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Forschungsarbeit

Modification of films made of spider silk proteins

By Elisabeth Schwab (26.01.2012)

One of most fascinating characteristics of living systems is their aptitude to self regenerate. The replacement of damaged/old structures allows not only the maintaining of life but also the growth of tissues and organs during the different development stages. In case of superior organisms, wound healing requires not only the restoring of the cellular population but also the renewal of the extracellular matrix surrounding them.

The extracellular matrix (ECM) is a complex network of proteins and polysaccharide chains surroundings the cells. It defines the extracellular micro-architecture in terms of adhesion and maintaining of tensile strength, facilitating cell migration, guiding tissue morphogenesis and repair, regulating activity of secreted proteins and being involved in cell-cell communication acting as co-receptors. Besides, other functions of ECM are the regulation of molecular diffusion of several molecules involved in cell metabolism and to act as reservoir for growth factors and several hormones related with cell proliferation and migration.

Because some types of wounds cannot be healed by self-regeneration, since beginning of the civilization humans have been searching for alternatives to improve tissue reparation by using natural/man-made materials, which could replace several properties of the natural ECM. Since the use of isolated natural ECM components (e.g. collagens, fibronectins, elastin) is extremely challenging and associated to the potential existence of biological risks (due to the presence of virus, bacteria, or prions among others), during the last decades the research of new synthetic polymers have become more important. Advantages in the use of synthetic biopolymers are their availability, consistence in their production, as well as the potentially modification of their biophysical characteristics in concordance with the physiological and biomedical necessities.

Figure 1: Experimental design and production of modified spider silk proteins. The genetic information (cDNA) of the spider silk protein is coupled to the genetic information of a special domain derived from other proteins within a plasmid vector.[Bildunterschrift / Subline]: Figure 1: Experimental design and production of modified spider silk proteins. The genetic information (cDNA) of the spider silk protein is coupled to the genetic information of a special domain derived from other proteins within a plasmid vector. This modified protein can be produced in bacteria and subsequently purified.

Since biopolymers used in tissue reparation should accomplish a series of biochemical and biophysical requirements (e.g. lack of toxicity and immune-response, the capability to support cell adhesion, adequate mechanical and textural properties, etc.), during the last decades multiple polymers have been tested with the purpose to substitute the natural ECM. One of these new biomaterials are silk proteins, which, due to the high immune-compatibility, the absence/low toxicity, morphological tuneability and the possibility to modify their primary structure with biological domains/motives, they present a high potential for being used in tissue engineering as replacement of the ECM.

In spite of these advantages, one of the drawbacks of scaffolds made of spider silk proteins is the lack/weak cell adhesion (Gao et al. 2008, Seo et al. 2008). Since spider silk proteins can be recombinantly produced in bacteria (Figure 1), one possibility to improve the cell attachment is by modifying the primary structure of these proteins, including specific motifs for cell adhesion.

During my Master’s thesis, the recombinant spider silk protein eADF4(C16) (= engineered Araneus diadematus fibroin), derived from the dragline-silk of orb-weaving spider A. diadematus, was chemically and genetically modified with a collagen binding domain (CBD) from von Willebrand factor (vWF). The peptide was successfully coupled to the amino terminus of eADF4(C16) in solution as well as to protein films.

The modified protein CBD-S3-C16 was used to cast films from different solvents. The films were further characterized using circular dichroism spectroscopy (CD) and Fourier transformed infrared spectroscopy (FTIR) in order to investigate the secondary structure of films of various thicknesses. After treatment with methanol or potassium phosphate, films of both, modified and non-modified, spider silk proteins showed a similar content of β-sheet structure. The wetting properties of different films, analysed via contact angle measurements, appeared to be dependent on film thickness, protein, and solvent used to cast the film. Above all the tryptophan side chain in the CBD sequence seems to have a big influence on surface properties.

Cell adhesion experiments with murine fibroblasts on different films were carried out. An improved adhesion was observed on functionalized films. The best results were seen with 1 mg protein/film (HFIP/MeOH) and 3 mg protein/film (aqueous solution/potassium phosphate). These results support the hypothesis that the process of cell adhesion can be improved by enhancing the affinity of collagen to silk films. In general, functionalized spider silk proteins could be the first step towards a new generation of biomaterials.


Stationen
  • Since 08/2011
  • PhD student at the University of Heidelberg, Department of Biophysical Chemistry (Prof. Dr. Joachim Spatz)
  • Since 10/2008
  • Elite Study Program Macromolecular Science
  • 10/2008 - 10/2010
  • Master student of “Biochemistry and molecular Biology” at the University of Bayreuth
  • 10/2005 - 08/2008
  • Bachelor student of Biochemistry at the University of Bayreuth

Berufliche Erfahrung
  • 02/2011 - 07/2011
  • Research Fellow, Department of Biophysical Chemistry, University of Heidelberg
  • 11/2010 - 12/2010
  • Research Fellow, Department of Biomaterials, University of Bayreuth

Stipendien und Auszeichnungen
  • Stipendium der Stiftung Industrieforschung