Chaga mushroom extract suppresses oral cancer cell growth via inhibition of energy metabolism.

Oral cancer stands as a prevalent maligancy worldwide; however, its therapeutic potential is limited by undesired effects and complications. As a medicinal edible fungus, Chaga mushroom (Inonotus obliquus) exhibits anticancer effects across diverse cancers. Yet, the precise mechanisms underlying its efficacy remain unclear. We explored the detailed mechanisms underlying the anticancer action of Chaga mushroom extract in oral cancer cells (HSC-4). Following treatment with Chaga mushroom extracts, we analyzed cell viability, proliferation capacity, glycolysis, mitochondrial respiration, and apoptosis. Our findings revealed that the extract reduced cell viability and proliferation of HSC-4 cells while arresting their cell cycle via suppression of STAT3 activity. Regarding energy metabolism, Chaga mushroom extract inhibited glycolysis and mitochondrial membrane potential in HSC-4 cells, thereby triggering autophagy-mediated apoptotic cell death through activation of the p38 MAPK and NF-κB signaling pathways. Our results indicate that Chaga mushroom extract impedes oral cancer cell progression, by inhibiting cell cycle and proliferation, suppressing cancer cell energy metabolism, and promoting autophagy-mediated apoptotic cell death. These findings suggest that this extract is a promising supplementary medicine for the treatment of patients with oral cancer.

Cyclic stretch induced epigenetic activation of periodontal ligament cells.

Periodontal ligament (PDL) cells play a crucial role in maintaining periodontal integrity and function by providing cell sources for ligament regeneration. While biophysical stimulation is known to regulate cell behaviors and functions, its impact on epigenetics of PDL cells has not yet been elucidated. Here, we aimed to investigate the cytoskeletal changes, epigenetic modifications, and lineage commitment of PDL cells following the application of stretch stimuli to PDL. PDL cells were subjected to stretching (0.1 Hz, 10 %). Subsequently, changes in focal adhesion, tubulin, and histone modification were observed. The survival ability in inflammatory conditions was also evaluated. Furthermore, using a rat hypo-occlusion model, we verified whether these phenomena are observed in vivo. Stretched PDL cells showed maximal histone 3 acetylation (H3Ace) at 2 h, aligning perpendicularly to the stretch direction. RNA sequencing revealed stretching altered gene sets related to mechanotransduction, histone modification, reactive oxygen species (ROS) metabolism, and differentiation. We further found that anchorage, cell elongation, and actin/microtubule acetylation were highly upregulated with mechanosensitive chromatin remodelers such as H3Ace and histone H3 trimethyl lysine 9 (H3K9me3) adopting euchromatin status. Inhibitor studies showed mechanotransduction-mediated chromatin modification alters PDL cells behaviors. Stretched PDL cells displayed enhanced survival against bacterial toxin (C12-HSL) or ROS (H2O2) attack. Furthermore, cyclic stretch priming enhanced the osteoclast and osteoblast differentiation potential of PDL cells, as evidenced by upregulation of lineage-specific genes. In vivo, PDL cells from normally loaded teeth displayed an elongated morphology and higher levels of H3Ace compared to PDL cells with hypo-occlusion, where mechanical stimulus is removed. Overall, these data strongly link external physical forces to subsequent mechanotransduction and epigenetic changes, impacting gene expression and multiple cellular behaviors, providing important implications in cell biology and tissue regeneration.

Antibacterial Activity and Sustained Effectiveness of Calcium Silicate-Based Cement as a Root-End Filling Material against Enterococcus faecalis.

Several calcium silicate cement (CSC) types with improved handling properties have been developed lately for root-end filling applications. While sealing ability is important, a high biocompatibility and antimicrobial effects are critical. This study aimed to conduct a comparative evaluation of the antimicrobial efficacy and sustained antibacterial effectiveness against Enterococcus faecalis (E. faecalis) of commercially available CSCs mixed with distilled water (DW) and chlorhexidine (CHX). Various products, viz., ProRoot mixed with DW (PRW) or with CHX (PRC), Endocem mixed with DW (EW) or with CHX (EC), and Endocem premixed (EP) syringe type, were used. While antibacterial activity against E. faecalis was evaluated using a direct contact method, the specimens were stored in a shaking incubator for 30 d for antibacterial sustainability. The cytotoxicity was evaluated using a cell counting kit-8 assay in human periodontal ligament stem cells. The antibacterial activities of EP, EW, and EC were greater than those of PRC and PRW (p < 0.05). The antibacterial sustainability of EP was the highest without cytotoxicity for up to 30 days (p < 0.05). In conclusion, the pre-mixed injectable type EP was most effective in terms of antibacterial activity and sustained antibacterial effectiveness without cytotoxicity.

Physicochemical, Biological, and Antibacterial Properties of Four Bioactive Calcium Silicate-Based Cements.

Calcium silicate-based cement (CSC) is a pharmaceutical agent that is widely used in dentistry. This bioactive material is used for vital pulp treatment due to its excellent biocompatibility, sealing ability, and antibacterial activity. Its drawbacks include a long setting time and poor maneuverability. Hence, the clinical properties of CSC have recently been improved to decrease its setting time. Despite the widespread clinical usage of CSC, there is no research comparing recently developed CSCs. Therefore, the purpose of this study is to compare the physicochemical, biological, and antibacterial properties of four commercial CSCs: two powder-liquid mix types (RetroMTA® [RETM]; Endocem® MTA Zr [ECZR]) and two premixed types (Well-Root™ PT [WRPT]; Endocem® MTA premixed [ECPR]). Each sample was prepared using circular Teflon molds, and tests were conducted after 24 h of setting. The premixed CSCs exhibited a more uniform and less rough surface, higher flowability, and lower film thickness than the powder-liquid mix CSCs. In the pH test, all CSCs showed values between 11.5 and 12.5. In the biological test, cells exposed to ECZR at a concentration of 25% showed greater cell viability, but none of the samples showed a significant difference at low concentration (p > 0.05). Alkaline phosphatase staining revealed that cells exposed to ECZR underwent more odontoblast differentiation than the cells exposed to the other materials; however, no significant difference was observed at a concentration of 12.5% (p > 0.05). In the antibacterial test, the premixed CSCs showed better results than the powder-liquid mix CSCs, and ECPR yielded the best results, followed by WRPT. In conclusion, the premixed CSCs showed improved physical properties, and of the premixed types, ECPR exhibited the highest antibacterial properties. For biological properties, none of these materials showed significant differences at 12.5% dilution. Therefore, ECPR may be a promising material with high antibacterial activity among the four CSCs, but further investigation is needed for clinical situations.

Physicochemical, Pre-Clinical, and Biological Evaluation of Viscosity Optimized Sodium Iodide-Incorporated Paste.

This study aimed to investigate the impact of different viscosities of silicone oil on the physicochemical, pre-clinical usability, and biological properties of a sodium iodide paste. Six different paste groups were created by mixing therapeutic molecules, sodium iodide (D30) and iodoform (I30), with calcium hydroxide and one of the three different viscosities of silicone oil (high (H), medium (M), and low (L)). The study evaluated the performance of these groups, including I30H, I30M, I30L, D30H, D30M, and D30L, using multiple parameters such as flow, film thickness, pH, viscosity, and injectability, with statistical analysis (p < 0.05). Remarkably, the D30L group demonstrated superior outcomes compared to the conventional iodoform counterpart, including a significant reduction in osteoclast formation, as examined through TRAP, c-FOS, NFATc1, and Cathepsin K (p < 0.05). Additionally, mRNA sequencing showed that the I30L group exhibited increased expression of inflammatory genes with upregulated cytokines compared to the D30L group. These findings suggest that the optimized viscosity of the sodium iodide paste (D30L) may lead to clinically favorable outcomes, such as slower root resorption, when used in primary teeth. Overall, the results of this study suggest that the D30L group shows the most satisfactory outcomes, which may be a promising root-filling material that could replace conventional iodoform-based pastes.

Photocatalytic effect-assisted antimicrobial activities of acrylic resin incorporating zinc oxide nanoflakes.

To overcome the deficiency of the antimicrobial effect of polymer, zinc oxide nanoparticles have been widely utilized as advanced nanofillers due to their antimicrobial and photocatalytic activity. However, the underlying antimicrobial mechanism has not been fully understood apart from topological and physical characteristics. In this study, we prepared zinc oxide nanoparticles-based acrylic resin to explore its antimicrobial mechanism under controlled mechanophysical conditions by using silane-treated zinc oxide nanoflakes (S-ZnNFs). S-ZnNFs incorporated acrylic resin (poly(methyl methacrylate), PMMA) composites up to 2 wt% were selected based on comparable mechanophysical properties (e.g., roughness, wettability, strength and hardness), possibly affecting antimicrobial properties beyond the zinc oxide nanoparticle effect, to bare PMMA. Antimicrobial adhesion results were still observed in 2 wt% S-ZnNFs incorporated PMMA using Candida albicans (C. albicans), one of the fungal infection species. In order to confirm the antimicrobial effects by photocatalysis, we pre-exposed the UV light on 2 wt% S-ZnNF composites before cell seeding, revealing synergetic antimicrobial effect via additional reactive oxygen species (ROS) generation to C. albicans over zinc oxide nanoparticle-induced one. RNA-seq analysis revealed distinguished cellular responses between zinc oxide nanoparticles and UV-mediated photocatalytic effect, but both linked to generation of intracellular ROS. Thus, the above data suggest that induction of high intracellular ROS of C. albicans was the main antimicrobial mechanism under controlled mechanophysical parameters and synergetic ROS accumulation can be induced by photocatalysis, recapitulating a promising use of a S-ZnNFs or possibly zinc oxide nanoparticles as intracellular-ROS-generating antimicrobial nanofillers in acrylic composite for biomedical applications.

Improvement of Biological Effects of Root-Filling Materials for Primary Teeth by Incorporating Sodium Iodide.

Therapeutic iodoform (CHI3) is commonly used as a root-filling material for primary teeth; however, the side effects of iodoform-containing materials, including early root resorption, have been reported. To overcome this problem, a water-soluble iodide (NaI)-incorporated root-filling material was developed. Calcium hydroxide, silicone oil, and NaI were incorporated in different weight proportions (30:30:X), and the resulting material was denoted DX (D5~D30), indicating the NaI content. As a control, iodoform instead of NaI was incorporated at a ratio of 30:30:30, and the material was denoted I30. The physicochemical (flow, film thickness, radiopacity, viscosity, water absorption, solubility, and ion releases) and biological (cytotoxicity, TRAP, ARS, and analysis of osteoclastic markers) properties were determined. The amount of iodine, sodium, and calcium ion releases and the pH were higher in D30 than I30, and the highest level of unknown extracted molecules was detected in I30. In the cell viability test, all groups except 100% D30 showed no cytotoxicity. In the 50% nontoxic extract, D30 showed decreased osteoclast formation compared with I30. In summary, NaI-incorporated materials showed adequate physicochemical properties and low osteoclast formation compared to their iodoform-counterpart. Thus, NaI-incorporated materials may be used as a substitute for iodoform-counterparts in root-filling materials after further (pre)clinical investigation.

Oral Microbiome Using Colocasia antiquorum var. esculenta Extract Varnish in a Mouse Model with Oral Gavage of P. gingivalis ATCC 53978.

Background and Objective: There is increasing interest in preventing periodontitis using natural products. The purpose of this study was to investigate the effect of Colocasia antiquorum var. esculenta (CA) varnish on the oral microbiome and alveolar bone loss in a mouse periodontitis model. Materials and Methods: Antibacterial activity against Porphyromonas gingivalis (P. gingivalis) ATCC 53978 and cell cytotoxicity using CCK-8 on L929 cells were measured. Balb/c mice were assigned into five groups (negative control, positive control, CA in drinking water, varnish, and CA varnish). P. gingivalis was administered to the mice by oral gavage three times. After sacrifice, the oral microbiome and the levels of the inflammatory cytokine IL-1ß and matrix metalloproteinase-9 were analyzed. Alveolar bone loss was measured using micro-computed tomography. Results: CA extract showed an antibacterial effect against P. gingivalis (p < 0.05) and showed no cytotoxicity at that concentration (p > 0.05). Although alpha diversity of the oral microbiome did not statistically differ between the groups (p > 0.05), the relative abundance of dominant bacteria tended to be different between the groups. The inflammatory cytokine IL-1ß was reduced in the CA varnish group (p < 0.05), and no difference was observed in MMP-9 expression and alveolar bone loss (p > 0.05). Conclusions: CA varnish did not affect the overall microflora and exhibited an anti-inflammatory effect, suggesting that it is possibility a suitable candidate for improving periodontitis.

Shear bond strengths of various resin cements between three types of adherends and bovine teeth with and without thermocycling.

This study evaluated the shear bond strengths of various types of resin cements between three types of adherends (composite resin, metal, and ceramic) and bovine teeth with and without thermocycling. A conventional resin cement (Variolink N), two adhesive resin cements (PANAVIA F 2.0, Multilink N), and three self-adhesive resin cements (MAXCEM ELITE, Rely X Unicem 2, Speed CEM) were used. The adherends were cemented on the superficial dentin of bovine incisors using each resin cement. Herein, 10 specimens from each group were thermocycled 5,000 times, and the other 10 were stored without thermocycling. With the resin and ceramic adherends, the shear bond strengths of Rely X Unicem 2 were significantly higher than those of the other resin cements both with and without thermocycling (p<0.05). With the metal adherend, the shear bond strengths were not significantly different among the cement groups, except MAXCEM ELITE, which showed the lowest strength.

Effects of Colocasia antiquorum var. Esculenta Extract In Vitro and In Vivo against Periodontal Disease.

Background and Objectives: Periodontal disease is a chronic inflammatory disease in which gradual destruction of tissues around teeth is caused by plaque formed by pathogenic bacteria. The purpose of this study was to evaluate the potential of 75% ethanol extract of Colocasia antiquorum var. esculenta (CA) as a prophylactic and improvement agent for periodontal disease in vitro and in vivo. Materials and Methods: The antimicrobial efficacy of CA against Porphyromonas gingivalis (P. gingivalis, ATCC 33277) was evaluated using minimum inhibitory concentration (MIC) and minimum bactericidal concentration (MBC) test, and cytotoxicity was confirmed by CCK-8 assay. For the in vivo study, P. gingivalis was applied by oral gavage to BALB/c mice. Forty-two days after the first inoculation of P. gingivalis, intraoral swabs were taken for microbiome analysis, and the mice were sacrificed to evaluate the alveolar bone loss. Results: The MIC of CA against P. gingivalis was 31.3 µg/mL, the MBC was 62.5 µg/mL, with no cytotoxicity. The diversity of the oral microbiome decreased in the positive control group, while those of the VA (varnish) and VCA (varnish added with CA) groups increased as much as in the negative control group, although the alveolar bone loss was not induced in the mouse model. Conclusions: CA showed antibacterial effects in vitro, and the VA and VCA groups exhibited increased diversity in the oral microbiome, suggesting that CA has potential for improving periodontal disease.

Inhibitory Effect of Asplenium incisum on Bacterial Growth, Inflammation, and Osteoclastogenesis.

Background and Objectives:Asplenium incisum, a natural plant, is known to possess numerous pharmacological and biochemical properties. However, the inhibitory effect of A. incisum against Porphyromonas gingivalis and other factors related to periodontal disease have not yet been demonstrated. This study aimed to investigate the potential of A. incisum extract as a phytotherapeutic candidate for improving periodontal diseases by assessing its antibacterial, anti-inflammatory, and anti-osteoclastogenic activities. Materials and Methods: The inhibition of proliferation of P. gingivalis by A. incisum and the sustainability of its antibacterial activity were evaluated in this study. The production of inflammatory cytokines (tumor necrosis factor-α (TNF-α) and interleukin-6 (IL-6)) and nitric oxide (NO) from lipopolysaccharide-stimulated RAW 264.7 cells was assessed using an enzyme-linked immunosorbent assay. To identify the anti-osteoclastogenic activity, tartrate-resistant acid phosphatase (TRAP) staining and TRAP activity analyses were performed on bone marrow macrophages. Results: The proliferation of P. gingivalis was significantly inhibited by A. incisum (p < 0.001), and the antibacterial activity was sustained for up to 3 days. A. incisum showed anti-inflammatory activities by significantly decreasing the release of TNF-α, IL-6 (p < 0.05), and NO (p < 0.01). In addition, A. incisum significantly suppressed TRAP-positive cells and TRAP activity (at 30 µg/mL, p < 0.01) without causing any cytotoxicity (p > 0.05). Conclusions:A. incisum showed antibacterial, anti-inflammatory, and anti-osteoclastogenic activities, suggesting it has strong therapeutic potential against periodontal diseases.

Antibacterial, anti-inflammatory, and anti-osteoclastogenic activities of Colocasia antiquorum var. esculenta: Potential applications in preventing and treating periodontal diseases.

This study aimed to investigate the inhibitory effects of Colocasia antiquorum var. esculenta (CA) on Porphyromonas gingivalis (P. gingivalis) growth, inflammation, and osteoclastogenesis. CA was effective in inhibiting the growth of P. gingivalis when applied together with an experimental fluoride varnish. CA also significantly decreased the release of interleukin-6, tumor necrosis factor-α, and nitric oxide from lipopolysaccharide-induced RAW 264.7 cells. No significant differences in viability were noted between the cells treated with CA and the controls. In addition, CA significantly attenuated osteoclast differentiation on bone marrow macrophages. In conclusion, CA inhibited the growth of P. gingivalis and showed anti-inflammatory and anti-osteoclastogenic effects. Therefore, CA may have the potential to act as a novel natural agent for preventing periodontitis.