Detection of oxidized low-density lipoproteins in gingival crevicular fluid from dental patients.

BACKGROUND AND OBJECTIVE: Oxidative modification of low-density lipoprotein (LDL) occurs in various diseased tissues and sites of local inflammation. For example, an increased plasma oxidized low-density lipoprotein (OxLDL) level is a well-known risk marker for cardiovascular diseases. Gingival crevicular fluid, the exudate from gingival tissues into the sulci, can be easily collected in a non-invasive manner. However, the possible presence of OxLDL in gingival crevicular fluid has not been studied. In this study, we established a procedure to measure OxLDL in human gingival crevicular fluid. MATERIAL AND METHODS: Human gingival crevicular fluid was sampled with paper points or paper strips. The gingival crevicular fluid samples from healthy gingival sulci (pocket depth < 4 mm, n = 14) were subjected to western blot and/or sandwich ELISA. The amounts of OxLDL and LDL were measured by sandwich ELISA using an anti-oxidized phosphatidylcholine monoclonal antibody and two anti-apolipoprotein B antibodies. Venous blood samples were analyzed biochemically. RESULTS: We tested two methods of gingival crevicular fluid collection, namely paper points and paper strips. Gingival crevicular fluid could be collected very safely with paper points and they showed good recovery of LDL and OxLDL throughout the analysis. Apolipoprotein B, the major protein component in LDL, was detected in gingival crevicular fluid by western blot, and OxLDL was found to be present in gingival crevicular fluid by ELISA. The OxLDL/LDL ratio in gingival crevicular fluid was 17.0 times higher than that in plasma. CONCLUSION: This is the first report to show the presence of apolipoprotein B and apolipoprotein B- oxidized phosphatidylcholine complex, which correspond to LDL and OxLDL, respectively, in gingival crevicular fluid.

Relationship between the gingival sulcus depth and interleukin-1 isoform concentrations within the adjacent gingival tissue.

BACKGROUND AND OBJECTIVE: While there is substantial information concerning the concentrations of interleukin-1 isoforms within gingival crevicular fluid, there is little information concerning their concentrations within either normal or diseased gingival tissues. Therefore, the aim of this study was to evaluate the relationship between the concentrations of gingival interleukin-1 isoforms and the adjacent sulcular depth. MATERIAL AND METHODS: Interdental gingival papillae were excised and grouped based on adjacent pocket depth and the presence of bleeding on probing. Gingiva adjacent to a sulcus of < or = 3 mm without bleeding on probing were classified as 'normal'; gingiva adjacent to a 3-mm sulcus with bleeding on probing were classified as 'diseased-slight'; gingiva adjacent to a 4-6-mm sulcus featuring bleeding on probing were classified as 'diseased-moderate'; and gingiva adjacent to a sulcus of > 6 mm featuring bleeding on probing were classified as 'diseased-severe'. Tissues were solublized and the concentrations of interleukin-1beta, interleukin-1alpha, interleukin-1 receptor antagonist and interleukin-6 were assessed by enzyme-linked immunosorbent assay. Data were compared by factorial analysis of variance, the post-hoc Tukey test and the Pearson's correlation test. RESULTS: Gingival concentrations of interleukin-6, interleukin-1 receptor antagonist, interleukin-1alpha- and interleukin-1beta were significantly greater at diseased-severe sites than at normal, diseased-slight, or diseased-moderate sites (p < 0.05); the gingival concentrations of interleukin-1 receptor antagonist and interleukin-1alpha were significantly greater at diseased-severe than at diseased-moderate sites (p < 0.05). Interleukin-1 receptor antagonist concentrations were significantly correlated with both interleukin-1alpha and interleukin-1beta concentrations. The ratios of concentrations of the interleukin-1 isoforms were different at the various stages of inflammation. CONCLUSION: Our data indicated a progressive increase in gingival concentrations of interleukin-1 isoforms with increased adjacent sulcular depth. However, within 'diseased' tissues, the proportional concentrations of interleukin-1alpha and -beta to interleukin-1 receptor antagonist were lowest within diseased-severe tissues.

Gingival crevicular fluid and serum leptin: their relationship to periodontal health and disease.

BACKGROUND & AIMS: Leptin is a pleiotrophic hormone produced by adipose tissue and it plays an important role in protection of the host from inflammation and infection. The purpose of this study is to determine the presence of leptin in gingival crevicular fluid (GCF) and serum samples and to find out their association, if any. METHODS: Forty two subjects were selected based on their body mass index and were divided into three groups of 14 each; healthy (Group I), chronic gingivitis (Group II) and chronic periodontitis (Group III). GCF samples (by microcapillary pipettes) and serum samples (by venipuncture) were collected to estimate the levels of leptin using enzyme linked immunosorbent assay kit. RESULTS: The highest mean leptin concentration in GCF was obtained for Group I (2658 pg/ml) and the least for Group III (1312 pg/ml). In contrast, the lowest serum leptin concentration was obtained for the Group I (8783 pg/ml), and the highest for Group III (12082 pg/ml). This suggests a negative correlation of GCF leptin concentration and a positive correlation of serum leptin concentration as the clinical attachment level progresses (p<0.05). CONCLUSION: These results suggest that greater the periodontal destruction, lesser is the GCF leptin concentration and greater the serum leptin concentration.

Cytokines, matrix metalloproteinases and tissue inhibitor of metalloproteinases-1 in gingival crevicular fluid from patients with Papillon-Lefèvre syndrome.

The aim of the present study was to compare concentrations of cytokines, matrix metalloproteinases (MMPs) and a metalloproteinase inhibitor (TIMP-1) in gingival crevicular fluids (GCF) from sites with gingival inflammation in 28 young patients with Papillon-Lefèvre syndrome (PLS), and in age- and gender-matched controls. Each group consisted of 17 females and 11 males with a mean age of 11.0 years (range 4-22 years). In both groups, anterior upper sites with a clinical diagnosis of gingival inflammation and with pockets < or = 3 mm were selected for sampling of GCF, which was carried out with filter disks inserted into the gingival crevice until saturated. The concentrations of cytokines (IL-1alpha, IL-1beta, TNF-alpha, and IL-8), matrix metalloproteinases (MMP-1, MMP-3, MMP-8, and MMP-9), and their tissue inhibitor (TIMP-1) were analysed using commercial ELISA kits. Significantly higher levels of IL-1beta (P < 0.001) and MMP-8 (P < 0.05) were disclosed among the PLS patients compared with their controls, while the opposite was found for IL-8 (P < 0.05) and MMP-1 (P < 0.001). The individual variations were considerable in both groups. When comparing the expression of cytokines, MMPs, and TIMP-1 in PLS patients with clinically active and non-active periodontitis, the non-active PLS patients showed significantly higher values of IL-1beta than the patients with active periodontal disease (ANOVA, P < 0.01). In conclusion, this study was unable to demonstrate a clear-cut pathognomonic expression of cytokines or MMPs in patients with PLS, but further studies on cytokine and MMP output are warranted.

Enhanced levels of prostaglandin E2, leukotriene B4, and matrix metalloproteinase-9 in gingival crevicular fluid from patients with Down syndrome.

The aim was to study the levels of prostaglandin E2 (PGE2), leukotriene B4 (LTB4), and matrix metalloproteinase-9 (MMP-9) in gingival crevicular fluid (GCF) from Down syndrome patients exhibiting gingival inflammation. The levels of PGE2, LTB4, and MMP-9 were determined in GCF from 18 Down syndrome patients and from 14 controls matched with respect to age and degree of gingival inflammation. Probing depth (PD) and gingival inflammation, assessed by bleeding on probing (BOP), were determined around all teeth. In each patient, GCF was collected from 6 sites (16m, 26m, 36m, 46m, 41m, 11d) using periopaper, and the volume was determined using Peritron 8000. The PGE2 and LTB4 levels were determined using RIA kits and MMP-9 level using ELISA kits. The degree of gingival inflammation, expressed as mean value of BOP (%) as well as the volume of GCF, was similar between Down syndrome patients and controls. The mean levels of PGE2, LTB4, and MMP-9 were significantly (P<0.05) higher in GCF from Down syndrome patients compared to controls. When comparing the two groups, the correlation coefficients for LTB4 to BOP and PD, respectively, as well as for MMP-9 to BOP significantly differed between Down syndrome and controls (P<0.05). The study supports the concept of an altered host response in periodontal tissue in Down syndrome subjects.

The effects of periodontal therapy on intracrevicular prostaglandin E2 concentrations and clinical parameters in pregnancy.

BACKGROUND: The increase in circulating levels of progesterone during pregnancy stimulates production of prostaglandins, especially prostaglandin E2, possibly resulting in pregnancy gingivitis. The purpose of this study is to evaluate the influence of prostaglandin E2 concentrations on gingival tissues in pregnancy and to assess its relationship to clinical parameters. METHODS: This study evaluates the effects of periodontal treatment on clinical indices including plaque index, gingival index, probing depth, and gingival crevicular fluid prostaglandin E2 levels of 22 pregnant women in their first, second, and third trimesters. Initial periodontal therapy consisting of scaling, root planing, and oral hygiene instruction was performed at the beginning of the first trimester and repeated each trimester. Prostaglandin E2 concentrations in gingival crevicular fluid were determined using a commercially available enzyme immunoassay kit. The statistical tests used were paired sample test and correlation analysis. RESULTS: The results of the study show that periodontal therapy has resulted in an improvement in clinical parameters (P<0.05). There is also a statistically significant decrease in levels of prostaglandin E2 at the second and third trimesters following periodontal therapy (P <0.001). The correlation between prostaglandin E2 concentrations and clinical parameters is found to be non-significant (P >0.05). CONCLUSIONS: Our data indicate that levels of prostaglandin E2 in gingival crevicular fluid may be used as a marker of gingival inflammation in order to determine the effects of periodontal therapy in pregnancy. Periodontal therapy that is performed throughout the entire pregnancy period may help prevent the threat of pregnancy gingivitis.

Effect of mechanical toothbrush stimulation on gingival microcirculatory functions in inflamed gingiva of dogs.

The effect of mechanical toothbrush stimulation on gingival microcirculatory functions was examined with and without removal of supragingival plaque in inflamed gingiva of 6 dogs. After removal of the ligatures, 4 treatment modalities: mechanical stimulation by vibration (MS), removal of supragingival plaque (PR), combination (MS+PR) and no treatment (NT), were administered to each quadrant for 2 weeks. Both quadrants with plaque removal showed a marked decrease in the gingival index score, while slight and moderate decreases were observed in NT and MS quadrants, respectively. Changes in gingival crevicular fluid flow, pocket oxygen tension and hemoglobin oxygen saturation in the gingiva were significant in the MS, PR and MS+PR quadrants. Significant treatment-by-time effects were found for all of the parameters of microcirculatory function between NT and MS quadrants, and gingival crevicular fluid flow between PR and MS+PR quadrants, respectively. These findings suggest that mechanical stimulation with a toothbrush may offer an additional benefit to gingival microcirculatory functions in inflamed gingiva.

Gingival crevicular fluid from patients with periodontitis contains bone resorbing activity.

Inflammatory processes occurring in the vicinity of bone tissue often result in stimulation of osteoclast activity and loss of skeletal mass. The aim of the current study was to determine if inflammatory exudates collected from gingival pockets in patients with periodontitis contain factors capable of stimulating resorptive activity. The degree of bone mineral mobilization and bone matrix degradation was assessed by analysis of the release of 45Ca and 3H from bones prelabelled with 45CaCl2 and [3H]proline, respectively. Gingival crevicular washings from six patients with signs of periodontitis stimulated 45Ca or 3H release from the calvarial bones. The stimulatory effect of the gingival crevicular washings on 45Ca release was concentration- and time-dependent, and reduced by calcitonin, a specific osteoclast inhibitor. These data demonstrate that crevicular fluid contains factor(s) which can stimulate osteoclastic degradation of bone in vitro. The bone resorbing activity was partially retained after extensive dialysis. Analysis of the concentrations of prostaglandin E2, interleukin-1alpha and interleukin-1beta in the crevicular fluids, and comparisons of these agents as stimulators of 45Ca release in the mouse calvarial assay, suggest that prostaglandin E2 is not the sole factor responsible for the bone resorbing activity of the exudates. The data indicate that other factors, such as IL-1, may play key roles in the stimulation of osteoclastic activity by gingival crevicular washings.

Substance P and neurokinin A in gingival crevicular fluid in periodontal health and disease.

The aims of the present study were to investigate whether the tachykinins substance P and neurokinin A were present in gingival crevicular fluid in both periodontal health and disease and to study the relationship with periodontal inflammation. Gingival crevicular fluid (GCF) was collected from a healthy, a gingivitis and a periodontitis site in 20 subjects with periodontitis and from a healthy site in 20 subjects without periodontitis. The volume of GCF was measured and each sample subsequently analysed for substance P and neurokinin A by radioimmunoassay. There were significantly increased levels of substance P-like immunoreactivity (SP-LI) and neurokinin A-like immunoreactivity (NKA-LI) in gingivitis and periodontitis sites compared with healthy sites. Both tachykinins were significantly elevated in periodontitis affected subjects, with significantly more tachykinin-like immunoreactivity at healthy sites in periodontitis affected compared with periodontally-healthy subjects. Despite the considerable individual variation in the levels of SP-LI and NKA-LI, both tachykinins were present at levels at which they could have biological activity. It is concluded that substance P and neurokinin A may have a rôle in the pathogenesis of periodontal disease and that further investigations could prove useful in clarifying the mechanisms through which neuropeptides could modulate periodontal health and disease.

A cross-sectional study on osteocalcin levels in gingival crevicular fluid from periodontal patients.

The purpose of the present study was to determine the levels of osteocalcin, a bone specific matrix protein, in gingival crevicular fluid (GCF) from periodontal disease patients and to investigate the relationship between GCF osteocalcin levels and clinical parameters. Nineteen initial visit patients, 5 patients with gingivitis and 14 patients with adult periodontitis, participated in this study. The clinical parameters including probing depth, attachment level, gingival index, and tooth mobility were recorded following careful sampling of GCF with a filter paper strip harvested for 3 minutes. Osteocalcin adsorbed on a strip was extracted in a plastic tube containing 150 microliters of 10 mM sodium phosphate buffer (pH 6.5). GCF osteocalcin was determined by a newly-developed, high sensitive enzyme immunoassay which could recognize the N-terminal 20 residue peptide. In gingivitis patients, no significant amounts of osteocalcin were detected. In periodontitis patients, on the other hand, osteocalcin levels were detected, ranging between 0 and 540 pg/tube and positively correlated with these clinical parameters (P < 0.01). Moreover, in several sites in GI = 3 group, extremely higher levels of GCF osteocalcin were detected. These results strongly suggest that in addition to the presence of GCF osteocalcin the levels of osteocalcin may reflect the degree of the periodontal inflammation at the sampled sites.

The ability of gingival crevicular fluid acute phase proteins to distinguish healthy, gingivitis and periodontitis sites.

3 acute phase proteins, from the local gingival inflammatory response, were examined for their ability to distinguish healthy, gingivitis and periodontitis sites. Indirect competitive immunoassays were developed for the quantification of alpha 2-macroglobulin (alpha 2-M) and transferrin (TF), and for alpha 1-antitrypsin (alpha 1-AT), a double antibody sandwich assay was produced. Healthy (25), gingivitis (31) and periodontitis (28) sites were sampled with filter paper strips (2 x 13 mm) and the volume assessed with the Periotron 6000. The samples were eluted in phosphate-buffered saline and analyzed for alpha 2-M, alpha 1-AT and TF. The results were expressed as absolute amounts per sample (ng/30 s) and on a concentration basis (ng/microliter of GCF). Higher GCF absolute amounts of alpha 2-M, alpha 1-AT and TF were consistently obtained from diseased (gingivitis and periodontitis) sites than healthy sites (p less than 0.005). Absolute amounts of GCF alpha 2-M, alpha 1-AT and TF were increased in periodontitis sites over gingivitis sites, although these differences were not statistically significant (p greater than 0.1). When the results were expressed on a concentration basis, alpha 2-M levels from diseased sites were significantly higher than healthy sites (p less than 0.01). In addition, GCF TF concentration was increased in periodontitis compared to healthy sites (p = 0.03).

Characterization of volatile sulphur compounds production at individual gingival crevicular sites in humans.

The present investigation describes a method for collection and analysis of volatile sulphur compounds (VSC) from gingival crevicular sites in humans. Tenax-GC trapping devices were used to adsorb and concentrate VSC from crevicular air at -55 degrees C, which were then thermally desorbed at 120 degrees C. Gas chromatographic (GC) analyses were performed using a Tracor 550 GC equipped with a flame-photometric detector and a Teflon column packed with 5% polyphenyl ether and 0.05% phosphoric acid on 30-40 mesh Teflon. Sulfides identified from crevicular sites include hydrogen sulfide [H2S], methyl mercaptan [CH3SH], dimethyl sulfide [(CH3)2S], and dimethyl disulfide [(CH3S)2]. Of the seventeen patients studied, crevicular sites that were either deep (P.D. > or = 4 mm) or inflamed (BoP = 1) exhibited significantly larger CH3SH to H2S ratios than corresponding crevicular shallow (P.D. < or = 3 mm) sites (p < .10) or noninflamed (BoP = 0) sites (p < .05). Similarly, total sulphur in deep and inflamed sites was significantly higher than in corresponding shallow (p < .01) and noninflamed (p < .05) sites. This is the first known in vivo study to quantitate VSC directly from individual gingival crevices.

Cathepsin B- and L-like activities at local gingival sites of chronic periodontitis patients.

The cysteine proteinases cathepsins B and L have the potential to degrade connective tissue in chronic periodontitis and this may progress episodically at individual tooth sites. The activities of cathepsin B- and L-like proteinases in homogenised gingival tissue from control and periodontitis patients were measured biochemically using the selective peptide substrate Z-Phe-Arg-AFC and the selective cathepsin L inhibitor Z-Phe-Phe-CHN2. Each tooth site was divided, where appropriate, into gingival tissue and granulomata. These were assayed separately and the measurements related to the DNA and protein contents of the tissues. Enzyme activity in healthy control tissue was significantly lower than in diseased tissue. Enzyme activity in gingival tissue and total tissue from periodontitis patients decreased with increasing pocket depth, clinical attachment level, gingival index and bleeding index whilst cathepsin B activity in granulomata increased with increasing pocket depth and clinical attachment level but not with increasing gingival index or gingival bleeding index. Mean enzyme activity in gingival tissue was 1.6-2.8 times greater than in granulomata. Mean patient enzyme activity in diseased patients did not correlate positively with their mean pocket depth, clinical attachment level, gingival index or gingival bleeding index. These results are best explained by the probable cellular origins of the enzymes and the likely influence of their serum and tissue inhibitors during the disease process.

Changes in hemoglobin concentration and oxygen saturation in human gingiva with decreasing inflammation.

The purpose of this study was to determine if functional changes in the human gingival vasculature were reversible following the resolution of gingival inflammation. Ten patients with 40 inflamed gingival sites were evaluated before and 2 weeks after the completion of treatment. We determined the hemoglobin concentration and the oxygen saturation of hemoglobin at each site by tissue reflectance spectrophotometry. With the use of treatment including motivation, oral hygiene instruction, and scaling, clinical parameters such as the gingival and plaque indices, the Periotron score, and the probing depth were altered toward a healthier state. With the resolution of gingival inflammation, the increased hemoglobin concentration and decreased oxygen saturation in the inflamed gingiva were restored to normal levels. These findings suggest that reversible changes in the local hemoglobin concentration and oxygen saturation are associated with decreasing gingival inflammation in human subjects.

Gingival crevicular fluid fibronectin degradation in periodontal health and disease.

The molecular forms of fibronectin (FN) in gingival crevicular fluid of five subjects with at least two sites exhibiting clinical signs of inflammation and pockets of at least 4 mm (test group) and five subjects with clinically healthy periodontium (control group) were investigated. Samples were collected with standard filter paper strips. In the test group samples from both diseased and healthy sites were collected. After collection the test group received one episode of periodontal treatment (scaling and root planning). The sampling and clinical recording were repeated for the diseased sites after about 2 wk. The crevicular fluid FN was analyzed using sodium dodecyl sulphate gel electrophoresis followed by western blotting with polyclonal antibodies against FN. Both intact FN and FN fragments were found in all samples. A larger proportion of FN was in degraded form in the diseased sites than in the healthy or the treated sites. FN was also degraded into smaller peptide fragments in the diseased than in the treated sites. These results suggest that crevicular fluid FN is partially degraded both in periodontal health and disease and that the degree of degradation of FN increases with periodontal inflammation and decreases with periodontal treatment.

[The concentration and duration of the action of metronidazole in the gingival pocket following local application]. / Untersuchungen zur Konzentration und Wirkungsdauer von Metronidazol in der Zahnfleischtasche nach lokaler Applikation.

Periodontitis is a locally limited disease caused by bacteria. The local application presents itself useful for the indicated medicamentous therapy of periodontitis by means of metronidazole. In preliminary tests the metronidazole liberation from hollow fibres, in gel and from polyvinyl alcohol platelets has been tested by ultraviolet absorption measurement. The hollow fibres have been evaluated no more because of too small agent absorption in the clinical test. In polyvinyl alcohol metronidazole acts definitely over 3 days. However, the prolonged action in the gel form is not definitely (45 min to 24 h). A single one or repeated twice metronidazole/polyvinyl alcohol platelet application results in a sufficient long and high metronidazole concentration in the gingival pocket.

Local immunoglobulin synthesis in juvenile periodontitis: initial findings.

An electro-immunoassay technique was used to determine simultaneously immunoglobulin G (IgG) and albumin concentrations in serum and extracts of gingival tissue comprising the pocket wall. Assays of samples obtained from seven patients with juvenile periodontitis (mean age, 18 years) indicated that local synthesis accounted for 72% of the IgG found in the gingiva.