Odontology DOI 10.1007/s10266-014-0157-2
ORIGINAL ARTICLE
Effects of microplasma irradiation on human gingival fibroblasts Ryoichi Takahashi • Kazuo Shimizu Yukihiro Numabe
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Received: 28 January 2014 / Accepted: 20 April 2014 Ó The Society of The Nippon Dental University 2014
Abstract The purpose of this research was to clarify the effects of microplasma irradiation on human gingival fibroblasts (HGF). Microplasma irradiation exposure for all HGF samples was limited to 30 s at an irradiation distance of 10 mm with a gas flow of 10 L/min. Three experimental groups were used: a 0 V control group (Control); a 650 V (low) microplasma irradiation group (LV); and a 975 V (high) irradiation group (HV). The following cellular characteristics were evaluated in order to analyze the effects of microplasma treatment; morphology, cell count, DNA content, metabolic activity, cell migration, fibroblast growth factor b (FGF-2) production, type I collagen secretion, and cytotoxic analysis. Cell count, DNA content and FGF-2 production have all been linked to wound healing and, interestingly, both the LV and HV groups showed significant (P \ 0.05) increases in these categories at 72 h after irradiation when compared to the control group. Cytotoxic effects were measured by determining the levels of lactate dehydrogenase, cell death, and DNA damage in HGF cells. In these analyses, the HV and LV groups were not statistically different when compared with the control group at 72 h post-irradiation. These findings suggest that microplasma irradiation activated HGF with no clear cell-damaging effects.
R. Takahashi (&) Y. Numabe Department of Periodontology, The Nippon Dental University School of Life Dentistry at Tokyo, 1-9-20 Fujimi, Chiyoda-ku, Tokyo 102-8159, Japan e-mail: [email protected]; [email protected] K. Shimizu Organization for Innovation and Social Collaboration, Shizuoka University, 3-5-1 Jyouhoku, Hamamatsu 432-8561, Japan e-mail: [email protected]
Keywords Microplasma Human gingival fibroblasts Growth factor Type I collagen Cell migration
Introduction In recent years, interest in the application of plasmas has increased, particularly in medical fields. By definition, plasmas are electrically neutral masses of particles containing approximately equal densities of positively and negatively charged ions. With recent advances in industrial technology, it is now possible to generate stable, micrometer-scale plasma, otherwise known as microplasma [1]. Importantly, microplasma and atmospheric pressure plasma of the character similar to a microplasma appear to be appropriate plasma for biomedical use and have become the primary type utilized in medical research [2–4]. Unlike conventional plasmas, which can only be created under certain conditions, microplasmas can be formed under atmospheric pressure without special gases at relatively low temperatures and voltages. In the field of medicine, these properties have led to their practical use in sterilization of medical implements, as well as to direct application to organisms and/or organic tissues. While research investigating plasma irradiation is ongoing [5–7], favorable effects have been reported for its use in sterilization [8–15], promotion of wound healing [16–18], promotion of cell proliferation [19–23], and blood coagulation [24–26]. These data have fueled higher expectations for additional clinical applications of plasma in medicine. One area of medical plasma research warranting further investigation is the treatment of periodontitis or the chronic inflammation of gingival tissue in the oral cavity resulting from prolonged exposure to dental plaque. Inflammation of
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the periodontal tissues leads to the formation of lesions, which are characterized by equal levels of local tissue destruction and repair. During the process of tissue repair, gingival fibroblasts play an important role [27]. In addition to being the main cell type found in gingival subepithelial connective tissue, they also proliferate during wound healing and play a key functional role in synthesis and secretion of collagen, a component of extracellular matrix important for tissue regeneration [28]. Research has indicated that gingival fibroblast proliferation requires the expression of fibroblast growth factor b (FGF-2). Unlike other growth factors in the FGF family, FGF-2 stimulates the biological activity of fibroblasts as well as various other cell types, such as vascular endothelial and epithelial cells [29–34]. Furthermore, increased levels of fibroblast proliferation and enhanced activation associated with increased expression of FGF-2 have been linked to inflammation and wound healing, and FGF-2 has been utilized clinically for tissue regeneration and repair. Among the studies on microplasma to date, few have investigated cellular irradiation, and even fewer studies have investigated the effect on cells from the oral cavity. In the present study, we irradiated human gingival fibroblasts (HGF) in vitro and then conducted biochemical and morphological analyses to investigate the effects of microplasma on wound healing in these cells.
Social Collaboration and Pulstec Industrial (Shizuoka, Japan). The power source driving the microplasma generator was a pulse wave (frequency 5 kHz; pulse width 1.0 ls). A dielectric barrier film was processed onto both electrodes and the gas flow rate during irradiation was 10 L/min. This configuration enables stable plasma formation in a microgap of 50–60 lm at atmospheric pressure [35– 38]. The irradiation distance, or the distance from the bottom of the plastic Petri dish to the plasma irradiation port, was 10 mm. Irradiation was performed for a period of 30 s using the following voltages: 0 V (non-irradiated control group); 650 V (low-voltage microplasma irradiated (LV) group); and 975 V [high-voltage microplasma irradiated (HV) group]. Evaluation of cell morphology and viability
Materials and methods
Morphological characteristics were observed in cells before, immediately after, and at 72 h after irradiation using a phase-contrast microscope (Diaphoto TMD300 multi-purpose inverted microscope; Nikon, Tokyo, Japan). The effects of microplasma irradiation on cell viability were determined by counting the number of living cells at 24, 48, and 72 h after irradiation. To do this, cells were detached using 0.2 % trypsin/EDTA and collected. Cells were then washed in prepared medium and stained with 0.4 % trypan blue to distinguish between living and dead cells. Living cells were counted on a hemocytometer.
Cell culture
DNA content analysis
In this study, normal human gingival fibroblasts (HGFs; DS Pharma Biomedical, Osaka, Japan) were grown in culture medium containing DMEM/F-12 with 10 % fetal bovine serum (Moregate Biotech, Brisbane, Australia), 50 U/mL penicillin G, 50 lg/mL streptomycin, and 0.25 lg/mL amphotericin B. Cells were cultured in 75 cm2 plastic flasks under a 5 % CO2 at 37 °C and culture medium was exchanged every 3 days. Fourth- to seventh-generation cells were subjected to irradiation and analysis. Cell counts and morphology were assessed before and after microplasma irradiation, and biochemical and cytotoxicity analyses of culture supernatants were performed with cells seeded at an initial concentration of 4,000 cells per 35 mm dish (Iwaki, Tokyo, Japan). The quantity of DNA and the cellular metabolic activity ratio were investigated using cells seeded in 96-well plates (Iwaki, Tokyo, Japan) at an initial concentration of 1,000 cells/well.
DNA contents were quantified using a Quant-iTTM PicoGreenÒ dsDNA Assay Kit (Life Technologies, Carlsbad, CA, USA). A 100-lL sample of isolated cells was mixed with an equal quantity of 0.1 % polyoxyethylene octylphenyl ether for 5 min at room temperature to rupture cell membranes. A 10-lL sample was then taken and reacted with an equal quantity of DNA reaction solution for 5 min at room temperature, followed by fluorometry and quantitation using a Corona Electric Microplate reader SH-9000 (Hitachi High-Technologies, Tokyo, Japan).
Conditions for microplasma irradiation The microplasma generator used was jointly developed by Shizuoka University Organization for Innovation and
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Biochemical analysis The cellular metabolic activity ratio was assessed using Cell Counting Kit-8 (Dojindo Molecular Technologies, Kumamoto, Japan). In brief, 10 ll of tetrazolium salt solution was added to each well containing approximately 1,000 cells, followed by incubation at 37 °C for 2 h. This was followed by colorimetric analysis at a wavelength of 450 nm on a Benchmark Plus (Bio-Rad, Tokyo, Japan). Control group activity was used as the baseline and activity
Odontology Table 1 Cell migration capacity was quantified using NIH Image-J image analysis software and analyzed for each group based on the rate of change in surface area covered between before irradiation and 8 h after Area ratio Cont
1.203 ± 0.028
LV
1.333 ± 0.078
HV
1.421 ± 0.121*
(n = 8; * P \ 0.05)
for each of the irradiated groups was calculated as a ratio against controls. Samples of culture supernatant were recovered before irradiation and at 72 h after irradiation and were used for analysis of FGF-2 and type I collagen production. Ability to produce FGF-2 was assessed via protein analysis using a colorimetric FGF-b enzyme-linked immunosorbent assay (ELISA) kit (Quantikine; R&D Systems, Minneapolis, MN, USA). A microplate reader was used to determine the absorbance at 450 nm. Type I collagen production was assessed using a colorimetric ELISA kit specific for human collagen 1 (ACBio-Technologies, Kanagawa, Japan) and the 450 nm absorbance was also analyzed on a microplate reader. Assessment of cell migration capacity Cell migration was analyzed using the scratch test. In 35 mm plastic petri dishes, HGF was cultured to confluence and a straight line of cells, approximately 1.5 mm in width, was then scraped off each plate using a pipette tip (Iwaki, Tokyo, Japan). Dishes were irradiated under the aforementioned conditions and cell migration into the scraped area from both edges was observed under a phasecontrast microscope. The rate of cell migration was calculated for each group by comparing the surface area covered by cells before irradiation to that 8 h after using NIH Image-J image analysis software (Table 1). Cytotoxicity analysis In order to measure the cytotoxic effects of microplasma irradiation, levels of lactate dehydrogenase (LDH), cell death, and DNA damage were investigated by comparing cell samples from before irradiation and at 72 h after irradiation. LDH levels were assessed in samples of isolated culture supernatant using colorimetric analysis of enzymatic activity with an LDH kit (Kainos, Tokyo, Japan). The cell death ratio was calculated from the proportion of dead cells observed among the total number of cells (living and dead) using 0.4 % trypan blue staining. Finally, the harmful effects of irradiation on DNA were
determined by analyzing DNA base restoration. After three washes in PBS, HGF cells were detached from the culture dish using 0.2 % trypsin, and DNA was then extracted with a QIAampÒ DNA Mini Kit (Qiagen, Venlo, Netherlands). To analyze the DNA samples, a DNA Damage Quantification Kit (Dojindo Molecular Technologies, Kumamoto, Japan) was used according to the manufacturer’s instructions. Briefly, an aliquot of DNA was mixed 1:1 with an equivalent amount of an aldehyde reactive probe (ARP) solution and incubated for 1 h at 37 °C. A 90-lL sample of the ARP-labeled DNA was then mixed with 100 lL of a colorimetric luminescent reagent, and absorbance was measured on a microplate reader. Statistical analysis Groups were compared using the Kruskal–Wallis H test and Mann–Whitney U test with Bonferroni correction using the analytical software SPSS ver. 15.0 J (IBM, Chicago, IL, USA). A 95 % significance threshold (p-values less than 0.05; P \ 0.05) was used to illustrate statistical significance.
Results In this study, HGF cells were subjected to low and high levels of microplasma irradiation (650 and 975 V, respectively) and compared to cells that were not irradiated (0 V). Changes in cell morphology, viability, biochemistry, and migration were then observed and are reported here. Microplasma irradiation does not appear to affect cell morphology A fusiform shape was observed for HGF cells in all groups before and immediately after irradiation, with no clear vacuolization or other changes in morphology. Furthermore, cellular organization was similar across all samples, with similar levels of floating cells after irradiation when compared to non-irradiated samples. At 72 h after irradiation, the HV group and the LV group tended to be higher in a cell count than non-irradiation group. However, the fusiform shape was retained and there were no noteworthy changes in cell morphology (Fig. 1). Cell count and DNA content levels are significantly higher post-irradiation Although cell morphology was not modified by microplasma irradiation, cell viability does appear to be altered. Observation of cell counts at 48 h following irradiation revealed higher numbers of living cells for samples in the
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Odontology Fig. 1 Morphological analysis of human gingival fibroblasts after microplasma irradiation, as compared to non-irradiated cell (control group). The irradiation group was divided into two groups by the difference in an output. They are a high-voltage group (HV group: 975 V) and a low-voltage group (LV group: 650 V). Morphological changes in cells before (BL), immediately after and at 72 h after irradiation were observed under a phase-contrast microscope Scale bar: 100 lm
LV and HV groups when compared to the Control group, but these differences were non-significant. At 72 h after microplasma irradiation, the higher cell count observed in both the LV and HV groups was significantly different from the Control group (Fig. 2a). Furthermore, at 72 h after irradiation, DNA contents were found to be significantly higher (P \ 0.05) in both the LV (333.2 ± 51.8 ng/mL) and HV groups (352.1 ± 55.7 ng/mL), as compared to the Control group (293.9 ± 38.1 ng/mL) (Fig. 2b). Cell biochemistry is significantly altered by microplasma irradiation Biochemical analysis of a cell after treatment is essential to understand the full effects of microplasma. Here, we were particularly interested in the metabolic activity and the presence of two proteins, FGF-2 and type I collagen, at 72 h after microplasma irradiation. At this time point, the cellular metabolic activity ratios for both the LV group (1.18 ± 0.1) and the HV group (1.21 ± 0.15) were found to be significantly higher (P \ 0.05) than that calculated for the Control group (1 ± 0.13) (Fig. 3a). With regard to FGF-2 production, at the 72-h time point, both the LV group (340 ± 61.9 pg/mL) and the HV group (333.9 ± 80.3 pg/mL) were found to have significantly higher (P \ 0.05) amounts of FGF-2 than the Control group (222.6 ± 65 pg/mL) (Fig. 3b). The HV group was found to have a significantly higher (P \ 0.05) amount of type I collagen (17.72 ± 4.54 lg/mL) than the Control
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group (12.32 ± 3.31 lg/mL) at 72 h post-irradiation, and the LV group (16.05 ± 4.31 lg/mL) also exhibited a tendency for higher levels when compared to the non-irradiated Control group (Fig. 3c). Assessment of cellular migration capacity Prior to irradiation, the boundaries between the empty surface and adhered cell regions were clear in all three groups. At 4 and 8 h after irradiation, some HGF migration from the edges of the scraped area was observed in each group (Fig. 4), with the area covered by cells tending to be larger at 8 h after irradiation in all cases. At this 8-h time point, the rate of migration (calculated from the change in surface area covered by cells) was significantly higher (P \ 0.05) in the HV group (1.421 ± 0.121), as compared with the Control group (1.203 ± 0.028) (Table 1). Microplasma irradiation does not show significant cytotoxic effects on HGF cells The cytotoxic effects of microplasma irradiation were determined by investigating three factors: LDH expression, cell death, and DNA damage. At 72 h after irradiation, neither the LV group (41 ± 15.9 IU/mL) nor the HV group (44 ± 13 IU/mL) was found to be significantly different from the Control group (32.3 ± 14.3 IU/mL) with regard to LDH levels (Fig. 5a). Similarly, the cell death ratios for the LV group (22.8 ± 4.85 %) and the HV group (25.6 ± 4.84 %) were not significantly different from the
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Fig. 2 a Prior culture of the cell was carried out at the concentration of 4,000 cells/35 mm dish. Cells were next washed in prepared medium and then stained with 0.4 % trypan blue to distinguish between living and dead cells. Living cell count was performed before and at 24, 48, and 72 h after irradiation (n = 8; * P \ 0.05). b Prior culture of the cell was carried out at the concentration of 1,000 cells/96 mm dish. 10 ll of the collected sample was analyzed using the DNA analysis kit. Quantity of DNA was performed before and 72 h after irradiation (n = 8; * P \ 0.05)
Control group (21.9 ± 8.3 %) (Fig. 5b). Finally, at 72 h after irradiation, the LV group (13 ± 3 sites/100,000 bp) and the HV group (12 ± 3 sites/100,000 bp) showed somewhat higher tendencies for DNA base restoration, used to indicate DNA damage, compared to the Control group (9 ± 3 sites/100,000 bp), but no significant differences were observed (Fig. 5c).
Discussion In the present study, the effects of non-thermal atmospheric-pressure dielectric barrier discharge microplasma irradiation on the biochemistry and morphology of HGF cells were analyzed to determine its potential for clinical application in dental care/treatment and periodontal tissue wound healing.
Fig. 3 Analysis of fibroblast biochemistry after microplasma irradiation. Metabolic activity ratio, FGF-2 production ability, and Type I collagen production ability were analyzed before and 72 h after irradiation. a Metabolic activity ratio was assessed using a Cell Counting Kit-8. Cont group activity was taken as the baseline, and activity in each of the irradiated groups was calculated as a ratio to that baseline. b FGF-2 production ability was assessed via protein analysis by ELISA kit on a microplate reader. c Type I collagen production ability was assessed via protein analysis by ELISA kit on a microplate reader. All experiments had n = 8 and asterisks indicate P \ 0.05
During wound healing, cell proliferation (particularly fibroblast proliferation) and migration not only provide excess cells at the injury site for extracellular matrix excretion (e.g., collagen), but the presence of these cells also
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Odontology Fig. 4 Cell migration capacity was determined by the scratch test, as follows: HGF was cultured to confluence on 35 mm plastic Petri dishes and a straight line of cells approximately 1.5 mm in width was then scraped off with a pipette tip. Dishes were irradiated under each set of conditions, and cell migration into the scraped area from both edges was observed under a phase-contrast microscope. Scrape test induced migration of fibroblasts at 8 h after microplasma irradiation, as observed under a microscope. Scale bar 1 mm
indicates the beginning of the proliferative stage associated with lower levels of tissue inflammation. When using microplasma irradiation at both low voltages and high voltages, we have shown that cell count was increased significantly when compared to the non-irradiated Control group. Similar increases in cell count following microplasma irradiation have been noted in several other reports on non-oral cavity cells, namely vascular endothelial cells and thymic endothelial cells [19, 20]. The increases observed in these studies were larger for cultures subjected to single-pass irradiation than for non-irradiated cultures and, along with the present study, point toward the possible use of microplasma irradiation as a treatment to promote cell proliferation. Importantly, cell proliferation not only involves an increase in cell number, but is also typically accompanied by increased amounts of genetic material and activated cell metabolism. Thus, we also investigated the effects of microplasma on DNA contents and cellular metabolic activity. The significantly higher levels of both components at 72 h after irradiation in both LV and HV samples support the hypothesis that microplasma treatment increases cell proliferation. Another characteristic of fibroblasts essential for wound healing is increased cell migration to the injury. Analysis of cell migration showed significantly higher levels for the HV group when compared to cells that were not treated with microplasma and a similar, although not statistically significant, trend was observed in the LV group. Taken together, these results suggest that microplasma irradiation does in fact act to promote HGF cell proliferation and migration. Furthermore, previous reports have indicated that HGF proliferation is linked to the expression of both FGF-2 and type I collagen [33]. Melcher et al. [27] noted that in the regeneration phase of gingival connective tissue wound
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healing, epithelial, fibroblast, and vascular endothelial cell proliferation is upregulated. This increased cell proliferation along with connective tissue formation lead to wound closure, vascularization, and substrate synthesis. It has also been reported that, in the regenerative phase, higher levels of HGF proliferation and vigorous increases in growth factor and type I collagen levels are strongly related to the promotion of wound healing [31, 32, 34]. Thus, treatment inducing additional increases in proliferation, FGF-2 production, and type I collagen expression would be expected to be beneficial during wound healing. In the present investigation, we found that FGF-2 production was significantly higher in both the LV and the HV groups at 72 h after irradiation. Type I collagen production was also found to be significantly higher in the HV group at 72 h, while a similar but not statistically significant tendency was found in the LV group. These observations indicate that HGF proliferation is related to FGF-2 and type I collagen production and our data suggest that microplasma irradiation potentially stimulates all three, which can then act in an autocrine manner on one another, as well as on neighboring cells, thus promoting wound healing. Although links between fibroblast proliferation, FGF-2, and type I collagen have been established, our study is one of only a few to have investigated the consequences of microplasma irradiation on these factors. Arjunan et al. [22], for example, analyzed the effects of microplasma irradiation on cell count, FGF-2 production, cellular metabolic activity, and cell migration capability of porcine aortic vascular endothelial cells and reported that all of these cellular processes were significantly increased by irradiation. This supports our results for HGF, and both studies suggest a favorable effect of microplasma irradiation on tissue wound healing.
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Fig. 5 Cytotoxicity analysis of fibroblast cells after microplasma irradiation. Cytotoxicity analysis of culture supernatant was performed with cells seeded in 35 mm dish at 4,000 cells/dish. a Cell culture supernatant was assessed for lactate dehydrogenase (LDH) content by colorimetric analysis of enzymatic activity using a lactate dehydrogenase kit. b Cell death ratio was calculated from the proportion of dead cells among the total (living and dead) cell count using 0.4 % trypan blue staining. c The collected cell was analyzed using a DNA damage quantification kit. All experiments had n = 8 and asterisks indicate P \ 0.05
Although microplasma irradiation appears to encourage wound healing, in order to be applied clinically, potential cytotoxic effects or changes in cell morphology or viability
need to be evaluated. Thus, the present study also included an investigation of cytotoxicity in terms of LDH levels, cell death ratio, and amount of DNA damage. At 72 h postirradiation, there were no significant differences between the Control group and the LV or HV groups for any of these processes. This was in agreement with Kalghatgi et al. [19] and Nosenko et al. [20], who previously reported that short-duration irradiation by low-voltage microplasma did not affect dead cell ratio, LDH levels, or the amount of DNA restoration in epithelial and vascular endothelial cells. However, they reported that microplasma irradiation appeared to increase cell death at higher voltages. The present study involved a different cell type under different conditions, and the results at both high and low voltages showed no indication of cytotoxic effects caused by microplasma irradiation. Moreover, in the present study, morphological evaluation of HGF using phase-contrast microscopy before, immediately after, and at 72 h after irradiation showed no notable morphological abnormalities in cell shape, ability to adhere (determined by number of cells floating in the media) or tendency for vacuolar degeneration. These data suggest that microplasma irradiation has little to no harmful effects on biological processes and it may be used for clinical applications. However, the microplasma irradiation environment, chiefly the voltage conditions, should be carefully evaluated before clinical use, as deviations have been shown to be detrimental in both normal [19, 20] and malignant cell types [39–43]. For example, in their investigations on melanoma irradiation by microplasma, Sensenig et al. [39] and Fridman et al. [40] found that irradiation resulted in a significant decrease in cell count over a 24-h time period, beginning at 1 h after irradiation. They also noted that DNA damage and the expression of the apoptosis factor caspase 3 were upregulated at 24 h after microplasma irradiation [42, 43]. We believe that these results, indicating that microplasma irradiation induces cell damage, are presumably attributable in large part to substantial differences in irradiation conditions when compared with the present study. For instance, Sensenig et al. [39] used a microplasma irradiation voltage setting 1.5–2 times as high as that applied in the present study, with an irradiation time of more than 1 min (longer than that used here), leading to cytotoxicity. These results further stress the need to consider proper irradiation conditions before clinical use. In addition, it is necessary to examine of voltage but which grade activated species are generated. The results of the present study indicate that HGF cell activation occurred because an appropriate level of stimulation was utilized, as shown by the microplasma irradiation-induced cell proliferation and increased production of growth factors. We hypothesize that the microplasmainduced HGF activation observed in the present study is
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due to the presence of reactive oxygen species (ROS). Microplasma irradiation ionizes oxygen in the air, thus generating ROS in large quantities. These variable levels of ROS exposure lead to the different effects observed in the irradiated object [23]. Extremely high levels can lead to oxidative stress and ultimately cell death, while sub-lethal levels act alongside the biologically produced ROS to restore cell signaling and homeostasis [21]. Further investigation is necessary to show the specific involvement of ROS in our system. In conclusion, the present results indicate that nonthermal atmospheric-pressure dielectric barrier discharge microplasma irradiation activates HGF with no clear celldamaging effects and suggest that this technique may be used to treat periodontitis and other dental diseases. The findings presented here represent the first step toward clinical application of microplasma in the treatment of oral tissue. Multifaceted assessment of the effects of microplasma irradiation on cells other than HGF, as well as the elucidation of the mechanisms of cell activation, will be necessary in the future. Conflict of interest The authors report no conflicts of interest. The authors alone are responsible for the content and writing of the paper.
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ORIGINAL ARTICLE
Effects of microplasma irradiation on human gingival fibroblasts Ryoichi Takahashi • Kazuo Shimizu Yukihiro Numabe
•
Received: 28 January 2014 / Accepted: 20 April 2014 Ó The Society of The Nippon Dental University 2014
Abstract The purpose of this research was to clarify the effects of microplasma irradiation on human gingival fibroblasts (HGF). Microplasma irradiation exposure for all HGF samples was limited to 30 s at an irradiation distance of 10 mm with a gas flow of 10 L/min. Three experimental groups were used: a 0 V control group (Control); a 650 V (low) microplasma irradiation group (LV); and a 975 V (high) irradiation group (HV). The following cellular characteristics were evaluated in order to analyze the effects of microplasma treatment; morphology, cell count, DNA content, metabolic activity, cell migration, fibroblast growth factor b (FGF-2) production, type I collagen secretion, and cytotoxic analysis. Cell count, DNA content and FGF-2 production have all been linked to wound healing and, interestingly, both the LV and HV groups showed significant (P \ 0.05) increases in these categories at 72 h after irradiation when compared to the control group. Cytotoxic effects were measured by determining the levels of lactate dehydrogenase, cell death, and DNA damage in HGF cells. In these analyses, the HV and LV groups were not statistically different when compared with the control group at 72 h post-irradiation. These findings suggest that microplasma irradiation activated HGF with no clear cell-damaging effects.
R. Takahashi (&) Y. Numabe Department of Periodontology, The Nippon Dental University School of Life Dentistry at Tokyo, 1-9-20 Fujimi, Chiyoda-ku, Tokyo 102-8159, Japan e-mail: [email protected]; [email protected] K. Shimizu Organization for Innovation and Social Collaboration, Shizuoka University, 3-5-1 Jyouhoku, Hamamatsu 432-8561, Japan e-mail: [email protected]
Keywords Microplasma Human gingival fibroblasts Growth factor Type I collagen Cell migration
Introduction In recent years, interest in the application of plasmas has increased, particularly in medical fields. By definition, plasmas are electrically neutral masses of particles containing approximately equal densities of positively and negatively charged ions. With recent advances in industrial technology, it is now possible to generate stable, micrometer-scale plasma, otherwise known as microplasma [1]. Importantly, microplasma and atmospheric pressure plasma of the character similar to a microplasma appear to be appropriate plasma for biomedical use and have become the primary type utilized in medical research [2–4]. Unlike conventional plasmas, which can only be created under certain conditions, microplasmas can be formed under atmospheric pressure without special gases at relatively low temperatures and voltages. In the field of medicine, these properties have led to their practical use in sterilization of medical implements, as well as to direct application to organisms and/or organic tissues. While research investigating plasma irradiation is ongoing [5–7], favorable effects have been reported for its use in sterilization [8–15], promotion of wound healing [16–18], promotion of cell proliferation [19–23], and blood coagulation [24–26]. These data have fueled higher expectations for additional clinical applications of plasma in medicine. One area of medical plasma research warranting further investigation is the treatment of periodontitis or the chronic inflammation of gingival tissue in the oral cavity resulting from prolonged exposure to dental plaque. Inflammation of
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the periodontal tissues leads to the formation of lesions, which are characterized by equal levels of local tissue destruction and repair. During the process of tissue repair, gingival fibroblasts play an important role [27]. In addition to being the main cell type found in gingival subepithelial connective tissue, they also proliferate during wound healing and play a key functional role in synthesis and secretion of collagen, a component of extracellular matrix important for tissue regeneration [28]. Research has indicated that gingival fibroblast proliferation requires the expression of fibroblast growth factor b (FGF-2). Unlike other growth factors in the FGF family, FGF-2 stimulates the biological activity of fibroblasts as well as various other cell types, such as vascular endothelial and epithelial cells [29–34]. Furthermore, increased levels of fibroblast proliferation and enhanced activation associated with increased expression of FGF-2 have been linked to inflammation and wound healing, and FGF-2 has been utilized clinically for tissue regeneration and repair. Among the studies on microplasma to date, few have investigated cellular irradiation, and even fewer studies have investigated the effect on cells from the oral cavity. In the present study, we irradiated human gingival fibroblasts (HGF) in vitro and then conducted biochemical and morphological analyses to investigate the effects of microplasma on wound healing in these cells.
Social Collaboration and Pulstec Industrial (Shizuoka, Japan). The power source driving the microplasma generator was a pulse wave (frequency 5 kHz; pulse width 1.0 ls). A dielectric barrier film was processed onto both electrodes and the gas flow rate during irradiation was 10 L/min. This configuration enables stable plasma formation in a microgap of 50–60 lm at atmospheric pressure [35– 38]. The irradiation distance, or the distance from the bottom of the plastic Petri dish to the plasma irradiation port, was 10 mm. Irradiation was performed for a period of 30 s using the following voltages: 0 V (non-irradiated control group); 650 V (low-voltage microplasma irradiated (LV) group); and 975 V [high-voltage microplasma irradiated (HV) group]. Evaluation of cell morphology and viability
Materials and methods
Morphological characteristics were observed in cells before, immediately after, and at 72 h after irradiation using a phase-contrast microscope (Diaphoto TMD300 multi-purpose inverted microscope; Nikon, Tokyo, Japan). The effects of microplasma irradiation on cell viability were determined by counting the number of living cells at 24, 48, and 72 h after irradiation. To do this, cells were detached using 0.2 % trypsin/EDTA and collected. Cells were then washed in prepared medium and stained with 0.4 % trypan blue to distinguish between living and dead cells. Living cells were counted on a hemocytometer.
Cell culture
DNA content analysis
In this study, normal human gingival fibroblasts (HGFs; DS Pharma Biomedical, Osaka, Japan) were grown in culture medium containing DMEM/F-12 with 10 % fetal bovine serum (Moregate Biotech, Brisbane, Australia), 50 U/mL penicillin G, 50 lg/mL streptomycin, and 0.25 lg/mL amphotericin B. Cells were cultured in 75 cm2 plastic flasks under a 5 % CO2 at 37 °C and culture medium was exchanged every 3 days. Fourth- to seventh-generation cells were subjected to irradiation and analysis. Cell counts and morphology were assessed before and after microplasma irradiation, and biochemical and cytotoxicity analyses of culture supernatants were performed with cells seeded at an initial concentration of 4,000 cells per 35 mm dish (Iwaki, Tokyo, Japan). The quantity of DNA and the cellular metabolic activity ratio were investigated using cells seeded in 96-well plates (Iwaki, Tokyo, Japan) at an initial concentration of 1,000 cells/well.
DNA contents were quantified using a Quant-iTTM PicoGreenÒ dsDNA Assay Kit (Life Technologies, Carlsbad, CA, USA). A 100-lL sample of isolated cells was mixed with an equal quantity of 0.1 % polyoxyethylene octylphenyl ether for 5 min at room temperature to rupture cell membranes. A 10-lL sample was then taken and reacted with an equal quantity of DNA reaction solution for 5 min at room temperature, followed by fluorometry and quantitation using a Corona Electric Microplate reader SH-9000 (Hitachi High-Technologies, Tokyo, Japan).
Conditions for microplasma irradiation The microplasma generator used was jointly developed by Shizuoka University Organization for Innovation and
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Biochemical analysis The cellular metabolic activity ratio was assessed using Cell Counting Kit-8 (Dojindo Molecular Technologies, Kumamoto, Japan). In brief, 10 ll of tetrazolium salt solution was added to each well containing approximately 1,000 cells, followed by incubation at 37 °C for 2 h. This was followed by colorimetric analysis at a wavelength of 450 nm on a Benchmark Plus (Bio-Rad, Tokyo, Japan). Control group activity was used as the baseline and activity
Odontology Table 1 Cell migration capacity was quantified using NIH Image-J image analysis software and analyzed for each group based on the rate of change in surface area covered between before irradiation and 8 h after Area ratio Cont
1.203 ± 0.028
LV
1.333 ± 0.078
HV
1.421 ± 0.121*
(n = 8; * P \ 0.05)
for each of the irradiated groups was calculated as a ratio against controls. Samples of culture supernatant were recovered before irradiation and at 72 h after irradiation and were used for analysis of FGF-2 and type I collagen production. Ability to produce FGF-2 was assessed via protein analysis using a colorimetric FGF-b enzyme-linked immunosorbent assay (ELISA) kit (Quantikine; R&D Systems, Minneapolis, MN, USA). A microplate reader was used to determine the absorbance at 450 nm. Type I collagen production was assessed using a colorimetric ELISA kit specific for human collagen 1 (ACBio-Technologies, Kanagawa, Japan) and the 450 nm absorbance was also analyzed on a microplate reader. Assessment of cell migration capacity Cell migration was analyzed using the scratch test. In 35 mm plastic petri dishes, HGF was cultured to confluence and a straight line of cells, approximately 1.5 mm in width, was then scraped off each plate using a pipette tip (Iwaki, Tokyo, Japan). Dishes were irradiated under the aforementioned conditions and cell migration into the scraped area from both edges was observed under a phasecontrast microscope. The rate of cell migration was calculated for each group by comparing the surface area covered by cells before irradiation to that 8 h after using NIH Image-J image analysis software (Table 1). Cytotoxicity analysis In order to measure the cytotoxic effects of microplasma irradiation, levels of lactate dehydrogenase (LDH), cell death, and DNA damage were investigated by comparing cell samples from before irradiation and at 72 h after irradiation. LDH levels were assessed in samples of isolated culture supernatant using colorimetric analysis of enzymatic activity with an LDH kit (Kainos, Tokyo, Japan). The cell death ratio was calculated from the proportion of dead cells observed among the total number of cells (living and dead) using 0.4 % trypan blue staining. Finally, the harmful effects of irradiation on DNA were
determined by analyzing DNA base restoration. After three washes in PBS, HGF cells were detached from the culture dish using 0.2 % trypsin, and DNA was then extracted with a QIAampÒ DNA Mini Kit (Qiagen, Venlo, Netherlands). To analyze the DNA samples, a DNA Damage Quantification Kit (Dojindo Molecular Technologies, Kumamoto, Japan) was used according to the manufacturer’s instructions. Briefly, an aliquot of DNA was mixed 1:1 with an equivalent amount of an aldehyde reactive probe (ARP) solution and incubated for 1 h at 37 °C. A 90-lL sample of the ARP-labeled DNA was then mixed with 100 lL of a colorimetric luminescent reagent, and absorbance was measured on a microplate reader. Statistical analysis Groups were compared using the Kruskal–Wallis H test and Mann–Whitney U test with Bonferroni correction using the analytical software SPSS ver. 15.0 J (IBM, Chicago, IL, USA). A 95 % significance threshold (p-values less than 0.05; P \ 0.05) was used to illustrate statistical significance.
Results In this study, HGF cells were subjected to low and high levels of microplasma irradiation (650 and 975 V, respectively) and compared to cells that were not irradiated (0 V). Changes in cell morphology, viability, biochemistry, and migration were then observed and are reported here. Microplasma irradiation does not appear to affect cell morphology A fusiform shape was observed for HGF cells in all groups before and immediately after irradiation, with no clear vacuolization or other changes in morphology. Furthermore, cellular organization was similar across all samples, with similar levels of floating cells after irradiation when compared to non-irradiated samples. At 72 h after irradiation, the HV group and the LV group tended to be higher in a cell count than non-irradiation group. However, the fusiform shape was retained and there were no noteworthy changes in cell morphology (Fig. 1). Cell count and DNA content levels are significantly higher post-irradiation Although cell morphology was not modified by microplasma irradiation, cell viability does appear to be altered. Observation of cell counts at 48 h following irradiation revealed higher numbers of living cells for samples in the
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Odontology Fig. 1 Morphological analysis of human gingival fibroblasts after microplasma irradiation, as compared to non-irradiated cell (control group). The irradiation group was divided into two groups by the difference in an output. They are a high-voltage group (HV group: 975 V) and a low-voltage group (LV group: 650 V). Morphological changes in cells before (BL), immediately after and at 72 h after irradiation were observed under a phase-contrast microscope Scale bar: 100 lm
LV and HV groups when compared to the Control group, but these differences were non-significant. At 72 h after microplasma irradiation, the higher cell count observed in both the LV and HV groups was significantly different from the Control group (Fig. 2a). Furthermore, at 72 h after irradiation, DNA contents were found to be significantly higher (P \ 0.05) in both the LV (333.2 ± 51.8 ng/mL) and HV groups (352.1 ± 55.7 ng/mL), as compared to the Control group (293.9 ± 38.1 ng/mL) (Fig. 2b). Cell biochemistry is significantly altered by microplasma irradiation Biochemical analysis of a cell after treatment is essential to understand the full effects of microplasma. Here, we were particularly interested in the metabolic activity and the presence of two proteins, FGF-2 and type I collagen, at 72 h after microplasma irradiation. At this time point, the cellular metabolic activity ratios for both the LV group (1.18 ± 0.1) and the HV group (1.21 ± 0.15) were found to be significantly higher (P \ 0.05) than that calculated for the Control group (1 ± 0.13) (Fig. 3a). With regard to FGF-2 production, at the 72-h time point, both the LV group (340 ± 61.9 pg/mL) and the HV group (333.9 ± 80.3 pg/mL) were found to have significantly higher (P \ 0.05) amounts of FGF-2 than the Control group (222.6 ± 65 pg/mL) (Fig. 3b). The HV group was found to have a significantly higher (P \ 0.05) amount of type I collagen (17.72 ± 4.54 lg/mL) than the Control
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group (12.32 ± 3.31 lg/mL) at 72 h post-irradiation, and the LV group (16.05 ± 4.31 lg/mL) also exhibited a tendency for higher levels when compared to the non-irradiated Control group (Fig. 3c). Assessment of cellular migration capacity Prior to irradiation, the boundaries between the empty surface and adhered cell regions were clear in all three groups. At 4 and 8 h after irradiation, some HGF migration from the edges of the scraped area was observed in each group (Fig. 4), with the area covered by cells tending to be larger at 8 h after irradiation in all cases. At this 8-h time point, the rate of migration (calculated from the change in surface area covered by cells) was significantly higher (P \ 0.05) in the HV group (1.421 ± 0.121), as compared with the Control group (1.203 ± 0.028) (Table 1). Microplasma irradiation does not show significant cytotoxic effects on HGF cells The cytotoxic effects of microplasma irradiation were determined by investigating three factors: LDH expression, cell death, and DNA damage. At 72 h after irradiation, neither the LV group (41 ± 15.9 IU/mL) nor the HV group (44 ± 13 IU/mL) was found to be significantly different from the Control group (32.3 ± 14.3 IU/mL) with regard to LDH levels (Fig. 5a). Similarly, the cell death ratios for the LV group (22.8 ± 4.85 %) and the HV group (25.6 ± 4.84 %) were not significantly different from the
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Fig. 2 a Prior culture of the cell was carried out at the concentration of 4,000 cells/35 mm dish. Cells were next washed in prepared medium and then stained with 0.4 % trypan blue to distinguish between living and dead cells. Living cell count was performed before and at 24, 48, and 72 h after irradiation (n = 8; * P \ 0.05). b Prior culture of the cell was carried out at the concentration of 1,000 cells/96 mm dish. 10 ll of the collected sample was analyzed using the DNA analysis kit. Quantity of DNA was performed before and 72 h after irradiation (n = 8; * P \ 0.05)
Control group (21.9 ± 8.3 %) (Fig. 5b). Finally, at 72 h after irradiation, the LV group (13 ± 3 sites/100,000 bp) and the HV group (12 ± 3 sites/100,000 bp) showed somewhat higher tendencies for DNA base restoration, used to indicate DNA damage, compared to the Control group (9 ± 3 sites/100,000 bp), but no significant differences were observed (Fig. 5c).
Discussion In the present study, the effects of non-thermal atmospheric-pressure dielectric barrier discharge microplasma irradiation on the biochemistry and morphology of HGF cells were analyzed to determine its potential for clinical application in dental care/treatment and periodontal tissue wound healing.
Fig. 3 Analysis of fibroblast biochemistry after microplasma irradiation. Metabolic activity ratio, FGF-2 production ability, and Type I collagen production ability were analyzed before and 72 h after irradiation. a Metabolic activity ratio was assessed using a Cell Counting Kit-8. Cont group activity was taken as the baseline, and activity in each of the irradiated groups was calculated as a ratio to that baseline. b FGF-2 production ability was assessed via protein analysis by ELISA kit on a microplate reader. c Type I collagen production ability was assessed via protein analysis by ELISA kit on a microplate reader. All experiments had n = 8 and asterisks indicate P \ 0.05
During wound healing, cell proliferation (particularly fibroblast proliferation) and migration not only provide excess cells at the injury site for extracellular matrix excretion (e.g., collagen), but the presence of these cells also
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Odontology Fig. 4 Cell migration capacity was determined by the scratch test, as follows: HGF was cultured to confluence on 35 mm plastic Petri dishes and a straight line of cells approximately 1.5 mm in width was then scraped off with a pipette tip. Dishes were irradiated under each set of conditions, and cell migration into the scraped area from both edges was observed under a phase-contrast microscope. Scrape test induced migration of fibroblasts at 8 h after microplasma irradiation, as observed under a microscope. Scale bar 1 mm
indicates the beginning of the proliferative stage associated with lower levels of tissue inflammation. When using microplasma irradiation at both low voltages and high voltages, we have shown that cell count was increased significantly when compared to the non-irradiated Control group. Similar increases in cell count following microplasma irradiation have been noted in several other reports on non-oral cavity cells, namely vascular endothelial cells and thymic endothelial cells [19, 20]. The increases observed in these studies were larger for cultures subjected to single-pass irradiation than for non-irradiated cultures and, along with the present study, point toward the possible use of microplasma irradiation as a treatment to promote cell proliferation. Importantly, cell proliferation not only involves an increase in cell number, but is also typically accompanied by increased amounts of genetic material and activated cell metabolism. Thus, we also investigated the effects of microplasma on DNA contents and cellular metabolic activity. The significantly higher levels of both components at 72 h after irradiation in both LV and HV samples support the hypothesis that microplasma treatment increases cell proliferation. Another characteristic of fibroblasts essential for wound healing is increased cell migration to the injury. Analysis of cell migration showed significantly higher levels for the HV group when compared to cells that were not treated with microplasma and a similar, although not statistically significant, trend was observed in the LV group. Taken together, these results suggest that microplasma irradiation does in fact act to promote HGF cell proliferation and migration. Furthermore, previous reports have indicated that HGF proliferation is linked to the expression of both FGF-2 and type I collagen [33]. Melcher et al. [27] noted that in the regeneration phase of gingival connective tissue wound
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healing, epithelial, fibroblast, and vascular endothelial cell proliferation is upregulated. This increased cell proliferation along with connective tissue formation lead to wound closure, vascularization, and substrate synthesis. It has also been reported that, in the regenerative phase, higher levels of HGF proliferation and vigorous increases in growth factor and type I collagen levels are strongly related to the promotion of wound healing [31, 32, 34]. Thus, treatment inducing additional increases in proliferation, FGF-2 production, and type I collagen expression would be expected to be beneficial during wound healing. In the present investigation, we found that FGF-2 production was significantly higher in both the LV and the HV groups at 72 h after irradiation. Type I collagen production was also found to be significantly higher in the HV group at 72 h, while a similar but not statistically significant tendency was found in the LV group. These observations indicate that HGF proliferation is related to FGF-2 and type I collagen production and our data suggest that microplasma irradiation potentially stimulates all three, which can then act in an autocrine manner on one another, as well as on neighboring cells, thus promoting wound healing. Although links between fibroblast proliferation, FGF-2, and type I collagen have been established, our study is one of only a few to have investigated the consequences of microplasma irradiation on these factors. Arjunan et al. [22], for example, analyzed the effects of microplasma irradiation on cell count, FGF-2 production, cellular metabolic activity, and cell migration capability of porcine aortic vascular endothelial cells and reported that all of these cellular processes were significantly increased by irradiation. This supports our results for HGF, and both studies suggest a favorable effect of microplasma irradiation on tissue wound healing.
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Fig. 5 Cytotoxicity analysis of fibroblast cells after microplasma irradiation. Cytotoxicity analysis of culture supernatant was performed with cells seeded in 35 mm dish at 4,000 cells/dish. a Cell culture supernatant was assessed for lactate dehydrogenase (LDH) content by colorimetric analysis of enzymatic activity using a lactate dehydrogenase kit. b Cell death ratio was calculated from the proportion of dead cells among the total (living and dead) cell count using 0.4 % trypan blue staining. c The collected cell was analyzed using a DNA damage quantification kit. All experiments had n = 8 and asterisks indicate P \ 0.05
Although microplasma irradiation appears to encourage wound healing, in order to be applied clinically, potential cytotoxic effects or changes in cell morphology or viability
need to be evaluated. Thus, the present study also included an investigation of cytotoxicity in terms of LDH levels, cell death ratio, and amount of DNA damage. At 72 h postirradiation, there were no significant differences between the Control group and the LV or HV groups for any of these processes. This was in agreement with Kalghatgi et al. [19] and Nosenko et al. [20], who previously reported that short-duration irradiation by low-voltage microplasma did not affect dead cell ratio, LDH levels, or the amount of DNA restoration in epithelial and vascular endothelial cells. However, they reported that microplasma irradiation appeared to increase cell death at higher voltages. The present study involved a different cell type under different conditions, and the results at both high and low voltages showed no indication of cytotoxic effects caused by microplasma irradiation. Moreover, in the present study, morphological evaluation of HGF using phase-contrast microscopy before, immediately after, and at 72 h after irradiation showed no notable morphological abnormalities in cell shape, ability to adhere (determined by number of cells floating in the media) or tendency for vacuolar degeneration. These data suggest that microplasma irradiation has little to no harmful effects on biological processes and it may be used for clinical applications. However, the microplasma irradiation environment, chiefly the voltage conditions, should be carefully evaluated before clinical use, as deviations have been shown to be detrimental in both normal [19, 20] and malignant cell types [39–43]. For example, in their investigations on melanoma irradiation by microplasma, Sensenig et al. [39] and Fridman et al. [40] found that irradiation resulted in a significant decrease in cell count over a 24-h time period, beginning at 1 h after irradiation. They also noted that DNA damage and the expression of the apoptosis factor caspase 3 were upregulated at 24 h after microplasma irradiation [42, 43]. We believe that these results, indicating that microplasma irradiation induces cell damage, are presumably attributable in large part to substantial differences in irradiation conditions when compared with the present study. For instance, Sensenig et al. [39] used a microplasma irradiation voltage setting 1.5–2 times as high as that applied in the present study, with an irradiation time of more than 1 min (longer than that used here), leading to cytotoxicity. These results further stress the need to consider proper irradiation conditions before clinical use. In addition, it is necessary to examine of voltage but which grade activated species are generated. The results of the present study indicate that HGF cell activation occurred because an appropriate level of stimulation was utilized, as shown by the microplasma irradiation-induced cell proliferation and increased production of growth factors. We hypothesize that the microplasmainduced HGF activation observed in the present study is
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due to the presence of reactive oxygen species (ROS). Microplasma irradiation ionizes oxygen in the air, thus generating ROS in large quantities. These variable levels of ROS exposure lead to the different effects observed in the irradiated object [23]. Extremely high levels can lead to oxidative stress and ultimately cell death, while sub-lethal levels act alongside the biologically produced ROS to restore cell signaling and homeostasis [21]. Further investigation is necessary to show the specific involvement of ROS in our system. In conclusion, the present results indicate that nonthermal atmospheric-pressure dielectric barrier discharge microplasma irradiation activates HGF with no clear celldamaging effects and suggest that this technique may be used to treat periodontitis and other dental diseases. The findings presented here represent the first step toward clinical application of microplasma in the treatment of oral tissue. Multifaceted assessment of the effects of microplasma irradiation on cells other than HGF, as well as the elucidation of the mechanisms of cell activation, will be necessary in the future. Conflict of interest The authors report no conflicts of interest. The authors alone are responsible for the content and writing of the paper.
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