Role of Piezo1 in modulating the RANKL/OPG ratio in mouse osteoblast cells exposed to Porphyromonas gingivalis lipopolysaccharide and mechanical stress.

AIMS: Excessive occlusal force with periodontitis leads to rapid alveolar bone resorption. However, the molecular mechanism by which inflammation and mechanical stress cause bone resorption remains unclear. We examined the role of Piezo1, a mechanosensitive ion channel expressed on osteoblasts, in the changes in the receptor activator of nuclear factor-kappa B ligand (RANKL)/osteoprotegerin (OPG) ratio in mouse MC3T3-E1 osteoblast-like cells under Porphyromonas gingivalis lipopolysaccharide (P.g.-LPS) and mechanical stress. METHODS: To investigate the effect of P.g.-LPS and mechanical stress on the RANKL/OPG ratio and Piezo1 expression, we stimulated MC3T3-E1 cells with P.g.-LPS. After 3 days in culture, shear stress, a form of mechanical stress, was applied to the cells using an orbital shaker. Subsequently, to investigate the role of Piezo1 in the change of RANKL/OPG ratio, we inhibited Piezo1 function by knockdown via Piezo1 siRNA transfection or by adding GsMTx4, a Piezo1 antagonist. RESULTS: The RANKL/OPG ratio significantly increased in MC3T3-E1 cells cultured in a medium containing P.g.-LPS and undergoing mechanical stress compared to cells treated with P.g.-LPS or mechanical stress alone. However, the expression of Piezo1 was not increased by P.g.-LPS and mechanical stress. In addition, phosphorylation of MEK/ERK was induced in the cells under P.g.-LPS and mechanical stress. MC3T3-E1 cells treated with P.g.-LPS and mechanical stress when cocultured with RAW264.7 cells induced their differentiation into osteoclast-like cells. The increased RANKL/OPG ratio was suppressed by either Piezo1 knockdown or the addition of GsMTx4. Furthermore, GsMTx4 inhibited the phosphorylation of MEK/ERK. CONCLUSION: These findings suggest that P.g.-LPS and Piezo1-mediated mechanical stress induce MEK/ERK phosphorylation and increase RANKL expression in osteoblasts. Consequently, this leads to the differentiation of osteoclast precursor cells into osteoclasts.

Effect of Periodontal Treatment on Reducing Chronic Inflammation in Systemically Healthy Patients With Periodontal Disease.

BACKGROUND: We determined the effects and an accurate marker of periodontal treatment on serum interleukin (IL)-6 and high-sensitivity C-reactive protein (HsCRP) levels in systemically healthy individuals with periodontal disease. METHODS: This multicenter study included systemically healthy individuals with periodontal disease who received initial periodontal treatment and had no periodontal treatment history. Periodontal parameters, including periodontal inflamed surface area, masticatory efficiency, and periodontal disease classification; serum IL-6 and HsCRP levels; and serum immunoglobulin (Ig)G titers against periodontal pathogens were evaluated at baseline and after treatment. Subjects were classified as low or high responders (group) based on periodontal inflamed surface area changes. RESULTS: There were 153 participants. Only periodontal inflamed surface area changes were markedly different between low and high responders. Periodontal treatment (time point) decreased both serum IL-6 and HsCRP levels. The interaction between group and time point was remarkable only for serum IL-6 levels. Changes in serum immunoglobulin (Ig)G titers against periodontal pathogens were not associated with IL-6 changes in high responders. We analyzed the indirect effect of serum anti-Porphyromonas gingivalis type 2 IgG titer changes using mediation analysis and found no significance. However, the direct effect of group (low or high responder) on IL-6 changes was considerable. CONCLUSIONS: Periodontal treatment effectively decreased serum IL-6 levels, independent of periodontal pathogen infection, in systemically healthy individuals with periodontal disease.

Implant Site Development by Orthodontic Extrusion and Buccal Root Torque at a Site Showing Severe Gingival Recession with Periodontitis: A Case Report.

This case report describes alveolar ridge augmentation around a hopeless maxillary canine with severe buccal gingival recession and periodontitis using orthodontic extrusion and a 90-degree buccal root torque in preparation for implant placement. After the orthodontic therapy, the palatal surface of the canine root reached the top of the alveolar bone, parallel to the occlusal plane, with newly formed bone and keratinized mucosa. An implant was successfully placed without combined bone augmentation. This technique may be a useful, minimally invasive approach for implant site development where hopeless teeth with severe buccal recession remain in the esthetic areas.

Effects of periodontal treatment on the medical status of patients with type 2 diabetes mellitus: a pilot study.

BACKGROUND: Studies have demonstrated that periodontal disease is associated with the development of systemic complications in patients with type 2 diabetes mellitus (T2DM). The purpose of this pilot study was to investigate which markers among various systemic disease parameters are affected by periodontal treatment in patients with T2DM. METHODS: Twelve patients with T2DM were given oral hygiene instructions and subsequent subgingival scaling and root planing. The periodontal status was recorded, and blood and urine samples were taken to measure various parameters of glucose control and systemic status at baseline and 1 month following the periodontal treatment. Serum concentrations of tumor necrosis factor-α and high-sensitivity C-reactive protein were measured by enzyme-linked immunosorbent assay. RESULTS: After the periodontal treatment, the glycated hemoglobin value was significantly improved. The levels of urinary N-acetyl-ß-D-glucosaminidase and albumin, which are markers of renal dysfunction, also decreased significantly after treatment. Among the parameters measured in serum, the γ-glutamyl transpeptidase level, which is usually interpreted as a marker of liver dysfunction, was significantly reduced. The serum concentrations of tumor necrosis factor-α and high-sensitivity C-reactive protein were also significantly reduced by periodontal treatment. CONCLUSION: Within the limitations of this pilot study, periodontal treatment may be effective not only in improving metabolic control, but also in reducing the risk of diabetic kidney and liver disease in patients with T2DM.

Prevalence and risk factors for peri-implant diseases in Japanese adult dental patients.

We investigated the prevalences and risk factors for peri-implant diseases in Japanese adult dental patients attending a follow-up visit at dental hospitals or clinics as part of their maintenance program. This cross-sectional multicenter study enrolled patients with dental implants who attended regular check-ups as part of a periodontal maintenance program during the period from October 2012 through September 2013. Patients with implants with at least 3 years of loading time were included in the study. The condition of peri-implant tissue was examined and classified into the following categories: healthy, peri-implant mucositis, and peri-implantitis. Patients were also evaluated for implant risk factors. A total of 267 patients (110 men, 157 women; mean age: 62.5 ± 10.7 years) were analyzed. The prevalence of patient-based peri-implant mucositis was 33.3% (n = 89), and the prevalence of peri-implantitis was 9.7% (n = 26). Poor oral hygiene and a history of periodontitis were strong risk factors for peri-implant disease. The present prevalences were lower than those previously reported. The quality of periodontal therapy before and after implant installation and patient compliance and motivation, as indicated by plaque control level, appear to be important in maintaining peri-implant tissue health.

Lysine-specific proteolytic activity responsible for forsythia detaching factor modification.

OBJECTIVES: The objective of the present study was to clarify the lysine-specific proteolytic activity derived from periodontal pathogens responsible for Forsythia detaching factor (FDF) modification. DESIGN: The activity responsible for FDF modification in Tannerella forsythia and Porphyromonas gingivalis were evaluated by colorimetric assay using Ac-Arg-Ala-Lys-p-nitroaniline as a substrate. FDF modification in T. forsythia and P. gingivalis were evaluated by Western blotting using recombinant FDF (rFDF) as a substrate. Furthermore, the activity in GCF of 20 patients with periodontitis and 10 healthy subjects was also evaluated by colorimetric assay. Bacteria in subgingival plaque were detected using polymerase chain reaction. RESULTS: The activity of both bacteria in colorimetric assay were 21.35 unit (P. gingivalis) and 3.61 unit (T. forsythia), respectively. Western blot analysis revealed that P. gingivalis was found to efficiently degrade rFDF and T. forsythia partially cleaved rFDF. The activity in GCF from patients with periodontitis (clinically healthy sites: CH, deep bleeding sites: DB and deep non-bleeding sites: DNB) was significantly higher than those from healthy subjects (healthy sites: H). Among the patients with periodontitis, the activity from CH was significantly lower than those from DB and DNB. T. forsythia was detected in 68.4% of DNB, in 78.4% of DB and in none of CH. P. gingivalis was detected in 63.2% of DNB, in 84.0% of DB and in 10.5% of CH. No bacterium was detected in healthy subjects. CONCLUSION: The lysine-specific proteolytic activity responsible for FDF modification correlates with the presence of major periodontal pathogens.

Modification of forsythia detaching factor by gingival crevicular fluid in periodontitis.

OBJECTIVES: Forsythia detaching factor (FDF) is a virulence factor of Tannerella forsythia detected as a mixture of the 60-kDa form of FDF and the 28-kDa C-terminal fragment (FDFc). The objective of the present study was to clarify the proteolytic activity of gingival crevicular fluid (GCF) from patients with periodontitis and healthy subjects using recombinant FDF (rFDF) as substrate. DESIGN: Eleven patients with periodontitis and 6 healthy subjects were recruited. Modification of rFDF and subsequent production of rFDFc by proteolytic activity of GCF was determined by Western blotting. Proteolytic activity of GCF was evaluated using an Ac-Arg-Ala-Lys-p-nitroaniline substrate. Correlation analysis between two different sets of variables was performed. Variables used in this analysis were proteolytic activity, clinical parameters, relative band density of rFDFc and those of rFDF. RESULTS: Proteolytic activity in GCF was significantly higher in patients with periodontitis than in healthy subjects. Production of rFDFc was determined by treatment of rFDF with GCF from patients with periodontitis and with GCF from healthy subjects. Correlations between clinical parameters and proteolytic activity in GCF were significantly positive. On the other hand, correlations between relative band density of rFDFc or rFDF on Western blot and cleaving activity or clinical parameters were significantly negative. CONCLUSION: The detected extend of GCF-activity generating rFDFc from rFDF and/or even further degrading rFDF correlates with severity of periodontitis.

The significance of podoplanin expression in human inflamed gingiva.

Podoplanin, a transmembrane glycoprotein, has been considered to be expressed specifically by lymphatic endothelial cells. However, recent studies have shown that the protein is expressed in a variety of normal as well as neoplastic tissues, and that its expression might be related to cell migration and invasion. In this study, we examined podoplanin expression in inflamed gingival tissues using an immunohistochemical method. Positive immunoreactivity for podoplanin was found in the cell membrane and cytoplasm of basal cells of oral gingival epithelium when severe inflammatory cell infiltration was present in the connective tissue just under the epithelium. When inflammatory changes were weak or absent, little or no reactivity for podoplanin in the basal cells was observed. Positive reactivity for podoplanin was also detected in basal cell extensions. Surprisingly, strong immunoreactivity for podoplanin was observed in all layers of oral sulcular and junctional epithelia associated with severe inflammatory reaction in the connective tissue. These findings suggest that increased expression of podoplanin in gingival epithelium is related to the progression of chronic periodontitis.

Hypoxia and reoxygenation augment bone-resorbing factor production from human periodontal ligament cells.

BACKGROUND: Oxygen deficiency caused by occlusal trauma and smoking may be associated with bone resorption in periodontitis. In the present study, the effects of hypoxia and reoxygenation on the production of bone-resorbing factors by cultured human periodontal ligament (PDL) cells were examined. METHODS: Human PDL cells were cultured in 1% O(2) (hypoxia), 20% O(2) (normal oxygen tension [normoxia]), or an oxygen concentration that went from 1% to 20% (reoxygenation). The concentrations of bone-resorbing factors, i.e., vascular endothelial growth factor (VEGF), interleukin (IL)-6 and -1beta, tumor necrosis factor-alpha (TNF-alpha), and prostaglandin E(2) (PGE(2)), in the cell culture supernatants were determined by enzyme-linked immunosorbent assay. Expression of the corresponding mRNAs was detected by reverse transcription-polymerase chain reaction. RESULTS: Significantly higher extracellular concentrations of VEGF and IL-6 were detected along with greater corresponding mRNA expression in the hypoxia group compared to the normoxia group. The protein production and mRNA expression of IL-1beta were observed only in the hypoxia group. Neither TNF-alpha nor PGE(2) was detectable in samples from either group, whereas cyclooxygenase-2 mRNA was detected. However, PGE(2) was detected after reoxygenation. Furthermore, VEGF and IL-6 and -1beta production also tended to increase in extracellular concentration and mRNA level after reoxygenation. CONCLUSION: Hypoxia and reoxygenation may stimulate the PDL to produce VEGF, IL-6 and -1beta, and PGE2, which could result in the resorption of alveolar bone in periodontitis.

The active metabolite of leflunomide, A771726, inhibits both the generation of and the bone-resorbing activity of osteoclasts by acting directly on cells of the osteoclast lineage.

Leflunomide is a disease-modifying antirheumatic drug that inhibits paw swelling and joint destruction in type II collagen-induced arthritis in mice and it also delays disease progression in patients with rheumatoid arthritis (RA), through inhibiting proliferation and cytokine production of T cells, via the blocking of de-novo pyrimidine biosynthesis by its active metabolite, A771726. However, the direct action of leflunomide on cells of osteoclast lineage responsible for bone destruction in RA remains to be clarified. In this study, we examined the effect of A771726 on osteoclast formation and bone-resorbing activity in vitro, using cultures of bone marrow-derived osteoclast progenitors and purified functionally mature osteoclasts, and then we elucidated the molecular mechanism of action of the effect of A771726 on osteoclasts. A771726 inhibited osteoclast formation from macrophage colony-stimulating factor (M-CSF)-dependent osteoclast progenitors in the presence of receptor activator of nuclear factor kappa B (NF-kappaB) ligand (RANKL), without any other types of cells present, in a dose-related manner, similar to the inhibition in cultures of unfractionated bone marrow cells. In addition, A771726 suppressed bone resorption by isolated mature osteoclasts. These results indicate that A771726 directly and intrinsically inhibited the differentiation and function of osteoclast lineage cells without any mediation by other cells. The inhibition by A771726 was not restored by the simultaneous addition of uridine, and may be independent of the blockade of NF-kappaB activation and the tyrosine phosphorylation of proteins. Thus, leflunomide, through its active metabolite, has the potential to prevent bone loss by directly inhibiting osteoclastogenesis and osteoclast function. This inhibition suggests a novel mechanism for leflunomide in the retardation of the joint destruction observed in RA patients.

Telomere length in leukocytes and cultured gingival fibroblasts from patients with aggressive periodontitis.

BACKGROUND: The association of genetic risk factors with the pathogenesis of aggressive periodontitis (AgP) has been a focus of attention. Telomeres, which are nucleoprotein complexes at the ends of chromosomes, could be a genetic marker for Down's syndrome and Hutchinson-Gilford progeria, in which patients' premature aging is involved in the pathogenesis. It has been reported that these patients tend to experience severe periodontitis. Therefore, we investigated the telomere length of peripheral blood leukocytes (PBL) obtained from patients with AgP and that in the patients' gingival fibroblasts undergoing cellular aging in vitro. METHODS: Twenty-one patients with AgP and 50 age-matched, periodontally healthy subjects (HS) participated in this study. Genomic DNA from PBL and from human gingival fibroblasts (HGF) was analyzed by Southern blotting for telomere length. The percentage of HGF positive for beta-galactosidase (beta-gal), a marker for cellular senescence, was also investigated. RESULTS: There was no significant difference in the telomere length (P = 0.20, Student's t test) between the two groups, and wide interindividual variation was found (5.93 to 11.4 kbp, average 8.35 +/- 1.19 kbp). The telomere length from PBL negatively correlated with donor age, but no significant difference in telomere loss between the two groups was observed. With HGF undergoing aging in culture, the mean telomere length of these cells from six patients with AgP and seven HS decreased an average of -67.5 bp and -81.0 bp, respectively. No association was found in the telomere length between PBL and HGF from the same donors (r = 0.56, P = 0.20). A significant association was found between the telomere length and the percentages of beta-gal-positive HGF during cell passages (r = 0.70, P < 0.001). CONCLUSIONS: These results indicate that patients with AgP do not have excessive telomere loss and thus do not support the notion of the occurrence of a generalized premature cellular aging in patients with AgP. Further studies are required to investigate the association between telomere length and beta-gal in HGF.

The influence of controlled occlusal overload on peri-implant tissue. part 4: a histologic study in monkeys.

PURPOSE: The purpose of this study was to observe, after removing occlusal trauma and conducting plaque control, possible macroscopic and histologic changes in peri-implant tissue that had deteriorated resulting from experimental peri-implantitis, and to investigate the necessity for treatment procedures for peri-implantitis. MATERIALS AND METHODS: Four monkeys (Macaca fascicularis) in good general health were used in this experiment. Three months after the second premolar and the first molar were extracted from the right mandible, 2 IMZ experimental implants were placed in each monkey. After a 3-month osseointegration period, a second surgery was conducted, followed by making an impression for fabrication of the prosthesis. Excessive occlusal height of the prosthesis was adjusted to 250 microm, and the experiment was continued for 8 weeks after placement of the prosthesis. Three models were created: (1) A superstructure with an excessive occlusal height was used for 8 weeks without any brushing (positive control, model P); (2) after the first 4 weeks with a prosthesis with excessive occlusal height and no brushing, the superstructure was removed and not used for the last 4 weeks while brushing was conducted (experimental model, model E); and (3) for 8 weeks, a prosthesis with an appropriate occlusal height was used with brushing (negative control, model N). RESULTS: When these 3 models were compared with each other, macroscopic findings indicated inflammation only in model P. Mobility of implants was not seen in any model. Histopathologic observations revealed a slight difference between model E and model P in terms of the degree of inflammatory cell infiltration in the connective tissue. DISCUSSION: No difference was found in the degree of bone resorption. Partial tearing was observed at the contact region between epithelial tissue and implant surfaces. CONCLUSIONS: (1) The contact between implants and epithelial or connective tissue is fragile; (2) inflammation and occlusion must be controlled more prudently than in the case of natural teeth; and (3) once peri-implantitis has progressed, the control of occlusion and inflammation is probably not sufficient to promote the healing mechanism.

Osteocalcin, deoxypyridinoline and interleukin-1beta in peri-implant crevicular fluid of patients with peri-implantitis.

The aim of the present study was to analyze the levels of osteocalcin, deoxypyridinoline (Dpd) and interleukin-1beta as markers of bone metabolism in peri-implant crevicular fluid (PICF) from peri-implantitis patients. PICF was sampled from a total of 34 endosseous titanium implants from 16 patients; nine females (mean age 52.8, range 40-62 years) and seven males (mean age 56.0, range 36-66 years). The implants had been in place for a period of 9-112 months (mean; 35.8 months) since the loading. These sites were categorized as six peri-implantitis, eight peri-implant mucositis and 20 healthy implant. PICF volume from peri-implantitis sites was significantly higher than mucositis and healthy implant sites (P < 0.01). Osteocalcin levels in PICF from mucositis sites were significantly higher than healthy implants (P < 0.05), whereas peri-implantitis sites were not significantly different from either mucositis or healthy implant sites. Dpd could not be detected in any of the samples examined. IL-1beta levels in PICF from peri-implantitis sites were significantly higher than levels from peri-implant mucositis (P < 0.05) and healthy implant sites (P < 0.01). In conclusion, osteocalcin in PICF may reflect increased local bone turnover around implants. Further, IL-1beta should be a useful marker for peri-implant inflammation.