Inflammatory and bone remodelling related biomarkers following periodontal transplantation of the tissue engineered biocomplex.

OBJECTIVES: To assess gingival crevicular fluid (GCF) levels of inflammatory and bone remodelling related biomarkers following transplantation of a tissue-engineered biocomplex into intrabony defects at several time-points over 12-months. MATERIALS AND METHODS: Group-A (n = 9) received the Minimal Access Flap (MAF) surgical technique combined with a biocomplex of autologous clinical-grade alveolar bone-marrow mesenchymal stem cells in collagen scaffolds enriched with an autologous fibrin/platelet lysate (aFPL). Group-B (n = 10) received the MAF surgery, with collagen scaffolds enriched with aFPL and Group-C (n = 8) received the MAF surgery alone. GCF was collected from the osseous defects of subjects via paper strips/30 sec at baseline, 6-weeks, 3-, 6-, 9-, 12-months post-surgery. Levels of inflammatory and bone remodelling-related biomarkers in GCF were determined by ELISA. RESULTS: Group-A demonstrated significantly higher GCF levels of BMP-7 at 6-9 months than baseline, with gradually decreasing levels of pro-inflammatory and pro-osteoclastogenic markers (TNF-α, RANKL) over the study-period; and an overall decrease in the RANKL/OPG ratio at 9-12 months than baseline (all p < 0.001). In comparison, only modest interim changes were observed in Groups-B and -C. CONCLUSIONS: At the protein level, the approach of MAF and biocomplex transplantation provided greater tissue regeneration potential as cell-based therapy appeared to modulate inflammation and bone remodelling in residual periodontal defects. CLINICAL RELEVANCE: Transplantation of a tissue engineered construct into periodontal intrabony defects demonstrated a biochemical pattern for inflammatory control and tissue regeneration over 12-months compared to the control treatments. Understanding the biological healing events of stem cell transplantation may facilitate the design of novel treatment strategies. CLINICAL DATABASE REGISTRATION: ClinicalTrials.gov ID: NCT02449005.

Characterization of the soft-tissue wall lining residual periodontal pockets and implications in periodontal wound healing.

AIM: To characterize the soft-tissue wall of remaining periodontal pockets for wound healing-related parameters versus healthy gingival crevices in the same individuals. MATERIALS AND METHODS: Gingival tissues collected from the diseased interface of pockets (GT biopsies) and from healthy gingival crevices (G biopsies) were subjected to RT2-profiler PCR Array for wound healing-related markers and network analysis of differentially expressed genes. Lymphangiogenesis-related gene expression was determined by qRT-PCR. The migration potential of mesenchymal stem cells isolated from GT biopsies (GT-MSCs) and G biopsies (G-MSCs) was evaluated by the scratch- and the transwell migration assays. The total collagen protein content was determined in GT-MSCs and G-MSCs homogenates. RESULTS: Gene-ontology analysis on significantly upregulated genes expressed in GT biopsies revealed enrichment of several genes involved in processes related to matrix remodeling, collagen deposition, and integrin signaling. No significantly expressed genes were seen in G biopsies. Regarding lymphangiogenesis-related genes, GT biopsies demonstrated greater expression for PROX1 than G biopsies (p = 0.05). Lower migration potential (p < 0.001), yet greater production of collagen protein (p = 0.05), was found for GT-MSCs over G-MSCs. CONCLUSION: Differential expression patterns of various molecular pathways in biopsies and cell cultures of diseased versus healthy gingival tissues indicate a potential of the former for tissue remodeling and repair. CLINICAL RELEVANCE: In the course of periodontitis, granulation tissue is formed within a periodontal defect in an attempt to reconstruct the site. Following treatment procedures periodontal granulation tissue remains inflamed but appears to retain healing potential.

Subgingival instrumentation.

The S3-level clinical guidelines for the treatment of patients with periodontitis stages I-III published by the European Federation of Periodontology in 2020, suggest a pre-established stepwise approach for oral-healthcare professionals with precise therapeutic pathways. The second step of this approach consists of the subgingival instrumentation of periodontal pockets by non-surgical means to disrupt the microbial biofilm and remove soft and mineralized deposits This step aims to resolve periodontal inflammation by closure of periodontal pockets (probing pocket depth ≤ 4 mm, absence of bleeding on probing) employing different types of instruments and treatment protocols toward this end. Novel non-surgical treatment approaches that adopt micro instruments or subgingival application of biological agents have been recently tested. Subgingival instrumentation has been shown to effectively restore the subgingival microbiota to one associated with periodontal health and to modulate the inflammatory response. The outcomes of the subgingival instrumentation have to be evaluated in order to guide the therapist in providing additional but focused treatment in the remaining pockets OR at sites with residual inflammation. Of great importance is the impact that non-surgical periodontal treatment has on the patient's well-being, based on evidence that emerges from studies evaluating patient related outcomes and quality of life.

Biological Effects of "Inflammageing" on Human Oral Cells: Insights into a Potential Confounder of Age-Related Diseases.

OBJECTIVES: The term "inflammageing" describes the process of inflammation-induced aging that leads living cells to a state of permanent cell cycle arrest due to chronic antigenic irritation. This in vitro study aimed to shed light on the mechanisms of "inflammageing" on human oral cells. METHODS: Primary cultures of human gingival fibroblasts (hGFs) were exposed to variable pro-inflammatory stimuli, including lipopolysaccharide (LPS), Tumor Necrosis Factor-alpha (TNFa), and gingival crevicular fluid (GCF) collected from active periodontal pockets of systemically healthy patients. Inflammageing was studied through two experimental models, employing either late-passage ("aged") cells (p. 10) that were exposed to the pro-inflammatory stimuli or early-passage ("young") cells (p. 1) continuously exposed during a period of several passages (up to p. 10) to the above-mentioned stimuli. Cells were evaluated for the expression of beta-galactosidase activity (histochemical staining), senescence-associated genes (qPCR analysis), and biomarkers related to a Senescence-Associated Secretory Phenotype (SASP), through proteome profile analysis and bioinformatics. RESULTS: A significant increase (p < 0.05) in beta-galactosidase-positive cells was observed after exposure to each pro-inflammatory stimulus. The senescence-associated gene expression included upregulation for CCND1 and downregulation for SUSD6, and STAG1, a profile typical for cellular senescence. Overall, pro-inflammatory priming of late-passage cells caused more pronounced effects in terms of senescence than long-term exposure of early-passage cells to these stimuli. Proteomic analysis showed induction of SASP, evidenced by upregulation of several pro-inflammatory proteins (IL-6, IL-10, IL-16, IP-10, MCP-1, MCP-2, M-CSF, MIP-1a, MIP-1b, TNFb, sTNF-RI, sTNF-RII, TIMP-2) implicated in cellular aging and immune responses. The least potent impact on the induction of SASP was provoked by LPS and the most pronounced by GCF. CONCLUSION: This study demonstrates that long-term exposure of hGFs to various pro-inflammatory signals induced or accelerated cellular senescence with the most pronounced impact noted for the late-passage cells. The outcome of these analyses provides insights into oral chronic inflammation as a potential confounder of age-related diseases.

The role of cigarette smoking in periodontal disease and treatment outcomes of dental implant therapy.

Tobacco smoking has been implicated in periodontal pathology through various mechanisms, including perturbations of the inflammatory and host responses to putative periodontal pathogens, alterations in the subgingival microbial communities, and a compromised healing potential of the tissues leading to imbalance of tissue homeostasis. This review provides the evidence for the relationship between cigarette smoking and periodontal disease in an attempt to explain possible mechanisms of how tobacco smoking may exert its negative effects on the periodontal tissues via systemic and localized pathways. Early and more recent studies explore cigarette smoking-induced changes in periodontal clinical indices; in subgingival microbial flora by employing traditional detection methods for selected microorganisms, in addition to modern techniques such as deep sequencing and bioinformatics analyses that are able to fully characterize the microbial communities; and in inflammatory and immune responses critically appraising study limitations and differences in study protocol designs. Periodontal treatment outcomes and implant therapy outcomes are reviewed in an attempt to shed light on possible mechanisms for the inferior treatment outcome noted in smokers. The potential harmful effects of passive smoking are also reviewed, providing evidence for the advantages of smoking cessation. Quitting cigarette smoking should be recommended by the dentist, and effort should be made to inform smokers about the negative effects of smoking on the periodontal status and implant therapy outcomes.

Microbial-stem cell interactions in periodontal disease.

Periodontitis is initiated by hyper-inflammatory responses in the periodontal tissues that generate dysbiotic ecological changes within the microbial communities. As a result, supportive tissues of the tooth are damaged and periodontal attachment is lost. Gingival recession, formation of periodontal pockets with the presence of bleeding, and often suppuration and/or tooth mobility are evident upon clinical examination. These changes may ultimately lead to tooth loss. Mesenchymal stem cells (MSCs) are implicated in controlling periodontal disease progression and have been shown to play a key role in periodontal tissue homeostasis and regeneration. Evidence shows that MSCs interact with subgingival microorganisms and their by-products and modulate the activity of immune cells by either paracrine mechanisms or direct cell-to-cell contact. The aim of this review is to reveal the interactions that take place between microbes and in particular periodontal pathogens and MSCs in order to understand the factors and mechanisms that modulate the regenerative capacity of periodontal tissues and the ability of the host to defend against putative pathogens. The clinical implications of these interactions in terms of anti-inflammatory and paracrine responses of MSCs, anti-microbial properties and alterations in function including their regenerative potential are critically discussed based on literature findings. In addition, future directions to design periodontal research models and study ex vivo the microbial-stem cell interactions are introduced.

A tissue-engineered biocomplex for periodontal reconstruction. A proof-of-principle randomized clinical study.

AIM: To assess the safety/efficacy of a tissue-engineered biocomplex in periodontal reconstruction. METHODS: Twenty-seven intrabony defects were block-randomized across three treatment groups: Group-A (NA  = 9) received autologous clinical-grade alveolar bone marrow mesenchymal stem cells (a-BMMSCs), seeded into collagen scaffolds, enriched with autologous fibrin/platelet lysate (aFPL). In Group-B (NB  = 10), the collagen scaffold/aFPL devoid of a-BMMSCs filled the osseous defect. Group-C (NC  = 8) received Minimal Access Flap surgery retaining the soft tissue wall of defects identically with Groups-A/-B. Subjects were clinically/radiographically assessed before anaesthesia (baseline) and repeatedly over 12 months. RESULTS: Quality controls were satisfied before biocomplex transplantation. There were no adverse healing events. All approaches led to significant clinical improvements (p < .001) with no inter-group differences. At 12 months, the estimated marginal means for all groups were as follows: 3.0 (95% CI: 1.9-4.1) mm for attachment gain; 3.7 (2.7-4.8) mm for probing pocket depth reduction; 0.7 (0.2-1.3) mm increase in recession. An overall greater mean reduction in the radiographic Cemento-Enamel Junction to Bottom Defect (CEJ-BD) distance was found for Groups-A/-C over Group-B (p < .023). CONCLUSION: Radiographic evidence of bone fill was less pronounced in Group-B, although clinical improvements were similar across groups. All approaches aimed to trigger the innate healing potential of tissues. Cell-based therapy is justified for periodontal reconstruction and remains promising in selected cases.

Evaluation of stemness properties of cells derived from granulation tissue of peri-implantitis lesions.

OBJECTIVES: Peri-implantitis (PI) is an inflammatory disease associated with peri-implant bone loss and impaired healing potential. There is limited evidence about the presence of mesenchymal stromal cells (MSCs) and their regenerative properties within the granulation tissue (GT) of infrabony peri-implantitis defects. The aim of the present study was to characterize the cells derived from the GT of infrabony PI lesions (peri-implantitis derived mesenchymal stromal cells-PIMSCs). MATERIAL AND METHODS: PIMSC cultures were established from GT harvested from PI lesions with a pocket probing depth ≥6 mm, bleeding on probing/suppuration, and radiographic evidence of an infrabony component from four systemically healthy individuals. Cultures were analyzed for embryonic (SSEA4, NANOG, SOX2, OCT4A), mesenchymal (CD90, CD73, CD105, CD146, STRO1) and hematopoietic (CD34, CD45) stem cell markers using flow cytometry. PIMSC cultures were induced for neurogenic, angiogenic and osteogenic differentiation by respective media. Cultures were analyzed for morphological changes and mineralization potential (Alizarin Red S method). Gene expression of neurogenic (NEFL, NCAM1, TUBB3, ENO2), angiogenic (VEGFR1, VEGFR2, PECAM1) and osteogenic (ALPL, BGLAP, BMP2, RUNX2) markers was determined by quantitative RT-PCR. RESULTS: PIMSC cultures demonstrated high expression of embryonic and mesenchymal stem cell markers with inter-individual variability. After exposure to neurogenic, angiogenic and osteogenic conditions, PIMSCs showed pronounced tri-lineage differentiation potential, as evidenced by their morphology and expression of respective markers. High mineralization potential was observed. CONCLUSIONS: This study provides evidence that MSC-like populations reside within the GT of PI lesions and exhibit a multilineage differentiation potential. Further studies are needed to specify the biological role of these cells in the healing processes of inflamed PI tissues and to provide indications for their potential use in regenerative therapies.

Understanding the microbial components of periodontal diseases and periodontal treatment-induced microbiological shifts.

In the mid-1960s the microbial aetiology of periodontal diseases was introduced based on classical experimental gingivitis studies . Since then, numerous studies have addressed the fundamental role that oral microbiota plays in the initiation and progression of periodontal diseases. Recent advances in laboratory identification techniques have contributed to a better understanding of the complexity of the oral microbiome in both health and disease. Modern culture-independent methods such as human oral microbial identification microarray and next-generation sequencing have been used to identify a wide variety of microbial taxa residing in the gingival sulcus and the periodontal pocket. The first theory of the 'non-specific plaque' hypothesis gave rise to the 'ecological plaque' hypothesis and more recently to the 'polymicrobial synergy and dysbiosis hypothesis'. Periodontitis is now considered to be a multimicrobial inflammatory disease in which the various bacterial species within the dental biofilm are in a dysbiotic state and this imbalance favours the establishment of chronic inflammatory conditions and ultimately the destruction of tooth-supporting tissues. Apart from the known putative periodontal pathogens, the whole biofilm community is now considered to play a role in the establishment of inflammation and the initiation and progression of periodontitis in a susceptible host. Treatment is unlikely to eliminate putative pathogens but, when it is thoroughly performed it has the potential to establish a healthy ecosystem by altering the microbial community in numbers and composition and also contribute to the maturation of the host immune response.

Interim tissue changes following connective tissue grafting and two-stage implant placement. A randomized clinical trial.

AIM: To determine tissue changes at implants placed either conventionally or in combination with a connective tissue graft (CTG). MATERIALS AND METHODS: Forty-eight partially edentulous subjects were randomized into two treatment Groups, and 46 completed the study. Group-A (NA  = 23) received crestal implant placement. In Group-B (NB  = 23), a CTG harvested from the palate was stabilized over the implant neck. At the time of implant placement (T0), Groups were categorized as having thin mucosa ≤ 2.5 mm at the surgical site (NSubgroup-AI  = 12, NSubgroup-BI  = 11) or thick mucosa > 2.5 mm (NSubgroup-AII  = 11, NSubgroup-BII  = 12). Mucosa thickness, width of keratinized tissue (WKT), crestal bone levels and relative bone thickness were determined at T0 and at the two-stage surgery (T1). RESULTS: At T1, on the alveolar crest, mucosa thickness significantly decreased in the thick mucosa Subgroups (-AII/-BII, both p = 0.001), but increased at thin mucosa grafted sites (p = 0.049). No significant changes were noted in the WKT for either group. Thin mucosa grafted Subgroups (-AI/-BI) demonstrated significant decreases in crestal bone levels (both p ≤ 0.008). Crestal relative bone thickness decreased in all Subgroups (p ≤ 0.027 for significant changes). CONCLUSIONS: Connective tissue grafting resulted in smaller reductions of mucosa thickness on the alveolar crest and appeared to have the greater effects at sites with initially thin mucosa.

Biomaterials and regenerative technologies used in bone regeneration in the craniomaxillofacial region: Consensus report of group 2 of the 15th European Workshop on Periodontology on Bone Regeneration.

BACKGROUND AND AIMS: To review the regenerative technologies used in bone regeneration: bone grafts, barrier membranes, bioactive factors and cell therapies. MATERIAL AND METHODS: Four background review publications served to elaborate this consensus report. RESULTS AND CONCLUSIONS: Biomaterials used as bone grafts must meet specific requirements: biocompatibility, porosity, osteoconductivity, osteoinductivity, surface properties, biodegradability, mechanical properties, angiogenicity, handling and manufacturing processes. Currently used biomaterials have demonstrated advantages and limitations based on the fulfilment of these requirements. Similarly, membranes for guided bone regeneration (GBR) must fulfil specific properties and potential biological mechanisms to improve their clinical applicability. Pre-clinical and clinical studies have evaluated the added effect of bone morphogenetic proteins (mainly BMP-2) and autologous platelet concentrates (APCs) when used as bioactive agents to enhance bone regeneration. Three main approaches using cell therapies to enhance bone regeneration have been evaluated: (a) "minimally manipulated" whole tissue fractions; (b) ex vivo expanded "uncommitted" stem/progenitor cells; and (c) ex vivo expanded "committed" bone-/periosteum-derived cells. Based on the evidence from clinical trials, transplantation of cells, most commonly whole bone marrow aspirates (BMA) or bone marrow aspirate concentrations (BMAC), in combination with biomaterial scaffolds has demonstrated an additional effect in sinus augmentation and horizontal ridge augmentation, and comparable bone regeneration to autogenous bone in alveolar cleft repair.

Clinical associations between acetylcholine levels and cholinesterase activity in saliva and gingival crevicular fluid and periodontal diseases.

AIM: The oral mucosa possesses a non-neuronal cholinergic system. This study aimed to determine clinical evidence for a role of cholinergic mechanisms in the pathogenesis of periodontal diseases. MATERIALS AND METHODS: Fifty healthy participants, 52 patients with gingivitis and 49 with periodontitis were recruited. Full periodontal parameters were recorded and saliva and gingival crevicular fluid (GCF) collected. Levels of acetylcholine and inflammatory mediators were quantified using commercially available assay kits. Acetylcholinesterase and butyrylcholinesterase activities were measured using a published biochemical assay. RESULTS: Acetylcholine levels are significantly elevated in saliva and GCF, whereas GCF levels of butyrylcholinesterase activity are significantly decreased, in patients with periodontal diseases. Acetylcholine levels in saliva and GCF correlated positively with clinical markers of disease severity and with increased levels of IL-17A and IL-17F. In contrast, butyrylcholinesterase activity levels in GCF showed significant negative correlations with clinical markers of disease severity and IL-17A and IL-17F levels. None of the findings were due to smoking. CONCLUSIONS: Elevated acetylcholine levels and reduced butyrylcholinesterase activity are clinically associated with periodontal diseases and elevated levels of IL-17A and IL-17F. Therefore, non-neuronal cholinergic mechanisms may influence IL-17 biology and the aetiopathogenesis of periodontal diseases and therefore are possible therapeutic targets.

Isolation and prolonged expansion of oral mesenchymal stem cells under clinical-grade, GMP-compliant conditions differentially affects "stemness" properties.

BACKGROUND: Development of clinical-grade cell preparations is central to meeting the regulatory requirements for cellular therapies under good manufacturing practice-compliant (cGMP) conditions. Since addition of animal serum in culture media may compromise safe and efficient expansion of mesenchymal stem cells (MSCs) for clinical use, this study aimed to investigate the potential of two serum/xeno-free, cGMP culture systems to maintain long-term "stemness" of oral MSCs (dental pulp stem cells (DPSCs) and alveolar bone marrow MSCs (aBMMSCs)), compared to conventional serum-based expansion. METHODS: DPSC and aBMMSC cultures (n = 6/cell type) were established from pulp and alveolar osseous biopsies respectively. Three culture systems were used: StemPro_MSC/SFM_XenoFree (Life Technologies); StemMacs_MSC/XF (Miltenyi Biotek); and α-MEM (Life Technologies) with 15% fetal bovine serum. Growth (population doublings (PDs)), immunophenotypic (flow cytometric analysis of MSC markers) and senescence (ß-galactosidase (SA-ß-gal) activity; telomere length) characteristics were determined during prolonged expansion. Gene expression patterns of osteogenic (ALP, BMP-2), adipogenic (LPL, PPAR-γ) and chondrogenic (ACAN, SOX-9) markers and maintenance of multilineage differentiation potential were determined by real-time PCR. RESULTS: Similar isolation efficiency and stable growth dynamics up to passage 10 were observed for DPSCs under all expansion conditions. aBMMSCs showed lower cumulative PDs compared to DPSCs, and when StemMacs was used substantial delays in cell proliferation were noted after passages 6-7. Serum/xeno-free expansion produced cultures with homogeneous spindle-shaped phenotypes, while serum-based expansion preserved differential heterogeneous characteristics of each MSC population. Prolonged expansion of both MSC types but in particular the serum/xeno-free-expanded aBMMSCs was associated with downregulation of CD146, CD105, Stro-1, SSEA-1 and SSEA-4, but not CD90, CD73 and CD49f, in parallel with an increase of SA-gal-positive cells, cell size and granularity and a decrease in telomere length. Expansion under both serum-free systems resulted in "osteogenic pre-disposition", evidenced by upregulation of osteogenic markers and elimination of chondrogenic and adipogenic markers, while serum-based expansion produced only minor changes. DPSCs retained a diminishing (CCM, StemPro) or increasing (StemMacs) mineralization potential with passaging, while aBMMSCs lost this potential after passages 6-7 under all expansion conditions. CONCLUSIONS: These findings indicate there is still a vacant role for development of qualified protocols for clinical-grade expansion of oral MSCs; a key milestone achievement for translation of research from the bench to clinics.

Microbiome of peri-implantitis affected and healthy dental sites in patients with a history of chronic periodontitis.

OBJECTIVE: To determine the composition of the microbiome of peri-implantitis sites and corresponding dental sites in subjects with a history of chronic periodontitis. DESIGN: Clinical and radiographic examination assessed the periodontal/peri-implant disease status. Plaque samples were collected from one diseased implant with peri-implantitis, functional for at least two years and healthy sites in ten non-smokers who had received periodontal treatment prior to implant placement. Following DNA extraction, the bacteria present in each sample were determined by high-throughput sequencing of V3-V4 region of the 16S rRNA gene using the Illumina MiSeq platform. OTUs were picked using QIIME. Differences between dental and implant sites were determined using linear discriminant analysis, effect size and diversity analyses were conducted using PAST v3.02. RESULTS: The microbiomes of healthy samples were more diverse than those found in disease, although disease was associated with a higher abundance of taxa relative to health. The genera Actinobacillus and Streptococcus were most closely associated with health, whereas Prevotella and Porphyromonas were most discriminative for disease. Synergistetes were highly associated with peri-implantitis. CONCLUSION: In patients with a history of periodontitis, putative periodontal pathogens prevailed in the microbiome of diseased implants. Diseased implants and corresponding healthy sites appear to have distinct microbiological ecosystems.

Control of dentin/root sensitivity during non-surgical and surgical periodontal treatment.

AIM: To determine the efficacy of a desensitizing regimen compared to a control in preventing the occurrence and/or alleviating dentin/root sensitivity (DRS) following non-surgical (NSPT) and surgical periodontal treatment (SPT). METHODS: Seventy-four chronic-periodontitis patients (CPP) were randomized into a test group (n = 38) using an in-office prophylaxis paste and a toothpaste at home both containing 8% arginine and calcium carbonate (Pro-Argin(™) Technology) or into a control group (n = 36) receiving a fluoride-free prophylaxis paste and a fluoride toothpaste. The examiner applied the assigned paste onto selected teeth for 3 s following NSPT and for 60 s before flap closure. Patients brushed with the assigned toothpaste twice daily throughout the study. DRS to air stimulus was assessed by the Schiff scale (0-3) and the Visual Analog Scale (VAS: 0-100 mm) six times over 17 weeks. RESULTS: In the test group, VAS scores significantly decreased at 8, 11 and 17 weeks from baseline (p ≤ 0.003) and Schiff scores at 8 and 11 weeks from baseline (p ≤ 0.014). The control group exhibited significant increases in VAS and Schiff during the study period (p ≤ 0.006). Marked inter-group differences were noted at all time points (p < 0.001). CONCLUSIONS: The combined use of desensitizing products (8% arginine and calcium carbonate) in-office and at-home prevented DRS development and maintained this effect for 17 weeks following NSPT and SPT.

Smoking, Porphyromonas gingivalis and the immune response to citrullinated autoantigens before the clinical onset of rheumatoid arthritis in a Southern European nested case-control study.

BACKGROUND: Antibodies to citrullinated proteins (ACPA) occur years before RA diagnosis. Porphyromonas gingivalis expresses its own peptidylarginine deiminase (PPAD), and is a proposed aetiological factor for the ACPA response. Smoking is a risk factor for both ACPA-positive RA and periodontitis. We aimed to study the relation of these factors to the risk of RA in a prospective cohort. METHODS: We performed a nested case-control study by identifying pre-RA cases in four populations from the European Prospective Investigation into Cancer and nutrition, matched with three controls. Data on smoking and other covariates were obtained from baseline questionnaires. Antibodies to CCP2 and citrullinated peptides from α-enolase, fibrinogen, vimentin and PPAD were measured. Antibodies to arginine gingipain (RgpB) were used as a marker for P.gingivalis infection and validated in a separate cohort of healthy controls and subjects with periodontitis. RESULTS: We studied 103 pre-RA cases. RA development was associated with several ACPA specificities, but not with antibodies to citrullinated PPAD peptides. Antibody levels to RgpB and PPAD peptides were higher in smokers but were not associated with risk of RA or with pre-RA autoimmunity. Former but not current smoking was associated with antibodies to α-enolase (OR 4.06; 95 % CI 1.02, 16.2 versus 0.54; 0.09-3.73) and fibrinogen peptides (OR 4.24; 95 % CI 1.2-14.96 versus 0.58; 0.13-2.70), and later development of RA (OR 2.48; 95 % CI 1.27-4.84 versus 1.57; 0.85-2.93), independent of smoking intensity. CONCLUSIONS: Smoking remains a risk factor for RA well before the clinical onset of disease. In this cohort, P.gingivalis is not associated with pre-RA autoimmunity or risk of RA in an early phase before disease-onset. Antibodies to PPAD peptides are not an early feature of ACPA ontogeny.

Oral hygiene reinforcement in the simplified periodontal treatment of 1 hour.

AIM: To compare the clinical and microbiological outcome of the 1-h ultrasonic debridement of chronic periodontitis patients (CPP) with and without frequent sessions of oral hygiene reinforcement. METHODS: Clinical measurements and subgingival plaque were collected from 44 CPP at baseline, 3- and 6-months. The control group received a single session of 1-h full-mouth ultrasonic debridement, while oral hygiene instructions (OHI) were reiterated over four visits. In the test group, OHI were limited in the 1-h treatment session. At 3-months, both groups received additional debridement and OHI. The "Checkerboard" DNA-DNA hybridization technique quantified Porphyromonas gingivalis, Tannerella forsythia and Treponema denticola in plaque. RESULTS: At three months, smaller reductions in plaque and bleeding indices, and in P. gingivalis numbers were noted in the test group, while these differences disappeared at six months. After the 3-month re-treatment visit, the test group presented with a greater probing pocket depth (PPD) reduction. Plaque negatively affected PPD in a similar manner after both treatment approaches. CONCLUSIONS: Lack of oral hygiene reinforcement in the 1-h full-mouth debridement resulted in higher plaque and bleeding scores and numbers of P. gingivalis at three months; professional removal of dental biofilm every three months is beneficial in subjects with compromised plaque control.

Influence of periodontal disease, Porphyromonas gingivalis and cigarette smoking on systemic anti-citrullinated peptide antibody titres.

BACKGROUND: Anti-citrullinated protein antibody (ACPA) responses may precede clinical onset of rheumatoid arthritis. Porphyromonas gingivalis peptidylarginine deiminase can citrullinate proteins possibly inducing autoimmunity in susceptible individuals. AIM: To determine whether periodontitis, carriage of P. gingivalis, smoking and periodontal therapy influence ACPA titres. METHODS: Serum and plaque samples were collected from 39 periodontitis patients before and after non-surgical periodontal treatment, and from 36 healthy subjects. Carriage of P. gingivalis was determined by PCR of plaque DNA. ACPA was determined by anti-cyclic citrullinated peptide (CCP) enzyme-linked immunosorbent assay (ELISA). Anti-P. gingivalis titres were determined by ELISA. RESULTS: Untreated periodontitis patients had higher anti-CCP antibody titres than healthy controls [three patients (8%) greater than manufacturer suggested assay diagnostic threshold (5 Assay Units/AU) versus none (0%); mean ± SEM: 1.37 ± 0.23 versus 0.40 ± 0.10 AU, p < 0.0001]. Periodontitis patients who smoked demonstrated lower anti-P. gingivalis (15956 ± 4385 versus 2512 ± 1290 Units/ml, p < 0.05), but similar anti-CCP than non-smoking periodontitis patients (smokers: 1.31 ± 0.35; non-smokers: 1.41 ± 0.32 AU). Healthy smokers demonstrated elevated anti-CCP titres (0.75 ± 0.19 AU), at levels between healthy non-smokers (0.15 ± 0.05 AU) and non-smoker periodontitis patients. Six months after periodontal treatment, there were significant reductions in anti-CCP (non-smokers p < 0.05) and anti-P. gingivalis (all participants p < 0.01). CONCLUSION: In subjects with periodontitis, P. gingivalis infection may be responsible for inducing autoimmune responses that characterize rheumatoid arthritis.

Modern approaches to non-surgical biofilm management.

The subgingival dental plaque is a microbial biofilm consisting of highly variable bacterial microcolonies embedded within a self-produced matrix of extracellular polymeric substance. In contrast to microorganisms growing in a planktonic state, the inhabitants of a biofilm are effectively protected within this dense structure from host defense mechanisms and from therapeutic agents, including antimicrobials. The mechanical removal of the microbial biofilm and the establishment of meticulous plaque control measures comprise the key elements for the success of non-surgical periodontal treatment. Ultrasonic devices are effective in disrupting the biofilm, and carefully remove soft and hard deposits from a root surface with minimal trauma to the tooth structure. Controversies and modern trends in non-surgical periodontal therapy - such as quadrant-wise treatment modalities versus full-mouth approaches, hand-versus power-driven instrumentation, and the time frame of non-surgical periodontal therapy - are discussed here in depth in order to provide an insight into modern approaches to non-surgical biofilm management. Clinical, microbiological and immunological findings following different treatment protocols, in addition to cost-effective benefits of these clinical modalities, are discussed.