Engineering Silk Materials: From Natural Spinning to Artificial Processing

Abstract

Silks spun by the arthropods are “ancient’ materials historically utilized for fabricating high-quality textiles. Silks are natural protein-based biomaterials with unique physical and biological properties, including particularly outstanding mechanical properties and biocompatibility. Current goals to produce artificially engineered silks to enable additional applications in biomedical engineering, consumer products, and device fields, have prompted considerable effort towards new silk processing methods using bio-inspired spinning and advanced biopolymer processing. These advances have redefined silk as a promising biomaterial past traditional textile applications and into tissue engineering, drug delivery, and biodegradable medical devices. In this review, we highlight recent progress in understanding natural silk spinning systems, as well as advanced technologies used for processing and engineering silk into a broad range of new functional materials.

I. Introduction

Silks are protein-based biopolymers spun by thousands of arthropod species, particularly silkworms and spiders, for various applications throughout their lifetime, including orb web construction for prey capture, housing for protection and reproduction. In general, silks possess extraordinary physical and biological properties including superior mechanical properties to biocompatibility, making them attractive materials.^1,2 Over thousands of years, silk fibers unraveled from cocoons spun by the silkworm Bombyx mori (B. mori) have been used on the commercial scale for fabricating soft and durable textiles for apparel and clinical sutures.

In recent decades, with an improved understanding of the physical properties of different silks from spiders to insects, spider silks have drawn considerable attention due to the superior mechanical properties compared to silks spun by other insects, for example, silkworms and the caddisfly.^3,4 Some of the silks provide an excellent combination of high tensile strength and extensibility and are able to absorb more energy per weight compared to synthesized industrial materials with high strength like Kevlar (Table 1). The dragline silk spun from spiders typically possesses a tensile strength >800 MPa and an astonishing toughness >110 MJ/m³.^5–7 These outstanding mechanical properties make spider silks one type of promising high-performance structural biomaterials for consideration in future applications. However, unlike silkworm silks, spiders cannot produce sufficient natural silk at a commercial scale that is required for most applications, although they were used by the ancient Greeks for wound treatments and by natives in New Guinea to make fishing nets and bags. To overcome the challenge of producing spider silk at a large scale, technologies are being developed to generate artificial spider silks with physical properties similar to natural silk by biomimicking the natural spinning process with silk-like recombinant proteins.^6,8

Table 1.

Mechanical properties of silk materials and other structural materials^5,7,13–16

Materials	Stiffness (GPs)	Strength (MPa)	Toughness (MJ/m3)
Spider dragline silk	3–14	140–1600	16–350
Bombyx mori silk	5–12	500–650	70–87
Collagen	0.0018–0.046	0.9–7.4	-
Elastin	0.001	2	2
Wool	0.5	200	60
High tensile steel	190–210	1650	6
Carbon fiber	300	4000	25
E-glass fiber	63–83	2200–3580	-
Nylon	1.8–5	430–950	80
Kevlar 49™	130	3600	50
Nylon	1.8–5	430–950	80
Poly (lactic acid)	0.35–3.5	21–60	1–3

Aside from using silks for textiles, since the late 1990s, considerable efforts have been made to extend the applications of silks into other fields such as biomedical engineering, tissue engineering, and biomedicine by taking advantages of the biocompatibility and enzymatically degradability of silk-based materials.^2,9–11 Towards these needs, silk-based materials, including natural and recombinant silks, have been fabricated into a variety of material forms, including films, sponges, scaffolds, fibers, tubes, and nano-/micro-particles for specific applications.^6,12 In this review, mechanisms of natural silk spinning and techniques developed to artificially transform silk into next-generation materials will be reviewed. Further, challenges and opportunities for future engineering of silk materials will be discussed.

II. Structure of natural silks

In nature, a large variety of silks with structures and properties are produced by different species of spiders and insects.^17–19 For example, silkworms primarily produce silk to construct cocoons for self-protection during metamorphosis. Female orb-weaving spiders can spin up to seven different types of silks that are specifically used for making webs, prey capture, and reproduction.¹⁷ Embioptera (Webspinners) produce nano-sized silk fibers for protection and breeding.²⁰ Trichoptera (Caddisfly) use silk to hunt and protect themselves in their aquatic environment.¹⁹ Surprisingly, in most structural silks across species, the molecular structure is conserved in high molecular weight amphiphilic sequences composed of highly repetitive amino acid motifs (Figure 1a).²¹ These repetitive motifs typically form into nanocrystalline β-sheet domains via strong and stable hydrogen bonding, while the nonrepetitive sequences form less structured regions, including helices, coils and turns. In this review, we primarily focus on cocoon silk from the silk moth B. mori and spider dragline silk from Nephila clavipes (N. clavipes) since the majority of silk-based studies are on these two types of silk. In general, cocoon silk and spider dragline silk are composed of hierarchical structures where semi-crystalline nanofibrils organize into fiber with typical diameters of 1–20 μm (Figure 1b). The molecular structure of cocoon silk protein (fibroin) consists of a heavy (H) chain of ~390 kDa and a light (L) chain of ~26 kDa, connected by a disulfide bond at the C-terminus of H-chain, forming a H-L complex.²² The highly repetitive GAGXGA (X=S, Y, V) sequence in the heavy chain makes up the bulk of the crystalline/semi-crystalline domains in the fibers. Spider dragline silk typically refers to major ampullate silk that most studies are carried out with towards fundamental understanding of the structure-property relationships as a guide to artificial production of synthetic spider silks using recombinant DNA technology.²³ The core constituents of spider major ampullate silk are two fibrous proteins called major ampullate spidroin 1 (MaSp1) and major ampullate spidroin 2 (MaSp2), each with a molecular weight of 250–350 kDa.²⁴ Both proteins contain large central, repetitive motifs of poly(Ala), poly(Gly-Ala), Gly-Gly-X or Gly-X-Gly (X=Gln, Leu, Tyr) where ploy(Ala) and ploy(Gly-Ala) form the β-sheet (dominated by antiparallel) structures organized into nanocrystallites with a approximate sizes of 2×5×7 nm.^4,25

Figure 1. Structures and hierarchy of silks.

(a) A schematic of silk protein: nonrepetitive N-terminus, repetitive region and non-repetitive C-terminus. (b) Structural illustrations of natural silk fibers: the raw silk is composed of silk fibrils with a combination of aligned nano-crystallites and less organized or amorphous domains in the structure. Reproduced with permission.³³ Copyright 2017, Wiley-VCH. Reproduced with permission.¹⁸ Copyright 2018, American Chemical Society.

A variety of techniques have been utilized to gain insight into molecular structures of silks, including X-ray diffraction (XRD), Fourier transform infrared spectroscopy (FTIR)/Raman spectroscopy, transmission electron microscopy (TEM), and NMR spectroscopy.^18,26–31 However, only a limited molecular-level picture has been obtained along with some remaining challenges such as the molecular structure of the amorphous regions in silk fibers and protein-protein interactions in the producing glands and the spun fibers. More effort is required to achieve complete understanding of silk structure-function relationships for providing an improved guide in the design and assembly of synthetic silks to recapitulate native properties where needed. Nevertheless, inspiration has driven a great deal of progress into silk-based biomaterials in recent biomedical research.³²

III. Natural Spinning of silks

Over millions of years, silkworms and spiders have developed sophisticated biological fiber spinning systems where silks are generally spun via a protein self-assembly process from highly concentrated protein solutions (known as ‘dope’) or gels into solid fibers. Specifically, the silk proteins are synthesized and stored in silk glands at high concentrations (~25–50 wt% for silkworms and spiders) (Figure 2a, 2b). Upon spinning, the dope is forced to flow through a specially shaped spinning duct and is subjected to a series of physiological changes such as shear, pH, metal gradient and salt gradients (Figure 2c).^34,35 During the process, the silk proteins undergo phase transitions along with self-assembly to form solid, semi-crystalline, insoluble fibers. Among the factors involved, pH gradients and shear forces are key in influencing the molecular structures and dynamics of the silk proteins. The flanking, non-repetitive domains (N- and C-termini) of the silk proteins are sensitive to pH (Figure 2d). Thus, for B. mori silk fibroin, the N-terminal domain undergoes a pH-sensitive conformational transition from random coil to well-ordered β-sheets at around pH 6.0, forming micelle-like oligomers.³⁶ For spider silk protein, the N- and C-termini generally possess well-defined structures with five helices.³⁷ In the gland, the C-terminal domains form into parallel-oriented dimers via a stable disulfide bond and electrostatic interactions.³⁸ Such dimers further form into micelle-like structures where hydrophobic repetitive domains are hidden within the cores. These micelle-like structures are stable under physiological conditions in the gland.³⁹ In contrast, the N-terminal domain is monomeric at physiological conditions in the gland and forms an antiparallel dimer when the pH level decreases in the duct that connects the gland to the spinneret.^37,40,41 Site-specific mutations revealed that the N-terminus prevents premature aggregation of the protein and enables rapid fiber formation.⁴²

Figure 2. Silk processing and assembly in fibers.

(a) Photograph of a B. mori silk gland with anterior silk gland (ASG), Funnel, middle silk gland (MSG) and posterior silk gland (PSG).^35,45 (b) Photograph of a N. clavipes spider major ampullate gland with sac region, tail, funnel, and duct.^35,46 Reproduced under the terms of the CC-BY Creative Commons Attribution 4.0 International Public License.^35,46 Reproduced with permission. ⁴⁵ Copyright 2015, Elsevier Ltd. (c) pH change inside gland in the natural spinning process for silkworms and spiders.⁴⁷ (d) Thermodynamic stabilities of N-terminus (NT) and C-terminus (CT) as a function of pH, measured as urea concentrations for half-denaturation.⁴⁷ Reproduced with permission.⁴⁷ Copyright 2015, Springer Nature. (e) Mechanism of the natural spinning to produce solid silk fibers from soluble protein dope.

Shear force also plays a crucial role in the self-assembly of silk proteins during fiber formation. Rheological studies of the flow characteristics of native silk dopes showed that a higher shear rate (>5 s⁻¹) led to instability of the proteins in silk dopes for both the silkworm and spider, leading to the formation of insoluble solid silk (Figure 2e).^43,44 Further analysis indicated that both native silk dopes responded to shear force like a typical molten polymer, which inspires the development of artificial spinning systems as mimics of the natural process.

IV. Artificial Processing of Silk

A. Bio-inspired Spinning

Inspired by the natural biological spinning process, different approaches have been developed to artificially spin silk fibers with the goal to generate the molecular and hierarchical structures found in natural spider and silkworm silks.⁴⁸ In general, artificial spinning systems can be classified into two categories: wet spinning and dry spinning systems. In wet spinning, the silk protein solution is extruded through a spinneret directly into a coagulation bath that initiates solidification into fibers via precipitation. In contrast, dry spinning solidification of the fiber occurs due to the evaporation of a volatile solvent. Three common types of spinning dope have been used in bio-inspired spinning including native liquid silk isolated from silkworm or spider glands, regenerated silk solution, and recombinant silk solution. Regenerated silk is generally referring to silk protein extracted from natural silk fibers. For instance, regenerated B. mori silk is typically obtained following a three-step process: fiber degumming by boiling the cocoons in basic solution; fiber dissolution by using denaturing agents (LiBr, HFIP, CaCl₂, Ca(NO₃)₂); solution purification by dialysis and centrifugation. Recombinant silk refers to the silk-inspired protein synthesized using recombinant DNA techniques. Several types of silk protein motifs or silk-inspired proteins have been expressed successfully by genetically modified organisms such as bacteria,^49,50 yeast,⁵¹ insect cells,⁵² plant cells,⁵³ and mammalian cells.⁵⁴ However, compared to natural silk proteins, the recombinant silk proteins are generally lower in molecular weight (<300 kDa) due to the challenges in replicating the full sequence of natural silk genes in a heterologous host system.^23,55

Various artificial spinning methods have been invented with an emphasis on mimicking the natural spinning system.^47,48 Some studies have shown that the artificially generated silks can exceed natural silk in terms of some of the mechanical properties.^56,57 Table 2 summarizes the properties of some artificial fibers produced with various spinning dopes and methods. The as-spun fibers normally show weak mechanical properties and require post-spinning treatments, including stretching and alcohol treatment to enhance the mechanical properties. Recently, a sophisticated artificial spinning system was developed by combining shear force and pH gradients (Figure 3).⁵⁸ Escherichia coli was used to express a water-soluble silk-like protein containing domains from the silks of two species of spider, Euprosthenops australis and Araneus ventricosus. The concentrated recombinant silk protein solutions were pumped through a glass capillary into an acidic bath, mimicking the conditions experienced by natural silk as it passes through a spider silk gland and ducts. The recombinant silk protein was solidified into fibers via self-assembly with a diameter of 10–20 micrometers and showed some physical properties comparable to natural silk fibers, but with lower toughness.

Table 2.

Summary of some best performing artificial fibers produced with various silk dopes and spinning methods

Spinning dope	Spinning type	Strength (MPa)	Stiffness (GPa)	Toughness (MJ/m3)	Reference
Regenerated B. mori silk 13% (w/v%)/95% formic acid	Wet	1077 ± 173	39.9 ± 6.1	257.8 (b)	63
Regenerated B. mori silk 13% (w/v%)/TFA	Wet	959 ± 149	43.2	156.7 (b)	63
Regenerated B. mori silk 15% (w/v%)/water	Wet	450 ± 20	12.5	100.6 ± 6.3	64
Regenerated B. mori silk 16% (w/v%)/water	Wet	390 ± 50	15.2 ± 3.3	109.1 ± 18.8	65
Regenerated B. mori silk 15% (w/v%)/CaCl2-water	Wet	314 ± 19	10.4	105.3 ± 10	66
Regenerated B. mori silk 12% (w/v%)/CaCl2-formic acid	Wet	470.4 ± 53.5	6.9 ± 2.1	105.3 ± 15.5	67
Regenerated B. mori silk 15% (w/v%)/water	Wet	450 ± 30	18.9 ± 1.1	91.0 ± 7.4	68
Regenerated B. mori silk 20% (w/v%)/water	Dry	301.5 ± 70.6	6.2 ± 1.7	104.8 ± 37.8	69
Regenerated B. mori silk 50% (w/v%)/CaCl2-water	Dry	614	19	136.4	60
Regenerated B. mori silk 5% (w/v%)/HFIP	Dry	109 ± 34	8 ± 1	13.9 ± 9.2	59
Regenerated N. clavipes dragline silk 2.5% (w/v%)/HFIP	Wet	320	8.0	-	70
60 kDa A. diadematus ADF3 10–28% (w/v%)/water	wet	269.6	13.2	101.4	54
284.9 kDa, Nephila clavipes MaSp1 20% (w/v%)/HFIP	Wet	508 ± 108	21 ± 4	-	56
70kDa, Nephila clavipes MaSp1 30% (w/v%)/HFIP	Wet	132.5 ± 49.2	5.7 ± 2.4	23.7 ± 18.5	71
66kDa, Nephila clavipes Flagelliform 15% (w/v%)/HFIP	Wet	150.6 ± 31.3	4	89.1 ± 23.9	72
366kDa, Nephila clavipes TuSp1/MiSp1 8–10% (w/v%)/HFIP	Wet	308 ± 57	9.3 ± 3.0	-	73
65kDa Nephila clavipes MaSp1/MaSp2 30% (w/v%)/HFIP	Wet	221.7 ± 11.0	-	102.5 ± 13.6	74
286 kDa, A. diadematus eADF3 20% (w/v%)/water	Wet	370 ± 59	4 ± 1	189 ± 33	75
47kDa, Nephila clavipes MaSp1 20% (w/v%)/NaCl-water	Wet	286 ± 138	8.4 ± 4.3	37.7 ± 28.8	76
33kDa, E. australis (MaSp1)/A. ventricosus (MiSp) 50% (w/v%)/aqueous buffer (pH 8)	Wet	162 ± 8	6 ± 0.8	45 ± 7	77
556 kDa, Nephila clavipes MsSp1 17% (w/v%)/HFIP	Wet	525 ± 83	7.8 ± 1.3	91 ± 30	57

TFA: trifluoroacetic acid; HFIP: hexafluoro-2-propanol; ADF3: Araneus diadematus Fibroin-3; eADF3: engineered Araneus diadematus Fibroin-3; TuSp1: tubuliform spidroin 1; MaSp1: Major ampullate spidroin 1; MiSp1: Minor ampullate spidroin 1.

Figure 3. Artificial systems to spin silk into fibers.

(a) Bio-inspired artificial spinning system for producing fibers rom recombinant spider silks. (b) Photograph of a fiber as spun into a low-pH aqueous bath. (c) Photograph of wet fiber nest in low-pH buffer. (d) Photograph of as-spun fibers on a frame. (e, f) SEM image of as-spun fibers, scale bars are 10 μm in (e) and 2 μm in (f). (g) Fraction of recombinant protein NT2RepCT (a mini-spidroin composed of an N-terminal domain from E. australis MaSp1 and a C-terminal domain from A. ventricosus MiSp bracketing a short repetitive region from E. australis) dimer over time at pH 7.5 (open symbols) and pH 5.5 (filled symbols). (h) Stress-strain curves for eight separate as-spun fibers. (i) FTIR spectra of NT2RepCT at 10 mg/ml (blue dashed line), NT2RepCT fibers (red continuous line), and native N. inaurata dragline silk (black line and dots).⁵⁸ Reproduced with permission.⁵⁸ Copyright 2017, Springer Nature.

A dry spinning method was developed to directly produce high performance regenerated silk fibers and involved spinning nematic silk microfibril solutions into fibers with direct extrusion into air (Figure 4a, 4b, 4c).⁵⁹ The nematic microfibril solution was prepared by partially dissolving native silkworm silk fibers into microfibrils, where the hierarchical structures in natural silk fibers were maintained in solution. The as spun regenerated silk fibers showed a maximum modulus of 11±4 GPa, higher than some natural spider silk.⁵ In addition, a microfluidic-based system was applied to mimic the natural spinning system (Figure 4d). The regenerated silk fibers were dry-spun from regenerated silk fibroin aqueous solution and showed higher toughness than degummed natural silk fibers, with a tensile strength of 614 MPa, a strain of 27% and a toughness of 101 kJ/kg (Figure 4e, 4f).^60,61

Figure 4. Microfluidic spinning of silk into fibers.

(a) Illustration of a bioinspired dry spinning process where the nematic silk microfibril solution was directly assembled into regenerated silk fibers without additional treatment. (b) Photograph of facile bioinspired spinning process. (c) High-resolution SEM images of a cross-section of RSF, scale bar is 20 μm (2 μm for the inset image).⁵⁹ (a-c) Reproduced under the terms of the CC-BY Creative Commons Attribution 4.0 International Public License.⁵⁹ (d) A microfluidic-based dry spinning system for producing regenerated silk fibers. (e, f) SEM images of the cross section (e) and the surface (f) of the spun fibers, scale bar is 10 μm.⁶² Reproduced with permission.⁶² Copyright 2014, Elsevier Ltd.

B. Electrospinning

Electrospinning has also been applied to produce artificial silk fibers with a wide range of diameters, from the nanoscale to microscale. The electrospun silk fibers h can used to construct biomimic scaffolds based on the diameters and overall morphology.^78–82 To fabricate the electrospun silk fibers, organic solvent-based and aqueous-based systems have been reported,^83–87 including an all aqueous solution at low silk concentration (<10%, w/v%).⁸⁸ The as-spun fibers showed good biocompatibility. While electrospun silk fibers possess structures and morphologies to mimic natural silk fibers⁸⁹, their mechanical properties are generally weak,^88,90 which limits applications for higher performance structural materials.

C. Multidimensional assembly

In nature, silk is primarily produced in fiber form, followed by further construction into multidimensional form such as orb webs and cocoons. In the previous section, artificial methods for spinning silk fibers were reviewed; here the focus is on techniques developed for generating multidimensional (2D, 3D, and 4D) silk-based materials. Most multidimensional silk-based materials are generated via self-assembly. For example, silk films are typically prepared by casting silk solution into a premade mold/surface with the subsequent slow evaporation of solvent (typically water). During the drying process, silk fibroin undergoes self-assembly where the final physical properties and molecular structures can be controlled by solvent, humidity and temperature, combined with the rate of drying.^91–93 Silk films prepared from aqueous silk solutions and cured at lower humidity (<75% RH) and ambient temperatures exhibit good water solubility, useful for fabricating silk-based bioresorbable devices (Figure 5a–d).^94,95 The silk film holds and transfers prefabricated electrical circuits to the biological system.⁹⁵ Further, by addition of salt and plasticizer, along with calcium chloride and glycerol, highly flexible silk films can be obtained.^96–98 The plasticization of silk into skin-like softness was achieved by the addition of Ca²⁺ and the subsequent hydration via ambient humidity (Figure 6).⁹⁹ The gold-based thin conductive layers on silk films form wrinkled structures during plasticization by ambient humidity, resulting in highly conductive and stretchable electrodes.⁹⁹ Additionally, to tune the mechanical properties and molecular structures, a variety of post-treatment methods were utilized, including water-annealing, methanol, and stretchingd.^91,100 In general, these treatments result in the formation of β-sheet structures (with variations in sizes and distribution depending on the treatment), which serve as the building blocks to form crystalline domains in the film.

Figure 5. Electronic devices generated with silk materials.

(a) Photograph and illustration of a silk-based transient electronics; transistors, diodes, inductors, capacitors, and resistors, with interconnects and interlayer dielectrics, on a thin silk substrate. (b) Photographs showing the time sequence of device dissolution in DI water. (c) Photograph of an implanted (left) and sutured (right) demonstration platform for transient electronics located in the subdermal dorsal region of a BALB/c mouse. (d) Photograph (left) and histological image (right) of tissue at the implant site excised after 3 weeks, showing a partially resorbed region of the silk film. (a-d) Reproduced with permission. ⁹⁵ Copyright 2012, American Association for the Advancement of Science. (e) Schematic representation of an on-skin electrode based on plasticized silk protein. (f) Photograph of plasticized silk electrodes conformably attached on the skin skin. (g, h) Cross-section SEM images of plasticized silk electrodes. (i) Photograph of an electromyography (EMG) measurement using plasticized silk electrodes laminated on a human forearm. (j) On-skin interfacial impedance of the plasticized silk electrodes in comparison with commercial gel electrodes. (k) EMG signals obtained by the plasticized silk electrodes and commercial gel electrodes. Two-direction arrows describe when motions happened.⁹⁹ (e-k) Reproduced with permission.⁹² Copyright 2018, Wiley-VCH.

Figure 6. Silk hydrogel-based materials.

(a) Schematic of fabrication of silk hydrogel with robust mechanical properties. (b) Compressive stress-strain curves of high strength silk hydrogels. (c) Photograph of various 3D structures made from high strength silk hydrogels. (d) Photograph of a 1.5 mm thick silk hydrogel tube. (e) The auxetic deformation of a reentrant honeycomb silk hydrogel fabricated by laser cutting. Top image shows he hydrogel before stretching and bottom image shows the hydrogel after stretching.¹⁰⁸ Reproduced with permission.¹⁰⁸ Copyright 2018, Wiley-VCH.

Hydrogels are another important form of biomaterials with appealing applications in tissue engineering.^101–104 Given the biocompatibility, biodegradability, and excellent mechanical properties of silk fibroin, processing silk into hydrogels has been explored.^105–108 Typical methods to obtain silk hydrogels are via sol-gel transition, where molecular network forms by controlling the formation of β-sheet structures as crosslinks for silk. A series of environmental factors including pH, salts, temperature, and solvents have been utilized towards this goal.¹⁰¹ Generally, a lower pH, higher salt concentration, and a higher incubation temperature facilitate increased kinetics towards the sol-gel transition.¹⁰¹ Recently, a robust silk-based hydrogel was generated via a combination of sol-gel transition and solvent-exchange (Figure 6).¹⁰⁸ In this process, the silk fibroin transitioned from random coils and/or helical structures to β-sheets, which served as the physical crosslinks. The hydrogels possessed combined high strength and toughness for machining, such as by laser cutting and mechanical turning.¹⁰⁸ Furthermore, silk hydrogels have been explored for various applications such as biocompatible scaffolds in cartilage regeneration and as biodegradable microfluidic devices.¹⁰⁹

Different types of silk-based micro-/nano-materials have also been developed with silk with promising applications from drug delivery to filtration to tissue engineering.^110–112 Microfluidics-based strategies were developed to spin liquid native silk, obtained directly from the silk gland of B. mori silkworms, into micron-scale capsules with controllable geometry and variable levels of intermolecular β-sheet content in their protein shells (Figure 7).¹¹³ Such microparticles enabled the encapsulation, storage and release of active biomolecules such as functional antibodies.¹¹³ Silk microfibers were fabricated by alkali hydrolysis of natural silk fibers, where alkali (sodium hydroxide) initiated hydrolysis of amide bonds and resulted in the chopping of the natural silk fibers into microfibers (Figure 8a–e).¹¹² The silk microfibers obtained in this process were used as reinforcements in fabricating 3D silk-based scaffolds with a combination of tunable mechanical properties, surface roughness, and porosity. Biological studies showed that the scaffolds supported human bone marrow-derived mesenchymal stem cell differentiation towards bone-like tissues in vitro and possessed minimal immunomodulatory responses in vivo, indicating potential utility as biomaterials for tissue engineering bone.¹¹²

Figure 7. Silk microdroplet formation.

(a) Schematic representation of the microfluidic-based processing of natural silk fibroin into microparticles. (b) Optical microscopy images of the silk microparticles formed at a single T-junction in the microfluidic device. Lower panels show micrographs of silk microparticles: (i) sphere, (ii) cylinder, (iii) short fiber, (iv) thin fiber, (v) thick fiber. Scale bar, 20 mm. (c) Release kinetics for C4scFv from different silk microparticles shapes.¹¹³ Reproduced under the terms of the CC-BY Creative Commons Attribution 4.0 International License.¹¹³

Figure 8. Processes to generate silk microfibers and nanoparticles.

(a) Schematic representation of processing raw silk fibers into silk microfibers. (b) SEM images of degummed silk fibers and molecular arrangements in the silk. (c-e) Silk microfibers with various lengths, scale bar is 400 μm. (f) Illustrations of different methods for generating silk nanoparticles. (g, h) SEM images of silk nanoparticles.^112,119 Reproduced with permission. ¹¹² Copyright 2012, National Academy of Sciences. Reproduced with permission. ¹¹⁹ Copyright 2016, American Chemical Society.

Silk nanoparticles are an interesting forms with potential in drug delivery.^110,114,115 These particles can be fabricated using a variety of methods, exploiting silk self-assembly.¹¹⁴ The most direct way to process silk into nanomaterials is physical milling, based on chopping and grinding natural silk fibers into nanoscale formats. However, this approach normally requires a long time frame and generally results in random nanoparticle aggregates with a broad size distribution.^116,117 An alternative process to generate silk nanoparticles is via wet chemistry using regenerated silk solution (Figure 8f). The self-assembly of silk in this process is controlled by monitoring environmental factors including pH, salt concentration, and solvent composition.^110,115 For example, in the presence of solvent such as methanol, ethanol, and dimethyl sulfoxide (DMSO), silk fibroin undergoes structural changes and phase separation, forming nanoparticles.¹¹⁰ In addition, salting out represents another approach for the production of silk nanoparticles, where the nanoparticles are formed due to hydrophobic interactions between silk protein chains and the decreased in water molecules in the high concentration salt bath.¹¹⁰ An intriguing strategy for preparing silk nanoparticles directly during the dissolution step (Figure 8f)¹¹⁵ utilizes a formic acid (FA)/lithium bromide (LiBr) dissolving system with optimized ratio to control the degree of dissolution degree of silk degummed fibers. After the usual dialysis and centrifugation processes that are used to prepare silk solutions, silk nanoparticles were formed directly and showed spherical shapes, no aggregation and dimensions of about 100–200 nm.¹¹⁵ In addition, silk-based ultrathin filtration membranes were formed with silk nanofibers generated directly from exfoliated natural silk fibers.^111,118 The membranes possessed a narrow distribution of pore sizes, ranging from 8 to 12 nm, a very high pure water flux, and high separation efficiency for dyes, proteins and colloids of nanoparticles (Figure 9).¹¹¹

Figure 9. Filtration membranes from silk nanofibers.

(a) Schematic of the process of producing silk nanofibers (SNF) and fabricating ultrathin SNF membranes. (b) Photograph of a free-standing SNF membrane with a thickness about 520 nm under visual light. (c) Pictures before and after filtering Rhodamine B aqueous solutions using SNF membranes with different thicknesses. The top and bottom images are under visual and UV light, respectively. (d-f) SEM images of SNF membranes with a thickness of 520 nm: (e) top view and (f) cross-sectional image. (g) Pore size distribution of the SNF membranes with thicknesses of 40, 60, and 120 nm.¹¹¹ Reproduced with permission.¹¹¹ Copyright 2016, American Chemical Society.

By controlled self-assembly, silk fibroin can form 3D bulk materials (silk monoliths), which can be further machined into predesigned shapes to fit various biomedical applications, such as orthopedic fixation devices.^120,121 Figure 10 illustrates the processing of silk fibroin into 3D bulk materials via different routes; controlled evaporation of water from aqueous silk solution results in a sol-gel-solid transition and solidification.¹²² An alternative route is to dissolve silk in organic solvent, such as HFIP, followed by long-term methanol treatment to induce β-sheet structures.¹²⁰ After removing the solvent by thorough drying, silk bulk materials were obtained. Recently, a heat compression-based method was developed to transform amorphous silk directly into compact bulk materials without added solvent.¹²³ This method is advantageous over traditional solution self-assembly methods based on the shortened time and improved efficiency, along with the flexibility of fabricating composite materials. In addition, the exploration of processing solid silk materials provides an important path to engineer silks with tools developed for commercial polymer processing; thus, a significant step towards industrial production of silk-based bulk materials.

Figure 10. Formation of bulk silk materials.

(a) Illustration of processing silk fibroin into bulk material via self-assembly. (b) Photograph of fabricated 3D silk constructs using the sol-gel-solid transition method. (c) Photograph of silk plate made by HFIP-based method. (d) Photograph of silk plate made by heat compression method. (e) Photograph of machined silk screws.^{121,123,124,125} Scale bars are 1 cm and 5 mm in (d) and (e) respectively. Reproduced with permission. ¹²¹ Copyright 2018, Wiley-VCH. Reproduced with permission.¹²³ Copyright 2019, Springer Nature. Reproduced with permission.¹²⁴ Copyright 2017, National Academy of Sciences. Reproduced with permission.¹²⁵ Copyright 2016, Elsevier Ltd.

To achieve high-performance biomaterials with desirable functions, artificial approaches inspired from natural material engineering processes to construct hierarchical structures is an intriguing strategy. Recently, a bio-inspired process was developed to generate hierarchically defined structures with multiscale morphology by using regenerated silk fibroin (Figure 11).¹²⁶ The combination of protein self-assembly and microscale mechanical constraints was used to form oriented, porous nanofibrillar networks within predesigned macroscopic structures. This approach provided a path to fabricate macroscale material geometries including anchors, cables, lattices, and webs with predefined mechanical and physical properties.¹²⁶

Figure 11. Hierarchical structures from silk.

(a) Schematic of the directed assembly of silk fibroin: aqueous silk solution premixed with crosslinkers (hydrogen peroxide and horseradish peroxidase) was infiltrated and gelled in predesigned PDMS molds. Mechanical tension was introduced by either contraction of the gel in mixtures of ethanol and water, or direct deformation of the elastomeric substrate. The resulting structures possessed tension-engineered nano-, micro- and macrostructures. (b) Birefringence and the corresponding internal nanofibrillar morphology of a ring-anchored fiber. (c) Photograph of structural web formed from 5% wt/v silk fibroin solution. (d) Birefringence of the web structure. (e) Images of a web taken between two polarizer films. (f, g) Six-anchor nanofibrillar web (~2.5 mg) supporting an 11 g point load. All scale bars are 1 mm.¹²⁶ Reproduced with permission.¹²⁶ Copyright 2017, Springer Nature.

D. Light-assisted processing

Femtosecond lasers have attracted attention for the precise, non-thermal processing of materials with control over structural damage.^127–129 Recently, this optical technique was been applied to processing silk hydrogels at ambient conditions with an emphasis on non-invasive shaping and heterostructuring of silk.¹³⁰ Based on the nonlinear multiphoton interactions of silk with a few-cycle femtosecond pulses, two approaches were proposed for optically processing silk: plasma-assisted ablation with higher laser intensity and photon-induced bulging with lower laser intensity (Figure 12). Plasma-assisted ablation facilitates non-invasive shaping of silk including localized nanocuting and micropatterning. Photon-induced bulging allows microwelding of silk with materials such as metal, glass and Kevlar, and with strength comparable to pristine silk.¹³⁰ Molecular structural analysis revealed that the polypeptide backbone remained intact while the s weak hydrogen bonds were disrupted. Using this approach, silk-based functional topological microstructures, such as Mobiüs strips, chiral helices and silk-based sensors were fabricated.¹³⁰

Figure 12. Optical processing of silk materials.

(a) Experimental set up of processing silk with femtosecond laser pulse: femtosecond pulses (7 fs, 800 nm, 2.2 nJ at 85 MHz repetition rate) were focused through a chirp-mirror-based dispersion-compensated objective with a numerical aperture of 0.2. S, shutter; ND, neutral density filter; CM, chirp mirror; M, Ag mirror; DM, dichroic mirror. (b) Periodic grooving on silk fibers with widths of 300nm (top) and 470nm (bottom) at 1 μm intervals. (c) SEM images of silk welded with silk, Kevlar, Cu and glass. (d) A silk-based trampoline force sensor: two identical (3 μm) silk threads were cross-welded on a substrate and a 1 mm² mirror was welded at the center. (e) Photograph of a contact lens suspended from a point welded silk fiber. (f) SEM images of welded silk on a PDMS surface.¹³⁰ Reproduced with permission.¹³⁰ Copyright 2017, Springer Nature.

E. Biomicrofabrication of silk

Inspired by advanced micro/nano-manufacturing technologies in the semiconductor industry, several multiscale manufacturing methods has been used to engineer silk-based materials, including photolithography,^131–135 soft lithography,^134–137 nanoimprinting lithography,^138–141 and scanning probe lithography¹⁴² covering 2D to 3D and nanoscale to macroscale systems. A combined electron-beam lithography (EBL) and ion-beam lithography (IBL) system was used to generate complex and arbitrary 3D silk-based microstructures (Figure 13).¹³³ Briefly, the IBL constructs the designed 3D structures from top to bottom, since the ions cross-link the silk protein while the EBL generates the structures from bottom to top since the electrons can penetrate and crosslink the silk protein. In addition, soft lithography techniques were applied to generate silk-based micro/nano-patterned structures, including 3D silk-based photonic crystals or silk inverse opals (SIOs) as biocompatible optical devices.¹⁴³ Generally, the SIOs were fabricated by infiltrating silk solution into a pre-templated 3D poly(methyl methacrylate) (PMMA)/polystyrene (PS) sphere array. Furthermore, the structure of SIOs could be tailored by water vapor annealing or exposure to ultraviolet (UV) radiation exposure to obtain structural color-coded patterns (Figure 14).^136,137

Figure 13. Silk nanostructures.

(a) Illustration of construction of 3D nanostructures with ion beam lithography (IBL) and electron beam lithography (EBL). (b) SEM images of 3D fabricated nanodesks.¹³³ Reproduced with permission.¹³³

Figure 14. Optical devices fabricated with silk.

(a) Schematic of free-standing silk film formation with patterned inverse opals. The SEM images show the green (left) and blue (right) silk opals. Scale bars are 500 nm.¹³⁶ (b) A 50 μm thick bent silk inverse opal (SIO) film showing different structural colors in different parts of the structure. (c) The SIO contracts uniformly anisotropically with water vapor (WV) treatment and non-uniformly anisotropically with UV irradiation. Left shows a floral pattern on the SIO by selectively exposing part of the SIO array to water vapor for different times. Right shows a butterfly pattern on SIO by exposing masked SIO to UV for different times. Reproduced with permission.¹³⁶ Copyright 2012, Springer Nature. Reproduced with permission.¹³⁷ Copyright 2017, Wiley-VCH.

F. 3D Bioprinting

Integrating advanced 3D printing technology with silk processing offers an enabling approach to generate different material platforms,^144,145 such as tissue engineering scaffolds and smart devices.¹⁴⁶ Silk-based “bioinks” can be either pure silk solutions or functional silk solutions tailored by chemical modifications, by doping or by mixing with functional components.^147–158 Direct-writing of concentrated regenerated silk fibroin (28–30 wt%) into a methanol-rich bath results in 3D, micro-periodic scaffolds.¹⁴⁷ The printed single filament is at diameter as small as 5 μm. The printed scaffold supports the adhesion and growth of human bone marrow-derived mesenchymal stem cells (hMSCs). In addition, the scaffold increases the production of glycosaminoglycan and the chondrogenic differentiation. This method has been further extended by integrating hydroxyapatite (HAP) into the silk for bone regeneration.¹⁴⁸ By incorporating functional materials/dopants such as polyol,¹⁴⁹ synthetic nanoclay,¹⁵⁰ gelatin,^151–153 PEG,¹⁵⁴ glycerol,¹⁵² and Konjac gum¹⁵⁵ into silk solution can further create a variety types of silk-based bioinks which allows free-standing bioprinting, and structural enhancement. Dopants can also include cells for the direct construction of 3D structures (Figure 15).¹⁵⁴ Silk/polyethylene glycol (PEG) bioink containing human bone marrow mesenchymal stem cells (hMSCs) was used to print a variety of tissue constructs with high resolution and homogeneity.¹⁵⁴ The cell-loaded constructs maintained their shape over at least 12 weeks in culture. Further, a higher concentration silk solution (10 wt%) facilitated cell growth, suggesting that these silk/PEG gels may provide suitable scaffold environments for cell printing.

Figure 15. 3D printing with silk.

(a) Schematic representation of 3D printing process: (i) Preparation of 3D printing bioink; (ii) Imaging and digital design; (iii) 3D bioprinting process. (b) Photograph of 3D printed construct loaded with hMSCs and fluorescence microscopy image after culturing for 2 days and after live/dead assay staining. (c) Fluorescence microscopy image of the construct after culturing for 2 days and after live/dead assay staining. (d) fluorescence microscopy image of the construct after culturing for 12 weeks and after live/dead assay staining.¹⁵⁴ Reproduced with permission.¹⁵⁴ Copyright 2018, Wiley-VCH.

Besides inject printing, light-based 3D printing technology has also been applied to construct silk-based 3D structures.^{133,156–158} Recently, a silk-based bioink was developed by functionalizing silk using glycidyl methacrylate (GMA), which was used to build complex organ structures, including the heart, vessel, brain, trachea and ear with the assistance of digital light processing (DLP) (Figure 16).¹⁵⁸ The printed structures showed excellent structural stability and cytocompatibility.

Figure 16. 3D printing with silk using digital light processing.

(a) Schematic representation for methacrylation of silk fibroin. (b) Schematic illustration of digital light processing (DLP) bioprinting procedure using methacrylated silk fibroin (Sil-MA). (c) Ear and brain mimicked shape: (left) CAD images depicting the ear and brain and (right) printed images. Printed products were not damaged when they were compressed by fingers tightly and they were back to their original shape when relaxed his fingers. (d) Cytocompatibility of methacrylated silk fibroin: (left) a Live and dead assay, cell viability of encapsulated NIH/3T3 for 14 days (live cells in green and dead cells in red). Scale bar indicates 500 μm; (right) CCK-8 assay, cell proliferation in the hydrogel for 14 days. The 30% Sil-MA hydrogel showed similar support of cell proliferation to t commercial GelMA (10%).¹⁵⁸ Reproduced with permission.¹⁵⁸ Copyright 2018, Springer Nature.

V. Perspective

Silk, a widely applied material for consumer use in textile applications for centuries has been reinvented and redefined in recent years as a versatile biomaterial for many biomedical applications and other utilities. With advanced spectroscopy and biotechnology, a lot of intriguing physical and biological properties of silks have been revealed, particularly, the outstanding mechanical properties and biocompatibility of the protein in material formats. These appealing properties have attracted interest from many fields including chemistry, physics and bioengineering. In general, two primary focuses have been established with the same goal of exploring broad applications of silk from high performance structural biomaterials to biomedical applications. One is to develop technologies to generate artificial fibers superior to the natural silk fibers. Based on the fundamental understanding of composition, molecular structure and self-assembly in natural silks, silk-like proteins (recombinant silk proteins) have been generated, particular spider silk-mimetic proteins and these have been used as spinning dopes to form artificial fibers. However, due to the complex physiology involved in the natural silk spinning process, full-mimic artificial spinning is difficult to achieve. More effort is needed to better understand the details of the natural process and then to optimize artificial spinning system. The other focus is to develop technologies to process silk into functional materials that can be used in biomedical applications, such as tissue engineering and implantable medical devices. In recent years, silk has been demonstrated as a versatile biomaterial that can be engineered using a variety of technologies, such as film casting, gelation, lithography, and 3D printing (Figure 17). However, most of these processing techniques rely on silk fibroin solution to generate the materials, which can be time and solvent intensive. The recent discovery of silk processing with classical polymer processing techniques such as thermal molding, promises to boost future industrialization of silk processing. Recent studies showed that regenerated amorphous silk fibroin can be directly molded into predesigned shapes or parts with heat compression, which provides a new path for engineering silk materials as is performed with synthetic polymers.

Figure 17.

Summary of the advanced technologies developed for processing silk into various forms of materials. Dry spinning: Reproduced under the terms of the CC-BY Creative Commons Attribution 4.0 International Public License.⁵⁹ Wet spinning: Reproduced with permission.⁵⁸ Copyright 2017, Springer Nature Electrospinning: Reproduced with permission.⁹⁰ Copyright 2004, American Chemical Society. Solidification: Reproduced with permission.¹²⁴ Copyright 2017, National Academy of Sciences. Inject printing: Reproduced with permission.¹⁵⁴ Copyright 2018, Wiley-VCH. Laser welding: Reproduced with permission.¹³⁰ Copyright 2017, Springer Nature. Filtration: Reproduced with permission.¹¹¹ Copyright 2016, American Chemical Society. Lithography: Reproduced with permission.¹³³ Copyright 2018, Wiley-VCH.