Palaeoproteomic investigation of an ancient human skeleton with abnormal deposition of dental calculus.

Detailed investigation of extremely severe pathological conditions in ancient human skeletons is important as it could shed light on the breadth of potential interactions between humans and disease etiologies in the past. Here, we applied palaeoproteomics to investigate an ancient human skeletal individual with severe oral pathology, focusing our research on bacterial pathogenic factors and host defense response. This female skeleton, from the Okhotsk period (i.e., fifth to thirteenth century) of Northern Japan, poses relevant amounts of abnormal dental calculus deposition and exhibits oral dysfunction due to severe periodontal disease. A shotgun mass-spectrometry analysis identified 81 human proteins and 15 bacterial proteins from the calculus of the subject. We identified two pathogenic or bioinvasive proteins originating from two of the three "red complex" bacteria, the core species associated with severe periodontal disease in modern humans, as well as two additional bioinvasive proteins of periodontal-associated bacteria. Moreover, we discovered defense response system-associated human proteins, although their proportion was mostly similar to those reported in ancient and modern human individuals with lower calculus deposition. These results suggest that the bacterial etiology was similar and the host defense response was not necessarily more intense in ancient individuals with significant amounts of abnormal dental calculus deposition.

Mechanical stretching determines the orientation of osteoblast migration and cell division.

Osteoblasts alignment and migration are involved in the directional formation of bone matrix and bone remodeling. Many studies have demonstrated that mechanical stretching controls osteoblast morphology and alignment. However, little is known about its effects on osteoblast migration. Here, we investigated changes in the morphology and migration of preosteoblastic MC3T3-E1 cells after the removal of continuous or cyclic stretching. Actin staining and time-lapse recording were performed after stretching removal. The continuous and cyclic groups showed parallel and perpendicular alignment to the stretch direction, respectively. A more elongated cell morphology was observed in the cyclic group than in the continuous group. In both stretch groups, the cells migrated in a direction roughly consistent with the cell alignment. Compared to the other groups, the cells in the cyclic group showed an increased migration velocity and were almost divided in the same direction as the alignment. To summarize, our study showed that mechanical stretching changed cell alignment and morphology in osteoblasts, which affected the direction of migration and cell division, and velocity of migration. These results suggest that mechanical stimulation may modulate the direction of bone tissue formation by inducing the directional migration and cell division of osteoblasts.

Neuropilin 1 (NRP1) Positively Regulates Adipogenic Differentiation in C3H10T1/2 Cells.

Neuropilin 1 (NRP1), a non-tyrosine kinase receptor for several ligands, is highly expressed in many kinds of mesenchymal stem cells (MSCs), but its function is poorly understood. In this study, we explored the roles of full-length NRP1 and glycosaminoglycan (GAG)-modifiable NRP1 in adipogenesis in C3H10T1/2 cells. The expression of full-length NRP1 and GAG-modifiable NRP1 increased during adipogenic differentiation in C3H10T1/2 cells. NRP1 knockdown repressed adipogenesis while decreasing the levels of Akt and ERK1/2 phosphorylation. Moreover, the scaffold protein JIP4 was involved in adipogenesis in C3H10T1/2 cells by interacting with NRP1. Furthermore, overexpression of non-GAG-modifiable NRP1 mutant (S612A) greatly promoted adipogenic differentiation, accompanied by upregulation of the phosphorylated Akt and ERK1/2. Taken together, these results indicate that NRP1 is a key regulator that promotes adipogenesis in C3H10T1/2 cells by interacting with JIP4 and activating the Akt and ERK1/2 pathway. Non-GAG-modifiable NRP1 mutant (S612A) accelerates the process of adipogenic differentiation, suggesting that GAG glycosylation is a negative post-translational modification of NRP1 in adipogenic differentiation.

The Impact of Oral Health Behaviors, Health Belief Model, and Absolute Risk Aversion on the Willingness of Japanese University Students to Undergo Regular Dental Check-Ups: A Cross-Sectional Study.

Oral health behaviors, risk aversion, and the health belief model are associated with health behaviors. However, there have been few studies that investigated the association between these factors and the willingness to undergo regular dental check-ups. The purpose of this cross-sectional study was to investigate the associations between the willingness of Japanese university students to undergo regular dental check-ups and oral health behaviors, the health belief model, and absolute risk aversion. An analysis was conducted with the cooperation of questionnaire respondents (n = 748) who underwent dental check-ups at Okayama University. The students answered questionnaires on oral health behaviors, the health belief model, absolute risk aversion, and willingness to undergo regular dental check-ups. The logistic regression analysis showed significant positive associations (p < 0.05) between oral health behaviors (use of the inter-dental brush and the dental floss) and the health belief model with the willingness to undergo regular dental check-ups. However, there was no significant association with absolute risk aversion (p > 0.05). These results suggest that willingness to undergo regular dental check-ups was associated with oral health behaviors and the health belief model, but not with absolute risk aversion.

Maternal Gut Microbiome Decelerates Fetal Endochondral Bone Formation by Inducing Inflammatory Reaction.

To investigate the effect of the maternal gut microbiome on fetal endochondral bone formation, fetuses at embryonic day 18 were obtained from germ-free (GF) and specific-pathogen-free (SPF) pregnant mothers. Skeletal preparation of the fetuses' whole bodies did not show significant morphological alterations; however, micro-CT analysis of the tibiae showed a lower bone volume fraction in the SPF tibia. Primary cultured chondrocytes from fetal SPF rib cages showed a lower cell proliferation and lower accumulation of the extracellular matrix. RNA-sequencing analysis showed the induction of inflammation-associated genes such as the interleukin (IL) 17 receptor, IL 6, and immune-response genes in SPF chondrocytes. These data indicate that the maternal gut microbiome in SPF mice affects fetal embryonic endochondral ossification, possibly by changing the expression of genes related to inflammation and the immune response in fetal cartilage. The gut microbiome may modify endochondral ossification in the fetal chondrocytes passing through the placenta.

Outer membrane vesicles of Porphyromonas gingivalis: Novel communication tool and strategy.

Extracellular vesicles (EVs) have been recognized as a universal method of cellular communications and are reportedly produced in bacteria, archaea, and eukaryotes. Bacterial EVs are often called "Outer Membrane Vesicles" (OMVs) as they were the result of a controlled blebbing of the outer membrane of gram-negative bacteria such as Porphyromonas gingivalis (P. gingivalis). Bacterial EVs are natural messengers, implicated in intra- and inter-species cell-to-cell communication among microorganism populations present in microbiota. Bacteria can incorporate their pathogens into OMVs; the content of OMVs differs, depending on the type of bacteria. The production of distinct types of OMVs can be mediated by different factors and routes. A recent study highlighted OMVs ability to carry crucial molecules implicated in immune modulation, and, nowadays, they are considered as a way to communicate and transfer messages from the bacteria to the host and vice versa. This review article focuses on the current understanding of OMVs produced from major oral bacteria, P. gingivalis: generation, characteristics, and contents as well as the involvement in signal transduction of host cells and systemic diseases. Our recent study regarding the action of P. gingivalis OMVs in the living body is also summarized.

Extracellular vesicles of P. gingivalis-infected macrophages induce lung injury.

Periodontal diseases are common inflammatory diseases that are induced by infection with periodontal bacteria such as Porphyromonas gingivalis (Pg). The association between periodontal diseases and many types of systemic diseases has been demonstrated; the term "periodontal medicine" is used to describe how periodontal infection/inflammation may impact extraoral health. However, the molecular mechanisms by which the factors produced in the oral cavity reach multiple distant organs and impact general health have not been elucidated. Extracellular vesicles (EVs) are nano-sized spherical structures secreted by various types of cells into the tissue microenvironment, and influence pathophysiological conditions by delivering their cargo. However, a detailed understanding of the effect of EVs on periodontal medicine is lacking. In this study, we investigated whether EVs derived from Pg-infected macrophages reach distant organs in mice and influence the pathophysiological status. EVs were isolated from human macrophages, THP-1 cells, infected with Pg. We observed that EVs from Pg-infected THP-1 cells (Pg-inf EVs) contained abundant core histone proteins such as histone H3 and translocated to the lungs, liver, and kidneys of mice. Pg-inf EVs also induced pulmonary injury, including edema, vascular congestion, inflammation, and collagen deposition causing alveoli destruction. The Pg-inf EVs or the recombinant histone H3 activated the NF-κB pathway, leading to increase in the levels of pro-inflammatory cytokines in human lung epithelial A549 cells. Our results suggest a possible mechanism by which EVs produced in periodontal diseases contribute to the progression of periodontal medicine.

Living with Family Is Directly Associated with Regular Dental Checkup and Indirectly Associated with Gingival Status among Japanese University Students: A 3-Year Cohort Study.

Although some studies showed that lifestyle was associated with oral health behavior, few studies investigated the association between household type and oral health behavior. The aim of this prospective cohort study was to investigate the association between household type, oral health behavior, and periodontal status among Japanese university students. Data were obtained from 377 students who received oral examinations and self-questionnaires in 2016 and 2019. We assessed periodontal status using the percentage of bleeding on probing (%BOP), probing pocket depth, oral hygiene status, oral health behaviors, and related factors. We used structural equation modeling to determine the association between household type, oral health behaviors, gingivitis, and periodontitis. At follow-up, 252 students did not live with their families. The mean ± standard deviation of %BOP was 35.5 ± 24.7 at baseline and 32.1 ± 25.3 at follow-up. In the final model, students living with their families were significantly more likely to receive regular dental checkup than those living alone. Regular checkup affected the decrease in calculus. The decrease in calculus affected the decrease in %BOP over 3 years. Living with family was directly associated with regular dental checkups and indirectly contributed to gingival status among Japanese university students.

Parafunctional masseter muscle activity during waking is related to periodontitis progression: A pilot prospective cohort study.

AIM: The purpose of this pilot prospective cohort study was to investigate the effects of parafunctional masseter muscle activity on periodontitis progression among patients receiving supporting periodontal therapy (SPT). MATERIALS AND METHODS: We collected data of patients treated at Okayama University Hospital from August 2014 to September 2018. The progression group was defined as patients with ≥2 teeth demonstrating a longitudinal loss of proximal attachment of ≥3 mm during the 3-year study period and/or at least one tooth extraction due to periodontitis progression. Surface electromyography of masseter muscles at baseline was continuously recorded while patients were awake and asleep. RESULTS: We analysed 48 patients (36 females) aged 66.8 ± 9.1 years (mean ± SD). The rate of parafunctional masseter muscle activity during waking hours and sleeping hours at baseline was 60.4% and 52.1%, respectively. Cox's proportional hazards regression model showed that the incidence of periodontitis progression was significantly associated with number of teeth present (p = 0.001) and parafunctional masseter muscle activity during waking hours (p = 0.041). CONCLUSION: Our results suggest that parafunctional masseter muscle activity during waking hours is a risk factor for periodontitis progression among patients receiving SPT.

Association between Household Exposure to Secondhand Smoke and Dental Caries among Japanese Young Adults: A Cross-Sectional Study.

The long-term effects of secondhand smoke (SHS) on dental caries among Japanese young adults remain unclear. The purpose of this cross-sectional study was to evaluate whether household exposure to SHS is associated with dental caries in permanent dentition among Japanese young adults. The study sample included 1905 first-year university students (age range: 18-19 years) who answered a questionnaire and participated in oral examinations. The degree of household exposure to SHS was categorized into four levels according to the SHS duration: no experience (-), past, current SHS < 10 years, and current SHS ≥ 10 years. Dental caries are expressed as the total number of decayed, missing, and filled teeth (DMFT) score. The relationships between SHS and dental caries were determined by logistic regression analysis. DMFT scores (median (25th percentile, 75th percentile)) were significantly higher in the current SHS ≥ 10 years (median: 1.0 (0.0, 3.0)) than in the SHS-(median: 0.0 (0.0, 2.0)); p = 0.001). DMFT ≥ 1 was significantly associated with SHS ≥ 10 years (adjusted odds ratio: 1.50, 95% confidence interval: 1.20-1.87, p < 0.001). Long-term exposure to SHS (≥10 years) was associated with dental caries in permanent dentition among Japanese young adults.

Outer membrane vesicles derived from Porphyromonas gingivalis induced cell death with disruption of tight junctions in human lung epithelial cells.

OBJECTIVE: Porphyromonas gingivalis (P. gingivalis) is a major bacterium responsible for the progression of periodontitis. P. gingivalis produces small vesicles called outer membrane vesicles (OMVs) containing virulence factors. Increasing evidence suggests a close relationship between periodontitis and respiratory system diseases, such as aspiration pneumonia. However, little is known about whether P. gingivalis OMVs give rise to the impediment of lung epithelial cells. We investigated the effect of the OMVs on cell viability and tight junctions of lung epithelial cells. DESIGN: Human lung epithelial A549 cells were treated with P. gingivalis OMVs. Cell viability was evaluated, and cell morphology was examined using scanning electron and phase contrast microscopies. To detect apoptosis induced by P. gingivalis OMVs, activation of caspase-3 and poly ADP-ribose polymerase (PARP) cleavage was examined by using Western blotting. Immunocytochemistry was performed to stain tight junction proteins. RESULTS: P. gingivalis OMVs decreased cell viability in A549 cells in a dose- and time-dependent manner. Microscopic analysis revealed that the OMVs induced morphological changes leading to irregular cell membrane structures. The OMVs caused cell shrinkage, membrane blebbing, and cytoplasmic expulsion in a dose-dependent manner. Western blot analysis showed the OMVs induced caspase-3 activation and PARP cleavage. Treatment with the OMVs disrupted the intact distributions of tight junction proteins. CONCLUSIONS: These results indicate that P. gingivalis OMVs induced cell death by destroying the barrier system in lung epithelial cells. Our present study raises the possibility that P. gingivalis OMVs is an important factor in the engagement of periodontitis with respiratory system diseases.

Relationship between oral hygiene knowledge, source of oral hygiene knowledge and oral hygiene behavior in Japanese university students: A prospective cohort study.

The aim of this prospective cohort study was to examine whether oral hygiene knowledge, and the source of that knowledge, affect oral hygiene behavior in university students in Japan. An oral exam and questionnaire survey developed to evaluate oral hygiene knowledge, the source of that knowledge, and oral hygiene behavior, such as the frequency of tooth brushing and regular dental checkups and the use of dental floss, was conducted on university student volunteers. In total, 310 students with poor tooth brushing behavior (frequency of tooth brushing per day [≤ once]), 1,963 who did not use dental floss, and 1,882 who did not receive regular dental checkup during the past year were selected. Among these students, 50, 364, and 343 in each respective category were analyzed in over the 3-year study period (follow-up rates: 16.1%, 18.5%, and 18.2%, respectively). The odds ratios (ORs) and 95% confidence intervals (CIs) for oral hygiene behavior were calculated based on oral hygiene knowledge and the source of that knowledge using logistic regression models. The results showed that dental clinics were the most common (> 50%) source of oral hygiene knowledge, and that a more frequent use of dental floss was significantly associated with dental clinics being a source of oral hygiene knowledge (OR, 4.11; 95%CI, 1.871-9.029; p < 0.001). In addition, a significant association was seen between dental clinics being a source of oral hygiene knowledge and more frequent regular dental checkups (OR, 13.626; 95%CI, 5.971-31.095; p < 0.001). These findings suggest the existence of a relationship between dental clinics being the most common source of oral hygiene knowledge and improved oral hygiene behavior in Japanese university students.

Association between Sleep Quality and Duration and Periodontal Disease among University Students: A Cross-Sectional Study.

The purpose of this cross-sectional study was to investigate the association between sleep quality and duration, and periodontal disease among a group of young Japanese university students. First-year students (n = 1934) at Okayama University who voluntarily underwent oral health examinations were included in the analysis. Sleep quality and duration were assessed by the Japanese version of the Pittsburgh Sleep Quality Index. Dentists examined Oral Hygiene Index-Simplified (OHI-S), probing pocket depth (PPD), and percentage of sites with bleeding on probing (BOP). Periodontal disease was defined as presence of PPD ≥ 4 mm and BOP ≥ 30%. Overall, 283 (14.6%) students had periodontal disease. Poor sleep quality was observed among 372 (19.2%) students. Mean (± standard deviation) sleep duration was 7.1 ± 1.1 (hours/night). In the logistic regression analysis, periodontal disease was significantly associated with OHI-S (odds ratio [OR]: 2.30, 95% confident interval [CI]: 1.83-2.90; p < 0.001), but not sleep quality (OR: 1.09, 95% CI: 0.79-1.53; p = 0.577) or sleep duration (OR: 0.98, CI: 0.87-1.10; p = 0.717). In conclusion, sleep quality and duration were not associated with periodontal disease among this group of young Japanese university students.

Caries Increment and Salivary Microbiome during University Life: A Prospective Cohort Study.

The purpose of this 3-year prospective cohort study was to explore the relationship between an increase in dental caries and oral microbiome among Japanese university students. We analyzed 487 students who volunteered to receive oral examinations and answer baseline (2013) and follow-up (2016) questionnaires. Of these students, salivary samples were randomly collected from 55 students at follow-up and analyzed using next-generation sequencing. Students were divided into two groups: increased group (Δdecayed, missing, and filled teeth (ΔDMFT) score increased during the 3-year period) and non-increased group (ΔDMFT did not increase). Thirteen phyla, 21 classes, 32 orders, 48 families, 72 genera, and 156 species were identified. Microbial diversity in the increased group (n = 14) was similar to that in the non-increased group (n = 41). Relative abundances of the family Prevotellaceae (p = 0.007) and genera Alloprevotella (p = 0.007) and Dialister (p = 0.039) were enriched in the increased group compared with the non-increased group. Some bacterial taxonomic clades were differentially present between the two groups. These results may contribute to the development of new dental caries prevention strategies, including the development of detection kits and enlightenment activities for these bacteria.