Fascinating Silk - Electrospinning, Contraction and Diffraction Experiments
- Fig. 1: a) Nephila clavata hanging from a silk dragline (from  with kind permission); b) electron diffraction pattern of the spidroin ß-sheet structure of Anelosimus eximius (silk courtesy of F. Vollrath, Oxford).
- Fig. 2: Stress-strain curves of silk from Bombyx mori reeled at different drawing speeds in comparison to natural cocoon silk, and of Nephila spider dragline silk (from  with kind permission).
- Fig. 3: Cocoons of the silkworm Bombyx mori, principle and experimental set-up of the electrospinning and continuous rotation collection of nanofibers from silk fibroin solution.
- Fig. 4: Contraction stress versus time of the cyclic response to wetting and drying of a fibroin fiber ribbon in the restrained state, and electron diffraction analysis of the participation of the oriented metastable silk-I structure (adapted from  with permission).
- Fig. 5: Super-contraction of highly crystalline restrained fibroin fibers at repeated wetting in ethanol, and partial relaxation during drying; electron diffraction confirms the pronounced crystalline ß-structure of the starting fibers (adapted from  with permission).
- The authors T. Yoshioka, A. Schaper, and Y. Kawahara have a wine-tasting session in Frankfurt/Main
Silk from spider and silkworm shows exceptional mechanical strength, toughness and extensibility if compared to man-made fibers. Other amazing properties are the occurrence of reversible cyclic contraction as well as of permanent super-contraction in fibroin nanofibers electrospun from regenerated Bombyx mori silk. We discovered the key role of the metastable silk-I modification and of the non-crystalline regions with oriented chains in the cyclic behavior under restrained conditions, and the role the silk-II ß-crystallinity plays in super-contraction.
Silk's Potentials as Functional Biomaterial
The interest in the physics and chemistry of natural silk is due to its extraordinary and versatile mechanical, chemical, and biological properties. The ‘cocktail' of protein sequences determines the fibers' special qualities along with a most complex nanoscale superstructure created by distinctive molecular organization principles during fiber formation under particular ambient conditions. Beside the silk's biocompatibility, its remarkable mechanical properties show promise of successful exploitation in biomimetic and other applications, such as artificial muscles or tendons, scaffolds for cell growth and tissue engineering, sutures for neurosurgery, or nanoscopic sensor devices.
Spider and Silkworm Silk
For spiders the dragline thread is a vital filament in that it supports the spider‘s weight during up and down movement, capturing prey, or building the web with an optimal safety coefficient (fig. 1a). The high mechanical stability and maximum efficiency of the thread is mainly provided by stiff nano-sized crystallites acting as reinforcing elements within the composite semicrystalline structure of the silk fibers. Low-dose single-fiber transmission electron diffraction enabled us to analyze the spidroin ß-structure that is formed by peptide chains with dominating alanine sequences in a pleated-sheet arrangement (fig. 1b).
Figure 2 illustrates the ultimate mechanical strength of spider dragline silk as opposed to the weaker pristine cocoon silk . The primary structure of silkworm fibroin differs from spider silk spidroin in the contributing amino acid sequences through the replacement of parts of the alanine repeats by glycine, leading to variations in the pleated-sheet ß-structure, to less stable crystal modifications with helical chain conformations, and thus to distinct changes in properties.
This is anything but surprising because evolution has assigned other functions to the silk produced by Bombyx mori: Spun into a cocoon the encapsulating silk serves as protection for the vulnerable pupal state of the larvae and its transformation into a moth. The cocoon is three to four centimeter in size and consists of one single filament up to one kilometer and a half in length. What figure 2 also shows is that silkworm silk artificially force-drawn under controlled drawing speed compare favorably with spider dragline silk. This evidences the crucial role spinning conditions play for adjusting the final mechanical properties . Other aspects of the mechanical behavior of silks are particularly amazing: one is irreversible super-contraction due to wetting of the fiber, the other is reversible cyclic contraction in response to alternating wetting and drying. Both phenomena were primarily assigned to spider silk only  but indications have been obtained recently with silkworm silk too . The fundamentals of the phenomena are far from being clear, more detailed studies appeared desirable.
Materials and Methods
The fabrication of micro- and nanofibers from regenerated Bombyx mori silk fibroin using electrospinning from solution followed by collection-induced structure orientation (fig. 3) enabled us systematically to study the effects of changes of the supermolecular structure on the complex contraction behavior . Raw Japanese silk was degummed in high-pressurized water, dissolved in CaCl2/CH3CH2OH/H2O (mole ratio 1 : 2 : 8), dialyzed, filtrated, and lyophilized. Nanofibers were e-spun from a 5 wt % solution in 1,1,1,3,3,3-hexafluoro-2-propanol (HFIP) at an applied voltage of 10 kV, and collected using a rotating disc or waterwheel-like collector at a take-up speed of 10 m/s. Variation of the super-molecular structure was achieved by post-treatment of the unrestrained fibers at 70°C in water vapor.
Main structure studies were performed using transmission electron microscopy and diffraction in a JEM-3010 microscope (Jeol Ltd., Japan) equipped with a slow scan CCD camera (Gatan Inc., USA). The combination of 300 kV accelerating voltage with high-sensitivity and fast image recording at low-dose large electron beam diameter illumination, along with eventual cryo-protection, is well-suited for reducing radiation damage at investigations of organic materials [6-10] like silk proteins. Additional data were obtained from X-ray diffraction investigations, preliminary in situ experiments on fiber deformation were performed at the electron synchrotron DESY (Hamburg, Germany) and at FRM-II (Garching, Germany) using neutron scattering of deuterated samples. Measurements of contraction stress and tensile properties were carried out in a tensile testing machine (Zwick Roell, Germany) with fiber ribbons.
Surprising Capabilities of Regenerated Fibroin Fibers
Silk which contained the metastable, water-soluble silk-I crystal phase and a high amount of molecular chains running parallel to the fiber axis showed cyclic contraction of its structure in dependence on the ambient humidity conditions (fig. 4). During the wetting treatment with ethanol the silk-I phase as well as hydrogen-bonded poorly defined crystalline linker regions become widely destroyed, but are re-formed during the drying stage. The numbered arrowheads in figure 4 indicate the successive steps of ethanol soaking. For the first time, this absolutely reversible behavior has been observed here in regenerated silkworm silk in its pure state. Dramatic changes of the mechanical properties were apparent when the starting structure has been changed (fig. 5): A fiber with high degree of silk-II ß-crystallinity shows strong irreversible super-contraction during wetting (see arrowheads) followed by slight relaxation in the drying stage. We assign the super-contraction to a raising crystallinity by lamellar overgrowth of the pre-existing ß-crystals.
Based on the results of mechanical tests and structure studies, a model of the contraction characteristics in regenerated silk has been developed .
The perspectives of application of silk-based materials mentioned in the introduction appear particularly reasonable in view of recent news about the successful production of silk analogue recombinant proteins by expression of synthetic genes in microbial systems. In this way, silk with specific properties should become available for a number of sophisticated applications sometime soon.
T.Y. is grateful to the Alexander von Humboldt Foundation for a fellowship. The authors thank Prof. Katsura Kojima and Prof. Tsunenori Kameda from the National Institute of Agrobiological Sciences, Tsukuba, for inspiring discussions.
 Osaki S.: Nature 384, 419 (1996)
 Shao Z. and Vollrath F.: Nature 418, 741 (2002)
 Blackledge T.A. et al.: J. Exp. Biol. 212, 1981-1989 (2009)
 Plaza G.R. et al.: Macromolecules 42, 8977-8982 (2009)
 Yoshioka T. et al.: Macromolecules 44, 7713-7718 (2011)
 Egerton R.F. et al.: Micron 43, 2-7 (2012)
 Schaper A.K. et al.: J. Microsc. 223, 88-95 (2006)
 Tsuji M.: Electron microscopy. Comprehensive Polymer Science, Pergamon, 785-839 (1988)
 Thomas E.L. and Ast D.G., Polymer 15, 37-41 (1974)
 Kobayashi K. and Ohara M.: Proc. 6th Int. Congr. Electr. Microsc., Kyoto, 579-580 (1966)
Dr. Andreas K. Schaper (corresponding author)
Materials Science Centre EM&Mlab
Philipps University of Marburg
Dr. Taiyo Yoshioka
Graduate School of Engineering
Toyota Technological Institute
Prof. Dr. Yutaka Kawahara
Department of Biological and Chemical Engineering