Isobavachin attenuates osteoclastogenesis and periodontitis-induced bone loss by inhibiting cellular iron accumulation and mitochondrial biogenesis.

As bone-resorbing cells rich in mitochondria, osteoclasts require high iron uptake to promote mitochondrial biogenesis and maintain a high-energy metabolic state for active bone resorption. Given that abnormal osteoclast formation and activation leads to imbalanced bone remodeling and osteolytic bone loss, osteoclasts may be crucial targets for treating osteolytic diseases such as periodontitis. Isobavachin (IBA), a natural flavonoid compound, has been confirmed to be an inhibitor of receptor activator of nuclear factor κB ligand (RANKL)-induced osteoclast differentiation from bone marrow-derived macrophages (BMMs). However, its effects on periodontitis-induced bone loss and the potential mechanism of its anti-osteoclastogenesis effect remain unclear. Our study demonstrated that IBA suppressed RANKL-induced osteoclastogenesis in BMMs and RAW264.7 cells and inhibited osteoclast-mediated bone resorption in vitro. Transcriptomic analysis indicated that iron homeostasis and reactive oxygen species (ROS) metabolic process were enriched among the differentially expressed genes following IBA treatment. IBA exerted its anti-osteoclastogenesis effect by inhibiting iron accumulation in osteoclasts. Mechanistically, IBA attenuated iron accumulation in RANKL-induced osteoclasts by inhibiting the mitogen-activated protein kinase (MAPK) pathway to upregulate ferroportin1 (Fpn1) expression and promote Fpn1-mediated intracellular iron efflux. We also found that IBA inhibited mitochondrial biogenesis and function, and reduced RANKL-induced ROS generation in osteoclasts. Furthermore, IBA attenuated periodontitis-induced bone loss by reducing osteoclastogenesis in vivo. Overall, these results suggest that IBA may serve as a promising therapeutic strategy for bone diseases characterized by osteoclastic bone resorption.

Anti-Porphyromonas gingivalis nanotherapy for maintaining bacterial homeostasis in periodontitis.

Periodontitis is caused by oral flora imbalance, which leads to immune imbalance. Porphyromonas gingivalis is a keystone pathogen in periodontitis, causing the blooming of inflammophilic microbes, and becoming dormant to resist antibiotics. Targeted interventions are needed to destroy this pathogen and collapse its inflammophilic flora. Therefore, a targeting nanoagent antibody-conjugated liposomal drug carrier with ginsenoside Rh2 (A-L-R) was developed for pleiotropic benefits. A-L-R showed high quality in high-performance liquid chromatography (HPLC), Fourier transform infrared (FTIR), and transmission electron microscope (TEM) detection. Only P. gingivalis was influenced by A-L-R, as shown by live/dead cell staining and a series of antimicrobial effects assays. With fluorescence in situ hybridization (FISH) staining and in propidium monoazide-quantitative polymerase chain reaction (PMA-qPCR), the clearance of P. gingivalis by A-L-R was more than for other groups, and only the proportion of P. gingivalis was reduced by A-L-R in monospecies culture. Moreover, in a periodontitis model, A-L-R targeted P. gingivalis with high efficiency and low toxicity, maintaining homeostasis with a relatively stable oral microflora. This targeting nanomedicine offers new strategies for periodontitis therapy, providing a foundation for the prevention and treatment of periodontitis.

Heat Shock Protein Inhibitors Show Synergistic Antibacterial Effects with Photodynamic Therapy on Caries-Related Streptococci In Vitro and In Vivo.

Caries are chronic infections in which the cariogenic biofilm plays a critical role in disease occurrence and progression. Photodynamic therapy (PDT) is a new effective treatment that is receiving wide attention in the antibacterial field, but it can lead to the upregulation of heat shock proteins (HSPs), which enhances bacterial resistance. Herein, we incorporated HSP inhibitors with PDT to evaluate the effect on Streptococcus mutans, Streptococcus sobrinus, and Streptococcus sanguinis under planktonic conditions and on cariogenic biofilms. Additionally, a model of caries was established in 2-week-old rats, and anticaries properties were evaluated by Keyes' scoring. Importantly, the combination of HSP inhibitors and PDT had outstanding efficiency in inhibiting the growth of tested Streptococcus strains and the formation of either monomicrobial or multispecies biofilms in vitro. In addition, the quantity of colonized streptococci and the severity of carious lesions were also distinctly suppressed in vivo. Overall, the synergistic application of HSP inhibitors and PDT has promising potential in the prevention and treatment of dental caries. IMPORTANCE Effective therapies for the prevention and control of caries are urgently needed. Cariogenic streptococci play a key role in the occurrence and progression of caries. Recently, photodynamic therapy has been demonstrated to have good antibacterial efficiency, but it can cause a heat shock response in bacteria, which may weaken its practical effects. We indicate here an effective therapeutic strategy of combining heat shock protein inhibitors and photodynamic therapy, which shows excellent inhibition toward three dominant streptococci related to caries and suppression of carious progression in a rat model. Further development for clinical application is promising.

Profiling the oral microbiomes in patients with Alzheimer's disease.

OBJECTIVES: To analyse the characteristics of the oral microbiomes and expected to find biomarkers about Alzheimer's disease (AD). SUBJECTS AND METHODS: AD patients (n = 26) and cognitive intact people (n = 26) were examined for cognition, depression, oral health and collected saliva and gingival crevicular fluid (GCF) in the morning. Full-length 16S rRNA gene was amplified and sequencing was performed using the PacBio platform. RESULTS: The predominant bacterium of salivary microbiome and periodontal microbiome from AD patients was Streptococcus oralis and Porphyromonas gingivalis, respectively. With respect to ß diversity analysis, there was a significance difference in periodontal microbiome between AD patients and cognitively intact subjects. The relative abundance of Veillonella parvula significantly increased in oral microbiomes from AD patients. Interestingly, the dominant species were different between early-onset AD and late-onset AD patients. Moreover, the predominant species were changed as the clinical severity of AD. Furthermore, the correlation analysis revealed that V. parvula was associated with AD in both saliva and GCF and that P. gingivalis was associated with AD only in GCF. CONCLUSIONS: In this study, the microbiome community of oral microbes was altered in AD patients and periodontal microbiome was sensitive to cognition changes. Moreover, V. parvula and P. gingivalis were associated with AD.

Ginsenoside Rb3 inhibits osteoclastogenesis via ERK/NF-κB signaling pathway in vitro and in vivo.

OBJECTIVE: The objective of the study was to determine the anti-osteoclastogenic potential of ginsenoside Rb3 for the treatment of periodontitis. METHODS: The anti-osteoclastogenic effect was determined using RANKL-induced RAW264.7 cells and murine bone marrow-derived macrophages followed by TRAP and phalloidin staining. Expression of osteoclastogenesis-related genes and proteins were examined by qPCR and WB. Activation of signaling pathways was detected by WB and IHC techniques. Experimental periodontitis rat model was built up by gingival injections of P. gingivalis LPS. After 21 days of Rb3 treatment, rats were sacrificed for micro-CT, IHC, H&E, and TRAP staining analyses. RESULTS: Rb3 dramatically inhibits RANKL-induced osteoclastogenesis. Nfatc1, Mmp9, Ctsk, Acp5 mRNA, and MMP9, CTSK proteins were dose-dependently downregulated by Rb3 pretreatment. WB results revealed that Rb3 suppressed activations of p38 MAPK, ERK, and p65 NF-κB, and the inhibition of ERK was most pronounced. Consistently, IHC analysis revealed that p-ERK was highly expressed in alveolar bone surface, blood vessels, odontoblasts, and gingival epithelia, which were notably suppressed by Rb3 treatment. H&E staining and micro-CT analyses showed that Rb3 significantly attenuated gingivitis and alveolar bone resorption in rats. CONCLUSION: Rb3 inhibits RANKL-induced osteoclastogenesis and attenuates P. gingivalis LPS-induced gingivitis and alveolar bone resorption in rats via ERK/NF-κB signaling pathway.

Strategies for dispersion of cariogenic biofilms: applications and mechanisms.

Bacteria residing within biofilms are more resistant to drugs than planktonic bacteria. They can thus play a significant role in the onset of chronic infections. Dispersion of biofilms is a promising avenue for the treatment of biofilm-associated diseases, such as dental caries. In this review, we summarize strategies for dispersion of cariogenic biofilms, including biofilm environment, signaling pathways, biological therapies, and nanovehicle-based adjuvant strategies. The mechanisms behind these strategies have been discussed from the components of oral biofilm. In the future, these strategies may provide great opportunities for the clinical treatment of dental diseases. Graphical Abstract.

Bioresponsive nanotherapy for preventing dental caries by inhibiting multispecies cariogenic biofilms.

Early childhood caries (ECC) is a public healthcare concern that greatly reduces the quality of life of young children. As a leading factor of ECC, cariogenic biofilms are composed of acidogenic/aciduric pathogens and extracellular polysaccharides (EPSs), creating an acidic and protected microenvironment. Antimicrobial photodynamic therapy (aPDT) is a noninvasive, painless, and efficient therapeutic approach that is suitable for treating ECC. However, due to the hyperfine structure of cariogenic biofilms, most photosensitizers (PSs) could not access and penetrate deeply in biofilms, which dramatically hamper their efficiency in the clinic. Herein, bioresponsive nanoparticle loaded with chlorin e6 (MPP-Ce6) is developed, which largely increases the penetration depth (by over 75%) and retention (by over 100%) of PS in the biofilm compared with free Ce6. Furthermore, MPP-Ce6-mediated aPDT not only kills the bacteria in preformed biofilms but also inhibits multispecies biofilm formation. A rampant caries model is established to mimic ECC in vivo, where the population of cariogenic bacteria is decreased to 10% after MPP-Ce6-mediated aPDT. Importantly, the number and severity of carious lesions are efficiently reduced via Keyes' scoring and micro-CT analysis. This simple but effective strategy can serve as a promising approach for daily oral hygiene in preventing ECC.

Water jet as a novel technique for enamel drilling ex vivo.

To investigate the usage of a water jet for enamel drilling ex vivo, 210 individual extracted molars without lesions or fillings were collected. Then, the specimens were drilled by a water jet or a high-speed dental drill. The cavities of 50 teeth were reconstructed digitally by micro-computed tomography (micro-CT) to measure the height and width. The cavities of 10 teeth were longitudinally incised and their surfaces were observed by scanning electronic microscopy (SEM). After the cavities were filled, 50 fillings were vertically incised. The bonding interface between tooth and filling was observed by SEM. 50 teeth with fillings were stained in 0.1% rhodamine B solution, and then the dye penetration between tooth and filling was observed under the stereomicroscope and confocal laser scanning microscopy (CLSM). The bonding strength between enamel and filling of 50 teeth was simulated and predicted with finite element analysis (FEA). At 140-150 MPa and for 2-3 s, cavities were made with a depth of approximately 764 µm in each tooth. SEM showed the cavity surface in the water jet group had a more irregular concave and convex structure than that in the high-speed dental drill group. There was a trend that the microleakage and bonding width was smaller in the water jet group than in the high-speed dental drill group. FEA indicated that the stress on the resin surface was greater than on the enamel surface in the water jet group. Compared with the tooth drilled by a high-speed dental drill, the tooth drilled by a water jet gained better retention of the filling material and suffered less bonding strength on the enamel surface. Water jet drilling is effective for enamel drilling.

Ginsenoside Rb3 Inhibits Pro-Inflammatory Cytokines via MAPK/AKT/NF-κB Pathways and Attenuates Rat Alveolar Bone Resorption in Response to Porphyromonas gingivalis LPS.

Conventional treatments for chronic periodontitis are less effective in controlling inflammation and often relapse. Therefore, it is necessary to explore an immunomodulatory medication as an adjuvant. Ginsenoside Rb3 (Rb3), one of the most abundant active components of ginseng, has been found to possess anti-inflammatory and immunomodulatory properties. Here, we detected the anti-inflammatory effect of Rb3 on Porphyromonas gingivalis LPS-stimulated human periodontal ligament cells and experimental periodontitis rats for the first time. We found that the expression of pro-inflammatory mediators, including IL-1ß, IL-6 and IL-8, upregulated by lipopolysaccharide (LPS) stimulation was remarkably downregulated by Rb3 treatment in a dose-dependent manner at both transcriptional and translational levels. Network pharmacological analysis of Rb3 showed that the mitogen-activated protein kinase (MAPK) signaling pathway had the highest richness and that p38, JNK, and ERK molecules were potential targets of Rb3 in humans. Western blot analysis revealed that Rb3 significantly suppressed the phosphorylation of p38 MAPK and p65 NF-κB, as well as decreased the expression of total AKT. In experimental periodontitis rat models, reductions in alveolar bone resorption and osteoclast generation were observed in the Rb3 treatment group. Thus, we can conclude that Rb3 ameliorated Porphyromonas gingivalis LPS-induced inflammation by inhibiting the MAPK/AKT/NF-κB signaling pathways and attenuated alveolar bone resorption in experimental periodontitis rats.

A new species of wedgefish, Rhynchobatus immaculatus (Chondrichthyes, Rhynchobatidae), from Taiwan.

A new species of wedgefish, Rhynchobatus immaculatus sp. nov., is described from a small collection of specimens obtained from fish markets in northern Taiwan. It is probably a medium-sized species (probably attaining ca. 1.5 m TL) because the largest known specimen, an immature male (ca. I m TL), has prolongated dorsal and caudal fins typical of adult wedgefishes. Rhynchobatus immaculatus is unique within the family in having a very high vertebral count (within the range of 165-170 total free centra) and in lacking a dark pectoral marking. Other Rhynchobatus species occurring in Taiwanese seas appear to attain a larger adult size, possess a dark pectoral marking at least in young, and have lower vertebral counts (fewer than 161 total fee centra). Rhynchobatus yentinesis, which was described from a specimen taken nearby at Wenzhou, China, has not yet been attributed to a currently recognised species. However, based on the illustration of the holotype, which reveals a broad-snouted species with a dark pectoral spot, it is closest to either R. palpebratus or R. springeri.