Effects of no-ozone cold plasma and mouse mesenchymal stem cell treatments on wound healing in a mouse skin model.

Skin wounds heal faster during stem cell differentiation. Cold plasma reportedly enhances cell proliferation and differentiation and enhances the efficacy of stem cell therapy. However, the exact mechanism of action involved remains unknown. Therefore, this study aimed to evaluate the effect of a combination therapy involving the transplantation of mouse mesenchymal stem cells (mMSCs) into mice with wounds followed by their activation using no-ozone cold plasma (NCP). Balb/c mMSCs were transplanted into BALB/c mice and treated with NCP for 5 min. The animals were divided into four groups based on treatments received: no treatment (Wound), mMSCs only (mMSC), NCP only (NCP), and both mMSC and NCP (mMSC + NCP). NCP treatment was administered six times over two weeks, and tissue samples were prepared by sacrificing the mice in the 1st and 2nd weeks. The wound healing efficacy was assessed using morphological, histological, and molecular approaches including wound healing length measurements, hematoxylin and eosin staining, Masson trichrome staining, immunofluorescence staining, immunohistochemistry, and real-time polymerase chain reaction. The wound healing effect was better in the mMSC + NCP group than that in the groups treated with either. Tracking the injected mMSCs in mice also revealed that the mMSC + NCP group had a greater survival rate. Furthermore, upon wound healing, the mMSC + NCP group exhibited elevated levels of growth factors, like platelet-derived growth factor, transforming growth factor-beta, and vascular endothelial growth factor. These results suggest that NCP stimulated transplanted mMSCs, resulting in faster wound healing. Therefore, further studies are warranted in preclinical and clinical studies to confirm this effect.

Effects of Different No-Ozone Cold Plasma Treatment Methods on Mouse Osteoblast Proliferation and Differentiation.

Background and Objectives: Enhanced osteoblast differentiation may be leveraged to prevent and treat bone-related diseases such as osteoporosis. No-ozone cold plasma (NCP) treatment is a promising and safe strategy to enhance osteoblast differentiation. Therefore, this study aimed to determine the effectiveness of direct and indirect NCP treatment methods on osteoblast differentiation. Mouse osteoblastic cells (MC3T3-E1) were treated with NCP using different methods, i.e., no NCP treatment (NT group; control), direct NCP treatment (DT group), direct NCP treatment followed by media replacement (MC group), and indirect treatment with NCP-treated media only (PAM group). Materials and Methods: The MC3T3-E1 cells were subsequently assessed for cell proliferation, alkaline phosphatase (ALP) activity, calcium deposition, and ALP and osteocalcin mRNA expression using real-time polymerase chain reaction. Results: Cell proliferation significantly increased in the NCP-treated groups (DT and PAM; MC and PAM) compared to the NT group after 24 h (p < 0.038) and 48 h (p < 0.000). ALP activity was increased in the DT and PAM groups at 1 week (p < 0.115) and in the DT, MC, and PAM groups at 2 weeks (p < 0.000) compared to the NT group. Calcium deposition was higher in the NCP-treated groups than in NT group at 2 and 3 weeks (p < 0.000). ALP mRNA expression peaked in the MC group at 2 weeks compared to the NP group (p < 0.014). Osteocalcin mRNA expression increased in the MC group at 2 weeks (p < 0.000) and was the highest in the PAM group at 3 weeks (p < 0.000). Thus, the effects of direct (DT and MC) and indirect (PAM) treatment varied, with MC direct treatment showing the most significant impact on osteoblast activity. Conclusions: The MC group exhibited enhanced osteoblast differentiation, indicating that direct NCP treatment followed by media replacement is the most effective method for promoting bone formation.

Antimicrobial Effects of Edible Mixed Herbal Extracts on Oral Microorganisms: An In Vitro Study.

Background and Objectives: The oral cavity is inhabited by pathogenic bacteria, whose growth can be inhibited by synthetic oral drugs, including antibiotics and other chemical compounds. Natural antimicrobial substances that elicit fewer negative side effects may serve as alternatives to synthetic agents for long-term use. Thus, the aim of this study was to evaluate the effects of edible mixed herbal extracts on the growth of oral pathogenic bacteria. Materials and Methods: The yield of each herbal extract was as follows: 5% Schizonepeta tenuifolia Briq (STB), 10.94% Mentha piperascens (MP), 5.47% Acanthopanax sessiliflorus Seem (AS), and 10.66% Glycyrrhiza uralensis (GU). The herbal extracts used included 0.5 mg/mL STB, 1.5 mg/mL MP, 1.5 mg/mL AS, and 2.0 mg/mL GU. Antimicrobial tests, morphological analyses (using scanning electron microscopy), microbial surface hydrophobicity measurements, and oral malodor reduction tests were performed using each extract. Statistical analyses were performed with IBM® SPSS® (version 24), using paired t-tests. Results: The mixed herbal extracts significantly inhibited the growth of Streptococcus mutans, Enterococcus faecalis, Candida albicans, and Porphyromonas gingivalis compared to the control (p < 0.001). Scanning electron microscopy results further revealed altered cellular morphology in the groups treated with the mixed herbal extracts. Additionally, the hydrophobicity assay results showed that the mixed herbal extracts reduced the oral adhesion capacities of bacteria (p < 0.001). Administration of the mixed herbal extracts also reduced the levels of volatile sulfur compounds, the main contributors to oral malodor (p < 0.001). Conclusions: Edible mixed herbal extracts can effectively eliminate oral pathogens and may be useful for improving oral health. The herbal extracts used were effective against all species of oral pathogens studied in this report.

Protective effects of non-thermal plasma on triethylene glycol dimethacrylate-induced damage in odontoblast-like MDPC-23 cells.

AIM: To evaluate whether the use of non-thermal plasma (NTP) could reduce triethylene glycol dimethacrylate (TEGDMA)-mediated damage in MDPC-23 cells. METHODOLOGY: The effects of NTP and TEGDMA on MDPC-23 cell proliferation were tested using WST-1 assays after pretreatment with NTP for 1 min and exposure to TEGDMA. Live/Dead assays were used to visualize cell death. To monitor the effects of NTP and TEGDMA on the cell cycle and apoptotic cell death, flow cytometry was performed. Western blotting was used to assess changes in protein levels mediated by NTP and TEGDMA treatment, and enzyme-linked immunosorbent assays were performed to evaluate the effects of NTP and TEGDMA on prostaglandin E2 (PGE2 ) expression. One-way analysis of variance and Duncan's post hoc tests were used for statistical analysis. RESULTS: NTP treatment effectively protected cells from TEGDMA-mediated cell damage and blocked TEGDMA-mediated cell growth inhibition (p < .05). NTP appeared to protect cells from death (p < .05) and blocked TEGDMA-mediated apoptotic cell death. Additionally, NTP reduced TEGDMA-mediated apoptotic activation of poly (ADP) ribose polymerase-1 and caspase-3 (p < .05). Furthermore, NTP effectively reduced TEGDMA-mediated expression of cyclooxygenase-2 and PGE2 proteins by inhibiting nuclear factor-κB protein expression (p < .05). CONCLUSIONS: NTP alleviated TEGDMA-mediated adverse effects by reducing cytotoxicity and inflammatory reactions in cells exposed to TEGDMA.

Increment of growth factors in mouse skin treated with non-thermal plasma.

Non-thermal plasma (NTP) has several beneficial effects, and can be applied as a novel instrument for skin treatment. Recently, many types of NTP have been developed for potential medical or clinical applications, but their direct effects on skin activation remain unclear. In this study, the effect of NTP on the alteration of mouse skin tissue was analyzed. After NTP treatment, there were no signs of tissue damage in mouse skin, whereas significant increases in epidermal thickness and dermal collagen density were detected. Furthermore, treatment with NTP increased the expression of various growth factors, including TGF-α, TGF-ß, VEGF, GM-CSF, and EGF, in skin tissue. Therefore, NTP treatment on skin induces the expression of growth factors without causing damage, a phenomenon that might be directly linked to epidermal expansion and dermal tissue remodeling.

Selective Killing of Melanoma Cells With Non-Thermal Atmospheric Pressure Plasma and p-FAK Antibody Conjugated Gold Nanoparticles.

Melanomas are fast growing high-mortality tumors, and specific treatments for melanomas are needed. Melanoma cells overexpress focal adhesion kinase (FAK) compared to normal keratinocytes, and we sought to exploit this difference to create a selectively lethal therapy. We combined gold nanoparticles (GNP) with antibodies targeting phosphorylated FAK (p-FAK). These conjugates (p-FAK-GNP) entered G361 melanoma cells and bound p-FAK. Treatment with p-FAK-GNP decreased the viability of G361 cells in a time dependent manner by inducing apoptosis. To maximize the preferential killing of G361 cells, non-thermal atmospheric pressure plasma was used to stimulate the GNP within p-FAK-GNP. Combined treatment with plasma and p-FAK-GNP showed much higher lethality against G361 cells than HaCaT keratinocyte cells. The p-FAK-GNP induced apoptosis over 48 hours in G361 cells, whereas plasma and p-FAK-GNP killed G361 cells immediately. This study demonstrates that combining plasma with p-FAK-GNP results in selective lethality against human melanoma cells.

Enhancement of the killing effect of low-temperature plasma on Streptococcus mutans by combined treatment with gold nanoparticles.

BACKGROUND: Recently, non-thermal atmospheric pressure plasma sources have been used for biomedical applications such as sterilization, cancer treatment, blood coagulation, and wound healing. Gold nanoparticles (gNPs) have unique optical properties and are useful for biomedical applications. Although low-temperature plasma has been shown to be effective in killing oral bacteria on agar plates, its bactericidal effect is negligible on the tooth surface. Therefore, we used 30-nm gNPs to enhance the killing effect of low-temperature plasma on human teeth. RESULTS: We tested the sterilizing effect of low-temperature plasma on Streptococcus mutans (S. mutans) strains. The survival rate was assessed by bacterial viability stains and colony-forming unit counts. Low-temperature plasma treatment alone was effective in killing S. mutans on slide glasses, as shown by the 5-log decrease in viability. However, plasma treatment of bacteria spotted onto tooth surface exhibited a 3-log reduction in viability. After gNPs were added to S. mutans, plasma treatment caused a 5-log reduction in viability, while gNPs alone did not show any bactericidal effect. The morphological changes in S. mutans caused by plasma treatment were examined by transmission electron microscopy, which showed that plasma treatment only perforated the cell walls, while the combination treatment with plasma and gold nanoparticles caused significant cell rupture, causing loss of intracellular components from many cells. CONCLUSIONS: This study demonstrates that low-temperature plasma treatment is effective in killing S. mutans and that its killing effect is further enhanced when used in combination with gNPs.

Enhancing Textile Water Repellency with Octadecyltrichlorosilane (OTS) and Hollow Silica Nanoparticles.

Superhydrophobic coatings have attracted substantial attention owing to their potential application in various industries. Conventional textiles used in daily life are prone to staining with water and household liquids, necessitating the development of water-repellent and stain-resistant coatings. In this study, we fabricated a highly water-repellent superhydrophobic PET fabric by using an eco-friendly water-based coating process. Fluorine-free octadecyltrichlorosilane (OTS) solutions with various wt.% of hollow silica (HS) nanoparticles were used to produce a superhydrophobic surface via a facile dip coating method. Our findings revealed that the incorporation of HS nanoparticles substantially increased the water contact angle, with higher concentrations resulting in enhanced water repellency and increased surface roughness. The treated fabrics had a remarkable water contact angle of 152.4° ± 0.8°, demonstrating their superhydrophobic fiber surface. In addition, the durability of these superhydrophobic properties was investigated via a laundry procedure, which showed that the fabrics maintained their water repellency even after 20 laundering cycles. EDX and XRD analyses confirmed that the morphological evaluations did not reveal any substantial structural alterations. Significantly, the fibers maintained their strength and durability throughout the testing, enduring only minor hollow SiO2 nanoparticle loss. This eco-friendly and cost-effective method holds great potential for application in apparel and other industries, offering an effective solution to resist water stains and improve performance in various contexts.

The Whitening Effect and Histological Safety of Nonthermal Atmospheric Plasma Inducing Tooth Bleaching.

Various light sources have been applied to enhance the bleaching effect. This study was to identify the histological evaluation in oral soft tissues, as well as tooth color change after tooth bleaching by nonthermal atmospheric pressure plasma (NAPP). Nine New Zealand adult female rabbits were randomly divided into three groups (n = 3): group 1 received no treatment; group 2 was treated with NAPP and 15% carbamide peroxide (CP), which contains 5.4% H2O2, and group 3 was treated with 15% CP without NAPP. Color change (ΔE) was measured using the Shade Eye NCC colorimeter. Animals were euthanized one day later to analyze the histological responses occurring in oral soft tissues, including pulp, gingiva, tongue, buccal mucosa, and hard and soft palates. Changes in all samples were analyzed by hematoxylin and eosin staining and Masson's trichrome. Teeth treated with plasma showed higher ΔE than that obtained with bleaching agents alone. Overall, the histological characteristics observed no appreciable changes. The combinational treatment of plasma had not indicated inflammatory responses as well as thermal damages. NAPP did not cause histological damage in oral soft tissues during tooth bleaching. We suggest that NAPP could be a novel alternative energy source to conventional light sources for tooth bleaching.

Enhancement of Osteoblast Differentiation Using No-Ozone Cold Plasma on Human Periodontal Ligament Cells.

Periodontitis is an inflammatory disease that leads to periodontal tissue destruction and bone resorption. Proliferation and differentiation of cells capable of differentiating into osteoblasts is important for reconstructing periodontal tissues destroyed by periodontitis. In this study, the effects of the nozone (no-ozone) cold plasma (NCP) treatment on osteoblastic differentiation in periodontal ligament (PDL) cells were investigated. To test the toxicity of NCP on PDL cells, various NCP treatment methods and durations were tested, and time-dependent cell proliferation was analyzed using a water-soluble tetrazolium salts-1 assay. To determine the effect of NCP on PDL cell differentiation, the cells were provided with osteogenic media immediately after an NCP treatment to induce differentiation; the cells were then analyzed using alkaline phosphatase (ALP) staining, an ALP activity assay, real time PCR, and Alizarin Red S staining. The NCP treatment without toxicity on PDL cells was the condition of 1-min NCP treatment immediately followed by the replacement with fresh media. NCP increased ALP, osteocalcin, osteonectin, and osteopontin expression, as well as mineralization nodule formation. NCP treatment promotes osteoblastic differentiation of PDL cells; therefore, it may be beneficial for treating periodontitis.

Gold nanoparticles conjugated with programmed death-ligand 1 antibodies induce apoptosis of SCC-25 oral squamous cell carcinoma cells via programmed death-ligand 1/signal transducer and transcription 3 pathway.

OBJECTIVE: Objective of this study is to test the anti-cancer effect of the gold nanoparticles conjugated with programmed death-ligand 1 (PD-L1) specific antibodies (PDL1-GNP), on oral squamous cell carcinoma. DESIGN: To test the effect of PDL1-GNP on oral squamous cell carcinoma, SCC-25 cells, a type of human oral squamous cell carcinoma which were isolated from human tongue, and HaCaT human keratinocytes as normal cell control, were used. Cell viability was tested by the water-soluble tetrazolium-1 and live/dead assays, while apoptotic cell death of SCC-25 cells were monitored by immunofluorescent staining and flow cytometry. The molecular changes during PDL1-GNP-mediated apoptosis were analyzed using Western blot analysis. RESULTS: PDL1-GNP treatment effectively decreased the growth of SCC-25 cells but not HaCaT cells. The results of the confocal microscopic assay showed that PDL1-GNP specifically bound to the SCC-25 cell membrane. Furthermore, the results of the live/dead, cytochrome c release assays and flow cytometry indicated PDL1-GNP-mediated apoptotic cell death of SCC-25 cells. PDL1-GNP-treated SCC-25 cells showed a phenotype with increased apoptotic proteins, including cleaved form of caspase-3, caspase-9, and poly (ADP-ribose) polymerase 1 (PARP1). PDL1-GNP treatment also effectively decreased B-cell lymphoma 2 (Bcl-2) and PD-L1 protein expression. Phosphorylation of signal transducer and transcription 3 (STAT3) was significantly increased after PDL1-GNP treatment on SCC-25 cells. CONCLUSIONS: PDL1-GNP treatment induced SCC-25 cell apoptosis possibly by inhibiting the function of the PD-L1 protein, since PD-L1 blocks STAT3 phosphorylation, which promotes apoptotic cell death.

Induction of Melanoma Cell-Selective Apoptosis Using Anti-HER2 Antibody-Conjugated Gold Nanoparticles.

PURPOSE: This study was conducted to verify the induction and mechanism of selective apoptosis in G361 melanoma cells using anti-HER2 antibody-conjugated gold nanoparticles (GNP-HER2). MATERIALS AND METHODS: Following GNP-HER2 treatment of G361 cells, cell cycle arrest and apoptosis were measured by WST-1 assay, Hemacolor staining, Hoechst staining, immunofluorescence staining, fluorescence-activated cell sorting analysis, and Western blotting. RESULTS: G361 cells treated with GNP-HER2 showed condensation of nuclei, which is an apoptotic phenomenon, and translocation of apoptosis-inducing factor and cytochrome c from mitochondria into the nucleus and cytoplasm, respectively. Increases in BAX in cells undergoing apoptosis, activation of caspase-3 and -9, and fragmentation of poly (ADP-ribose) polymerase and DNA fragmentation factor 45 (inhibitor of caspase-activated DNase) were observed upon GNP-HER2 treatment. Following GNP-HER2 treatment, an increase of cells in sub-G1 phase, which is a signal of cell apoptosis, was observed. This resulted in the down-regulation of cyclin A, cyclin D1, cyclin E, cdk2, cdk4, and cdc2 and the up-regulation of p21. Thus, GNP-HER2 treatment was confirmed to induce the cessation of cell cycle progression. Also, decreases in phospho-focal adhesion kinase and phospho-human epidermal growth factor receptor, which activate cellular focal adhesion, and decreases in phospho-paxillin, which stimulates the disassembly of filamentous actin, were observed. Reduced cell adhesion and disassembly of the intracellular structure indicated cell deactivation. CONCLUSION: GNP-HER2 can selectively kill G361 melanoma cells without affecting normal cells. The mechanism of G361 cell death upon treatment with GNP-HER2 was apoptosis accompanied by activation of caspases.

The effect of biomechanical stimulation on osteoblast differentiation of human jaw periosteum-derived stem cells.

BACKGROUND: This study was to investigate the effect of biomechanical stimulation on osteoblast differentiation of human periosteal-derived stem cell using the newly developed bioreactor. METHODS: Human periosteal-derived stem cells were harvested from the mandible during the extraction of an impacted third molar. Using the new bioreactor, 4% cyclic equibiaxial tension force (0.5 Hz) was applied for 2 and 8 h on the stem cells and cultured for 3, 7, and 14 days on the osteogenic medium. Biochemical changes of the osteoblasts after the biomechanical stimulation were investigated. No treatment group was referred to as control group. RESULTS: Alkaline phosphatase (ALP) activity and ALP messenger RNA (mRNA) expression level were higher in the strain group than those in the control group. The osteocalcin and osteonectin mRNA expressions were higher in the strain group compared to those in the control group on days 7 and 14. The vascular endothelial growth factor (VEGF) mRNA expression was higher in the strain group in comparison to that in the control group. Concentration of alizarin red S corresponding to calcium content was higher in the strain group than in the control group. CONCLUSIONS: The study suggests that cyclic tension force could influence the osteoblast differentiation of periosteal-derived stem cells under optimal stimulation condition and the force could be applicable for tissue engineering.

Targeting NEU Protein in Melanoma Cells with Non-Thermal Atmospheric Pressure Plasma and Gold Nanoparticles.

Non-thermal atmospheric pressure plasma effectively kills cancer cells, but it cannot selectively kill cancer cells. The authors targeted NEU (human epidermal growth factor receptor 2) protein, which is frequently over-expressed in the cell membrane of melanoma cells, using anti-NEU antibody-labeled gold nanoparticles. The labeled nanoparticles preferentially targeted melanoma cells rather than normal keratinocytes. After the addition of labeled gold nanoparticles to melanoma and normal keratinocyte cells, both cells were exposed to non-thermal atmospheric pressure plasma. The death rate of melanoma cells was significantly higher than that of normal keratinocyte cells; many vacuoles, indicative of cell death, were observed in melanoma cells treated with anti-NEU antibody labeled gold nanoparticles and plasma. This selective cancer cell death was attributed to the selective destruction of NEU protein and a downstream effector of NEU. Our study findings show that treatment with a combination of non-thermal atmospheric pressure plasma and anti-NEU antibody-labeled gold nanoparticles effectively and selectively kills melanoma cells.

Scutellariae radix induces apoptosis in chemoresistant SCC-25 human tongue squamous carcinoma cells.

Scutellariae radix is one of the most widely used anticancer herbal medicines in several Asian countries, including Korea, Japan, and China. Squamous cell carcinoma (SCC) is one of the most common head and neck carcinomas, which is highly invasive and metastatic, and can potentially develop chemoresistance. Therefore, new effective treatment methods are urgently needed. We determined the effects of Scutellariae radix on SCC-25 cells using the WST-1 assay, F-actin staining, flow cytometry analysis, immunofluorescence staining, and western blot analysis. Scutellariae radix treatment inhibited SCC-25 cell growth in a dose- and time-dependent manner, but it did not inhibit HaCaT (human keratinocyte) cell growth. Changes in cell morphology and disruption of filamentous (F)-actin organization were observed. Scutellariae radix-induced apoptosis as indicated by the translocation of cytochrome c and apoptosis-inducing factor (AIF) into the nucleus and cytosol. Scutellariae radix-induced an increase in cells with sub-G1 DNA content, and increased Bax, cleaved caspase-3, caspase-7, caspase-9, DNA fragmentation factor 45 (DFF 45), and poly(ADP-ribose) polymerase-1 (PARP-1) expression levels. Furthermore, increased expression of phosphorylated mitogen-activated protein kinase (MAPK)-related proteins was detected. The antitumor effect of Scutellariae radix was due to decreased cell proliferation, changes in cell morphology, and the activation of caspase and MAPK pathways. Taken together, the findings of this study highlight the anticancer activity of Scutellariae radix in chemoresistant SCC-25 oral squamous carcinoma cells.