Cytotoxicity of intracanal dressings on apical papilla cells differ upon activation with E. faecalis LTA

Abstract Objective The aim of this study was to investigate the cytotoxic effects of modified triple antibiotic paste and an experimental composition using calcium hydroxide on lipoteichoic acid (LTA)-primed apical papilla cells (APC). Material and Methods Human APC were tested for in vitro cytotoxicity of modified Triple Antibiotic Paste (mTAP - Ciprofloxacin, Metronidazole and Cefaclor at 1:1:1) and of a paste of Ciprofloxacin, Metronidazole and Calcium hydroxide (CMC - 1:1:2) and modified CMC (mCMC - 2:2:1) by using MTT assay. The substances were reconstituted in DMEM at 1,000 µg/mL and » serially diluted before being kept in contact with cells for 1, 3, 5 and 7 days. Further, cells were primed with 1 µg/mL of Enterococcus faecalis LTA for 7 days prior to the viability test with 1,000 µg/mL of each substance. Statistical analysis was performed using one-way analysis of variance (ANOVA) and two-way ANOVA respectively followed by Tukey's post-test. Significance levels were set at p<0.05. Results In the first assay, the higher cytotoxic rates were reached by mTAP for all experimental periods. CMC was found toxic for APC at 5 and 7 days, whereas mCMC did not affect the cell viability. Only CMC and mCMC were able to induce some cellular proliferation. In the second assay, when considering the condition with medium only, LTA-primed cells significantly proliferated in comparison to LTA-untreated ones. At this context, mTAP and CMC showed similar cytotoxicity than the observed for LTA-untreated cells, while mCMC was shown cytotoxic at 7 days only for LTA-primed APC. Comparing the medications, mTAP was more cytotoxic than CMC and mCMC. Conclusion mTAP showed higher cytotoxicity than CMC and mCMC and the effect of topic antimicrobials might differ when tested against apical papilla cells under physiological or activated conditions.

Influência de medicações intracanal utilizadas em procedimentos endodônticos regenerativos na sobrevivência de células da papila apical in vitro / Influence of intracanal medications used in regenerative endodontic procedures on survival of apical papilla cells in vitro

Procedimentos endodônticos regenerativos proporcionaram mudanças no tratamento de pacientes com dentes imaturos e periodontite apical possibilitando desenvolvimento radicular completo e menor incidência de fratura dental. Medicações intracanal são utilizadas para a realização da desinfecção; entretanto, o efeito delas sobre às células da papila apical é pouco elucidado. Adicionalmente, pouco se conhece a respeito do efeito destas substâncias sobre células previamente submetidas à condição pró- inflamatória. O objetivo deste estudo foi avaliar a citotoxicidade de medicações intracanal empregadas em procedimentos regenerativos em Endodontia sobre células de papila humana em cultura em condição fisiológica e ativada. Cultura de células foi estabelecida a partir da papila apical removida de um terceiro molar imaturo extraído. As substâncias estudadas foram a pasta tripla antibiótica modificada: ciprofloxacina, metronidazol e cefalosporina (1:1:1); CFC: ciprofloxacina, metronidazol e hidróxido de cálcio (1:1:2) e CFC modificado: ciprofloxacina, metronidazol e hidróxido de cálcio (2:2:1). Parte das células foram estimuladas previamente por ácido lipoteicóico (LTA) de Enterococcus faecalis por 7 dias. Após plaqueamento, células foram expostas a concentrações crescentes das medicações por 1, 3, 5 e 7 dias. Foram avaliados viabilidade celular por meio de brometo de 3-(4,5-dimetiliazol-2-il)-2,5-difeniltetrazólio (MTT) e liberação de óxido nítrico (NO) pelo método de Griess

A análise estatística foi realizada por meio de análise de variância a 1 critério (ANOVA) seguida de pós teste de Tukey, com nível de significância de 5%. O CFC modificado foi a medicação que demonstrou menor efeito citotóxico sobre a viabilidade celular nos tempos experimentais estudados, o CFC promoveu queda da viabilidade celular especialmente após 7 dias de contato. A pasta tripla antibiótica modificada resultou em comprometimento importante da viabilidade podendo ser considerada a mais citotóxica. A ativação celular por LTA resultou em níveis aumentados de atividade mitocondrial para todas as medicações sendo mais evidente nos períodos experimentais mais longos. A ativação celular também contribuiu para níveis maiores de óxido nítrico. Conclui-se que o efeito citotóxico das medicações testadas é dependente de sua concentração, tempo de contato e condição celular, sendo a pasta tripla antibiótica modificada a mais citotóxica em concentrações elevadas podendo implicar clinicamente na diminuição da viabilidade das células da papila apical podendo diminuir o sucesso dos procedimentos regenerativos.

Regenerative Endodontic procedures have provided changes in treatment of patients with immature teeth and apical periodontitis enabling full root development and lower incidence of dental fracture. Intracanal dressings are used for disinfection; however, their effect on apical papilla cells is poorly elucidated. Additionally, the effect of these substances on cells previously subjected to proinflammatory condition is still unknown. The aim of this study was to evaluate the cytotoxicity of intracanal dressings used in Endodontics regenerative procedures on cultured human apical papilla cells at physiologic and activated condition. Cell culture was established from the apical papilla removed from an extracted immature third molar. The substances studied were triple antibiotic modified paste: ciprofloxacin, metronidazole and cephalosporin (1:1:1); CFC: ciprofloxacin, metronidazole and calcium hydroxide (1:1:2) and modified CFC: ciprofloxacin , metronidazole and calcium hydroxide (2:2:1). Part of the cells was stimulated previously with lipotheichoic acid (LTA) of Enterococcus faecalis por 7 days. Once plated, cells were exposed to increasing concentration of the medications for 1, 3, 5 and 7 days. Cell viability was evaluated by means of 3-bromide (4.5-dimetiliazol-2-yl) -2.5-difeniltetraze (MTT) and Nitric Oxide (NO) release was assessed by the Griess method.

The statistical analysis was done through analysis of variance with 1 criteria (ANOVA) followed by Tukey test with 5% of significance level. Modified CFC was the medication that demonstrated the less cytotoxic effect on cell viabilityat the experimental periods studied while CFC promoted significant decrease on cell viability specially after 7 days of contact. The modified triple antibiotic paste resulted in important alteration of cell viability being considered the most citotoxic. Cellular activation by LTA resulted in increased levels of mitochondrial activity for all medications being more evident at the longer experimental periods. Cellular activation also contributed to higher levels of nitric oxide release. In conclusion, the cytotoxic effect of the tested medications is dependent on concentration, time of contact and cellular condition, being the triple antibiotic modified paste the most cytotoxic in high concentrations leading clinically in the decreased of the cells viability of the apical papilla, decreasing the success of regenerative procedures.