by Diabetic wounds often become chronically infected and are notoriously difficult to treat. Two primary reasons for this include the formation of a bacterial biofilm and high levels of oxidative stress. A novel hydrogel dressing was developed recently to combat both these undesirable characteristics and tested for its effects on diabetic-infected wound healing. The report appeared in Nature Communications.
Background
Wound healing is recognized to have four stages, namely, coagulation, inflammation, proliferation, and maturation. When this doesn’t happen, chronic wounds result. Most occur due to prolonged inflammation triggered by competing pro- and anti-inflammatory signals leading to loss of redox homeostasis.
Chronic inflammation attracts leukocytes that secrete reactive oxygen species (ROS), a defense against microbial invasion. However, these ROS also antagonize wound healing by damaging living tissues and cells at various levels and promoting breakdown and further inflammation.
In the worst cases, cells die within and around the wound site by apoptosis and other modes of programmed cell death because of excessively high ROS levels. Neighboring cells react to this and eventually die themselves, accounting for the severe necrosis, or tissue death, common to such wounds. This means that tissue debridement or even amputations, at times, becomes necessary to treat these wounds.
Biofilm formation by microbes is another complication that gives rise to chronic wounds, preventing topical antioxidants from acting on the wound surface. Biofilms use up nutrients from the wound bed and secrete extracellular polymeric substances (EPS) that form a protective barrier against immune cells, antibiotics, and other antimicrobials. Moreover, they remain stable on the wound surface until medically removed.
Biofilm microbes are, in fact, the primary species found in chronic wounds and are resistant to treatment in many cases. Most commonly, these are methicillin-resistant Staphylococcus aureus (MRSA) or carbapenem-resistant Pseudomonas aeruginosa (CRPA).
Chronic wounds cost the economy over USD 50 billion in just the USA, in just one year. And this is only expected to increase as the population grows around the world. Diabetic wounds are among the most common types of chronic wounds and have, unfortunately, as high a risk of death as cancer, at about 31%.
The effectiveness of ordinary wound dressings in chronic wounds is small. Dressings designed for chronic wounds have so far not been developed as stand-alone treatments. At present, specialized chronic wound dressings require the additional use of photothermal irradiation or release and leave significant amounts of antibiotics or metal ions in the wound.
The current study was motivated by the need for improved chronic wound dressings that would be adequate by themselves, would not contaminate the wound, and would not produce unwanted discharge and moist wound matter.
The researchers used a hydrogel, PPN, formed by crosslinked polyethylene glycol (PEG) hydrogel tethered with highly potent antibacterial cationic polymer, polyimidazolium (PIM), and the antioxidant N-acetylcysteine (NAC). The cationic hydrogel kills bacteria by absorbing them into its pore spaces and then contact killing by the pore walls.
PPN was designed to have dual functionality, opposing both biofilm formation and oxidative stress in the wound bed. Both properties would act together in synergy to promote the healing of infected diabetic wounds.
Very little of this hydrogel leaches into the wound, and it contains neither antibiotic nor metal compounds, ensuring the wound is uncontaminated by any of these once the dressing is taken off.
What did the study show?
PPN showed high antibacterial efficacy in vitro. The hydrogel formulations swelled up, absorbing 10-12 times their original weight of water within an hour. In two days, when tested in infected wounds on murine models, the hydrogels became dirty yellow, probably because of the absorption of fluid and dead bacteria in the wound. They remained structurally stable, however, indicating that they do not break down in the presence of infected wounds.
The researchers tested these hydrogels on a human skin model that was grown in a 3D structure. This demonstrated improved keratinocyte differentiation in the presence of NAC. In addition, it speeded up re-epithelialization and, thus, wound closure. Notably, silver dressings have been shown to retard keratinocyte proliferation in chronic wounds.
Subsequently, they applied the dual-functionality hydrogel on infected wounds in diabetic rats, which closely resembled diabetic wounds in humans. The wounds were coated with a biofilm containing either MRSA or CRPA.
The hydrogels showed excellent biocompatibility compared to silver dressings in current use. The infected wounds treated with the hydrogel showed rapid healing compared to those in control animals. Bacterial counts fell rapidly and steeply over the first three days and remained low over the next two weeks.
In contrast, bacterial reduction was lower for both silver dressings and control dressings. The wounds were smaller and sloughing minimal in PPN-treated wounds compared to silver or control dressings or no treatment. In fact, untreated wounds showed biofilm formation and pus discharge with sloughing wounds, with evidence of reinfection.
Wound healing factors were also found at higher levels in PPN-treated wounds than in untreated or control-treated wounds. More mature collagen was found in the PPN-treated wound, indicating better regeneration of skin structure. Both components of PPN were found to contribute to the improved results compared to only one.
The hydrogel can be formulated in different ways for application to the healing of superficial or deep wounds. Its advantages include the absence of wound contamination and the fact that it does not require the use of photothermal irradiation or other healing modalities.
What are the implications?
PPN first removes bacteria from the wound site, allowing the number of inflammatory cells to drop. ROS levels are reduced by the NAC component, which allows them to diffuse into the hydrogel, providing an immune boost while relieving oxidative stress. Also, it encourages the release of wound-healing factors.
Finally, the NAC stimulates keratinocyte differentiation and the restoration of a normal epithelial covering over the wound. All these promote wound healing.
This PPN dressing is more potently antibacterial than silver dressing, with activity against MRSA and CRPA. It also does not cause further inflammation and accelerates wound healing. The feasibility of multiple formats for meeting different needs and its possible extension to other biomedical needs make this hydrogel a promising alternative for the treatment of chronic infected diabetic wounds.
