Danae Apatzidou

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.

Nonsurgical Mechanical Treatment Strategies for Periodontal Disease

The comparison of the efficacy of surgical and nonsurgical procedures revealed that scaling and root planing alone or in combination with flap procedures are effective methods for the treatment of chronic periodontitis. Also, the consistent message is that in treating deep pockets, open-flap debridement results in greater probing pocket depth reduction and clinical attachment gain than nonsurgical modalities. Nonsurgical modalities in shallower pockets consistently involve less post-therapy recession and are clearly recognized as being more conservative. Research is still needed on the clinical benefit of the granulation tissue removal that is a feature of periodontal surgical therapy and, to a lesser extent, occurs through indirect trauma in nonsurgical therapy.

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

BackgroundDevelopment 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.MethodsDPSC 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 (β-galactosidase (SA-β-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.ResultsSimilar 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.ConclusionsThese 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.

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.

Stem cell-like populations and immunoregulatory molecules in periodontal granulation tissue

BACKGROUND AND OBJECTIVES Determine the presence of mesenchymal stem cells (MSCs) in healthy periodontal tissue and periodontal granulation tissue (GT) and explore associations between immuno-regulatory molecules and selected subgingival microorganisms. MATERIAL AND METHODS Mesenchymal stem cells were isolated, propagated and characterised by flow cytometry from a region of healthy gingival tissue and inflamed GT of 10 systemically healthy non-smokers with chronic periodontitis. Tissue levels of immunoregulatory molecules were determined by qPCR and Gingival Crevicular Fluid (GCF) levels by ELISA. Subgingival plaque levels of periodontal pathogens were determined by qPCR RESULTS: Cells with MSC-properties were isolated from both inflamed GT and healthy gingival (G) tissue. A pro-inflammatory process predominated in GT which was partly reflected in GCF and putative periodontal pathogens were higher at diseased sites. However, there was no significant difference in surface levels of mesenchymal (CD90, CD73, CD146, CD271, STRO-1), endothelial (CD105, CD106), hematopoietic (CD34, CD45) and embryonic (SSEA-4) stem cell markers between MSCs isolated from GT and G tissue. CONCLUSION Periodontal lesions, albeit inflamed, retain healing potential as inferred by the presence of MSC-like cells with similar immunophenotypic characteristics to those found in healthy periodontal tissue. Therefore, there might be merits for healing in preserving sufficient GT in-situ during periodontal surgery.

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.

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.

Microbiome associated with peri-implantitis versus periodontal health in individuals with a history of periodontal disease

ABSTRACT The microbiome of peri-implantitis sites and healthy dental sites was determined in subjects with a history of periodontitis. Ten systemically healthy non-smokers received periodontal treatment before implant placement. Plaque samples were collected from four sites of one implant with peri-implantitis (bone loss ≥2mm; PPD ≥6mm; BOP/suppuration; >1year loading) and from one dental site per quadrant with periodontal health (PPD ≤3mm, CAL <4mm, absence of BOP and bone loss). Following DNA extraction, the bacteria present in each sample were determined by high-throughput sequencing of the 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. The microbiomes of diseased samples were less diverse than those found in health, 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. Other disease associated genera were Peptostreptococcus, Tannerella, Treponema, TG5 and Atopobium. Diseased peri-implant and healthy periodontal tissues in the same individual appear to harbour distinct microbiological ecosystems with putative periodontal pathogens predominating in the peri-implantitis-associated microbiome.