Antonio J. Moretti

Alteration of PTGS2 Promoter Methylation in Chronic Periodontitis

Levels of prostaglandin E2 and the prostaglandin-endoperoxide synthase-2 (PTGS2, or COX-2) increase in actively progressing periodontal lesions, but decrease in chronic disease. We hypothesized that chronic inflammation is associated with altered DNA methylation levels within the PTGS2 promoter, with effects on COX-2 mRNA expression. PTGS2 promoter methylation levels from periodontally inflamed gingival biopsies showed a 5.06-fold increase as compared with non-inflamed samples (p = 0.03), and the odds of methylation in a CpG site in the inflamed gingival group is 4.46 times higher than in the same site in the non-inflamed group (p = 0.016). The level of methylation at −458 bp was inversely associated with transcriptional levels of PTGS2 (RT-PCR) (p = 0.01). Analysis of the data suggests that, in chronically inflamed tissues, there is a hypermethylation pattern of the PTGS2 promoter in association with a lower level of PTGS2 transcription, consistent with a dampening of COX-2 expression in chronic periodontitis. These findings suggest that the chronic persistence of the biofilm and inflammation may be associated with epigenetic changes in local tissues at the biofilm-gingival interface.

Interferon‐gamma promoter hypomethylation and increased expression in chronic periodontitis

AIM The goal of this investigation was to determine whether epigenetic modifications in the IFNG promoter are associated with an increase of IFNG transcription in different stages of periodontal diseases. MATERIALS AND METHODS DNA was extracted from gingival biopsy samples collected from 47 total sites from 47 different subjects: 23 periodontally healthy sites, 12 experimentally induced gingivitis sites and 12 chronic periodontitis sites. Levels of DNA methylation within the IFNG promoter containing six CpG dinucleotides were determined using pyrosequencing technology. Interferon gamma mRNA expression was analysed by quantitative polymerase chain reactions using isolated RNA from part of the biological samples mentioned above. RESULTS The methylation level of all six analysed CpG sites within the IFNG promoter region in the periodontitis biopsies {52% [interquartile range, IQR (43.8%, 63%)]} was significantly lower than periodontally healthy samples {62% [IQR (51.3%, 74%)], p=0.007} and gingivitis biopsies {63% [IQR (55%, 74%)], p=0.02}. The transcriptional level of IFNG in periodontitis biopsies was 1.96-fold and significantly higher than tissues with periodontal health (p=0.04). Although the mRNA level from experimental gingivitis samples exhibited an 8.5-fold increase as compared with periodontally healthy samples, no significant methylation difference was observed in experimental gingivitis sample. CONCLUSIONS A hypomethylation profile within IFNG promoter region is related to an increase of IFNG transcription present in the chronic periodontitis biopsies, while such an increase of IFNG in experimentally induced gingivitis seems independent of promoter methylation alteration.

Epigenetic Regulation of TNFA Expression in Periodontal Disease

BACKGROUND Tumor necrosis factor-α (TNF-α) plays a central role in the molecular pathogenesis of periodontal disease. However, the epigenetic regulation attributable to microbial and inflammatory signals at the biofilm-gingival interface are poorly understood. In this study, the DNA methylation alteration within the TNFA promoter in human gingival biopsies from different stages of periodontal disease is investigated and the regulatory mechanism of TNFA transcription by DNA methylation is explored. METHODS Gingival biopsies were obtained from 17 patients with chronic periodontitis (CP) and 18 periodontally healthy individuals. Another 11 individuals participated in an experimentally induced gingivitis study, and gingival biopsies were collected at the baseline, induction, and resolution phase. To confirm that TNFA promoter methylation modulated TNFA transcription, THP.1 cells were treated with a DNA methyltransferase inhibitor, 5-Aza-2-deoxycytidine (5-Aza-2dC), and an RAW294.7 cell line transfected with a TNFA promoter-specific luciferase reporter system with or without methylation was used. RESULTS In gingival biopsies from individuals with severe CP, two individual cytosine-guanine dinucleotides (CpG sites) within the TNFA promoter (at -163 and -161 bp) displayed increased methylation in CP samples compared to those with gingival health (16.1% ± 5.1% versus 11.0% ± 4.6%, P = 0.02 and 19.8% ± 4.1% versus 15.4% ± 3.6%, P = 0.04, respectively). The methylation level at -163 bp was inversely associated with the transcription level of TNFA (P = 0.018). However, no significant difference in the TNFA promoter methylation pattern was observed in samples biopsied during the induction or resolution phase of experimentally induced gingivitis, which represented a reversible periodontal lesion. THP.1 cells treated with 5-Aza-2dC demonstrated a time-dependent increase in TNFA messenger level. It was also found that the luciferase activity decreased 2.6-fold in a construct containing an in vitro methylated TNFA promoter when compared to the unmethylated insert (P = 0.03). CONCLUSION Although the biopsy samples represented a mixed cell population, the change in promoter methylation status in chronic periodontal disease suggested that DNA methylation may be an important regulatory mechanism in controlling TNFA transcriptional expression in periodontal disease.

Bone healing in critical-size defects treated with new bioactive glass/calcium sulfate: a histologic and histometric study in rat calvaria.

This study analyzed histologically the influence of new spherical bioactive glass (NBG) particles with or without a calcium sulfate (CS) barrier on bone healing in surgically created critical-size defects (CSD) in rat calvaria. A CSD was made in each calvarium of 60 rats, which were divided into three groups: C (control): the defect was filled with blood clot only; NBG: the defect was filled with NBG only; and NBG/CS: the defect was filled with NBG covered by CS barrier. Subgroups were euthanized at 4 or 12 weeks. Amounts of new bone and remnants of implanted materials were calculated as percentages of total area of the original defect. Data were statistically analyzed. In contrast to Group C, thickness throughout defects in Groups NBG and NBG/CS was similar to the original calvarium. At 4 weeks, Group C had significantly more bone formation than Group NBG/CS. No significant differences were found between Group NBG and either Group C or Group NBG/CS. At 12 weeks, Group C had significantly more bone formation than Group NBG or NBG/CS. NBG particles, used with or without a CS barrier, maintained volume and contour of area grafted in CSD. Presence of remaining NBG particles might have accounted for smaller amount of new bone in Groups NBG and NBG/CS at 12 weeks post-operative.

In Vivo Assessment of Osseous Wound Healing Using a Novel Bone Putty Containing Lidocaine in the Surgical Management of Tooth Extractions

Objective. This preclinical pilot study evaluated the systemic, radiographic, and histological responses to bone putty containing lidocaine in a canine tooth extraction model. Methods. In five beagle dogs the right mandibular premolars were extracted and sockets grafted with (1) xenograft particulate bone and a collagen sponge plug (control), (2) bone putty alone, (3) bone putty mixed with xenograft (3 : 1), or (4) xenograft sandwiched between bone putty. At 6 weeks post-op, the systemic and local responses were evaluated using a blood chemistry panel, micro-CT, and histological analyses. Results. No significant differences in blood chemistries were noted at 6 weeks postgrafting compared to baseline. Sockets grafted with either bone putty formulation demonstrated comparable radiographic and histologic evidence of bone healing compared to control sockets. Conclusions. Our preclinical results indicate that this bone putty appears to be a safe biocompatible device that may be useful in the postoperative management of tooth extractions.

Potential Correlation between Statins and Pulp Chamber Calcification

INTRODUCTION 3-Hydroxy-3-methylglutaryl-coenzyme A reductase inhibitors (statins) are the first-line pharmaceuticals for the prevention and treatment of dyslipidemia. A recent investigation has shown that statins induced odontoblastic differentiation of dental pulp stem cells. Statins enhance the differentiation of human dental pulp cells by up-regulating mineralization nodules and odontogenic markers. This study tested the hypothesis that the systemic administration of statins results in increased dental pulp calcification. METHODS This retrospective case-control study used digital bitewing radiographs of mandibular molars. Subjects (N = 90) aged ≥60 years were assigned to either test (n = 45) or control (n = 45) groups based on the systemic use of statins. The dimensions of the pulp chambers were measured using a standardized method for height and mesiodistal distances. The chi-square test was used to analyze the data. Multiple linear regression model analysis was performed to explore the association between statin intake and pulp calcification. RESULTS Three of the 45 mandibular molars in the test group exhibited almost complete pulp chamber obliteration. There was a significant reduction in pulp chamber height ratio shown in the statin group compared with the control group (P < .0001). When the mesiodistal width was compared between the 2 groups, there was no significant difference (P = .3730). CONCLUSIONS The significant increase of calcification and loss of vertical height of the pulp chamber observed in mandibular molars in patients on statin medication indicated a possible increased odontoblastic activity. Therefore, systemic statins could be a contributing factor for pulp chamber calcification.