Team:CCA San Diego/Description

From cosmetics to supplements to prosthetics, collagen has countless applications, and is prevalent in a wide variety of products. The most prevalent protein in the human body, it is present in bones, connective tissue, ligaments, and the skin. In the human body, collagen is the most prevalent protein as it is essential for wound healing. On damaged connective tissues, collagen folds into a natural scaffold-like shape that is completely nontoxic and biodegradable. It also attracts fibroblasts, which assists in creating scars to cover the wounds. Collagene is an effective, efficient, and ethical approach to producing bacterial collagen in yeast.

Collagen: the Binding of the Present

From cosmetics to supplements to prosthetics, collagen has countless applications, and is prevalent in a wide variety of products. The most prevalent protein in the human body, it is present in bones, connective tissue, ligaments, and the skin. It is a hard, fibrous, and resistant protein thanks to its triple helical shape (Shoulders et al 09). These properties make it a great biomaterial, applicable to skin meshes, bone grafts, and beauty appliances. It has also produced exceptional results in the meat, food, and beverage industries (Kolar et al 90).

In the human body, collagen is the most prevalent protein as it is essential for wound healing. On damaged connective tissues, collagen folds into a natural scaffold-like shape that is completely nontoxic and biodegradable. It also attracts fibroblasts, which assists in creating scars to cover the wounds (Rangaraj et al 11). Due to its ability to create new connective tissues to cover wounded skin, scientists have taken steps to create surgical biomaterials.

Problems in the Status Quo

Current methods of collagen extraction are questionable from an ethical perspective, as they often involve the harvesting of calf tendons and hides for collagen samples. Alternative methods of production are shown to have dramatically lower yields, or are much less efficient - and therefore more expensive. While the current highest yields of collagen may come from animals, the derivation process is both unethical and immoral; said animals (typically cows) are treated very inhumanely, and they are raised only to ultimately be slaughtered (Noorzai et al 20).

Additionally, the byproducts of bovine collagen harvesting pose significant health concerns to humans. The Food and Drug Administration (FDA) has found that - due to contaminants - several collagen materials have been the cause of serious problems, such as arsenic poisoning and mad cow disease (Regenstein et al 07). These contaminations often occur in slaughterhouses, where it has been shown to be a breeding ground for zoonotic diseases.

There have also been numerous discussions regarding livestock’s effect on the environment. The amount of space necessary, the amount of food and energy, and resources necessary to grow livestock is much less efficient, and producing in a lab setting is likely cheaper and more compact. To bypass the ethical, medical, and financial concerns, we propose that we express collagen through yeast.

Producing the Collagen

We needed to find a way to produce collagen in an efficient and ethical manner, and doing so would be through recombinant expression in E.Coli or through yeast. There also needed to be a balance on how affordably the collagen could be extracted, so after reading through existing literature, we decided that yeast would be the perfect balance between monetary accessibility and efficiency.

To find alternative methods of yielding larger quantities of collagen, we considered multiple bacterial collagen-like proteins as well, such as Bcl (Bacillus collagen-like), Pcl (Pneumococcal collagen-like), Bucl (Burkholderia collagen-like), and Scl (Streptococcal collagen-like) proteins (Lukomski et al 17). Of these, our team chose to work with Scl, as it is the most well-known and well-documented collagen-like protein (though still relatively new, allowing for creativity and novelty on our part).

Our method of extraction is to use a Scl2 expression gene and express it via S. cerevisiae, a type of yeast. Scl2 has demonstrated qualities similar to collagen with the similar triple helical structures - yet with the benefit of not requiring a post-translational modification mechanism (a step necessary for most mammalian collagen).

The Blank Slate

Unlike other approaches to the problem which also utilise synthetic biology, our yeast-expressed collagen can be used as an engineering template for future research; due to the absence of hydroxyproline and binding sequences, it can be used and tested many different ways. The novelty of our product lies in the simple versatility of it: ours is the first to express bacterial collagen in yeast, the first to have gap repair cloning, and since we are not expressing proxyl hydroxylase, we have effectively cut both costs and oxygen.

Applications

Due to the versatile nature of collagen and its natural ability to repair itself, our yeast-expressed version can be used in a variety of different instances, with a primary focus on its tissue engineering capabilities. We created three products to demonstrate a few of its many applications, which can be reviewed in depth on our Project Implementation

Thread

This thread could be used as a substitute for stents in cardiac bypass surgery - serving as a graft - with mechanical properties mimicking that of blood vessels (Zhang et al 19). Additionally, it could potentially connect neurons, serving as a structural basis for the regeneration of neuronal connections (Xiao et al 11). Other applications might include textiles, sutures, tissue grafts, and more.

Gel

Collagen gel has various applications in tissue engineering research, and if developed further, could potentially be used as a bioink for 3D-printing artificial tissues and organs including, but not limited to: skin, bone, cartilage, as well as liver, neural, corneal, and cardiovascular tissue (Olegovich Osidak et al 20). It could also be used as a stabilising agent in vein grafts (Atala et al 08), or a component in topical solutions for both medical and cosmetic use.

Felt

Presently, traditional wound dressings (i.e. plasters, bandages, gauze, etc.) make it difficult for the healing process to be seamless, introducing complications like easy infection, discomfort, irritation, excessive drainage, and the simple lack of an environment conducive to the healing itself. Collagen felt could be used as a wound dressing, or even a surgical material in medical settings; a facilitator of wound healing processes, its characteristics are extremely versatile and applicable in many instances (Hoque et al 18).

Collagene: the Building Blocks of the Future

Numerous types of biomaterials can be formed using the collagen fiber creation method that we propose, from wound dressings, skin grafts, vascular grafts, artificial corneas, and more. The use of collagen in the biomedical industry has endless possibilities and we hope that our protein can act as the base of many more biomaterials to come.

References

Shoulders, M. D., & Raines, R. T. (2009). Collagen structure and stability. Annual review of biochemistry, 78, 929-958.

Kolar, K. (1990). Colorimetric determination of hydroxyproline as measure of collagen content in meat and meat products: NMKL collaborative study. Journal of the Association of official Analytical Chemists, 73(1), 54-57.

Cavallaro, J. F., Kemp, P. D., & Kraus, K. H. (1994). Collagen fabrics as biomaterials. Biotechnology and Bioengineering, 43(8), 781–791. https://doi.org/10.1002/bit.260430813.

Lukomski, S., Bachert, B. A., Squeglia, F., & Berisio, R. (2017). Collagen‐like proteins of pathogenic streptococci. Molecular microbiology, 103(6), 919-930.

Rangaraj, A., Harding, K., & Leaper, D. (2011). Role of collagen in wound management. Wounds uk, 7(2), 54-63.

Noorzai, S., Verbeek, C. J. R., Lay, M. C., & Swan, J. (2020). Collagen extraction from various waste bovine hide sources. Waste and Biomass Valorization, 11(11), 5687-5698.

Regenstein, J. M., & Zhou, P. (2007). Collagen and gelatin from marine by-products. In Maximising the value of marine by-products (pp. 279-303). Woodhead Publishing.

Zhang, F., Xie, Y., Celik, H., Akkus, O., Bernacki, S. H., & King, M. W. (2019). Engineering small-caliber vascular grafts from collagen filaments and nanofibers with comparable mechanical properties to native vessels. Biofabrication, 11(3), 035020. https://doi.org/10.1088/1758-5090/ab15ce.

Xiao, T., Staub, W., Robles, E., Gosse, N. J., Cole, G. J., & Baier, H. (2011). Assembly of Lamina-specific neuronal connections by slit bound to Type IV COLLAGEN. Cell, 146(1), 164–176. https://doi.org/10.1016/j.cell.2011.06.016.

Olegovich Osidak, E., Igorevich Kozhukhov, V., Sergeevna Osidak, M., & Petrovich Domogatskiy, S. (2020). Collagen as Bioink for bioprinting: A comprehensive review. International Journal of Bioprinting, 6(3). https://doi.org/10.18063/ijb.v6i3.270.

Atala, A., Lanza, R., Thomson, J. A., & Nerem, R. M. (Eds.). (2008). Principles of regenerative medicine. Elsevier - Academic Press.

Hoque, A., & Mahmood, I. (2018, March 11). Collagen: Changing the course of the future. Textile Study Center. https://textilestudycenter.com/collagen-changing-course-future/.