II. Background Investigation

As a result of our investigation, the reasons for the difficulty in diabetic wound healing include: a sugar-rich tissue environment provides a medium for bacterial proliferation, the chronic release of pro-inflammatory factors, ischemia and hypoxia in the wound due to peripheral vascular disease, and neuropathy leading to poor patient awareness of the extent of wound development. [6]

We hope to gain a clearer understanding of the hierarchical relationship between the various factors that contribute to non-healing diabetic wounds and provide patients with treatment options that address both the symptoms and the root cause. After our further investigation, we targeted the key class of molecules, AGEs, which have been shown to be associated with vascular/neuropathy, excessive inflammatory response, and lack of growth factors at the wound [7] [8], and reducing the effective concentration of AGEs at the wound can alleviate the vascular neuropathy [8]. We, therefore, hope to use AGEs inhibitors, growth factors and antimicrobial substances acting together to promote wound healing.

III. Main Module Design

Next, our exploration proceeded in parallel with the selection of chassis cells and effector molecules. For the chassis cells, we proposed the idea of stem cells combined with exosomes for drug delivery, but the idea was abandoned due to the difficulty of obtaining stem cells and the harsh preservation conditions, which posed a challenge to the mass production of our products. After that, Chlamydomonas reinhardtii came into our vision. The advantage of the algal chassis is autotrophy, which means we do not need to add nutrients to the dressing, making it less susceptible to bacterial attack; in addition, the oxygen produced by the photosynthesis of C.reinhardtii can alleviate wound hypoxia.

For effector molecules, it has been shown that soluble AGEs receptor (sAGER) expression in mice significantly reduces inflammation and vascular complications [9], and we decided to use sAGER as a blocker of AGEs-receptor binding. Since various types of growth factors play redundant roles in wound healing and inflammatory responses, we selected a representative one, hEGF. For antimicrobial substances, we choose AMP LL37 for its promising behavior in fighting bacterial infections.

We encapsulated the above components into a therapeutic module. We tried to construct a negative feedback release regulation mechanism, but the idea was abandoned because the pathway design for sensing the concentration of AGEs in the environment was too complex, and the constitutive promoter was used instead to control the expression of the three effector molecules.

IV. Reporting and Biosafety Design

After completing the design of the main module, we started to think about the functionality and biosafety of the final product and designed the reporting module and the biosafety module.

The idea of the reporting module was initially proposed because of the patient's unclear understanding of the disease process due to possible neuropathy. At first, we wanted to report to the patient on the treatment of the wound by detecting the concentration of inflammatory factors at the wound, but again due to the difficulty of implementing the pathway, we switched the idea to have the dressing report only two states: a signal when the dressing is working and another signal when the wound deteriorates due to severe bacterial infection. We linked the luciferase gene downstream of the promoter of the therapeutic module and designed a sensing system to detect the AHL signal of P. aeruginosa at the wound to achieve our goal.

However, the ensuing problem is that only a few cells in the lower layer of the dressing can contact the wound and the concentration of reporter molecules produced when an infection occurs is too low to be detected. Therefore, we designed a quorum sensing module, where just a few cells receiving AHL molecules will cause all cells to initiate reporting together, giving a strong danger indication to the patient, which greatly enhances the sensitivity of the reporting module.

For biosafety, our initial design is to prevent the leakage of the engineered bacteria by gel-embedding the engineered bacteria, but for insurance purposes, we hope to further improve safety by allowing the engineered bacteria to initiate suicide under specific conditions. We initially designed a photosensitive signaling pathway to allow the chassis cells to initiate suicide under the irradiation of blue light, which is provided by an LED in the hardware.

However, we found that this approach introduced a larger amount of genetic engineering and that actively killing the engineered bacteria with light was not as efficient as treating them directly with physical methods. So, we switched our idea and introduced a Fe3+-sensitive promoter into the toxin-antitoxin system and added Fe3+ to the gel so that the engineered bacteria leaving the gel cannot survive.

V. Hardware Design

To achieve the desired results, the hardware design focused on two main issues. The first is the choice of hydrogel material, which should be biocompatible and provide the required culture environment for C.reinhardtii; the second is how to express the feedback signal.

For the first problem, we compared various materials and chose a hydrogel material based on polyvinyl alcohol with the addition of filamentous protein and hemoglobin. This material not only ensures the normal culture of C.reinhardtii but also improves the mechanical strength of the gel due to the addition of serine protein.

For the expression of feedback signals, our initial idea was to determine the wound condition by visual observation of the color change of the dressing. Considering the needs of the elderly patient group and the fact that the wound location is mostly in the foot which is not easy to observe, we designed a wraparound brace for this purpose. Based on an everyday ankle brace, we modified it by adding flexible OLEDs to provide light for the C.reinhardtii and implanting a color recognition sensor. Since the wraparound brace provides an excellent darkroom environment, we were able to display the dressing color change on the feedback component of the brace in real time through the sensor and keep the patient informed of the wound condition through the change of light in the center of the brace and the buzzer alarm.

After solving these two key issues, we focused on the design of the dressing. We designed a three-layer structure for the dressing. The first layer is an antibacterial and waterproof PP non-woven fabric to protect the wound from external bacteria; the second layer is a hydrogel layer; and the third layer is a composite porous absorbent pad to prevent the gel liquid from spilling or embedding in the wound causing a secondary infection while protecting the skin around the wound.

Later we will focus on intelligent design and try to combine hardware and software to give real-time feedback on wound conditions to cell phones to enhance patients' experience.