Quantitative proteomic analysis of gingival crevicular fluids to identify novel biomarkers of gingival recession in orthodontic patients.

OBJECTIVE: To identify gingival recession-related biomarkers in orthodontic patients, we compared the proteome of gingival crevicular fluids (GCF) from healthy gingiva without orthodontic treatment (GH), healthy gingiva undergoing orthodontic treatment (OGH), and recessed gingiva undergoing orthodontic treatment (OGR). METHODS: GCF samples were obtained from the anterior teeth of 15 volunteers (n = 5/group). Quantitative proteomic analysis was performed using DIA-based liquid chromatography-tandem mass spectrometry (LC-MS/MS). Gene Ontology (GO) terms and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathways were used to annotate differentially expressed proteins (DEPs). Receiver-operating characteristic (ROC) analysis was performed to detect and filter biomarker candidates, while Protein-Protein Interaction (PPI) Networks were utilized to determine the interactions between these DEPs. RESULTS: A total of 253, 238, and 101 DEPs were found in OGR vs. OGH, OGR vs. GH, and OGH vs. GH groups, respectively. Based on the Venn diagram of three groups, 128 DEPs in OGR vs. OGH group were identified as specific proteins associated with progressive gingival recession (GR) during orthodontic treatment. Molecular function analysis showed that 128 DEPs were enriched in "molecular binding", including antigen binding, RNA binding, double-stranded RNA binding, cadherin binding involved in cell-cell adhesion, vinculin binding, S100 protein binding, and Ral GTPase binding. The majority of these DEPs were also involved in cytoskeletal regulation. In addition, biological process analysis showed an enrichment in translation, while cellular component analysis indicated that 128 DEPs were related to extracellular exosome. Furthermore, Ribosome and Phagosome were the top two terms in KEGG analysis. The results of ROC analysis demonstrated that 26 proteins could be potential biomarker candidates for GR. PPI networks analysis predicted that IQGAP1, ACTN1, TLN1, VASP, FN1, FERMT3, MYO1C, RALA, RPL35, SEC61G, KPNB1, and NPM1 could be involved in the development of GR via cytoskeletal regulation. CONCLUSIONS: In summary, we identified several GCF proteins associated with GR after orthodontic treatment. These findings could contribute to the prevention of GR in susceptible patients before the initiation of orthodontic treatment. SIGNIFICANCE: Orthodontic patients with GR often report esthetic defects or root hypersensitivity during orthodontic treatment, especially at the anterior teeth site. GCF, rich in protein, is an easily accessible source of potential biomarkers for the diagnosis of periodontal diseases; however, little is known about the changes in GCF proteome associated with GR in orthodontic patients. In this study we firstly used DIA-based LC-MS/MS to evaluate the proteome and to identify the biomarker candidates for GR in orthodontic patients. These findings will improve our understanding of GR during orthodontic treatment, and could contribute to an earlier diagnosis, or even prevention, of GR in susceptible populations before orthodontic treatment.

Dynamics of Matrix Metalloproteinase-1 and -8 Secretion in Gingival Crevicular Fluid after Gingival Recession Therapy via MCAT with Either Subepithelial Connective Tissue Graft or Collagen Matrix.

OBJECTIVES: The objective of this study was to determine and estimate the changing levels of matrix metalloproteinases 1 and 8 (MMP-1 and MMP-8) in GCF at consecutive stages of healing after root coverage procedure via modified coronally advanced tunnel (MCAT) combined with either sub-epithelial connective tissue graft (SCTG) or collagen matrix (CM) and also to relate those changes to clinical outcomes of both therapeutic approaches. MATERIALS AND METHODS: The study involved 20 patients with a total of 91 recessions. Those on one side of the mandible received MCAT plus CM while the contralateral ones MCAT plus SCTG. The evaluation of MMP-1 and MMP-8 concentrations in Gingival Crevicular Fluid (GCF) took place at baseline, then at 1, 2, and 4 weeks, and finally at 3 months after surgery. Elisa protocol was applied to determine the levels of MMP-1 and MMP-8 in GCF. RESULTS: Three-month observation revealed statistically significant changes in MMP-1, MMP-8 and Sulcus Fluid Flow Rate (SFFR) values after implementation of both techniques. A correlation was found between a difference in MMP-1 concentrations and gain in Keratinized Tissue (KT) after SCTG and CM. MMP-8 levels and a Gingival Thickness (GT) gain observed after CM was also correlated. CONCLUSIONS: A type of augmentative material does appear to determine the dynamics of MMP-1 secretion.

Preservation of Natural Gingival Pigmentation When Treating Multiple Gingival Recession Defects.

Proper surgical techniques can permit treatment of multiple gingival recession defects without altering the visible gingival pigmentation. In this case report, a patient presented with multiple adjacent sites displaying gingival recession. Natural pigmentation was present in the interdental papillae and in the zone of keratinized gingiva. This patient wanted his natural pigmentation to be preserved. A modified tunnel technique was used to avoid traumatizing the pigmented tissues and allow placement of a large graft. A double layer of an acellular dermal matrix (ADM) was implanted and the gingival complex was relocated coronally. Root coverage and preservation of the natural gingival pigmentation were achieved. The use of the modified tunnel technique with a double layer of ADM can preserve natural gingival pigmentation when treating adjacent gingival recession defects.

Síndrome de boca ardiente secundario a pregabalina en un paciente con atrofia frontal leve / Burning mouth syndrome secondary to pregabaline in a patient with mild frontal lobes atrophy

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C-Reactive Protein in Peripheral Blood of Patients with Chronic and Aggressive Periodontitis, Gingivitis, and Gingival Recessions.

CRP is a plasma protein that reflects a measure of the acute phase response to inflammation and is one of the markers of choice in monitoring this response. CRP can be used for the prediction and early detection of periodontal disease. The aim of this study was to compare and evaluate the systemic levels of CRP in the peripheral blood samples of patients with chronic and aggressive periodontitis, gingivitis, and gingival recessions and compare them with periodontal clinical parameters. All patients (N = 158) were examined prior to the initiation of periodontal treatment. Patients were divided into four groups. Group A consisted of 26 patients with aggressive periodontitis, Group B consisted of 111 patients with chronic periodontitis, Group C consisted of 13 patients with gingivitis, and Group D consisted of 8 patients with gingival recessions. Our study results indicate that CRP levels increase subsequently with the severity of the periodontal disease and that the bleeding on probing index showed much better positive correlation with the CRP levels compared to the pocket depth index in both periodontitis patients groups, especially in aggressive periodontitis patients.

Concentrated growth factor in the treatment of adjacent multiple gingival recessions: a split-mouth randomized clinical trial.

AIM: The aim of this study was to determine the clinical effect of concentrated growth factor (CGF) in combination with coronally advanced flap (CAF) compared to CAF alone for the treatment of multiple adjacent gingival recessions (GRs). MATERIALS AND METHODS: Twenty patients with a total of 119 Miller Class I and II GRs in the maxilla were included to this study. Recessions were randomly treated according to a split-mouth design by means of CAF + CGF (test; 60 defects) or CAF (control; 59 defects). Clinical outcomes were evaluated at baseline and 6 months after surgery. RESULTS: The mean root coverage (MRC) was 82.06% and 86.67%, complete root coverage (CRC) was 45.8% (27/59) and 56.7% (34/60) for CAF and CAF + CGF, respectively at 6th month. Statistically no difference was demonstrated between the two groups in terms of recession depth (RD), MRC and CRC at 6th month. The increase in width of keratinized gingiva (KGW) and gingival thickness (GT) were statistically significant in the CAF + CGF group compared to the CAF group at 6th month. CONCLUSIONS: The use of CGF in combination with CAF did not provide additional benefits in RD, CRC and MRC. This study suggests that use of CGF + CAF may increase the success of GRs because of a significant increase in KGW and GT.

Assessment of oxygen saturation in dental pulp of permanent teeth with periodontal disease.

INTRODUCTION: In individuals with periodontal disease, dental pulp status should be determined before a treatment plan is made. Pulse oximeters are promising diagnostic tools to evaluate pulp vascularization. This study used pulse oximetry to determine the level of oxygen saturation in dental pulp of intact permanent teeth with periodontal attachment loss (PAL) and gingival recession (GR) and to evaluate the correlation between periodontal disease and level of oxygen saturation in the pulp. METHODS: This study included 67 anterior teeth of 35 patients; all teeth showed intact crowns, PAL, a periodontal pocket (PP), and GR. The teeth underwent periodontal examination, cold and electric pulp testing, and pulse oximetry measurements. The Pearson correlation coefficient and a linear regression coefficient were calculated to evaluate the degree of correlation between periodontal disease markers (PAL, PP, and GR) and the level of oxygen saturation in dental pulp. These tests also evaluated possible associations between oxygen saturation and cold and electric pulp testing. RESULTS: PAL, PP, and GR had negative correlations with oxygen saturation in dental pulp. Conversely, no statistically significant association was found between oxygen saturation in dental pulp and the response to electric sensibility testing. CONCLUSIONS: Oxygen saturation was lower in the pulp of permanent teeth with PAL, PP, and GR, indicating that periodontal disease correlates with the level of oxygen saturation in the pulp.

Comparisons between two biochemical markers in evaluating periodontal disease severity: a cross-sectional study.

BACKGROUND: The purpose of this study was to compare two biochemical markers, which have been previously used to determine the degrees of alveolar bone destruction, in evaluating periodontal disease severity. METHODS: The WF6 epitope of chondroitin sulfate (CS) and the alkaline phosphatase (ALP) levels were determined in gingival crevicular fluid (GCF) samples collected from patients with various degrees of disease severity, including ten patients with gingivitis (50 gingivitis sites) and 33 patients with chronic periodontitis (including gingivitis, slight, moderate, and severe periodontitis sites; n = 50 each), as well as from ten healthy volunteers (50 healthy sites) by Periopaper strips. The levels of CS and ALP were measured by an ELISA and a fluorometric assay, respectively. RESULTS: The results demonstrated low levels of CS and ALP in non-destructive and slightly destructive periodontitis sites, whereas significantly high levels of these two biomolecules were shown in moderately and severely destructive sites (p < 0.05). Although a significant difference in CS levels was found between moderate and severe periodontitis sites, no difference in ALP levels was found. Stronger correlations were found between CS levels and periodontal parameters, including probing depth, loss of clinical attachment levels, gingival index and plaque index, than between ALP levels and these parameters. CONCLUSIONS: It is suggested that the CS level is a better diagnostic marker than the ALP level for evaluating distinct severity of chronic periodontitis.

Coronally advanced flap with and without a xenogenic collagen matrix in the treatment of multiple recessions: a randomized controlled clinical study.

Multiple adjacent recession defects were treated in 32 patients using a coronally advanced flap (CAF) with or without a collagen matrix (CM). The percentage of root coverage was 81.49% ± 23.45% (58% complete root coverage) for CAF sites (control) and 93.25% ± 10.01% root coverage (72% complete root coverage) for CM plus CAF sites (test). The results achieved in the test group were significantly greater than in the control group, indicating that CM plus CAF is a suitable option for the treatment of multiple adjacent gingival recessions.

Salivary antioxidants in patients with type 1 or 2 diabetes and inflammatory periodontal disease: a case-control study.

BACKGROUND: The purpose of this study was to evaluate and compare salivary concentrations of reduced, oxidized glutathione, uric acid, ascorbic acid, and total antioxidant capacity in subjects with diabetes and systemically healthy subjects with inflammatory periodontal disease. METHODS: Sixteen patients with type 1 diabetes mellitus (DM), 25 patients with type 2 DM, and 24 systemically healthy patients, all with inflammatory periodontal disease, were recruited. Whole-saliva samples were obtained, and full-mouth clinical periodontal measurements, including plaque index, probing depth, gingival recession, clinical attachment level, and bleeding on probing, were recorded at six sites per tooth. Saliva flow rate and salivary levels of reduced and oxidized glutathione, vitamin C, uric acid, and total antioxidant capacity were determined. Data were analyzed statistically by non-parametric tests. RESULTS: The subjects with type 2 DM had fewer teeth and more sites with probing depths >4 mm than the patients with type 1 DM (both P <0.01). The mean salivary reduced-glutathione concentration was lower in patients with type 1 DM than in the other two groups (both P <0.05). No significant differences in the salivary concentrations of the other antioxidants measured were found among the groups (P >0.05). Oxidized glutathione levels in the patients with type 1 DM were significantly lower than in the systemically healthy group (P = 0.007). In both groups with diabetes, salivary reduced-glutathione levels correlated positively with probing depth, and total antioxidant capacity correlated with salivary flow rate (P <0.01). CONCLUSION: The decrease in salivary reduced-glutathione levels in patients with type 1 DM may have a role in periodontal tissue destruction by predisposing tissues to oxidative stress.

Comparative immunohistochemical study of the distribution of fibronectin in healthy and diseased root surfaces.

BACKGROUND: Periodontal disease is an inflammatory process which may result in damage to and/or loss of tooth-supporting tissues, including bone, cementum, and periodontal ligament. Significant evidence supports a strong correlation between periodontitis and diseased or altered cementum. The aim of this study was to investigate the presence and distribution of fibronectin in normal human cementum and to determine whether its distribution is altered in periodontitis. METHODS: Five healthy and 10 periodontally affected teeth were collected. Following fixation and demineralization, specimens were embedded in paraffin, sectioned, and exposed to antibodies against fibronectin. Stained sections were assessed using light microscopy. RESULTS: The distribution of fibronectin, in the form of fibrils, in normal cementum was uniform in the whole cementum mass. In recession cementum, fibronectin appeared to lose its fibrillar morphology or to be completely amorphous on the whole cementum mass. Fibronectin showed variation in its distribution and fibrillar structure in pocket cementum; its absence from the cementum surface is characteristic. In the cementum apical to the pocket, fibronectin showed normal fibril structures, similar to normal cementum. CONCLUSIONS: Changes in cementum due to periodontitis include changes in the distribution and morphology of fibronectin. These changes may influence the ability for regeneration and connective tissue attachment onto periodontally affected root surfaces.

Application of the checkerboard immunoblotting technique to the quantification of host biomarkers in gingival crevicular fluid.

BACKGROUND: The aim of this study was to describe the development and validation of the checkerboard immunoblotting (CBIB) technique for the high-throughput quantification of multiple inflammatory mediators in gingival crevicular fluid (GCF) samples. METHODS: Monoclonal antibodies were used to bind GCF interleukin (IL)-1beta and -8 and matrix metalloproteinase (MMP)-8 to the surface of membranes. Biotinylated antibodies were used to detect bound antigens in a checkerboard format. Signals were developed using chemiluminescence, captured on film, and quantified using software for array analysis. The assay was tested for potential cross-reactions among the three pairs of antibodies. Eleven CBIBs were processed to determine the analytical sensitivity of the assay. Forty GCF samples were analyzed using CBIB and enzyme-linked immunosorbent assay (ELISA) in parallel, and the significance of the correlations among the results was tested using the Pearson correlation coefficient. Nine hundred thirty-one GCF samples were collected from 20 periodontally healthy subjects and 20 periodontitis subjects and analyzed using CBIB to test the assay's sensitivity and dynamic ranges using clinical samples. RESULTS: The CBIB was capable of distinguishing among the three analytes. The sensitivity and dynamic ranges of the assay were suitable for the detection of the three targets in the majority of GCF samples. There were highly statistically significant (P <0.0001) positive correlations between CBIB and ELISA data for all three biomarkers. The periodontitis subjects had statistically significantly higher mean levels of IL-1beta and -8 compared to healthy subjects. CONCLUSION: The CBIB technique is a sensitive and specific assay for the high-throughput quantification of MMP-8 and IL-8 and -1beta in GCF.

Inflammatory mediator release following bone grafting in humans: a pilot study.

AIM: The aim of this pilot study was to track markers of periodontal inflammation and bone resorption associated with decalcified freeze-dried bone allografts. MATERIAL AND METHODS: Eleven subjects completed standardized treatment of intrabony defects > or =3 mm with allografts. Gingival crevicular fluid was collected from the defect site and an adjacent interproximal site within the surgical field at baseline, 2, 4, and 8 weeks post-operatively, and analysed for biochemical markers of inflammation/bone resorption. Probing depth, recession, bleeding on probing, plaque, and 6-month radiographic bone height change were measured. RESULTS: Both prostaglandin E(2) (p=0.007) and bone-specific type 1 collagen (p=0.01) increased in crevicular fluid after 2 weeks in the bone graft sites. Matrix metalloproteinase-9 levels remained constant over time. There were positive correlations between prostaglandin levels during the first 8 weeks and bone height change over 6 months. CONCLUSIONS: Periodontal bone grafts stimulate an inflammatory response during the first 2 weeks post-operatively, and the potential negative effects of inhibiting prostaglandins post-operatively should be investigated further.

Initial apical migration of junctional epithelium in rats following application of lipopolysaccharide and proteases.

BACKGROUND: Apical migration of junctional epithelium (JE) occurs in association with periodontal pocket formation. The aim of this study was to investigate the gingival changes occurring during apical migration of the JE following application of factors associated with inflammatory periodontal disease pathogenesis. METHODS: Six-week-old male Wistar rats were divided into six groups: three experimental groups to investigate gingival changes following 2, 4, and 8 weeks topical application of lipopolysaccharide (LPS) and proteases and three control groups using pyrogen-free water. After 2, 4 or 8 weeks, nuclear DNA fragmentation was detected in periodontal ligament (PDL) fibroblasts using the terminal deoxynucleotidyl transferase-mediated dUTP-biotin nick end-labeling (TUNEL) method, and proliferative activities of the basal cells and fibroblasts were evaluated through expression of proliferating cell nuclear antigen (PCNA). Collagen destruction was examined histologically. RESULTS: Gingiva treated with LPS and proteases showed an increase in PCNA-positive basal cells but not the fibroblasts. Collagen destruction was observed at 2 weeks; apical migration of the JE and TUNEL-positive fibroblasts was seen at 4 weeks. CONCLUSIONS: Following application of LPS and proteases to rat gingival sulci, the apical migration of the JE appears to occur simultaneously with the apoptosis of PDL fibroblasts, which in turn follows proliferation of the basal cells and collagen destruction.

Calcium and phosphorus content of roots exposed to the oral environment.

This study analyzed the calcium and phosphorus content of extracted tooth roots exposed to the in vivo oral environment. 20 teeth were obtained from 16 patients and divided into two groups of 10 teeth each. In group 1, the teeth had gingival probing depths of 5 mm or more, and teeth of group 2 had gingival recessions of 3 mm or more. Prior to extraction, the gingival margin location was recorded by placing a groove on the tooth surface. After extraction, the teeth were sectioned coronal-apically, air dried and coated with carbon. Energy dispersive X-ray spectra, excited in a scanning electron microscope, were analyzed to measure relative calcium and phosphorus contents and for calculation of their ratios. X-rays were collected from two positions on the sectioned root. Experimental positions were selected within the exposed portion of the roots of groups 1 and 2, and unexposed positions were selected from that portion of the same root with attached periodontal membrane. At each position, calcium and phosphorus content was measured at 4 depths into the root surface: in cementum, in dentin three-quarters of the distance to the pulp chamber, and at 2 locations in between on either side of the cemento-dentinal junction. Analysis of data demonstrated large variations in calcium and phosphorus content from surface to surface of individual teeth and from tooth to tooth in a subject. No statistically significant differences were found between experimental and unexposed locations. Calcium and phosphorus contents were greater in roots exposed to pockets when compared to roots exposed by recession at both experimental and unexposed locations.(ABSTRACT TRUNCATED AT 250 WORDS)