Human gingival crevicular fluid contains MRP8 (S100A8) and MRP14 (S100A9), two calcium-binding proteins of the S100 family.

Human gingival crevicular fluid contains unidentified proteins which might play a role as markers in periodontal diseases. Therefore, low-molecular-weight proteins found in human gingival crevicular fluid (GCF), but absent from serum, were identified in the present study by means of two-dimensional electrophoresis (2-D PAGE) analysis. GCF, serum, and whole saliva were collected from periodontitis and healthy subjects, as well as from edentulous and newborn subjects. Protein samples were separated by two-dimensional polyacrylamide gel electrophoresis, stained with silver, and compared with reference protein maps in the SWISS-2D PAGE database. In GCF and saliva from periodontitis patients and healthy subjects, four dominant low-molecular-mass (from 8 to 14 kDa) acidic spots were observed. They were not found in serum and were less visible in saliva from edentulous and newborn subjects. From N-terminal amino acid sequencing, the two 2-D protein spots of 8 kDa and isoelectric points between 6.5 and 7.0 were both identified as protein MRP8 (SI00A8), a member of the S100 family of calcium-binding proteins. Using peptide mass fingerprinting and matrix-assisted laser desorption ionization time of flight mass spectrometry (MALDI-TOF-MS), we identified the other two protein spots, with mass of 14 kDa and isoelectric points between 5.5 and 6.0, as protein MRP14 (S100A9), also belonging to the S100 family. The presence of MRP8 and MRP14 in GCF was confirmed by Western blot, with monoclonal antibodies. The two polypeptides, MRP8 and MRP14, identified in GCF represent the major difference between the 2-D PAGE patterns of serum and GCF, and we hypothesize that they may play an important role in the gingival sulcus and could represent possible markers for periodontal diseases.

Effects of nicotine on periodontal ligament fibroblasts in vitro.

Cigarette smoking is associated with increased incidence of periodontal disease and poor response to therapy. In the present study, we examined the effects of nicotine on several functions of periodontal ligament fibroblasts (PDLF): proliferation, attachment, alkaline phosphatase production and chemotaxis. Nicotine concentrations varying from 5 ng/ml to 250 microg/ml were tested. Proliferation of cells was studied by the incorporation of 3H-thymidine, and a dose-dependent inhibition was observed with concentrations > or =100 ng/ml. Similar results were observed when studying the attachment of the cells on plastic surfaces, using a colorimetric method. The inhibition of attachment was even more evident after 6 h incubation of the cells with nicotine. The activity of alkaline phosphatase, as determined with the substrate p-nitrophenyl phosphate, in both conditioned medium (CM) and cellular extract (CE), was also significantly decreased in a concentration-related fashion. Finally, the chemotaxis of PDLE as examined by a modification of the Boyden's blind-well chamber technique, was inhibited in a dose-dependent manner. The degree of inhibition varied from 15% with the lowest concentration of nicotine (50 ng/ml), to almost 90% with the highest (5 microg/ml). The results show that nicotine can have direct adverse effects on various functions of the periodontal cells.

Radiographic measurement of alveolar bone loss in the living rat.

We describe a method which allows longitudinal measurements of alveolar bone loss in the living rat. The anesthetized animal is kept in a fixed and reproducible position and radiographs of an upper molar arch are taken with a mammography. In a preliminary study, 4 rats were repeatedly radiographed. In the main study, 10 rats were radiographed at day 0 and after 63 days of diet known to cause periodontitis. The radiographs were enlarged and the amount of bone lost calculated by comparing the weights of the interdental areas reproduced on standard paper. In the preliminary study, the coefficient of variation (C.V.) of the average weights of paper were fairly low (4 to 9%) in the first 3 rats, and amounted to 17% in the fourth. A statistically significant average loss of bone (16%) was found in the 10 rats in the main study when comparing the weights of papers at days 0 and 63.

Salivary albumin of edentulous patients.

In order to evaluate the contribution of the periodontal sulcus or pocket to the presence of albumin in the mouth, this protein was analysed in whole saliva of 20 completely dentate adults, aged between 63 and 83 years, and in 23 edentulous patients of similar age (51-88 years). In spite of the considerable intra- and interindividual variations revealed by a preliminary trial, the concentration of salivary albumin was significantly higher (range 60-1080 mg/l) in the dentate than in edentulous individuals (range 2-690 mg/l). The low albumin content in saliva of old edentulous people was similar to that in a group of younger individuals with a healthy periodontium.

A longitudinal study of various crevicular fluid components as markers of periodontal disease activity.

In order to examine the relationship of possible crevicular biochemical parameters to attachment loss (ALOSS), 330 sites from 8 untreated adult patients were monitored longitudinally at 3-month intervals, for up to 1 year. Attachment levels were measured with a force-sensing probe and an acrylic stent in duplicates at each study point. Crevicular samples were collected and used for the determination of the following 11 markers: number of polymorphonuclear leukocytes (PMNs), prostaglandin E2 (PGE2), osteocalcin (OC), alkaline phosphatase (ALP), collagenase (COL), beta-glucuronidase (BG), antigenic and functional elastase (AEL and FEL), alpha-1 antitrypsin (a1AT), alpha-2 macroglobulin (a2M) and aspartate aminotransferase (AST). 10 sites with ALOSS of > or = 1.5 mm per 3 months (active sites) and 43 sites with negligible changes (inactive sites) were identified. Total amounts of ALP, BG and COL were found to be significantly higher in active as compared to inactive sites, prior to significant ALOSS, without any significant differences in crevicular fluid volume and clinical indices. When biochemical parameters were expressed as ratios to the number of PMNs, PGE2/ PMNs was significantly elevated in active sites. The capacity of such individual parameters to distinguish between active and inactive sites was limited. However, linear discriminant analysis using total amounts of PGE2, COL, ALP, a2M, OC and AEL showed more significant diagnostic values (sensitivity: 80%, specificity: 91%). These findings suggest that the combination of several biochemical parameters in crevicular fluid could give more information to predict future clinical ALOSS.

Bone density of class II furcation lesions treated by guided tissue regeneration. A follow-up study by digital analysis of superimposable radiographs.

12 buccal or lingual class II furcation lesions were selected in the lower molars of 7 patients after treatment by routine scaling. In each of the 7 patients, 1 lesion was treated by guided tissue regeneration (GTR) with expanded polytetrafluoroethylene membranes, while 5 neighbouring molars underwent conventional flap operations. The index of gingival inflammation, the plaque index, the depths of the pockets, the horizontal and vertical attachment levels were measured before and 6 months, 1 year and 2 years after surgery. Standardized reproducible radiographs were taken before treatment, post-operatively (at the time of membrane removal for the GTR treated sites), at 6 months, 1 year and 2 years after treatment. The density of bone was quantified in several areas by using high-resolution digital analysis. With the exception of the higher gain in horizontal attachment observed in the GTR treated furcations, the improvement of clinical parameters was similar in all sites during the 2 years of follow-up. The radiographical analysis showed no statistically significant changes neither in the GTR nor in the conventionally treated lesions over this period of time, even when they were compared to untreated bone control areas. However, in this study limited to 7 patients, the interdental bone next to the GTR treated furcations showed significant signs of remodeling.

Low molecular-weight proteins in human gingival crevicular fluid.

Samples of gingival crevicular fluid (GCF) were collected in 30 volunteers with inflamed gingiva, using either capillary tubes (cGCF) or Durapore strips (sGCF). They were examined, together with samples of serum from the same patients, by sodium dodecyl sulphate-polyacrylamide gel electrophoresis and/or by two-dimensional electrophoresis, followed by silver staining. The results confirmed that the distribution of the major proteins in GCF is similar to that found in serum. However, an 8.5 kDa protein was found in gingival fluid but not in serum. The low molecular-weight protein appears to decrease with time of fluid sampling with both techniques, and does not originate either from blood or from the cellular fraction of GCF. Two-dimensional electrophoretic analysis suggested that it may consist of several polypeptides.

A case of localized juvenile periodontitis: treatment and 3 years follow-up with superimposable radiographs.

A 17-year-old male patient with localized juvenile periodontitis was treated by subgingival instrumentation with full thickness flap on the lower molars, combined with a 3-week course of systemic tetracycline, and a programme of supervised oral hygiene. The treatment was rapidly followed by dramatic clinical and microbiological improvement. However, despite good oral hygiene, gingival inflammation recurred at regular intervals. It was necessary to maintain the clinical results by periodic subgingival instrumentation with an ultrasonic scaler. Healing of alveolar bone was monitored in the lower 1st molar regions over 3 years by using superimposable radiographs. Quantitative analysis of bone density performed with a high-resolution digitalisation technique showed a considerable improvement 1 year after therapy. However, continuous remodelling, probably related to variations in inflammation, occurred during the 3 postoperative years.

Functional characteristics of gingival and periodontal ligament fibroblasts.

In periodontal surgery, healing after guided tissue regeneration (GTR) may be explained by differences in functional activities of gingival and periodontal ligament fibroblasts (GF and PDLF). Several studies in vitro have supported this hypothesis, but much remains to be defined. In the present work, gingival and periodontal ligament fibroblasts derived from five healthy subjects were isolated and compared in vitro. The morphology of the cells was observed under scanning electron microscopy (SEM). Several extracellular matrix components (ECM) were studied to compare the effects on fibroblast attachment, proliferation, and protein synthesis. Several biochemical markers were examined in both cellular extract (CE) and conditioned medium (CM). We also examined the muscle differentiation markers alpha-smooth muscle actin, desmin, and smooth-muscle myosin. Finally, we studied the effects of epithelial cells on the proliferation and protein synthesis of the two types of fibroblasts. GF and PDLF appeared identical under the SEM. All ECM components enhanced attachment; however, while collagen types I and IV promoted the attachment of GF, gelatin, laminin, and vitronectin promoted that of PDLF. Most ECM components increased the proliferation rate of GF and the biosynthetic activity of PDLF. The biochemical markers were similarly distributed between the two cell types, except for alkaline phosphatase, which was detected only in the CE of PDLF. Both GF and PDLF strongly expressed alpha-smooth-muscle actin and were negative for desmin; only PDLF were positive for smooth-muscle myosin. Epithelial cells increased the proliferation of both GF and PDLF but had no effect on their biosynthetic activity. These in vitro results may better explain the in vivo functional differences between GF and PDLF.

Reliability of a paralleling instrument for dental radiographs.

The reliability of a paralleling instrument for dental radiographs was retrospectively evaluated in the course of a 6-month clinical study. The angular variation between successive exposures was precisely quantified. Ninety-one percent of the angular variations were below a 1.4 degree threshold, but some paired images were found unsuitable for quantitative analysis. A mathematic procedure to assess the global angular projection error of any similar coupled x-ray cone-film holder device is proposed. This mathematic procedure should be routinely used to check superimposability of serial radiographs before density quantification or subtraction.

Kinetics of spiramycin/metronidazole (Rodogyl) in human gingival crevicular fluid, saliva and blood.

The peripheral distribution of spiramycin and metronidazole, which are combined in the proprietary drug "Rodogyl", has been studied in gingival fluid, saliva and blood after a single administration to 12 healthy volunteers and after repeated administration to 4 patients with recurring severe periodontitis. Analysis of the 2 antibiotics have been performed at regular intervals during the 24-h period immediately following the administration to the volunteers and after the 1st and the 15th days of repeated administration to the patients. The results show that gingival fluid contains concentrations of spiramycin and metronidazole higher than those needed to inhibit the growth of periodontopathic bacteria. Spiramycin was found at higher concentrations in GCF than in blood, although this feature was not found for metronidazole, which was administered simultaneously and showed similar concentrations in both fluid and serum. Such high concentrations persist for a long time, and suggest the potential of this compound in the treatment of severe cases of periodontitis.

Elastase release from gingival crevicular and peripheral neutrophils in periodontitis and health.

The activity of elastase increases significantly in the gingival sulcus during inflammation. The release of this enzyme from crevicular and peripheral polymorphonuclear leucocytes (PMNs) was studied in seven patients with gingivitis, six with adult periodontitis, seven with rapidly progressive periodontitis and in nine healthy subjects. Peripheral PMNs were isolated from blood and crevicular PMNs from gingival washings. After preincubation with cytochalasin B, the same numbers of crevicular and peripheral cells were incubated either in phosphate-buffered saline (spontaneous release) or in the same buffer containing formyl methionyl-leucyl-phenyl-alanine (stimulated release). Elastase activity was measured in the supernatant by a fluorimetric technique. The results confirm that compared to peripheral PMNs, crevicular cells show a higher spontaneous release of elastase and a lower stimulated release. The activity of elastase released either spontaneously or after stimulation of crevicular cells was significantly lower in the controls as compared with cells obtained from patients with gingivitis or periodontitis. Crevicular PMNs collected from patients with deeper pockets appear to release larger amounts of elastase than those from controls.

The recovery efficiency of various materials for sampling enzymes and polymorphonuclear leukocytes from gingival crevices.

The present investigation was designed to assess the effects of strips made of different materials on the recovery of enzymes in gingival crevicular fluid (GCF) (Experiment 1), and to examine a possible relationship between lysosomal enzyme activities and number of crevicular polymorphonuclear leukocytes (PMNs) (Experiment 2). In Experiment 1, GCF was collected with capillaries from 14 patients with periodontal disease, and applied on various test strips in microcentrifuged tubes or directly into tubes (controls). Strips were then shaken and centrifuged to elute GCF enzymes. The supernate was used for determinations of alkaline phosphatase (ALP), beta-glucuronidase (BG) and aspartate aminotransferase (AST) activities. The % recoveries were calculated as relative percentages to controls. When using saline as eluent, 90% or more ALP, BG and AST were found to be recovered from strips of durapore and papers. More than 35% of ALP and BG was found to remain on paper points. However, the % recoveries from paper points were improved using eluent with 0.1% (w/v) cetyl pyridinium chloride. In Experiment 2, 54 GCF samples were collected from 3 periodontitis patients by using durapore strips, in order to measure both PMN numbers and BG activities in the same samples. The 2 parameters showed strong and positive correlation with 0.847 (p < 0.001) of the Pearson's correlation coefficient. These findings suggest that durapore is a useful material not only for counting PMNs but also for measuring activities of GCF enzymes. Elevation of BG activities in GCF can be due to increasing numbers of PMNs.

Osteocalcin, prostaglandin E2 and alkaline phosphatase in gingival crevicular fluid: their relations to periodontal status.

Prostaglandin E2 (PGE2) and alkaline phosphatase (ALP) have often been measured in gingival crevicular fluid (GCF) as possible indicators of gingival inflammation and bone metabolism. Osteocalcin (OC), a major component of bone matrix, is mainly produced by osteoblasts, and could also be considered as a marker of bone turnover. The aims of this preliminary study were to examine if OC was present in GCF and to assess the relationships of OC, PGE2 and ALP in GCF to periodontal conditions. GCF samples were collected with durapore strips from 34 healthy, 72 gingivitis and 118 periodontitis sites in 17 subjects. ELISA techniques were used for the determinations of OC and PGE2. ALP was measured spectrophotometrically by using p-nitrophenyl phosphate as substrate. Total amounts and concentrations of PGE2 and ALP were significantly higher in periodontitis as compared to healthy and gingivitis sites, and were significantly and positively correlated with probing depth (PD) and gingival index (GI). OC was present in GCF from both healthy and diseased sites with mean concentrations more than ten times greater than normal serum levels. Total OC amounts from strips soaked with GCF from periodontitis sites were significantly higher than those found in healthy and gingivitis sites. When the data were expressed as concentrations, OC showed significantly positive correlations with GI, but not with PD. However, total amounts of OC significantly correlated with both clinical parameters. OC, PGE2 and ALP were found to have significantly positive correlations with each other, both when expressed as total amounts and concentrations. These data suggest that a significant amount of OC present in GCF is produced locally by periodontal tissues.(ABSTRACT TRUNCATED AT 250 WORDS)

[Gingival hyperplasia of drug origin. A review of the literature]. / Les hyperplasies gingivales d'origine médicamenteuse. Une revue de la littérature.

The frequency of gingival overgrowth is increasing, due to the more and more frequent use of cyclosporin and calcium antagonists. Such lesions are the same as those known to occur in patients treated with phenytoin. They appear after a few months of treatment and are favoured by inflammation. Marginal gingiva starts to grow in excess, especially in the anterior region, and might end up covering the teeth entirely, with major occlusal problems. The hyperplasia occurs in about 50% of the phenytoin treated patients and in about 20% of those treated with cyclosporin or calcium antagonists. A genetic predisposition has been thought to be present in such susceptible people. However, no precise pathogenic pathway, possibly common for the three types of drugs, has been clearly proposed. As for therapy, the most severe lesions are treated by surgery but, in general, the dentist can be very helpful by simply stressing gingival hygiene.

A rapid and simple method for counting crevicular polymorphonuclear leucocytes.

The techniques presently available for counting crevicular polymorphonuclear leucocytes (CPMNs) require special equipment and are only semi-quantitative. The present new and simple method is based on the property that Durapore strips have to harvest a maximum number of crevicular cells and to release them when shaken in a suitable buffer. Durapore strips were inserted into periodontal crevices or pockets, shaken in PBS and the liberated PMNs counted in a Neubauer chamber. The validity of the method was ascertained in 5 experiments. First, it was shown that 90% of the harvested cells were released from Durapore strips after being shaken in buffer. Second, decreasing numbers of CPMNs were found when performing sequential samplings. Third, when counting CPMNs before and after therapy, a significant decrease in the number of cells was found. Fourth, we could confirm that sites with bleeding on probing did contain a much higher number of CPMNs as compared to non-bleeding sites. Finally, our counting technique confirmed that deeper pockets contain a significantly higher number of CPMNs.

[The passage of antibiotics into human ginigival fluid. A review of the literature]. / Passage d'antibiotiques dans le fluide gingival humain. Une revue de la littérature.

Many antibiotics are utilized in dentistry, particularly in periodontology, to treat aggressive forms of periodontitis. In order to be efficient, both on a clinical and on a microbiological point of view, these substances must be present in sufficient concentrations in the gingival sulcus. The aim of the present literature review was to summarize studies concerning the passage of the various families of antibiotics into the gingival fluid, together with their spectrum and clinical efficacy. Whereas certain antibiotics, such as natural or semisynthetic tetracyclines or spiramycin, are able to accumulate in gingival fluid comparatively to serum, others, like metronidazole, tinidazole and rifampicin, have been found in similar concentrations in the fluid as in serum. Some other antibiotics, such as erythromycin and ampicillin, are, on the contrary, less concentrated in crevicular fluid as compared to blood. One has to remember that any systemic antibiotic treatment will be useful, clinically and microbiologically speaking, only when a mechanical therapy is also applied.