Transdentinal Diffusion of New Intracanal Formulations Containing Nitrofurantoin or Doxycycline

against E. faecalis of new intracanal medications on the external root surface. The medications tested were a placebo gel (PC); the new formulations with either 3% nitrofurantoin (NIT) or 3% doxycycline hydrochloride (DX) and 2% chlorhexidine (CHX) gel as positive control. The new formulations were tested using the traditional agar diffusion test (ADT) and an adapted agar diffusion method (AADM), where the teeth were filled with the medications and left to diffuse on agar surface seeded with E. faecalis. In the ADT, the larger zones of microbial growth inhibition were seen in DX, followed by CHX and NIT. In the AADM test only DX and CHX showed antimicrobial effect. Statistically significant differences between groups were observed by the Kruskal-Wallis test (χ2=47.126; p<0.001). The new intracanal formulations with DX and NIT have demonstrated antimicrobial effect against E. faecalis, but only DX was able to diffuse through the dentinal tubules and exert antimicrobial effect outside the roots. A n t i m i c r o b i a l E f f e c t a n d Transdentinal Diffusion of New Intracanal Formulations Containing Nitrofurantoin or Doxycycline


Introduction
Endodontic infections are essentially polymicrobial and in most cases the microorganisms grow in the root canal system as sessile biofilms.A variety of bacterial species may be isolated from primary periapical infections.In the secondary infections, one of the most often isolated species is Enterococcus faecalis, which is seen as an opportunistic pathogen and a natural inhabitant of the oral cavity that survives as a mono-infection (1).
Despite the action of the chemomechanical preparation with antimicrobial irrigating solutions, some microorganisms, including E. faecalis, may survive in the root canal system.The use of an intracanal dressing that could act through the root canal system would certainly improve the outcome of the endodontic treatment (2).
The intracanal medication should have a broad antimicrobial spectrum, should be able to diffuse through the dentinal tubules and all ramifications of the root canal system including the areas that neither the instruments nor the irrigating solution can reach, and should not be cytotoxic to live tissues (3).Several substances have been tested and improved in order to achieve the ideal results.Calcium hydroxide (Ca(OH) 2 ) is believed to have most of the properties of an ideal root canal dressing due to its high pH.However, it is not equally effective against all species of microorganism found in the root canal system (4).
Chlorhexidine gluconate has been used in endodontics as an irrigant.Recently, due to its antimicrobial effect and adsorption to dental hard tissues, it has also been suggested as an intracanal medication (5,6).Authors have demonstrated that chlorhexidine is more effective than Ca(OH) 2 when used against E. faecalis (5), and that the use of intracanal medication with chlorhexidine alone or associated with calcium hydroxide reduces the microbial factors responsible for pain and inflammation after endodontic treatment (7).
Antibiotics have been used in endodontics and for dental traumas systemically and locally.Although local application of antibiotics could cause side effects such as discoloration (8), this may be a more effective way for delivering the antibiotics (9) and reducing the bacterial load inside the root canal system, since the antibiotic agents increase significantly the antibacterial properties of endodontic materials (10).This effectiveness is due to the fact that it does not depend on circulation to reach the infected sites with compromised vasculature (11).
However, regarding the antimicrobial component of MTAD (doxycycline), some authors claim that nitrofurantoin, a synthetic nitrofuran commonly used in urinary tract infections where E. faecalis is usually involved (17,18), has demonstrated better efficacy than doxycicline against the same bacteria (18).
The purpose of this experiment was to create an intracanal medication based on the formulation of MTAD.The better efficacy of nitrofurantoin to treat some E. faecalis infections when compared with doxycycline led the authors to choose it for the new formulation.The purpose of this study was to compare the antimicrobial effect of each new formulation against E. faecalis and its ability to diffuse through dentin.

Material and Methods
The antibacterial effect of the new formulations against E. faecalis was determined by an agar diffusion test (ADT) and an adapted agar diffusion method (AADM) through the external surface of the roots.

Adapted Agar Diffusion Method (AADM)
The methodology used for AADM was a modification of the one previously described by Gomes et al. 2003 and2009.Fifty extracted human canine teeth were selected and the single root canal was confirmed radiographically.Curettes were used to remove soft tissues and debris from tooth surfaces and if any kind of anomaly, such as a fracture were detected, the tooth was excluded from the study.Teeth crowns were removed 3 mm above the cementoenamel junction and the roots were standardized to a length of 15 mm with a water-cooled diamond saw.A size 10 K-file (Dentsply/Maillefer, Ballaigues, Switzerland) was introduced into each canal until it could be seen at the apical foramen.The working length was established subtracting 1 mm from this measurement (14 mm).In order to preserve patency, the same K-file was used to recapitulate the canal 1 mm beyond its length between each file.The anatomic diameter of apical foramen was sized using manual K-files, beginning with size 10 files, inserted until the working length was reached.File sizes were progressively increased until an instrument that bound at the working length was obtained.The size of the obtained instrument was recorded for each root.The roots were enlarged with K-files three numbers up to apical file size determined at the working length The teeth were instrumented using the crown-down principles, combining Gates Glidden drills (Dentsply/ Maillefer) with K-files.The cervical two-thirds (10 mm) of the canals were prepared using K-files 25 to 35, followed by a size 2 Gates Glidden bur and then by a size 3, 1 mm shorter.The apical stop was established using files three numbers up to the anatomic diameter file.At each file change the root canals were irrigated with 1 mL of 5,25% NaOCl.After mechanical preparation, the teeth were rinsed with 5 mL of 17% ethylenediamine tetra-acetic acid (EDTA) solution for 3 min, to remove the smear layer, followed by 1 mL of 5,25% NaOCl and finally with 5mL of saline.
The teeth were sterilized in individual tubes containing 5 mL of Brain Heart Infusion Broth (BHI) and the process confirmed by overnight incubation of the specimens at 36 ºC.Then, they were divided in 5 groups of 10 teeth each, according to the substance used as intracanal dressing (20), which were the same described in the agar diffusion test (ADT): a) Teeth sealed without medication (WM -negative control); b) CHX -positive control, c) PC; d) NIT and e) DX.
Immediately after filling the root canals, the foramen and the pulp chamber were sealed with wax.The Muller Hinton agar plates were first seeded by a swab with the E. faecalis 10 8 cells/mL suspension and the teeth were then placed inside.The plates were kept for 2 h at room temperature to allow the diffusion of the agents through the dentinal tubules and the agar, and then incubated at 36 ºC for 48 h.After this period the zones of microbial growth inhibition were measured, considering the shortest distance New intracanal dressings study from the outer margin of the tooth to the initial growth.
The non-parametric Kruskal-Wallis test was used to verify any significant difference between groups.In the post-hoc procedures the Tamhane test was used for multiple group comparisons, allowing the assumption that variances are not equal and adjustment for type I errors.

Agar Diffusion Test (ADT)
Figure 1 and Table 1 show that, with the exception of the PC, all tested formulations had potential to eliminate E. faecalis.DX was the one that exhibited the largest inhibition zone followed by NIT and CHX.

Adapted Agar Diffusion Method (AADM)
Figure 2 shows the area of microbial inhibition of the different intracanal medications through the external root surface against E. faecalis.Only CHX and DX showed inhibition zones, measuring 11.17 (±2.17) mm and 5.52(±2.9)mm respectively, while PC and NIT did not inhibit E. faecalis growth as well as the negative control (WM).The Kruskal-Wallis test (χ2=47.126;p<0.001) revealed statistical differences between the groups (Fig. 3).In a multiple comparisons post-hoc procedure it was possible to observe statistically significant differences between CHX and DX and the other groups.

Discussion
In both tests (ADT and AADM) DX presented the largest zones of microbial growth inhibition, demonstrating a better antibacterial effect and/or a better diffusion ability compared with NIT and CHX.NIT was not able to exert any antimicrobial action on the external root surface (AADM) during the incubation period.With the methodology used in this study, when the mean area of microbial inhibition is equal to zero, it means either the medication did not present any antibacterial effect or the substances were not able to diffuse through the agar or through the dentinal tubules.When ADT was applied, DX, NIT and CHX were effective in inhibiting E. faecalis growth, as also shown  by other authors (17).
Although the size of the inhibition zones would indicate activity against the microorganisms, their effectiveness depends on many factors, like the molecular size, solubility and diffusion of the materials through the agar medium, sensitivity of the drug, bacterial source, the number of inoculated bacteria, pH of the substrate in plates, agar viscosity, storage conditions of the agar plates, time of incubation and the metabolic activity against the microorganisms (4,21).
In the AADM test the cementum layer could affect the diffusion of substances to the agar.However, some authors observed that presence or absence of cementum layer does not interfere significantly in the inhibition zones (20).
The NIT formulation presented a higher viscosity than DX.Although it demonstrated antimicrobial effect against E. faecalis in the ADT, this factor could have prevented it from reaching the external root surface, explaining the lack of diffusion observed in AADM.Therefore, it was understood that the methods applied in the present study could be used to analyze and verify the existence of antibacterial activity and diffusion ability trough dentinal tubules, but further studies are required.
The detergent used in intracanal formulations, Tween 80, reduces the surface tension allowing an increased wetting effect, and consequently a better penetration of intracanal medication into dentinal tubules and irregular spaces with difficult access (22).Since NIT presented still a high viscosity, the authors believe that it may be beneficial to increase the detergent weight percentage in the medication to observe better antimicrobial results outside the roots.
Since the low pH of MTAD induces dentin demineralization (23), in this study a phosphate buffer was used to induce a neutral pH, with the goal of keeping the new formulations inside the root canals for longer periods.
The main purpose of this study was to create an intracanal medication based on MTAD with good activity against E. faecalis (13,14), respecting the requirements of an ideal medication.
Chlorhexidine 2% is used as the gold standard for inhouse assay due to its broad spectrum and effectiveness against E. faecalis (24).For the CHX group, concentration of 2% was chosen because its effectiveness is well established.In the new formulations containing DX or NIT, concentration of 3% was used since they were based on MTAD solution.
The use of antibiotics in irrigant solutions made it possible creating new formulations of intracanal dressings using these products.
In the present study, the formulation with DX was the most effective against E. faecalis, showing larger inhibition zones than NIT.Nitrofurantoin is effective against many Gram positive and negative bacteria, including E. faecalis (17,18) and doxycycline is a tetracycline broad-spectrum antibiotic, effective against a wide range of microorganisms (22).In some studies, DX showed low activity against most strains of E. faecalis (25).These results could also be explained by the use of a strain more susceptible to doxycycline than nitrofurantoin and this characteristic deserves further investigation.
Despite the limitations of this study, such as the fact that in vitro models present different conditions from in vivo infections and the fact that a mono-infection assay (E.faecalis) was used to test the intracanal medication, the results are promising and indicate the efficacy of a new intracanal formulation proposed by the authors.Further studies are required to corroborate the present findings and expand the efficacy of these formulations.
The findings of this study suggest that both the new formulations with DX or NIT presented antimicrobial effect against E. faecalis.However, only DX was able to reach the dentinal tubules.These new formulations may be promising as intracanal dressings, increasing the outcome of