Cytotoxicity and bioactive potential of new root repair materials for use with BMP-2 transfected human osteoblast cells.

Modified formulations of calcium silicate repair materials with additives have been developed to enhance handling, consistency, biocompatibility and bioactivity. Considering the relevance of osteoblastic cell response to mineralized tissue repair, human osteoblastic cells (Saos-2 cells overexpressing BMP-2) were exposed to mineral trioxide aggregate (MTA) (with calcium tungstate - CaWO4), MTA HP Repair, Bio-C Repair and Bio-C Pulpo. Cell viability was assessed by 3-(4,5-dimethylthiazol)-2,5-diphenyltetrazolium bromide (MTT) and neutral red (NR), and cell death, by flow cytometry. Gene expression of bone morphogenetic protein 2 (BMP-2), runt-related transcription factor 2 (RUNX-2), and alkaline phosphatase (ALP) osteogenic markers were evaluated by real-time polymerase chain reaction (RT-qPCR). ALP activity and alizarin red staining (ARS) were used to detect mineralization nodule deposition. Bioactive cements presented no cytotoxic effect, and did not induce apoptosis at the higher dilution (1:12). MTA, Bio-C Repair and Bio-C Pulpo exhibited higher ALP activity than the control group (P < 0.05) after 7 days. MTA, MTA HP and Bio-C Pulpo affected the formation of mineralized nodules (p < 0.05). Exposure to all cement extracts for 1 day increased BMP-2 gene expression. RUNX-2 mRNA was greater in MTA, MTA HP and Bio-C Repair. MTA, MTA HP and Bio-C Pulpo increased the ALP mRNA expression, compared with BMP-2 unexposed cells (P < 0.05). Calcium silicate cements showed osteogenic potential and biocompatibility in Saos-2 cells transfected BMP-2, and increased the mRNA expression of BMP-2, RUNX-2, and ALP osteogenic markers in the BMP-2 transfected system, thereby promoting a cellular response to undertake the mineralized tissue repair.

Different formulations of peracetic acid: effects on smear layer removal, dentine erosion, cytotoxicity and antibiofilm activity.

OBJECTIVE: To assess the effects of different peracetic acid (PAA) formulations on smear layer (SL) removal, dentine erosion, cytotoxicity, and antibiofilm activity. METHODOLOGY: SL removal and dentine erosion were assessed using 90 premolars, distributed into six groups, according to final irrigation: PAA formulations (1% Sigma, 1% Bacterend OX, 1% Arposept, and 0.09-0.15% Anioxyde), 17% ethylenediaminetetraacetic acid (EDTA), and water (control). Cytotoxicity was assessed by methyl-thiazol-tetrazolium (MTT) and neutral red assays. Antibacterial and antibiofilm effectiveness was evaluated against Enterococcus faecalis. For cytotoxicity and antibiofilm activity assessment, the 2.5% NaOCl was also included. RESULTS: EDTA, Sigma, and Bacterend OX removed more SL than Arposept, Anioxyde, and water (p<0.05). EDTA caused more severe dentine erosion than Sigma and Bacterend OX (p<0.05). Sigma and Bacterend OX had higher cytotoxicity than the other solutions (p<0.05). NaOCl, Bacterend OX, Sigma, and Anioxyde significantly reduced E. faecalis colony-forming units (CFU) (p<0.05). The 2.5% NaOCl solution promoted greater biofilm biomass reduction (p<0.05) than the other solutions. All PAA formulations promoted greater biomass reduction than 17% EDTA (p<0.05). CONCLUSIONS: Although Sigma and Bacterend OX had higher cytotoxicity, they had a SL removal capability similar to that of EDTA, were as effective as NaOCl against E. faecalis biofilm, and promoted less dentine erosion than EDTA. Arposept and Anioxyde failed to remove the SL, had lower cytotoxicity, and showed less bacterial activity than NaOCl.

Cytotoxicity and bioactive potential of new root repair materials for use with BMP-2 transfected human osteoblast cells

Abstract: Modified formulations of calcium silicate repair materials with additives have been developed to enhance handling, consistency, biocompatibility and bioactivity. Considering the relevance of osteoblastic cell response to mineralized tissue repair, human osteoblastic cells (Saos-2 cells overexpressing BMP-2) were exposed to mineral trioxide aggregate (MTA) (with calcium tungstate - CaWO4), MTA HP Repair, Bio-C Repair and Bio-C Pulpo. Cell viability was assessed by 3-(4,5-dimethylthiazol)-2,5-diphenyltetrazolium bromide (MTT) and neutral red (NR), and cell death, by flow cytometry. Gene expression of bone morphogenetic protein 2 (BMP-2), runt-related transcription factor 2 (RUNX-2), and alkaline phosphatase (ALP) osteogenic markers were evaluated by real-time polymerase chain reaction (RT-qPCR). ALP activity and alizarin red staining (ARS) were used to detect mineralization nodule deposition. Bioactive cements presented no cytotoxic effect, and did not induce apoptosis at the higher dilution (1:12). MTA, Bio-C Repair and Bio-C Pulpo exhibited higher ALP activity than the control group (P < 0.05) after 7 days. MTA, MTA HP and Bio-C Pulpo affected the formation of mineralized nodules (p < 0.05). Exposure to all cement extracts for 1 day increased BMP-2 gene expression. RUNX-2 mRNA was greater in MTA, MTA HP and Bio-C Repair. MTA, MTA HP and Bio-C Pulpo increased the ALP mRNA expression, compared with BMP-2 unexposed cells (P < 0.05). Calcium silicate cements showed osteogenic potential and biocompatibility in Saos-2 cells transfected BMP-2, and increased the mRNA expression of BMP-2, RUNX-2, and ALP osteogenic markers in the BMP-2 transfected system, thereby promoting a cellular response to undertake the mineralized tissue repair.

Different formulations of peracetic acid: effects on smear layer removal, dentine erosion, cytotoxicity and antibiofilm activity

Abstract Objective: To assess the effects of different peracetic acid (PAA) formulations on smear layer (SL) removal, dentine erosion, cytotoxicity, and antibiofilm activity. Methodology: SL removal and dentine erosion were assessed using 90 premolars, distributed into six groups, according to final irrigation: PAA formulations (1% Sigma, 1% Bacterend OX, 1% Arposept, and 0.09-0.15% Anioxyde), 17% ethylenediaminetetraacetic acid (EDTA), and water (control). Cytotoxicity was assessed by methyl-thiazol-tetrazolium (MTT) and neutral red assays. Antibacterial and antibiofilm effectiveness was evaluated against Enterococcus faecalis. For cytotoxicity and antibiofilm activity assessment, the 2.5% NaOCl was also included. Results: EDTA, Sigma, and Bacterend OX removed more SL than Arposept, Anioxyde, and water (p<0.05). EDTA caused more severe dentine erosion than Sigma and Bacterend OX (p<0.05). Sigma and Bacterend OX had higher cytotoxicity than the other solutions (p<0.05). NaOCl, Bacterend OX, Sigma, and Anioxyde significantly reduced E. faecalis colony-forming units (CFU) (p<0.05). The 2.5% NaOCl solution promoted greater biofilm biomass reduction (p<0.05) than the other solutions. All PAA formulations promoted greater biomass reduction than 17% EDTA (p<0.05). Conclusions: Although Sigma and Bacterend OX had higher cytotoxicity, they had a SL removal capability similar to that of EDTA, were as effective as NaOCl against E. faecalis biofilm, and promoted less dentine erosion than EDTA. Arposept and Anioxyde failed to remove the SL, had lower cytotoxicity, and showed less bacterial activity than NaOCl.

Physicochemical, biological, and antibacterial evaluation of tricalcium silicate-based reparative cements with different radiopacifiers.

OBJECTIVE: To evaluate tricalcium silicate-based (TCS) experimental materials, associated with zirconium oxide (ZrO2), calcium tungstate (CaWO4) or niobium oxide (Nb2O5) radiopacifiers, in comparison with MTA Repair HP (Angelus). METHODS: Physicochemical tests: setting time, radiopacity, pH and solubility. In vitro assays: cytotoxicity: MTT and Neutral Red - NR; cell bioactivity: alkaline phosphatase activity (ALP), Alzarin red staining (ARS) and real time PCR (qPCR). Antibacterial activity: direct contact on Enterococcus faecalis in the planktonic form. Physicochemical and ARS data were submitted to ANOVA/Tukey tests; antibacterial activity, to Kruskall-Wallis and Dunn tests; MTT, NR, ALP and qPCR were analyzed by ANOVA/Bonferroni tests (α = 0.05). RESULTS: TCS + CaWO4 presented the longest setting time and MTA HP the shortest. Except for TCS, all the materials presented radiopacity above 3 mmAl. The cements had alkaline pH, antibacterial activity, low solubility and no cytotoxic effects. The highest ALP activity occurred in 14 days, especially to TCS, TCS + ZrO2 and TCS + CaWO4. TCS + ZrO2, TCS + Nb2O5 and MTAHP had higher mineralized nodule formation than those of the negative control (NC). After 7 days, there was no difference in mRNA expression for ALP, when compared to NC. However, after 14 days there was no overexpressed ALP mRNA, especially TCS + Nb2O5, in relation to the CN. All the materials presented antimicrobial action. SIGNIFICANCE: The pure tricalcium silicate associated with ZrO2, CaWO4 or Nb2O5 had appropriate physicochemical properties, antibacterial activity, cytocompatibility and induced mineralization in Saos-2, indicating their use as reparative materials.

In vivo and in vitro anti-inflammatory and pro-osteogenic effects of citrus cystatin CsinCPI-2.

Cystatins are natural inhibitors of cysteine peptidases. Recently, cystatins derived from plants, named phytocystatins, have been extensively studied. Among them, CsinCPI-2 proteins from Citrus sinensis were identified and recombinantly produced by our group. Thus, this study described the recombinant expression, purification, and inhibitory activity of this new phytocystatin against human cathepsins K and B and assessed the anti-inflammatory effect of CsinCPI-2 in vitro in mouse and in vivo in rats. In addition, the pro-osteogenic effect of CsinCPI-2 was investigated in vitro. The inflammatory response of mouse macrophage cells stimulated with P. gingivalis was modulated by CsinCPI-2. The in vitro results showed an inhibitory effect (p < 0.05) on cathepsin K, cathepsin B, IL-1ß, and TNF-α gene expression. In addition, CsinCPI-2 significantly inhibited in vivo the activity of TNF-α (p < 0.05) in the blood of rats, previously stimulated by E. coli lipopolysaccharide (LPS). CsinCPI-2 had a pro-osteogenic effect in human dental pulp cells, demonstrated by the increase in alkaline phosphatase (ALP) activity, deposition of mineralized nodules, and the gene expression of the osteogenic markers as bone morphogenetic protein 2 (BMP-2), runt-related transcription factor 2 (Runx-2), ALP, osteocalcin, and bone sialoprotein (BSP). These preliminary studies suggested that CsinCPI-2 has a potential anti-inflammatory, and at the same time, a pro-osteogenic effect. This may lead to new therapies for the control of diseases where inflammation plays a key role, such as periodontal disease and apical periodontitis.

Influência da proporção pó-líquido nas propriedades físico-químicas do cimento MTA Repair HP / Influence of powder-liquid ratio on the physicochemical properties of MTA Repair HP cement

Introdução: o MTA Repair HP é um material à base de MTA, com modificações do radiopacificador e veículo, em relação ao MTA Angelus. Objetivo: avaliar o tempo de presa, a radiopacidade e solubilidade do MTA Repair HP na proporção pó-líquido indicada pelo fabricante (MTA HP+, sendo 0,8g de pó e 320 µl de líquido) ou com menor quantidade de pó (MTA HP-, sendo 0,7g de pó e 320 µl de líquido), em comparação ao MTA Angelus. Métodos: a radiopacidade foi avaliada por radiografias dos materiais, em comparação a uma escala de alumínio. O tempo de presa foi avaliado por agulhas de Gilmore e a solubilidade, após imersão dos materiais em água destilada (7 dias). Os dados foram analisados por meio dos testes ANOVA e Tukey, com um nível de significância de 5%. Resultados: os tempos de presa inicial e final foram maiores para o MTA HP- do que para o MTA HP+ e o MTA Angelus (p < 0,05). O MTA HP+ e o MTA HP- apresentaram maior solubilidade do que o MTA Angelus (p < 0,05). Os valores de radiopacidade do MTA HP+ e do MTA HPforam menores do que do MTA Angelus (p < 0,05). Conclusão: a diminuição da quantidade de pó-líquido do MTA HP resulta em tempo de presa mais longo, sem alteração das demais propriedades avaliadas. O MTA HP apresenta menor radiopacidade do que o MTA Angelus.

Effect of rotary instrument associated with different irrigation techniques on removing calcium hydroxide dressing.

Calcium hydroxide [Ca(OH)2 ] residues in root canals may compromise sealing of filling and endodontic treatment success. The aim of this study was to compare the efficacy of using rotary instrument associated with EndoActivator, EndoVac, passive ultrasonic irrigation (PUI), and conventional needle irrigation (CNI), in Ca(OH)2 removal from root canal, by means of scanning electron microscopy (SEM) images. Sixty-six human canines were prepared with the Protaper system up to F5 and filled with Ca(OH)2 . After 7 days, Ca(OH)2 was removed with rotary instrument F5 associated with the irrigation techniques used in each group (n = 15): GI (CNI), GII (EndoVac), GIII (EndoActivator) and GIV (PUI). In all groups 15 mL of 2.5% NaOCl and 3 mL of 17% EDTA were used for Ca(OH)2 removal. The Ca(OH)2 residues was evaluated by SEM in the middle and apical third using a system of scores. The results were analyzed by the Kruskal-Wallis and Dunn tests (α = 0.05). None of the techniques completely removed the Ca(OH)2 from root canals. There was no difference between EndoActivator, EndoVac and PUI (P > 0.05), but the three techniques removed more Ca(OH)2 than the CNI (P < 0,05), in the middle and apical thirds of the root canal. It was concluded that the rotary instrument combined with EndoActivator, EndoVac, and PUI was shown to be more efficient than the rotary instrument combined with the CNI in removing Ca(OH)2 from the root canal.

Evaluation of the pH, calcium release and antibacterial activity of MTA Fillapex / Avaliação do pH, liberação de cálcio e atividade antibacteriana do MTA Fillapex

Objetivo: O estudo avaliou, em diversos períodos de análise, o pH e liberação de cálcio e a atividade antibacteriana proporcionada pelo MTA Fillapex, em relação ao Sealapex e AH Plus. Material e Método: Tubos de polietilenos foram preenchidos com um dos cimentos e imersos em água destilada. Após de 24 horas, 14 e 28 dias, o valor do pH e o cálcio liberado pelos cimentos foram avaliados diretamente na água destilada em que os espécimes permaneceram imersos. A atividade antibacteriana dos cimentos foi avaliada em culturas de Enterococcus faecalis ou Staphylococcus aureus, por meio do teste de difusão em ágar. Os dados obtidos foram submetidos aos testes de ANOVA e Tukey (α=0.05). Resultado: Em todos os períodos analizados, o Sealapex proporcionou os maiores valores de pH (p<0,05) em relação aos demais cimentos e o MTA Fillapex proporcionou maiores valores que o dos AH Plus (p<0,05). Em 14 dias, o MTA Fillapex promoveu maior liberação de cálcio que o proporcionado pelo Sealapex (p<0,05). Em 28 dias, o Sealapex proporcionou maior liberação de cálcio que o MTA Fillapex (p<0,05). Em todos os períodos, o AH Plus apresentou a menor liberação de cálcio em relação aos outros cimentos (p<0,05). Em relação ao E. faecalis, não foram observadas diferenças (p>0,05) entre as zonas de inibição de crescimento bacteriano produzidas pelos diveros cimentos. Em relação ao S. aureus, o Sealapex apresentou maior atividade antibacteriana que o MTA Fillapex e o AH Plus (p<0,05), que por sua vez foram semelhantes entre si (p>0,05). Conclusão: Após o período final de avaliação, o pH e a liberação de cálcio proporcionado pelo MTA Fillapex foram menores que os do Sealapex e maiores que os do AH Plus. A atividade antimicrobiana do MTA Fillapex não diferiu dos demais cimentos endodônticos.

Objective: This study evaluated, in several analysis periods, pH and calcium release and antibacterial activity provided by MTA Fillapex sealer compared to Sealapex and AH Plus sealers. Material and Method: Polyethylene tubes were filled with a sealer and immersed in distilled water. After 24 hours, 14 and 28 days, pH and calcium release by endodontic sealers were evaluated directly in water which the tubes were stored. Sealers antibacterial activity was evaluated against Enterococcus faecalis and Staphylococcus aureus by means of agar diffusion test. All data were submitted to ANOVA and Tukey tests (α=0.05). Result: In all periods evaluated, Sealapex had the highest pH value (p<0.05) in comparison to other sealers and MTA Fillapex provided higher pH values than AH Plus (p<0.05). In 14-days period, MTA Fillapex had greater calcium release value than Sealapex (p<0.05). In 28-days period, Sealapex provided higher calcium release than MTA Fillapex (p<0.05). In all periods, AH Plus provided lower calcium release than other sealers (p<0.05). In relation to E. faecalis, there were no differences among the sealers, in relation to antibacterial activity (p>0.05). In relation to S. aureus, Sealapex presented better antibacterial effectiveness than the MTA Fillapex and AH Plus (p<0.05), which were similar each other (p>0.05). Conclusion: In final evaluation period, pH values and calcium release provided by MTA Fillapex were lower than provided by Sealapex and higher than provided by AH Plus. The MTA Fillapex antimicrobial action was similar to other endodontic sealers.

Carcinoma de lábio com prognóstico sombrio devido conduta médica inadequada / Lip carcinoma with unfavorable prognostic due inadequate medical conduct

O carcinoma de células escamosas é o que mais ocorre entre os cânceres de boca, estimando se sua incidência em mais de 90%. Como importante fator prognóstico concorrem o diagnóstico precoce e a imediata instituição do tratamento, que pode envolver quimio/radioterapia além da cirurgia. As lesões iniciais não oferecem grandes dificuldades diagnósticas e atualmente exames complementares de laboratório definem o diagnóstico prontamente, No presente relato, a conduta médica inicial deixou a desejar, com interpretação incompleta do resultado laboratorial e adoção de conduta terapêutica inadequada, o que resultou em evolução constante da lesão. Com essa história médica pregressa é que a paciente se apresentou à consulta, após seis meses do primeiro atendimento realizado em outro centro. Ao exame físico, observou-se lesão ulcerada infiltrativa no lábio inferior, com acentuada perda de tecido superficialmente. A paciente acusava dor espontânea e fazia uso de uma pomada, receitada há seis meses por ocasião do primeiro atendimento. Como conduta diagnóstica foi indicada a realização de citologia esfoliativa e biópsia, definindo-se o diagnóstico de Carcinoma de Células Escamosas. A paciente foi imediatamente encaminhada ao serviço médico de seu município para gerenciamento do tratamento oncológico especializado. A paciente foi submetida a cirurgia seguida de radioterapia, constatando-se envolvimento de outras estruturas por contiguidade. Atualmente, a paciente se encontra acamada, sem perspectivas viáveis de cura. O presente relato enfatiza a importância do conhecimento clínico e responsabilidade profissional no manuseio de casos dessa natureza

The Squamous Cell Carcinoma is what more happens among the oral cancers, being considered sweats incidence in more than 90%. As important prognostic factor competes the early diagnosis and the immediate institution of the treatment, that it can involve radiotherapy and chemotherapy besides the surgery. The initial lesions don’t offer great diagnostic difficulties and now complemental laboratory exams define the diagnosis quickly, In the present report, the initial medical conduct was inadequate, with incomplete interpretation of the laboratorial result and adoption of inadequate therapeutic conduct, what resulted in constant evolution of the lesion. The patient came to the consultation with that medical history, after six months of the first attendance accomplished in another center. To the physical exam, infiltrative ulcerated lesion was observed in the lower lip, with having accentuated tissue loss superficially. The patient accused spontaneous pain and she did use an ointment, recommended six months ago for occasion of the first attendance. As diagnostic conduct was indicated the accomplishment of cytology and biopsy, being defined the diagnosis of Squamous Cell Carcinoma. The patient was immediately guided to the medical service of its municipal district for management of the specialized oncologic treatment. The patient was submitted the surgery followed by radiotherapy, being verified involvement of another structures by contiguity. Now, the patient meets abed, without viable perspectives of cure. The present report emphasizes the importance of the clinical knowledge and professional responsibility in attendance of cases of that nature