Development and Application of Reversible and Irreversible Covalent Probes for Human and Mouse Cathepsin-K Activity Detection, Revealing Nuclear Activity.

Cathepsin-K (CTSK) is an osteoclast-secreted cysteine protease that efficiently cleaves extracellular matrices and promotes bone homeostasis and remodeling, making it an excellent therapeutic target. Detection of CTSK activity in complex biological samples using tailored tools such as activity-based probes (ABPs) will aid tremendously in drug development. Here, potent and selective CTSK probes are designed and created, comparing irreversible and reversible covalent ABPs with improved recognition components and electrophiles. The newly developed CTSK ABPs precisely detect active CTSK in mouse and human cells and tissues, from diseased and healthy states such as inflamed tooth implants, osteoclasts, and lung samples, indicating changes in CTSK's activity in the pathological samples. These probes are used to study how acidic pH stimulates mature CTSK activation, specifically, its transition from pro-form to mature form. Furthermore, this study reveals for the first time, why intact cells and cell lysate exhibit diverse CTSK activity while having equal levels of mature CTSK enzyme. Interestingly, these tools enabled the discovery of active CTSK in human osteoclast nuclei and in the nucleoli. Altogether, these novel probes are excellent research tools and can be applied in vivo to examine CTSK activity and inhibition in diverse diseases without immunogenicity hazards.

In Vitro Effect of Streptococcus mutans Biofilm Produced in Sugar-Free Coca-Cola on Enamel.

OBJECTIVE: Sugary drinks such as Coca-Cola may expedite dental caries. For this reason, sugar-free drinks like Coca-Cola Zero Sugar (CZ) may be considered advantageous. This research aims to evaluate in vitro the CZ effect in the presence of Streptococcus mutans (S. mutans) biofilm on enamel demineralization. METHODS: Ninety-six human enamel slabs (4 × 4 mm) were used. S. mutans UA-159 72-hour biofilm was created over enamel surfaces. The specimens were soaked in CZ, HCl, or 10% sucrose in PBS solution, 3 times a day for 15 minutes over the course of 4 days. Viable counts (CFU/mL) and biofilm biomass (Crystal Violet staining) were evaluated. pH was measured after each exposure. After 4 days, Demineralization was evaluated clinically and by Vickers microhardness tests. Slabs were photographed using a stereomicroscope before and after exposure to caries-promoting conditions. RESULTS: Slabs that were soaked in CZ showed an increase in viable counts compared to control and almost similar counts with 10% sucrose in PBS solution exposures (1010and109CFUmL, respectivly). Biofilm biomass tests showed a 25% higher bacterial growth in the CZ group. CZ pH measures were the lowest and the only group to show a decrease in pH over time (pH ∼3). Enamel slabs that were evaluated clinically in the stereomicroscope postexposures had a chalky and matt appearance as opposed to their shiny appearance in the baseline evaluation. CONCLUSIONS: CZ creates a favourable environment for the growth of S. mutans. It may be suggested that even though CZ is sugar free it has a cariogenic effect on enamel. CLINICAL SIGNIFICANCE: Clinicians need to educate patients that sugar-free carbonated drinks may be just as harmful as regular carbonated drinks, and hence avoided. This research emphasizes the harmful effect sugar-free carbonated drinks on teeth and sheds new light on their cariogenic potential.

Trans-Cinnamaldehyde-Fighting Streptococcus mutans Using Nature.

Streptococcus mutans (S. mutans) is the main cariogenic bacterium with acidophilic properties, in part due to its acid-producing and -resistant properties. As a result of this activity, hard tooth structures may demineralize and form caries. Trans-cinnamaldehyde (TC) is a phytochemical from the cinnamon plant that has established antibacterial properties for Gram-positive and -negative bacteria. This research sought to assess the antibacterial and antibiofilm effects of trans-cinnamaldehyde on S. mutans. TC was diluted to a concentration range of 156.25-5000 µg/mL in dimethyl sulfoxide (DMSO) 0.03-1%, an organic solvent. Antibacterial activity was monitored by testing the range of TC concentrations on 24 h planktonic growth compared with untreated S. mutans. The subminimal bactericidal concentrations (MBCs) were used to evaluate the bacterial distribution and morphology in the biofilms. Our in vitro data established a TC MBC of 2500 µg/mL against planktonic S. mutans using a microplate spectrophotometer. Furthermore, the DMSO-only controls showed no antibacterial effect against planktonic S. mutans. Next, the sub-MBC doses exhibited antibiofilm action at TC doses of ≥625 µg/mL on hydroxyapatite discs, as demonstrated through biofilm analysis using spinning-disk confocal microscopy (SDCM) and high-resolution scanning electron microscopy (HR-SEM). Our findings show that TC possesses potent antibacterial and antibiofilm properties against S. mutans. Our data insinuate that the most effective sub-MBC of TC to bestow these activities is 625 µg/mL.

Impact of dietary interventions on pre-diabetic oral and gut microbiome, metabolites and cytokines.

Diabetes and associated comorbidities are a global health threat on the rise. We conducted a six-month dietary intervention in pre-diabetic individuals (NCT03222791), to mitigate the hyperglycemia and enhance metabolic health. The current work explores early diabetes markers in the 200 individuals who completed the trial. We find 166 of 2,803 measured features, including oral and gut microbial species and pathways, serum metabolites and cytokines, show significant change in response to a personalized postprandial glucose-targeting diet or the standard of care Mediterranean diet. These changes include established markers of hyperglycemia as well as novel features that can now be investigated as potential therapeutic targets. Our results indicate the microbiome mediates the effect of diet on glycemic, metabolic and immune measurements, with gut microbiome compositional change explaining 12.25% of serum metabolites variance. Although the gut microbiome displays greater compositional changes compared to the oral microbiome, the oral microbiome demonstrates more changes at the genetic level, with trends dependent on environmental richness and species prevalence in the population. In conclusion, our study shows dietary interventions can affect the microbiome, cardiometabolic profile and immune response of the host, and that these factors are well associated with each other, and can be harnessed for new therapeutic modalities.

Biocompatibility, Surface Morphology, and Bacterial Load of Dental Implant Abutments following Decontamination Protocols: An In-Vitro Study.

The long-term success of dental implant rehabilitation depends significantly on proper peri-implant soft tissue integration. Therefore, decontamination of abutments prior to their connection to the implant is beneficial to enhance soft tissue attachment and to aid in maintaining marginal bone around the implant. Consequently, different implant abutment decontamination protocols were evaluated regarding biocompatibility, surface morphology, and bacterial load. The protocols evaluated were autoclave sterilization, ultrasonic washing, steam cleaning, chlorhexidine chemical decontamination, and sodium hypochlorite chemical decontamination. The control groups included: (1) implant abutments prepared and polished in a dental lab without decontamination and (2) unprepared implant abutments obtained directly from the company. Surface analysis was performed using scanning electron microscopy (SEM). Biocompatibility was evaluated using XTT cell viability and proliferation assays. Biofilm biomass and viable counts (CFU/mL) (n = 5 for each test) were used for surface bacterial load evaluation. Surface analysis revealed areas of debris and accumulation of materials, such as iron, cobalt, chromium, and other metals, in all abutments prepared by the lab and with all decontamination protocols. Steam cleaning was the most efficient method for reducing contamination. Chlorhexidine and sodium hypochlorite left residual materials on the abutments. XTT results showed that the chlorhexidine group (M = 0.7005, SD = 0.2995) had the lowest values (p < 0.001) (autoclave: M = 3.6354, SD = 0.1510; ultrasonic: M = 3.4077, SD = 0.3730; steam: M = 3.2903, SD = 0.2172; NaOCl: M = 3.5377, SD = 0.0927; prep non-decont.: M = 3.4815, SD = 0.2326; factory: M = 3.6173, SD = 0.0392). Bacterial growth (CFU/mL) was high in the abutments treated with steam cleaning and ultrasonic bath: 2.93 × 109, SD = 1.68 × 1012 and 1.83 × 109, SD = 3.95 × 1010, respectively. Abutments treated with chlorhexidine showed higher toxicity to cells, while all other samples showed similar effects to the control. In conclusion, steam cleaning seemed to be the most efficient method for reducing debris and metallic contamination. Bacterial load can be reduced using autoclaving, chlorhexidine, and NaOCl.

High-fat diet and oral infection induced type 2 diabetes and obesity development under different genetic backgrounds.

BACKGROUND: Type 2 diabetes (T2D) is an adult-onset and obese form of diabetes caused by an interplay between genetic, epigenetic, and environmental components. Here, we have assessed a cohort of 11 genetically different collaborative cross (CC) mouse lines comprised of both sexes for T2D and obesity developments in response to oral infection and high-fat diet (HFD) challenges. METHODS: Mice were fed with either the HFD or the standard chow diet (control group) for 12 weeks starting at the age of 8 weeks. At week 5 of the experiment, half of the mice of each diet group were infected with Porphyromonas gingivalis and Fusobacterium nucleatum bacteria strains. Throughout the 12-week experimental period, body weight (BW) was recorded biweekly, and intraperitoneal glucose tolerance tests were performed at weeks 6 and 12 of the experiment to evaluate the glucose tolerance status of mice. RESULTS: Statistical analysis has shown the significance of phenotypic variations between the CC lines, which have different genetic backgrounds and sex effects in different experimental groups. The heritability of the studied phenotypes was estimated and ranged between 0.45 and 0.85. We applied machine learning methods to make an early call for T2D and its prognosis. The results showed that classification with random forest could reach the highest accuracy classification (ACC = 0.91) when all the attributes were used. CONCLUSION: Using sex, diet, infection status, initial BW, and area under the curve (AUC) at week 6, we could classify the final phenotypes/outcomes at the end stage of the experiment (at 12 weeks).

The Collaborative Cross-Mouse Population for Studying Genetic Determinants Underlying Alveolar Bone Loss Due to Polymicrobial Synergy and Dysbiosis.

Dysbiosis of oral microbiota is associated with the initiation and progression of periodontitis. The cause-and-effect relationship between genetics, periodontitis, and oral microbiome dysbiosis is poorly understood. Here, we demonstrate the power of the collaborative cross (CC) mice model to assess the effect of the genetic background on microbiome diversity shifts during periodontal infection and host suitability status. We examined the bacterial composition in plaque samples from seven different CC lines using 16s rRNA sequencing before and during periodontal infection. The susceptibility/resistance of the CC lines to alveolar bone loss was determined using the micro-CT technique. A total of 53 samples (7 lines) were collected before and after oral infection using oral swaps followed by DNA extraction and 16 s rRNA sequencing analysis. CC lines showed a significant variation in response to the co-infection (p < 0.05). Microbiome compositions were significantly different before and after infection and between resistant and susceptible lines to periodontitis (p < 0.05). Gram-positive taxa were significantly higher at the resistant lines compared to susceptible lines (p < 0.05). Gram-positive bacteria were reduced after infection, and gram-negative bacteria, specifically anaerobic groups, increased after infection. Our results demonstrate the utility of the CC mice in exploring the interrelationship between genetic background, microbiome composition, and periodontitis.

Effects of a ZnCuO-Nanocoated Ti-6Al-4V Surface on Bacterial and Host Cells.

This study aims to investigate the effects of a novel ZnCuO nanoparticle coating for dental implants-versus those of conventional titanium surfaces-on bacteria and host cells. A multispecies biofilm composed of Streptococcus sanguinis, Actinomyces naeslundii, Porphyromonas gingivalis, and Fusobacterium nucleatum was grown for 14 days on various titanium discs: machined, sandblasted, sandblasted and acid-etched (SLA), ZnCuO-coated, and hydroxyapatite discs. Bacterial species were quantified with qPCR, and their viability was examined via confocal microscopy. Osteoblast-like and macrophage-like cells grown on the various discs for 48 h were examined for proliferation using an XTT assay, and for activity using ALP and TNF-α assays. The CSLM revealed more dead bacteria in biofilms grown on titanium than on hydroxyapatite, and less on sandblasted than on machined and ZnCuO-coated surfaces, with the latter showing a significant decrease in all four biofilm species. The osteoblast-like cells showed increased proliferation on all of the titanium surfaces, with higher activity on the ZnCuO-coated and sandblasted discs. The macrophage-like cells showed higher proliferation on the hydroxyapatite and sandblasted discs, and lower activity on the SLA and ZnCuO-coated discs. The ZnCuO-coated titanium has anti-biofilm characteristics with desired effects on host cells, thus representing a promising candidate in the complex battle against peri-implantitis.

Correlation Between the Tragus-Incisal Distance and Interocclusal Rest Distance.

PURPOSE: To evaluate correlations of arch size and sex with the interocclusal rest distance (IORD), as well as to estimate proportional variance. MATERIALS AND METHODS: A total of 106 participants were examined. The participants, 38 men and 68 women, were aged 22 to 30 years, were fully dentate, had no signs of abnormal abrasion, and had intact posterior occlusal contacts. Measurements of interocclusal rest distance and tragus-incisal distance were recorded, and the rest angle created between the tragus-incisal distance in maximum intercuspation and in resting vertical dimension were calculated according to the cosine formula. Correlation between the size of the mandible (tragus-incisal distance, mean of left and right sides) and the IORD were calculated using Pearson correlation coefficient. Correlations for sex (calculated separately for male and female) and rest angle were also assessed. RESULTS: The mean (SD) tragus-incisal distance values were 123.38 (6.77) mm for all participants, 120.01 (4.64) mm for women, and 130.72 (5.24) mm for men. The mean (SD) IORD values were 2.76 (1.3) mm for all participants, 2.13 (0.9) mm for women, and 3.87 (1.17) mm for men. The mean (SD) rest angle values were 1.26 (0.55) degrees for all participants, 1.02 (0.41) degrees for women, and 1.7 (0.49) degrees for men. Pearson correlation coefficient between IORD and tragus-incisal distance was significant (P < .05). According to t test, there was a significant difference between men and women for IORD, tragus-incisal distance, and rest angle (P < .01). CONCLUSION: A correlation exists between IORD and arch size. A statistically significant difference was found between men and women for IORD and arch size values.

Phage Targeting Streptococcus mutans In Vitro and In Vivo as a Caries-Preventive Modality.

Dental caries is a common infectious disease worldwide. Current conventional therapies lack specific antimicrobial effects against Streptococcus mutans, a key bacterium that induces caries. A promising alternative approach is bacteriophage (phage) therapy. Recently, SMHBZ8 phage targeting S. mutans was isolated and characterized. The aim of this study was to evaluate the caries-prevention efficacy of SMHBZ8 using in vitro and in vivo caries models. Hemi-mandibles dissected from euthanized healthy mice were subjected to caries-promoting conditions in vitro. Jaws treated with phage therapy in suspension and in formulation with a sustained-release delivery system showed no carious lesions, similar to control and chlorhexidine-treated jaws. Subsequently, SMHBZ8 phage suspension also prevented carious lesion development in a murine caries model in vivo. In both models, caries lesions were analyzed clinically and radiographically by µCT scans. This study shows how SMHBZ8 phage therapy targeting S. mutans can serve as an efficient caries-prevention modality, in suspension or with a sustained-release delivery system, by in vitro and in vivo mouse models.

Studying host genetic background effects on multimorbidity of intestinal cancer development, type 2 diabetes and obesity in response to oral bacterial infection and high-fat diet using the collaborative cross (CC) lines.

Background: Multimorbidity of intestinal cancer (IC), type 2 diabetes (T2D) and obesity is a complex set of diseases, affected by environmental and genetic risk factors. High-fat diet (HFD) and oral bacterial infection play important roles in the etiology of these diseases through inflammation and various biological mechanisms. Methods: To study the complexity of this multimorbidity, we used the collaborative cross (CC) mouse genetics reference population. We aimed to study the multimorbidity of IC, T2D, and obesity using CC lines, measuring their responses to HFD and oral bacterial infection. The study used 63 mice of both sexes generated from two CC lines (IL557 and IL711). For 12 weeks, experimental mice were maintained on specific dietary regimes combined with co-infection with oral bacteria Porphyromonas gingivalis and Fusobacterium nucleatum, while control groups were not infected. Body weight (BW) and results of a intraperitoneal glucose tolerance test (IPGTT) were recorded at the end of 12 weeks, after which length and size of the intestines were assessed for polyp counts. Results: Polyp counts ranged between 2 and 10 per CC line. The combination of HFD and infection significantly reduced (P < .01) the colon polyp size of IL557 females to 2.5 cm2, compared to the other groups. Comparing BW gain, IL557 males on HFD gained 18 g, while the females gained 10 g under the same conditions and showed the highest area under curve (AUC) values of 40 000-45 000 (min mg/dL) in the IPGTT. Conclusion: The results show that mice from different genetic backgrounds respond differently to a high fat diet and oral infection in terms of polyp development and glucose tolerance, and this effect is gender related.

TNFα expression by Porphyromonas gingivalis-stimulated macrophages relies on Sirt1 cleavage.

OBJECTIVE: Periodontitis is one the most common chronic inflammatory conditions, resulting in destruction of tooth-supporting tissues and leading to tooth loss. Porphyromonas gingivalis activates host macrophages to secrete pro-inflammatory cytokines and elicit tissue damage, in part by inducing NF-kappa-B transactivation. Since NFκB transactivation is negatively regulated by the Nicotinamide adenine dinucleotide (NAD)-dependent deacetylase enzyme Sirt1, we sought to assess if RAW264.7 macrophages exposed to P. gingivalis demonstrate impaired Sirt1 activity, to ultimately induce a pro-inflammatory response. METHODS: RAW264.7 macrophages were incubated with heat- killed P. gingivalis for 2, 4, 8, and 24 h. Stimulated RAW264.7 were assessed for TNFα expression via PCR, ELISA, and ChIP analysis. Following the activation of RAW264.7 macrophages, immunoblot analysis was executed to detect modifications in Sirt1 and the NFκB subunit RelA that is essential for NFκB transcriptional activity. RESULTS: TNFα expression was elevated 4 h after exposure to P. gingivalis. ChIP confirmed that RelA was enriched in the mouse TNFα promoter 4 h following stimulation, which correlated with the increased TNFα mRNA levels. Preceding TNFα expression, we detected Phosphoserine 536 and acetylated lysine 310 of RelA after 2 hours exposure with P. gingivalis. Moreover, reduced Sirt1 activity was associated with its cleavage in RAW264.7 protein extracts, after 2 hours of P. gingivalis exposure. Blocking TLR2/4 signaling prevented Sirt1 cleavage, loss of deacetylase activity, and TNFα secretion, while co-administering CA074Me (a cathepsin B inhibitor) with P. gingivalis reduced RelA promoter enrichment, resulting in impaired TNFα expression. CONCLUSIONS: Together, the results suggest that P. gingivalis induces TNFα expression, at least in part, by enhancing cleavage of Sirt1 via a TLR-dependent signaling circuit.

Assessing the host genetic background effects on type 2 diabetes and obesity development in response to mixed-oral bacteria and high-fat diet using the collaborative cross mouse model.

BACKGROUND: Host genetic background and sex, play central roles in defining the pathogenesis of type 2 diabetes (T2D), obesity and infectious diseases. Our previous studies demonstrated the utilization of genetically highly diverse inbred mouse lines, namely collaborative cross (CC), for dissecting host susceptibility for the development of T2D and obesity, showing significant variations following high-fat (42% fat) diet (HFD). Here, we aimed to assessing the host genetic background and sex effects on T2D and obesity development in response to oral-mixed bacterial infection and HFD using the CC lines. MATERIALS AND METHODS: Study cohort consists of 97 mice from 2 CC lines (both sexes), maintained on either HFD or Standard diet (CHD) for 12 weeks. At week 5 a group of mice from each diet were infected with Porphyromonas gingivalis (Pg) and Fusobacterium nucleatum (Fn) bacteria (control groups without infection). Body weight (BW) and glucose tolerance ability were assessed at the end time point of the experiment. RESULTS: The CC lines varied (P < .05) at their BW gain and glucose tolerance ability (with sex effect) in response to diets and/or infection, showing opposite responses despite sharing the same environmental conditions. The combination of diet and infection enhances BW accumulation for IL1912, while restraints it for IL72. As for glucose tolerance ability, only females (both lines) were deteriorated in response to infection. CONCLUSIONS: This study emphasizes the power of the CC mouse population for the characterization of host genetic makeup for defining the susceptibility of the individual to development of obesity and/or impaired glucose tolerance.

Translation of mouse model to human gives insights into periodontitis etiology.

To suggest candidate genes involved in periodontitis, we combined gene expression data of periodontal biopsies from Collaborative Cross (CC) mouse lines, with previous reported quantitative trait loci (QTL) in mouse and with human genome-wide association studies (GWAS) associated with periodontitis. Periodontal samples from two susceptible, two resistant and two lines that showed bone formation after periodontal infection were collected during infection and naïve status. Differential expressed genes (DEGs) were analyzed in a case-control and case-only design. After infection, eleven protein-coding genes were significantly stronger expressed in resistant CC lines compared to susceptible ones. Of these, the most upregulated genes were MMP20 (P = 0.001), RSPO4 (P = 0.032), CALB1 (P = 1.06×10-4), and AMTN (P = 0.05). In addition, human orthologous of candidate genes were tested for their association in a case-controls samples of aggressive (AgP) and chronic (CP) periodontitis (5,095 cases, 9,908 controls). In this analysis, variants at two loci, TTLL11/PTGS1 (rs9695213, P = 5.77×10-5) and RNASE2 (rs2771342, P = 2.84×10-5) suggested association with both AgP and CP. In the association analysis with AgP only, the most significant associations were located at the HLA loci HLA-DQH1 (rs9271850, P = 2.52×10-14) and HLA-DPA1 (rs17214512, P = 5.14×10-5). This study demonstrates the utility of the CC RIL populations as a suitable model to investigate the mechanism of periodontal disease.

Killing mechanism of bacteria within multi-species biofilm by blue light.

Objectives: The aim of the study was to characterize the immediate and delayed effects of non-coherent blue-light treatment on the composition and viability of an in vitro biofilm composed of anaerobic multispecies, as well as the mechanisms involved. Methods: A multispecies biofilm was constructed of Streptococcus sanguinis, Actinomyces naeslundii, Porphyromonas gingivalis and Fusobacterium nucleatum, test groups were exposed to blue light. The multispecies biofilm was explored with a newly developed method based on flow cytometry and confocal microscopy. The involvement of the paracrine pathway in the phototoxic mechanism was investigated by a crossover of the supernatants between mono-species P. gingivalis and F. nucleatum biofilms. Results: Blue light led to a reduction of about 50% in the viable pathogenic bacteria P. gingivalis and F. nucleatum, vs that in the non-exposed biofilm. Biofilm thickness was also reduced by 50%. The phototoxic effect of blue light on mono-species biofilm was observed in P. gingivalis, whereas F. nucleatum biofilm was unaffected. A lethal effect was obtained when the supernatant of P. gingivalis biofilm previously exposed to blue light was added to the F. nucleatum biofilm. The effect was circumvented by the addition of reactive oxygen species (ROS) scavengers to the supernatant. Conclusion: Blue-light has an impact on the bacterial composition and viability of the multispecies biofilm. The phototoxic effect of blue light on P. gingivalis in biofilm was induced directly and on F. nucleatum via ROS mediators of the paracrine pathway. This phenomenon may lead to a novel approach for 'replacement therapy,' resulting in a less periodonto-pathogenic biofilm.

Phages in a thermoreversible sustained-release formulation targeting E. faecalis in vitro and in vivo.

INTRODUCTION: Enterococcus faecalis is a key pathogen recovered from root canals when conventional treatment fails. Phage therapy has generated new interest in combating pathogens. A sustained-release formulation using specific phages against E. faecalis may offer an alternative approach. OBJECTIVES: To evaluate the efficacy of anti-E. faecalis phages formulated in a thermo- sustained-release system against E. faecalis in vitro and in vivo. METHODS: EFDG1 and EFLK1 phages were formulated with poloxamer P407. Gelation time, phage survival, activity and toxicity were evaluated. Lytic activity was evaluated in vitro against E. faecalis at various growth phases, including anti-biofilm activity. Methods included viable bacterial count (CFU/mL), biofilm biomass determination and electron microscopy (live/dead staining). Further evaluation included infected incisors in an in vivo rat model. Anti-E. faecalis phage-cocktail suspension and sustained-release phage formulation were evaluated by viable bacterial count (CFU/mL), histology, scanning electron microscopy (SEM) and 16S genome sequencing of the microbiota of the root canal. RESULTS: Gelation time for clinical use was established. Low toxicity and a high phage survival rate were recorded. Sustained-release phages reduced E. faecalis in logarithmic (4 logs), stationary (3 logs) and biofilm (4 logs) growth phases. Prolonged anti-biofilm activity of 88% and 95% reduction in biomass and viable counts, respectively, was recorded. Reduction of intracanal viable bacterial counts was observed (99% of enterococci) also seen in SEM. Phage treatment increased Proteobacteria and decreased Firmicutes. Histology showed reduced periapical inflammation and improved healing following phage treatment. CONCLUSION: Poloxamer P407 formulated with phages has an effective and long-lasting effect in vitro and in vivo targeting E. faecalis.

Peri-implant alveolar bone resorption in an innovative peri-implantitis murine model: Effect of implant surface and onset of infection.

PURPOSE: To compare the difference in alveolar bone resorption around implants after immediate placement in a bacterial induced experimental periimplantitis murine model. The various conditions that were examined were: Effect of implant surface characteristics and the onset of the induced infection. MATERIALS AND METHODS: Screw-shaped titanium implants, smooth-surface or sand-blasted large-grit acid-etched (SLA) coated, were inserted immediately after extraction of the first upper left molar, in 90 5-6-week-old BALB/c mice. The mice were infected with Porphyromonas gingivalis and Fusobacterium nucleatum 21 (early infection) or 42 days (delayed infection) after implantation. Six weeks post infection, bone volume around inserted implants was measured using micro-CT, and was compared to alveolar bone level around teeth. Histological analysis was also performed. RESULTS: The level of bone loss was significantly higher around the implants compared to the teeth, for smooth surface implants the bone loss was higher than of the SLA surface in both control and infected groups with no statistical significance. The survival rate of the implants in immediate infection was 75% compared of the 100% survival of the delayed infection and control mice. There is no significant difference between the early and the delayed infection in alveolar bone loss level around the implants. CONCLUSIONS: This model can assist in studying the differences in alveolar bone resorption in different implants and their effect on the development of the disease.

Antibacterial Orthodontic Cements and Adhesives: A Possible Solution to Streptococcus mutans Outgrowth Adjacent to Orthodontic Appliances.

PURPOSE: To assess the antibacterial effect of orthodontic cements containing 1% insoluble antibacterial polycat-ionic nanoparticles against Streptococcus mutans. MATERIALS AND METHODS: Polycationic polyethyleneimine (PEI)-based nanoparticles were incorporated into GC Fuji Ortho LC (GC), CX-plus (Shofu) orthodontic cements and in Neobond Transbond plus (Denstply) and Transbond XT (3M) orthodontic adhesives. The samples were evaluated immediately after setting, as well as after two weeks and one month of aging. The antibacterial effect against S. mutans was evaluated with the direct contact test and the agar diffusion test. In addition, the antibacterial properties of the eluate from the examined materials was tested. Four-way ANOVA followed by Tukey's test was used to determine bacterial growth rates. RESULTS: S. mutans outgrowth was substantially reduced (p < 0.05) following direct contact with the surface of Neobond adhesives (95%, i.e. 5-6 log reduction) and GC Fuji Ortho LC cement samples (97% reduction) containing PEI nanoparticles. CX-plus cement, Transbond plus and Transbond XT adhesives with and without PEI showed no antibacterial effect, and S. mutans outgrowth was similar to that of the controls. CONCLUSIONS: Neobond adhesive and GC Fuji Ortho LC cement with 1% incorporated insoluble antibacterial polycat-ionic nanoparticles exhibited stable antibacterial properties, particularly after immediate contact between the cement and the adhesive, and thus may prevent S. mutans outgrowth adjacent to orthodontic appliances. Further studies are needed to evaluate the efficacy, physical properties and possible side effects of the PEI nanoparticles in vivo.

High-resolution novel method for tracking bacteria in a multi-species biofilm.

The aim of this study is to establish a novel high resolution tracking ability of a specific bacterium in multispecies biofilm. A periodontal multispecies biofilm was constructed with Streptococcus sanguis, Actinomyces naeslundii, Porphyromonas gingivalis and Fusobacterium nucleatum. A single species was stained with fluorescein isothiocyanate (FITC). The mature biofilm was stained for viability (propidium iodide) and analysis was performed with flow cytometry. The sensitivity of the assay was compared with colony forming units (CFU) counts. A single cell suspension of P. gingivalis was grown in broth and biofilm to identify the location of these events on side scatter and forward scatter. The sensitivity of the assay was comparable to that of the CFU counts. The assay allows quantification of the ratio of a single bacterium within the biofilm, and its viable proportion. The described method is reproducible and of high resolution, and allows the examination of microbes' composition and viability within a biofilm structure.

Antibacterial Activity of Orthodontic Cement Containing Quaternary Ammonium Polyethylenimine Nanoparticles Adjacent to Orthodontic Brackets.

Enamel demineralization is a common problem found in patients using orthodontic devices, such as orthodontic braces. It was found that Streptoccocus mutans growth increases adjacent to orthodontic devices, which may result in caries development. Incorporated antibacterial quaternary ammonium polyethylenimine (QPEI) nanoparticles were previously shown to be highly efficacious against various bacteria. Combining antibacterial materials in orthodontic cement may be advantageous to prevent bacterial outgrowth adjacent to orthodontic brackets. The aim was to evaluate the efficiency of orthodontic cement containing QPEI nanoparticles in reducing S. mutans and Lactobacillus casei outgrowth adjacent to orthodontic brackets. Orthodontic brackets were bonded to the buccal surfaces of extracted lower incisors. The antibacterial effect on S. mutans and L. casei outgrowth of Neobond bracket adhesive orthodontic cement with and without QPEI nanoparticles was compared. The antibacterial effect was evaluated using crystal violet staining and bacterial count (CFU/mL). The teeth in the experimental group, with the QPEI nanoparticles cement, showed significantly lower optical density (OD) values and CFU counts of S. mutans and L. casei than the teeth in the control group (p < 0.05). Based on the results, it can be concluded that orthodontic cement containing QPEI nanoparticles significantly inhibits S. mutans and L. casei growth around orthodontic brackets.