The microbial causation of apical periodontitis has been well-documented. ¹ , ² Dentinal tubules and apical irregularities of root canal anatomy such as deltas and isthmuses may harbor tenacious biofilms, contributing to the failure of endodontic treatment. ³ Conventional mechanical instrumentation and irrigation with sodium hypochlorite (NaOCl) would not predictably make root canals free of bacteria. ⁴ , ⁵

Calcium hydroxide (CH), as an interappointment medicament, reduces bacteria in dentinal tubules. ⁶ However, a systematic review concluded that CH has limited antibacterial effectiveness, ⁷ and the remnants of it can affect the sealing ability of root canal sealers. ⁸

Knappwost has introduced a technique named depotphoresis for the disinfection of root canal system (RCS) using CH with copper ions (cupral). ⁹ Cupral is placed in the coronal third of the RCS, and the ions are activated and moved to the apical third of the RCS by electric current. It has been shown that depotphoresis eradicates viable bacteria in dentinal tubules to a depth of 500 μm. ⁶ , ¹⁰ It has been claimed by the manufacturer of the depotphoresis system that “the electrical field transports cupral even in the mechanically not reachable apical part of the canal.” ¹¹

The purpose of this study was to evaluate the efficacy of depotphoresis method in the disinfection of accessible and inaccessible RCS.

The methods of this in vitro study were approved by the Research Ethics Committee of Mashhad University of Medical Sciences (Approval ID: IR.MUMS.DENTISTRY.REC.1399.027).

Forty freshly extracted noncarious mature maxillary anterior teeth were collected and kept in saline. After placing the teeth in 5.25% NaOCl for 30 min to detach residual debris from the root surfaces, a diamond disc was used to cut the crowns perpendicular to the long axis of the teeth to have the same length of roots (16 mm). A #10 K-type file was placed into each root canal to confirm apical patency.

Then, the roots were divided into two phases: the specimens without canal obstruction and the specimens with canal obstruction.

Phase 1: The specimens without canal obstruction

The root canals were instrumented to F3 (ProTaper Universal; Dentsply Sirona, Ballaigues, Switzerland) 0.5 mm beyond the apical foramen. Irrigation with 5.25% NaOCl was performed between each file insertion. To remove the smear layer, a final irrigation sequence of 5 ml of 17% EDTA for 5 min, 5 ml of 5.25% NaOCl for 5 min, and 10 ml of normal saline was done according to Lin et al.'s study. ¹⁰ The roots were placed in Eppendorf tubes containing 2 ml of Tryptic Soy Broth (TSB) medium and then autoclaved for 30 min at a pressure of 15 PSI and temperature of 121°C. Then, the tubes were incubated at 37°C for 24 h.

A sterile micropipette was used to aspirate TSB and deliver 1 ml of Enterococcus faecalis suspension with a standard concentration of 0.5 McFarland (1.5 × 10 ⁸colony-forming units [CFU]/ml) into the root canal and around the root so as to immerse the root in the E. faecalis suspension. Thereafter, the tubes were incubated at 37°C for 24 h, and bacterial viability was determined by CFU count. Subsequently, the specimens were incubated at 37°C for 21 days. To prohibit dehydration of the specimens during incubation, the TSB medium was refreshed every 3 days.

The roots were randomly divided into NaOCl plus passive ultrasonic agitation (PUA) group (n = 10) and depotphoresis disinfection group (n = 10). As shown in Figure 1, each root was fixed in a 6-cm high round pot which was half-filled with 0.9% sterile saline and had two holes in its lid, one to place the root, and the other to place the lip clip of Depotphorese ^®-system Original II. Figure 1

The experimental model (a) lip clip; (b) electrode; (c) lid; (d) tooth; (e) normal saline.

Phase 1

One of 10 depotphoresis specimens and four of 10 NaOCl plus PUA specimens showed no bacterial growth in any of their two samples. The means and standard deviations of each group are reported in Table 1. Using the Kruskal–Wallis test, the Log CFU of the depotphoresis group was significantly lower than NaOCl plus PUA group (P = 0.006).{Table 1}

Phase 2

The bacterial growth of two of the 10 specimens in the NaOCl plus PUA group was negative. The means and standard deviations of each group are reported in Table 2. Using the Kruskal–Wallis test, the Log CFU of the depotphoresis group was significantly lower than the NaOCl plus PUA group (P = 0.009).{Table 2}

In either phase of the current study, depotphoresis treatment had a significantly greater reduction in Log CFU of E. faecalis than NaOCl plus PUA treatment.

Owing to the high prevalence of E. faecalis in persistent endodontic infections, ¹² , ¹³ , ¹⁴ its single-species biofilm was used in this study. The difficulty of E. faecalis biofilm eradication is supported by the studies that have shown its ability to invade dentinal tubules ¹⁵ and form biofilms in medicated root canals. ¹⁶ Benefits of monospecies biofilm model systems are simplicity, standardization, and control, ¹⁷ although they are more susceptible to antimicrobial treatment than multispecies biofilms. ¹⁸ , ¹⁹ , ²⁰ , ²¹

According to similar studies, ²² , ²³ standardization of the specimens was done by shaping the root canals 0.5 beyond the apical foramen. Removal of the smear layer was performed to get the maximum penetration of E. faecalis biofilm. Because there were no similar studies for the second phase of the present study, it seems, the best possible method that could simulate the clinical situation was adopted to allow sampling from the apical portion of the obstructed canal.

A low current electric field of about 5 mA was used to mobilize ions derived from cupral paste according to Fuss et al.'s study, ⁶ which eliminated all bacteria to a depth of 500 μm in dentinal tubules. Lin et al. applied a current of 10 mA that was more effective than pure calcium hydroxide in depths of 200–500 μm. ¹⁰ One study used a current of 15 mA that was the most effective against Gram-positive cocci. ²⁴ The manufacturer's instructions of Depotphorese ^®-systems Original II recommended 5 mA in each session of treatment. ¹¹

It has been demonstrated that the maturity of the biofilm increases its resistance to NaOCl. ²⁰ , ²⁵ Because E. faecalis biofilm gets mature in 14–21 days, ²⁶ a 3-week incubation was considered in the majority of studies. ¹⁷

Zou et al. noted that NaOCl penetration depth in the dentinal tubules was 77–300 μm. ²⁷ Haapasalo and Orstavik reported that after 3 weeks, E. faecalis caused dense infection of dentinal tubules to a depth of 400 μm, and the front of infection could reach 800–1000 μm. ¹⁵ Furthermore, bacteria can reside in uninstrumented recesses of the main canal. ²⁸ 6% NaOCl eradicated 99.99% of E. faecalis biofilm bacteria in vitro; ²⁹ however, its anti-biofilm efficacy was lower in vivo. ²⁸ , ³⁰ In the present study, 5.25% NaOCl plus PUA could not make all the root canals free of bacteria which could be related to NaOCl penetration depth, inaccessible areas of the canal, and decreased NaOCl anti-bacterial efficacy in the root canal.

Laser activation of irrigation has been shown to increase NaOCl penetration in dentinal tubules, ³¹ and NaOCl irrigation plus photon-induced photoacoustic streaming was more effective than NaOCl irrigation alone in the eradication of E. faecalis biofilm. ³² However, iontophoresis using Cu was more effective than laser therapy against both Gram-positive and Gram-negative bacteria. ²⁴

It has been claimed by the manufacturer that inaccessible areas of the root canal can be disinfected by depotphoresis. ¹¹ Owing to fractured file conductivity, an electric current can be established in the apical portion of the canal, and the presence of the copper, calcium, and hydroxide ions can lead to disinfection. The quantity of the copper ion can be determined by spectrophotometry, which can be conducted in future studies. ³³

Intracanal sampling technique was one of the limitations of this study as passive sampling may not detect the bacteria that reside in inaccessible areas of the root canal which can cause false-negative results. ³⁴ Future in vitro and in vivo studies can be conducted using newer methods of spectrophotometry like microspectroscopy, ³⁵ and confocal laser scanning microscopy can be used in future in vitro studies.

Treatment with depotphoresis was significantly more effective than NaOCl irrigation plus PUA treatment in either teeth with canal obstruction or teeth without canal obstruction.

Acknowledgments

This research has been supported by the Vice Chancellor for Research of Mashhad University of Medical Sciences, Mashhad, Iran. The authors deny any conflicts of interest related to this research.

Financial support and sponsorship

This study was supported by Mashhad University of Medical Sciences.

Conflicts of interest

The authors of this manuscript declare that they have no conflicts of interest, real or perceived, financial or non-financial in this article.