Nuclear Factor Erythroid 2-Related Factor 2 Down-Regulation in Oral Neutrophils Is Associated with Periodontal Oxidative Damage and Severe Chronic Periodontitis.

The balance between reactive oxygen species and antioxidants plays an important role in periodontal health. We previously demonstrated that high reactive oxygen species production by oral polymorphonuclear neutrophils (oPMNs) in chronic periodontitis (CP) refractory to conventional therapy is associated with severe destruction of periodontium. Herein, we show that inhibition of antioxidant production through down-regulation of nuclear factor erythroid 2-related factor 2 (Nrf2) pathway in oPMN, despite enhanced recruitment in the oral cavity, is associated with severe CP. Twenty-four genes in the Nrf2-mediated oxidative stress response pathway were down-regulated in PMNs of diseased patients. Downstream of Nrf2, levels of oPMN superoxide dismutase 1 and catalase were decreased in severe CP, despite increased recruitment. Nrf2(-/-) mice had more severe loss of periodontium in response to periodontitis-inducing subgingival ligatures compared with wild-types. Levels of 8-hydroxy-deoxyguanosine were increased in periodontal lesions of Nrf2(-/-) mice, indicating high oxidative damage. We report, for the first time, Nrf2 pathway down-regulation in oPMNs of patients with severe CP. PMNs of CP patients may be primed for low antioxidant response in the context of high recruitment in the oral cavity, resulting in increased oxidative tissue damage.

Salivary Cytoprotective Proteins in Inflammation and Resolution during Experimental Gingivitis--A Pilot Study.

OBJECTIVE: The protective mechanisms that maintain periodontal homeostasis in gingivitis and prevent periodontal tissue destruction are poorly understood. The aim of this study was to identify changes in the salivary proteome during experimental gingivitis. STUDY DESIGN: We used oral neutrophil quantification and whole saliva (WS) proteomics to assess changes that occur in the inflammatory and resolution phases of gingivitis in healthy individuals. Oral neutrophils and WS samples were collected and clinical parameters measured on days 0, 7, 14, 21, 28, and 35. RESULTS: Increased oral neutrophil recruitment and salivary cytoprotective proteins increased progressively during inflammation and decreased in resolution. Oral neutrophil numbers in gingival inflammation and resolution correlated moderately with salivary ß-globin, thioredoxin, and albumin and strongly with collagen alpha-1 and G-protein coupled receptor 98. CONCLUSIONS: Our results indicate that changes in salivary cytoprotective proteins in gingivitis are associated with a similar trend in oral neutrophil recruitment and clinical parameters. CLINICAL RELEVANCE: We found moderate to strong correlations between oral neutrophil numbers and levels of several salivary cytoprotective proteins both in the development of the inflammation and in the resolution of gingivitis. Our proteomics approach identified and relatively quantified specific cytoprotective proteins in this pilot study of experimental gingivitis; however, future and more comprehensive studies are needed to clearly identify and validate those protein biomarkers when gingivitis is active.

Oral neutrophil transcriptome changes result in a pro-survival phenotype in periodontal diseases.

BACKGROUND: Periodontal diseases are inflammatory processes that occur following the influx of neutrophils into the periodontal tissues in response to the subgingival bacterial biofilm. Current literature suggests that while neutrophils are protective and prevent bacterial infections, they also appear to contribute to damage of the periodontal tissues. In the present study we compare the gene expression profile changes in neutrophils as they migrate from the circulation into the oral tissues in patients with chronic periodontits and matched healthy subjects. We hypothesized that oral neutrophils in periodontal disease patients will display a disease specific transcriptome that differs from the oral neutrophil of healthy subjects. METHODS: Venous blood and oral rinse samples were obtained from healthy subjects and chronic periodontitis patients for neutrophil isolation. mRNA was isolated from the neutrophils, and gene expression microarray analysis was completed. Results were confirmed for specific genes of interest by qRT-PCR and Western Blot analysis. RESULTS AND DISCUSSION: Chronic periodontitis patients presented with increased recruitment of neutrophils to the oral cavity. Gene expression analysis revealed differences in the expression levels of genes from several biological pathways. Using hierarchical clustering analysis, we found that the apoptosis network was significantly altered in patients with chronic inflammation in the oral cavity, with up-regulation of pro-survival members of the Bcl-2 family and down-regulation of pro-apoptosis members in the same compartment. Additional functional analysis confirmed that the percentages of viable neutrophils are significantly increased in the oral cavity of chronic periodontitis patients. CONCLUSIONS: Oral neutrophils from patients with periodontal disease displayed an altered transcriptome following migration into the oral tissues. This resulted in a pro-survival neutrophil phenotype in chronic periodontitis patients when compared with healthy subjects, resulting in a longer-lived neutrophil. This is likely to impact the severity and length of the inflammatory response in this oral disease.

Oral neutrophils display a site-specific phenotype characterized by expression of T-cell receptors.

BACKGROUND: Neutrophils, key cells of the innate immune system, were previously thought to be terminally differentiated cells, incapable of altering their gene expression after differentiation and maturation in the bone marrow. Only recently has it been shown that neutrophils perform rapid and complex changes in gene expression during inflammatory responses. Previous work by the authors has demonstrated differences in reactive oxygen species production between oral and peripheral blood neutrophils isolated from patients with chronic periodontitis, suggesting that oral neutrophils present with a unique oral phenotype. Understanding differences in the neutrophil transcriptome after transit from circulation into the site of inflammation will give new insights into how these innate immune cells function during inflammation. METHODS: Venous blood and oral rinse samples were obtained from five healthy participants. Blood neutrophils were isolated using a standard gradient method. Oral neutrophils were isolated through nylon mesh filters of different pore sizes (40 to 10 µm). RNA was purified from isolated neutrophils, and gene expression microarray analysis was completed. Results were confirmed by quantitative reverse transcription-polymerase chain reaction and immunofluorescence microscopy. RESULTS: Oral neutrophil isolation, which is critical when analyzing gene expression with samples clear of epithelial cell contamination, was optimized. It was also demonstrated that oral neutrophils present with a significant increase in T-cell receptor expression compared with circulating neutrophils, suggesting a role for oral neutrophils in crosstalk between the innate and adaptive immune system in the mouth. CONCLUSION: To the best of the authors' knowledge, it is demonstrated for the first time that, compared with circulating neutrophils, oral neutrophils present a site-specific gene expression profile in healthy individuals.

A non-invasive oral rinse assay predicts bone marrow engraftment and 6 months prognosis following allogeneic hematopoietic stem cell transplantation.

BACKGROUND: We have previously shown in a pediatric Hematopoietic stem cell transplant (HSCT) population that a non-invasive oral rinse can be used to monitor engraftment, neutrophil tissue delivery and susceptibility to infection post-HSCT. METHODS: Using the same oral rinse protocol, we studied neutrophil tissue delivery kinetics and its relationship to clinical parameters and outcomes following HSCT in 29 adult patients. Oral neutrophil counts were compared to circulating neutrophil levels, oral mucositis scores and patient health status at 6 months post-HSCT. RESULTS: Neutrophils were detected on average 8.4 ± 3.4 SD days earlier in the oral tissues than in the blood circulation, enabling us to confirm successful engraftment more than one week earlier than when using blood neutrophil counts alone. As well, in this population the time-span between oral engraftment (OE) and blood engraftment (BE) was a consistent predictor of treatment outcome at 6 months following HSCT where a BE-OE of <6 days resulted in 100% of patients having a negative outcome. CONCLUSION: We conclude that monitoring the timing of neutrophil delivery to the oral tissues with a non-invasive oral rinse has the potential to allow the physician to identify those patients who are at a high risk of HSCT failure within just a few weeks of the initiation of treatment.

Diabetes and periodontal diseases: interplay and links.

The association between diabetes and periodontal diseases is well-established. Diabetes is a risk factor for periodontal disease, with diabetic patients exhibiting an increased prevalence, extent and severity of gingivitis and perio- dontitis compared to healthy adults. Several mechanisms involved in the pathogenesis of diabetes have also been associated with periodontal disease progression. It is recognized today that there is a bidirectional relationship between diabetes and periodontal disease, with recent research showing that periodontal disease may affect the metabolic control of diabetes in diabetic patients. In this review, we present the current knowledge of the interplay between periodontal diseases and diabetes through the evaluation of randomized control and longitudinal cohort studies published in the past 15 years. Current data support the conclusion that diabetic patients are at increased risk for periodontal diseases, and that patients with poorly controlled diabetes are at risk for severe periodontitis. This results in the destruction of oral connective tissue and generalized bone loss, leading ultimately to tooth loss. Although the effect of periodontal disease on glycemic control in type 1 diabetic patients is controversial, evidence does show a direct correlation between periodontal health and glycemic control in type 2 diabetic patients. Furthermore, several studies have demonstrated the beneficial effect of periodontal treatment on metabolic control of type 2 diabetic patients.

Refractory periodontitis population characterized by a hyperactive oral neutrophil phenotype.

BACKGROUND: Neutrophils, in addition to being the primary protective component of the innate immune system, also contribute to periodontal destruction through production of reactive oxygen species (ROS), which cause damage to connective tissues and extracellular matrix after neutrophil activation. We have previously shown that hyperactive neutrophils are present in peripheral blood samples of patients diagnosed with refractory periodontitis. To test the hypothesis that oral neutrophil hyperactivity is related to periodontal disease severity, we used a flow cytometric approach to isolate and analyze oral neutrophil ROS (oROS) production in a refractory periodontal disease patient population. METHODS: Oral rinse samples and venous blood were obtained from 13 patients diagnosed with refractory periodontitis. After isolation of neutrophils from both samples, dihydrorhodamine 123 was used as a fluorescent probe for phorbol 12-myristate 13-acetate-mediated ROS production as assessed through flow cytometry. For each patient, oROS production levels were expressed as a percentage of their baseline to maximal peripheral blood neutrophil ROS production range. RESULTS: Two distinct groups of refractory patients were identified based on levels of phorbol 12-myristate 13-acetate-stimulated oROS production. The patient group with high oROS production had significantly more clinical attachment loss (AL) compared to the patient group with low oROS production. CONCLUSIONS: Our findings demonstrate that a group of refractory patients with increased clinical AL present a hyperactive oral neutrophil phenotype characterized by increased potential for ROS production. Identification of this exaggerated oral neutrophil phenotype could allow clinicians to identify which patients are more susceptible to rapid disease progression.

Bio-degradation of a resorbable collagen membrane (Bio-Gide) applied in a double-layer technique in rats.

OBJECTIVE: The aim of this study was to evaluate histologically the bio-degradation of two layers of Bio-Gide((R)) (BG) membrane, as compared with that of a single layer. MATERIAL AND METHODS: Two circular calvarial bony defects, 5 mm in diameter, were made in 24 Wistar rats. BG membrane, labeled with biotin, was cut into 5-mm-diameter disks, and placed in defects either as a mono-layer membrane (MLM) or as a double-layer membrane (DLM). Rats were sacrificed after 4 or 9 weeks and histology was performed. Membranes were stained with horseradish peroxidase-conjugated streptavidin and aminoethyl carbazole as a substrate for detection of biotinylated collagen. The area of collagen and thickness of the residual membranes were measured by image analysis software. Statistical analysis was performed using the non-parametric Wilcoxon's signed-ranks test. RESULTS: At 4-week collagen area per measurement window within the DLM sites (0.09+/-0.05 mm(2)) was significantly greater (P<0.01) than that in the MLM sites (0.047+/-0.034 mm(2)). At 9 weeks, the collagen area was also greater in the DLM sites (0.037+/-0.026 mm(2)) compared with that of the MLM sites (0.025+/-0.016 mm(2)); however, this difference did not reach statistical significance. The rate of membrane degradation, calculated as percent membrane lost compared with baseline, was similar for the DLM and MLM at both time points ( approximately 60% at 4 weeks and approximately 80% at 9 weeks). In addition, the residual DLM thickness at 4 weeks (475.5+/-73.77 mum) was significantly (P<0.01) greater than that of MLM (262.38+/-48.01 mum). At 9 weeks, membrane thickness was also greater in the DLM sites (318.22+/-70.45 mum) compared with that of the MLM sites (183.32+/-26.72 mum); however, this difference did not reach statistical significance. The reduction in thickness between 4 and 9 weeks was 30% for MLM and 33% for DLM. DISCUSSION: The use of a double layer of BG membrane results in a barrier of increased collagen area and thickness, compared with application of a single layer.

Systemic tetracycline delays degradation of three different collagen membranes in rat calvaria.

OBJECTIVES: The aim of this study was to quantitatively evaluate the effect of systemic tetracycline (TTC) on the degradation of three different collagen membranes. MATERIALS AND METHODS: Collagen membranes were cut into 5 mm diameter membrane discs and labeled with aminohexanoyl-biotin-N-hydroxy-succinimide ester. One membrane disc each of a non-cross-linked [BioGide (BG)], glutaraldehyde cross-linked [BioMend Extend (BM)], and ribose cross-linked [Ossix (OS)] was implanted on the calvaria of 40 Wistar rats. Another 10 biotinylated collagen membrane discs from each membrane type were processed for histologic observation and served as baseline; half of them (five from each group) were also treated with formic acid to inspect possible interference with biotinilazation of collagen by formic acid used during the decalcification process. A 10 mg/kg dose of TTC (50% of the minimal recommended antibacterial dose) to the experimental (20 animals) and saline to the control (20 animals) group was administered intramuscularly every 3 days. From each group, block sections were retrieved in half of the animals after 14 days and in the remaining after 28 days. Decalcified tissue histology was stained with streptavidin horseradish peroxidase. A computer-assisted program measured the membranes' collagen contents. Statistical analysis consisted of analysis of variance (ANOVA) with repeated measures. RESULTS: No statistically significant differences in collagen contents were appreciated between biotinylated non-implanted membranes treated or not treated by formic acid. Systemic TTC had a different effect on the bio-degradation of the membranes: while it significantly decreased the resorption of two of the membranes (BG and BM), it had minimal influence on the ribose cross-linked membrane (OS). ANOVA with repeated measures, tests of within-subjects effects, showed a statistically significant difference between the membranes (P<0.001), within the membranes at the different time-points (P<0.001), a significant interaction between membranes and time and between the membranes and administered TTC (P<0.001). Test of between-subject effects revealed a statistically significant interaction with time and with TTC (P<0.001). CONCLUSIONS: Systemically administered TTC in sub-antibacterial doses may offer a possible treatment alternative to reduce bio-degradation and enhance bio-durability of certain collagen membranes. The findings of the present study could have clinical application in large non-self-contained bone defects, where prolonged membrane barrier functions are desirable.

Biodegradation of three different collagen membranes in the rat calvarium: a comparative study.

BACKGROUND: Collagen barrier membranes are commonly applied in periodontal and bone-regenerative procedures. Membranes differ in their resorption pattern following implantation, thus influencing clinical outcome. The purpose of this study was to quantitatively evaluate the biodegradation of three different commercially available collagen membranes. METHODS: Collagen membranes were cut into 5-mm-diameter disks and labeled with aminohexanoyl-biotin-N-hydroxy-succinimide ester. One membrane disk of each type (non-cross-linked [NCL], glutaraldehyde cross-linked [GCL], and ribose cross-linked [RCL]) was implanted on the calvaria of 20 Wistar rats. Block sections were retrieved after 2 days (baseline, two animals), 14 days (10 animals), or 28 days (eight animals). Decalcified histologic sections were stained with streptavidin horseradish peroxidase. Residual membrane thickness and area were measured. Statistical analysis consisted of analysis of variance (ANOVA) with repeated measures. RESULTS: Statistically significant differences in the amount of residual membrane material were recorded within each membrane (among different time points) and among different membranes at the same time points (P <0.001). At 28 days, the least amount of residual collagen area, expressed as the percentage of baseline, was observed in the NCL group (13.9% +/- 10.25%), followed by the GCL (24.7% +/- 35.11%) and RCL (91.3% +/- 10.35%) groups. Residual membrane thickness, expressed as the percentage of baseline thickness, presented a similar pattern (31% +/- 16.55%, 37% +/- 41.90%, and 94.1% +/- 12.22%, respectively). ANOVA with repeated measures showed a significant interaction between membranes and time (P <0.001). CONCLUSIONS: The tested membranes differed in their degradation patterns and collagen contents. Membranes should be chosen for each clinical case according to the desired biodegradation characteristics.