Natural biomaterials to support wound healing

The project led by Dr. Mateusz Rybka, titled “The effect of keratin dressing enriched with M-CSF on wound healing in a type 2 diabetes model in rats: a pilot study”, received funding from the National Science Center in the PRELUDIUM 23 competition [grant number UMO-2024/53/N/NZ4/03697].    

Why do wounds in diabetes heal more poorly? 

The proper wound healing process consists of four phases: hemostasis (stopping bleeding), inflammation (cleaning and removing necrotic tissues), proliferation (rebuilding damaged tissues), and remodeling (tissue restructuring and scar formation). If any of these phases is disrupted, the wound will not heal properly. Such chronic wounds significantly reduce quality of life and sometimes even its length. On the national scale, patients with chronic wounds represent a major challenge for the healthcare system. 

One of the diseases in which wound healing is impaired is type 2 diabetes. The most affected is the inflammatory phase, although it should be emphasized that abnormalities occur at each stage of healing. Damage associated with type 2 diabetes, such as blood vessel injury (angiopathy), peripheral nerve injury (neuropathy), and chronic hyperglycemia (an increased glucose concentration in blood serum), negatively impacts the function of cells involved in body regeneration. This often leads to the formation of characteristic chronic wounds known as diabetic foot syndrome. Diabetic foot is one of the most serious complications of diabetes. It often leads to limb amputation, greatly reducing quality of life, and increases the risk of severe infections that may threaten health or even life.

Unfortunately, the incidence of diabetes continues to grow, and available methods of wound treatment in these patients are often insufficient. This is why it is so important to seek new, bioactive dressings that could be used in chronic wound therapy for this condition.    

Keratins – proteins with many faces    

Keratin dressings are attracting growing interest among researchers studying wound healing. Although keratin biomaterials have been used for decades, only recently have they begun to be enriched with additional substances that can support regenerative processes. 

Keratins form a large protein family with more than 40 different types. They naturally occur in structures such as hair and nails, and in animals also in fur. 

Different keratins play distinct roles in wound healing, and their presence depends on their location in the skin as well as the healing phase. Some keratins support cell growth and proliferation (multiplication), while others participate in cell migration and proper adhesion (attachment). Good cooperation between these proteins is key to tissue regeneration. Chronic wounds are often characterized by a deficiency of specific keratins, particularly those responsible for stimulating cell proliferation through the regulation of cyclin activity. 

The role of immune cells in wound healing

Alongside keratinocytes (epidermal cells) and fibroblasts (connective tissue cells that bring wound edges together), immune cells such as neutrophils, lymphocytes, and macrophages (the latter being crucial in my project) play a critical role in healing.
In the early inflammatory phase, neutrophils enter the wound, where they initiate inflammation and remove necrotic cells from the wound bed. Over time, they are replaced by lymphocytes and macrophages, which modulate the intensity of the inflammatory response depending on the body’s needs, allowing the healing process to transition into the proliferative phase. 

In difficult-to-heal diabetic wounds, an increased number of neutrophils and other acute inflammatory cells persist for too long. Breaking this pattern is essential for effective therapy of chronic wounds.

Macrophages are among the most important cells, coordinating subsequent stages of tissue repair. They are divided into two main polarizations: M1 and M2. Although this division somewhat simplifies the reality, it is sufficient from the perspective of chronic wound therapy to understand their role in the healing process. 

M1 macrophages support neutrophils in inducing a short-term, controlled inflammatory response. They phagocytose necrotic cells and prepare tissue for further healing stages. M2 macrophages, on the other hand, produce anti-inflammatory cytokines such as TGF-β and IL-10, promote angiogenesis, and support scar formation. Proper activity of M2 macrophages is often decisive in whether a wound heals effectively or turns into a chronic, non-healing lesion.

Modern biomaterials and their unique properties    

Given the role of immune cells in healing, it is also important to consider the properties of the dressing itself, which should provide optimal conditions for their activity. In 1989, Terence D. Turner proposed a set of criteria for the ideal dressing, which still guides the development of modern medical materials. 

According to Turner, a dressing should create a moist wound environment conducive to regeneration while also absorbing excess exudate to prevent skin damage. The biomaterial must be well tolerated by the body and must not trigger any inflammation. Additionally, it should allow for air and vapor permeability to ensure proper gas exchange and maintain stable wound temperature. The dressing must also be designed so that it can be changed without pain or risk of damaging fragile, newly formed tissue. Biomaterials made in line with these principles make healing more efficient and effective.

Keratin has proven to be a particularly promising material for the manufacture of modern dressings. Researchers worldwide have developed various hydrogel and hydrocolloid dressings based on soluble keratin fractions. Much less attention, however, has been given to insoluble keratin fractions, which can be used to create bioactive dressings with unique structures. Such dressings, often in the form of scaffolds, provide a framework in the wound bed that supports cell migration along keratin fibers and accelerates healing. Importantly, insoluble keratin fractions can be obtained without toxic solvents harmful to the environment, which are commonly used for soluble keratin fractions.

Animal model studies have confirmed the effectiveness of insoluble keratin-based dressings in treating surgical wounds in both healthy animals and those with type 1 diabetes. The potential of these biomaterials, however, is much broader. Thanks to the unique architecture of the dressing, it is possible to enrich it with additional substances that modify its properties depending on the patient’s needs. Keratin scaffolds have successfully been developed, containing silver nanoparticles with antibacterial effects, sodium butyrate with anti-inflammatory properties, and even opioid analgesics. All these substances were released from the dressing in a predictable and consistent manner over several days.

In chronic wounds, keratin biomaterials regulate the local immune response. Wounds treated with them contain more lymphocytes and macrophages, cells essential for controlling chronic inflammation. Particularly significant was the rapid appearance of M2 macrophages, which were present after only a few days in treated wounds, while the same process in untreated wounds took up to two weeks. Accelerating this phenomenon has been shown to be critical for effective therapy of diabetic wounds.

When a dressing becomes targeted therapy – keratin and M-CSF

In the skin, macrophage polarization toward the M2 phenotype occurs mainly under the influence of cytokines such as IL-4 and IL-13. Another key factor in this process is macrophage colony-stimulating factor (M-CSF, CSF-1). The primary function of M-CSF in the body is to regulate the proliferation, differentiation, and activation of monocytes, i.e., the precursors of macrophages. In the context of wound healing, this is particularly important as it leads to faster cell renewal and modulation of immune responses. 

In my project, I plan to create an innovative dressing based on insoluble keratin fractions enriched with macrophage colony-stimulating factor (M-CSF), and then test its effectiveness in the treatment of wounds in rats with iatrogenically induced type 2 diabetes. The use of such dressings still represents only a small fraction of research on keratin biomaterials. Most existing studies have focused on simpler models, such as wounds in healthy rodents or animals with type 1 diabetes. Although chronic wounds can occur in patients with both type 1 and type 2 diabetes, the latter is more common and has a greater impact on quality of life.

In patients with type 2 diabetes, many signaling pathways essential for wound healing, such as PI3K/AKT and JAK/STAT, are impaired. Their activity can be enhanced by cytokines such as M-CSF. Nonetheless, the mechanisms of the activity of M-CSF in skin regeneration remain poorly understood and require further research, especially in complex models such as animals with type 2 diabetes.

The main goal of the project is to develop a new class of dressings capable of modulating the impaired immune processes observed in chronic wounds. In addition to evaluating the effectiveness of the dressing in stimulating healing, we will also conduct histological, immunohistochemical, and molecular studies. Confirming the effectiveness of the dressing will help elucidate the mechanisms by which keratin and M-CSF influence the immune response at the wound site.

Demonstrating the effectiveness of such a dressing will be an important step forward in the development of biomaterials whose mechanism of action targets specific processes and signaling pathways. We hope that the results of our research will accelerate the creation of a new class of dressings that can be successfully applied in clinical practice in the future.