Team:Greatbay SCIE/Human Practices

Human Practices

Interview with Doctor Huang from


Peking University Shenzhen Hospital

To learn about the current bioadhesives the hospital is using, the deficiencies of them and to get some suggestions on our project’s product design, we interviewed a doctor majored in cardiovas-cular surgery from Peking University Shenzhen Hospital.

When and how do they use it?

The bioadhesives are generally used when doctors need to glue the blood vessels, which have been cut during the surgery, for controlling the loss of blood after the surgery, and there are 400-500 places need to be glued in one operation. When using it, they need to mix the fibrinogen and iodine glycerol together to make the glue sticky, and it takes 5-10 minutes for the glue to fully stanch bleeding, but generally, they would not wait for it. That type of glue works outside the blood vessels, and is called exogenous soft glue. Besides it, there is a type of glue works inside the blood vessel. However, in cardiovascular surgery, they generally use exogenous soft glue, while bioadhesives like Olix and glubran which work inside blood vessels are generally used in aneu-rism and bleeding in the brain. Further, Doctor Huang told us that one operation usually con-sumed 2 bioadhesives, and neurosurgery actually demanded much the use of bioadhesives than cardiovascular surgery, which only used it in major operation.

How do the bioadhesives work?

The bioadhesives can reduce bleeding after surgery effectively as compared with previous years when there was no bioadhesives, and the doctor generally left the blood vessels to recover natu-rally. The exogenous soft glue used in surgery works to stanch bleeding by combing with the blood vessel wall. Since it’s a kind of soft glue, and is made up of fibers from animals like pigs, it could expand as the blood vessels expand, having high elasticity as compared with another kind of hard glue which lacks elasticity, constraining the movement of blood vessels while having high strength, which makes the hard glue useful in coronary artery bypass surgery. Those types of bioadhesives have greatly improved the bleeding after surgery since it was invented, but Doctor Huang suggested that bleeding after surgery still existed, so he told us that it would be great if our bioadhesives can reach stronger stickiness.

What happens to the bioadhesive after the surgery is finished?

Although the bioadhesives can stanch bleeding, it shouldn’t stay in the patient’s body for a long time. It has to degrade after a short time when the human body is working to recover the wound at the same time. The home-made bioadhesives like Hugulai-shi ( http://www.nuancebiotech.com/index.php?c=article&a=type&tid=278 ) are used the most in Chi-nese hospital as compared with imported bioadhesives like Olix and Bioscal, taking about 2 weeks to fully degrade. Doctor Huang suggested that it would be great if the degradation time is shorter, since if the bioadhesives stay in human body over a certain time, infection might occur. The reason why they generally use the home-made bioadhesives, rather than the better imported bioadhesives, is about the price. Generally, one home-made bioadhesive costs about 3000 RMB while imported one like Bioscal costs 1,2000 to 1,5000 RMB. Thus, price is an important point worth considering while we are designing our product.

Exogenous fibrin bioadhesives

Glubran, a kind of bioadhesives working inside the blood vessels

Conclusion of suggestions from Doctor Huang on our product design:

1. We should consider that where and how would our bioadhesives work: inside the blood vessel or outside the blood vessel? Working as soft glue or hard glue?

2. In terms of packing, doctor Huang told us that we can use hard tip (which is pinpoint) or soft tip (flexible), and since the protein glue used inside blood vessels demand hard work to inject, we can add pump to the hypodermic syringe to save work.

3. In terms of ingredients which we can add to our product, Doctor Huang suggested us to put in-dicator with color to our adhesive protein so if the patients’ condition get wrong after surgery, we can use X-ray to check where have we used the bioadhesives, and whether it is causing problems.

Interview with Doctor Yingqi Chen


from Shenzhen Institutes of Advanced Technology

To learn about the potential of our adhesive protein to be applied for use in orthopedic biomateri-als, and suggestions on product design, we visited Dr.Chen’s lab.

Dr. Yingqi Chen works in Shenzhen Institutes of Advanced Technology and has engaged in the research about the application of orthopedic biomaterials and developed personalized multi-parameter regu-lated biomaterial precision molding technology based on 3D printing. He discovered the regulation of key factors for bone repair materials such as the shape, mechanics, microscopic three-dimensional structure and the degradation cycle and developed a series of active bone repair biomaterials. He and his team have already registered a company for converting their work in the lab to product and they had already built collaboration with hospitals for their product. Homepage of their company: http://jcmed.com/.

Their lab had started to use 3D-printer to manufacture materials for repairing bon defect.

Feedback

Dr.Chen told us that our adhesive protein might be useful in repairing large broken bone defect, where patients lost a big part of their bone. Our adhesive protein is supposed to have good biocom-patibility since it comes from marine creatures, which makes it great to be used inside human’s body. Besides that, Dr.Chen pointed several points writhing considering for our product design:

1. The location on bones where our product is used should be specified because different parts of bones sustain different levels of force and pressure. We need to make sure our product can with-stand the forces.

2. The rate of decomposition should be carefully considered because our final goal is to assist the proper growth of bones instead of replacing the bones and the bone requires enough time to grow back as well.

3. Sufficient force of adhesion should be reached to stick the chips of bone together, balancing the adhesion, ductility and biosecurity well.

4. For ingredients what we can add to our adhesive protein, Dr.Chen suggested us to use magnesi-um ions, RGD sequence and BMP2 to promote the growth of bones.

Interview with Professor Tim Wong


from Shenzhen University

To learn about barnacle cement that is used in our project and find out the potential of adhesive protein to be applied for use in anti-fouling, we visited Professor Wong.

Professor Wong’s work mainly focuses on the gene regulation in marine creatures’ larva, which combines bioinformatics with practical experiments. In the area of anti-fouling, he is trying to accelerate and deceler-ate the adherence of larva.

Barnacle

There are many types of barnacles: mobile barnacles and immobile barnacles; stalk barnacles and acorn barnacles, differing in their shape. Unlike mussels, barnacles are crustaceans instead of mollusks but they get to convergent evolution with foot mussels since they both can produce sticky underwater adhesives. Mussel’s shell belong to its head.

Life cycle of barnacle

Barnacles are hermaphrodite but the fertilization involves two individual barnacles. This is the reason why we always find them congregating together. After the fertilized egg developed inside adult barnacles, barnacle cyprid is released. The released larva explore the surfaces it meets and find a spot to adhere and create temporary larva cement. Permanent adult cement is produced by cement gland of barnacles when they reach juvenile stage after metamorphosis.

As to the proteins in the cement, Professor Wong and his team have found that the proteins are consist of lots of lysine and there is lysine oxidase enzyme in barnacles’ body so it’s possible that the adhesion is af-fected by the cross link of the side chain of lysine. However, Professor Wong told us that our genetic engi-neering project might not have to consider the modification if the protein we make is sticky.

In addition, Professor Wong introduced another finding to us: The barnacles which adhere to the shells of turtles, can move constantly on the shells, instead of sticking to one place.

Professor Wong gave us abundant knowledge about the barnacles, although it isn’t necessarily related with our product design, it’s a pleasure to learn about the creature we are studying in.

Life cycle of Barnacles.

Anti-fouling

We discussed the potential of adhesive protein to be used in anti-fouling, for example the mechanism of anti-fouling, which might help us to better understand how would our adhesive protein work in anti-fouling.

On the surface under the sea, organic molecules can accumulate, attracting bacteria to adhere, which leads to the formation of biofilm. After this, microalgae grows on the biofilm. Eventually, the larva of sea creatures comes to adhere on the surface.

Currently, there are chemical, physical and material methods to deal with anti-fouling. Chemical method works by killing organisms on the surface but pollutes the sea water. Physical method can prevent adher-ence by creating nanostructure on the surface or using ultrasound to expel organisms, but is too costly to cover a huge ship. Material method is relatively feasible but the hydrophilic painting used for anti-fouling may be eaten by bacteria.

In his opinion, we can try to use the hydrophilic attribute of our adhesive protein to achieve anti-fouling. For example, we can focus on the hydrophobic lipid on the outermost layer of barnacle’s cement and try to avoid the adherence of it by putting a layer of bacteria on the surface of ships to produce hydro-philic adhesive protein. However, if we take this method, we should be aware that our bacteria may be re-placed by the wild bacteria in the sea. Besides, we have to consider the environment which our bacteria is going to live in, for example, is it going to be sea water or fresh water?

Except for antifouling, Professor Wong suggested another application for our adhesive protein: in works of underwater archeology. Underwater Archeologists can use our adhesives to stick pieces of the object they discovered together under the water, preserving the object’s structure.

Conclusion

In conclusion, Professor Wong didn’t suggest us to use the adhesive protein in anti-fouling for the diverse sea environment will inhibit the work of our product in many ways like the replacement of bacteria from the sea and the unsuitable living environment for our bacteria. Instead, Professor Wong guided us to apply our adhesive protein to underwater archeology.

Reference

Okano, Keiju & Hunter, Ewan. (2000). Bryozoa and barnacles.

Integrated Human Practice with Professor Zhong

The 73rd Academic Seminar of the Institute of Synthetic Biology

We attended this seminar to learn about Professor Zhong’s recent work on adhesive protein in Shenzhen.

Amyloid proteins, which are useful in building blocks for natural materials and architectures, are the main focus of Professor Zhong’s research and have the following excellent attributes:

• Hierarchical assembly across several length scales
• ultra thermal, enzymatic and chemical stability
• outstanding mechanical properties
• Self-healing

Curli amyloid subunit in E.coli like CsgA has been found to have huge potential in building co-hesion for substances like adhesives. CsgA is the major subunit of curly fiber, and other subu-nits include the minor subunits of curli fiber like CsgB, CsgC, CsgE, CsgF etc.

Professor Zhong mentioned the concept of “Living Functional Materials” based on amyloids. It’s a conceptually new type of materials having multifunctional, self-healing, self-regenerating, and adaptable properties, and is organized in a distributed, bottom-up, autonomously assembled, and environmentally sustainable manner. According to Professor Zhong, the living functional materials can be used to create biofilms for bioremediation and to constitute living functional glues.

We also learnt an exciting news which was the adhesives Professor Zhong’s lab had made could now finish the self-healing process within 30 seconds, using blood vessels as the example. What we learnt from the interview with Doctor Huang from PUSH was that the current adhesives took 5-10 minutes to fully stanch bleeding. See HP with Doctor Huang. Besides this, the living functional glue is responsive to stimuli and is able to reach autonomous repairs, which is truly a huge leap from current adhesives.

Interview with Professor Zhong

In order to get suggestions on the issues we encountered during the experiment of cloning and pro-tein purification, and to better introduce the overall progress of our project, we conducted an online interview with Professor Zhong on August 7, 2019. Professor Zhong together with his students at-tended this interview.

In the experiment of protein purification, we only managed to produce an exceptionally low yield of protein and Professor Zhong suggested us to use yeast to produce adhesive protein for the follow-ing reasons:

1. Yeast is eukaryotic, which means it’s more genetically related with marine creatures like bar-nacles, so it might express the adhesive protein more easily.
2. Professor Zhong had already used yeasts to produce adhesive protein, finding this method could help to reach higher output, although he didn’t publish the results.
3. Using yeast to produce adhesive protein makes the protein purification process easier. In yeasts, the adhesive protein are released to the exterior of them through exocytosis, so there’s no need to break the cells to get the adhesive protein.

To see integration of this advice to our project: Click here.

In terms of the stickiness of the adhesive protein, Professor Zhong pointed a new design to us: We can merge mfp5 and mfp3 together, which imitates the structure of adhesive protein in barna-cles. Before the interview, we made a design to link multiple mfp5 together. However, Professor Zhong told us that mfp5 might affect the assembly of CsgA, and since mfp3 is smaller than mfp5, stronger stickiness might be reached.

see integration of this advice to our project:Click here.

In terms of product design, Professor Zhong gave the following advice:

1. Bacteria which can produce adhesive protein are useful in repairing concrete's cracks.
2. We can research on the potential of adhesive protein to be used in submarine which stays in ex-treme environment, making adhesive protein beneficial to military.

Further contact

We set up a WeChat group with Professor Chao Zhong, and we were uploading our experiment’s results to the WeChat group all the time.

CCiC

CCiC is one of the biggest student academic meetings in China, providing a platform for Chinese iGEM teams to communicate through presentation and poster session. Every year, they would invite teams with excellent results to make speeches and experts in synthetic biology to give iGEM teams comments, helping to improve their projects.

We attended the Sixth CCiC took place from 2019.8.19 to 2019.8.23.

Feedback

The major concern of our project is the low yield of adhesive protein, and in order to give a direct signal of our production, judges suggested us to link GFP to our adhesive protein. Click here