Evaluating the effect of cold plasma on the healing of gingival wound

Abstract

Background

This is the first study that conducted to the effect of cold plasma on the healing of gingival wound in diabetic rabbits.

Material and method

Eighteen healthy rabbits is purched from pastor institute. The aloxan was injected to the rabbits. After induction of anesthesia the gum tissue is removed. The rabbits were treated by the plasma jet for 3, 5 and 10 days each day 30 s and they were considered histological.

Results and discussion

After 3 days the plasma jet treatment, the production of collagen and fibroblast and migration of epithelial cells is observed. As can be seen from the results after 5 days the cold plasma treatment the increase of neovascularation, collagen and inflammatory infiltration is seen in gum tissue. Formation of granulation tissue is seen after 10 days the plasma jet treatment.

Conclusion

The cold plasma treatment is an effective way for gingival wound treatment. Cold plasma treatment resulted in reduction of inflammatory phase and accelerates the recovery phase by increase neovascularation and collagen production.

Introduction

In recent years, many researchers have paid attention to efficiency of cold atmospheric plasma (CAP) for the treatment of wounds. The effects of cold plasma treatment on gingival wounds have not been covered previously in any study. The treatment of such wounds takes long time which involves healing of the skin and soft tissues after an injury. The various stages of healing comprise coagulation, inflammation, granulation, fibroplasia, collagenase, wound constriction, and epithelialization [1] Cold atmospheric plasma is known as an effective tool for wound healing. The plasmas produce reactive and natural spices, UV and photons. For applying cold plasma in human body pre-clinical experiments should be done [2]. There are some studies about the effect of cold plasma on wound healing. Isbary et al., [3] show that cold plasma can accelerate the healing of chronic wounds. Elizabeth Garcıa-Alcantara [4] demonstrated that the combined treatment of argon and helium plasma is the effective method for acute wounds. It was previously shown that plasma jet could eliminate the Aspergillus keratitis in eyes [5]. Amini et al., 2020 compared the effect of cold plasma, uv and infrared on wound healing and they concluded that the cold plasma treatment is the most effective method [6]. To date there are no study about effect of cold plasma treatment on gingival wounds. Because of saliva, moist external environment and cell phenotypes, the healing of gingival wound is different [7].

In this study the gum wound was treated by cold plasma at 10 kV. Amini et al., 2021 show that the voltage of cold plasma is an important factor for wound healing. They also demonstrated that plasma jet treatment at 10 kv led to healing of tendon injury but the wound was not healed after the cold plasma treatment at 5 kv [8]. In this research, we applied the cold plasma to the animals for 30 s.

Pervious researchers demonstrated that 30s treatment of cold plasma treatment led to endothelial proliferation and human keratinocyte improvement [9, 10].

Materials and methods

Cold plasma therapy

The cold air plasma (Plasma Teb Afranil co; model: SAION 8.1 m) is used for this study. The setup is driven by 15 kV and 30 kHz AC high voltage. The 1 cm circular copper sheet was used as an electrode (According to Fig. 1).

Fig. 1.

Shows the schematic of the plasma treatment

Design of experiment

Eighteen healthy rabbits (male, white New Zealand, (20-week-old) and 2.5 -3.5 kg weight) is purched from pastor institute. The aloxan (150 mg/kg) was injected to the rabbits.

Anesthesia induced (ketamine hydrochloride 10% (50 mg/kg) and Xylazine hydrochloride 2%(5 mg/kg)) and the gum tissue is removed by A punchibiopsy instrument with 3 mm diameter. The rabbits was treated by the cold plasma for 3, 5 and 10 days, each day 30 s, according to Fig. 1. Then the gums were analyzed histologically.

Ethic statement

The experiments were approved by the ethics committee in Islamic Azad University.

Optical emission spectroscopy

For transferring the light of plasma to a computer controlled spectrometer (Ocean Optics HR 2000). The wavelengths of 200 and 1000 nm with an integration time of 0.1 s were recorded. The peaks was determined by NIST Atomic Spectra Database.

Results and discussion

Figure 2 shows the OES analysis of the cold plasma system. The OII, OIII, OIV, NII, NIV peaks was observed in the analysis.

Fig. 2.

The OES analysis of the cold plasma

According to Figs. 3 and 6, the production of collagen and fibroblast and migration of epithelial cells after 3 days the plasma jet treatment is observed. As can be seen from the results of Figs. 4 and 6, after 5 days the cold plasma treatment the increase of neovascularation, collagen and inflammatory infiltration is seen in gum tissue. Formation of granulation tissue is seen (Figs. 5 and 6) after 10 days the plasma jet treatment.

Fig. 3.

Histological aspect of the rabbit’s tissue after 3 days. a control group. b the treated tissue. a: The tissue is extremely inflammatory and there is no evidence of repair. b (inflammatory cells are highly observed and but there are reduced compared to the control group

Fig. 6.

The score of the wound after the cold plasma treatment. The score of the wound after the cold plasma treatment (a): after 3 days, b: after 5 days, c: after 10 days

Fig. 4.

Histological aspect of the rabbit’s tissue after 5 days. a control group. b the treated tissue. a The inflammatory cells are low and the tissues under repair are observed, and there are sections of the vessel. b The inflammatory cells are heavily reduced and the tissue of the broth bud is formed

Fig. 5.

Histological aspect of the rabbit’s tissue after 10 days. a control group. b the treated tissue. a The budding tissue is formed, but there is still a small amount of inflammatory cells. b An evolved bud tissue is better and more evolved than the control group. The vein sections are well visible and well visible

Large full-fledged epidermis layer is confirmed that the wound completely healed after 10 days treatment. Comparing the results of the figs show that in the plasma treated group the Inflammatory infiltration is stopped after 5 days but it is increased in control group. The rate of gingival wound healing in control groups is less in control group. This is the first study that evaluates the effects of cold atmospheric plasma treatment on the healing of gingival wound.

Eroglu et al., 2018 showed that ozone therapy is effective method for healing of oral wound [11].

Evaluating the results show the less inflammatory cells in the cold plasma treated wound than control, which confirm the reduction of inflammation phase. This phase is known as the first phase of wound healing. In this phase reactive oxygen species (ROS) is produced by generation of excessive nitric oxide [12].

Production of lipids, fibrosis and proteins and DNA damage is done by oxidative stress of ROS [13–16]. Cuzzocrea et al., 2004 show that Reactive oxygen spices are responsible for inflammatory response [17] .

New vascularization was increased at first and then decreased and it was significantly more evident in plasma group in compare with control group.

The literature showed that the neovascularization is an important parameter in wound healing [18]. The reactive particle in plasma cause activation of growth factors accomplished by angiogenes [19, 20].

One of the important factors responsible for wound healing is blood circulation, which causes nutrients and oxygen to reach tissue [21]. Then Neovascularization is the important parameter in the wound healing [18]. As can been from the results the neovascularization is increased after cold plasma treatment.

Cold plasmas generate UV, photons, free radicals, reactive ions and natural spices.

Reactive spices such as ions and radicals led to interaction of cells and plasma for example NO activated the TGF-β 1 cytokine and the MAPK pathway [12] The MAPK pathway led to the cellular proliferation and inflammation process. The literature shows that the free radicals of cold plasma treatment eliminate microbial cells and decontaminate wounds [22].

In the cold plasma treatment the in spite of neutral radicals and UV the electric fields affect the cells [20, 23].

The mitochondria and the potential of the membrane are affected by electric fields.

The results show that the cold plasma treatment led to increase of Collagen formation.

Amini et al., 2020 compared the effect of direct and indirect plasma and showed that UV and electric field of the cold plasma is not responsible for the healing and they also concluded that uncharged spices and ROS of floating discharge plasma affect the wound [24].

Conclusion

The cold plasma treatment is a very effective way for gingival wound treatment.

Cold plasma treatment resulted in reduction of inflammatory phase and accelerate the recovery phase by increase neovascularation and collagen production.

Declarations

Conflict of interest

None.