Can keratin proteins and seracina from silk help treat diabetic wounds?

Dr Marek Konop received 3,950,734.00 PLN of funding from the National Science Center as part of the OPUS competition for the implementation of the project “The role of cytokeratins 16 and 17 in wound healing in diabetic rats – research into molecular mechanisms using keratin-sericin biomaterial.”

Why do wounds heal more poorly in people with diabetes?

Wound healing is a precisely programmed process. First, the body needs to stop bleeding and remove damaged cells and microorganisms. Then, it begins the reconstruction of the blood vessels, connective tissue and epidermis. The individual stages should follow each other in the right order.

In diabetes, this mechanism is disturbed. Persistent chronic inflammation, oxidative stress, poorer perfusion, and hypoxia cause a wound to remain in the inflammatory stage. Cells responsible for skin reconstruction act more slowly, and the new epidermis does not migrate  properly over the wound surface. Macrophages, cells of the immune system present in the wound, play a special role here. Initially, they remove damaged tissues and combat threats. Later, some of them should change the way they act and start supporting regeneration. In diabetic wounds, this change is often disturbed. Therefore, we will check if the new biomaterial may help "switch" the wound environment from chronic inflammation to tissue regeneration.

Keratin and sericin – natural proteins in a new role

Keratin is a protein that builds such structures as hair, wool, nails and the outer layers of the skin. However, it is not only a passive construction material. When properly prepared, it may create an environment promoting the adhesion, growth, and migration of cells that regenerate the epidermis.

Sericin is derived from the cocoons of domestic silk moth, Bombyx mori. It surrounds the fibroin fibers which form silk. For years, it has been treated mainly as a by-product of the silk industry. Today, it is known to have interesting biological properties: it is tissue-compatible, biodegradable, it can reduce oxidative stress and inflammation and support blood vessel formation.

In the project, we will combine both proteins into one composite dressing material. Keratin is intended to form the structure and support the cells that regenerate the epidermis, and sericin is intended to enhance the protective and regenerative effects. We will also examine modifications of the material containing selected compounds with anti-inflammatory, antioxidant and tissue-repair and regeneration-supporting potential. Each variant will be assessed separately in terms of its safety, physical properties and biological activity.

Cytokeratins 16 and 17 as indicators of the initiation of the epidermal regeneration program

Understanding the role of cytokeratins 16 and 17 (KRT16/17) is the most important element of the project. These are proteins produced by keratinocytes, the basic cells of the epidermis. The expression of cytokeratins 16 and 17 is relatively low in the healthy skin.  

Following an injury, keratinocytes enter the "repair mode" – they change their gene expression profile, reorganize the cytoskeleton, move towards the defect and rebuild the continuity of the epidermis. The rapid increase in the expression of KRT16 and KRT17 is one of the characteristic features of this activation. These cytokeratins play a dual role: they participate in changing the properties and behavior of keratinocytes and, at the same time, they are biological indicators of triggering a repair response. Thus, their presence makes it possible to assess whether keratinocytes have been activated and have started the process of re-epithelialization.
However, the activity of cytokeratins 16 and 17 must be properly regulated in epidermal regeneration. An insufficient response to injury may delay wound closure, but improper or prolonged activation also prevents appropriate epidermal regeneration. Therefore, we want to determine how KRT16 and KRT17 behave in diabetic wounds and whether keratin and sericin biomaterials and their modifications can restore a more physiological course of healing.

From cell cultures to a diabetic wound model

The research will be carried out by an interdisciplinary team combining experience in physiology, molecular biology, dermatology and dermatopathology as well as biomaterial engineering, in cooperation with scientists from the United States, Spain and the United Kingdom.

The first stage of the project involves the preparation of various variants of the dressing and examining their structure, porosity, water absorption capacity, durability and release of the tested substances. Material intended for contact with the wound must not only exert biological effects, but also have appropriate functional properties.

Next, we will conduct in vitro testing on cell cultures. We will assess whether the studied materials are safe, whether they support cell growth and migration and how they influence inflammation, oxidative stress and macrophage activity. In the laboratory, we will also recreate conditions of elevated glucose levels to better mimic the environment in patients with diabetes.

To confirm that the observed effects are indeed dependent on cytokeratins 16 and 17, we will temporarily limit their production in keratinocytes. As a result, we will check whether cells deprived of one of these proteins still react to the biomaterial in the same way. We will also analyze mechanisms by which cells receive signals to grow, migrate and initiate regeneration.

Another stage will involve studies using a full-thickness wound model in rats with type 2 diabetes. We will assess the rate of healing, the regeneration of the epidermis and dermis, blood vessel formation, the composition of the inflammatory infiltration and proteins associated with regeneration. Light microscopy, fluorescence microscopy and molecular biology techniques will be used for this purpose.

What do we already know?

The project does not start from scratch. My team has developed and modified methods to obtain keratin and sericin biomaterials using simple, non-toxic reagents. Our previous research showed that keratin and sericin biomaterials we had developed were non-toxic and could accelerate wound healing. In wounds treated with keratin-derived powder, we observed more macrophages with a regeneration-promoting profile as well as an increase in the expression of KRT16 and KRT17.

Preliminary research in sericin is also promising. Sericin supports the viability of fibroblasts and influences genes associated with inflammation, antioxidant protection and keratinocyte activation. In the epidermal cells, we observed, for example, an increase in the expression of KRT16 and KRT17 as well as an enhanced response associated with the SOD2 enzyme, which helps protect cells against oxidative stress. 

It should be emphasized that these are preliminary results. Therefore, they do not yet conclusively determine the effectiveness of the future dressing, but provide a solid foundation for further research.

Will the project lead to the development of a new dressing?

The primary aim of the project is to elucidate molecular mechanisms discussed in the literature only briefly. We want to determine why a particular material works or does not work, rather than simply noting that the wound healed faster. Such knowledge is necessary in order to design dressings in a rational way in the future and to select their composition to match the problems occurring in a chronic wound.

If our hypotheses are confirmed, the results obtained may become the basis for the development of a safe, biodegradable and relatively inexpensive material supporting the treatment of wounds, especially in patients with diabetes. 

However, the path from laboratory testing to patient use is long. It requires material optimization, the confirmation of the safety profile, standardization of production as well as pre-clinical and clinical trials. 

Therefore, we do not promise a product that is ready to use after completing the project. However, we are expecting insights that will help us identify the most promising solutions.

Natural materials and medicine of the future

Keratin and sericin are natural materials, available and obtainable using methods that limit the use of toxic solvents. Their use fits into the development of sustainable biomaterials, in which the material of biological origin receives a new medical application.

The most important outcome of the project, however, will be a better understanding of how skin cells behave in diabetes.

Understanding the role of KRT16 and KRT17 may explain why the epidermis cannot effectively complete the repair process in a chronic wound. Combining this knowledge with biomaterial technology may help create dressings that will not only cover the wound, but actively influence its biological environment.