title: Biofabrication using recombinant spider silk proteins
Creators: name: Liensdorf, Elise
Publisher: name: University of Bayreuth
Year: 2018
Subjects: subject: recombinant spider silk, biofabrication, 3D bioprinting, electrospinning
subject: 500
subject: 620

description: For medicine to advance such that it would be possible to regenerate tissue after illness or injury, it is necessary both to achieve a better understanding of human physiology and to apply engineering techniques. The field dedicated to these goals and this type of approach is tissue engineering. The most common approach to forming tissues in vitro is by creating scaffolds with specific characteristics and then seeding the surface with selected cell types. As the field of tissue engineering has progressed, and there is more evidence indicating that this approach alone is not satisfactory, more elegant tactics have arisen. Biofabrication is the simultaneous processing of biomaterials, cells and other biologically active agents to form constructs that have biological functions. However, one of the major bottlenecks for biofabrication is appropriate biomaterials, which has made biomaterial development for biofabrication of significant relevance. Spider silk is a valuable natural resource for high-performance textiles due to its mechanical toughness and stability. More recently, it has not only been valued for its use as a textile, but for its use as a biomaterial. However, natural spider silk suffers from batch-to-batch variability, and farming of spiders is difficult as they are cannibalistic. A solution to this problem is to produce recombinant spider silk protein. The key characteristics of natural spider silk are captured in the sixteen repeats of a spider silk protein amino acid sequence, the C module, that constitute the engineered spider silk protein Araneus diadematus fibroin 4 (eADF4(C16)), which exhibits similar toughness to natural spider silk, hypoallergenicity and biocompatibility, and can be produced consistently in large quantities. Moreover, it can be modified with the cell binding peptide RGD to promote cell attachment onto various scaffolds produced of eADF4(C16)-RGD. For this reason, this protein was investigated for use as a cell-loaded hydrogel for 3D bioprinting, that is, as a bioink. This approach proved to be promising and inspired subsequent work with these proteins as a biomaterial for biofabrication. The purpose of this work was to develop different biofabrication techniques using the recombinant spider silk protein eADF4(C16), in particular to develop bioinks for 3D printing and for biologically-friendly dopes for electrospinning. The motivation for combining these two approaches is that they complement each other. 3D bioprinting allows for precise deposition of cell-loaded hydrogels into complex macrostructures whereas electrospinning produces fibers in the nano- to micron- range. These two approaches together, therefore, can cover a broad spectrum of scaffold features. Although eADF4(C16) has already been used to produce electrospun mats for fine particle filters and in vitro cell culture, the processing conditions have not been biocompatible. Therefore, an aqueous electrospinning dope was developed using highly concentrated eADF4(C16) solution and 400 kDa poly(ethylene oxide) (PEO) as an additive. Furthermore, the post-treatment method was modified from ethanol vapor treatment at 60 °C to water vapor treatment at 37 °C. Using green fluorescent protein (GFP) as a model for a biological active agent, it was demonstrated that GFP remained fluorescent using the all-aqueous processing route. However, fluorescence activity was diminished when added to the traditional spinning dope containing hexafluoroisopropanol (HFIP), or when post-treated by ethanol, thereby demonstrating the significance of the all-aqueous electrospinning route. eADF4(C16) and eADF4(C16)-RGD bioinks were also developed and characterized in this work. By simple observation, it was clear that both the addition of cell culture media and the RGD peptide sequence have an effect on the final properties of the bioinks. It was found that RGD increases the stiffness and the gelation rate of the bioinks, when compared to the same concentration of eADF4(C16), however the addition of cell culture media had a more pronounced effect in terms of increasing the gelation rate and stiffness. After the bioinks were characterized based on their formulation, they were optimized for 3D cell culture. By changing the seeding regime, it was possible to have 100 % cell viability after encapsulation, and the cells were also able to proliferate in eADF4(C16)-RGD bioinks. By simply blending with a low amount of unmodified gelatin the resolution of the printed bioinks were improved, although the cells had reduced viability and proliferation post-printing. Overall, through this work it was shown that the recombinant spider silk protein eADF4(C16) is a versatile biomaterial for biofabrication. In particular, it was successfully used for electrospinning biologically active nonwovens and as a platform for 3D cell culture. Possible future work could include using other variants of the protein to tailor the release of biologicals from electrospun nonwovens, or to promote certain cell behaviors, or to adapt bioink properties. Furthermore, these two types of processing could be used together to create composite scaffolds with variable morphologies.