Total Eradication of Bacterial Infection in Root ... - ACS Publications

Article Cite This: ACS Biomater. Sci. Eng. XXXX, XXX, XXX−XXX

Total Eradication of Bacterial Infection in Root Canal Treatment: An Electrochemical Approach Abhijith Segu,† Divya Bijukumar,† Tina Trinh,†,‡ Manila Nuchhe Pradhan,‡ Qian Xie,‡ Sukotjo Cortino,‡ and Mathew T. Mathew*,†,‡ †

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Regenerative Medicine and Disability Research Lab, Department of Biomedical Sciences, University of Illinois College of Medicine, 1601 Parkview Avenue, Rockford, Illinois 61107, United States ‡ College of Dentistry, University of Illinois, 801 S. Paulina Street, Chicago, Illinois 60612, United States

ABSTRACT: According to the American Association of Endodontists, currently 22.3 million endodontic procedures are being performed annually with the success rate of 70−95% and the average survival rate of the root canal procedure is approximately 67% after 5 years and 56% after 8 years. One of the major reason for the failure is relapse of infection. Hence, it is imperative to develop an assistive or alternative method to eradicate the bacterial infection effectively without affecting patient compliance. The application of electrochemistry has been used previously to disinfect catheters and implant disinfection. Hence, the aim of this study is to utilize the principles of electrochemistry to develop a microelectronic device to eradicate bacterial infection for root canal treatment. The electrochemical protocol includes open circuit potential (60 s) and potentiostatic scan at varying voltage (−9 to +2 V) at a different time duration (1−5 min). Enterococcus faecalis in the form of planktonic and biofilm was used in this study. After electrochemical treatment, the bacterial viability was evaluated using alamarBlue assay, colony forming units, confocal microscopy, and scanning electron microscopy. Cytotoxicity evoked by electrochemical voltage in comparison to NaOCl solution was performed using osteoblasts in 2D and 3D cell culture systems. The results of the study show that the application of −2 to +2 V at 1−5 min did not show any significant reduction in bacterial growth. However, the cathodic voltage of −9 V for 5 min showed a significant reduction (p < 0.001) in the bacterial count (80−95%). Similar results were obtained from biofilm study, which is more realistic to the in vivo condition. In contrast, the method did not induce cytotoxicity to the cells in 3D culture system (65% viability) in comparison to the highly toxic nature (0% viability) of NaOCl, indicating better patient compliance. Hence, the study provides supporting evidence to develop an electrochemically driven microelectronic device that can be a potential assistive dental instrument for endodontic procedures. KEYWORDS: root canal infection, electrochemical treatment, Enterococcus faecalis, osteoblasts, 3D culture

1. INTRODUCTION From the survey conducted by the American Association of Endodontists in the year 2005−2006, there were 15.1 million patients who had undergone the root canal treatment, out of which 72% were performed by general dentists, and 28% were done by endodontists. The procedural error due to lack of sufficient practical and technical skill by the practitioner may also encompass treatment failure. It was reported from a recent a retrospective clinical study that the success rate of a root canal is 88%.1,2 Additionally, the average survival rate of the tooth which has undergone a root canal procedure is approximately 67% after 5 years and 56% after 8 years.2,3 © XXXX American Chemical Society

The current root canal treatment method includes drilling an access hole into the crown of the tooth mechanically and then cleaning the canal space with chemical irrigants followed by refilling the canal space.4−7 The technique demands high skill the lack of which may eventually lead to the failure of the endodontic procedure.8 In addition, the residual bacteria can flourish and may cause the spread of bacterial infection in the patient.9 It was reported that primary root canal infections are Received: February 4, 2018 Accepted: June 4, 2018 Published: June 4, 2018 A

DOI: 10.1021/acsbiomaterials.8b00136 ACS Biomater. Sci. Eng. XXXX, XXX, XXX−XXX

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ACS Biomaterials Science & Engineering

Figure 1. (a) Stage 1: Feasibility of the system and free potential monitoring. (b) Stage 2: With remote center and potentiostatic condition. (c) Stage 3: Miniature version of remote center and prototype for biological validation tests. (d) Stage 4: Final remote center eliminated miniature version of the setup.

The use of electrochemistry for catheter disinfection was reported by Liu et al.21 In dentistry, electrochemistry has been used for implant disinfection.21−23 Recently, Canty et al. reported the application of cathodic voltage-controlled electrical stimulation to eradicate the implant associated biofilm infections after 4−8 h stimulation. There are several reports highlighting the effect of low-level DC and AC currents and pulsed electric fields on microbes.22,24−27 It was also reported that the antibacterial effect was dose- and time-dependent, where the increase in current increases the bacterial cell death.28 Cathodic potential such as −1.8 V for 1 h significantly reduced the bacterial growth by 80−98%. More importantly, the method is very efficient for bacteria that are highly tolerant to antibiotics.29−32 However, the application of electrochemistry to eradicate bacteria within the root canal system has not been explored. The purpose of the current study was to develop a microelectronic device and evaluate the efficacy of electrochemistry in root canal disinfection. An in vitro tooth model was designed, and various potentials ranging from anodic to cathodic voltage (vs Pt wire) were applied to optimize the procedure for complete eradication of bacterial growth using E. faecalis as the model bacterial culture. Here, we chose voltage ranging from −9 to +2 V primarily to understand the effect of different voltage on bacterial eradication in short duration (1−5 min) for the application of root canal treatment.

often polymicrobial, which includes 10−30 different bacterial species, whereas secondary infection is primarily due to the development of resistant species, Enterococcus faecalis, an anaerobic Gram-positive coccus, which forms biofilm with the capacity to invade dentinal tubules.10 There are various measures currently in use to eliminate the growth of microorganisms, which include different instrumentation techniques, intracanal medicaments such as chlorhexidine, Ca(OH)2,11 as well as chemical agents called irrigants. A variety of chemicals has been used as irrigants such as sodium hypochlorite (NaOCl), chlorhexidine (CHX), ethylenediaminetetraacetic acid (EDTA), MTAD (mixture of tetracycline, acid, and detergent),12 etc. The most widely used chemical irrigant is sodium hypochlorite. However, toxicity and adverse effects due to accidental penetration of irrigants to the oral tissue have been reported in several studies.13−15 Although the traditional method has proven to be quite successful, there is an increase in the number of reports of relapse of infection a few years after the procedure.16,17 This is primarily due to the presence of resistant bacteria inside the dentinal tubules as well as due to the mature mineralized E. faecalis biofilm after several weeks of biofilm formation.5,18,19 Therefore, it is important to develop an effective, noninvasive, nontoxic assistive strategy for the complete elimination of antibiotic resistant20 biofilm to decrease the secondary infection with better patient compliance. B


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the platinum wire (RE). The WE was same as the previous stage which is the reamer file, connected to the potentiostat through a copper wire. An OCP test was conducted to monitor the evolution of potential, and a potentiostatic test was used to determine the controlled/stable current flow as well as the amount of current that was being generated with the various potentials (−9, −6, 0, and +2 V) that were applied. Each potential was tested for 1800 s (30 min) for 3 trials (N = 3). 2.2. Temperature Measurement. In a 96-well plate, 100 μL of different types of cell culture media and bacterial culture media such as MEM, BHI, and PBS was added onto the plate, which was then subjected to potential ranging from −9 to +2 V for a period of 5 min in pentacles, and the temperature was recorded before and after the electrochemical treatment using an infrared temperature gun. 2.3. Bacterial Planktonic and Biofilm Culture. E. faecalis ATCC 29212 was used in this study. The strain was routinely inoculated on brain heart infusion (BHI, BD Difco, Sparks, MD, United States) agar and cultured aerobically at 37 °C for 24 h. A single bacterial colony was inoculated into 5 mL of BHI medium and grown overnight. The preparation of planktonic bacteria for the alkaline challenge assay was done by adding 50 mL of BHI broth to 500 μL of overnight E. faecalis culture, followed by an additional 12 h of culture. The concentration of E. faecalis was adjusted to an OD600 value of 0.8, which corresponded to approximately 109 colony-forming units per milliliter (CFU/mL). E. faecalis culture (approximately 109 CFU/mL) and TSB containing 1% glucose at a ratio of 1:100 were added to 48-well plates (1 mL per well). After 24 h of incubation, the supernatant and nonadhesive cells were discarded, and the biofilm on the bottom of the well was gently washed with sterile PBS. 2.4. Electrochemical Treatment to Planktonic and Biofilm Culture. One-hundred microliters of 1:8 dilution in BHI media of 0.8 OD600 bacteria was inoculated onto an appropriate number of wells in a 96-well plate and subjected to different treatment parameters ranging from −9 to +2 V for a time period of 1, 3, and 5 min, and a notreatment group was considered as a negative control for the study in both planktonic and biofilm culture. To maintain patient compliance, treatment time was kept to a minimum. Then, alamarBlue assay was performed as previously described to determine the viability of bacterial cell culture based on the protocol reported elsewhere.33,34 Briefly, 1:8 dilution of the E. faecalis with OD600 value of 0.8 was taken and serially diluted using BHI media. Ninety microliters of each dilution was added to a 96-well plate, and 10 μL of commercially available alamarBlue was added, making it 10% v/v. Plane BHI medium was used as blank to measure the background. The plate was incubated for 30 min, and absorbance was read at 575 and 600 nm. A standard curve was plotted, and R2 was calculated using the graph. Also, the bacterial cell suspension was serially diluted 5 times; 2 μL of the bacterial suspension was plated onto a checkered BHI 1% agar plate, and the results from both alamarBlue and CFU were correlated. A confocal laser scanning microscope (CLSM) was used to evaluate the E. faecalis biofilm that had been subjected to electrochemical treatment. For the E. faecalis biofilms, E. faecalis cultures (approximately 109 CFU/mL) and TSB containing 1% glucose at a ratio of 1:100 were added to 8-well chamber slides. After incubation for 24 h,35 the biofilms on the bottom were subjected to electrochemical treatment at −6 and −9 V for 5 min. E. faecalis biofilms were stained with a mixture of 6 μM SYTO 9 stain and 30 μM PI at room temperature in the dark for 15 min according to the specifications of the Live/Dead BacLight Bacterial Viability Kit (L13152; Molecular Probes, Invitrogen, Inc., Eugene, OR, United States). Images were then captured using an Olympus Fluoview confocal laser scanning microscope (Olympus Fv10i Fluoview confocal microscope). SYTO 9 and PI were excited at 488 and 543 nm, respectively. The viable cells were stained green, and the dead cells were stained red. Five random (four angular vertexes and a central point in the square) areas were captured from each treatment group. The viable and dead cells were analyzed based on fluorescence intensity. 2.5. Cell Culture (2D and 3D). MG-63 human osteoblast-like cells, purchased from ATCC, were used in this study. The cells were maintained in minimum essential medium supplemented with 10%

2. EXPERIMENTAL METHODS 2.1. Development and Validation of Electrochemical Tool. To develop an electrochemical setup for the treatment of root canal infection, a tooth model attached to a resin base was designed. The mounted tooth was then subjected to different electrochemical conditions at each stage. The following are the stages of the development of the electrochemical setup. 2.1.1. Stage 1: Feasibility of the System and Free Potential Monitoring. A tooth model with an access orifice already drilled into the crown was used as the test subject (Figure 1A). A platinum wire, about 2 in. long, and a root canal file with a copper wire attached on the opposite end were carefully placed inside of the access orifice so that they were not in contact with one another. One-hundred microliters of phosphate buffer saline (PBS) was pipetted into the orifice. The reamer file connected with a copper wire was taken as working electrode and platinum wire as the reference electrode. The counter electrode was not used at this stage. The electrodes were connected to a potentiostat (Gamry Interface 1000). A potential open circuit (OCP) test was used to monitor the evolution of the free potential as a function of time to see the feasibility and electrochemical stability of the system. The OCP test was conducted for a period 24 h for three trials (N = 3) and the electrochemical potential as a function of time was recorded. 2.1.2. Stage 2: With Remote Center and Potentiostatic Condition. An updated tooth model with an access orifice already drilled into the crown (naturally extracted teeth) was used as the test subject (Figure 1B). Hence, to conduct the potentiostatic experiments at controlled potential, a three-electrode system was used, namely, a working electrode (WE), reference electrode (RE), and counter electrode (CE), onnected with Gamry made potentiostat (PA, United States). However, to achieve miniature size for the medical application, an approach similar to classical salt bridge method in electrochemistry was employed where the RE and CE were immersed in PBS in an additional glass beaker (remote location). Further, a copper wire was used to interface between the tooth orifice and electrolyte in the beaker (see Figure 1B). Reamer file with attached copper wire used in stage 1 was maintained as a WE in this stage. An OCP and potentiostatic test were used to measure the free potential as well as the current generated as a function of time, respectively. The potentiostatic test was conducted at different potentials starting from −0.9, −0.6, −0.3, 0, and +0.05 V (vs SCE). Each test was run for 1800 s (30 min) and repeated for N = 3. 2.1.3. Stage 3: Miniature Version of Remote Center and Prototype for Biological Validation Tests. A tooth model with an access orifice already drilled into the crown of the extracted tooth was used as the test subject (Figure 1C). The purpose of this stage was to minimize the setup and increase the flexibility of usage for a clinician. Hence, the SCE/graphite rod/beaker assembly was replaced with a mini capped tube (3 mL electrolyte). The capped tube was filled with PBS and essentially takes the place of the beaker from Stage 2. Three holes were made into the cap of the Eppendorf tube so that a platinum wire (RE), a gold wire (CE), and a platinum wire can each fit through an orifice and suspend in the PBS solution without touching each other. A platinum wire (to avoid any corrosion issue with copper wire) was used to connect with electrolytes in the capped tube and tooth orifice (Figure 1C). The components in the access hole should not touch, and 2−3 drops were, again, added to the environment. The WE was the reamer file, which connected to the potentiostat through a copper wire. An OCP test was conducted to monitor the evolution of potential, and a potentiostatic test was used to determine the controlled/stable current flow as well as the amount of current that was being generated with the various potentials (−9, −6, −3, 0, and +2 V) that were applied. Each potential was tested for 1800 s (30 min) for 3 trials (N = 3). 2.1.4. Stage 4: Further Simplification of Remote Center and Biological Validation Test. A tooth model with an access orifice already drilled into the crown of the extracted tooth was used as the test subject (Figure 1D). The purpose of this stage was to further simplify the connections to obtain a simple and ready device. To do that, we tried to eliminate the remote center by connecting the CE to C


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Figure 2. (a) Graph representing the OCP obtained with the electrochemical setup at Stage 1. It represents the data points from n = 2. (b) The graph represents the current produced, which was obtained with the electrochemical setup at Stage 2. It represents the data points from n = 3 at different voltages ranging from −0.9 to +0.05 V. (c) Graph representing the OCP obtained with the electrochemical setup at Stage 3. It represents the data points from n = 2 at different voltages ranging from −0.9 to +0.05 V. fetal bovine serum and penicillin/streptomycin solution. The cells were incubated in a CO2 incubator with 5% CO2. After reaching confluency, the cells were detached from the flask with trypsin− ethylenediaminetetraacetic acid (EDTA) (Sigma-Aldrich, United States). The cell suspension was centrifuged at 3000 rpm for 3 min; cell density was counted using the hemocytometer, and then the desired density of cells was resuspended in growth medium for further studies. The cells were maintained in minimum essential medium supplemented with 10% fetal bovine serum. The cells were plated onto a 96- and 24-well plates with a seeding density of 8000 and 15 000 cells/well respectively, according to the requirement of the experiment. Then, the plate was left in the incubator for 48 h, after which the cells were subjected to the appropriate treatment in triplicates (N = 3). For 3D cell culture, 2.5% medium viscosity alginate (Sigma-Aldrich) solution was prepared in normal saline by overnight stirring. MG63 cells were cultured based on the standard protocol reported previously.36 After 85% confluency, the cells were trypsinized, counted by hemocytometer, and resuspended in alginate solution such that the final concentration was 1 × 105 cells/mL of 1.5% alginate (SigmaAldrich) solution. The alginate/cell suspension was then dropped into 200 mM CaCl2 solution prepared in basal medium with antibiotics and incubated for 30 min at 37 °C. After incubation at 37 °C, the crosslinked alginate beads encapsulated with MG63 cells were washed with PBS for removing the CaCl2 solution. After, 1 mL of 20% MEM (Sigma-Aldrich) was dispensed in the well plate, which resulted in the medium covering the beads. Media change was done every 24 h to maintain the viability of the cells. 2.6. Cell Viability of 3D Encapsulated MG63 Cells. The cell viability of MG63 cell-encapsulated alginate beads were evaluated using an alamarBlue assay. Briefly, cells were encapsulated in alginate beads at a density of 1 × 105 cells/bead and placed in a 24-well plate and cultured for up to 4 days in MEM with 10% FBS under standard culture conditions. Cells with similar seeding density culture on cell culture 2D well plate were considered as control. After 24 h of incubation at 37 °C, the cells were incubated with 10% alamarBlue (Invitrogen, United States) in complete medium for 6 h. After incubation, the medium was pipetted into 96-well plates, and the optical density was recorded using a microplate spectrophotometer at 575 nm with 600 nm set as the reference wavelength. Results are presented as the mean ± standard deviation of three independent analyses performed in triplicate. 2.7. Electrochemical Treatment to MG63 Cells in 2D and 3D Culture. After the cells were cultured in a 96-well plate and achieved a proper confluency, they were subjected to the electrochemical treatment ranging from −9 to −6 V for 5 min. The viability of the cells was assessed with 10% alamarBlue after 4 h, and the absorbance was read at 575 and 600 nm. The MG63 cells were successfully encapsulated in sodium alginate as mentioned in Section 2.5, and then, 3−5 beads were transferred to an appropriate number of wells in a 96-well plate. One-hundred

microliters of culture media was added, after which the cells were subjected to the electrochemical treatment ranging from −9 to −6 V for 5 min. The viability of the cells were assessed with 10% alamarBlue after 4 h, and the absorbance was read at 575 and 600 nm. Approximately 15 000 osteoblast cells were seeded onto a coverslip and incubated for 48 h to ensure the adhesion of the cells onto the coverslip. The cells on the coverslip were then subjected to −6 V for 5 min, and then the coverslips were stained with Annexin-V-FITC and PI to test for apoptosis. 2.8. Statistics. All experiments were done in triplicate (N = 3), and ANOVA + Tukey was performed on IBM-SPSS v22 (UIC Licensed) to obtain the statistical significance. P < 0.05 was considered as significant. Several intergroup significances were observed, but because we are interested in comparing the significance levels with the control group, only those have been reported.

3.0. RESULTS AND DISCUSSION 3.1. Initial Stages of Development: Proof of Concept. To evaluate the feasibility of electrochemical protocol for root canal treatment, we developed a miniature form of the electrochemical setup. The developed microelectronic device was modified, updated, and validated experimentally in three different stages. Open circuit potential was used for initial screening of the applicability of the method. To assess the stability of the stage 1 setup, OCP test ran for 86 400 s (24 h) for 2 trials, and the electric potential as a function of time was recorded and is depicted in Figure 2a. From the result obtained, it was evident that the OCP was stable for 35 000 s, and then the stability of the connection was altered in both the trials, indicating that the setup is not stable for long durations. Hence, we proceeded to the next stage of development, where our main aim was to develop a prototype which will produce a stable potential and current. The second stage (stage II) of development is shown in Figure 1b. Due to the changes done from the previous stage, we were able to produce a stable OCP and record the current obtained when different potential was applied. The current generated was stable for both anodic and cathodic voltages which ranged from −0.9 to +0.05 V, suggesting that the electrochemical setup which was designed had a capability to produce a stable voltage which in turn could generate current, as shown in Figure 2b. To miniaturize the setup to make it clinician-friendly and increase the applicability of the same, we updated the setup as shown in Figure 1c. We were successfully able to miniaturize the setup and yet retain the capability of the device to maintain the efficiency which is indicative in Figure 2c, which was recorded when the constant potential was applied. It is evident from the graph which shows the device is capable of applying a D


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min. In addition, there is no significant difference (±1 °C) in temperature change between the test solutions such as brain heart infusion medium (bacterial culture medium), PBS, and minimum essential medium (cell culture medium). Even though ±1 °C can cause anomalies in growth of cells in a longer duration, because the treatment time is momentary, it should not affect any surrounding tissue. The study further confirms the fact that the results obtained from other experiments in this study are primarily due to the effect of applied voltage and not due to the heat generation from the electrochemical experiments. To translate this into a clinical scenario, the applied potential will not cause any abrupt changes in the temperature which may cause burning of the tissue. 3.3. Bacterial Cell Viability Assay. E. faecalis is a Grampositive, commensal bacterium inhabiting the gastrointestinal tracts of humans and other mammals.37 Like other species in the genus Enterococcus, E. faecalis can cause life-threatening infections in humans, especially in the nosocomial (hospital) environment,38 where the naturally high levels of antibiotic resistance found in E. faecalis contribute to its pathogenicity. E. faecalis has frequently been found in root canal-treated teeth in prevalence values ranging from 30 to 90% of the cases.19,37 Root canal-treated teeth are about nine times more likely to harbor E. faecalis than cases of primary infections. Hence, in this study, we employed the use of E. faecalis to study the applicability of the technique we developed. Two factors were considered while performing the experiments: translational capability (miniature size, cost, clinicianfriendliness) and time duration of the electrochemical treatment (low time duration is favorable for the patients). Initially,

constant potential which in turn produces current. This setup (stage 3) was then used to conduct the experiments on biological samples, the results of which are shown below. 3.2. Temperature Change with Voltage. The device was successful in applying stable voltage for about 2000 s, which is indicated in the previous experimental trials (Figure 2). However, it is important to analyze the increase in temperature upon applied potential on different types of solutions before continuing with actual experiments with bacterial and mammalian cell culture; the purpose of this experiment was to show that there is no temperature build up in the solution due to the application of the potential, and this also aids in final interpretation of the results. The results (Figure 3) of the study

Figure 3. Graph representing the change in temperature when 100 μL of different solutions were exposed to a voltage varying from −9 to +2 V for 5 min.

showed that there is no significant difference in change in potential in all the potential applied in the current study for 5

Figure 4. (a) Graph representing the bacterial viability when the potential applied was varied; CFU for the same is shown in panels c and d. Panel b represents the viability of the bacterial cells when −9 V was applied and the treatment time was varied from 5 to 1 min, and CFU of the same is shown in panel e. E


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Figure 5. (a and b) Confocal microscopic images of (a) control group cells, which had less apoptotic and necrotic activity, whereas (b) treatment group was exposed for 3 min, and −9 V shows apoptotic and necrotic activity. (c) Unstained cells and (d) stained cells encapsulated in sodium alginate, indicating the viability of osteoblast cells. (e) Stained beads without cells using alamarBlue. Panel f is the live/dead imaging of the sodium alginate beads embedded with the cells; panel g shows the 3D nature of the bead, and panel h is the live/dead imaging of the sodium alginate beads after treatment of −9 V for 5 min. Panel i shows the 3D nature of the bead. Panel j indicates the percentage viability of osteoblast cells after treatment in 2D and 3D culture.

tooth surface and create a biofilm. As the biofilm grows, an anaerobic environment is formed that leads to oxygen depletion. Microbes use sucrose and other dietary sugars as a food source. The dietary sugars go through anaerobic fermentation pathways, producing lactate. The lactate is excreted from the cell onto the tooth enamel and then ionizes. The lactate ions demineralize the hydroxyapatite crystals, causing the tooth to be degraded. After the degradation of the tooth, the bacteria enter into the soft tissue part of the tooth and begin utilizing the nutrients which are there for the survival of the dentine and pulp tissue, which leads to necrosis of the tissue. When root canal procedure is performed, the healing and complete restoration time of the tooth may go up to 15 months due to the loss of dentine and pulp tissue during the root canal procedure.40 To decrease the healing time of the tooth, viability of the surrounding cells after the treatment is a key factor to be considered. Osteoblast cells were considered for the purpose of experimentation because they are cellular structures and have connective tissue between the bone and musculature similar to those of dentine; they also play a major role in bone formation.41 Dentine has very similar characteristics to osteoblasts: both dentine and osteoblasts express dentin matrix protein 1 (DMP1), which is a noncollagenous protein important for the mineralization of bones and teeth.42 Mineralization is a key factor for complete recovery of the tooth. Hence, it is important to maintain the viability of the connective tissue while killing the bacterial cells. So, the following experiments were performed to confirm that the cathodic potential applied to the cells was less toxic to the connective tissue (osteoblast cells in this case). Initial experimentation was done on a 2D monolayer culture of the osteoblast cells in 96-well plate, as described above. Cathodic potential varying from −9 to −6 V was used to show

we experimented with the potential applied to the bacterial cells. We exerted a potential ranging from −9 to +2 V using stage 3 experimental setup, the methodology of which is stated above. The results obtained are shown in Figure 4. Initially, we used lower potential for experiments. Potential ranging from −2 to +2 V for 5 min was applied. But, no significant difference in bacterial death was observed from Alamar blue assay compared to the control (Figure 4b). Corresponding CFU values are shown in Figure 4e. The viability of the bacterial cells did not go below 88%. Cathodic potential showed a more promising effect than anodic. Hence, in further experimentation, cathodic potential ranging from −9 to −6 V for 5 min was applied. It was found that at the applied potential −6 V for 5 min, bacterial viability reduced to 82%. However, at the cathodic potential of −9 V for 5 min, there is a significant reduction in viability, viz. 25.20%. The results were further correlated and confirmed with the CFU analysis, as shown in Figure 4c. Similar experimentation was done using the stage 4 experimental setup for the purpose of validation. The cathodic potential of −9 V showed a translational capability. The maximum reduction in the viability of cells was seen in −9 V. Hence, we conducted experiments at different time intervals such as 1, 3, and 5 min. When the same cathodic potential of −9 V was applied for 1 and 3 min, we could see the viability of bacterial cells at 39 and 37%, which was comparable to −7 V for 5 min, which is shown in Figure 4a, and CFU is shown in Figure 4c. The maximum reduction of bacterial cell viability was shown at −9 V and 5 min. Hence, it was considered for further experimentation. 3.4. Osteoblast Cell Viability: 2D Culture. The tooth is a complex structure of inner pulp tissue which is encapsulated in a connective dentine tissue and then covered with a hard enamel shell which is the protective inherit layer.39 The process in which a root of a tooth is infected is a gradual one. The initial stage of infection is caries, where these microbes attach to the F


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ACS Biomaterials Science & Engineering the maximal reduction in bacterial cell viability when compared to other potentials. In addition, NaOCl (0.1%) was also used as a control in this study to compare the toxic effect of irrigant to that of the electrochemical treatment. Cathodic potential of −9 V for 5 min gave a reduction in bacterial cell viability up to 80% and also showed a toxic effect against 2D monolayer culture of osteoblast cells, as shown in Figure 5h. However, the lower voltages of −8, −7, and −6 V for 5 min indicated a viability of 4, 59, and 55%, respectively; positive control was the treatment of 0.1% NaOCl for 5 min, and the negative control was untreated cells, which are also shown in Figure 5h. Osteoblast cells also showed apoptotic activity when −9 V for 3 min was applied, which is shown in Figure 5g when compared to untreated cells Figure 5f. 3.5. Osteoblast Cell Viability: 3D Culture. Because the cells in the human body are in a 3D microenvironment, we further evaluated the cytotoxicity of electrochemical protocol in a 3D environment. To obtain results with better translational capability in 3D, sodium alginate culture technique was used. The osteoblast cells were encapsulated in acellular 3D sodium alginate beads. These encapsulated cells were then exposed to different electrochemical potential. Osteoblast cells in 3D culture indicated a positive result of 50 ± 15% viability even after exposure to the cathodic potential of −9 V for 5 min, which are shown in Figure 5h. Cell viability of osteoblasts was retained at 97 ± 10, 81 ± 9.7, and 47 ± 30.7% when the cathodic potential of −6, −7, and −8 V for 5 min was applied, respectively. However, the complete toxicity was observed in NaOCl treated cells both in 2D and 3D culture conditions, which revealed the potential of the method for the current treatment. These results were promising as it had a better translational capability than the ones obtained in 2D culture. This study can also show that the treatment will not affect the cells encapsulated in the 3D extracellular matrix. Maintaining the viability of the cells even after the treatment will help to improve the recovery time for a patient from 15 months40 to less than 4 months as the number of cells in the dentine will be higher and will collectively contribute to the mineralization of the tooth. 3.6. Validation of Experimental Setup (Stage 4) with Antibacterial Studies. In this stage, we again considered the factor, translational capability (miniature size, cost, and clinician-friendliness) of the instrument. Based on the results from the stage 3 experimental setup, we selected the voltages −9, −6, 0, and +2 V for validation of stage 4. The potentials were applied to the bacterial cells, and the results indicated that there were not many changes in bactericidal effect at voltages −6, 0, and +2 V. However, stage 3 indicated a bactericidal activity of 80% at −9 V for 5 min, but stage 4 has a bactericidal effect of 65% (Figure 6). Hence, we concluded that multiple treatments of −9 V for 5 min will result in complete eradication of bacteria, and also the stage 4 setup will be more feasible for the clinical translation. 3.7. Confocal Microscopy of Bacterial Biofilm. To consider the method for clinical application of root canal treatment, we further evaluated the effectiveness of the method on bacterial biofilm, which is a more analogous in vivo environment compared to bacterial suspension. Further, the results provided a visual and qualitative evidence of the eradication of bacteria. Figure 7A represents the confocal microscopic images of the Syto-PI stained the E. faecalis before and after the electrochemical treatment at two different conditions −6 and −9 V for 5 min. Viable cells were stained

Figure 6. Graph representing the bacterial viability using the stage 4 setup after the applied potential of −9, −6, 0, and +2 V for 5 min.

by green Syto stain, and the DNA of the dead cells was stained by PI, indicative of the compromised cell wall of the bacteria. In the control image (Figure 7A(a)), most of the cells are visibly viable because the cells are stained in green, whereas the cells which were subjected to electrochemical treatment are colored red due to PI stain; the same possibility can be seen in both the cases −6 and −9 V for 5 min. The quantitative estimation of the viable bacteria in the previous experiment after treatment of −6 V for 5 min was 82%. A similar result can be seen in the confocal microscopic image where a few of the cells were stained red, hence the overlap image of viable (green) and nonviable (red) give a yellow color, which is the confirmation of the previous result. 3.8. SEM Images of Bacterial Biofilm. To further confirm the result from both planktonic cell suspension experiment as well as the confocal microscopy, we subjected the untreated bacterial cells and the cells treated with −9 V for 5 min to SEM imaging. SEM image of control group can be seen in Figures 7B(a−c). All the three images are the viable cells. Figure 7B(c) shows that the structural integrity of the bacterial cell is maintained. In Figure 7B(f), the bacterial cells shrunk after being subjected to the treatment, i.e., 9 V for 5 min. 3.9. Possible Mechanisms under Electrochemical Exposure. A schematic diagram is proposed to explain the possible mechanistic pathways of the biological cells under electrochemical exposure. A viable cell has an intact cell wall and is structurally stable. To facilitate the transport of proteins and metabolites into the cell and excrete the waste out of the cell, there are designated pores and channel proteins.43 These channel proteins act as selective protein transport membranes, whereas the pores completely rely on the difference in the osmotic potential or the membrane potential inside and outside the cell. In general, the membrane potential across the cell membrane varies anywhere between −20 and −200 mV,43 which varies between different cells and organisms. The stability of the membrane potential is very much necessary to maintain the integrity of the cell wall of the bacterial cell. When the potential of −9 V is applied, there is a very high chance that the microenvironment inside the cell is being altered and triggering the pores to excrete the cellular content (Figure 8) out to the external environment. Once the cellular contents are excreted outside the cell, shrinkage of the cell might occur, which is probably what is being seen in the SEM images. Also, it is important to maintain a stable membrane potential in bacteria to facilitate cell division.44 When such intervention is occurring, it may delay the recovery; in the meantime, external G


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Figure 7. (A) Confocal microscopic images of Syto-PI staining of E. faecalis after application of the cathodic potential. (a) Control showing viable cells, (b) −6 V for 5 min showing partially viable bacterial culture, and (c) −9 V for 5 min, showing the maximum death of bacterial cells with less viability. (B) SEM images of control and −9 V, 5 min treated E. faecalis at different magnifications (50, 5, and 1 μm).

Figure 8. Schematic representation of the root canal treatment procedure with the developed device and possible mechanism of action, which takes place to eradicate the bacterial infection.

are concerns in translation to a product or clinical device. In the future, a realistic tooth model will be used as a better experimental strategy for such evaluation; in this current study, we concentrated in terms of treatment to the molar tooth or multirooted tooth, but in the future, different types of tooth (monorooted) will be used for study to give a better understanding, applicability, and penetrability of the voltage into the dentine tubules to completely eradicate the bacteria. Also, it is key to simplify and miniaturize the device further (hardware and display unit) and develop the applicability of using this system as a treatment tool for a dental practitioner.

antibiotics will be sufficient to completely eradicate the bacteria and eliminate the chance of relapse of the infection. Also, there is an increase in microbes becoming resistant to certain strains of antibiotics, and in such situations, the developed method can be a better treatment strategy.

4.0. LIMITATIONS AND FUTURE DIRECTIONS The study reports the proof of concept of the development of new microelectronic device for the total eradication of infection during the root canal treatment. The work has several limitations. The current study used an in vitro model; there H


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(9) Siqueira, J. F.; Rôca̧ s, I. N. Clinical Implications and Microbiology of Bacterial Persistence after Treatment Procedures. J. Endod. 2008, 34 (11), 1291. (10) Chiniforush, N.; Pourhajibagher, M.; Shahabi, S.; Bahador, A. Clinical Approach of High Technology Techniques for Control and Elimination of Endodontic Microbiota. J. Lasers Med. Sci. 2015, 6 (4), 139−150. (11) Atila-Pektaş, B.; Yurdakul, P.; Gülmez, D.; Görduysus, Ö . Antimicrobial Effects of Root Canal Medicaments against Enterococcus Faecalis and Streptococcus Mutans. Int. Endod. J. 2013, 46 (5), 413−418. (12) Iqbal, A. Antimicrobial Irrigants in the Endodontic Therapy. Int. J. Health Sci. 2012, 6 (2), 186−192. (13) Jaju, S.; Jaju, P. P. Newer Root Canal Irrigants in Horizon: A Review. Int. J. Dent. 2011, 2011, 1−9. (14) Gernhardt, C. R.; Eppendorf, K.; Kozlowski, A.; Brandt, M. Toxicity of Concentrated Sodium Hypochlorite Used as an Endodontic Irrigant. Int. Endod. J. 2004, 37 (4), 272−280. (15) Anderson, A. C.; Hellwig, E.; Vespermann, R.; Wittmer, A.; Schmid, M.; Karygianni, L.; Al-Ahmad, A. Comprehensive Analysis of Secondary Dental Root Canal Infections: A Combination of Culture and Culture-Independent Approaches Reveals New Insights. PLoS One 2012, 7 (11), e49576. (16) Haapasalo, M.; Udnaes, T.; Endal, U. Persistent, Recurrent, and Acquired Infection of the Root Canal System Post-Treatment. Endod. Top. 2003, 6 (1), 29−56. (17) Al-Ahmad, A.; Pelz, K.; Schirrmeister, J. F.; Hellwig, E.; Pukall, R. Characterization of the First Oral Vagococcus Isolate from a RootFilled Tooth with Periradicular Lesions. Curr. Microbiol. 2008, 57 (3), 235−238. (18) Molander, A.; Reit, C.; Dahlén, G.; Kvist, T. Microbiological Status of Root-Filled Teeth with Apical Periodontitis. Int. Endod. J. 1998, 31 (1), 1−7. (19) Ng, Y.-L.; Mann, V.; Gulabivala, K. Tooth Survival Following Non-Surgical Root Canal Treatment: A Systematic Review of the Literature. Int. Endod. J. 2010, 43 (3), 171−189. (20) Jungermann, G. B.; Burns, K.; Nandakumar, R.; Tolba, M.; Venezia, R. A.; Fouad, A. F. Antibiotic Resistance in Primary and Persistent Endodontic Infections. J. Endod. 2011, 37 (10), 1337−1344. (21) Liu, W. K.; Brown, M. R.; Elliott, T. S. Mechanisms of the Bactericidal Activity of Low Amperage Electric Current (DC). J. Antimicrob. Chemother. 1997, 39 (6), 687−695. (22) Mohn, D.; Zehnder, M.; Stark, W. J.; Imfeld, T. Electrochemical Disinfection of Dental Implants−a Proof of Concept. PLoS One 2011, 6 (1), e16157. (23) Pileggi, R. Ozonization and Electrochemical Root Canal Disinfection. Disinfection of Root Canal Systems: The Treatment of Apical Periodontitis 2014, 239. (24) Sandvik, E. L.; McLeod, B. R.; Parker, A. E.; Stewart, P. S. Direct Electric Current Treatment under Physiologic Saline Conditions Kills Staphylococcus Epidermidis Biofilms via Electrolytic Generation of Hypochlorous Acid. PLoS One 2013, 8 (2), e55118. (25) Sahrmann, P.; Zehnder, M.; Mohn, D.; Meier, A.; Imfeld, T.; Thurnheer, T. Effect of Low Direct Current on Anaerobic Multispecies Biofilm Adhering to a Titanium Implant Surface. Clin. Implant Dent. Relat. Res. 2014, 16 (4), 552−556. (26) Stoodley, P.; Lappin-Scott, H. M. Influence of Electric Fields and pH on Biofilm Structure as Related to the Bioelectric Effect. Antimicrob. Agents Chemother. 1997, 41 (9), 1876−1879. (27) Ercan, B.; Kummer, K. M.; Tarquinio, K. M.; Webster, T. J. Decreased Staphylococcus Aureus Biofilm Growth on Anodized Nanotubular Titanium and the Effect of Electrical Stimulation. Acta Biomater. 2011, 7 (7), 3003−3012. (28) Niepa, T. H. R.; Wang, H.; Gilbert, J. L.; Ren, D. Eradication of Pseudomonas Aeruginosa Cells by Cathodic Electrochemical Currents Delivered with Graphite Electrodes. Acta Biomater. 2017, 50, 344− 352. (29) Maisonneuve, E.; Gerdes, K. Molecular Mechanisms Underlying Bacterial Persisters. Cell 2014, 157 (3), 539−548.

Further, different 3D matrixes, cell types, and a mouse model will be used to further evaluate the effect of the treatment on the mammalian cells or tissue.

5.0. CONCLUSIONS This study provides supporting evidence that electrochemical technique can be used as an effective tool for the total eradication of bacteria by maintaining viable dentine cells after treatment. This research provided the proof of concept with different stages of development by considering the miniature size and feasibility for a dental practitioner. This technique has shown promising results in the in vitro model. Hence, the developed electrochemistry-based microelectronic device can be considered for the next generation of oral clinical instruments. However, further studies are required to develop the miniature sized and user-friendly hardware to control and monitor the electrochemical parameters. In addition, the technique has to be validated through an in vivo model before any further clinical trials.

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AUTHOR INFORMATION

Corresponding Author

*E-mail: [email protected]. ORCID

Abhijith Segu: 0000-0002-1620-3588 Divya Bijukumar: 0000-0001-8114-5444 Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS The authors acknowledge the Blazer Foundation for providing funding for the project. We also thank the Guaranteed Paid Internship Program (GPIP) offered by the UIC College of Engineering. The authors are thankful to Dr. Vishal Khatri and Dr. Nikhil Chauhan for their valuable suggestions in this study. We thank Dr. Karl W Hagglund, NUANCE, Northwestern University for SEM analysis. We also would like to thank Ms. Laura Sykaluk for technical assistance.

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REFERENCES

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