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Background: Previously, we showed that systemic metronidazole and amoxicillin significantly improved the outcomes of non-surgical debridement in generalized aggressive periodontitis patients. This study aimed to observe whether re-treatment with adjunctive antimicrobials would give the placebo group benefits comparable with the test group., Methods: Thirty-eight of 41 subjects, from the initial 6-month trial, completed the second phase, re-treatment of sites with remaining pockets 5 mm. Subjects on placebo in phase one, received adjunctive antibiotics for 7 days. Clinical parameters were collected at 2 months posttreatment (8 months from baseline)., Results: Patients who received antibiotics at initial therapy, showed statistically significant improvement in pocket depth reduction and in the % of sites improving above clinically relevant thresholds, compared with patients who received antibiotics at re-treatment. In deep pockets (7 mm), the mean difference was 0.9 mm (p=0.003) and in moderate pockets (4-6 mm) it was 0.4 mm (p=0.036). For pockets converting from 5 to 4 mm, this was 83% compared with 67% (p=0.041) and pockets converting from 4 to 3 mm was 63% compared with 49% (p=0.297)., Conclusions: At 8 months, patients who had antibiotics at initial therapy showed statistically significant benefits compared with those who had antibiotics at re-treatment., (© 2010 John Wiley & Sons A/S.)

The molecular mechanisms underlying sperm penetration of the physical barriers surrounding the oocyte have not been completely delineated. Although neutral-active or "reproductive" hyaluronidases (hyases), exemplified by Sperm Adhesion Molecule 1 (SPAM1), are thought to be responsible for hyaluronan digestion in the egg vestments and for sperm-zona binding, their roles in mouse sperm have been recently questioned. Here we report that acidic "somatic" Hyaluronidase 3 (HYAL3), a homolog of SPAM1 with 74.6% structural similarity, exists in two isoforms in human ( approximately 47 and approximately 55 kDa) and mouse ( approximately 44 and approximately 47 kDa) sperm, where it resides on the plasma membrane over the head and midpiece. Mouse isoforms are differentially distributed in the soluble (SAP), membrane (MBP), and acrosome-reacted (AR) fraction where they are most abundant. Comparisons of zymography of Hyal3 null and wild-type (WT) AR and MBP fractions show significant HYAL3 activity at pH 3 and 4, and less at pH 7. At pH 4, a second acid-active hyase band at approximately 57 kDa is present in the AR fraction. HYAL3 activity was confirmed using immunoprecipitated HYAL3 and spectrophotometry. In total proteins, hyase activity was higher at pH 6 than at 4, where Spam1 nulls had significantly (P < 0.01) diminished activity implicating an acidic optima for murine SPAM1. Although fully fertile, Hyal3 null sperm showed delayed cumulus penetration and reduced acrosomal exocytosis. HYAL3 is expressed in epididymal tissue/fluid, from where it is acquired by caudal mouse sperm in vitro. Our results reveal concerted activity of both neutral- and acid-active hyaluronidases in sperm., ((c) 2010 Wiley-Liss, Inc.)

Background: Changes in cell adhesion and migration in the tumor microenvironment are key in the initiation and progression of metastasis. R-Ras is one of several small GTPases that regulate cell adhesion and migration on the extracellular matrix, however the mechanism has not been completely elucidated. Using a yeast two-hybrid approach we sought to identify novel R-Ras binding proteins that might mediate its effects on integrins., Methods and Findings: We identified Filamin A (FLNa) as a candidate interacting protein. FLNa is an actin-binding scaffold protein that also binds to integrin beta1, beta2 and beta7 tails and is associated with diverse cell processes including cell migration. Indeed, M2 melanoma cells require FLNa for motility. We further show that R-Ras and FLNa interact in co-immunoprecipitations and pull-down assays. Deletion of FLNa repeat 3 (FLNaDelta3) abrogated this interaction. In M2 melanoma cells active R-Ras co-localized with FLNa but did not co-localize with FLNa lacking repeat 3. Thus, activated R-Ras binds repeat 3 of FLNa. The functional consequence of this interaction was that active R-Ras and FLNa coordinately increased cell migration. In contrast, co-expression of R-Ras and FLNaDelta3 had a significantly reduced effect on migration. While there was enhancement of integrin activation and fibronectin matrix assembly, cell adhesion was not altered. Finally, siRNA knockdown of endogenous R-Ras impaired FLNa-dependent fibronectin matrix assembly., Conclusions: These data support a model in which R-Ras functionally associates with FLNa and thereby regulates integrin-dependent migration. Thus in melanoma cells R-Ras and FLNa may cooperatively promote metastasis by enhancing cell migration.

Large amounts of biological samples are usually required to measure multiple components by the enzyme-linked immunosorbant assay. However, the amounts of many tissue extracts and fluids, including gingival crevicular fluid (GCF), are generally extremely small. The aim of this study was, therefore, to develop and validate a novel multiplex bead assay (MBA) to simultaneously measure a profile of healing-related mediators in the GCF of treated periodontal wounds. An MBA was developed and validated by assessment of assay selectivity, recovery, precision and sensitivity, using eight recombinant human growth mediators as assay standards. GCF samples were collected on paper strips from healing wound (test) and healthy unaffected (control) sites of 15 patients with periodontitis, seven days post-periodontal surgery. Each GCF sample was eluted and the levels of the mediators measured using the MBA and antibody pairs specific for angiopoietin-1, vascular endothelial growth-factor, bone morphogenetic protein-2, osteoprotegerin, tissue inhibitor of metalloprotease-1 (TIMP-1), basic fibroblast growth-factor, keratinocyte growth-factor, and platelet derived growth-factor. Less than 1.8% of cross-reactivity was observed between antibodies and the eight different analytes, for which the recovery was more than 85%. Mean intra- and inter-assay precision were within the acceptance criteria of 20% and 25%, respectively. Detection of all mediators was highly sensitive (

Glycosyl phosphatidylinositol (GPI)-linked proteins, which are involved in post-testicular maturation of sperm and have a role in fertilization, are acquired on the sperm surface from both vesicular and membrane-free soluble fractions of epididymal luminal fluid (LF) and uterine LF. Herein, we investigate the mechanism of uptake of these proteins from the soluble fraction of LFs using sperm adhesion molecule 1 (SPAM1) as a model. Ultracentrifugation and native Western blot analysis of the soluble fraction revealed that SPAM1 is present in low-molecular-weight (monomeric) and high-molecular-weight (oligomeric) complexes. The latter are incapable of transferring SPAM1 and may serve to produce monomers. Monomers are stabilized by hydrophobic interactions with clusterin (CLU), a lipid carrier that is abundantly expressed in LFs. We show that CLU is involved in the transfer of SPAM1 monomers, whose delivery was decreased by anti-CLU antibody under normal and apolipoprotein-enhanced conditions. Coimmunoprecipitation revealed an intimate association of CLU with SPAM1. Both plasma and recombinant CLU had a dose-related effect on transfer efficiency: high concentrations reduced and low concentrations enhanced delivery of SPAM1 to human and mouse sperm membranes, reflecting physiological states in the epididymal tract. We propose a lipid exchange model (akin to the lipid-poor model for cholesterol efflux) for the delivery of GPI-linked proteins to sperm membranes via CLU. Our investigation defines specific conditions for membrane-free GPI-linked protein transfer in vitro and could lead to technology for improving fertility or treating sperm pathology by the addition of relevant GPI-linked proteins critical for successful fertilization in humans and domestic animals.

We recently reported an association between interleukin-6 (IL6) polymorphisms (SNPs) and haplotypes and aggressive periodontitis (AgP). The aim of this study was to investigate this association in a larger cohort of subjects, affected by either aggressive or chronic periodontitis. Five IL6 SNPs were analyzed in 765 subjects (167 generalized aggressive periodontitis, 57 localized aggressive, 310 chronic periodontitis and 231 periodontally healthy). Among Caucasians (n=454) there were moderate associations for -1363T allele (p=0.011) and for -174GG and -1363GG genotypes with diagnosis of periodontitis (respectively, p=0.044, OR=1.6, 95% CI=1.0-2.4, and p=0.017, OR=1.8, 95% CI=1.1-2.8, adjusted for age, gender and smoking). Haplotypes containing the -174G>C, -1363G>T and -1480C>G polymorphisms were associated with diagnosis of periodontitis (p=0.02). Subgroup analysis by disease phenotype showed associations for the localized AgP (LAgP) group and -1480C>G and -6106A>T SNPs (p=0.007 and 0.010, respectively). Among Caucasians the genotypes IL6 -1480 CC and -6106 TT increased the adjusted OR for LAgP (OR=3.09 and 2.27, respectively). This study supports the hypothesis that IL6 polymorphisms and haplotypes are moderately associated with periodontitis, possibly acting through influencing tissue levels of IL6. This association is stronger for LAgP than for other periodontal disease phenotypes.

Background and Objective: Family history is a primary diagnostic criterion for current classification of aggressive periodontitis (AgP). However, results of previous studies have shed controversy over the degree of familiarity of AgP and its possible inheritance mechanisms. The aims of this study were to estimate the percentage of affected relatives of AgP individuals, to analyse the disease phenotypes in relatives and to explore the distributions of genetic polymorphisms of interleukin-6 (IL-6) in AgP patients and in diseased and healthy relatives., Material and Methods: Patients with AgP were clinically examined and asked to provide relatives for examination. First-degree relatives were clinically and radiographically diagnosed. Blood samples were collected, DNA was extracted and analysis of single nucleotide polymorphisms of IL-6 (at positions -174, -1363 and -1480) by polymerase chain reaction was performed in patients and relatives., Results: Fifty-five AgP patients provided relatives for examination. A total of 100 first-degree relatives were assessed and 10 of them (10%) were found to have AgP. All relatives diagnosed with AgP had the same disease as the corresponding proband (localized AgP/localized AgP or generalized AgP/generalized AgP). The same IL-6 genotypes (-174 GG, -1480 CC) previously associated with AgP showed a tendency for association with AgP in relatives., Conclusion: This pilot study confirmed a relatively high risk for relatives of AgP patients to have AgP (10%). Genetic polymorphisms in the IL-6 gene may have an impact in aetiopathogenesis. This study provides a sample size calculation for a novel study design using healthy relatives as control subjects.

Sperm uptake of glycosyl phosphatidylinositol (GPI)-linked proteins from luminal fluids has been shown to occur in male and estrous female reproductive tracts. In males, this is attributed to membranous vesicles secreted into the epididymis and prostate. While epididymosomes have been characterized, there have been no reports of the presence of vesicles in female luminal fluids. Here we report the presence of vesicles, characterized as "uterosomes," in the murine estrous female reproductive fluid; and use Sperm Adhesion Molecule 1 (SPAM1/PH-20), a well-known hyaluronidase found in male and female fluids, as a model to investigate vesicle-mediated GPI-linked protein transfer to sperm. Epididymosomes and uterosomes isolated after ultracentrifugation of epididymal (ELF) and uterine luminal fluid (ULF) were analyzed by electron microscopy and shown to be approximately 10-70 and approximately 15-50 nm in diameter. The structural integrity of uterosomes was confirmed by their resistance to hypo-osmotic and freeze/thaw stresses; and immunogold labeling localized SPAM1 to their outer membrane surface, as was the case for epididymosomes. SPAM1 was acquired by caudal sperm during incubation in epididymosomes and uterosomes; uptake was abolished when the GPI anchor was enzymatically cleaved. Sperm analyzed by confocal and transmission electron microscopy (TEM) after incubation in fluorescently labeled vesicles revealed the label on the membrane over the acrosome and midpiece of the flagella, where SPAM1 normally resides. High magnification TEM images demonstrated vesicles juxtaposed to the sperm plasma membrane potentially transferring SPAM1. Taken together, these results implicate vesicular docking as the mechanism of vesicle-mediated GPI-linked protein transfer to sperm from murine reproductive fluids.

Aim: The aim of this analysis was to investigate the relationship between a vitamin D receptor (VDR) polymorphism and the diagnosis and progression of periodontitis., Material and Methods: Data were derived from two different studies, including 231 subjects with healthy periodontium, 224 aggressive periodontitis and 79 chronic periodontitis (CP) patients in a case-control investigation. Sixty-one of these CP patients also took part in an observational study with a 1-year follow-up, in which progression of periodontitis was determined at the subject level. All 534 subjects provided a blood sample from which genomic DNA was extracted to study VDR -1056 TaqI polymorphism., Results: The interaction between smoking and VDR polymorphism was associated with the diagnosis of periodontitis in Caucasians [p=0.001, odds ratio (OR)=1.33, 95% confidence intervals (CI)=1.12-1.57] and all subjects (p=0.033, OR=1.60, 95% CI=1.04-2.48). In the longitudinal study, subjects were divided into two clusters at 1 year according to the median number of progressing sites (Delta cumulative attachment loss >2 mm). Logistic regression analysis revealed that the interaction between VDR Taq-I polymorphism and smoking showed limited evidence of association with the "severe progression" cluster (p=0.033, OR=15.24, 95% CI=1.24-187.42)., Conclusions: Vitamin D receptor Taq-I TT polymorphism was moderately associated with both the presence and the progression of periodontitis in smokers, while no association was detected in non-smoking individuals. VDR genetic factors may interact with smoking in the pathogenesis of periodontitis.

Sperm uptake of epididymal sperm adhesion molecule 1 (SPAM1) in vitro has recently been shown to be a marker of sperm maturation, since acquisition of this surface hyaluronidase increases cumulus dispersal efficiency. Here, we demonstrate that this glycosyl phosphatidylinositol-linked sperm antigen, previously shown to be expressed during estrous in the female reproductive tract, is secreted in the uterine and oviductal fluids (ULF and OF respectively) in a 67 kDa form, which can bind to sperm. We show that it can be acquired by caudal sperm from Spam1 null, Spam1-deficient mutant, and wild-type (WT) mice in vitro during incubation in ULF or OF at 37 degrees C, as detected by immunocytochemistry and flow cytometry. SPAM1 binding after ULF incubation was localized predominantly to the acrosome and the mid-piece of the flagella of Spam1 null sperm in a pattern identical to that of WT sperm. After ULF incubation, WT sperm demonstrated a significantly (P<0.001) enhanced hyaluronic acid-binding ability, and the involvement of SPAM1 in this activity was shown by a significant (P<0.001) decrease in binding when sperm were exposed to SPAM1 antiserum-inhibited ULF. Importantly, when Spam1 null sperm were exposed to ULF with SPAM1 accessible (in the presence of pre-immune serum) or inaccessible (in the presence of SPAM1 antiserum) for uptake, there was a significant difference in cumulus dispersal efficiency. Taken together, these results suggest that in the sperm surface remodeling that occurs prior to and during capacitation, the fertilizing competence of sperm is increased via acquisition of SPAM1, and likely other hyaluronidases, from the female tract.

Background: Interleukin-6 (IL-6) polymorphisms have been shown to affect IL-6 promoter activity. This study investigated the possible role of IL-6 genetic polymorphisms and haplotypes in the predisposition to aggressive periodontitis (AgP)., Material and Methods: A case-control association study on 224 AgP patients and 231 healthy controls was performed in order to detect differences in genotype distributions of five single nucleotide polymorphisms (SNPs) located in the promoter region of the IL-6 gene., Results: The IL-6 -1363 polymorphism was associated with a diagnosis of AgP in subjects of all ethnicities (p=0.006, adjusted logistic regression). The -1480 SNP was associated with LAgP in subjects of all ethnicities (p=0.003). The -1480 and -6106 polymorphisms were associated with Localized AgP in Caucasians (n=24) (p=0.007 and 0.010, respectively). Haplotypes determined by the -1363 and -1480 polymorphisms were also associated with LAgP (p=0.001) in Caucasians., Conclusions: This study supports the hypothesis of a link between IL-6 genetic factors and AgP and highlights the importance of two IL-6 polymorphisms (-1363 and -1480) in modulating disease phenotype and susceptibility.

Growing evidence suggests that individual genetic susceptibility may influence the host's response to infections. The aim of this project was to study whether gene polymorphisms of inflammatory markers are associated with the presence of viable periodontopathogenic bacteria. We extracted genomic DNA from 45 young adults diagnosed with generalized aggressive periodontitis to study Fc receptors, formyl peptide receptor, Interleukin-6, tumor necrosis factor-alpha, and vitamin D receptor polymorphisms. The presence and viable numbers of Actinobacillus actinomycetemcomitans, Porphyromonas gingivalis, and Tannerella forsythensis were determined by culture, and their identities confirmed by PCR. Multiple logistic regressions revealed that both Fcgamma receptor and IL-6 -174 polymorphisms were associated with increased odds of detecting A. actinomycetemcomitans, P. gingivalis, and T. forsythensis after adjustment for age, ethnicity, smoking, and periodontitis extent. These findings support the hypothesis that complex interactions between the microbiota and host genome may be at the basis of susceptibility to aggressive periodontitis.

Background: A role for Sperm Adhesion Molecule 1 (SPAM1) hyaluronidase in murine kidney, where Spam1 transcript levels have been reported to be higher in males, has not been clarified., Methods: Spam1 RNA and protein were studied using RT-PCR, in situhybridization, Western blotting, immunohistochemistry and hyaluronic acid substrate gel electrophoresis. Urine volume and osmolality were studied in wild-type and Spam1 null mice., Results: While RT-PCR supported a tendency of higher RNA expression in males, no sex difference for the protein was detectable in the cortex, medulla, and urine. Transcripts were predominantly localized in the proximal tubules and glomeruli, with lower levels in the medulla. Similarly, Western blotting and immunohistochemistry revealed that SPAM1 is more abundant in the cortex. Hyaluronidase activity was absent at neutral and acidic pH: suggesting non-enzymatic role(s) for SPAM1. Wild-type and Spam1 null mice given free access to water showed significantly reduced urine volumes (p < 0.01; n = 12) in the latter. Baseline urine osmolality was similar in both, leading to a significantly (p < 0.05) lower osmolar output in the nulls. After water deprivation (24 h), a significant (p < 0.01) increase in urine osmolality was seen only for wild-type mice., Conclusion: SPAM1 is implicated in fluid reabsorption and urine concentration., (Copyright 2007 S. Karger AG, Basel.)

Introduction: Neutrophils (PMN) in aggressive periodontitis (AgP) patients have been reported to be hyperactive especially with regards to superoxide production. Polymorphisms in genes influencing PMN function have been proposed as candidate risk factors for AgP. The aim of this study was to test the association of specific gene polymorphisms affecting PMN functions with AgP., Materials and Methods: Two hundred and twenty-four patients with confirmed diagnosis of AgP and 231 subjects with healthy periodontium took part in the study. A blood sample was collected from subjects and genotypes for p22phox (CYBA) NADPH oxidase, FP, Fcalpha and Fcgamma receptors were analysed in a blind fashion., Results: The C242T p22phox NADPH oxidase T allele was significantly associated with AgP in a multiple logistic regression model adjusting for confounders, and this was observed for all subjects [p = 0.002, odds ratio (OR) = 1.87, 95% confidence interval (CI) = 1.27-2.83] and Caucasians (p = 0.009, OR=2.07, 95% CI = 1.20-3.59). Concomitant presence of C242T p22phox NADPH oxidase T allele and FcgammaRIIIb NA1 homozygosity was associated with the generalized AgP phenotype in Caucasians (p = 0.001, OR = 30.35, 95% CI = 3.81-241.97)., Conclusions: C242T p22phox NADPH oxidase and FcgammaR polymorphisms may predispose to AgP through a modulation of neutrophil superoxide production.

Objectives: To assess the periodontal status of relatives of Aggressive Periodontitis (AgP) patients, and to evaluate the reliability of the family history report as provided by the proband., Material and Methods: Data from 54 AgP patients were gathered along with a family history report for each of their relatives. Only 27 patients (probands) had relatives willing to be examined. This yielded a total of 61 relatives from whom the periodontal status was obtained. The family history report for each examined relative was compared with the periodontal diagnosis made at examination to assess reliability., Results: Eight percentage of the examined relatives, aged between 12-76, were diagnosed with AgP, while chronic periodontitis was present in 39%, gingivitis in 38% and 15% were healthy. If the report provided by the proband was positive, the likelihood of finding any type of periodontitis in that relative was 85.7%, whereas if the report was negative the likelihood of the absence of periodontitis was 70.6%., Conclusion: The percentage of examined relatives who were affected with AgP (8%), although lower than percentages reported in other AgP family studies, was still higher than the prevalence of the condition in random populations. Reliability of periodontal family history was considered good and more reliable when it was positive.

There is strong evidence that genetic as well as environmental factors affect the development of periodontitis, and some suggestion that aggressive and chronic forms of the disease share the same genetic predisposition. This study addresses the hypothesis that there are both shared and unique genetic associations in these forms of periodontitis. A sample of 51 patients with aggressive disease, 57 patients with chronic disease, and 100 healthy controls was recruited for this study. Ten functional polymorphisms in 7 candidate genes were genotyped. The results show statistically significant (p

Background: The objective of this study was to assess the adjunctive clinical effect of the administration of systemic amoxicillin and metronidazole in the non-surgical treatment of generalized aggressive periodontitis (GAP)., Methods: Forty-one systemically healthy subjects with GAP were included in this 6-month double-blind, placebo-controlled, randomized clinical trial. Patients received a course of full-mouth non-surgical periodontal treatment delivered over a 24 h period using machine-driven and hand instruments. Test subjects received an adjunctive course of systemic antibiotic consisting of 500 mg amoxicillin and 500 mg metronidazole three times a day for 7 days. Clinical parameters were collected at baseline, and at 2 and 6 months post-treatment., Results: In both the test and the placebo groups, all clinical parameters improved at 2 and 6 months. In deep pockets (> or =7 mm), the test treatment resulted in an additional 1.4 mm (95% confidence interval 0.8, 2.0 mm) in full-mouth probing pocket depth (PPD) reduction and 1 mm (0.7, 1.3 mm) of life cumulative attachment loss (LCAL) gain at 6 months. In moderate pockets (4-6 mm), the adjunctive benefit was smaller in magnitude: PPD reduction was 0.4 mm (0.1, 0.7 mm) and LCAL gain was 0.5 mm (0.2, 0.8 mm). In addition, the 6-month data showed LCAL gains > or =2 mm at 25% of sites in test patients compared with 16% in placebo (p=0.028). Similarly, PPD reductions of 2 mm or more were observed in 30% of sites in test and 21% of sites in placebo patients. Seventy-four percent of pockets with PPD > or =5 mm at baseline were 4 mm or shallower at 6 months in the test group. This compared with 54% in the placebo group (p=0.008). Disease progression at 6 months was observed at 1.5% of test and 3.3% of sites in test and placebo, respectively (p=0.072)., Conclusions: These data indicate that a 7-day adjunctive course of systemic metronidazole and amoxicillin significantly improved the short-term clinical outcomes of full-mouth non-surgical periodontal debridement in subjects with GAP.

Background: Inflammation of periodontal tissues during postoperative wound healing is mediated by cell surface adhesion molecules. Soluble forms of these antigens have also been identified and shown to be important in immunoregulatory processes, but have previously not been investigated during periodontal repair and regeneration. The present study has examined the presence and possible changes in soluble intercellular adhesion molecule-1 (sICAM-1; CD54) and lymphocyte function-associated antigen-3 (sLFA-3; CD58) in gingival crevical fluid (GCF) following periodontal surgery., Methods: GCF samples were collected from four groups: 1) a guided tissue regeneration (GTR) test; 2) a GTR control, at least one complete tooth unit away from the periodontal defect; 3) a conventional flap (CF) surgery; and 4) a crown lengthening (CL). Sandwich enzyme-linked immunosorbent assay (ELISA) was used to measure the levels of sICAM-1 and sLFA-3 in the GCF samples., Results: A marked increase in GCF volumes was found in all sites after surgery, although a persistent increase was associated only with the period of membrane retention at the GTR test sites. In addition, sICAM-1 and sLFA-3 were found in the GCF of healthy as well as diseased sites prior to treatment and the total amounts of both increased transiently following surgical intervention, especially sLFA-3. However, the concentrations of these GCF components, particularly sICAM-1, tended to decrease., Conclusions: The temporal decrease in the concentration of sICAM-1 and sLFA-3 in GCF may serve to enhance inflammatory reactions at surgically-treated periodontal sites, thereby limiting repair and regeneration in the periodontium. These soluble adhesion molecules may thereby be of potential therapeutic value and might also be useful markers for monitoring periodontal wound healing.

Objectives: Growth factors play a major part in wound healing, including in the periodontium. However, the presence of growth factors in gingival crevicular fluid (GCF) in humans during periodontal wound healing has not yet been determined. Our hypothesis is that such factors are present in GCF and that changes in their levels might be of value as a prognostic marker of wound-healing activity and therapeutic progress following periodontal surgery. The aim of this study was therefore to measure transforming growth factor-beta1 (TGF-beta1) in GCF collected from sites that have undergone guided tissue regeneration (GTR) and conventional flap (CF) surgery and to compare these with GCF collected from unaffected healthy sites., Materials and Methods: GCF samples were collected, using filter paper strips, at baseline (pre-surgical) and then at intervals up to 26 weeks from 16 patients undergoing GTR and from 11 patients undergoing CF surgery. After elution and acid treatment, TGF-beta1 levels were measured by ELISA., Results: Treatment of periodontal defect sites significantly reduced the mean probing pocket depth (PPD) and improved the mean lifetime cumulative attachment loss (LCAL). Average GCF volumes also significantly increased at all sites at 2 weeks post-surgery and thereafter declined to baseline levels, except at the GTR test sites that were still elevated at 7 weeks. TGF-beta1 could be detected in almost all GCF samples, and 2 weeks after surgery, the average levels increased two-fold at the surgically treated but not at the control sites, which remained unchanged., Conclusion: TGF-beta1 is readily detectable in GCF and increases transiently following periodontal surgery. This suggests that changes in the levels of this growth factor in GCF might be useful for monitoring the progress of periodontal repair and regeneration.

Objectives: Previous periodontal literature has shown that there is a strong relationship between treatment effects, such as guided tissue regeneration (GTR), and baseline disease severity. However, relating change to baseline values using correlation or regression is methodologically flawed due to mathematical coupling, where the statistical procedure of testing the null hypothesis-that the coefficient of correlation or slope of regression is equal to zero-becomes erroneous. The aim of this study is to investigate if baseline disease severity is genuinely associated with the treatment outcome of intrabony defects using GTR after adjustment for mathematical coupling. In particular, we seek to demonstrate the potential effect that mathematical coupling has in distorting the results from the statistical analyses of trials of dental treatment, using data from the periodontal literature on GTR. The erroneous results arising from the use of simple correlation and regression techniques to analyse this association will be demonstrated, also the methodological flaw where the statistical procedure tests the null hypothesis-that the coefficient of correlation or the slope of regression is equal to zero., Methods: Three main periodontal journals were electronically and manually searched to extract the data for the clinical outcomes of pocket probing depth (PPD) and lifetime cumulative attachment loss (LCAL) in the studies using GTR. The relationship between clinical outcomes and baseline measurements were reanalysed using Oldham's method and the variance ratio test., Results: The results of these analyses were compared with those from the papers where the authors used the standard approach of correlation or regression. This shows that mathematical coupling caused spurious correlations between baseline disease severity and treatment effect. Ten out of 12 studies for PPD and nine out of 14 for LCAL initially claimed a significant positive relationship; after using either of the more appropriate statistical methods of adjustment, only three correlations in each group of studies remained significant., Conclusions: Previous evidence suggesting an association between baseline disease severity and treatment effect for GTR is challenged and therefore needs to be critically reviewed. All future clinical research should avoid using mathematically coupled data in correlation or regression analysis. In seeking to examine the bivariate association between baseline and subsequent change, Oldham's method is recommended.

Background: Statistical analyses of periodontal data that average site measurements to subject mean values are unable to explore the site-specific nature of periodontal diseases. Multilevel modeling (MLM) overcomes this, taking hierarchical structure into account. MLM was used to investigate longitudinal relationships between the outcomes of lifetime cumulative attachment loss (LCAL) and probing depth (PD) in relation to potential risk factors for periodontal disease progression., Methods: One hundred males (mean age 17 years) received a comprehensive periodontal examination at baseline and at 12 and 30 months. The resulting data were analyzed in two stages. In stage one (reported here), the absolute levels of disease were analyzed in relation to potential risk factors; in stage two (reported in a second paper), changes in disease patterns over time were analyzed in relation to the same risk factors. Each approach yielded substantially different insights., Results: For absolute levels of disease, subject-level risk factors (covariates) had limited prediction for LCAL/PD throughout the 30-month observation period. Tooth position demonstrated a near linear relationship for both outcomes, with disease increasing from anterior to posterior teeth. Sites with subgingival calculus and bleeding on probing demonstrated more LCAL and PD, and supragingival calculus had an apparently protective effect. Covariates had more "explanatory power" for the variation in PD than for the variation in LCAL, suggesting that LCAL and PD might be generally associated with a different profile of covariates., Conclusion: This study provides, for a relatively young cohort, considerable insights into the factors associated with early-life periodontal disease and its progression at all levels of the natural hierarchy of sites within teeth within subjects.

Background: The aim of this study was to investigate the longitudinal relationships between the outcome measurements of changes in lifetime cumulative attachment loss (cLCAL) and changes in probing depth (cPD) in relation to potential risk factors or other risk markers for periodontal disease progression from a cohort of 100 young males. In order to account for the hierarchical data structure, and to explore explicitly the site, tooth, and subject levels simultaneously, multilevel modeling was undertaken., Methods: The analyses were undertaken in two parts. Within a previous article, the absolute levels of disease were analyzed in relation to potential risk factors; within this article, changes in disease are analyzed in relation to these factors. Each analytical approach yielded substantively different insights., Results: Subject-level risk factors had limited predictive value for cLCAL/cPD throughout the 30-month observation period. Tooth position demonstrated a near linear relationship for both outcomes, with disease increasing from anterior to posterior teeth. Supragingival plaque had no significant effect on cLCAL/cPD, while subgingival calculus and bleeding on probing were negatively associated with cLCAL/cPD. In contrast to the outcomes LCAL/PD, supragingival calculus had no significant protective effect on cLCAL/cPD. There was no significant influence of smoking in this cohort., Conclusions: This study provides, for a relatively young cohort, considerable insights into the factors associated with longitudinal patterns of early-life periodontal disease at all levels of the natural hierarchy of sites within teeth within subjects. Furthermore, it is demonstrated how multilevel modeling can provide considerable insight into some of the inconsistencies and controversies found in the previous periodontal literature.

Objectives: The aim of this study was to investigate high-frequency ultrasound imaging for periodontal assessment., Material and Methods: A newly developed ultrasonic scanner with a frequency of 20 MHz was used. Pig jaws were selected as the experimental model. Three teeth per jaw were imaged with the scanner and duplicate measurements were made of the distance from a fixed landmark on the teeth to the alveolar bone crest. These measurements were compared to transgingival and direct measurements of the same teeth following reflection of the soft tissues. One further jaw was used for histological comparison with the ultrasound image., Results: Using ultrasonography, it was possible to image the main periodontal structures. The ultrasound measurements showed better repeatability than either of the other two methods (repeatability coefficient: 0.44 mm for ultrasound, 0.93 mm for transgingival probing and 0.6 mm for direct measurements). Also, ultrasound was in better agreement with direct, open probing measurements (0.004 +/- 0.58 mm) than transgingival probing with direct measurements (0 +/- 0.7 mm)., Conclusions: Ultrasonography provides a highly accurate and repeatable technique for periodontal assessment in this model.

Previously, burst and linear theories for periodontal disease progression were proposed based on different but limited statistical methods of analysis. Multilevel modeling provides a new approach, yielding a more comprehensive model. Random coefficient models were used to analyze longitudinal periodontal data consisting of repeated measures (level 1), sites (level 2), teeth (level 3), and subjects (level 4). Large negative and highly significant correlations between random linear and quadratic time coefficients indicated that subjects and teeth with greater-than-average linear change experienced decelerated variation. Conversely, subjects and teeth with less-than-average linear change experienced accelerated variation. Change therefore exhibited a dynamic regression to the mean at the tooth and subject levels. Since no equilibrium was attained throughout the study, changes were cyclical. When considered as a multilevel system, the "linear" and "burst" theories of periodontal disease progression are a manifestation of the same phenomenon: Some sites improve while others progress, in a cyclical manner.

Previous studies using correlation or regression analysis have showed that treatment effects measured by the change in clinical parameters are often associated with baseline values of the same parameters. These studies, however, have a methodological weakness. Correlation/regression between baseline measures and the derived change variable invalidates the statistical procedures of testing the null hypothesis: that the coefficient of correlation/regression is zero. This is due to the phenomenon of mathematical coupling. To investigate the impact that this has on the observed correlation/regression coefficient when in reality this is zero, we used random simulations of hypothetical data to model the treatment of periodontal pockets. Results showed a strong probability of obtaining statistically significant correlation/regression coefficients. To separate this artificial effect of mathematical coupling from the true underlying biological relationship, one must apply appropriate analytical strategies to re-evaluate previous evidence within the periodontal literature.

Aim: The aims of this study were to monitor the prevalence and progression of lifetime cumulative attachment loss (LCAL) in a group of young British male military recruits over a 3-year period, and to determine the relationship between signs of LCAL and selected periodontal variables., Methods: 100 subjects, aged 16-20 years (mean 17 years) at baseline, were examined at 0 (baseline), 12 and 30 months. LCAL, probing depth, plaque, bleeding on probing, gingival colour and supra- and subgingival calculus were assessed on the mesio-buccal, disto-buccal, mesio-lingual and disto-lingual surfaces of all teeth present, excluding third molars. Data were analysed cross-sectionally at each examination., Results: Over the period of the study, the prevalence of LCAL > or =1 and 2 mm ranged from 95-100%, whereas LCAL > or =3 mm ranged from 40-47%. The extent of LCAL > or =1 mm ranged from 76-86%. However, the extent of LCAL > or =2 mm was dramatically lower (10.5-12.7%), and LCAL > or =3 mm was uncommon (0.5-0.9%). Examining the number of subjects according to the number of sites affected above a threshold, showed that a small number of subjects have a large number of sites above threshold. Using Pearson's rank correlation coefficient a significant correlation (p<0.05) was found between LCAL and the periodontal variables of gingival bleeding and supra- and subgingival calculus., Conclusions: These data suggest that the onset and progression of chronic periodontitis can be seen in young adults, and in this group gingival bleeding and supra- and subgingival calculus are the variables most strongly associated with early periodontitis.

Aim: In the present 6-month multicentre trial, the outcome of 2 different approaches to non-surgical treatment of chronic periodontitis, both involving the use of a locally delivered controlled-release doxycycline, was evaluated., Material and Methods: 105 adult patients with moderately advanced chronic periodontitis from 3 centres participated in the trial. Each patient had to present with at least 8 periodontal sites in 2 jaw quadrants with a probing pocket depth (PPD) of > or =5 mm and bleeding following pocket probing (BoP), out of which at least 2 sites had to be > or =7 mm and a further 2 sites > or =6 mm. Following a baseline examination, including assessments of plaque, PPD, clinical attachment level (CAL) and BoP, careful instruction in oral hygiene was given. The patients were then randomly assigned to one of two treatment groups: scaling/root planing (SRP) with local analgesia or debridement (supra- and subgingival ultrasonic instrumentation without analgesia). The "SRP" group received a single episode of full-mouth supra-/subgingival scaling and root planing under local analgesia. In addition, at a 3-month recall visit, a full-mouth supra-/subgingival debridement using ultrasonic instrumentation was provided. This was followed by subgingival application of an 8.5% w/w doxycycline polymer at sites with a remaining PPD of > or =5 mm. The patients of the "debridement" group were initially subjected to a 45-minute full-mouth debridement with the use of an ultrasonic instrument and without administration of local analgesia, and followed by application of doxycycline in sites with a PPD of > or =5 mm. At month 3, sites with a remaining PPD of > or =5 mm were subjected to scaling and root planing. Clinical re-examinations were performed at 3 and 6 months., Results: At 3 months, the proportion of sites showing PPD of < or =4 mm was significantly higher in the "debridement" group than in the "SRP" group (58% versus 50%; p<0.05). The CAL gain at 3 months amounted to 0.8 mm in the "debridement" group and 0.5 mm in the "SRP" group (p=0.064). The proportion of sites demonstrating a clinically significant CAL gain (> or =2 mm) was higher in the "debridement" group than in the "SRP" group (38% versus 30%; p<0.05). At the 6-month examination, no statistically significant differences in PPD or CAL were found between the two treatment groups. BoP was significantly lower for the "debridement" group than for the "SRP" group (p<0.001) both at 3- and 6 months. The mean total treatment time (baseline and 3-month) for the "SRP" patients was 3:11 h, compared to 2:00 h for the patients in the "debridement" group (p<0.001)., Conclusion: The results indicate that simplified subgingival instrumentation combined with local application of doxycycline in deep periodontal sites can be considered as a justified approach for non-surgical treatment of chronic periodontitis.

Background: Expanded polytetrafluoroethylene (ePTFE) barrier membranes have been widely used for guided tissue regeneration (GTR) of the human periodontal ligament (PL). However, the precise cellular and molecular events involved in the re-growth of the new tissue are still unclear., Methods: Retrieved membranes and the newly-regenerated soft tissue (RT) underlying the membranes were used to examine the cells associated with GTR compared with normal human PL and gingival cells. Flow cytometry (FCM) was used, for the first time, to analyze the spindle-shaped fibroblast-like cells which were adherent to these membranes and the cells which grew out of the RT., Results: The results showed that the membrane-associated (M) cells had the lowest rate of proliferation and appeared to be larger and more granular than the other types of cell. Moreover, both the M- and RT-derived cells were found to express higher levels of the extracellular matrix (ECM) proteins collagen type 1, fibronectin, tenascin, and decorin. In addition, evidence based on FCM profiles identified distinct sub-populations of GTR cells in which fibronectin expression was markedly up-regulated compared with normal PL cells and which also differed in size and granularity., Conclusions: The results of this study show that cells associated with GTR barrier membranes and with the underlying tissue appear to have distinct phenotypic and functional activities consistent with the production of new periodontal connective tissue and periodontal regeneration.

Objective: To introduce the concepts of random coefficient multilevel models through an application to periodontal research data., Basic Research Design: Multilevel models with random coefficients are illustrated using periodontal data that comprise four levels: repeated measurements at level-1, sites at level-2, teeth at level-3, and subjects at level-4. The study explores random coefficient models--where random variation occurs about explanatory variable coefficients. Outcomes considered are lifetime cumulative attachment loss and pocket probing depth., Participants: The study data were taken from a survey of periodontal disease involving 100 white male trainee engineers aged between 16 and 20 entering the apprentice training school at the Royal Air Force-Halton, UK., Results: The application of multilevel modelling to longitudinal data provides a new way of exploring old problems. The multilevel random coefficient models provide an opportunity to examine the 'linear' and 'burst' theories of periodontal disease progression, leading to the postulation that both can be unified within the multilevel framework., Conclusions: The multilevel methodology illustrates how advances in the understanding of oral health can be achieved with the advent of new statistical methods

Background: Previous studies have shown that the use of index teeth may underestimate the prevalence of chronic periodontitis in adults. However, there is little information on the effect of using index teeth to estimate the prevalence of early periodontitis in younger adults and the effect this may have on planning treatment needs and health care resources. The aim of this study was to compare full mouth examination with partial examination using index teeth in a group of young British males., Methods: One hundred subjects aged between 16 and 20 years (mean 17 years) on entry to the study were examined at baseline, 12 months later, and 30 months later. Lifetime cumulative attachment loss (LCAL) > or =1 mm was measured on the mesio-buccal, disto-buccal, mesio-lingual, and disto-lingual surfaces of all teeth, excluding third molars. All data were entered into a database. The indices used to express LCAL were prevalence, defined as the percentage of subjects with LCAL > or =1 mm, 2 mm, or 3 mm, and extent, defined as the percentage of sites with LCAL > or =1 mm, 2 mm, or 3 mm. Two sets of index teeth were chosen to compare with full mouth recordings, Ramfjord index teeth and the Periodontal Index for Treatment (PIT) teeth., Results: The prevalence of LCAL > or =1 mm was similar (approaching 100%) for the full mouth and both partial mouth recordings. However, as LCAL increased from a minimum of 1 to 3 mm, partial mouth recording resulted in an underestimation of the prevalence of disease. LCAL > or =2 mm was underestimated by up to 22% and LCAL > or =3 mm by up to 36%. The extent of LCAL was less affected by partial mouth recording, in that the percentage of sites with no sign of early attachment loss was underestimated by up to 11%. However, the percentage of sites with LCAL > or =1 mm and 2 mm were overestimated by 11% and, 7% respectively., Conclusions: These data indicate that the use of index teeth in epidemiological studies which include young adults may result in an underestimation of the prevalence of early periodontitis and an overestimation of the extent.

Background, Aims: Conventional treatment of chronic periodontitis involves mechanical debridement of periodontal pockets. Recently, subgingival antimicrobials have been used adjunctively following such debridement. This 2-centre study compared the clinical effects of subgingival scaling (SRP) with SRP plus subgingival application of 25% metronidazole gel, Elyzol (SRP+gel), in patients with chronic adult periodontitis., Method: Voluntary informed written consent was obtained from 45 subjects at the Eastman (mean age 46, range 34-63) and 43 subjects at RAF Halton (mean age 47, range 34-71) who participated in this blind, randomised split-mouth design study. All had at least 2 sites in each quadrant with probing pocket depth (PPD) > or = 5 mm. PPD, bleeding on probing (BOP), and clinical probing attachment levels (CAL) measured using a stent, were recorded at baseline and at 1, 3, 6 and 9 months post-therapy. After subgingival scaling of all quadrants, 2 quadrants were randomly selected to be treated with metronidazole gel., Results: A paired t-test on baseline values showed no bias between groups. Both treatments effectively reduced the signs of periodontitis. At each follow-up visit, reduction in PPD, CAL and BOP after the combined treatment was greater than for SRP alone. Paired t-tests showed that the improvement in the SRP+gel group was statistically significantly better (p<0.001) than for SRP alone (mean 0.5 +/- 0.6 mm. 95% CI 0.4-0.6 mm.) Similarly, the % of sites which improved to a final pocket depth of < or = 3 mm and the % of sites which improved over the 9 months of the study by as much as > or = 2 mm were greater for SRP+gel than for SRP alone., Conclusions: At the end of the study, the mean reductions for PPD were 1.0 mm (SRP) compared to 1.5 mm (SRP+gel), and for CAL they were 0.4 mm (SRP) compared to 0.8 mm (SRP+gel), with mean difference for CAL between treatments of 0.4 +/- 0.6 mm (95% confidence intervals of 0.3-0.6 mm). The combination therapy of SRP+gel was superior to the conventional treatment of SRP alone, and these differences were maintained for 9 months.

Objective: To explain the theory of multilevel modelling and demonstrate its application in the analysis of dental research data., Basic Research Design: Multilevel modelling was introduced using dental data comprising four levels: repeated measurements at level-1, sites at level-2, teeth at level-3, and subjects at level-4. Variance components models (which have no explanatory variables) were evaluated for all outcome measures. Explanatory variables were added to the models with outcomes for both lifetime cumulative attachment loss and pocket probing depth. Salient features of the multilevel models were discussed., Participants: Research data were obtained from a longitudinal survey of periodontal disease conducted on 100 white male trainee engineers aged between 16 and 20 years entering the apprentice training school at Royal Air Force Halton, England., Results: The statistical methods revealed that periodontal measures demonstrate considerable variation at all levels of the multilevel structure. Models for lifetime cumulative attachment loss and pocket probing depth illustrated that risk factors operated at more than one level. Supragingival calculus was a risk factor at the subject-level (subjects experiencing more sites with the condition had greater attachment loss and greater pocketing) whilst there was apparently a protective effect occurring at the site (sites with the condition had less attachment loss and less pocketing)., Conclusions: This study demonstrates that multilevel modelling is a more powerful research tool than single-level techniques for the analysis of hierarchical dental data. Researchers using these techniques are well equipped to analyse complex hierarchical data structures, such as those often found within dentistry.

The activity of alkaline phosphatase (ALP) is considered to indicate the presence of osteoblast cells and the formation of new bone. In the present study this enzyme was investigated in cells obtained from retrieved polytetrafluoroethylene membranes (M cells) of periodontal disease patients treated by guided tissue regeneration (GTR) and from the regenerated tissue underlying the membrane (RT cells). Normal periodontal ligament (PL) and gingival cells were also grown from the corresponding healthy tissues of human subjects. ALP activity was measured colourimetrically, using paranitrophenyl phosphate as the substrate, after 4 and 7 d of culture in the absence and presence of dexamethasone (DEX), a synthetic glucocorticoid which induces osteoblast differentiation. The results showed that basal levels of ALP activity were expressed by all the cells and that DEX upregulated ALP levels in the M, RT and PL cells but not in the gingival cells. Moreover, both the basal and DEX-induced ALP activities were statistically significantly higher in the RT cells than in any of the other cells. Our results suggest that both the GTR-associated and normal PL cells express osteoblast-like characteristics and, furthermore, that the RT cultures in particular contain a high proportion of osteoprogenitor cells.

This paper reports the detailed calibration of the new Periotron 8000 with different fluids and uses the method of least squares to derive polynomial regression equations up to the 6th order, to investigate the most accurate descriptor of the resulting calibration lines. The use of a 4th order polynomial regression equation (recommended by the manufacturer) provided better coefficients of determination (R2: 0.999) and root mean square errors (RMSE = 1.6) than either linear regression (R2: 0.986, RMSE = 10.9) or quadratic models (R2: 0.998, RMSE = 3.2). Data derived using the manufacturer's MLCONVERT software program lacked accuracy and incurred large errors for volumes > 0.5 microliter. Calibrations performed on one day could be used with accuracy to derive volumes > 0.1 microliter collected on subsequent days, when using the same machine (s.d. for residuals plot = 2.49 Periotron units), but this was not the case for different machines (s.d. = 9.57 Periotron units). Varying serum protein concentration by up to 500% had a negligible effect on calculated volumes above 0.1 microliter. We conclude that the Periotron 8000 is at least as reliable a machine as the Periotron 6000, and that the calibration lines for both machines are best described using 4th order polynomial regression equations and "look-up" tables, rather than quadratic (Periotron 6000) or the manufacturer's software (Periotron 8000). Serum seems to be an acceptable GCF substitute for calibrations, which can be performed 1 day, and used on subsequent days for a given machine and for volumes above 0.1 microliter.

Osteocalcin (Oc) and the collagen cross-links pyridinoline (Pyr) and deoxypyridinoline (dPyr) are used as markers of bone turnover in metabolic bone diseases. The aims of this study were: 1) to establish if Oc, Pyr and dPyr can be detected in GCF and 2) using the orthodontic tooth movement model of alveolar bone resorption to evaluate GCF levels of osteocalcin and these collagen cross-links as markers of bone breakdown. Plaque, colour and bleeding indices, probing measurements and GCF samples were collected at two sites in each of 20 adolescents, during 4 stages of fixed appliance therapy: (1) prior to appliance fit, (2) post appliance fit, (3) during active retraction of the maxillary canines, (4) during retention. GCF was collected onto filter paper strips and the volume determined by weighing. An ELISA kit was used for the detection of osteocalcin, whereas Pyr and dPyr were assayed using high performance liquid chromotography (HPLC). Wilcoxon signed ranks test and Bonferroni correction revealed statistically significant increases in plaque (p= 0.012), GCF volume (p=0.024) and osteocalcin concentration (p=0.012), between stages 1 and 2. There were no statistically significant differences between the other variables at this stage or between any of the variables at stages 2 and 3, or between stages 3 and 4. All but 3 of the GCF samples yielded detectable osteocalcin, with large site and subject variation. The median values of osteocalcin and osteocalcin concentration of all the samples were 87.5 pg and 66 pg/microl, with a range of 0-1,248 pg, 0-1,572 pg/microl. The detection of osteocalcin in GCF during every stage, the wide variation between subjects, and the lack of a consistent pattern related to stages of orthodontic treatment, suggests that osteocalcin may merely be a constituent of GCF associated with the developing dentition, which would reduce its potential as a marker of bone turnover in this group. None of the 16 GCF samples analysed for Pyr and dPyr gave a positive result. This study confirms that fitting an orthodontic appliance results in plaque accumulation and increased gingival inflammation, and that GCF volume is the most sensitive indicator of that inflammation. Osteocalcin was detected in GCF collected from adolescents, whereas Pyr and dPyr could not be detected. Further work is required to establish whether GCF osteocalcin levels can be used as a marker of bone turnover, and whether improvements in the sensitivity of detecting Pyr & dPyr make further study of these promising bone markers worthwhile.

Gingival and periodontal ligament (PDL) fibroblasts are the major cellular components of periodontal soft connective tissues, but the precise differences between these cells are not yet known. In the present study, we have therefore examined the phenotypic and functional features of the cells obtained from gingival and PDL biopsy samples. Spindle-shaped cells characteristic of fibroblasts were the main cell type observed in vitro, although epithelial cells were also present in primary gingival cell cultures. Flow cytometry was used to measure the size and granularity of the cultured cells, and showed that the gingival fibroblasts were smaller and less granular compared with the PDL cells. The expression of certain key extracellular matrix (ECM) proteins, fibronectin, collagen type I, and tenascin was measured by flow cytometry. Analysis of the fluorescence profiles of these cultures showed that the majority of cells expressed fibronectin and that the average fluorescence intensity of this antigen in the PDL cells was higher than that in the gingival fibroblasts. Moreover, the fibronectin-positive PDL cells apparently comprised two subpopulations which expressed fibronectin at different levels, suggesting that the cells in the PDL cultures were functionally heterogeneous. The level of collagen type I was also found to be up-regulated in the PDL compared with the gingival cells and, as with fibronectin, was expressed at two different levels by subsets of the PDL cells. In contrast, tenascin was expressed at very similar levels by both the gingival fibroblasts and PDL cells. In addition, measurement of alkaline phosphatase, a marker enzyme for mineralized tissue-forming cells, showed that the PDL cells had higher activity than the gingival fibroblasts and that the alkaline phosphatase activity in the PDL cells was far more markedly up-regulated by dexamethasone. Our findings demonstrate that, despite their similar spindle-shaped appearance, fibroblasts derived from gingival and PDL tissues appear to display distinct functional activities which are likely to play a vital part in the maintenance of tissue integrity and regenerative processes.

The present clinical trial was designed to evaluate the effects of a bioactive glass, Perioglas, in the treatment of periodontal intrabony defects. 20 patients, 23-55 years of age (44 sites), with intrabony defects completed the 1-year study. Teeth with furcation involvement were excluded. After completion of initial therapy, defects were randomly assigned to either a test or control procedure. Following flap reflection, root planing and removal of chronic inflammatory tissue in both groups, the test defects were restored with the bioactive glass particulate material. Mucoperiosteal flaps were replaced, sutured and a periodontal dressing was used. All the patients received postoperative antibiotics and analgesics and were seen at 1 week for suture removal. Follow-up was then carried out weekly and at 3 months, 6 months, 9 months and 1 year post-surgery. Plaque score, bleeding score, probing pocket depth (PPD), probing attachment level (PAL) and gingival recession were recorded at baseline, 3 months and 1 year. Standardised radiographs for computer-assisted densitometric image analysis (CADIA) were taken at baseline, immediately post-operatively and at 1 year. The CADIA data showed a significant increase (F-ratio: 15.67, p < 0.001) in radiographic density and volume between the defects treated with the Perioglas when compared to those treated with surgical debridement only. PPD and PAL showed significant improvements in both experimental and control sites, with a greater trend to improvement in the experimental sites. It was concluded that this bioactive glass is effective as an adjunct to conventional surgery in the treatment of intrabony defects.

The aim of this study was to investigate the accuracy and reproducibility of experienced and inexperienced examiners using 3 automated periodontal probes (Florida Pocket Probe, Florida Disk Probe, Peri Probe) in comparison with 3 conventional periodontal probes (Marquis, Williams and EN-15 probes). Test blocks of aluminium had 30 holes of diameter 1.10 mm and depths ranging from 2.75 to 10.0 mm. machined with a tolerance of +/- 0.01 mm. 8 experienced examiners and 8 inexperienced examiners were selected to perform duplicate measurements on the blocks over 6 visits using each of the 6 probes. 1 automated and 1 conventional probe were used at each examination. The % accuracy and reproducibility for each of the duplicate measurements was calculated and analysed using Friedman 2-way analysis of variance and the Wilcoxon matched pairs test. On average, all probes showed high reproducibility, with the Florida Disk Probe, the Florida Pocket Probe and the Williams probe ranked best and the other 3 probes were less reproducible. On average, all probes showed a high degree of accuracy, automated probes were ranked best and were significantly better than conventional probes. Experience had little effect on reproducibility, with only the Peri Probe showing significant differences at the 5% level between the groups. Experience appeared to be more important for accuracy, as experienced examiners were more accurate than inexperienced examiners, with significant differences at the 5% level for the EN-15, Florida Disk Probe and Peri Probe. However, inexperienced examiners were significantly more accurate using the Williams probe. This in vitro study has shown that automated probes offer increased accuracy over conventional probes and the Florida Pocket and disk probes compare well with conventional probes for reproducibility.

Perioscan requires a plaque sample to detect the presence of enzymes capable of degrading N-benzoyl-DL-arginine-2-naphthylamide (BANA) from relatively few anaerobic periodontal pathogens. Periocheck assays the presence of neutral proteases in crevicular fluid. The aim of this study was to compare these test kits with traditional clinical methods of detecting periodontal disease and to monitor the ability of the kits to reflect the response to initial therapy. 19 patients with moderately severe chronic periodontitis were seen before and after a course of oral hygiene and root instrumentation consisting of 4 appointments. Clinical measurements and test assays were collected at 5 diseased sites and 2 healthy sites in each subject. Complete data from 125 sites were available for statistical analysis. At baseline Periocheck had a sensitivity of 88% and a specificity of 61% whereas Perioscan had a sensitivity of 99% and a specificity of 55%, when related to the clinical diagnosis. A composite clinical assessment, based on improvement or deterioration of one whole unit change of the subjective clinical indices and 2 mm changes or greater in probing depth or probing attachment level, revealed 75 sites which improved following treatment, whereas 45 sites did not change and 5 sites deteriorated. The probability that the tests agreed with the clinical outcome after treatment, was calculated as 50.4% for Periocheck and 52% for Perioscan. The diagnostic kits did not reliably reflect the clinical assessment of periodontal disease in the cross sectional study, or the outcome following treatment.

Changes in the permeability of the underlying vasculature of the gingival crevice region may occur either as part of an inflammatory response during gingivitis or as a response to trauma during collection of gingival crevicular fluid IgM was selected as a marker of permeability and the concentration in gingival crevicular fluid was determined immunochemically by dot blotting, using rabbit antihuman IgM specific for mu chain. Gingival crevicular fluid samples were derived from experimental (non-cleaning) and control (cleaning) sites in six individuals participating in a 28-day experimental gingivitis study using a split-mouth protocol. The fluid was collected by a filter-paper method. At each site, four gingival crevicular fluid samples were collected for 5 s each over a 3-min period and a fifth sample was collected after a further 6 min. IgM was detected in 7/30 experimental strips at baseline (after intensive professional prophylaxis) and this increased to 21/30 by 24 days: control values were 13/30 and 12/30, respectively. The data indicate that IgM was detected with greater frequency as gingivitis becomes evident, suggesting increased permeability in response to plaque accumulation. IgM was detected in 17/120 of the first strip samples, collected over the 66-day study period, whereas in the fifth strip samples IgM was detected in 68/120, and the concentration of IgM in gingival crevicular fluid also increased, indicating that sequential collection induced permeability. It is concluded that the permeability of the gingiva changes in response to both an inflammatory stimulus and trauma due to collection.

Objective: To assess the use of an internal reference when performing histogram analyses in digital subtraction images and to determine the ability of the method to detect initial bone lesions., Methods: Fifty-one Royal Air Force recruits had standardized vertical bitewing radiographs and clinical assessment of attachment level recorded annually over three years. Subtraction analyses of crestal bone changes at the mesial surface of the upper right first molar were compared with changes at the mesial surface of the same tooth. Changes over the periods from age 17 to 18 years and age 18 to 20 years were monitored using two subtraction procedures., Results: Reproducibility studies revealed that Pearson correlations between duplicate measurements of the test site alone (16 crest; r = 0.74) were lower than those between duplicate measurements of changes where misalignment was controlled for, either as a difference (16 crest-16 tooth; r = 0.93) or ratio (16 crest/16 tooth; r = 0.93). We used the differences between the mean subtraction density for 16 crest and 16 tooth as our measure of change in bone density. For the duplicate measurements, the standard deviation of these differences was 3.9: a difference of +/- 7.8 was therefore taken as a threshold value for evidence of real change. Bone gain was noted between the ages of 17 and 18 years (16/21 subjects), but some early bone loss was seen between 18 and 20 years (12/21 subjects), with four subjects showing changes significantly greater than the method error. There were no associations between the clinical and radiographic observations., Conclusion: Use of a control site in subtraction radiography improves the reproducibility; such systems can detect small changes in alveolar bone which may assist in early diagnosis of the initial periodontal lesion which may precede observable clinical changes.

2 periodontal dressings, Coe-pak and RD8 (a product under development), were assessed following periodontal surgery, using a double-blind study of randomised split-mouth design, with each patient acting as their own control. Matched sites were selected for periodontal flap surgery under local analgesia. Sites were randomly allocated to each group. The dressings were applied by a single operator and both patients and clinical assessors were blind as to the dressing employed. The dressings were assessed as to their handling properties, in addition to a 7-day post-operative assessment of the surgical sites and a subjective patient report. Maximum pain levels were relatively low (4 in a scale 0-10); subjects reported more pain and analgesics required with RD8 than with Coe-pak. This was reflected in the results of the patient assessment which indicated that 50% preferred Coe-pak, compared to 25% who preferred the RD8 dressing. The results showed RD8 to be an acceptable periodontal dressing when compared to Coe-pak. There were no statistically significant differences between treated sites in both groups with regard to post-operative plaque scores, bleeding scores or tissue healing.

Gingival crevicular fluid (GCF) sampling was performed on 2 occasions separated by 1 year, at 2 sites in the mouths of 102 male adolescents, mean age 17.85 years. Samples were collected onto 5 filter paper strips which were sequentially applied to the mouth of the crevice over a 9-min collection period. Volume and flow rates of GCF were determined for each site and were compared with clinical measurements of plaque, gingival colour, bleeding, gingival index (GI) and pocket depth, using a general linear models (GLM) procedure. While the initial volume of GCF showed no association with any clinical measurement, there was an association between flow rate of GCF and gingival colour. The volume of GCF collected in the final, 5th sample was associated with the GI. The sample site strongly influenced all measures of GCF volume. It is proposed that the flow rate of GCF may be a better indicator of gingival inflammation than the more imprecise clinical assessments of inflammation, since GCF flow rates more precisely reflect changes in tissue permeability. The association between the final sample, collected after 9 min, and clinical measurements, was probably a reflection of the association between clinically-detectable inflammation and the susceptibility of the site to mild irritation.

Dental plaque and saliva are both possible contaminants of gingival crevicular fluid (GCF). Plaque samples from 12 sites in four subjects who had allowed plaque to accumulate for 24-48 h were quantified using the Plaque Index and then transferred to filter paper strips for fluid volume determination using the Periotron 6000. The mean volume of fluid for plaque scores of 0 was 0.02 (+/- 0.01) microliter, whereas for plaque scores of 3 the mean volume was 0.15 (+/- 0.07) microliters. In a clinical study, GCF samples, from 19/184 subjects (10.4%) were assessed as contaminated or suspected to be contaminated with saliva, but only 28/1740 strips (1.6%) were placed in these categories of contamination. An assay to confirm salivary contamination was established, using an immunochemical, double-antibody method with sheep anti-human salivary alpha-amylase followed by peroxidase-conjugated rabbit anti-sheep immunoglobulin. The detection threshold of the assay was 7.5 ng of alpha-amylase, which represents approx. 15-25 nl of saliva. The assay was evaluated on 90 GCF samples categorized as 'known' (n = 16), 'suspected' (n = 16), or 'not' contaminated (n = 58) with saliva; 81.25, 50-61.5 and 5.2-8.6%, respectively, were positive for salivary alpha-amylase. In some GCF samples salivary contamination was in excess of 50%. It was concluded that GCF samples seen to be contaminated with saliva should be discarded, whereas samples considered free from contamination may be used with confidence. Samples suspected of contamination may require an alpha-amylase assay before further analysis.

We have estimated the levels of Interleukin-1 beta (IL-1 beta) by ELISA in gingival crevicular fluid (GCF) at 58 sites from 37 patients with adult periodontitis. GCF was collected for 5 s on filter papers and a 2nd sample was collected for 30 s 1 min later. 68/116 strips yielded detectable levels of IL-1 beta. IL-1 beta was present in both the 1st and 2nd samples at 28 sites, in the 1st only at 4 sites and in the 2nd only at 8 sites; 18 sites were below the level of detection for the assay. When the concentrations of IL-1 beta were calculated in the original volume of GCF on each strip, the mean value for positive strips was 34.16 +/- 29.45 (SD) pg/microliters with a range from 1.75 to 97.13 pg/microliters. There were no statistically significant correlations with the plaque index, bleeding index or probable crevice depth (pocket depth). The results indicate that IL-1 is present in the GCF from a proportion of sites with evidence of previous periodontal destruction.

We have reviewed the recent literature on the humoral immune responses to a variety of subgingival plaque bacterial species in patients with destructive periodontal diseases. We do not feel that the information presently available on the specific antibody responses to proposed pathogens such as Bacteroides gingivalis and Actinobacillus actinomycetemcomitans allows antibody responses to be diagnostic. All control subjects without periodontal destruction have antibodies to candidate pathogens but the generally higher levels in patients are not sufficiently elevated to be diagnostic. Nor can they be used to predict the initiation of disease or the onset of new episodes of destruction where disease had previously occurred. Successful treatment of patients may lead to lower levels of antibodies to some organisms, including possible pathogens, and thus support a given species in the aetiopathogenesis of disease. It appears that unsuccessful treatment may be accompanied by continuing high antibody levels to some organisms and further studies may enable this observation to be used to monitor therapy. There is some evidence from serological studies that each destructive episode may be induced by a different bacterial species or consortium. The start of studies using single antigens and the techniques of molecular biology will provide not only antibody-based diagnostic methods but also allow us to determine which bacterial antigens are virulence factors and thus the role of the antibody responses, whether protective or damaging, in the periodontal diseases.

The histological evaluation of surgical biopsies from affected tissues is a standard way of assessing pathological change and determining treatment in many diseases. In most forms of periodontal disease, however, this approach finds limited application. Here, we review what uses the histopathological approach has in the study and evaluation of the periodontal diseases. Current understanding of the changes in epithelial anatomy during pocket formation, the cellular composition and dynamics of the inflammatory infiltrate and the mechanisms of bone resorption and repair are reviewed from the perspective of the information available from microscopical investigation, including the uses and potential application of modern immunocytochemical methods to these questions. The usefulness of histological study of biopsy material is reassessed in the light of advances made in immunohistochemical techniques and their application to gingival inflammatory infiltrates and epithelia. Such techniques offer immediately valuable research opportunities with potential for diagnostic applications, noteably the recognition of phases of destructive activity and their differentiation from periods of effective host defence.

Many factors have been hypothesised either to characterise groups and individuals at risk for periodontal disease or to be markers of periodontal breakdown. In order to identify these as associated either with disease status or progression, a statistical association between the factor and a measure of disease will have to be demonstrated. The statistical modelling of data arising from periodontal research presents special problems. These include the large number of measurements made in each subject, the large magnitude of measurement error compared to the changes in attachment level, the analysis of longitudinal studies, the lack of a measure of instantaneous rate of attachment loss and controversies over the nature of the progression of the disease. We consider statistical methods currently available in the light of these difficulties and identify areas in which further research is necessary.

Periodontal microbiology is reviewed with regard to the potential of certain characteristics to serve as markers of high risk groups or individuals for periodontal diseases. The generally accepted associations between particular organisms and the various periodontal diseases are discussed. The usefulness of various clinical study designs is reviewed. The ecology of the subgingival plaque microflora is discussed and a number of suggestions for future research are made. We have concluded that there is no monospecific aetiology to any of the various periodontal conditions. Nevertheless, we give particular attention to the role of the black-pigmented bacteroides based upon our belief that they, and Bacteroides gingivalis in particular, are fundamental to our understanding of the biology of periodontal diseases in humans and other animals. Consequently, the contribution of its various virulence factors and their potential as markers of disease susceptibility and activity is addressed.