title: Spider Silk Scaffolds as a Material for new Biomedical Applications
Creators: name: Aigner, Tamara Bernadette
Publisher: name: University of Bayreuth
Year: 2020
Subjects: subject: spider silk
subject: biomedical engineering
subject: tissue engineering
subject: bioactive agent
subject: heart muscle regeneration
subject: nerve regeneration
subject: enzyme confinement
subject: 500

description: Biomedical engineering arose out of the collaboration of medical doctors, biologists and engineers and substantially accelerated advances in medicine. Researchers in this field strive to improve medical care through the development of diagnostic tools and devices, prosthetics, surgical tools and robots, and tissue engineering. The latter one seeks regeneration or restoration of a damaged or diseased tissue. Materials used on or in human body must fulfill several divergent requirements. Firstly, they should provide mechanical support by offering sufficient mechanical strength, on a scale commonly found in synthetic polymers. Secondly, they should be biologically compatible and for instance trigger cell recognition usually attributed to natural polymers. One material type that captures both requirements are spider silk-based materials. Their unique chemistry and structure give spider silk fibers extraordinary mechanical properties and no immune response is induced in the body. Inherent cell recognition (eukaryotic and prokaryotic) of most native spider silks is poor, but if required, cell binding motives can be added using a biotechnological approach to produce recombinant spider silk proteins, which can further be processed into various morphologies. The objective of this dissertation was to exploit the beneficial properties of coatings, films, nonwovens and self-rolling bilayers made of recombinant spider silk proteins to widen the scope of their use in biomedical engineering. Spider silk scaffolds were investigated for loading with bioactive agents, use in heart muscle and nerve regeneration and as enzyme container. Accurate delivery of sensitive biological substances can improve cell behavior on scaffolds for tissue engineering. In the first project electrospun nanofibers with their inherent advantages of high porosity and surface-to-volume ratio were loaded with green fluorescent protein (GFP) as a bioactive agent model. Its fluorescence is structure dependent and common solvents used for electrospinning and post-treatment of silk destroyed GFP’s structure. Therefore, an aqueous electrospinning and post-treatment process was developed, which allowed a mild encapsulation and kept the structural integrity of GFP. The fast release of GFP could be inhibited or slowed down by genetic modification giving the system a broader application window. Post-surgical infections are a life-threatening risk and the increase in antibiotic resistant bacteria prove the need for alternatives. The second project investigated the antibacterial properties of spider silk coated selenium nanoparticles alone and encapsulated in silk films. Selenium nanoparticles are effective against gram-positive bacteria. By applying a coating of spider silk, the scope could be widened to gram-negative bacteria. Low doses of the coated nanoparticles killed Escherichia coli without impairing eukaryotic cell viability. The body’s capability of cardiac repair after a myocardial infarct is poor and materials to assist cardiac regeneration are desperately needed. Thus, in the third project, the behavior of primary cardiac cells on spider silk films was investigated. The secondary structure content and the water contact angle of these films was found to be in a suitable range. Cell studies showed that silk films are non-toxic and provoke no pharmacologic effect. Furthermore, cardiac cells grown on these silk films showed required cell-to-cell communication and responded properly to extracellular stimuli, thus, laying the foundation for use of silk in cardiac tissue engineering. Injuries of the peripheral nerve system still show an inacceptable recovery rate. Thus, in the fourth project, bilayers of spider silk and chitosan were self-rolled into tubular structures to act as nerve guidance conduits. The tubes were either lined with a silk film containing a cell-recognition motif or aligned silk nanofibers, or an anisotropic collagen cryogel was encapsulated filling out the luminal space. The mechanical properties of the collagen cryogel are in the range of healthy peripheral nerves. Nerve cells could be entrapped by the gentle rolling process and their differentiation was achieved inside of the tubes, allowing the formation of neurite outgrowths. The aligned structures even triggered orientation of these neurite outgrowths in longitudinal direction, as is required in nerve repair. In the fifth project, these self-rolling tubular structures were optimized to encapsulate enzymes. They were shown to be stable in relevant organic and aqueous solutions and to possess a molecular weight cut-off above 20 kDa. Hence, these enzyme containers allowed the entrapment of enzymes, while substrates, intermediates and/or products can diffuse freely through the tube wall. This allowed to exchange the surrounding media without removing the enzymes, thereby saving often costly enzymes and enabling the design of a flow-through system. Reaction rates were slowed down, but longer reaction times were observed. Thus, this system can protect enzymes and may be useful for applications in biodiagnostics. Through this work, we showed that recombinant spider silk-based materials have a high potential in several fields of biomedical engineering. The processability into various morphologies and the precise control over the protein sequence makes it an interesting option for diverse applications. This thesis only gives a small glimpse at the scope of possibilities and more is yet to come.