Temple University School of Podiatric Medicine Journal Review Club

Editor's note: This post is part of the Temple University School of Podiatric Medicine (TUSPM) journal review club blog series. In each blog post, a TUSPM student will review a journal article relevant to wound management and related topics and provide their evaluation of the clinical research therein.

Article title: Investigation on the effects of the atmospheric pressure plasma on wound healing in diabetic rats.
Authors: Sara Fathollah, Shahriar Mirpour, Parvin Mansouri, Ahmad Reza Dehpour, Mahmood Ghoranneviss, Nastaran Rahimi, Zahra Safaie Naraghi, Reza Chalangari and Katalin Martits Chalangari.
Journal name and issue: Scientific Reports, 6 Article Number 19144, 2016.
Reviewed by: Farheen Iqbal, Class of 2019, Temple University School of Podiatric Medicine.

Introduction:

Wound healing occurs in four phases: hemostasis, inflammation, tissue growth, and tissue remodeling. The process of wound healing can be affected by multiple factors, including oxygenation, age, stress, and disease such as diabetes. Wound healing in patients with diabetes can be further complicated by neuropathy and vascular disease. Typical treatments for diabetic wounds, like wound dressings, cell therapy, and oxygen therapy, are not entirely successful because diabetes slows and impairs the healing process. The authors of this study propose the use of a non-thermal atmospheric pressure plasma treatment as a novel therapy for diabetic wounds. Unlike conventional methods, this treatment can accelerate tissue repair without negatively affecting normal tissue. Using atmospheric plasma accelerates tissue repair because the plasma produces reactive oxygen and nitrogen species, which are key for the inflammatory response. It also induces neovascularization and enhances epidermal layer formation.

Materials and Methods:

The study was conducted using 25 male rats that were divided into diabetic and non-diabetic groups. The diabetic group contained 3 subdivisions: plasma treatment, helium gas treatment, and no treatment (control). The non-diabetic group contained 2 subdivisions: plasma treatment and no treatment (negative control). Diabetes was induced in the rats through intravascular injection of streptozotocin. The circular, full-thickness skin wounds on the dorsum of the rat were 2 mm in diameter, and were created after general anesthesia was administered.

The plasma used in this study contained ionized helium gas produced by a high-voltage (8 kV) and high-frequency (6 kHz) power supply. The plasma jet was made of a Pyrex tube wrapped with copper wire. The 10 mm wide jet nozzle was placed 20 mm from the wound surface, and each treatment lasted 10 minutes. Tissue specimens were subject to histological analysis and immunochemistry assays. Optical emission spectroscopy was also performed on the plasma.

Results:

The findings indicate that in the absence of any treatment, diabetic wounds healed at a significantly slower rate compared to non-diabetic wounds. The use of non-thermal atmospheric pressure plasma significantly increased the rate of wound healing in both non-diabetic and diabetic animals compared to non-treated animals.

With plasma treatment, the diabetic wound healed completely in 30 days, indicating a significant acceleration in healing time compared to the helium treatment.

Histological analyses showed that the plasma treatment induced the formation of a new epidermal layer, enhanced fibroblast cell proliferation, and produced neovascularization in the diabetic animals. While the helium treatment also increased cell proliferation and regrowth of the epidermis, it was significantly less than the plasma treatment.

Immunohistochemical analyses revealed that plasma treatment activated TGF-β1 cytokine significantly more than the helium treatment. A significant increase in TGF-β1 can be seen 7 days after plasma treatment. The cytokines were released by blood plasma cells, and cytokine levels showed a significant decrease 15 days after treatment. Using optical emission spectroscopy, it was discovered that the plasma had produced ions and radicals such as hydroxyl and nitric oxide molecules.

Discussion:

A major issue with diabetic wound healing is poor blood circulation. Without proper blood circulation, oxygen and nutrients are unable to reach the wound site, therefore making neovascularization crucial for healing. The non-thermal atmospheric pressure plasma treatment used in this study produced neovascularization 7 days after treatment. Another factor that can impair wound healing is a lack of the growth factor TGF-β1, which is often seen in diabetes mellitus as a result of cell dysfunction. Typically in diabetic patients, elevated glucose levels prevent the increase in TGF-β1 levels, and this halts the migration of keratinocytes to the wound site. In the current study, however, the plasma treatment enhanced the release of TGF-β1 7 days after treatment. Therefore, it is likely that this would further enhance the migration of keratinocytes, and lead to the formation of the keratin layer above the epidermis. The authors observed that most of the TGF-β1growth factor clustered around endothelial cells, and this is thought to further increase angiogenesis.

Plasma also produces reactive oxygen and nitrogen species. These reactive species play a crucial role in wound healing by inducing inflammation in the epidermis layer. Furthermore, reactive free radicals stimulate vascular endothelial growth factors that are important for inducing angiogenesis. Reactive species are also involved in wound sterilization by eliminating microbial agents at the wound site.

In conclusion, the use of non-thermal atmospheric pressure plasma accelerated diabetic wound healing through many processes. The treatment induced neovascularization, cell proliferation, keratinocyte migration, epidermal layer formation, and the release of TGF-β1 cytokines. Upon further investigation, the plasma treatment proposed in this study has the potential to be effective for the treatment of diabetic wounds in humans.

About the Authors:
Farheen Iqbal is a second-year student at Temple University School of Podiatric Medicine. She graduated from Towson University with a B.S. in biology, and minors in chemistry and cultural studies. At TUSPM, she is an officer of Dermatology Club and Journal Club.

Dr. James McGuire is the director of the Leonard S. Abrams Center for Advanced Wound Healing and an associate professor of the Department of Podiatric Medicine and Orthopedics at the Temple University School of Podiatric Medicine in Philadelphia.