Team:HZAU-China/Human Practices
Human Practices
Overview
Tooth Fairy aims to efficiently repair the nano-scale structural damage on the surface of enamel while eliminating the interference of Streptococcus mutans. To fully realize this vision, the project taking "from laboratory to public life" as its tenet, closely focusing on three aspects of sterilization method, repair method and product safety, and a series of human practice activities have been conducted. Then, the feedback of the activities was summarized, to improve the project design and to achieve a seamless connection between experiment and real life. Considering the security needs of both parties during the COVID-19 pandemic, all offline activities regarding "enamel" and the "Teeth braces" were transferred to E-mails, online surveys and telephone interviews. Until September, 2020, various evidences showing that most provinces including Wuhan in China have become a low-risk area for the epidemic (Click here to get the document). After the Tooth Fairy matured in experiments and hardware design, we launched a small-scale offline activity in order to raise the public's attention to enamel protection and verify the acceptance of future products. Additionally, under the premise of ensuring safety, HZAU-China combined its WeChat tweets conducted several synthetic biology and iGEM sharing sessions for college freshmen (Figure 1).
Impact on us
Nano-scale enamel repair is an emerging proposition, and its large amount of uncertainty will bring many challenges in the design and experiments of Tooth Fairy. In terms of technology, enamel repair is by no means equivalent to a simple covering of hydroxyapatite. The biomimetic properties and mechanical strength of the calcified layer should be considered during restoration. In terms of working environment, the microbial composition of the oral cavity is complex, so the microenvironment on the surface of enamel is easily affected by bacterial activities that will hinder the repair process. In terms of future market competition, there have been many enamel repair methods and products. Only by improving the repair efficiency on nano-scale damage of Tooth Fairy and highlighting its non-invasive repair characteristics can we gain a place in the market. In terms of biosafety, Tooth Fairy's working environment has already involved the external environment, and its biosafety will be subject to more stringent and comprehensive requirements. In addition, public acceptance is also an issue that needs to be considered. In order to better solve the challenges and uncertainties that will be faced in the progress of the project, we need to "go out" of the laboratory to communicate with experts in related fields, and "integrate into" the public to gather opinions.
Interview with Professor Yuqing Li
After completing the preliminary design of Tooth Fairy, in order to verify the feasibility and market value of the project, as well as to understand the various factors that cause enamel damage and the current mainstream repair methods. We conducted some interviews with Professor Yuqing Li of Sichuan University, West China School of Stomatology, State Key Laboratory of Oral Disease Research via emails (Figure 2).
1. What factors may cause enamel damage?
Developmental factors. Mainly due to hypoplasia of enamel. Clinically, it is common that the enamel of bilateral permanent teeth is soft, the overall color of the teeth is yellow, and the acid resistance of the teeth is weak.
Bacterial infection factors. Caries are the main source. Early caries are manifested by demineralization of enamel, which is common clinically as chalky demineralized areas with rough feeling and has no X-ray film. Patients usually have no symptoms. If early treatment or prevention measures are not taken, caries will continue to progress.
Trauma factors. Mainly seen in various types of maxillofacial trauma, predominantly anterior teeth. Damage is usually not limited to enamel, and there might be visible pulp exposure and even secondary pulp inflammation.
Chemical factors. Mainly seen in fluorosis and tetracycline teeth. Dental fluorosis often occurs in areas with high fluoride water quality and is manifested by excessive fluoridation of teeth, which can be yellowish brown or dark brown. Tetracycline teeth often occur in children who have used tetracycline antibiotics in infancy and appear as yellow to dark brown.
2. What are the current mainstream enamel repair methods?
For enamel damage from developmental and chemical sources, bleaching is usually used to remove tooth staining. Some patients with relatively serious conditions can apply crown restoration (mainly all-ceramic veneers of anterior teeth). For enamel damage from the source of infection, because its early manifestations are not obvious, and most patients have no symptoms, it is recommended to use routine oral examinations for early diagnosis and topical application of fluorine preparations to prevent the aggravation of caries. For the source of trauma, since the damage is often not limited to the enamel, the treatment should give priority to the health of the dental pulp and periodontal tissue. Follow-up methods such as aesthetic resin and anterior veneers can be used to restore the tooth shape.
3. Compared with traditional enamel repair methods, what are the advantages of Tooth Fairy?
Non-traumatic enamel repair has considerable development prospects. The repair methods used at the present stage focus on resin filling and crown restoration. Due to the retention requirements, tooth tissues have a certain degree of grinding, and during the grinding process of both mechanical stimulation and heat will irritate the dental pulp, which is not conducive to the health of the dental pulp.
4. Do you have any suggestions or comments on the current design of Tooth Fairy and its supporting hardware?
First is the material selection of Teeth braces. The double-layer space design determines the relatively large volume of this brace. When the upper and lower jaws are worn, the movement space of soft tissues such as the labial frenulum and tongue body will be greatly reduced, thereby affecting comfort level. In addition, if it needs to be worn for a long time, the jaw bones will be open for a long time, which may cause fatigue of bilateral joints and ascender muscles, causing bilateral muscle soreness and other adverse effects. It is recommended to start with the material and thickness of the interlayer, or improve the comfort level by reducing the wearing time. For the material part, try to use Polyethylene terephthalate (PET) and Thermoplastic polyurethanes(TPU). Furthermore, there are a large number of pits, fissures and hidden gaps in the oral cavity. Meanwhile, the moist environment and rich nutrients in mouth are suitable for bacterial growth. What's more, the way to prevent engineered bacteria from leaking to the oral cavity requires serious consideration. The method of adding a new filter device to isolate bacteria, allowing only signal molecules and secreted proteins to pass can be considered.
In the early design stage of Tooth Fairy, Professor Li helped us tremendously. He not only established our confidence in the project, but also pointed out the potential bottlenecks and highlights of Tooth Fairy. More importantly, Professor Li's detailed explanation of the factors of enamel damage made us pay attention to the serious threat of bacterial infection to enamel protection. Compared with the damage caused by other factors, bacterial infection as a biological factor is most suitable to be solved by synthetic biology. At the same time, due to the colonization of the bacteria on the surface of enamel, its bacterial biofilm and metabolites will to a certain extent hinder the repair process. After many discussions with Professor Li, we focused our attention on the main pathogenic bacteria S. mutans in the early stages of caries [1]. To eliminate the interference of early cariogenic bacteria on enamel repair, the Detection & Report module that can sense the S. mutans and the Sterilization module are added to the Tooth Fairy. In other words, the Tooth Fairy has the ability to repair nano-scale enamel damage and also has the effect of preventing caries. In the choice of brace materials, we also adopted Professor Li's suggestion and tried to use PET or TPU that meets FDA's standards. Moreover, it benefited from Professor Li’s reminder on biosafety, in the process of conceiving the safety evaluation of Tooth Fairy we referred to the suggestion of SZPT-China, which is also committed to improving dental health, changing to use the harmless Escherichia coli Nissle 1917 as chassis.
Questionnaire released to the public
After adding a Detection & Report module that can sense the concentration of S. mutans and a Sterilization module to the basis of the repair module, Tooth Fairy will be presented in the "Teeth braces" with the function of preventing bacterial infection in the process of efficient enamel repair, together with an auxiliary APP (software) that records and reports the health condition of oral cavity. As a widely available oral health product, we wish that it could suit for everyone. To this end, we need to ascertain the public's attitude towards their enamel health and their health condition of enamel. Furthermore, as the main part of Tooth Fairy, the acceptance of "Teeth braces" will determine the size of its future potential market. Therefore, we need to be clear about the public's first impression of "Teeth braces", gathering their requirements and expectations, and trying to meet the public's needs or standards through design optimization.
1. Are you concerned about the health condition of your enamel?
Referring to the third national oral health epidemiological survey report (2007) and the fourth national oral health epidemiological survey report (2017), in China, people's cognition of oral diseases and awareness of oral health protection has improved significantly in that decade. At the same time, with the continuous improvement of living standards, people pay more and more attention to their health, and under the influence of the COVID-19 epidemic, health has become a hot word nowadays. The general public's concern about the health of enamel will benefit the promotion of Tooth Fairy (Figure 3).
2. What is the condition of your enamel health?
The overall result is in line with our expectations. The majority of interviewees are 18-30 years old, and the proportion of slightly and seriously damaged increases with age. The phenomenon of enamel damage is widespread, and it often continues to increase with age. The result shows that Tooth Fairy has a broad potential market (Figure 4).
3. When your enamel is slightly damaged, are you willing to use "Teeth braces" to repair it?
At the bottom of the questionnaire's headline, we briefly introduced the working principle of Tooth Fairy, which uses the method of synthetic biology, that the protein secreted by engineering bacteria is used to construct the hydroxyapatite network, so as to guide the biomimetic remineralization of apatite crystal. Therefore, we are worried that a small number of people will not accept the project due to their concern about genetically modified technology and biosafety. Fortunately, people have become more open in the face of science and technology. All interviewees showed a certain degree of interest in the concept of the "Teeth braces" and nearly 30 percent were willing or very willing to use it. The public's positive attitude towards Tooth Fairy has to a certain extent dispelled our worries about product promotion, and also made us determined to focus our future work on how to make the product meet the public's needs or standards rather than persuade them to try it (Figure 5).
4. When you use "Teeth braces", what are your top three concerns?
Perhaps because Tooth Fairy has not yet materialized, the public's expectations or requirements for "Teeth braces" tend to be conceptualized. They mainly focus on the safety, repair efficiency and cost of it. But, the public's great attention to product safety coincides with Professor Li's suggestion (Figure 6). We began to try to add a kill switch to the engineered bacteria, and set the environmental restriction factor as the excitation factor to meet the working characteristics of the Tooth Fairy.
5. If we improve the following attributions of "Teeth braces", will it increase your acceptance of Tooth Fairy?
The results showed that the increasing comfort level and safety of the "Teeth braces" can promote the public's acceptance of Tooth Fairy (Figure 7). Therefore, on the basis of comprehensively considering the comfort level and cost of "Teeth braces", we decided to further simplify the internal structure of "Teeth braces" and use TPU as the raw material to reduce its volume and quality. In terms of biosecurity, we decided to add a kill switch that relies on Fe2+ to the engineering bacteria [2]. Besides, in subsequent improvements, we would try to introduce a module with the function of producing fragrance into the engineered bacteria (Figure 8).
Offline investigation
In the earlier questionnaire survey, we found that the interviewees were unable to make specific and practical requirements for "Teeth braces" because they may be unable to contact the project entity. In the follow-up interview, we tried to design the problem from the user's angle, in order to verify the accuracy of the result getting from the online survey and compare the public focus before and after the substantiation of "Teeth braces" and fluorescence detection box. We gathered 14 students from Huazhong Agricultural University with different professional backgrounds as the interviewees of the offline survey (Figure 9).
First, we briefly described the working principle of Tooth Fairy to them and asked them for their first impression of the project.
Despite varying degrees, all interviewees are willing to use the "Teeth braces" when enamel is damaged, and this result was consistent with earlier online surveys' (Figure 10). In addition, compared to the average level of society, young people receiving higher education may have stronger understanding, acceptance and courage to experience new technologies. Therefore, when answering the same question (Are you willing to try the "Teeth braces"?), the proportion of undergraduates who chose "Willingness" and "Very willing" was significantly higher than the results of the online survey that included all age groups.
Afterwards, we showed them a schematic diagram of the "Teeth braces" and its matching fluorescence detection box, and asked "what are the three things you are most concerned about if you use the "Teeth braces"?
Although there were differences in the proportions, the most concerned ones were still the "Safety", "Repair effect" and "Costs" of the "Teeth braces". However, in the offline survey, we found for the first time that an interviewee regarded "Comfort level" as one of the three most concerned items of the "Teeth braces" (Figure 11). In the follow-up conversation with him, we learned that he was the only person among the 14 interviewees who had used braces.
After the statistics were completed, we showed the interviewees the "Teeth braces" model and the entity of the fluorescence detection box, and asked the question again.
When the interviewees directly observed and touched Tooth Fairy's "Teeth braces" model and the entity of the fluorescence detection box, we found that their focus had shifted, with the most significant increase in the percentage of interviewees considering "Comfort level" as the most important items of "Teeth braces" to 57.14%, up from 7.14% previously (Figure 12). This may be due to cost constraints, and the two actualized hardware were relatively crude in appearance. Besides, the condensation shaping property of TPU makes it difficult for us to ensure a high degree of fit between the tooth model and the "Teeth braces" during the continuous solidification of the material. Fortunately, the results of this survey showed us that the public's attention to the "Teeth braces'" "Comfort level" was no less than that of "Repair effect" and "Costs", and the result can also support that improving the comfort level of the "Teeth braces" can increase the public's acceptance of Tooth Fairy.
Long-term cooperation with SZPT-China
Among any three people walking, I will find something to learn for sure. SZPT-China's Toothkeeper and our Tooth Fairy both use the ComDE two-component system to detect S. mutans, and we also have certain similarities in model like detecting the S. mutans and potential users. In order to optimize the design of our projects and realize the intercommunication of related resources, the two teams have carried out in-depth cooperation in the experiments, modeling and Human Practices since May 2020. In addition, in order to share experience and build communication among iGEM teams in different provinces in China, we hosted an online Friend Team Meetup involving 9 teams on August 15, 2020 (Figure 13).
Participate in CCiC
The Conference of China iGEMer Community (CCiC) was first held by HZAU-China in 2013, and has gradually grown into the largest and highest-level communication meeting among iGEMers in China. This year, due to the COVID-19 pandemic, the meeting was held online, but we still actively participated in it with the original intention of extensive exchanges and mutually learning. In the conference, a judge raised questions about the setting of the switching thresholds of Tooth Fairy's three response patterns and suggested that the conversion threshold should be clarified by establishing the relationship between the ComDE two-component system and the concentration of CSP or S. mutans. With this reminder, we began to collect literature data and tried to establish the relationship between the ComDE two-component system and the concentration of S. mutans to adjust the affinity of PnlmC and PnlmAB to P-ComE.
Interview with a professor from West China School of Stomatology
After two exchange meetings, we gathered a lot of opinions from experts and iGEMers, most of which have been resolved in our discussions with the instructors and have contributed to the overall improvement of the project. However, Tooth Fairy, as an in-situ repair method for nano-enamel damage, its clinical value still needs to be recognized by doctors with rich clinical experience. With the help of SCU-China, we contacted a professor who has been committed to clinical diagnosis and treatment, and had many discussions with her via emails.
1. From our previous investigation, we found that the colonization of S. mutans in the oral cavity can cause enamel damage and hinder the work of Repair module. Therefore, we added Detection & Report module and Sterilization module to the Repair module to improve Tooth Fairy's enamel repair efficiency. What do you think the three modules in our circuit should be improved?
The organic combination of "Detection & Report", "Sterilization" and "Repair" is the highlight of the project. But there are still a few points worth noting.
In terms of "Detection & Report": It is necessary to clarify the relationship between the ComDE two-component system and the concentration of S. mutans, as well as the relationship between the concentration of S. mutans and the degree of damage to enamel. In the past, similar problems were mainly evaluated by the acid production of S. mutans.
In terms of "Repair": The conversion of the three response patterns realized by the P-ComE concentration must meet the relationship between the ComDE two-component system and the concentration of S. mutans.
2. Do you think Tooth Fariy has clinical application value?
In general, in vitro experiments have proved the relationship between the ComDE two-component system and the concentration of S. mutans, the enamel repair ability of LRAP, and the specific sterilization and anti-caries effect of the ClyR. If the Tooth Fairy achieves precise regulation of the three functions, it will be beneficial to its clinical application.
The professor also suggested that we should clarify the relationship between the ComDE two-component system and the concentration of S. mutans, in order to achieve a reasonable conversion of the three response patterns. At the same time, she affirmed the clinical application value of Tooth Fairy, which is based on the precise regulation of the three modules. The professor's suggestion has once again clarified the focus of the experiments and models, but due to time constraints, we have not been able to realize the unity of actual conversion thresholds and model recommended thresholds of the three response patterns before the wiki freezes.
Impact on society
A series of multidisciplinary tweets featuring synthetic biology
HZAU-China has long been committed to the dissemination and sharing of knowledge on synthetic biology. With the help of the WeChat official account, we regularly publish tweets and improve the forms of sharing based on feedback. This year we tried to expand the contents to other scientific fields. Because Tooth Fairy aims to repair nano-scale structural damage on the surface of tooth enamel, we have set up a special column for dental health, focusing on various common dental diseases for the public. In the end, we hope that readers can understand synthetic biology from our previous tweets, and are used to looking at any new technology in an objective manner. Besides, we also hope that readers can realize the importance of dental health by reading the dental health column, master some basic knowledge of dental protection, understand and accept Tooth Fairy (Figure 14).
The "Love Tooth Encyclopedia" series of popular science videos
During the investigation, we found that the public are unfamiliar with some basic oral health knowledge and lack regular dental examinations. In order to further raise the public's awareness of oral health care and make the project more acceptable, we have made a series of popular science videos "Love Tooth Encyclopedia" aiming at the public's knowledge blindness, which has received a good response (Figure 15).
The Teeth Defense War
We asked some volunteers to try out our self-made turn-based card game "Teeth Defense War" in groups of two to collect suggestions for modification (Figure 16). The game takes the complex oral microbial environment as the background, and sets up two opposing camps "tooth protector" and "tooth aggressor" around the contradiction between tooth protection methods and tooth damage factors such as bacterial infection (Figure 17). The original intention of designing this game is to popularize the methods of tooth protection and the importance of oral health to more young people through the way of entertaining and learning.
Synthetic biology and iGEM sharing session
Science has never been a castle in the sky or a world unique to geniuses. On the contrary, it comes from life, serves life, and is inextricably linked to everyone. And synthetic biology is just one of the many branches of science, and what it enjoys is nothing but the mystery that laymen cannot understand. As for our mission, it is not to rely on superficial knowledge and only experience to be deliberately mystifying, but to show the wonders of a new world to the latecomers in popular language and appropriate metaphors. We renamed the traditional "synthetic biology and iGEM lecture" to "synthetic biology and iGEM sharing session", changing the form from unilateral explanation to the interaction and experimental experience. We hope that through sharing and communication, the latecomers in synthetic biology will build up their initial interest in the subject and try to understand the daily life of researchers (Figure 18).
After ten months, with the attention and participation of the public as well as the guidance and help of two professors of Sichuan University, West China School of Stomatology, Tooth Fairy was finally presented to the world. In our process of "go out" of the laboratory and "integrate into" the public, Professor Yuqing Li has established our confidence in the project and pointed out the superiority of using TPU or PET as the "Teeth braces'" material. At the same time, he inspired us to pay attention to the damage of bacterial infection to enamel, and provided the initial idea for the design of the Sterilization module. The public's requirements for efficiency, cost and comfort level of the "Teeth braces" made us clarify the optimization direction of Tooth Fairy. Public's high degree of concern for bio-safety is the main reason why we changed to E. cN as the chassis and introduced kill switch. Through the Friend Team Meetup and CCiC, we deepened our understanding of synthetic biology and accumulated experience in circuit design and experimental operation. The professor, from the perspective of a clinician, shared her views on Tooth Fairy with us, and affirmed the working principle of the three modules in our circuit.
The generous help from the society has made Tooth Fairy born, and we also hope to do our best to give back to the society. In the Human Practice of "Impact on Society", we focused on the popularization of oral health knowledge and the promotion of synthetic biology, and hoped to increase the public's attention to oral health and to allow synthetic biology to enter their vision and life. Online we launched tweets and the "Love Tooth Encyclopedia" series of popular science videos, both about synthetic biology and oral health. Offline we conducted several Synthetic Biology and iGEM Sharing Sessions and a "Teeth Defense War" game demo activity.
References
[1] Loesche W J. Role of Streptococcus mutans in human dental decay[J]. Microbiological reviews, 1986, 50(4): 353.
[2] Engelberg-Kulka, Hanna, Ronen Hazan, and Shahar Amitai. "mazEF: a chromosomal toxin-antitoxin module that triggers programmed cell death in bacteria." Journal of cell science 118.19 (2005): 4327-4332.
