Spider Silk For Future Scaffolds Heidi Bringhurst, R. Decker, S. Frisby, C. Tucker and Dr. Randolph Lewis Department of Biological Engineering, Utah State University, Logan, UT Abstract Spider silk, an ancient biomaterial, has many qualities worth replicating. With the use of genetic modification, relatively large amounts of the spider silk protein have been produced through goat milk. With access to this protein we have worked to create spider silk films and hydrogels. Through chemical and mechanical means, we are discovering treatments that maximize cell growth and cell attachment on spider silk films and hydrogels. Results Cell Growth on Spider Silk Films Cell Growth on Spider Silk Hydrogels Effects of Formic Acid Effects of Isopropanol Treatment Pre Isopropanol Treatment Post Isopropanol Treatment Control MaSP1 with 0% Formic Acid MaSP1 with 1% Formic Acid Introduction Although spider silk has been around for 100s of millions of years, we are just starting to understand its many uses. The previous lack of understanding was due to a lack of technology sufficient to obtain amounts large enough to experiment with. Since the creation of synthetic spider silk protein using transgenic goats, much research has been done with silk fibers, films, and now hydrogels. Applications for spider silk films and hydrogels are broad, ranging from physical protection to biocompatible materials. Examples of these applications include: high performance helmets, coatings for medical devices, and lightweight UV protection. Spider silk’s potential to serve as a cellular scaffold holds great promise for the medical industry. Spider silk can easily be impregnated with nanotubes, enzymes, and other substances making it highly customizable. Surface Surface Cross Section Cross Section Comparison of Growth on Plate vs. Hydrogel Methods Comparison of Growth on Collagen vs. Spider Silk and RGD Sequence Control Collagen MaSP1 silk film Chemical Multiple spider silk solutions (dopes) were created by dissolving silk protein powder in water. Protein powder was obtained from transgenic goats expressing the spider silk protein (MW approx. 65kDa) in their milk. Further experimentation has included the addition of chemicals which may increase strength in films and hydrogels and increase cell attachment. Future use of hydrogels as a scaffolds will require strength to withstand pressure experienced in vivo. Mechanical Films: Dope solutions are poured in molds made from polymethylsiloxane and left under the hood to dry. Films are then extracted from the mold and put into wells for cell seeding. Hydrogels: After hydrogels are poured into their respective wells ample time is allowed for them to set. Each gel is treated with isopropanol for varying amounts of time to ensure insolubility. Hydrogels are then washed with DPBS prior to seeding with CHO (chinese hamster ovarian) cells and media a top the hydrogels. Control Plate 0.1% Formic Acid Hydrogel MaSP1 silk film + RGD Non Acidic Hydrogel 1% Imidizole Hydrogel Conclusion Cell Growth on Films Mammalian cells are biocompatible with spider silk protein Cell growth on films with 0.5% formic acid produces optimal growth Cell growth and attachment on films is optimized with the addition of RGD, which is illustrated by the visual assessment of growth compared to the dramatic inverse in cell count recorded by the ViCell Cell Growth on Hydrogels Pores shrink upon isopropanol treatment Pore size in the hydrogels is adequate for CHO cells to navigate through, even after isopropanol treatment Preliminary research has revealed that even after multiple washes with DPBS, hydrogels with just a 0.1% acid concentration are still unfit for cell growth. Further research is needed to either create a more stable non-acidic hydrogel or a wash protocol that will allow for optimal cell growth throughout the gel as has been determined with films. Acknowledgements Thanks to Justin Jones of Dr. Lewis’ Lab.