Team:Groningen/Project/Silk

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<h1>Silk Silk</h1>
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<h1>Spider Silk</h1>
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Spider silk is regarded by many as the bio-material of the future, this due to is incredible high tensile strength combined with its elasticity. This makes spider silk as strong as steel and tougher than kevlar. However it is way less known that spider silk also has some impressive medical properties. Foremost the fact that spider silk (and silk in general) doesn’t cause a immune response in the human body. Immune response obviously being one of the major concerns when placing implants inside patients, so our iGEM team saw a great opportunity to combine the spider silk with the implants.
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<h1>Silk</h1>
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<p>
<p>
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Silk is a natural protein fibre that is known for its use in textiles. The best known silk comes from the silk moth pupa but a lot of animals such as arthropods are also capable of producing silk. One of the arthropods well known for its silk is the spider and spider silk is our material of choice for our implant coating.
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Spider silk is a biomaterial with great potential. On one hand it has incredible high tensile strength combined with elasticity, which makes spider silk as strong as steel and tougher than kevlar [1]. On the other hand spider silk also has some peculiar medical properties. Foremost the fact that spider silk (and silk in general) does not cause a strong immune response in the human body [7]. Immune response obviously being one of the major concerns when placing implants inside patients, so our iGEM team saw a great opportunity to combine the spider silk with the implants.
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</p>
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<h2>Background</h2>
<p>
<p>
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The spider silk we used is the so called dragline silk from the Argiope aurantia (MaSp2)  from Brooks et al (2008).  It is the strongest silk produced inside the spider. The spider silk consists out of a big repetitive domain (around 2500 base pairs) with an N and a C terminus (figure 1 b).  
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 +
There are many arthropods that have the ability to produce silk, most popular being the <i>Bombyx mori</i> silk worm used in the production of silk for clothing. However the spider easily takes the crown in terms of applications. The spider uses its silk as their Swiss Army Knife, some of these uses include, spinnig webs for catching preys, Dragline's for better movement, and for reproduction [2]. Over the 400 million years of evolution the silk is optimized in many aspects. Like with many fascinating phenomena from nature, humans have learned to utilize the spider webs. The Nephila spiders in tropical rain forests (of Papua New Guinea) have powerful webs to catch flying birds, and ancient cultures have used these webs for fishing purposes [13].</p>
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<br>
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<p>
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The medical properties of spider silk have been known for a long time; there are many records of uses throughout history.
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<br>The oldest known application of a spider web dates back to from Ancient Greece were it was used as wound dressing [13]. Some other historical mentions date from around 1600. In the Polish book ‘With Fire and Sword’:
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<br><i>‘ “This is nothing, nothing at all” said he, feeling the wounds with his fingers. “He will be well to-morrow. I will take care of him. Mix up bread and spider-webs for me! ’</i>
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<br>
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<br>
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And in the famous Shakespearian  comedy ‘Midsummer Night’s Dream’:
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<br><i>“I shall desire you of more acquaintance, good master cobweb,
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If I cut my finger, I shall make bold of you.”</i>
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<br>
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<p>
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Apparently the medical properties of spider webs have been known for a long time. Its use as wound cover improves the recovery without causing problems. What is it that gives the spider silk such excellent healing properties?</p>
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<br>
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<h2>Biomedical properties</h2>
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<img src="https://static.igem.org/mediawiki/2013/c/c9/Schematic_silk_protein.jpg" width="100%" >
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<A href="http://www.flickr.com/photos/basf/5412675961/in/set-72157624601397168"><img src="http://farm5.staticflickr.com/4110/5412675961_5defa98b10.jpg" width="100%"></a>
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<font size="1">Figure 1: schematic drawing of natural spider silk (Lukas Eisoldt et al. 2011)</font>
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<font size="1">Figure 1, Cells growing under a spider silk non-woven bandage. Magnification 3200:1.<b> [11]</b></i></font>
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<h2> The properties of silk </h2>
 
<p>
<p>
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The unique properties of silk are a result of its highly constant and repetitive amino-acid structure. The sequence of amino-acids determines what secondary structures will arise, and thus the final preferred protein conformation. The secondary structures may be beta sheets, beta-spirals, and beta-helices, of which the sheets realize the silk's amazing tensile strength, and the spirals and helices its elongation.
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Normal cells need a scaffold to adhere to. If there is a wound the tissue is repaired very slowly, and only after that the cells from the wound are able to completely recover. When a wound is covered with a good biomedical material, the cells will start to spread, proliferate and differentiate more quickly. On an unsuitable material, the interaction can lead to immune responses and cell death.
</p>
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In the figure 2 below a stress-strain diagram can be found. where Clavipus </p>
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Spider silk is suitable as biomedical material because:
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<p>
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<ol type="numbers">
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A final general property of silk it that it can be integrated with the human body, it will not induce an immune response, making it an ideal choice for many biomedical applications. Its compatibility extends to the gastrointestinal tract, that is, it is even safe to eat.
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<li>
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<b>It has no toxicity [3-5]</b>. Short-term and long-term tests with various cell lines gave no indication of toxicity.
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</li>
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<li>
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<b>No/low immune response [7-9]</b>. Different types of cells were examined for immunological response to silk. One study compared collagen, which is used as biomaterial for coating, to silk. The results favoured silk, and deemed silk as being biocompatible [7].</li><li>
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<b>No inflammation</b>. The body will not detect the silk as a foreign material, as it consists of a smooth protein layer.</li><li>
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<b>Cell adherence [6]</b>. This depends on the processing of the silk. With small modifications (for instance, genetic addition of an RGD motif) cell adhesion can be improved, to stimulate tissue recovery of a wound. However, for the use of implants, cell adhesion is undesired, as fibrosis can occur.</li><li>
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<b>Biodegradable</b>. Spider silk has a slow biodegradability, which is useful for medical applications.</li><li>
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<b>Elasticity and mechanical properties</b>. The spider silk material is strong and stable material. The elasticity of a biomedical material has shown to influence cell growth and proliferation, and therefore the flexible and adjustable spider silk is very potent.</li><li>
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<b>Does not swell</b>. Due to the nature of the protein it is both hydrophilic and hydrophobic, the latter preventing any major uptake of water.</li><li>
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<b>No bacterial or fungal degradation [7]</b>; this is credited to the long evolution. One can imagine that if the base of a spiders nature, its web, would be easily torn apart by the hundreds of bacteria and fungi around, then they would not have gotten this far. The silk typically has a very flat surface that does not allow cells to grow on it very easily, nor be identified by antibodies.</li></ol>
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<img src="https://static.igem.org/mediawiki/2013/5/52/Stressstrain.JPG" width="100%" >  
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<img src="http://img819.imageshack.us/img819/9168/1o11.png" width="100%"></a>
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<font size="1">Figure 2: Properties of spidersilk versus other materials (Frank K. Ko, et at. 2001). Spider silk is compared to, Kevlar 29, normal silkworm silk, polyethylene terephthalate (PET), Nylon 6, and Merino wool. The stress-strain diagram relates the degree of deformation to the amount of energy absorbed.</font>
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<font size="1">Figure 2, Overview of biomedical advantages </i></font>
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<h2> Silk as a coating material</h2>
 
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<p>
 
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Silk can be regarded as a superb version of collagen. Collagen is a material used a lot for the coating of implants.
 
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Although collagen is a naturally occurring protein found inside the human body, the implants are coated with proteins harvested from cows, which can give an immune response. On the other hand spider silk is an in allergic material and will never give an immune response. 
 
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Spider Silk is biodegradable, has a low bacterial adherence and  can rival any artificial material with the respect of cell growth (1-3). Therefore spider silk is a good candidate for the coating of medical implants.
 
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</p>
 
<p>
<p>
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For example one can think of a cartilage implant. The implant consists of a biodegradable polymer coated with spider silk. The human cells will grow into the implant, slowly degrading the implant.  After a certain amount of time the implant is fully degraded, leaving only a new piece of cartilage in for example a knee.  
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Studies performed with coated implants in mice and pigs support these medical properties. Also spider silk coated breast implants are tested in preclinical trials, and have so far shown promising results [10].
</p>
</p>
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<br>
<br>
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<h2> References </h2>
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<div>
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(1) Silk-based materials for biomedical applications, A.Leal-Egana (2010)
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<table  id="normal" width=60%>
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<br>
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<tr>
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(2) Silk as biomaterial, C. Vepari (2007)
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<th>Biomedical material</th>
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<br>
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<th>Spider Silk</th>
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(3) Interactions between spider silk and cells, J.W. Kuhlier (2010)
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<th>Collagen</th>
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<tr>
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<td>No inflammation</td>
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<br>
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<td></html>[[File:Green_tick.gif | 25px]]<html></td>
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<u>Alternative (just an idea)</u>
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<td></html>[[File:Green_tick.gif | 25px]]<html></td>
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<br>
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</tr>
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<h1>History</h1>
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<td>No immune response</td>
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<td></html>[[File:Green_tick.gif | 25px]]<html></td>
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<td></html>[[File:Green_tick.gif | 25px]]<html></td>
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<td>High tensile strength</td>
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<td></html>[[File:Green_tick.gif | 25px]]<html></td>
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<td></html>[[File:Green_tick.gif | 25px]]<html></td>
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</tr>
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<tr>
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<td>Low bacterial adherence</td>
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<td></html>[[File:Green_tick.gif | 25px]]<html></td>
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<td></td>
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</tr>
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<tr>
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<td>Stable, slow-degrading</td>
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<td></html>[[File:Green_tick.gif | 25px]]<html></td>
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<td></td>
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</tr>
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<tr>
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<td>No disease transmission</td>
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<td></html>[[File:Green_tick.gif | 25px]]<html></td>
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<td></td>
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</tr>
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<tr>
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<td>Promotes cell adherence</td>
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<td></html>[[File:Green_tick.gif | 25px]]<html></td>
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<td></html>[[File:Green_tick.gif | 25px]]<html></td>
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</tr>
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<tr>
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<td>Promotes regeneration</td>
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<td></html>[[File:Green_tick.gif | 25px]]<html></td>
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<td></td>
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<tr>
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<td colspan="3"><font size="1">Table 1: Comparison of spider silk with another biomedical material: collagen. Collagen is a mammalian fibrous protein, and in many respects similar to spider silk. However, there are still limits to collagen, and some aspects where spider silk is superiour [8]</font></td>
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</tr>
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</table>
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</div>
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<p>
<p>
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There are many anthropoids that have the ability to produce silk, most popular being the <i>Bombyx mori</i> silk worm used in the production of silk for clothing. However the spider easily takes the crown in terms of applications.
+
Spider silk is our choice to approach the problems of medical implants. However, so far no large scale production of spider silk is achieved, and production, purification and processing are challenging. Why is the production of the spider silk protein so difficult? How can we make a coating from the spider silk? </p>
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The Spider uses its silk as their Swiss Army Knife,some of these uses include, spinnig webs for catching preys, Dragline's for better movement, and for reproduction. Over the 400 million years of evolution the silk is optimized in many aspects. Like with many fascinating phenomena from nature, humans have learned to utilize the spider webs. Giant spiders() in tropical rain forests() have powerful webs to catch flying birds, and old cultures have used these webs for fishing purposes.
 
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</p>
 
<br>
<br>
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[Picture cartoon of spider/swiss knife]
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<h2>Silk protein</h2>
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<br><br>
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<p>
<p>
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Remarkably, the biomedical properties of spider silk have been recognized for a long time. The oldest known application of a spider web as wound dressing is from Ancient Greece. Some other historical mentions date from around 1600. In the Polish book ‘With Fire and Sword’:
+
The spider silk protein is a fibrous protein. It does not have a folded state on its own; it is able to assemble (multimerize) with multiple identical proteins to form the silk material. The protein consists of roughly 3 motifs, each featuring a particular secondary structure in the assembly (Table 2).  
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<br><i>‘ “This is nothing, nothing at all” said he, feeling the wounds with his fingers. “He will be well to-morrow. I will take care of him. Mix up bread and spider-webs for me! ’</i>
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</p>
</p>
<br>
<br>
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[Picture cartoon of Shakespearian character with wound and web]
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<br><br>
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<table id="normal">
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<tr>
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<th>Amino acid sequence</th>
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<th>Secondary structure</th>
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<th>Properties</th>
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</tr>
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<tr>
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<td>AAAAAAAA</td>
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<td>β-sheet</td>
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<td>Tensile strenght, rigidity, hydrophobicity</td>
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</tr>
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<tr>
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<td>GPG(AG)QQ / GPG(SGG)QQ / GPGGX</td>
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<td>β-spiral / β-turn</td>
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<td>Extensibility, elasticity</td>
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</tr>
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<tr>
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<td>GGX</td>
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<td>3<sub>10</sub> helix</td>
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<td>Link, alignment, flexibility</td>
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</tr>
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</table>
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<font size="1">Table 2, Spider silk protein motifs</font><br><br>
<p>
<p>
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And in Shakespeare’s comedy ‘Midsummer Night’s Dream’:
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Depending on the processing of the silk proteins the material can have a degree of these secondary structures, defining its properties (Table 2). The processing inside a spider is still not completely elucidated. The spider has a special duct in which many processes take place, among which are drastic decrease in pH, and change in salts [14]. The latter is also utilized in the processing of recombinant spider silk. It was found that kosmotropic ions ("salting-out"), such as phosphate and potassium, induce beta-sheet formation and thus rigidity and hydrophobicity of the silk.</p><p>
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<br><i>“I shall desire you of more acquaintance, good master cobweb,
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<br>
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If I cut my finger, I shall make bold of you.”</i>
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This is because the assembly of the silk proteins is induced by depletion of solvent (generally water). Any action or substance that can contribute to the removal of water, can also contribute to polymerization and structure of the spider silk. Often methanol is used to improve the formation of beta-sheets[14].
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<br><br>
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Apparently the spider webs are suitable as wound cover: they improve the recovery without giving problems. What gives the spider silk such excellent healing properties?
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</p>
</p>
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<h1>Biomedical properties</h1>
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<br>
<p>
<p>
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In order to make our spider silk coating material, a large amount of these proteins are required. The protein has a very repetitive nature (fig. 2), with these motifs (table 2) recurring within the protein. This makes is difficult to translate the protein, because it requires presence of the same tRNAs in a large amount. This can be solved with codon optimization. See <A href="https://2013.igem.org/Team:Groningen/Navigation/Codonoptimization">codon optimization</a> at the modelling section for the explanation of this approach.
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</p>
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Shielding, preventing bacteria, improve wound repair,  
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<font size="1">Figure 2, Major ampullate Spidroin 2 (MaSp2) from <i>Argiope aurantia</i></font>
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<br>
<br>
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No toxicity, no immune response, elasticity, cell adhesion, biodegradation
 
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<br><br>
 
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(See Review 'Silk-based materials for biomedical applications' from Aldo Leal-Egana and Thomas Scheibel, 2010, Biotech. Appl. Biochem)
 
<br>
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</p>
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<h2>References</h2></P>
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<font size="1,5">
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[1] &nbsp;&nbsp;&nbsp; Griffiths, J. R., and V. R. Salanitri. "The Strength of Spider Silk." Journal of Materials Science 15.2 (1980): 491-96<br>
 +
[2] &nbsp;&nbsp;&nbsp; Foelix, Rainer F. Biology of Spiders. New York: Oxford UP, 1996.<br>
 +
[3] &nbsp;&nbsp;&nbsp; Liu, Tie-lian, Jing-cheng Miao, Wei-hua Sheng, Yu-feng Xie, Quan Huang, Yun-bo Shan, and Ji-cheng Yang. "Cytocompatibility of Regenerated Silk Fibroin Film: A Medical Biomaterial Applicable to Wound Healing." Journal of Zhejiang University SCIENCE B 11.1 (2010): 10-16<br>
 +
[4] &nbsp;&nbsp;&nbsp; Allmeling, Christina. "Use of Spider Silk Fibres as an Innovative Material in a Biocompatible Artificial Nerve Conduit." Journal of Cellular and Molecular Medicine 10.3 (2006): n. pag.<br>
 +
[5] &nbsp;&nbsp;&nbsp; Acharya, C., B. Hinz, and S. Kundu. "The Effect of Lactose-conjugated Silk Biomaterials on the Development of Fibrogenic Fibroblasts." Biomaterials 29.35 (2008): 4665-675. Print.<br>
 +
[6] &nbsp;&nbsp;&nbsp; Seo, Y. K., H. H. Yoon, Y. S. Park, K. Y. Song, W. S. Lee, and J. K. Park. "Correlation between Scaffold in Vivo Biocompatibility and in Vitro Cell Compatibility Using Mesenchymal and Mononuclear Cell Cultures." Cell Biology and Toxicology 25.5 (2009): 513-22<br>
 +
[7] &nbsp;&nbsp;&nbsp; Meinel, L., S. Hofmann, V. Karageorgiou, C. Kirkerhead, J. Mccool, G. Gronowicz, L. Zichner, R. Langer, G. Vunjaknovakovic, and D. Kaplan. "The Inflammatory Responses to Silk Films in Vitro and in Vivo." Biomaterials 26.2 (2005): 147-55<br>
 +
[8] &nbsp;&nbsp;&nbsp; MacIntosh, Ana C., Victoria R. Kearns, Aileen Crawford, and Paul V. Hatton. "Skeletal Tissue Engineering Using Silk Biomaterials." Journal of Tissue Engineering and Regenerative Medicine 2.2-3 (2008): 71-80<br>
 +
[9] &nbsp;&nbsp;&nbsp; Panilaitis, B. "Macrophage Responses to Silk." Biomaterials 24.18 (2003): 3079-085<br>
 +
[10] &nbsp;&nbsp;&nbsp; AMSilk GmbH, http://www.amsilk.com/en/products/implant-coating.html, Copyright © 2013 AMSilk GmbH<br>
 +
[11] &nbsp;&nbsp;&nbsp; BASF The Chemical Company, January 26 2011, http://www.flickr.com/photos/basf/5412675961/in/set-72157624601397168, Copyright 2011 BASF<br>
 +
[12] &nbsp;&nbsp;&nbsp; Hayashi, Cheryl Y., Nichola H. Shipley, and Randolph V. Lewis. "Hypotheses That Correlate the Sequence, Structure, and Mechanical Properties of Spider Silk Proteins." International Journal of Biological Macromolecules 24.2-3 (1999): 271-75<br>
 +
[13] &nbsp;&nbsp;&nbsp; Scheibel T., ETSI Caminos, "Silk structure, silk protein folding and assembly", http://www.youtube.com/watch?feature=player_detailpage&v=vIcMd9BjSFo#t=211<br>
 +
[14]  &nbsp;&nbsp;&nbsp; Spiess, Kristina, Andreas Lammel, and Thomas Scheibel. "Recombinant Spider Silk Proteins for Applications in Biomaterials." Macromolecular Bioscience 10.9 (2010): 998-1007<br>
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Latest revision as of 03:35, 5 October 2013

Spider Silk

Spider silk is a biomaterial with great potential. On one hand it has incredible high tensile strength combined with elasticity, which makes spider silk as strong as steel and tougher than kevlar [1]. On the other hand spider silk also has some peculiar medical properties. Foremost the fact that spider silk (and silk in general) does not cause a strong immune response in the human body [7]. Immune response obviously being one of the major concerns when placing implants inside patients, so our iGEM team saw a great opportunity to combine the spider silk with the implants.

Background

There are many arthropods that have the ability to produce silk, most popular being the Bombyx mori silk worm used in the production of silk for clothing. However the spider easily takes the crown in terms of applications. The spider uses its silk as their Swiss Army Knife, some of these uses include, spinnig webs for catching preys, Dragline's for better movement, and for reproduction [2]. Over the 400 million years of evolution the silk is optimized in many aspects. Like with many fascinating phenomena from nature, humans have learned to utilize the spider webs. The Nephila spiders in tropical rain forests (of Papua New Guinea) have powerful webs to catch flying birds, and ancient cultures have used these webs for fishing purposes [13].


The medical properties of spider silk have been known for a long time; there are many records of uses throughout history.
The oldest known application of a spider web dates back to from Ancient Greece were it was used as wound dressing [13]. Some other historical mentions date from around 1600. In the Polish book ‘With Fire and Sword’:
‘ “This is nothing, nothing at all” said he, feeling the wounds with his fingers. “He will be well to-morrow. I will take care of him. Mix up bread and spider-webs for me! ’

And in the famous Shakespearian comedy ‘Midsummer Night’s Dream’:
“I shall desire you of more acquaintance, good master cobweb, If I cut my finger, I shall make bold of you.”


Apparently the medical properties of spider webs have been known for a long time. Its use as wound cover improves the recovery without causing problems. What is it that gives the spider silk such excellent healing properties?


Biomedical properties

Figure 1, Cells growing under a spider silk non-woven bandage. Magnification 3200:1. [11]

Normal cells need a scaffold to adhere to. If there is a wound the tissue is repaired very slowly, and only after that the cells from the wound are able to completely recover. When a wound is covered with a good biomedical material, the cells will start to spread, proliferate and differentiate more quickly. On an unsuitable material, the interaction can lead to immune responses and cell death.

Spider silk is suitable as biomedical material because:

  1. It has no toxicity [3-5]. Short-term and long-term tests with various cell lines gave no indication of toxicity.
  2. No/low immune response [7-9]. Different types of cells were examined for immunological response to silk. One study compared collagen, which is used as biomaterial for coating, to silk. The results favoured silk, and deemed silk as being biocompatible [7].
  3. No inflammation. The body will not detect the silk as a foreign material, as it consists of a smooth protein layer.
  4. Cell adherence [6]. This depends on the processing of the silk. With small modifications (for instance, genetic addition of an RGD motif) cell adhesion can be improved, to stimulate tissue recovery of a wound. However, for the use of implants, cell adhesion is undesired, as fibrosis can occur.
  5. Biodegradable. Spider silk has a slow biodegradability, which is useful for medical applications.
  6. Elasticity and mechanical properties. The spider silk material is strong and stable material. The elasticity of a biomedical material has shown to influence cell growth and proliferation, and therefore the flexible and adjustable spider silk is very potent.
  7. Does not swell. Due to the nature of the protein it is both hydrophilic and hydrophobic, the latter preventing any major uptake of water.
  8. No bacterial or fungal degradation [7]; this is credited to the long evolution. One can imagine that if the base of a spiders nature, its web, would be easily torn apart by the hundreds of bacteria and fungi around, then they would not have gotten this far. The silk typically has a very flat surface that does not allow cells to grow on it very easily, nor be identified by antibodies.


Figure 2, Overview of biomedical advantages

Studies performed with coated implants in mice and pigs support these medical properties. Also spider silk coated breast implants are tested in preclinical trials, and have so far shown promising results [10].


Biomedical material Spider Silk Collagen
No inflammation 25px 25px
No immune response 25px 25px
High tensile strength 25px 25px
Low bacterial adherence 25px
Stable, slow-degrading 25px
No disease transmission 25px
Promotes cell adherence 25px 25px
Promotes regeneration 25px
Table 1: Comparison of spider silk with another biomedical material: collagen. Collagen is a mammalian fibrous protein, and in many respects similar to spider silk. However, there are still limits to collagen, and some aspects where spider silk is superiour [8]

Spider silk is our choice to approach the problems of medical implants. However, so far no large scale production of spider silk is achieved, and production, purification and processing are challenging. Why is the production of the spider silk protein so difficult? How can we make a coating from the spider silk?


Silk protein

The spider silk protein is a fibrous protein. It does not have a folded state on its own; it is able to assemble (multimerize) with multiple identical proteins to form the silk material. The protein consists of roughly 3 motifs, each featuring a particular secondary structure in the assembly (Table 2).


Amino acid sequence Secondary structure Properties
AAAAAAAA β-sheet Tensile strenght, rigidity, hydrophobicity
GPG(AG)QQ / GPG(SGG)QQ / GPGGX β-spiral / β-turn Extensibility, elasticity
GGX 310 helix Link, alignment, flexibility
Table 2, Spider silk protein motifs

Depending on the processing of the silk proteins the material can have a degree of these secondary structures, defining its properties (Table 2). The processing inside a spider is still not completely elucidated. The spider has a special duct in which many processes take place, among which are drastic decrease in pH, and change in salts [14]. The latter is also utilized in the processing of recombinant spider silk. It was found that kosmotropic ions ("salting-out"), such as phosphate and potassium, induce beta-sheet formation and thus rigidity and hydrophobicity of the silk.


This is because the assembly of the silk proteins is induced by depletion of solvent (generally water). Any action or substance that can contribute to the removal of water, can also contribute to polymerization and structure of the spider silk. Often methanol is used to improve the formation of beta-sheets[14].


In order to make our spider silk coating material, a large amount of these proteins are required. The protein has a very repetitive nature (fig. 2), with these motifs (table 2) recurring within the protein. This makes is difficult to translate the protein, because it requires presence of the same tRNAs in a large amount. This can be solved with codon optimization. See codon optimization at the modelling section for the explanation of this approach.


Figure 2, Major ampullate Spidroin 2 (MaSp2) from Argiope aurantia


References

[1]     Griffiths, J. R., and V. R. Salanitri. "The Strength of Spider Silk." Journal of Materials Science 15.2 (1980): 491-96
[2]     Foelix, Rainer F. Biology of Spiders. New York: Oxford UP, 1996.
[3]     Liu, Tie-lian, Jing-cheng Miao, Wei-hua Sheng, Yu-feng Xie, Quan Huang, Yun-bo Shan, and Ji-cheng Yang. "Cytocompatibility of Regenerated Silk Fibroin Film: A Medical Biomaterial Applicable to Wound Healing." Journal of Zhejiang University SCIENCE B 11.1 (2010): 10-16
[4]     Allmeling, Christina. "Use of Spider Silk Fibres as an Innovative Material in a Biocompatible Artificial Nerve Conduit." Journal of Cellular and Molecular Medicine 10.3 (2006): n. pag.
[5]     Acharya, C., B. Hinz, and S. Kundu. "The Effect of Lactose-conjugated Silk Biomaterials on the Development of Fibrogenic Fibroblasts." Biomaterials 29.35 (2008): 4665-675. Print.
[6]     Seo, Y. K., H. H. Yoon, Y. S. Park, K. Y. Song, W. S. Lee, and J. K. Park. "Correlation between Scaffold in Vivo Biocompatibility and in Vitro Cell Compatibility Using Mesenchymal and Mononuclear Cell Cultures." Cell Biology and Toxicology 25.5 (2009): 513-22
[7]     Meinel, L., S. Hofmann, V. Karageorgiou, C. Kirkerhead, J. Mccool, G. Gronowicz, L. Zichner, R. Langer, G. Vunjaknovakovic, and D. Kaplan. "The Inflammatory Responses to Silk Films in Vitro and in Vivo." Biomaterials 26.2 (2005): 147-55
[8]     MacIntosh, Ana C., Victoria R. Kearns, Aileen Crawford, and Paul V. Hatton. "Skeletal Tissue Engineering Using Silk Biomaterials." Journal of Tissue Engineering and Regenerative Medicine 2.2-3 (2008): 71-80
[9]     Panilaitis, B. "Macrophage Responses to Silk." Biomaterials 24.18 (2003): 3079-085
[10]     AMSilk GmbH, http://www.amsilk.com/en/products/implant-coating.html, Copyright © 2013 AMSilk GmbH
[11]     BASF The Chemical Company, January 26 2011, http://www.flickr.com/photos/basf/5412675961/in/set-72157624601397168, Copyright 2011 BASF
[12]     Hayashi, Cheryl Y., Nichola H. Shipley, and Randolph V. Lewis. "Hypotheses That Correlate the Sequence, Structure, and Mechanical Properties of Spider Silk Proteins." International Journal of Biological Macromolecules 24.2-3 (1999): 271-75
[13]     Scheibel T., ETSI Caminos, "Silk structure, silk protein folding and assembly", http://www.youtube.com/watch?feature=player_detailpage&v=vIcMd9BjSFo#t=211
[14]     Spiess, Kristina, Andreas Lammel, and Thomas Scheibel. "Recombinant Spider Silk Proteins for Applications in Biomaterials." Macromolecular Bioscience 10.9 (2010): 998-1007

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