroin and a structural change of fibroin in the silk fibroin material. (Item 2) (i) selecting an elevated temperature and pressure to produce a desired silk fibroin article of desired crystallinity and desired material properties; (ii) applying said elevated temperature and said elevated pressure to a silk fibroin material comprising a substantially amorphous structure to form a silk fibroin article, wherein said silk fibroin article has said desired crystallinity and said desired material properties. (Item 3) Item 3. The method of claim 2, wherein the applying step induces fusion between at least a portion of the silk fibroin and a structural change of fibroin in the silk fibroin material. (Item 4) 10. The method of claim 1, wherein the applying step includes applying both elevated temperature and pressure to the silk fibroin material. (Item 5) 10. The method of any one of the preceding items, wherein the silk fibroin article is substantially homogeneous. (Item 6) 10. The method of any one of the preceding items, wherein the silk fibroin article comprises silk in an amount of about 10% (w / w) or more. (Item 7) 10. The method of any one of the preceding items, wherein the applying step is or includes a heat press. (Item 8) 8. The method according to item 7, wherein the heat pressing is carried out at a pressure of at least 1 MPa. (Item 9) 9. The method according to item 7 or 8, wherein the heat pressing is carried out at a temperature between 25°C and 200°C. (Item 10) 10. The method of claim 1, further comprising selecting an amount of plasticizer to include in the silk fibroin material to produce a desired crystallinity and desired material properties, and including the plasticizer in the silk fibroin material in the selected amount, wherein the plasticizer is optionally water. (Item 11) 10. The method of any one of the preceding items, wherein the elevated temperature and pressure are applied simultaneously. (Item 12) 10. The method of any one of the preceding items, wherein high pressure is applied to the silk fibroin material followed by high temperature. (Item 13) 10. The method of any one of the preceding items, wherein the applying step is performed in a mold. (Item 14) 13. The method of any one of items 1 to 12, wherein the applying step is not performed in a mold. (Item 15) 10. The method of any one of the preceding items, further comprising processing the silk fibroin article. (Item 16) Item 16. The method of item 15, wherein the processing step is or includes at least one of machining, turning, rolling, thread rolling, drilling, milling, sanding, punching, die cutting, extruding, chemical etching, coating, molding, and any combination thereof. (Item 17) 10. The method of any one of the preceding items, wherein the silk fibroin article is or comprises a film, fiber, mesh, needle, tube, plate, screw, rod, or any desired shape. (Item 18) 2. The method of claim 1, wherein the applying step results in a structural change in silk fibroin, inducing a beta-sheet structure with an increase of at least 1% compared to the level of beta-sheet in the silk fibroin material before the applying step. (Item 19) 10. The method of any one of the preceding items, wherein the silk fibroin article is substantially transparent. (Item 20) 2. The method of any one of the preceding items, wherein the silk fibroin article has been subjected to at least one of macropatterning, micropatterning, and nanopatterning. (Item 21) 2. The method of any one of the preceding items, wherein the silk fibroin article comprises at least one additive. (Item 22) 22. The method of claim 21, wherein the silk fibroin material is mixed with at least one additive to form a composite silk fibroin article prior to the applying step. (Item 23) 23. The method according to item 21 or 22, wherein the additive is or comprises at least one of organic or inorganic molecules, organic polymers, inorganic polymers, biopolymers such as peptides and proteins; conductive materials, carbon-based materials, antibodies and antigen-binding fragments thereof; antigens; nucleic acids; nucleic acid analogs and derivatives; saccharides; immunogens; naturally occurring compounds and extracts from biological systems such as cells, bacteria, or tissues; synthetic materials; metallic materials; alloys; hydrophobic materials; hydrophilic materials; nanomaterials; and any combination thereof. (Item 24) 24. The method of claim 23, wherein the organic polymer is or comprises at least one enzyme, such as a protease. (Item 25) 25. The method of claim 24, wherein the activity of the enzyme is stabilized by silk under elevated temperature and pressure. (Item 26) 26. The method of item 24 or 25, wherein the protease is or comprises one or more of protease XIV, proteinase K, alpha-chymotrypsin, collagenase, matrix metalloproteinase-1 (MMP-1), and MMP-2. (Item 27) 24. The method according to item 23, wherein the conductive material is or comprises an inorganic conductive material (e.g., silver, gold, iron oxide), an organic conductive material (e.g., graphene), a metal, an alloy, a semiconductor material and / or a conjugated polymer. (Item 28) 28. The method according to any one of items 22 to 27, wherein the additive is mixed with the silk fibroin material in a proportion between 0.001% and 95.0% by weight. (Item 29) 2. The method of any one of the preceding items, wherein the silk fibroin article degrades by at least 1% by weight after being exposed to an aqueous environment at 37° C. for 30 days. (Item 30) 2. The method of any one of the preceding items, wherein the silk fibroin article is bioabsorbable. (Item 31) 10. The method of any one of the preceding items, wherein the silk fibroin article has thermoformable properties and can be reshaped into a desired shape at elevated temperature or pressure. (Item 32) 2. The method of claim 1, wherein the beta-sheet content of the silk fibroin material comprising a substantially amorphous structure is 60% or less, 50% or less, 40% or less, 30% or less, 20% or less, 10% or less, 5% or less, or 1% or less. (Item 33) 33. A silk fibroin article obtained by the method according to any one of items 1 to 32. (Item 34) 34. The article of claim 33, wherein the silk fibroin article is or comprises a packaging material. (Item 35) 35. The article of item 34, wherein the packaging material is suitable for use in the manufacture of electronic devices, drug delivery systems, patterning, molding, and any combination thereof. (Item 36) 10. A silk fibroin article made by a method comprising the steps of any one of the preceding items; wherein the silk fibroin material has been converted into a solid silk article that has undergone a silk conformational change, the solid silk article comprising at least some of the silk fibroin that have been fused together. (Item 37) A silk fibroin article comprising semi-crystalline silk fibroin, wherein the silk fibroin article has a glass transition temperature between about 40°C and 120°C. (Item 38) 38. The silk fibroin article of any one of items 33 to 37, wherein the silk fibroin article is substantially homogeneous. (Item 39) The flexural strength of the silk fibroin article is at least 5 MPa, and the substantially overall density of the silk fibroin article is at least 1.20 g / cm 3 39. The silk fibroin article according to any one of Items 33 to 38, wherein

Claims

[Claim 1] The invention described in this specification.

Images