Biomedicine, bioengineering or Biomimetics and biomaterials

Presentation on Spider Silk,Spring 2015

Facts about spiders

There are more than 150,000 different species of spider about 35,000 are identified. Elsewhere I read that as of 2008 43,678 spider species had been identified, but dissension over classification of what is a spider

Some are small, some large enough to eat birds in Australia

Some bite and some are poisonous

They have 8 legs

Introduction

Spiders have many uses for the silk:

webs or other structures to catch prey

nests or cocoons to protect their young

suspension to hang from

food source

Ballooning Many small spiders extrude several threads (gossamer) in the air & let themselves be carried by the wind. Also called kiting

Other Ways Spiders Use their Silk

Prey immobilisation

As guidelines for a trail to lead back to the nest

Drop lines or Anchors an emergency line in case of falling or they can drop down on them if alarmed. Some hang from them while feeding

Alarm lines rather than traps, an alarm line can be used to rush out and secure a meal of something small e.g. an ant

Pheromonal trail for the opposite sex to find a mate

Different Types of Silk required for all these ecological uses

These different types of silk are produced in different glands

The silk from a particular gland can be linked to its use by a spider

A single spider can produce up to 7 different types of silk each with different properties to match their functions

Some silks are primitive and some are very complex

Types of Spider Silk

Dragline Silk or Ampullate (major) for a web’s outer rim, spokes and lifeline

Dragline Silk or Ampullate (minor) for temporary scaffolding during web construction

Flagelliform spiral capture silk – used for capturing lines of web

Tubuliform – Egg cocoon silk –used to protect the egg sacs

Types of Spider Silk contd.

Aciniform – used to wrap & secure freshly captured prey, used in male sperm webs and as stabilimenta (a conspicuous ultraviolet-light reflecting silken structure like a decoration in the orb web if certain spiders.)

Aggregate – a silk glue of sticky globules

Piriform – used to form bonds between separate threads for attachment points.

Properties of Spider Silk - 1

Mechanical properties – Each spider AND each type of silk has a set of mechanical properties optimized for their biological function.

Most silks especially dragline silks have exceptional mechanical properties with a unique combination of high tensile strength and extensibility (ductibility.) This enables a silk fibre to absorb a lot of energy before breaking (toughness.)

Weight for weight, silk is stronger than steel, but not as strong as Kevlar. Silk is however tougher than both.

Properties of Spider Silk - 2

Strength – in detail, a dragline’ silk’s tensile strength (TS) is equal to that of a high-grade steel alloy and about ½ as strong as aramid filaments such as Twaron (also called Arenka) and Kevlar (Dupont.)

TS is the maximum stress that a material can withstand before failing or breaking and is NOT the same as compressive strength and the ability to withstand loads tending to reduce size.

A note on aramid fibers

These are a class of heat-resistant, strong synthetic fibres used in aerospace and military applications. They are man made and high performance characterized by relatively rigid polymer chains. They are a man made organic polymer, produced by spinning a solid fibre from a liquid chemical, it’s a type of nylon, with high strength, low density and a very high specific strength.

Properties of Spider Silk - 3

Toughness – The combination of strength and ductibility gives dragline silks a very high toughness (or work to fracture) which ‘equals that of commercial polyaramid (aromatic nylon) filaments which themselves are bench marks of modern polymer fibre technology.’

An illustration of the Difference between toughness and strength.

Structure of Kevlar – a para-aramid

The chemical structure of spider silk

Weak hydrogen bonds

Properties of Spider Silk - 4

Density – Consisting mainly of protein, silks are about 1/6th the density of steel. As a result, a strand long enough to circle the earth would weigh less than 500 grams (18ozs.) Spider dragline silk has a tensile strength (TS) of roughly 1.3 GPa (Giga-Pascal). The TS listed for steel might be slightly higher e.g. up to 1.65 GPa, but spider silk is a much less dense material. This means that a given weight of spider silk is 5 times as strong as the same weight of steel

Properties of Spider Silk - 4

Energy density – Silks are also extremely ductile (some can stretch to up to 5 times their relaxed length without breaking.) This is an aspect of silk’s plasticity i.e the extent to which it can be plastically deformed without fracture.

Properties of Silk - 5

Temperature – While unlikely to be relevant in nature, dragline silks can hold their strength below –40°c and up to 220°C (428F)

Supercontraction – When exposed to water, dragline silks undergo super-contraction, shrinking up to 50% in length and behaving like a weak rubber under tension. (Theory is that this can automatically tension webs built during the night using the morning dew.)

Macroscopic (visible to the eye) composition

Silk as well as many other biomaterials have a hierarchical structure (e.g. cellulose, hair.) The primary structure is its amino acid sequence of mainly highly repetitive glycine and adenine blocks. Hence silks are often referred to as a block co-polymer. On a secondary structure level, the short-sided chin alanine is mainly found in crystalline domains (beta sheets) of the nanofibril. Glycine is mostly found in the so-called amorphous matrix consisting of helical and beta twin structures.

Macroscopic composition Contd

It is the interplay between the hard crystalline segments, and the strained elastic semi-amorphous regions, that gives spider silk its extraordinary properties.

Various compounds other than protein are used to enhance the fibre’s properties. (See next slide)

Other compounds in Spider Silk

Pyrrolidine – has hygroscopic properties that keep silk moist and also wards off ant invasion. It occurs in especially high concentrations in glue threads (aggregate.)

Potassium hydrogen phosphate – this releases protons in aqueous solution, resulting in a pH of about 4. This makes silk acidic and protects it from fungi and bacteria that would otherwise digest the protein

Other compounds in silk contd.

Potassium nitrate – is believed to prevent the protein from denaturing in the acidic milieu

Models of Silk

The very first basic model of silk was introduced by Termonia in 1994 and suggested crystallines embedded in an amorphous matrix interllnked with hydrogen bonds. This model has been refined over the years. Semi-crystalline were also found in a fibrillar skin core model that was suggested for spider silk later. These are nanofibrillar structures and Atomic Force Microscopy (AFM) and Transmission electron microscopy beams (TEM) were used to visualize them using neutron scattering.

Structure of spider silk. Inside a typical fibre there are crystalline regions separated by amorphous linkages.

The crystals are beta-sheets that have assembled

together

The structure of spider silk

Non-protein composition of spider silk

Various compounds other than protein are found such as sugars, lipids, ions and pigments that might affect the aggregation behaviour and act as a protection layer in the final fibre.

Biosynthesis

The production of silk differs in an important respect from the production of other fibrous biological materials. They are not continuously grown as keratin (hair) or cellulose in the cell walls of plants, or even the fibres formed from the compacted faecal matter of beetles. Silk is spun on demand from the liquid silk precursor, sometimes referred to as unspun silk dope. It occurs by pultrusion by pulling rather than squeezing. It is pulled through silk glands of which there may be duplicates and differing types in the same spider

The Silk Gland

This gland is visible and the external part is called a spinneret. Depending on their complexity spiders will have 2 to 8 sets of spinnerets, usually found in pairs.

Behind each spinneret visible on the surface of the spider, lies a gland – see schematic diagram which is based on the major ampullate gland from a golden orb weaving spider, as they are thee most studied and presumed to be thee most complex.

Schematic of a generalised gland of a Golden silk orb-weaver. Each differently coloured section highlights a

discrete section of the gland

Human Uses of Silk

Biomedical – peasants in the Southern Carpathian mountains cut up the tubes made by Atypus spiders to cover wounds. Reportedly they facilitate healing and even connected skin.

This is believed due to their antiseptic qualities & the silk is rich in vitamin K which helps blood to clot.

Nephila spider silk has been used recently to help mammalian neuronal regeneration.

Biomedical Uses continued

Hanover Medical School Germany – exploring the use of spider silk to treat wounds & repair torn tendons and nerves. Recently succeeded in bridging a 6 centimetre (2-4 inch) tibial nerve defect with spider silk in a large animal model. The nerves regenerated in just 10 months.Bred their own spiders & so limited commercial use

German firm – AM Silk makes skin implant coating made from recombinant spider silk proteins and sold in the cosmetics industry since November 2013.

Biomedical Uses contd

Spider silk can be produced that is good enough to be used in ligament repair without side effects of inflammation

Note again some Spider Silk properties

Low immunogenicity and has a remarkable effect on healing problems.

Antimicrobial biocompatiblity and completely biodegradable

Human Uses of Silk contd

Fishermen – In Indo-Pacific use the web of Nephila Clavipes spiders to catch small fish.

In the past spider silk was commonly used as a thread for cross-hairs in optical instruments such as, microscopes, telescopes, and rifle sights.

In 2011 spider silk fibres were used in the field of optics to generate very fine diffraction patterns over N-slit interferometric signals used in optical communications,

In 2012 spider silk fibres were used to create a set of violin strings.

Human Uses of Silk contd

Spider silk is used to suspend inertial confinement fusion targets during laser ignition, as it remains considerably elastic and has a high energy to break at temperatures as low as 10 – 20K (Kelvin). In addition it is made from ‘light’ atomic number elements that won’t emit X-rays during irradiation and so avoids preheating targets and thereby upsetting the pressure differential required for fusion .

Hurdles to greater Human Use

How to produce spider silk in enough volume?

Spiders are cannabalistic and cannot be raised in colonies like silk worms and their silk output is low. For example, it took 4 years and 82 people to collect the silk for the Madagascar –made spider silk jacket shown earlier. (Displayed in London’s Victoria & Albert Museum in 2012.)

One spider can provide up to 200 metres (656 feet) of silk in a single thread, but this is not enough for commercial use.

Artificial Synthesis

Various organisms have been used as a basis for attempts to replicate components of all or of some of the proteins involved in making spider silk. These proteins must then be extracted, purified and then spun before their properties can be tested.

See diagram of organisms used.

Goats have been genetically modified to secrete silk proteins in their milk which can then be purified.

Their average maximum breaking stress MPa (mega-pascals) was 285 – 250. Whereas the average stress is 30 – 40. This silk fibre is very strong.

More on this later

The Work on Goats

Synthetic Spider Silk

In 2000 the Canadian biotechnology company Nexia successfully produced spider silk in transgenic goats that carried the gene for spider silk; the milk of these goats contained significant quantities of the protein and 1-2 grams of silk proteins per litre of milk. Attempts to spin the protein into a fibre similar to natural spider silk, resulted in fibres with tenacities of 2 –3 grams per denier. Reportedly 7 to 10 times as strong as steel if compared for the same weight and it can stretch up to 20 times its unaltered size without losing its strength properties. It also holds its properties within -20°C to 330°C.

More about Nexia’s synthetic spider silk - 1

The company had created lines of goats to produce recombinant versions of either the major ampullate spidron 1 or dragline 1 from the golden orb weaver spider (Nephila clavipes), or major ampullate spidron 2 or dragline 2 from the same species of spîder. When the female goats lactated, their milk was harvested & subjected to chromatographic techniques to purify the recombinant silk proteins. These were then dried, dissolved using solvents (DOPE formation) and transformed into microfibres, using web-spinning fiber production methods.

More about Nexia’s synthetic spider silk - 2

The spun fibres had tenacities in the range of

2 – 3 grams/denier and an elongation range of 25 – 40% (denier to measure fineness of silk, rayon or hosiery)

This Biotech polymer has been transformed into nanofibres and nanomeshes using the electrospinning technique

Extrapolating from creating goat’s milk spider silk

The cut and coding technique used on goats to create spider milk can now be used to splice bacteria and to study and experiment on damaged bits of genetic code in cancer treatment to kill cancer cells and preserve healthy cells. They use the logic of computer circuits. See also BioBricks international bio-brick competition. Scientists from very diverse backgrounds compete in this.

Looking to the future

Synthetic biology has the potential to create a new Industrial revolution. It is perhaps the defining technology for the 21st Century and it needs an informed public discourse.

Bioengineering, or Biomimetics & Silk

The work of Markus J Buehler & his team at MIT

The exceptional strength of silkworm and spider silks arises from ß-sheet nanocrystals that consist of highly-conserved poly(Gly-Ala) and Poly-Ala domains. This is counterintuitive as the key molecular interactions in ß-sheet nanocrystals are hydrogen bonds, one of the weakest chemical bonds known. However Buehler et al report that using ß-sheet nanocrystals confined to a few nanometers achieves higher stiffness than larger nanocrystals. He demonstrates how size effects can be exploited to create bioinspired materials with superior mechanical properties in spite of relying on mechanically inferior hydrogen bonds.

Work of Buehler’s team contd. - 2

Computer models were used to assist in this analysis. They were able to use models that could simulate not just the structures of the molecules, but also how they move and interact in relation to each other

Work of Buehler’s team contd - 3

Silks are made from proteins, including some that form thin planar crystals called ß –sheets. These sheets are connected to each other through hydrogen bonds which are weak chemical bonds. Through atomic-level computer simulations, Bueher’s team investigated the molecular failure mechanisms in silk

Work of Buehler’s team contd - 4

Findings: The small yet rigid crystals showed the ability to quickly re-form their broken bonds, and as a result fail ‘gracefully’ – that is they gradually rather then suddenly fail.

Work of Buehler’s team contd – 5

Implications: In most engineering materials, high strength comes with brittleness, as with ceramics. Visually when ductibility is introduced materials become weak – but NOT silk.

Silk has high strength, because the tiny ß-sheet crystals, as well as the filaments that join them –are arranged in a structure that resembles a stack of pancakes, but with crystal structures within each pancake alternating their orientations

Work of Buehler’s team contd 6

This geometry of the tiny silk nanocrystals allows hydrogen bonds to work cooperatively, reinforcing adjacent chains against external forces which leads to their outstanding extensibility & the strength of spider silk

You also do not have to create high temperatures to produce it.

Work of Buehler’s team contd 7

A surprising finding is the critical dependence of the properties of silk on the exact size of these ß-sheet crystals within the fibres. When crystal size is about 3 nanometers (billionth of a meter,) the material has its ultra-strong, ductile characteristics. BUT, let those crystals grow to 5 nanometers & the material becomes weak and brittle.

Implications for using other biological materials

Other biological materials such as wood, plant fibres could be used to develop highly functional materials out of abundant inexpensive materials.

Another use of spider silk

As non-polluting pesticide Rondonin is an antifungal peptide derived from spiders

Deriving Silk from Fermented Bacteria

Randy Lewis of Utah State university isolated the gene that produces spider silk proteins and transferred the gene to other organisms (including transgenic goats) while at the University of Wyoming.

Lewis & his team also transferred it to fermented bacteria.

Lewis & Team contd

They had 2 breakthroughs in 2014:

1. They achieved a 4 – 6 fold increase in the amount of bacteria they could produce in a fermentation vessel

2. They could quadruple the amount of protein each bacterium produced by a factor of 10.

Work at Keio University in Japan

Also worked on producing spider silk from fermented bacteria is the start-up company Spiber

The artificial protein they have derived is calledQmonos.These have produced a class of proteins that form natural silk, including spider silk and silk worm silk in a microbial fermentation process. This silk can be fabricated into fibres, films, gels,sponges & powders & is being developed for use in the automotive, aerospace and medical industries.

More on Qmonos

There are more than 400 variations of Qmonos to date.

In 2013 the first prototype production plant was created and the first Qmonos products can be expected on the market in a few years.

Other transgenic silk products

Kraig Biocraft Labs in Michigan inserted specific spider genes into silk worm chromosomes and produced threads very like spider silk. They made monster silk worms with red eyes to distinguish them

They can vary the flexibility, strength & toughness of this silk by moving around the DNA sequence.

They are getting close to commercialisation

Biomaterials

Spider silk will see increased usage in textiles blends in the near future for shirts, neck ties etc

Military applications – flak jackets, parachute cords, tethers for planes and aircraft carriers. Fire-resistant underwear from fake spider silk for body armour, as the silk does not melt on the skin when exposed to heat. It also resists penetration by finer particles like sand & dirt & so keeps wounds clean.

Spider Silk used to make ‘Bulletproof’ Human Skin U. of Puerto Rico used Darwin bark spider’s silk,

as more elastic & 10 times better fibre than Kevlar