RESUMO
OBJECTIVES: the objective of the present exploratory study was to determine bacterial diversity and endotoxin levels in deep carious lesions of teeth presenting symptoms of reversible pulpitis. MATERIALS AND METHODS: Twenty patients with deep carious lesions, reporting clinical symptomatology compatible with reversible pulpitis (n = 10) or not reporting clinical symptomatology (n = 10), were selected. Carious dentin samples were obtained with the aid of sterile and pyrogen-free spoon excavators and harvested in two steps: before and after infected dentin removal. Samples were collected for checkerboard and for kinetic chromogenic LAL assay for determination of microbial profile and quantitation of endotoxin, respectively. Data were analyzed by Mann Whitney for bacteria and two-way ANOVA for endotoxins (5%). RESULTS: No difference on the studied bacteria was detected between the superficial and deep dentin layers. Symptomatic teeth showed greater presence of Lactobacillus species, Capnocytophaga sputigena, and Leptotrichia buccalis. For the endotoxins, symptomatic teeth resulted in greater quantity of endotoxins (p = 0.047), being 4.13 log10 EU/mL/µg dentin and 3.45 log10 EU/mL/µg dentin, for symptomatic and asymptomatic teeth, respectively. Dentin collected in different areas presented similar number of endotoxins (p = 0.139). CONCLUSION: The amount of the studied bacteria does not seem to be related to reported symptomatology of deep carious lesions, while endotoxins quantity is greater in symptomatic scenarios, regardless of the harvesting area. CLINICAL RELEVANCE: The understanding of bacterial amount in reversible pulpitis is important to establish a clinical protocol of treatment.
Assuntos
Cárie Dentária , Pulpite , Bactérias , Capnocytophaga , Dentina , Endotoxinas , Humanos , LeptotrichiaRESUMO
OBJECTIVES: To compare the microbial load and composition and to determine the lipopolysaccharides (LPS) and lipoteichoic acid (LTA) concentrations found in primary apical periodontitis (PAP) and post-treatment apical periodontitis (PTAP), correlating these findings with clinical/tomographic features. MATERIAL AND METHODS: Sixty patients with PAP (31) and PTAP (29) were submitted to clinical and tomographic assessment. Samples were collected from each root canal using paper points for microbiological assessment (culture technique and Checkerboard DNA-DNA hybridization) and determination of LPS and LTA levels (limulus amebocyte lysate and enzyme-linked immunosorbent assays, respectively). Data were correlated with clinical/tomographic findings and statistically analyzed using the Mann-Whitney and Pearson correlation tests (α = 5%). RESULTS: A higher number of cultivable bacteria and LPS were found in PAP (p < 0.05). The median number of species per root canal found in PAP and PTAP was 9 and 22, respectively (p < 0.05). LPS was positively correlated with a larger periapical lesion volume (p < .05). LTA levels were similar in both infections and had no correlation with signs and symptoms. In PAP, gram-positive bacteria were correlated with spontaneous pain (p < .05) and exudate (p < .05). Tenderness to percussion and pain on palpation were correlated to the presence of both gram-positive and negative bacteria. In PTAP, a positive correlation was observed between both gram-positive and gram-negative bacteria with exudate and periapical lesion volume (p < .05). CONCLUSIONS: PAP had higher contents of microbial load and LPS compared with PTAP. However, PTAP presented a more diverse microbiota compared with PAP. Higher content of LPS was positively correlated with larger periapical bone destruction, whereas signs and symptoms with specific microorganisms. CLINICAL RELEVANCE: It was verified that PAP and PTAP are polymicrobial infections with predominance of gram-negative bacteria and a more diverse bacterial population found in PTAP. A wide interaction of specific microbial species resulted in different clinical features in both infections.
Assuntos
Lipopolissacarídeos , Periodontite Periapical , Antibacterianos , Cavidade Pulpar , Bactérias Gram-Negativas , Bactérias Gram-Positivas , Humanos , Periodontite Periapical/complicações , Periodontite Periapical/metabolismo , Periodontite Periapical/microbiologia , Ácidos TeicoicosRESUMO
Biological effects of titanium (Ti) alloys were analyzed on biofilms of Candida albicans, Enterococcus faecalis, Pseudomonas aeruginosa, Staphylococcus aureus, Streptococcus mutans, and Streptococcus sanguinis, as well as on osteoblast-like cells (MG63) and murine macrophages (RAW 264.7). Standard samples composed of aluminum and vanadium (Ti-6Al-4V), and sample containing niobium (Ti-35Nb) and zirconium (Ti-13Nb-13Zr) were analyzed. Monomicrobial biofilms were formed on the Ti alloys. MG63 cells were grown with the alloys and the biocompatibility (MTT), total protein (TP) level, alkaline phosphatase (ALP) activity, and mineralization nodules (MN) formation were verified. Levels of interleukins (IL-1ß and IL-17), tumor necrosis factor alpha (TNF-α), and oxide nitric (NO) were checked, from RAW 264.7 cells supernatants. Data were statically analyzed by one-way analysis of variance (ANOVA) and Tukey's test, or T-test (P ≤ 0.05). Concerning the biofilm formation, Ti-13Nb-13Zr alloy showed the best inhibitory effect on E. faecalis, P. aeruginosa, and S. aureus. And, it also acted similarly to the Ti-6Al-4V alloy on C. albicans and Streptococcus spp. Both alloys were biocompatible and similar to the Ti-6Al-4V alloy. Additionally, Ti-13Nb-13Zr alloy was more effective for cell differentiation, as observed in the assays of ALP and MN. Regarding the stimulation for release of IL-1ß and TNF-α, Ti-35Nb and Ti-13Nb-13Zr alloys inhibited similarly the synthesis of these molecules. However, both alloys stimulated the production of IL-17. Additionally, all Ti alloys showed the same effect for NO generation. Thus, Ti-13Nb-13Zr alloy was the most effective for inhibition of biofilm formation, cell differentiation, and stimulation for release of immune mediators.
Assuntos
Ligas/farmacologia , Materiais Biocompatíveis/farmacologia , Biofilmes/efeitos dos fármacos , Diferenciação Celular/efeitos dos fármacos , Osteogênese/efeitos dos fármacos , Titânio/farmacologia , Ligas/química , Animais , Materiais Biocompatíveis/química , Biofilmes/crescimento & desenvolvimento , Candida albicans/efeitos dos fármacos , Candida albicans/fisiologia , Células Cultivadas , Teste de Materiais , Camundongos , Testes de Sensibilidade Microbiana , Osteoblastos/citologia , Osteoblastos/efeitos dos fármacos , Osteoblastos/fisiologia , Pseudomonas/efeitos dos fármacos , Pseudomonas/fisiologia , Células RAW 264.7 , Staphylococcus aureus/efeitos dos fármacos , Staphylococcus aureus/fisiologia , Streptococcus/efeitos dos fármacos , Streptococcus/fisiologia , Propriedades de Superfície , Titânio/químicaRESUMO
This study evaluated the prophylactic effects of the live or heat-killed probiotic strain Lactobacillus rhamnosus ATCC 7469 in Galleria mellonella, inoculated with Staphylococcus aureus or Escherichia coli. L. rhamnosus suspension was prepared and a part of it was autoclaved to obtain heat-killed lactobacilli. The larvae were inoculated of these suspensions and pathogenic. The survival of the larvae was observed during 7 days and after 24 h of inoculation haemocytes counted, melanization and nitric oxide production were analyzed. Larvae survival rate increased in the group inoculated with heat-killed L. rhamnosus, however, with no statistical difference. There was a significant increase in total haemocyte counts in all test groups. Haemolymph melanization and nitric oxide production were higher in the group inoculated with L. rhamnosus and infected with S. aureus. It was concluded that, in this model of infection, heat-killed L. rhamnosus ATCC 7469 promoted greater protection in Galleria mellonella infected with S. aureus or E. coli.
Assuntos
Escherichia coli/imunologia , Lacticaseibacillus rhamnosus/imunologia , Mariposas/imunologia , Staphylococcus aureus/imunologia , Animais , Escherichia coli/fisiologia , Hemolinfa/metabolismo , Interações Hospedeiro-Patógeno , Larva/imunologia , Larva/microbiologia , Mariposas/microbiologia , Probióticos , Staphylococcus aureus/fisiologiaRESUMO
INTRODUCTION: This study evaluated the biocompatibility of 5 and 10 µg/mL LL-37 in vitro and its effect on the differentiation of human dental pulp stem cells (DPSCs) into odontoblast-like cells. METHODS: Cell viability, genotoxicity, nitric oxide production, cell cycle, dentine sialophosphoprotein (DSPP) production, and DSPP gene expression. RESULTS: Concentrations of 5 and 10 µg/mL of LL-37 were not cytotoxic and generally increased cell viability, especially on the third day (P < .05). The tested concentrations did not induce genotoxicity (P < .05). LL-37 did not significantly alter nitrite production at either concentration. Cell cycle analysis revealed that 10 µg/mL of LL-37 arrested cells in G0/G1 (P < .05). The control group exhibited higher numbers of cells in other phases of the cell cycle (P < .05). The expression of the DSPP protein and gene was also higher in the 10 µg/mL of LL-37 group (P < .05). CONCLUSIONS: These results demonstrated that LL-37 was biocompatible at these concentrations and increased the number of viable cells, especially during the initial period. The 10 µg/mL concentration arrested the cell cycle and increased expression of the DSPP protein and gene, which indicates that this peptide contributes to odontoblastic differentiation.
Assuntos
Catelicidinas/farmacologia , Diferenciação Celular/efeitos dos fármacos , Proliferação de Células/efeitos dos fármacos , Polpa Dentária/citologia , Polpa Dentária/efeitos dos fármacos , Células-Tronco/citologia , Células-Tronco/efeitos dos fármacos , Peptídeos Catiônicos Antimicrobianos , Células Cultivadas , HumanosRESUMO
R. officinalis L. is an aromatic plant commonly used as condiment and for medicinal purposes. Biological activities of its extract were evaluated in this study, as antimicrobial effect on mono- and polymicrobial biofilms, cytotoxicity, anti-inflammatory capacity, and genotoxicity. Monomicrobial biofilms of Candida albicans, Staphylococcus aureus, Enterococcus faecalis, Streptococcus mutans and Pseudomonas aeruginosa and polymicrobial biofilms composed of C. albicans with each bacterium were formed in microplates during 48 h and exposed for 5 min to R. officinalis L. extract (200 mg/mL). Its cytotoxic effect was examined on murine macrophages (RAW 264.7), human gingival fibroblasts (FMM-1), human breast carcinoma cells (MCF-7), and cervical carcinoma cells (HeLa) after exposure to different concentrations of the extract, analyzed by MTT, neutral red (NR), and crystal violet (CV) assays. The anti-inflammatory activity was evaluated on RAW 264.7 non-stimulated or stimulated by lipopolysaccharide (LPS) from Escherichia coli and treated with different concentrations of the extract for 24 h. Interleukin-1 beta (IL-1ß) and tumor necrosis factor alpha (TNF-α) were quantified by ELISA. Genotoxicity was verified by the frequency of micronuclei (MN) at 1000 cells after exposure to concentrations of the extract for 24 h. Data were analyzed by T-Test or ANOVA and Tukey Test ( P ≤ 0.05). Thus, significant reductions in colony forming units per milliliter (CFU/mL) were observed in all biofilms. Regarding the cells, it was observed that concentrations ≤ 50 mg/mL provided cell viability of above 50%. Production of proinflammatory cytokines in the treated groups was similar or lower compared to the control group. The MN frequency in the groups exposed to extract was similar or less than the untreated group. It was shown that R. officinalis L. extract was effective on mono- and polymicrobial biofilms; it also provided cell viability of above 50% (at ≤ 50 mg/mL), showed anti-inflammatory effect, and was not genotoxic. Impact statement Rosmarinus officinalis L. extract effectively contributed to in vitro control of important species of microorganisms such as Candida albicans, Staphylococcus aureus, Enterococcus faecalis, Streptococcus mutans, and Pseudomonas aeruginosa in mono- and polymicrobial biofilms that are responsible for several infections in oral cavity as in other regions of the body. Furthermore, this extract promoted also cell viability above 50% at concentrations ≤ 50 mg/mL, excellent anti-inflammatory effect, showing inhibition or reduction of the synthesis of proinflammatory cytokines, being also non-genotoxic to cell lines studied. Thus, this extract may be a promising therapeutic agent that can be added in some medical and dental formulations such as toothpastes, mouthwashes, irrigating root canals, ointments, soaps, in order to control pathogenic microorganisms and biofilms, with anti-inflammatory effect and absence of cytotoxic and genotoxic.
Assuntos
Extratos Vegetais/farmacologia , Rosmarinus , Animais , Antibacterianos/farmacologia , Anti-Inflamatórios/farmacologia , Biofilmes/efeitos dos fármacos , Candida albicans/efeitos dos fármacos , Citotoxinas/farmacologia , Relação Dose-Resposta a Droga , Enterococcus faecalis/efeitos dos fármacos , Fibroblastos/efeitos dos fármacos , Células HeLa/efeitos dos fármacos , Humanos , Células MCF-7/efeitos dos fármacos , Camundongos , Testes de Sensibilidade Microbiana , Folhas de Planta/química , Pseudomonas aeruginosa/efeitos dos fármacos , Células RAW 264.7/efeitos dos fármacos , Rosmarinus/química , Staphylococcus aureus/efeitos dos fármacos , Streptococcus mutans/efeitos dos fármacosRESUMO
This study evaluated the action of Pfaffia paniculata K., Juglans regia L., and Rosmarius officinalis L. extracts against planktonic form and biofilm of Klebsiella pneumoniae (ATCC 4352). Minimum inhibitory concentration (MIC) and minimum microbicidal concentration (MMC) values were determined for each extract by microdilution broth method, according to Clinical and Laboratory Standards Institute. Next, antimicrobial activity of the extracts on biofilm was analyzed. For this, standardized suspension at 107 UFC/mL of K. pneumoniae was distributed into 96-well microplates (n = 10) and after 48 h at 37°C and biofilm was subjected to treatment for 5 min with the extracts at a concentration of 200 mg/mL. ANOVA and Tukey tests (5%) were used to verify statistical significant reduction (p < 0.05) of planktonic form and biofilm. P paniculata K., R. officinalis L., and J. regia L. showed reductions in biomass of 55.6, 58.1, and 18.65% and cell viability reduction of 72.4, 65.1, and 31.5%, respectively. The reduction obtained with P. paniculata and R. officinalis extracts was similar to the reduction obtained with chlorhexidine digluconate 2%. In conclusion, all extracts have microbicidal action on the planktonic form but only P. paniculata K. and R. officinalis L. were effective against biofilm.
Assuntos
Antibacterianos/farmacologia , Biofilmes , Klebsiella pneumoniae/efeitos dos fármacos , Extratos Vegetais/farmacologia , Amaranthaceae/química , Juglans/química , Klebsiella pneumoniae/crescimento & desenvolvimento , Testes de Sensibilidade Microbiana , Rosmarinus/químicaRESUMO
This study aimed to evaluate the capacity of Lactobacillus rhamnosus and/or its products to induce the synthesis of cytokines (TNF-α, IL-1ß, IL-4, IL-6, IL-10, and IL-12) by mouse macrophages (RAW 264.7). Three microorganism preparations were used: live L. rhamnosus (LLR) suspension, heat-killed L. rhamnosus (HKLR) suspension, and the supernatant of a heat-killed L. rhamnosus (SHKLR) suspension, which were cultured with macrophages (37°C, 5% CO2) for 2 h and 30 min. After that, cells were cultured for 16 h. The supernatants were used for the quantitation of cytokines, by ELISA. The results were compared with the synthesis induced by lipopolysaccharide (LPS) and analysed, using ANOVA and Tukey test, 5%. LLR and HKLR groups were able to significantly increase the production of TNF-α, IL-6, and IL-10 (P < 0.05). SHKLR also significantly increased the production of TNF-α and IL-10 (P < 0.05) but not IL-6 (P > 0.05). All the L. rhamnosus suspensions were not able to produce detectable levels of IL-1ß or significant levels of IL-4 and IL-12 (P > 0.05). In conclusion, live and heat-killed L. rhamnosus suspensions were able to induce the synthesis of different cytokines with proinflammatory (TNF-α and IL-6) or regulatory (IL-10) functions, suggesting the role of strain L. rhamnosus ATCC 7469 in the modulation or in the stimulation of immune responses.