Periodontal diseases are inflammatory diseases resulting in the destruction of tissues of the periodontium. Although bacteria must be present for periodontal disease to occur, a susceptible host is also required, which is determined by genetic, environmental, and acquired factors. One such factor, autoimmunity, may play a role in the tissue destruction. Data indicate that some antibodies that occur in the gingival lesion are directed to host tissue components, such as type I collagen, although investigations of other periodontal autoimmune targets are limited.
Histologic sections and extracts from periodontally healthy teeth and the associated soft tissues were probed with serum from localized aggressive periodontitis (LAgP), chronic periodontitis (CP), and periodontally healthy subjects to determine autoreactivity to components of the periodontium. Any autoreactivity observed was characterized further by mass spectrometry and enzyme-linked immunosorbent assay.
Autoreactivity to components of the periodontium was observed in CP and LAgP. Known autoimmune targets, such as collagen and heat shock protein, were identified along with multiple potential autoimmune targets, including members of the extracellular matrix, such as vimentin, spectrin, filamin, actin, lamin, keratin, and tubulin. Finally, it was determined that the autoreactivity observed in LAgP was more severe and diverse than that observed in CP.
These data demonstrated that autoimmune reactivity can play a role in the tissue destruction of periodontal disease but that the nature of the autoreactivity may differ based on the type and/or stage of periodontal disease.
Antibody; autoimmunity; collagen; periodontal diseases
Reports from studies of twins, disease aggregation in families, animal models for periodontal disease, and various genetic-analysis studies have determined that genetics plays a role in the susceptibility to periodontal disease. The purpose of this pilot study was to evaluate the effect of genetics on periodontal disease by evaluating the heritability of alveolar bone loss in a captive baboon population.
A collection of baboon skulls from a pedigreed colony (for which scientists and veterinarians maintain complete genealogical and veterinary records) were obtained from the Southwest National Primate Research Center and used in this pilot study. Measurements of alveolar bone loss were performed on 390 dry baboon skulls. A periodontal probe was used to measure alveolar bone loss. Maximum likelihood methods (designed to handle complex genealogies) were used to determine the heritability of alveolar bone loss. This software utilized known pedigrees in the captive baboon sample and tested the relationship between pairwise kinship and alveolar bone loss data to determine the heritability of alveolar bone loss from periodontal disease.
Genetic data were available for 347 of the 390 specimens. Using age and sex as covariates, genetic analysis indicated a heritability of 35% (standard error=20%, p=0.01). While sex was not a significant factor in periodontal disease (p=0.96), age was highly significantly associated with periodontal disease (p<0.0001).
In this pilot study, analysis of alveolar bone loss measurements from captive baboons indicates that bone loss increases with age and that a portion of periodontal disease risk may be due to genetic variance. These findings provide evidence that periodontal disease is heritable in captive baboons and indicate that a larger, more-detailed study is warranted.
periodontitis; alveolar bone; genetics; baboons
Current scientific evidence addressing the relation between periodontitis and hypertension is limited to a few studies producing inconsistent results.
All participants of an on-going representative cohort of Puerto Rican elderly who were 70 years and older and residing in San Juan metropolitan area were invited to this cross-sectional study. Periodontal probing depth (PD) and attachment loss (AL) were summarized using CDC-AAP definition for severe periodontitis (≥2 teeth with AL ≥6mm and ≥1 tooth with PD ≥5mm). We averaged three repeated blood pressure (BP) measurements taken using a standardized auscultatory method. Information on hypertension history, use of anti-hypertensive medications and potential confounders (age, gender, smoking, heavy and binge drinking, diabetes, utilization of preventive dental services, flossing, body mass index, fruit and vegetable, whole wheat bread and high-fiber cereal consumption) was collected during in-person interviews. High BP was defined as average systolic BP ≥140 mmHg or diastolic ≥90 mmHg. Multivariate logistic regression models were used to study the relation between severe periodontitis, hypertension history and high BP.
The study population comprised 182 adults. In multivariate analysis, there was no association between severe periodontitis and hypertension history (OR=0.99, 95% CI: 0.40–2.48). Severe periodontitis was associated with high BP, with OR of 2.93 (95% CI: 1.25–6.84), after adjusting for age, gender, smoking, and binge drinking. This association was stronger when restricted to those with hypertension or taking anti-hypertensive medications: OR=4.20 (95% CI: 1.28–13.80).
Our results suggest that periodontitis may contribute to poor blood pressure control among older adults.
Periodontal diseases; periodontitis; hypertension; blood pressure
The periodontal pathogen Actinobacillus actinomycetemcomitans produces a leukotoxin that is considered a primary virulence factor in localized juvenile Periodontitis (LJP). Select strains of the bacterium contain a 530-bp deletion in the promoter region of the leukotoxin gene operon which results in enhanced transcription of the leukotoxin. DNA hybridization and polymerase chain reaction (PCR) were used to examine genetic variants of A. actinomycetemcomitans in 24 LJP-susceptible children from 21 families having a history of the disease and 34 control children from non-LJP families. A significant association was found between the detection of variants that had a deletion in the leukotoxin promoter region, indicative of a high level expression leukotoxin genotype, and conversion from a healthy periodontal status to disease. Subjects harboring A. actinomycetemcomitans of this genotype were more likely to convert to LJP than those subjects who had variants containing the full length leukotoxin promoter region (odds ratio = 22.50, 95% C.I.). These findings support the concept that highly virulent strains or clonal types of periodontal pathogens play a major role in the initiation of periodontal disease in susceptible hosts.
Periodontitis; juvenile/epidemiology; Actinobacillus actinomycetemcomitans; leukotoxin; epidemiology; polymerase chain reaction; polymorphism; restriction fragment length
Biofilm-induced inflammatory osteolytic oral infections, such as periodontitis and peri-implantitis, have complex etiology and pathogenesis. A significant obstacle to research has been the lack of appropriate animal models where the inflammatory response to biofilms can be investigated. The aim of this study is to develop a novel animal model to study the host response to Aggregatibacter actinomycetemcomitans (previously Actinobacillus actinomycetemcomitans)–biofilm colonizing titanium implants.
Titanium implants were inoculated in vitro with A. actinomycetemcomitans, establishing a biofilm for 1 to 3 days. Biofilm-inoculated and control implants were transmucosally placed into rat hard palate or alveolar ridge. Analysis included documentation of clinical inflammation, polymerase chain reaction, and culture detection of A. actinomycetemcomitans and microcomputed tomography quantitation of peri-implant bone volume.
Viable A. actinomycetemcomitans biofilm was successfully established on titanium implants in vitro, detected by confocal laser scanning microscopy. An inflammatory response characterized by clinical inflammation, bleeding, ulceration, hyperplasia, and necrosis was observed around biofilm-inoculated implants. A. actinomycetemcomitans was detected by polymerase chain reaction and culture analysis on 100%of biofilm-inoculated implants for up to 3 weeks and 25%for up to 6 weeks. Microcomputed tomography analysis demonstrated significantly lower bone volume (P <0.05) around biofilm-inoculated implants (29.6% ± 7.6%) compared to non-inoculated implants (50.5% ± 9.6%) after 6 weeks.
These results describe a novel animal model where A. actinomycetemcomitans biofilm was established in vitro on titanium implants before placement in rat oral cavity, leading to an inflammatory response, osteolysis, and tissue destruction. This model may have potential use for investigation of host responses to biofilm pathogens and antibiofilm therapy.
Aggregatibacter actinomycetemcomitans; biofilms; dental implants; infection; models; animal; rats
Dental biofilms contain a protein that inhibits mammalian cell growth, possibly lysine decarboxylase from Eikenella corrodens. This enzyme decarboxylates lysine, an essential amino acid for dentally attached cell turnover in gingival sulci. Lysine depletion may stop this turnover, impairing the barrier to bacterial compounds. The aims of this study were to determine biofilm lysine and cadaverine contents before oral hygiene restriction (OHR), and their association with plaque index (PI) and gingival crevicular fluid (GCF) after OHR for a week.
Laser-induced fluorescence after capillary electrophoresis was used to determine lysine and cadaverine contents in dental biofilm, tongue biofilm and saliva before OHR and in dental biofilm after OHR.
Before OHR, lysine and cadaverine contents of dental biofilm were similar and 10-fold greater than in saliva or tongue biofilm. After a week of OHR, the biofilm content of cadaverine increased and that of lysine decreased, consistent with greater biofilm lysine decarboxylase activity. Regression indicated that PI and GCF exudation were positively related to biofilm lysine post-OHR, unless biofilm lysine exceeded the minimal blood plasma content in which case PI was further increased but GCF exudation was reduced.
After OHR, lysine decarboxylase activity seems to determine biofilm lysine content and biofilm accumulation. When biofilm lysine exceeds minimal blood plasma content after OHR, less GCF appeared despite more biofilm. Lysine appears important for biofilm accumulation and the epithelial barrier to bacterial proinflammatory agents.
Inhibiting lysine decarboxylase may retard the increased GCF exudation required for microbial development and gingivitis.
Gingival crevicular fluid; Gingivitis; Oral hygiene; Pathogenesis of periodontal disease(s); Microbiology
We previously reported in a cross-sectional study that patients who were in periodontal maintenance programs and were taking vitamin D and calcium supplementation had a trend for better periodontal health compared with patients not taking supplementation. The objective of the present study was to determine, for the same group of subjects, whether there was a difference in periodontal health over a one–year period.
Fifty-one patients enrolled in maintenance programs from two dental clinics were recruited. Twenty-three were taking vitamin D (≥400 international units/day) and calcium (≥1000mg/day) supplementation, and twenty-eight were not taking supplementation. All subjects had ≥2 interproximal sites with ≥3 mm clinical attachment loss. For mandibular-posterior teeth, these clinical parameters were recorded: gingival index, plaque index, probing depth, attachment loss, bleeding upon probing, calculus index and furcation involvement. Photostimulable-phosphor, posterior bitewing radiographs were taken to assess alveolar bone. Daily vitamin D and calcium intakes were estimated by nutritional analysis. Data were collected at baseline, 6 months, and 12 months.
Clinical parameters improved with time in both groups (p<0.01). When clinical measures were considered collectively, the results were borderline significant at baseline (p=0.061), significant at 6 months (p=0.049) but not significant at 12 months (p=0.114). After adjusting for covariates, the effect of supplements was significant at baseline (p=0.037), borderline at 6 months (p=0.058) and not significant at 12 months (p=0.142)
Calcium and vitamin D supplementation has a modest positive effect on periodontal health, and consistent dental care improves clinical parameters of periodontal disease regardless of such supplements. Calcium and vitamin D supplementation has a modest positive effect on periodontal health, and consistent dental care improves clinical parameters of periodontal disease regardless of such supplements. Our findings raise the possibility that vitamin D, perhaps at higher doses, may positively impact on periodontal disease severity.
vitamin D; calcium; chronic periodontitis; alveolar bone
Inflammatory periodontal diseases are a leading cause of tooth loss and are linked to multiple systemic conditions, such as cardiovascular disease and stroke. Reconstruction of the support and function of affected tooth-supporting tissues represents an important therapeutic endpoint for periodontal regenerative medicine. An improved understanding of periodontal biology coupled with current advances in scaffolding matrices has introduced novel treatments that use cell and gene therapy to enhance periodontal tissue reconstruction and its biomechanical integration. Cell and gene delivery technologies have the potential to overcome limitations associated with existing periodontal therapies, and may provide a new direction in sustainable inflammation control and more predictable tissue regeneration of supporting alveolar bone, periodontal ligament, and cementum. This review provides clinicians with the current status of these early-stage and emerging cell- and gene-based therapeutics in periodontal regenerative medicine, and introduces their future application in clinical periodontal treatment. The paper concludes with prospects on the application of cell and gene tissue engineering technologies for reconstructive periodontology.
Macrolide antibiotics yield high concentrations in inflamed tissue, suggesting that their levels in gingival crevicular fluid (GCF) could be increased at gingivitis sites. However, the increased volume of GCF associated with gingivitis could potentially dilute macrolides. To determine whether these assumptions are correct, the bioavailability of systemically-administered azithromycin was compared in GCF from healthy and gingivitis sites.
Materials and methods
Experimental gingivitis was induced in one maxillary posterior sextant in nine healthy subjects. Contralateral healthy sextants served as controls. Subjects ingested 500 mg of azithromycin followed by a 250 mg dose 24 hours later. Four hours after the second dose, plaque was removed from experimental sites. GCF was collected from 8 surfaces in both the experimental and control sextants and pooled separately. GCF samples were subsequently collected on the 2nd, 3rd, 8th and 15th days and azithromycin content was determined by agar diffusion bioassay.
On days 2 and 3, the pooled GCF volume at experimental sites was significantly higher than at control sites (P <0.01) and the total azithromycin mass in 30 second GCF samples pooled from experimental sites was significantly higher than at control sites (P < 0.02). However, there were no significant differences in azithromycin concentration between the experimental and control pools at any point. Concentrations exceeded 7.3 μg/ml on day 2 and 2.5 μg/ml on day 15.
Azithromycin concentrations are similar in GCF from gingivitis sites and healthy sites, suggesting that the processes that regulate GCF azithromycin concentration can compensate for local inflammatory changes.
Anti-infective agents; inflammation; pharmacokinetics
Understanding the molecular features of bone repair and osseointegration may aid in the development of therapeutics to improve implant outcomes. The purpose of this investigation is to determine the gene expression dynamics during alveolar bone repair and implant osseointegration.
An implant osseointegration preclinical animal model was used whereby maxillary defects were created at the time of oral implant placement, while a tooth extraction socket healing model was established on the contralateral side of each animal. The surrounding tissues in the zone of the healing defects were harvested during regeneration for temporal evaluation using histology, immunohistochemistry, laser capture microdissection, and quantitative reverse transcription–polymerase chain reaction for the identification of a panel of 17 putative genes associated with wound repair.
In both models, three distinct expression patterns were displayed: 1) genes that are slowly increased during the healing process, such as bone morphogenetic protein 4, runt-related transcription factor 2, and osteocalcin; 2) genes that are upregulated at the early stage of healing and then downregulated at later stages, such as interleukin and chemokine (C-X-C motif) ligands 2 and 5; and 3) genes that are constitutively expressed over time, such as scleraxis. Although some similarities between osseointegration and tooth extraction socket were seen, distinct features developed and triggered a characteristic coordinated expression and orchestration of transcription factors, growth factors, extracellular matrix molecules, and chemokines.
Characterization of these events contributes to a better understanding of cooperative molecular dynamics in alveolar bone healing, and highlights potential pathways that could be further explored for the enhancement of osseous regenerative strategies.
Analysis of samplings from periodontal pockets is important in diagnosis and therapy control of periodontitis. In this study, three different sampling techniques were compared to determine if one method can yield samples suitable for reproducible and simultaneous determination of bacterial load, cytokines, neutrophil elastase, and Arg-specific gingipains. R-gingipains are an important virulence factor of Porphyromonas gingivalis, the exact concentration of which in gingival crevicular fluid (GCF) has not yet been quantified.
GCF was sampled from four sites per patient (each two sites one method) in 36 chronic periodontitis patients. One week later, the procedure was repeated with alternative methods. The variables that had been determined were: loads of Aggregatibacter actinomycetemcomitans and P. gingivalis, levels of interleukin-6 and interleukin-8, activity of neutrophil elastase and level of R-gingipains.
The detected cytokine levels were higher using paper strips compared to paper points. Bacteria were found in similar loads from the paper strips and paper points. R-gingipains were detectable in high quantities only by washing of the periodontal pocket. The level of R-gingipains correlated with the load of P. gingivalis.
The use of paper strips is suitable for simultaneous determination of microbial and immunological parameters. Obtaining GCF by washing can be useful for special purposes. Gingipain concentration in periodontal pockets was directly determined to be up to 1.5 μM. This value indicates that most of so far identified substrates of these proteases by in vitro assays can be easily degraded in P. gingivalis infected sites.
gingival crevicular fluid; sampling techniques; Porphyromonas gingivalis; Arg-gingipain; cytokines
Fluoxetine, a selective serotonin reuptake inhibitor, has recently been found to possess anti-inflammatory properties. The present study investigated the effects of fluoxetine on inflammatory tissue destruction in a rat model of ligature-induced periodontitis (PD).
Male Wistar rats were randomly assigned into three groups (n=10 animals/group): 1) Control rats (without ligature); 2) rats with ligature + placebo (saline; oral gavage); 3) rats with ligature + fluoxetine (20 mg/kg/day in saline; oral gavage). Histological analyses were performed on the furcation region and mesial of mandibular first molars of rats sacrificed at 15 days after ligature-induced PD. Reverse transcriptase-polymerase chain reaction (RT-PCR) and zymography were performed to analyze the mRNA expression of interleukin (IL)-1β, cyclooxygenase (COX)-2, matrix metalloproteinase (MMP)-9 and inducible nitric oxide synthase (iNOS), and the MMP-9 activity, respectively, in gingival tissues samples.
Compared to the ligature + placebo group, alveolar bone loss was reduced in the fluoxetine group (P < 0.05), and the integrity of collagen fibers in the gingival tissue was maintained. Moreover, in gingival tissue sampled 3 days after ligature attachment, fluoxetine administration reduced IL-1β and COX-2 mRNA expression. Fluoxetine down-regulated MMP-9 activity, without affecting MMP-9 mRNA expression induced by ligature, compared to the ligature + placebo group (P < 0.05). These data suggested that fluoxetine suppressed proinflammatory responses, as well as proteolytic enzyme activity, induced by ligature.
In the present study, fluoxetine suppressed the inflammatory response and protected against periodontal bone resorption and destruction of collagen fibers, suggesting that fluoxetine can constitute a promising therapeutic approach for periodontal diseases.
Fluoxetine; inflammation; periodontitis; bone resorption; collagen
The proinflammatory chemokine interleukin-8 (IL8) is important in the regulation of the inflammatory response. Single nucleotide polymorphism (SNP) rs4073 have shown that the A allele up regulates the IL8 levels after stimulation with lipopolysaccharides. We investigated the association of the SNP rs4073 with chronic periodontitis.
Genotyping was performed by standard polymerase chain reaction-restriction fragment length polymorphism assay in 289 genomic DNA samples of healthy control and chronic periodontitis patients; analyses were adjusted by the multivariate logistic regression modeling. Real-time PCR performance was used to detect the levels of the IL8 mRNA.
Analysis points to a statistical significant association of chronic periodontitis with the heterozygous TA genotype (p = 0.001); the results showed an increase in the frequency of the A allele in the diseased group (36% in the control × 48% in the periodontitis group). The higher levels of the IL8 mRNA were found in the periodontitis group, mainly in individuals who presented the TA genotype (p=0.03).
The SNP rs4073 is associated with chronic periodontitis, in non-smoker Brazilian subjects, since the frequency of A allele is higher in the disease than control group and the TA genotype was associated with increased levels of IL8 mRNA transcripts.
periodontitis; polymorphism; single nucleotide; Interleukin-8; genotype; alleles
Inflammatory stimuli activate inducible nitric oxide synthase (iNOS) in a variety of cell types, including osteoclasts (OC) and osteoblasts, resulting in sustained NO production. In this study, we evaluate the alveolar bone loss in rats with periodontitis under long-term iNOS inhibition, and the differentiation and activity of OC from iNOS-knockout (KO) mice in vitro.
Oral aminoguanidine (an iNOS inhibitor) or water treatment was started 2 weeks before induction of periodontitis. Rats were sacrificed 3, 7, or 14 days after ligature placement, and alveolar bone loss was evaluated. In vitro OC culture experiments were also performed to study the differentiation of freshly isolated bone marrow cells from both iNOS KO and wild-type C57BL/6 mice. OC were counted 6 days later after tartrate-resistant acid phosphatase staining (a marker of osteoclast identity), and bone resorption activity was assessed by counting the number of resorption pits on dentin disks.
Rats with ligature showed progressive and significant alveolar bone loss compared to sham animals, and aminoguanidine treatment significantly inhibited ligature-induced bone loss at 7 and 14 days after the induction. In comparison to bone marrow cells from wild-type mice, cells from iNOS KO mice showed decreased OC growth and the resulting OC covered a smaller culture dish area and generated fewer resorption pit counts.
Our results demonstrate that iNOS inhibition prevents alveolar bone loss in a rat model of ligature-induced periodontitis, thus confirming that iNOS-derived NO plays a crucial role in the pathogenesis of periodontitis, probably by stimulating OC differentiation and activity.
Animal experimental use; bone resorption; iNOS enzyme; nitric oxide; osteoclasts; periodontitis
A major cause of chronic inflammatory periodontal diseases is Porphyromonas gingivalis (P. gingivalis), a non-motile, gram-negative, rod-shaped, anaerobic bacterium. Within gingival tissue, both macrophages and fibroblasts participate in the immune response to foreign entities by releasing cytokines and expressing molecules to recruit and activate lymphocytes. However, the contribution of gingival macrophages and fibroblasts to the immune response to P. gingivalis infection is not fully known.
The AMJ2-C8 cell line (AM cells), a mouse alveolar macrophage cell line, and ESK-1 cells, a mouse gingival fibroblast cell line made in our laboratory, were treated with lipopolysaccharide (LPS) from either P. gingivalis or Escherichia coli (E. coli). The expression of immune response molecules was quantified by real-time PCR and enzyme-linked immunoassay.
AM and ESK-1 cells responded differently to P. gingivalis and E. coli LPS stimulation. The ESK-1 gingival fibroblast cell line was more responsive to E. coli LPS stimulation as seen by elevated levels of interleukin (IL)-6, inducible nitric oxide (iNOS), and monocyte chemotactic protein-1 (MCP-1) expression relative to stimulation by P. gingivalis LPS. Conversely, the AM macrophage cell line was more responsive to P. gingivalis LPS stimulation, particularly for interleukin IL-1β, IL-6, and MCP-1, relative to stimulation by E. coli LPS.
These findings demonstrate that E. coli LPS induces a stronger cytokine/chemokine response in gingival fibroblasts, while P. gingivalis LPS induces a stronger response in macrophages.
cytokines; fibroblasts; host response; immunology; inflammation and innate immunity
This report is a further analysis of a study designed to determine clinical and microbial risk indicators for progressing periodontitis.
MATERIAL AND METHODS
One hundred and ninety subjects periodontally healthy or adults with early signs of periodontitis (20–40 years) were monitored clinically at 6-month intervals followed by supragingival cleaning. At each visit, GCF and blood were collected for determination of IL-1β content (GCF) and IL-1 genotype (blood). Inter-proximal sites with >1.5 mm increase in clinical attachment over 18 months were considered disease active. Characteristics were compared between active and inactive subjects.
IL-1β levels in GCF increased with severity of disease and correlated well with clinical signs of incipient disease. However, IL-1 genotype did not show any significant associations with disease or extent of disease.
Indicators of inflammation may be important clinical determinants of future periodontal disease progression, but IL-1 genotype was not a risk indictor for early (slight) periodontitis as defined in this subject population.
genotype; gingival crevicular fluid; health; Periodontitis; race/ethnicity smoking
Obesity is increasing in prevalence and is a major contributor to worldwide morbidity. One consequence of obesity might be an increased risk for periodontal disease, although periodontal inflammation might, in turn, exacerbate the metabolic syndrome, of which obesity is one component. This review aims to systematically compile the evidence of an obesity–periodontal disease relationship from epidemiologic studies and to derive a quantitative summary of the association between these disease states.
Systematic searches of the MEDLINE, SCOPUS, BIOSIS, LILACS, Cochrane Library, and Brazilian Bibliography of Dentistry databases were conducted with the results and characteristics of relevant studies abstracted to standardized forms. A meta-analysis was performed to obtain a summary measure of association.
The electronic search identified 554 unique citations, and 70 studies met a priori inclusion criteria, representing 57 independent populations. Nearly all studies matching inclusion criteria were cross-sectional in design with the results of 41 studies suggesting a positive association. The fixed-effects summary odds ratio was 1.35 (Shore-corrected 95% confidence interval: 1.23 to 1.47), with some evidence of a stronger association found among younger adults, women, and non-smokers. Additional summary estimates suggested a greater mean clinical attachment loss among obese individuals, a higher mean body mass index (BMI) among periodontal patients, and a trend of increasing odds of prevalent periodontal disease with increasing BMI. Although these results are highly unlikely to be chance findings, unmeasured confounding had a credible but unknown influence on these estimates.
This positive association was consistent and coherent with a biologically plausible role for obesity in the development of periodontal disease. However, with few quality longitudinal studies, there is an inability to distinguish the temporal ordering of events, thus limiting the evidence that obesity is a risk factor for periodontal disease or that periodontitis might increase the risk of weight gain. In clinical practice, a higher prevalence of periodontal disease should be expected among obese adults.
Body weight; obesity; overnutrition; periodontal diseases; review
Azithromycin is a macrolide antibiotic that is active against several periodontal pathogens. Macrolides are taken up and concentrated inside gingival fibroblasts, which could influence their pharmacokinetics. This study tested the hypothesis that steady-state levels of azithromycin are higher and more sustained in gingival crevicular fluid (GCF) than in serum.
Four healthy subjects received an initial dose of 500 mg azithromycin followed by 250 mg doses on each of the next 2 days. Serum and GCF samples were obtained 2 hours after the last dose (day 2) and on days 4 and 7. GCF samples were collected from maxillary posterior sites with paper strips. The strips were pooled and eluted with high purity water. After extraction, the azithromycin content of the serum samples and GCF eluates was determined with an agar diffusion bioassay.
On days 2, 4 and 7, the concentrations of azithromycin in blood serum were 0.22 ± 0.02, 0.08 ± 0.02 and 0.04 ± 0.01 μg/ml, respectively. The concentrations in GCF were 8.82 ± 1.25, 7.90 ± 1.72 and 7.38 ± 1.15 μg/ml, respectively. Mean GCF levels were significantly higher than mean serum levels (P ≤ 0.02, paired t-test).
The findings demonstrate that the pharmacokinetic profiles of azithromycin are different in GCF and serum. At steady state, azithromycin concentrations in GCF were higher and more sustained than those in serum. Based on previous studies, the levels observed in GCF were above the MIC for Aggregatibacter actinomycetemcomitans, Porphyromonas gingivalis, and Prevotella intermedia.
Anti-infective agents; Periodontitis; Tissue distribution
Clarithromycin inhibits several periodontal pathogens and is concentrated inside gingival fibroblasts and epithelial cells by an active transporter. We hypothesized that polymorphonuclear leukocytes (PMNs) and less mature myeloid cells possess a similar transporter for clarithromycin. It is feasible that clarithromycin accumulation inside PMNs could enhance their ability to kill Aggregatibacter actinomycetemcomitans.
To test the first hypothesis, purified PMNs and cultured HL-60 cells were incubated with [3H]-clarithromycin. Clarithromycin transport was assayed by measuring changes in cell-associated radioactivity over time. The second hypothesis was examined with PMNs loaded by incubation with clarithromycin (5 μg/ml). Opsonized bacteria were incubated at 37°C with control and clarithromycin-loaded PMNs.
Mature human PMNs, HL-60 cells differentiated into granulocytes and undifferentiated HL-60 cells all took up clarithromycin in a saturable manner. The kinetics of uptake by all yielded linear Lineweaver-Burk plots. HL-60 granulocytes transported clarithromycin with a Km of approximately 250 μg/ml and a Vmax of 473 ng/min/106 cells, which were not significantly different from the values obtained with PMNs. At steady state, clarithromycin levels inside HL-60 granulocytes and PMNs were 28- to 71-fold higher than extracellular levels. Clarithromycin-loaded PMNs killed significantly more A. actinomycetemcomitans and achieved shorter half times for killing than control PMNs when assayed at a bacteria-to-PMN ratio of 100:1 (P < 0.04). At a ratio of 30:1, these differences were not consistently significant.
PMNs and less mature myeloid cells possess a transporter that takes up and concentrates clarithromycin. This system could help PMNs cope with an overwhelming infection by A. actinomycetemcomitans.
macrolides; neutrophils; granulocyte precursor cells; periodontitis
Analysis of biomarkers in gingival crevicular fluid (GCF) may be helpful in forecasting vulnerability to future attachment loss. This paper sought to correlate GCF biomarkers of inflammation and bone resorption with subsequent periodontal attachment and bone loss in a longitudinal trial of a matrix metalloproteinase (MMP)-inhibitor.
GCF was collected from two periodontal pockets (mean 5.1 ± SD 1.0 mm) at baseline and annually in postmenopausal females with moderate/advanced periodontitis undergoing periodontal maintenance every 3–4 months during a two-year double-blind, placebo-controlled, randomized clinical trial of subantimicrobial-dose doxycycline (SDD, 20 mg bid). Subjects were randomized to SDD (n = 64) or placebo (n = 64). GCF was analyzed for inflammation markers interleukin-1β (enzyme-linked immunosorbent assay), total collagenase activity (hydrolysis of synthetic octapeptide), MMP-8 (Western blot), and bone resorption marker carboxyterminal telopeptide cross-link fragment of type I collagen (ICTP; radioimmunoassay). Generalized estimating equations were used to associate these biomarkers, categorized into tertiles, with subsequent clinical attachment (automated disk probe) or interproximal bone loss (radiography). Odds ratio (OR) values compared highest to lowest tertile groups.
Increases in GCF IL-1β and MMP-8 during the first year of periodontal maintenance were associated with increased odds of subsequent (year 2) periodontal attachment loss (OR = 1.67, p = 0.01 and OR = 1.50, p = 0.02, respectively), driven by the placebo group. Elevated baseline ICTP also was associated with increased odds of 1- and 2-year alveolar bone density loss (OR = 1.98, p = 0.0001) in placebo, not SDD, and bone height loss (OR = 1.38, p = 0.06), again driven by placebo.
These data support the hypothesis that elevated GCF biomarkers of inflammation and bone resorption from a small number of moderate/deep sites hold promise in identifying patients vulnerable to progressive periodontitis, and SDD may modify that risk.
Gingival crevicular fluid; doxycycline; periodontitis; type I collagen; interleukin-1; matrix metalloproteinase-8
Chronic kidney disease (CKD) is a worldwide health problem with increasing prevalence and poor outcomes including severe cardiovascular disease and renal osteodystrophy. With advances in medical treatment, CKD patients are living longer and require oral care. The aim of this study was to determine the effects of CKD and dietary phosphate on mandibular bone structure using a uremic mouse model.
Uremia (U) was induced in female DBA/2 mice by partial renal ablation. Uremic mice received either a normal phosphate (NP) or a high phosphate (HP) diet. Sham surgeries were performed in a control group of mice, and half received either a NP or a HP diet. At termination, animals were sacrificed and mandibles collected for microcomputed tomography (micro-CT) and histological analysis.
Sera levels of BUN, PTH and alkaline phosphatase were all significantly increased in U/NP and U/HP vs. Sham controls, while serum calcium was increased in the U/HP group and no differences were noted in serum phosphate levels between groups. Micro-CT analyses revealed a significant reduction in cortical bone thickness and an increase in trabecular thickness and trabecular bone volume/tissue volume in U/NP and U/HP groups compared to Sham/NP. A significant reduction in cortical bone thickness was also found in the Sham/HP vs. Sham/NP group. Histological evaluation confirmed increased trabeculation in the U groups.
CKD in mice, especially under conditions of high phosphate feeding, results in marked effects on alveolar bone homeostasis.
Bone biology; periodontal-systemic disease interactions; risk factor(s)
The objectives were to measure the levels of gingival crevicular fluid (GCF) biomarkers and subgingival bacterial species in periodontally healthy and periodontitis subjects in order to explore relations among these biomarkers, the subgingival microbiota, and clinical parameters of periodontal disease.
Material and methods
Clinical periodontal parameters were measured at 6 sites per tooth in 20 periodontitis and 20 periodontally healthy subjects. GCF and subgingival plaque samples were obtained from the mesiobuccal aspect of every tooth. GCF levels of interleukin-1β (IL-1β), matrix metalloproteinase-8 (MMP-8) and IL-8 were measured using checkerboard immunoblotting and the levels of 40 bacterial taxa quantified using checkerboard DNA-DNA hybridization. A subset of “clinically healthy” (CH) sites from each group was analyzed separately. Significance of differences between groups was determined using the unpaired t-test or the Mann-Whitney test. Correlations among immunological, microbiological and clinical data were determined using the Spearman rank correlation coefficient.
There were positive correlations among mean clinical parameters and mean levels of the 3 biomarkers and proportions of Orange and Red complex species (p<0.05). CH sites from periodontitis subjects had higher levels of IL-1β and IL-8 and higher proportions of Orange and Red complex species (p<0.05) than CH sites from periodontally healthy subjects. Red complex species were positively associated with the expression of all biomarkers (p<0.05), while Purple and Yellow complex species had negative correlations with IL-1β and IL-8 (p<0.05).
CH sites from periodontitis subjects present higher levels of GCF biomarkers and periodontal pathogens than CH sites from periodontally healthy subjects. Different microbial complexes demonstrated distinct associations with specific GCF biomarkers.
Periodontal diseases; Microbiology; Gingival crevicular fluid; Cytokines; Matrix metalloproteinases
Few studies have specifically examined the relationship between periodontal disease and gestational diabetes mellitus (GDM). The objective of this study was to examine whether maternal periodontal disease is associated with GDM.
A case-control study was conducted of 53 pregnant women with GDM and 106 pregnant women without GDM at Woman’s Hospital, Baton Rouge, USA. The periodontal examinations were performed by a calibrated dentist who was blinded on the diabetic status of the pregnant women. Periodontitis was defined as the presence of any site with a probing depth (PD) ≥ 4 mm or a clinical attachment loss (CAL) ≥ 4 mm. The severity of periodontal disease was measured in quartiles of PD and CAL. Univariable analysis and multivariable logistic regression were used to examine the relationships between periodontal disease and GDM.
The percentage of periodontitis was 77.4% in women with GDM and 57.5% in pregnant non-GDM women, with an odds ratio (OR) and 95% confidence interval (CI) of 2.5 (1.2–5.3). After adjusting for confounding variables of maternal age, parity, race, marital status, education, family income, smoking, alcohol consumption, systemic antibiotics in pregnancy, family history of diabetes, income, dental insurance coverage and body mass index, the adjusted OR (95% CI) was 2.6 (1.1–6.1). The adjusted ORs (95% CIs) of GDM comparing the highest-to-lowest quartiles of PD and CAL were 3.8 (1.0–14.0) and 4.5 (1.2–16.9).
This study supports the hypothesis of an association between periodontal disease and GDM.
Case-control study; gestational diabetes mellitus; periodontal disease; pregnancy
To determine: 1) if periodontal treatment in pregnant women before 21 weeks of gestation alters levels of inflammatory mediators in serum; and 2) if changes in these mediators are associated with birth outcomes.
823 pregnant women with periodontitis were randomly assigned to receive scaling and root planing before 21 weeks of gestation or after delivery. Serum obtained between 13 weeks and 16 weeks 6 days (study baseline) and 29–32 weeks gestation was analyzed for C-reactive protein, prostaglandin E2, matrix metalloproteinase-9, fibrinogen, endotoxin, interleukin (IL)-1β, IL-6, IL-8, and tumor necrosis factor-α. Cox regression, multiple linear regression, t-tests, chi-square and Fisher’s exact tests were used to examine associations between the biomarkers, periodontal treatment, and gestational age at delivery and birthweight.
796 women had baseline serum data; 620 had baseline and follow-up serum and birth data. Periodontal treatment did not significantly alter the level of any biomarker (P>0.05). Neither baseline levels nor change from baseline in any biomarker was significantly associated with preterm birth or infant birthweight (P>0.05). In treatment subjects, change in endotoxin was negatively associated with change in probing depth (P<0.05).
Non-surgical mechanical periodontal treatment in pregnant women delivered before 21 weeks of gestation did not reduce systemic (serum) markers of inflammation. In pregnant women with periodontitis, levels of these markers at 13–17 weeks and 29–32 weeks gestation were not associated with risk for preterm birth or with infant birthweight.
Pregnancy; preterm birth; periodontitis; inflammation; cytokines
Cementogenesis is sensitive to altered local phosphate levels; thus, we hypothesized a cementum phenotype, likely of decreased formation, would be present in the teeth of X-linked hypophosphatemic (Hyp) mice. Mutations in the phosphate-regulating gene with homologies to endopeptidases on the X chromosome (Phex) cause X-linked hypophosphatemia, characterized by rickets, osteomalacia, and hypomineralized dentin formation, a phenotype recapitulated in the Hyp mouse homolog. Here, we report a developmental study of tooth root formation in Hyp mouse molars, focusing on dentin and cementum.
Light and transmission electron microscopy were used to study molar tissues from wild-type (WT) and Hyp mice. Demineralized and hematoxylin and eosin–stained tissues at developmental stages 23 to 96 days postcoital (dpc) were examined by light microscopy. Immunohistochemistry methods were used to detect bone sialoprotein (BSP) distribution in Hyp and WT mouse molar tissues, and transmission electron microscopy was used to study similar molar tissues in the non-demineralized state.
Dentin in Hyp mice exhibited mineralization defects by 33 dpc, as expected, but this defect was partially corrected by 96 dpc. In support of our hypothesis, a cementum phenotype was detected using a combination of immunohistochemistry and transmission electron microscopy, which included thinner BSP-positive staining within the cementum, discontinuous mineralization, and a globular appearance compared to WT controls.
Mutations in the phosphate-regulating Phex gene of the Hyp mouse resulted in defective cementum development.
Cementum; dentin; metabolism; Phex; phosphate; X-linked hypophosphatemic