1. 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.