Molecular size distribution analysis of human gingival proteoglycans and glycosaminoglycans in specific periodontal diseases

Abstract. In order to determine the molecular-size distribution of gingival proteoglycans (PGs) and glycosaminoglycans (GAGs) both in periodontal health and disease states, gingival tissues were obtained from patients with early onset periodontally (EOP) and adult periodontitis (AP) and also from periodontally healthy subjects. Gel filtration chromatography of gingival PGs revealed different profiles for periodontally diseased and healthy gingiva. Healthy gingiva was mainly composed of high-molecular size proteins and PGs. while diseased gingival tissue presented a decrease in high-molecular size PG forms and a shift towards low-molecular size proteins and PGs. This indicates the degradation of PG macromolecules during periodontal disease activity. Furthermore, this shift towards low-molecular size forms was more intense in EOP patients when compared to AP patients. Gel filtration of gingival GAGs also demonstrated depolymerization of GAGs, with low-molecular size GAGs being more intense in periodontally diseased gingival, while healthy gingival GAGs profile was mainly composed of high-molecular size GAGs. Similar to the profile of gingival PGs. low-molecular size gingival GAGs. were more prominent in gingival tissue from patients with EOP, These findings suggest that both PGs and GAGs. essential components of the extracellular matrix (ECM). are depolmerized during eriodontal disease activity, which is more prominent in EOP, Since the basie feature of periodontal disease is matrix degradation. ECM components, more specifically PGs and GAGs, are likely to provide valuable information for a better understandi ng of periodontal disease activity.     

Collagenolytic fragments and active gelatinase complexes in periodontitis

Background: Periodontal tissues remodel rapidly, which enables quick adaptation to mechanical changes. Matrix metalloproteinases (MMPs) are involved in these remodeling processes under control of tissue inhibitor of metalloproteinases (TIMPs). In periodontitis, overactivity of MMPs results in pathologic tissue degradation. The aim of this study was to analyze MMPs and TIMPs in healthy and diseased gingiva, periodontal ligament (PDL), and gingival crevicular fluid (GCF). Methods: Samples of gingiva, PDL, and GCF were obtained from healthy controls (gingiva: n = 18; PDL: n = 15; GCF: n = 8) and subjects with periodontitis (gingiva: n = 11; PDL: n = 18; GCF: n = 12). MMPs and TIMPs were analyzed by gelatin-, collagen-, and reverse zymography and by Western blotting. Total MMP activity was analyzed using a fluorogenic substrate. Results: TIMP-1 and -2, active and pro-MMP-2 and -9, and active MMP-1 and -8 were present in all samples. Large amounts of active MMP-2 complexes and collagenolytic fragments were also found. Their levels were higher in PDL and GCF from subjects with periodontitis. In general, TIMP levels were lower in diseased periodontal tissues. Especially diseased GCF contained more MMPs. Surprisingly, some MMPs were more abundant in healthy gingiva and PDL than in diseased tissue. Conclusions: Unexpected variations in MMP and TIMP levels in gingiva, PDL, and GCF may result from differences in subject characteristics and disease activity. The levels of active MMP-2 complexes and collagenolytic fragments are higher in the periodontium of subjects with periodontitis and might contribute significantly to periodontal destruction.     

Analysis of superoxide dismutase activity levels in gingiva and gingival crevicular fluid in patients with chronic periodontitis and periodontally healthy controls

OBJECTIVES: Superoxide dismutase (SOD) is an antioxidant enzyme that acts against superoxide, an oxygen radical, released in inflammatory pathways and causes connective tissue breakdown. In this study, SOD activities in gingiva and gingival crevicular fluid (GCF) from patients with chronic periodontitis (CP) and periodontally healthy controls were compared. MATERIAL AND METHODS: Twenty-six CP patients and 18 controls were studied. In patients, teeth with moderate-to-severe periodontal breakdown and > or =5 mm pockets that required full-thickness flap surgery in the right or left maxillary quadrant, and in controls, teeth scheduled for extraction for orthodontic reasons were studied. After the clinical measurements (probing depth, clinical attachment level, gingival index, gingival bleeding index, plaque index), GCF samples were collected. Tissue samples were harvested from the same teeth, during flap operation in patients and immediately after tooth extraction in controls. SOD activities were spectrophotometrically assayed.The results were statistically analysed. RESULTS: Gingival SOD activity was significantly higher in the CP group than in controls (p<0.05). No significant difference was found in GCF SOD activity between the groups (p>0.05). Correlations between gingival and GCF SOD activities were not statistically significant in CP and control groups (p>0.05). CONCLUSION: In CP, SOD activity seems to increase in gingiva, probably as a result of a higher need for SOD activity and protection in gingiva in CP than in periodontal health, while not significantly changing in GCF, suggesting a weak SOD activity in GCF in periodontal disease state. The weak correlation between gingival and GCF SOD activities suggests distinct actions of these SODs.     

Analysis of human gingival tissue and gingival crevicular fluid beta-glucuronidase activity in specific periodontal diseases

BACKGROUND: Beta-glucuronidase (betaG) is one of the enzymes involved in the destruction of non-collagenous components of the extracellular matrix. It is also considered an indicator or predictor of periodontal disease activity. The present study was conducted to determine the presence and the levels of betaG activity in gingival tissue and gingival crevicular fluid (GCF) in periodontal disease and health status. The validity of 2 expressions of data, total betaG activity versus betaG concentration, and the correlations between clinical periodontal status and betaG profile was also evaluated. METHODS: betaG activities in gingival tissues and GCF samples from 57 individuals, divided into 3 equal groups of adult periodontitis (AP), early-onset periodontitis (EOP), and periodontally healthy subjects were spectrophotometrically examined. RESULTS: Both patient groups had higher betaG levels in both gingiva and GCF than controls. Significant differences were observed among all groups when total GCF betaG activities were examined (P <0.05). However, the difference between AP and controls was not significant when concentration values were compared (P >0.05). The highest GCF betaG activity, with both expressions, was detected in EOP group. No absolute correlations between clinical parameters and betaG activity were observed, except for random correlations in the patient groups with mean total betaG activities. Also GCF/gingiva betaG levels and the 2 expressions did not show absolute correlations. CONCLUSIONS: The findings of the present study confirm the relationship between betaG activity and periodontal diseases. The differences in data concerning GCF total betaG activity and betaG concentration may suggest that they are not matching measures. Data presentation seems to be an important factor in GCF/enzyme profile studies.     

Relationship of the substance P to indicators of host response in human gingival crevicular fluid

BACKGROUND: The substance P (SP) level in human gingival crevicular fluid (GCF) was studied in relation to clinical periodontal variables and to various indicators of host response in the GCF. METHODS: GCF was collected from periodontal sites with gingival inflammation and shallow or moderately deep pocket in 48 subjects. The total amount of SP and the substances based on host response factors in a 30-s sample were determined by ELISA and enzymatic methods. RESULTS: Significant correlation was found between SP and probing depth (r= 0.637, p<0.001), while correlation was weak between SP and either gingival (r= 0.177, p=0.23) or plaque index (r=0.008, p=0.96). SP also showed significant correlation with the indicators of host response: prostaglandin E2, aspartate aminotransferase, alkaline phosphatase, myeloperoxidase, interleukin-1beta, tumor necrosis factor-alpha, interleukin-8 and monocyte chemoattractant protein-1 (r=0.434-0.867, p<0.01-0.001). CONCLUSION: These results indicate that neuropeptide SP in GCF may have a potential as an indicator of periodontal inflammation and the host response.     

Gingival tissue proteoglycan and chondroitin-4-sulphate levels in cyclosporin A-induced gingival overgrowth and the effects of initial periodontal treatment

OBJECTIVES: Cyclosporin A (CsA) is a potent immunosuppressive drug used in organ transplant patients to prevent graft rejection. CsA-induced gingival overgrowth is one of the side effects of this drug and its pathogenesis is still unclear. The present study was planned to comparatively analyse total proteoglycan (PG) and chondroitin-4-sulphate (C4S) levels in CsA-induced overgrown gingival tissue samples obtained before and after initial periodontal treatment and to compare these findings with the situation in healthy gingiva. MATERIAL AND METHODS: Gingival tissue samples were obtained from nine patients with CsA-induced gingival overgrowth before and 4 weeks after initial periodontal treatment including oral hygiene instruction and scaling and also from 10 healthy control subjects. Total PG and C4S levels were determined by biochemical techniques. PG levels were analysed using modified Bitter and Muir method. C4S assay was carried out using chondroitin sulphate lyase AC and chondroitin-6 sulphate sulphohydrolase enzymes. The results were tested statistically using non-parametric tests. RESULTS: All clinical measurements in the CsA-induced gingival overgrowth group demonstrated significant reductions 4 weeks after initial periodontal treatment (p<0.05). There was no significant difference between the levels of baseline total PG in CsA-induced gingival overgrowth and healthy control groups (p>0.05). The gingival tissue levels of PG in CsA-induced gingival overgrowth group decreased significantly 4 weeks after treatment (p=0.043). Gingival tissue C4S levels in the overgrowth group were significantly higher than the healthy control group at baseline (p=0.000). C4S levels of the overgrowth group were significantly reduced after treatment (p=0.033), but these levels were still significantly higher than the healthy control group (p=0.000). CONCLUSION: The observed prominent increase in gingival tissue C4S levels may be interpreted as a sign of an increase in C4S synthesis in CsA-induced gingival overgrowth. Furthermore, remission of clinical inflammation by means of initial periodontal treatment had a positive effect on tissue levels of these extracellular matrix molecules.     

Identification of the osteoprotegerin/receptor activator of nuclear factor-kappa B ligand system in gingival crevicular fluid and tissue of patients with chronic periodontitis

BACKGROUND AND OBJECTIVE: Recent findings have suggested that osteoclastogenesis is directly regulated by receptor activator of nuclear factor-kappa B ligand (RANKL) and its decoy receptor, osteoprotegerin (OPG). However, no studies have described interactions of OPG/RANKL and the gp130 cytokine family in periodontal disease. This study aimed to identify and quantify OPG/RANKL in the gingival crevicular fluid (GCF) and connective tissue of patients with periodontitis, and to clarify possible correlations with disease severity and interleukin-6 (IL-6) cytokines. MATERIAL AND METHODS: Ninety-five sites in 20 patients with generalized chronic periodontitis were divided into four groups by site based on probing depth (PD) and bleeding on probing (BOP). In periodontitis patients, GCF was obtained using sterile paper strips from clinically healthy sites (PD 6 mm with BOP, n = 27). Fourteen clinically healthy sites from four periodontally healthy individuals were used as the control group. The levels of OPG, RANKL and two gp130 cytokines - IL-6 and oncostatin M (OSM) - in the GCF were determined by an enzyme-linked immunosorbent assay (ELISA) and are expressed as total amounts (pg/site). Immunohistochemical localization of OPG- and RANKL-positive cells was also performed on gingival connective tissues harvested from patients with periodontitis (inflammatory group, n = 8 biopsies) and from non-diseased individuals (healthy group, n = 8 biopsies). RESULTS: GCF RANKL, but not OPG, was elevated in diseased sites of patients with periodontitis. However, the expressions of OPG and RANKL showed no correlation with disease severity (r = 0.174 and 0.056, respectively), but the content of RANKL in the GCF was significantly positively correlated with those of IL-6 (r = 0.207) and OSM (r = 0.231) (p < 0.01). Immunohistochemical staining showed that RANKL-positive cells were significantly distributed in the inflammatory connective tissue zone of diseased gingiva, compared with those of samples from non-diseased persons (p < 0.01). However, few OPG-positive cells were found in connective tissue zones of either the diseased gingiva or healthy biopsies. CONCLUSION: These findings imply that in this cross-sectional study of GCF, RANKL, IL-6 and OSM were all prominent in periodontitis sites, whereas OPG was inconsistently found in a few samples of diseased sites but was undetectable in any of the control sites. The results also imply that the expression of RANKL was positively correlated with IL-6 and OSM in the GCF.     

龈沟液中神经肽P物质与慢性牙周炎关系临床研究

目的:探讨龈沟液中神经肽P物质(SP)与慢性牙周炎发生发展的关系.方法:研究组48 例,对照组45 例,研究组口内各选1 个牙周炎牙位、对照组各选1 个健康牙位收集龈沟液样本,采用放射免疫分析技术分别测定2 组龈沟液SP含量;记录牙龈指数(GI)、牙周袋探诊深度(PD)、附着丧失(AL)、牙槽骨吸收(ABL)情况,并进行与SP的相关性分析.结果:研究组SP含量显著高于对照组(t＝9.92,P<0.01);轻度牙周炎SP含量及牙周临床指标与对照组相比,均有显著差异(P<0.01);轻度牙周炎龈沟液SP含量与GI无显著相关性,但与PD、AL、ABL之间,以及中、重度牙周炎龈沟液SP含量与GI之间,均呈显著性相关(P＜0.05);中、重度牙周炎SP含量与PD、AL、ABL之间均呈非常显著性相关(P<0.01).结论:龈沟液中SP含量与慢性牙周炎的发生发展密切相关,可以反映牙周组织的破坏程度;测定患者龈沟液中SP含量,对于判断病情、指导治疗具有重要意义.     

Changes in vasoactive intestinal peptide in gingival crevicular fluid in response to periodontal treatment

AIMS: To evaluate the role of the anti-inflammatory neuropeptide vasoactive intestinal peptide (VIP) in periodontal health and disease and to determine the effects of periodontal treatment, resulting in a return to periodontal health, on the levels of VIP in gingival crevicular fluid (GCF). METHODS: At baseline, 10 subjects with periodontitis (nine females, one male, mean age 43.0, SD 7.3) started a course of non-surgical periodontal treatment. Clinical indices were measured at one periodontitis and one clinically healthy site at an initial visit and at 8 weeks after the completion of treatment in each subject. A 30-s sample of GCF was collected from each test site using perio paper strips. The volume of GCF was measured and each sample subsequently analysed for VIP by radioimmunoassay. One healthy site was sampled from each member of a control group (10 females, mean age 29.9, SD 8.2 years) with clinically healthy gingiva and no periodontitis. RESULTS: The clinical condition of all periodontitis sites improved as a result of periodontal treatment. The levels of VIP (pg/30 s sample) in periodontitis-affected sites fell significantly from 302.0 (SD 181.2) at the initial visit to 78.0 (54.4) after treatment, p = 0.007. The reduction in the concentration of VIP (pg/ micro L) in GCF from 524.3 (322.3) to 280.8 (280.2) was not statistically significant. The levels of VIP in clinically healthy sites fell from 115.5.5 (74.3) to 77.8 (32.3), n.s. and the concentration changed little from 883.8 (652.1) to 628.7 (323.3), n.s. There were substantially smaller amounts of VIP (25.8, SD 12.8) pg in healthy sites sampled from control subjects. CONCLUSIONS: VIP is present in GCF in greater quantities in periodontitis-affected than clinically healthy sites. In addition, the reduction in inflammation resulting from effective periodontal treatment is associated with a reduction in the levels of VIP in gingival crevicular fluid.     

Relative conversion of arachidonic acid through lipoxygenase and cyclooxygenase pathways by homogenates of diseased periodontal tissues

The present study shows that ¹⁴C-arachidonic acid metabolism in gingiva of patients with periodontal disease is mainly via the lipoxygenase pathway. In two pools of gingival tissue homogenates the lipoxygenase products contained 22.65% and 23.38%, while the prostaglandins (PGs), products of the cyclooxygenase pathway, contained only 4.85% and 3.98% of the total radioactivity incubated. 12-hydroxy-eicosatetraenoic acid (12-HETE), a lipoxygenase product, was detected as a major metabolite of arachidonic acid in gingiva.     

Cigarette smoking, salivary/gingival crevicular fluid cotinine and periodontal status. A 10-year longitudinal study

BACKGROUND, AIMS: The primary purpose of this study was to determine the association of salivary and gingival crevicular fluid (GCF) cotinine levels with periodontal disease status in smokers and non-smokers. METHODS: 147 male smokers and 30 male non-smokers were included in the current longitudinal study. The 177 individuals were part of a group of 200 subjects (89%) seen 10 years previously for a baseline survey. Oral hygiene indices, probing depth and attachment loss were recorded. Salivary and GCF cotinine levels of 58 smokers were determined by means of ELISA. RESULTS: Results indicated that no significant difference was found in subjects who smoked, when compared to subjects who did not smoke with respect to plaque accumulation and calculus deposits. Smokers, however, had fewer gingival bleeding sites. Cigarette smoking was associated with a greater increase in probing depth and attachment loss, as well as greater tooth loss at an earlier age. There was greater tooth loss in smokers than non-smokers (p < 0.001). 11 smokers became edentulous, while only 1 non-smoker lost all his teeth within 10 years. The degree of periodontal tissue breakdown was different in each age group with greater periodontal deterioration as age increased. All smokers had detectable salivary and GCF cotinine. Mean GCF cotinine was about 4x higher than mean salivary cotinine levels. Individuals who smoked > or = 20 pack years when compared to <20 pack years, had significantly higher saliva and GCF cotinine levels (p < or = 0.05). CONCLUSION: Neither salivary cotinine nor GCF cotinine was significantly correlated with probing depth, attachment loss and tooth loss (p > 0.05).     

Changes in substance P and neurokinin A in gingival crevicular fluid in response to periodontal treatment

BACKGROUND: The study of periodontitis provides a unique model for assessing the involvement of neuropeptides in inflammatory disease. AIM: To investigate the effects of periodontal treatment, resulting in a return to periodontal health, on the levels of substance P (SP) and neurokinin A (NKA) in gingival crevicular fluid (GCF). METHOD: We completed a cause of non-surgical treatment for 8 subjects with periodontitis (6 females 2 males, mean age 45.1, range 38-67 years) started a course of non-surgical periodontal treatment. Clinical indices were measured at 2 periodontitis sites at the initial visit and at 8 weeks after the completion of treatment in each subject. A 30-s sample of GCF was collected from each test site using perio paper strips. Each strip was placed into 500 microl of ice cold 0.1 M PBS, pH 7.4, vortex mixed for 30 s, and then stored at -70 degrees C until analysed by radioimmunoassay. RESULTS: The clinical condition of all test sites improved as a result of the periodontal treatment. The levels (pg/30 s sample) of SP fell from 56.3 (SD 66.0) at the initial visit to 4.2 (3.1) after treatment, p=0.017. The concentration (pg/microl) of SP in GCF fell from 140.6 (175.6) to 24.2 (11.1), p=0.036. The levels of NKA fell from 30.5 (17.1) to 10.6 (4.9), p=0.012 whereas the concentration changed little from 85.4 (43.5) to 61.6 (15.1), p=0.41. CONCLUSION: The reduction in inflammation resulting from effective periodontal treatment is associated with a reduction in the levels of tachykinins in gingival crevicular fluid.     

Carboxypeptidase‐mediated metabolism of calcitonin gene‐related peptide in human gingival crevicular fluid – a rôle in periodontal inflammation?

Background: Metabolism by peptidases plays an important rôle in modulating the levels of biologically‐active neuropeptides. The metabolism of the anti‐inflammatory neuropeptide calcitonin gene‐related peptide (GCRP), but not the pro‐inflammatory neuropeptides substance P (SP) and neurokinin A (NKA) by components of the gingival crevicular fluid (GCF), could potentiate the inflammatory process in periodontitis. Aims: To characterise the extracellular hydrolysis of CGRP as a mechanism for the selective inactivation of this neuropeptide in GCF from periodontitis sites. Methods: Samples of GCF from periodontitis patients and periodontally‐healthy subjects were incubated with synthetic human SP, NKA or CGRP. Reaction between the GCF constituents and synthetic peptides was allowed to progress from 0–180 min. Results of neuropeptide metabolism at each time were analysed by matrix‐assisted laser desorption/ionisation mass spectrometry. Results: There was no evidence of metabolism of SP, NKA or CGRP by constituents of healthy GCF. Metabolism of synthetic SP and NKA was minimal even after extensive incubation with periodontitis GCF. However, loss of carboxy‐terminal amino acids was evident after only 1 min incubation with periodontitis GCF. The pattern of CGRP metabolism, which proceeded from the C‐terminus, indicated that the neuropeptide was degraded by a carboxypeptidase. After 180 min, there was extensive carboxypeptidase degradation of CGRP to an 11 amino acid peptide. Conclusions: It is concluded that carboxypeptidase activity in GCF from periodontitis patients is responsible for rapid breakdown of CGRP but not SP or NKA. The rapid action of this carboxypeptidase on the anti‐inflammatory neuropeptide CGRP is suggestive of a pathophysiological rôle for the enzyme in selectively degrading CGRP, thereby potentiating periodontal inflammation.     

Vascular endothelial growth factor in gingival tissues and crevicular fluids of diabetic and healthy periodontal patients

BACKGROUND: Periodontal disease is one of the major oral problems encountered in patients with diabetes mellitus (DM). Vascular changes, neutrophil dysfunction, altered collagen synthesis, and genetic predisposition observed in DM may contribute to periodontitis; and the vascular alterations observed in such patients may depend on vascular endothelial growth factor (VEGF) actions. Few reports are available about the mechanism of neovascularization and the angiogenic factors that contribute to the periodontal pathology and the role of VEGF in periodontal diseases. The aim of this study is to compare VEGF expression in healthy and periodontally diseased tissues with gingival crevice fluid (GCF) of healthy persons and diabetic patients. METHODS: Gingival tissue and GCF samples were collected from sites of periodontitis in 10 healthy subjects and in 10 type 2 diabetic patients, and from the sites of healthy gingiva within the same groups. Therefore, each patient became his/her own control. Additionally, 10 people without any systemic or periodontal diseases were enrolled, forming a negative control group. Thus, a total of 50 tissue and 50 GCF samples were provided. RESULTS: No VEGF staining was observed in the negative control group or in the systemically healthy people's healthy tissue samples, whereas four samples of diabetic patients showed positive staining (P < 0.05). However, VEGF was revealed in two tissue samples of periodontal sites of systemically healthy people and in six samples of the diabetic patients (P > 0.05). In all test groups, GCF VEGF levels were higher in periodontal sites (P < 0.05) than in healthy sites. CONCLUSION: The results of this study showed that VEGF is increased in all periodontal tissues of both groups and in the healthy sites of diabetic patients. Additionally, GCF VEGF values increased in periodontal sites of all test groups.     

牙周治疗前后龈沟液中白细胞介素-6水平的变化

目的：探讨牙周洁利治对患牙龈沟液中IL-6水平的影响。方法：选取12例成人牙周炎患者的重度牙周炎换牙12颗,采集治疗前患牙的龈沟液并记录相关的临床指标。然后对患牙进行龈上洁治和龈下刮治,两周后,再次采集患牙的龈沟液并记录相关的临床指标。采用双抗体夹心ELISA法对牙周炎患牙龈沟液中IL-6水平进行检测。比较牙周洁刮治前后龈沟液中IL-6水平的差异。结果：经过牙周洁刮治,患牙龈沟中IL-6的水平明显降低,同时患牙的牙周临床指标也有明显的改善。结论：牙周基础治疗在缓解牙周炎患牙局部炎症的同时,也对患牙局部的IL-6水平产生明显影响。     

Levels of platelet activating factor in gingival crevice fluid following periodontal surgical therapy

BACKGROUND AND OBJECTIVE: Elevated levels of platelet activating factor (PAF), a potent inflammatory phospholipid mediator, have been previously detected in gingival tissues and gingival crevice fluid (GCF) in periodontal disease. However, the role of this mediator during wound healing after periodontal surgery remains unclear. The hypothesis, a relationship between PAF levels and periodontal healing, was tested by measuring PAF levels in GCF samples collected from sites that had undergone guided tissue regeneration (GTR) or flap surgery. MATERIAL AND METHODS: Using a split-mouth design, 30 intrabony defects were randomly assigned to treatment with GTR (group 1) or to flap surgery (group 2). GCF was sampled pre-operatively and at 6-, 12- and 24-wk follow-up evaluation visits. PAF levels in GCF were analyzed by high-performance liquid chromatography (HPLC). RESULTS: Both treatment modalities significantly reduced the probing pocket depth and improved the clinical attachment level (p < 0.01). Compared with pre-operative values, the GCF volume and PAF levels were significantly decreased at postoperative weeks 6, 12 and 24 in both groups (p < 0.01). There were also significant differences in GCF volume and PAF levels at all time points up to 24 wks in both groups (p < 0.01). No statistically significant differences were observed in any of the parameters investigated between the two groups (p > 0.05). CONCLUSION: PAF is detectable in GCF by HPLC and showed a continuous decrease at all the time points monitored following periodontal surgical therapy. This suggests that changes in the levels of this mediator in GCF might be useful for monitoring the progress of periodontal repair and regeneration.     

Measurement of interleukin-1α and -1β in gingival crevicular fluid: Implications for the pathogenesis of periodontal disease

Samples of gingival crevicular fluid (GCF) were harvested from sites manifesting features characteristic of active disease including inflammation, periodontal attachment loss, and radiographic signs of alveolar bone destruction in untreated patients with advanced periodontitis. The presence and concentrations of interleukin-1 alpha (IL-1 alpha) and interleukin-1 beta (IL-1 beta) were measured using ELISAs specific for these cytokine molecules. IL-1 alpha and/or IL-1 beta were identified in the GCF of 15 of 15 patients having untreated periodontitis. Ninety percent (71 of 79) of the sites tested contained measureable amounts of IL-1, with IL-1 beta as the more frequently occurring form. IL-1 alpha levels ranged from 0.23 nM to 13.9 nM in the GCFs. IL-1 beta levels were between 0.04 nM and 5.28 nM. Marked reductions of total IL-1 levels were observed following effective treatment. Both forms of IL-1 messenger RNA (mRNA) were detected in 17 of 17 gingival tissue samples from 6 patients. These results demonstrate that IL-1 is produced and released locally in periodontal disease at concentrations sufficient to mediate tissue inflammation and bone resorption. IL-1 may serve as a marker of periodontal tissue destruction.     

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.     

Experimental tooth pain elevates substance P and matrix metalloproteinase-8 levels in human gingival crevice fluid

Objective. Tooth pain can induce a neurogenic inflammatory reaction in gingiva in association with local elevations of matrix metalloproteinase (MMP)-8, which is considered the major tissue destructive protease in gingival crevice fluid (GCF). The pro-inflammatory neuropeptides released by sensory nerves coordinate the activities of the immuno-effector cells and may influence the secretion of MMP-8. With this background, we studied whether experimental tooth pain can trigger changes in GCF levels of the neuropeptide substance P (SP) and MMP-8. Material and methods. The GCF SP levels of stimulated and non-stimulated teeth were analyzed for SP using a competitive enzyme immunoassay (EIA). The GCF MMP-8 levels were determined by quantitative immunofluorometric assay (IFMA). Results. Painful stimulation of the upper central incisor caused significant elevations in GCF SP and MMP-8 levels of the stimulated tooth. At the same time, the GCF SP and MMP-8 levels of non-stimulated control teeth were unchanged. Conclusions. These data indicate that experimental tooth pain can induce local elevations of SP and MMP-8 levels in GCF simultaneously. This supports the possibility of a local neurogenic spread of inflammatory reactions from intrapulpal to surrounding periodontal tissues.