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.

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.

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.

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 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.

Comparison of enamel matrix derivative (Emdogain) and subepithelial connective tissue graft for root coverage in patients with multiple gingival recession defects: A randomized controlled clinical study.

OBJECTIVE: The purpose of the present study was to compare the clinical efficiency of enamel matrix derivative (EMD) placed under a coronally advanced flap (CAF; test group), to a connective tissue graft (CTG) placed under a CAF (control group), in patients with multiple recession defects. METHOD AND MATERIALS: Twelve patients with multiple Miller's Class I or II gingival recessions in contralateral quadrants of the maxilla were selected. The primary outcome variable was the change in depth of the buccal recession (REC), at 6 months (T6) after surgery. The secondary outcome parameters included the clinical attachment level (CAL), the probing pocket depth (PPD), and the width of keratinized gingiva (WKT) apical to the recession. Recession defects were randomly divided to the test or control group by using a computer-generated randomization list. Data were analyzed within the frame of Mixed Linear Models with the ANOVA method. RESULTS: There were no statistically significantly differences observed between test and control groups in regards with the depth of buccal recession with a mean REC of 1.82 mm (CTG) and 1.72 mm (EMD) respectively. Similarly the mean PPD value was 1.3 mm for both groups at T6, while the respective value for CAL was 1.7 mm (EMD) and 1.8 mm (CTG). Statistically significant differences were observed only for the WKT, which were 3.0 mm and 3.6 mm for the test and control groups respectively (P < .001) at T6. CONCLUSION: The use of EMD in conjunction with a CAF resulted in similar results as compared to the CTG plus CAF.

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.

Influence of placement depth and abutment connection pattern on bone remodeling around 1-stage implants: a prospective randomized controlled clinical trial.

OBJECTIVES: The aim of this study is to evaluate peri-implant marginal bone level changes in relation to crestal or subcrestal implant placement and type of fixture/abutment connection 3 months after implant placement. MATERIALS AND METHODS: The duration of the study was 3 months. A total of 105 implants were placed in 81 subjects following a one-stage surgical procedure and assigned into four groups. In the first and second groups, implants with a screwed tapered internal connection were placed subcrestally and crestally, respectively, while in the third and fourth groups, implants with an internal conical seal connection were similarly placed. Clinical recordings and standardized periapical digital radiographs were taken the day of implantation and 3 months later, before placement of the final prosthetic restoration. The modified plaque index (mPLI), modified gingival index (mGI), and probing depths (PD) were recorded at four sites around each implant, and the vertical distance between fixture/abutment junction and alveolar crest at the mesial and distal sites of each implant utilizing subtractive radiography were all measured on placement and at 3 months. RESULTS: There was no statistically significant difference between the four groups for PD. The highest values of mPLI and mGI were recorded for Group 2. The mean (±SE) peri-implant bone loss was recorded as follows: Group 1: 0.68 ± 0.07 mm, Group 2: 0.79 ± 0.06 mm, Group 3: 0.49 ± 0.06 mm, and Group 4: 0.40 ± 0.07 mm. The statistical analysis revealed significant differences in bone resorption between groups with different abutment connections. CONCLUSIONS: The connection between fixture/abutment rather than vertical implant placement in relation to alveolar bone level seems to affect peri-implant marginal bone resorption.

The utilization of a diode laser in the surgical treatment of peri-implantitis. A randomized clinical trial.

OBJECTIVES: A comparison of different treatment modalities of peri-implantitis can lead to the development and application of more effective and efficient methods of therapy in clinical practice. This study compares the effectiveness of open flap debridement used alone, with an approach employing the additional use of a diode laser for the treatment of peri-implantitis. MATERIALS AND METHODS: Nineteen patients were divided into two groups and treated for peri-implantitis. In the control group (C group), the therapy utilized access flaps, plastic curettes, and sterilized gauzes soaked in saline. The test group (L group) was treated similarly but with additional irradiation using a diode laser. The parameters studied were pocket depth (PD) as the primary outcome variable, clinical attachment level (CAL), bleeding on probing (BOP), and plaque index (PI) as secondary variables. Measurements were performed at three different time points, baseline (BSL), 3 months, and 6 months after treatment. Three months after treatment, a mean PD reduction of 1.19 mm for the control group and 1.38 mm for the laser group was recorded. The corresponding BOP changes were 72.9 and 66.7%, respectively. These changes were significant and remained at the same levels at the 6-month examination (p < 0.05). CAL was reduced significantly only in group L from 5.25 mm at baseline to 4.54 mm at 3 months, remaining at this level at 6 months (p < 0.05). PI was reduced significantly in group C at 3 months from 37.5 to 6.3%. The 6-month data showed no statistically significant difference (p < 0.05) from the 3-month measurements. RESULTS: The two methods of therapy for peri-implantitis examined seemed to be equally efficient in the reduction of the PD and BOP 3 months after surgery, with the results sustained at the same levels after 6 months. CAL significantly improved only in the test group after 3 months. PI was reduced and maintained at low levels in both groups. CONCLUSIONS: Surgical treatment of peri-implantitis by access flaps leads to improvement of all clinical parameters studied while the additional use of diode laser does not seem to have an extra beneficiary effect. CLINICAL RELEVANCE: The additional use of a diode laser in the surgical treatment of peri-implantitis offers a limited clinical benefit.

Effects of local application of simvastatin on bone regeneration in femoral bone defects in rabbit.

Simvastatin (SIM), which is widely used in hyperlipidemia treatment, has also attracted attention due to its anabolic effects on bones. This study is designed to investigate the effectiveness of 2 mg SIM combined with 3 different carriers as delivery systems. Bone defects were surgically created in the femoral bones of 14 New Zealand white rabbits. The carriers used were the inorganic bovine bone graft (BOS), the hydroxyapatite combined with calcium sulfate (HACS), and the collagen sponge (COS). The bone defects were divided for each time period into 7 groups, as follows: passive control group (CONT), active control groups (BOS), (HACS) and (COS) (no simvastatin), and groups (BOS + SIM), (HACS + SIM) (carrier and simvastatin combination). Animal were sacrificed at 4 and 8 weeks postoperatively, and bone defects areas were prepared for histological examination and histomorphometric evaluation. Analysis of variance demonstrated statistically significant differences between groups depending on the carrier used. At 4 weeks, the BOS + SIM group presented higher rates of new bone formation, whereas at 8 weeks more new bone formation was noted for the HACS + SIM group. This study suggests that local application of simvastatin, combined with an appropriate carrier, can promote new bone formation.

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.

Immunological and microbiological findings after the application of two periodontal surgical techniques: a randomized, controlled clinical trial.

AIM: To examine microbiological and immunological alterations following two periodontal surgical techniques, over a 6-month period. MATERIALS AND METHODS: A total of 30 chronic periodontitis patients participated in the present randomized controlled clinical trial and were randomized in two groups. Modified Widman flap (MWF) was applied in the control group and apically positioned flap (APF), without intervention to the bone, in the experimental group. Gingival crevicular fluid samples and subgingival plaque samples from the operated sites were collected at baseline, 6th, 12th and 24th post-operative week. RESULTS: No major differences were noticed in immunological and microbiological profile of patients receiving either modified MWF or APF, for a period of 6 months. CONCLUSIONS: The choice of the periodontal surgical procedure does not seem to affect the immunological and the microbiological profile of patients with chronic periodontitis.

Clinical, radiographic, microbiological, and immunological outcomes of flapped vs. flapless dental implants: a prospective randomized controlled clinical trial.

OBJECTIVES: The objective of this study was to compare the placement of flapped vs. flapless dental implants utilizing clinical, radiographic, microbiological, and immunological parameters. MATERIAL AND METHODS: A total of 20 patients received 30 dental implants following a one-stage protocol. The patients were randomly assigned into two study groups: control group with 15 flapped implants and test group with 15 flapless implants. Follow-up examinations were carried out after 1, 2, 6, and 12 weeks. Clinical recordings, sulcular fluid sampling, microbiological analysis, and digital subtraction radiography were utilized to compare the two surgical approaches. RESULTS: Peri-implant sulcus depth was significantly greater in flapped implants at both 6 and 12 postsurgical weeks (P < 0.001). Flapped implants showed crestal bone loss (0.29 ± 0.06 mm), whereas no bone resorption was detected around flapless implants. Matrix metalloproteinase-8 values were higher to a statistically significant level in the control group at 1 (P = 0.003) and 6 weeks (P = 0.007) after placement. In the test group, the presence of Porphyromonas gingivalis was significantly higher at the 2nd postoperative week (P = 0.005), whereas the counts of Tannerella forsythia were significantly elevated at the 1st (P = 0.005), 2nd (P = 0.001), and 12th (P = 0.002) postoperative weeks, possibly indicating an earlier formation and maturation of the peri-implant sulcus. Patients reported more pain after flapped implant placement. CONCLUSIONS: Flapless implant placement yielded improved clinical, radiographic, and immunological outcomes compared with flapped implantation. In addition, patients seem to better withstand flapless implant placement.

Prevalence and distribution of Aggregatibacter actinomycetemcomitans serotypes and the JP2 clone in a Greek population.

AIM: to investigate the distribution of Aggregatibacter actinomycetemcomitans serotypes and the prevalence of the JP2 clone in subgingival samples of Greek subjects. MATERIALS AND METHODS: two hundred and twenty eight subjects participated in the present study. Each contributed with one pooled subgingival sample from the mesiobuccal surface of the four first molars. Samples were analysed using polymerase chain reaction for five serotypes of A. actinomycetemcomitans and the JP2 clone, using primers and conditions described previously. Subjects were stratified according to periodontal status (untreated periodontitis, non-periodontitis and periodontitis patients receiving supportive treatment). Comparisons between and within groups were performed by applying non-parametric tests (Kruskall-Wallis, Pearson χ(2) , z-test with Bonferroni's corrections and Kramer's V-test) at p=0.05 level. RESULTS: a. actinomycetemcomitans was detected statistically more frequently in untreated patients (27.5%) compared with the other two groups (11.7% for non-periodontitis and 10% for periodontitis patients receiving supportive treatment). No statistical differences were observed concerning the distribution of serotypes among groups (z-test with Bonferroni's corrections p>0.05). Serotype c was more predominant within the periodontally diseased groups (Kramer's V-test p<0.05). The JP2 clone was not detected. CONCLUSIONS: a. actinomycetemcomitans serotype b was not statistically correlated with periodontal disease in the investigated sample and the utility of microbiological testing before antimicrobial administration is emphasized.

The effect of locally delivered doxycycline in the treatment of chronic periodontitis. A clinical and microbiological cohort study.

OBJECTIVES: To evaluate the efficacy of locally delivered doxycycline as an adjunct to non-surgical treatment with the use of an ultrasonic device compared to scaling and root planing using hand instruments, by means of clinical and microbiological criteria. MATERIAL AND METHODS: Thirty three patients with chronic periodontitis participated in this cohort study and were divided into two groups. Patients in control group received scaling and root planing using hand instruments, whereas patients in control group received ultrasonic debridement and 8.8% doxycycline gel was applied after initial therapy and at 3 months at preselected sites. Clinical recordings concerning probing pocket depth, clinical attachment level, plaque index and gingival bleeding index were performed at baseline, 3 and 6 months after baseline. Subgingival samples were analysed using the "checkerboard" DNA-DNA hybridisation technique for Porphyromonas gingivalis, Tannerella forsythia and Treponema denticola. RESULTS: Both treatments resulted in significant improvement in all clinical recordings. Six months after the treatment a statistically significant decrease was observed for Porphyromonas gingivalis in both of groups and Treponema denticola in the control group (P < 0.05). No inter-group differences were observed (P < 0.05). CONCLUSIONS: Both treatment modalities provided comparable clinical and microbiological results in the treatment of chronic periodontitis.

Clinical and microbiological effects of adjunctive, locally delivered chlorhexidine on patients with chronic periodontitis.

OBJECTIVE: The impact of a locally delivered chlorhexidine chip (Periochip) on clinical and microbiological parameters of chronic periodontitis requires further documentation. The aim of the present study was to investigate the effects of the chip as an adjunct to mechanical treatment of chronic periodontitis. METHODS: Fifty patients with chronic periodontitis were randomized into two groups. The test group (n = 25) received scaling and root planing and adjunctive Periochip in four pockets. The control group (n = 25) received scaling and root planing only. Clinical indices (probing depth, probing attachment level, bleeding on probing) were assessed at baseline, three and six months. Subgingival samples were analyzed at baseline, three weeks, three and six months after treatment for levels of eight bacterial species using "checkerboard" DNA-DNA hybridization. RESULTS: The targeted difference of probing depth of 2 mm between groups was not observed. Both treatments resulted in improvement of clinical indices and non-statistically significant differences were observed between the two groups at any time point, with the exception of bleeding on probing at three months (ANOVA, p < 0.05). No major differences were observed concerning levels of important periodontal pathogens (Mann-Whitney test, p < or = 0.05). CONCLUSIONS: In this small, six-month, phase 4 trial, no differences in mean probing depth reduction or "red-complex" periodontal pathogens were detected for patients with chronic periodontitis treated with adjunctive chlorhexidine chip (single administration) as compared to patients treated with scaling and root planing alone.

Relationships among gingival crevicular fluid biomarkers, clinical parameters of periodontal disease, and the subgingival microbiota.

BACKGROUND: The objectives of this study were to measure levels of gingival crevicular fluid (GCF) biomarkers and subgingival bacterial species in periodontally healthy subjects and subjects with periodontitis to explore the relationships among these biomarkers, the subgingival microbiota, and the clinical parameters of periodontal disease. METHODS: Clinical periodontal parameters were measured at six sites per tooth in 20 subjects with periodontitis and 20 periodontally healthy subjects. GCF and subgingival plaque samples were obtained from the mesio-buccal aspect of every tooth. GCF levels of interleukin (IL)-1beta and IL-8 and matrix metalloproteinase 8 were measured using checkerboard immunoblotting, and the levels of 40 bacterial taxa were quantified using checkerboard DNA-DNA hybridization. A subset of "clinically healthy" sites from each group was analyzed separately. The significance of the differences between groups was determined using the unpaired t test or the Mann-Whitney test. Correlations among immunologic, microbiologic, and clinical data were determined using the Spearman rank correlation coefficient. RESULTS: There were positive correlations among mean clinical parameters, mean levels of the three biomarkers, and the proportions of orange and red complex species (P <0.05). Clinically healthy sites from subjects with periodontitis had higher levels of IL-1beta and IL-8 and higher proportions of orange and red complex species (P <0.05) than clinically healthy sites from periodontally healthy subjects. Red complex species were positively associated with the expression of all biomarkers (P <0.05), whereas purple and yellow complex species had negative correlations with IL-1beta and IL-8 (P <0.05). CONCLUSIONS: Clinically healthy sites from subjects with periodontitis have higher levels of GCF biomarkers and periodontal pathogens than clinically healthy sites from periodontally healthy subjects. Different microbial complexes demonstrated distinct associations with specific GCF biomarkers.

Cigarette smoking associated with advanced periodontal destruction in a Greek sample population of patients with periodontal disease.

OBJECTIVES: The objective of this study was to evaluate the possible risk factor related to the severity of periodontal destruction in an adult Greek population and to determine possible risk factors of chronic periodontal disease. METHODS: The 115 participants (mean age 47.5, range 28-74 years) were referred for periodontal treatment in two high-standard therapeutic centers. All individuals were clinically examined and answered a detailed questionnaire. The sociodemographic characteristics and periodontal findings were recorded and statistically analyzed. RESULTS: The prevalence of periodontal destruction was significantly high, as 91.3% of the participants had at least one tooth with attachment loss > or = 7 mm and 73% presented with mean loss of attachment > 4 mm. In this subject cohort, 31.3% had never smoked, 15.7% had quit smoking, and 53% were currently smokers (heavy, moderate, or occasional). Heavy smokers exhibited worse periodontal tissue breakdown and less bleeding tendency compared to moderate, infrequent, or never smokers. Among the other investigated parameters, age and customary oral hygiene practices were correlated with periodontal pocket formation and clinical attachment loss. CONCLUSIONS: The results of this study suggest that smoking appears to be a major environmental factor associated with accelerated periodontal destruction in an adult urban Greek population with regular oral hygiene habits.

Prevalence of tetM, tetQ, nim and bla(TEM) genes in the oral cavities of Greek subjects: a pilot study.

AIM: To investigate the prevalence of tetM, tetQ, nim and bla(TEM) antimicrobial resistance genes in subgingival and tongue samples of Greek subjects. MATERIALS AND METHODS: Fifty-four subjects participated in the present study. Participants each contributed with one pooled subgingival sample from the mesiobuccal surface of the four first molars and one sample from the tongue. Samples were analysed using polymerase chain reaction for tetM, tetQ, nim and bla(TEM) genes using the primers and conditions described previously. Subjects were stratified according to periodontal status (health, gingivitis or periodontitis). Intake of any antibiotic for medical or dental reasons during the previous 12 months was also recorded (self-reported). Comparisons within and between groups were performed by applying non-parametric tests (z-test with Bonferroni corrections). RESULTS: A high prevalence of tetM, tetQ and bla(TEM) genes was detected in both tongue and subgingival samples (48.1-82.2%). No differences were observed across genes between periodontally healthy, gingivitis or periodontitis cases, and no statistical correlation was observed between the presence of the bla(TEM) gene and the intake of beta-lactams during the last 12 months (Fisher's exact test, p>0.05). CONCLUSIONS: Findings from the present study suggest a high prevalence of tetM, tetQ and bla(TEM), but not nim resistance genes in subgingival and tongue samples from Greek subjects.

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.