Tendon repair by plasma jet treatment

Abstract

Objective

In recent years many researchers applied cold plasma for wound healing. The cold plasma is irradiated on the surface of wound. In this paper the effect of irradiation of cold plasma on the skin for healing of injured tissue which is located inside body, such as tendon, is evaluated.

Methods

The male, white New Zealand, (20-week-old) were selected. Aloxan injection induced for diabetes induction and a week later the blood glucose level was measured. The standard tendon injury was created. The rabbits was divided in 3 groups. Control group, Plasma treated group at 5 kv, plasma treated group at 10 kv. Cold plasma was applied to the rabbits for 21 days.

Results

After 21 days the tendon tissue were considered histologically. The results show that inflammatory cells were significantly lower in the tendon treated with cold plasma at 10 kv than the others, which confirms that cold plasma treatment reduce the inflammation phase. Cold plasma treatment led to increase neovascularation and collagen production.

Conclusion

The results of this study confirm that the cold plasma treatment of skin has positive effect on healing of tissue inside body.

Introduction

In recent years cold atmospheric plasma (CAP) usage become to a new treatment method [4, 5]. Many researchers applied cold plasma for wound healing. For examples: Isbary et al., in 2013 by a retrospective study showed that CAP could be an effective method especially in chronic wounds [18]. Elizabeth Garcıa-Alcantara et al. [14] showed combined treatment of Argon and Helium could accelerate skin acute wounds healing.

Cheng et al. [7] show that argon plasma jet enhanced the wound healing in diabetic rats. They show that the plasma treatment led to increase of superoxide dismutase (SOD), TGF-β, catalase (CAT) and glutathione peroxidase. The literatures focus on applying cold plasma on the surface of wound. All injuries is not occur on skin and some of them is occur in tissues which is located inside body. For healing of some of this wound surgery is needed. Healing of this injuries without surgery is important and could help many patient. In this paper we try to show if irradiation of plasma on the skin could affect the healing processes of the inside injuries. For this aim the tendon tissue are selected. Tendon injuries are a common problem during sports. Among soft tissues tendon heals slowly. Tendon healing include tree stages, inflammatory, a proliferative and remodeling stage, which take slowly [15]. The main parameter for treatment of injured tendons is return to normal tensile strength. Some therapies such as tissue engineering, gene therapy, physical therapy and laser therapy were applied for fast healing [33]. This is the first study that evaluated the effect of cold plasma on tendon tissue. For this aim the rabbits were divided in 3 groups: control, plasma treated group at 5kv, plasma treated group at 10kv. the rabbits were treated by the cold plasma for 30s. some literature showed that 30 s plasma treatment is required for migration of fibroblasts and keratinocyte [2, 31].

Materials and methods

Cold plasma therapy

The plasma system consisted of two electrodes. One of them is powered by pulse DC high voltage power supply. The other one is wrapped around the tube. The distance between the rabbit and electrode was about 1 cm. The voltage of the plasma was adjusted to 5 and 10kv. The frequency is adjusted to 10 Kh.

Ethics statement

The experiment of this study was done according to the guidelines of the animal care which approved by the ethics committee in Islamic Azad University.

Design of experiment

The healthy rabbits were purchased from the Pasteur Institute of Iran. All of them was male, white New Zealand, (20-week-old) and the body weight of them was about 2.5–3.5 kg. Aloxan injection induced for diabetes induction and a week later the blood glucose level was measured. The rabbits were divided in 3 groups of 6 rabbits each: (Control: animals with tendon injuries without any treatment (Fig. 1).

Fig. 1.

The plasma jet treatment of rabitts

Cold plasma at 5 kv: animals with tendon injuries was treated by the cold plasma).

Cold plasma at 10 kv: animals with tendon injuries was treated by the cold plasma). At first Anesthesia induced with combination of Xylazine hydrochloride 2%(8 mg/kg) and ketamine hydrochloride 10% (35 mg/kg).

After anesthesia and clipping, the skin of rabbits was disinfected by antiseptic povidone-iodine solution. The skin (2 mm) was cut and the right tendons were freed from surrounding tissues. Then the sharply incision was made.

The skin and tissue were sutured. Cold plasma was applied to the rabbits for 21 days (30 s each day). The rabbits were kept in standard condition: standard cages, filtered tap water, standardized food, room temperature: 18–22 °C, humidity: of 40–50%, 12 h/12 h light/dark cycle. After 21 days the tendons was analyzed histologically. For Histological examination: at first tendons was washed in physiological solution then they were fixed using 10% formalin and embedded in paraffin.

The sections were kept under light microscopy and stained with hematoxylin and eosin (H&E*4). The inflammatory phase, production and arrangement of collagen were considered with a specific grading. The score of the tendon was determined according to Loppini et al. [20].

Optical emission spectroscopy

A wide range of various excited gaseous species is produced during cold plasma generation. Optical spectroscopy of emitted light from plasma generation gap was employed to study the plasma discharge characteristics and to identify the produced species. A fiber cable was used to transfer the light of discharge to a computer controlled spectrometer (Ocean Optics HR 2000) and the emissions were recorded between 200 and 1000 nm wavelengths. The recorded spectra with an integration time of 0.1 s were averaged and after noise reduction, identification of active chemical species was performed by comparing peaks to the data of the NIST Atomic Spectra Database (version 5.1; [19]).

Determination of voltage and current

The voltage and current was determined using a Tektronix P6015A high-voltage probe and a Tektronix DPO digital oscilloscope.

Results and discussion

Figure 2 Shows that the current of the plasma as a function of different voltage.

Fig. 2.

The current and voltage of the plasma jet

Figure 3 shows that the OES analysis of the plasma at 5 and 10 kv.

Fig. 3.

The OES analysis of cold plasma system

The analysis shows that the nitrogen and oxygen peaks increase when voltage increases.

Figure 4 shows the histological image of the wound after 21 days of the cold plasma treatment.

Fig. 4.

The histophatological images of tendon. c: control group after 21 days, b: plasma treated group at 5 kv after 21 days, c: plasma treated group at 10 kv after 21 days. Arrows: cell rounding, the tip of arrows: inflammation cell

To the best of our knowledge, the data obtained in this report reveal some new aspects among the few study designed to compare the effect of non-thermal atmospheric pressure plasma at different voltage for tendon injury. The results show that the plasma treatment at 5 kv had no any effect on tendon healing but 10 kv is effective for healing of tendon injury.

Tendon repair divided into three main phases: inflammatory phase, a proliferative or repair phase, and a matrix remodeling phase [15]. The sampling results (Fig. 4) indicate that the formation of the epithelium increased during healing, which confirms the success of the diagnosis. The inflammatory cells were significantly lower in the tendon treated with cold plasma than control, which confirms that cold plasma treatment reduce the inflammation phase (according to Fig. 5).

Fig. 5.

The score of the control and the treated tendon. The score of the structural cell, arrangement of cell, cell rounding, neo vasularization, cell density, inflammation of control and the treated tendon was measured

Cold plasma treatment resulted in reduction of inflammatory phase and accelerates the recovery phase by increase neovascularation and collagen production. There are no studies about tendon healing by cold plasma for comparison. Also there are no study about the applying cold plasma on skin for treatment of inside wound such as tendon. In this term the ionization wave does not touching the wound. Touching plasma could generate charging of surface. In the non-touching cold plasma production of neutral radicals, UV and electric fields led to some effect on cells. For example: [24, 32].

Electric fields affect the potential of the membrane and mitochondria of cells.

The Fig. 2 shows that increasing the voltage of the plasma had no any effect on the current of the plasma. As the electric field is proportional to current, the electric field of the plasma at 5 and 10 kv is the same. Then the electric field has no any effect on the healing.

The cold plasma treatment resulted in reduction of inflammation phase. In this phase generation of excessive nitric oxide led to increase the production of reactive oxygen species (ROS) [10].

ROS induce oxidative stress which cause lipids, proteins and DNA damages and fibrosis ([16]; 13. [1, 13, 21]). Some reports showed that low-level laser therapy has positive effect on tendon healing ([12, 27]; 19. [3]). It has been demonstrated that low-level laser therapy could enhance ROS. There are number of report that ROS play an important role in the inflammatory response [10]. This shows that the healing process in the plasma group was faster than the control group. Increasing trends in the plasma group was quicker, and always one step ahead of the control group, which indicates that helium plasma improved the healing compared to the control group due to the inhibition of the infection by helium plasma. Collagen formation in the plasma group was always faster than the control group, which illustrates the positive effects of helium plasma in treating tendon. Recent reports showed that low level laser therapy accelerate tendon healing by increasing collagen types I and III [15, 29]. In this study, cold plasma treatment of the tendon resulted in increased production of collagen. Production of collagen are important to the repair strength of the tendons [28]. One of the primary factors responsible for the slow healing of a tendon is poor blood circulation, which leads to low levels of oxygen and nutrients in the region of the wound [9]. Neovascularization is another important factor in the healing of a wound [8]. The results show that plasma treatment increases the neovascularization of the wounds as compared to the control group. Hirata et al. [17] and Ye et al. [34] hypothesized that the reactive particle present in plasma can cause the activation of growth factors accomplished by angiogenesis. The results also indicate that the keratinocyte cell migrates in case of wounds treated with cold plasma after a few days of the treatment; this is not observed in the rabbits of the control group. Plasma produces certain spices, such as free radicals. Reactive ions and radicals play an important role in the interaction between the plasma and cells. It was reported that NO activated the TGF-β 1 cytokine by S-nitrosation of the latency-associated peptide and the MAPK pathway [22]. The MAPK pathway is known to play an important role in the healing of wounds as well as to be involved in the cellular inflammation–proliferation process [23, 25]. Chen [6] reported that reactive free radicals are involved in the angiogenesis process by activating the vascular endothelial growth factors. In addition, reactive free radicals are responsible for the sterilization of the wounds [6]. In this study cold plasma treatment on skin led to reduction of the inflammation phase and acceleration of the recovery phase in tendon tissue inside body. This study demonstrated that the effect of cold plasma is not limited to the irradiated surface. In this terms electric fields could affect the cells [11, 26, 30].

This study show that the electric field had no any effect on the healing.

The optical emission spectroscopy of the plasma (Fig. 4) showed the emission species: O (108, 216, 324, 325, 328, 436, 1170, 1189, 1195, 2900, 2907), N (1600, 1670).

The OES analysis of the cold plasma at 5 and 10 kv show that the ROS of the cold plasma at 10kv is higher than 5 kv. Then the ROS of the cold plasma is the main parameter for the healing.

Conclusion

The results show that the voltage of cold plasma is an important factor for wound healing. The results show that the plasma treatment at 10 kv is effective for healing of the internal injury but the treatment at 5 kv had no any effect on the injury.

Declarations

Conflict of interest

None.