Tough Silk Research Articles

Superstrong and tough silk fibers cross-linked with functionalized graphene

Natural silkworm silks with an intriguing integration of good strength, modulus, toughness and biocompatibility, have been widely used in textile and medical industry. However, the strength of silkworm silks is inferior to that of spider dragline silks, which are the strongest natural silks but extremely low yield. Herein, we develop a feasible strategy to continuously produce superstrong and tough regenerated silk fibers (RSFs) through the reconstruction of hierarchical and cross-linked architectures. Amine functionalized graphene (NH2-G) has been introduced to improve the content of β-sheet and crystalline orientation, and glutaraldehyde (GA) has been used to bridge NH2-G and silk micro/nanofibrils (SNFs) to form a highly interconnected and closely packed network, resulting in the remarkably improvement of mechanical properties. The resultant fibers exhibit an excellent tensile strength of 1029.6 ± 144.8 MPa, a Young's modulus of 16.8 ± 1.5 GPa, and a toughness of 277.4 ± 17.0 MJ/m3, exceeding those of the natural silkworm silks, spider dragline silks and most other previously reported regenerated silk fibers. We believe that the high-performance silk-based fibers are promising applications in lightweight and advanced structural materials.

Biomechanics: Rain yields tougher spider silks

Broad ecological sampling of spider silks from multiple species shows that the biomechanical properties of spider silk reflect the habitat in which their orb webs are built. Silk toughness is highest in habitats with dense rain.

Domain swap facilitates structural transitions of spider silk protein C-terminal domains.

Domain swap is a mechanism of protein dimerization where the two interacting domains exchange parts of their structure. Web spiders make use of the process in the connection of C-terminal domains (CTDs) of spidroins, the soluble protein building blocks that form tough silk fibers. Besides providing connectivity and solubility, spidroin CTDs are responsible for inducing structural transitions during passage through an acidified assembly zone within spinning ducts. The underlying molecular mechanisms are elusive. Here, we studied the folding of five homologous spidroin CTDs from different spider species or glands. Four of these are domain-swapped dimers formed by five-helix bundles from spidroins of major and minor ampullate glands. The fifth is a dimer that lacks domain swap, formed by four-helix bundles from a spidroin of a flagelliform gland. Spidroins from this gland do not undergo structural transitions whereas the others do. We found a three-state mechanism of folding and dimerization that was conserved across homologues. In chemical denaturation experiments the native CTD dimer unfolded to a dimeric, partially structured intermediate, followed by full unfolding to denatured monomers. The energetics of the individual folding steps varied between homologues. Contrary to the common belief that domain swap stabilizes protein assemblies, the non-swapped homologue was most stable and folded four orders of magnitude faster than a swapped variant. Domain swap of spidroin CTDs induces an entropic penalty to the folding of peripheral helices, thus unfastening them for acid-induced unfolding within a spinning duct, which primes them for refolding into alternative structures during silk formation.

Highly strong and tough silk by feeding silkworms with rare earth ion-modified diets

Nature-derived silk fibers possess excellent biocompatibility, sustainability, and mechanical properties, yet producing strong and tough silk fibers in a facile and large-scale manner remains a significant challenge. Herein, we report a simple method for preparing strong and tough silk fibers by feeding silkworms rare earth ion-modified diets. The resulting silk fibers exhibit significantly increased tensile strength and toughness, with average values of 0.85 ± 0.07 GPa and 156 ± 13 MJ m−3, respectively, and maximum values of 0.97 ± 0.04 GPa and 188 ± 19 MJ m−3, approaching those of spider dragline silk. Our findings suggest that the incorporation of rare earth ions (La3+ or Eu3+) into the silk fibers contributes to this enhancement. Structure analysis reveals a reduction in content and an improvement in orientation of β-sheet nanocrystals in silk fibers. X-ray photoelectron spectroscopy analysis confirms the chemical interaction between rare earth ions with β-sheet nanocrystals. The structural evolution and chemical interactions lead to the simultaneous enhancement in both strength and toughness. This work presents a simple, scalable, and effective strategy for producing ultra-strong and tough silk fibers with potential applications in areas requiring super structural materials, such as personal protection and aerospace.

Tough silk fibroin hydrogel via polypropylene glycol (PPG) blending for wearable sensors

AbstractRegenerated silk fibroin is a promising bio‐material because of its sustainability, biocompatibility, degradability, and eco‐friendliness. However, silk fibroin hydrogel's intrinsic hard and brittle mechanical properties hinder its application on human skin as a wearable sensor. We used polypropylene glycol (PPG) as a plasticizer to physically blend with a silk fibroin solution, and designed a set of green and environmentally friendly preparation methods without organic solvents to obtain the hydrogel for wearable sensors. PPG molecules will aggregate into clusters to act as a plasticizer. Meanwhile, a large number of hydrogen bonds are formed between the hydroxyl groups in PPG and silk fibroin molecular chains. When the hydrogel is stretched, the hydrogen bond in the silk fibroin chain will release heat in an irreversible form to increase the toughness. The PPG/silk fibroin hydrogel exhibits mechanical strength of 16.6 MPa, breaking elongation of 160%, and toughness of 17.7 MJ/m3. The Ca2+ introduced during the sample preparation process gives the hydrogel ionic conductivity, which provides the possibility of using the hydrogel as a human skin sensing material.

Nacre-inspired, strong, tough silk fibroin hydrogels based on biomineralization and the layer-by-layer assembly of ordered silk fabric

Hydrogels are attractive materials with structures and functional properties similar to biological tissues and widely used in biomedical engineering. However, traditional synthetic hydrogels possess poor mechanical strength, and their applications are limited. Herein, a multidimensional material design method is developed; it includes the in situ gelation of silk fabric and nacre-inspired layer-by-layer assembly, which is used to prepare silk fibroin (SF) hydrogels. The in situ gelation method of silk fabric introduces a directionally ordered fabric network in a silk substrate, considerably enhancing the strength of hydrogels. Based on the nacre structure, the layer-by-layer assembly method enables silk hydrogels to break through the size limit and increase the thickness, realizing the longitudinal extension of the hydrogels. The application of the combined biomineralization and hot pressing method can effectively reduce interface defects and improve the interaction between organic and inorganic interfaces. The multidimensional material design method helps increase the strength (287.78 MPa), toughness (18.43 MJ m−3), and fracture energy (50.58 kJ m−2) of SF hydrogels; these hydrogels can weigh 2000 times their own weight. Therefore, SF hydrogels designed using the aforementioned combined method can realize the combination of strength and toughness and be used in biological tissue engineering and structural materials.

Tough Porous Silk Nanofiber-Derived Cryogels with Osteogenic and Angiogenic Capacity for Bone Repair.

Tough porous cryogels with angiogenesis and osteogenesis features remain a design challenge for utility in bone regeneration. Here, building off of the recent efforts to generate tough silk nanofiber-derived cryogels with osteogenic activity, deferoxamine (DFO) is loaded in silk nanofiber-derived cryogels to introduce angiogenic capacity. Both the mechanical cues (stiffness) and the sustained release of DFO from the gels are controlled by tuning the concentration of silk nanofibers in the system, achieving a modulus above 400kPa and slow release of the DFO over 60 days. The modulus of the cryogels and the released DFO induce osteogenic and angiogenic activity, which facilitates bone regeneration in vivo in femur defects in rat, resulting in faster regeneration of vascularized bone tissue. The tunable physical and chemical cues derived from these nanofibrous-microporous structures support the potential for silk cryogels in bone tissue regeneration.

Restructuring the Interface of Silk-Polycaprolactone Biocomposites Using Rigid-Flexible Agents.

Natural fiber-reinforced biocomposites with excellent mechanical and biological properties have attractive prospects for internal medical devices. However, poor interfacial adhesion between natural silk fiber and the polymer matrix has been a disturbing issue for such applications. Herein, rigid-flexible agents, such as polydopamine (PDA) and epoxy soybean oil (ESO), were introduced to enhance the interfacial adhesion between Antheraea pernyi (Ap) silk and a common medical polymer, polycaprolactone (PCL). We compared two strategies of depositing PDA first (Ap-PDA-ESO) and grafting ESO first (Ap-ESO-PDA). The rigid-flexible interfacial agents introduced multiple molecular interactions at the silk-PCL interface. The "Ap-PDA-ESO" strategy exhibited a greater enhancement in interfacial adhesion, and interfacial toughening mechanisms were proposed. This work sheds light on engineering strong and tough silk fiber-based biocomposites for biomedical applications.

Robust Silk Protein Hydrogels Made by a Facile One-Step Method and Their Multiple Applications.

Silk fibroin is a natural polymer that has various material forms and wide applications. Hydrogel is one of the most attractive silk materials because of its hydrophilicity, biocompatibility, and flexibility. However, its applications are still quite limited because they have a complicated preparation process and/or low mechanical strength. Herein, a simple way to prepare tough silk fibroin hydrogels via a solvent-exchange method is introduced. The degummed silk fiber was directly dissolved in a calcium chloride/formic acid solution and then water was used to replace the solvent. The silk fibroin hydrogel that was obtained using this facile method exhibited even better mechanical properties than most silk fibroin hydrogels that have been reported in the literature. Also, the silk fibroin hydrogel maintained biocompatibility that was as good as that prepared via other methods. Finally, the possibility of using this regenerated silk fibroin hydrogel as a multi-functional platform (such as a catalyst carrier, photothermal agent, and underwater adhesive) has been discussed. Therefore, such a natural, sustainable, robust, and good biocompatible silk fibroin hydrogel that is prepared by an improved method may have great potential for further applications.

Engineered Tough Silk Hydrogels through Assembling β-Sheet Rich Nanofibers Based on a Solvent Replacement Strategy.

β-Sheet rich silk nanofiber hydrogels are suitable scaffolds in tissue regeneration and carriers for various drugs. However, unsatisfactory mechanical performance limits its applications. Here, insight into the silk nanofibers stimulates the remodeling of previous solvent systems to actively regulate the assembly of silk nanofibers. Formic acid, a solvent of regenerated silk fibroin, is used to shield the charge repulsion of silk nanofibers to facilitate the nanofiber assembly under concentrated solutions. Formic acid was replaced with water to solidify the assembly, which induced the formation of a tough hydrogel. The hydrogels generated with this process possessed a modulus of 5.88 ± 0.82 MPa, ultimate stress of 1.55 ± 0.06 MPa, and toughness of 0.85 ± 0.03 MJ m-3, superior to those of previous silk hydrogels prepared through complex cross-linking processes. Benefiting from the dense gel network and high β-sheet content, these silk nanofiber hydrogels had good stability and antiswelling ability. The modulus could be modulated via changing the silk nanofiber concentration to provide differentiation signals to stem cells. Improved mechanical and bioactive properties with these hydrogels suggest utility in biomedical and engineering fields. More importantly, our present study reveals that the in-depth understanding of silk nanofibers could infuse power into traditional fabrication systems to achieve more high performance biomaterials, which is seldom considered in silk material studies.

Darwin's bark spider shares a spidroin repertoire with Caerostris extrusa but achieves extraordinary silk toughness through gene expression.

Spider silk is a protein-based material whose toughness suggests possible novel applications. A particularly fascinating example of silk toughness is provided by Darwin's bark spider (Caerostris darwini) found in Madagascar. This spider produces extraordinarily tough silk, with an average toughness of 350 MJ m-1 and over 50% extensibility, and can build river-bridging webs with a size of 2.8 m2. Recent studies have suggested that specific spidroins expressed in C. darwini are responsible for the mechanical properties of its silk. Therefore, a more comprehensive investigation of spidroin sequences, silk thread protein contents and phylogenetic conservation among closely related species is required. Here, we conducted genomic, transcriptomic and proteomic analyses of C. darwini and its close relative Caerostris extrusa. A variety of spidroins and low-molecular-weight proteins were found in the dragline silk of these species; all of the genes encoding these proteins were conserved in both genomes, but their genes were more expressed in C. darwini. The potential to produce very tough silk is common in the genus Caerostris, and our results may suggest the existence of plasticity allowing silk mechanical properties to be changed by optimizing related gene expression in response to the environment.

Biomechanical Properties of 3D-Printed Cervical Interbody Fusion Cage With Novel SF/nHAp Composites

Anterior cervical discectomy and fusion (ACDF) is a commonly used surgical method for the treatment of cervical spondylosis. As ACDF surgery is widely used in clinics, identifying suitable materials to design and prepare cervical interbody fusion cages is a hot research topic. Here, we describe a new three-dimensional (3D) printing approach to create stretchable and tough silk fibroin/nano-hydroxyapatite (SF/nHAp) composites with tunable mechanical properties. The compressive strength of the novel composites with biomimetic structure could reach more than 128 MPa. More importantly, the composites were prepared using 30% silk fibroin and 70% hydroxyapatite, a composition similar to the human bone tissue. Finite element analysis results indicate that the stress distribution of SF/nHAp composite cervical interbody fusion cages in vivo is more uniform than that of commercial Ti alloy cages. This study evaluates the effectiveness of SF/nHAp composites for application in cervical interbody fusion cages and in the field of bone tissue engineering.

Transcriptomic analysis of the bagworm moth silk gland reveals a number of silk genes conserved within Lepidoptera.

Lepidopteran insects produce cocoons with unique properties. The cocoons are made of silk produced in the larval tissue silk gland and our understanding of the silk genes is still very limited. Here, we investigated silk genes in the bagworm moth Eumeta variegata, a species that has recently been found to produce extraordinarily strong and tough silk. Using short-read transcriptomic analysis, we identified a partial sequence of the fibroin heavy chain gene and its product was found to have a C-terminal structure that is conserved within nonsaturniid species. This is in accordance with the presence of fibroin light chain/fibrohexamerin genes and it is suggested that the bagworm moth is producing silk composed of fibroin ternary complex. This indicates that the fibroin structure has been evolutionarily conserved longer than previously thought. Other than fibroins we identified candidates for sericin genes, expressed strongly in the middle region of the silk gland and encoding serine-rich proteins, and other silk genes, that are structurally conserved with other lepidopteran homologues. The bagworm moth is thus considered to be producing conventional lepidopteran type of silk. We further found a number of genes expressed in a specific region of the silk gland and some genes showed conserved expression with Bombyx mori counterparts. This is the first study allowing comprehensive silk gene identification and expression analysis in the lepidopteran Psychidae family and should contribute to the understanding of silk gene evolution as well as to the development of novel types of silk.

Strong and Tough Silk for Resilient Attachment Discs: The Mechanical Properties of Piriform Silk in the Spider Cupiennius salei (Keyserling, 1877)

Spiders are able to produce different types of silk with different mechanical and biological properties. Piriform silk is produced to secure spiders and their webs to surfaces by using a multi-fibrils system embedded in a cement-like matrix. Despite their fundamental role, the mechanical properties and function of these anchorages are still poorly understood due to the practical difficulties in sample preparation, the complexity of the system and the high variation of attachment disc structures. Here we estimated the mechanical properties of silk and those of the whole silk membrane in the large wandering spider Cupiennius salei through a combination of nanoindentation and nanotensile techniques and with the support of a simple analytical model. The results highlight the mechanical properties of the piriform silk, facilitating the modelling of silk composite mechanics. This could inspire the design of more efficient bio-inspired adhesives and fabrics.

NMR assignments of a dynamically perturbed and dimerization inhibited N-terminal domain variant of a spider silk protein from E. australis.

Web spiders use specialized glands to produce silk proteins, so-called spidroins, which assemble into extraordinarily tough silk fibers through tightly regulated phase and structural transitions. A crucial step in the polymerization of spidroins is the pH-triggered assembly of their N-terminal domains (NTDs) into tight dimers. Major ampullate spidroin NTDs contain an unusually high content of the amino acid methionine. We previously showed that the simultaneous mutation of the six hydrophobic core methionine residues to leucine in the NTD of the major ampullate spidroin 1 from Euprosthenops australis, a nursery web spider, yields a protein (L6-NTD) retaining a three-dimensional fold identical to the wildtype (WT) domain, yet with a significantly increased stability. Further, the dynamics of the L6-NTD are significantly reduced and the ability to dimerize is severely impaired compared to the WT domain. These properties lead to significant changes in the NMR spectra between WT and L6-NTD so that the previously available WT-NTD assignments cannot be transferred to the mutant protein. Here, we thus report the de novo NMR backbone and side chain assignments of the major ampullate spidroin 1 L6-NTD variant from E. australis as a prerequisite for obtaining further insights into protein structure and dynamics.

Small behavioral adaptations enable more effective prey capture by producing 3D-structured spider threads

Spiders are known for producing specialized fibers. The radial orb-web, for example, contains tough silk used for the web frame and the capture spiral consists of elastic silk, able to stretch when prey impacts the web. In concert, silk proteins and web geometry affects the spider’s ability to capture prey. Both factors have received considerable research attention, but next to no attention has been paid to the influence of fiber processing on web performance. Cribellate spiders produce a complex fiber alignment as their capture threads. With a temporally controlled spinneret movement, they connect different fibers at specific points to each other. One of the most complex capture threads is produced by the southern house spider, Kukulcania hibernalis (Filistatidae). In contrast to the so far characterized linear threads of other cribellate spiders, K. hibernalis spins capture threads in a zigzag pattern due to a slightly altered spinneret movement. The resulting more complex fiber alignment increased the thread’s overall ability to restrain prey, probably by increasing the adhesion area as well as its extensibility. Kukulcania hibernalis' cribellate silk perfectly illustrates the impact of small behavioral differences on the thread assembly and, thus, of silk functionality.

Molecular Interactions and Toughening Mechanisms in Silk Fibroin-Epoxy Resin Blend Films.

Natural silkworm silks have been applied to reinforce epoxy resin to achieve sub-ambient and impact toughness in the composite. However, the molecular interactions at the silk fiber-matrix interface of the composite are poorly understood. In this work, silk fibroin extracted from Bombyx mori silk is blended with an epoxy resin polymer system to study the molecular interactions between silk fibroin, epoxy compounds, and hardeners. The effects of chemical crosslinks between epoxy groups and hardeners or silk fibroin, as well as physical crosslinks in the β-sheet structure of silk fibroin, were discussed on the thermal stability, glass transition behavior, and mechanical properties of the blend films. A relationship between the crosslinking structure and mechanical properties for the films is proposed to enlighten on the toughening mechanisms. The findings would provide insights into forming strong and tough silk fibroin material as well as understanding the interface interactions of silk-epoxy composites.

A study of the extraordinarily strong and tough silk produced by bagworms

Global ecological damage has heightened the demand for silk as ‘a structural material made from sustainable resources’. Scientists have earnestly searched for stronger and tougher silks. Bagworm silk might be a promising candidate considering its superior capacity to dangle a heavy weight, summed up by the weights of the larva and its house. However, detailed mechanical and structural studies on bagworm silks have been lacking. Herein, we show the superior potential of the silk produced by Japan’s largest bagworm, Eumeta variegata. This bagworm silk is extraordinarily strong and tough, and its tensile deformation behaviour is quite elastic. The outstanding mechanical property is the result of a highly ordered hierarchical structure, which remains unchanged until fracture. Our findings demonstrate how the hierarchical structure of silk proteins plays an important role in the mechanical property of silk fibres.

Black widow spiders,Latrodectusspp. (Araneae: Theridiidae), and other spiders feeding on mammals

A survey of reports on spiders preying on small, non-flying mammals (i.e., mice, deer mice, voles, rats, heteromyid rodents, shrews) published in the literature and in the social media yielded a total of 42 naturally occurring incidents. Spiders from five families (Agelenidae, Ctenidae, Sparassidae, Theraphosidae, and Theridiidae) were reported capturing small mammals under natural conditions. Additionally, spiders from four more families (Atracidae, Lycosidae, Pisauridae, and Porrhothelidae) are known to kill small mammals in captivity. Approximately 80% of the reported incidents were attributable to theridiid spiders, especially the Australian redback spider (Latrodectus hasselti Thorell, 1870) and three species of North American widow spiders (Latrodectus geometricus C.L. Koch, 1841, Latrodectus hesperus Chamberlin & Ivie, 1935, and Latrodectus mactans (Fabricius, 1775)) that have been shown to be expert mouse-catchers. The success of widow spiders in subduing small mammals can be explained by their ability to spin strong webs made up of tough silk, and producing a very potent toxin (α-latrotoxin) specifically targeting the vertebrate nervous system.

Integration of stiff graphene and tough silk for the design and fabrication of versatile electronic materials.

The production of structural and functional materials with enhanced mechanical properties through the integration of soft and hard components is a common approach to Nature's materials design. However, directly mimicking these optimized design routes in the lab for practical applications remains challenging. For example, graphene and silk are two materials with complementary mechanical properties that feature ultrahigh stiffness and toughness, respectively. Yet no simple and controllable approach has been developed to homogeneously integrate these two components into functional composites, mainly due to the hydrophobicity and chemical inertness of the graphene. In this study, well-dispersed and highly stable graphene/silk fibroin (SF) suspension systems were developed, which are suitable for processing to fabricate polymorphic materials, such as films, fibers, and coatings. The obtained graphene/SF nanocomposites maintain the electronic advantages of graphene, and they also allow tailorable mechanical performance to form including ultrahigh stretchable (with a strain to failure to 611±85%), or high strength (339 MPa) and high stiffness (7.4 GPa) material systems. More remarkably, the electrical resistances of these graphene/SF materials are sensitive to material deformation, body movement, as well as humidity and chemical environmental changes. These unique features promise their utility as wearable sensors, smart textiles, intelligent skins, and human-machine interfaces.