Anti-biofilm effect of salivary histatin 5 on Porphyromonas gingivalis.

This study aimed to investigate the effects of salivary histatin 5 (Hst5) on Porphyromonas gingivalis (P. gingivalis) biofilms in vitro and in vivo and the possible mechanisms. In in vitro experiments, P. gingivalis biomass was determined by crystal violet staining. Polymerase chain reaction, scanning electron microscopy, and confocal laser scanning microscopy were used to determine the Hst5 concentration. A search for potential targets was performed using transcriptomic and proteomic analyses. In vivo experimental periodontitis was established in rats to evaluate the effects of Hst5 on periodontal tissues. Experimental results showed that 25 µg/mL Hst5 effectively inhibited biofilm formation, and increased concentrations of Hst5 increased the inhibitive effect. Hst5 might bind to the outer membrane protein RagAB. A combination of transcriptomic and proteomic analyses revealed that Hst5 could regulate membrane function and metabolic processes in P. gingivalis, in which RpoD and FeoB proteins were involved. In the rat periodontitis model, alveolar bone resorption and inflammation levels in periodontal tissues were reduced by 100 µg/mL Hst5. This study showed that 25 µg/mL Hst5 inhibited P. gingivalis biofilm formation in vitro by changing membrane function and metabolic process, and RpoD and FeoB proteins might play important roles in this process. Moreover, 100 µg/mL Hst5 inhibited periodontal inflammation and alveolar bone loss in rat periodontitis via its antibacterial and anti-inflammatory effects. KEY POINTS: • Anti-biofilm activity of histatin 5 on Porphyromonas gingivalis was investigated. • Histatin 5 inhibited Porphyromonas gingivalis biofilm formation. • Histatin 5 showed inhibitory effects on the occurrence of rat periodontitis.

Porphyromonas gingivalis can invade periodontal ligament stem cells.

BACKGROUND: Porphyromonas gingivalis is strongly associated with the development, progression, severity and recurrence of periodontitis. Periodontal ligament stem cells (PDLSCs) play an important role in the maintenance of periodontal tissue self-renewal and repair. The purpose of this study was to investigate the ability of P. gingivalis to infect PDLSCs using an in vitro monolayer model. METHODS: We separated and cultured primary PDLSCs using the tissue block with limiting dilution method. The efficiency of P. gingivalis (ATCC 33277) infection of PDLSCs was measured using agar plate culture and quantitative polymerase chain reaction (q-PCR) methods. PDLSCs infected with P. gingivalis were also observed by transmission electron microscopy. RESULTS: We assessed stem cell properties including cell morphology, clone formation, growth activity, cell surface antigens and multiple differentiation capacity. The infection rates of P. gingivalis in PDLSC at MOIs of 50, 100, 200, and 500 were 5.83%, 8.12%, 7.77% and 7.53% according to the agar plate culture method. By q-PCR, the efficiencies of P. gingivalis infection of PDLSCs at MOIs of 50, 100, 200, and 500 were 6.74%, 10.56%, 10.36% and 9.78%, respectively. Overall, the infection efficiency based on q-PCR was higher than that according to agar plate culture. Using transmission electron microscopy, we verified that P. gingivalis (ATCC 33277) could infect and invade PDLSCs after 2 h of incubation, and endocytic vacuoles were not found surrounding the internalized bacteria. CONCLUSIONS: In conclusion, our data demonstrate that P. gingivalis can invade PDLSCs.

Molecular pathways underlying inhibitory effect of antimicrobial peptide Nal-P-113 on bacteria biofilms formation of Porphyromonas gingivalis W83 by DNA microarray.

BACKGROUND: Wound-related infection remains a major challenge for health professionals. One disadvantage in conventional antibiotics is their inability to penetrate biofilms, the main protective strategy for bacteria to evade irradiation. Previously, we have shown that synthetic antimicrobial peptides could inhibit bacterial biofilms formation. RESULTS: In this study, we first delineated how Nal-P-113, a novel antimicrobial peptide, exerted its inhibitory effects on Porphyromonas gingivalis W83 biofilms formation at a low concentration. Secondly, we performed gene expression profiling and validated that Nal-P-113 at a low dose significantly down-regulated genes related to mobile and extrachromosomal element functions, transport and binding proteins in Porphyromonas gingivalis W83. CONCLUSIONS: These findings suggest that Nal-P-113 at low dose is sufficient to inhibit the formation of biofilms although Porphyromonas gingivalis W83 may maintain its survival in the oral cavity. The newly discovered molecular pathways may add the knowledge of developing a new strategy to target bacterial infections in combination with current first-line treatment in periodontitis.

Preventive effects of the novel antimicrobial peptide Nal-P-113 in a rat Periodontitis model by limiting the growth of Porphyromonas gingivalis and modulating IL-1ß and TNF-α production.

BACKGROUND: P-113 (AKRHHGYKRKFH-NH2) is a 12-amino-acid histidine-rich peptide derived from histatin 5 that is highly degradable in high salt concentrations and biological fluids such as serum, plasma and saliva. Nal-P-113, a novel antimicrobial peptide whose histidine residues are replaced by the bulky amino acids ß-naphthylalanine, causes the antimicrobial peptide to retain its bactericidal activity even in physiological environments. This study evaluated the effect of the novel antimicrobial peptide Nal-P-113 in a rat periodontitis model and the mechanisms of action of Nal-P-113 for suppressing periodontitis. METHODS: Periodontitis was induced in mandibular first molars in rats receiving a ligature and infected with Porphyromonas gingivalis. Animals were randomly divided into six groups: a, P. gingivalis W83 alone; b, P. gingivalis W83 with 6.25 µg/mL of Nal-P-113; c, P. gingivalis W83 with 25 µg/mL of Nal-P-113; d, P. gingivalis W83 with 100 µg/mL of Nal-P-113; e, P. gingivalis W83 with 400 µg/mL of Nal-P-113; and f, control without P. gingivalis W83 or Nal-P-113. Morphometric analysis was used to evaluate alveolar bone loss. Microbiological assessment of the presence of Porphyromonas gingivalis and total bacteria was performed using absolute quantitative real-time PCR and scanning electron microscopy. Gingival tissue was collected for western blot and immunohistochemical assays of IL-1ß and TNF-α levels. RESULTS: Alveolar bone loss was inhibited by 100 µg/mL or 400 µg/mL of Nal-P-113 compared to the control group (P < 0.05). Lower amounts of P. gingivalis and total bacteria were found in groups d and e compared with group a (P < 0.05). A decrease in the levels of IL-1ß and TNF-α was detected in group d and group e compared to the control group (P < 0.05). The amount of P. gingivalis was positively correlated with IL-1ß and TNF-α expression in periodontal tissue (P < 0.05). CONCLUSIONS: Nal-P-113 exhibited protective effects on Porphyromonas gingivalis-induced periodontitis in rats by limiting the amount of bacteria and modulating IL-1ß and TNF-α production. The use of Nal-P-113 in vivo might serve as a beneficial preventive or therapeutic approach for periodontitis.

Gene expression changes in Porphyromonas gingivalis W83 after inoculation in rat oral cavity.

BACKGROUND: The development of chronic periodontitis was due to not only periodontal pathogens, but also the interaction between periodontal pathogens and host. The aim of this study is to investigate the alterations in gene expression in Porphyromonas gingivalis (P.gingivalis) W83 after inoculation in rat oral cavity. RESULTS: P.gingivalis W83 inoculation in rat oral cavity caused inflammatory responses in gingival tissues and destroyed host alveolar bone. Microarray analysis revealed that 42 genes were upregulated, and 22 genes were downregulated in the detected 1786 genes in the inoculated P.gingivalis W83. Real-time quantitative PCR detection confirmed the expression alterations in some selected genes. Products of these upregulated and downregulated genes are mainly related to transposon functions, cell transmembrane transportation, protein and nucleic acid metabolism, energy metabolism, cell division and bacterial pathogenicity. CONCLUSIONS: P.gingivalis W83 has a pathogenic effect on host oral cavity. Meanwhile, inflammatory oral environment alters P.gingivalis W83 gene expression profile. These changes in gene expression may limit the proliferation and weaken the pathogenicity of P.gingivalis W83, and favor themselves to adapt local environment for survival.

P. gingivalis alters lung microbiota and aggravates disease severity of COPD rats by up-regulating Hsp90α/MLKL.

Background: Epidemiological evidence has confirmed that periodontitis is an essential and independent risk factor of chronic obstructive pulmonary disease (COPD). Porphyromonas gingivalis, a major pathogen implicated in periodontitis, may make a vital contribution to COPD progression. However, the specific effects and molecular mechanism of the link between P. gingivalis and COPD are not clear. Methods and Results: A COPD rat model was constructed by smoke exposure combined intratracheal instillation of E. coli-LPS, then P. gingivalis was introduced into the oral cavity of COPD rats. This research observed that lower lung function, more severe alveolar damage and inflammation occurred in COPD rats with P. gingivalis group. Meanwhile, P. gingivalis/gingipains could colonize the lung tissues and be enriched in bronchoalveolar lavage fluid (BALF) of COPD rats with P. gingivalis group, along with alterations in lung microbiota. Proteomic analysis suggested that Hsp90α/MLKL-meditated necroptosis pathway was up-regulated in P. gingivalis-induced COPD aggravation, the detection of Hsp90α and MLKL in serum and lung tissue verified that Hsp90α/MLKL was up-regulated. Conclusion: These results indicate that P. gingivalis could emigrate into the lungs, alter lung microbiota and lead to aggravation of COPD, which Hsp90α/MLKL might participate in.

Relationship among clinical periodontal, microbiologic parameters and lung function in participants with chronic obstructive pulmonary disease.

BACKGROUND: The aim of this study was to evaluate the relationship among clinical periodontal, microbiologic parameters and lung function in participants with chronic obstructive pulmonary disease (COPD). METHODS: A total of 160 participants were recruited, including 80 participants with COPD (COPD group) and 80 participants without COPD (control group). All participants completed questionnaires and underwent clinical periodontal and lung function examinations. Subgingival plaques were obtained to determine the prevalence of selected oral and respiratory bacterial species. RESULTS: 1) Significant relationships were noted in the participants among oral hygiene index-simplified (OHI-S), clinical attachment level (CAL) and forced expiratory volume in one second (FEV1%). 2) Porphyromonas gingivalis (Pg), Klebsiella pneumonia (Kp), Pseudomonas aeruginosa (Pa) and Streptococcus pneumonia (Sp) prevalence was increased in participants with COPD compared with control participants. 3) A significant negative association was noted between the relative content of Pg and FEV1% in participants with COPD. CONCLUSION: The results of this study confirm that periodontal destruction and oral pathogens are associated with lung function.

Assessment of Alveolar Bone Status in Middle Aged Chinese (40-59 Years) with Chronic Periodontitis--Using CBCT.

OBJECTIVE: This study used con-beam computed tomography (CBCT) to investigate the prevalence and severity of alveolar bone loss in middle-aged (40-59 years) Chinese with chronic periodontitis. MATERIALS AND METHODS: The study group comprised 145 dentate individuals aged 40 to 59 years residing in China who suffered from chronic periodontitis. CBCT and the application of NNT software were used to examine the level and location of alveolar bone loss. RESULTS: The study revealed that 40-59 year old patients with chronic periodontitis had severe bone loss. At 5,286 sites (34.7%), alveolar bone loss was mild; severe alveolar bone loss was found at 5,978 sites (39.2%). A comparison of bone loss in different jaws revealed that the area with the highest degree of bone loss was on the lingual side of the maxillary molar (56.3 ± 7.2%), and that the area with the lowest degree was primarily on the lingual side of the mandibular canine (27.5 ± 6.3%). There was a lower degree of alveolar bone loss in males than females. Differences were observed when comparing the incidence of bone loss between males and females (P < 0.05). Menopause in females and smoking in both genders may affect the level of bone loss. Male smokers experienced a greater degree of bone loss (41.67 ± 5.76%) than male non-smokers (32.95 ± 4.31%). A 42.23 ± 6.34% bone loss was found in menopausal females versus 31.35 ± 3.62% in non-menopausal females. CONCLUSIONS: The study revealed that different sites and teeth exhibited a diverse degree of bone loss. In middle-aged patients with chronic periodontitis, the highest degrees of bone loss in the incisors, premolars, and molars were on the lingual side, mesial side and lingual side, respectively. Menopause in females and smoking may affect the level of bone loss.

[Effect of smokers'sera on Porphyromonas gingivalis internalizing KB cells and the expression of matrix metalloproteinase-1, -9 and tissue inhibitor of metalloproteinase-1].

OBJECTIVE: To investigate the effects of serum from smoking individuals or non-smoking individuals with periodontitis on Porphyromonas gingivalis (Pg) internalizing KB cells, and the expression of matrix metalloproteinase(MMP)-1, MMP-9, tissue inhibitor of metalloproteinase-1 (TIMP-1) in the culture supernatant of KB cells. METHODS: The venous blood of 20 periodontitis patients' (10 smoking and 10 non-smoking) was extracted under the informed consent and centrifuged for serum. The smoking-individual serum (Y group) and non-smoking-individual (N group) serum were added to the model of Pg internalizing KB cells for 12 hours, plated on brain-heart infusion (BHI) and incubated anaerobically at 37 °C for 5 days. The colony forming units (CFU) of cell-invasive bacteria were estimated by colony counting. MMP-1, MMP-9 and TIMP-1 protein levels in culture supernatant were determined by enzyme-linked immunosorbent assay(ELISA) in the two groups following co-culture of Pg with KB cells for 12 hours. RESULTS: The CFU were (11.2 ± 1.1)×10(4), (12.6 ± 1.2)×10(4), (44.7 ± 1.3)×10(4) CFU/ml when adding 200, 400, 800 µl Y-group serum to the model of Pg co-culture with KB cells and when the serum was extracted from N group, the CFU were (33.6 ± 1.4)×10(4),(38.9 ± 1.1)×10(4), (11.2 ± 1.2)×10(4) CFU/ml respectively. When 200, 400, 800 µl Y group-serum was added to co-culture fluid of Pg internalizing KB cells, the concentrations of MMP-1 secreted from KB cells were (107.2 ± 21.5), (165.9 ± 20.2), (434.4 ± 48.0) µg/L respectively, the concentrations of MMP-9 were (3.99 ± 0.29), (4.21 ± 0.61), (5.62 ± 0.47) µg/L respectively, the concentrations of TIMP-1 were (401.3 ± 12.7), (418.3 ± 28.5), (637.3 ± 37.3) µg/L. When the serum (200, 400, 800 µl) extracted from N group, the concentration of MMP-1 and MMP-9 secreted by KB cell were (77.6 ± 10.8), (84.7 ± 10.2) and (98.2 ± 9.7) µg/L and (3.84 ± 0.52), (4.02 ± 0.68), (4.25 ± 0.37) µg/L, respectively. The concentration of TIMP-1 were (67.3 ± 26.9) , (89.4 ± 22.7) and (78.2 ± 16.5) µg/L secreted by KB cells in the course of Pg internalized KB cell. With the increasing of Y group-serum, the more MMP-1, MMP-9 and TIMP-1 were secreted by KB cells(P < 0.05). When 800 µl Y group-serum was added compared with N group-serum to the Pg co-culture with KB model, the more MMP-1, MMP-9 and TIMP-1 were secreted by KB cells(P < 0.05), when 400 µl Y group-serum was added compared with N group-serum to the Pg co-culture with KB model, the more MMP-1 and TIMP-1 were secreted by KB cells (P < 0.05). CONCLUSIONS: The smoking-serum might enhance Pg internalizing KB cells and enhance the expression of MMP-1, MMP-9 and TIMP-1 secreted from KB cells. The local microenvironment of smoking individual may contribute to the recurrence and progression of chronic periodontitis.

The sociodemographic characteristics, periodontal health status, and subgingival microbiota of patients with chronic periodontitis and type 2 diabetes mellitus: a case-control study in a Chinese population.

BACKGROUND: In China, chronic periodontitis (CP) is common in patients with type 2 diabetes mellitus (T2DM). The purpose of this study is to identify the sociodemographic characteristics associated with such patients and to assess the periodontal health status and subgingival microbiota of patients with CP and T2DM (T2DMCP) in the Chinese population. METHODS: A total of 150 patients with T2DMCP and 306 patients with CP without any systemic disease completed questionnaires, underwent clinical periodontal examinations and participated in diabetes-related parameter examinations. Subgingival plaques were obtained to determine the prevalence and amounts of selected oral bacterial species using polymerase chain reaction (PCR) and real-time PCR, respectively. RESULTS: The income level and mean body mass index (BMI) of the patients with T2DMCP were significantly higher than those of the patients with CP. Additionally, the patients with T2DMCP were more likely to be urban residents, and they had significantly more severe periodontitis than did the patients with CP. In the patients with T2DMCP, the prevalence and amounts of Treponema denticola and Tannerella forsythia were significantly higher than those in the patients with CP. Finally, compared with the patients with CP, the patients with T2DMCP had a significantly lower prevalence and amount of Prevotella intermedia. CONCLUSIONS: Compared with the patients with CP, the patients with T2DMCP were more likely to be urban residents and generally had higher incomes, higher mean BMI, and poorer periodontal health status. Higher levels of T. denticola and T. forsythia and lower levels of P. intermedia were identified in the subgingival plaque of the patients with T2DMCP.

[A study of frequency of TNF alpha gene with type 2 diabetes mellitus with chronic periodontitis].

PURPOSE: To detect the frequency of TNF alpha gene in patients of type 2 diabetes mellitus with chronic periodontitis, periodontitis without any systemic diseases and healthy controls. METHODS: The case series were consisted of 112 patients with moderate, severe type 2 diabetes mellitus with chronic periodontitis, 99 patients with moderate, severe periodontitis without any systemic disease, 50 age- and gender-matched subjects with healthy periodontal conditions were enrolled. Clinical parameters were measured and recorded including probing depth(PD), clinical attachment loss(CAL), bleeding index(BI), and tooth movement(TM). The polymorphism of TNF-α-308 genotype (TNF1/2) was examined after electrophoresis on agarose gel and ethidium bromide staining. The difference between the case and healthy groups was analysed by Chi-square test, the difference in clinical index among groups which had different allele was analyzed for ANOVA with SPSS13.0 software package. RESULTS: We divided DM and CP groups into moderate and severe groups. There were significant difference between severe DM group and severe, moderate CP group, moderate DM group and chronic periodontitis of severe,moderate group. The probing depth and clinical attachment loss of the patients who took TNF-α-308 allele II were significantly higher than the patients who took TNF-α-308 allele I in DM and CP group. CONCLUSIONS: TNF-α-308 allele II might increase the susceptivity of periodontitis in population. TNF-α-308 allele II may play an important role in synergistic reaction of periodontitis and type 2 diabetes.

[A survey on the periodontal status in type 2 diabetic patients].

OBJECTIVE: To investigate the type 2 diabetic patient's periodontal condition and to analyze the influencing factors of periodontitis. METHODS: A total of 182 type 2 diabetic patients were included in the survey and requested to fill out a questionnaire, and their periodontal status was evaluated by measuring probing depth (PD), attachment level (AL), sulcus bleeding index (SBI), simplified oral hygiene index (OHI-S). RESULTS: The prevalence of periodontitis in this group of patients was 96.7% (176/182), including 20 patients with mild periodontitis, 156 with moderate to advanced periodontitis. The mean PD and AL of the 182 patients were (2.92 +/- 0.67) mm and (2.87 +/- 1.31) mm. At least one tooth was lost in 57.1% (104/182) of the patients. The factors related to periodontitis were age, gender, smoking, living in town or country, and 2 h plasma glucose of oral glucose tolerance test (OGTT). There was no relationship between the severity of periodontitis and education level. The majority of patients did not receive any periodontal therapy. CONCLUSIONS: Periodontal status was bad in patients with type 2 diabetes. It is important to develop an education program on oral health for type 2 diabetic patients.

[Combination use of rh-bFGF and Bio-Oss collagen to enhance periodontal regeneration: an experimental study in dogs].

PURPOSE: To evaluate the effectiveness of combined use of rh-bFGF and Bio-Oss collagen compounds on regeneration of the canine periodontal tissues. METHODS: Four adult hybrid canine were selected and bone defect of 5mmx5mmx5mm in size was created in the mesio and distal alveolar crest where the premolars were lost in both sides of the maxilla and mandible. Three methods were used in 3 groups,in the experimental group 1, rh-bFGF and Bio-Oss collagen compounds were implanted,Bio-Gide were overlaid; in the experimental group 2,Bio-Oss collagen compounds were implanted, Bio-Gide were overlaid;in the control group only Bio-Gide were overlaid. The therapeutic results were evaluated by clinical and imaging examination as well as histological index. SAS 6.2 software package was used for paired t test. RESULTS: 8 weeks post operation, there were various amounts of newly formed alveolar bone,cement and connective tissues in each 3-wall intrabony defect. There was significant difference in alveolar osteogenesis between the control group(1.2mm+/-0.1mm) and the 2 experimental groups (3.7mm+/-0.3mm, 2.3mm+/-0.2mm).The difference was also significant between the two experimental groups (P<0.01). There was no significant difference in newly formed cement and connective tissues among the 3 groups (P>0.05). CONCLUSIONS: Application of rh-bFGF/ Bio-Oss collagen compounds benefits to the regeneration of the alveolar bone and avoid the side effects of root resorption ,bone malunion which often occur after conventional periodontal regeneration therapy.Addition of rh-bFGF/ Bio-Oss collagen compounds was useless to cement genesis and periodontal ligament formation.