After assembling our team, we spent the first two months in brainstorming sessions, researching potential topics for this year’s competition. A team member presented us the field of bionic prosthesis, and we were all shocked by the number of people who were suffering from an amputation. His first idea was to use the capacity of bacteria to drive an electrical current to amplify the signal between the patient’s nerves and electrical sensors linked to a prosthesis. This way, redirection of nerves would not be necessary and the patient would be able to accomplish more natural actions.

When we started to look more in details the field of prosthesis and implants, we realized that along with device loosening or malfunctions and foreign-material reactions, infection remains the most serious problems encountered with surgical implants. We quickly learned that biofilm formation is common to all types of implanted foreign-body infections. Indeed, the high susceptibility of implanted devices to infection is due to a locally acquired host defense deficiency. Thus, this persistence at a specific site is mainly caused by the rapid formation of a biofilm, which is resistant to host defense and antimicrobial agents as a result of reduced access and diffusion characteristics within it. We then decided to integrate this aspect inside our project: while favoring the growth of the nerve and the conduction of a signal through a bacterial interface, we could also diminish the risk of infection. At first, we wanted to find a system to directly kill the Staphylococcus aureus, but after talking with Dr. Jean-Marc Ghigo (Genetics of Biofilm Unit, Institut Pasteur), we realized that the biofilm configuration would give us some hard time and require extra steps to manipulate. We decided to shift our goal, and rather than killing S. aureus, we would limit the virulence of the bacteria and restrict its ability to form a biofilm. This could be achieved by subverting the quorum sensing of the pathogenic bacteria and blocking the signal. In this way, S. aureus could be handled by the host’s immune system and by the patient’s doctor with a normal dosage of antibiotics.

In order to learn more about the significance of these issues in the medical field, we interviewed many professionals working either directly with patients, industries working on high tech metallic or ceramic implants, and amputees through the contact of ADEPA « Association for the Defense and Study of Amputated People ». Through all this work, we were invited for many tours, first at the European Hospital George-Pompidou where we had the chance to observe Dr. Benjamin Bouyer M.D., a lumbar rachis surgeon, during surgery to see the procedures put in place to diminish the risk of infection during the integration of an implant inside the body.

The first thing we needed to do was to narrow down the pathogenic bacteria we would work on. After researching in the literature and talking with experts in the fields of prosthetics, we found out that S. aureus and S. epidermidis are the two most frequently found bacterial infectious agents on implants. We decided to use the biobrick designed by the iGEM team SDU-Denmark 2009 who worked on a genetic circuit enabling Escherichia coli to actively detect S. aureus .

Our bacterial chassis of choice, which would inhibit the quorum sensing of S. aureus, needed to be a bacterium naturally occurring inside the human body. Indeed, we didn’t want to introduce a new strain inside the human body. The chassis also needed to have a good secretion system, as well as a good tolerance for the immune system. We narrowed down our choices between B. subtilis, which the iGEM LMU Munich 2014 worked on, with the same problematic, and E. coli, naturally living inside the gastrointestinal tract of humans with mostly no problem except for some strains. Because we wanted to improve the construction of iGEM SDU Denmark 2009 and because of convenience inside our lab, since we already had competent E. coli cells, we decided to use this strain. We optimized our sequences for an E. coli expression, and adapted them to its secretion system by adding export signals. This way, our construction could be broadened and used in a different type of bacterium. To replicate our plasmids, we used E. coli DH5 alpha and to secrete our proteins, E. coli BL21De3 pLys S.

When conceptualizing our proof of concept device, we decided to design a microfluidic chip to simulate the actions that would occur inside the patient’s body. The chip would be capable of measuring the neuronal signal as well as the conductivity of the biofilm, letting us know whether our system would work correctly inside a prosthetic. After talking with many professionals such as Dr. Heng Lu (ESPCI) and Dr. Ayako Yamada (ENS, Ecole Normale Supérieure, Paris), we first designed a PDMS microfluidic chip with a vitreous carbon electrode to measure the signals. After talking with Dr. Catherine Villard (Institut Curie, Paris) and Dr. Frederic Khanoufi (University Paris Diderot—ITODYS), an electrochemist, we realized this type of electrode was highly sensitive, perhaps too sensitive for our type of device, and not adaptable to the size of our microfluidic chip. Guided by their advice, we switched to gold electrodes and started the fabrication process of the different type of chips at the Pierre Gilles de Gennes Institute. After attending the iCOE 2018, we learned about PEDOT (poly(3,4-ethylene dioxythiophene) polystyrene sulfonate ), and its conductive properties. As we also wanted to confine our bacteria so they would not harm our neuronal cells, we partnered with Sterlitech, and tested nanoporous polycarbonate membranes coated in gold as well as nanoporous alumina oxide membrane coated in PEDOT: PSS, PEDOT: CL and PEDOT: TS. This way, we could still measure the neuronal signal and the conductivity of the biofilm while protecting the cells from getting eaten by the bacteria.

When we started to think about the scientific aspect of our project, we also started to design and think about how our biofilm would integrate into a physical medical device. First, we wanted to design a full prosthesis. We realized that it wasn’t the core of the problem. Indeed, the technology missing in this field was the actual interface between the prosthesis and the osseointegrated steel/titanium/ceramic stem inside the human body, limiting the field of the bionic prosthesis. We decided to focus on this interface and started to talk with Dr. Benjamin Bouyer, a lumbar rachis surgeon, on how we could deposit the biofilm on or in the osseointegrated stem. We realized that the stem would be in direct contact with the surgeon meaning that if we coated the whole structure with our biofilm, it would probably be stripped off, get deposited on the gloves and contaminate other areas. We also spoke with one member of the board of directors of ADEPA, “Association de Défense et d’Etude des Personnes Amputés », which translates to « Association for the Defense and Study of Amputated Persons” who is himself an amputee. He gave us great advice on the designing phase of this interface and raised the issue of the socket causing excessive sudation and discomfort for the patient. After meeting with experts from i.CERAM (Ceramic medical devices company, Limoges, France) and the CERAH (Center for Studies and Research on the Equipment for the Handicapped), we learned more and more about the different materials used in the making of prostheses. At this point, we realized that doing a full stainless-steel interface would stimulate the growth of bacteria in a biofilm structure. Therefore, we decided to switch and do the part in direct contact with the patient in ceramic, knowing that we still have the same problem that i.CERAM and the CERAH were facing currently. Indeed, steel doesn’t last as much as ceramic, which could be a problem when creating an interface composed of both materials. We knew then that the patient would probably need corrective surgeries to fix his osseointegrated stem. Having considered these parameters, we then decided to start modeling our prototype by integrating those different aspects as much as possible. Because we wanted it to be cost-effective and injection-moldable, we want to build our future prototypes in ABS, a thermoplastic polymer that could be molded by injection. We ordered the electronic parts composed of a charger, battery, and amplifier, and assembled it into its current state as our POC.

While we were doing adjustments to our design in the lab, our team of jurists also researched how our device could be integrated into society given the current political and economic landscape in France. The use of GMOs, for the environment, food industry or medicine, is highly regulated in France and Europe. It is limited to design animal models of diseases and to produce large quantities of molecules for the pharmaceutical industry. The use of genetically modified bacteria inside the human body is not the subject of specific laws, which made our research on the subject difficult. We focused our research on the medical device’s regulation in order to see if our project could be marketed. At first, it appeared almost naturally that our project was a medical device, but as it has to be understood as a legal term, it responds to a specific definition. Working on medical devices’ regulations helped us to better define our project, classify it and analyze our project in the scope of European regulations. We also tried to cover some legal questions related to our project and tried to find answers from other legal systems such as the American one.

All these feedback from scientific experts, association members representing patients, and physicians/surgeons, allowed us to develop our project NeuronArch to what it is today. Without them, our project would not have been the same, and we really want to thank them for that.

In medicine, surgery is a very common procedure. According to the data available, gathered by the Royal College of Surgeons, around 4.7 million surgical procedures were performed in 2014 in England; meaning that 7% of the total population is impacted by surgery. In the United States of America, according again to data gathered by the 2010 Ambulatory Medical Care Survey (NHAMCD), 48.23 million surgical procedures were performed in 2010. That’s around 14.8% of the total population. 5% of patients undergoing surgery develop surgical site infections (SSIs), which may cause substantial morbidity that can endanger a patient’s life, increase the number of days in the hospital and increase healthcare costs. SSIs are defined as infections that occur within 1 year if an implant is being placed.

We wanted to understand what goes on during a surgical procedure, what types of precautions were taken and what were the impacts of an infection for the patient. Thus, we began contacting surgeons and associations to learn more.

Interview with Dr. Bernard Zalc

Dr. Bernard Zalc is co-leader of the oligodendrocyte development and neurovascular interactions department. He studies oligodendroglial cell development in the embryonic brain and the interactions between neural cells and the cerebral vascular network.

Dr. Bernard Zalc facilitated our understanding of the nerve growth and explained to us some issues that we could encounter. He gave us some tips regarding nerve growth and the connection between the nerve and our interface. Indeed, for him, other neurotrophins (like BDNF and VEGFA) and chemoattractant molecules are needed in order to attract and direct the growth toward the implanted interface of our system. We exposed to him our experimental approach for the proof of concept and he talked about the importance of the Schwann cells and the myelinization for the conduction. Moreover, he shared contacts which can help us regarding the modeling parts and the prosthesis.

Interview with Dr. Eric DENES, Dr. Evelyn Poli and Dr. Christelle Arico

Dr. Eric Denes is an infectious diseases specialist at the University Hospital of Limoges and is the scientific director of i.CERAM. Dr. Evelyn Poli is a chemistry research and development engineer and Dr. Christelle Arico is a project manager at i.CERAM.

Dr. Eric Denes, as well as Dr. Evelyn Poli and Dr. Christelle, were very interested in our project. They are currently designing new materials in ceramic, titanium, and stainless-steel to test the bacterial adhesion on their prostheses. They warned us about the use of metals for the prostheses since S. aureus and P. aeruginosa tend to form biofilms easily on metals rather than on ceramic. They are also using Gentamicin and Vancomycin charged implants to cure infections. Their system does have a long half-life time (4 days) whereas our biofilm could be a longer-term solution.

IRAMIS is at the Saclay Institute of Matter and Radiation, and is the second institute in size of the CEA Fundamental Research Division. Inside IRAMIS there are projects directly linked to nanoscience for the technology of information and health, the interaction between matter, radiation and low-carbon energy applications.

Contact with Cyril Cartron and Marion Liard

Cyril Cartron and Marion Liard are two members of the Paris City Hall. Cyril Cartron works for the DICOM (Direction of Information and Communication) and deals with all events organized by the City Hall, especially sporting events. Marion Liard is a project manager in the Town Communication department. Her actions are particularly devoted to handicap.

Since NeuronArch is a project directly linked to handicap, we decided to get involved with the Mairie de Paris to make Paris a more accessible town for disabled people by creating new markings and signs in direct connection with the Paralympic games. For this, we contacted the Paris City Hall to make this project in collaboration, especially in preparation for 2024 Paralympics in Paris.

First, we contacted Cyril Cartron by phone. He was determined to help us. He gave us the contact of Marion Liard, in charge of handicap events. After some emails, she put us in contact with the cabinet of Nicolas Nordman, deputy mayor of the Paris City Hall, responsible for all issues concerning disabled people and their accessibility. This cabinet will contact us for a half-day conciliation meeting, during autumn, to discuss 2024 Paralympics. Thus, we are eager to do this meeting with them!

Jean Marc Victor is a researcher at the Université Pierre et Marie Curie (UPMC). He helped us a lot for the modeling of the neurons growth when we faced a problem. He also is Director of Research at the CNRS and works at the laboratory of Theorical Physics of soft matter in the group "Multiscale Modeling of Living Matter". His dynamism and interest for the our project really helped us to finish our modeling.

The project NeuronArch is also based on a survey that gives us information to orient, improve and integrate our ideas. This survey has three main goals. They concern both our project and synthetic biology in general:

- Vulgarize synthetic biology
- Introduce our project to the general public
- Validate our project by health professionals

To achieve those goals, we had to think about the survey’s framework. For that, we decided to categorize two population types: the general public (children, students, parents…) and health professionals. Then, we conceived a map of our survey which is connected to our goals and our different target populations.

As shown by the map above, there are two common sections : the first part includes general questions, synthetic biology, and biofilms for all, and the second part to collect feedback and opinions. Then, a specific section about infections aimed at health professional in the field of prostheses. This choice enabled us to collect relevant information concerning the project NeuronArch and to achieve the third goal of this survey. Besides the distinction between general public and medical professionals, we wanted to differentiate their various levels in biology . Therefore, we asked a question on their science education level. This question helped us to analyze answers about synthetic biology for example. After defining goals, different populations and framework of our survey, we had to think on how to analyze the results.

As a consequence, we adapted our explanation section making it more readable. We added some questions: for example, we decided to ask biology levels to improve our analysis and to better understand comments and feedback. We also modified some questions:
- About the age range
- About the infection rate in prosthesis: we discussed among ourselves and took into account comments that mentioned that the question was too confusing. Moreover, we received other comments about the project and the scientific part. Following a suggestion in the comments, we thought about developing a collaborative approach allowing the public to get an answer to their questions, and hence make it more interactive. This is an aspect that we will consider in the future. This process through trial and error enabled us to improve the survey and obtain relevant answers.

See all our data in this Excel file.

Our survey was launched on August 21st, 2018 on the platform Google Form and was destined to the French public. We received 205 answers from 80 men and 125 women.

In the end, although we still have a majority of young people between 16 and 25. However, we had an improvement of almost 1/3 of the answers coming from other age ranges.

With the first version of our survey, we did not ask details about the scientific background, and especially the background in biology. However, we quickly realized while reading some comments and seeing the first results that feedback on the survey, and on what people understood or already knew, was highly related to their background in biology. For example, we were surprised at some of the first people who answered saying that the survey did not help them to better understand synthetic biology but with this question about the background in biology, we discovered that some of them had a Ph.D. or were College students in biology and therefore did not get any new information from our explanation.