Periodontitis is associated with altered plasma fatty acids and cardiovascular risk markers.

BACKGROUND AND AIMS: In periodontitis it has been found that some perturbation exists in lipid biomarkers, such as increased serum total cholesterol and low-density lipoprotein cholesterol. Nevertheless, the relationship between fatty acids and periodontitis has been demonstrated only in a few studies and remains controversial. The aim of this investigation was to explore the effects of periodontitis on a cluster of traditional and novel cardiovascular risk factors such as plasma-lipids profile, types of plasma fatty acids, adhesion molecules and systemic inflammatory markers. METHODS AND RESULTS: At a university dental school, 56 patients all over 35 years old were enrolled and invited to participate in the study. Total plasma fatty acids, saturated, n-6 polyunsaturated and monounsaturated fatty acids, peroxidability index, soluble VCAM, TNF-alpha, cholesterol, triacylglycerols, and VLDL-c were significantly higher in the periodontitis group compared to the non-periodontitis group. CONCLUSIONS: This close association found between plasma triacylglycerols, LDL-c, saturated fatty acids, polyunsaturated fatty acids, total amount of fatty acids and coenzyme Q(10) with some periodontal data such as periodontal probing depth, recession of the gingival margin and clinical attachment level (Pearson correlation between 0.3 and 0.6), leads to the conclusion that there is an inter-relationship between periodontitis, plasma fatty acids profile and the increase in metabolic risk factors for cardiovascular diseases.

Nifedipine and cyclosporin affect fibroblast calcium and gingiva.

It has been stated that cyclosporin and nifedipine produce gingival overgrowth. However, the specific pathogenic mechanism remains uncertain. We used an experimental rat model to test the hypothesis that changes in collagen metabolism and numbers of gingival blood vessels are not mediated by intracellular calcium concentration (ratiometric Fura-2 AM measurement) in gingival fibroblasts. In the cyclosporin group, both width (364.2 +/- 67.5 mum) and microvessel density (number of vessels/mm(2), stained with anti-CD34 antibody) (41.6 +/- 5.1) of gingiva were statistically different when compared with those in the control group (width = 184.3 +/- 35.2 mum, microvessel density = 19.6 +/- 2.4). The nifedipine group showed the highest content of collagen (proportion of total stroma occupied by collagen, stained with Picro-Mallory) (nifedipine group = 66.3 +/- 9.4, cyclosporin group = 55.2 +/- 7.9, control group = 30.1 +/- 10.2). Freshly cultured fibroblasts from the cyclosporin group exhibited higher ratiometric values of fluorescence than did both the control and nifedipine groups (p = 0.03). Our results support the hypothesis that changes in gingival collagen metabolism are not mediated by calcium intracellular oscillations.

Adsorption/desorption of amine fluorides to hydroxyapatite.

This study concerned the adsorption and desorption of commercial amine fluoride (AmF) preparations to hydroxyapatite (HA). The influence of pH, ionic strength, temperature, saliva and albumin, the latter as a gingival crevicular fluid analogue, on adsorption/desorption was investigated. AmF levels were determined using a surfactant electrode. AmFs 297 and 335 were found to bind immediately and irreversibly to HA in water over a range of pH values, ionic strengths and temperatures, the amounts increasing with concentration. More monovalent AmF 335 was absorbed than divalent AmF 297. Any AmF desorbed by water from HA was at the lowest end of the minimum inhibitory concentration for oral bacteria. AmF 297 was desorbed by CaCl2, and to a lesser extent by H+, OH-, NH4+, La3+, EDTA, Triton X100 and ethanol, whereas AmF 335 was only slightly desorbed by ethanol. Preadsorption of proteins on HA had little effect on subsequent adsorption or desorption of either AmF. It is postulated that both AmF 297 and AmF 335 are inactivated by an excess of proteins in the surrounding medium, supra- or subgingivally, and not by such proteins preventing or altering the mode or rate of adsorption, or interfering with antibacterial activity, when the AmFs contact a protein-coated tooth surface.

Effect of repeated high dose prophylaxis with amoxycillin on the resident oral flora of adult volunteers.

Healthy adult volunteers received either single or repeated 3-g doses of amoxycillin by mouth at weekly intervals on three occasions. The salivary flora of each volunteer was monitored before, during and up to 11 weeks after the final dose of antibiotic. Viable counts of anaerobic bacteria, streptococci and streptococci resistant to amoxycillin 2 mg/L and 40 mg/L were determined in samples of saliva. All 20 volunteers harboured low numbers of streptococci resistant to amoxycillin 2 mg/L (mean count = 6.57 X 10(3) cfu/ml of saliva) before administration of the antibiotic; much lower carriage rates (45%) were observed for bacteria resistant to amoxycillin 40 mg/L (mean count = 116 cfu/ml of saliva). Each dose of amoxycillin had a rapid but transient effect on the numbers of salivary bacteria. A placebo lacking the antibiotic had no effect. A single 3-g dose of amoxycillin had little or no effect on the numbers of resistant streptococci and, therefore, it was concluded that in patients at risk of infective endocarditis a second prophylactic dose would not be invalidated. The numbers of resistant streptococci increased significantly after the second and third doses of amoxycillin, and persisted for 4-7 weeks. Consequently, in at-risk patients requiring repeated dental procedures liable to produce bacteraemia, either alternative antibiotic regimens should be used each time or intervals of at least 4 weeks should be left between treatment sessions.

Serum antibody response to surface-associated material from periodontopathogenic bacteria.

Saline extracts of Actinobacillus actinomycetemcomitans, Porphyromonas gingivalis and Eikenella corrodens contain surface-associated components of these bacteria. It has been shown that these extracts are potent stimulators of bone resorption in vitro. The possibility that the components of these surface-associated materials (SAM) could contribute to the serum immune response in patients with juvenile or adult onset forms of rapidly progressive periodontitis were investigated by direct binding ELISA. Very high titres of serum IgG antibodies to SAM from A. actinomycetemcomitans were detected in patients with localized juvenile periodontitis (LJP). Patients with adult onset rapidly progressive periodontitis (RPP) had significantly raised antibody levels to SAM from P. gingivalis. Both groups of patients had significantly raised levels of antibodies to SAM from E. corrodens compared with control sera. Thus, not only does solubilized SAM have the capacity to induce bone resorption, but it also contributes to the antigenic load on the immune system in LJP and RPP.

Attrition, eruption, and the periodontium.

Features of natural masticatory function, of physiological attrition, both occlusal and approximal, and of continuing tooth eruption in adult life need to be borne in mind in considering how the chronic inflammatory periodontal diseases and dental caries have become so widespread. Evidence is reviewed showing that teeth continue to erupt in adulthood, that natural masticatory function prevented plaque accumulation at the approximal risk site of onset of both CIPDs and caries, that epithelial attachment to cementum may be physiological, and that periodontal attachment studies must be age-related.

Keratinocyte growth factor receptor is up-regulated in cyclosporin A-induced gingival hyperplasia.

Keratinocyte growth factor stimulates the growth and activity of epithelial cells via the keratinocyte growth factor receptor. We have recently shown that the growth factor is markedly elevated in cyclosporin A-induced gingival hyperplasia tissue in vivo, but the effects of cyclosporin A on the receptor are not yet known. The present study was therefore carried out to determine whether expression of the keratinocyte growth factor receptor is up-regulated in gingival hyperplasia compared with normal gingiva. Using immunohistochemistry and the reverse-transcribed polymerase chain-reaction, we obtained results which showed that receptor antigen and gene transcript levels were both elevated in gingival hyperplasia tissue. In addition, flow cytometry and the reverse-transcribed polymerase chain-reaction showed that the receptor and mRNA were also higher in gingival epithelial cells following incubation with cyclosporin A in vitro. These findings suggest that the keratinocyte growth factor-receptor pathway of mesenchymal-epithelial interaction could play an important part in the molecular pathogenesis of gingival hyperplasia.

Flow cytometry analysis of gingival and periodontal ligament cells.

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.

A microbiological study of early caries of approximal surfaces in schoolchildren.

A cross-sectional epidemiological study has been undertaken to relate the bacterial composition of approximal dental plaque with the earliest stages of caries development in schoolchildren. Small samples of plaque were removed from multiple sites around the contact areas of 42 premolars extracted for orthodontic reasons from 29 schoolchildren (mean age = 13.5 yr). Caries diagnosis was based on polarized light microscopy and contact microradiography of thin sections cut through the sample sites. Fifty-seven percent of sites (37/60) showed histological evidence of demineralization. Both the isolation frequency and the mean percentage viable count of mutans streptococci and Actinomyces viscosus were higher at sites with early caries, although mutans streptococci could not be detected at 37% of sites with early caries. At these latter sites, the proportions of Veillonella were markedly reduced. Lactobacilli were rarely isolated and were never recovered from caries-free surfaces. Analysis of the data shows that the relationship between plaque bacteria and enamel is neither merely passive nor indifferent, and that particular stages of lesion formation may be associated with different combinations of bacteria.

Inhibition of microflora associated with oral malignancy.

Changes in the microflora on oral carcinoma surfaces may lead to both local and systemic infections, which may complicate the morbidity of the patient suffering from oral malignant neoplasms. Thus, anticancer therapy, irradiation, chemotherapy or surgery impairs the defence mechanism of the oral mucosa and is accompanied by proliferation of the mucosal biofilm with overgrowth of yeast and bacteria. This study investigates the inhibition of the biofilm present on the surface of oral squamous cell carcinomas. Biofilm samples were obtained from the central surface (1 cm2) of each lesion in 10 patients (eight male, two female; mean age: 47.6 years; SD +/- 7.6) before any antibiotherapy or tumour treatment. Patients were randomly divided into two groups and were rinsed with Meridol mouthrinse (amine fluoride) or placebo (saline solution) for 7 days. Samples were repeatedly taken from the same site after rinsing. Samples were transported in pre-reduced brain heart infusion broth and cultured within 1 h of removal, using aerobic and anaerobic complete and selective media. Total aerobic and anaerobic counts were determined and isolated bacteria were identified. The median counts of colony forming units (CFU/ml) after rinsing with Meridol were significantly lower for both aerobes and anaerobes than before rinsing with Meridol. (For aerobes before rinsing: 1.35 x 10(6), after rinsing: 7.55 x 10(5); p = 0.025; for anaerobes before rinsing: 1.39 x 10(6), after rinsing: 7.15 x 10(5); p = 0.011. Rinsing with placebo: no significant difference was found. Aerobe median counts before rinsing: 1.17 x 10(6), after rinsing: 1.03 x 10(5), and for anaerobes: before rinsing 1.75 x 10(6), after rinsing: 1.51 x 10(6); p > 0.05 [Wilcoxon test].) It was concluded that 7-days (three times a day) Meridol rinsing significantly reduced the surface biofilm of oral carcinoma compared to rinsing with placebo. Clinical examination indicated no irritation of the mucosa. The mouthrinse was well tolerated by the patients, who commented on a reduction in burning sensation and bad breath. Besides routine oral hygiene, rinsing itself could reduce patient morbidity. The findings of the present study indicate that in addition to any other oral focus, the lesion itself, when ulcerated, should receive direct antimicrobial treatment so as to reduce patient morbidity and enhance quality of life.

The microflora associated with human oral carcinomas.

Both local and systemic infections may complicate the morbidity of patients with oral malignant neoplasms, particularly those presenting intraorally. This study investigated the microbial contents of the biofilms present on the surfaces of oral squamous cell carcinomas. Biofilm samples were obtained from the central surface of the lesions in 21 patients (20 male, 1 female) aged 52.8 (+/- 8.2) years, and from contiguous healthy mucosa, before any antibiotic therapy or any tumour treatment. All lesions were keratinising squamous cell carcinomas with surface ulceration. Samples were transported in reduced brain heart infusion (BHI) broth and cultured within 1 h of removal, using aerobic and anaerobic complete and selective media. The median number of anaerobic colony forming units (CFU/ml) at the tumour sites (1.6 x 10(8)) was significantly higher than for the healthy (control) mucosa (3.0 x 10(7); P = 0.0001, Wilcoxon); the same was true for aerobes at the tumour sites (1.51 x 10(8)) relative to the controls (2.8 x 10(7); P = 0.0008, Wilcoxon). The species isolated in increased numbers at tumour sites were Veillonella, Fusobacterium, Prevotella, Porphyromonas, Actinomyces and Clostridium (anaerobes), and Haemophilus, Enterobacteriaceae and Streptococcus spp. (aerobes). Candida albicans was found at eight of the 21 tumour sites, but never at control sites. It was concluded that human oral carcinoma surface biofilms harbour significantly increased numbers of aerobes and anaerobes as compared with the healthy mucosal surface of the same patient. Candida albicans can also be present in these biofilms. These findings must be considered in relation to the known predisposition of such patients to systemic infections, and to the unpleasant complications of oral morbidity due to infected lesions.

Neutrophils and IgG at the host-plaque interface on children's teeth.

This study is part of an investigation aimed at discovering how subcontact area plaque, unlike corresponding embrasure plaque, survives the host response and enters the gingival crevice. Approximal gingival margin plaques incorporating these two sites were obtained from children's teeth and subjected to transmission electron and immunoelectron microscopy. The specimens were examined for the main components of the initial host response at the plaque-host interface, namely polymorphonuclear leukocytes and IgG. Both were found preferentially at the actual apical border of plaque, especially in embrasures. Some polymorphs showed evidence of phagocytosis. Most of those in contact with plaque showed lysosome and glycogen loss and occasional phagocytosis. It was concluded that at least one important mechanism by which chronic gingivitis and approximal caries are initiated, involves lack of polymorph activity apical to the contact area. Possible explanations include the stagnation of gingival fluid so that neutrophils do not reach the plaque, failure of neutrophils to adhere to tapering border plaque, lysis of polymorphonuclear leukocytes (PMN) or elaboration of antichemotactic or other factors inhibiting PMN function by the colonizers of the gingival crevice.

Calcium, matrix polymers and plaque formation.

This study explores the relationship between calcium ions, matrix polymers and the formation of natural plaque on the children's teeth, using direct observation, scanning, transmission and freeze-etching electron microscopy techniques. Plaques were observed either in situ or after removal from enamel with a chelating agent, EDTA. Plaque was not disrupted by EDTA. Matrix polymers in outer plaque were less abundant than in deeper layers of specimens exposed to EDTA before embedding. The several cationic electron-histochemical reagents used generally disclosed more matrix than routine contrasting. The various polymers, other than discrete structures such as fimbriae, may derive from saliva and gingival fluid as well as bacteria. It is concluded that calcium ions are of importance in the early phase of bacterial attachment to the host organic enamel integument, but that calcium plays no part in the formation or maintenance of the strong bonds that link organisms in established plaque.

Gingival crevice neutrophil function in periodontosis.

This study examined gingival crevicular polymorphonuclear leucocyte function in periodontosis patients. Cells were examined for viability, function and ultrastructure. Eighty percent or more of the cells in each sample were viable as assessed by the fluorescein diacetate technique, but the test organism, Candida guillermondiae, was not phagocytosed. Gingival crevicular fluid contained many lysing neutrophils and nonphagocytosed organisms. Recognizable polymorphs contained Gram-negative and Gram-positive organisms. On the basis of this and previous studies it is concluded that gingival crevice neutrophils from periodontosis sites show reduced phagocytic function compared with cells from normal or periodontitis-affected gingival crevices. It is possible that the behavior of neutrophils from gingival crevices may be irrelevant. Original changes by that stage may have obscured their capabilities.

Ultrastructure and histochemistry of the dental cuticle in adult periodontitis.

This study examined the dental cuticle (DC) at the interface with cementum surface, as well as its relationship to the overlying subgingival plaque (SP), the so-called plaque-free zone (PFZ), the junctional epithelium (JE), and the coronal fibers of the residual periodontal ligament (PL) by transmission electron microscopy (TEM) and histochemistry. Material comprised of 41 extracted, adult periodontitis-affected teeth (AP). Following extraction, 20 teeth were fixed in 3% glutaraldehyde in 0.1M sodium cacodylate, post-fixed in 1% osmium tetroxide, embedded in araldite, decalcified in EDTA, re-embedded in araldite, and sectioned. En bloc histochemistry was undertaken on the remaining 21 teeth, using ruthenium red, alcian blue-lanthanum nitrate, or safranin-O, and processed as above. Results show that the DC covered the cementum surface from the SP to the JE, and formed an interface with these structures. No DC was observed at the interface with PL. Morphological variations in DC surface were observed at the interface with the SP and at the so-called PFZ where bacteria were always in close contact with or surrounded by the DC. At the interface with JE, the DC appeared homogeneous, although layers varying in electron density were distinguishable. Teeth treated histochemically revealed a positive reaction of DC and bacteria to the three methods, suggesting the presence of anionic polymers including glycoproteins in the DC. It was concluded that on adult periodontitis affected teeth, the DC always covers exposed cementum and may mediate bacterial adhesion, and adsorb components from the periodontal pocket.

Bacterial penetration of the pocket tissues in juvenile/postjuvenile periodontitis after the presurgical oral hygiene phase.

Previous ultrastructural investigations of untreated sites of both adult and juvenile periodontitis have shown bacteria within the periodontal soft tissues. In the present study biopsies of the soft tissue walls of deep pockets from seven patients with juvenile (JP) or postjuvenile periodontitis (PJP) were removed at the end of the presurgical oral hygiene phase of treatment and examined in the transmission electron microscope. Bacteria were sparse, regardless of the level of tissue breakdown, both on the surface and within the superficial layers of the epithelium, deep to the basement membrane and throughout the underlying connective tissue. Of the 140 blocks from 20 biopsies, only two revealed intratissue accumulations of microorganisms. The organisms observed were gram-positive or gram-negative and appeared to be exclusively coccoid or rod-shaped. It is suggested that the reduced tissue content of bacteria reflects the establishment of adequate oral hygiene. Evidently either the tissue content of bacteria is less than has been reported previously or the host response is able to cope with residual bacteria that have penetrated the soft tissue.

Subgingival metronidazole in acrylic resin vs. chlorhexidine irrigation in the control of chronic periodontitis.

After initial scaling, root planing and cleaning of subcontact areas, chronic periodontitis patients received oral hygiene instruction consisting of Bass toothbrushing using a sodium fluoride toothpaste, but without stress on interdental cleaning. They were then assigned to two groups, one of which self-administered 0.2% chlorhexidine solution subgingivally, once daily for 4 weeks, while the other was subjected to subgingival application of acrylic resin strips containing 40% metronidazole once weekly for 4 weeks. Plaque Index, Sulcus Bleeding Index, probeable pocket depth and gingival shrinkage were assessed for this treatment period and for a further 8 weeks. Highly significant improvements compared with baseline values were achieved and maintained to the end of the study. There were no significant differences between the groups at any time, except for Sulcus Bleeding Index which at Days 56 and 84 showed metronidazole to be superior. It was concluded that the regimens selected were equally effective in improving periodontal health over a 3-month period.

Susceptibility of oral bacteria to phenoxyethanol and phenoxyethanol/chlorhexidine combinations.

A total of 57 bacterial strains (26 different species) which may be isolated from subgingival plaque were tested for their in vitro susceptibility to phenoxycthanol, a commonly-used antiseptic and preservative. Ninety-five percent of the strains, including those associated with chronic inflammatory periodontal disease, were susceptible to concentrations of phenoxyethanol used topically (2% w/v). Phenoxyethanol at a concentration of 1% (w/v) was also found that to have a rapid bactericidal effect achieving a 99.9% kill in 5 minutes or less for species such as Bacterodides gingivalis, Fusobacterium nucleatum, Eikenella corrodens, and Wolinella recta. Addition of chlorhexidine to phenoxyethanol resulted in a mixture with increased antibacterial activity. For most bacterial strains, the presence of chlorhexidine resulted in at least a four-fold decrease in the concentration of pheoxyethanol required for inhibition of growth. These results imply that phenoxyethanol may be useful in the treatment of chronic inflammatory periodontal disease either by itself or in combination with chlorhexidine.

Susceptibility and resistance of plaque bacteria to minocycline.

The in vitro susceptibility of 55 strains of subgingival plaque bacteria to minocycline was determined. A concentration of 1 microgram/ml minocycline was found to inhibit 85% of the strains tested and the MIC ranged from 0.03 to 32 micrograms/ml. For 71% of the strains tested the MBC was at least 4 times greater than the corresponding MIC, suggesting a bacteriostatic activity for minocycline. A concentration of 20 mg/ml of magnesium ions was capable of neutralizing 8 micrograms/ml of minocycline and was used to eliminate "carry-over" effects inherent in the experimental procedure. After 6 to 7 weeks exposure to sub-lethal concentrations of minocycline there was no appreciable increase in the MICs of most organisms with the exception of Actinobacillus actinomycetemcomitans NCTC 10981 and Campylobacter concisus NCTC 11485. Short term (6 hour exposure of bacteria to minocycline (8 micrograms/ml) markedly reduced the viability of a number of periodontopathogens but had little effect on the viability of Veillonella parvula NCTC 11456 and Fusobacterium nucleatum NCTC 11326. These in vitro investigations have demonstrated that minocycline is capable of inhibiting most of the periodontitis-associated bacteria tested and can kill some of these bacteria after a comparatively short exposure time. However, some of the organisms tested exhibited a low susceptibility to minocycline and others became less susceptible following exposure to low concentrations of the antibiotic for several weeks.

The apical border plaque in chronic adult periodontitis. An ultrastructural study. I. Morphology, structure, and cell content.

THIS STUDY CONCERNS THE APICAL BORDER (AB) plaque in relation to chronic adult periodontitis (AP). Fifty-six teeth from 24 patients with AP were examined by transmission electron microscopy (TEM). The AB was not discrete with islands of bacteria in the so-called plaque-free zone (PFZ). Coronal to the AB, the established plaque commonly consisted of three to four layers of Gram-positive and Gram-negative cocci, rods, filaments, and spirochetes and a superficial layer, mainly of spirochetes, but including filaments, "test tube brush," and "corn-cob" formations. The most apical apparently intact organisms in the PFZ were in bacterial islands or in isolation and were predominantly Gram-negative cocci and rods, with occasional other morphotypes. The most apical microorganisms were invariably ghost cells. A cuticle of varying thickness and structure was present at the plaque/tooth interface. It was concluded that there was a limited range of intact bacterial morphotypes in the apical border plaque in chronic periodontitis.