Dr Chris Holland, Department of Materials Science and Engineering, University of Sheffield, UK
When asked by my Dphil supervisor if I would like to look into silk rheology I didn’t know what rheology was and how it would shape my research career. Five years on when asked what I do for a living I now reply with enthusiasm “I study how spiders and silkworms spin silk”
Rheology concerns the study of the flow and deformation of matter. Here I will discuss how we have found use for this technology to study features of the natural world, specifically in understanding how Nature produces one of her finest materials, silk. More usual for this technique are questions about engineering materials such as the visco-elastic properties of paints, polymers and food.
Silk is a biological material, a protein fibre produced through a process called spinning. It is unique to the arthropods, where it is wide spread. The most famous silk producers are the spider and many moths, foremost among them the silkworm. However it may surprise you that silk is spun by some bee’s and ants too! The evolutionary success of silk can be attributed to its versatility, being used for protection from predators to capturing of prey with reproduction and dispersal of young in-between. Spiders are exceptional in producing more than one type of silk. Indeed they are able to make up to seven different types of silk, with each fibre possessing mechanical properties specifically tuned to their biological function.
Whilst silk is clearly an attractive material in nature man has also been captured by its qualities. The silk of the Chinese silkworm (Bombyx mori) is the world’s oldest commercial fibre, dating back over 5000 years. Being one of only two truly domesticated insects (bees being the other) today Bombyx are reared in the millions and current worldwide production of silk is in excess of 100,000 metric tonnes with a value of over $1bn (before the added value of high-end textiles). However the reasons for the commercial success of silk go way beyond simple aesthetics. Spider silk in particular is weight for weight stronger than steel and it is much tougher than Kevlar. It could be considered the pinnacle of any fibre production process and quite unlike its industrial counterparts silk is ‘green’, being produced sustainably at room temperatures, using only water as a solvent and being completely biodegradable.
Yet despite our fascination with this supermaterial for five millennia we know surprisingly little about its evolutionary origins and synthesis. However because silk has been selected primarily for its physical properties and to perform outside the body, it is possible to translate the testing methodologies we use to characterise our man-made materials to try to understand why silk is so interesting and important.
One such technique applied to silk has been straightforward tensile testing. By stretching silk fibres we are able to determine many of their physical properties, from how stiff they are to how much energy they can absorb (toughness). The most informative animal to use in this approach is the multi-talented spider. By carefully ‘silking’ the spider onto a reel we are able to control the production rate (reeling speed) of a silk and in conjunction with varying the temperature and humidity it is possible to produce silks from the same feedstock with a wide range of mechanical properties. By repeating this for different spiders we begin to see overlaps in these properties, to the extent that even silks with different amino acid compositions can be tuned to have almost identical material properties. This implies that the processing of a silk is as important as the feedstock in defining a silk fibre’s performance.
Therefore a pertinent question to ask is how is silk spun? Quite unlike other biological materials which are grown slowly over months and years (like hair bone and feathers), silk is by definition spun, in seconds transforming from gel to fibre. In the spider and silkworm the silk protein feedstock is stored as a gel, potentially for extended periods of time, in specialised organs called silk glands. Upon spinning these proteins travel down an elongated tapering tubular duct (shaped like an industrial hyperbolic dye) during which they undergo a transition into a solid fibre.
Examining these events on molecular scale reveals that in order to spin you need a source of energy. This energy fuels the removal of water from the silk proteins and the subsequent alignment and formation of a network of strong amide-amide intra and intermolecular hydrogen bonds. This network consists of ordered (high H-bonding) and disordered (low H-bonding) regions. Whilst it may be thought that a fully ordered crystalline material may be preferable, it is this careful balance of order and disorder at the nano-meter level that is responsible for silk’s superior mechanical properties. When silk deformed (i.e. stretched) stress is concentrated in the ordered regions (the stiffest parts) and then quickly and efficiently transferred to the disordered regions which are able to dissipate this excess energy as heat. Because silk is excellent at dissipating energy it is very tough, making it the ideal material to absorb impact (from a fly hitting a web to a bird attempting to break a cocoon).
But what is the main source of energy that fuels the transition from gel to fibre? Here we draw inspiration from an unlikely source: fishermen. Prior to the introduction of nylon fishing lines in the second half of last century fishermen took the silk glands of Bombyx and repeatedly drew them through their teeth, much like pulling chewing gum. If sufficiently skilled the fishermen could produce fibres that were very strong and in fact superior to the first plastic alternative lines that came onto the market. We now know that quite like the spider and the silkworm, the fishermen were using the same method of introducing energy into silk, by shearing the silk proteins. Here we can use rheology to further our understanding. By characterising exactly how these materials respond to shear deformation (mechanical energy input), we can begin to determine how silk is spun. Through rheology, the study of flow and deformation of matter, we are able to apply these shearing conditions to silk in vitro, thus providing us with a window into the silk production process.
Whilst fortunately my research does not involve me pulling silk trough my teeth, there have been moments where studying this material has felt like pulling teeth.
One such moment arose at the start of this work: We quickly discovered that preparing unspun silk for rheological testing requires incredible care and attention. Unspun silk is maintained at the precipice of the transition from gel to fibre, so much so that the smallest introduction of energy initiates this process. Thus when handling these materials the slightest shearing of the material through pinch of the forceps or accidental bump leads to massive variations in the data (over two orders of magnitude for viscosity). This can essentially mask any intended induced experimental variation. Thus during the first year I had to develop new ways of handling silk feedstocks and loading them onto the rheometer before we were able to even start investigating them.
In practice rheological testing involves placing the fresh silk feedstock between two metal plates (8mm in diameter) and moving the top relative to the bottom, thus subjecting the sample to different combinations of stress and strain. From this we are able to get information regarding the materials ability to handle energy over different timescales (oscillation tests) or the degree of intermolecular associations (viscosity tests).
Once the methodology was mastered we were keen to examine differences between the flow properties of silk feedstocks. Given tens of thousands of silks to choose form we confined our initial studies to comparing the silks of the golden orb weaving spider (Nephila edulis) and the commercial Chinese silkworm (Bombyx mori). Our work confirmed previous rheological studies on silkworm dope in that it is a weak gel with Non-Newtonian properties, therefore what about the spider?
The results were quite surprising. We found that despite hundreds of millions of years of evolutionary separation, using unrelated proteins and being selected to produce materials for completely different purposes (protection, silkworm, and capture, spider) the rheological properties of these two silks were practically identical. From a biological perspective this is important as it is a perfect example of evolutionary convergence, and something that is as yet invisible to molecular approaches. Hence we concluded that in order for Nature to spin a high performance fibre these feedstocks are under evolutionary constraints to have a particular set of rheological properties.
Most surprising however was not that these two materials behaved the same as one another, but that their rheological properties were akin to classic molten polymers! This has opened the door to using tools and techniques developed for the polymer sciences to be adapted to study silk. On the flip-side, because of a similar approach to processing our materials we can now learn from silk to improve our current polymeric fibre production methods.
Hence biotechnologically, the spinning pathway of the spider and silkworm is a highly advanced fibre production system. Through it the animal is able to control the energetically efficient production of fibres, with a wide variation of mechanical properties, using the same feedstock, by just altering the spinning conditions. These properties, perhaps even above the high performance characteristics, makes silk such a highly desirable material.
Therefore it is not surprising that many attempts have been made to recreate silk. Typically such artificial silk fibres are spun from natural silk that is “reconstituted” (chemically broken down) and then respun using processing conditions ranging from classical alcohol baths to electrospinning. However the tensile mechanical properties of these reconstituted fibres pale in comparison to their natural progenitors. Thus we were faced by a problem; by testing the properties of the end product alone it becomes difficult to determine what requires improvement, the feedstock or the processing.
Here we took the evolutionary constraints from our first study and turned them into design criteria for artificial silks. Our hypothesis was simple, if you could match the rheological properties of the reconstituted silk feedstock to the native feedstock then you should be able to process them like a silk into a silk-like fibre. Thus our rheological characterisation methods would help us to determine whether reconstituted silk dope actually has the potential (or not) to form a fibre with the qualities of its natural predecessor and model.
Consequently in our second study we compared the rheological properties of artificial and natural silk feedstocks over a wide range of concentrations (0.1-30% dry weight). Our findings were astonishing. For the same amount of silk protein present in the feedstocks there was over five orders of magnitude – a 10000 fold – difference between native and reconstituted silks in their ability to absorb energy (as defined by the plateau modulus) and the strength of their intermolecular associations (as defined by the zero shear viscosity). Put in real world terms the viscosity differences are akin to comparing lard to that of olive oil. We concluded that the act of reconstitution severely damages the silk proteins and that it will not be possible to spin biomimetically until the quality of the feedstock can be improved although rheology now holds the key to unlocking both the secrets of the feedstock and as a QA tool to improve our methods of reconstitution.
Where are we headed in the future? I have shown that rheology has created two new pathways for the field of studying silk, understanding the processing and development of a next generation artificial silk feedstock. From a scientific perspective we must start integrating new tools with rheology to characterise the responses of the silk proteins to shear on a molecular scale. One such approach is our work with the ISIS pulsed Neutron source at the Rutherford Appleton Laboratories where we are now beginning to combine state-of-the-art rheometry with small angle neutron scattering to understand how the conformation of the silk proteins change as they are being spun. From an industrial perspective things are already moving quickly in interesting directions demonstrating silk is more than just a fibre. Because silk can be chemically “unspun” back into a feedstock is may be reprocessed in new and exciting ways, creating structures from films to foams. The initial use of these materials is found in biomedical devices and implants that make use of silks excellent mechanical properties, biocompatibility and its ability to be chemically functionalised.
And what is the final lesson: stop during your spring-cleaning and spare a thought for the spider and its amazing technology.
Title image: Nathan Dumlao on Unsplash
