Identification and synthesis of novel biomaterials based on spider structural silk fibers

The diversity in function and mechanical behavior of spider silks, and the ability to produce these silks recombinantly, have tremendous potential in creating a new class of biomimetic materials. Here we investigate the structural and mechanical properties of pyriform silks from the golden orb-weaver, Nephila clavipes. Nanoscale indentation measurements using atomic force microscopy on natural pyriform silk suggests that this biomaterial has high toughness that may be suitable for dissipating high amounts of mechanical energy. We also observed the occurrence of highly organized nanocrystals within the pyriform silk fibers that may contribute to the remarkable energy dissipation capability of these silks. It has been demonstrated that poly-(Gly–Ala) and poly-Ala stretches within the internal block repeat modules of dragline silk fibroins form nanocrystals, and these nanocrystalline structures may be responsible for the high extensibility of the dragline silks. In contrast, amino acid sequence analysis shows that PySp2 does not contain the same motifs. In the absence of poly-(Gly–Ala) and poly-Ala repeats, we hypothesized that PySp2 contains new protein motifs sufficient to polymerize into functional structures. To investigate the functional contributions of these novel motifs during pyriform fiber formation, we expressed different recombinant PySp2 fibroins with various segments spanning its block repeat units. We demonstrate that PySp2 recombinant proteins with the Pro-rich sub-block domain (PXP motifs, where X= sub-set of the amino acids A, L, or R) and/or the Ser + Gln + Ala-rich sub-block domain (QQSSVAQS motifs) are sufficient for artificial fiber formation. Moreover, we show that recombinant PySp2 proteins that contain a single block repeat unit can self-assemble into foam-like nanostructures. Collectively, our findings support the use of PySp2 recombinant proteins for a wide range of biomimetic materials with morphologies ranging from fibers to porous structures.     

Origin of the variability of the mechanical properties of silk fibres: 2 The nanomechanics of single silkworm and spider fibres

The variability in mechanical stress–strain behaviour of various silks obtained from Bombyx mori silkworm and Nephila madagascarensis spider fibres has been studied by high resolution Raman analysis using the Raman shift induced by application of a controlled strain on the νN–H mode as a probe. Silk fibres exhibiting typical 1, 2, 3 and 4 Types have been selected from their characteristic tensile stress–strain behaviour. A perfect relationship between the nanomechanic (at the scale of the chemical bond) and macroscopic (silk single fibre) behaviour is observed as in the case of other polyamide fibres (natural keratin and synthetic PA66). Copyright © 2012 John Wiley & Sons, Ltd.     

Preparation and mechanical properties of layers made of recombinant spider silk proteins and silk from silk worm

Layers of recombinant spider silks and native silks from silk worms were prepared by spin-coating and casting of various solutions. FT-IR spectra were recorded to investigate the influence of the different mechanical stress occurring during the preparation of the silk layers. The solubility of the recombinant spider silk proteins SO1-ELP, C16, AQ24NR3, and of the silk fibroin from Bombyx mori were investigated in hexafluorisopropanol, ionic liquids and concentrated salt solutions. The morphology and thickness of the layers were determined by Atomic Force Microscopy (AFM) or with a profilometer. The mechanical behaviour was investigated by acoustic impedance analysis by using a quartz crystal microbalance (QCMB) as well as by microindentation. The density of silk layers (d<300 nm) was determined based on AFM and QCMB measurements. At silk layers thicker than 300 nm significant changes of the half-band-half width can be correlated with increasing energy dissipation. Microhardness measurements demonstrate that recombinant spider silk and sericine-free Bombyx mori silk layers achieve higher elastic penetration modules E_EP and Martens hardness values H_M than those of polyethylenterephthalate (PET) and polyetherimide (PEI) foils.     

Affinity of protein fibres towards sulfation

Wool, Bombyx mori and Antheraea pernyi (Tussah) silk fibres were treated with chlorosulfonic acid in pyridine and investigated by FT‐IR and FT‐Raman spectroscopies as well as mechanical measurements. The reactivity towards sulfation was found to decrease along the series: wool > Bombyx mori silk fibroin > Tussah silk fibroin, in agreement with weight gain which decreased along the same series. Accordingly, Tussah silk maintained its intrinsic tensile properties essentially unchanged upon the treatment, while for Bombyx mori silk fibroin, the tensile performance decreased sharply especially at longer reaction times. Sulfated wool was characterized by an increased fibre extensibility. New IR and Raman bands attributable to various vibrations of sulfated groups were detected in sulfated wool and to a lower extent in Bombyx mori silk fibroin fibres; all the fibres underwent conformational rearrangements upon sulfation, independent of the sulfation yield. Wool fibres treated with chlorosulfonic acid in pyridine bound considerable amounts of sulfate mainly through the hydroxyl groups of serine, threonine and tyrosine. Also, tryptophan and basic amino acids were found to participate to the reaction. B. mori silk fibroin fibres appeared to bind a minor amount of sulfate groups mainly trough the hydroxyl groups of Ser. Weight gain, spectroscopic and mechanical data are discussed in relation to the difference in fibre morphology, structure and crystallinity, as well as to the amount and accessibility of potentially reactive amino acids. Copyright © 2012 John Wiley & Sons, Ltd.     

Rheological characterization of hydrogels formed by recombinantly produced spider silk

Many fibrous proteins such as spider silks exhibit impressive mechanical properties and are highly biocompatible leading to many potential biomaterial applications. For applications such as tissue engineering, polymer hydrogels have been proposed as an effective means of producing porous but stable scaffolds. Here, nanofiber-based hydrogels were produced from engineered and recombinantly produced spider silk proteins. The silk nanofibers are stable semi-flexible polymers which assemble into hydrogel networks. We studied the hydrogel rheology and determined the concentration dependence of the elastic modulus. AFM images indicate that the nanofibers might assemble into branch-like structures, which would also be consistent with the measured rheological behavior. Since the developed spider silk hydrogels are stable over weeks and show a high elastic modulus at low volume fractions, they are well suited for a broad variety of applications.     

Predicting the mechanical properties of spider silk as a model nanostructured polymer

Spider silk is attractive because it is strong and tough. Moreover, an enormous range of mechanical properties can be achieved with only small changes in chemical structure. Our research shows that the full range of thermo-mechanical properties of silk fibres can be predicted from mean field theory for polymers in terms of chemical composition and the degree of order in the polymer structure. Thus, we can demonstrate an inherent simplicity at a macromolecular level in the design principles of natural materials. This surprising observation allows in depth comparison of natural with man-made materials.     

Statistical approaches for investigating silk properties

Amino acid repeats or motifs have engendered interest because of their significance for protein physical characteristics as well as folding properties. Spider dragline silk proteins are unique because they are composed of long repetitive sections and relatively short non-repetitive sections that are known to interact to generate the very peculiar mechanical and solubility properties of silk. Computational analysis compared with in vitro measurements suggest that the silks achieve their unique pattern of extreme solubility inside the spider glands/complete insolubility outside by correlating their repetitive hydrophobic regions through a type of stochastic resonance, generated by the addition of the non-repetitive sequences to a basically periodic hydrophobicity pattern. PACS 87.14.Ee; 87.15.Cc; 87.15.He; 02.50.Ey; 05.40.Ca     

Processing and modification of films made from recombinant spider silk proteins

Protein films represent an interesting class of materials with various possibilities for applications. We investigated films made of two different synthetic spider silk proteins derived from the garden spider’s (Araneus diadematus) two dragline silk proteins ADF-3 and ADF-4. Protein films cast from hexafluoroisopropanol solutions displayed a predominantly α-helical secondary structure. Processing such films with potassium phosphate or methanol resulted in a transition to a β-sheet rich structure. While as-cast films could be dissolved in water, processed β-sheet rich films were water insoluble. The chemical stability of processed films depended on the amino acid sequence of the respective protein employed. As a proof of principle, fluorescent probes or enzymes were covalently attached to the film surface. The presented approach provides a basis for designing tailor-made protein films using silk proteins as scaffold, in which the film properties can be controlled by genetic engineering of the underlying silks. PACS 68.55.Jk; 81.15.Lm; 87.14.Ee     

Resilient silk captures prey in black widow cobwebs

The gumfoot thread of a black widow (Latrodectus hesperus) spider’s cob web is a spring-loaded trap that yanks walking insects into the web. Since spider silks are known as energy dissipating materials, we investigated this trap to find out where the energy is stored. Using previously measured material properties, we modeled the gumfoot thread as a damped harmonic oscillator and compared it to high speed video analysis of prey capture. These measurements show that the gumfoot thread is plastically deformed during prey capture and cannot be the site of energy storage. We then measured the material properties of scaffolding silk that makes up the upper portion of the cob web. Scaffolding silk is highly resilient (90%) at strains less than 3%. This energy storage is sufficient to drive the oscillations seen in prey capture and is consistent with the measured kinematics. This study is the first demonstration of energy-storage as a primary biological function for spider silk. PACS 87.15.La; 81.40.Jj; 81.05.Lg     

Crystallinity and degradation of silk: correlations between analytical signatures and physical condition on ageing

Adequately characterising the physical condition of historic textiles, and understanding the microstructural changes that occur in these materials, is essential when considering appropriate conservation, display and storage strategies. Our work has concentrated on developing non-destructive or micro-destructive methodologies that will permit this for one of the most important historic fibres, silk. We have been able to demonstrate that correlations can be drawn between the physical deterioration of silk samples and certain measurable spectroscopic, chromatographic and chemical signatures. Understanding the way in which these signatures arise then allows the microstructural changes within the crystalline and amorphous content of the fibres to be investigated and more fully interpreted. The techniques developed by our group and by other researchers in the field include polarised FTIR-ATR (Pol-ATR) and near infrared (NIR) spectroscopy, HPLC microsampling analyses and pH measurements. The results of these analyses correlate to measurable mechanical properties and thus suggest that the physical state of historic silk fabrics might be adequately characterised for conservation purposes by such indirect micromethodology. PACS 81.05.Lg; 82.80.Gk; 87.14.Ee     

Ultrasound regulated flexible protein materials: Fabrication,structure and physical-biological properties

Ultrasound can be used in the biomaterial field due to its high efficiency, easy operation, no chemical treatment, repeatability and high level of control. In this work, we demonstrated that ultrasound is able to quickly regulate protein structure at the solution assembly stage to obtain the designed properties of protein-based materials. Silk fibroin proteins dissolved in a formic acid-CaCl₂ solution system were treated in an ultrasound with varying times and powers. By altering these variables, the silks physical properties and structures can be fine-tuned and the results were investigated with Fourier transform infrared spectroscopy (FTIR), X-ray diffraction (XRD), scanning electron microscopy (SEM), differential scanning calorimetry (DSC), thermogravimetric analysis (TGA), dynamic mechanical analysis (DMA), gas permeability and water contact angle measurements. Ultrasonic treatment aids the interactions between the calcium ions and silk molecular chains which leads to increased amounts of intermolecular β-sheets and α-helix. This unique structural change caused the silk film to be highly insoluble in water while also inducing a hydrophilic swelling property. The ultrasound-regulated silk materials also showed higher thermal stability, better biocompatibility and breathability, and favorable mechanical strength and flexibility. It was also possible to tune the enzymatic degradation rate and biological response (cell growth and proliferation) of protein materials by changing ultrasound parameters. This study provides a unique physical and non-contact material processing method for the wide applications of protein-based biomaterials.     

Effect of degumming time on silkworm silk fibre for biodegradable polymer composites

Recently, many studies have been conducted on exploitation of natural materials for modern product development and bioengineering applications. Apart from plant-based materials (such as sisal, hemp, jute, bamboo and palm fibre), animal-based fibre is a kind of sustainable natural materials for making novel composites. Silkworm silk fibre extracted from cocoon has been well recognized as a promising material for bio-medical engineering applications because of its superior mechanical and bioresorbable properties. However, when producing silk fibre reinforced biodegradable/bioresorbable polymer composites, hydrophilic sericin has been found to cause poor interfacial bonding with most polymers and thus, it results in affecting the resultant properties of the composites. Besides, sericin layers on fibroin surface may also cause an adverse effect towards biocompatibility and hypersensitivity to silk for implant applications. Therefore, a proper pre-treatment should be done for sericin removal. Degumming is a surface modification process which allows a wide control of the silk fibre's properties, making the silk fibre possible to be used for the development and production of novel bio-composites with unique/specific mechanical and biodegradable properties. In this paper, a cleaner and environmentally friendly surface modification technique for tussah silk in polymer based composites is proposed. The effectiveness of different degumming parameters including degumming time and temperature on tussah silk is discussed through the analyses of their mechanical and morphological properties. Based on results obtained, it was found that the mechanical properties of tussah silk are affected by the degumming time due to the change of the fibre structure and fibroin alignment.     

Nanomechanics of new materials — AFM and computer modelling studies of trichoptera silk

Caddisfly (Trichopera) can glue diverse material underwater with a silk fiber. This makes it a particularly interesting subject for biomimetcs. Better understanding of silk composition and structure could lead to an adhesive capable to close bleeding wounds or to new biomaterials. However, while spiderweb or silkworm secretion is well researched, caddisfly silk is still poorly understood. Here we report a first nanomechanical analysis of H. Angustipennis caddisfly silk fiber. An Atomic Force Microscope (AFM) imaging shows dense 150 nm bumps on silk surface, which can be identified as one of features responsible for its outstanding adhesive properties. AFM force spectroscopy at the fiber surface showed, among others, characteristic saw like pattern. This pattern is attributed to sacrificial bond stretching and enhances energy dissipation in mechanical deformation. Similarities of some force curves observed on Tegenaria domestica spiderweb and caddisfly silk are also discussed. Steered Molecular Dynamics simulations revealed that the strength of short components of Fib-H HA species molecules, abundant in Trichoptera silk is critically dependent on calcium presence.     

Origin of the variability of the mechanical properties of silk fibres: 1 ‐ The relationship between disorder,hydration and stress/strain behaviour

The causes of the variability in mechanical behaviour of various silks obtained from silkworms (Bombyx mori, Antheraea/Tussah) and spiders (Nephila madagascarensis) have been studied by tensile uniaxial tests, differential scanning calorimetry and Raman microspectrometry. Analyses of tensile stress–strain curves recorded for different silkworm and spider baves and single fibres, undergoing different histories (age, degumming, thermal/chemical treatments), allow the behaviour to be classified into five groups and correlated with the fibroin/spidroin structural status: (1) Type I exhibits linear elastic behaviour and then a quasi plateau, (2) Type II has a two‐step linear elastic behaviour with kinks then a quasi plateau, (3) Type III exhibits a smooth transition from a linear behaviour to a plateau, (4) Type IV has a behaviour rather similar to that of Type I but above ~8–12% hardening occurs, and (5) Type V results in the breakage of fibres during the first elastic stage. Unambiguously, Type IV is more frequent for degummed, very dried fibres and Type III for water‐saturated fibres. The most striking Raman signature differences are observed in the Raman intensity of the amorphous/‘ordered’ νN–H and H₂O band components and correlated to the water content and disorder degree according to the calorimetry study. Types I and II are frequently observed for fresh (largely amorphous) spun fibres/baves. Type V is characteristic of degraded fibres. Type IV represents the most ordered state. The similar stress–strain types for dried silkworm and spider fibres show that the general difference in amino acid contents is not the most pertinent parameter for the mechanical behaviour. Copyright © 2011 John Wiley & Sons, Ltd.     

Origin of the variability of the mechanical properties of silk fibres: 3. Order and macromolecule orientation in Bombyx mori bave,hand‐stretched strings and Nephila madagascarensis spider fibres

The comparison of the low wavenumber of polarized Raman spectra (50–300 cm^–1) from Bombyx mori (fresh cocoons fibres, hand‐stretched ‘Crins de Florence’ strings from the gland content, dried gland, regenerated silk films) and Nephila madagascarensis silks reveals the high polarisation of fibre modes and the absence of polarisation for dried gland and regenerated silk films. This is consistent with X‐ray diffraction measurements. The orientation of the fibroin/spidroin chains is due to the stretching during production, as for advanced synthetic fibres. The bandwidth of the ‘ordered chains’ signature is almost the same for the different fibres. However, the degree of polarisation seems to be higher in the case of spider fibre. The huge bandwidth of low wavenumber components of regenerated films indicates high disorder. Measurements along the fibre point out conformation changes with a periodicity (~20 mm) related to the silkworm head motion during the fabrication of the cocoon. Copyright © 2012 John Wiley & Sons, Ltd.     

Mechanical properties of silk: interplay of deformation on macroscopic and molecular length scales

Using an in situ combination of tensile tests and x-ray diffraction, we have determined the mechanical properties of both the crystalline and the disordered phase of the biological nanocomposite silk by adapting a model from linear viscoelastic theory to the semicrystalline morphology of silk. We observe a strong interplay between morphology and mechanical properties. Silk's high extensibility results principally from the disordered phase; however, the crystals are also elastically deformed.     

Effect of low-temperature plasma treatment on tailorability and thermal properties of wool fabrics

Dielectric barrier discharge type of plasma reactor was used for the low-temperature plasma (LTP) treatment of the wool fabrics. Air was used as the non-polymerizing gas for the plasma treatment at different time intervals. Low-stress mechanical properties of the treated and untreated wool fabrics were evaluated using Siro-fast technique which revealed that the tensile, bending, compression, shear, dimensional stability and surface properties were altered after the LTP treatment. Other properties such as thermal conductivity, thermal resistance and pilling propensity were also evaluated. The surface topographical changes of the wool fibres after LTP treatment were analysed by scanning electron microscopy. The changes in these properties are supposed to be related closely to the interfibre and interyarn frictional force and increased surface area of the fibres induced by the etching effect of plasma.     

Minerals discovered in paleolithic black pigments by transmission electron microscopy and micro-X-ray absorption near-edge structure

Spiders produce high performance fibers that compare favorably with the best manmade fibers in strength and toughness. The amino acid sequences of silk proteins have been determined for a number of silk types and species, revealing extensive variation. This variation in sequence is hypothesized to confer different material properties. However, the material properties of silk have been characterized from only a few ecologically similar species, even though spiders are extremely diverse. Using a Nano Bionix® tensile tester, we measured mechanical properties of one type of silk, the dragline, from a broad sample of spider species. These taxa included orb-weavers and representatives of other lineages of true spiders that do not spin aerial capture webs. We found that all of the species sampled produce high-performance dragline fibers, suggesting that the remarkable properties of dragline silk predate the origin of the aerial orb-web. However, we report significant variation in all of the material properties measured. Furthermore, material properties tend not to be correlated, implying that different properties may have been selected upon in different spider lineages. We suggest that the spectrum of dragline silk sequences and material properties that have been produced over evolutionary time provides a rich resource for the design of biomimetic silk fibers.     

Mechanical properties of thin films of graphene materials: A study on their structural quality and functionalities

Several studies have been done on physiochemical properties of thin films of graphene materials, but less on their mechanical properties. The mechanical properties such as tensile and storage modulus of films of graphene oxide (GO), different reduced graphene oxides (rGO), functionalised reduced graphene oxide (frGO) and a few layers graphene (graphene) were analysed in this study. During syntheses processes, a range of variations occurs due to different reducing agents and functionalising components used; this affects or changes the mechanical properties of the materials. In addition, it has become vital to comprehend the mechanical properties of these films as the potential applications such as sensor and electrodes demand extended life cycles or lifetime. It has been found that the ultimate tensile strength (UTS), tensile modulus, and storage modulus vary across all the samples that highly depend on nature/efficiency of reducing agent used, amount of impurities such as oxygen functional groups and defect density such as discrepancies/holes in the aromatic structure. The highest UTS and modulus have been identified with a few layers graphene and with hydroiodic acid reduced GO among the rGOs. The frGO shows almost similar properties to that of graphene.     

Polypropylene-natural fibre composites. Analysis of fibre structure modification during compounding and its influence on the final properties

The final properties of composite materials are highly dependent on the residual geometrical parameters (length, diameter, aspect ratio), orientation and distribution of the fibres in the matrix, which in turn are related to the processing conditions. This study analysed the fibre structure variation during the processing of a polypropylene matrix reinforced with cellulose flax pulp for different reinforcement concentrations. The fibre's geometrical parameters, length, diameter and aspect ratio have been measured and their statistical distributions have been assessed for each concentration. Furthermore, the effect of the microstructure variation on the final mechanical properties was analysed. In particular, changes in the interfacial area were evaluated based on the hypothesis that the fibres were cylindrical in shape and considering the average values of the diameters and the lengths calculated using a statistical distribution approach. The fibre interfacial area after the process decreases as the fibre concentration increases and this evaluation explains how the adhesion methods that are used for fibre surface modification fail because of the decrement in the modifier interfacial density. The Halpin–Tsai approach was used to model the experimental data obtained from tensile tests for different composites, so as to confirm the effect of fibre parameters, such as aspect ratio and interfacial area values, in the PP/cellulose blends final properties.