Team:CCA San Diego/Human Practices

Part of the reason why we chose to work on synthetically produced collagen this year is because of the unethical methods of extracting collagen from animals. Our human practices approach was centered around the conversations with our community, industry experts, an ethicist, and professors, as well as the input we received from these parties

Overview

Collagen is an advancing biomaterials with many potential applications within the medical field, most notably as tissue grafts. Despite these benefits, the current methods for harvesting the bovine and porcine collagen for research are highly unethical, requiring the slaughter of young animals to obtain hoof tendons. Parades of cows are led into a chamber where they are zapped and killed in a routine like an assembly line. Our team seeks to provide a non-animal derived alternative to currently used products in the medical field, ending the unethical practice through a bacterial collagen. In order for our project to be successful, our team needed to meet with different communities and have conversations about the ethical, technical, and scientific values of our solution. Throughout this project, our team consulted the community, professors, and industry experts for input on our solution. These conversations were crucial to ensuring our solution is feasible and actually benefits communities in need of our collagen products. We needed to incorporate this crucial advice and knowledge into our project design to best reflect the public.

Beginning with our brainstorming, we consulted professors from diverse backgrounds about their research into the field and how we can best approach this imminent problem. We kindly received advice from these interviews that shaped our final project design, from discussing various proposals to creating concrete ideas for our collagen solution. Another important facet of our IHP this year was the proposed implementation and considerations of how our solution works in the real world. We began by talking with a bioethicist about patient beliefs and the influence it may have on their medical decision making, specifically for animal-derived products. After weeks of lab work, we had a discussion with a doctor about their ideas for improving our collagen products, both for patient safety and concerns. Through our Human Practices work, we hope to reflect our communities and display the collaborative effort this project was.

Our Values

During the initial research phase of our project, we noticed the inhumane practices required for bovine collagen harvesting. Collagene seeks to use bacterial collagen derived from yeast, eliminating the unethical practices previously used for the collagen industry. By incorporating the ideas brought forth through our public survey and the interviews, our project design is best with our scientific, safety, and ethical standards.

Our Three-Step Human Practices Work

Our Human Practices was centered on the conversations we had regarding our project and incorporating the input into our solution. Each step in our Human Practices work was vital towards aligning our project with the values we set at the beginning of the season.

Stage 1 - Background of the Problem

Dr. Samuel Hudson

Background: Once our team decided on collagen fibers for our project, we reached out to Dr. Samuel Hudson, a professor Emeritus of Polymer and Color Chemistry at North Carolina State University. He worked on biopolymers, particularly cellulose, chitosan, and silk-like proteins all of his career. Additionally, Dr. Hudson is a co-founder of Karamedica, Inc., where he is Chief Scientific Officer.

Takeaways: From Dr. Samuel Hudson, we were able to gain some insight into a multitude of problems. Dr. Hudson recommended the expression of a single protein and until we are about what material we intend on using it is best to rank and take a single material to the next step. He explained how making a film will be much more beneficial and easier than making filaments and also explained how to make films. He also described some issues with using sericin and helped our team better understand the material.

Moving Forward: After we concluded this meeting, our team determined we needed to discuss our project with an engineer. Our team needs to continue working on the wet lab procedures to work in the lab over the summer on schedule. While materials are being finalized, our team should find a couple of sources of collagen, as well as various materials that can be crosslinked and narrowed down.

Dr. John Cavallaro

Background: To better understand the potential implementations of collagen, our team met with Dr. John Cavallaro, the Senior Director and Head of Pipeline Analytics at Bristol Myers Squibb. His group, part of Strategic Options and Assessment function within the division of Business Insights and Analytics, uses advanced analytical techniques to advise product development teams on high-value strategies to advance clinical trials for potential new therapeutics to treat diseases such as cancer, cardiovascular disease, fibrosis, and auto-immune disorders.

Takeaways: From Dr. John Cavallaro, we were able to gain some insight on the specific collagen our team might want to target, specifically the Type 1 collagen, which has various desirable properties. He explained how high tensile strength would possibly be bad for the native cells. We learned how making a monofilament thread is not all it takes to make cloth, which led us to a sound bound felt-like fabric. While continuing to look at crosslinking, he explained how crosslinking fabric reduces biocompatibility.

Moving Forward: After our meeting, our lab team needed to determine whether crosslinking is necessary by looking at the pros and cons. Our team began brainstorming some possible implementations of our project, which included coronary grafts, various fabrics, or ropes.

Stage 2 - Designing our Solution

Deciding Values to Prioritize

Our project design was shaped around our three main values of safety, scientific, and ethics. Through all the information we gathered from the public survey and our professor interviews, our team carefully solidified the most important aspects of our solution. Prioritization is the basis for our proposed implementation and project design. Without proper prioritization we will not achieve our goals and a responsible product.

The human practices survey we conducted on the ethics and environmental impacts of our project reached an international audience and was integral to our values. The questions pointed towards peoples’ opinions on the current situation with biomedical products, as well as key issues in our project design such as the crosslinking reagent. At the end, there was a section for additional suggestions which our lab team looked through thoroughly before proceeding. A significant portion of our respondents, 30.7% , had an education level of master’s degree or higher. As a result, most of the suggestions and responses come from people educated in the fields of science, providing more specific and accurate data about the scientific portion of our project. Additionally, the sample of our survey covered a wide demographic of people in education and age, providing the best summary of the general public.

From the survey, our team concluded that our community is highly concerned with the safety of our products in vivo, the unethical practices of collagen harvesting, and the actual technicality of our solution.

Patient safety from crosslinking reagents

By altering the methods and technology of collagen harvesting, our project is able to not only reassure patients with their environmental benefits of reducing biomedical waste and diminishing the harm of animals, but patients will also feel safer about their own personal employment of synthetically produced collagen.

According to our ethical surveys which discuss the non-harmful substances within the production of synthetic collagen, it is evident that the majority were open to the usage of collagen without the use of non-toxic chemicals. This may suggest that through the new utilization of crosslinking reagents, patients be reassured of their investment in collagen as well as maintain a secure fiber structure.

Ethics of current collagen harvesting techniques

Replacing the current inhumane techniques for collagen harvesting is highly important in our project design. Our team wanted to advance research into collagen biomaterials, while reversing the unethical practices.

According to our ethical survey, we can conclude that 61% of participants recognized the drawbacks that come from the process of extracting collagen from bovine tendons. Our solution needs to provide an equal alternative for collagen products that bypasses the process of extraction, so we planned to use yeast to express the protein.

Concerns of patients about our products

For our proposed implementation, we need to consider the ethical concerns patients may have about using bacterial collagen. Through our Human Practices survey, we gathered that participants were somewhat comfortable with the production of collagen using yeasts, with the average rating of 3.97 out of a total of 5. We recognize that GMOs are still controversial in society and not easily accepted, so the comfort and acceptance of the public has been a high priority value. We hope to assuage these hesitations through synthetic biology education and clearly describing our methodology.

Technicality of our collagen products

Our team believes that our final solution should achieve the same properties and maintain the structure of current collagen products. The new bacterial collagen should not function worse compared to harvested bovine collagen. We incorporated this into our solution design by crosslinking the expressed collagen to strengthen the material.

One of our survey respondents suggested considering the highly variable nature of yeast expression, resulting in less stable production of collagen. As a result, in our proposed implementation we seeked to create a bioreactor to better grow yeast colonies for mass production.

Dr. Oded Shoseyov

Background: While designing our wet lab procedure, Dr. Oded Shoseyov provided assistance on our wet spinning apparatus and the crosslinking reagents. He is a professor at the Hebrew University of Jerusalem, focusing on nanotechnology and plant molecular biology.

Takeaways: From Dr. Oded Shoseyov, our team gained insight on how different draw ratios will change fiber properties and molecular order. He also explained how telopeptides are not integral for collagen assembly and properties. We also learned that the time for the process will depend on the organism being used

Moving Forward: Our HP team needs to work on the proposed implementation of our project, as the various issues with materials are being sorted out and concluded. Our team also needs to continue working on and engineering the spinning apparatus. Now that project design was finalized, we were advised to find a source for the collagen testing.

Dr. Greta Faccio

Background: To narrow down our crosslinking reagents, our team scheduled a consultation with Dr. Greta Faccio, who is currently active in the fields of intellectual property protection and as an independent scientist. Her expertise in protein science has been applied to food science, biomaterials design, medtech devices, biosensing, and recently, cosmetics.

Takeaways: Throughout the meeting, Dr. Faccio discussed the benefits of different crosslinking reagents, as well as the differences between chemical and enzymatic crosslinking. Enzymes are used in small amounts, and the process occurs relatively quickly. She also explained that UV crosslinking is not ideal because of its weakness and excessive time needed. For the wet lab procedure, she informed our team to be careful with the formation of dimers.

Moving Forward: Dr. Faccio recommended designing a smart material with application in many fields and not limiting ourselves to a biomedical thread. Our team will try creating a collagen thread, gel, and felt for multiple implementations. Moreover, we will use codon optimization for our plasmids and order directly from IDT.

Mr. Leonard Sebastian Fresenborg

Background: As a former iGEM alum for Goethe University Frankfurt in 2012, he is now a PhD student with a Bachelor’s and Master's degrees in Biochemistry. During his time at Frankfurt, his team used Saccharomyces cerevisiae to express their plasmid and had successful results, which gave motivation for CCA’s iGEM to reach out for advice.

Takeaways: During our discussion about expressing our plasmid through yeast assembly, we clarified the pros and cons of transferring the plasmids to E.coli to get a higher yield of Scl2 before transferring it back into the yeast. After a quick look at our plasmid, we also talked about using His-tags and ways to simplify our project, especially since our project is not trying to produce larger, commercial-level yields. Lastly, Leonard gave us advice on using BioBricks and how CCA can document different plasmid parts for the parts documentation criteria.

Moving Forward: We plan to continue our project with Leonard’s ideas in mind, especially with the additional step of transferring the plasmid into the E.coli instead of a one-step yeast assembly, which can lead to lower yields. We also have to decide whether to use His-tags in the near future.

Dr. Yu Qiao

Background: To understand the feasibility of modeling mechanical properties of our collagen and possible avenues we could take, we met with Dr. Yu Qiao, professor of materials science engineering at UCSD.

Takeaways: Dr. Yu Qiao provided valuable insights on factors that should be tested to determine the quality of our collagen. Dr. Qiao stressed the importance of strength and provided variables that aid in determining the strength of materials (e.g. chain length, secondary bonds, polarity, etc.). He also taught us methods for testing strength as well as these variables. The proposed tests are carried out either by testing melting temperature or by observing the creep of our collagen by utilizing a weight.

Moving Forward: Our lab team will test the collagen’s strength using Dr. Qiao’s ideas for assessment. The modeling department will focus on the factors of strength and create equations for modeling the strength and Young’s modulus of the collagen.

Global Survey Analysis

The survey results helped us understand our project and public perceptions about the topics that our project covers better. For instance, the survey indicated that most respondents believed that they are relatively concerned with biomedical waste, but understand that it is necessary. Such responses reaffirmed and validated our solutions as an effective solution for helping various issues relating to our project. The survey also showed that the majority of people would be comfortable with using genetically modified products and that our project addresses many of the top concerns that people have regarding genetic modification. All of this information helped further reaffirm our project and showed that our method was relevant. Finally, we did correlation analysis with gender and age to assess whether any confounding variables affected our results drastically. While there were some differences in results between males/females and older/younger participants, we believe that we had a wide variety of responses that fairly represented public opinions on our topic. We used these responses to further improve our project so that it will have the greatest positive impact on the people.

Survey Analysis

Stage 3 - Real World Implementation

Proposed Implementation

After designing our solution and testing the procedure through wet labs, our team began considering the proposed implementation into the real world. We designed our collagen solution with safety and ethics in mind, but we need to understand the issues that may occur from implementing the project into hospitals. To gather information on this topic, we reached out to a bioethicist and a nurse practitioner to understand the process of introducing a new biomaterial into the medical field.

First, for our proposed implementation we contacted Mr. Daniel Rodgers, a bioethicist with interests in perioperative care and patient treatment. Our team wanted to ensure we were addressing any patient moral qualms about our collagen products that would prevent its usage. He advised us to clearly articulate the process for producing the collagen from bacteria to patients and the non animal-derived materials. This informed our patient safety and ethical values.

Additionally, we reached out to Dr. Athena Mohebbi, who works as a nurse practitioner in regenerative medicine. When we asked about the properties required for biomedical products, she explained the hypoallergenic properties needed. This led us to research the biocompatibility of the collagen products and the process of FDA approval. She also described the many potential applications of our collagen because of its three materials, but we need to ensure it functions. Through this interview we better understood the technical aspects of our collagen products and its applications in the biomedical field.

Check out our Proposed Implementation page for the specific details and considerations

Closing the Loop between what was designed and what was desired

We made multiple changes to our desired product to our designed product. To begin with, we modified the TCA Extraction procedure. Our collagen would denature a bit over body temperature so we modified the TCA extraction such that it didn’t cross the temperature threshold.

We ordered different fragments that ended up wasted as our DNA design was changed. Initially we were doing primers with overhangs instead we changed to fragments with overhangs in order to maximize efficiency. In addition, we made plasmids with 2 and 3 fragments which required multiple reiterations. Also, we changed medium from Ampicillin to carbenicillin as our bacteria gained Ampicillin resistance. Finally, we abandoned our original gelification procedure and used a PG based buffer for new gelification.

Mr. Daniel Rodger

Background: Our team reached out to Mr. Daniel Rodger for advice on our proposed implementation and the ethics of animal-derived constituents in the medical field. He is a registered Operating Department Practitioner (ODP) and a Senior Lecturer in Perioperative Practice at London South Bank University. His research interests broadly include areas within bioethics, medical ethics, and perioperative care.

Takeaways: Mr. Rodger elaborated on the issues of current medical practice and how our solution can be implemented into the real world ethically. Since Collagene uses collagen derived from yeast, practically all secular and religious qualms disappear. There is no legal obligation to inform patients about the origins of our synthetic medical device because of the astronomically low concerns. However, people may forgo their ethical concerns if the cost of our product is too high. Additionally, he explained that currently there is no fully vegan medical device because animal clinical testing is mandatory.

Moving Forward: Our team needs to better understand the incentives for patients to switch to the Collagene products. Elaborating on the benefits of yeast-derived collagen and the harmless properties of GMOs is vital. Mr. Rodger also advised us to look for precedents and whether our concerns were issues in the past.

Dr. Athena Mohebbi

Background: To better understand the potential applications of Collagen and concerns patients may have with our products, we interviewed Dr. Athena Mohebbi for her input. She is a cell and regenerative medicine nurse practitioner with over a decade of experience in the field.

Takeaways: From Dr. Athena Mohebbi, our team learned that the product can have various applications in spinal injuries, burn and trauma, or other orthopedic fields. Since our team is producing three materials from the expressed collagen, we can easily modify the collagen for different applications. In general, the products should have hypoallergenic properties to ensure public safety and alignment with our project values.

Moving Forward: We were advised to better educate our community about bacterial expression because patients may be averted by bacterial products. Bacteria has a negative connotation and is only viewed as infections, many patients may have hesitancy with using novel biomedical products. Our team needs to ensure Collagene is not too costly and we clarify the production methods.

Ethics and Environment

Based on feedback and concerns we got from the public - via class presentations and surveys we decided to write an environmental and ethical report to ensure our project was safe to the environment and considered all possible ethics issues. This report is influenced by our meeting with an ethicist as well as other research papers addressing potential problems with our project. We did this very early on in our project and our lab work, modeling, hardware, and scicomm are influenced by this. For example, we made use of a bioreactor to contain the yeast so that the yeast cannot enter the environment. Even if they do, we modified our lab work and plasmid so that none of the protein they are expressing will be harmful to the other biota.

	Extracting collagen from animals	Bioengineering collagen
Humane practices	No, extracting collagen from animals is not humane at all and is far less humane than bioengineering collagen, like our project topic. In order to gain a large amount of collagen to be applied to biomedical purposes, a large amount of animals would have to be killed as the yield of by-products is low and only a portion of those by-products contain collagen.	Yes, bioengineering collagen is more humane than extracting collagen from animals. Since we are bioengineering collagen and do not need to kill animals (mostly pigs and cows), the production of bioengineered collagen will be completely humane.
Time (how long the process of extracting collagen takes)	Extracting collagen from animal hooves and other parts and humans is the most time efficient as it would require time to kill the animal, gather the by-products, determine which of the by-products contain collagen, and prepare the collagen.	Bioengineering collagen is more time efficient than extracting collagen from animals because bioengineering methods will be utilized which will produce the collagen itself. Taking steps to prepare the animal will not be needed.
Efficiency/effectiveness	The process is not very efficient or effective. Variability in preparation of the collagen extracted from animals can lead to difficulties. Animal extracted collagen can transmit diseases, as well as some people may experience allergic reactions to animal extracted collagen. The yield also depends on the species of animal, and on physical properties of the animal itself.	More efficient and effective. Since bioengineered collagen will be manufactured in a lab and will be able to be controlled.
Consistency	This method of extracting collagen from animals and humans is not very consistent. The specific types of collagen can slightly vary from species to species and sources in general. This creates some problems in the engineering process of making things out of collagen currently since each type is slightly different at the molecular level so they all react in different ways.	The process of bioengineering collagen is more consistent than naturally extracting collagen from species of animals and different sources. Since bioengineered collagen is more monitored in the engineering process and follows strict lab protocol, all of the bioengineered collagen will be exactly the same at a molecular level meaning that there will be fewer, if any, variations in how the collagen reacts with things. To conclude, bioengineering collagen would give the collagen a more consistent finish with fewer variations of the same thing.
Waste	Extracting collagen from animal species and humans creates a great deal of waste since the animals must essentially be dissected to an extremely small scale in order to extract the collagen from them and the remains of the different animal species, and sometimes humans, are left without a use so they create waste.	Bioengineering collagen in a lab would create less hazardous waste for the environment. This is due to the fact that there are fewer items and parts that cannot be used and only classic lab waste is left while mutilated and dissected animals are left in the process that is used currently, extracting it from dead animal species and humans.
Biomaterial use	When extracting collagen from animals and humans, the collagen cannot be used to deliver drugs or develop the bioengineering of human tissue. It is not heavily regulated and the molecular structure cannot be controlled when using the method which simply extracts collagen from animals.	Biomedical engineering collagen in a lab rather than extracting the collagen from animals means that the collagen which is biomedically engineered can be used to deliver drugs to various parts of the body and more. Since the lab procedures are much more regulated than the current method, which is extracting collagen from deceased animals and humans, the molecular structures of each collagen molecule will be much more similar to each other so they can perfectly be recreated every time in order to gie drugs to people. Additionally, bioengineered collagen can be used to create fake human tissue which can be used on any part of the body.
Ability to be manipulated	Since this process of extracting the collagen from animals simply extracts the collagen, the specific structure of the collagen is already predetermined and cannot be manipulated into being used for a certain or specific cause that it may be needed for.	Bioengineering collagen creates the collagen from yeast from scratch, or the very beginning of the process. This is advantageous because it can be manipulated in a large variety of ways throughout the entire experiment to be suited for the specific cause or purpose that people want to use the bioengineered collagen for and people can use it for so many things if it is slightly modified including tissue development, as well as being a drug deliverer. Different drugs could be delivered based on how the collagen structure is modified during the bioengineering process.

Environmental Analysis Ethical Concerns Review