Altered composition of the oral microbiome in integrin beta 6-deficient mouse.

In periodontal disease (PD), bacterial biofilms suppress ß6 integrin expression transforming growth factor-ß1 signaling, resulting in gingival inflammation and bone loss. ß6 integrin-null (Itgb6-/- ) mice develop spontaneous PD. The aim of this study was to unravel potential differences in oral microbiome in wild-type (WT) and Itgb6-/- FVB mice. Mouse oral microbiome was analyzed from 3- and 6-month-old WT and Itgb6-/- . The periodontal inflammation and spontaneous bone loss were present in 3-month-old and advanced in 6-month-old Itgb6-/- mice. The observed amplicon sequence variants (ASVs) of alpha diversity showed close similarity in 3-month-old and 6-month-old Itgb6-/- mice. Chao1 and ACE methods revealed that the microbiome in Itgb6-/- mice showed less diversity compared to the WT. UniFrac Principal Coordinate analyses (PCoA) showed a clear spatial segregation and clustering between Itgb6-/- and WT mice in general, and between 3-month- and 6-month-old WT mice. Weighted PCoA showed the tight clustering and distinct separation of individual mouse samples within Itgb6-/- and WT. The most abundant microbial classes varied between the sample groups. However, the genus Aggregatibacter significantly increased in the 6-month-old Itgb6-/- mice. ß6 integrin-deficient mice develop periodontal inflammation that may relate to dysbiosis in the microbiome that further promotes the disease process.

Seasonal Variation of Nonsyndromic Orofacial Clefts in Northern Chinese Population.

BACKGROUND: Nonsyndromic orofacial clefts (NSOC) comprise a range of disorders affecting the lips and oral cavity. Various authors evaluated seasonal influence on the occurrence of NSOC differently. The aim of present study was to investigate seasonal variation in birth of individuals with orofacial cleft in northern China. METHODS: A retrospective study comprised 499 cases and 452 controls were conducted. Children with NSOC operated in The First Affiliated Hospital of Harbin Medical University from 2009 to 2015 were investigated. Controls were children patients with trauma and bone fracture from the same hospital during the same period. Data on sex, birth time, area of residence was retrospectively collected from patients' records. Chi-squared test was used for comparisons. P values less than or equal to 0.05 were considered to be significant. RESULTS: Seasonal distribution was significantly different between cases and controls (P < 0.05). Birth time peaks of cases, especially males, occurred in winter. Furthermore, compared with controls, more cases with cleft lip/palate were born in winter (P < 0.05). There was no significant seasonal difference between female cases and controls (P > 0.05), and no statistical difference was found between cases with cleft palate and controls (P > 0.05). CONCLUSIONS: Our study revealed the presence of seasonal variation in individuals with orofacial cleft in northern Chinese population. We found a peak incidence of birth time for NSOC during winter.

Exploring the Imbalance of Periodontitis Immune System From the Cellular to Molecular Level.

Periodontitis is a common chronic inflammatory disease of periodontal tissue, mostly concentrated in people over 30 years old. Statistics show that compared with foreign countries, the prevalence of periodontitis in China is as high as 40%, and the prevalence of periodontal disease is more than 90%, which must arouse our great attention. Diagnosis and treatment of periodontitis currently rely mainly on clinical criteria, and the exploration of the etiologic criteria is relatively lacking. We, therefore, have explored the pathogenesis of periodontitis from the perspective of immune imbalance. By predicting the fraction of 22 immune cells in periodontitis tissues and comparing them with normal tissues, we found that multiple immune cell infiltration in periodontitis tissues was inhibited and this feature can clearly distinguish periodontitis from normal tissues. Further, protein interaction network (PPI) and transcription regulation network have been constructed based on differentially expressed genes (DEGs) to explore the interaction function modules and regulation pathways. Three functional modules have been revealed and top TFs such as EGR1 and ETS1 have been shown to regulate the expression of periodontitis-related immune genes that play an important role in the formation of the immunosuppressive microenvironment. The classifier was also used to verify the reliability of periodontitis features obtained at the cellular and molecular levels. In conclusion, we have revealed the immune microenvironment and molecular characteristics of periodontitis, which will help to better understand the mechanism of periodontitis and its application in clinical diagnosis and treatment.

Influence of photodynamic therapy on the periodontitis-induced bone resorption in rat.

This study aimed to evaluate the effects of toluidine blue-mediated photodynamic therapy (TB-PDT) on the periodontitis-induced bone resorption in periodontitis in rats. Periodontal disease was induced by cotton ligature around the right second maxillary molar in 64 rats. After 4 weeks, the rats were randomly divided into four groups: sterile saline solution (control group); laser therapy (laser group); TB (100 µg/mL); TB plus laser (0.15 W/cm2) irradiation every other day for 240 s (PDT group). All rats were euthanized at 15 days postoperatively. Eight gingival tissue samples were collected from each group. The expressions of receptor activator of nuclear factor kappa-Β ligand (RANKL) and osteoprotegerin (OPG) in gingival tissue samples were detected by real-time quantitative polymerase chain reaction (RT-qPCR). The maxillae from the rest of the rats were taken for histological examination. In the PDT group, the analysis revealed less bone loss than in the control treatment (P < 0.05). No significant difference was found among the control group, TB group, and laser group (P > 0.05). Significantly higher and lower expressions of RANKL and OPG were revealed in the PDT group than that in control group, respectively (P < 0.01). When compared with the control group, the expression of RANKL was significantly reduced by 40.0% in periodontitis in rats treated with TB-PDT for 15 days (P < 0.01). The expression of OPG was increased in the PDT group with TB-PDT for 15 days, when compared with the control group (P < 0.05). TB-PDT treatment significantly reverses the abnormal expression of RANKL and OPG in periodontitis in rats.

Super Activated Platelet Lysate, a Novel Autologous Platelet Lysate, Regulates the Expression of Inflammasome and Cytokine in the Experimental Periodontitis in Rats.

PURPOSE: The aim of the present study was to evaluate the expression of inflammasome and cytokine on experimental periodontitis with super activated platelet lysate (SPL) in rats. METHODS: Periodontitis was induced by submerging cotton ligatures on the right side of the maxillary second molar in 36 Wistar rats. The rats were divided into 3 groups randomly: the rats received no treatment (control group); local injection with sterile saline (ligature+saline group) and local injection with SPL (ligature+SPL group). After treatments, the alveolar bone level and inflammation of periodontal tissue were evaluated by micro-computed tomography (micro-CT) scanning and histological examination, respectively. The expression of inflammasome and cytokine was evaluated by real-time quantitative polymerase chain reaction (RT-qPCR) assay. RESULTS: Compared with the control group, the bone loss significantly increased by 0.9 mm in the ligature+saline group and 0.4 mm in the ligature+SPL group (P < 0.001). 0.5 mm reduction in the bone loss was founded in the ligature+SPL group compared with the ligature+saline group (P < 0.001). The gene expression of CCL2, CXCL2, IL-6, IL-18, IL-1α, IL-1ß, CXCL10, CXCL16, CCL5 was significantly reduced in the ligature+SPL group compared with the ligature+saline group (P < 0.05). Compared with the ligature+saline group, the expression for inflammasome NLRP3, AIM2, CASP1 was both downregulated in the ligature+SPL group (P < 0.05). CONCLUSION: Our present study demonstrated local injection of SPL regulated the expression of inflammasome and cytokine and had a visible effect of relieving inflammation in the experimental periodontitis in rats.

Hematoporphyrin monomethyl ether mediated sonodynamic antimicrobial chemotherapy on porphyromonas gingivalis in vitro.

This study aimed to evaluate the efficacy of hematoporphyrin monomethyl ether (HMME)-mediated sonodynamic antimicrobial chemotherapy (SACT) on Porphyromonas gingivalis (P. gingivalis). P. gingivalis (ATCC 33277) was used in the present study. The bacterial suspension was randomly divided into five groups: Group 1 was incubated for 2 h in the dark with HMME in various concentrations (10, 20, 30 and 40 µg/mL). Then exposed to 1 MHz ultrasound frequency with 3 W/cm2 ultrasound intensity for 10 min. Group 2 was incubated with 40 µg/mL HMME and then irradiated with 2, 4, 6, 8 and 10 min ultrasonic time. Group 3 received different HMME concentration (10, 20, 30 and 40 µg/mL) treatment alone with no ultrasound as the HMME control group. Group 4 received ultrasound treatment alone in different ultrasonic time (2, 4, 6, 8 and 10 min) with no HMME as the ultrasound control group. Group 5 received no treatment as the no treatment control group. After the SACT, the bactericidal effect was determined by the colony forming unit assay. The intracellular content of reactive oxygen species (ROS) was detected using the laser scanning confocal microscope based on DCFH-DA. 4.7 lg reduction in CFU, When P. gingivalis was treated with ultrasound (3 W/cm2 for 10 min) at 40 µg/mL HMME concentration (P < 0.01). The intracellular ROS in SDT group had a significant difference in comparison with the no treatment control group (P < 0.01). HMME mediated SACT can be a potential antibacterial therapy to significantly inhibit P. gingivalis growth.

Hematoporphyrin monomethyl ether-mediated sonodynamic therapy induces A-253 cell apoptosis.

It has been found that >90% of oral cancer patients suffer from squamous cell carcinoma (SCC). The 5-year survival rate of SCC is ~50%, despite the availability of different treatments. Sonodynamic therapy (SDT) has been developed as a novel therapy for cancer, resisting bacterial infection and inhibiting atherosclerotic plaque progression. The present study investigated the efficacy of hematoporphyrin monomethyl ether (HMME)-mediated SDT on the A-253 epidermoid cancer cell line. The cytotoxicity of HMME and the survival rate of cells following SDT were examined by the MTT assay. Apoptosis and necrosis of cells were detected using flow cytometry with Annexin V and propidium iodide (PI) staining, and fluorescence microscopy with Hoechst 33258 and PI staining. Intracellular reactive oxygen species (ROS) and Ca2+ levels were measured using a fluorescence microscope based on 2',7'-dichlorofluorescein diacetate and fluo-3/acetoxymethylester, respectively. Results of the MTT assay demonstrated that a lower concentration (<10 µg/ml) of HMME had no significant effect on the A-253 cells, but SDT combined with ultrasonic treatment for 1 min and 10 µg/ml HMME decreased the cell survival rate by 27%. Flow cytometry analysis revealed that A-253 cells in the SDT group had a higher rate of late apoptosis compared with the control group. Furthermore, fluorescence quantitation of apoptotic A-253 cells demonstrated that the percentages of apoptotic cells were increased in the ultrasound and SDT group compared with those in the control group. In the present study, the ROS level in the SDT group was elevated compared with that in the control group. The Ca2+ levels were increased to 181.2 and 268.7% in the ultrasound and SDT groups, respectively, relative to the control group. Taken together, the findings of the present study demonstrated that HMME-SDT significantly induces the apoptosis of A-253 cells together with intracellular ROS generation and Ca2+ overload. Thus, HMME-SDT may be a promising treatment option for patients with SCC.

Epidermal growth factor receptor signaling suppresses αvß6 integrin and promotes periodontal inflammation and bone loss.

In periodontal disease (PD), bacterial biofilms cause gingival inflammation, leading to bone loss. In healthy individuals, αvß6 integrin in junctional epithelium maintains anti-inflammatory transforming growth factor-ß1 (TGF-ß1) signaling, whereas its expression is lost in individuals with PD. Bacterial biofilms suppress ß6 integrin expression in cultured gingival epithelial cells (GECs) by attenuating TGF-ß1 signaling, leading to an enhanced pro-inflammatory response. In the present study, we show that GEC exposure to biofilms induced activation of mitogen-activated protein kinases and epidermal growth factor receptor (EGFR). Inhibition of EGFR and ERK stunted both the biofilm-induced ITGB6 suppression and IL1B stimulation. Furthermore, biofilm induced the expression of endogenous EGFR ligands that suppressed ITGB6 and stimulated IL1B expression, indicating that the effects of the biofilm were mediated by autocrine EGFR signaling. Biofilm and EGFR ligands induced inhibitory phosphorylation of the TGF-ß1 signaling mediator Smad3 at S208. Overexpression of a phosphorylation-defective mutant of Smad3 (S208A) reduced the ß6 integrin suppression. Furthermore, inhibition of EGFR signaling significantly reduced bone loss and inflammation in an experimental PD model. Thus, EGFR inhibition may provide a target for clinical therapies to prevent inflammation and bone loss in PD.

Low-Intensity Ultrasound Combined with Hematoporphyrin Monomethyl Ether in the Treatment of Experimental Periodontitis in Rats.

Objectives. This study aims to evaluate the efficacy of hematoporphyrin monomethyl ether- (HMME-) mediated sonodynamic therapy (SDT) on experimental periodontal disease in rats. Methods. Periodontal disease was induced by submerging ligatures at the first maxillary molar subgingival region in forty-eight male SD rats. After 30 days, the ligatures were removed. The rats were randomly allocated into four groups; the experimental SDT group was treated through hypodermic injection of 40 µg/mL HMME and 3 W/cm2 low-intensity ultrasound irradiation (1 MHz, 600 s). Those in control groups received 40 µg/mL HMME alone (control 1 group) or 3 W/cm2 ultrasound irradiation alone (control 2 group) or were subjected to neither HMME nor ultrasound (control 3 group). After 10 days of treatment, all rats were euthanized, the maxilla was obtained for histological examination, and the alveolar bone level was evaluated by histometric analysis. Results. The control groups showed more bone loss (P < 0.05) after 10 days of treatment than the SDT group. There is no significant difference among the control groups (P > 0.05). Conclusions. HMME mediated SDT was an effective therapy of experimental periodontal tissue in rats.

Association of Parental Environmental Exposures and Supplementation Intake with Risk of Nonsyndromic Orofacial Clefts: A Case-Control Study in Heilongjiang Province, China.

The aim of present study was to check the possible association of potential parental environmental exposures and maternal supplementation intake with the risk of nonsyndromic orofacial clefting (NSOC). A retrospective study comprised 499 cases and 480 controls was conducted in Heilongjiang Province. Chi-square analysis and unconditional multiple logistic regression were used in the study. The results showed that maternal history of fever and the common cold without fever (ORCL/P = 3.11 and 5.56, 95%CI: 1.67-5.82 and 2.96-10.47, ORCPO = 3.31 and 8.23, 95%CI: 1.58-6.94 and 4.08-16.95), paternal smoking and alcohol consumption (ORCL/P = 2.15 and 5.04, 95%CI: 1.37-3.38 and 3.00-8.46, ORCPO = 1.82 and 4.40, 95%CI: 1.06-3.13 and 2.50-7.74), maternal exposure to organic solvents, heavy metals, or pesticides (ORCL/P = 6.07, 5.67 and 5.97, 95%CI: 1.49-24.76, 1.34-24.09 and 2.10-16.98, ORCPO = 10.65, 7.28 and 3.48, 95%CI: 2.54-44.67, 1.41-37.63 and 1.06-11.46) and multivitamin use during the preconception period (ORCL/P = 0.06, 95%CI: 0.02-0.23, ORCPO = 0.06, 95%CI: 0.01-0.30) were associated with cleft lip or without cleft palate (CL/P) and cleft palate only (CPO). Maternal history of skin disease and negative life events (ORCL/P = 12.07 and 1.67, 95%CI: 1.81-80.05 and 1.95-2.67) were associated with CL/P. Some potential parental hazardous exposures during the periconception period and maternal use of multivitamins during the preconception period were associated with risk of NSOC.

Sonodynamic effect of hematoporphyrin monomethyl ether on ligature-induced periodontitis in rats.

OBJECTIVES: The aim of this study was to perform a histological evaluation of sonodynamic therapy (SDT) of hematoporphyrin monomethyl ether (HMME) on artificially induced periodontal disease in rats. METHODS: Submerging ligatures were placed at the subgingival region of the first maxillary molar in rats. Eighty rats were randomly assigned into four groups: group 1 received no treatment; group 2 was subjected to 50 µg/mL HMME alone; group 3 was treated with low-intensity ultrasound alone (1 W/cm(2)); and group 4 was treated with 50 µg/mL HMME plus ultrasound irradiation (1 MHz, 30 minutes). Ten rats in each group were euthanized at 7 and 15 days, and periodontal tissue samples were taken for histological examination. RESULTS: The animals treated by SDT showed less bone loss (P<0.05) at all experimental periods than the other three groups. No significant differences were found between the control and HMME groups (P>0.05). CONCLUSION: Our results suggest that HMME-mediated SDT can effectively alleviate the periodontal tissue destruction in artificially induced periodontitis in rats. Hence, SDT may have good clinic potential as a noninvasive treatment of periodontal diseases.

Association of JARID2 polymorphisms with non-syndromic orofacial clefts in northern Chinese Han population.

OBJECTIVES: Non-syndromic orofacial clefts (NSOC) are the most common human craniofacial malformation in all worldwide populations. Recently, the jumoji AT-rich interaction domain 2 (JARID2) had been reported to be a novel candidate gene for non-syndromic cleft lip with or without cleft palate (CL/P). The SNPs rs2076056, rs2237138 and rs2299043 in JARID2 were highly significant in Italian families. MATERIAL AND METHODS: In the current research, a case-control study was conducted to examine the association between these three SNPs and NSOC in a northern Chinese Han population. Genotyping of the three SNPs were performed using SNaPshot minisequencing technique. RESULTS: Distribution of rs2237138 genotypes in CL/P group was different from those in the control group (P = 0.04), but significant results did not persist after Benjamini and Hochberg false discovery rate (FDR) correction for multiple tests. Further logistic regression analysis showed that rs2237138 GG genotypes were associated with decreased CL/P susceptibility (OR = 0.34, 95% CI = 0.13-0.84), compared with the AA wild-type homozygote. For the haplotype CGT, a statistically difference was identified between the CL/P group and controls (P = 0.04). And carriers of GAT haplotype were considered to be less frequent among cleft palate only group as compared to controls (P = 0.02). However, both of the haplotypes association did not remain statistically significant after Benjamini and Hochberg FDR correction. CONCLUSION: We got a weak association between these polymorphisms and NSOC in both single-marker and haplotype analyses. Our data further strengthen the conclusion that JARID2 polymorphisms are associated with NSOC susceptibility.

Sonodynamic effects of hematoporphyrin monomethyl ether on Staphylococcus aureus in vitro.

This study aims to investigate the effect of hematoporphyrin monomethyl ether (HMME)-mediated sonodynamic antimicrobial chemotherapy (SACT) on Staphylococcus aureus. SACT was carried out using HMME and 1 MHz ultrasound irradiation. The bactericidal effect was evaluated by the counting colony-forming units (CFU), and important SACT parameters including ultrasound intensity and HMME concentration were determined. More than 95% of the bacteria colonies were effectively killed in the SACT group by 50 µg mL(-1) HMME combined with 6 W cm(-2) tone-burst ultrasound at 1 MHz, but this ultrasound level without HMME only reduced CFU by 38%. In the sonodynamic treatment, higher HMME concentrations and higher ultrasound intensities caused more death of bacteria. Incubation with different HMME concentrations without ultrasound showed no effect. Our results show that the HMME-mediated SACT can be significantly in killing S. aureus.