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Monitoring bacterial-demineralization of human dentine by electrochemical impedance spectroscopy
journal of dentistry 38 (2010) 138–148
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Monitoring bacterial-demineralization of human dentine by electrochemical impedance spectroscopy Zhang Xu a, Koon Gee Neoh b, Bennett Amaechi c, Anil Kishen d,* a
Department of Restorative Dentistry, Faculty of Dentistry, National University of Singapore, 5 Lower Kent Ridge Road, 119074 Singapore, Singapore b Department of Chemical & Biomolecular Engineering, National University of Singapore, Kent Ridge 119260, Singapore c Department of Community Dentistry, University of Texas Health Science Center at San Antonio, 7703 Floyd Curl Drive, San Antonio, TX 78229, USA d Discipline of Endodontics, Faculty of Dentistry, University of Toronto, 124 Edward Street, Toronto, ON, Canada
article info
abstract
Article history:
Objective: The purpose of this study was two-fold: (1) to monitor bacterial biofilm formation
Received 28 May 2009
and bacteria-induced demineralization of dentine in situ by using electrochemical impedance
Received in revised form
spectrum (EIS); (2) to examine the relationship between EIS findings and changes in the
21 September 2009
chemical composition and ultrastructure of dentine during bacteria-induced demineralization.
Accepted 27 September 2009
Methods: In this study, dentine demineralization was induced by Streptococcus mutans (ATCC 25175) in the presence of sucrose in culture medium and was monitored using two EIS measurement systems (Type A with a working electrode and Type B without a working
Keywords:
electrode). Scanning electron microscopy (SEM), field emission scanning electron micro-
Electrochemical impedance
scopy (FESEM), energy dispersive X-ray (EDX) and X-ray diffraction (XRD) were employed to
spectroscopy (EIS)
examine the morphology, element contents and crystallinity of hydroxyapatite (HAP) on the
Bacterial-induced demineralization
dentine surface. Transverse microradiography (TMR) was used to characterize the lesion
Dentine
depth and degree of mineral loss during demineralization. Results: The resistance of the bulk dentine (Rd) and the apparent resistance of dentine (Ra) measured from the Type A and Type B EIS systems, respectively, decreased gradually with demineralization. The resistance of the biofilm formed on dentine surface was determined by fitting the EIS data with equivalent circuits. The presence of biofilm slightly increased Ra of dentine before demineralization. However, the electrochemical behavior of biofilm did not affect the decreasing impedance of dentine with demineralization. The SEM, EDX, XRD and TMR results demonstrated that the surface and bulk dentine gradually became more porous due to the loss of minerals during demineralization, which in turn resulted in the decrease in Rd and Ra values obtained from EIS systems. Conclusions: This investigation highlighted EIS as a potential technique to monitor biofilm formation and bacterial-induced demineralization in situ. # 2009 Elsevier Ltd. All rights reserved.
1.
Introduction
Electrical impedance spectroscopy (EIS), a non-destructive and sensitive method, is widely used to characterize bulk and
interfacial electrical properties of solid or liquid materials.1 EIS has been used to measure electrical resistance of biological tissues such as dental tissues,2–7 bone,8 skin9 and blood cells.10 In clinical dentistry, visual inspection and bitewing radio-
* Corresponding author. E-mail address: [email protected] (A. Kishen). 0300-5712/$ – see front matter # 2009 Elsevier Ltd. All rights reserved. doi:10.1016/j.jdent.2009.09.013
journal of dentistry 38 (2010) 138–148
graphy are used to detect early caries, but they are based on subjective evaluation, and only a limited portion of early caries can be detected.11 EIS technology seems to have the potential to diagnose early caries reliably and accurately.12 Some studies have been conducted to detect early caries based on the porosity of enamel as indicated by EIS data5–7; however, EIS application has seldom focused on monitoring the demineralization of dentine. It is significant to characterize dentinal demineralization using EIS, because dentine demineralizes more rapidly than enamel and thus in many cases show severe demineralization underlying enamel, which is difficult to detect using conventional methods in clinical practice.13 The application of EIS technology to dental tissue depends on the penetration of electrolyte into specimens. Subsequently, the tissue will be conductive, allowing EIS measurements. Since conductive properties are affected by the microstructure of the tissue, impedance spectra usually contain features that can be directly related to the changes in the microstructure of the tissue.14 Smear layers on dentine have been detected according to this theory using EIS technology.4 In fact, these changes in the microstructure are usually associated with variation in the chemical constituents. Therefore, for studying dentine demineralization it is necessary to characterize the change in ultrastructure and chemical components by using SEM, FSEM, EDX, XRD and TMR. This strategy can provide comprehensive and integral insight into microstructural changes of dental tissue compared to the application of a single technique. Dentine has a regularly tubular structure, which is composed of intertubular and peritubular dentine. Intertubular dentine consists of a fibrous network of collagen on which hydroxyapatite (HAP) crystals are deposited, while peritubular dentine is a more highly mineralized tissue without collagen fibrils.15 EIS has been used to characterize porous ceramics, the structure of which is similar to that of dentine.16 It has also been used to monitor the degradation process of a polymer that is similar to the demineralization process of dentine.17 Consequently, based on the regular porous structure of dentine, it is possible to use EIS in situ to study the changes in the chemical components and ultrastructure of dentine caused by demineralization under simulated carious conditions. In our previous work,18 acid-induced demineralization of dentine was monitored in situ using EIS, and the EIS findings correlated well with the changes in the chemical composition and ultrastructure of dentine. It is well known that bacterial biofilm, or dental plaque, is involved in the demineralization of dental hard tissues. Thus, in order to simulate a more clinically realistic model, bacterialinduced demineralization of dentine will be monitored by EIS technique in this investigation. In contrast to acid-induced demineralization, biofilm formed on dentine may result in more complex EIS readings. Recently, EIS technique has been applied to characterize and detect bacterial biofilm.19–22 Equivalent circuits were used to characterize biofilm and its resistance was obtained by fitting EIS data.23 The hypothesis of this study was that bacteria-induced demineralization of dentine in situ and biofilm formation can induce chemical and ultrastructural changes in the dentine, which in turn could be monitored by using EIS technology. In this study, besides the one-chamber and three-electrode configuration (Type A),18 a
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two-chamber and two-electrode configuration (Type B) was also used to measure impedance of the dentine sections with or without bacterial biofilm. The Type B EIS system has been employed previously to test the micro-leakage between dental tissues and restorative materials.24 The apparent resistance of the specimen can be directly obtained by measuring the resistance between two electrodes without fitting the EIS data with equivalent circuits. Combination of these two types of EIS measurement system could provide more complete understanding of the change in the EIS reading of dentine during bacterial-induced demineralization, and the influence of bacterial biofilm on the EIS results.
2.
Materials and methods
2.1.
Preparation of specimens and culture medium
The collection and use of extracted human teeth for all the experiments conducted in this study has been approved by the institutional review board of the National University of Singapore. Extracted non-carious human third molars without visible evidence of cracks, and maintained in phosphate buffered saline (PBS) were used in this study. The roots and pulps of these teeth were removed, and then serially sectioned perpendicular to their long axes by means of Microslice 2 (Metals Research Limited, Cambridge, England) with a rotating, internal-cutting, copper disc impregnated with diamonds (Buehler1, diamond wafering blade, 7.6 cm diameter 0.15 mm width, USA). Subsequently, the sections were ground into 5 mm 5mm 1 mm square samples using Phoenix Beta Grinder/Polisher (Wirtz, Buehler, USA) with special silicon carbide grinding papers (Carbimet1, P1200, Buehler, USA). These dentine sections were obtained from the same position along the long axes of different molars, which enables us to obtain dentine specimen with approximately similar diameter and number of dentinal tubules for the same batch of measurement. The smear layers formed on these dentine sections were not removed. Six dentine sections were used to fabricate working electrodes for the Type A EIS measurement and nine were randomly and averagely divided into three groups for the Type B EIS measurement. In SEM, EDX, and XRD characterization, fifteen dentine sections were randomly and averagely divided into five groups corresponding to different periods (2, 4, 6, 10 and 14 days) and they were sealed with varnish on all surfaces except the surface perpendicular to dentinal tubules exposed to the bacterial culture. The same grouping method and treatment was applied on the specimens in TMR characterization, but the roots of these specimens remained as holders for the convenience of further cutting. The culture medium is mainly composed of sterile brainheart infusion broth (BHI) (Merck KGaA, Germany, Lot 811893) with or without 5% sucrose (w/v). After 18 h growth of pure cultures with 1–2 108 CFU/ml determined by measuring the absorption intensity at 660 nm using a UV–vis spectrophotometer (UV-1700, SHIMADZU, Japan), 100 ml of a cell suspension of Streptococcus mutans (ATCC 25175) was inoculated in 30 ml of culture medium for each dentine section.25 The culture medium was replenished every 48 h in order to remove
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Fig. 1 – (A) Cell for the Type A EIS system; (B) bacterial culture set; 1: counter electrode; 2: reference electrode; 3: working electrode; 4: specimen of dentine slice; 5: cell chamber; 6: 50 ml polypropylene conical centrifuge tube (Falcon). Schematic diagram of the Type A EIS measurement system.
dead cells and allow better biofilm growth.26 Contamination was verified in the medium each day by means of Gram staining.
2.2.
EIS system and measurement
Two types of EIS measurement system were used in this study. Type A contains a one-chamber cell and three electrodes: a reference Ag/AgCl electrode (Metrohm, Switzerland), a platinum counter electrode (Metrohm, Switzerland) and a working electrode as shown in Fig. 1. The fabrication of working electrodes with dentine sections was the same as that of our previous study.18 The cell and the electrodes were sterilized using autoclaving and UV radiation for 3 h before use, respectively. During demineralization process, the working electrodes with dentine samples were immersed in a bacterial culture medium and placed in a shaking incubator (QT
Instruments Pte. Ltd., Singapore) at 37 8C and the measurements were conducted with EIS at different intervals (Fig. 1). In total, six working electrodes with similar baseline data (i.e., initial impedance spectrum in Nyquist plots) were chosen for treatment with the bacterial medium and EIS measurement. The Type B system was specially designed and fabricated according to the principle in Nelly’s study24 as shown in Fig. 2. In this configuration, a cell and a 50 ml centrifuge tube (Falcon, BD Biosciences, San Jose, CA, USA) constitute the two chambers; two platinum electrodes (Metrohm, Switzerland) were placed in each one of them, respectively. First, the dentine section was fixed on the 3 mm diameter hole on the cap of the tube by spreading wax around it. Then, the leakage of the samples was checked by measuring the resistance between two chambers. Finally, the dentine samples along with plastic support were sterilized using UV radiation for 3 h. During demineralization process, the bacterial culture med-
Fig. 2 – (A) Cell for the Type B EIS system; (B) bacterial culture set; 1: platinum electrode; 2: plastic beaker; 3: 50 ml polypropylene conical centrifuge tube (Falcon); 4: cap with a dentine section; 5: the cell chamber. Schematic diagram of the Type B EIS measurement system.
journal of dentistry 38 (2010) 138–148
ium was taken in a 50 ml centrifuge tube, which was kept in a glass beaker enveloped by parafilm (Pechiney Plastic Packaging Company, Chicago, IL, USA) to prevent contamination. The culture was also placed in the shaking incubator at 37 8C. As shown in Fig. 2, the cap were first taken off and then placed on the 50 ml centrifuge tube in the cell for the EIS measurement at different intervals. All the operations and measurements were performed within a work station (NuAire, Inc., Plymouth, MN, USA). In the Type B EIS system, dentine sections with or without smear layers were used. The smear layers on both sides of dentine section were removed. In order to characterize the effect of biofilm on impedance of dentine, the dentine sections with smear layers were treated with BHI medium with or without sucrose (three sections in each group); the three dentine sections without smear layers were treated with the medium without sucrose. The smear layer of dentine section was removed by etching the specimen surface with phosphoric acid (35%, w/v) for 15 s.4 Each treatment group comprised three dentine sections. The mechanism of measurement using the Type B EIS system was introduced by Nelly et al.24 Herein, the shape of the impedance spectra is ‘‘V’’ shape in Nyquist plane (i.e., complex plane) as shown in Fig. 3. At the frequency where the value of G is the lowest (around 1000 Hz), the contribution of the impedance of the electrodes is negligible and the real part of the impedance R corresponds to the sum of the resistance of electrolyte (Re) and the apparent resistance of specimen (Ra). Re was obtained by measuring the impedance at the same frequency, with the same cap without the dentin section (Rd = 0). Therefore, Ra of specimens undergoing different demineralization periods could be monitored and calculated using the Type B EIS system. In both the Type A and Type B EIS systems, sterilized 0.01 M PBS was used as electrolyte in the chamber. The EIS readings would be recorded at different periods (2, 4, 6, 10 and 14 days). The EIS analysis was carried out using an Autolab PGSTAT 100 (Metrohm, Switzerland), while FRA4.9 software (Metrohm, Switzerland) was used to control and record measurements. The frequency range was 1 mHz–100 kHz which will take about 20 min and potential balance before EIS measurement will take 5 min. A Faraday cage that isolated the EIS systems
from noise was used to improve the reliability of results from a high impedance sample at typical AC amplitudes. It should be noted that leads themselves can act as a capacitor, the leads of the reference and counter electrodes are shorted and placed outside the Faraday cage. The leads of working electrode is shorted and placed inside the Faraday cage. These controls can minimize the artifactual drift in EIS measurement. The amplitude of the sinusoidal voltage signal was 10 mV. The FRA software was also used to analyze the experimental data.
2.3.
SEM and EDX characterization
In each group (each treatment period) three samples were first characterized by XRD and then by SEM and EDX, because of the need to coat the specimens with platinum for the latter. After different periods of immersion in the bacterial culture medium, the specimens were removed and then rinsed three times with deionized water (3 min each). Next they were immersed in a fixative solution containing 4% gluteraldehyde in sodium cacodylate buffer at 4 8C for 3 h.27 Finally, the specimens were dehydrated using ethanol (at gradient concentration of 70%, 80%, 90% and 100% for 20 min), and stored in a desiccator. Fracture surfaces were prepared using two pliers after the dehydration procedure. The specimens were sputter-coated with platinum, and then visualized and analyzed with SEM and EDX (JSM-5600LV models, JEOL Company, Tokyo, Japan) with a beam voltage at 15 kV. A quantitative elemental analysis of Ca and P was carried out using EDX. Three randomly selected areas were analyzed at a magnification of 1500. The average concentration of detected elements was recorded.
2.4.
XRD characterization
The structure and crystallinity of different dentine samples were investigated by a XRD-6000 X-ray diffractometer (SHIMADZU, Tokyo, Japan) with Cu Ka radiation. The data were collected in the 2u range of 20–408 at a scan rate of 28 per min. The value of the percentage of intensity (I%) which can characterize the crystallinity was calculated using the software Jade 5 (MDI, Materials Data. Inc., USA), and represents the ratio of the area of any other peak to that of the peak with a maximum area. The amount of HAP crystal on the surface of the specimens were evaluated from I% of HAP at 2u = 25.78 position that was assigned to the (0 0 2) plane.7
2.5.
Fig. 3 – Typical impedance curves of a specimen treated with Streptococcus mutans (ATCC 25175) in the medium enriched with sucrose. Re: resistance of electrolyte; Ra1: apparent resistance of specimen before treatment; Ra2: apparent resistance of specimen after treatment.
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TMR characterization
After different treatment intervals of demineralization, specimens were rinsed with deionized water for 5 min and then exposed to UV radiation for 15 min. Next, the specimens were sectioned perpendicularly to the lesion surface using a watercooled, Silverstone–Taylor hard tissue microtome to obtain the 100 mm-thick dentine sections for TMR characterization. The sections were placed in a specially fabricated radiographic plate-holding cassette, incorporating an aluminum step wedge (10 steps of 24.5 mm thickness). The cassette was loaded with type lA high resolution glass X-ray plates (Microchrome Technology, CA, USA) and placed into a Phillips X-ray generator system set up for this purpose. This apparatus
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Fig. 4 – (a and b) Typical experimental EIS data (symbol) and their fitted data (solid line) for dentine after different intervals of demineralization in total impedance Bode plot (a) and phase angle Bode plot (b). &: control; &: 2-day; ^: 4-day; ^: 6-day; ~: 10-day; ~: 14-day; *: bare electrode.
is equipped with a copper target and nickel filter, producing monochromatic radiation of wavelength appropriate for ˚ ). The plates were exposed for 10 min hydroxyapatite (184 A at an anode voltage of 20 kV and a tube current of 10 mA, and then processed. Processing consisted of a 5 min development in Kodak HR developer and 15 min fixation in Kodak Rapidfixer before a final 30 min wash period. After drying, the microradiographs were subjected to visualization and image analysis using a computer program, TMR2006 version 3.0.0.6 (Inspektor Research, Amsterdam, Netherlands). The hardware was a Leica DMR optical microscope linked via a Sony model XC-75CE CCTV camera to a 90 MHz DellTM Pentium Personal Computer. The enhanced image of the microradiographs were analyzed under standard conditions of light intensity and magnification and processed, along with data from the image of the step wedge, by the TMR program. By this method, the parameters of integrated mineral loss, Dz (vol% mm) and lesion depth, LD (mm) were quantified for each demineralized (carious) lesion.
3.
Results
3.1.
EIS spectra
Impedance spectra are generally displayed either in Nyquist (i.e., complex plane) or in Bode plot. The impedance spectra displayed in Bode plot can give more information including frequency and phase angle than only in Nyquist plot. In this study, therefore, the representative EIS data obtained through the Type A system was displayed in total impedance Bode plot (logjZj vs. log( f)) and phase angle Bode plot (u vs. log( f)) (Fig. 4). The respective total impedance plot (Fig. 4(a)) shows that the impedance initially decreased more in the low-frequency region than in the high-frequency region with increasing time of demineralization. The impedance finally decreased over the whole frequency region and the impedance spectra gradually approached that of bare electrode. Regarding the phase angle Bode plot, as shown in Fig. 4(b), the maximum phase angle at the high-frequency zone decreased gradually with time and increased at the low-frequency zone.
The impedance spectra were fitted to circuit parameters using the non-linear least square method in the program EQUIVCRT by Boukamp.28 Two equivalent circuits (Fig. 5(a) and (b)) were used to fit the EIS data. The quality of the fit to the equivalent circuit was judged by the x2-value, comparing experimental and simulated data. Fig. 4 shows the typical EIS experimental data (symbol) and the fitted data (curve) for dentine after different intervals of demineralization. The values of the fitted parameters are reported in Table 1. Herein, the equivalent circuit (a) in Fig. 5 is used to fit the EIS data for the specimens with either biofilm or smear layer, while the equivalent circuit (b) in Fig. 5 is for the specimens with smear layer and biofilm simultaneously. For non-homogeneous materials such as dentine, in order to compensate for its heterogeneity a constant-phase element (CPE) is usually used in place of capacitor in an equivalent
Fig. 5 – (a and b) Equivalent circuits for fitting EIS data, Re: resistance of electrolyte; Rs: resistance of smear layer; Qs: constant-phase element (CPE) of smear layer; Rf: resistance of biofilm; Qf: constant-phase element (CPE) of biofilm; Rd: resistance of dentine layer; Qd: CPE of dentine layer; Rct: charge transfer resistance associated with the penetration of the electrolyte into the specimens; Qct: CPE of the substrate/electrolyte interface; W: Warburg impedance.
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Table 1 – EIS fitting parameters of Fig. 4 (x2 < 9 T 10S3). Control
2-day
4-day
6-day
10-day
14-day
Re (V) Rf (V) Qf Y0 (V1 sn) n
19.70 –
17.46 –
17.85 –
18.60 330
16.89 353
17.78 571
– –
– –
– –
4.43 107 0.792
4.83 106 0.689
3.83 106 0.619
Rs (V) Qs Y0 (V1 sn) n
2673
1250
1046
338
2.60 107 0.830
4.40 107 0.811
2.21 107 0.582
1.05 108 0.423
– –
– –
88.40
55.79
18.35
12.55
10.21
9.32
5.07 106 0.212
1.10 105 0.284
3.98 106 0.329
2.46 105 0.286
3.18 106 0.334
2.38 106 0.380
Rd (kV) Qd Y0 (V1 sn) n
–
Rct (kV) Qct Y0 (V1 sn) n
34.6
14.33
4.57
2.92
2.67
3.56
9.68 108 0.818
1.87 107 0.773
9.85 106 0.653
1.55 107 0.831
2.63 107 0.689
3.34 107 0.732
W (V1
4.51 105
3.24 106
5.50 105
1.52 104
1.29 104
2.49 104
s0.5)
circuit where CPE is denoted with Q.29 The CPE is a function of the angular frequency, v, and whose phase is independent of the frequency. Its impedance components are as follows: ZCPE ¼
1 Y 0 ð jvÞn
Here, j is the imaginary number, j = (1)1/2, v (=2pf) is the angular frequency.30 The factor n, defined as the CPE power, is an adjustable parameter that lies between 0 and 1. For n = 1, the CPE describes an ideal capacitor with Y0 equal to the capacitance C, for n = 0, the CPE is an ideal resistor; when n = 0.5, the CPE represents a Warburg impedance, W, which is introduced to represent the diffusion process; for 0.5 < n < 1, the CPE describes a frequency dispersion of time constants due to local heterogeneity on the surface. For the Type B EIS system, the average values of Ra of specimens in the three treatment groups at different intervals of demineralization were recorded in Fig. 6. The Ra of the
specimens decreased with increasing time when the medium was enriched with sucrose and increased slightly when the medium did not contain sucrose.
3.2.
The typical cross-section and longitudinal SEM micrographs of dentine specimens which have undergone different periods of demineralization including the control are shown in Fig. 7. With increasing time of demineralization, the smear layer disappeared and the dentinal tubules were gradually exposed with their diameters increasing (Fig. 7(a)– (h)). After the specimen was sonicated, the transition state of the smear layer disappeared and the orifices of dentinal tubules were gradually exposed as can be observed in Fig. 7(d). At the same time, from a comparison of Fig. 7(a)– (c), (e) and (g), it can be seen that the density of biofilm increased with time. The bacterial cells penetrated into dentinal tubules after 14 days (Fig. 7(h) insert). After 14-day demineralization the smear layer and peritubular dentine not only completely disappeared comparing Fig. 7(g) and (h) with Fig. 7(i) and (j), both the network of collagen fibrils were also exposed on the surface of intertubular dentine (Fig. 7(g) insert), both of which caused the specimens to become more porous. The results of the EDX analysis summarized in Fig. 8 showed that the content of Ca and P decreased with increasing time.
3.3. Fig. 6 – Change in apparent resistance of the dentine specimens measured by the Type B EIS system. ^: specimens with smear layer treated with Streptococcus mutans (ATCC 25175) in medium enriched with sucrose; &: specimens without smear layer treated with Streptococcus mutans (ATCC 25175) in the medium without sucrose; ~: specimens without smear layer treated with Streptococcus mutans (ATCC 25175) in the medium without sucrose.
SEM and EDX
XRD
A series of typical XRD spectra of the specimens is shown in Fig. 9. The XRD analysis indicated that the main crystal composition on the surface of dentine specimens in the control group was hydroxyapatite (HAP). As the time of demineralization increased, the diffractions peaks at 25.788 and 338 broadened gradually. The I% of HAP in the control, 2and 4-day-demineralization group was 43%, 27%, and 18%, respectively. The specimens in the 10- and 14-day-deminer-
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journal of dentistry 38 (2010) 138–148
Fig. 7 – SEM micrographs of cross-section and longitudinal dentine specimens after different periods of demineralization: (a) 2-day; (b) 4-day; (c) and (d) 6-day; (e) and (f) 10-day; (g) and (h) 14-day; (i) and (j) control. The specimens in (d) and (f) underwent sonication treatment to remove the biofilm. The insert in (g) is a FSEM micrograph showing network of collagen
journal of dentistry 38 (2010) 138–148
Fig. 8 – Calcium and phosphorus content (wt%) of the surface of dentine specimens after different intervals of demineralization.
Fig. 9 – XRD pattern of the dentine specimens after various periods of demineralization: (a) control; (b) 2-day; (c) 4-day; (d) 6-day; (e) 10-day; (f) 14-day.
alized groups showed no significant diffraction peaks in their patterns, i.e., an ‘amorphous’ pattern, indicating the loss of crystallinity of dentine due to the dissolution of HAP crystal structure of by the acid ions produced by the bacterial mediated reaction.
3.4.
TMR
The result of TMR is presented in Fig. 10 showed that both the mineral loss and lesion depth increased with increasing demineralization time. Thus, the specimens gradually became porous from the surface to inner region.
4.
Discussion
In this study, two types of EIS system were used to characterize the bacterial-induced demineralization of dentine specimens. For the Type A EIS system, the impedance
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Fig. 10 – Change in mineral loss and lesion depth of the specimens measured by TMR with increasing time.
information could be obtained by fitting the EIS data with equivalent circuits and more information could be found when the EIS measurements are carried out over an appropriate frequency range. However, the quality of fabrication of working electrode, such as leakage and degree of contact between specimen and surface of electrode, may affect the EIS measurements. Although the smear layer and biofilm could be detected by fitting the EIS data, this system did not seem to differentiate between them. For the Type B EIS system, preparation of specimens is relatively simple and the specimen is located between two chambers without contacting the electrodes. The apparent resistance of the specimen can be measured directly from the change in resistance between two electrodes without the need to fit the EIS data with equivalent circuits. As shown in Table 1 and Fig. 6, both Rd and Ra decreased with increase in duration of demineralization. However, their absolute values were quite different, which could be attributed to the dentine section with the electrode in the Type A EIS system. Aziza et al.31 have reported similar findings by contacting specimens with electrodes. Nelly et al.32 have also obtained data similar to ours by using the Type B EIS system. Although different EIS systems were used in their study, their results indicated that the smear layer could increase the resistance of dentine specimen. In this study, the impact of biofilm on the resistance of dentine was investigated by using both types of EIS system. It has been reported that biofilm on metal surface could be fitted as a RQ element in the equivalent circuit.23 Table 1 shows that besides the dentine layer, other RQ elements could be attributed to the smear layer and biofilm due to their electrical properties, while the result in Fig. 6 directly indicated that biofilm on the surface of dentine and bacterial cells penetrating into dentinal tubules (Fig. 7) increased the apparent resistance of dentine regardless of the presence of the smear layer. Therefore, biofilm may retard the diffusion of ions and thus cause an increase in Ra. This physical barrier-effect of biofilm is similar to that of smear layer; however this effect did not significantly alter the decreasing trend of resistance of dentine when the medium was enriched with sucrose, which metabolized to
fibrils (arrow) on the surface of intertubular dentine. The insert in (h) is a magnified image of the left of this SEM micrograph showing the bacterial cells (arrow) penetrating into dentinal tubules. ODT: orifice of dentinal tubule DT: dentinal tubule; ID: intertubular dentine; PD: peritubular dentine.
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organic acid that demineralized dentine and render dentine porous. Our previous study18 focused on the choice of equivalent circuits for fitting EIS data and the EIS data was only presented in a Nyquist plot. Nevertheless, these spectra provide information on the impedance of the specimen only. In this study, the EIS data was displayed in a Bode plot (Fig. 4), which would give us other information such as (1) frequency of a point and (2) ability to visualize large ranges of gain value. It was found that the Bode plot of dentine demineralization is similar to that of polymer degradation monitored by EIS technique.33 The reason might be that their change in compositions would result in an increase porosity, which would cause deeper penetration of the electrolyte into the specimen. The impedance spectra of the polymer showed capacitive behavior at a high-frequency region (i.e., the phase angle was close to 908), indicating good insulating properties.34 Thus, as shown in Fig. 4(b), the decline of the phase angle in a high-frequency indicated that the dentine gradually showed less capacitive behavior and became more and more conductive. Therefore, it is reasonable to conclude that the electrical behavior of the working electrode with dentine section would approach that of the bare electrode, as the dentine underwent demineralization. The findings obtained from the Type A and B systems support our hypothesis that the bacteria-induced demineralization of dentine in situ and biofilm formation could be monitored by using EIS technology. Although EIS can work as a simple and sensitive method to monitor the process of demineralization, it can only indicate porosity without reflecting other ultrastructural changes in the dentine. Therefore, it is necessary to employ other techniques to characterize demineralization of dentine to affirm the EIS results. In this study, SEM, FSEM, EDX, XRD and TMR were used to characterize specimens. The SEM images (Fig. 7) show the change in ultrastructure of dentine and formation of biofilm prior to the process of demineralization, and also confirms that the specimens became more porous with demineralization. Subsequently, electrolyte could penetrate into the dentinal tubules and intertubular dentine more readily, inducing a decrease in both Rd and Ra values. Furthermore, since the network of collagen fibrils lacked minerals, the intertubular dentine on the surface of the specimen tended to shrink after dehydration as shown in Fig. 7(h). EDX and XRD are powerful methods to detect elements and identify crystal structure. In this study, they were used to characterize the changes in chemical composition on the surface of specimens. By combining the EIS and EDX results, it could be concluded that the loss of Ca and P ions from the HAP lattice on the surface of dentine resulted in the destruction of the crystal structure of HAP which is then converted into an amorphous state with increasing extent of demineralization. This is the primary reason for the increase in porosity of dentine undergoing demineralization. It is worth noting that the XRD can reflect the crystallinity of HAP, which is significant in demineralization and remineralization study.35 Since the EDX and XRD techniques provide analytical information on the surface of specimens only36,37 they could
Fig. 11 – (a and b) Analysis of correlation between EIS and TMR results. (a) &: Rd vs. LD; ^: Rd vs. ML; (b) ~: Ra vs. LD; &: Ra vs. ML; Rd: resistance of dentine layer obtained from the Type A EIS system; Ra: apparent resistance of dentine specimen obtained from the Type B EIS system; LD: lesion depth; ML: mineral loss.
not characterize the region inside dentine. TMR, an accepted method to characterize the degree of demineralization, was used to complement the EDX and XRD method. The mineral loss and lesion depth of specimens could be related to the porosity of specimens of dental tissue.38 In this study the correlation between EIS and TMR results was investigated. As shown in Fig. 11, the results from the Type B EIS system are more associated with mineral loss and lesion depth than that from the Type A EIS system. Although the present EIS technology cannot completely replace TMR for characterization of demineralization, it could be a convenient and reliable method to monitor the process of demineralization or even remineralization of dentine in acid model and bacterial model in vitro. The particular advantage of EIS is that since its measurement is based on the movement of ions, it could directly estimate the effect of treatment for tooth hypersensitivity and the leakage at the restorative material and dental tissue interface. The fabrication of more reliable and standard working electrodes and further work to relate EIS result and TMR result will be focused in the future.
5.
Conclusion
This study focused on monitoring the process of demineralization of dentine induced by bacteria using EIS technique together with other morphology and composition characterization methods. The EIS findings indicated that bacterial biofilm and bacterial-induced dentine demineralization can be monitored by the Type A and Type B EIS systems. Although bacterial biofilm showed changing electrochemical behavior,
journal of dentistry 38 (2010) 138–148
it did not affect the decrease in impedance measured by EIS. The results from SEM, EDX, XRD and TMR analyses demonstrate that the surface and bulk of dentine gradually became more porous during demineralization and thus causing the values of bulk resistance of dentine (Rd) and apparent resistance of dentine (Ra) to decrease.
Acknowledgement The authors wish to acknowledge funding from the Academic Research Funding (NUS) R-224-000-029-112.
references
1. Bard AJ, Faulkner LR. Electrochemical methods: fundamental and applications. New York, USA: Wiley; 1980. 2. Levinkind M, Vandernoot TJ, Elliott JC. Electrochemical impedance characterization of human and bovine enamel. Journal of Dental Research 1990;69:1806–11. 3. Levinkind M, Vandernoot TJ, Elliott JC. Evaluation of smear layers on serial sections of human dentin by means of electrochemical impedance measurements. Journal of Dental Research 1992;71:426–33. 4. Eldarrat AH, High AS, Kale GM. In vitro analysis of smear layer on human dentine using ac-impedance spectroscopy. Journal of Dentistry 2004;32:547–54. 5. Huysmans MC, Longbottom C, Pitts NB, Los P, Bruce PG. Impedance spectroscopy of teeth with and without approximal caries lesions—an in vitro study. Journal of Dental Research 1996;75:1871–8. 6. Longbottom C, Huysmans MC, Pitts NB, Los P, Bruce PG. Detection of dental decay and its extent using a.c. impedance spectroscopy. Nature Medicine 1996;2:235–7. 7. Liao YM, Feng ZD, Chen ZL. In situ tracing the process of human enamel demineralization by electrochemical impedance sepectroscopy (EIS). Journal of Dentistry 2007;35:425–30. 8. Behari J, Singh S. Bioelectric characteristics of unstressed in vivo bone. Medical and Biological Engineering and Computing 1981;19:49–54. 9. Ollmar S, Nyren M, Nicander I, Emtestam L. Electrical impedance compared with other non-invasive bioengineering techniques and visual scoring for detection of irritation in human skin. British Journal of Dermatology 1994;130:29–36. 10. Ackmann JJ, Seitz MA, Dawson CA, Hause LL. Specific impedance of canine blood. Annals of Biomedical Engineering 1996;24:58–66. 11. Selwitz RH, Ismail AI, Pitts NB. Dental caries. The Lancet 2007;369:51–9. 12. Pretty IA. Caries detection and diagnosis: Novel technologies. Journal of Dentistry 2006;34:727–39. 13. Chan SH, Su YC. A self-adaptive fluidic probe for electrical caries detection. Biomedical Micordevices 2008;10:447–57. 14. Licˇina V, Gajovic´ A, Mogusˇ-Milankovic´ A, Djerdj I, Tomasˇic´ N, Su D. Correlation between the microstructure and the electrical properties of ZrTiO4. Ceramics 2007;91:178–86. 15. Gotliv BA, Veis A. Peritubular dentin, a vertebrate apatitic mineralized tissue without collagen: role of a phospholipid– proteolipid complex. Calcified Tissue International 2007;81:191–205.
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16. Banarji Behera P, Nayak RNP, Choudhary. Structural and impedance properties of KBa2V5O15 ceramics. Materials Research Bulletin 2008;43:401–10. 17. Amirudin A, Thierry D. Application of electrochemical impedance spectroscopy to study the degradation of polymer-coated metals. Progress in Organic Coatings 1995;26:1–28. 18. Zhang X, Koon GN, Anil K. Monitoring aciddemineralization of human dentine by electrochemical impedance spectroscopy (EIS). Journal of Dentistry 2008;36:1005–12. 19. Yang L, Ruan C, Li Y. Detection of viable Salmonella typhimurium by impedance measurement of electrode capacitance and medium resistance. Biosensors and Bioelectronics 2003;19:495–502. 20. Yang L, Li Y, Griffis CL, Johnson MG. Interdigitated microelectrode (IME) impedance sensor for the detection of viable Salmonella typhimurium. Biosensors and Bioelectronics 2004;19:1139–47. 21. Yang L, Li Y. Detection of viable Salmonella using microelectrode-based capacitance measurement coupled with immunomagnetic separation. Journal of Microbiological Methods 2006;64:9–16. 22. Yang L, Li Y. AFM and impedance spectroscopy characterization of the immobilization of antibodies on indium–tin oxide electrode through self-assembled monolayer of epoxysilane and their capture of Escherichia coli O157:H7. Biosensors and Bioelectronics 2005;20:1407–16. 23. Sheng XX, Ting YP, Simo OP. The influence of sulphatereducing bacteria biofilm on the corrosion of stainless steel AISI 316. Corrosion Science 2007;49:2159–76. 24. Nelly PP, Francois W, Bernard P, Pierre C. Evaluation of microleakage of composite resin restorations by an electrochemical technique: the impedance methodology. Dental Materials 2004;20:425–34. 25. Boeckh C, Schumacher E, Podbielski A, Haller B. Antibacterial activity of restorative dental biomaterials in vitro. Caries Research 2002;36:101–7. 26. Edmunds DH, Whittaker DK, Green RM. Suitability of human, bovine, equine and ovine tooth enamel for studies of artificial bacterial carious lesions. Caries Research 1988;22:327–36. 27. George S, Kishen A, Song KP. The role of environmental changes on monospecies biofilm formation on root Canal Wall by Enterococcus faecalis. Journal of Endodontics 2005;31:867–72. 28. Boukamp BA. A microcomputer based system for frequency dependent impedance/admittance measurements. Solid State Ionics 1986;11:339–46. 29. Macdonald JR, Franceschetti DR. In: MacDonald JR, editor. Impedance spectroscopy: emphasizing solid materials and systems. New York: Wiley & Sons Inc.; 1987. p. 84. 30. Tan YS, Strinivasan SO, Pehkonen. Simon YM. Effects of ring substituents on the protective properties of selfassembled benzenethiols on copper. Corrosion Science 2006;48:840–62. 31. Eldarrat AH, Wood DJ, Kale GM, High AS. Age-related changes in ac-impedance spectroscopy studies of normal human dentine. Journal of Material Science Material Medicine 2007;18:1203–10. 32. Nelly PP, Francois W, Pierre C. Effect of conditioners on dentin permeability using an impedance method. Journal of Dentistry 2002;30:251–7. 33. Cesar FS, Calum JM, Keith R. Electrochemical impedance spectroscopy studies of polymer degradation: application to biosensor development. Trends in Analytical Chemistry 2005;24:37–48. 34. Mcneil CJ, Athey D, Ball M, Wah OH, Steffi K, Armstrong RD, et al. Electrochemical sensors based on impedance
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measurement of enzymel catalyzed polymer dissolution: theory and applications. Analytical Chemistry 1995;67:3928–35. 35. Low IM. Depth-profiling of crystal structure, texture, and microhardness in a functionally graded tooth enamel. Journal of the American Ceramic Society 2004;87:2125–31. 36. Wong HS, Buenfeld NR. Monte Carlo simulation of electron– solid interactions in cement-based materials. Cement and Concrete Research 2006;36:1076–82.
37. Gerhard T, Faschinger W, Landwehr G. Depth-sensitive Xray diffraction using extremely asymmetrical reflections by variation of the wavelength. Semiconductor Science and Technology 1998;13:583–9. 38. Jing W, Yoshiko S, Daisuke I, Chris L, Hideo M. Relationship between electrical resistance measurements and microradiographic variables during remineralization of softened enamel lesions. Caries Research 2005;39:60–4.
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Monitoring bacterial-demineralization of human dentine by electrochemical impedance spectroscopy Zhang Xu a, Koon Gee Neoh b, Bennett Amaechi c, Anil Kishen d,* a
Department of Restorative Dentistry, Faculty of Dentistry, National University of Singapore, 5 Lower Kent Ridge Road, 119074 Singapore, Singapore b Department of Chemical & Biomolecular Engineering, National University of Singapore, Kent Ridge 119260, Singapore c Department of Community Dentistry, University of Texas Health Science Center at San Antonio, 7703 Floyd Curl Drive, San Antonio, TX 78229, USA d Discipline of Endodontics, Faculty of Dentistry, University of Toronto, 124 Edward Street, Toronto, ON, Canada
article info
abstract
Article history:
Objective: The purpose of this study was two-fold: (1) to monitor bacterial biofilm formation
Received 28 May 2009
and bacteria-induced demineralization of dentine in situ by using electrochemical impedance
Received in revised form
spectrum (EIS); (2) to examine the relationship between EIS findings and changes in the
21 September 2009
chemical composition and ultrastructure of dentine during bacteria-induced demineralization.
Accepted 27 September 2009
Methods: In this study, dentine demineralization was induced by Streptococcus mutans (ATCC 25175) in the presence of sucrose in culture medium and was monitored using two EIS measurement systems (Type A with a working electrode and Type B without a working
Keywords:
electrode). Scanning electron microscopy (SEM), field emission scanning electron micro-
Electrochemical impedance
scopy (FESEM), energy dispersive X-ray (EDX) and X-ray diffraction (XRD) were employed to
spectroscopy (EIS)
examine the morphology, element contents and crystallinity of hydroxyapatite (HAP) on the
Bacterial-induced demineralization
dentine surface. Transverse microradiography (TMR) was used to characterize the lesion
Dentine
depth and degree of mineral loss during demineralization. Results: The resistance of the bulk dentine (Rd) and the apparent resistance of dentine (Ra) measured from the Type A and Type B EIS systems, respectively, decreased gradually with demineralization. The resistance of the biofilm formed on dentine surface was determined by fitting the EIS data with equivalent circuits. The presence of biofilm slightly increased Ra of dentine before demineralization. However, the electrochemical behavior of biofilm did not affect the decreasing impedance of dentine with demineralization. The SEM, EDX, XRD and TMR results demonstrated that the surface and bulk dentine gradually became more porous due to the loss of minerals during demineralization, which in turn resulted in the decrease in Rd and Ra values obtained from EIS systems. Conclusions: This investigation highlighted EIS as a potential technique to monitor biofilm formation and bacterial-induced demineralization in situ. # 2009 Elsevier Ltd. All rights reserved.
1.
Introduction
Electrical impedance spectroscopy (EIS), a non-destructive and sensitive method, is widely used to characterize bulk and
interfacial electrical properties of solid or liquid materials.1 EIS has been used to measure electrical resistance of biological tissues such as dental tissues,2–7 bone,8 skin9 and blood cells.10 In clinical dentistry, visual inspection and bitewing radio-
* Corresponding author. E-mail address: [email protected] (A. Kishen). 0300-5712/$ – see front matter # 2009 Elsevier Ltd. All rights reserved. doi:10.1016/j.jdent.2009.09.013
journal of dentistry 38 (2010) 138–148
graphy are used to detect early caries, but they are based on subjective evaluation, and only a limited portion of early caries can be detected.11 EIS technology seems to have the potential to diagnose early caries reliably and accurately.12 Some studies have been conducted to detect early caries based on the porosity of enamel as indicated by EIS data5–7; however, EIS application has seldom focused on monitoring the demineralization of dentine. It is significant to characterize dentinal demineralization using EIS, because dentine demineralizes more rapidly than enamel and thus in many cases show severe demineralization underlying enamel, which is difficult to detect using conventional methods in clinical practice.13 The application of EIS technology to dental tissue depends on the penetration of electrolyte into specimens. Subsequently, the tissue will be conductive, allowing EIS measurements. Since conductive properties are affected by the microstructure of the tissue, impedance spectra usually contain features that can be directly related to the changes in the microstructure of the tissue.14 Smear layers on dentine have been detected according to this theory using EIS technology.4 In fact, these changes in the microstructure are usually associated with variation in the chemical constituents. Therefore, for studying dentine demineralization it is necessary to characterize the change in ultrastructure and chemical components by using SEM, FSEM, EDX, XRD and TMR. This strategy can provide comprehensive and integral insight into microstructural changes of dental tissue compared to the application of a single technique. Dentine has a regularly tubular structure, which is composed of intertubular and peritubular dentine. Intertubular dentine consists of a fibrous network of collagen on which hydroxyapatite (HAP) crystals are deposited, while peritubular dentine is a more highly mineralized tissue without collagen fibrils.15 EIS has been used to characterize porous ceramics, the structure of which is similar to that of dentine.16 It has also been used to monitor the degradation process of a polymer that is similar to the demineralization process of dentine.17 Consequently, based on the regular porous structure of dentine, it is possible to use EIS in situ to study the changes in the chemical components and ultrastructure of dentine caused by demineralization under simulated carious conditions. In our previous work,18 acid-induced demineralization of dentine was monitored in situ using EIS, and the EIS findings correlated well with the changes in the chemical composition and ultrastructure of dentine. It is well known that bacterial biofilm, or dental plaque, is involved in the demineralization of dental hard tissues. Thus, in order to simulate a more clinically realistic model, bacterialinduced demineralization of dentine will be monitored by EIS technique in this investigation. In contrast to acid-induced demineralization, biofilm formed on dentine may result in more complex EIS readings. Recently, EIS technique has been applied to characterize and detect bacterial biofilm.19–22 Equivalent circuits were used to characterize biofilm and its resistance was obtained by fitting EIS data.23 The hypothesis of this study was that bacteria-induced demineralization of dentine in situ and biofilm formation can induce chemical and ultrastructural changes in the dentine, which in turn could be monitored by using EIS technology. In this study, besides the one-chamber and three-electrode configuration (Type A),18 a
139
two-chamber and two-electrode configuration (Type B) was also used to measure impedance of the dentine sections with or without bacterial biofilm. The Type B EIS system has been employed previously to test the micro-leakage between dental tissues and restorative materials.24 The apparent resistance of the specimen can be directly obtained by measuring the resistance between two electrodes without fitting the EIS data with equivalent circuits. Combination of these two types of EIS measurement system could provide more complete understanding of the change in the EIS reading of dentine during bacterial-induced demineralization, and the influence of bacterial biofilm on the EIS results.
2.
Materials and methods
2.1.
Preparation of specimens and culture medium
The collection and use of extracted human teeth for all the experiments conducted in this study has been approved by the institutional review board of the National University of Singapore. Extracted non-carious human third molars without visible evidence of cracks, and maintained in phosphate buffered saline (PBS) were used in this study. The roots and pulps of these teeth were removed, and then serially sectioned perpendicular to their long axes by means of Microslice 2 (Metals Research Limited, Cambridge, England) with a rotating, internal-cutting, copper disc impregnated with diamonds (Buehler1, diamond wafering blade, 7.6 cm diameter 0.15 mm width, USA). Subsequently, the sections were ground into 5 mm 5mm 1 mm square samples using Phoenix Beta Grinder/Polisher (Wirtz, Buehler, USA) with special silicon carbide grinding papers (Carbimet1, P1200, Buehler, USA). These dentine sections were obtained from the same position along the long axes of different molars, which enables us to obtain dentine specimen with approximately similar diameter and number of dentinal tubules for the same batch of measurement. The smear layers formed on these dentine sections were not removed. Six dentine sections were used to fabricate working electrodes for the Type A EIS measurement and nine were randomly and averagely divided into three groups for the Type B EIS measurement. In SEM, EDX, and XRD characterization, fifteen dentine sections were randomly and averagely divided into five groups corresponding to different periods (2, 4, 6, 10 and 14 days) and they were sealed with varnish on all surfaces except the surface perpendicular to dentinal tubules exposed to the bacterial culture. The same grouping method and treatment was applied on the specimens in TMR characterization, but the roots of these specimens remained as holders for the convenience of further cutting. The culture medium is mainly composed of sterile brainheart infusion broth (BHI) (Merck KGaA, Germany, Lot 811893) with or without 5% sucrose (w/v). After 18 h growth of pure cultures with 1–2 108 CFU/ml determined by measuring the absorption intensity at 660 nm using a UV–vis spectrophotometer (UV-1700, SHIMADZU, Japan), 100 ml of a cell suspension of Streptococcus mutans (ATCC 25175) was inoculated in 30 ml of culture medium for each dentine section.25 The culture medium was replenished every 48 h in order to remove
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Fig. 1 – (A) Cell for the Type A EIS system; (B) bacterial culture set; 1: counter electrode; 2: reference electrode; 3: working electrode; 4: specimen of dentine slice; 5: cell chamber; 6: 50 ml polypropylene conical centrifuge tube (Falcon). Schematic diagram of the Type A EIS measurement system.
dead cells and allow better biofilm growth.26 Contamination was verified in the medium each day by means of Gram staining.
2.2.
EIS system and measurement
Two types of EIS measurement system were used in this study. Type A contains a one-chamber cell and three electrodes: a reference Ag/AgCl electrode (Metrohm, Switzerland), a platinum counter electrode (Metrohm, Switzerland) and a working electrode as shown in Fig. 1. The fabrication of working electrodes with dentine sections was the same as that of our previous study.18 The cell and the electrodes were sterilized using autoclaving and UV radiation for 3 h before use, respectively. During demineralization process, the working electrodes with dentine samples were immersed in a bacterial culture medium and placed in a shaking incubator (QT
Instruments Pte. Ltd., Singapore) at 37 8C and the measurements were conducted with EIS at different intervals (Fig. 1). In total, six working electrodes with similar baseline data (i.e., initial impedance spectrum in Nyquist plots) were chosen for treatment with the bacterial medium and EIS measurement. The Type B system was specially designed and fabricated according to the principle in Nelly’s study24 as shown in Fig. 2. In this configuration, a cell and a 50 ml centrifuge tube (Falcon, BD Biosciences, San Jose, CA, USA) constitute the two chambers; two platinum electrodes (Metrohm, Switzerland) were placed in each one of them, respectively. First, the dentine section was fixed on the 3 mm diameter hole on the cap of the tube by spreading wax around it. Then, the leakage of the samples was checked by measuring the resistance between two chambers. Finally, the dentine samples along with plastic support were sterilized using UV radiation for 3 h. During demineralization process, the bacterial culture med-
Fig. 2 – (A) Cell for the Type B EIS system; (B) bacterial culture set; 1: platinum electrode; 2: plastic beaker; 3: 50 ml polypropylene conical centrifuge tube (Falcon); 4: cap with a dentine section; 5: the cell chamber. Schematic diagram of the Type B EIS measurement system.
journal of dentistry 38 (2010) 138–148
ium was taken in a 50 ml centrifuge tube, which was kept in a glass beaker enveloped by parafilm (Pechiney Plastic Packaging Company, Chicago, IL, USA) to prevent contamination. The culture was also placed in the shaking incubator at 37 8C. As shown in Fig. 2, the cap were first taken off and then placed on the 50 ml centrifuge tube in the cell for the EIS measurement at different intervals. All the operations and measurements were performed within a work station (NuAire, Inc., Plymouth, MN, USA). In the Type B EIS system, dentine sections with or without smear layers were used. The smear layers on both sides of dentine section were removed. In order to characterize the effect of biofilm on impedance of dentine, the dentine sections with smear layers were treated with BHI medium with or without sucrose (three sections in each group); the three dentine sections without smear layers were treated with the medium without sucrose. The smear layer of dentine section was removed by etching the specimen surface with phosphoric acid (35%, w/v) for 15 s.4 Each treatment group comprised three dentine sections. The mechanism of measurement using the Type B EIS system was introduced by Nelly et al.24 Herein, the shape of the impedance spectra is ‘‘V’’ shape in Nyquist plane (i.e., complex plane) as shown in Fig. 3. At the frequency where the value of G is the lowest (around 1000 Hz), the contribution of the impedance of the electrodes is negligible and the real part of the impedance R corresponds to the sum of the resistance of electrolyte (Re) and the apparent resistance of specimen (Ra). Re was obtained by measuring the impedance at the same frequency, with the same cap without the dentin section (Rd = 0). Therefore, Ra of specimens undergoing different demineralization periods could be monitored and calculated using the Type B EIS system. In both the Type A and Type B EIS systems, sterilized 0.01 M PBS was used as electrolyte in the chamber. The EIS readings would be recorded at different periods (2, 4, 6, 10 and 14 days). The EIS analysis was carried out using an Autolab PGSTAT 100 (Metrohm, Switzerland), while FRA4.9 software (Metrohm, Switzerland) was used to control and record measurements. The frequency range was 1 mHz–100 kHz which will take about 20 min and potential balance before EIS measurement will take 5 min. A Faraday cage that isolated the EIS systems
from noise was used to improve the reliability of results from a high impedance sample at typical AC amplitudes. It should be noted that leads themselves can act as a capacitor, the leads of the reference and counter electrodes are shorted and placed outside the Faraday cage. The leads of working electrode is shorted and placed inside the Faraday cage. These controls can minimize the artifactual drift in EIS measurement. The amplitude of the sinusoidal voltage signal was 10 mV. The FRA software was also used to analyze the experimental data.
2.3.
SEM and EDX characterization
In each group (each treatment period) three samples were first characterized by XRD and then by SEM and EDX, because of the need to coat the specimens with platinum for the latter. After different periods of immersion in the bacterial culture medium, the specimens were removed and then rinsed three times with deionized water (3 min each). Next they were immersed in a fixative solution containing 4% gluteraldehyde in sodium cacodylate buffer at 4 8C for 3 h.27 Finally, the specimens were dehydrated using ethanol (at gradient concentration of 70%, 80%, 90% and 100% for 20 min), and stored in a desiccator. Fracture surfaces were prepared using two pliers after the dehydration procedure. The specimens were sputter-coated with platinum, and then visualized and analyzed with SEM and EDX (JSM-5600LV models, JEOL Company, Tokyo, Japan) with a beam voltage at 15 kV. A quantitative elemental analysis of Ca and P was carried out using EDX. Three randomly selected areas were analyzed at a magnification of 1500. The average concentration of detected elements was recorded.
2.4.
XRD characterization
The structure and crystallinity of different dentine samples were investigated by a XRD-6000 X-ray diffractometer (SHIMADZU, Tokyo, Japan) with Cu Ka radiation. The data were collected in the 2u range of 20–408 at a scan rate of 28 per min. The value of the percentage of intensity (I%) which can characterize the crystallinity was calculated using the software Jade 5 (MDI, Materials Data. Inc., USA), and represents the ratio of the area of any other peak to that of the peak with a maximum area. The amount of HAP crystal on the surface of the specimens were evaluated from I% of HAP at 2u = 25.78 position that was assigned to the (0 0 2) plane.7
2.5.
Fig. 3 – Typical impedance curves of a specimen treated with Streptococcus mutans (ATCC 25175) in the medium enriched with sucrose. Re: resistance of electrolyte; Ra1: apparent resistance of specimen before treatment; Ra2: apparent resistance of specimen after treatment.
141
TMR characterization
After different treatment intervals of demineralization, specimens were rinsed with deionized water for 5 min and then exposed to UV radiation for 15 min. Next, the specimens were sectioned perpendicularly to the lesion surface using a watercooled, Silverstone–Taylor hard tissue microtome to obtain the 100 mm-thick dentine sections for TMR characterization. The sections were placed in a specially fabricated radiographic plate-holding cassette, incorporating an aluminum step wedge (10 steps of 24.5 mm thickness). The cassette was loaded with type lA high resolution glass X-ray plates (Microchrome Technology, CA, USA) and placed into a Phillips X-ray generator system set up for this purpose. This apparatus
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Fig. 4 – (a and b) Typical experimental EIS data (symbol) and their fitted data (solid line) for dentine after different intervals of demineralization in total impedance Bode plot (a) and phase angle Bode plot (b). &: control; &: 2-day; ^: 4-day; ^: 6-day; ~: 10-day; ~: 14-day; *: bare electrode.
is equipped with a copper target and nickel filter, producing monochromatic radiation of wavelength appropriate for ˚ ). The plates were exposed for 10 min hydroxyapatite (184 A at an anode voltage of 20 kV and a tube current of 10 mA, and then processed. Processing consisted of a 5 min development in Kodak HR developer and 15 min fixation in Kodak Rapidfixer before a final 30 min wash period. After drying, the microradiographs were subjected to visualization and image analysis using a computer program, TMR2006 version 3.0.0.6 (Inspektor Research, Amsterdam, Netherlands). The hardware was a Leica DMR optical microscope linked via a Sony model XC-75CE CCTV camera to a 90 MHz DellTM Pentium Personal Computer. The enhanced image of the microradiographs were analyzed under standard conditions of light intensity and magnification and processed, along with data from the image of the step wedge, by the TMR program. By this method, the parameters of integrated mineral loss, Dz (vol% mm) and lesion depth, LD (mm) were quantified for each demineralized (carious) lesion.
3.
Results
3.1.
EIS spectra
Impedance spectra are generally displayed either in Nyquist (i.e., complex plane) or in Bode plot. The impedance spectra displayed in Bode plot can give more information including frequency and phase angle than only in Nyquist plot. In this study, therefore, the representative EIS data obtained through the Type A system was displayed in total impedance Bode plot (logjZj vs. log( f)) and phase angle Bode plot (u vs. log( f)) (Fig. 4). The respective total impedance plot (Fig. 4(a)) shows that the impedance initially decreased more in the low-frequency region than in the high-frequency region with increasing time of demineralization. The impedance finally decreased over the whole frequency region and the impedance spectra gradually approached that of bare electrode. Regarding the phase angle Bode plot, as shown in Fig. 4(b), the maximum phase angle at the high-frequency zone decreased gradually with time and increased at the low-frequency zone.
The impedance spectra were fitted to circuit parameters using the non-linear least square method in the program EQUIVCRT by Boukamp.28 Two equivalent circuits (Fig. 5(a) and (b)) were used to fit the EIS data. The quality of the fit to the equivalent circuit was judged by the x2-value, comparing experimental and simulated data. Fig. 4 shows the typical EIS experimental data (symbol) and the fitted data (curve) for dentine after different intervals of demineralization. The values of the fitted parameters are reported in Table 1. Herein, the equivalent circuit (a) in Fig. 5 is used to fit the EIS data for the specimens with either biofilm or smear layer, while the equivalent circuit (b) in Fig. 5 is for the specimens with smear layer and biofilm simultaneously. For non-homogeneous materials such as dentine, in order to compensate for its heterogeneity a constant-phase element (CPE) is usually used in place of capacitor in an equivalent
Fig. 5 – (a and b) Equivalent circuits for fitting EIS data, Re: resistance of electrolyte; Rs: resistance of smear layer; Qs: constant-phase element (CPE) of smear layer; Rf: resistance of biofilm; Qf: constant-phase element (CPE) of biofilm; Rd: resistance of dentine layer; Qd: CPE of dentine layer; Rct: charge transfer resistance associated with the penetration of the electrolyte into the specimens; Qct: CPE of the substrate/electrolyte interface; W: Warburg impedance.
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Table 1 – EIS fitting parameters of Fig. 4 (x2 < 9 T 10S3). Control
2-day
4-day
6-day
10-day
14-day
Re (V) Rf (V) Qf Y0 (V1 sn) n
19.70 –
17.46 –
17.85 –
18.60 330
16.89 353
17.78 571
– –
– –
– –
4.43 107 0.792
4.83 106 0.689
3.83 106 0.619
Rs (V) Qs Y0 (V1 sn) n
2673
1250
1046
338
2.60 107 0.830
4.40 107 0.811
2.21 107 0.582
1.05 108 0.423
– –
– –
88.40
55.79
18.35
12.55
10.21
9.32
5.07 106 0.212
1.10 105 0.284
3.98 106 0.329
2.46 105 0.286
3.18 106 0.334
2.38 106 0.380
Rd (kV) Qd Y0 (V1 sn) n
–
Rct (kV) Qct Y0 (V1 sn) n
34.6
14.33
4.57
2.92
2.67
3.56
9.68 108 0.818
1.87 107 0.773
9.85 106 0.653
1.55 107 0.831
2.63 107 0.689
3.34 107 0.732
W (V1
4.51 105
3.24 106
5.50 105
1.52 104
1.29 104
2.49 104
s0.5)
circuit where CPE is denoted with Q.29 The CPE is a function of the angular frequency, v, and whose phase is independent of the frequency. Its impedance components are as follows: ZCPE ¼
1 Y 0 ð jvÞn
Here, j is the imaginary number, j = (1)1/2, v (=2pf) is the angular frequency.30 The factor n, defined as the CPE power, is an adjustable parameter that lies between 0 and 1. For n = 1, the CPE describes an ideal capacitor with Y0 equal to the capacitance C, for n = 0, the CPE is an ideal resistor; when n = 0.5, the CPE represents a Warburg impedance, W, which is introduced to represent the diffusion process; for 0.5 < n < 1, the CPE describes a frequency dispersion of time constants due to local heterogeneity on the surface. For the Type B EIS system, the average values of Ra of specimens in the three treatment groups at different intervals of demineralization were recorded in Fig. 6. The Ra of the
specimens decreased with increasing time when the medium was enriched with sucrose and increased slightly when the medium did not contain sucrose.
3.2.
The typical cross-section and longitudinal SEM micrographs of dentine specimens which have undergone different periods of demineralization including the control are shown in Fig. 7. With increasing time of demineralization, the smear layer disappeared and the dentinal tubules were gradually exposed with their diameters increasing (Fig. 7(a)– (h)). After the specimen was sonicated, the transition state of the smear layer disappeared and the orifices of dentinal tubules were gradually exposed as can be observed in Fig. 7(d). At the same time, from a comparison of Fig. 7(a)– (c), (e) and (g), it can be seen that the density of biofilm increased with time. The bacterial cells penetrated into dentinal tubules after 14 days (Fig. 7(h) insert). After 14-day demineralization the smear layer and peritubular dentine not only completely disappeared comparing Fig. 7(g) and (h) with Fig. 7(i) and (j), both the network of collagen fibrils were also exposed on the surface of intertubular dentine (Fig. 7(g) insert), both of which caused the specimens to become more porous. The results of the EDX analysis summarized in Fig. 8 showed that the content of Ca and P decreased with increasing time.
3.3. Fig. 6 – Change in apparent resistance of the dentine specimens measured by the Type B EIS system. ^: specimens with smear layer treated with Streptococcus mutans (ATCC 25175) in medium enriched with sucrose; &: specimens without smear layer treated with Streptococcus mutans (ATCC 25175) in the medium without sucrose; ~: specimens without smear layer treated with Streptococcus mutans (ATCC 25175) in the medium without sucrose.
SEM and EDX
XRD
A series of typical XRD spectra of the specimens is shown in Fig. 9. The XRD analysis indicated that the main crystal composition on the surface of dentine specimens in the control group was hydroxyapatite (HAP). As the time of demineralization increased, the diffractions peaks at 25.788 and 338 broadened gradually. The I% of HAP in the control, 2and 4-day-demineralization group was 43%, 27%, and 18%, respectively. The specimens in the 10- and 14-day-deminer-
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Fig. 7 – SEM micrographs of cross-section and longitudinal dentine specimens after different periods of demineralization: (a) 2-day; (b) 4-day; (c) and (d) 6-day; (e) and (f) 10-day; (g) and (h) 14-day; (i) and (j) control. The specimens in (d) and (f) underwent sonication treatment to remove the biofilm. The insert in (g) is a FSEM micrograph showing network of collagen
journal of dentistry 38 (2010) 138–148
Fig. 8 – Calcium and phosphorus content (wt%) of the surface of dentine specimens after different intervals of demineralization.
Fig. 9 – XRD pattern of the dentine specimens after various periods of demineralization: (a) control; (b) 2-day; (c) 4-day; (d) 6-day; (e) 10-day; (f) 14-day.
alized groups showed no significant diffraction peaks in their patterns, i.e., an ‘amorphous’ pattern, indicating the loss of crystallinity of dentine due to the dissolution of HAP crystal structure of by the acid ions produced by the bacterial mediated reaction.
3.4.
TMR
The result of TMR is presented in Fig. 10 showed that both the mineral loss and lesion depth increased with increasing demineralization time. Thus, the specimens gradually became porous from the surface to inner region.
4.
Discussion
In this study, two types of EIS system were used to characterize the bacterial-induced demineralization of dentine specimens. For the Type A EIS system, the impedance
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Fig. 10 – Change in mineral loss and lesion depth of the specimens measured by TMR with increasing time.
information could be obtained by fitting the EIS data with equivalent circuits and more information could be found when the EIS measurements are carried out over an appropriate frequency range. However, the quality of fabrication of working electrode, such as leakage and degree of contact between specimen and surface of electrode, may affect the EIS measurements. Although the smear layer and biofilm could be detected by fitting the EIS data, this system did not seem to differentiate between them. For the Type B EIS system, preparation of specimens is relatively simple and the specimen is located between two chambers without contacting the electrodes. The apparent resistance of the specimen can be measured directly from the change in resistance between two electrodes without the need to fit the EIS data with equivalent circuits. As shown in Table 1 and Fig. 6, both Rd and Ra decreased with increase in duration of demineralization. However, their absolute values were quite different, which could be attributed to the dentine section with the electrode in the Type A EIS system. Aziza et al.31 have reported similar findings by contacting specimens with electrodes. Nelly et al.32 have also obtained data similar to ours by using the Type B EIS system. Although different EIS systems were used in their study, their results indicated that the smear layer could increase the resistance of dentine specimen. In this study, the impact of biofilm on the resistance of dentine was investigated by using both types of EIS system. It has been reported that biofilm on metal surface could be fitted as a RQ element in the equivalent circuit.23 Table 1 shows that besides the dentine layer, other RQ elements could be attributed to the smear layer and biofilm due to their electrical properties, while the result in Fig. 6 directly indicated that biofilm on the surface of dentine and bacterial cells penetrating into dentinal tubules (Fig. 7) increased the apparent resistance of dentine regardless of the presence of the smear layer. Therefore, biofilm may retard the diffusion of ions and thus cause an increase in Ra. This physical barrier-effect of biofilm is similar to that of smear layer; however this effect did not significantly alter the decreasing trend of resistance of dentine when the medium was enriched with sucrose, which metabolized to
fibrils (arrow) on the surface of intertubular dentine. The insert in (h) is a magnified image of the left of this SEM micrograph showing the bacterial cells (arrow) penetrating into dentinal tubules. ODT: orifice of dentinal tubule DT: dentinal tubule; ID: intertubular dentine; PD: peritubular dentine.
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organic acid that demineralized dentine and render dentine porous. Our previous study18 focused on the choice of equivalent circuits for fitting EIS data and the EIS data was only presented in a Nyquist plot. Nevertheless, these spectra provide information on the impedance of the specimen only. In this study, the EIS data was displayed in a Bode plot (Fig. 4), which would give us other information such as (1) frequency of a point and (2) ability to visualize large ranges of gain value. It was found that the Bode plot of dentine demineralization is similar to that of polymer degradation monitored by EIS technique.33 The reason might be that their change in compositions would result in an increase porosity, which would cause deeper penetration of the electrolyte into the specimen. The impedance spectra of the polymer showed capacitive behavior at a high-frequency region (i.e., the phase angle was close to 908), indicating good insulating properties.34 Thus, as shown in Fig. 4(b), the decline of the phase angle in a high-frequency indicated that the dentine gradually showed less capacitive behavior and became more and more conductive. Therefore, it is reasonable to conclude that the electrical behavior of the working electrode with dentine section would approach that of the bare electrode, as the dentine underwent demineralization. The findings obtained from the Type A and B systems support our hypothesis that the bacteria-induced demineralization of dentine in situ and biofilm formation could be monitored by using EIS technology. Although EIS can work as a simple and sensitive method to monitor the process of demineralization, it can only indicate porosity without reflecting other ultrastructural changes in the dentine. Therefore, it is necessary to employ other techniques to characterize demineralization of dentine to affirm the EIS results. In this study, SEM, FSEM, EDX, XRD and TMR were used to characterize specimens. The SEM images (Fig. 7) show the change in ultrastructure of dentine and formation of biofilm prior to the process of demineralization, and also confirms that the specimens became more porous with demineralization. Subsequently, electrolyte could penetrate into the dentinal tubules and intertubular dentine more readily, inducing a decrease in both Rd and Ra values. Furthermore, since the network of collagen fibrils lacked minerals, the intertubular dentine on the surface of the specimen tended to shrink after dehydration as shown in Fig. 7(h). EDX and XRD are powerful methods to detect elements and identify crystal structure. In this study, they were used to characterize the changes in chemical composition on the surface of specimens. By combining the EIS and EDX results, it could be concluded that the loss of Ca and P ions from the HAP lattice on the surface of dentine resulted in the destruction of the crystal structure of HAP which is then converted into an amorphous state with increasing extent of demineralization. This is the primary reason for the increase in porosity of dentine undergoing demineralization. It is worth noting that the XRD can reflect the crystallinity of HAP, which is significant in demineralization and remineralization study.35 Since the EDX and XRD techniques provide analytical information on the surface of specimens only36,37 they could
Fig. 11 – (a and b) Analysis of correlation between EIS and TMR results. (a) &: Rd vs. LD; ^: Rd vs. ML; (b) ~: Ra vs. LD; &: Ra vs. ML; Rd: resistance of dentine layer obtained from the Type A EIS system; Ra: apparent resistance of dentine specimen obtained from the Type B EIS system; LD: lesion depth; ML: mineral loss.
not characterize the region inside dentine. TMR, an accepted method to characterize the degree of demineralization, was used to complement the EDX and XRD method. The mineral loss and lesion depth of specimens could be related to the porosity of specimens of dental tissue.38 In this study the correlation between EIS and TMR results was investigated. As shown in Fig. 11, the results from the Type B EIS system are more associated with mineral loss and lesion depth than that from the Type A EIS system. Although the present EIS technology cannot completely replace TMR for characterization of demineralization, it could be a convenient and reliable method to monitor the process of demineralization or even remineralization of dentine in acid model and bacterial model in vitro. The particular advantage of EIS is that since its measurement is based on the movement of ions, it could directly estimate the effect of treatment for tooth hypersensitivity and the leakage at the restorative material and dental tissue interface. The fabrication of more reliable and standard working electrodes and further work to relate EIS result and TMR result will be focused in the future.
5.
Conclusion
This study focused on monitoring the process of demineralization of dentine induced by bacteria using EIS technique together with other morphology and composition characterization methods. The EIS findings indicated that bacterial biofilm and bacterial-induced dentine demineralization can be monitored by the Type A and Type B EIS systems. Although bacterial biofilm showed changing electrochemical behavior,
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it did not affect the decrease in impedance measured by EIS. The results from SEM, EDX, XRD and TMR analyses demonstrate that the surface and bulk of dentine gradually became more porous during demineralization and thus causing the values of bulk resistance of dentine (Rd) and apparent resistance of dentine (Ra) to decrease.
Acknowledgement The authors wish to acknowledge funding from the Academic Research Funding (NUS) R-224-000-029-112.
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