Protein kinase D3 conditional knockout impairs osteoclast formation and increases trabecular bone volume in male mice.

Studies using kinase inhibitors have shown that the protein kinase D (PRKD) family of serine/threonine kinases are required for formation and function of osteoclasts in culture. However, the involvement of individual protein kinase D genes and their in vivo significance to skeletal dynamics remains unclear. In the current study we present data indicating that protein kinase D3 is the primary form of PRKD expressed in osteoclasts. We hypothesized that loss of PRKD3 would impair osteoclast formation, thereby decreasing bone resorption and increasing bone mass. Conditional knockout (cKO) of Prkd3 using a murine Cre/Lox system driven by cFms-Cre revealed that its loss in osteoclast-lineage cells reduced osteoclast differentiation and resorptive function in culture. Examination of the Prkd3 cKO mice showed that bone parameters were unaffected in the femur at 4 weeks of age, but consistent with our hypothesis, Prkd3 conditional knockout resulted in 18 % increased trabecular bone mass in male mice at 12 weeks and a similar increase at 6 months. These effects were not observed in female mice. As a further test of our hypothesis, we asked if Prkd3 cKO could protect against bone loss in a ligature-induced periodontal disease model but did not see any reduction in bone destruction in this system. Together, our data indicate that PRKD3 promotes osteoclastogenesis both in vitro and in vivo.

Depleting CD103+ resident memory T cells in vivo reveals immunostimulatory functions in oral mucosa.

The oral mucosa is a frontline for microbial exposure and juxtaposes several unique tissues and mechanical structures. Based on parabiotic surgery of mice receiving systemic viral infections or co-housing with microbially diverse pet shop mice, we report that the oral mucosa harbors CD8+ CD103+ resident memory T cells (TRM), which locally survey tissues without recirculating. Oral antigen re-encounter during the effector phase of immune responses potentiated TRM establishment within tongue, gums, palate, and cheek. Upon reactivation, oral TRM triggered changes in somatosensory and innate immune gene expression. We developed in vivo methods for depleting CD103+ TRM while sparing CD103neg TRM and recirculating cells. This revealed that CD103+ TRM were responsible for inducing local gene expression changes. Oral TRM putatively protected against local viral infection. This study provides methods for generating, assessing, and in vivo depleting oral TRM, documents their distribution throughout the oral mucosa, and provides evidence that TRM confer protection and trigger responses in oral physiology and innate immunity.

Intracellular calprotectin (S100A8/A9) facilitates DNA damage responses and promotes apoptosis in head and neck squamous cell carcinoma.

OBJECTIVES: In head and neck squamous cell carcinoma (HNSCC), poor prognosis and low survival rates are associated with downregulated calprotectin. Calprotectin (S100A8/A9) inhibits cancer cell migration and invasion and facilitates G2/M cell cycle arrest. We investigated whether S100A8/A9 regulates DNA damage responses (DDR) and apoptosis in HNSCC after chemoradiation. MATERIALS AND METHODS: Human HNSCC cases in TCGA were analyzed for relationships between S100A8/A9 and expression of apoptosis-related genes. Next, S100A8/A9-expressing and non-expressing carcinoma lines (two different lineages) were exposed to genotoxic agents and assessed for 53BP1 and γH2AX expression and percent of viable/dead cells. Finally, S100A8/A9-wild-type and S100A8/A9null C57BL/6j mice were treated with 4-NQO to induce oral dysplastic and carcinomatous lesions, which were compared for levels of 53BP1. RESULTS: In S100A8/A9-high HNSCC tumors, apoptosis-related caspase family member genes were upregulated, whereas genes limiting apoptosis were significantly downregulated based on TCGA analyses. After X-irradiation or camptothecin treatment, S100A8/A9-expressing carcinoma cells (i.e., TR146 and KB-S100A8/A9) showed significantly higher 53BP1 and γH2AX expression, DNA fragmentation, proportions of dead cells, and greater sensitivity to cisplatin than wild-type KB or TR146-S100A8/A9-KD cells. Interestingly, KB-S100A8/A9Δ113-114 cells showed similar 53BP1 and γH2AX levels to S100A8/A9-negative KB and KB-EGFP cells. After 4-NQO treatment, 53BP1 expression in oral lesions was significantly greater in calprotectin+/+ than S100A8/A9null mice. CONCLUSIONS: In HNSCC cells, intracellular calprotectin is strongly suggested to potentiate DDR and promote apoptosis in response to genotoxic agents. Hence, patients with S100A8/A9-high HNSCC may encounter more favorable outcomes because more tumor cells enter apoptosis with increased sensitivity to chemoradiation therapy.

In vitro physicochemical characterization of five root canal sealers and their influence on an ex vivo oral multi-species biofilm community.

AIM: To evaluate the physicochemical properties of five root canal sealers and assess their effect on an ex vivo dental plaque-derived polymicrobial community. METHODOLOGY: Dental plaque-derived microbial communities were exposed to the sealers (AH Plus [AHP], GuttaFlow Bioseal [GFB], Endoseal MTA [ESM], Bio-C sealer [BCS] and BioRoot RCS [BRR]) for 3, 6 and 18 h. The sealers' effect on the biofilm biomass and metabolic activity was quantified using crystal violet (CV) staining and MTT assay, respectively. Biofilm community composition and morphology were assessed by denaturing gradient gel electrophoresis (DGGE), 16S rRNA sequencing and scanning electron microscopy. The ISO6876:2012 specifications were followed to determine the setting time, radiopacity, flowability and solubility. Obturated acrylic teeth were used to assess the sealers' effect on pH. Surface chemical characterization was performed using SEM with coupled energy-dispersive spectroscopy. Data normality was assessed using the Shapiro-Wilk test. One-way anova and Tukey's tests were used to analyze data from setting time, radiopacity, flowability and solubility. Two-way anova and Dunnett's tests were used for the data analysis from CV, MTT and pH. 16S rRNA sequencing data were analyzed for alpha (Shannon index and Chao analysis) and beta diversity (Bray-Curtis dissimilarities). Differences in community composition were evaluated by analysis of similarity (p < .05). RESULTS: The sealers significantly influenced microbial community composition and morphology. All sealers complied with ISO6876:2012 requirements for setting time, radiopacity and flowability. Although only AHP effectively reduced the biofilm biomass, all sealers, except BRR, reduced biofilm metabolic activity. CONCLUSION: Despite adequate physical properties, none of the sealers tested prevented biofilm growth. Significant changes in community composition were observed. If observed in vivo, these changes could affect intracanal microbial survival, pathogenicity and treatment outcomes.

In vitro efficacy of a non-instrumentation technique to remove intracanal multispecies biofilm.

AIM: The aim of this study was to assess the efficacy of a non-instrumentation technique to disinfect root canals infected by a human dental plaque-derived multispecies biofilm. METHODOLOGY: Twenty-two mandibular incisors were accessed, autoclaved and inoculated with dental plaque. The Center for Disease Control biofilm reactor was used to promote contamination of the root canal space. In the conventional technique (control), the specimens were instrumented until size 35/04 and irrigated with 6% NaOCl. In the non-instrumentation technique, a glide path was established using K-files size 10-20 and specimens were immediately cleaned with the GentleWave System. Samples were obtained for culture and 16S rRNA gene sequencing. Differences in abundances of genera were evaluated using Kruskal-Wallis test, and differences in alpha diversity were compared using anova. Alpha and beta diversity indices were calculated using mothur. The Shannon and Chao1 indices were used to measure alpha diversity. The Bray-Curtis dissimilarity was used to measure beta diversity. Differences in community composition were evaluated using analysis of similarity with Bonferroni correction for multiple comparisons. RESULTS: The total numbers of reads in biological samples ranged from 126 to 45 286. Significantly fewer reads were obtained from samples following cleaning by either method (p < .0001), and significantly fewer reads were obtained in post-cleaning samples following conventional versus non-instrumentation cleaning regiment (p = .002). Communities in pre-treatment samples were similar in both groups; however, significantly greater relative abundances of Streptococcus, Veillonella and Campylobacter were observed following cleaning using non-instrumentation technique (Kruskal-Wallis p = .009, .033, and .001, respectively). Whilst no significant differences were observed in Shannon alpha diversity, the Chao1 index was significantly lower in post-cleaning samples. CONCLUSIONS: Significant shifts in composition were observed following cleaning by using both regimens, but the impact of this change was greater following a conventional cleaning technique.

Investigation of a modified hydraulic calcium silicate-based material - Bio-C Pulpo.

This study evaluated the physicochemical, biological, and antimicrobial properties of a new hydraulic calcium silicate-based modified material, and compared it with MTA Repair HP and MTA Angelus. The materials were assessed regarding color luminosity (L), color change, radiopacity, setting time, and ISO 6876:2012 linear flow. Volumetric filling and volume change were evaluated using microcomputed-tomography (µCT). Chemical characterization after 28 days in Hank's Balanced Salt Solution (HBSS) and pH analysis were also assessed. Biological characterization of cytotoxicity and microbiological assessment were also undertaken. Shapiro-Wilk, ANOVA, Levene and post hoc analyses with Bonferroni correction were performed, adopting a 5% significance level (p <0.05). Bio-C Pulpo exhibited the highest L values after 90 days. All tested materials demonstrated color change during the analyses, and had radiopacity above 5 mm Al. MTA Repair HP set faster than Bio-C Pulpo, whereas the latter had the highest linear flow. MTA Repair HP had the highest volumetric filling in µCT analysis. Bio-C Pulpo showed the highest alkalinity during all tested periods, and the highest volumetric loss (above 9%), in comparison with MTA Repair HP and MTA Angelus. Bio-C Pulpo did not form calcium hydroxide after hydration. MTA Repair HP demonstrated the highest cytocompatibility, and Bio-C Pulpo, the highest cytotoxicity. No inhibition halos were observed for any material, and similar higher turbidity values were seen after direct contact. Composition additives used in Bio-C Pulpo modified its properties, and both the absence of calcium hydroxide deposition after hydration, and the related cytotoxicity of this material are of particular concern.

Physicochemical, antimicrobial, and biological properties of White-MTAFlow.

OBJECTIVES: To evaluate a new material containing tantalum oxide as an alternative radiopacifier, and a water-based gel for hydration, in comparison with two calcium silicate-based cement: ProRoot MTA and Biodentine. MATERIALS AND METHODS: ProRoot MTA (Dentsply), Biodentine (Septodont), and a new hydraulic calcium silicate cement White-MTAFlow (Ultradent) (in 'thin' consistency) were characterized using X-ray diffraction (XRD), scanning electron microscopy (SEM), and energy-dispersive spectroscopy (EDS). The interaction with dentin was also assessed using SEM and EDS. Physical and chemical properties radiopacity, setting time, linear flow, volumetric central filling, and lateral flow, pH, and volume change were investigated together with the color luminosity (L) and color change (ΔE). The agar diffusion and direct contact antimicrobial activity, and methylthiazolyldiphenyl-tetrazolium-bromide (MTT) cytotoxicity using human fibroblast cells were also evaluated. Data were statistically analyzed at a 5% significance level. RESULTS: All materials were composed of tricalcium and dicalcium silicate but had different radiopacifiers, and calcium hydroxide (portlandite) deposition was detected in XRD analysis. White-MTAFlow exhibited radiopacity values in accordance with ISO standard, and the longest setting time. The water-based gel provided the highest linear flow, a comparable cavity central filling, and the highest groove-lateral flow in the volumetric flow analysis. White-MTAFlow exhibited an alkalinity reduction, and Biodentine, a progressive increase of pH values after 28 days. However, similar volume loss for White-MTAFlow was assessed in comparison to Biodentine after the 28-day immersion. White-MTAFlow showed the highest L value (91.5), and ProRoot MTA the lowest (78.1) due to dentin staining caused by bismuth migration. None of the materials exhibited inhibition halos against the tested bacteria, and similar turbidity values were obtained after 48 h in direct contact with E. faecalis, indicating an upregulation to bacterial growth. White-MTAFlow showed MTT cytocompatibility similarly to the control group. CONCLUSIONS: White-MTAFlow in 'thin' consistency presents comparable physicochemical, biological, and antimicrobial properties to ProRoot MTA and Biodentine, and does not cause color alteration in dentin. CLINICAL RELEVANCE: White-MTAFlow is a suitable material for use as reparative endodontic cement. Further studies considering its biocompatibility are necessary.

Investigation of a modified hydraulic calcium silicate-based material - Bio-C Pulpo

Abstract This study evaluated the physicochemical, biological, and antimicrobial properties of a new hydraulic calcium silicate-based modified material, and compared it with MTA Repair HP and MTA Angelus. The materials were assessed regarding color luminosity (L), color change, radiopacity, setting time, and ISO 6876:2012 linear flow. Volumetric filling and volume change were evaluated using microcomputed-tomography (µCT). Chemical characterization after 28 days in Hank's Balanced Salt Solution (HBSS) and pH analysis were also assessed. Biological characterization of cytotoxicity and microbiological assessment were also undertaken. Shapiro-Wilk, ANOVA, Levene and post hoc analyses with Bonferroni correction were performed, adopting a 5% significance level (p <0.05). Bio-C Pulpo exhibited the highest L values after 90 days. All tested materials demonstrated color change during the analyses, and had radiopacity above 5 mm Al. MTA Repair HP set faster than Bio-C Pulpo, whereas the latter had the highest linear flow. MTA Repair HP had the highest volumetric filling in µCT analysis. Bio-C Pulpo showed the highest alkalinity during all tested periods, and the highest volumetric loss (above 9%), in comparison with MTA Repair HP and MTA Angelus. Bio-C Pulpo did not form calcium hydroxide after hydration. MTA Repair HP demonstrated the highest cytocompatibility, and Bio-C Pulpo, the highest cytotoxicity. No inhibition halos were observed for any material, and similar higher turbidity values were seen after direct contact. Composition additives used in Bio-C Pulpo modified its properties, and both the absence of calcium hydroxide deposition after hydration, and the related cytotoxicity of this material are of particular concern.

Dental discoloration caused by Grey-MTAFlow cement: analysis of its physicochemical, biological and antimicrobial properties.

Tricalcium silicate-based cement are materials used in reparative and regenerative procedures in endodontics. A recently proposed formulation aimed to enhance handling during clinical use with a versatile material applicable by syringe. Although, the use of bismuth oxide as radiopacifier and grey raw powder are drawbacks considering aesthetics. Objectives Evaluate physicochemical, biological, and antimicrobial properties of Grey-MTAFlow (Ultradent) and assess whether the addition of zinc oxide (ZnO) prevents dentinal discoloration caused by bismuth oxide. Methodology Grey-MTAFlow was manipulated in 'thin' consistency for all tests. Luminosity, color change, ion migration to dentine, radiopacity, setting time, ISO 6876:2012 linear flow, volumetric lateral flow and central filling of simulated grooves scanned using micro-computed tomography (µCT), pH, calcium release, volumetric change using µCT, chemical characterisation, cytotoxicity, and antimicrobial activity were assessed. Addition of 5% ZnO to Grey-MTAFlow and a bismuth-containing experimental composition were comparatively tested. Statistical analyses used Shapiro-Wilk, T-test, ANOVA, and Kolmogorov-Smirnov (p<0.05). Results The addition of ZnO to Grey-MTAFlow prevented dentine darkening after 90 days due to bismuth migration reduction, although no statistical difference was found (p=0.863). ZnO addition significantly enhanced Grey-MTAFlow radiopacity without differences in initial setting time. Grey-MTAFlow presented an ISO linear flow of 10.9 mm and a balanced volumetric lateral flow with central filling in µCT evaluation. All compositions presented an alkaline pH after immersion. Grey-MTAFlow had a significantly higher calcium ion release after 28 days in comparison to 24 hours (p=0.011) and volumetric expansion of 0.4±1.8% after immersion. ZnO addition altered the hydrated cement matrix once calcium hydroxide (portlandite) could not be detected in characterisation. Neither of the materials produced inhibition halos nor reduced bacterial turbidity, but all presented cytocompatibility above 100%. Conclusion Grey-MTAFlow expanded after immersion and exhibited higher luminosity values after the evaluation period when ZnO was added, but chemical modifications after this addition occurred.

Dental discoloration caused by Grey-MTAFlow cement: analysis of its physicochemical, biological and antimicrobial properties

Abstract Tricalcium silicate-based cement are materials used in reparative and regenerative procedures in endodontics. A recently proposed formulation aimed to enhance handling during clinical use with a versatile material applicable by syringe. Although, the use of bismuth oxide as radiopacifier and grey raw powder are drawbacks considering aesthetics. Objectives Evaluate physicochemical, biological, and antimicrobial properties of Grey-MTAFlow (Ultradent) and assess whether the addition of zinc oxide (ZnO) prevents dentinal discoloration caused by bismuth oxide. Methodology Grey-MTAFlow was manipulated in 'thin' consistency for all tests. Luminosity, color change, ion migration to dentine, radiopacity, setting time, ISO 6876:2012 linear flow, volumetric lateral flow and central filling of simulated grooves scanned using micro-computed tomography (μCT), pH, calcium release, volumetric change using μCT, chemical characterisation, cytotoxicity, and antimicrobial activity were assessed. Addition of 5% ZnO to Grey-MTAFlow and a bismuth-containing experimental composition were comparatively tested. Statistical analyses used Shapiro-Wilk, T-test, ANOVA, and Kolmogorov-Smirnov (p<0.05). Results The addition of ZnO to Grey-MTAFlow prevented dentine darkening after 90 days due to bismuth migration reduction, although no statistical difference was found (p=0.863). ZnO addition significantly enhanced Grey-MTAFlow radiopacity without differences in initial setting time. Grey-MTAFlow presented an ISO linear flow of 10.9 mm and a balanced volumetric lateral flow with central filling in μCT evaluation. All compositions presented an alkaline pH after immersion. Grey-MTAFlow had a significantly higher calcium ion release after 28 days in comparison to 24 hours (p=0.011) and volumetric expansion of 0.4±1.8% after immersion. ZnO addition altered the hydrated cement matrix once calcium hydroxide (portlandite) could not be detected in characterisation. Neither of the materials produced inhibition halos nor reduced bacterial turbidity, but all presented cytocompatibility above 100%. Conclusion Grey-MTAFlow expanded after immersion and exhibited higher luminosity values after the evaluation period when ZnO was added, but chemical modifications after this addition occurred.

Diferenciação entre granulomas e cistos periapicais por meio de imagens de tomografia computadorizada: revisão de literatura / Differentiation between granulomas and periapical cysts by computed tomography images: literature

As lesões periapicais são formadas em resposta a um processo inflamatório que envolve a região periodontal de dentes necrosados. Assim, o tratamento endodôntico frequentemente está relacionado ao tratamento dessas lesões, as quais podem se manifestar na forma de cistos ou granulomas periapicais. Entretanto, ainda restam muitas dúvidas quanto ao diagnóstico diferencial. A avaliação histopatológica de uma amostra submetida à biópsia ainda é considerada o padrão-ouro para determinação do tipo da lesão periapical. A fim não só de proporcionar um tratamento endodôntico o menos invasivo possível, como também com o objetivo de otimizá-lo, é de suma importância que seja identificado um método não cirúrgico capaz de distinguir entre cistos periapicais e granulomas. Buscando um novo método diagnóstico, tem sido proposta a utilização de Tomografias Computadorizadas de Feixe Cônico (TCFC). Apesar de elas serem capazes de identificar lesões periapicais, ainda existe discordância sobre o seu real potencial de proporcionar a distinção entre cistos e granulomas. Assim, o objetivo dessa revisão de literatura foi proporcionar uma visão abrangente e crítica sobre o potencial da utilização de TCFC para discernir lesões periapicais como cistos ou granulomas. Pôde-se concluir que ainda são necessárias mais pesquisas sobre métodos alternativos não invasivos para a realização do diagnóstico diferencial entre cistos periapicais e granulomas. Apesar disso, a partir do conhecimento transmitido pelos estudos realizados até hoje, a tomografia computadorizada pode ser considerada como uma ferramenta em potencial para realizar o diagnóstico diferencial não cirúrgico de lesões periapicais

Comparative evaluation of push-out bond strength of a MTA-based root canal sealer

AIM: To evaluate the bond strength to root dentin of three root canal sealers: a mineral trioxide aggregate (MTA)-based sealer (MTA Fillapex(r)), an epoxy resin-based sealer (AH Plus(r)), and a zinc oxide eugenol-based sealer (EndoFill(r)). METHODS: Thirty extracted single-root human teeth of similar sizes and circular canals were prepared using #3 and #2 Gates Glidden drills in the cervical portion of the canal and K3(r) rotary instruments to a size #25/0.06 to working length. Irrigation with 0.5 mL 2% chlorhexidine gel was used before and 1 mL saline after each instrument.The smear layer was removed with 3 mL 17% EDTA for 3 min. The samples were sectioned horizontally into eight 1±0.1 mm-thick serial slices and then the push-out test was carried out. Two-way analysis of variance (ANOVA) and the post-hoc Tukey test were used for the analysis of the data with a significance level of 5%. RESULTS: AH Plus presented significantly higher bond strengths (p<0.05) than the other sealers, while MTA Fillapex showed the lowest bond strengths (p<0.05). CONCLUSIONS: The present study concluded that EndoFill(r) sealer and MTA FillApex(r) core combination were not superior to AH Plus(r) sealer and gutta-percha core combination...

Endodontists perceptions of single and multiple visit root canal treatment: a survey in Florianópolis - Brazil

Introduction: One of the main debates that have occurred in Endodontics is about the amount of sessions required to complete an appropriate treatment. There are very different philosophies regarding this matter. Objective: to the aim of this study was to investigate the Endodontists' point of view regarding single and multiple visit root canal treatment, identifying the basis on which the choice is made and how the information necessary for the choice is acquired. Material and methods: Endodontists registered in the dental practice board of Florianópolis/SC were contacted, and if they agreed to participate, they were interviewed using a questionnaire. The following topics were addressed: demographics, current clinical procedures, treatment rationales and preferences. Forty-three endodontists agreed to participate in the study. Results: Single visit endodontic treatment is carried out in 59.5% of biopulpectomy cases, 31.0% of necropulpectomy cases without lesion and only 11.9% in necropulpectomy cases with periapical lesion. The presence of vital pulp (81.4%) and a canal without exudate (93.0%) are the most important criteria for carrying out single visit endodontic treatment. The most used intracanal medication was calcium hydroxide. The lack of studies comparing long-term success on single visit endodontic treatment is considered an important issue to determine this therapy. Conclusion: It can be concluded that Florianopolis-Brazil endodontists prefer multiple visit over single visit root canal treatment in pulp necrosis cases. When pulp vitality is not compromised there is an increase in the number of endodontists who choose single visit root canal treatment.