CoordenaÃÃo de AperfeiÃoamento de Pessoal de NÃvel Superior / There is an agreement that in dental treatments with incomplete rizogenese it is necessary an intracanal medication with high antibacterial effect because the use of files and standard protocol based on instrumentation must be avoided to protect the fragile dental element from extra hazard. Several endodontic dressings have been used with this purpose, alone or associated to a copious irrigation procedure. However, it is not yet standardized in the literature a unique medication with protracted and excellent antimicrobial effect, associated to a feasible handling and insertion into the duct. The purpose of this study was to evaluate the in vitro effectiveness of different antimicrobial pastes currently used as intracanal medication against the pathogen Enterococcus faecalis by means of agar disk diffusion method. Accordingly, it was created a polypropylene device to simulate the release characteristics of the main root canal. The efficacy tests were performed over a period of 30 days. The formulations studied were distributed in triplicate just as follows: Triantibiotic Paste (metronidazole, ciprofloxacin and minocycline); Biantibiotic Paste (metronidazole and ciprofloxacin); Ciprofloxacin Paste; Amoxilin Paste; Calcium hydroxide paste; 0.9% saline (control group). All pastes were still allotted into 2 categories: open and closed apex. The devices remained in the medium at 37ÂC over 30 days (static). Samples used for the measurement of biologic response were collected in pre-fixed times: H1(1st hour), H6(6th hour), H24(24th hour), D3(3rd day), D7(7th day), D14(14th day) and D30(30th day) in the course of thirty days. At every settled time, 20μL of solution was removed from each device and embedded on sterile paper discs with 6mm of diameter, being the equivalent volume replaced with fresh release medium. After, the discs were transferred with sterile tweezersg to the surface of agar-BHI medium previously inoculated with bacteria over petri plaques. From the devices with closed apex, it was removed 20μL of solution from above the paste surface, avoiding any directly touch on it. This collect aimed to evaluate the antimicrobial effect of different preparations inside the root canal. From the devices with open apex it was removed 20μL of the solution in which the plastic device was submerged, in order to measure the action of the diffused antibiotic to the periapical region through the apex. From the inhibition halos obtained in each case it was possible to rank the formulations in order of effectiveness. The Amoxicillin paste presented an initial antimicrobial effect more robust, keeping it throughout the experiment. However from the second week there was no more significant difference between this and the other antibiotic pastes (triantibiotic and biantibiotic). These pastes had an initial effect less representative than amoxicillin, however the effect became similar from the second week. Nevertheless, calcium hydroxide paste had a discreet effect initially and it was completely ceased already after the 3rd day. Even if Amoxicillin paste presented a superior result among the studied pastes, it was noticed color changes associated to degradation signs after two weeks. The findings show that these antibiotics pastes posed an excellent pharmacological effect, apart from calcium hydroxide. It can be concluded that all the antibiotics pastes, among them the pastes of amoxicillin, triantibiotic and biantibiotic were effective against E. faecalis. As a result its use as dressing for teeth with incomplete risogenesis in may be a good alternative. Among the advantages it is the microbial effectiveness, as well as the feasible manipulation during the clinical procedure. When comparing biantibiotic and triantibiotic pastes, the first showed to be almost equally effective, being a great choice for the clinical treatments of anterior teeth, where the pigmentation caused by minocycline antibiotic could present a drawback in the final result of the treatment. / Existe um consenso de que no tratamento de dentes com rizogÃnese incompleta à necessÃria uma medicaÃÃo intracanal com mÃximo efeito antibacteriano, pois o uso de limas e o protocolo convencional de instrumentaÃÃo devem ser evitados para que nÃo torne o elemento dental ainda mais frÃgil. Diversos curativos endodÃnticos tÃm sido utilizados com este objetivo, bem como o uso de uma irrigaÃÃo mais copiosa e com irrigante mais efetivo. No entanto, ainda nÃo està padronizada na literatura uma medicaÃÃo com efeito antibiÃtico prolongado e efetivo, alÃm de fÃcil manipulaÃÃo e inserÃÃo no conduto. O objetivo deste trabalho foi avaliar in vitro a eficÃcia de diferentes pastas antimicrobianas utilizadas como medicaÃÃo intracanal sobre o patÃgeno Enterococcus faecalis utilizando o mÃtodo de disco - difusÃo em Ãgar. Para isso, foi confeccionado um dispositivo de polipropileno que possui caracterÃsticas semelhantes ao canal radicular principal. Os testes de eficÃcia foram realizados por um perÃodo de 30 dias. Foram analisadas as seguintes formulaÃÃes: Pasta triantibiÃtica (metronidazol, ciprofloxacino e minociclina); Pasta biantibiÃtica (metronidazol e ciprofloxacino); Pasta de Ciprofloxacino; Pasta de Amoxicilina; Pasta de hidrÃxido de cÃlcio; SoluÃÃo fisiolÃgica 0,9% (grupo controle). Todas as pastas foram ainda alocadas em duas categorias: Ãpice aberto e Ãpice fechado e ensaiadas em triplicata. Os dispositivos permaneceram em estufa a 37ÂC durante 30 dias (sem agitaÃÃo), sendo as coletas para aferiÃÃo da resposta biolÃgica realizadas em perÃodos preestabelecidos: H1(1 hora), H6(6 hora), H24(24 hora), D3(3 dia), D7(7 dia), D14(14 dia) e D30(30 dia) no decorrer de 30 dias. A cada coleta foram retirados de cada dispositivo 20ÂL de soluÃÃo e depositados sobre discos de papel de filtro estÃreis com 6 mm de diÃmetro. Em seguida, os discos de papel embebidos foram transferidos com pinÃa estÃril para a superfÃcie das placas de petri previamente semeadas com a bactÃria. Dos dispositivos com Ãpice fechado foram removidos 20ÂL da soluÃÃo que estava sobre a superfÃcie da pasta, tendo o cuidado para nÃo tocÃ-la. Este procedimento teve o intuito de medir a aÃÃo antimicrobiana direta dos diferentes preparados no interior do conduto radicular. A partir dos dispositivos com Ãpice aberto foram retirados 20ÂL da soluÃÃo na qual o dispositivo plÃstico encontrava-se imerso, com o intuito de medir a aÃÃo do preparado difundido à regiÃo periapical. A partir dos halos de inibiÃÃo obtidos, foi possÃvel observar quais preparados foram mais eficazes com respeito ao seu efeito antibacteriano. A pasta de amoxicilina apresentou o maior efeito antimicrobiano inicial, mantendo-o durante todo o experimento, no entanto, a partir da segunda semana nÃo houve mais diferenÃa estatÃstica entre as pastas antibiÃticas e o efeito tornou-se semelhante. As pastas triantibiÃtica e biantibiÃtica tiveram efeito inicial menor do que o da Amoxicilina, porÃm o efeito foi igualado a partir da segunda semana. Por outro lado, a pasta de hidrÃxido de cÃlcio teve um efeito discreto inicialmente, o qual foi totalmente cessado jà apÃs o 3 dia. Ainda que a pasta de Amoxicilina tenha apresentado os melhores resultados dentre as pastas estudadas, foi evidenciado o aparecimento de cor escurecida associada a sinais de degradaÃÃo apÃs duas semanas. Este estudo mostrou que as pastas antibiÃticas tiveram um excelente efeito farmacolÃgico, ao contrÃrio da pasta de hidrÃxido de cÃlcio. Pode-se concluir que as pastas antibiÃticas, dentre elas as pastas de Amoxicilina, triantibiÃtica e biantibiÃtica apresentaram um excelente efeito sobre o micro-organismo E. faecalis. Portanto, seu uso como medicaÃÃo intracanal nos casos de dentes com rizogÃnese incompleta pode ser uma Ãtima alternativa, estando ainda a facilidade de manipulaÃÃo durante o procedimento clÃnico atrelada à eficÃcia microbicida. Quando comparadas, a pasta biantibiÃtica mostrou-se quase que igualmente efetiva a triantibiÃtica, podendo ser uma Ãtima escolha em casos clÃnicos de tratamento de dentes anteriores, sempre e quando a pigmentaÃÃo causada pelo fÃrmaco Minociclina possa comprometer o resultado final do tratamento. PÃde-se concluir que as pastas antibiÃticas, dentre elas as pastas de amoxicilina, triantibiÃtica e biantibiÃtica apresentaram excelente efeito inibitÃrio sobre o micro-organismo E. faecalis. Portanto, o seu uso como medicaÃÃo intracanal nos casos de dentes com rizogÃnese incompleta pode ser uma excelente alternativa.

Indiana University-Purdue University Indianapolis (IUPUI) Endodontic Regeneration Graduate Endodontic Department / Introduction: Regenerative endodontic procedures are used to treat immature teeth with pulpal necrosis in order to control infection, enable continued root development and enhance formation of a pulp like tissue in the canal. Canal disinfection is an integral part the regenerative endodontic process. Double antibiotic paste (DAP; i.e., equal parts of ciprofloxacin and metronidazole) has been successfully used for canal disinfection in regenerative endodontics. A comparison of the residual antibacterial effect of dentin treated with various dilutions of DAP pastes on biofilm formation has not yet been investigated thoroughly. Objectives: The aims of this in-vitro study were to investigate how concentration and time of treatment affect the residual antibacterial properties of DAP in preventing E. faecalis biofilm formation on human dentin. Materials and Methods: Extracted human teeth were used to obtain 4x4mm radicular dentin specimens. Each specimen was pretreated for 1 or 4 weeks with the 77 clinically used concentration of DAP (500 mg/mL), low concentrations of DAP (1, 5 or 50 mg/mL) loaded into a methylcellulose system, calcium hydroxide (Ca(OH)2), or placebo paste. After treatment, samples were rinsed and placed in sterile phosphate buffered saline (PBS) for three weeks. Samples were then inoculated with cultured E. faecalis and incubated in anaerobic conditions for three weeks to allow mature biofilm formation. The dentin samples were rinsed and biofilms detached. The detached biofilm cells were then diluted and spirally plated for enumeration on blood agar plates. The plates were then incubated for 24 h and the number of CFUs/mL was determined using an automated colony counter. Data was analyzed using Fisher’s Exact and Wilcoxon rank sum tests were used for statistical comparisons (α=0.05). Results: Dentin pretreatment for 4 weeks with 5, 50 or 500 mg/mL of DAP demonstrated significantly higher residual antibacterial effects and complete eradication of E. faecalis biofilms in comparison to a 1 week pretreatment with similar concentrations. However, dentin pretreated with 1 mg/mL of DAP or Ca(OH)2 did not provide a substantial residual antibacterial effect regardless of the application time. Conclusion: Dentin treated with 500, 50, or 5 mg/mL of DAP for 4 weeks was able to completely prevent the colonization of bacterial biofilm. Four-week treatment of dentin with DAP offers superior residual antibacterial effect in comparison to a one-week treatment. Intracanal application of DAP for 4 weeks during endodontic regeneration may offer an extended residual antibacterial effect.

Indiana University-Purdue University Indianapolis (IUPUI) Indiana University School of Dentistry Indiana University Endodontic Department / The residual antibacterial effects of antimicrobials used in endodontic regeneration against biofilm bacteria obtained from immature and mature teeth Jordon C. Jacobs DDS, Richard L Gregory PhD, Ygal Ehrlich DMD, Kenneth Spolnik DDS, MS, Josef S. Bringas DMD, DDS, MS, and Ghaeth Yassen BDS, MSD, PhD We explored the residual antibacterial properties of dentin pretreated with low concentrations of double antibiotic paste (DAP) against biofilm bacteria obtained from different clinical sources. Dentin blocks were sterilized and randomized into 4 treatment groups and 2 control groups (n=20). Blocks from treatment groups were pretreated with DAP (1 or 5 mg/ml) loaded into methylcellulose, calcium hydroxide (Ca(OH)2), or methylcellulose paste. After one week, the treatment pastes were removed and all blocks were immersed in PBS. The dentin blocks from treatment groups and one of the control groups were then inoculated with bacterial isolates obtained from immature or mature teeth with pulpal necrosis(n=10). The remaining control group received no bacteria and was used as a sterile control. Blocks were then incubated anaerobically for 3 weeks. Biofilm disruption assays were conducted for all samples. Two-way ANOVA and pair-wise comparisons were used for statistical analyses. The residual antibacterial effect of dentin pretreated with 5 mg/ml of DAP was significantly higher than all other groups regardless of the source of biofilm. Dentin pretreated with 1 mg/ml of DAP demonstrated significantly higher residual antibacterial effects in comparison to dentin pretreated with placebo paste and Ca(OH)2 only in bacterial isolates obtained from mature teeth with pulpal necrosis. Dentin pretreated with Ca(OH)2 did not demonstrate any residual antibacterial effects. Dentin pretreated with 1 or 5 mg/ml of DAP demonstrated significantly better residual antibacterial effects against biofilm bacteria obtained from mature teeth with pulpal necrosis in comparison to bacterial isolates obtained from immature teeth with pulpal necrosis.

Low concentrations (1-10mg/mL) of double antibiotic paste (DAP) have demonstrated antibacterial properties in regenerative endodontics. The aim of this study was to evaluate if DAP made radiopaque (RoDAP) with barium sulfate has antibacterial effects against bacterial isolates from a mature and immature tooth with necrotic pulp. Clinical bacterial isolates were obtained from the canals of mature and immature teeth with necrotic pulps during root canal therapy or a regenerative procedure, respectively. Bacterial isolates were grown anaerobically for three weeks on 4x4mm dentin specimens prepared from extracted human teeth (n=48 per biofilm type). The dentin specimens were allocated into six groups and treated as follows: 1mg/mL RoDAP, 10mg/mL RoDAP, calcium hydroxide (UltraCal), placebo (barium sulfate in methylcellulose), no treatment, and no bacteria or treatment (sterile control). After one week of treatment the biofilm was detached and biofilm disruption assays were conducted to determine the bacterial numbers (CFUs/mL). The data was analyzed using Wilcoxon Rank Sum tests followed by pairwise comparisons. 1 and 10 mg/mL RoDAP as well as calcium hydroxide demonstrated significant antibacterial effects against the tested bacterial isolates. The placebo paste did not demonstrate any significant antibacterial effects. No significant difference in antibacterial effects was found against isolates from both mature and immature teeth regardless of the type of treatment. Both 1 and 10 mg/mL RoDAP demonstrated significant antibacterial effects against bacterial isolates from mature and immature teeth with necrotic pulps. RoDAP can be beneficial clinically since its adequate placement within the canal system can be confirmed radiographically.

Indiana University-Purdue University Indianapolis (IUPUI) / Introduction: Some intracanal medicaments used in regenerative endodontics may compromise the bond strength of root cements and lead to tooth discoloration. Objectives: To evaluate the effects of 1) low concentrations of TAP and DAP (1 mg/mL) on push-out bond strength of various root cements, and 2) low concentrations of TAP and DAP (1 mg/mL and 10 mg/mL) on crown discoloration. Materials and Methods: Single rooted human teeth (n = 144) were horizontally decoronated and instrumented according to standardized protocol. The samples were randomized into six experimental groups (Ca(OH)2, 1000 mg/mL TAP and DAP, 1 mg/mL TAP and DAP, and no medicament control group. After four weeks, the medicaments were removed and each group was divided into three subgroups to receive MTA cement, Biodentine cement, or Endosequence Bioceramic putty cement for two weeks. Then, two root cylinders were obtained from each root and push-out bond strength testing was performed. For the crown discoloration experiment, 160 crowns were obtained from intact human molars and randomized into experimental groups as described earlier with the addition of two groups (10 mg/mL TAP and DAP). The pulp chambers in half of the samples from each group were coated with an adhesive bonding agent before receiving the assigned intracanal medicament. Color changes (ΔE) were detected by spectrophotometer at 1 day, 1 week, and 4 weeks after application, as well as after thermocycling. Results: In the push-out bond strength experiment, 1 mg/mL DAP generally demonstrated significantly higher bond strength of root cements compared with the other treatment groups. For the crown discoloration experiment, when an adhesive bonding agent was used prior to (10 mg/mL or 1000 mg/mL) TAP, the crowns had significantly less discoloration than those without adhesive. DAP 10 mg/mL had the least significant color change at all time points regardless of whether adhesive was used. Conclusion: 1) 1 mg/mL DAP and Ca(OH)2 did not have significant negative effect on the bond strength of calcium-silicate-based cement to radicular dentin. 2) 1 mg/mL and 10 mg/mL of DAP and Ca(OH)2 had significantly less effect on the color change of the human tooth crown than all intracanal medicaments used in this study. / 2020-08-13

Indiana University-Purdue University Indianapolis (IUPUI) / Introduction: Regenerative endodontic procedures are an effective treatment option for immature teeth with infected necrotic pulps to allow for healing and potential continued root development, yet challenges to ideal treatment outcomes remain. Consistent development of root length and width of dentin remains a challenge, as does development of the pulp-dentin complex. Previous in vitro studies have assessed the role of different growth factors and bioactive molecules in combination with scaffolds to potentially facilitate continued development of the pulp-dentin complex using dental pulp stem cells (DPSCs). The proline-rich tyrosine kinase 2 (Pyk2) is linked with osteoblast activity and the regulation of bone mass. Further, the Pyk2 inhibitor PF-4618433 (PF-46) has been shown in previous studies to enhance osteoblast activity and mineral deposition in vitro. However, whether Pyk2 targeting promotes the osteogenic differentiation of DPSCs remains unknown. Objective: The purpose of this study was to investigate the effect of a Pyk2 inhibitor, PF-46, on the proliferation, differentiation, and mineralization of human DPSCs. Materials and Methods: Human DPSCs were cultured in 24-well plates with α-MEM with 10% FBS, and containing 0 μM (vehicle control) or 0.1 μM, 0.3 μM, or 0.6 μM PF-46. Fresh media and treatments were replaced every 2-3 days. After 1 day incubation, cytotoxic effects were evaluated by using an MTS proliferation assay. After 4 days of treatment, direct cell counting was performed. To induce osteogenic differentiation, ascorbic acid and β-glycerol phosphate were added to the culture media and the DPSCs were cultured with PF-46 for 14 days. Then, an alkaline phosphatase (ALP) assay and mineral deposition assay were performed. Differences between treatment groups were analyzed by a one-way ANOVA followed by pair-wise tests conducted using Tukey’s multiple comparisons procedure with a 5% significance level. Results: The 0.6 μM PF-46 group had a significantly higher cell count, ALP activity and mineral deposition when compared to 0 μM PF-46. The 0.1 and 0.3 μM PF-46 groups also had significantly higher ALP activity compared to the 0 μM PF-46 group after 14 days of incubation. There was a general trend of increased differentiation and mineral deposition as the concentration of PF-46 increased from 0.1 μM to 0.6 μM. Conclusion: There was a general concentration-dependent increase in cell count, differentiation, and mineral deposition by human DPSCs as the concentration of PF-46 increased from 0 μM up to 0.6 μM, with the highest activity observed with 0.6 μM PF-46. Although further research is needed, these results suggest that strategies that target Pyk2 may potentially be used to improve the osteogenic differentiation of DPSCs to aid endodontic regeneration.

Indiana University-Purdue University Indianapolis (IUPUI) / Endodontic regeneration (ERP) has been successfully used in the treatment of traumatized immature teeth. The procedure has three essential steps: disinfecting the root canal (i.e. triple antibiotic paste (TAP) or double antibiotic paste (DAP)), provoking bleeding inside the canal to form a scaffold upon which pulp stem cells will be deposited and continue root growth, and creating a good coronal seal. Previous research has reported that antibiotic pastes (TAP and DAP) are cytotoxic to stem cells in the concentrations commonly used in endodontic regeneration (1000 mg/mL). To decrease the adverse effects on stem cells and increase the rate of success of the regeneration, defining appropriate antibiotic concentrations for ERP is critical. In this project, five in-vitro experiments were conducted to determine the breakpoint dilutions of both TAP and DAP medicaments, and to prepare a suitable novel pastes containing diluted TAP or DAP medicaments for ERP. In the first experiment, we compared the antibacterial effect of TAP, and DAP against early biofilm formation of Enterococcus faecalis (E. faecalis) and Porphyromonas gingivalis bacteria. In the second study, we investigated the antibacterial effect of various dilutions of TAP and DAP antibiotic medicaments against established E. faecalis biofilm. In the third experiment, we investigated longitudinally the residual antibacterial activity of human radicular dentin treated with 1000, 1 or 0.5 mg/ml of TAP and DAP. In the fourth study, we investigated the cytotoxic effect of various dilutions of TAP and DAP antibiotic medicaments on the survival of human dental pulp stem cells (DPSC). And in the fifth experiment, we investigated the antibacterial and cytotoxic effect of novel intracanal medicaments consisting of methylcellulose (MC) and/or propylene glycol (PG) mixed with 1mg/ml of TAP or DAP. 1 mg/ml of DAP or TAP medicaments had a significant antibacterial effect against early bacterial biofilm formation, and established bacterial biofilm. Furthermore, 1 mg/ml had a residual antibacterial activity comparable to 1000 mg/ml. The novel intracanal medicaments had comparable antibacterial effect to currently used medicaments (1000 mg/ml). Additionally, the novel intracanal medicaments significantly enhanced DPSC metabolic activity, compared to currently used medicaments in endodontic regeneration procedures.