"

From 2013.igem.org

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. 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. 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

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. 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

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[1][2][3]. Short-term and long-term test with various cell lines have given no indication of toxicity.
2. No/low immune response[5][6][7]. 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[5].
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. 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[5]; 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.

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[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. The protein consists of roughly 3 motifs, each featuring a particular secondary structure in the assembly (table 1) (ref 11 paper recomb biomaterials).

Depending on the processing of the silk proteins, it can have a degree of these secondary structures, defining its properties (table 1). This characteristic of the silk proteins will be discussed in detail in the ‘Silk protein’(link) section.

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 1, Spider silk protein motifs

In order to make the silk material a large amount of these proteins are required. The protein has a very repetitive nature (fig. 2), with these motifs (table 1) recurring within the protein. This is difficult to produce, because it requires presence of the same tRNAs in a large amount. This can be solved with codon optimization. See ‘Codon optimization’ (link) at the modelling section for the explanation of this approach.

References

[1]     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
[2]     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.
[3]     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.
[4]     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
[5]     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
[6]     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
[7]     Panilaitis, B. "Macrophage Responses to Silk." Biomaterials 24.18 (2003): 3079-085
[8]     AMSilk GmbH, http://www.amsilk.com/en/products/implant-coating.html, Copyright © 2013 AMSilk GmbH [9] BASF The Chemical Company, January 26 2011, http://www.flickr.com/photos/basf/5412675961/in/set-72157624601397168, Copyright 2011 BASF