The Association between Salivary Metabolites and Gingival Bleeding Score in Healthy Subjects: A Pilot Study.

Periodontal diseases, including gingivitis and periodontitis, are among the most prevalent diseases in humans. Gingivitis is the mildest form of periodontal disease, characterized by inflammation of the gingiva caused by the accumulation of dental plaque. Salivary diagnostics are becoming increasingly popular due to the variation in saliva composition in response to pathological processes. We used a metabolomics approach to investigate whether a specific saliva metabolic composition could indicate preclinical stage of gingivitis. 1H-NMR spectroscopy was used to obtain the salivary metabolite profiles of 20 healthy subjects. Univariate/multivariate statistical analysis evaluated the whole saliva metabolite composition, and the Full-Mouth Bleeding Score (FMBS) was employed as a classification parameter. Identifying a signature of specific salivary metabolites could distinguish the subjects with high FMBS scores but still within the normal range. This set of metabolites may be due to the enzymatic activities of oral bacteria and be associated with the early stages of gingival inflammation. Although this analysis is to be considered exploratory, it seems feasible to establish an FMBS threshold that distinguishes between the absence and presence of early inflammatory alterations at the salivary level.

Assessment of Myeloperoxidase and Nitric Levels around Dental Implants and Natural Teeth as a Marker of Inflammation: A Comparative Study.

INTRODUCTION: Dental implants form the mainstay of dental treatment involving rehabilitation of missing teeth. One of the major concerns for the clinicians doing dental implants is the postsurgical failure of dental implants. Success of dental implants is dependent upon the skills of the surgeon and the amount and quality of the bone remaining at the edentulous area where dental implant has to be placed. Myeloperoxidase (MPO) and nitrites are few of the enzymes and molecules which are said to be altered in inflammation. However, their exact role in the inflammatory processes around natural tooth and dental implant is still unclear. Hence we comparatively evaluated the levels of MPO and nitrites in the areas around the dental implants and natural teeth. MATERIALS AND METHODS: The present study comprises 42 patients who underwent prosthetic rehabilitation by dental implants from 2011 to 2014. Depth of probing value (DP), score of plaque index (SPI), gingival index (GI), and index of gingival bleeding time (GBT) were evaluated for the assessment of the periimplant soft tissue changes. Assessment of inflammation around the dental implant surface and around natural tooth was done based on the readings of these parameters. For the measurement of the MPO levels, spectrophotometric MPO assay was used. All the results were analyzed by Statistical Package for the Social Sciences (SPSS) software. RESULTS: The mean plaque index values were 1.56 and 0.97 in periodontitis cases of natural teeth and inflamed cases of dental implants respectively. While comparing mean plaque index, mean probing depth, and mean gingival bleeding index in between the two groups, significant difference was obtained. Mean MPO concentration in periodontitis and gingivitis cases in natural teeth were 0.683 and 0.875 U/µL, while in inflamed dental implant cases, the mean value was 0.622 U/µL. While comparing the total MPO levels, total nitrite levels, and total nitrite concentration in between two study groups, significant difference was obtained. On comparing the healthy and periodontitis cases in natural teeth, significant difference was obtained. CONCLUSION: In the inflammatory processes occurring around dental implant and natural teeth, MPO and NO make some amount of significant contribution. CLINICAL SIGNIFICANCE: The present study enforces on the role of MPO and nitrite as diagnostic and prognostic marker.

Response of chronic gingivitis to hygiene therapy and experimental gingivitis. Clinical, microbiological and metabonomic changes.

PURPOSE: To compare the clinical, microbiological and metabonomic profiles of subjects with high and low levels of chronic gingival bleeding during a controlled oral hygiene regimen intervention including sequential phases of rigorous therapeutic oral hygiene followed by experimental gingivitis (EG). METHODS: Two cohorts of qualified study subjects with differences in gingival bleeding on probing levels at their baseline clinical examination were entered into the study. These two cohorts were followed through three separate study phases including a 1-week baseline phase, a 2-week phase of rigorous oral hygiene including dental prophylaxis, and a 3-week EG phase of no oral hygiene to encourage relapse of gingivitis. The 58 subjects were assessed during each phase of the study for clinical presentation of gingivitis and concurrently had plaque sampled for real-time polymerase chain reaction (RTPCR) microbiological characterization and salivary lavage samples for 'systems biology' metabonomics assessment by 1H-NMR. RESULTS: Subjects presenting with different levels of gingival bleeding on probing when they entered the study responded differently to rigorous oral hygiene and EG. Specifically, the high bleeding cohort responded sluggishly to rigorous oral hygiene and exhibited markedly greater relapse to gingivitis during EG. RTPCR analysis showed changes in bacterial populations that were associated with study phases, particularly the increases in putative periodontal pathogens during EG. However, the microbiological profiles of high- and low-susceptibility gingival bleeding patients were largely similar. Metabonomic analysis likewise revealed significant changes in metabolite composition during study phases associated with differences in plaque toxicity, especially the short chain carboxylic acids propionate and n-butyrate, which tracked clinical changes in gingivitis severity. Systems analysis of metabonomic changes suggested differences between cohorts, although analysis to date has not elucidated whether these differences are causative (population predictive) or simply diagnostic of clinical status within populations.

Assessment of tissue oxygenation of periodontal inflammation in smokers using optical spectroscopy.

BACKGROUND: We have recently developed a periodontal diagnostic tool that was validated in non-smokers with periodontitis. Tobacco smoking is a recognized risk factor for periodontal diseases that can mask gingival bleeding and lead to a false negative diagnosis. Therefore, the purpose of current study is to further validate this instrument in smokers with periodontal diseases. METHODS: Using a portable optical near-infrared spectrometer, optical spectra were obtained, processed and evaluated from healthy (n = 108), gingivitis (n = 100), and periodontitis (n = 79) sites of 54 systemically healthy smokers. A modified Beer-Lambert unmixing model that incorporates a non-parametric scattering loss function was used to determine the relative contribution of deoxygenated haemoglobin (Hb) and oxygenated haemoglobin (HbO2 ) to the overall spectrum. The balance between tissue oxygen delivery and utilization in periodontal tissues was then assessed. RESULTS: Tissue oxygen saturation was significantly decreased in the gingivitis (p = 0.016) and periodontitis (p = 0.007) sites, compared to the healthy sites. There was a trend towards increased concentration of Hb and decreased concentration of HbO2 from healthy to diseased sites, without statistical significance (p > 0.05). CONCLUSIONS: Optical spectroscopy can determine tissue oxygenation profiles of healthy and diseased sites in smokers. The spectral profile of periodontal sites in smokers generally resembles those from non-smoking patients.

Altered relationship between MMP-8 and TIMP-2 in gingival crevicular fluid in adolescents with Down's syndrome.

BACKGROUND AND OBJECTIVE: Periodontitis is more frequently found in subjects with Down's syndrome. The aim was to investigate whether the relationship between MMPs and TIMPs) in the gingival crevicular fluid of subjects with Down's syndrome is altered compared with controls. MATERIAL AND METHODS: Twenty-one adolescents with Down's syndrome and gingivitis (DS-G), 12 subjects with Down's syndrome and periodontitis (DS-P), 26 controls with gingivitis (HC-G) and eight controls with periodontitis (HC-P) were clinically examined. All patients were between 11 and 20 years of age. Gingival crevicular fluid was collected from each subject and the concentrations of MMPs (2, 3, 8, 9 and 13) and TIMPs (1, 2 and 3) (expressed as pg/µL adjusted for volume of gingival crevicular fluid) were determined using multianalyte kits from R&D Systems. RESULTS: The concentrations of MMP-2, MMP-3, MMP-8, MMP-9 and TIMP-2 in gingival crevicular fluid were significantly higher (p < 0.005) in the DS-G group compared with the HC-G group. The correlation coefficient between MMP-8 and TIMP-2 differed significantly (p = 0.006) between the DS-G group and the HC-G group. On the contrary, the correlation coefficients between MMPs and TIMPs did not differ significantly between the DS-P group and the HC-P group. However, the DS-P group exhibited a significantly lower concentration of TIMP-2 in the gingival crevicular fluid compared with the HC-P group. CONCLUSION: Down's syndrome subjects with gingivitis exhibit higher concentrations of MMPs in gingival crevicular fluid with an altered relationship between MMP-8 and TIMP-2, which might impair the periodontal tissue turnover.

Influence of smoking on interleukin-1beta level, oxidant status and antioxidant status in gingival crevicular fluid from chronic periodontitis patients before and after periodontal treatment.

BACKGROUND AND OBJECTIVE: The aim of this study was to evaluate the impact of smoking on the relationship between interleukin-1 (IL-1ß) and oxidation in patients with periodontitis and response to nonsurgical periodontal therapy. MATERIAL AND METHODS: Data were obtained from 30 patients with generalized chronic periodontitis (15 smokers and 15 nonsmokers) and from 10 periodontally healthy controls. IL-1ß level, total oxidant status (TOS) and total antioxidant status (TAS) were recorded in gingival crevicular fluid. Probing depth, clinical attachment level, gingival and plaque indices and bleeding on probing were also measured. The gingival crevicular fluid and clinical parameters were recorded at baseline and 6 wk after periodontal treatment. RESULTS: The study showed statistically significant improvement of clinical parameters in both smokers and nonsmokers after periodontal treatment. Moreover, the baseline IL-1ß levels were significantly higher in smokers compared with nonsmokers (p < 0.05). After periodontal treatment, the IL-1ß levels were significantly reduced in both smokers and nonsmokers (p < 0.05). There were no significant differences in TOS and TAS between periodontitis patients and healthy controls at baseline and 6 wk after periodontal treatment. The level of IL-1ß in gingival crevicular fluid was positively correlated with TOS in both smokers and nonsmokers. CONCLUSIONS: Periodontal treatment improved the clinical parameters in both smokers and nonsmokers. The results confirm that periodontal therapy has an effect on IL-1ß levels in gingival crevicular fluid, but not on TOS and TAS.

Bone remodeling-associated salivary biomarker MIP-1α distinguishes periodontal disease from health.

BACKGROUND AND OBJECTIVE: The field of salivary diagnostics lacks an accepted and validated biomarker of alveolar bone remodeling. To address this, we examined levels of salivary biomolecules specifically associated with biological aspects of bone remodeling in subjects with chronic periodontitis in a case-control study. MATERIAL AND METHODS: Levels of macrophage inflammatory protein-1α (MIP-1α), osteoprotegerin, C-telopeptide pyridinoline cross-links of type I collagen and ß-C-terminal type I collagen telopeptide in unstimulated whole saliva of 80 subjects (40 subjects with moderate to severe chronic periodontitis and 40 sex- and age-matched healthy control subjects) were measured using enzyme immunosorbent assays. Saliva was collected before clinical examination, which included probing depth, clinical attachment loss and bleeding on probing. RESULTS: The mean level of MIP-1α in subjects with periodontitis was 18-fold higher than in healthy subjects (p < 0.0001). Clinical periodontal indices correlated significantly with MIP-1α levels (p < 0.0001). Of the biomolecules examined, MIP-1α demonstrated the greatest ability to discriminate between periodontal disease and health as determined by the area under the curve (0.94) and classification and regression tree analysis (sensitivity 94% and specificity 92.7%). Osteoprotegerin levels were elevated 1.6-fold (p = 0.055), whereas C-telopeptide pyridinoline cross-links of type I collagen and ß-C-terminal type I collagen telopeptide levels were below the level of detection in the majority of subjects. CONCLUSION: These findings suggest that the chemokine MIP-1α may aid in identifying periodontitis. Future longitudinal studies are warranted to determine whether this biomarker can help in ascertaining the progression of bone loss in subjects with periodontal disease.

Elevated levels of salivary lactoferrin, a marker for chronic periodontitis?

BACKGROUND AND OBJECTIVE: Whole saliva is a complex mixture of fluids essential for the well-being of the oral hard and soft tissues. Saliva contains numerous antimicrobial proteins that help protect the oral ecosystem from infectious agents. Chronic periodontitis is an infectious chronic inflammatory condition that affects the tooth-supporting structures and leads to their destruction. The aim of the present study was to investigate differences in concentrations of salivary lactoferrin in subjects with and without periodontal disease and correlate these values with clinical variables associated with periodontal disease. MATERIAL AND METHODS: Stimulated whole saliva was collected from 17 subjects with chronic periodontitis and 17 periodontally healthy control subjects. Data relating to bleeding on probing, probing pocket depth and horizontal bone loss were registered. Concentrations of lactoferrin, lysozyme and IgA in stimulated whole saliva were quantified using ELISA. RESULTS: Subjects with chronic periodontits showed higher concentrations of lactoferrin in stimulated whole saliva compared with periodontally healthy control subjects (p < 0.05). Salivary concentrations of lactoferrin were positively correlated with bleeding on probing (p < 0.001) and the number of sites with probing pocket depth ≥ 6 mm (p < 0.001). CONCLUSION: Lactoferrin is raised in stimulated whole saliva in subjects with chronic periodontitis and is correlated with probing pocket depth ≥ 6 mm.

Relationship between salivary antioxidant capacity and phases of the menstrual cycle.

BACKGROUND AND OBJECTIVE: Reactive oxygen species and free radicals are involved in the pathogenesis of periodontal disease. Previous studies have shown that the stage of the menstrual cycle is associated with the levels of gingival inflammation and discomfort. This study examined changes in salivary antioxidant activities, clinical parameters and bacterial levels during the menstrual cycle. MATERIAL AND METHODS: The study group consisted of 16 women with periodontitis and 12 healthy women. Clinical and bacterial measurements were performed for all subjects during the ovulatory and follicular phases. RESULTS: Salivary antioxidant activity during the ovulatory phase was significantly lower than during the follicular phase in the women with periodontitis. The antioxidant activity in all subjects during the ovulatory phase was negatively correlated with Prevotella intermedia (r = -0.430; p = 0.023) and total bacterial counts (r = -0.496; p = 0.007); however, these correlations were not significant for subjects in the follicular phase. CONCLUSION: This study showed that salivary antioxidant capacity decreased, while bleeding on probing and P. intermedia increased, over the course of the menstrual cycle in women with periodontitis. Antioxidant capacity could be involved in the pathogenesis of periodontitis.

Smoking influences salivary histamine levels in periodontal disease.

OBJECTIVES: Histamine, a potent vasoactive amine, is increased in saliva of periodontitis patients. The present study aimed to further investigate the diagnostic potential of histamine for periodontal disease and assessed smoking, a major risk factor of periodontitis, as a possible influencing factor. METHODS: Salivary and serum samples of 106 participants (60 periodontitis patients, 46 controls) were collected. Salivary histamine was determined by a commercially available ELISA kit, and serum C-reactive protein was measured by a routine laboratory test. Cigarettes per day and packyears were assessed as smoking exposure parameters. RESULTS: Statistically significantly increased levels of salivary histamine and serum C-reactive protein were detected between the patient and control group (P = 0.022 and P = 0.001). Salivary histamine levels were significantly higher in smoking compared with non-smoking patients (P < 0.001), and salivary histamine as well as serum C-reactive protein correlated significantly positively with smoking exposure parameters (P < 0.05). CONCLUSIONS: Smoking, an established and common risk factor of periodontitis, was assessed as a possible influencing factor for salivary histamine. Most interestingly, salivary histamine differed highly significantly between smoking and non-smoking periodontitis patients. Our results suggest a possible involvement of histamine in tobacco-exacerbated periodontal disease, but do not suggest salivary histamine as a reliable diagnostic marker for periodontitis.

The gingiva contains leptin and leptin receptor in health and disease.

The presence of leptin (OB) and soluble OB receptor (s-OB-R) in gingival tissue extract and gingival crevicular fluid has led the studies investigating the relationship between OB and periodontal diseases. This study aims to investigate the levels of OB and s-OB-R in serum and their presence in gingiva of healthy controls (HC), gingivitis (G), aggressive periodontitis (AP), and chronic periodontitis (CP) patients; and whether correlations exist between clinical and serum parameters, OB and s-OB-R. Seventy-seven subjects [HC (n = 20), G (n = 20), CP (n = 21), and AP (n = 16)] were included in this study. After the clinical periodontal parameter recordings and venous blood sampling, gingival tissues obtained. Serum parameters' levels determined with enzyme linked immune sorbent assay; and OB and OB-R in gingiva immunohistochemically. No significant differences were observed regarding the serum parameters [high sensitivity C-reactive protein (hs-CRP), lipids, OB, and s-OB-R] when the groups were compared (P > 0.0125). The serum OB has positive correlations with hs-CRP in the G group (P < 0.05), and s-OB-R has presented significant negative correlations with BOP in HC group (P < 0.05), with hs-CRP in G (P < 0.05) and AP groups (P < 0.05). The positive correlations were observed between the serum OB and HDL and body mass index in the CP group (P < 0.05). In all of the tissue samples of all groups, there was positive OB and OB-R immunoreactivity in the gingival epithelium. The gingival tissues contain both OB and OB-R. The serum levels of OB and s-OB-R do not vary between patients and with different periodontal conditions.

Correlations between pentraxin 3 or cytokine levels in gingival crevicular fluid and clinical parameters of chronic periodontitis.

The gingival crevicular fluid (GCF) contains various biomarkers, such as interleukin (IL)-1ß, IL-6, IL-8, tumor necrosis factor-α (TNF-α), and IL-10, among others. These cytokines have been reported to correlate with gingival inflammation and periodontal status. Therefore, the analysis of GCF may be useful for the diagnosis of periodontal status. Pentraxin 3 (PTX3) is the first identified long pentraxin, and is released by several cell types in response to proinflammatory signals. The aim of this study was to determine the levels of IL-1ß, IL-6, IL-8, TNF-α, IL-10 and PTX3 in GCF from diseased and healthy sites in patients with chronic periodontitis. Cross-sectional clinical data were obtained from 50 patients with chronic periodontitis. GCF samples were collected with paper strips from one periodontal diseased site and one periodontally healthy site per subject. The levels of IL-1ß, IL-6, IL-8, IL-10 and TNF-α were determined using a multiplexed bead immunoassay, and the PTX3 level was measured using an enzyme-linked immunosorbent assay. Mean clinical parameters were significantly higher at diseased sites (P < 0.01) as compared to healthy sites, and the mean levels of PTX3, IL-1ß, IL-6, IL-8, IL-10 and TNF-α were higher in diseased sites (P < 0.01) than in healthy sites. There were strong correlations between PTX3 or IL-1ß and periodontal status. These results suggest that GCF PTX3 levels might be useful as a diagnostic marker for periodontal disease.

Determination of salivary levels of mucin and amylase in chronic periodontitis patients.

BACKGROUND AND OBJECTIVE: Patients with periodontal disease show differences in the profile of proteins in whole saliva. This profile reflects the nature and amplitude of the host response to a periodontal microbial challenge. Since periodontitis is a chronic inflammatory disease with different progression stages, the aim of the study was to evaluate the host response in these different clinical stages by assessing salivary flow rate, the concentrations of proteins and mucin and the amylase activity. MATERIAL AND METHODS: Sixty adult subjects were clinically examined and distributed into four groups (n = 15) according to the periodontal status, namely, healthy, mild, moderate and severe periodontitis. Whole saliva was collected for 5 min, followed by a second 5 min sampling period with stimulation by chewing a paraffin block, and flow rate was determined. Salivary proteins, amylase and mucin were determined by colorimetric methods. RESULTS: The concentrations of proteins, amylase and mucin increased in subjects with moderate and severe periodontal disease in unstimulated saliva, while flow rate decreased. A positive correlation was found between proteins and amylase or mucin concentrations among the different groups, indicating that the concentrations changed in the same way, being the response of salivary glands to the disease, possibly to enhance the protective potential of saliva. Mucin concentration was lower in the mild periodontitis group. Mechanical stimulation induced an increase in flow rate and output of proteins, amylase and mucin. CONCLUSION: Periodontitis induces an increase in the output of proteins, including mucin and amylase, thereby enhancing the protective potential of saliva, but this is accompanied by a decrease in flow rate.

Expression of transient receptor potential vanilloid receptor 1 and toll-like receptor 4 in aggressive periodontitis and in chronic periodontitis.

BACKGROUND AND OBJECTIVE: The objective of the present study was to evaluate the expression and the distribution of the transient receptor potential vanilloid receptor 1 (TRPV1) and of toll-like receptor 4 (TLR4) in tissue samples from patients with periodontal disease (aggressive periodontitis and chronic periodontitis) and from healthy controls. MATERIAL AND METHODS: Ten tissue samples from each disease group (aggressive periodontitis and chronic periodontitis) and from healthy subjects were obtained during routine oral surgical procedures. Subgingival specimens were collected from sites with advanced loss of support (probing depth>5mm) and specimens from the corresponding healthy controls were obtained during tooth extraction for orthodontic reasons or following surgical extraction of an impacted third molar. The distribution of TRPV1 and TLR4 receptors in human gingival tissue was studied by immunohistochemistry. RESULTS: Both TLR4 and TRPV1 were detected in gingival tissues from healthy subjects, and from patients with chronic periodontitis and aggressive periodontitis, particularly in gingival keratinocytes, fibroblasts, inflammatory cells and the endothelial lining of capillaries in connective tissues. Histologic examination of the samples from healthy controls disclosed that clinically healthy gingiva does not correspond to histologically healthy gingiva. Subsequently, these samples were redesignated as gingivitis samples. TRPV1 was down-regulated in all cell types in samples obtained from patients with chronic periodontitis compared to samples obtained from patients with gingivitis, whereas TLR4 was down-regulated only in the epithelium and in gingival fibroblasts. In contrast, the levels of these markers in patients with aggressive periodontitis were similar to those in healthy patients. CONCLUSION: Local expression of TRPV1 and TLR4 in gingival tissues may contribute to both physiological and pathological processes in the periodontium. Our data suggest that TRPV1 and TLR4 may play a role specifically in the pathophysiology of chronic periodontitis.

Proteome analysis of proteins related to aggressive periodontitis combined with neutrophil chemotaxis dysfunction.

AIM: Some patients suffering from aggressive periodontitis (Ag-P) also display neutrophil chemotaxis dysfunction. In this study, we attempted to identify the proteins involved in Ag-P associated with neutrophil chemotaxis dysfunction using proteome analysis. MATERIAL AND METHODS: A two-dimensional fluorescence difference gel electrophoresis system was used to detect differences in protein expression between neutrophils from four patients suffering from Ag-P combined with neutrophil chemotaxis dysfunction and those from four controls. Moreover, the mRNA levels of the proteins identified by the above method were examined in neutrophils from four types of subjects using the real-time polymerase chain reaction: twenty patients suffering from Ag-P with or without the dysfunction, 15 patients with chronic periodontitis, and 15 controls. RESULTS: Four proteins, lactoferrin, caldesmon, heat shock protein 70, and stac, displayed a higher protein expression level in the neutrophils from the patients suffering from Ag-P combined with the neutrophil dysfunction than in those from the control group. The caldesmon mRNA levels in the neutrophils from the patients suffering from Ag-P combined with the neutrophil dysfunction were high compared with those in the neutrophils from the patients suffering from the other two types of periodontitis and those from the control group. CONCLUSION: Caldesmon may be a marker of Ag-P combined with neutrophil chemotaxis dysfunction.

Myeloid-related protein (MRP8/14) expression in gingival crevice fluid in periodontal health and disease and after treatment.

BACKGROUND AND OBJECTIVE: Myeloid-related protein (MRP8/14) and its subunits are biomarkers of inflammation. The present study evaluated whether gingival crevice fluid levels of these markers discriminate periodontitis from healthy sites in patients with chronic periodontitis or diseased from healthy subjects, and whether these biomarkers detect longitudinal changes after therapy. MATERIAL AND METHODS: Levels of MRP8/14, MRP14 and total protein were quantified in 19 periodontitis patients before non-surgical periodontal therapy, after 3 and 6 mo of treatment, and were measured once in 11 periodontally healthy subjects. In total, diseased subjects contributed 59 sites with probing depths >4 mm (PP) and 21 sites <4 mm (PH); healthy subjects contributed 91 sites (HH). RESULTS: Overall, in diseased subjects, MRP8/14, MRP14 and total protein were not significantly different between PP and PH sites. However, at baseline, MRP8/14 and total protein had significantly higher values at sites in periodontally diseased than in healthy subjects. Clinical improvement was associated with a significant decrease of MRP8/14 and MRP14 from baseline to month 6 in PP sites. Interestingly, a similar decrease was observed in PH sites for all three markers. At 6 mo, however, levels of MRP8/14 and protein in PP and PH sites of patients were still significantly higher than in healthy subjects. CONCLUSION: Gingival crevice fluid levels of MRP8/14 did not differentiate between clinically diseased and healthy sites in patients with chronic periodontitis. However, this marker was elevated in periodontally diseased compared with healthy subjects, and its values decreased following therapy. MRP8/14 may be used to monitor the response to treatment.

Periodontitis-specific molecular signatures in gingival crevicular fluid.

BACKGROUND AND OBJECTIVE: Periodontitis is currently diagnosed almost entirely on gross clinical manifestations that have been in situ for more than 50 years without significant improvement. The general objective of this study was, therefore, to evaluate whether mid-infrared spectroscopy can be used to identify disease-specific molecular alterations to the overall biochemical profile of tissues and body fluids. MATERIAL AND METHODS: A total of 190 gingival crevicular fluid samples were obtained from periodontitis (n = 64), gingivitis (n = 61) and normal sites (n = 65). Corresponding infrared absorption spectra of gingival crevicular fluid samples were acquired and processed, and the relative contributions of key functional groups in the infrared spectra were analysed. The qualitative assessment of clinical relevance of these gingival crevicular fluid spectra was interpreted with the multivariate statistical analysis-linear discriminant analysis. RESULTS: Using infrared spectroscopy, we have been able to identify four molecular signatures (representing vibrations in amide I, amide II/tyrosine rings and symmetric and asymmetric PO2- stretching vibrations of phosphodiester groups in DNA) in the gingival crevicular fluid of subjects with periodontitis or gingivitis and healthy control subjects that clearly demarcate healthy and diseased periodontal tissues. Furthermore, the diagnostic accuracy for distinction between periodontally healthy and periodontitis sites revealed by multivariate classification of gingival crevicular fluid spectra was 98.4% for a training set of samples and 93.1% for a validation set. CONCLUSION: We have established that mid-infrared spectroscopy can be used to identify periodontitis-specific molecular signatures in gingival crevicular fluid and to confirm clinical diagnoses. Future longitudinal studies will assess whether mid-infrared spectroscopy represents a potential prognostic tool, recognized as key to advancement of periodontics.

Host beta-globin gene fragments in crevicular fluid as a biomarker in periodontal health and disease.

BACKGROUND AND OBJECTIVE: Leukocytes and epithelium are the first line of defense in preventing bacterial invasion into periodontium. Some of these cells die in gingival crevicular fluid, whereupon their DNA is spilled out. The present study was designed to investigate the profile of host beta-globin gene fragments in the gingival crevicular fluid of various periodontal conditions. MATERIAL AND METHODS: Gingival crevicular fluid from 40 teeth with chronic periodontitis, 30 with gingivitis and 22 that were clinically healthy were centrifuged (3,000 g, 10 min). The supernatant (cell-free gingival crevicular fluid) was centrifuged again (13,000 g, 10 min), resulting in the pellet and the supernatant as debris and debris-free fractions, respectively. Specific primers for amplifying 110 bp, 536 bp and 2 kb amplicons of human beta-globin gene were used to investigate host DNA by quantitative and qualitative polymerase chain reaction. RESULTS: The periodontitis group showed the largest amount of host beta-globin gene fragments, while the healthy group had the lowest. In the debris and debris-free fractions, the 536 bp and 2 kb amplicons were more often detected in the periodontitis group than in the other groups. Interestingly, the presence of 2 kb amplicon in the debris fraction could be used to discriminate periodontitis from gingivitis and healthy groups because we found it in 85% of periodontitis samples but only in 13% of gingivitis samples, and it was absent in the healthy group. CONCLUSION: This study shows the different DNA profiles of cell-free gingival crevicular fluid in periodontal health and disease. It suggests that the quantity and quality of host DNA are dependent on the disease conditions. Therefore, the beta-globin gene fragments in cell-free gingival crevicular fluid may be a potential biomarker of periodontal disease progression.

Gingival crevicular fluid can degrade Emdogain and inhibit Emdogain-induced proliferation of periodontal ligament fibroblasts.

BACKGROUND AND OBJECTIVE: Emdogain (EMD), consisting mostly of amelogenin, is used in periodontal therapy to regenerate lost connective tissue. Emdogain is applied onto periodontally affected root surfaces, where it becomes exposed to proteolytic enzymes. In this study, we aimed to find out whether gingival crevicular fluid or matrix metalloproteinases (MMPs) could degrade EMD, and whether this degradation has consequences for in vitro cell proliferation. MATERIAL AND METHODS: We studied the effects of 156 gingival crevicular fluid samples collected from subjects with different stages of periodontal disease and from healthy control subjects and the effects of MMP-1, -2, -8, -9, -13 and -14 on the degradation of EMD using EMD-embedded zymography. The effects of gingival crevicular fluid with or without EMD and the effects of amelogenin on the proliferation of cultured periodontal ligament fibroblasts were studied by cell proliferation enzyme-linked immunosorbent assay kit. RESULTS: Degradation of Emdogain induced by gingival crevicular fluid was greater in samples from all stages of periodontal diseases compared with healthy control samples. Of the MMPs studied, only MMP-2 and MMP-8 showed limited EMD-degrading activities. One hundred micrograms per millilitre of EMD increased proliferation of periodontal ligament fibroblasts on average by 24% (confidence interval 0.60-0.64) and at 200 microg/mL by 30% (confidence interval 0.62-0.68) compared with control fibroblasts (confidence interval 0.48-0.52). However, gingival crevicular fluid (10 microg/mL) together with 100 microg/mL EMD induced the proliferation only by 6% (confidence interval 0.51-0.55) and with 200 microg/mL EMD by 12% (confidence interval 0.54-0.58). Amelogenin at 200 microg/mL decreased the proliferation of periodontal ligament fibroblasts by 54% (confidence interval 0.22-0.25). CONCLUSION: We suggest that diseased gingival crevicular fluid containing various proteases leads to degradation of EMD and decreased proliferation of periodontal ligament fibroblasts.

Crevicular fluid glutathione levels in periodontitis and the effect of non-surgical therapy.

AIM: To quantify reduced and oxidized glutathione (GSH and GSSG) levels in gingival crevicular fluid (GCF) of periodontitis patients pre-therapy (versus periodontally healthy controls) and ascertain whether successful non-surgical therapy alters glutathione levels. MATERIALS AND METHODS: Thirty-second GCF samples (6/subject) were collected on Periopaper() strips from starved, non-smokers (n=20; mean age 43.6 years) with chronic periodontitis, before and 3 months after non-surgical therapy, and periodontally healthy, age- and gender-matched controls (n=20). GSH and GSSG levels were determined using reversed-phase high-performance liquid chromatography with fluorescence detection. RESULTS: Lower concentrations of GSH (p<0.01) and GSSG (p<0.05) were detected in GCF from patients (pre- and post-therapy) than controls and treatment had no significant effect. Amounts per 30-second sample did not differ between patients and controls. However, the amount of GSSG per 30-second sample decreased in patients after therapy (p<0.05). Consequently, therapy increased the GSH:GSSG ratio (p<0.05) in patients compared with the controls (p=0.8). CONCLUSION: These data demonstrate high concentrations of GSH within GCF, which are compromised in chronic periodontitis. While therapy does not appear to fully restore GSH concentrations in GCF, it does restore the redox balance (GSH:GSSG ratio), suggesting that the abnormal redox balance arises secondary to oxidative stress resulting from periodontal inflammation.