Effect of An Atmospheric Plasma Jet on the Differentiation of Melanoblast Progenitor.

OBJECTIVE: Melanoblasts are the cell source of regeneration for pigment restoration. The ability to differentiate into mature melanocytes is the essential feature of melanoblasts in depigmentation diseases. Cold atmospheric plasma is an ionized gas with near-room temperature and highly reactive species that has been shown to induce stem cell differentiation. The aim of the study was to explore the effect of cold atmospheric plasma on the differentiation of melanoblast progenitor cells. METHODS: In this study, melanoblasts were exposed to the plasma jet and the cell morphology was observed. The cell cycle and cell proliferation were detected. Furthermore, the cell immunofluorescence and the detection of melanin particle and nitric oxide were carried out to investigate the differentiation of melanoblast progenitor cells. RESULTS: Cells that were treated with the plasma had longer and more synaptic structures, and the G1 phase of cell cycle was prolonged in the treated group. More melanin synthesis-related proteins and melanin particles were produced after plasma treatment. Nitric oxide was one of the active components generated by the plasma jet, and the nitric oxide content in the cell culture medium of the treated group increased. CONCLUSION: These results indicate that an increase in nitric oxide production caused by a plasma jet can promote cell differentiation. The application of plasma provides an innovative strategy for the treatment of depigmentation diseases.

Nerve Stem Cell Differentiation by a One-step Cold Atmospheric Plasma Treatment In Vitro.

As the development of physical plasma technology, cold atmospheric plasmas (CAPs) have been widely investigated in decontamination, cancer treatment, wound healing, root canal treatment, etc., forming a new research field named plasma medicine. Being a mixture of electrical, chemical, and biological reactive species, CAPs have shown their abilities to enhance nerve stem cells differentiation both in vitro and in vivo and are becoming a promising way for neurological disease treatment in the future. The much more exciting news is that using CAPs may realize one-step, and safely directed, differentiation of neural stem cells (NSCs) for tissue transportation. We demonstrate here the detailed experimental protocol of using a self-made CAP jet device to enhance NSC differentiation in C17.2-NSCs and primary rat neural stem cells, as well as observing the cell fate by inverted and fluorescence microscopy. With the help of immunofluorescence staining technology, we found both the NSCs showed an accelerated differential rate than the untreated group, and ~75% of the NSCs selectively differentiated into neurons, which are mainly mature, cholinergic, and motor neurons.

Cold Atmospheric Plasmas: A Novel and Promising Way to Treat Neurological Diseases.

Cold atmospheric plasmas (CAPs) can enhance neural cell differentiation into neurons both in vitro and in vivo, which is of great interest for medical treatment of neurodegenerative diseases like Alzheimer's disease and traumatic injuries of the central nervous system. CAPs represent a promising method for future neurological disease therapy.

Effects of non-equilibrium plasma in the treatment of ligature-induced peri-implantitis.

AIM: To evaluate the effects of non-equilibrium plasma in the treatment of ligature-induced peri-implantitis in beagle dogs. MATERIALS AND METHODS: Six beagles received 12 implants installed in the position of the fourth mandibular premolars. Ligature-induced peri-implantitis was initiated at 3 months post-implantation. When approximately 40% of the supporting bone was lost, the ligatures were removed. The implants were subjected to the muco-periosteal scaling and chlorhexidine irrigation with or without plasma irrigation. Three months later, clinical, radiographic and microbiological analyses were performed. Block biopsies were prepared for micro-CT and histomorphometric analysis. The primary outcome was the difference in bone healing of peri-implant sites, and the secondary outcomes included changes in clinical parameters (SBI, PD) and bacterial detection. RESULTS: At baseline, no significant differences were observed between the two groups. At 3 months post-treatment, the plasma group showed a significantly higher bone level than the control group (p < 0.05), a significantly decreased detection of bacteria (Porphyromonas gingivalis and Tannerella forsythia) (p < 0.05), and a significant improvement in clinical examination (p < 0.05). CONCLUSIONS: Within the limits of this study, non-equilibrium plasma treatment as an adjunct to the conventional therapy is a feasible approach for the treatment of peri-implantitis.

Effect of modified nonequilibrium plasma with chlorhexidine digluconate against endodontic biofilms in vitro.

INTRODUCTION: Nonequilibrium plasma has been reported to effectively kill Enterococcus faecalis in endodontic biofilm compared with chlorhexidine digluconate (CHX). The purpose of this study was to evaluate the antimicrobial in vitro activity of modified nonequilibrium plasma with CHX against E. faecalis and multispecies biofilms on bovine dentin discs. METHODS: Sterile bovine dentin discs were incubated with E. faecalis or a mixture of bacteria from human dental root canal infections to form 1- and 3-week-old biofilms. The specimens were subjected to nonequilibrium plasma, modified nonequilibrium plasma with CHX, and 2% CHX for 2- and 5-minute exposure. After treatment, the biofilms were stained with viability dyes and examined by confocal laser scanning microscopy and 3-dimensional reconstruction analysis. The proportions of bacterial cells killed by the treatments were calculated. RESULTS: The 3-dimensional reconstruction images showed that 1- and 3-week-old biofilms adhered to bovine dentin discs. The proportions of dead cells increased significantly with the longer exposure in each treatment group (P < .05). Modified nonequilibrium plasma was the most effective in killing bacteria in E. faecalis and multispecies biofilms at both 2 and 5 minutes (P < .05). No significant difference was detected between nonequilibrium plasma and CHX groups (P > .05). Significantly more cells were killed in 1-week-old biofilms than in 3-week-old biofilms in all groups (P < .05). CONCLUSIONS: The modified nonequilibrium plasma killed more bacteria than conventional nonequilibrium plasma and 2% CHX in E. faecalis and multispecies endodontic biofilms in vitro and thus shows promise as an additional tool in infection control during endodontic treatment.

Atmospheric pressure room temperature plasma jets facilitate oxidative and nitrative stress and lead to endoplasmic reticulum stress dependent apoptosis in HepG2 cells.

Atmospheric pressure room temperature plasma jets (APRTP-Js) that can emit a mixture of different active species have recently found entry in various medical applications. Apoptosis is a key event in APRTP-Js-induced cellular toxicity, but the exact biological mechanisms underlying remain elusive. Here, we explored the role of reactive oxygen species (ROS) and reactive nitrogen species (RNS) in APRTP-Js-induced apoptosis using in vitro model of HepG2 cells. We found that APRTP-Js facilitated the accumulation of ROS and RNS in cells, which resulted in the compromised cellular antioxidant defense system, as evidenced by the inactivation of cellular antioxidants including glutathione (GSH), superoxide dismutase (SOD) and catalase. Nitrotyrosine and protein carbonyl content analysis indicated that APRTP-Js treatment caused nitrative and oxidative injury of cells. Meanwhile, intracellular calcium homeostasis was disturbed along with the alteration in the expressions of GRP78, CHOP and pro-caspase12. These effects accumulated and eventually culminated into the cellular dysfunction and endoplasmic reticulum stress (ER stress)-mediated apoptosis. The apoptosis could be markedly attenuated by N-acetylcysteine (NAC, a free radical scavenger), which confirmed the involvement of oxidative and nitrative stress in the process leading to HepG2 cell apoptosis by APRTP-Js treatment.

Evaluation of antibacterial effects by atmospheric pressure nonequilibrium plasmas against Enterococcus faecalis biofilms in vitro.

INTRODUCTION: The aim of this study was to evaluate the in vitro antibacterial activity by atmospheric pressure nonequilibrium plasmas (APNPs) as an effective approach against bacterial biofilms in root canal systems during endodontic therapy. METHODS: Sterile cover slips were placed into the wells of tissue culture plates to permit the formation of Enterococcus faecalis biofilms. Biofilms were treated for 5 minutes with APNPs or 2% chlorhexidine digluconate (CHX). The viability of biofilm bacteria was analyzed by staining and confocal laser scanning microscopy. In addition, infected single-rooted teeth were exposed to APNPs or 2% CHX for 5, 10, and 15 minutes. After treatment, the root canals were flushed, and the resulting suspensions were inoculated onto brain-heart infusion agar to assess bacterial survival. Finally, micro-computed tomography scanning was used to observe and verify the root canal systems relative to the antibacterial effects obtained. RESULTS: Treatment for 5 minutes with APNPs or 2% CHX killed the majority of bacteria in the E. faecalis biofilms. Moreover, APNP treatment was as effective as 2% CHX for inactivating bacteria in infected root canals (P > .05). Bacterial survival after treatment with APNPs or 2% CHX remarkably reduced with increasing exposure times (P < .05). There was no significant difference between bacterial survival in complex root canal systems and simple straight canals (P > .05). CONCLUSIONS: APNPs can be an effective adjunct to standard endodontic antimicrobial treatment.

Bacterial-killing effect of atmospheric pressure non-equilibrium plasma jet and oral mucosa response.

Recently, plasma sterilization has attracted increasing attention in dental community for the atmospheric pressure non-equilibrium plasma jet (APNPs), which is driven by a kilohertz pulsed DC power, may be applied to the dental and oral diseases. However, it is still in doubt whether APNPs can effectively kill pathogenic bacteria in the oral cavity and produce no harmful effects on normal oral tissues, especially on normal mucosa. The aim of this study was to evaluate the bacterial-killing effect of APNPs in the biofilms containing a single breed of bacteria (Porphyromonas gingivalis, P.g.), and the pathological changes of the oral mucosa after treatment by APNPs. P.g. was incubated to form the biofilms in vitro, and the samples were divided into three groups randomly: group A (blank control); group B in which the biofilms were treated by APNPs (the setting of the equipment: 10 kHz, 1600 ns and 8 kV); group C in which the biofilms were exposed only to a gas jet without ignition of the plasma. Each group had three samples and each sample was processed for up to 5 min. The biofilms were then fluorescently stained, observed and photographed under a laser scanning confocal microscope. In the animal experiment, six male Japanese white rabbits were divided into two groups randomly (n=3 in each group) in terms of the different post-treatment time (1-day group and 5-day group). The buccal mucosa of the left side and the mucosa of the ventral surface of the tongue were treated by APNPs for 10 min in the same way as the bacterial biofilm experiment in each rabbit, and the corresponding mucosa of the other sides served as normal control. The clinical manifestations of the oral mucosa were observed and recorded every day. The rabbits were sacrificed one or five day(s) after APNPs treatment. The oral mucosa were harvested and prepared to haematoxylin and eosin-stained sections. Clinical observation and histopathological scores were used to assess mucosal changes. The results showed the obvious P.g. biofilms were formed at 10 days, and most of the bacteria in groups A and C were alive under a laser scanning confocal microscope, but the bacteria in the group B were almost all dead. In animal experiment, no ulcers, anabrosis and oral mucositis were found in both the 1-day and 5-day groups. The average mucous membrane irritation index was -0.83 and -0.67 in the 1-day and 5-day groups, respectively, suggesting that no intense mucosal membrane irritation responses occurred. It was concluded that APNPs could effectively kill P.g. in the biofilms and did not cause any pathological changes in the normal mucosa, suggesting that the plasma jet (APNPs) may be applied to oral diseases as a novel sterilization device in the future.

Bacterial-killing Effect of Atmospheric Pressure Non-equilibrium Plasma Jet and Oral Mucosa Response / 华中科技大学学报（医学）（英德文版）

Recently,plasma sterilization has attracted increasing attention in dental community for the atmospheric pressure non-equilibrium plasma jet (APNPs),which is driven by a kilohertz pulsed DC power,may be applied to the dental and oral diseases.However,it is still in doubt whether APNPs can effectively kill pathogenic bacteria in the oral cavity and produce no harmful effects on normal oral tissues,especially on normal mucosa.The aim of this study was to evaluate the bacterial-killing effect of APNPs in the biofilms containing a single breed of bacteria (Porphyromonas gingivalis,Pg.),and the pathological changes of the oral mucosa after treatment by APNPs.Pg.was incubated to form the biofilms in vitro,and the samples were divided into three groups randomly:group A (blank control);group B in which the biofilms were treated by APNPs (the setting of the equipment:10 kHz,1600 ns and 8 kV); group C in which the biofilms were exposed only to a gas jet without ignition of the plasma.Each group had three samples and each sample was processed for up to 5 min.The biofilms were then fluorescently stained,observed and photographed under a laser scanning confocal microscope.In the animal experiment,six male Japanese white rabbits were divided into two groups randomly (n=3 in each group) in terms of the different post-treatment time (1-day group and 5-day group).The buccal mucosa of the left side and the mucosa of the ventral surface of the tongue were treated by APNPs for 10 min in the same way as the bacterial biofilm experiment in each rabbit,and the corresponding mucosa of the other sides served as normal control.The clinical manifestations of the oral mucosa were observed and recorded every day.The rabbits were sacrificed one or five day(s) after APNPs treatment.The oral mucosa were harvested and prepared to haematoxylin and eosin-stained sections.Clinical observation and histopathological scores were used to assess mucosal changes.The results showed the obvious P.g.biofilms were formed at 10 days,and most of the bacteria in groups A and C were alive under a laser scanning confocal microscope,but the bacteria in the group B were almost all dead.In animal experiment,no ulcers,anabrosis and oral mucositis were found in both the 1-day and 5-day groups.The average mucous membrane irritation index was -0.83 and -0.67 in the 1-day and 5-day groups,respectively,suggesting that no intense mucosal membrane irritation responses occurred.It was concluded that APNPs could effectively kill P.g.in the biofilms and did not cause any pathological changes in the normal mucosa,suggesting that the plasma jet (APNPs) may be applied to oral diseases as a novel sterilization device in the future.