Artificial Silk Research Articles

Degree of Biomimicry of Artificial Spider Silk Spinning Assessed by NMR Spectroscopy.

Biomimetic spinning of artificial spider silk requires that the terminal domains of designed minispidroins undergo specific structural changes in concert with the β-sheet conversion of the repetitive region. Herein, we combine solution and solid-state NMR methods to probe domain-specific structural changes in the NT2RepCT minispidroin, which allows us to assess the degree of biomimicry of artificial silk spinning. In addition, we show that the structural effects of post-spinning procedures can be examined. By studying the impact of NT2RepCT fiber drying, we observed a reversible beta-to-alpha conversion. We think that this approach will be useful for guiding the optimization of artificial spider silk fibers.

Degree of Biomimicry of Artificial Spider Silk Spinning Assessed by NMR Spectroscopy

AbstractBiomimetic spinning of artificial spider silk requires that the terminal domains of designed minispidroins undergo specific structural changes in concert with the β‐sheet conversion of the repetitive region. Herein, we combine solution and solid‐state NMR methods to probe domain‐specific structural changes in the NT2RepCT minispidroin, which allows us to assess the degree of biomimicry of artificial silk spinning. In addition, we show that the structural effects of post‐spinning procedures can be examined. By studying the impact of NT2RepCT fiber drying, we observed a reversible beta‐to‐alpha conversion. We think that this approach will be useful for guiding the optimization of artificial spider silk fibers.

The Development of Fibers That Mimic the Core–Sheath and Spindle‐Knot Morphology of Artificial Silk Using Microfluidic Devices

Spider and silkworm produce diverse silk fibers from spinning dopes through smart spinnerets. Spider's capture silk is composed of core thread and periodic spindle‐knots, while silkworm silk consists of fibroin core and sericin outer layer. To mimic the morphologies of natural heterostructured silks, artificial fibers are dry‐spun using a multichannel microfluidic chip, served with a highly viscous core solution of regenerated silk fibroin and low viscosity sheath solution of sericin. Silk fibers with core–sheath, groove, and spindle‐knot structures are obtained by controlling the flow rates and viscosities of the two microfluids depending on the laminar flow, Kelvin–Helmholtz instability, or Plateau–Rayleigh instability.

Effect of Draw Ratio on the Microstructure of Silk Fibroin/Graphene Oxide Hybrid Fibers

In order to fabricate high performance artificial silk, hybrid fibers were dry spun from regenerated silk fibroin (RSF) aqueous solution mixed with graphene oxide (GO) nanosheets. The influence of draw ratio on the microstructure and mechanical properties of RSF/GO hybrid fibers was studied through Fourier transform infrared spectroscopy (FT-IR), synchrotron radiation wide-angle X-ray diffraction, Raman spectroscopy and mechanical testing. The results showed that with increasing draw ratio, both amorphous phase and mesophase of the dry-spun artificial fiber gradually converted to crystals. The orientation of crystals and mesophase changed slightly at low draw ratio, but increased significantly at draw ratios of 3 and 4X. The β-sheet content of silk fibroin and the disorder degree of GO sheets in the fibers also increased with the increase of draw ratio. The initial modulus, breaking strength and breaking energy of the hybrid fibers achieved great improvement at a draw ratio of 4X. The structure evolution of the RSF/GO hybrid fibers may benefit the understanding of the structure-property relationship of other composite fibers.

Introducing biomimetic shear and ion gradients to microfluidic spinning improves silk fiber strength

Silkworm silk is an attractive biopolymer for biomedical applications due to its high mechanical strength and biocompatibility; as a result, there is increasing interest in scalable devices to spin silk and recombinant silk so as to improve and customize their properties for diverse biomedical purposes (Vepari and Kaplan 2007 Prog. Polym. Sci. 32). While artificial spinning of regenerated silk fibroins adds tunability to properties such as degradation rate and surface functionalization, the resulting fibers do not yet approach the mechanical strength of native silkworm silk. These drawbacks reduce the applicability and attractiveness of artificial silk (Kinahan et al 2011 Biomacromolecules 12). Here, we used computational fluid dynamic simulations to incorporate shear in tandem with biomimetic ion gradients by coupling a modular novel glass microfluidic device to our previous co-axial flow device. Fibers spun with this combined apparatus demonstrated a significant increase in mechanical strength compared to fibers spun with the basic apparatus alone, with a three-fold increase in Young’s modulus and extensibility and a twelve-fold increase in toughness. These results thus demonstrate the critical importance of ionic milieu and shear stress in spinning strong fibers from solubilized silk fibroin.

A directed self-assembly quasi-spider silk protein expressed in Pichia pastoris

ABSTRACTThe spider silk protein has distinctive physical, chemical, mechanical and biological properties. As a functional material, the application value of spider silk has increased in many fields. Consequently, considerable progress has been made in the expression of recombinant spider silk proteins through many host systems by gene engineering techniques. However, the mechanical properties of the silk fibre spun with the recombinant spider silk proteins are unsatisfactory because the recombinant spider silk proteins have too low of a molecular weight and do not have molecular orientation. This paper describes the construction and expression of a quasi-spider silk protein composed of spider silk protein and collagen-like peptides with the Pichia pastoris expression system. The quasi-spider silk protein is an ‘ABA-type’ triblock copolymer composed of triple helix-forming A blocks at both ends of the middle section. The triple helix-forming A blocks at both ends of the triblock copolymers consist of (Pro–Gly–Pro)n homopolymeric stretches, and the middle section of the molecule (B section) contains a spider silk protein that has been optimally designed. The supramolecular structure created by the three-block copolymers through directed self-assembly ensures that the artificial spider silk fibres will meet the requirement for molecular weight and definite molecular orientation, thus promoting the formation of silk fibres. The authors hope that this project will contribute to the study of materials science and biomedical engineering in regards to the huge potential of spider silk protein in these fields.

Progress and Trends in Artificial Silk Spinning: A Systematic Review.

More than 400 million years of natural selection acting throughout the arthropoda has resulted in highly specialized and energetically efficient processes to produce protein-based fibers with properties that are a source of inspiration for all. As a result, for over 80 years researchers have been inspired by natural silk production in their attempts to spin artificial silks. While significant progress has been made, with fibers now regularly outperforming silkworm silks, surpassing the properties of superior silks, such as spider dragline, is still an area of considerable effort. This review provides an overview of the different approaches for artificial silk fiber spinning and compares all published fiber properties to date which has identified future trends and challenges on the road towards replicating high performance silks.

Biomimetic spinning of artificial spider silk from a chimeric minispidroin.

Herein we present a chimeric recombinant spider silk protein (spidroin) whose aqueous solubility equals that of native spider silk dope and a spinning device that is based solely on aqueous buffers, shear forces and lowered pH. The process recapitulates the complex molecular mechanisms that dictate native spider silk spinning and is highly efficient; spidroin from one liter of bacterial shake-flask culture is enough to spin a kilometer of the hitherto toughest as-spun artificial spider silk fiber.

Preparation and Characterization of Artificial Spider Silk Produced through Microchannel Techniques

Spider silk (SS) is naturally tough; however, it turns soft when wet by water. Spiders produce high-quality silk threads by adjusting the molecular assemblage of SS-proteins and the arrangements structure of threads and recombinant spider dragline silk (RSDS). The general wet spinning techniques for producing recombinant spidroins results in uncorrected explanation to the natural spinning technique. In this study, we use tailored-SS with relative low molecular weight of 47 kD to produce a water-soluble RSDS protein. We built a microfluidic ship and used it to spun SS using aqueous solutions-micro-technique (wet spinning). This was done in order to mimic the spider-spinning processes using a steady post-spin drawing process. We succeeded to produce assemblies of spidroins with fibril structure. Then, compact constituting of micro-threads followed these wet spinning processes. Wet spinning was followed by improving the orientation, crystalline structure, and fibril melting of the hierarchical structure. The initial mechanical characterization (tensile strength) of the RSDSs attained about 510 MPa with respective extension 44%.

Bioinspired Special Wettability Surfaces: From Fundamental Research to Water Harvesting Applications.

Nowadays, the pollution of water has become worse in many parts of the world, which causes a severe shortage of clean water and attracts widespread attention worldwide. Bioinspired from nature, i.e. spider silk, cactus, Namib desert beetle, Nepenthes alata, special wettability surfaces have attracted great interest from fundamental research to water-harvesting applications. Here, recently published literature about creatures possessing water-harvesting ability are reviewed, with a focus on the corresponding water-harvesting mechanisms of creatures in dry or arid regions, consisting of the theory of wetting and transporting. Then a detailed account of the innovative fabrication technologies and bionic water-harvesting materials with special wetting are summarized, i.e. bio-inspired artificial spider silk, bio-inspired artificial cactus-like structures, and bio-inspired artificial Namib desert beetle-like surfaces. Special attentions are paid to the discussion of the advantages and disadvantages of the technologies, as well as factors that affect the amount of water-harvesting. Finally, conclusions, future outlooks and the current challenges for future development of the water-harvesting technology are presented and discussed.

Recombinant spider silk from aqueous solutions via a bio-inspired microfluidic chip.

Spiders achieve superior silk fibres by controlling the molecular assembly of silk proteins and the hierarchical structure of fibres. However, current wet-spinning process for recombinant spidroins oversimplifies the natural spinning process. Here, water-soluble recombinant spider dragline silk protein (with a low molecular weight of 47 kDa) was adopted to prepare aqueous spinning dope. Artificial spider silks were spun via microfluidic wet-spinning, using a continuous post-spin drawing process (WS-PSD). By mimicking the natural spinning apparatus, shearing and elongational sections were integrated in the microfluidic spinning chip to induce assembly, orientation of spidroins, and fibril structure formation. The additional post-spin drawing process following the wet-spinning section partially mimics the spinning process of natural spider silk and substantially contributes to the compact aggregation of microfibrils. Subsequent post-stretching further improves the hierarchical structure of the fibres, including the crystalline structure, orientation, and fibril melting. The tensile strength and elongation of post-treated fibres reached up to 510 MPa and 15%, respectively.

Crystal Structure of the Nephila clavipes Major Ampullate Spidroin 1A N-terminal Domain Reveals Plasticity at the Dimer Interface

Spider dragline silk is a natural polymer harboring unique physical and biochemical properties that make it an ideal biomaterial. Artificial silk production requires an understanding of the in vivo mechanisms spiders use to convert soluble proteins, called spidroins, into insoluble fibers. Controlled dimerization of the spidroin N-terminal domain (NTD) is crucial to this process. Here, we report the crystal structure of the Nephila clavipes major ampullate spidroin NTD dimer. Comparison of our N. clavipes NTD structure with previously determined Euprosthenops australis NTD structures reveals subtle conformational alterations that lead to differences in how the subunits are arranged at the dimer interface. We observe a subset of contacts that are specific to each ortholog, as well as a substantial increase in asymmetry in the interactions observed at the N. clavipes NTD dimer interface. These asymmetric interactions include novel intermolecular salt bridges that provide new insights into the mechanism of NTD dimerization. We also observe a unique intramolecular "handshake" interaction between two conserved acidic residues that our data suggest adds an additional layer of complexity to the pH-sensitive relay mechanism for NTD dimerization. The results of a panel of tryptophan fluorescence dimerization assays probing the importance of these interactions support our structural observations. Based on our findings, we propose that conformational selectivity and plasticity at the NTD dimer interface play a role in the pH-dependent transition of the NTD from monomer to stably associated dimer as the spidroin progresses through the silk extrusion duct.

Silk Spinning in Silkworms and Spiders.

Spiders and silkworms spin silks that outcompete the toughness of all natural and manmade fibers. Herein, we compare and contrast the spinning of silk in silkworms and spiders, with the aim of identifying features that are important for fiber formation. Although spiders and silkworms are very distantly related, some features of spinning silk seem to be universal. Both spiders and silkworms produce large silk proteins that are highly repetitive and extremely soluble at high pH, likely due to the globular terminal domains that flank an intermediate repetitive region. The silk proteins are produced and stored at a very high concentration in glands, and then transported along a narrowing tube in which they change conformation in response primarily to a pH gradient generated by carbonic anhydrase and proton pumps, as well as to ions and shear forces. The silk proteins thereby convert from random coil and alpha helical soluble conformations to beta sheet fibers. We suggest that factors that need to be optimized for successful production of artificial silk proteins capable of forming tough fibers include protein solubility, pH sensitivity, and preservation of natively folded proteins throughout the purification and initial spinning processes.

전처리가 오배자 추출물에 의한 여름용 인견직물의 염색 및 기능성 향상에 미치는 영향

Viscose rayon filament fabrics have been called 'artificial silk' and beloved as summer clothing materials for a long time in Korea. This is because the viscose rayon filament fabrics reveals glossy surface and cool touch feeling compared to other conventional fabrics composed of staple fibers. Therefore, we tried to prepare the higher value added viscose rayon filament fabrics for summer textile products. In this study, we applied gallnut extract to the viscose rayon filament fabric to develop summer fabrics with natural color and multi-functions such as antibacterial and antioxidant properties. This process also pursue eco-friendly and multi-functional fabric finishing from the natural material "gallnut". In addition, various pre-treatment with cationizer, chitosan, or chito-oligomer was applied to the finishing process to improve the finishing efficacy and durability. Consequently, it was found that the active component of gallnut extract was successfully incorporated to the viscose rayon filament fabric through a pad-dry-cure process. And, the treated viscose rayon filament fabrics showed excellent antibacterial and antioxidant properties. Therefore, it was expected that the rayon filament fabrics treated by gallnut extract could be used as effective summer fabrics preventing the growth of bacteria and skin ageing as well as providing cool touch feeling. However, the pre-treatments were not that meaningful on the functionalities but effective on coloring.

Chimeric spider silk proteins mediated by intein result in artificial hybrid silks.

Hybrid silks hold a great potential as specific biomaterials due to its controlled mechanical properties. To produce fibers with tunable properties, here we firstly made chimeric proteins in vitro, called W2C4CT and W2C8CT, with ligation of MaSp repetitive modules (C) with AcSp modules (W) by intein trans splicing technology from smaller precursors without final yield reduction. Intein mediated chimeric proteins form fibers at a low concentration of 0.4 mg/mL in 50 mM K3 PO4 pH 7.5 just drawn by hand. Hybrid fibers show smoother surface, and also have stronger chemical resistance as compared with fibers from W2CT (W fibers) and mixture of W2CT/C8CT (MHF8 fibers). Fibers from chimeric protein W2C4CT (HFH4) have improved mechanical properties than W fibers; however, with more C modules W2C8CT fibers (HFH8) properties decreased, indicates the length proportion of various modules is very important and should be optimized for fibers with specific properties. Generally, hybrid silks generated via chimeric proteins, which can be simplified by intein trans splicing, has greater potential to produce fibers with tunable properties. Our research shows that intein mediated directional protein ligation is a novel way to make large chimeric spider silk proteins and hybrid silks. © 2016 Wiley Periodicals, Inc. Biopolymers 105: 385-392, 2016.

Transmissible amyloid.

There are around 30 human diseases associated with protein misfolding and amyloid formation, each one caused by a certain protein or peptide. Many of these diseases are lethal and together they pose an enormous burden to society. The prion protein has attracted particular interest as being shown to be the pathogenic agent in transmissible diseases such as kuru, Creutzfeldt-Jakob disease and bovine spongiform encephalopathy. Whether similar transmission could occur also in other amyloidoses such as Alzheimer's disease, Parkinson's disease and serum amyloid A amyloidosis is a matter of intense research and debate. Furthermore, it has been suggested that novel biomaterials such as artificial spider silk are potentially amyloidogenic. Here, we provide a brief introduction to amyloid, prions and other proteins involved in amyloid disease and review recent evidence for their potential transmission. We discuss the similarities and differences between amyloid and silk, as well as the potential hazards associated with protein-based biomaterials.

Highly Efficient Fog Collection Unit by Integrating Artificial Spider Silks

Highly Efficient Fog Collection Unit by Integrating Artificial Spider Silks

Hybrid Silk Fibers Dry-Spun from Regenerated Silk Fibroin/Graphene Oxide Aqueous Solutions.

Regenerated silk fibroin (RSF)/graphene oxide (GO) hybrid silk fibers were dry-spun from a mixed dope of GO suspension and RSF aqueous solution. It was observed that the presence of GO greatly affect the viscosity of RSF solution. The RSF/GO hybrid fibers showed from FTIR result lower β-sheet content compared to that of pure RSF fibers. The result of synchrotron radiation wide-angle X-ray diffraction showed that the addition of GO confined the crystallization of silk fibroin (SF) leading to the decrease of crystallinity, smaller crystallite size, and new formation of interphase zones in the artificial silks. Synchrotron radiation small-angle X-ray scattering also proved that GO sheets in the hybrid silks and blended solutions were coated with a certain thickness of interphase zones due to the complex interaction between the two components. A low addition of GO, together with the mesophase zones formed between GO and RSF, enhanced the mechanical properties of hybrid fibers. The highest breaking stress of the hybrid fibers reached 435.5 ± 71.6 MPa, 23% improvement in comparison to that of degummed silk and 72% larger than that of pure RSF silk fiber. The hybrid RSF/GO materials with good biocompatibility and enhanced mechanical properties may have potential applications in tissue engineering, bioelectronic devices, or energy storage.

Optimizing structural and mechanical properties of recombinant spider wrapping silk fibres

Event Abstract Back to Event Optimizing structural and mechanical properties of recombinant spider wrapping silk fibres Nathan Weatherbee-Martin1, Lingling Xu1, Andre Hupe2, Douglas S. Fudge2, Laurent Kreplak3, Xiang-Qin Liu1 and Jan K. Rainey1, 4 1 Dalhousie University, Department of Biochemistry & Molecular Biology, Canada 2 University of Guelph, Department of Integrative Biology, Canada 3 Dalhousie University, Department of Physics & Atmospheric Science, Canada 4 Dalhousie University, Department of Chemistry, Canada Introduction: Spider silks are extraordinary biomaterials with diverse and impressive mechanical properties. This, along with their excellent bioresponse properties, makes them highly sought after for industrial and biomedical applications. However, it is infeasible to harvest large amounts of silk from spiders, due at least in part to their cannibalistic and territorial nature. Spiders produce up to seven types of silk, serving different biological functions such as locomotion (dragline silk) or wrapping of prey (wrapping silk). To date, artificial spider silk production has been problematic since limited information is available on spider silk fibre formation and structure, most of which pertains to dragline silk (the strongest), not wrapping silk (the toughest). In this study, we developed a fibre production method for recombinant wrapping silk protein and characterized the biophysical, mechanical and morphological properties of these fibres. Materials and Methods: Native wrapping silk protein in Argiope trifasciata consists of a core domain with at least 14 consecutive modular 200 amino acid repeat (“W”) units comprising a beads-on-a-string structure[1]. We have shown that recombinant 3 W-unit proteins (W3) produced in Escherichia coli can be manually pulled into fibres[2]. Automated fibre production (“spinning”) was achieved by solubilization of W3 in two different solvent-based spinning “dopes”, followed by extrusion, dehydration, and post-spin stretching. The secondary structure and aggregation state of W3 in each spinning dope were characterized by far-UV circular dichroism spectroscopy and dynamic light scattering, respectively, following similar characterization of W1[3]. Mechanical properties of each W3 fibre type were correlated to the overall molecular orientation assessed by birefringence and W3 secondary structure assessed by Raman spectromicroscopy. Results and Discussion: Recombinantly-produced W3 proteins were successfully solubilized into two different dopes and wet-spun into fibres. Solubilized W3 in both spinning dopes exhibited α-helical behaviour and evidence of self-assembly. Following wet-spinning and post-spin stretching, the mechanical strength of fibres formed from both spinning dopes dramatically improved, corresponding to an increase in β-sheet conformation and in birefringence. Interestingly, fibre extensibility differed dramatically as a function of spinning dope composition. These differences likely result from differential structuring and pre-assembly of W3 in the dopes Conclusion: The importance of spinning dope composition on mechanical properties and the importance of wet-spinning and post-spin stretching procedures in producing fibres of desirable structural and/or mechanical properties is clear. High-throughput synthetic recombinant wrapping silk fibre production and linking of molecular-level structure[1] to properties will facilitate tailoring of this material to a wide variety of applications. NSERC Discovery Grant funded; NSERC CGS-M Award; Bruce Stewart for technical assistance; Dr. J. Michael Lee for polarizing microscope access

In-vivo analysis of nerve regeneration after sciatic nerve injury in a rat model

We investigated the effectiveness of artificial silk-based nerve graft in nerve recovery and in helping regain motor functionality in rats. The purpose of this study is to test two questions: (1) Can nerve growth after transection be improved using artificial nerve guides? (2) Does nerve recovery have an impact on the walking patterns of the rat during the recovery period? Rats underwent sciatic nerve sectioning followed by reconstructive surgery. In these experiments, the sciatic nerve was substituted with an artificial silk guide that was biofunctionalized with growth factors and tested in vitro, as described previously. Three-dimensional motion capture and histological analyses were performed. After recovery periods of three and six months, the rats’ kinematics were analyzed during an over-ground walking task to evaluate the nerve recovery progress. The functional nerve recovery showed significant differences between the operated and non-operated sides of the rat, but no differences in their walking patterns during the recovery period. The histological analysis showed the reorganization of nerve tissue into the nerve as well as the presence of multiple neuronal extensions. Here, we have shown histological improvement in the absence of functional restoration after sciatic nerve transection and reconstruction at six months.