The Reproducibility of Tooth Impedance Spectroscopy Measurements: an in vitro Study

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1 ORIGINAL PAPERS Dent. Med. Probl. 2007, 44, 1, ISSN X Copyright by Silesian Piasts University of Medicine in Wrocław and Polish Stomatological Association JOANNA WOŹNIAK, URSZULA KACZMAREK, DAGMARA PIESIAK PAŃCZYSZYN, AGNIESZKA CZAJCZYŃSKA, PRZEMYSŁAW ŁOŚ The Reproducibility of Tooth Impedance Spectroscopy Measurements: an in vitro Study Powtarzalność pomiarów impedancji spektroskopowej zębów w badaniu in vitro Department of Conservative Dentistry and Paedodontics Silesian Piasts University of Medicine in Wroclaw, Poland Abstract Background. Dental tissues differ in their electrical conductivity due to their different molecular composition. Mature enamel is less conductive than immature and dentin is more conductive than enamel. The increase in poros ity of dental hard tissues due to the demineralization process decreases their electrical resistance, or impedance. Objectives. The purpose of this study was to evaluate the reproducibility of tooth impedance spectroscopy mea surements under in vitro conditions. Material and Methods. The material comprised 48 extracted third molars. A complex of impedance spectra from 100 Hz to 400 khz was measured using an Autolab 30 analyzer. The data were analyzed using Zplot and Zview software of Solatron Instruments Ltd. Four impedance parameters were considered: Rs, T, ϕ, and Rb. The mea surements were carried out by two examiners to assess intra examiner and inter examiner reproducibility, which were assessed using kappa statistics. Results. Maximum errors of the parameters Rs, T, ϕ, and Rb were 7.0%, 6.6%, 6.2%, and 4.3%, respectively. No significant differences between inter examiner and intra examiner measurements using the Wilcoxon pair test at p < 0.05 were found. Kappa values for intra examiner and inter examiner measurements indicated very high repro ducibility of the parameters Rb (0.81) and T (0.75) and high reproducibility of the parameter Rs (0.67). Conclusions. The high reproducibility of the tooth impedance spectroscopy measurements in vitro encourages the continuation of research directed towards early carious lesion detection (Dent. Med. Probl. 2007, 44, 1, 11 17). Key words: tooth, impedance spectroscopy, measurement, reproducibility. Streszczenie Wprowadzenie. Tkanki zęba wykazują różne przewodnictwo elektryczne spowodowane zróżnicowanym składem molekularnym. Dojrzałe szkliwo ma mniejsze przewodnictwo niż niedojrzałe, a zębina większe niż szkliwo. Wzrost porowatości twardych tkanek zęba spowodowany demineralizacją wywołaną procesem próchnicowym zmniejsza ich oporność elektryczną lub impedancję. Cel pracy. Porównanie powtarzalności pomiarów impedancji spektroskopowej zębów w badaniu in vitro. Materiał i metody. Zbadano 48 usuniętych zębów trzecich trzonowych. Spektrum impedancji mierzono w zakre sie Hz za pomocą analizatora Autolab 30. Dane analizowano z użyciem oprogramowania Zplot and Zview, Solatron Instruments LTD. Uwzględniono 4 parametry impedancji: Rs, T, ϕ i Rb. Pomiary przeprowadza li dwaj badacze w celu oceny powtarzalności wyników uzyskanych przez jednego badacza i między badaczami, przy czym wiarygodność wyników oceniano za pomocą statystyki kappa. Wyniki. Maksymalny błąd mierzonych parametrów Rs, T, ϕ i Rb wynosił odpowiednio 7,0; 6,6; 6,2 i 4,3%. Stosując test par Wilcoxona, nie stwierdzono istotnych różnic na poziomie p < 0,05 między pomiarami tego samego badacza i między badaczami. Wartości kappa dla pomiarów tego samego badacza i pomiarów między badaczami wykazały bardzo wysoką powtarzalność, dla parametru Rb 0,81 i T 0,75, a wysoką dla parametru Rs 0,67. Wniosek. Wysoka powtarzalność pomiarów impedancji spektroskopowej w badaniu in vitro zachęca do kontynuo wania badań ukierunkowanych na wykrywanie wczesnych zmian próchnicowych (Dent. Med. Probl. 2007, 44, 1, 11 17). Słowa kluczowe: ząb, impedancja spektroskopowa, pomiar, powtarzalność.

2 12 Dental tissues differ in their electrical conduc tivity due to different molecular composition. Mature enamel is less conductive than immature and dentin is more conductive than enamel. When a current is applied by placing two electrodes on a tooth surface, the electrical conductance of all the material between them can be measured. As all of these materials except enamel have high con centrations of electrolytes, the measured conduc tance is mainly that of the enamel. When hard den tal tissues demineralize in the process of caries formation, the loss of mineral content leads to an increase in electrical conductivity or, conversely, increased porosity leads to decreased electrical resistance, or impedance [1]. Electrical conduc tance through hard dental tissue in mainly the result of ionic migration through the electrolyte solution contained within the enamel and dentin pores [2, 3]. Devices have been developed in which a single fixed frequency alternating current is used to mea sure the bulk resistance of the tooth (Vanguard Electronic Caries Detector, Caries Meter L, Vanguard Caries Detector, Electronic Caries Meter, Electronic Caries Monitor, Cariometer 800). The Electronic Caries Monitor III (ECM III) device is currently commercially available. Electrical impedance spectroscopy has an advantage over fixed frequency measurements because materials reveal various electrical responses at different fre quencies. Therefore, impedance spectroscopy is able to determine more precisely the various para meters that reveal these differences. This is based on the measurement of electrical resistance (the real part of impedance) during the flow of an alter nating current. It is one of the most promising diag nostic methods used in carious lesions at present. Studies on the characteristics of hard dental tissues during the flow of alternating current with a wide frequency range showed that it was possible to detect slight increases in the porosity of tissue in early carious lesions. From a clinical point of view it is essential that the results obtained using a given diagnostic tool be reproducible, i.e. that the measurements per formed by the same operator at different times (intra examiner reproducibility) and by different operators (inter examiner reproducibility) be the same. The value of electrical conductance mea surements by the ECM in caries diagnosis has been the subject of many in vitro and in vivo stud ies [4 16]. The principles of electrical impedance have been applied in research to detect carious lesions at approximal sites of teeth [16 20]. The purpose of this study was to evaluate the repro ducibility of spectroscopy measurements on molar occlusal surfaces in vitro conditions. Material and Methods J. WOŹNIAK et al. The material comprised 48 third molars extracted from subjects aged from 23 to 35 years. The teeth were obtained for research purpose with the patients written consent. The protocol was approved by the Bioethics Commission of the Silesian Piasts University of Medicine in Wroclaw. Inclusion criteria for the teeth in this study were the absence of restorations and appar ent hypoplastic pits, fractures, or frank cavitation. After cleaning to remove any debris, the teeth were stored in thymolized physiological saline before and between measurements. Immediately before a measurement, a tooth was taken from the saline and blotted dry with tissue paper. Then it was positioned by a holder in a special plastic chamber to enable good contact to a working elec trode with the tooth surface and to provide repro ducible measurements. The working electrode consisted of a metal conical rod 0.1 mm in diame ter which made direct contact with the occlusal fis sure. The counter electrode was a strip of platinum foil placed next to the roots of the examined tooth, which was dipped in a conducting gel (Ky Jelly, Johnson & Johnson). Measurements were carried out at room temperature. The electrical measurements were performed using the two electrode system connected to an Autolab 30 analyzer (ECO Chemie, The Nederland) used both for measurement and data collection. A complex of impedance spectra was measured from 100 Hz to 400 khz at an amplitude of the applied potential of E(t) = 50 mv and ampli tude of the measured current of I(t) = 50 na to 50 µa. The impedance data were analyzed by commer cially available Zplot and Zview software (Solatron Instruments Ltd.) [21]. The obtained data were interpreted using a suitable equivalent circuit (Fig. 1). In the analysis, four impedance parameters were considered: Rs [Ω], the resistance of the probe at a frequency of ω 0 (direct current resis tance); T [Ω 1 ϕ], a capacitance related parameter with measured impedance, ϕ [rad], the phase angle, i.e. the slope of the curve to the Z axis; and Rb [Ω], the resistance of the test probe at a frequency of ω 0 (resistance at high frequencies). The measurements were carried out by two examiners. To verify intra examiner reproducibili ty, an examiner performed all examinations again after a period of 14 days. Reproducibility was assessed using unweighted kappa statistics. This was performed for the repeated readings carried out by each examiner (intra examiner repro ducibility) and for the pair of examiners (inter examiner reproducibility). According to the Landis and Koch criteria [22], kappa values denote

3 Reproducibility of Tooth Impedance Spectroscopy Measurements 13 ideal (κ = 1), very high (κ > 0.8), high (0.8 κ> 0.6), moderate (0.4 < κ 0.6), and poor (κ 0.4) agreement. Significance of the difference between examiners was assessed by the Wilcoxon pair test at a level of p < Results Rather high variability (the value of the stan dard deviation over the mean value), particularly for the parameters T and Rb, was found (Table 1). The main source of variation in the measured para meters was possibly the repositioning of the tooth between each measurement, which could lead to a change in the surface contact between the tooth and the working electrode. However, comparing values of measurements performed by the two examiners in sequence, i.e. measurement 1 exam iner 1, measurement 1 examiner 2, measurement 2 examiner 1, measurement 2 examiner 2, the maxi mal measurement errors for the impedance para meters Rs, T, Rb, and ϕ were 6.9, 6.2, 6.0 and 4.0%, respectively. In the re performed measure ments (measurement 1 examiner 1, measurement 2 examiner 1, measurement 1 examiner 2, measure ment 2 examiner 2) the maximal measurement errors for the impedance parameters were 7.0, 6.6, 6.2, and 4.3%, respectively. No significant differ ences between inter examiner and intra examiner measurements using the Wilcoxon pair test at p < 0.05 were found (Table 2). Kappa values for intra examiner and inter examiner measurements pointed to very high reproducibility for the parameters Rb (0.81) and T (0.75) and high reproducibility for the parameter Rs (0.67). Values of the impedance measurements are presented in Figures 1 and 2 and their numeri cal data in Tables 3 and 4. Discussion In the last years, many papers have been pub lished on measurements of tooth resistance. The studies were performed to assess their possible use in the diagnosis of dental caries on occlusal [3, 4, 8, 9, 11, 15] and approximal surfaces [6, 17, 19, 20] as well as in the prognosis of caries develop Rb R CPE Fig. 1. Pattern of equivalent circuit; Rb bulk resis tance, R resistance of studied teeth at frequency ω 0, CPE constant phase element Ryc. 1. Schemat obwodu zastępczego: Rb opór całkowity, R opór dla częstości ω 0, CPE element stałofazowy Table 1. Mean values and standard deviations of impedance spectroscopy parameters obtained by two examiners in two measurements Tabela 1. Średnie i odchylenia standardowe impedancji spektroskopowej uzyskane przez dwóch badających w dwóch pomiarach Measurement Examiner Rs 10 5 [Ω] T 10 8 [Ω 1 s ϕ] Rb 10 5 [Ω] ϕ [rad] (Pomiar) (Badający) x ± SD x ± SD x ± SD x ± SD ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± 0.23 Table 2. Values of the Wilcoxon pair test in comparison of inter examiner and intra examimer measurements Tabela 2. Wartość par Wilcoxona do porównania pomiarów między badającymi i między badaniami u tego samego badającego Comparison Values at p < 0.05 (Porównanie) (Wartości przy p < 0,05) Rs T Rb ϕ Examiner 1 vs. examiner (Między badającymi) Measurement 1 vs. measurement (Między pomiarami)

4 14 J. WOŹNIAK et al. Z [Ω] a b c d Z[ Ω] Fig. 2. Examplary graphs of impedance measurements on occlusal tooth surface per formed by 2 examiners in 2 following measurements; single points are experimental results and lines are approximation of equivalent circuit a study 1, examiner 1, measurement 1; data approximation b study 1, examiner 1, measurement 2; data approximation c study 1, examiner 2, measurement 1; data approximation d study 1, examiner 2, measurement 2; data approximation Ryc. 2. Przykładowy wykres pomiarów impedancji na powierzchni żującej zęba przeprowadzonych przez dwóch badaczy w dwóch kolejnych pomiarach; pojedyncze punkty są wynikami eksperymentalnymi, a linie aproksymacją do obwodu zastępczego a badanie 1, badający 1, pomiar 1; aproksymacja danych b badanie 1, badający 1, pomiar 2; aproksymacja danych c badanie 1, badający 2, pomiar 1; aproksymacja danych d badanie 1, badający 2, pomiar 2; aproksymacja danych ment [8, 23]. Prototype and commercial devices as sources of alternating current with different fre quencies were used [2 8, 10, 12, 17 20]. The pre liminary experiments of the present study were carried out using a wider frequency range of alter nating current, i.e. from 0.5 Hz to 900 khz. However, after analyzing the data, the frequency range was restricted to the 100 Hz to 400 khz range due to the better reproducibility of the mea surements as well as the potential of using imped ance spectroscopy in vivo studies, which con strains the minimum frequency to about 100 Hz. Lower frequencies would be too similar to the rhythm of the heart. Despite the fact that in this method only a closed circuit was used (in the case of studying hard dental tissues, only inside the oral cavity), precautions should be undertaken to elim inate the possibility of cardiac disorders due to background resonance, as they might cause car diac arrest. The next aspect which requires consid eration in the use of electric current on the human organism is the level of the pain threshold. According to Valentinuzzi [24], the flow of an alternating current with a frequency of 100 Hz passing through an organism can be felt by man when its intensity is in the range from 0.3 to l ma. At a higher alternating current, from 10 to 100 ma, intensive stimulation of nerves and muscles may appear which is felt as muscular contractions, pain, and fatigue. The levels of current which are threshold values for feeling and pain depend on the current s frequency. The dependence of thresh old sensitivity on the frequency of the applied cur rent for electrodes placed on both sides of the chest is presented in Fig. 4. Values of current determining threshold sensitivity increase with current frequency. These values are higher for teeth due to the insulating properties of enamel [25]. Therefore, in vivo conditions it is best to apply a high frequency alternating current for rea sons of patient comfort and safety. The maximum values of the applied current in the present study at 100 Hz frequency was 5 µa and at 400 khz 50 µa. In the present study, similarly to that of Huysman et al. [17], complex impedance spec troscopy was used in a setup that could, in its basic form, be applied clinically. The gel in which the tooth rested represents the part of the body before the tooth and the counter electrode, which may be hand held or lie on the oral mucosa. Electrical impedance spectroscopy has been used before for the evaluation of electrical para meters of healthy and carious teeth [2, 17, 18], but mainly bulk resistance (Rb) was analyzed. In this

5 Reproducibility of Tooth Impedance Spectroscopy Measurements 15 Table 3. Values of the impedance spectroscopy parameters presented in Figure 1 Tabela 3. Wartości pomiarów impedancji spektroskopowej przedstawione na ryc. 1 Measurement Examiner Measurement Rs 10 5 T10 8 Rb 10 5 ϕ (Pomiar) (Badający) number [Ω] [Ω 1 s ϕ] [Ω] [rad] (Numer pomiaru) Tables 4. Values of the impedance spectroscopy parameters presented in Figure 2 Tabela 4. Wartości pomiarów impedancji spektroskopowej przedstawione na ryc. 2 Measurement Examiner Measurement Rs 10 5 T10 8 Rb 10 5 ϕ (Pomiar) (Badający) number [Ω] [Ω 1 s ϕ] [Ω] [rad] (Numer pomiaru) Z [Ω] c d b a Z[ Ω] Fig. 3. Examplary graphs of impedance measurements on occlusal tooth surface per formed by the same examiner in 2 following measurements; single points are experi mental results and lines are approximation of equivalent circuit a study 1, examiner 1, measurement 1; data approximation b study 1, examiner 1, measurement 2; data approximation c study 2, examiner 1, measurement 1; data approximation d study 2, examiner 1, measurement 2; data approximation Ryc. 3. Przykładowy wykres pomiarów impedancji na powierzchni żującej zęba przeprowadzonych przez tego samego badacza w dwóch kolejnych pomiarach; poje dyncze punkty są wynikami eksperymentalnymi, a linie aproksymacją do obwodu zastępczego a badanie 1, badający 1, pomiar 1; aproksymacja danych b badanie 1, badający 1, pomiar 2; aproksymacja danych c badanie 2, badający 1, pomiar 1; aproksymacja danych d badanie 2, badający 1, pomiar 2; aproksymacja danych study, apart from Rb, three additional parameters, Rs, T, and ϕ, were analyzed. The level of varia tions, especially for the phase angle ϕ, reflected the reproducibility of the measurements more accurately. Diagnostic methods should be valid and reli able. Validity means that the test actually measures what it is supposed to measure, while reproducibil ity, also termed repeatability, reliability, or consis tency, means that the test can be used repeatedly

6 16 J. WOŹNIAK et al. current intensity [ma] natężenie prądu [ma] frequency [Hz] częstotliwość [Hz] Fig. 4. Threshold of sense for alternating current flow ing between two electrodes placed in both sides of chest (TT) and between electrodes situated on patient s neck (N) and abdomen (A) (according to [24]) Ryc. 4. Próg odczucia dla prądu zmiennego płynącego między dwiema elektrodami umieszczonymi po bokach klatki piersiowej (TT) i między elektrodami umiesz czonymi na szyi (N) i brzuchu (A) pacjenta (wg [24]) with the same result. If the same examiner repeats a test and obtains the same result, this is called intra examiner reproducibility. When several examiners repeat the same test and obtain the same result, this is called inter examiner reproducibility [26]. In the present study, very good inter examin er and intra examiner repeatability of the measure ments were obtained. This could suggest that the technique would be reproducible under ex vivo conditions. It could also be concluded that the mea surements of impedance spectroscopy are promis ing for application in vivo conditions. The obtained high reproducibility of tooth impedance spectroscopy measurements in this in vitro study encourages continuing research direct ed towards early carious lesion detection. References [1] LONGBOTTOM C., HUYSMAN M.C.: Electrical measurements for use in caries clinical trials. J. Dent. Res. 2004, 83, Spec. Iss. C, C76 C79. [2] HUYSMAN M.C., LONGBOTTOM C., CHRISTIE A.M., BRUCE P.G., SHELLIS R.P.: Temperature dependence of the elec trical resistance of sound and carious teeth. J. Dent. Res. 2000, 79, [3] RICKETTS D.N.J.: Electrical conduction detection methods. In: Early detection of dental caries. Ed. G.K. Stookey, Indiana University School of Dentistry 1996, [4] WHITE G.E., TSAMTSOURIS A., WILLIAMS D.L.: Early detection of occlusal caries by measuring the electrical resis tance of the tooth. J. Dent. Res. 1978, 57, [5] VERDONSCHOT E.H., WENZEL A., TRUIN G.J., KONIG K.G.: Performance of electrical resistance measurements adjunct to visual inspection in the early diagnosis in occlusal caries. J. Dent. Res. 1993, 21, [6] HUYSMANS M.C., VERDONSCHOT E.H., RONDEL P.: Electrical conductance and electrode area on sound smooth enamel in extracted teeth. Caries Res. 1995, 29, [7] ASHLEY P.F., BUNKHORN A.S., DAVIES R.M.J.: Occlusal caries diagnosis: an in vitro histological validation of the Electronic Caries Monitor (ECM) and others methods. J. Dent. Res. 1998, 26, [8] ASHLEY P.F., ELWOOD R.P., WORTHINGTON H.V., DAVIES R.M.J.: Predicting occlusal caries using the Electronic Caries Monitor. Caries Res. 2000, 34, [9] HUYSMAN M.C., LONGBOTTOM C., PITTS N.B.: Electrical methods in occlusal caries diagnosis; an in vitro com parison with visual inspection and bite wing radiography. Caries Res. 1998, 32, [10] HUYSMAN M.C., KUHNISCH J., TEN BOSCH J.J.: Reproducibility of electrical caries measurements: a technical prob lem? Caries Res. 2005, 39, [11] HUYSMAN M.C., LONGBOTTOM C., HINTZE H., VENDONSCHOT E.H.: Surface specific electrical occlusal caries diag nosis: reproducibility, correlation with histological lesion depth, and tooth type dependence. Caries Res. 1998, 32, [12] WANG J., SOMEYA Y., INABA D., LONGBOTTOM C., MIYAZAKI H.: Relationship between electrical resistance mea surements and microradiographic variables during remineralization of softened enamel lesions. Caries Res. 2005, 39, [13] RICKETTS D.N., KIDD E/A., LIEPINS P.J., WILSON R.F.: Histological validation of electrical resistance measure ments in the diagnosis of occlusal caries. Caries Res. 1996, 30, [14] ROCK W.P., KIDD E.A.M.: The electronic detection of demineralization in occlusal fissures. Br. Dent. J. 1988, 23, 164, [15] KUHNISCH J., HEINRICH WELTZIEN R., TABATABAIE M., STOSSER L., HUYSMANS M.C.: An in vitro comparison between two methods of electrical resistance measurement for occlusal caries detection. Caries Res. 2006, 40, [16] PRETTY I.A.: Caries detection and diagnosis: novel technologies. J. Dent. 2006, 34, [17] HUYSMAN M.C., LONGBOTTOM C., PITTS N.B., LOS P., BRUCE P.G.: Impedance spectroscopy of teeth with and with out approximal caries lesions an in vitro study. J. Dent. Res. 1996, 75, [18] LONGBOTTOM C., HUYSMAN M.C., PITTS N.B., LOS P., BRUCE P.G.: Detection of dental decay and its extent using a.c. impedance spectroscopy. Nat. Med.1996, 2,

7 Reproducibility of Tooth Impedance Spectroscopy Measurements 17 [19] KACZMAREK U., PIESIAK D., WOŹNIAK J., CZAJCZYŃSKA WASZKIEWICZ A., ŁOŚ P., PITTS N.B., LONGBOTTOM C.: Charakterystyka właściwości przewodnictwa elektrycznego powierzchni zębowych metodą spektroskopii impe dancyjnej badania wstępne. Czas. Stomat. 2001, 9, [20] KACZMAREK U., PIESIAK D., WOŹNIAK J., CZAJCZYŃSKA WASZKIEWICZ A., ŁOŚ P., PITTS N.B., LONGBOTTOM C.: Porównanie pomiarów przewodnictwa elektrycznego metodą impedancji spektroskopowej w warunkach poko jowych i symulujących środowisko jamy ustnej. Czas. Stomat. 2002, 4, [21] MCDONALD J.R., SCHOONMANN J., LEHNER A.P.: The applicability and power of complex nonlinear least squares for the analysis of impedance and admittance data. J. Electroanal. Chem. 1982, 131, [22] LANDIS J.R., KOCH G.G.: The measurement of observer agreement of categorical data. Biometrics 1997, 33, [23] IE Y.L., VERDONSCHOTE.H., SCHAEKENM.M., HOF van t M.A.: Electrical conductance of fissure enamel in recent ly erupted molar teeth as related to caries status. Caries Res. 1995, 29, [24] VALENTINUZZI M.E.: Bioelectrical impedance techniques in medicine. Part I: Bioimpedance measurement. First section: General concepts. Crit. Rev. Biomed. Eng. 1996, 24, [25] LEVINKIND M.: Electrochemical impedance strategies for early caries detection In: Early detection of dental caries. Ed. G.K. Stookey, Indiana University School of Dentistry 1996, [26] KIDD E.A.M., MEJARE I., NYVAD B.: Clinical and radiographic diagnosis. In: Dental caries. The disease and its clinical management. Ed. O. Fejerskov, E. Kidd. Blackwell Munksgaard 2003, 112. Address for correspondence: Urszula Kaczmarek Department of Conservative Dentistry and Paedodontics Silesian Piasts University of Medicine Krakowska Wrocław Poland Tel.: E mail: ukaczm@stom.am.wroc.pl Received: Revised: Accepted: Praca wpłynęła do Redakcji: r. Po recenzji: r. Zaakceptowano do druku: r.