Although the use of skin scaffolds and dressings for the treatment of large wounds such as burns has been widely applied in the clinic, these materials involve direct contact with the wound and long-term presence in the wound for treatment. No matter how safe the material is, the potential infection and rejection still exist. Therefore, the biological safety and degradability of skin scaffolds are very important. Through our data search and interviews with doctors (For more details: Integrated human practice), we found that no matter what kind of skin stent is implanted into the human body for wound treatment, it must be combined with wound debridement. In addition, many issues such as wound protection and regular debridement are also under our consideration. In order to solve above problems, we designed a hardware, which has the characteristics of portability, light shielding, detachability and biodegradability. Meanwhile, it can timely monitor patients wounds and handle unexpected conditions to ensure the effective treatment.

We first conducted research on competitive products in the market to determine the advantages and application of our products. We have designed the application process of our products (For more details: Implementation) by referring to a variety of wound treatment products including Integra and Pelnac, which are well-known in present market^1,2. Initially, we decided to cover another layer of film on the Bacterial Cellulose(BC) film as protection and fixation, and designed a hardware with protection and monitoring functions as observation and control measures, so as to form a three-layer structure to ensure the successful performance of our products.

During the follow-up development of hardware, we continued to communicate with relevant manufacturers and doctors of burn and plastic surgery department (For more details: Integrated human practice) in order to better improve our products and hardware. Especially during the communication with Dr. Li, we confirmed that it is necessary to clean the wound exudate in time during the treatment of wounded skin stent. Inspired by this incident, we improved the hardware design in the early stage.

Optogenetics technology, with its advantages such as long-range, traceless and space-time specificity, can accurately control biological processes such as cell proliferation, localization, and signal pathway transduction^3-5, it has attracted much attention in the fields of gene editing, gene therapy and cell therapy. However, optogenetic technology has some disadvantages that are easily ignored in practical applications. On the one hand, some photosensitive proteins are highly sensitive to specific wavelengths of light, and the light control system can be easily activated. On the other hand, it is difficult for people to avoid various lights in the environment. Some lights may accidentally activate the light control system. This runs counter to the original intention of optogenetics to accurately regulate cellular biological processes.

With the study of related research papers, we gained a deeper understanding of the sensitivity of photosensitive proteins. When designing the hardware system, our team fully considered the disadvantage of the light control system which is easy to be started by mistake, hoping to improve the feasibility of our optogenetics hardware. In addition, we hope that our efforts can provide effective ideas and references for other teams who mean to use optogenetic technology.

The shell of our hardware is designed in the form of two pieces of arc covers that can open and close. Its core function is to protect the engineered cells from light, which prevents the accidental activation of the light control system. On this basis, we have installed LED lights with three different colors on the hardware, which can be easily and accurately controlled to turn on or off the light control system. We also embedded a micro-camera in the hardware, which can be wirelessly connected to the mobile phone and makes it easy to observe the recovery of the wound. We hope that our work can provide valuable ideas on improving user experience of optogenetic hardware.

Two light control systems are applied in this study. The function of the blue light control system is to initiate the expression of cellulase to degrade BC membrane (Fig.1). Another one is the red light control system, whose function is to activate the suicide switch and promote the apoptosis of engineered cells (Fig.2).

Fig.1 Blue light activates the production of cellulase of the engineered fibroblast ATCC CRL-2522(BJ) to degrade BC membrane

Fig.2 Red light initiates the suicide of the engineered BJ cells.

When two light control systems are applied in one project, it becomes particularly important to accurately control the light. Otherwise, the false activation of the light control system will cause irreversible consequences. In order to accurately control the light control system, we installed red and blue LED lights in the hardware. The red and blue LED light activate the red and blue light control system, respectively. The reason for choosing red and blue lights is that their wavelengths are extremely different and do not overlap, which can effectively avoid unintended activation.

How can we illuminate the interior of the hardware for the camera to take images without starting the two light control systems? We believe that such light should not overlap with red light (625~740 nm) and blue light (440~475 nm) in wavelength and should be bright enough. We choose yellow light (570~585 nm) as the lighting light, which meets these two criteria. Yellow light is an ideal lighting light. We continued to add yellow LED lights to the hardware. Therefore, there are red, blue, and yellow LED lights in the hardware.

The basic function of our hardware is to avoid light and prevent the light control system from being started by mistake. After discussion and brainstorming within the team, we decided to design a cylinder-shaped product to wrap the hurt limbs of patients.

In order to implement and improve our design, we used 3D printing technology to produce our products. 3D printing technology allows us to use various materials to make products, which can be plastic or even metal. When selecting materials, we noticed the new biodegradable material polylactic acid (PLA). PLA has the advantages of good biocompatibility, safety, environmental friendliness and low price⁶. Therefore, we decided to use PLA to make the shell. The use of degradable and renewable materials conforms to the concept of environmental protection and sustainability of iGEM. At the same time, PLA is also a light material, which will not bring too much burden to patients.

We modeled the shell on the computer and then used the 3D printing technology to make the shell. We also designed two semi cylindrical shells that can be quickly disassembled and assembled by Velcro. The function of hardware is not only to avoid light but also to cooperate with the treatment process. The shell can be heated to place the embedded nuts and connect with tiny hinges. In order to observe the state of the engineered cells to understand the treatment progress and adjust the treatment plan at any time, we have installed a small camera on the hardware. In order to control the light control system of the engineered cells more conveniently, we have installed a printed circuit board (PCB) on the hardware. The red, blue, and yellow LED lights on the PCB can emit light of specific wavelength to control the light control systems. After discussion and research, we made two holes on the cylindrical shell. The camera and PCB were connected to the upper part of the shell through screws.

Frequent opening and closing of the hardware shell may affect the state of the engineered cell and the recovery process of the wound. And it may even lead to unexpected startup of the light control systems. In order to avoid such adverse effects, we installed a camera on the hardware. At the same time, the camera can be connected to the mobile phone wirelessly. With this camera, you can remotely observe the state of fibroblasts growing on the BC membrane, the state of the BC membrane, and the process of wound recovery on the mobile App.

We intended to develop a functional circuit board with red, blue, and green LED lamps on the circuit board. Different lamps can emit light of specific wavelength to control the light control systems. The first version of the circuit board design has several defects (Fig.3). Firstly, we wrongly used green light to provide the lighting required by the camera. Secondly, the brightness of the light cannot be adjusted. In addition, the switch and the LED light are facing the same side. In this way, we cannot achieve the two purposes of avoiding light and controlling the LED light at the same time. Therefore, we avoided these shortcomings in the subsequent iterations of the product.

Fig.3 The first version of the LED circuit board

We learned lessons from the first LED circuit board. In the actual operation, we found that the lighting capacity of green light seemed to be unsatisfactory, so we began to seek a new lighting light. We believe that such light should not overlap with red light (625~740 nm) and blue light (440~475 nm) in wavelength and should be bright enough. Finally, we choose yellow light (570~585 nm) as the lighting light, which is an ideal lighting light. We removed the green LED from the hardware and added the yellow LED.

Based on the above ideas, we decided to develop a new functional circuit board (Fig.4). Compared with first circuit board, the second version of the LED circuit board replaced the green lights with yellow lights. What’s more, the board is smaller.

Fig.4 The second version of the LED circuit board a. Front b. Back

Some members thought that the previous generations of products were not beautiful enough, and they hope to replace the raw materials or improve manufacturing methods. At the same time, we noticed that the brightness of the LED cannot be adjusted. So, we made changes in the design of the circuit board to make sure that he brightness of the LED can be adjusted.

So, we did another product iteration. Compared with the previous products, the third version of LED circuit board is based on PCB. The appearance is more beautiful. In addition, the LED light brightness is adjustable.

Fig.5 Schematic diagram of the third version of the LED circuit board

The LED light emitting panel uses MicroUSB to provide 5V power supply. Because all LEDs work at 2-3V, R1-R6 is used for voltage division to make LEDs work at normal voltage. R4-R6 is an adjustable potentiometer, which can fine tune the brightness of LED lamp. U1-U3 (yellow LED) lights up immediately when the power cord is plugged in. When KEY1 is pressed, U4-U6 (blue LED) or U7-U9 (red LED) will be lit. KEY2 will select the part to be lit. U7-U9 (red LED) will be lit when the button is pressed, and U4-U6 (blue LED) will be lit when it pops up.

Fig.6 The PCB drawing of the third version of LED circuit board

Fig.7 The Physical photos of the third version of LED circuit board (already embedded in the shell and in working condition). a. Front b. Back

The main design process of the device occurs during 3D printing. First, the Soildworks software is used for modeling. The cylindrical shell is divided into two semi cylindrical shells, which are connected by hinges. The shell is built to enclose the arm, which can be quickly disassembled and assembled.

Fig.8 Schematic of modeling with Soildworks

To conveniently observe the state of the engineered cells and the wound, we installed a small camera on the hardware. To conveniently control the light control systems, we installed a PCB on the hardware. There are red, blue, and yellow LED lights on the PCB. We made two holes on the hardware cylindrical shell. So, the camera and PCB can be connected to the upper part of the shell through screws.

Fig.9 Add camera and PCB

After the model is completed, the 3D printing process of FDM is adopted. We used PLA to produce the shell, which is environmentally friendly. Meanwhile, we used black PLA to avoid light. Embedded nuts were placed onto the two semi cylindrical shells by heating (Fig.11a). The two semi cylindrical shells were then connected with tiny hinges. The shell can be quickly assembled and disassembled by Velcro (Fig.11b).

Fig.10 The scene of 3D printing

Fig.11 a. Nut and hinge b. The quick assembling with Velcro

The upper half of the shell is heated using the air gun and finally the camera and PCB are placed and fixed on the shell with screws (Fig.12a). In addition, the sleeves are used to furtherly block the light (Fig.12b). Our design can achieve a light free environment inside the hardware.

Fig.12 Some representative parts of our hardware. a. The camera and PCB are fixed on the shell b. The sleeve is light shielded to achieve a light free environment

Based on the digital documents needed for 3D printing and manufacturing hardware, four views of parts are derived and displayed (Fig.13), which can help other engineers understand our products better and make our product design more standardized.

Fig.13 Four views of parts.

We have placed a video here to show the functions of the hardware.

1. Turn on the camera power and the circuit board power.

2. Open the mobile phone app and connect with the camera through the WLAN according to the prompts in the app.

3. When the camera is connected successfully, exchange data with the app and adjust the working state of the camera.

4. Turn on the yellow LED on the circuit board for lighting.

5. Check the image data sent back by the camera on the mobile app to observe the healing of the wound.

6. Turn on the blue or red LED lights when the wound recovers properly. The blue and red LED lights activate the function of the blue and red light control systems, respectively, which induce the degradation of BC membrane and the apoptosis of engineered cells.

Before installing the hardware, determine whether the device is in right conditions, and whether the camera, red and blue light and lighting can be started normally.

Install the hardware and fix it on the patient's limbs.

Regularly check the wound healing. When checking, connect the mobilephone and hardware. Operate software on the mobile phone to control LED lights and cameras and view the wound image transferred by the hardware.

Treat the wound exudate regularly. When processing, turn on the hardware and close it again after processing.

When the wound recovers well, turn on the blue and red LED lights of the hardware to make the engineering cells apoptosis and let BC membrane degradation.

After treatment, remove the hardware. The hardware can be recycled.

We have developed a multifunctional app. This App can connect camera and transmit images to understand the recovery of wounds, save captured images and medical record.

In the future, more functions will be developed to make our equipment more intelligent and convenient.

For more details, please click on the following link:

https://2022.igem.wiki/bnuzh-china/software.

1 Cytal® Burn Matrix.

2 Xiang, X. Y., Zhou, G. F., WANG-Jun & Chen, L. The Use of PELNAC in Repairing the Wounds Caused by Plastic Surgery. West China Medical Journal (2013).

3 Yousefi, O. S. et al. Optogenetic control shows that kinetic proofreading regulates the activity of the T cell receptor. eLife 8 (2019).

4 Ma, G. et al. Optogenetic engineering to probe the molecular choreography of STIM1-mediated cell signaling. Nat Commun 11, 1039 (2020).

5 Tkatch, T. et al. Optogenetic control of mitochondrial metabolism and Ca(2+) signaling by mitochondria-targeted opsins. Proceedings of the National Academy of Sciences of the United States of America 114, e5167-e5176 (2017).

6 Fang, X., Chen, S. & Wu, Z. Research progress of modification and application of polylactic acid. Speciality Petrochemicals 28, 71-76 (2011).