Short and long range order of the morphology of silk from Latrodectus hesperus (Black Widow) as characterized by atomic force microscopy.

The surfaces of both stretched and unstretched silk threads from the cobweb weaver, Latrodectus hesperus (Black Widow) have been examined by atomic force microscopy (AFM). AFM images of cobweb scaffolding threads show both unordered and highly ordered regions. Two types of fibers within the threads were observed: thicker (approximately 300 nm in diameter) fibers oriented parallel to the thread axis and thinner (10-100 nm) fibrils oriented across the thread axis. While regions which lacked parallel fibers or fibrils were observed on threads at all strain values, the probability of observing fibers and/or fibrils increased with strain. High-resolution AFM images show that with increasing strain, both mean fiber and fibril diameters decrease and that fibrils align themselves more closely with the thread axis. The observation of fibers and fibrils within the cobweb threads has implications for current models of the secondary and tertiary structure and organization of spider silk.     

The common house spider alters the material and mechanical properties of cobweb silk in response to different prey

Many spiders depend upon webs to capture prey. Web function results from architecture and mechanical performance of the silk. We hypothesized that the common house spider, Achaearanea tepidariorum, would alter the mechanical performance of its cobweb in response to different prey by varying the structural and material properties of its silk. We fed spiders either large, high kinetic energy crickets or small, low kinetic energy pillbugs for 1 week and then examined their freshly spun silk. We separated mechanical performance into structural and material effects. We measured both types of properties for silk threads collected directly from cobwebs to test for "tuning" of silk performance to different aspects of prey capture. We compared silk from two different functional regions of the cobweb-sticky gumfooted threads that adhere directly to prey and supporting threads that maintain web integrity. Supporting threads from cricket-fed spiders were stiffer and tougher than supporting threads from pillbug-fed spiders. Both types of silk from cricket-fed spiders broke at higher loads than silk from pillbug-fed spiders. We explain this variation using a simple model of forces exerted by prey and spiders on single threads and propose potential mechanisms for this change in material properties. Two alternative, nonexclusive, hypotheses are suggested by our data. Spiders may tune silk to different types of prey by spinning threads that are able to hold prey without deforming permanently. Alternatively, as spider's body mass differed dramatically between the two feeding regimes, spiders may tune silk to their own body mass.     

Mussel collagen molecules with silk-like domains as load-bearing elements in distal byssal threads

Mechanically stressed biological materials like tendon, spider silk or mussel byssal threads are typically composite materials comprising multi-domain proteins, in which molecular building blocks contribute to overall material function. Mussel byssal threads are the anchorage of sessile mytilid mussels, which withstand recurring external loads from waves and tides. A single thread is elastic and ductile proximally, while the distal portion exhibits an extraordinary stiffness and toughness with a transient gradient of both mechanical features along the thread. The main components of byssal threads include a set of various collagen-like structural proteins (preCols) consisting of a collagenous core sequence flanked by globular domains. Here, structural analysis using polarized Fourier-transform infrared spectroscopy (FTIR) on stretched distal portions of mussel byssal threads determines the impact of external linear load on various molecular moieties. It is concluded that the preCol collagenous core domain is the main load-bearing element in distal byssal threads, while polyalanine beta-sheets in the flanking domains, similar to those found in spider silk proteins, provide high stiffness at low strains. Load dissipation is mediated by domain stretching of amorphous glycine-rich helical moieties followed by complete unfolding of the preCol flanking domains.     

Ultrastructure of spider thread anchorages

Spiders attach silken threads to substrates by means of glue-coated nanofibers (piriform silk), spun into disc-like structures. The organization and ultrastructure of this nano-composite silk are largely unknown, despite their implications for the biomechanical function and material properties of thread anchorages. In this work, the ultrastructure of silken attachment discs was studied in representatives of four spider families with Transmission Electron Microscopy to facilitate a mechanistic understanding of piriform silk function across spiders. Based on previous findings from comparative studies of piriform silk gland morphology, we hypothesized that the fibre-glue proportion of piriform silk differs in different spiders, while the composition of fibre and glue fractions is consistent. Results confirmed large differences in the relative proportion of glue with low amounts in the orb weaver Nephila senegalensis (Araneidae) and the hunting spider Cupiennius salei (Ctenidae), larger amounts in the cobweb spider Parasteatoda tepidariorum (Theridiidae) and a complete reduction of the fibrous component in the haplogyne spider Pholcus phalangioides (Pholcidae). We rejected our hypothesis that glue ultrastructure is consistent. The glue is a colloid with polymeric and fluid fractions that strongly differ in proportions and assembly. We further confirmed that in all species studied both dragline and piriform silk fibres do not make contact with the environmental substrate. Instead, adhesion is established by a thin dense skin layer of the piriform glue. These results advance our understanding of piriform silk function and the interspecific variation of its properties, which is significant for spider biology, web function and the bioengineering of silk.     

Molecular and mechanical properties of major ampullate silk of the black widow spider, Latrodectus hesperus

Molecular and material properties of major ampullate silk were studied for the cobweb-building black widow spider Latrodectus hesperus. Material properties were measured by stretching the silk to breaking. The strength was 1.0 +/- 0.2 GPa, and the extensibility was 34 +/- 8%. The secondary structure of the major ampullate silk protein was studied using carbon-13 NMR spectroscopy. Alanine undergoes a transition from a coiled structure in pre-spun silk to a beta sheet structure in post-spun silk. We have also isolated two distinct cDNAs (both about 500 bp) which encode proteins similar to major ampullate spidroin 1 and 2 (MaSp1 and MaSp2). The MaSp1-like silk contains polyalanine runs of 5-10 residues as well as GA and GGX motifs. The MaSp2-like silk contains polyalanine runs of varying length as well as GPG(X)(n) motifs. L. hesperus major ampullate silk is more like major ampullate silk from other species than other L. hesperus silks.     

Biomechanical variation of silk links spinning plasticity to spider web function

Spider silk is renowned for its high tensile strength, extensibility and toughness. However, the variability of these material properties has largely been ignored, especially at the intra-specific level. Yet, this variation could help us understand the function of spider webs. It may also point to the mechanisms used by spiders to control their silk production, which could be exploited to expand the potential range of applications for silk. In this study, we focus on variation of silk properties within different regions of cobwebs spun by the common house spider, Achaearanea tepidariorum. The cobweb is composed of supporting threads that function to maintain the web shape and hold spiders and prey, and of sticky gumfooted threads that adhere to insects during prey capture. Overall, structural properties, especially thread diameter, are more variable than intrinsic material properties, which may reflect past directional selection on certain silk performance. Supporting threads are thicker and able to bear higher loads, both before deforming permanently and before breaking, compared with sticky gumfooted threads. This may facilitate the function of supporting threads through sustained periods of time. In contrast, sticky gumfooted threads are more elastic, which may reduce the forces that prey apply to webs and allow them to contact multiple sticky capture threads. Therefore, our study suggests that spiders actively modify silk material properties during spinning in ways that enhance web function.     

Study of protein conformation and orientation in silkworm and spider silk fibers using Raman microspectroscopy

Raman microspectroscopy has been used for the first time to determine quantitatively the orientation of the beta-sheets in silk monofilaments from Bombyx mori and Samia cynthia ricini silkworms, and from the spider Nephila edulis. It is shown that, for systems with uniaxial symmetry such as silk, it is possible to determine the order parameters P2 and P4 of the orientation distribution function from intensity ratios of polarized Raman spectra. The equations allowing the calculation of P2 and P4 using polarized Raman microspectroscopy for a vibration with a cylindrical Raman tensor were first derived and then applied to the amide I band that is mostly due to the C=O stretching vibration of the peptide groups. The shape of the Raman tensor for the amide I vibration of the beta-sheets was determined from an isotropic film of Bombyx mori silk treated with methanol. For both the Bombyx mori and Samia cynthia ricini fibroin fibers, the values of P2 and P4 obtained are equal to -0.36 +/- 0.03 and 0.19 +/- 0.02, respectively, even though the two types of silkworm fibroins strongly differ in their primary sequences. For the Nephila edulis dragline silk, values of P2 and P4 of -0.32 +/- 0.02 and 0.13 +/- 0.02 were obtained, respectively. These results clearly indicate that the carbonyl groups are highly oriented perpendicular to the fiber axis and that the beta-sheets are oriented parallel to the fiber axis, in agreement with previous X-ray and NMR results. The most probable distribution of orientation was also calculated from the values of P2 and P4 using the information entropy theory. For the three types of silk, the beta-sheets are highly oriented parallel to the fiber axis. The orientation distributions of the beta-sheets are nearly Gaussian functions with a width of 32 degrees and 40 degrees for the silkworm fibroins and the spider dragline silk, respectively. In addition to these results, the comparison of the Raman spectra recorded for the different silk samples and the polarization dependence of several bands has allowed to clarify some important band assignments.     

Use of spider silk fibres as an innovative material in a biocompatible artificial nerve conduit

Defects of peripheral nerves still represent a challenge for surgical nerve reconstruction. Recent studies concentrated on replacement by artificial nerve conduits from different synthetic or biological materials. In our study, we describe for the first time the use of spider silk fibres as a new material in nerve tissue engineering. Schwann cells (SC) were cultivated on spider silk fibres. Cells adhered quickly on the fibres compared to polydioxanone monofilaments (PDS). SC survival and proliferation was normal in Live/Dead assays. The silk fibres were ensheathed completely with cells. We developed composite nerve grafts of acellularized veins, spider silk fibres and SC diluted in matrigel. These artificial nerve grafts could be cultivated in vitro for one week. Histological analysis showed that the cells were vital and formed distinct columns along the silk fibres. In conclusion, our results show that artificial nerve grafts can be constructed successfully from spider silk, acellularized veins and SC mixed with matrigel.     

Behavioural and biomaterial coevolution in spider orb webs

Mechanical performance of biological structures, such as tendons, byssal threads, muscles, and spider webs, is determined by a complex interplay between material quality (intrinsic material properties, larger scale morphology) and proximate behaviour. Spider orb webs are a system in which fibrous biomaterials—silks—are arranged in a complex design resulting from stereotypical behavioural patterns, to produce effective energy absorbing traps for flying prey. Orb webs show an impressive range of designs, some effective at capturing tiny insects such as midges, others that can occasionally stop even small birds. Here, we test whether material quality and behaviour (web design) co‐evolve to fine‐tune web function. We quantify the intrinsic material properties of the sticky capture silk and radial support threads, as well as their architectural arrangement in webs, across diverse species of orb‐weaving spiders to estimate the maximum potential performance of orb webs as energy absorbing traps. We find a dominant pattern of material and behavioural coevolution where evolutionary shifts to larger body sizes, a common result of fecundity selection in spiders, is repeatedly accompanied by improved web performance because of changes in both silk material and web spinning behaviours. Large spiders produce silk with improved material properties, and also use more silk, to make webs with superior stopping potential. After controlling for spider size, spiders spinning higher quality silk used it more sparsely in webs. This implies that improvements in silk quality enable ‘sparser’ architectural designs, or alternatively that spiders spinning lower quality silk compensate architecturally for the inferior material quality of their silk. In summary, spider silk material properties are fine‐tuned to the architectures of webs across millions of years of diversification, a coevolutionary pattern not yet clearly demonstrated for other important biomaterials such as tendon, mollusc byssal threads, and keratin.     

Spider silk fibres in artificial nerve constructs promote peripheral nerve regeneration

Abstract.  Objective : In our study, we describe the use of spider silk fibres as a new material in nerve tissue engineering, in a 20-mm sciatic nerve defect in rats. Materials and methods : We compared isogenic nerve grafts to vein grafts with spider silk fibres, either alone or supplemented with Schwann cells, or Schwann cells and matrigel. Controls, consisting of veins and matrigel, were transplanted. After 6 months, regeneration was evaluated for clinical outcome, as well as for histological and morphometrical performance. Results : Nerve regeneration was achieved with isogenic nerve grafts as well as with all constructs, but not in the control group. Effective regeneration by isogenic nerve grafts and grafts containing spider silk was corroborated by diminished degeneration of the gastrocnemius muscle and by good histological evaluation results. Nerves stained for S-100 and neurofilament indicated existence of Schwann cells and axonal re-growth. Axons were aligned regularly and had a healthy appearance on ultrastructural examination. Interestingly, in contrast to recently published studies, we found that bridging an extensive gap by cell-free constructs based on vein and spider silk was highly effective in nerve regeneration. Conclusion : We conclude that spider silk is a viable guiding material for Schwann cell migration and proliferation as well as for axonal re-growth in a long-distance model for peripheral nerve regeneration.     

Spider silks and their applications

Spider silks are characterized by remarkable diversity in their chemistry, structure and functions, ranging from orb web construction to adhesives and cocoons. These unique materials have prompted efforts to explore potential applications of spider silk equivalent to those of silkworm silks, which have undergone 5,000 years of domestication and have a variety of uses, from textiles to biomedical materials. Recent progress in genetic engineering of spider silks and the development of new chimeric spider silks with enhanced functions and specific characteristics have advanced spider silk technologies. Further progress in yields of expressed spider-silk proteins, in the control of self-assembly processes and in the selective exploration of material applications is anticipated in the future. The unique features of spider silks, the progress and challenges in the cloning and expression of these silks, environmentally triggered silk assembly and disassembly and the formation of fibers, films and novel chimeric composite materials from genetically engineered spider silks will be reviewed.     

Interactions between Spider Silk and Cells – NIH/3T3 Fibroblasts Seeded on Miniature Weaving Frames

Background

Several materials have been used for tissue engineering purposes, since the ideal matrix depends on the desired tissue. Silk biomaterials have come to focus due to their great mechanical properties. As untreated silkworm silk has been found to be quite immunogenic, an alternative could be spider silk. Not only does it own unique mechanical properties, its biocompatibility has been shown already in vivo. In our study, we used native spider dragline silk which is known as the strongest fibre in nature.

Methodology/Principal Findings

Steel frames were originally designed and manufactured and woven with spider silk, harvesting dragline silk directly out of the animal. After sterilization, scaffolds were seeded with fibroblasts to analyse cell proliferation and adhesion. Analysis of cell morphology and actin filament alignment clearly revealed adherence. Proliferation was measured by cell count as well as determination of relative fluorescence each after 1, 2, 3, and 5 days. Cell counts for native spider silk were also compared with those for trypsin-digested spider silk. Spider silk specimens displayed less proliferation than collagen- and fibronectin-coated cover slips, enzymatic treatment reduced adhesion and proliferation rates tendentially though not significantly. Nevertheless, proliferation could be proven with high significance (p<0.01).

Conclusion/Significance

Native spider silk does not require any modification to its application as a biomaterial that can rival any artificial material in terms of cell growth promoting properties. We could show adhesion mechanics on intracellular level. Additionally, proliferation kinetics were higher than in enzymatically digested controls, indicating that spider silk does not require modification. Recent findings concerning reduction of cell proliferation after exposure could not be met. As biotechnological production of the hierarchical composition of native spider silk fibres is still a challenge, our study has a pioneer role in researching cellular mechanics on native spider silk fibres.     

Alteration of tubuliform silk gland cytoarchitecture with the reproductive cycle of the Western black widow spider,Latrodectus hesperus

The protein synthetic and secretory activity of spider tubuliform glands is known to be coordinated with the reproductive stage of the spider. For spiders that produce multiple egg cases, such as the black widow Latrodectus hesperus, this means that the cells that make up the tubuliform gland cycle from minimal to maximal silk protein synthesis and exocytosis as the spider transitions from early vitellogenesis to a gravid state and back. The impact of these transitions on the cells that form the tubuliform gland has yet to be characterized. The entire tubuliform gland undergoes an elastic deformation, doubling in size in response to the accumulation and depletion of egg case silk proteins within its lumen. Similarly, the diversity and organization of organelles within the cytoplasm of the secretory epithelial cells that make up the wall of the tubuliform gland change with the reproductive stage of the spider. Progression of a spider from early to late vitellogenesis is accompanied by decondensed nucleoli and distention of the rough endoplasmic reticulum, markers of protein synthetic activity. The presumed silk proteins that fill the lumen of the tubuliform gland of a gravid spider include a fibrous matrix with homogeneous spherical inclusions. These components are also present within the cytoplasm of the cell; however, only the fibrous material appears to be enclosed by membranous organelles. Transition of the tubuliform gland from peak silk synthesis back to a quiescent state is marked by the appearance of multivesicular bodies and organelles resembling phagophores and autophagosomes, suggestive of a role for autophagy in the process of recovery. The reproducible cellular dynamics of the tubuliform silk gland of the black widow spider makes it a potential model system for study of the regulation of silk gene expression, endomembrane transport, and exocytosis of silk proteins and autophagy.     

Mechanical testing of spider silk at cryogenic temperatures

A method and results for mechanical testing of spider silk in extreme environments is presented. In particular, silk from the spider Steatoda triangulosa is harvested, and samples are subjected to cryogenic temperatures by means of liquid nitrogen submersion. Samples are destructively tested while immersed in liquid nitrogen, and the stress-strain characteristics are compared to those of silk at room temperature. The strength, elasticity, and toughness of the cryogenically submersed silk are determined. It is found that on average, silk is 64% stronger while immersed in liquid nitrogen (i.e., at -196°C). The testing method could also be used for testing of silk in chemically hostile environments.     

Spider silks from plants – a challenge to create native-sized spidroins

Silk threads from spiders exhibit extraordinary mechanical properties, such as superior toughness and elasticity. Spider silks consist of several different large repetitive proteins that act as the basic materials responsible for these outstanding features. The production of spider silk protein variants in plants opens up new horizons in the production and functional investigation that enable the use of spider silks in innovative material development, nanotechnology and biomedicine in the future. This review summarizes and discusses production of spider silk protein variants in plants, especially with regards to plant expression systems, purification strategies, and characteristics of spider silk variants. Furthermore, the challenge of producing native-sized recombinant spidroins in planta is outlined, presenting three different strategies for achieving these high repetitive proteins with the help of non-repetitive C-terminal domains, crosslinking transglutaminase, and self-linking inteins. The potential of these fascinating proteins in medicine is also highlighted.     

Modeling of mechanical properties and structural design of spider web

With a unique combination of strength and toughness among materials, spider silk is the model for engineering materials. This paper presents the stress-strain behavior of Nephila clavipes spider silk under tension, transverse compression, and torsional deformation obtained by a battery of micro testing equipment. The experimental results showed significantly higher toughness than the state-of-the-art fibers in tension and in transverse compression. Higher shear modulus was also observed for the spider silk comparing to other liquid crystalline fibers such as aramid fibers. On the basis of the experimental results finite element analysis is used to simulate static and dynamic properties of spider web and to explore the role of both material properties and architectural design in its structural integrity and mechanical performance.     

Design, expression and solid-state NMR characterization of silk-like materials constructed from sequences of spider silk, Samia cynthia ricini and Bombyx mori silk fibroins

Silk has a long history of use in medicine as sutures. To address the requirements of a mechanically robust and biocompatible material, basic research to clarify the role of repeated sequences in silk fibroin in its structures and properties seems important as well as the development of a processing technique suitable for the preparation of fibers with excellent mechanical properties. In this study, three silk-like protein analogs were constructed from two regions selected from among the crystalline region of Bombyx mori silk fibroin, (GAGSGA)(2), the crystalline region of Samia cynthia ricini silk fibroin, (Ala)(12), the crystalline region of spider dragline silk fibroin, (Ala)(6), and the Gly-rich region of spider silk fibroin, (GGA)(4). The silk-like protein analog constructed from the crystalline regions of the spider dragline silk and B. mori silk fibroins, (A(6)SCS)(8), that constructed from the crystalline regions of the S. c.ricini and B. mori silk fibroins, (A(12)SGS)(4), that constructed from and the crystalline region of S. c.ricini silk fibroin and the glycine-rich region of spider dragline silk fibroin, (A(12)SGS)(4),were expressed their molecular weights being about 36.0 kDa, 17.0 kDa and 17.5 kDa, respectively in E. coli by means of genetic engineering technologies. (A(12)SCS)(4) and (A(12)SGS)(4 )undergo a structural transition from alpha-helix to beta-sheet on a change in the solvent treatment from trifluoroacetic acid (TFA) to formic acid (FA). However, (A(6)SCS)(8) takes on the beta-sheet structure predominantly on TFA treatment and FA treatment. Structural analysis was performed on model peptides selected from spider dragline and S. c.ricini silks by means of (13)C CP/MAS NMR.     

High-Toughness Silk Produced by a Transgenic Silkworm Expressing Spider (Araneus ventricosus) Dragline Silk Protein

Spider dragline silk is a natural fiber that has excellent tensile properties; however, it is difficult to produce artificially as a long, strong fiber. Here, the spider (Araneus ventricosus) dragline protein gene was cloned and a transgenic silkworm was generated, that expressed the fusion protein of the fibroin heavy chain and spider dragline protein in cocoon silk. The spider silk protein content ranged from 0.37 to 0.61% w/w (1.4–2.4 mol%) native silkworm fibroin. Using a good silk-producing strain, C515, as the transgenic silkworm can make the raw silk from its cocoons for the first time. The tensile characteristics (toughness) of the raw silk improved by 53% after the introduction of spider dragline silk protein; the improvement depended on the quantity of the expressed spider dragline protein. To demonstrate the commercial feasibility for machine reeling, weaving, and sewing, we used the transgenic spider silk to weave a vest and scarf; this was the first application of spider silk fibers from transgenic silkworms.