Integrated Human Practices

Overview:

During the brainstorm, our team leader Zhanning Lin share a news: On Dec.11th, 2021, a piece of news indeed made many Hangzhou citizens heartbroken: a seven-year-old girl named Qiqi passed away due to the striking e-bike explosion accident happened on July.18th in which both Qiqi and her father got severely burned. 95% of Qiqi’s skin was severely burned and the girl tragically died despite thorough efforts and treatment from medical workers 4 months later. As a team from Hangzhou, this tragedy really shocked us a lot. Therefore, artificial skin attracts more attention recently and we are determined to make artificial skin via synthesis biotechnology to help burn patients.
To better understand the status quo of burns and trauma repair, we researched many news, reports, papers and books and also visited relevant experts. We learned that the partial or complete loss of skin functions caused by severe burns or trauma can bring unbearable and long-lasting physical and mental pain for patients. Due to limited medical technology, patients cannot get enough skin resource as a result of large skin grafting area and long treatment cycle, exerting severe impact on their daily life.
We interviewed some experts to prove our feasibility when figuring out our project significance. During our experiment, we kept close contact with several experts and visited two relevant companies to make further improvements. In addition, we communicated with Prof.Tan to share our phase results and get more guidance and inspirations.
Apart from technological communications with medical workers and enterprises, we also explore the feasibility of the following sections of our project. Since our mission is to optimize medical instrument and medicine via synthesis biology, the beneficiary group also turns to be an important factor. So we specially designed a questionnaire to acquire suggestions from the general public. We have received 357 feedback that can help guide our future plan.
The holistic process of our human practice in terms of project design, implementation, rectification and execution is listed in the following table:

    Part 1: Problem investigation and project direction setting

  1. Project inspiration

    On Dec.11th, 2021, a piece of news indeed made many Hangzhou citizens heartbroken: a seven-year-old girl named Qiqi passed away due to the striking e-bike explosion accident happened on July.18th in which both Qiqi and her father got severely burned. 95% of Qiqi's skin was severely burned and the girl tragically died despite thorough efforts and treatment from medical workers 4 months later . The live video of the news is as follows .As a team from Hangzhou, this tragedy really shocked us a lot.
    As a matter of fact, such tragedy is not rare in China. It is said that about 26m people suffer from burns with various extent annually, accounting for 2% of the total population with the death toll only second to that of traffic accidents. According to the statistics, kids from 0-12 years old take up 30-50% which is showing positive growth trend. Burns cause significantly serious consequences among kids with high lethal rates. About 96,000 kids die of burns annually around the globe and many suffer from partially malformation, appearance destruction and mental trauma. At the same time, burns also exert overwhelming financial and mental pressure on families.
    Skin grafting is an important treatment for burns that is conducive to the recovering of body functions with less pain and contributes to a good-looking wound that alleviate mental trauma. However, obtaining healthy skin for grafting is limited. First, patients themselves have difficulties in providing enough skin and have to rely on donations. Therefore, artificial skin attracts more attention recently and we are determined to make artificial skin via synthesis biotechnology to help burn patients.

  2. Interview with doctor Zhaofan Xia
    Expert background: Zhaofan Xia, Expert in burn surgery.
    Objective: Understand the current situation and treatment of burns

    Key points:
    1.Experts confirm the significance of our solution to provide dressings or artificial skin for the treatment process of burns.
    2.The cost of the skin production method and its means will determine whether it can be mass-produced and used.
    3.3D printing technology is very forward-looking and requires consultation with material science experts and visits to relevant 3D printing companies.
    Expert in traumatic surgery, mainly dedicated in clinical and basic research of burn and trauma diseases.


    Communication details:

    We interviewed Dr. Xia by telephone. Dr. Xia told us that the skin, as the most prominent tissue and organ of the human body, is crucial in maintaining the stability of the body's environment and resisting external bacterial infections. According to statistics, millions of people in China suffer from burns yearly, leading to an annual cost of over 1 trillion yuan for skin trauma repair and reproduction. In some explosive accidents, most burn patients require skin dressing during debridement. However, there is a limited amount of skin remaining to meet the dressing and skin grafting needs. Artificial skin has also become quite a valued resource when many healthcare professionals collect allogeneic skin to treat patients, so Dr. Xia recognized the significance of our project. Dr. Xia said, "Every year in China, tens of millions of patients need medical treatment for burns, mechanical injuries or chronic skin ulcers. Nearly 100,000 patients with severe skin defects die due to lack of timely and effective treatment and skin resources." Dr. Xia suggested us to focus on two aspects when determining the method: to control the developmental cost and to create a function similar to human skin to make sure that it can be more widely used in clinical surgery.

    Our response:

    We were honored to receive Dr. Xia's recognition and conducted a series of research. If we want to control the cost of obtaining mass-produced artificial skin, 3D printing technology is a good choice. We found in our online research that 3D bio-printing already has a relatively mature application in printing biological stents (such as heart stents). We believe that if synthetic biology and 3D bio-printing can be combined to produce artificial skin, it will be easier to achieve mass production and be more targeted. The cost of research and development can be controlled with synthetic biology methods, which may provide an easy way to large-scale skin reproduction and show a promising application prospect. When meeting an emergency, artificial skin that can be quickly mass-produced and it can meet the demand for high-volume burn treatment to solve the emergency.

  3. Investigate the 3D technology related to iGEM competition, find the technical papers related to 3D printing artificial skin, and communicate with the tutor about the feasibility of the technology

    The 2015 PacificU-Oregon team used 3D printing technology in their project. The 2019 BrownStanfordPrinctn team also used 3D printing technology to apply their project to the production and purification of drugs on 3d printed expression and PDMS purification microfluidic chips. 2017 Warwick team used 3D printing technology in their project: Application of optogenetic control mechanisms for manipulating biopolymer synthesis in advancing 3D printing technologies
    We researched a paper entitled "Simple synthesis of soft, tough, and cytocompatible biohybrid composites," in which a team of researchers from Cornell University created a new biomaterial that can be used to create bionic skin. The bio-hybrid composites developed by the research team have a unique composition of collagen mixed with "amphiphilic" hydrogel. This soft and biocompatible material is flexible enough to withstand continuous deformation. While the scientists' development project is still ongoing, they say this bio-ink could one day be the basis for 3D printing patient cell scaffolds to heal wounds effectively.
    In addition, we learned through the online search that the cosmetics company L'Oreal plans to use Organovo's NovoGen 3D printing platform. This technology will analyze the skin tissue architecture and all important cell information to create a unique "bio-ink" (bio-ink) composite cellular tissue and print with layers of skin tissue. This technology has been used to repair facial wounds and burns.

  4. visit 3D printing biotechnology company

    Company name: Regenovo Biotechnology Co., Ltd.
    Interviewees: Mr. Fuwei Li and Ms. Man Jiang

    Key points :

    1. 3D bioprinting technology is relatively mature. The company had already conducted some programs that needed artificial skin printing. They introduced an expert in this field to us.
    2. The composition of bio-ink is the core of the printing process, directly relating to the later molding of the produce. Choosing proper printing machine and the setting are also important.

    Communication details :

    They told us their company had received many projects for 3D printing biomaterials, such as printing skin, bone organs, etc. There are also projects for 3D-printed skin scaffolds. 3D-printed artificial skin has many advantages. Not only can it reduce the time and cost of skin cell culture, but also, through 3D scanning, the artificial skin fits the wound better than the traditional autograft, which can improve the speed of wound healing and shorten the treatment cycle. Nevertheless, the point is, what is the printed skin's application direction? Skin implants? A dressing? Or a scaffold. Since the configuration of 3D printing bio-ink directly relates to the material that is later molded, choosing a printer and setting parameters will also affect the application of the product.

    Our response:

    1. Knowing that our idea was supported by technology and papers bring us more confidence.
    2. Moreover, implementing a more detailed direction, the selection and optimization of the bio-ink for printing artificial skin have been the focus of our project.
    3. With the results of our phase, we communicated again with our adviser, Prof. Tan, and implemented the next experimental plan and human social practice work under his guidance.

    5. Part 2: determine project focus

  5. Guidance from Prof. Tan

    Key points :

    1. We need to confirm the current artificial skin production method and learn the structure of human skin.
    2. Learn the common technological means for 3D bio-printing and their application in human skin replacement.
    3. We need to survey the general public to know their cognition level and acceptance of this new product.

    Communication Details:

    Based on the interviews and communication with 3D printing companies in the first phase, we focused our project on bio-ink applicable to artificial skin. We created a mind map of our work under the guidance of Mr. Tan and divided our team members into three groups to investigate different background information as follows :
    The skin stem cell extraction technology and 3D scanning modelling technology that were mentioned in one reference . We found that by 3D scanning, fully customized skin can be printed to match the specific 3D features of specific parts of the human body, improving the fit and success rate of artificial skin to the wound. However, the problems such as immune rejection and incomplete differentiation of foreign stem cells were not considered when the stem cells were extracted. The artificial skin using collagen sponge mentioned in another reference can shorten the wound healing cycle and enhance the skin elasticity after wound healing. However, there is bacterial contamination in the printing process.

    Our Response:

    1. The materials of 3D-printed skin are becoming more and more extensive. There are many papers and patents on bio-inks for 3D-printed artificial skin in China, but the existing bio-inks can not fully meet the needs of users. Structural proteins play a key role in bio-ink.
    2. After referring to papers and brainstorming, we plan to find high-quality structural proteins and spider silk protein to configure bio-ink gel and apply them to 3D printing artificial skin. In one paper, we found that spider silk protein is anti-bacterial and anti-immune, which solves the problems in the above existing techniques.
    3. Nevertheless, we still need to find a dermatologist or surgeon to learn more about the feasibility.

  6. Interviewing Mr. Cai

    Expert background: Xianlei Cai, a surgeon, specializes in transplant surgery
    Objective: According to the previous work in human social practice, we determined to use spider silk protein to 3D print artificial skin. With this idea in mind, we interviewed Dr. Cai, a surgeon who is good at transplantation surgery.

    Key points:

    1. Experts are generally supportive and believe the idea is feasible and offer advice on methods of making artificial skin.
    2. It affirmed clinical practicability, reminded us of the necessary clinical experimental steps before clinical implementation and introduced the structural characteristics of skin tissue to us.
    3. Application of 3D printing technology in the production of artificial skin This method has significant advantages, which can be customized and designed according to the needs of skin grafting patients.
    4. It reminds us of the technical difficulty of printing complete artificial skin and inspires us to focus on evaluating the printing potential of spider silk protein bio-gel.

    Communication Details:

    Mr Cai told us after hearing our ideas that:

    1. Seed cells, scaffolds and growth factors are the main components of artificial skin, and if any of these three cannot be completed, the probability of artificial skin production is minuscule. We can think about researching wound dressings or narrowing the project goals.
    2. Our project aimed to use spider silk protein's excellent mechanical strength and biocompatibility, but the problem of biodegradability is not explicitly mentioned.After discussing with teachers, we plan to solve this problem by mathematical modeling.
    3. To increase the project's innovation, we can look for spider silk protein with good performance, which has less natural production and is hardly ever researched.

    Our Response:

    According to Dr Cai's suggestion, we found a reference to a protein called pyriform gland filament silk (PySp1) of the Araneus ventricosus. So far, PySp1 is the least researched of the seven spider silks because it is produced in small quantities and mixed with other silk proteins in the attachment disc. In contrast to other spider silk family members, PySp1 may have evolved to possess unique molecular properties that can be optimized for spinning into a rapidly solidifying liquid gelatinous substance. This finding provides a valid theoretical basis for the configuration of excellent bio-inks. The synthetic biological approach, using E. coli prokaryotic expression of PySp1, can help us achieve large-scale production and reduce the cost. Furthermore, considering the different applicability, we decided to add spider silk protein repeating units to explore its mechanical properties, one repeat unit is R, and two repeat units are 2R.

  7. Survey on general public

    In the initial phase of problem inquiry and technique feasibility testing, we spoke with experts, referred to scholarly articles, and engaged in extensive conversations under Professor Tan's guidance. Finally, we established that the project's primary goal would be the prokaryotic production of spider silk protein in a bio-ink gel applied to 3D-printed human skin. We also conducted a questionnaire survey for the general public because the project would benefit them, and their opinions are crucial to our project since their responses are relevant to the use and acceptance of the product.
    When designing the survey, we strictly follow the criteria of human practice and try our best to protect the privacy of interviewees. We invite non-group members to voluntarily review the survey. Our public survey received 357 responses from all walks of life: the participants included people of all ages, from primary school to PhD students, covering most of the country. In conclusion, the results of this survey can represent the general attitude of the Chinese public (Figure 1).

    Figure 1 Distribution and age of respondents


    We asked the participants about their cognition level on synthetic biology, spidroin, and artificial skin and the result is shown below(Figure 2), classified by the education levels of the interviewees. We divide the understanding level into three categories: basic understanding,vague impression, and zero understanding. The ideal cognition level we set for the public is to have a vague impression (or to say that they have heard it before). The result shows that the public has a relatively high understanding of what artificial skin is and how people use it. It also indicates that spidroin, as a material, is not that well-acknowledged by the public. We sort the percentage of each choice by education level and find that the cognition level is generally getting higher as the education level goes up. The research found that the trust one puts into a product will increase as one knows more about it through whatever method. Thus, the high cognition level that our survey finds suggests an ideal start to our project from the perspective of public trust .



    Figure2: cognition level on synthetic biology, spidroin, and artificial skin

    In order to see if artificial skin can replace traditional skin grafting skin from the perspective of social morality, we ask our respondents to show their preference for materials used in skin grafting. The result is shown below:


    In the form, we can see that people have almost equal acceptance toward planting patient’s skin and artificial skin made with synthetic biology and 3D printing, leading to the conclusion that on the level of public acceptance, artificial skin can be a perfect substitute for the traditional self-skin grafting, along with a shorter recovery cycle and a broader range of application.

    The public's high expectation toward the general product that we are working on definitely provides our project with a promising future. However, it is not enough. The next question that we include is to test if the participants trust our project or not.

    There were about 73% of the participants declared that they are paying high expectations for our project, and here is the reason:

    This result narrows down our project's aim, which is to lower the price and increase the performance by using PySp1 in artificial skin production. A low proportion of people think negatively of our project, however. We also ask them why they think so, and we find out that the worries mostly come from the maturity of the method. We relate the response to their basic information and answer to the previous questions and find out that most do not have a clear understanding of synthetic biology. These phenomena is due to a lack of knowledge on the method itself, which can be solved by increasing the general cognition level on this field through public education.

    8. Part 3: Detail Design and Project Optimizing

  8. Visiting Hangzhou Singclean Medical Products Company
    Interviewee: Xijiang Feng Head of national scientific research project in Hangzhou Singclean Medical Products Company

    Key point:

    1. The team shall test the biocompatibility of the products. The test should cover both the intended application and evaluation. The team should test the product for cytotoxic, chronic toxic, carcinogenic and other hazards.
    2. The proposed applications could extend beyond 3D printing inks for artificial skin.
    3. It is suggested to design R+GFP gene pathway instead of 2R gene pathway.


    Communication details:
    In order to better understand where our products can be applied, we visited Hangzhou United Medical Supplies Co., LTD. Mr Feng and one of his colleagues told us about their company, whose products are related to hyaluronic acid. Mr Feng has been engaged in hyaluronic acid medical beauty and skin care products for over ten years. After listening to the introduction of our expected products, Mr Feng gave a suggestion for our products and several points we should consider.
    The first suggestion is about biosafety. We should ensure that our products are safe, so biocompatibility testing is crucial. According to Mr Feng's suggestion, we can obtain laws and standards related to biomedical devices from GB/T 16886. We can entrust medical device testing institutions for clinical testing to ensure that our products are safe. We should also consider exclusion ability testing of our artificial skin to ensure that our artificial skin can function and work as well as normal skin.
    The second suggestion is about the application areas of our products. Mr Feng told us that our products could be applied in many areas. It is not limited to printing artificial skin. If high-purity spider silk protein can be obtained, the cost is low and can also be applied in other aspects, such as wound dressings.
    The third suggestion is about the proportion of ingredients in artificial skin. Feng believes that hyaluronic acid has a shallow rejection rate and can be used as one of the raw materials for our artificial skin. Mr Feng also told us that the ratio of raw materials is essential for us to consider. The difference between good medical products and poor wound dressings lies in the proportion. In addition, we can consider adding some visual elements to observe the distribution of raw materials of spider silk protein in the printing solution to adjust our ratio better. Therefore, we plan to add a reporter gene EGFP before the R protein to build gene circuit instead of 2R gene pathway.

  9. Suggestions from BNUZH-China team

    Key point:

    1. Purification and purity of PySp1 are vital, and an effective purification protocol is required, and its purity is determined. This brings us to the direction of the concept certification.
    2. It is suggested to start with one repeat R (PySp1), and after confirming that the ink ratio can be successfully applied to 3D printing, do two repeat 2R extensions.

    Communication details:

    The BNUZH-China team is committed to developing a biodegradable skin regeneration scaffold system (BCAID) based on bacterial cellulose, which can promote the healing of severe skin wounds. This system has a similar application to our project, giving us a lot of practical guidance and help. We had a pleasant project exchange with their team, as shown on the collaborations page

    After we shared the project, they gave a high recognition and evaluation, confirming that spider silk protein's large intestine sensory production is feasible to reduce the cost. The bio-ink of 3D printing technology can be customized for human skin in the future and has practical application value.

    When it comes to our genetic design pathways and our ideas, they gave us some practical 3D biological printing ink. Therefore, we can strengthen it in depth. If a PySp1 repetitive sequence R can optimize the effect of ink, we can consider the plan of 2 R.
    In addition, they tell us that spider silk protein purification is the highlight of our project and we must ensure the quality of the protein and be careful in the verification experiment. It is suggested that we should carry on the proving of the concept by mass spectrometry and nucleic acid sequencing process step by step. It is better to print a preliminary test material to evaluate the physical properties.

    Our response:

    According to the suggestions of the BNUZH-China team and after in-depth communication with Professor Tan, we determined that the core of our project was about the expression, purification and identification of PySp1 protein. In concept certification, the potential of PySp1 protein applied in 3D-printed bio-gels was initially explored.

    References: