Characterization of oral microbiota in 6-8-month-old small breed dogs.

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.

The association between trypsin-like protease activity in the oral cavity and kidney function in Japanese workers.

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 m2 . RESULTS: The prevalence for each eGFR category was as follows: ≥90 (31.6%), 60-89 (63.8%) and <60 mL/min/1.73 m2 (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.

The Ability of a Novel Trypsin-like Peptidase Activity Assay Kit to Detect Red-Complex Species.

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.

The Association between Tannerella forsythia and the Onset of Fever in Older Nursing Home Residents: A Prospective Cohort Study.

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.

Mechanisms involved in suppression of osteoclast supportive activity by transforming growth factor-ß1 via the ubiquitin-proteasome system.

Orthodontic treatment requires the regulation of bone remodeling in both compression and tension sides. Transforming growth factor-ß1 (TGF-ß1) is an important coupling factor for bone remodeling. However, the mechanism underlying the TGF-ß1-mediated regulation of the osteoclast-supporting activity of osteoblasts and stromal cells remain unclear. The current study investigated the effect of TGF-ß1 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-ß1 downregulated the expression of RANKL induced by D3 and Dex in mouse bone marrow stromal lineage, ST2 cells. Co-culture system revealed that TGF-ß1 suppressed osteoclast differentiation from bone marrow cell induced by D3 and Dex-activated ST2 cells. The inhibitory effect of TGF-ß1 on RANKL expression was recovered by inhibiting the interaction between TGF-ß1 and the TGF-ß type I/activin receptor or by downregulating of smad2/3 expression. Interestingly, TGF-ß1 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-ß1 was recovered by a ubiquitin-proteasome inhibitor. We also observed that poly-ubiquitination of RXR-α protein was induced by TGF-ß1 treatment. These results indicated that TGF-ß1 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.

Sleep duration and severe periodontitis in middle-aged Japanese workers.

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.

Characterization and Study of Gene Expression Profiles of Human Periodontal Mesenchymal Stem Cells in Spheroid Cultures by Transcriptome Analysis.

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.

Interruption of regular dental visits during the COVID-19 pandemic due to concerns regarding dental visits was associated with periodontitis in Japanese office workers.

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.

Evaluation of the ability of the trypsin-like peptidase activity assay to detect severe periodontitis.

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.

Validation of a self-report questionnaire for periodontitis in a Japanese population.

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.

Odontoblast differentiation is regulated by an interplay between primary cilia and the canonical Wnt pathway.

Primary cilium is a protruding cellular organelle that has various physiological functions, especially in sensory reception. While an avalanche of reports on primary cilia have been published, the function of primary cilia in dental cells remains to be investigated. In this study, we focused on the function of primary cilia in dentin-producing odontoblasts. Odontoblasts, like most other cell types, possess primary cilia, which disappear upon the knockdown of intraflagellar transport protein 88. In cilia-depleted cells, the expression of dentin sialoprotein, an odontoblastic marker, was elevated, while the deposition of minerals was slowed. This was recapitulated by the activation of canonical Wnt pathway, also decreased the ratio of ciliated cells. In dental pulp cells, as they differentiated into odontoblasts, the ratio of ciliated cells was increased, whereas the canonical Wnt signaling activity was repressed. Our results collectively underscore the roles of primary cilia in regulating odontoblastic differentiation through canonical Wnt signaling. This study implies the existence of a feedback loop between primary cilia and the canonical Wnt pathway.

Anti-inflammatory effect of glycyrrhizin with Equisetum arvense extract.

Periodontal disease is the most prevalent infectious disease, and inflammatory mediators play critical roles in its progression. Therefore, controlling pro-inflammatory cytokine production, especially at initial disease stages, is essential to maintaining gingival and periodontal health. Glycyrrhizin (GL) has an anti-inflammatory effect and has been added to toothpaste and mouth rinse to prevent periodontal disease. However, there is a maximum dose for the use of GL. The aim of the present study is to screen plant extracts which can effectively enhance the effects of GL. The effects of extracts from six different plants on GL-suppressed TNF-α expression in Aggregatibacter actinomycetemcomitans (A.a.)-LPS-stimulated human oral keratinocytes (RT7) were examined. Results demonstrated that Equisetum arvense (EA) extract had the strongest additive effect on the suppression of TNF-α by GL at both mRNA and protein levels. In addition, GL downregulated the production of TNF-α by suppressing NF-κB p65 phosphorylation, but not JNK or p38 phosphorylation. In contrast, EA decreased JNK phosphorylation but not NF-κB p65 or p38 phosphorylation. The combination of GL and EA effectively attenuated A.a.-LPS-induced phosphorylation of NF-κB p65 and JNK. Furthermore, an LPS-induced periodontitis rat model showed that GL with EA supplementation significantly downregulated TNF-α mRNA in the gingival tissue. These results indicate that EA can suppress A.a.-LPS-induced pro-inflammatory cytokine production by inhibiting JNK activation and can promote the anti-inflammatory effects of GL. Our findings suggest that a combination of GL and EA may improve the development of new oral hygiene products aimed at enhancing periodontal health.

Dectin-1-mediated suppression of RANKL-induced osteoclastogenesis by glucan from baker's yeast.

Immunoreceptors expressed on osteoclast precursor cells modify osteoclast differentiation and bone resorption activity. Dectin-1 is a lectin receptor of ß-glucan and is specifically expressed in osteoclast precursor cells. In this study, we evaluated the bioactivity of ß-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.

Schizophyllum commune ß-glucan: Effect on interleukin-10 expression induced by lipopolysaccharide from periodontopathic bacteria.

ß-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 ß-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.

A preliminary study of the scope of practice of dental hygienists and oral health providers in Asia.

The aim of the present study was to gather information to clarify the scope of oral health services in Asia, focusing particularly on the role of dental hygienists, anticipating that international exchange of data on oral health would benefit the establishment of such systems in other countries. Data on oral health providers were obtained from reliable sources, cited reports and professional websites, or from embassies of Asian countries located in Japan. The embassies were contacted by email, telephone, post, or interview. The survey was conducted from February 1, 2015, until June 18, 2019. Twenty-eight countries in Asia were selected according to their classification by the United Nations. Among them, 14 countries were found to recognize the professional status of dental hygienists. Nine of the 28 countries had no working dental hygienists, and their role was taken by other oral hygiene providers. It was unclear whether oral hygiene providers were operating in 5 of those countries. Many countries were found to lack formal oral health systems, and some had limited systems in rural areas. Dental hygienists work to prevent oral diseases in order to protect teeth and gums, as is the case in Japan. However, other oral health providers offer other dental services in other Asian countries, probably depending on individual national circumstances.

A Preliminary Study on the Ability of the Trypsin-Like Peptidase Activity Assay Kit to Detect Periodontitis.

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.

3D spheroid culture models for chondrocytes using polyethylene glycol-coated microfabricated chip.

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.

Comparison of dental plaque reduction after use of electric toothbrushes with and without QLF-D-applied plaque visualization: a 1-week randomized controlled trial.

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.

Co-cultured spheroids of human periodontal ligament mesenchymal stem cells and vascular endothelial cells enhance periodontal tissue regeneration.

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.