Nanotopography and oral bacterial adhesion on titanium surfaces: in vitro and in vivo studies

Abstract The present study aimed to evaluate the influence of titanium surface nanotopography on the initial bacterial adhesion process by in vivo and in vitro study models. Titanium disks were produced and characterized according to their surface topography: machined (Ti-M), microtopography (Ti-Micro), and nanotopography (Ti-Nano). For the in vivo study, 18 subjects wore oral acrylic splints containing 2 disks from each group for 24 h (n = 36). After this period, the disks were removed from the splints and evaluated by microbial culture method, scanning electron microscopy (SEM), and qPCR for quantification of Streptococcus oralis, Actinomyces naeslundii, Fusobacterium nucleatum, as well as total bacteria. For the in vitro study, adhesion tests were performed with the species S. oralis and A. naeslundii for 24 h. Data were compared by ANOVA, with Tukey's post-test. Regarding the in vivo study, both the total aerobic and total anaerobic bacteria counts were similar among groups (p > 0.05). In qPCR, there was no difference among groups of bacteria adhered to the disks (p > 0.05), except for A. naeslundii, which was found in lower proportions in the Ti-Nano group (p < 0.05). In the SEM analysis, the groups had a similar bacterial distribution, with a predominance of cocci and few bacilli. In the in vitro study, there was no difference in the adhesion profile for S. oralis and A. naeslundii after 24 h of biofilm formation (p > 0.05). Thus, we conclude that micro- and nanotopography do not affect bacterial adhesion, considering an initial period of biofilm formation.

Culturable microbiological profile of a non-sterile drugs pharmaceutical production environment

Abstract For places where non-sterile drug production occurs, regulatory bodies recommend monitoring of the environmental bioburden. This procedure provides information regarding possible microbiological risks to which the products may be exposed, so that subsequent action measures may be implemented. The aim of the present work was to quantify and characterize the microorganisms present in Grade D (ISO 8) cleanrooms of a Brazilian pharmaceutical industry, identifying any possible seasonal climatic influences on these environments. Sampling was performed by surface and air monitoring, over 12 months during the year 2019, in rooms that were in operation. For both sampling methods, no statistically significant differences in bacteria and fungi counts were found between months or seasonal periods. Microorganisms that presented higher incidence included Staphylococcus epidermidis (15%) and Micrococcus spp. (13%), common to the human microbiota, and the fungi Cladosporium sp. (23%) and Penicillium sp. (21%), typical of the external environment. The results showed that microbial contamination in the Grade D cleanrooms was within the permissible maximum levels and remained similar throughout the year. Microbiological quality control in the clean areas of the pharmaceutical industry investigated was considered effective, with regular maintenance being necessary to keep bioburden levels controlled.

The Self-assembling peptide P11-4 influences viability and osteogenic differentiation of stem cells of the apical papilla (SCAP).

OBJECTIVE: To analyze the effect of P11-4 self-assembly peptide on cell viability and osteogenic capacity of SCAPs through mineral deposition and gene expression of osteogenic markers. METHODS: SCAPs were seeded in contact with P11-4 (10 µg/ml, 100 µg/ml and 1 mg/ml) solution. Cell viability was evaluated using a colorimetric assay MTT: 3-(4,5-dimethyl-thiazolyl-2)-2,5- diphenyltetrazolium bromide) in an experimental time of 24, 48 and 72 h (n = 7). Mineral deposition and quantification provided by the cells was tested using the Alizarin Red staining and Cetylpyridinium Chloride (CPC), respectively, after 30 days (n = 4). Gene expression of Runt-related transcription factor 2 (RUNX2), Alkaline phosphatase (ALP) and Osteocalcin (OCN) was quantified using quantitative polymerase chain reaction (RT-qPCR), at 3 and 7 days with Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) as the housekeeping gene, and relative gene expression was measured using the ΔΔCq method. Data were analyzed using Kruskall-Wallis followed by multiple comparisons, and T-test for gene expression with α=0.05. RESULTS: All tested concentrations (10 µg/ml, 100 µg/ml and 1 mg/ml) were not cytotoxic at time 24 and 48 h. After 72 h, a slight decrease in cell viability was observed for the lowest concentration (10 µg/ml). The concentration of 100 µg/ml P11-4 showed the highest mineral deposition. However, qPCR analysis of P11-4 (10 µg/ml) showed upregulation of RUNX2 and OCN at 3 days, with downregulation of ALP at 3 and 7d CONCLUSION: P11-4 did not affect cell viability, induced mineral deposition in SCAPs, and upregulated the expression of RUNX2 and OCN genes at 3 days, while downregulating ALP expression at 3 and 7 days. CLINICAL SIGNIFICANCE: Based on the results obtained in this study it can be stated that self-assembling peptide P11-4 is a potential candidate to induce mineralization on dental stem cells for regenerative purposes and also for a clinical use as a capping agent without compromising the cells health.

IL10 promoter rs6667202 polymorphism is functional in health but not in grade c periodontitis patients: A pilot study.

BACKGROUND AND OBJECTIVE: Previous studies have demonstrated an association between the IL10 promoter rs6667202 (C > A) single-nucleotide polymorphism (SNP) and grade C, stage 3 or 4 periodontitis (Perio4C) in the Brazilian population, where the altered A allele was detected more frequently in these patients. However, no functional analysis of this variation has yet been performed. Thus, the objective of this preliminary study was to evaluate the functionality of rs6667202 in gingival fibroblasts (GFs) of individuals with Perio4C and with periodontal health (PH) stimulated with Aggregatibacter actinomycetencomitans protein extract (AaPE). METHODS: Patients with PH and Perio4C were segregated according to their genotype (AA, AC, or CC), and a biopsy was performed to establish the culture of the GFs. After GFs exposure to AaPE at 5 µg/ml for 1.5 h, RNA was extracted to analyze IL10 expression by qPCR. Aliquots of the cell's supernatant were subjected to immunoenzymatic analysis (MAGpix) to detect interleukin-10 (IL-10). RESULTS: In PH, the genotypes AA and AC are related to less expression of IL10 (p = 0.027 and p < 0.0001) and less production of IL-10 (p = 0.002 and p = 0.001), when compared to CC. In Perio4C, there was no statistical difference between the genotypes (p > 0.05), although a lower IL-10 expression and release compared with PH CC was seen (p = 0.033 and p < 0.001). CONCLUSION: The rs6667202 SNP is functional in PH, as it decreases the expression and production of IL-10. In Perio4C, other factors may be masking its action by altering the IL-10's response.

Statins with potential to control periodontitis: From biological mechanisms to clinical studies.

BACKGROUND: Statins are widely used for the treatment of hyperlipidemia. However, these drugs have pleiotropic effects that can be promising for the prevention and treatment of oral diseases, such as periodontitis. HIGHLIGHT: This review aimed to identify preclinical, observational, and clinical studies that evaluate the effects and biological mechanisms of statins on oral cells and tissues and those using these drugs to treat periodontitis. A literature survey has been conducted in PubMed using combinations of the uniterms: "statins," "dentistry," "periodontal disease," and "periodontal treatment." In vitro findings showed positive statin results in cell lines related to alveolar bone metabolism by altering the signaling pathway Osteoprotegerin/Receptor Activator of Nuclear Factor Kappa B/Receptor Activator of Nuclear Factor Kappa B Ligand (OPG/RANK/RANKL), stimulating the production of alkaline phosphatase and osteocalcin, and reducing the production of matrix metalloproteinases (MMPs). Animal studies have shown a reduction in alveolar bone loss and osteoclastic activity, in addition to a reduction in inflammatory markers, such as IL-1, IL-6, and TNF-α, when statins were used prophylactically. Clinical trials showed a positive impact on clinical parameters, leading to a higher reduction in probing depth and gain in clinical attachment when a local statin was adjunctively associated with mechanical therapy. CONCLUSION: Statins were shown to be promising for regenerating and stimulating bone activity, with great potential for treating chronic periodontitis. However, further studies are required to confirm its effectiveness.

Máscaras caseiras na pandemia de COVID-19: recomendações, características físicas, desinfecção e eficácia de uso / Mascarillas caseras en la pandemia de COVID-19: recomendaciones, características físicas, desinfección y eficacia de uso / Homemade masks in the COVID-19 pandemic: recommendations, physical characteristics, disinfection and efficacy

Objetivo: Descrever as recomendações, características físicas, métodos de desinfecção e eficácia de uso de máscaras caseiras na redução da transmissão da COVID-19. Métodos: Realizou-se busca nas bases de dados MEDLINE, SciELO e Google Scholar, além das recomendações oficiais de uso. Resultados: Foram incluídas 31 referências. A capacidade de filtração de tecidos variou entre 5% e 98%. Tecidos 100% algodão em duas ou três camadas apresentaram eficácia de filtração entre 70% e 99% em estudos in vitro. Máscaras caseiras, cirúrgicas e respiradores apresentaram respirabilidade entre 2,2 e 3,0 Pascal. A capacidade de redução da propagação de microrganismos por pessoas usando máscaras caseiras foi três vezes menor do que usando máscaras cirúrgicas, embora tenha sido superior ao não uso de máscaras. Conclusão: A respirabilidade de máscaras caseiras mostrou-se adequada, enquanto a capacidade de filtração parece ser inferior à das máscaras cirúrgicas, mas superior a não se usar máscara. Não há evidências que respaldem a eficácia e efetividade das máscaras caseiras.

Objetivo: Describir las recomendaciones, características físicas, métodos de desinfección y efectividad de mascarillas caseras para reducir la transmisión de COVID-19. Métodos: La búsqueda se realizó en las bases de datos MEDLINE, SciELO y Google Scholar, además de las recomendaciones oficiales de uso. Resultados: Se incluyeron 31 referencias. La capacidad de filtración de los tejidos varió entre 5% y 98%. Los tejidos al 100% de algodón, en dos o tres capas, mostraron eficiencia de filtración entre 70% y 99%, en estudios in vitro. Mascarillas caseras, quirúrgicas y de respiradores mostraron respirabilidad entre 2,2 y 3,0 Pascal. La capacidad de reducir la propagación de microorganismos por personas que usan máscarillas caseras fue tres veces menor que cuando usaban mascarillas quirúrgicas, pero superior a no usarlas. Conclusión: La respirabilidad de las mascarillas caseras puede ser adecuada, mientras que la eficiencia de filtración parece ser inferior a la de las mascarillas quirúrgicas, pero superior a no utilizar mascarilla. No hay evidencia que respalde su eficacia y efectividad.

Objective: To describe the recommendations, physical characteristics, disinfection methods and efficacy of the use of homemade face masks to reduce COVID-19 transmission. Methods: We searched MEDLINE, SciELO, and Google Scholar, in addition to the official recommendations for the use of masks. Results: Thirty-one references were included. Fabric filtration efficiency ranged from 5% to 98%. The filtration efficacy of three layered 100% cotton fabric face masks ranged from 70% and 99% in vitro studies. Homemade, surgical, and respirator masks showed breathability between 2.2 and 3.0 Pascal. The capacity to reduce the spread of microorganisms by people wearing homemade face masks was three times lower when compared to those wearing surgical masks, although this capacity was higher when compared to those who did not wear masks. Conclusion: The breathability of homemade masks proved to be adequate, while the filtration ability seemed to be lower than that of surgical masks, but it was better than not wearing any masks at all. There is no evidence to support the efficacy and effectiveness of homemade masks.

Stress-related salivary proteins affect the production of volatile sulfur compounds by oral bacteria.

OBJECTIVE: To determine whether stress-related substances and sex hormones influence the growth and in vitro production of volatile sulfur compounds (VSCs) by Solobacterium moorei and Fusobacterium nucleatum. MATERIALS AND METHODS: Bacteria growth and VSCs production were evaluated in the presence of alpha-amylase, beta-defensin-2, mucin, estradiol, and progesterone. Growth was evaluated by colony counting, and the production of the VSCs hydrogen sulfide (H2 S) and methyl mercaptan (CH3 SH) was measured using the Oral Chroma™ instrument. RESULTS: Mucin induced the production of H2 S by both bacteria, but had a slight inhibitory effect on CH3 SH production by F. nucleatum. It also increased the viability of F. nucleatum. Alpha-amylase increased H2 S production by S. moorei and CH3 SH production by F. nucleatum, but had no effect on H2 S production by F. nucleatum. No substance altered the viability of S. moorei. No effects of beta-defensin-2, estradiol, or progesterone were observed. CONCLUSION: The salivary stress-related proteins mucin and alpha-amylase altered VSCs production by F. nucleatum and S. moorei, favoring H2 S production. These findings are a step toward understanding the relation between stress and increased amounts of H2 S.

Effects of chlorhexidine preprocedural rinse on bacteremia in periodontal patients: a randomized clinical trial

Abstract Objective: Single dose of systemic antibiotics and short-term use of mouthwashes reduce bacteremia. However, the effects of a single dose of preprocedural rinse are still controversial. This study evaluated, in periodontally diseased patients, the effects of a pre-procedural mouth rinse on induced bacteremia. Material and Methods: Systemically healthy individuals with gingivitis (n=27) or periodontitis (n = 27) were randomly allocated through a sealed envelope system to: 0.12% chlorhexidine pre-procedural rinse (13 gingivitis and 13 periodontitis patients) or no rinse before dental scaling (14 gingivitis and 15 periodontitis patients). Periodontal probing depth, clinical attachment level, plaque, and gingival indices were measured and subgingival samples were collected. Blood samples were collected before dental scaling, 2 and 6 minutes after scaling. Total bacterial load and levels of P. gingivalis were determined in oral and blood samples by real-time polymerase chain reaction, while aerobic and anaerobic counts were determined by culture in blood samples. The primary outcome was the antimicrobial effect of the pre-procedural rinse. Data was compared by Mann-Whitney and Signal tests (p<0.05). Results: In all sampling times, polymerase chain reaction revealed higher blood bacterial levels than culture (p<0.0001), while gingivitis patients presented lower bacterial levels in blood than periodontitis patients (p<0.0001). Individuals who experienced bacteremia showed worse mean clinical attachment level (3.4 mm vs. 1.1 mm) and more subgingival bacteria (p<0.005). The pre-procedural rinse did not reduce induced bacteremia. Conclusions: Bacteremia was influenced by periodontal parameters. In periodontally diseased patients, pre-procedural rinsing showed a discrete effect on bacteremia control.

Silver nanoparticles in dentistry.

OBJECTIVE: Silver nanoparticles (AgNPs) have been extensively studied for their antimicrobial properties, which provide an extensive applicability in dentistry. Because of this increasing interest in AgNPs, the objective of this paper was to review their use in nanocomposites; implant coatings; pre-formulation with antimicrobial activity against cariogenic pathogens, periodontal biofilm, fungal pathogens and endodontic bacteria; and other applications such as treatment of oral cancer and local anesthesia. Recent achievements in the study of the mechanism of action and the most important toxicological aspects are also presented. METHODS: Systematic searches were carried out in Web of Science (ISI), Google, PubMed, SciFinder and EspaceNet databases with the keywords "silver nano* or AgNP*" and "dentist* or dental* or odontol*". RESULTS: A total of 155 peer-reviewed articles were reviewed. Most of them were published in the period of 2012-2017, demonstrating that this topic currently represents an important trend in dentistry research. In vitro studies reveal the excellent antimicrobial activity of AgNPs when associated with dental materials such as nanocomposites, acrylic resins, resin co-monomers, adhesives, intracanal medication, and implant coatings. Moreover, AgNPs were demonstrated to be interesting tools in the treatment of oral cancers due to their antitumor properties. SIGNIFICANCE: The literature indicates that AgNPs are a promising system with important features such as antimicrobial, anti-inflammatory and antitumor activity, and a potential carrier in sustained drug delivery. However, there are some aspects of the mechanisms of action of AgNPs, and some important toxicological aspects arising from the use of this system that must be completely elucidated.

In vitro effects of Melaleuca alternifolia essential oil on growth and production of volatile sulphur compounds by oral bacteria

ABSTRACT Objective Halitosis can be caused by microorganisms that produce volatile sulphur compounds (VSCs), which colonize the surface of the tongue and subgingival sites. Studies have reported that the use of natural products can reduce the bacterial load and, consequently, the development of halitosis. The aim of this study was to evaluate the antimicrobial activity of the essential oil of Melaleuca alternifolia on the growth and volatile sulphur compound (VSC) production of oral bacteria compared with chlorhexidine. Material and Methods The effects of these substances were evaluated by the Minimum Inhibitory Concentration (MIC) and Minimum Bactericidal Concentration (MBC) in planktonic cultures of Porphyromonas gingivalis and Porphyromonas endodontalis. In addition, gas chromatography analyses were performed to measure the concentration of VSCs from bacterial cultures and to characterize M. alternifolia oil components. Results The MIC and MBC values were as follows: M. alternifolia - P. gingivalis (MIC and MBC=0.007%), P. endodontalis (MIC and MBC=0.007%=0.5%); chlorhexidine - P. gingivalis and P. endodontalis (MIC and MBC=1.5 mg/mL). M. alternifolia significantly reduced the growth and production of hydrogen sulfide (H2S) by P. gingivalis (p<0.05, ANOVA-Dunnet) and the H2S and methyl mercaptan (CH3SH) levels of P. endodontalis (p<0.05, ANOVA-Dunnet). Chlorhexidine reduced the growth of both microorganisms without altering the production of VSC in P. endodontalis. For P. gingivalis, the production of H2S and CH3SH decreased (p<0.05, ANOVA-Dunnet). Conclusion M. alternifolia can reduce bacterial growth and VSCs production and could be used as an alternative to chlorhexidine.

In vitro effects of Melaleuca alternifolia essential oil on growth and production of volatile sulphur compounds by oral bacteria.

OBJECTIVE: Halitosis can be caused by microorganisms that produce volatile sulphur compounds (VSCs), which colonize the surface of the tongue and subgingival sites. Studies have reported that the use of natural products can reduce the bacterial load and, consequently, the development of halitosis. The aim of this study was to evaluate the antimicrobial activity of the essential oil of Melaleuca alternifolia on the growth and volatile sulphur compound (VSC) production of oral bacteria compared with chlorhexidine. MATERIAL AND METHODS: The effects of these substances were evaluated by the Minimum Inhibitory Concentration (MIC) and Minimum Bactericidal Concentration (MBC) in planktonic cultures of Porphyromonas gingivalis and Porphyromonas endodontalis. In addition, gas chromatography analyses were performed to measure the concentration of VSCs from bacterial cultures and to characterize M. alternifolia oil components. RESULTS: The MIC and MBC values were as follows: M. alternifolia - P. gingivalis (MIC and MBC=0.007%), P. endodontalis (MIC and MBC=0.007%=0.5%); chlorhexidine - P. gingivalis and P. endodontalis (MIC and MBC=1.5 mg/mL). M. alternifolia significantly reduced the growth and production of hydrogen sulfide (H2S) by P. gingivalis (p<0.05, ANOVA-Dunnet) and the H2S and methyl mercaptan (CH3SH) levels of P. endodontalis (p<0.05, ANOVA-Dunnet). Chlorhexidine reduced the growth of both microorganisms without altering the production of VSC in P. endodontalis. For P. gingivalis, the production of H2S and CH3SH decreased (p<0.05, ANOVA-Dunnet). CONCLUSION: M. alternifolia can reduce bacterial growth and VSCs production and could be used as an alternative to chlorhexidine.

Downregulation of Proteinase-Activated Receptor-2, Interleukin-17, and Other Proinflammatory Genes by Subantimicrobial Doxycycline Dose in a Rat Periodontitis Model.

BACKGROUND: Subantimicrobial dose doxycycline (SDD) has been used as an adjunct in periodontal treatment because of its matrix metalloproteinase inhibition properties. Although the benefits of SDD therapy, such as improvement in the parameters of periodontal probing depth and clinical attachment level, have been proven in multiple clinical studies, the comprehension of other biologic mechanisms of action on periodontitis remains poorly investigated. Therefore, this animal-model study evaluated the effects of SDD monotherapy on the expressions of the following key proinflammatory genes: proteinase-activated receptor-2 (PAR2), tumor necrosis factor (TNF)-α, interleukin (IL)-17, and IL-1ß. METHODS: Male Wistar rats were assigned randomly to the following: 1) control group: no ligature-induced periodontitis and no treatment; 2) ligature group: ligature-induced periodontitis and placebo treatment; and 3) ligature + doxycycline group: ligature-induced periodontitis and SDD treatment. After the experimental time, animals were sacrificed, and reverse transcription-polymerase chain reaction was performed to analyze the mRNA expression of IL-1ß, IL-17, TNF-α, and PAR2 in gingival tissue samples. Histologic analyses were performed on the furcation region and mesial gingiva of mandibular first molars to measure periodontal bone loss and collagen content. RESULTS: SDD administration significantly downregulated PAR2, IL-17, TNF-α, and IL-1ß mRNA expressions (P <0.05). In addition, SDD treatment was accompanied by lower rates of alveolar bone loss (P <0.05) and maintenance of the amount of gingival collagen fibers. CONCLUSION: These findings reveal new perspectives regarding SDD efficacy because it can be partially related to proinflammatory gene expression modulation, even considering PAR2 and IL-17, which has not been investigated thus far.