Clinical models that objectively evaluate interproximal plaque based on total protein concentration.

AIM: To establish clinical models that accurately and objectively assess the levels of interproximal plaque (IPP) by measuring the total protein concentration. MATERIAL AND METHODS: The multiple treatment (MTM) and single-treatment models (STM) were designed as randomized, double-blinded, crossover studies whereby IPP was collected using floss. The total protein concentration in the plaque was then measured using a fluorescence microplate assay. To demonstrate sensitivity, the levels of residual IPP were compared for two distinctly different plaque removal routines: brushing versus brushing+flossing. RESULTS: Eighteen subjects completed the study for the MTM, while 16 participants completed the study for the STM. The results showed that both models were able to distinguish between treatments. Moreover, fundamental profiles were established that describe the mean changes in the IPP concentration over time and by treatment. Finally, a comparison between our results and a direct measure of IPP, i.e., plaque weight, demonstrated the potential of using protein as a quantitative means to assess IPP levels. CONCLUSION: The MTM and STM provide an objective, quantitative assessment of IPP based on the total protein concentration in plaque.

Effects of periodontal therapy during pregnancy on periodontal status, biologic parameters, and pregnancy outcomes: a pilot study.

BACKGROUND: Few studies have examined the potential effects of periodontal treatment during pregnancy on pregnancy outcomes, periodontal status, and inflammatory biomarkers. METHODS: A randomized, delayed-treatment, controlled pilot trial was conducted to evaluate the effects of second-trimester scaling and root planing and the use of a sonic toothbrush on the rate of preterm delivery (<37 weeks gestation). Secondary outcome measures included changes in periodontal status, levels of eight oral pathogens, levels of gingival crevicular fluid (GCF) interleukin-1beta (IL-1beta), prostaglandin E(2) (PGE(2)), 8-isoprostane (8-iso), and IL-6, and serum levels of IL-6, soluble intercellular adhesion molecule 1 (sICAM1), 8-isoprostane, soluble glycoprotein 130 (sGP130), IL-6 soluble receptor (IL-6sr), and C-reactive protein (CRP). Logistic regression models were used to test for effects of treatment on preterm delivery. Secondary outcomes were analyzed by analysis of covariance adjusting for subject baseline values. RESULTS: Periodontal intervention resulted in a significantly decreased incidence odds ratio (OR) for preterm delivery (OR = 0.26; 95% confidence interval = 0.08 to 0.85), adjusting for baseline periodontal status which was unbalanced after randomization. Pregnancy without periodontal treatment was associated with significant increases in probing depths, plaque scores, GCF IL-1beta, and GCF IL-6 levels. Intervention resulted in significant improvements in clinical status (attachment level, probing depth, plaque, gingivitis, and bleeding on probing scores) and significant decreases in levels of Prevotella nigrescens and Prevotella intermedia, serum IL-6sr, and GCF IL-1beta. CONCLUSIONS: Results from this pilot study (67 subjects) provide further evidence supporting the potential benefits of periodontal treatment on pregnancy outcomes. Treatment was safe, improved periodontal health, and prevented periodontal disease progression. Preliminary data show a 3.8-fold reduction in the rate of preterm delivery, a decrease in periodontal pathogen load, and a decrease in both GCF IL-1beta and serum markers of IL-6 response. However, further studies will be needed to substantiate these early findings.

Gemcitabine combined with cisplatin as first-line treatment in patients 60 years or older with epithelial ovarian cancer: a phase II study.

This phase II study evaluated the activity and toxicity of gemcitabine plus cisplatin as first-line treatment of patients with advanced ovarian cancer. Chemonaive patients >/=60-year-old with FIGO stage IIIC or IV epithelial ovarian carcinoma were enrolled. Patients received cisplatin 75 mg /m2 on day 1 and gemcitabine 1250 mg /m2 on day 1 (before cisplatin) and day 8 of a 21-day cycle. Of 44 female patients (median age, 70 years), 72.7% had stage IIIC disease and 67.4% had a Karnofsky performance status >/=80. Of the 37 response-evaluable patients (35 with measurable lesion[s] >/=2 cm), there were seven (18.9%) pathologic complete responses, two (5.4%) pathologic partial responses, two (5.4%) clinical complete responses, and 12 (32.4%) clinical partial responses, for an overall response rate of 62.2% (95% CI, 44.8%-77.5%), and a pathologic response rate of 24.3% (95% CI, 11.8%-41.2%). Median survival was 27.7 months (95% CI, 14.3-40.8 months). Grade 3/4 neutropenia and thrombocytopenia occurred in 59.5% and 30.2% of patients, respectively, with neutropenic fever in one patient. Grade 3 nausea /vomiting and alopecia occurred in 25.6% and 9.5% of patients, respectively. We conclude that gemcitabine plus cisplatin is active and feasible as first-line treatment of advanced epithelial ovarian cancer in patients >/=60 years. Further clinical trials adding gemcitabine to current standard, first-line treatment seem warranted in younger as well as older patients.

Subantimicrobial dose doxycycline as an adjunct to scaling and root planing: post-treatment effects.

BACKGROUND/OBJECTIVE: Subantimicrobial dose doxycycline (SDD 20 mg bid) plus scaling and root planing (SRP) significantly improved clinical attachment level (CAL) and reduced probing depth (PD) compared with placebo plus SRP in a double-blind, placebo-controlled, multicenter study of patients with adult periodontitis (AP). In a study conducted as a follow-up, the post-treatment effects of SDD were assessed in patients who completed the SRP study. METHODS: The SRP study was a 9-month, active-treatment study and the follow-up was a 3-month, no-treatment study. In the SRP study, tooth sites in qualifying quadrants were scaled and root planed and patients were randomized to receive twice daily SDD 20 mg or placebo. In the follow-up, patients received no study drug; investigators and patients remained blinded to the previous treatment group assignments. Efficacy measures included the change in CAL and PD from baseline values determined at the start of the SRP study in tooth sites stratified by baseline PD (i.e., 0-3 mm, 4-6 mm, > or =7 mm). Safety was evaluated using adverse event data and the results of clinical laboratory tests, oral pathology examinations, and microbiological assessments. RESULTS: Within each disease stratum, the incremental improvements in PD and CAL demonstrated in the SDD group over 9 months of active treatment were maintained through 3 additional months of no treatment. Treatment cessation did not result in an accelerated regression of periodontal health. No differences in the incidence of adverse events (including those related to infection) or laboratory or microbiological parameters were noted between the SDD group and the placebo group. CONCLUSIONS: The administration of SDD 20 mg bid for a period of up to 9 months is not associated with rebound effects or delayed or negative after-effects for a 3-month period after cessation of therapy.

Adult mice deficient in actinin-associated LIM-domain protein reveal a developmental pathway for right ventricular cardiomyopathy.

Although cytoskeletal mutations are known causes of genetically based forms of dilated cardiomyopathy, the pathways that link these defects with cardiomyopathy are unclear. Here we report that the alpha-actinin-associated LIM protein (ALP; Alp in mice) has an essential role in the embryonic development of the right ventricular (RV) chamber during its exposure to high biomechanical workloads in utero. Disruption of the gene encoding Alp (Alp) is associated with RV chamber dilation and dysfunction, directly implicating alpha-actinin-associated proteins in the onset of cardiomyopathy. In vitro assays showed that Alp directly enhances the capacity of alpha-actinin to cross-link actin filaments, indicating that the loss of Alp function contributes to destabilization of actin anchorage sites in cardiac muscle. Alp also colocalizes at the intercalated disc with alpha-actinin and gamma-catenin, the latter being a known disease gene for human RV dysplasia. Taken together, these studies point to a novel developmental pathway for RV dilated cardiomyopathy via instability of alpha-actinin complexes.

Confocal laser scanning microscopy and scanning electron microscopy of tissue Ti-implant interfaces.

Microscopic inspection of heterogenous three-dimensional (3D) objects such as oral implants, or implants in general, is conventionally performed either on ground sections of methyl-metacrylate-embedded material, at the cellular level by histologic analysis of the peri-implant tissue by light microscopy (LM), or at the supramolecular level by transmission electron microscopy (TEM). Alternatively, the architecture of the tissue/implant interface is visualized by scanning electron microscopy (SEM). The two approaches exclude each other because of the sample preparation.We elaborate conditions for the non-invasive analysis of tissue/implant interfaces by confocal laser scanning microscopy (CLSM) in buffer, hoping to obtain a 3D view of fluorescently labeled tissue constituents at the tissue implant interface and, through subsequent SEM, of the metal surface. The use of water-immersion objectives, originally developed for high LM under physiological conditions is essential. In an exploratory approach, the tissue/Ti-interfaces of two retrieved dental implants were analyzed. One was a step-cylinder used for orthodontic anchoring and the other was an endosseous step-screw implant retrieved after infection-related loosening prior to load. The adhering tissue fragments were fluorescently triple-labeled for actin, fibronectin, and sm-alpha-actin. Optical sections for fluorescent images and for the laser reflection map were registered concomitantly. This approach allowed the labeled structures to be located on the metal surface. Subsequently, the same implants were prepared for SEM of the tissue/implant interface, and upon removal of the adhering structures, of the underlying metal surface. Thus, specific proteins can be identified and their spatial architecture as well as that of the underlying metal surface can be visualized for one and the same implant. The immediate visualization after fluorescence labeling in buffer by means of water immersion objective lenses proved most critical.

Acoustic energy affects human gingival fibroblast proliferation but leaves protein production unchanged.

BACKGROUND, AIMS: Sonic toothbrushes are well-established in oral home care for plaque removal; however, the effects of low frequency acoustic (sonic) energy released from sonic toothbrushes to the cells of the periodontium have not been investigated. The purpose of this study was to evaluate the effects of sonic energy on human gingival fibroblast proliferation and protein production in cell culture. METHODS: Direct and indirect transfer calibration studies found the fundamental frequency of the Sonicare sonic toothbrush to be 261 hertz (Hz) with amplitudes ranging from 70 to 104 decibels (dB) in the human periodontium. Using an in vitro delivery system, which coupled a signal-wave generator with a bone transducer to mimic the energy delivered by the Sonicare toothbrush, the effects of signal, amplitude and duration were evaluated longitudinally using a gingival fibroblast cell culture model. 8 strains of fibroblasts isolated from healthy human gingiva were seeded at 30,000 cells/35 mm culture dish in minimum essential medium supplemented with 10% fetal bovine serum. To ascertain the relationship of the amplitude and the duration of sonic stimulation to cellular proliferation, gingival fibroblasts were subjected 2x daily to 261 Hz sound at various amplitudes (67-97 dB) for 0, 15, 30, 60, and 120 s on days 1, 3, 5, 7, and 10. RESULTS: It was found that either 30 or 120 s of sound exposure for 10 days of treatment had significant effects on cell proliferation in comparison to control cultures. Specifically, at day 10, 87 dB at 261 Hz for 30 s 2x daily resulted in a 25.5% increase in cell number (p<0.001), whereas 87 dB at 261 Hz for 120 s twice daily caused a 30.9% decrease in cell number (p<0.001) when compared to control cultures. When cells are stimulated under optimum acoustic conditions for 10 days, there was no difference between the treatment and control groups for collagen (p=0.897) or noncollagen (p=0.697) protein production. CONCLUSIONS: Sonic energy has been shown to both increase and decrease cellular proliferation depending on exposure time; however, during optimum sound-induced conditions for cellular proliferation, sonic energy had no effect on fibroblast protein production. These data suggest that sonic energy can affect the behavior of cells in culture. Further research into the mechanisms of these changes will provide important information for manipulating cellular behavior.

Treatment with subantimicrobial dose doxycycline improves the efficacy of scaling and root planing in patients with adult periodontitis.

BACKGROUND: In a previous study, subantimicrobial dose doxycycline (SDD) significantly improved clinical parameters associated with periodontal health in patients with adult periodontitis (AP) when used as an adjunct to a maintenance schedule of supragingival scaling and dental prophylaxis. In this double-blind, placebo-controlled, parallel-group, multicenter study, the efficacy and safety of SDD were evaluated in conjunction with scaling and root planing (SRP) in patients with AP. METHODS: Patients (n = 190) received SRP at the baseline visit and were randomized to receive either SDD 20 mg bid or placebo bid for 9 months. Efficacy parameters included the per-patient mean changes in clinical attachment level (CAL) and probing depth (PD) from baseline, the per-patient percentages of tooth sites with attachment loss (AL) > or = 2 mm and > or = 3 mm from baseline, and the per-patient percentage of tooth sites with bleeding on probing. Prior to analysis, tooth sites were stratified by the degree of disease severity evident at baseline RESULTS: In tooth sites with mild to moderate disease and severe disease (n = 183, intent-to-treat population), improvements in CAL and PD were significantly greater with adjunctive SDD than with adjunctive placebo at 3, 6, and 9 months (all P <0.05). In tooth sites with severe disease, the per-patient percentage of sites with AL > or = 2 mm from baseline to month 9 was significantly lower with adjunctive SDD than with adjunctive placebo (P<0.05). Improvements in clinical outcomes occurred without detrimental shifts in the normal periodontal flora or the acquisition of doxycycline resistance or multiantibiotic resistance. SDD was well tolerated, with a low incidence of discontinuations due to adverse events. CONCLUSIONS: The adjunctive use of SDD with SRP is more effective than SRP alone and may represent a new approach in the long-term management of AP.

Power toothbrushes, gender, and dentin hypersensitivity.

Power toothbrushes require less force for plaque removal than manual brushes. In addition, in vitro studies have indicated that brushing with low force could occlude patent dentin tubules by formation of a smear layer. Hence, lessening the force necessary to remove plaque may reduce dentin hypersensitivity. However, it was recently suggested that the use of an oscillating/rotating power toothbrush could decrease tooth sensitivity as compared to a sonic power toothbrush. Therefore, the objective of the present research was to compare the effect on dentin hypersensitivity of two different types of power brushes, the Optiva Sonicare and the Braun Oral B Ultra Plaque Remover. The null hypothesis was tested in an 8-week, randomized, parallel group, examiner-blind clinical trial. Fifty-nine subjects with a history of dentin hypersensitivity participated. Dentin hypersensitivity-associated pain was elicited using tactile and evaporative stimuli and assessed using a visual analog scale (VAS) instrument. Clinical examinations were carried out at screening and baseline and repeated after 8 weeks of twice daily use of the power brushes. Data analysis was performed on VAS scores obtained at the final visit following adjustment for group differences at baseline. A 35% to 40% reduction in pain as compared to baseline was observed in both treatment groups. Treatment-related differences were not statistically significant. A gender-related effect on dentin hypersensitivity was observed using the tactile stimulus and may merit further investigation.

Ectosomes released by human neutrophils are specialized functional units.

Here we show that human polymorphonuclear leukocytes (PMN) release ectosomes independently of complement attack during their activation both in vitro and at the site of inflammation in vivo. Patterns of biotinylated proteins on the surface of PMN and on PMN-derived ectosomes indicated a specific sorting of cell surface proteins into and out of ectosomes. Ectosomes expressed clusters of complement receptor 1 (CR1), which allowed them to bind efficiently to opsonized bacteria. Myeloperoxidase and human leukocyte elastase, both stored within the azurophilic granules of PMN, were found to colocalize on ectosomes with CR1. Furthermore, myeloperoxidase colocalized with human leukocyte elastase. In contrast, not present on CR1-expressing ectosomes were CD63, a selective marker for the azurophilic granules, and CD14, which is located within the same granules and the secretory vesicles as CR1. Of the other complement regulatory proteins expressed by PMN, only CD59 colocalized with CR1, while CD55 and CD46 were almost absent. Ectosomes released by activated PMN at the site of inflammation may function as a well organized element (ecto-organelle), designed to focus antimicrobial activity onto opsonized surfaces.

Measurement of clinical attachment levels using a constant-force periodontal probe modified to detect the cemento-enamel junction.

The handpiece of a Florida sleeve probe was modified to create a flange with the capability to detect the cemento-enamel junction (CEJ). This new instrument (the Pressure-controlled, Automated, Standardised Handpiece or Florida PASHA probe) was used to determine whether (a) the CEJ could be reproducibly detected in dried, human skulls and (b) clinical attachment levels could be reliably measured in human subjects. When using the Florida PASHA probe to detect the CEJ at 157 different sites in four dried, human skulls, there were no statistically significant (p > or = 0.15) differences in mean CEJ detection measurements for any of the three participating examiners; either when the CEJ was visualised or obscured. The mean differences between first and second replicates ranged from 0.00 to 0.08 mm. Intraclass correlation coefficients (ICCs) of repeated measures in both conditions ranged from 0.70 to 0.83 for tactile CEJ detection (CEJ obscured), and from 0.95 to 0.96 for visual detection (CEJ visible). In human studies, the Florida PASHA probe was used by two examiners to determine clinical attachment levels (CAL) at 660 sites in 5 human subjects undergoing supportive periodontal therapy. Intra-examiner agreement of replicate measurements recorded by the probe, as measured by calculating ICCs, ranged from 0.79 to 0.85 for the 2 examiners, respectively. A statistically significant inter-examiner difference in mean CAL measurements when using the Florida PASHA probe was found (p<0.001). Notwithstanding this difference, inter-examiner agreement was good, with an ICC of 0.83. These data suggest that the Florida PASHA probe can reproducibly detect the CEJ and is proposed as a tool for measuring CAL in humans.

Cost-effective design for dental randomized clinical trials with longitudinal observations.

In general, randomized clinical trials (RCT) in dentistry involve longitudinal observations. In such studies, the total cost is a function of the number of study subjects and visits, the study duration, and the type and number of examinations at each visit. In this paper, we derived the minimum cost design for longitudinal RCTs with 2 treatment arms and multiple visits. We optimized the number of subjects, visits and repeated measurements under the constraints of the requirements for statistical significance, power and minimum total study cost. A SAS macro was written and made available on the World Wide Web, so interested clinical investigators can easily find optimal designs. The application of the program is illustrated using an example.

Option-3 median measurement scheme in outlier reduction.

When measurement are subject to rare but large errors, it is better to measure twice instead of once, and, if the two measurements differ too much, to take a third measurement. This is called the option-3 scheme. This paper shows that when the measurement error is the mixture of two normal random variables, use of the median of the three measurements is better than use of the average of the two closest. To reach the maximum sample size benefit, the threshold for taking the third measurement is approximately three times the measurement error standard deviation.

Cyclosporin A and hydroxycyclosporine (M-17) affect the secretory phenotype of human gingival fibroblasts.

The responsiveness of human gingival fibroblast populations to cyclosporin A (CsA) and its principal metabolite, hydroxycyclosporine (M17), was evaluated in cell culture. Gingival fibroblasts exhibited a dose-dependent accumulation and bell-shaped distribution of dansylated CsA. A 100-fold excess of non-labeled CsA prevented the accumulation of the fluorescent probe in the fibroblasts. Both CsA (400 ng/ml) and M17 (100 ng/ml) stimulated mean gingival fibroblast cell number to 23.2% and 36.7% above controls, and reduced mean collagen production by 37.7% and 37.4% below controls, respectively; however, neither CsA nor M17 affected mean protein production in comparison to control cultures. Analyses of responses to CsA and M17 by ligand-accumulating and non-accumulating fibroblasts sorted out from the parent cultures did not provide consistent interstrain responses either by cells representing the upper quartile of fluorescence or cells representing the bottom quartiles of fluorescence. These data demonstrate that CsA is accumulated by gingival fibroblasts and that CsA and M17 are potent modulators of gingival fibroblast phenotype.

Cytotoxicity of endodontic materials.

An in vitro cell culture model of human gingival fibroblasts and L-929 cells was used to measure the cytotoxicity of currently used root canal sealers Endomet, CRCS, and AH26 and root-end filling materials Amalgam, Gallium GF2, Ketac Silver, mineral trioxide aggregate (MTA), and Super-EBA. Cytotoxic effects were assessed using the MTT assay for mitochondrial enzyme activity and the CV assay for cell numbers. Using inserts culture and L-929 fibroblasts. All-Bond-2 was also evaluated. The statistical analysis of results showed that CRCS was the least cytotoxic sealer followed by Endomet and AH26. Among root-end filling materials, MTA was not cytotoxic; Gallium GF2 displayed little cytotoxicity; and Ketac Silver, Super-EBA, and Amalgam showed higher levels of cytotoxicity. All Bond-2 also displayed a high degree of cytotoxicity. CRCS was the best root canal sealer and MTA the best root-end filling material. The outcome was favorable also for Gallium GF2 as a retrofilling material.

Reconstitution, characterisation and mass analysis of the pentacylindrical allophycocyanin core complex from the cyanobacterium Anabaena sp. PCC 7120.

The phycobilisome (PBS) of Anabaena sp. PCC 7120 was allowed to dissociate into its constituents and the resulting allophycocyanin (AP) fraction was purified. Its reconstitution yielded a complex which according to negative stain electron microscopy and spectral analysis was identical to the native pentacylindrical PBS core domain. Each cylinder of the central tricylindric unit was comprised of four AP (alphabeta)3 disks. Mass analysis using the scanning transmission electron microscope (STEM) showed the presence of 16 AP trimers in the intact reconstitute, which had a total mass of 1966(+/-66) kDa. Composition analysis indicated an AP trimer distribution of (AP-II):(AP-LCM):(AP-B):(AP-I)=6:2:2:6, i.e. an addition of two AP-I and two AP-II complexes compared to a tricylindrical PBS core domain. Therefore, we suggest that each supplementary half-core cylinder found in pentacylindrical AP core domains is comprised of one AP-I and one AP-II trimer, in agreement with the current model. The structural significance of the 127 kDa core membrane linker polypeptide was further investigated by subjecting the AP core reconstitute to mild chymotryptic degradation. After isolation, the digested complex exhibited a tricylindrical appearance while STEM mass analysis confirmed the presence of only 12 AP complexes. Polypeptide analysis by SDS-PAGE and Edman degradation related the half-cylinder loss to cleavage of the Rep4 domain of the core membrane linker polypeptide. On the basis of these data, a general model for the assembly of the three hemidiscoidal PBS types known to date is discussed.

Severe osteolysis after third-body wear due to hydroxyapatite particles from acetabular cup coating.

One concern about the fixation of HA-coated implants is the possible disintegration of the surface, with the migration of HA granules into the joint space, producing third-body wear. We report a study of six revisions of HA-coated polyethylene RM cups at 9 to 14 years after successful primary arthroplasty. In all six hips, we found HA granules embedded in the articulating surface of the polyethylene, with abrasive wear of the cup and the metal femoral head. The cup had loosened in four hips and three showed severe osteolysis of the proximal femur. Third-body wear due to HA particles from implant coating may produce severe clinical problems with few early warning signs. Further clinical, radiological and histological observations are needed to determine the possible incidence of this late complication in the various types of coating of a variety of substrates.

Evaluating the merits of option-3 methods in relative attachment level measurement in a healthy population.

Attachment loss is the principal clinical indicator for periodontal disease progression. However, the attachment measurement can be inaccurate due to outliers. A remedial scheme, the option-3 scheme, has been suggested in the literature. This scheme involves first measuring the relative attachment twice at any given site. A third measurement is required if there is a large disagreement between the first two probings. When the third measurement is taken, there are several possible ways to make the final assessment of the true attachment level. Among them are the average of the 3 measurements, the average of the closest 2, and the median of the 3. This report evaluates the merits of the option-3 scheme by comparing it with other schemes to reduce measurements variance and outliers. Since true attachment levels are unknown, we chose to check accuracy through measurement consistency between visits in healthy subjects using the fact that such subjects should have little real change in attachment levels. 12 healthy subjects participated in the study. They visited the clinic 4 times in 4 months. Two types of the Florida stent probe were used by 2 calibrated examiners. It was found that option-3 schemes were more accurate than a single probing or duplicate probings in estimating the relative attachment level. The 3 final assessment schemes performed almost equally well from a practical standpoint, but the median was the best among them in a statistical sense.

Periodontal probing.

For decades, probing clinical pocket depth and attachment level have been recognized as the dentist's most important tools in diagnosing periodontal health and disease. They are physical methods to measure the distance from the bottom of a pocket to a reference line, usually the gingival margin or the cemento-enamel junction. Probing accuracy and precision are affected by factors like the design of the probe, probing force, probe position, pocket depth, or tissue inflammation. Recently, several new electronic periodontal probes have been developed. They feature high instrument precision, allowing for measurements to the nearest tenth of a millimeter. They control for probing force and permit data to be collected and stored electronically. The purpose of this review paper is to summarize various aspects of periodontal probing. First, the history of periodontal probes will be briefly recollected, and interesting and significant inventions of the past and the present emphasized. Then, the importance of the periodontal tissues relative to probe tip penetration will be reviewed, and the probing performance will be discussed. The paper will conclude with notes on selected statistical issues.