The effect of non‐thermal atmospheric pressure plasma application on wound healing after gingivectomy

Abstract

Recent studies have indicated the potential benefits of Non‐thermal atmospheric pressure plasma (NTAPP) as a novel therapeutic approach. The purpose of the current study was thus to assess the effect of NTAPP on gingival wound healing. Fifteen patients with bilaterally symmetrical gingival hyperplasia were included in the study. After gingivectomy and gingivoplasty, the left‐hand side of the symmetrical surgical area was irradiated with NTAPP (plasma jet kINPen 11). Digital photographs of the gingival wounds were taken at baseline and days 3, 7, and 14. Wound epithelialisation was evaluated. Landry Wound Healing Index (WHI) scores and visual analogue scale (VAS) scores were also recorded. There were significant differences between the epithelialisation of the NTAPP‐treated sites and the control sites after the surgical procedures. The NTAPP‐treated sites had significantly smaller stained surface areas compared with the control sites on the 3rd, 7^th, and 14th days (P < .05). The NTAPP‐treated sites had better WHI scores than the control sites throughout the follow‐up period (P < .05). It can be concluded that NTAPP enhances epithelialisation and stimulates wound healing after gingivectomy and gingivoplasty. However, further clinical studies with larger sample sizes are needed to determine the exact benefits of NTAPP for gingival wound healing.

1. INTRODUCTION

Gingivectomy is used to remove diseased gingiva or hyperplasia of the gingiva to establish normal gingival anatomy and improve aesthetics. Various aetiological factors may cause gingival enlargements, such as gingival inflammation, mouth breathing, and the use of certain drugs. ¹ Wound healing after oral surgery is a complicated and active process of restoring cellular structures and gingival tissue and involves the following stages: inflammation, reepithelialisation, granulation tissue formation, matrix formation, and tissue remodelling. ² Reepithelialisation is a critical phase that includes interactions between the extracellular matrix and keratinocytes that migrate, proliferate, and differentiate, thereby restoring gingival tissue function and structure. ² , ³ In addition to the complex wound healing process, the bleeding and pain that are frequently seen after gingivectomy may be a problem. Several studies have been carried out to determine how to overcome these problems and to try to control the cellular activity of the gingiva by techniques, such as the use of growth factors, ⁴ enamel matrix proteins, ⁵ periodontal ligament cells,⁶ and laser applications. ⁷ However, the options for controlling the cellular activity of the gingiva remain limited, and there is no technique for regenerating the damaged periodontal tissues. ⁸

Recent studies have indicated the potential benefits of Non‐thermal atmospheric pressure plasma (NTAPP) as a novel therapeutic approach. NTAPP, which is also known as low‐temperature atmospheric pressure plasma or cold atmospheric plasma, has the benefit of allowing the application of plasma treatment to living tissue. Plasma has been described as a completely or partly ionised gas and as the fourth state of matter. Electrons, neutral atoms, positive and negative ions, and neutral or charged molecules can be detected in plasma. ⁹ There are two types of plasma: “hot or thermal” and “cold or non‐thermal” plasma. The production of plasma at atmospheric pressure and low temperature (30°C‐40°C) allows its application to living cells. ¹⁰ Previous studies have found that NTAPP stimulates the proliferation and migration of keratinocytes and fibroblasts. ¹¹ , ¹² This is hypothesised to lead to wound healing. NTAPP also inactivates bacteria and improves wound healing by modulating adhesion molecules and matrix metalloproteinase‐9 and increasing integrin expression. ¹³ , ¹⁴ It has been reported that NTAPP application enhances the release of cytokines associated with wound healing, including Interleukin 8 (IL‐8), in primary human THP‐1 monocytes, keratinocytes, and fibroblasts. ¹¹ , ¹⁵ There is evidence that IL‐8 plays an important role in the wound healing process. The results of a previous study showed that NTAPP irradiation modulates angiogenesis and may be used to affect angiogenesis during wound healing. ¹⁶ It also has the potential to be used for haemostasis in different surgical areas. ¹⁷ Despite the increased application of Plasma technology in medicine, there is no direct evidence or consensus that NTAPP could play a basic role in the oral wound healing process. The purpose of the present study was to evaluate the effects of NTAPP on the healing of the gingiva after gingivoplasty and gingivectomy.

2. MATERIALS AND METHODS

Patients (4 men and 11 women, between the ages of 19 and 43 years with a mean age of 27.93 ± 7.14 years) with bilaterally symmetrical gingival hyperplasia on their maxillary anterior region were included in the study. The study sample was selected from patients who were referred to the Department of Periodontology, Faculty of Dentistry, Osmangazi University. Written informed consent was obtained from all patients, and ethical clearance was obtained from the institutional ethical committee with approval number 25403353‐050.99‐E.42818.

2.1. Surgical procedure

Patients who had systemic disease (ie, HIV, cancer, uncontrolled diabetes mellitus, etc.) that could damage the wound healing process, or who had been treated with systemic corticosteroids or immunosuppressive agents, or who had used any tobacco product within the past 3 months, or who had had chemotherapy and/or radiation therapy prescribed or received within the past 2 months, were excluded from the study. Initial probing depths were recorded from the mesial and distal interproximal surfaces and mid‐buccal of each tooth using William's periodontal probe (Hu‐Friedy). In all patients, scaling and root planning and oral hygiene strategies were carried out in preparation before surgery. Surgery was performed under local anaesthesia. During surgery, an external bevel incision and then a sulcular incision were made and the hyperplasic gingiva was extracted. Gingivectomy and gingivoplasty were carried out using blade and gingivectomy knives.

2.2. Non‐thermal atmospheric pressure plasma application

After surgery, the left segment of the symmetrical surgical area was irradiated using NTAPP. The NTAPP used for this study was the kINPen 11 plasma jet developed by the Leibniz Institute for Plasma Science and Technology, Greifswald, Germany, in cooperation with Neoplas GmbH, Greifswald, Germany. The plasma jet comprises a base station, including the control unit and the power supply, together with a handpiece containing the electrical discharge system and the gas flow. The discharge system is supplied with argon plasma, with the pulses being generated at a frequency of 21 kHz with an applied voltage of 5 kV. The plasma stream is approximately 10 mm long and has a plasma–tissue interaction zone of about 1.5 mm in diameter. Argon gas was used as the carrier gas at a flow rate of 5 L/min at 2.5 Bar. The distance between nozzle and tissue was approximately 5 mm. The device kINPen 11 has European Conformity certification for fulfilling the electrical safety standards required for use on humans.

The wounds were treated with the plasma jet moving in three different directions (vertical, horizontal, and diagonal) over the wound surface. Approximately 1 cm² of the wound surface was treated for 1 minute. All operations and NTAPP treatments were carried out by the same surgeon (B.S.) to avoid confounding (Figure 1). NTAPP application was performed in the same way in the control group for preventing the placebo effect. However, the application did not occur because the start button was not pushing. The wound was not closed with a periodontal dressing, and the patients were prescribed analgesic for use when they had pain. Also, the patients were informed not to take analgesics within 24 hours before visual analogue scale (VAS) evaluations.

FIGURE 1.

Preoperative photograph of bilaterally hyperplasic gingiva before gingivectomy and gingivoplasty surgery (A), and immediately after surgery (B). Application of the kINPen 11 plasma jet (Blue arrow: Plasma jet) (C). Postoperative determination of epithelialisation of the non‐thermal atmospheric pressure plasma (NTAPP)‐treated and control sites on the 3rd (D), 7th (E), and 14th (F) days

2.3. Epithelialisation and subjective assessments

After the gingivectomy procedure, wound epithelialisation was assessed using a plaque disclosing solution (Mira‐2‐tone, GmbH & Co., Duisburg, Germany). Mira‐2‐Tones has been used previously for scanning of spaces where the oral epithelial layer has been abraded, and this technique has been recommended for the determination of segments without epithelium. ¹⁸ Epithelialisation of the gingiva was assessed using the method previously described by Esen et al. ¹⁹ Photographs of the gingival wounds were taken preoperatively and on the 3rd, 7th, and 14th day postoperatively with a digital camera (Nikon D 7100). The digital camera was positioned 20 to 25 cm from the labial surface of the maxillary anterior teeth. ²⁰ The photographic images were analysed using image analysis software (Image J 1.31o, National Institutes of Health, Bethesda, Maryland). The dark‐coloured areas were considered to be areas without epithelium that were still undergoing wound healing, and these were identified using a pointer on the screen. The software calculated the wound size (area) automatically in square millimetres (mm²). The dark‐coloured areas on the NTAPP‐treated wounds and the controls were assessed on the 3rd, 7th, and 14th day after surgery (Figure 2) and were compared by statistical analysis.

FIGURE 2.

Comparison of stained surface areas (non‐epithelialisation) between non‐thermal atmospheric pressure plasma (NTAPP)‐treated sites and control sites. The figure demonstrates faster re‐epithelialization on the NTAPP‐treated sites at all time points.

The amount of epithelialisation, the Landry Wound Healing Index (WHI), pain, and burning sensations according to a VAS and bleeding (yes/no) were recorded. The wounds of both NTAPP‐treated sites and the controls were scored using the Landry WHI on the 3rd, 7th, and 14th day. ²¹ This index system evaluates healing using a five‐level index that includes granulation tissue, response to palpation, tissue colour, and incision margin parameters. The scores range from one to five and correspond to very poor to excellent healing. Postoperative pain was measured using a VAS [no pain (0) to unbearable pain (100)]. ²² The degree of pain perception on the right‐hand and left‐hand sides was recorded by patients on the day of surgery and then daily until day 14.

2.4. Statistical analysis

The Shapiro–Wilk test was used to determine the normality of the measurements. Repeated measures analysis of variance (ANOVA) with adjustment for multiple comparisons using the Sidak test was used to compare the VAS, Landry WHI, and reepithelialisation measurements between groups and days. Continuous variables are presented as mean and SD. Categorical variables are presented as frequencies and percentiles. All analyses were performed using IBM SPSS Statistics version 22. A P value of less than .05 was considered significant.

3. RESULTS

All patients completed the study course and attended the follow‐up visits. No side effects were detected in the NTAPP‐treated group or control group after surgery and NTAPP application. Thirty percent of segments in the control site and 13.3% of segments in the NTAPP‐treated sites reported bleeding within the first 3 days, but the difference between the two sites was not statistically significant (P > .05).

The comparison of the epithelialisation of the wounds revealed significant differences between the dark‐coloured areas of the NTAPP‐treated sites and the control areas after gingivectomy and gingivoplasty. NTAPP‐treated sites had significantly smaller dark‐coloured areas compared with the control sites on the 3rd, 7th, and 14th day postoperatively (P < .05), and these findings demonstrated more rapid reepithelialisation on the NTAPP‐treated sites at all‐time points (Figure 2).

The epithelialisation of wounds was also assessed using the WHI and was compared at 3rd, 7th, and 14th day postoperatively. The results showed that the NTAPP‐treated sites had better WHI scores than the control sites throughout the follow‐up period (P < .05) (Table 1).

TABLE 1.

Comparison of Landry Wound Healing Index (WHI) scores between non‐thermal atmospheric pressure plasma (NTAPP)‐treated sites and control sites

Group	Landry WHI scores
	3rd day	7th day	14th day
NTTAP‐treated (mean ± SD)	2.00 ± 0.00	3.53 ± 0.51	4.73 ± 0.45
Control (mean ± SD)	1.73 ± 0.45	2.93 ± 0.45	4.20 ± 0.41
P value	0.032*	0.002*	0.002*

Note: Repeated measures ANOVA with adjustment for multiple comparisons: Sidak test.

^* P < .05.

The NTAPP‐treated sites displayed lower VAS scores than the control sites on the 1st, 3rd, 7th, and 14th day when the two sites were compared but this difference was not significant (P > .05) (Figure 3). Also, according to the intragroup comparison, the analysis showed there was a significant difference in VAS scores on all days within both NTAPP‐treated and control sites (P < .05) (Table 2 and Table 3).

FIGURE 3.

Comparison of visual analogue scale (VAS) scores between non‐thermal atmospheric pressure plasma (NTAPP)‐treated sites and control sites. The NTAPP‐treated sites had better VAS scores than the control sites on the 3rd, 7th and 14th days post‐operatively

TABLE 2.

Intragroup comparison of visual analogue scale (VAS) scores in control sites

Comparison	VAS	P value
1st day vs 3rd day	25.00 ± 4.25	.000*
1st day vs 7th day	47.66 ± 3.25	.000*
1st day vs 14th day	69.00 ± 2.91	.000*
3rd day vs 7th day	22.66 ± 4.90	.000*
3rd day vs 14th day	44.00 ± 3.08	.000*
7th day vs 14th day	21.33 ± 2.26	.000*

Note: Repeated measures ANOVA with adjustment for multiple comparisons: Sidak test (P < .05*).

TABLE 3.

Intragroup comparison of visual analogue scale (VAS) scores in non‐thermal atmospheric pressure plasma (NTAPP)‐treated sites

Comparison	VAS	P value
1st day vs 3rd day	24.66 ± 4.38	.000*
1st day vs 7th day	49.33 ± 2.55	.000*
1st day vs 14th day	66.33 ± 2.57	.000*
3rd day vs 7th day	24.66 ± 3.38	.000*
3rd day vs 14th day	41.66 ± 4.67	.000*
7th day vs 14th day	17.00 ± 2.97	.000*

Note: Repeated measures ANOVA with adjustment for multiple comparisons: Sidak test.

^* P < .05.

4. DISCUSSION

In this clinical study, wound healing was assessed after gingivectomy over a number of days to determine whether NTAPP treatment could promote gingival wound healing and to observe patient comfort after surgery. NTAPP is a new method not only for the treatment of wounds but with a wide range of other applications, such as the treatment of cancer or treatment of other skin diseases with microbial involvement. ²³ , ²⁴ However, there is a lack of information in the literature regarding the efficacy of NTAPP application to oral wounds.

The present study showed that NTAPP stimulates gingival wound healing after gingivoplasty and gingivectomy surgery. Wound healing is a complex biological process. First, epithelial cells migrate to the wound, and fibroblasts proliferate to form a matrix and deposit new connective tissue below the epithelial covering. ²⁵ Throughout this period, growth factors and cytokines are released by immune cells, such as neutrophils and macrophages that promote wound healing. ²⁶ Kalghatgi et al showed that NTAPP exposure improves the proliferation of cells, for example, endothelial and mammalian breast epithelial cells. ²⁷ Previous in‐vivo studies of gingival mucosa fibroblasts indicated the high capacity of these cells to reorganise extracellular matrix molecules, and this feature is directly associated with their high migratory capacity. ²⁸ , ²⁹ Epithelial cell migration is a key stage of wound healing. The proliferation and migration of keratinocytes and the connection between the collagen of the new connective tissue and the migrant cells determine the degree of reepithelialisation of the wound. ³ Kang et al revealed that treatment with NTAPP increased the levels of T‐EGFR (Truncated Epidermal Growth Factor Receptor), p‐STAT3 (Phospho‐Signal transducer and activator of transcription 3), and Type I collagen in normal fibroblasts. ³⁰ For successful wound healing, mucosal cells also have to migrate to the wound to enable closure, which is associated with the secretion of extracellular matrix proteins, like type I collagen. The results of this study show that, in all periods, better healing was achieved in the NTAPP‐treated surgical areas than in the control group, probably because of improved collagen production and the proliferation of epithelial cells.

It has previously been shown that NTAPP supports pro‐inflammatory cytokines and growth factors. ¹⁵ Furthermore, it has recently been indicated that NTAPP application enhances the secretion of cytokines associated with wound healing, such as IL‐8, THP‐1 monocytes, and keratinocytes. ¹¹ , ¹⁵ As reported by Kwon et al, exposure of gingival fibroblasts to NTAPP improved the mRNA synthesis of two growth factors, vascular endothelial growth factor (VEGF) and TGF‐β (transforming growth factor‐beta). ³¹ TGF‐β plays a major role in the healing process by directly regulating fibroblast expression, and controls the cellular proliferation and ECM (extracellular matrix) turnover through the ECM protein synthesis and SMAD signalling pathway. ³² Furthermore, it influences the synthesis of matrix ingredients and collagen by fibroblasts. ²⁵ VEGF directly affects inflammation and the healing process via endothelial cell differentiation and proliferation. ³³ Hence, improved expression of both VEGF and TGF‐β induces the cellular activity of gingival fibroblasts that is associated with the formation of scaffold and matrix. ²⁵

The pain scores after surgery were higher for the control areas than for the wounds treated with NTAPP, but the differences were not statistically significant. The clinical assessment of the wounds revealed better healing of wounds treated with NTAPP according to mucosa colour and contour. This outcome indicates the advanced wound healing of NTAPP‐treated wounds. This is significant for the comfort of the patient after surgery as it leads to lower pain for wounds treated with NTAPP.

Additional wound care and antimicrobial treatment increased the rate of healing, as a result of infection control. Reactive species occur following irradiation with NTAPP, which inactivates bacteria and inhibits bacterial growth on the wound surface. ³⁴ Other studies have also reported inactivation of microorganisms with NTAPP treatment and advanced wound healing through the induction of integrin expression and regulation of matrix metalloproteinase‐9 and adhesion molecules. ¹³ , ¹⁴ In this way, NTAPP can be used to control the bacteria that directly or indirectly affect epithelial and connective tissue cells and can stimulate oral wound healing.

Non‐thermal atmospheric pressure plasma has no toxic effect on cells, demonstrating the same level of high cell viability after irradiation that resulted in growth‐related gene expression. ³¹ According to previous findings, plasma produces several kinds of ROS (reactive oxygen species), ³⁵ including O₃ (ozone) ³⁶ and NO (nitric oxide). ³⁷ These reactive species have many effects on living cells, some of which are positive. Furthermore, these oxygen species may enter the cells via diffusion or induce new species within the cells. It has been hypothesised that the improved healing observed with ozone therapy might be associated with the activation of local antioxidant mechanisms, which stimulate the release of growth factors and support tissue repair, as well as its antimicrobial effect. ³⁸ In addition, NO plays a significant role in inflammatory and healing processes as an important signalling molecule. ³⁹ NO stimulates the proliferation of fibroblasts, which is required for collagen deposition and also accelerates reepithelialisation. ⁴⁰ Further signalling effects of NO, such as inflammatory cell and cytokine modulation, are equally important. ⁴¹ Despite these findings, the exogenous NO and O₃ produced by NTAPP resulted in positive cellular activity in the wound healing process.

Angiogenesis is a significant and physiological process in wound healing and should be promoted. The formation of new blood vessels is induced by a lack of oxygen and other endogenous proangiogenic factors, such as cytokines (IL‐1, 2, 6, 8; TGF, tumor necrosis factor (TNF)), growth factors (epidermal growth factor (EGF), VEGF, fibroblast growth factor (FGF)), ROS, and NO. Plasma is thought to provoke angiogenesis because of the formation of different ROS and NO. The patients in the current study reported less bleeding at the NATTP‐treated sites during the first 3 days postoperatively. This difference between the NTAPP‐treated and control wounds may be attributed to the haemostatic capacity of plasma. NTAPP can be used to control bleeding with enhanced blood coagulation, and this can minimise complications. ⁴² Haemostasis with NTAPP application is ordinarily achieved by blood coagulation, which involves clot formation, platelet aggregation, and coagulation factors. ⁴³

Given all these positive effects on wound healing, treatment time, and dose are directly related to the effect of plasma on living tissues. Short application times and low plasma doses have stimulating effects (improvement of migration and proliferation, induction of DNA repair) and improve wound healing. However, long application times and high doses provoke lethal effects (including halting proliferation and causing cell death by apoptosis, cell cycle arrest, and DNA damage) that can be used to attack cancer cells. ¹⁰ Therefore, in this study, the dose and treatment time of NTAPP followed the manufacturer's recommendations (5 ± 1 L/min and 60 s/cm²) for the promotion of wound healing. In addition, the lowest temperature threshold for thermal injury to soft tissues was described by Moritz et al, who advised to avoid long‐term exposure to temperatures exceeding 44°C. ⁴⁴ It has been shown that the kinPen 11 plasma jet causes 35°C to 38°C on the working surface, which is acceptable temperature ranges for application on living tissues. ⁴⁵

Within the limitations of this study, the final results indicate that NTAPP could be used as a supportive method for wound healing after periodontal surgery. Clinical studies are needed to determine the most appropriate treatment options for patients. Further clinical studies with large sample sizes are also needed to determine the exact advantages of NTAPP in gingival wound healing.

CONFLICT OF INTEREST

The authors declare no conflict of interest. The funders had no role in the design of the study; in the collection, analyses, or interpretation of data; in the writing of the manuscript, or in the decision to publish the results.

Kusakci‐Seker B, Demirayak‐Akdemir M. The effect of non‐thermal atmospheric pressure plasma application on wound healing after gingivectomy. Int Wound J. 2020;17:1376–1383. 10.1111/iwj.13379