Natural Silk Spinning Research Articles

Bridging Biocondensation and Biomimetic Fibrillation by Regulating Intra- And Interchain Hydrogen Bonds in Artificial Biomacromolecular Condensates.

Utilizing biocondensates as feedstocks can be a state-of-the-art strategy for emulating natural silk spinning. Although current biocondensates can form solid fibers using a biomimetic draw spinning method, the fibrillation is primarily achieved through evaporation of highly concentrated biocondensates rather than structural conversion in natural spinning. Current artificial biocondensates lack biomimetic features of stress-induced fibrillation since they are unable to replicate the structural complexity of native proteins in the dope. Herein, we successfully achieved biomimetic fibrillation at significantly reduced concentrations by constructing artificial biocondensates using naturally derived silk fibroin. The biomimetic features of stress-induced fibrillation in native proteins are replicated in our artificial biocondensates by tailoring multivalent interactions in biocondensation. Our findings unravel the fundamental correlations between biocondensation and stress-induced fibrillation. This work can not only provide a framework for designing artificial biocondensates in biomimetic spinning but also improve the molecular insights into natural spinning.

Complexity of Spider Dragline Silk.

The tiny spider makes dragline silk fibers with unbeatable toughness, all under the most innocuous conditions. Scientists have persistently tried to emulate its natural silk spinning process using recombinant proteins with a view toward creating a new wave of smart materials, yet most efforts have fallen short of attaining the native fiber’s excellent mechanical properties. One reason for these shortcomings may be that artificial spider silk systems tend to be overly simplified and may not sufficiently take into account the true complexity of the underlying protein sequences and of the multidimensional aspects of the natural self-assembly process that give rise to the hierarchically structured fibers. Here, we discuss recent findings regarding the material constituents of spider dragline silk, including novel spidroin subtypes, nonspidroin proteins, and possible involvement of post-translational modifications, which together suggest a complexity that transcends the two-component MaSp1/MaSp2 system. We subsequently consider insights into the spidroin domain functions, structures, and overall mechanisms for the rapid transition from disordered soluble protein into a highly organized fiber, including the possibility of viewing spider silk self-assembly through a framework relevant to biomolecular condensates. Finally, we consider the concept of “biomimetics” as it applies to artificial spider silk production with a focus on key practical aspects of design and evaluation that may hopefully inform efforts to more closely reproduce the remarkable structure and function of the native silk fiber using artificial methods.

Theoretical rheo-physics of silk: Intermolecular associations reduce the critical specific work for flow-induced crystallization

Silk is a semidilute solution of randomly coiled associating polypeptide chains that crystallize following the stretch-induced disruption, in the strong extensional flow of extrusion, of the solvation shell around their amino acids. We propose that natural silk spinning exploits both the exponentially broad stretch distribution generated by associating polymers in extensional flow and the criterion of a critical concentration of sufficiently stretched chains to nucleate flow-induced crystallization. To investigate the specific-energy input needed to reach this criterion in start-up flow, we have coupled a model for the Brownian dynamics of a bead-spring-type chain, whose beads represent coarse-grained Gaussian chain segments, to the stochastic, strain-dependent binding and unbinding of their associations. We have interpreted the simulations with the aid of analytic calculations on simpler, tractable models with the same essential physical features. Our simulations indicate that the associations hamper chain alignment in the initial slow flow, but, on the other hand, facilitate chain stretching at low specific work at later, high rates. We identify a minimum in the critical specific work at a strain rate just above the stretch transition (i.e., where the mean stretch diverges), which we explain in terms of analytical solutions of a two-state master equation. We further discuss how the silkworm appears to exploit the chemical tunability of the associations to optimize chain alignment and stretching in different locations along the spinning duct: this delicate mechanism also highlights the potential biomimetic industrial benefits of chemically tunable processing of synthetic association polymers.

Spinning Beta Silks Requires Both pH Activation and Extensional Stress

AbstractSynthetic silk production has undergone significant technological and commercial advances over the past 5 years, with fibers from most labs and companies now regularly matching the properties of natural silk by one metric or another. Yet the fundamental links between silk protein processing and performance remain largely unresolved and fiber optimization is commonly achieved through non‐natural methods. In an effort to address this challenge, data that closes this loop of processing and performance is presented by spinning a native silk feedstock ex vivo into a near‐native fiber using just two naturally occurring parameters; pH activation and extensional flow (i.e., spinning rate). This allows us to link previous experimental and modelling hypothesis surrounding silk's pH responsiveness directly to multiscale hierarchical structure development during spinning. Finally, fibers that match, and then exceed, natural silk's mechanical properties are spun and understood by rate of work input. This approach not only provides energetic insights into natural silk spinning and controlled protein denaturation, but is believed will help interpret and improve synthetic silk processing. Ultimately, it is hoped that these results will contribute towards novel bioinspired energy‐efficient processing strategies that are driven by work input optimization and where excellent mechanical properties are self‐emergent.

Bioactive hierarchical silk fibers created by bioinspired self-assembly

Artificial recapitulation of the hierarchy of natural protein fibers is crucial to providing strategies for developing advanced fibrous materials. However, it is challenging due to the complexity of the natural environment. Inspired by the liquid crystalline spinning of spiders, we report the development of natural silk-like hierarchical fibers, with bundles of nanofibrils aligned in their long-axis direction, by self-assembly of crystallized silk fibroin (SF) droplets. The formation of self-assembled SF fibers is a process of coalesced droplets sprouting to form a branched fibrous network, which is similar to the development of capillaries in our body. The as-assembled hierarchical SF fibers are highly bioactive and can significantly enhance the spreading and growth of human umbilical vein endothelial cells compared to the natural SF fibers. This work could help to understand the natural silk spinning process of spiders and provides a strategy for design and development of advanced fibrous biomaterials for various applications.

Engineering Silk Materials: From Natural Spinning to Artificial Processing.

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.

Direct Observation of Native Silk Fibroin Conformation in Silk Gland of Bombyx mori Silkworm.

To understand the natural silk spinning mechanism, synchrotron Fourier transform infrared (S-FTIR) microspectroscopy was employed in this study to monitor the conformation changes of silk protein in the silk gland of Bombyx mori silkworm. The ultrahigh brightness of S-FTIR microspectroscopy allowed the imaging of the silk gland with micrometer-scale spatial resolution. Herein, tissue sections of a silk gland, including cross-section slices and longitudinal-section slices, were characterized. The results obtained clearly confirm that the conformation of the silk fibroin changes gradually along the silk gland from the tail to the spinneret. In the middle silk gland, silk fibroin mainly contains random coil/helix conformation. When it comes to the spinneret through the anterior silk gland, the content of β-sheet increases, but the content of random coil/helix instead reduces gradually. Further, the β-sheet distribution in the cross-section of the anterior silk gland was imaged using S-FTIR mapping technique. The results show that the structural distribution of the silk fibroin in cross-section is uniform without significant shell-core structure, which implies that the primary driving force to induce the conformation transition of silk fibroin from random coil/helix to β-sheet during the spinning process is elongational flow of silk fibroin in the silk gland and not the shear force between the silk fibroin and the lumen wall of silk gland. These direct pieces of evidence of silk fibroin structure in the silk gland would definitely promote a deeper understanding of the natural spinning process.

Disruption of the Metal Ion Environment by EDTA for Silk Formation Affects the Mechanical Properties of Silkworm Silk.

Silk fiber has become a research focus because of its comprehensive mechanical properties. Metal ions can influence the conformational transition of silk fibroin. Current research is mainly focused on the role of a single ion, rather than the whole metal ion environment. Here, we report the effects of the overall metal ion environment on the secondary structure and mechanical properties of silk fibers after direct injection and feeding of silkworms with EDTA. The metal composition of the hemolymph, silk gland, and silk fiber changed significantly post EDTA treatment. Synchrotron FTIR analysis indicated that the secondary structure of silk fiber after EDTA treatment changed dramatically; particularly, the β-sheets decreased and the β-turns increased. Post EDTA treatment, the silk fiber had significantly decreased strength, Young’s modulus, and toughness as compared with the control groups, while the strain exhibited no obvious change. These changes can be attributed to the change in the metal ion environment in the silk fibroin and sericin in the silk gland. Our investigation provides a new theoretical basis for the natural silk spinning process, and our findings could help develop a method to modify the mechanical properties of silk fiber using metal ions.

The Energy Requirements for Flow-Induced Solidification of Silk.

Natural silk spinning has undergone strong selection for resource efficiency and thus presents a biomimetic ideal for fiber production. Industrial replication of natural silk fibers would enable access to low energy, cost-efficient processing, but is hampered by a lack of understanding surrounding the conversion of liquid feedstock into a solid fiber as a result of flow. Previously, shear stress, shear rate, or time have been presented as criteria for silk fiber formation, but here it is proposed that spinning requires carefully balancing all three, and is a result of controlled energy accumulation in the feedstock. To support this hypothesis, rheology is used to probe the energy required for conversion, compare differences between amorphous solid and ordered fiber production and explain the energetic penalty the latter demands. New definitions of what constitutes an artificial silk fiber are discussed, along with methods to ensure that each spinning criterion is met during biomimetic spinning.

Using hydrodynamic focusing to predictably alter the diameter of synthetic silk fibers.

Spiders and silkworms provide a model of superior processing for multifunctional and highly versatile high-performance fibers. Mimicking the spider’s complex control system for chemical and mechanical gradients has remained an ongoing obstacle for synthetic silk production. In this study, the use of hydrodynamic fluid focusing within a 3D printed biomimetic spinning system to recapitulate the biological spinneret is explored and shown to produce predictable, small diameter fibers. Mirroring in silico fluid flow simulations using a hydrodynamic microfluidic spinning technique, we have developed a model correlating spinning rates, solution viscosity and fiber diameter outputs that will significantly advance the field of synthetic silk fiber production. The use of hydrodynamic focusing to produce controlled output fiber diameter simulates the natural silk spinning process and continues to build upon a 3D printed biomimetic spinning platform.

Silk nanofibril self-assembly versus electrospinning.

Natural silk fibers represent one of the most advanced blueprints for (bio)polymer scientists, displaying highly optimized mechanical properties due to their hierarchical structures. Biotechnological production of silk proteins and implementation of advanced processing methods enabled harnessing the potential of these biopolymer not just based on the mechanical properties. In addition to fibers, diverse morphologies can be produced, such as nonwoven meshes, films, hydrogels, foams, capsules and particles. Among them, nanoscale fibrils and fibers are particularly interesting concerning medical and technical applications due to their biocompatibility, environmental and mechanical robustness as well as high surface-to-volume ratio. Therefore, we introduce here self-assembly of silk proteins into hierarchically organized structures such as supramolecular nanofibrils and fabricated materials based thereon. As an alternative to self-assembly, we also present electrospinning a technique to produce nanofibers and nanofibrous mats. Accordingly, we introduce a broad range of silk-based dopes, used in self-assembly and electrospinning: natural silk proteins originating from natural spinning glands, natural silk protein solutions reconstituted from fibers, engineered recombinant silk proteins designed from natural blueprints, genetic fusions of recombinant silk proteins with other structural or functional peptides and moieties, as well as hybrids of recombinant silk proteins chemically conjugated with nonproteinaceous biotic or abiotic molecules. We highlight the advantages but also point out drawbacks of each particular production route. The scope includes studies of the natural self-assembly mechanism during natural silk spinning, production of silk fibrils as new nanostructured non-native scaffolds allowing dynamic morphological switches, as well as studying potential applications. This article is categorized under: Biology-Inspired Nanomaterials > Peptide-Based Structures Nanotechnology Approaches to Biology > Nanoscale Systems in Biology Biology-Inspired Nanomaterials > Protein and Virus-Based Structures.

The rheological properties of native sericin

Unlike spider silk, spinning silkworm silk has the added intricacy of being both fibre and micron-thick glue-like coating. Whilst the natural flow properties of the fibre feedstock fibroin are now becoming more established, our understanding of the coating sericin is extremely limited and thus presents both a gap in our knowledge and a hindrance to successful exploitation of these materials. In this study we characterise sericin feedstock from the silkworm Bombyx mori in its native state and by employing both biochemical, rheological and spectroscopic tools, define a natural gold standard. Our results demonstrate that native sericin behaves as a viscoelastic shear thinning fluid, but that it does so at a considerably lower viscosity than its partner fibroin, and that its upper critical shear rate (onset of gelation) lies above that of fibroin. Together these findings provide the first evidence that in addition to acting as a binder in the construction of the cocoon, sericin is capable of lubricating the flow of fibroin within the silk gland, which has implications for future processing, modelling and biomimetic use of these materials. Statement of SignificanceThis study addresses one of the major gaps in our knowledge regarding natural silk spinning by providing rigorous rheological characterisation of the other major protein involved – sericin. This allows progress in silk flow modelling, biomimetic system design, and in assessing the quality of bioinspired and waste sericin materials by providing a better understanding of the native, undegraded system.

Sericin Promotes Fibroin Silk I Stabilization Across a Phase-Separation.

Natural silk spinning offers several advantages over the synthetic fiber spinning, although the underlying mechanisms of this process are yet to be fully elucidated. Silkworm silks, specifically B. mori, comprise two main proteins: fibroin, which forms the fiber, and sericin, a coextruded coating that acts as a matrix in the resulting nonwoven composite cocoon. To date, most studies have focused on fibroin's self-assembly and gelation, with the influence of sericin during spinning receiving little to no attention. This study investigates sericin's effects on the self-assembly of fibroin via their natural phase-separation. Through changes in sample opacity, FTIR, and XRD, we report that increasing sericin concentration retards the time to gelation and β-sheet formation of fibroin, causing it to adopt a Silk I conformation. Such findings have important implications for both the natural silk spinning process and any future industrial applications, suggesting that sericin may be able to induce long-range conformational and stability control in silk fibroin, while being in a separate phase, a factor that would facilitate long-term storage or silk feedstocks.

Spider silk as a blueprint for greener materials: a review

Spider silk exhibits remarkable properties, especially its well-known tensile performances. They rely on a complex nanostructured hierarchical organisation that studies progressively elucidate. Spider silk encompasses a vast range of fibres that exhibit diverse and captivating physical and biological characteristics. The full understanding of the relation between structure and properties may lead in the future to the design of a variety of high-performance, tailored materials and devices. Reknown for being produced in mild and benign conditions, this outstanding biological material constitutes one of the more representative example of biomimetism. In addition, silk's structure is produced with limited means, i.e. low energy and relatively simple renewable constituents (silk proteins). Then, if successfully controlled and adequately transposed in biomaterials, some properties of natural silk could lead to innovative green materials that may contribute to reduce the ecological footprint of societies. In fact, striking recent advanced applications made with B. mori silk suggest that spider silk-based materials may lead to advanced resistant and functional materials, then becoming among the most promising subject of study in material science. However, several challenges have to be overcome, especially our ability to produce native-like silk, to control biomaterials’ structure and properties and to minimise their ecological footprint. This paper reviews the characteristics of spider silk that make it so attractive and that may (or may not) contribute to reduce ecological footprint of materials and the challenges in producing innovative spider silk-based materials. First, from a biomimetic perspective, the structure and models that explain the tensile resistance of natural silk are presented, followed by the state of knowledge regarding natural silk spinning process and synthetic production methods. Biocompatibility (biosafety and biofunctionality) as well as biodegradability issues are then addressed. Finally, examples of applications are reviewed. Features that may lead to the design of green materials are emphasised throughout.

Shotgun proteomic analysis of the Bombyx mori anterior silk gland: An insight into the biosynthetic fiber spinning process

The Bombyx mori anterior silk gland (ASG) is a natural fiber manipulator for the material provided by the middle and posterior silk glands. In view of the significant role of the ASG in the liquid-crystal spinning process, a shotgun proteomics approach was taken to study the relationship between the function of proteins in the silkworm ASG and the spinning mechanism. A total of 1132 proteins with 7647 unique peptides were identified in the ASG dataset including some involved in the cuticle, ion transportation, energy metabolism, and apoptosis. Two putative cuticle-specific proteins were highly and specifically expressed in the ASG; therefore, the ASG dataset could provide clues for comprehensive understanding of the natural silk spinning mechanism in the silkworm. All MS data have been deposited in the ProteomeXchange with identifier PXD000090.

Distinct structural and optical regimes in natural silk spinning

This study investigates the relationship between birefringence and mechanical properties in the dragline silk of the gold orb weaving spider Nephila edulis. Using a custom birefringence-tensile testing device, we probed the orientation and water-induced swelling of fibers spun at variety of drawing rates ranging from 0.003 to 400 mm s(-1). Our results indicate that based upon drawing rate, silk fibers fall into three distinct regimes each with characteristic orientation and swelling properties. Further investigation using in situ tensile testing reveals interactions between a fiber's drawing speed, mechanical properties, and orientation that support previous model predictions. We propose that simultaneous birefringence-tensile testing provides a unique and readily accessible insight into the structural behavior of this interesting and important biomaterial.

The properties of native silk fibroin (SF) solution/gel from bombyx mori silkworms during the full fifth instar larval stage

The properties of native silk fibroin (SF) solution in the gland of silkworms during the full fifth instar larval stage were examined in an attempt to elucidate the mechanism of natural silk spinning in the silkworm. The flow and gelation behavior, birefringence phenomenon, rheological properties, specific viscosity and conformation of SF solutions from the gland of silkworms were measured by polarized light microscope, HAKKE rheometer, Ubbelohde viscometer and Solid-state 13C NMR, respectively. After comparing their properties with regenerated SF solutions from natural silk fibers, it is believed that there exists a progressive maturation process favorable to spin silk fibers with excellent properties from native SF solution and a weak bonded and highly oriented SF gel network with SF molecules partly extended in α-helix conformation is formed in the middle section of the gland of silkworms. This suggests that a biomimetic maturation process for making spinnable solution might be necessary for artificial silk fiber spinning in order to obtain improved fiber properties.

Acceleration effect of sericin on shear-induced β-transition of silk fibroin

Abstract Although silk sericin (SS) occupies 25% of silk protein, its importance has often been overlooked in the natural silk spinning process and in the formation of the crystalline structure of silk fibroin (SF). In this study, we elucidated the role of SS in the crystallization process of SF under shear using SF/SS blend solutions. In order to apply shear stress to the blend solution, a rotating glass rod was inserted into a glass tube filled with the solution and the shear rate was determined to be in the range of 598–724 s−1. After shearing, SF aggregates were formed and the amount of the aggregates increased with shearing time. Additionally, it was observed that the aggregate formation and β-sheet transition of SF were enhanced when a proper amount of SS was in the blend solution. Consequently, the SS considerably contributes to the structural transition of SF under shear. The SS can improve the shear-induced β-sheet transition and crystallization of SF.

Conformational transition and liquid crystalline state of regenerated silk fibroin in water

The conformational transition of molecular chains of regenerated silk fibroin (SF) aqueous solution is systematically investigated by circular dichroism, Raman, IR, and UV-vis spectroscopies. It is found that an initial random coil conformation of the SF can be readily changed into an ordered beta-sheet structure by optimizing the solution conditions, such as the SF concentration, pH, temperature, or metal-ion content. Circular dichroic spectra quantitatively confirm a steadily decreased content of the random coil conformation but a significantly increased beta-sheet content after an ultrasonic or extruding treatment. Furthermore, the extrusion is more powerful to achieve high beta-sheet content than the ultrasonic. It is interesting that the polarized optical micrographs of the SF aqueous solution extruded by injection illustrate the formation and existence of liquid crystalline state. A study of extrusion in vitro could be used as a model system to understand the natural silk spinning process in silkworm.

Conformation transition kinetics of Bombyx mori silk protein

Time-resolved FTIR analysis was used to monitor the conformation transition induced by treating regenerated Bombyx mori silk fibroin films and solutions with different concentrations of ethanol. The resulting curves showing the kinetics of the transition for both films and fibroin solutions were influenced by the ethanol concentration. In addition, for silk fibroin solutions the protein concentration also had an effect on the kinetics. At low ethanol concentrations (for example, less than 40% v/v in the case of film), films and fibroin solutions showed a phase in which beta-sheets slowly formed at a rate dependent on the ethanol concentration. Reducing the concentration of the fibroin in solutions also slowed the formation of beta-sheets. These observations suggest that this phase represents a nucleation step. Such a nucleation phase was not seen in the conformation transition at ethanol concentrations > 40% in films or > 50% in silk fibroin solutions. Our results indicate that the ethanol-induced conformation transition of silk fibroin in films and solutions is a three-phase process. The first phase is the initiation of beta-sheet structure (nucleation), the second is a fast phase of beta-sheet growth while the third phase represents a slow perfection of previously formed beta-sheet structure. The nucleation step can be very fast or relatively slow, depending on factors that influence protein chain mobility and intermolecular hydrogen bond formation. The findings give support to the previous evidence that natural silk spinning in silkworms is nucleation-dependent, and that silkworms (like spiders) use concentrated silk protein solutions, and careful control of the pH value and metallic ion content of the processing environment to speed up the nucleation step to produce a rapid conformation transition to convert the water soluble spinning dope to a tough solid silk fiber.