Iain L. C. Chapple

Periodontitis in systemic rheumatic diseases

Periodontitis is a chronic inflammatory disease that is characterized by loss of the periodontal ligament and alveolar bone, and is a major cause of tooth loss. Results from clinical and epidemiologic studies have suggested that periodontitis and tooth loss are more prevalent in individuals with rheumatoid arthritis (RA). However, the strength and temporality of the association are uncertain. Several biologically plausible causal and noncausal mechanisms might account for this association between periodontitis and RA. There is evidence to suggest that periodontitis could indeed be a causal factor in the initiation and maintenance of the autoimmune inflammatory response that occurs in RA. If proven, chronic periodontitis might represent an important modifiable risk factor for RA. In addition, patients with RA might show an increased risk of developing periodontitis and tooth loss through various mechanisms. Moreover, exposure to common genetic, environmental or behavioral factors might contribute to a noncausal association between both conditions.

Clinical research on peri-implant diseases: consensus report of Working Group 4.

BACKGROUND Two systematic reviews have evaluated the quality of research and reporting of observational studies investigating the prevalence of, the incidence of and the risk factors for peri-implant diseases and of experimental clinical studies evaluating the efficacy of preventive and therapeutic interventions. MATERIALS AND METHODS For the improvement of the quality of reporting for both observational and experimental studies, the STROBE and the Modified CONSORT recommendations were encouraged. RESULTS To improve the quality of research in peri-implant diseases, the following were recommended: the use of unequivocal case definitions; the expression of outcomes at the subject rather than the implant level; the implementation of study validation tools; the reporting of potential sources of bias; and the use of appropriate statistical methods. CONCLUSIONS In observational studies, case definitions for peri-implantitis were agreed. For risk factor determination, the progressive use of cross-sectional and case-control studies (univariate analyses), to prospective cohorts (multilevel modelling for confounding), and ultimately to intervention studies were recommended. For preventive and interventional studies of peri-implant disease management, parallel arm RCTs of at least 6-months were encouraged. For studies of non-surgical and surgical management of peri-implantitis, the use of a composite therapeutic end point was advocated. The development of standard control therapies was deemed essential.

Diabetes and periodontal diseases: consensus report of the Joint EFP/AAP Workshop on Periodontitis and Systemic

BACKGROUND Diabetes and periodontitis are complex chronic diseases with an established bidirectional relationship. There is long-established evidence that hyperglycaemia in diabetes is associated with adverse periodontal outcomes. However, given the ubiquity of periodontal diseases and the emerging global diabetes epidemic, the complications of which contribute to significant morbidity and premature mortality, it is timely to review the role of periodontitis in diabetes. AIMS To report the epidemiological evidence from cross-sectional, prospective and intervention studies for the impact of periodontal disease on diabetes incidence, control and complications and to identify potential underpinning mechanisms. EPIDEMIOLOGY Over the last 20 years, consistent and robust evidence has emerged that severe periodontitis adversely affects glycaemic control in diabetes and glycaemia in non-diabetes subjects. In diabetes patients, there is a direct and dose-dependent relationship between periodontitis severity and diabetes complications. Emerging evidence supports an increased risk for diabetes onset in patients with severe periodontitis. BIOLOGICAL MECHANISMS Type 2 diabetes is preceded by systemic inflammation, leading to reduced pancreatic β-cell function, apoptosis and insulin resistance. Increasing evidence supports elevated systemic inflammation (acute-phase and oxidative stress biomarkers) resulting from the entry of periodontal organisms and their virulence factors into the circulation, providing biological plausibility for the effects of periodontitis on diabetes. AGE (Advanced Glycation Endproducts)-RAGE (Receptor for AGEs) interactions and oxidative-stress-mediated pathways provide plausible mechanistic links in the diabetes to periodontitis direction. INTERVENTIONS Randomized controlled trials (RCTs) consistently demonstrate that mechanical periodontal therapy associates with approximately a 0.4% reduction in HbA1C at 3 months, a clinical impact equivalent to adding a second drug to a pharmacological regime for diabetes. RCTs are needed with larger numbers of subjects and longer term follow-up, and if results are substantiated, adjunctive periodontal therapies subsequently need to be evaluated. There is no current evidence to support adjunctive use of antimicrobials for periodontal management of diabetes patients. GUIDELINES Given the current evidence, it is timely to provide guidelines for periodontal care in diabetes patients for medical and dental professionals and recommendations for patients/the public.

Glutathione in gingival crevicular fluid and its relation to local antioxidant capacity in periodontal health and disease

Aims: To determine possible changes in gingival crevicular fluid (GCF) antioxidant defence in chronic adult periodontal disease and to investigate the nature of the local radical scavenging mechanisms, with particular reference to glutathione. Methods: GCF and plasma were collected from patients with chronic periodontitis and age and sex matched control subjects (n = 10). Polymorphonuclear leucocytes (PMNLs) were prepared and gingival epithelial cells (GECs) were collected by conventional methods from periodontally healthy subjects. PMNL were stimulated with F-Met-Leu-Phe after cytochalasin B treatment. Enhanced chemiluminescence was used to determine the total antioxidant capacity and to investigate the activity of cell fractions and reducing agents. GCF concentrations of reduced (GSH) and oxidised (GSSG) glutathione were determined by high performance liquid chromatography. Results: Plasma and GCF from patients contained lower mean (SD) total antioxidant capacity (501.8 (123) μM Teq/litre and 658.3 (392) μM Teq/litre, respectively) compared with controls (577.9 (99.8) and 1351.5 (861) μM Teq/litre, respectively). Antioxidant light recovery profiles for GCF demonstrated a stepped response, not seen in plasma, which was inhibited by N-ethylmaleimide. This response was also detected in the cytosolic fraction of GEC and anaerobically stimulated PMNL. Similar antioxidant profiles, inhibitable by N-ethylmaleimide, were obtained with cysteamine, cysteine, and GSH. Control GCF contained high mean (SD) concentrations of glutathione (GSH, 1899.8 (494.4)μM; GSSG, 256.8 (152.4)μM). GCF from patients with periodontitis contained significantly lower amounts of GSH (mean, 1183.1; SD, 580.3μM) and GSSG (mean, 150.1; SD, 44.9μM). Conclusions: GSH values and total antioxidant capacity are reduced in chronic periodontal disease. The high concentrations of GSH present in GCF in health are similar to those found extracellularly in the lung and may represent an important antioxidant and anti-inflammatory defence strategy common to exposed epithelial surfaces.

Enhanced chemiluminescent assay for measuring the total antioxidant capacity of serum, saliva and crevicular fluid.

This paper reports the development of an enhanced chemiluminescent (ECL) assay for measuring the total antioxidant (AO) capacity of serum, saliva and a fluid collectable from the gum margin called gingival crevicular fluid (GCF). The theory behind the assay is explained, and the optimum conditions for the assay, and for storage of reagents and clinical samples is described. Calibration lines were linear (R≥0·99; P<0·0001) and the within batch coefficient of variations for a water soluble vitamin E analogue (Trolox), serum and saliva samples were <5%. In saliva and GCF, a characteristic AO response not seen in serum of the same patients, was identified. Total peripheral (serum) and local (saliva) AO capacities (μmol/L Trolox) were investigated in patients with (n = 18) and without (n = 16) adult periodontitis. Serum AO status did not differ between groups. Salivary total AO concentrations were lower in the periodontitis (P) group [175 (53)μmol/L] than in the nonperiodontitis (NP) group [254 (110)μmol/L1: P<0·01], as were saliva:serum AO ratios [0·37 (0·11) versus 0·5 (0·18): P<0·01]. Periodontitis patients may have a reduced salivary AO concentration, which could result from, or predispose to, the damaging effects of reactive oxygen species (ROS). The potential for ROS production in the oral and periodontal environment may explain the presence of a specific antioxidant in oral fluids that is not detectable in serum. The ECL assay described provides a rapid, simple and reproducible method of measuring total antioxidant defence in small volumes of biological fluids.

Hypochlorous acid regulates neutrophil extracellular trap release in humans

Neutrophil extracellular traps (NETs) comprise extracellular chromatin and granule protein complexes that immobilize and kill bacteria. NET release represents a recently discovered, novel anti‐microbial strategy regulated non‐exclusively by nicotinamide adenine dinucleotide phosphate (NADPH) oxidase generation of reactive oxygen intermediates (ROIs), particularly hydrogen peroxide. This study aimed to characterize the role of ROIs in the process of NET release and to identify the dominant ROI trigger. We employed various enzymes, inhibitors and ROIs to record their effect fluorometrically on in vitro NET release by human peripheral blood neutrophils. Treatment with exogenous superoxide dismutase (SOD) supported the established link between hydrogen peroxide and NET production. However, treatment with myeloperoxidase inhibitors and direct addition of hypochlorous acid (HOCl; generated in situ from sodium hypochlorite) established that HOCl was a necessary and sufficient ROI for NET release. This was confirmed by the ability of HOCl to stimulate NET release in chronic granulomatous disease (CGD) patient neutrophils which, due to the lack of a functional NADPH oxidase, also lack the capacity for NET release in response to classical stimuli. Moreover, the exogenous addition of taurine, abundantly present within the neutrophil cytosol, abrogated NET production stimulated by phorbol myristate acetate (PMA) and HOCl, providing a novel mode of cytoprotection by taurine against oxidative stress by taurine.

Neutrophil Hyper-responsiveness in Periodontitis

Peripheral neutrophil hyper-responsiveness in chronic periodontitis leads to excessive reactive oxygen species (ROS) production. We aimed to determine whether neutrophil hyper-responsiveness was constitutive or reactive, and to discover the effect of non-surgical therapy. Peripheral blood neutrophils from patients (n = 19), before and 3 months after therapy, and matched control individuals were Fcγ-receptor-stimulated with/without priming with P. gingivalis and F. nucleatum. Total and extracellular ROS were determined by luminol/isoluminol chemiluminescence. The high total ROS generation of patients’ neutrophils compared with that of control individuals (P = 0.016) continued at a reduced level post-therapy (P = 0.059). Reduced activity post-therapy was also seen with priming. Unstimulated total ROS levels did not differ between patients and control individuals before or after therapy. However, the high unstimulated, extracellular ROS production by patients’ neutrophils compared with control individuals (P < 0.05) continued post-therapy and was unaffected by priming. Therapy reduced Fcγ-receptor-stimulated total ROS production, but not unstimulated extracellular radical release, suggesting that constitutive and reactive mechanisms underlie neutrophil hyper-responsiveness.

Differential activation of NF-κB and gene expression in oral epithelial cells by periodontal pathogens

To investigate the molecular effects of the periodontopathogens Fusobacterium nucleatum (FN) and Porphyromonas gingivalis (PG) on the oral epithelium, the H400 oral epithelial cell line was cultured in the presence of non‐viable bacteria. Following confirmation of the presence of transcripts for the bacterial pattern recognition receptors in H400 cells, Toll‐like receptors ‐2, ‐4 and ‐9, and components of the NF‐κB signalling pathway, immunocytochemical analyses were performed showing that NF‐κB was activated within 1 h of exposure to both periodontopathogens. A significantly greater number of NF‐κB nuclear translocations were apparent following H400 cell exposure to FN as compared with PG. Gene expression analyses indicated that transcripts known to be regulated by the NF‐κB pathway, including cytokines/chemokines TNF‐α, IL‐1β, IL‐8, MCP‐1/CCL2 and GM‐CSF, were up‐regulated following 4 and 24 h of exposure to both periodontopathogens. In addition, H400 periodontopathogen exposure resulted in differential regulation of transcripts for several cytokeratin gene family members. Consistent with the immunocytochemical data, microarray results indicated that FN induced a greater number of gene expression changes than PG following 24 h of exposure, 609 and 409 genes, respectively. Ninety‐one genes were commonly differentially expressed by both periodontopathogens and represented biological processes commonly associated with periodontitis. Gene expression analyses by reserve transcriptase‐polymerase chain reaction (RT‐PCR) of molecules identified from the microarray data sets, including Heme oxygenase‐1, lysyl oxidase, SOD2, CCL20 and calprotectin components, confirmed their differential expression profiles induced by the two periodontopathogens. FN and PG have clearly different molecular effects on oral epithelial cells, potentially highlighting the importance of the composition of the plaque biofilm in periodontitis pathogenesis.

Localized delivery of growth factors for periodontal tissue regeneration: Role, strategies, and perspectives

Difficulties associated with achieving predictable periodontal regeneration, means that novel techniques need to be developed in order to regenerate the extensive soft and hard tissue destruction that results from periodontitis. Localized delivery of growth factors to the periodontium is an emerging and versatile therapeutic approach, with the potential to become a powerful tool in future regenerative periodontal therapy. Optimized delivery regimes and well‐defined release kinetics appear to be logical prerequisites for safe and efficacious clinical application of growth factors and to avoid unwanted side effects and toxicity. While adequate concentrations of growth factor(s) need to be appropriately localized, delivery vehicles are also expected to possess properties such as protein protection, precision in controlled release, biocompatibility and biodegradability, self‐regulated therapeutic activity, potential for multiple delivery, and good cell/tissue penetration. Here, current knowledge, recent advances, and future possibilities of growth factor delivery strategies are outlined for periodontal regeneration. First, the role of those growth factors that have been implicated in the periodontal healing/regeneration process, general requirements for their delivery, and the different material types available are described. A detailed discussion follows of current strategies for the selection of devices for localized growth factor delivery, with particular emphasis placed upon their advantages and disadvantages and future prospects for ongoing studies in reconstructing the tooth supporting apparatus.

Impaired neutrophil extracellular trap formation: a novel defect in the innate immune system of aged individuals

Neutrophil extracellular traps (NETs) are a recently discovered addition to the defensive armamentarium of neutrophils, assisting in the immune response against rapidly dividing bacteria. Although older adults are more susceptible to such infections, no study has examined whether aging in humans influences NET formation. We report that TNF‐α‐primed neutrophils generate significantly more NETs than unprimed neutrophils and that lipopolysaccharide (LPS)‐ and interleukin‐8 (IL‐8)‐induced NET formation exhibits a significant age‐related decline. NET formation requires generation of reactive oxygen species (ROS), and this was also reduced in neutrophils from older donors identifying a mechanism for reduced NET formation. Expression of IL‐8 receptors (CXCR1 and CXCR2) and the LPS receptor TLR4 was similar on neutrophils from young and old subjects, and neutrophils challenged with phorbol‐12‐myristate‐13‐acetate (PMA) showed no age‐associated differences in ROS or NET production. Taken together, these data suggest a defect in proximal signalling underlies the age‐related decline in NET and ROS generation. TNF‐α priming involves signalling through p38 MAP kinase, but activation kinetics were comparable in neutrophils from young and old donors. In a clinical setting, we assessed the capacity of neutrophils from young and older patients with chronic periodontitis to generate NETs in response to PMA and hypochlorous acid (HOCL). Neutrophil extracellular trap generation to HOCL, but not PMA, was lower in older periodontitis patients but not in comparison with age‐matched controls. Impaired NET formation is thus a novel defect of innate immunity in older adults but does not appear to contribute to the increased incidence of periodontitis in older adults.