Levels of leukotriene B4 and platelet activating factor in gingival crevicular fluid in renal transplant patients receiving cyclosporine A

BACKGROUND: Cyclosporine A (CsA) is a potent immunosuppressant effectively used to prevent organ transplant rejection and also to treat several systemic diseases. CsA-induced gingival overgrowth (CsA GO) is the most widely seen side effect of this drug; its pathogenesis is not completely understood. The aim of the present study was to identify the role of leukotriene B4 (LTB4) and platelet activating factor (PAF) in the pathogenesis of CsA GO. METHODS: LTB4 and PAF levels were detected in gingival crevicular fluid (GCF) samples from renal transplant patients receiving CsA therapy and exhibiting CsA GO, from patients with gingivitis and from periodontally healthy subjects. Plaque index, papilla bleeding index, and hyperplastic index were recorded at each study site. GCF samples and clinical data were obtained from: 2 sites exhibiting CsA GO (CsA GO+) and 2 sites not exhibiting CsA GO (CsA GO-) in each CsA-treated patient; 2 diseased sites in each patient with gingivitis; and 2 healthy sites in each subject with clinically healthy periodontium. LTB4 was extracted from the samples by solid-phase method using C18 cartridge and purified by high-performance liquid chromatographic (HPLC) method and analyzed by radioimmunoassay (RIA). PAF was extracted from GCF samples passing through amberlit resin columns, purified by HPLC, and analyzed by RIA. RESULTS: Total amounts of LTB4 and PAF in GCF were higher in CsA GO+ sites compared to the healthy sites from healthy controls. However, the amount of LTB4 and PAF elevation in CsA GO+ sites was not significantly higher than those in diseased sites. Clinical degrees of gingival inflammation were also similar between CsA GO+ and diseased sites. LTB4 and PAF total amounts in GCF were higher in CsA GO+ sites compared to CsA GO- sites in the same subjects, but this difference just failed to reach significance. Similar findings were obtained with concentration data. CONCLUSIONS: The results of this study indicate that CsA therapy does not have a significant effect on GCF LTB4 and PAF levels and that gingival inflammation seems to be the main reason for their elevation. In CsA-treated patients, alterations in LTB4 and PAF levels might play a role in CsA GO through some asyet unknown mechanism. To our knowledge, this is the first report describing the levels of lipid mediators in GCF of CsA-treated patients. We assume that further studies will contribute to the understanding of the pathogenesis of CsA-induced gingival overgrowth.     

Evaluation of transforming growth factor-beta 1 level in crevicular fluid of cyclosporin A-treated patients

BACKGROUND: The aim of the present study was to investigate the level of transforming growth factor-beta 1 (TGF-beta 1) in gingival crevicular fluid (GCF) samples of cyclosporin A (CsA)-treated patients and to compare the results with control groups. METHODS: Fourteen renal transplant patients exhibiting severe CsA-induced gingival overgrowth, 10 patients with chronic gingivitis, and 10 subjects with clinically healthy periodontium were included in the study. In CsA-treated patients, GCF samples were harvested from sites exhibiting gingival overgrowth (CsA GO+) and sites not exhibiting gingival overgrowth (CsA GO-). The TGF-beta 1 levels in a total of 96 GCF samples from the 34 participants were analyzed by enzyme-linked immunosorbent assay. The results were expressed in terms of total amount (pg/2 sites) and concentration (ng/ml). RESULTS: TGF-beta 1 total amounts in CsA GO+ and CsA GO- sites were similar and significantly higher than that of healthy sites (P < 0.02 and P < 0.01, respectively). The total amount of TGF-beta 1 was also higher in gingivitis sites compared to the healthy sites, but the difference was not statistically significant (P > 0.05). CsA GO+ and CsA GO- sites exhibited higher total amount and concentration of TGF-beta 1 than that of gingivitis sites, but the differences were insignificant (P > 0.05). CONCLUSIONS: The results of the present study support the theory that CsA increases the synthesis of TGF-beta 1 in GCF. However, since the difference between CsA GO+ and CsA GO- sites was not statistically significant, it seems unlikely that GCF TGF-beta 1 level is the sole factor responsible for the CsA-induced gingival overgrowth. Complex interactions between various mediators of inflammation and tissue modeling are possibly involved in the pathogenic mechanisms of this side effect.     

Evaluation of p53, bcl-2, and interleukin-15 levels in gingival crevicular fluid of cyclosporin A-treated patients

BACKGROUND: Apoptosis plays an important role in the maintenance of tissue homeostasis. Considering that apoptosis mediators may play a role in the pathogenesis of drug-induced gingival overgrowth, this study was conducted to evaluate p53, bcl-2, and interleukin-15 (IL-15) levels in gingival crevicular fluid (GCF) of cyclosporin A (CsA)-treated patients. METHODS: Twenty renal transplant patients exhibiting CsA-induced gingival overgrowth and 15 systemically healthy gingivitis patients were included in the study; 15 systemically and periodontally healthy volunteer subjects served as the healthy control group. GCF samples were obtained from one interdental site with gingival overgrowth (GO+) and one site without (GO-) from each CsA-treated patient; hyperplasia index, probing depth, papilla bleeding index, and plaque presence were recorded. One site from each gingivitis patient and healthy control was selected, GCF samples were obtained, and the same clinical parameters were recorded. GCF p53, bcl-2, and IL-15 levels were analyzed by enzyme-linked immunosorbent assay. The results were tested statistically. RESULTS: p53 and bcl-2 levels were below the minimum detectable level in all GCF samples analyzed. CsA GO+ and CsA GO- sites, as well as gingivitis sites, exhibited significantly higher GCF levels of IL-15 compared to healthy controls (P<0.05). The difference between CsA GO+ sites and gingivitis sites was not statistically significant, although the total amount of IL-15 in CsA GO+ sites was lower than gingivitis sites (P>0.05). The total amount of IL-15 in CsA GO- sites was significantly lower than gingivitis sites (P<0.05). No significant correlation was found between the clinical parameters and GCF IL-15 levels (P>0.05). CONCLUSIONS: The pathogenesis of CsA-induced gingival overgrowth is multifactorial. The findings of the present study indicate that IL-15 may play a role in the pathogenesis of CsA-induced gingival overgrowth due to its interactions with CsA and its role in apoptosis and inflammation.     

Plasminogen activators and plasminogen activator inhibitors in gingival crevicular fluid of cyclosporin A-treated patients

BACKGROUND: The plasminogen activator (PA) system plays many roles in the inflammatory process and tissue remodelling and repair and is considered to play a significant role in periodontal tissue destruction and healing. The aim of this study was to evaluate the role of the PA system in cyclosporin A (CsA)-induced gingival overgrowth in renal transplant patients. METHODS: Eighteen renal transplant patients exhibiting moderate to severe CsA-induced gingival overgrowth, 10 other renal transplant patients receiving CsA therapy but showing no sign of CsA-induced gingival overgrowth (CsA-H), 16 chronic gingivitis patients (CG) and 16 systemically and periodontally healthy control subjects (H) were included in the study. Gingival crevicular fluid (GCF) samples were obtained from four randomly selected sites in each subject with the exception of the CsA-induced gingival overgrowth group, where four GCF samples were harvested from sites with severe overgrowth (CsA GO+) and from four sites without any gingival overgrowth (CsA GO-). The GCF levels of albumin, tissue-type plasminogen activator (t-PA), urokinase-type plasminogen activator (u-PA), plasminogen activator inhibitor 1 (PAI-1) and plasminogen activator inhibitor 2 (PAI-2) were analysed by enzyme-linked immunosorbent assay. The results were tested for statistical differences. RESULTS: In CsA GO+ sites t-PA levels were significantly elevated in comparison with gingivitis and healthy sites, while PAI-2 levels in these sites showed statistically significant differences only with CsA-H and gingivitis sites (p<0.05). The levels of t-PA and PAI-2 were significantly higher in CsA GO- sites compared with those of CsA-H, gingivitis and healthy sites (p<0.05). The levels of u-PA and PAI-1 failed to show significant differences between the study groups. CONCLUSIONS: The findings of the present study indicate alterations in GCF t-PA and PAI-2 levels in CsA-induced gingival overgrowth and might suggest involvement of the plasminogen activating system in the pathogenesis of this side-effect of CsA therapy. However, to what extent these molecules contribute to the pathogenesis of CsA-induced gingival overgrowth remains to be determined.     

Gingival crevicular fluid and serum matrix metalloproteinase-8 and tissue inhibitor of matrix metalloproteinase-1 levels in renal transplant patients undergoing different immunosuppressive therapy

Aim: We investigated gingival crevicular fluid (GCF) and serum matrix metalloproteinase‐8 (MMP‐8) and tissue inhibitor of matrix metalloproteinase‐1 (TIMP‐1) levels from renal transplant patients receiving cyclosporine‐A (CsA) and having gingival overgrowth (GO), from patients receiving CsA therapy and having no GO and patients receiving tacrolimus therapy. Material and Methods: GCF samples were collected from sites with GO (GO+) and without GO (GO?) in CsA patients having GO; and GO? sites in CsA patients having no GO; sites from tacrolimus, gingivitis and healthy subjects. GCF and serum MMP‐8 and TIMP‐1 levels were determined by a time‐resolved immunofluorometric assay (IFMA) and enzyme‐linked immunosorbent assay. Results: GO+ sites in CsA patients having GO had elevated GCF MMP‐8 levels compared with those of CsA patients having no GO, tacrolimus and healthy subjects (p<0.005), but these levels were similar to those of gingivitis. The GCF MMP‐8 level was higher in GO+ compared with GO? sites in CsA patients having GO (p<0.05). GCF TIMP‐1 levels were similar between groups. Tacrolimus patients had lower GCF MMP‐8 levels than gingivitis (p<0.005), but levels similar to the healthy group. Conclusion: These results show that CsA and tacrolimus therapy has no significant effect on GCF MMP‐8 levels, and gingival inflammation seems to be the main reason for their elevations.     

GCF and serum myeloperoxidase and matrix metalloproteinase-13 levels in renal transplant patients

Aim

The rationale of this study was to address whether local or systemic changes reflect proteolytic (matrix metalloproteinase-13) or oxidative (myeloperoxidase) stress in renal transplant patients receiving cyclosporine-A (CsA) and having gingival overgrowth (GO), in patients receiving CsA therapy and having no GO and patients receiving tacrolimus therapy.

Material and methods

Gingival crevicular fluid (GCF) samples were collected from sites with (GO+) and without GO (GO−) in CsA patients having GO; GO− sites in CsA patients having no GO; sites from tacrolimus, gingivitis and healthy subjects. GCF and serum myeloperoxidase (MPO) and matrix metalloproteinase-13 (MMP-13) levels were determined by ELISA.

Results

GO+ sites in CsA patients having GO had elevated GCF MPO levels than those of CsA patients having no GO, tacrolimus and healthy subjects (p < 0.005), but comparable to those of gingivitis. GCF MPO levels were higher in GO+ compared to GO− sites in CsA patients having GO (p < 0.05). Patient groups had similar, but higher GCF MMP-13 levels than healthy group.

Conclusions

These results show that CsA and tacrolimus therapy have not a significant effect on GCF MPO and MMP-13 levels, and gingival inflammation seems to be the main reason for their elevations.     

Gingival crevicular fluid osteocalcin, N-terminal telopeptides, and calprotectin levels in cyclosporin A-induced gingival overgrowth

Background: The aim of this cross‐sectional study is to investigate gingival crevicular fluid (GCF) osteocalcin, cross‐linked N‐terminal telopeptide (NTx), and calprotectin levels in cyclosporin A (CsA)–induced gingival overgrowth (GO). Methods: Forty medicated patients with CsA including 20 with GO (CsA GO+), 10 without GO (CsA GO?), 10 with GO and chronic periodontitis (CsA CP) and 60 patients with CP alone, 20 patients with gingivitis, and 20 healthy patients were enrolled. Probing depth, clinical attachment level, plaque index, and papillary bleeding index were recorded. GCF calprotectin, osteocalcin, and NTx levels were analyzed by enzyme‐linked immunosorbent assay. Parametric tests were used for statistical analysis. Results: The CsA GO+ and CP groups had significantly lower GCF osteocalcin levels and osteocalcin/NTx ratio than the healthy group, whereas GCF osteocalcin levels and osteocalcin/NTx ratio in the gingivitis group were higher than the CsA GO+, CsA GO?, CsA CP, and CP groups (P <0.05). The CP group had elevated GCF calprotectin levels compared to the other study groups (P <0.05). The CsA GO+ and CsA GO? groups also had higher GCF calprotectin levels compared to the CsA CP, gingivitis, and healthy groups (P <0.05). Conclusions: Increased GCF calprotectin and decreased GCF osteocalcin levels in the CsA GO+ and CsA GO? groups might suggest that CsA plays a role on the levels of these markers. The similarity of GCF osteocalcin, NTx, and calprotectin levels in the CsA GO+ and CsA GO? groups might suggest that these molecules are not involved in the pathogenesis of GO.     

Gingival crevicular fluid transforming growth factor-beta1 in cyclosporine and tacrolimus treated renal transplant patients without gingival overgrowth

BACKGROUND: Gingival crevicular fluid (GCF) levels of transforming growth factor-beta(1) (TGF-beta(1)) have been previously investigated in relation to the pathogenesis of cyclosporine-A (CsA)-induced gingival overgrowth (GO) but no clinical data are available regarding the GCF levels of TGF-beta(1) in patients treated with tacrolimus (Tac). However, as gingival inflammation is pronounced at sites of GO and this consequently may lead to an elevation in TGF-beta(1) levels the present study aimed to evaluate gingival crevicular fluid (GCF) TGF-beta(1) levels in renal transplant patients using CsA or Tac without GO. METHODS: GCF TGF-beta(1) levels were investigated in 30 renal transplant patients without GO medicated with either CsA (n=15) or Tac (n=15). Sixteen gingivitis patients and 15 periodontally healthy subjects were selected as controls. Periodontal status was evaluated by measuring probing depth, plaque index and papilla bleeding index. The TGF-beta(1) levels were analysed by enzyme-linked immunosorbent assay. RESULTS: Both CsA and Tac groups had significantly elevated GCF TGF-beta(1) total amount compared to gingivitis and healthy groups (p<0.008). GCF TGF-beta(1) total amount of CsA and Tac groups was similar (p>0.008). Gingivitis and healthy groups had also similar GCF TGF-beta(1) total amount (p>0.008). CONCLUSIONS: Within the limits of the present data it is unlikely that TGF-beta(1) is an exclusive mediator of CsA- or Tac-induced GO. However, pathogenesis of GO is multifactorial and contribution of TGF-beta(1) to the interrelations between cytokines and growth factors with fibrogenic potential cannot be disregarded.     

Interleukin‐6 Family of Cytokines in Crevicular Fluid of Renal Transplant Recipients With and Without Cyclosporine A–Induced Gingival Overgrowth

Background: Interleukin (IL)‐6 family of cytokines, including IL‐6, oncostatin M (OSM), leukemia inhibitory factor (LIF), and IL‐11, have fibrogenic features. The current study determines gingival crevicular fluid (GCF) levels of fibrosis‐related IL‐6–type cytokines in cyclosporine A (CsA)–induced gingival overgrowth (GO). Methods: Eighty non‐smokers were included (40 CsA‐medicated renal transplant patients with GO [GO + ; n = 20] or without GO [GO?; n = 20], 20 individuals with gingivitis, and 20 healthy participants). Probing depth and plaque, papilla bleeding, and hyperplastic index scores were recorded. GCF samples were obtained from the mesio‐buccal aspects of two teeth. GCF IL‐6, IL‐1β, OSM, LIF, and IL‐11 levels were analyzed by enzyme‐linked immunosorbent assay. Results: The GO+ and GO? groups had higher IL‐6 total amounts than the healthy group (P <0.008). IL‐1β total amounts in the GO+ group were significantly higher than in both the healthy and GO? groups (P <0.008). OSM total amount was elevated in the GO+ and GO? groups compared with both the gingivitis and healthy groups (P <0.008). All groups had similar LIF and IL‐11 total amounts (P >0.008). Moderate positive correlations were detected among IL‐6, IL‐1β, OSM, and IL‐11 total amount in GCF and clinical parameters (P <0.05). Conclusions: IL‐6 and OSM increases in GCF as a result of CsA usage or an immunosuppressed state irrespective of the severity of inflammation and the presence of GO. The IL‐6 family of cytokines might not be directly involved in biologic mechanisms associated with CsA‐induced GO. Lack of an association between assessed IL‐6 cytokines and CsA‐induced GO might indicate distinct effects of these cytokines on fibrotic changes of different tissues.     

Evaluation of matrix metalloproteinase-1 and tissue inhibitor of metalloproteinase-1 levels in gingival fibroblasts of cyclosporin A-treated patients

BACKGROUND: Cyclosporin A (CsA) is a potent immunosuppressant used to prevent organ transplant rejection and to treat various autoimmune diseases. CsA-induced gingival overgrowth (CsA GO) is the most widely seen side effect of this drug; its pathogenesis is not completely understood. The aim of this study was to identify and compare matrix metalloproteinase-1 (MMP-1) and tissue inhibitor of metalloproteinase-1 (TIMP-1) levels in gingival fibroblast cultures of tissues derived from renal transplant patients receiving CsA and exhibiting gingival overgrowth and from periodontally healthy control subjects. METHODS: Gingival overgrowth samples were obtained from patients undergoing therapy with CsA, and control tissues were obtained from systemically healthy donors. Gingival fibroblasts were grown using explant cultures. Three different study groups were identified: 1) CsA GO fibroblast culture; 2) CsA-treated healthy gingival fibroblast culture (H+CsA); and 3) healthy gingival fibroblast culture (H). The levels of MMP-1 and TIMP-1 in these groups of gingival fibroblasts were analyzed by enzyme-linked immunoabsorbent assay (ELISA). RESULTS: The levels of TIMP-1 were significantly lower in CsA GO than H (P < 0.05). There was no statistically significant difference in the levels of MMP-1 between H and CsA GO (P = 0.505). The ratio of MMP-1 to TIMP-1 was significantly higher in CsA GO than H (P < 0.05). CONCLUSIONS: The results of this study indicate that CsA therapy does not have a significant effect on MMP-1 levels. However, low TIMP-1 levels can be an important factor in the pathogenesis of CsA GO, since the balance between MMP-1 and TIMP-1 levels was changed by CsA.     

Gingival crevicular fluid collagenase-2 (MMP-8) test stick for chair-side monitoring of periodontitis

BACKGROUND: A rapid chair-side test based on the immunological detection of elevated levels of collagenase-2 (matrix metalloproteinase-8, MMP-8) in gingival crevicular fluid (GCF) was developed to identify and monitor the course and treatment of adult periodontitis. METHODS: MMP-8 was determined in GCF from periodontitis (11 patients, 90 sites), gingivitis (10 patients, 58 sites) and healthy control (8 patients, 59 sites) sites (i) by a test stick incorporating monoclonal antibodies to two epitopes on MMP-8 and (ii) by measuring MMP-8 concentration by a quantitative immunofluorometric assay. Patients with adult periodontitis were treated by scaling and root planing (SRP) and received oral hygiene instructions. GCF MMP-8 testing and clinical measurements were done before and after SRP. RESULTS: MMP-8 GCF levels and chair-side test differentiated periodontitis from gingivitis and healthy control sites. MMP-8 GCF levels > 1 mg/l and positive chair-side test identified especially severe periodontitis sites. A positive and negative test stick result, the outcome of which was rapidly detectable in 5 mins, in GCF correlated well with MMP-8 immunofluorometric assay analysis from the collected GCF samples and the severity of periodontitis. Scaling and root planing reduced the MMP-8 levels in severe periodontitis sites with positive MMP-8 test and gingival probing pocket depth (PD) > 5 mm before treatment. The test stick result and the quantitative assay were discrepant in only 18 of the 207 sites tested, thus agreement was very good (kappa = 0.81). With a threshold of 1 mg/l MMP-8 activity the chair-side test provided a sensitivity of 0.83 and specificity of 0.96 (n = 207). CONCLUSION: The MMP-8 test can be used to differentiate periodontitis from gingivitis and healthy sites as well as to monitor treatment of periodontitis. A reduction in GCF MMP-8 levels and a change in test stick result provide a means to optimize patient control during maintenance of periodontal treatment.     

Analysis of the inflammatory process around endosseous dental implants and natural teeth: myeloperoxidase level and nitric oxide metabolism

PURPOSE: The aim of the present study was to analyze the 2 molecular measures of inflammation: (1) the nitrite, an end metabolite of nitric oxide (NO) oxidation and (2) myeloperoxidase (MPO). Both are found in peri-implant sulcus fluid (PISF) of implants and gingival crevicular fluid (GCF) of natural teeth in healthy or diseased states. MATERIALS AND METHODS: A total of 109 tooth or dental implant sites, either healthy/noninflamed, inflamed (Gingival Index [GI] > 0), or affected by periodontitis, were classified, and GCF/PISF samples were obtained. GCF/PISF MPO and nitrite levels were spectrophotometrically determined. For comparison of clinical parameters and PISF/GCF nitrite and MPO levels, Kruskal-Wallis analysis followed by Mann-Whitney test with Bonferroni correction was performed. Healthy/noninflamed, slightly inflamed, moderate/severely inflamed sites were also analyzed using the Kruskal-Wallis test followed by the Mann-Whitney test with Bonferroni correction. The correlation between nitrite and MPO levels and clinical inflammatory status were analyzed with Spearman's correlation coefficient. RESULTS: Clinical parameters, including both the GCF and PISF volumes, demonstrated gradual increases with the presence of gingival/peri-implant inflammation (P < .05). Despite the higher PISF than GCF volume at healthy sites (P = .001), there were no volumetric differences at inflamed sites (P = .771). PISF from inflamed sites (P = .025) and GCF from gingivitis and periodontitis sites presented higher total MPO levels (P < .05) than samples from noninflamed sites. Despite the relatively stable GCF nitrite levels at healthy and diseased sites, PISF from inflamed sites had higher nitrite content than noninflamed sites (P < .05). CONCLUSIONS: The present study demonstrated the volumetric similarities of PISF and GCF in terms of response to inflammation. However, some differences between the 2 biochemical measures of inflammation and their presence in PISF and GCF were also observed. PISF is likely to have a considerable diagnostic potential for reflecting the biologic changes around load-bearing endosseous dental implants. (Cohort Study) (More than 50 references.)     

Gingival Crevicular Fluid and Plasma Levels of Transglutaminase‐2 and Oxidative Stress Markers in Cyclosporin A‐Induced Gingival Overgrowth

Background: Transglutaminase (TGM)‐2 has been shown to contribute to fibrosis by extracellular matrix accumulation in some organs and is activated by intracellular reactive oxygen species. The aim of this study is to investigate levels of gingival crevicular fluid (GCF) and plasma TGM‐2 and oxidative stress markers (OSMs) in cyclosporin A (CsA)‐induced gingival overgrowth (GO). Methods: The study enrolled 20 healthy (H) individuals; 20 patients with gingivitis (G); 20 CsA‐medicated patients with GO (CsA GO+); and 20 CsA‐medicated patients without GO (CsA GO?). GCF and plasma levels of TGM‐2 were analyzed by enzyme‐linked immunosorbent assay. Spectrofluorometry was used to analyze thiobarbituric acid reactive substance (TBARS); ferric‐reducing antioxidant power (FRAP); total oxidant status (TOS); and total antioxidant capacity (TAC). Results: GCF TGM‐2 level was elevated in CsA GO+ compared with G (P = 0.048) and H (P = 0.001) groups. GCF TBARS level was elevated in CsA GO+ compared with other groups (CsA GO? group: P = 0.003; G group: P <0.001; and H group: P <0.001) and was higher in CsA GO? than in H (P = 0.048). GCF FRAP level was lower in CsA GO? than in H (P = 0.04). Both CsA GO+ and CsA GO? groups had lower GCF TOS levels than H (P <0.001 and P = 0.002) and G (P = 0.003 and P = 0.04). GCF TAC was higher in CsA GO+ than in H (P = 0.02). Plasma TGM‐2 level was elevated in CsA GO+ compared with G (P = 0.048) and H (P = 0.002). Plasma FRAP level was higher in H and CsA GO? than in CsA GO+ (P = 0.008 and P = 0.02). Conclusions: CsA use significantly alters GCF and plasma levels of TGM‐2 and OSMs. TGM‐2 may contribute to CsA‐induced GO in CsA‐treated patients by changing GCF and plasma levels of OSMs. Further studies are needed to prove causality and its direction.     

Crevicular fluid myeloperoxidase at healthy, gingivitis and periodontitis sites

The volume and myeloperoxidase (MPO) activity of gingival crevicular fluid (GCF) collected with filter paper strips for 30 s from the sulcus of healthy, gingivitis and periodontitis sites of Chinese subjects were measured. MPO/site and MPO/microliter GCF were both greater at gingivitis and periodontitis sites than at healthy sites. Enzyme activity was similar at the 2 categories of diseased sites. Mean GCF volume and MPO activity were calculated for all samples from healthy, gingivitis and periodontitis sites with GI 0, 1, 2 and 0 + 1. GCF volume, MPO/site and MPO/microliter GCF all were greater at GI 2 than GI 0 or 0 + 1. These data indicate that increased GCF MPO previously observed at periodontitis sites is not specific to such sites. Rather increased GCF MPO likely occurs when additional polymorphonuclear leukocytes enter the sulcus as a result of gingival inflammation. A second sample was obtained from 22 sites 4 weeks after the initial collection. These samples were collected for 5 s rather than 30 s. The GCF volume, MPO/site and MPO/microliters GCF were each greater in samples collected for 30 s rather than 5 s. Correlation coefficients showed that the amount of GCF and MPO activity of the fluid collected for 5 s and 30 s was dependent upon the site even though the 5-s and 30-s samples were collected 4 weeks apart.     

Quantitative analysis of MRP-8 in gingival crevicular fluid in periodontal health and disease using microbore HPLC

BACKGROUND: The protein components of GCF can be separated by reverse-phase microbore HPLC on a C18 column with detection on the basis of 214 nm absorbance. A single major symmetrical protein peak eluting with a retention time of 26 min (50% acetonitrile) was evident in gingival crevicular fluid (GCF) from periodontitis patients but not in healthy GCF. This protein was identified as human MRP-8 by N-terminal amino acid sequencing and liquid chromatography quadropole mass spectrometry. AIMS: To quantify the amount of MRP-8 detectable in GCF from individual healthy, gingivitis and periodontitis affected sites and to study the relationship, if any, between the levels of this responsive protein and periodontal health and disease. METHODS: GCF was sampled (30 s) from healthy, gingivitis, and periodontitis sites in peridontitis subjects (n=15) and from controls (n=5) with clinically healthy gingiva and no periodontitis. Purified MRP-8 was sequenced by Edmann degradation and the phenylthiohydantoin (PTH) amino acid yield determined (by comparison of peak area with external PTH amino acid standards). This value was subsequently used to calculate the relative amount of protein in the peak eluting with a retention time of 26.0 min (MRP-8) in individual GCF chromatograms. RESULTS: Higher levels of MRP-8 were detected in inflammatory sites: periodontitis 457.0 (281.0) ng; gingivitis 413.5 (394.5) ng compared with periodontally healthy sites in diseased subjects 14.6 (14.3) ng and in controls 18.6 (18.5) ng, p=0.003. There was at least 20-fold more MRP-8 in the inflammatory compared with the healthy sites studied. CONCLUSIONS: The preliminary data indicate that MRP-8 is present in GCF, with significantly greater amounts present at diseased than healthy sites. A systematic study of the relationship of this protein to periodontal disease could prove useful in further clarifying whether MRP-8 could be a reliable GCF biomarker of gingivitis and periodontitis.     

The effect of subgingival controlled-release delivery of chlorhexidine chip on clinical parameters and matrix metalloproteinase-8 levels in gingival crevicular fluid

BACKGROUND: The present study evaluated the efficacy of controlled-release delivery of chlorhexidine gluconate (CHX) on clinical parameters and on gingival crevicular fluid (GCF) matrix metalloproteinase (MMP)-8 levels in chronic periodontitis patients. METHODS: Twenty patients with chronic periodontitis were screened for 6 months. Two interproximal sites were selected from mesial surfaces of anterior teeth with probing depths of 6 to 8 mm that bled on probing in each patient. There were at least 2 teeth between the selected sites. CHX chip was inserted into a randomly selected site following scaling and root planing (SRP+CHX), while the other selected site received only SRP in each patient. Probing depth (PD), clinical attachment level (CAL), plaque index (PI), and papilla bleeding index (PBI) were recorded at baseline and at 1, 3, and 6 months. GCF MMP-8 levels were analyzed at baseline; 2 and 10 days; and at 1, 3, and 6 months by immunofluorometric assay (IFMA). RESULTS: At baseline, there were no statistically significant differences in the mean PD, CAL, PBI, and PI scores between SRP+CHX and SRP alone groups. At 1, 3, and 6 months, all clinical parameters in each group significantly decreased (P <0.0167) when compared to baseline. The reduction of PD and improvement in CAL were higher in the SRP+CHX group compared to SRP alone at 3 and 6 months. However, the differences between the 2 groups were not statistically significant. PBI and PI scores were not significantly different between SRP+CHX and SRP alone groups at any visit. GCF MMP-8 levels were similar in both groups at baseline. Intragroup analysis showed significant decreases in the GCF MMP-8 level for the SRP+CHX group between baseline and 1, 3, and 6 months (P<0.01). Intergroup analysis demonstrated significantly lower mean levels of GCF MMP-8 at 1 month in the SRP+CHX group compared to the SRP alone group (P <0.05). CONCLUSIONS: These data suggest that CHX chip application following SRP is beneficial in improving periodontal parameters and reducing GCF MMP-8 levels for 6 months' duration. The use of a chairside MMP-8 dipstick periodontitis test might be a useful adjunctive diagnostic tool when monitoring the course of CHX chip treatment.     

Concentrations of fibronectin in the sera and crevicular fluid in various stages of periodontal disease

While fibronectin (FN) has previously been demonstrated to be present in gingival crevicular fluid (GCF), its quality and quantity has not been reported. Since this information is relevant for ongoing studies on the use of FN for gingival reattachment, we performed these measurements and compared plasma levels in healthy subjects, patients with gingivitis and periodontitis, and in patients undergoing maintenance therapy. Plasma and GCF samples were obtained from 4 sites in each subject using a Periotron to permit quantification of samples. FN concentrations were determined in a microELISA using hyperimmune anti-FN antibody. Purified FN served as a reference for quantification. The functional activity of each sample was assessed by examining the natural affinity of FN for gelatin. Subjects with gingivitis and those in maintenance had significantly depressed levels of plasma fibronectin. While little fibronectin could be detected in the GCF of healthy sites regardless of patient category, examination of the most diseased sites in each group revealed that the concentration of FN in the GCF was highest in health and reduced when there was gingival inflammation. In no case was GCF FN found to be biologically active.     

Crevicular fluid myeloperoxidase and periodontitis disease

The volume and myeloperoxidase (MPO) activity of gingival crevicular fluid (GCF) collected with filter paper strips for 30 sec from the sulcus of healthy, gingivitis and periodontitis sites of Chinese subjects were measured. MPO/site and MPO/microliter GCF were both greater at gingivitis and periodontitis sites than healthy sites. Enzyme activity was similar at the 2 categories of diseased sites. Mean GCF volume and MPO activity of the samples were calculated for all sites with GI 0, 1, 2 and 0 + 1 irrespective of experimental group assignment. GCF volume MPO/site and MPO/microliter GCF all were greater at GI 2 than GI 0 or 0 + 1. These data indicate that increased GCF MPO previously observed at periodontitis sites is not specific to these sites. Rather, increased GCF MPO likely occurs when additional polymorphonuclear leukocytes enter the sulcus as a result of gingival inflammation. A second sample was obtained from 22 sites 4 weeks after the initial collection. These samples were collected for 5 rather than 30 sec. The GCF volume, MPO/site and MPO/microliter GCF each were greater in samples collected for 30 rather than 5 sec. Correlation coefficients showed that the amount of GCF and MPO activity of the fluid collected for 5 and 30 sec was dependent upon the site even though the samples were collected at different times.     

Variations in crevicular fluid elastase levels in periodontitis patients on long-term maintenance

Granulocyte elastase was determined in the gingival crevicular fluid (GCF) of 18 periodontitis patients. They initially had similar severity of disease but had responded differently to 5-yr maintenance, 13 responders and 5 non-responders. A total of 102 sites were investigated and categorized as: i) consistently healthy, ii) healthy after treatment, iii) gingivitis, and iiii) periodontitis, according to clinical criteria. GCF elastase activity was determined with a granulocyte-specific substrate. The sites from non-responders had consistently higher elastase levels than the corresponding category of sites from responders, despite similar gingival inflammation and periodontal destruction, with the exception of consistently healthy sites. Within the non-responders, the periodontitis sites had higher elastase levels than the gingivitis sites commensurate with probing depth, while no difference existed between gingivitis sites and sites healthy after treatment, despite a difference in probing depth. In contrast, in the responders similar elastase levels were found at the periodontitis sites and gingivitis sites despite difference in probing depth, while both diseased sites had higher elastase levels than the sites healthy after treatment, commensurate with probing depth. This study suggests that increased granulocyte-specific elastase levels in GCF may serve as a diagnostic marker for refractory periodontitis patients.