Proteomics, Lipidomics, Metabolomics, and 16S DNA Sequencing of Dental Plaque From Patients With Diabetes and Periodontal Disease.

Oral microbiome influences human health, specifically prediabetes and type 2 diabetes (Pre-DM/DM) and periodontal diseases (PDs), through complex microbial interactions. To explore these relations, we performed 16S rDNA sequencing, metabolomics, lipidomics, and proteomics analyses on supragingival dental plaque collected from individuals with Pre-DM/DM (n = 39), Pre-DM/DM and PD (n = 37), PD alone (n = 11), or neither (n = 10). We identified on average 2790 operational taxonomic units and 2025 microbial and host proteins per sample and quantified 110 metabolites and 415 lipids. Plaque samples from Pre-DM/DM patients contained higher abundance of Fusobacterium and Tannerella than plaques from metabolically healthy patients. Phosphatidylcholines, plasmenyl phosphatidylcholines, ceramides containing non-OH fatty acids, and host proteins related to actin filament rearrangement were elevated in plaques from PD versus non-PD samples. Cross-omic correlation analysis enabled the detection of a strong association between Lautropia and monomethyl phosphatidylethanolamine (PE-NMe), which is striking because synthesis of PE-NMe is uncommon in oral bacteria. Lipidomics analysis of in vitro cultures of Lautropia mirabilis confirmed the synthesis of PE-NMe by the bacteria. This comprehensive analysis revealed a novel microbial metabolic pathway and significant associations of host-derived proteins with PD.

Cariogenic potential of oral nutritional supplements measured by intraoral plaque pH telemetry.

BACKGROUND & AIMS: Oral nutritional supplements (ONS) administered to malnourished elders and other patients contain high-levels of carbohydrates that could be a potential risk factor for dental caries. This study aimed to evaluate the cariogenic potentials of ONS using intraoral plaque telemetry. METHODS: Ten ONS were tested on five healthy volunteers (mean age: 76.8 ± 9.15 years). Participants were requested to refrain from performing oral hygiene 3-7 days prior to testing. The pH-value below the dental plaque on the tooth was measured while the ONS was being consumed. After neutralizing the participant's saliva, a control solution (10% sucrose) was administered and telemetry measurements were repeated. Mean relative cariogenicity (RC) was calculated for each ONS. ANOVA and post hoc tests were used for statistical analyses (p < 0.05). RESULTS: All ten ONS were potentially cariogenic on enamel with an overall RC of 0.519 ± 0.35 (Range: Min = 0.31 ± 0.16; Max = 1.00 ± 0.34). RC differed significantly between the ONS (p = 0.002). RC was lower in ONS that contained high-protein (p = 0.018). RC was not influenced by other factors such as readily consumable (p = 0.102), flavor (p = 0.869), consistency (p = 0.126), fiber containing (p = 0.134), style (p = 0.112), and age of plaque (p = 0.339). CONCLUSIONS: The ONS administered to elders and malnourished patients are potentially cariogenic. It is imperative that the administration of ONS must be based on individual needs to potentiate a maximum benefit. Wherever possible, an attempt to limit the use of high-carbohydrate containing ONS must be practiced along with the adoption of suitable preventive measures to arrest the development and progression of caries.

Enhanced purification coupled with biophysical analyses shows cross-ß structure as a core building block for Streptococcus mutans functional amyloids.

Streptococcus mutans is an etiologic agent of human dental caries that forms dental plaque biofilms containing functional amyloids. Three amyloidogenic proteins, P1, WapA, and Smu_63c were previously identified. C123 and AgA are naturally occurring amyloid-forming fragments of P1 and WapA, respectively. We determined that four amyloidophilic dyes, ThT, CDy11, BD-oligo, and MK-H4, differentiate C123, AgA, and Smu_63c amyloid from monomers, but non-specific binding to bacterial cells in the absence of amyloid precludes their utility for identifying amyloid in biofilms. Congo red-induced birefringence is a more specific indicator of amyloid formation and differentiates biofilms formed by wild-type S. mutans from a triple ΔP1/WapA/Smu_63c mutant with reduced biofilm forming capabilities. Amyloid accumulation is a late event, appearing in older S. mutans biofilms after 60 hours of growth. Amyloid derived from pure preparations of all three proteins is visualized by electron microscopy as mat-like structures. Typical amyloid fibers become evident following protease digestion to eliminate non-specific aggregates and monomers. Amyloid mats, similar in appearance to those reported in S. mutans biofilm extracellular matrices, are reconstituted by co-incubation of monomers and amyloid fibers. X-ray fiber diffraction of amyloid mats and fibers from all three proteins demonstrate patterns reflective of a cross-ß amyloid structure.

The adhesive PitA pilus protein from the early dental plaque colonizer Streptococcus oralis: expression, purification, crystallization and X-ray diffraction analysis.

PitA is the putative tip adhesin of the pilus islet 2 (PI-2)-encoded sortase-dependent pilus in the Gram-positive Streptococcus oralis, an opportunistic pathogen that often flourishes within the diseased human oral cavity. Early colonization by S. oralis and its interaction with Actinomyces oris seeds the development of oral biofilm or dental plaque. Here, the PI-2 pilus plays a vital role in mediating adherence to host surfaces and other bacteria. A recombinant form of the PitA adhesin has now been produced and crystallized. Owing to the large size (∼100 kDa), flexibility and complicated folding of PitA, obtaining diffraction-quality crystals has been a challenge. However, by the use of limited proteolysis with α-chymotrypsin, the diffraction quality of the PitA crystals was considerably enhanced to 2.16 Šresolution. These crystals belonged to space group P1, with unit-cell parameters a = 61.48, b = 70.87, c = 82.46 Å, α = 80.08, ß = 87.02, γ = 87.70°. The anomalous signal from the terbium derivative of α-chymotrypsin-treated PitA crystals prepared with terbium crystallophore (Tb-Xo4) was sufficient to obtain an interpretable electron-density map via terbium SAD phasing.

Lipidomic profiling of non-mineralized dental plaque and biofilm by untargeted UHPLC-QTOF-MS/MS and SWATH acquisition.

Dental plaque is a structurally organized biofilm which consists of diverse microbial colonies and extracellular matrix. Its composition may change when pathogenic microorganisms become dominating. Therefore, dental biofilm or plaque has been frequently investigated in the context of oral health and disease. Furthermore, its potential as an alternative matrix for analytical purposes has also been recognized in other disciplines like archeology, food sciences, and forensics. Thus, a careful in-depth characterization of dental plaque is worthwhile. Most of the conducted studies focused on the screening of microbial populations in dental plaque. Their lipid membranes, on the other hand, may significantly impact substance (metabolite) exchange within microbial colonies as well as xenobiotics uptake and incorporation into teeth. Under this umbrella, a comprehensive lipidomic profiling for determination of lipid compositions of in vivo dental plaque samples and of in vitro cultivated biofilm as surrogate matrix to be used for analytical purposes has been performed in this work. An untargeted lipidomics workflow utilizing a ultra-high-performance liquid chromatography (UHPLC)-quadrupole-time-of-flight (QTOF) platform together with comprehensive SWATH (sequential window acquisition of all theoretical fragment ion mass spectra) acquisition and compatible software (MS-DIAL) that comprises a vast lipid library has been adopted to establish an extensive lipidomic fingerprint of dental plaque. The main lipid components in dental plaque were identified as triacylglycerols, followed by cholesterol, cholesteryl esters as well as diacylglycerols, and various phospholipid classes. In vivo plaque is a rare matrix which is usually available in very low amounts. When higher quantities for specific research assays are required, efficient ways to produce an appropriate surrogate matrix are mandatory. A potential surrogate matrix substituting dental plaque was prepared by cultivation of in vitro biofilm from saliva and similarities and differences in the lipidomics profile to in vivo plaque were mapped by statistical evaluation post-analysis. It was discovered that most lipid classes were highly elevated in the in vitro biofilm samples, in particular diacylglycerols, phosphatidylglycerols, and phosphatidylethanolamines (PEs). Furthermore, an overall shift from even-chain lipid species to odd-chain lipids was observed in the cultivated biofilms. On the other hand, even-chain phosphatidylcholines (PCs), lysoPCs, cholesteryl esters, and cholesterol-sulfate were shown to be specifically increased in plaque samples. Graphical abstract.

Chemical and structural composition of black pigmented supragingival biofilm of bovines with periodontitis / Composição química e estrutural do biofilme supragengival pigmentado de bovinos com periodontite

Bovine periodontitis is a multifactorial disease primarily associated with a potentially pathogenic microbiota housed in the oral biofilm of animals. Biofilms are organized structures, in which the constituents coexist in symbiosis, already described as a predisposing factor to periodontitis in other species. The objective of the present study was to characterize the structure and chemical aspects of the bovine black pigmented supragingival biofilm using scanning electron microscopy (SEM) and energy dispersive spectroscopy (EDS), respectively, and determine its relationship with bovine periodontitis. Eleven premolar teeth from different animals were evaluated; five non-pigmented samples and six samples with black pigmented biofilms were initially submitted to SEM, and three areas of these samples were selected for EDS. The structure of the pigmented biofilm was more complex and irregular because of a higher content of mineral elements. The semi-quantitative EDS data indicated an association of iron (p<0.014) and magnesium (p<0.001) with the occurrence of periodontitis, whereas carbon, phosphorus, calcium, manganese, sodium, and potassium were not associated with the disease. Carbon (p<0.039), manganese (p<0.007), and iron (p<0.015) were associated with pigmentation, whereas phosphorus, calcium, and magnesium were not associated with it. Spearman correlation test showed the relationships between calcium and phosphorus, and iron and silicon. The strong association of iron in the pigmented supragingival biofilm and with the occurrence of periodontitis suggests the presence of microorganisms that use this element in their metabolism and that are also associated with bovine periodontitis. This study suggests that the pigmented deposits in the crown of the teeth of cattle are an true biofilm with the deposition of iron, and it indicates that the presence of iron and magnesium in these formations may be involved in the metabolism of some microorganisms associated with the etiology of bovine periodontitis.(AU)

A periodontite bovina é uma infecção multifatorial associada primariamente à microbiota potencialmente patogênica presente no biofilme bucal. Biofilmes são estruturas organizadas, nas quais os constituintes convivem em simbiose, descritos em outras espécies como um fator predisponente à periodontite. O objetivo do presente estudo foi caracterizar estrutural e quimicamente o biofilme supragengival pigmentado de preto em bovinos, utilizando-se as técnicas de microscopia eletrônica de varredura (MEV) e espectroscopia de dispersão de energia (EDS), respectivamente, correlacionando os elementos identificados à ocorrência de periodontite e pigmentação. Foram avaliados 11 dentes primeiro-molares; cinco amostras sem pigmentação visível e seis amostras com biofilme pigmentado de preto, que foram submetidas inicialmente à MEV; posteriormente foram selecionadas três áreas aleatórias de cada dente para realização da EDS. A estrutura do biofilme pigmentado revelou formações irregulares e mais complexas, provavelmente devido ao maior acúmulo de elementos minerais. Os resultados semi-quantitativos da EDS apontaram associações entre a presença de ferro (p<0,014) e magnésio (p<0,001) com a ocorrência de periodontite. Carbono, fósforo, cálcio, manganês, sódio e potássio não apresentaram associação com a periodontite. Em relação à pigmentação, carbono (p<0,039), manganês (p<0,007) e ferro (p<0,015) foram os elementos estatisticamente significantes, enquanto fósforo, cálcio e magnésio não apresentaram associação com a pigmentação. O teste de correlação de Spearman demonstrou associações entre os elementos cálcio e fósforo, e ferro e silício. A forte associação do ferro presente no biofilme supragengival com a ocorrência de periodontite, sugere a presença de micro-organismos que utilizam este elemento em seu metabolismo e que possivelmente tenham envolvimento com o desenvolvimento da periodontite bovina. Os resultados inéditos do presente trabalho sugerem que os depósitos pigmentados que se formam na coroa dos dentes de bovinos são um biofilme verdadeiro com deposição de ferro, e indicam que a presença de ferro e magnésio nestas formações pode estar envolvida no metabolismo de alguns dos principais micro-organismos associados à etiologia da periodontite bovina.(AU)

Fluoride Levels in Saliva and Plaque following the Use of High Fluoride and Conventional Dentifrices- a Triple Blinded Randomised Parallel Group Trial.

CONTEXT: The comparison of fluoride levels in saliva and plaque following the use of conventional, 2800 and 5000 ppm dentifrices for different time intervals up to 24 hours has not been explored. AIM: The aim of the present study was to assess salivary and plaque fluoride levels at different time intervals following the use of high fluoride dentifrices. STUDY DESIGN: This randomised control trial was conducted on sixty adolescents between the age group of 16 and 18 years divided into three groups A, B, and C. INTERVENTION: Subjects were asked to brush the occlusal surfaces of posterior teeth with one of the dentifrices for 2 min. Unstimulated whole saliva and plaque samples were collected at different time intervals. Fluoride levels were determined using SPADNS method. Data was collected and statistically analyzed using SPSS 20 Inc. by mixed repeated measure ANOVA. RESULTS: A significant difference in fluoride levels was observed at different time intervals in both saliva (p=0.048) and plaque (p=0.03). The variance was low with time and concentration of the dentifrice used in saliva, whereas the magnitude of treatment was large for time (>0.25) but small for (<0.09) concentration in plaque. CONCLUSION: A significant difference in fluoride levels was observed at different time intervals in both saliva and plaque among the three groups. There was a positive correlation between fluoride levels in saliva and plaque. Both high fluoride dentifrices were effective in maintaining higher intraoral fluoride levels up to 12 hours and 5000 ppm up to 24 hours compared to conventional dentifrice.

Micro-Raman Spectroscopy Reveals the Presence of Octacalcium Phosphate and Whitlockite in Association with Bacteria-Free Zones Within the Mineralized Dental Biofilm.

Through a correlative analytical approach encompassing backscattered electron scanning electron microscopy (BSE-SEM), energy dispersive X-ray spectroscopy (EDX), and micro-Raman spectroscopy, the composition of the mineralized biofilm around a dental implant, retrieved due to peri-implantitis, was investigated. The mineralized biofilm contains two morphologically distinct regions: (i) bacteria-containing zones (Bact+), characterized by aggregations of unmineralized and mineralized bacteria, and intermicrobial mineralization, and (ii) bacteria-free zones (Bact-), comprised mainly of randomly oriented mineral platelets. Intramicrobial mineralization, within Bact+, appears as smooth, solid mineral deposits resembling the morphologies of dental plaque bacteria. Bact- is associated with micrometer-sized Mg-rich mineral nodules. The Ca/P ratio of Bact+ is higher than Bact-. The inorganic phase of Bact+ is carbonated apatite (CHAp), while that of Bact- is predominantly octacalcium phosphate (OCP) and whitlockite (WL) inclusions. Compared with native bone, the inorganic phase of Bact+ (i.e., CHAp) exhibits higher mineral crystallinity, lower carbonate content, and lower Ca/P, C/Ca, Mg/Ca, and Mg/P ratios. The various CaPs found within the mineralized dental biofilm (CHAp, OCP, and WL) are related to the local presence/absence of bacteria. In combination with BSE-SEM and EDX, micro-Raman spectroscopy is a valuable analytical tool for nondestructive investigation of mineralized dental biofilm composition and development.

Influence of restorative margins position on one-stage laser-microgrooved implants-supported single screwed crowns: A clinical, biochemical, and microbiological analysis.

AIM: To clinically, biochemically, and microbiologically evaluate the influence of crown margins position on one-stage laser-microgrooved implants. MATERIALS AND METHODS: Twenty-one-stage titanium implants with a laser-microgrooved collar surface, supporting screwed, single crown restorations, were placed in 20 partially edentulous patients and evaluated. Clinical parameters included modified plaque index, modified gingival index, peri-implant probing pocket depth, bleeding on probing, and distance between implant shoulder and mucosal margin. The parameters were recorded at baseline (crowns delivery) and at every 6-month recall visit, until the end of the 3 years follow-up period. At the same time intervals, radiographic marginal bone levels were assessed at the mesial and distal aspect of the implant sites. For biochemical analysis, the volume of the peri-implant sulcus fluid, and its levels of interleukin-1beta (IL-1ß), interleukin-6 (IL-6), and of tumor necrosis factor-α, were utilized to evaluate the peri-implant health conditions at the end of the 3-year follow-up period. At the same time, microbiological analysis, including the concentration of five putative periodontal pathogens (Aggregatibacter actinomycetemcomitans, Porphyromonas gingivalis, Prevotella intermedia, Treponema denticola, and Tannerella forsythensis), were assessed. The crown margins positions were classified into four groups (A = intracrevicular position >2 mm, B = intracrevicular position ≤2 mm/<1 mm, C = intracrevicular position ≤1 mm/<0 mm, and D = extracrevicular position), and the biochemical, and microbiological parameters were evaluated at 3 years. RESULTS: No statistical differences of clinical and biochemical parameters were found between the four groups. In group A, compared to groups B, C, and D, a statistically significant higher concentration of putative periodontal pathogens was found. CONCLUSIONS: Results showed that the intracrevicular deeper position of the restoration margin does not influence the clinical and biochemical peri-implant parameters. The deeper position of the crown margin is associated with a greater amount of putative periodontal pathogenic microflora colonization.

Clinical peri-implant parameters and inflammatory cytokine profile among smokers of cigarette, e-cigarette, and waterpipe.

BACKGROUND: To date, there has been no study identified that assessed peri-implant parameters and local levels of proinflammatory cytokines between waterpipe smokers (WS) and electronic cigarette users (VS). To evaluate the true effect of the risk indicator (WS and VS), a positive control (cigarette smokers [CS]) was added in the study design. PURPOSE: To compare clinical and radiographic peri-implant parameters and proinflammatory cytokine profile in the peri-implant sulcular fluid (PISF) among CS, WS, VS, and nonsmokers (NS). MATERIALS AND METHODS: Forty CS, WS, VS, and NS were included. Details regarding demographics, duration of implants, and habits were recorded. Clinical examination for periodontal and peri-implant plaque index (PI), bleeding on probing (BOP), and probing depth (PD ≥ 4 mm) were assessed. Radiographic bone loss (RBL) was assessed using digital periapical radiographs and viewed on a calibrated computer screen using an image analyzer. Levels of tumor necrosis factor-alpha (TNF-α), interleukin (IL)-6, and IL-1ß in PISF were evaluated using enzyme-linked immunosorbent assay. RESULTS: Mean peri-implant PI (P < .05), PD ≥ 4 mm (P < .05), and total RBL (P < .01) was significantly higher among CS, WS, and VS compared with NS. Statistical differences in BOP were observed in NS (P < .01) compared to CS, WS, and VS. CS and WS showed significantly higher PD ≥ 4 mm and RBL compared with VS (P < .05). Levels of TNF-α, IL-6, and IL-1ß were significantly higher in CS, WS, and VS compared to NS. There were no statistical differences in the mean levels of all proinflammatory cytokines among individuals in CS and WS. CONCLUSION: Tobacco smoking is associated with poor peri-implant health. The effect of waterpipe smoking was of largely the same extent as that of cigarette smoking. Higher levels of inflammatory cytokines in tobacco smokers may propose increased peri-implant inflammatory process which could play a principal role in the progression of peri-implant tissue damage.

Titanium as a modifier of the peri-implant microbiome structure.

BACKGROUND: Recent data support the implication of accelerated titanium dissolution products in peri-implantitis. It is unknown whether these dissolution products have an effect on the peri-implant microbiome, the target of existing peri-implantitis therapies. PURPOSE: This study assessed the relationship between the peri-implant microbiome, dissolved titanium levels, and peri-implantitis. MATERIALS AND METHODS: Clinical, microbiome, and titanium data were collected from a periodontal population having implants in function for 10 years. Clinical examinations were performed, and submucosal plaque samples were collected from the deepest site per implant. An aliquot of the sample was used for 16S rRNA gene sequencing, with the remainder analyzed for titanium quantity using mass spectrometry. Sequences were clustered into taxonomic units at 97% minimum sequence similarity using the QIIME pipeline approach. RESULTS: Fifteen implants were assessed. According to established case definitions, six had a diagnosis of peri-implantitis; nine were healthy. The genera Streptococcus, Prevotella and Haemophilus characterized peri-implant health. Peri-implantitis was associated with a marked increase in Veillonella. Quantities of dissolved titanium were identified in 40% of sites. Titanium presence was associated with peri-implant disease status (P = .02) and correlated to the first principal component of the microbiome (rho = 0.552) and its alpha-diversity (rho = -0.496). Canonical correlation analyses found that titanium levels, but not health or disease status of the implant, were significantly associated with the microbiota composition (P = .045). CONCLUSIONS: These findings suggest an association between titanium dissolution products and peri-implantitis and support a role for these products in modifying the peri-implant microbiome structure and diversity.

In situ Effect of Arginine-Containing Dentifrice on Plaque Composition and on Enamel Demineralization under Distinct Cariogenic Conditions.

There is limited evidence that arginine-containing fluoridated dentifrices (AFD) have a better anticaries effect than regular fluoridated dentifrices (FD), especially in subjects at a higher risk for caries development. This study aimed to assess the effect of AFD on enamel demineralization and on the microbial and biochemical compositions of biofilm formed under different frequencies of sucrose exposure. It consisted of an in situ split-mouth design, where 12 adult volunteers who used FD for at least 2 months prior to the beginning of this study wore acrylic palatal appliances containing 4 bovine enamel specimens (1 pair at each side of the appliance) during 2 phases of 14 days each. FD slurry (3×/day) and 20% sucrose solution (4× and 8×/day) were dripped on the specimens during the first experimental phase. The same volunteers then used AFD during a 2-month washout period, followed by a second experimental phase where the AFD slurry and sucrose solution were applied onto a new subset of specimens. The percentage of enamel surface hardness loss (%SHL), the lesion depth (LD), the integrated mineral loss (IML), microbial counts on biofilms, the biomass, and inorganic and insoluble extracellular polysaccharide (IEPS) biofilm concentrations were determined. Higher %SHL, biomass, and IEPS and lower fluoride values were found at sucrose 8×/day exposure. Lower IEPS were found in the presence of AFD compared to FD. Similar %SHL, LD, and IML values were found between FD and AFD, irrespectively of the cariogenic challenge. The results suggest that AFD have an anticaries effect similar to that of regular FD.

Can oral ADS activity or arginine levels be a caries risk indicator? A systematic review and meta-analysis.

OBJECTIVES: The objective of this study was to evaluate the association between salivary and plaque arginine levels/ADS activities with dental caries. MATERIALS AND METHODS: A systematic search was performed as per PRISMA statement using PubMed, Scopus, Cochrane Library, and Web of Science. Published studies that investigated adults and children (P) with caries-active status (E) and caries-free status (C), whereby arginine levels/ADS activity (O) was measured in saliva/plaque to analyze exposure-outcome association compared to the control group were deemed eligible for inclusion. Quality assessment was performed using combined Newcastle-Ottawa Scale and Modified RTI Item Bank scale. Meta-analysis was performed for effect size, precision estimation, and subgroup effects analysis. RESULTS: Of 233 records identified, seven (κ = 1.00) were included for qualitative synthesis (systematic review) and four for quantitative synthesis (meta-analysis). No specific bias could be identified in five studies assessed as per the Modified RTI Item Bank scale. Two studies received lower scores on the Newcastle Ottawa scale. Plaque ADS activity in adults (effect size = 0.93, p = 0.008), salivary ADS activity in adults and children (effect size = 0.85, p < 0.001), and salivary ADS activity in adults (effect size = 0.87, p < 0.001) identified a statistically significant effect size. Subgroup analysis demonstrated non-significant variance (Q value = 0.042, p = 0.838) between saliva and plaque ADS activities of adults. CONCLUSIONS: The results of this review suggest the salivary and plaque ADS activities appear to be promising caries risk indicators for adults, while results remain inconclusive in children. CLINICAL RELEVANCE: Measuring ADS activities (saliva or plaque) can be a potential caries risk indicator in adults. The protocol was registered on PROSPERO database: CRD42017060701.

Full validation of a method for the determination of drugs of abuse in non-mineralized dental biofilm using liquid chromatography-tandem mass spectrometry and application to postmortem samples.

Alternative matrices play a major role in postmortem forensic toxicology, especially if common matrices (like body fluids or hair) are not available. Incorporation of illicit and medicinal drugs into non-mineralized dental biofilm (plaque) seems likely but has not been investigated so far. Analysis of plaque could therefore extend the spectrum of potentially used matrices in postmortem toxicology. For this reason, a rapid, simple and sensitive method for the extraction, determination and quantification of ten drugs of abuse from plaque using liquid chromatography-tandem mass spectrometry (LC-MS/MS) was developed and fully validated. Amphetamine, methamphetamine, 3,4-methylenedioxymethamphetamine (MDMA), 3,4-methylenedioxy-N-ethylamphetamine (MDEA), 3,4-methylenedioxyamphetamine (MDA), cocaine, benzoylecgonine, morphine, codeine and 6-acetylmorphine were extracted from 2mg of dried and powdered plaque via ultrasonication with acetonitrile. The extracts were analyzed on a triple-quadrupole linear ion trap mass spectrometer in scheduled multiple reaction monitoring mode (sMRM). The method was fully validated and proved accurate, precise, selective and specific with satisfactory linearity within the calibrated ranges. The lower limit of quantification was 10-15pgmg-1 for all compounds except for MDA (100pgmg-1) and amphetamine (200pgmg-1). The method has been successfully applied to three authentic postmortem samples with known drug history. Amphetamine, MDMA, cocaine, benzoylecgonine, morphine and codeine could be detected in these cases in concentrations ranging from 18pgmg-1 for cocaine to 1400pgmg-1 for amphetamine.

Fluoride Uptake Profiles of Selected European Toothpastes into Hard Tissues and Plaque.

OBJECTIVES: To compare the fluoridating potential of selected European toothpastes using a combination of enamel, dentin, and plaque in vitromodels. METHODS: Four in vitromodels were included: 1) Enamel Fluoride (F) Uptake (EFU); 2) Dentin F Uptake (DFU); 3) Enamel Solubility Reduction (ESR); and 4) Plaque F Uptake (PFU). A core set of marketed products was included in all studies, plus a standard toothpaste (1100 ppm F as NaF/silica) and placebo control (the PFU study did not include a placebo control). Test dentifrices: [A] Fluocaril® Bi-Fluoré 250 (1500 ppm F as NaF+1000 ppm F as SMFP); [B] LacerAnticaries (2500 ppm F as SMFP); [C] Elmex® Caries Professional™ (1450 ppm F as SMFP+1.5% arginine); [D] Colgate® Triple Action (1450 ppm F as SMFP); [E] Placebo (0 ppm F); and [F] standard toothpaste (1100 ppm as NaF/silica). In all studies (EFU, DFU, ESR, and PFU), assessments were compared for each pair using the Tukey-Kramer HSD test (p < 0.05). RESULTS: In all studies of fluoride uptake, the Fluocaril dentifrice [A] provided the greatest numerical benefit, regardless of the substrate. Statistical groupings were EFU: A > F = B = C = D > E; DFU: A > F = B = C = D = E; PFU: A = B > F = C = D). In demineralization prevention, the Fluocaril dentifrice [A] also provided the greatest benefit (ESR: A = F = C = B = D > E). In all studies that included a placebo control, all of the F-containing dentifrices performed better than the placebo control. CONCLUSIONS: While these results demonstrate that all of the marketed products tested provide effective anticaries benefits, the Fluocaril Bi-Fluoré 250 dentifrice consistently delivered unsurpassed performance. It delivered the highest level of F to plaque, provided greater measures of efficacy in both remineralization and inhibition of demineralization, and delivered substantial improvement in fluoridation of dentin, suggesting the potential for delivering both coronal and root caries benefits.

The strip method and the microelectrode technique in assessing dental plaque pH.

BACKGROUND: The pH strip method has been proposed for use in a clinical setting for the evaluation of dental plaque pH. The aim of this in-vivo study was to compare the reliability of the pH-indicator strip method to the microelectrode technique in plaque pH measurement. METHODS: 136 subjects (7-9 years) were enrolled and, for each subject, measurements of plaque pH were made at two interproximal sites; a total of 272 sites were analyzed. Plaque pH was assessed in triplicate using pH-indicator strips (pH range: 4.0-7.0) and an iridium microelectrode with a diameter of 0.1 mm. The caries status of 544 proximal surfaces between two primary molars, in proximity at the plaque pH sites, was assessed. All measurements were performed before and 2, 5, 10, 15, 20, and 30 minutes after a mouth rinse with water solution of sucrose (10%). One-way ANOVA was performed to analyze statistically significant differences between the two techniques. Instrument reliability was measured by assessing the intraclass correlation coefficient. RESULTS: Comparable, not statistically significant pH values (P values range from 0.98 to 1.00) were obtained by the two methods for AUC5.7, AUC6.2, maximum pH fall and minimum pH. High intraclass coefficients were recorded (Pearson's r=0.96, Yule's Q=0.99), implying an almost perfect association when the measurements were grouped as a carious lesion being present or not on the surface near the site of measurement. CONCLUSIONS: The use of the pH strip method has high validity and may replace the traditional microtouch electrode technique for clinical use.

Investigation of a Novel Predictive Biomarker Profile for the Outcome of Periodontal Treatment.

BACKGROUND: An ability to predict the response to conventional non-surgical treatment of a periodontal site would be advantageous. However, biomarkers or tests devised to achieve this have lacked sensitivity. The aim of this study is to assess the ability of a novel combination of biomarkers to predict treatment outcome of patients with chronic periodontitis. METHODS: Gingival crevicular fluid (GCF) and subgingival plaque were collected from 77 patients at three representative sites, one healthy (probing depth [PD] ≤3 mm) and two diseased (PD ≥6 mm), at baseline and at 3 and 6 months after treatment. Patients received standard non-surgical periodontal treatment at each time point as appropriate. The outcome measure was improvement in probing depth of ≥2 mm. Concentrations of active enzymes (matrix metalloproteinase [MMP]-8, elastase, and sialidase) in GCF and subgingival plaque levels of Porphyromonas gingivalis, Tannerella forsythia, and Fusobacterium nucleatum were analyzed for prediction of the outcome measure. RESULTS: Using threshold values of MMP-8 (94 ng/µL), elastase (33 ng/µL), sialidase (23 ng/µL), and levels of P. gingivalis (0.23%) and T. forsythia (0.35%), receiver operating characteristic curves analysis demonstrated that these biomarkers at baseline could differentiate healthy from diseased sites (sensitivity and specificity ≥77%). Furthermore, logistic regression showed that this combination of these biomarkers at baseline provided accurate predictions of treatment outcome (≥92%). CONCLUSION: The "fingerprint" of GCF enzymes and bacteria described here offers a way to predict the outcome of non-surgical periodontal treatment on a site-specific basis.

CPP-ACP: Effect on Dental Plaque Acidity after Water Rinsing Following Topical Fluoride Therapy.

OBJECTIVES: There is some evidence that water rinsing immediately after topical fluoride therapy has the potential to reduce the effectiveness of fluoride. The aim was to determine if covering fluoridated teeth with a layer of mousse containing CPP-ACP could prevent the adverse effect of rinsing on fluoride and consequently its buffering effect on dental plaque pH during cariogenic challenge. STUDY DESIGN: This randomized, controlled, crossover, in situ study was conducted on 25 participants. The participants were subjected to acidulated phosphate fluoride (APF) application followed by five treatment protocols: (1) water rinsing after 30 minutes (APF-30) or (2) immediate water rinsing (APF-0); (3) using CPP-ACP immediately before water rinsing (F-CPP-ACP); and two control groups: (4) no fluoride therapy (No-F) and (5) using CPP-ACP and immediate water rinsing (CPP-ACP-0). After 48 hours, teeth were rinsed with 10% sucrose solution and plaque pH was measured before and after 5, 10, 15, 20 and 30 minutes. RESULTS: The least pH changes, the lowest pH drop, and the quickest pH recovery were found in the APF-30 and F-CPP-ACP groups. APF-0 ranked in the middle and the highest values were in the control groups. CONCLUSIONS: The results show that in the case using CPP-ACP on fluoridated teeth, water rinsing immediately after topical fluoride therapy did not seem to influence the inhibitory effect of fluoride on plaque acidity.

Increased Levels of Dissolved Titanium Are Associated With Peri-Implantitis - A Cross-Sectional Study.

BACKGROUND: Peri-implantitis represents a disruption of the biocompatible interface between the titanium dioxide layer of the implant surface and the peri-implant tissues. Increasing preclinical data suggest that peri-implantitis microbiota not only triggers an inflammatory immune response but also causes electrochemical alterations of the titanium surfaces, i.e., corrosion, that aggravate this inflammatory response. Thus, it was hypothesized that there is an association between dissolution of titanium from dental implants, which suggests corrosion, and peri-implantitis in humans. The objective of this study is to compare levels of dissolved titanium in submucosal plaque collected from healthy implants and implants with peri-implantitis. METHODS: Submucosal plaque from 20 implants with peri-implantitis and 20 healthy implants was collected with sterile curets from 30 participants. Levels of titanium were quantified using inductively coupled plasma mass spectrometry and normalized for mass of bacterial DNA per sample to exclude confounding by varying amounts of plaque per site. Statistical analysis was performed using generalized estimated equations to adjust for clustering of implants per participant. RESULTS: Implants with peri-implantitis harbored significantly higher mean levels of titanium (0.85 ± 2.47) versus healthy implants (0.07 ± 0.19) after adjusting for amount of plaque collected per site (P = 0.033). CONCLUSIONS: Greater levels of dissolved titanium were detected in submucosal plaque around implants with peri-implantitis compared with healthy implants, indicating an association between titanium dissolution and peri-implantitis. Factors triggering titanium dissolution, as well as the role of titanium corrosion in the peri-implant inflammatory process, warrant further investigation.