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The objective of this study was to examine whether native low-density lipoprotein (LDL) induces foam cell formation by macrophages and to examine the effect of lipopolysaccharide (LPS) on native LDL-induced foam cell formation by macrophages in vitro. RAW 264.7 cells were cultured with LDL or high-density lipoprotein (HDL) in the presence of LPS derived from Aggregatibacter actinomycetemcomitans. Foam cell formation was determined by staining with Oil-red-O to visualize cytoplasmic lipid droplet accumulation. The expression of LDL-receptor and the degree of internalization of FITC-conjugated LDL in RAW 264.7 cells were examined by immunofluorescence microscopy. The images were digitally recorded and analyzed with Image J software. Statistical analysis was performed by JMP software. Foam cell formation was induced by the addition of native LDL in dose- and time-dependent manners, whereas HDL showed no effect. LPS enhanced the foam cell formation induced by native LDL. In addition, LPS stimulated the expression of LDL-receptor protein on RAW 264.7 cells and enhanced the internalization of LDL. The enhancement of foam cell formation induced by LPS and LDL was inhibited by the depolymerizing agent nocodazole and amiloride analog 5-(N-ethyl-N-isoprophyl) amiloride (EIPA). Our findings indicate that LPS plays an important role in foam cell formation by LDL-stimulated macrophages. [ABSTRACT FROM PUBLISHER]

Porphyromonas gingivalis is the most pathogenic periodontal bacterium in the world. Recently, P. gingivalis has been considered responsible for dysbiosis during the development of periodontitis. This study aimed to evaluate a novel immunochromatographic device using monoclonal antibodies against P. gingivalis in subgingival plaques. A total of 72 patients with chronic periodontitis and 53 periodontally healthy volunteers underwent clinical and microbiological examinations. Subgingival plaque samples were analyzed for the presence of P. gingivalis and compared using real-time polymerase chain reaction (PCR). In the periodontitis group, a significant positive correlation was observed between the test device scores and the real-time PCR results. The specificity, positive predictive value, negative predictive value, and accuracy of the test device for P. gingivalis , as determined by real-time PCR, were 98%, 94%, 89%, and 90%, respectively. There were significant differences in bacterial counts by real-time PCR among the groups with different ranges of device scores. Additionally, there was a significant positive correlation between the device scores for P. gingivalis and periodontal parameters. These results suggest that this novel immunochromatographic device can be effectively used for rapid detection and semi-quantification of P. gingivalis in subgingival plaques.

Human dental pulp stem cells are a potentially useful resource for cell-based therapies and tissue repair in dental and medical applications. However, the primary culture of isolated dental pulp stem cells has notably been limited. A major requirement of an ideal human dental pulp stem cell culture system is the preservation of efficient proliferation and innate stemness over prolonged passaging, while also ensuring ease of handling through standard, user-friendly culture methods. In this study, we have engineered a novel human dental pulp stem cell line, distinguished by the constitutive expression of telomerase reverse transcriptase (TERT), and the conditional expression of the R24C mutant cyclin-dependent kinase 4 (CDK4 R24C ) and Cyclin D1. We have named this cell line Tet-off K4DT hDPSCs. Furthermore, we have conducted a comprehensive comparative analysis of their biological attributes in relation to a previously immortalized human dental pulp stem cells, hDPSC-K4DT, which were immortalized by the constitutive expression of CDK4 R24C , Cyclin D1 and TERT. In Tet-off K4DT cells, the expression of the K4D genes can be precisely suppressed by the inclusion of doxycycline. Remarkably, Tet-off K4DT cells demonstrated an extended cellular lifespan, increased proliferative capacity, and enhanced osteogenic differentiation potential when compared to K4DT cells. Moreover, Tet-off K4DT cells had no observable genomic aberrations and also displayed a sustained expression of stem cell markers even at relatively advanced passages. Taken together, the establishment of this new cell line holds immense promise as powerful experimental tool for both fundamental and applied research involving dental pulp stem cells., (© 2024 John Wiley & Sons Ltd.)

Background: Periodontitis is the most common oral disease in dogs, and its progression and severity are influenced by risk factors, such as age and body size. Recent studies have assessed the canine oral microbiota in relation to different stages of periodontitis and niches within the oral cavity. However, knowledge of the bacterial composition at different ages and body sizes, especially in puppies, is limited. This study aimed to characterize the oral microbiota in the healthy gingiva of small breed puppies using next-generation sequencing. Additionally, we assessed the impact of dental care practices and the presence of retained deciduous teeth on the oral microbiota., Results: In this study, plaque samples were collected from the gingival margin of 20 small breed puppies (age, 6.9 ± 0.6 months). The plaque samples were subjected to next-generation sequencing targeting the V3-V4 region of the 16 S rRNA. The microbiota of the plaque samples was composed mostly of gram-negative bacteria, primarily Proteobacteria (54.12%), Bacteroidetes (28.79%), and Fusobacteria (5.11%). Moraxella sp. COT-017, Capnocytophaga cynodegmi COT-254, and Bergeyella zoohelcum COT-186 were abundant in the oral cavity of the puppies. In contrast, Neisseria animaloris were not detected. The high abundance of Pasteurellaceae suggests that this genus is characteristic of the oral microbiota in puppies. Dental care practices and the presence of retained deciduous teeth showed no effects on the oral microbiota., Conclusions: In this study, many bacterial species previously reported to be detected in the normal oral cavity of adult dogs were also detected in 6-8-month-old small breed dogs. On the other hand, some bacterial species were not detected at all, while others were detected in high abundance. These data indicate that the oral microbiota of 6-8-month-old small breed dogs is in the process of maturating in to the adult microbiota and may also have characteristics of the small dog oral microbiota., (© 2024. The Author(s).)

Aim: To evaluate the association between trypsin-like protease (TLP) activity in the oral cavity as an indicator of periodontal health status and kidney function in Japanese workers., Materials and Methods: This cross-sectional study included 1117 Japanese workers (mean age = 43.8 years). Tongue-swab TLP activity was quantified as a* value (the redness intensity of the matrix disc of the TLP activity assessment kit; a larger value indicates more intense enzymatic activity in the samples and poorer periodontal health status). Kidney function was assessed using the estimated glomerular filtration rate (eGFR; a lower value indicates poorer kidney function). We performed ordinal logistic regression analyses to assess the association of the a* value with three eGFR categories: ≥90, 60-89 and <60 mL/min/1.73 m 2 ., Results: The prevalence for each eGFR category was as follows: ≥90 (31.6%), 60-89 (63.8%) and <60 mL/min/1.73 m 2 (4.6%). After adjusting for potential confounders, the a* value was found to be significantly associated with reduced kidney function. The multivariable-adjusted odds ratio (95% confidence interval) for reduced kidney function was 1.12 (1.02-1.22) per unit increase in the a* value., Conclusions: Higher TLP activity was associated with reduced kidney function in Japanese workers., (© 2023 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.)

Objectives: Medicinal herbs are plants with potential medicinal and health benefits. In recent years, they are being increasingly used as a treatment alternative owing to their effectiveness against various diseases. In this study, we investigated the inhibitory effects of 15 medicinal herbs on causative bacteria for dental caries and periodontal disease., Methods: This study evaluated the effects of the extracts of 15 medicinal herbs on growth and biofilm formation in five oral pathogenic bacterial strains. The herbs were processed into extracts, and bacterial strains were cultured. Then, bacterial growth and biofilm formation were assessed using various methods. Finally, the extract of the herb Hibiscus sabdariffa (hibiscus) was analyzed using high-performance liquid chromatography., Results: Incubation of bacteria with the herbal extracts showed that hibiscus exerted a significant inhibitory effect on all the oral pathogenic bacterial strains evaluated in this study. In addition, the pigment delphinidin-3-sambubioside, which is found in hibiscus extract, was identified as a particularly important inhibitory component., Conclusions: These results lay the ground work for the potential development of novel therapeutic or preventive agents against dental caries and periodontal disease, two major oral diseases., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 Japanese Association for Oral Biology. Published by Elsevier B.V. All rights reserved.)

Cancer stem cells (CSCs) are considered to be responsible for recurrence, metastasis, and resistance to treatment in many types of cancers; therefore, new treatment strategies targeting CSCs are attracting attention. In this study, we fabricated a polyethylene glycol-tagged microwell device that enabled spheroid formation from human oral squamous carcinoma cells. HSC-3 and Ca9-22 cells cultured in the microwell device aggregated and generated a single spheroid per well within 24-48 h. The circular shape and smooth surface of spheroids were maintained for up to five days, and most cells comprising the spheroids were Calcein AM-positive viable cells. Interestingly, the mRNA expression of CSC markers ( Cd44 , Oct4 , Nanog , and Sox2 ) were significantly higher in the spheroids than in the monolayer cultures. CSC marker-positive cells were observed throughout the spheroids. Moreover, resistance to cisplatin was enhanced in spheroid-cultured cells compared to that in the monolayer-cultured cells. Furthermore, some CSC marker genes were upregulated in HSC-3 and Ca9-22 cells that were outgrown from spheroids. In xenograft model, the tumor growth in the spheroid implantation group was comparable to that in the monolayer culture group. These results suggest that our spheroid culture system may be a high-throughput tool for producing uniform CSCs in large numbers from oral cancer cells.

The differentiation and function of osteocytes are controlled by surrounding cells and mechanical stress; however, the detailed mechanisms are unknown. Recent findings suggest that IL-33 is highly expressed in periodontal tissues in orthodontic tooth movement. The present study aimed to elucidate the effect of IL-33 on the expression of regulatory factors for bone remodeling and their molecular mechanisms in the osteocyte-like cell line MLO-Y4. MLO-Y4 cells were treated with IL-33, and the activation of intracellular signaling molecules and transcriptional factors was determined using Western blot analysis and chromatin immunoprecipitation assay. IL-33 treatment enhanced the expression of IL-6 in MLO-Y4 cells, which was suppressed by the knockdown of the IL-33 receptor ST2L. Additionally, IL-33 treatment induced activation of NF-κB, JNK/AP-1, and p38 MAPK signaling pathways in MLO-Y4 cells. Moreover, pretreatment with specific inhibitors of NF-κB, p38 MAPK, and JNK/AP-1 attenuated the IL-33-induced expression of IL-6. Furthermore, chromatin immunoprecipitation indicated that IL-33 increased c-Jun recruitment to the IL-6 promoter. Overall, these results suggest that IL-33 induces IL-6 expression and regulates osteocyte function via activation of the NF-κB, JNK/AP-1, and p38 MAPK pathways through interaction with ST2L receptors on the plasma membrane.

Chondrogenesis is strictly regulated by several factors, including cytokines, hormones, and extracellular matrix proteins. Mouse teratocarcinoma-derived lineage cells, differentiate into chondrocytes in the presence of insulin. Although ascorbic acid promotes chondrogenic differentiation, the detailed regulative mechanisms underlying its role in chondrogenesis remain unclear. Therefore, in this study, we evaluated the effects of ascorbic acid on insulin-induced chondrogenic differentiation of ATDC5 cells and the underlying intracellular signaling. The results revealed that insulin-stimulated collagen deposition, matrix formation, calcification, and expression of chondrogenic differentiation marker genes in ATDC5 cells. This enhancement by insulin was amplified with the addition of ascorbic acid. Molecular analysis revealed that the activation of insulin-induced phosphoinositide 3-kinase (PI3K)/Akt signaling was enhanced in the presence of ascorbic acid. In contrast, Wnt/β-catenin signaling was suppressed during chondrocyte differentiation via upregulation of the Wnt agonist, secreted Frizzled-related protein 1 (sFRP-1) and 3 (sFRP-3). Notably, ascorbic acid upregulated the expression of insulin receptors and their substrates (IRS-1 and IRS-2). Furthermore, ascorbic acid reversed the suppression of IRS-1 and IRS-2 protein by insulin. These results indicate that ascorbic acid positively regulates the chondrogenic differentiation of ATDC5 cells via enhancement of insulin signaling. Our findings provide a substantial basis for further elucidation of the regulatory mechanisms of chondrocyte differentiation and the pathophysiology of OA, thus aiding in development of effective treatment strategies., (© 2023 International Federation of Cell Biology.)

The effect of hydrogen peroxide, an antiseptic dental treatment, on Aggregatibacter actinomycetemcomitans , the main causative agent of localized invasive periodontitis, was investigated. Hydrogen peroxide treatment (0.06%, 4× minimum inhibitory concentration) resulted in the persistence and survival of approximately 0.5% of the bacterial population. The surviving bacteria did not genetically acquire hydrogen peroxide resistance but exhibited a known persister behavior. Sterilization with mitomycin C significantly reduced the number of A. actinomycetemcomitans persister survivors. RNA sequencing of hydrogen peroxide-treated A. actinomycetemcomitans showed elevated expression of Lsr family members, suggesting a strong involvement of autoinducer uptake. In this study, we found a risk of A. actinomycetemcomitans persister residual from hydrogen peroxide treatment and hypothesized associated genetic mechanisms of persister from RNA sequencing.

In recent years, magnesium hydroxide has been widely studied due to its bioactivity and biocompatibility. The bactericidal effects of magnesium hydroxide nanoparticles on oral bacteria have also been reported. Therefore, in this study, we investigated the biological effects of magnesium hydroxide nanoparticles on inflammatory responses induced by periodontopathic bacteria. Macrophage-like cells, namely J774.1 cells, were treated with LPS derived from Aggregatibacter actinomycetemcomitans and two different sizes of magnesium hydroxide nanoparticles (NM80/NM300) to evaluate their effects on the inflammatory response. Statistical analysis was performed using an unresponsive Student's t-test or one-way ANOVA followed by Tukey's post hoc test. NM80 and NM300 inhibited the expression and secretion of IL-1β induced by LPS. Furthermore, IL-1β inhibition by NM80 was dependent on the downregulation of PI3K/Akt-mediated NF-κB activation and the phosphorylation of MAPK molecules such as JNK, ERK1/2, and p38 MAPK. By contrast, only the deactivation of the ERK1/2-mediated signaling cascade is involved in IL-1β suppression by NM300. Although the molecular mechanism involved varied with size, these results suggest that magnesium hydroxide nanoparticles have an anti-inflammatory effect against the etiologic factors of periodontopathic bacteria. These properties of magnesium hydroxide nanoparticles can be applied to dental materials.

Although various caries-preventive agents have been developed, dental caries is still a leading global disease, mostly caused by biological factors such as mutans streptococci. Magnesium hydroxide nanoparticles have been reported to exhibit antibacterial effects; however, they are rarely used in oral care practical applications. In this study, we examined the inhibitory effect of magnesium hydroxide nanoparticles on biofilm formation by Streptococcus mutans and Streptococcus sobrinus -two typical caries-causing bacteria. Three different sizes of magnesium hydroxide nanoparticles (NM80, NM300, and NM700) were studied, all of which inhibited biofilm formation. The results showed that the nanoparticles were important for the inhibitory effect, which was not influenced by pH or the presence of magnesium ions. We also determined that the inhibition process was mainly contact inhibition and that medium (NM300) and large (NM700) sizes were particularly effective in this regard. The findings of our study demonstrate the potential applications of magnesium hydroxide nanoparticles as caries-preventive agents.

The trypsin-like peptidase activity assay kit measures the trypsin-like protease produced by three red-complex species, Porphyromonas gingivalis, Tannerella forsythia, and Treponema denticola, causing periodontitis, and detects the presence of these bacteria in samples. The purpose of this study was to investigate the relationship between the detection of TLPs by a novel TLP-AA, ADCHECK and the detection of red-complex pathogens by real-time PCR using tongue swabs from patients with periodontitis. The detection limit of trypsin-like protease activity by ADCHECK was validated using the culture supernatants of two different Porphyromonas gingivalis bacterial strains. Real-time PCR was performed to determine the number of red-complex species in the tongue coatings of patients with periodontal disease. Trypsin-like protease activity in tongue-swab samples was scored using ADCHECK. ADCHECK successfully detected trypsin-like protease activity in 103 Porphyromonas gingivalis bacterial strains. The specificity, positive predictive value, negative predictive value, and accuracy of ADCHECK for the presence of red-complex pathogens determined by real-time PCR were 90%, 97%, 98%, and 92%, respectively. ADCHECK is an effective tool for the detection of red-complex pathogens.

Background: Periodontal pathogens are related to the incidence of systemic diseases. This study aimed to examine whether periodontal pathogen burden is associated with the risk of fever onset in older adults. Methods: Older adults in nursing homes, aged ≥65 years, were enrolled. The study was set in Kitakyushu, Japan. The body temperatures of participants were ≥37.2 °C and were recorded for eight months. As periodontal pathogens, Porphyromonas gingivalis, Treponema denticola, and Tannerella forsythia were qualified by a real-time polymerase chain reaction at the baseline. For statistical analysis, the number of bacterial counts was logarithmically conversed to 10 as a base. Results: Data from 56 participants with a median age of 88 (62−98) years were available for analysis. The logarithmic-conversed bacterial counts of T. forsythia, but not P. gingivalis or T. denticola, were associated with the onset of fever in older residents. The Kaplan−Meier method revealed that the group with <104 of T. forsythia had significantly less cumulative fever incidence than the group with ≥104 of T. forsythia. The group with ≥104 of T. forsythia was associated with an increased risk of fever onset (hazard ratio, 3.7; 98% confidence interval, 1.3−10.2; p = 0.012), which was adjusted for possible confounders. Conclusions: Bacterial burden of T. forsythia in the oral cavity was associated with the risk of the onset of fever in older nursing homes residents.

Orthodontic treatment requires the regulation of bone remodeling in both compression and tension sides. Transforming growth factor-β1 (TGF-β1) is an important coupling factor for bone remodeling. However, the mechanism underlying the TGF-β1-mediated regulation of the osteoclast-supporting activity of osteoblasts and stromal cells remain unclear. The current study investigated the effect of TGF-β1 on receptor activator of nuclear factor kappa-B ligand (RANKL) expression in stromal cells induced by 1α,25(OH)2D3 (D3) and dexamethasone (Dex). TGF-β1 downregulated the expression of RANKL induced by D3 and Dex in mouse bone marrow stromal lineage, ST2 cells. Co-culture system revealed that TGF-β1 suppressed osteoclast differentiation from bone marrow cell induced by D3 and Dex-activated ST2 cells. The inhibitory effect of TGF-β1 on RANKL expression was recovered by inhibiting the interaction between TGF-β1 and the TGF-β type I/activin receptor or by downregulating of smad2/3 expression. Interestingly, TGF-β1 degraded the retinoid X receptor (RXR)-α protein which forms a complex with vitamin D receptor (VDR) and regulates transcriptional activity of RANKL without affecting nuclear translocation of VDR and phosphorylation of signal transducer and activator of transcription3 (STAT3). The degradation of RXR-α protein by TGF-β1 was recovered by a ubiquitin-proteasome inhibitor. We also observed that poly-ubiquitination of RXR-α protein was induced by TGF-β1 treatment. These results indicated that TGF-β1 downregulates RANKL expression and the osteoclast-supporting activity of osteoblasts/stromal cells induced by D3 and Dex through the degradation of the RXR-α protein mediated by ubiquitin-proteasome system., Competing Interests: The authors have declared that no competing interests exist.

Soluble tumor necrosis factor receptors 1 and 2 (sTNF-R1 and sTNF-R2) are reported to protect against excessive TNF-α, a primary mediator of systemic responses to infection. This study aimed to investigate the levels of TNF-α, sTNF-R1, and sTNF-R2 in saliva and to verify whether their dynamics are associated with periodontal health. The study population comprised 28 adult patients. Probing pocket depth, clinical attachment level, and bleeding on probing were assessed, and periodontal inflamed surface area (PISA) was calculated. Stimulated saliva was collected before the oral examinations. The levels of TNF-α, sTNF-R1, sTNF-R2, and total protein (TP) in saliva samples were determined. There were significant positive correlations between TNF-α, sTNF-R1, and sTNF-R2 to TP (/TP) in stimulated saliva. Moreover, there were significant positive correlations between PISA and sTNF-R2/TP. Stepwise multiple regression analysis revealed that PISA was significantly associated with sTNF-R2/TP in saliva; however, TNF-α/TP was not significantly associated with PISA. In conclusion, this study demonstrates that significant relationships exist between the salivary levels of TNF-α and sTNF-R1, and that salivary sTNF-R2 is associated with the expansion of inflamed periodontal tissue.

Aim: To evaluate the association between sleep duration and severe periodontitis in Japanese workers., Materials and Methods: This cross-sectional study included 1130 workers (mean age 43.0 years) who underwent full-mouth periodontal examinations and health check-ups and completed a self-administered questionnaire that included questions on sleep duration. Logistic regression and a restricted cubic spline model were used to analyse the data., Results: Severe periodontitis was identified in 6.3% of the study population. Those with <5, 5-5.9, 6-6.9, 7-7.9, and ≥8 hr of sleep were 6.7%, 17.4%, 40.3%, 26.3%, and 8.9%, respectively. After adjusting for potential confounders, study participants who slept <5 hr were more likely to have severe periodontitis (adjusted odds ratio = 2.64; 95% confidence interval = 1.06-6.60) than those who slept 7-7.9 hr. The spline model, with a reference value of 399 min (the median sleep duration), showed a non-linear association between sleep duration and severe periodontitis, where an increased prevalence of severe periodontitis was observed only among those with a shorter sleep duration. The prevalence of severe periodontitis did not increase with longer sleep duration., Conclusions: Short sleep duration was associated with severe periodontitis in this cohort of Japanese adults., (© 2021 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.)

Objective: To investigate the interrelationships among concerns regarding dental visits, the status of regular dental visits, and periodontal health during the coronavirus disease 2019 (COVID-19) pandemic., Background: Continuous oral health care and regular dental visits are important for maintaining periodontal health. Due to the possibility of contracting COVID-19, individuals have been reluctant to visit medical institutions. It is unclear how the periodontal health of the Japanese population has been affected by the interruption of regular dental visits during the COVID-19 pandemic and how concerns regarding dental visits have affected attendance at regular dental visits., Methods: This study included 199 Japanese office workers in one municipal office at Fukuoka Prefecture, Japan (average age = 42.6 years; age range = 19-77 years; 123 men and 76 women). Periodontitis was defined based on a full-mouth periodontal examination. The status of regular dental visits during the COVID-19 pandemic and concerns regarding dental visits were obtained via questionnaire. We tested the hypothesis that concerns regarding dental visits would indirectly affect periodontal health through the interruption of regular dental visits during the COVID-19 pandemic. We used mediation analysis, in which concerns regarding dental visits (present or absent) were set as the exposure, periodontitis (present or absent) was set as the outcome, and the status of regular dental visits (continued during the COVID-19 pandemic or not) was set as the mediator., Results: Of the 199 study participants, 108 had a habit of attending regular dental visits. Of these, 31 (28.7%) discontinued regular dental visits during the COVID-19 pandemic. Compared to the individuals who continued regular dental visits, those who discontinued regular dental visits had a higher prevalence of periodontitis (49.4% vs 77.4%, p < 0.05) and concerns regarding dental visits (22.1% vs 64.5%, p < 0.05). Discontinuing regular dental visits significantly mediated the association between concerns regarding dental visits and periodontitis (natural indirect effect: odds ratio = 1.68, 95% confidence interval = 1.02-2.79, proportion mediated = 64.3%)., Conclusion: The study results showed that individuals who discontinued regular dental visits during the COVID-19 pandemic due to concerns regarding dental visits had relatively poor periodontal health., (© 2021 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.)

Periodontal disease is an inflammatory disease caused by periodontopathogenic bacteria, which eventually leads to bone tissue (alveolar bone) destruction as inflammation persists. Periodontal tissues have an immune system against the invasion of these bacteria, however, due to the persistent infection by periodontopathogenic bacteria, the host innate and acquired immunity is impaired, and tissue destruction, including bone tissue destruction, occurs. Osteoclasts are essential for bone destruction. Osteoclast progenitor cells derived from hematopoietic stem cells differentiate into osteoclasts. In addition, bone loss occurs when bone resorption by osteoclasts exceeds bone formation by osteoblasts. In inflammatory bone disease, inflammatory cytokines act on osteoblasts and receptor activator of nuclear factor-κB ligand (RANKL)-producing cells, resulting in osteoclast differentiation and activation. In addition to this mechanism, pathogenic factors of periodontal bacteria and mechanical stress activate osteoclasts and destruct alveolar bone in periodontitis. In this review, we focused on the mechanism of osteoclast activation in periodontitis and provide an overview based on the latest findings., (© 2021 The Authors.)

A spheroid is known as a three-dimensional culture model, which better simulates the physiological conditions of stem cells. This study is aimed at identifying genes specifically expressed in spheroid-cultured human periodontal ligament mesenchymal stem cells (hPDLMSCs) using RNA-seq analysis to evaluate their functions. Transcriptome analysis was performed using spheroid and monolayer cultures of hPDLMSCs from four patients. Cluster and Gene Ontology analyses revealed that genes involved in cell-cell adhesion as well as the G2/M and G1/S transitions of mitotic cell cycles were strongly expressed in the monolayer culture group. However, genes involved in the negative regulation of cell proliferation, histone deacetylation, and bone morphogenetic protein signaling were strongly expressed in the spheroid culture group. We focused on the transcription factor nuclear receptor subfamily 4 group A member 2 ( NR4A2 ) among the genes that were strongly expressed in the spheroid culture group and analyzed its function. To confirm the results of the transcriptome analysis, we performed real-time polymerase chain reaction and western blotting analyses. Interestingly, we found that the mRNA and protein expressions of NR4A2 were strongly expressed in the spheroid-cultured hPDLMSCs. Under osteogenic differentiation conditions, we used siRNA to knock down NR4A2 in spheroid-cultured hPDLMSCs to verify its role in osteogenesis. We found that NR4A2 knockdown significantly increased the levels of mRNA expression for osteogenesis-related genes alkaline phosphatase ( ALP ), Osteopontin ( OPN ), and type 1 collagen ( COL1 ) (Student's paired t -test, p < 0.05). ALP activity was also significantly increased when compared to the negative control group (Student's paired t -test, p < 0.05). Additionally, spheroid-cultured hPDLMSCs transfected with siNR4A2 were cultured for 12 days, resulting in the formation of significantly larger calcified nodules compared to the negative control group (Student's paired t -test, p < 0.05). On the other hand, NR4A2 knockdown in hPDLMSC spheroid did not affect the levels of chondrogenesis and adipogenesis-related genes under chondrogenic and adipogenic conditions. These results suggest that NR4A2 negatively regulates osteogenesis in the spheroid culture of hPDLMSCs., Competing Interests: The authors declare no conflicts of interest associated with this manuscript., (Copyright © 2021 Takenori Suga et al.)

Objectives: N-benzoyl-DL-arginine peptidase (trypsin-like peptidase) is specifically produced by certain strains of periodontitis-associated bacteria. We aimed to examine the effectiveness of an objectively quantified trypsin-like peptidase activity assay (TLP-AA) for detecting severe periodontitis., Methods: The study population included 347 adults (108 men and 239 women; average age, 43.3 years) who underwent a full-mouth periodontal examination. Specimens for the TLP-AA were obtained using tongue swabs. Using a color reader, the TLP-AA results were obtained as a* values, with higher positive a* values indicating an increased intense enzymatic activity. The predictive validity of the TLP-AA results for severe periodontitis was assessed using receiver operating characteristic curve analysis and the periodontitis case definition provided by the Centers for Disease Control and Prevention/American Academy of Periodontology as the gold standard. Furthermore, multivariable logistic regression analyses were performed to predict severe periodontitis using the TLP-AA results and health characteristics, as the exposure variables., Results: Severe periodontitis was observed in 5.2% of the participants. TLP-AA had high diagnostic accuracy for severe periodontitis, with an area under the curve of 0.83 (95% confidence interval [CI]: 0.75-0.92). The cut-off score for the a* value that best differentiated individuals with severe periodontitis was 0.09, with a sensitivity of 83% and specificity of 77%. Multivariable logistic regression analyses revealed that the TLP-AA results were significantly associated with severe periodontitis after adjusting for health characteristics (adjusted odds ratios: 1.90 [95% CI: 1.37-2.62] for the a* value)., Conclusions: Objectively quantified TLP-AA results are potentially useful for detecting severe periodontitis in epidemiological surveillance., Competing Interests: The authors report no conflicts of interest related to this study.

We aimed to assess the validity of the self-report questionnaire for periodontitis in a Japanese population. A Japanese 9-item self-report questionnaire, developed by translating English-version questions that were used to detect periodontitis, was validated against full-mouth clinically-assessed periodontitis in 949 Japanese adults (average age = 43.2 years). Multivariable logistic regression modeling was used to calculate the area under the receiver operating characteristic curve (AUC), wherein the periodontitis case definition of the Centers for Disease Control and Prevention/American Academy of Periodontology was considered the gold standard. Severe, moderate, and mild periodontitis were identified in 6.2%, 30.0%, and 6.7% of the study population, respectively. Self-reported oral health questions combined with socio-demographic and health-related variables had an AUC > 0.70 (range, 0.71-0.87) for any periodontitis category. Four oral health questions ("have gum disease," "loose tooth," "lost bone," and "bleeding gums") were selected in the parsimonious model for severe periodontitis. The periodontitis screening score generated by the responses to these four questions had an AUC, sensitivity, and specificity of 0.82, 73.1%, and 74.3%, respectively, where the cut-off was set at 2 points. In conclusion, a locally adapted version of the self-report questionnaire had an acceptable diagnostic capacity for the detection of periodontitis in this study population., (© 2021. The Author(s).)

Immunoreceptors expressed on osteoclast precursor cells modify osteoclast differentiation and bone resorption activity. Dectin-1 is a lectin receptor of β-glucan and is specifically expressed in osteoclast precursor cells. In this study, we evaluated the bioactivity of β-glucan on receptor activator of nuclear factor-kappa B ligand (RANKL)-induced osteoclastogenesis and observed that glucan from baker's yeast inhibited this process in mouse bone marrow cells and dectin-1-overexpressing RAW264.7 (d-RAW) cells. In conjunction, RANKL-induced nuclear factor of activated T cell c1 expression was suppressed, subsequently downregulating TRAP and Oc-stamp. Additionally, nuclear factor-kappa B activation and the expression of c-fos and Blimp1 were reduced in d-RAW cells. Furthermore, glucan from baker's yeast induced the degradation of Syk protein, essential factor for osteoclastogenesis. These results suggest that glucan from baker's yeast suppresses RANKL-induced osteoclastogenesis and can be applied as a new treatment strategy for bone-related diseases., (© 2020 Wiley Periodicals LLC.)

Magnesium hydroxide nanoparticles are widely used in medicinal and hygiene products because of their low toxicity, environment-friendliness, and low cost. Here, we studied the effects of three different sizes of magnesium hydroxide nanoparticles on antibacterial activity: NM80, NM300, and NM700. NM80 (D 50 = 75.2 nm) showed a higher bactericidal effect against Escherichia coli than larger nanoparticles (D 50 = 328 nm (NM300) or 726 nm (NM700)). Moreover, NM80 showed a high bactericidal effect against not only exponential cells but also persister cells, which are difficult to eliminate owing to their high tolerance to antibiotics. NM80 eliminated strains in which magnesium-transport genes were knocked out and exhibited a bactericidal effect similar to that observed in the wild-type strain. The bactericidal action involved physical cell damage, as confirmed using scanning electron microscopy, which showed that E. coli cells treated with NM80 were directly injured.

Although the anti-tumor and anti-infective properties of β-glucans have been well-discussed, their role in bone metabolism has not been reviewed so far. This review discusses the biological effects of β-glucans on bone metabolisms, especially on bone-resorbing osteoclasts, which are differentiated from hematopoietic precursors. Multiple immunoreceptors that can recognize β-glucans were reported to be expressed in osteoclast precursors. Coordinated co-stimulatory signals mediated by these immunoreceptors are important for the regulation of osteoclastogenesis and bone remodeling. Curdlan from the bacterium Alcaligenes faecalis negatively regulates osteoclast differentiation in vitro by affecting both the osteoclast precursors and osteoclast-supporting cells. We also showed that laminarin, lichenan, and glucan from baker's yeast, as well as β-1,3-glucan from Euglema gracilisas, inhibit the osteoclast formation in bone marrow cells. Consistent with these findings, systemic and local administration of β-glucan derived from Aureobasidium pullulans and Saccharomyces cerevisiae suppressed bone resorption in vivo. However, zymosan derived from S. cerevisiae stimulated the bone resorption activity and is widely used to induce arthritis in animal models. Additional research concerning the relationship between the molecular structure of β-glucan and its effect on osteoclastic bone resorption will be beneficial for the development of novel treatment strategies for bone-related diseases.

β-glucans are potent immunomodulators, with effects on innate and adaptive immune responses via dectin-1 as the main receptor. In this study, we investigated the biological effect of β-glucan from Schizophyllum commune, called Schizophyllan (SPG) on Interleukin-10 (IL-10) expression induced by a lipopolysaccharide (LPS) from Aggregatibacter actinomycetemcomitans in murine macrophages (J774.1). SPG and dectin-1 interaction up-regulates LPS-induced IL-10 expression. The regulative effect of SPG on IL-10 expression is dependent on prolongation of nuclear translocation activity of nuclear factor-kappa B (NF-κBα) pathway induced by LPS. We also found that LPS-induced phosphorylation of mitogen- and stress-activated protein kinase 1 (MSK1) and cAMP-responsive-element-binding protein (CREB), followed by up-regulation of IL-10, was stimulated by SPG priming via activation of the spleen tyrosine kinase (Syk). Our data indicate that SPG augments the anti-inflammatory response in murine macrophages which can be useful to create an intervention for periodontal disease treatment., (Copyright © 2020 Elsevier Ltd. All rights reserved.)

Background: Bacteria survive in various environments by forming biofilms. Bacterial biofilms often cause significant problems to medical instruments and industrial processes. Techniques to inhibit biofilm formation are essential and have wide applications. In this study, we evaluated the ability of two types of biosurfactants (rhamnolipids and surfactin) to inhibit growth and biofilm formation ability of oral pathogenic bacteria such as Aggregatibacter actinomycetemcomitans, Streptococcus mutans, and Streptococcus sanguinis., Results: Rhamnolipids inhibited the growth and biofilm formation ability of all examined oral bacteria. Surfactin showed effective inhibition against S. sanguinis ATCC10556, but lower effects toward A. actinomycetemcomitans Y4 and S. mutans UA159. To corroborate these results, biofilms were observed by scanning electron microscopy (SEM) and confocal microscopy. The observations were largely in concordance with the biofilm assay results. We also attempted to determine the step in the biofilm formation process that was inhibited by biosurfactants. The results clearly demonstrated that rhamnolipids inhibit biofilm formation after the initiation process, however, they do not affect attachment or maturation., Conclusions: Rhamnolipids inhibit oral bacterial growth and biofilm formation by A. actinomycetemcomitans Y4, and may serve as novel oral drug against localized invasive periodontitis.

Persister cells are difficult to eliminate because they are tolerant to antibiotic stress. In the present study, using artificially induced Escherichia coli persister cells, we found that reactive oxygen species (ROS) have greater effects on persister cells than on exponential cells. Thus, we examined which types of ROS could effectively eliminate persister cells and determined the mechanisms underlying the effects of these ROS. Ultraviolet (UV) light irradiation can kill persister cells, and bacterial viability is markedly increased under UV shielding. UV induces the production of ROS, which kill bacteria by moving toward the shielded area. Electron spin resonance-based analysis confirmed that hydroxyl radicals are produced by UV irradiation, although singlet oxygen is not produced. These results clearly revealed that ROS sterilizes persister cells more effectively compared to the sterilization of exponential cells ( ** p < 0.01). These ROS do not injure the bacterial cell wall but rather invade the cell, followed by cell killing. Additionally, the sterilization effect on persister cells was increased by exposure to oxygen plasma during UV irradiation. However, vapor conditions decreased persister cell sterilization by reducing the levels of hydroxyl radicals. We also verified the effect of ROS against bacteria in biofilms that are more resistant than planktonic cells. Although UV alone could not completely sterilize the biofilm bacteria, UV with ROS achieved complete sterilization. Our results demonstrate that persister cells strongly resist the effects of antibiotics and starvation stress but are less able to withstand exposure to ROS. It was shown that ROS does not affect the cell membrane but penetrates it and acts internally to kill persister cells. In particular, it was clarified that the hydroxy radical is an effective sterilizer to kill persister cells., (Copyright © 2020 Kawano, Yamasaki, Sakakura, Takatsuji, Haruyama, Yoshioka and Ariyoshi.)

This study aimed to explore whether the Trypsin-Like Peptidase Activity Assay Kit (TLP-AA-Kit), which measures the activity of N-benzoyl-dl-arginine peptidase (trypsin-like peptidase), can be used as a reliable tool for periodontitis detection in population-based surveillance. In total, 105 individuals underwent a full-mouth periodontal examination and provided tongue swabs as specimens for further analyses. The results of the TLP-AA-Kit were scored between 1 and 5; higher scores indicated higher trypsin concentrations. Receiver operating characteristic analyses were used to evaluate the predictive validity of the TLP-AA-Kit, where the periodontitis case definition provided by the Centers for Disease Control/American Academy of Periodontology served as the reference. Severe and moderate periodontitis were identified in 4.8% and 16.2% of the study population, respectively. The TLP-AA-Kit showed high diagnostic accuracy for severe periodontitis, with an area under the curve of 0.93 (95% confidence interval = 0.88-0.99). However, the diagnostic accuracy of the TLP-AA-Kit for moderate/severe periodontitis was not reliable. While further studies are necessary to validate our results, the results provided herein highlight the potential of the TLP-AA-Kit as a useful tool for the detection of periodontitis, particularly in severe cases, for population-based surveillance.

Background: To evaluate the efficacy of a newly developed electric toothbrush in reducing dental plaque via a quantitative light-induced fluorescence-digital (QLF-D)-applied visualisation system in the brush head., Methods: Participants included 20 adults aged 19 to 28 years. Participants were randomly assigned either (i) an electric toothbrush with a monitor to visualise red-fluorescent dental plaque via a camera built into the brush head (monitor usage group, n = 10) or (ii) an electric toothbrush without a monitor (monitor-non-use group, n = 10). The amount of dental plaque was assessed by personal hygiene performance (PHP) at baseline and 1 week later., Results: In the monitor-usage group, PHP score was significantly lower at the 1-week follow-up than at baseline (6 vs 16; range, 0-12 vs 13-21; P = 0.029). This change was not observed in the monitor-non-use group (14 vs 13; range, 6-21 vs 2-26; P = 0.778). After 1 week, the change in PHP scores in the monitor usage group was significantly greater than that in the monitor non-use group (- 10 vs 0; range, - 21 to 9 vs - 8 to 16; P = 0.021)., Conclusions: Our results clearly demonstrate that brushing teeth while looking at a monitor that depicts red-autofluorescent dental plaque via application of QLF-D improved the efficacy of dental-plaque removal relative to brushing teeth without a monitor., Trial Registration: Trial registration number: UMIN000033699. Name of registry: Study on effect of new devise for oral care on dental plaque clearance. Date of registration: 8th September 2018. Status of registration: Completed.

Introduction: Human periodontal ligament mesenchymal stem cells (hPDLMSCs) have been known that they play important roles in homeostasis and regeneration of periodontal tissues. Additionally, spheroids are superior to monolayer-cultured cells. We investigated the characteristics and potential of periodontal tissue regeneration in co-cultured spheroids of hPDLMSCs and human umbilical vein endothelial cells (HUVECs) in vitro and in vivo ., Methods: Co-cultured spheroids were prepared with cell ratios of hPDLMSCs: HUVECs = 1:1, 1:2, and 2:1, using microwell chips. Real-time polymerase chain reaction (PCR) analysis, Enzyme-Linked Immuno Sorbent Assay (ELISA), and nodule formation assay were performed to examine the properties of co-cultured spheroids. Periodontal tissue defects were prepared in the maxillary first molars of rats and subjected to transplantation assay., Results: The expression levels of stemness markers, vascular endothelial growth factor ( VEGF ), osteogenesis-related genes were up-regulated in co-cultured spheroids, compared with monolayer and spheroid-cultured hPDLMSCs. The nodule formation was also increased in co-cultured spheroids, compared with monolayer and spheroid cultures of hPDLMSCs. Treatment with co-cultured spheroids enhanced new cementum formation after 4 or 8 weeks of transplantation, although there was no significant difference in the new bone formation between co-cultured spheroids and hPDLMSC spheroids., Conclusions: We found that co-cultured spheroids enhance the periodontal tissue regeneration. Co-cultured spheroids of hPDLMSCs and HUVECs may be a useful therapy that can induce periodontal tissue regeneration., Competing Interests: The authors declare no competing interests., (© 2020 The Japanese Society for Regenerative Medicine. Production and hosting by Elsevier B.V.)

As chondrocytes fail to retain their chondrogenic potential in two-dimensional monolayer cultures, several three-dimensional culture systems have been employed for investigating the physiology and pathophysiology in articular cartilage tissues. In this study, we introduced a polyethylene glycol-coated microfabricated chip that enables spheroid formation from ATDC5 cell line, commonly used as a model for in vitro chondrocyte research. ATDC5 cells cultured in our devices aggregated immediately and generated a single spheroid per well within 24 h. Most cells in spheroids cultured in differentiation medium were viable and the circular shape and smooth surface of the spheroid were maintained up to 14 d in culture. We also detected potent hypoxia conditions, a key factor in chondrogenesis, in whole lesions of ATDC5 spheroids. Expression of chondrogenesis-related genes and type X collagen protein was significantly increased in ATDC5 spheroids grown in differentiation medium, compared with monolayer-cultured ATDC5 cells. We also demonstrated that the differentiation medium-induced Akt protein phosphorylation was upregulated in ATDC5 cells cultured in our spheroid device, suggesting that enhancement of chondrogenic potential in ATDC5 spheroids results from PI3/Akt signaling activation. These results indicated that our spheroid culture system could constitute a high-throughput strategy approach towards elucidating the molecular mechanisms that regulate chondrogenesis.

Interleukin-17A (IL-17A), a major effector cytokine secreted by T helper 17 (Th17) cells, is elevated in atherosclerosis lesions. The purpose of this study was to assess the role of IL-17A in the pathogenesis of atherosclerosis. To measure the expression of adhesion molecules, human umbilical vein endothelial cells (HUVECs) and U937 cells were stimulated with IL-17A. Western blot and real-time polymerase chain reaction analyses revealed that intercellular adhesion molecule-1 (ICAM-1) and vascular cell adhesion molecule-1 (VCAM-1) expression in HUVECs, and very late antigen-4 (VLA-4), lymphocyte function-associated antigen-1 (LFA-1) and macrophage-1 antigen (MAC-1) expression in U937 cells was upregulated by IL-17A. Furthermore, IL-17A stimulation resulted in mRNA and protein expression of scavenger receptor (LOX-1) in phorbol 12-myristate 13-acetate (PMA)-activated U937 cells. Oil Red O also demonstrated that IL-17A enhanced foam cell formation by PMA-activated U937 cells induced by oxidized low-density lipoprotein (ox-LDL), and this enhancement of ox-LDL-induced foam cell formation in IL-17A-treated U937 cells was downregulated by transfection of LOX-1 siRNA. These results indicated that IL-17A induced the expression of adhesion molecules, promoted the adherence of monocytes to vascular endothelial cells. IL-17A also stimulated ox-LDL-induced foam cell formation via upregulation of LOX-1 in activated macrophages. IL-17A may be responsible for the pathogenesis of atherosclerosis by inducing the adhesion of leukocytes to vascular endothelium and foam cell formation.

Nanoparticles are used in industry and medicine, because of their physiochemical properties, such as size, charge, large surface area and surface reactivity. Recently, metal nanoparticles were reported to show cell toxicity on cancer cells. In this study, we focused novel platinum nanoparticles-conjugated latex beads (P2VPs), platinum nanocomposite (PtNCP) beads, and investigated the possibility to incorporate novel anti-cancer effect of these combined nanoparticles. Oral squamous cell carcinoma cell lines, HSC-3-M3 cells were injected subcutaneously into the back of nude mice to produce a xenograft model. PtNCP beads were injected locally and examined by measuring tumor volume and comparing pathological histology. PtNCP beads treatment suppressed tumor growth and identified increasing pathological necrotic areas, in vivo. PtNCP beads inhibited the cell viability of HSC-3-M3 cells in dose-dependent manner and induced the cytotoxicity with extracellular LDH value, in vitro. Furthermore, SEM images were morphologically observed in PtNCP beads-treated HSC-3-M3 cells. The aggregation of the PtNCP beads on the cell membrane, the destructions of the cell membrane and globular structures were observed in the SEM image. Our results indicated that a potential anti-cancer effect of the PtNCP beads, suggesting the possibility as a therapeutic tool for cancer cell-targeted therapy. © 2019 Wiley Periodicals, Inc. J Biomed Mater Res Part B: Appl Biomater 107B: 2281-2287, 2019., (© 2019 Wiley Periodicals, Inc.)

Macrophages, critical modulators of the immune response, polarize into various phenotypes, including M1 and M2. M1 macrophages are typically activated by lipopolysaccharide and produce proinflammatory cytokines. Conversely, M2 macrophages are activated by stimulation with interleukin 4 (IL)-4 and promote tissue remodeling and anti-inflammatory reactions. Recently, polyunsaturated fatty acids (PUFAs) have been shown to play important roles in the regulation of inflammation. Docosahexaenoic acid (DHA), a PUFA, has anti-inflammatory effects on chronic inflammatory disease, but its role in macrophage polarization remains unclear. In this study, we clarified the effects of DHA on macrophage polarization using U937 cells. Treatment with DHA resulted in upregulation of M2 macrophage markers and increased secretion of anti-inflammatory cytokines by U937 cells. IL-4, but not DHA, triggered phosphorylation of signal transducer and activator of transcription 6 (STAT6). DHA enhanced the expression of krüppel-like factor-4 (KLF4), a transcription factor involved in the regulation of macrophage polarization and increased the phosphorylation of p38 mitogen-activated protein kinase (MAPK). A selective inhibitor of p38 MAPK downregulated the expression of CD206 in DHA-treated U937 cells. Moreover, inhibitors of autophagy suppressed the phosphorylation of p38 MAPK and the expression of CD206 in DHA-treated U937 cells. Expression of microtubule-associated protein light chain 3-II, which is involved in autophagosome formation, was enhanced in DHA-treated U937 cells. Taken together, these results indicated that DHA enhanced the expression of M2 macrophage markers through the p38 MAPK signaling pathway and autophagy, suggesting that DHA regulates M2 macrophage polarization and plays an important role in innate immunity., (© 2019 Wiley Periodicals, Inc.)