SCANNING VOL. 9999, 1–7 (2015) © Wiley Periodicals, Inc.
Caries-Removal Effectiveness of a Papain-Based Chemo-Mechanical Agent: A Quantitative Micro-CT Study ALINE A. NEVES,1 ROSEANE A. LOURENSc O,2 HAIMON D. ALVES,3 RICARDO T. LOPES,3 AND LAURA G. PRIMO1 1
Departamento de Odontopediatria e Ortodontia, Universidade Federal do Rio de Janeiro, Rio de Janeiro, Brazil Escola de Ci^encias da Saude, Universidade do Grande Rio, Duque de Caxias, Rio de Janeiro, Brazil 3 Laboratorio de InstrumentaSc ~ao Nuclear – COPPE – Universidade Federal do Rio de Janeiro, Rio de Janeiro, Brazil 2
Summary: The aim of this study was to access the effectiveness and specificity of a papain-based chemomechanical caries-removal agent in providing minimum residual caries after cavity preparation. In order to do it, extracted carious molars were selected and scanned in a micro-CT before and after caries-removal procedures with the papain-based gel. Similar parameters for acquisition and reconstruction of the image stacks were used between the scans. After classification of the dentin substrate based on mineral density intervals and establishment of a carious tissue threshold, volumetric parameters related to effectiveness (mineral density of removed dentin volume and residual dentin tissue) and specificity (relation between carious dentin in removed volume and initial caries) of this caries-removal agent were obtained. In general, removed dentin volume was similar or higher than the initial carious volume, indicating that the method was able to effectively remove dentin tissue. Samples with an almost perfect accuracy in carious dentin removal also showed an increased removal of caries-affected tissue. On the contrary, less or no affected dentin was removed in samples where some carious tissue was left in residual dentin. Mineral density values in residual dentin were always higher or similar to the threshold for mineral density values in carious dentin. In conclusion, the papain-based gel was effective in removing carious dentin up to a conservative in vitro threshold. Lesion characteristics, such as activity and morphology of enamel lesion, may also influence caries-removal
Address for reprints: Aline de Almeida Neves, Universidade Federal do Rio de Janeiro, Departamento de Odontopediatria e Ortodontia, Rua Prof. Rodolpho Palo Rocco, 325, Cidade Universitaria, Rio de Janeiro 21941-617, Brazil E-mail: [email protected] Received 7 February 2015; revised 25 February 2015; Accepted with revision 10 March 2015 DOI: 10.1002/sca.21206 Published online XX Month Year in Wiley Online Library (wileyonlinelibrary.com).
properties of the method. SCANNING 9999:1–7, 2015. © 2015 Wiley Periodicals, Inc. Key words: residual caries, cariesremoving techniques, micro-CT, Papacarie
Introduction The concept of minimally invasive dentistry, among broader definitions, precludes a systematical and conservative removal of decayed tissue and its replacement by adhesive restorations to appropriately restore the integrity and aesthetics of the tooth (Banerjee, 2013). This approach means removing only infected and irreversibly destroyed dentin and leaving, as residual substrate for further adhesive procedures, a slightly demineralized caries-affected dentin (Kidd, 2010). Based on this philosophy, chemo-mechanical cariesremoving agents have been developed (Neves et al., 2011a). These products act on the degradation of the partially demineralized and altered dentin matrix which has been previously exposed to bacterial action (infected dentin), allowing its removal and preventing damage to the underlying remineralizable tissue (affected dentin) (Ericson et al., ’99). Among them, CarisolvTM (RLS Global AB, Gothenburg, Sweden), a sodium hypochloride-based gel added of an amino acid mixture became very popular among general dentists around the world. CarisolvTM has already proved its effectiveness and accuracy in the conservative removal of dentin carious tissue by different microscopy methods (Banerjee et al., 2000; Yazici et al., 2003; Neves et al., 2011b). More recently, a papain-based gel, also intended for chemomechanical caries removal has been developed and marketed (PapaCarieTM, Formula e ASc ~ao, S~ao Paulo, SP, Brazil) (Gianini et al., 2010; Khattab and Omar, 2012). This product contains the enzyme papain, a protease of broad proteolytic activity extracted from the latex of leaves and fruits of Carica papaya tree. It is
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suggested that this product also acts exclusively on breaking down the partially degraded collagen, without damaging intact collagen fibrils (Bussadori et al., 2005). The selective interaction of the papain enzyme with the affected components of the carious dentin is attributed to the lack of an antiprotease a-1-anti-trypsin, which inhibits protein digestion in sound collagenbased tissues, although this statement has recently been partially contested (Bertassoni and Marshall, 2009). In Brazil and other South American countries, PapaCarieTM has found a broad range of applications in dentistry, due to its clinical applicability, market availability, and relative low cost (Lopes et al., 2007), being nowadays one of the most used to conservatively remove carious dentin using a chemomechanical method in those regions. Its clinical effectiveness has also already been studied, and apparently, the use of the papain gel results in residual dentin with similar microbiological characteristics than that excavated by means of conventional burs (Kotb et al., 2009; Bussadori et al., 2011; Singh et al., 2011). In terms of lesion histopathology, however, the laboratorial micro-CT technique has a clear application in the study of caries-removing methods by allowing nondestructive analysis of samples (longitudinal evaluation), as recently demonstrated (Neves et al., 2010). Although some studies have been published on this respect for conventional and other improved cariesremoving methods (Neves et al., 2011; Ahmed et al., 2012; Zhang et al., 2013), caries-removal effectiveness of a papain-based caries-removing gel has not been accessed longitudinally in vitro. Thus, the aim of the present study was to access volumetric parameters related to effectiveness (mineral density of removed volume and residual dentin tissue) and specificity (relation between carious dentin in removed volume and initial caries) of dentin cariesremoval procedures performed by a papainbased chemo-mechanical caries-removal agent.
Materials and Methods Sample Selection and Preparation
Extracted human molar teeth presenting occlusal carious lesions were selected from a pool of extracted elements stored for less than 6 months in 1% aqueous chloramine solution under refrigeration. The teeth were collected in a faculty practice after written consent given by the patient. This studied complied with all ethical aspects of donation and use of biological human tissue and has been approved by the local university ethical committee. After ultrasonic cleaning of plaque, calculus, and other debris, a digital radiograph was obtained, so that
teeth without carious involvement in dentin and those in which the carious lesion was approaching the pulp chamber, were excluded. Ten teeth were initially selected and mounted by the roots in gypsum stubs to facilitate further manipulation. After that, the occlusal surface was submitted to a perpendicular flat cut with a diamond disk on a low speed cutting machine (Isomet, Buehler, Lake Bluff, IL) to expose the subjacent carious dentin and exclude any carious enamel. This procedure was performed to exclude possible areas of carious enamel, which by having similar micro-CT gray values than dentin, could impair some of the following proposed quantitative micro-CT analysis, as previously described (Neves et al., 2010).
Micro-CT Acquisition, Reconstruction, and Calibration
The teeth were subjected to a baseline scanning procedure using a Skyscan 1173 micro-CT system (Bruker micro-CT, Kontich, Belgium) (baseline stacks). The acquisition settings used were 70 kV, 114 mA, 14.6 mm pixel size, 260 ms exposure, frame averaging of 5 and rotation step of 0.5˚ through 360˚. A flatfield reference was obtained before the first scan and the random-movement amplitude was set to 20 lines to reduce ring artefacts. A 1-mm aluminium filter was also used to reduce the beam hardening effect. After cariesremoval procedures, the micro-CT scan was repeated for all studied samples using the same parameters described above (treated stacks). The acquired micro-CT projections were reconstructed into cross-section slices using a proprietary software interface (NRecon v.1.6.9, Bruker micro-CT). Specific reconstruction settings included a 50% beamhardening correction, ring artifact correction factor of 5 and input of optimal contrast limits (0–0.075) for all stack reconstructions. For calibration of micro-CT gray values into mineral density (MD) values of hydroxyapatite (HAp), three phantoms made of fine calcium hydroxyapatite powder embedded in epoxy resin, with different mineral densities (0.25, 0.75, and 3.14 g/cm3), were scanned and reconstructed using the same parameters described above. Gray values obtained by micro-CT were further converted into MD values of HA (g/cm3) after definition of a calibration curve using a linear equation fit. Caries-Removal Procedures
The teeth were treated with the papain-based cariesremoval gel (PapaCarie Duo1, Formula e ASc ~ao, S~ao Paulo, Brazil) following the manufacturer’s instructions. In short, the gel was dispensed over the carious lesion, and after 30 s the carious tissue was gently
A. A. Neves et al.: Micro-CT Investigation of Papain Caries Removal
scrapped with a blunt spoon excavator. When the gel turned grayish, the cavity was cleaned with a stream of water, air-dried, a new batch of gel was applied, and excavation procedure was started. The whole treatment was repeated until no more dentin tissue could be removed by the gel-excavation combination.
Image Registration of Baseline and Treated Image Stacks
After cross-section slices reconstruction, each treated stack was registered using its corresponding baseline stack as the template, by means of the Rigid Registration plugin implemented in the FIJI software interface (Schindelin et al., 2012). The rigid registration method defines six degrees of freedom for matching boundary surfaces in both images, and for this reason, this method was chosen since image boundaries were similar before and after caries-removal procedures. The similarity metric was based on the maximized mutual information algorithm, a registration method based on finding the alignment, or pairing of points, which match both histograms as closely as possible (Berry, 2008). The optimization steps of the registration algorithm were repeated until image stacks did not differ by more than 0.4 tolerance. In fact, after the stacks were placed at the same spatial position, further image operations allowed calculation of caries-removal parameters.
Quantitative Evaluation of Caries Removal
Pre-processing of digital images included a tridimensional median filter with a 2 2 2 kernel (8-voxel). The filtering algorithm used was part of the 3D ImageJ Suite built in the Process menu of the Fiji open-source software implementation (Ollion et al., 2013). After definition of cut-off points and arithmetic operations, the segmented
3
TABLE I Classification of dentin substrate based on MD value intervals Type of dentin substrate Sound dentin (x)
Affected dentin Carious dentin
Range of MD values used to classify the dentin substrate Mean MD over a circular 100 pixel-radius ROI interpolated over 20 slices in unaffected dentin areas >1.11 g/cm3 HA x 0–1.11 g/cm3 HA
volumes were quantified by means of the 3D Object Counter plugin, after proper calibration of the resolution (Bolte and Cordelieres, 2006). For definition of the carious lesion volume, the cutoff point corresponding to a MD of 1.11 g/cm3 HA was defined in both baseline (initial carious volume) and treated stacks (residual dentin volume), based on previously published studies on microhardness (Kinney et al., ’94) and micro-CT (Neves et al., 2010; Neves et al., 2011c) of dentinal carious tissue. Tooth tissue presenting values that were lower or equal than the selected threshold were considered as carious tissue. Tooth tissue belonging to higher MD values than the threshold, but lower than sound dentin values obtained for the specific sample were considered as affected dentin (Table I). Removed (excavated) volume was obtained for each treated sample after an arithmetic XOR operation between baseline and treated stacks and volumetric quantification. From the removed tissue volumes, the carious and affected dentin contributions were calculated by defining specific MD thresholds, as described in Table I.
Results From 10 initial selected samples, four were excluded after the baseline scan due to an initial carious volume
Fig 1. Stereomicroscopic images of the studied samples 1–6 (a–f) before and after caries removal. Arrow in (d) indicates a dark pigmented arrested lesion, while arrow in (e) indicates residual enamel areas.
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lower than 0.5 mm3, which was considered too low to allow appropriate quantitative measurements, resulting in a final sample of six teeth included in the analysis. Figure 1 shows occlusal surface images of each studied sample before and after caries removal with the papain-based agent aided by a conventional spoon excavator. The initial carious dentin volume varied from 0.616 to 5.196 mm3, as seen in Figure 2(A) (dark gray bars). The respective excavated (removed) volumes (light gray bars on top) are also shown. Except for sample 5, in all other studied teeth, the removed dentin volume was similar or higher than the initial carious volume, indicating that the papain-based method was able to effectively remove dentin tissue. For the lesion in
sample 5, further removal of dentin could have been impaired by the remaining enamel area remaining over the lesion (red arrow, Fig. 1(E)). The specificity of carious tissue removal can be inferred by examining the volumetric difference between initial carious volume (dark gray bars, Fig. 2 (A)) and carious volume in excavated dentin (black dots, Fig. 2(A)). The difference between these two parameters results in the volume of carious tissue left at the cavity (Table II). It is possible to note that in samples in which an almost perfect accuracy in carious dentin removal was observed (samples 2, 4, and 6) were, in fact, followed by increased removal of caries-affected tissue (Fig. 2(B), dark gray bars). On the contrary, in those samples where apparently some carious tissue was left (samples 1, 3, and 5) less or no affected dentin was removed at the expense of some carious tissue in residual dentin. Figure 2(C) shows mean MD values in sound dentin for each studied sample (black lines). Mean MD values in residual dentin were always higher or, in case of sample 5, similar to the used threshold of 1.11 g/cm3 HA for carious dentin (dark gray line). Indeed, samples showing higher volumes of carious tissue left in the cavity (1, 3, and 5) showed also the higher differences between MD values in sound dentin and MD at residual dentin. Figure 3 shows volumetric renderings and selected micro-CT slices of baseline and treated stacks (sample 2) highlighting the effectiveness of carious dentin removal.
Discussion The present results show that excavation with the papain-based chemo-mechanical agent (PapaCarieTM) was able to reach an acceptable in vitro threshold, as seen by the MD in residual dentin (Fig. 2(C)). In all studied samples, carious tissue comprised most of the removed tissue after cavity preparation. In fact, mean MD in residual dentin and within excavated tissue were similar to those found in the literature (ClementinoLuedemann et al., 2006; Neves et al., 2011c; Ahmed et al., 2012).
TABLE II Carious dentin volume left after excavation with the papain-based gel Sample
Fig 2. Caries-removal parameters evaluated in each studied sample. (a) Initial caries volume, removed (excavated) tissue volume and carious tissue volume in removed tissue; (b) contributions of carious and affected dentin in removed volume; (c) mineral density values in sound and residual dentin and mean values in removed tissue volume.
1 2 3 4 5 6
Carious volume left at the cavity bottom 1.21 mm3 0.23 mm3 0.68 mm3 0.05 mm3 0.99 mm3 0.05 mm3
A. A. Neves et al.: Micro-CT Investigation of Papain Caries Removal
5
Fig 3. Volume renderings and selected micro-CT slices of baseline and treated stacks (sample 2) calibrated for dentin mineral density values (g/cm3). (a) 3D volume rendering of baseline tooth; (b) 3D volume rendering of treated tooth; (c) micro-CT slice of baseline tooth; (d) micro-CT slice of treated (excavated) tooth. Arrow point to locations of carious dentin at the threshold for removal, suggesting a conservative excavation result.
Methodologically, the micro-CT technique in association with digital image analysis has a clear advantage over conventional microscopy methods for the study of caries-removing techniques as follows: it is nondestructive, allowing comparison of results obtained from the same sample before and after caries removal; its tridimensional nature allows obtaining volumetric results from the whole bulk of the specimen and also allows calibration against HA phantoms (g/cm3 of HA) into actual dentin mineral density data (Zou et al., 2009; Neves et al., 2010). To further increase the accuracy of the technique, in the present study, carious enamel areas were mechanically removed from the samples by a flat cut through the occlusal surface. This was performed to overcome the limitation to a fully quantitative volumetric approach on dentin caries removal related to the similarity in gray values between carious enamel and sound dentin (Neves et al., 2010; Taylor et al., 2010). However, this procedure also resulted in elimination of
some carious dentin areas, increasing the need to exclude some samples in the present study. Indeed, samples presenting less than 0.5 mm3 of initial carious volume were excluded since accurate results could not be achieved, as the micro-CT system used has limitations regarding spatial resolution. In fact, it has been already shown that chemomechanical methods resulted in the best agreement in conservative caries removal compared to conventional (burs) and laser-assisted methods, following similar mineral density parameters as used in the present study (Neves et al., 2011c). Even when a lower threshold for carious tissue is used, chemo-mechanical methods have proven to be more conservative than burs in establishing the endpoint of caries removal (Ahmed et al., 2012). The more specificity of chemo-mechanical methods is probably due to the specific chemical dissolution caused by the active component in these products, combined to the limited mechanical action of a hand instrument more conservatively used.
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Regarding the results showing some carious tissue at the cavity bottom for some samples (Table II), it is important to stress that the threshold for carious tissue used in the present study is originally obtained from in vitro measurements of mechanical properties of different zones of carious dentin (Pugach et al., 2009) and may not fully represent the more clinically relevant threshold of “infected dentin” (Kuboki et al., ’77). Moreover, some carious tissues eventually left at the bottom of a sealed cavity do not pose harm to longterm pulp vitality, as shown by recently controlled clinical trials (Alves et al., 2010; Maltz et al., 2012). Other specific discrepancies in caries removal, as seen for sample 5, where apparently not all carious tissue was removed (Fig. 2(A)) and, for sample 4, where MD in removed volume were slightly higher than the threshold for carious tissue (Fig. 2(C)) are related some factors as follows: in sample 5, some residual enamel originated from the fissures can still be identified, and this could have impaired further subjacent carious dentin removal (arrow, Fig. 1(E)). In sample 4, the higher mean MD value in removed tissue compared to the carious tissue threshold (1.37 g/cm3 against 1.11 g/cm3) can be explained by the presence of an arrested, dark colored lesion (arrow, Fig. 1(D)). These lesions normally present higher MD values (Bjorndal and Mjor, 2001; Neves et al., 2011c) due to formation of tertiary dentine, wearing off soft and necrotic tissue, and external pigmentation (Bjorndal and Darvann, ’99). These factors, however, can be promptly identified by the clinician during cavity preparation procedures and should not be considered to impair adequate caries removal by the papain-gel method.
Conclusion Dentin caries removal aided by a papain-based gel (PapaCarieTM) was effective in removing carious dentin up to a conservative in vitro threshold considered as endpoint of caries removal. Lesion characteristics, such as activity and morphology of enamel lesion, may also influence the caries-removal properties of the method.
Acknowledgements This study was supported by FAPERJ (APQ1 E-26/ 110.771/2012). Roseane de Araujo LourenSc o was supported by a research grant from Santander Universidades.
References Ahmed M, Davis GR, Wong FS. 2012. X-ray microtomography study to validate the efficacies of caries removal in primary
molars by hand excavation and chemo-mechanical technique. Caries Res 46:561–567. Alves LS, Fontanella V, Damo AC, Ferreira de Oliveira E, Maltz M. 2010. Qualitative and quantitative radiographic assessment of sealed carious dentin: a 10-year prospective study. Oral Surg Oral Med Oral Pathol Oral Radiol Endod 109:135–141. Banerjee A, Kidd EAM, Watson TF. 2000. In vitro evaluation of five alternative methods of carious dentine excavation. Caries Res 34:144–150. Banerjee A. 2013. Minimal intervention dentistry: part 7. Minimally invasive operative caries management: rationale and techniques. Br Dent J 214:107–111. Berry E. 2008. Image registration. A practical approach to medical image processing. Boca Raton: Taylor & Francis. pp. 161–185. Bertassoni LE, Marshall GW. 2009. Papain-gel degrades intact nonmineralized type I collagen fibrils. Scanning 31:253–258. Bjorndal L, Darvann T. 1999. A light microscopic study of odontoblastic and non-odontoblastic cells involved in tertiary dentinogenesis in well-defined cavitated carious lesions. Caries Res 33:50–60. Bjorndal L, Mjor IA. 2001. Pulp-dentin biology in restorative dentistry. Part 4: dental caries-characteristics of lesions and pulpal reactions. Quintessence Int 32:717–736. Bolte S, Cordelieres FP. 2006. A guided tour into subcellular colocalization analysis in light microscopy. J Microsc 224:213–232. Bussadori SK, Castro LC, Galvao AC. 2005. Papain gel: a new chemo-mechanical caries removal agent. J Clin Pediatr Dent 30:115–119. Bussadori SK, Guedes CC, Bachiega JC, Santis TO, Motta LJ. 2011. Clinical and radiographic study of chemicalmechanical removal of caries using Papacarie: 24month follow up. J Clin Pediatr Dent 35:251–254. Clementino-Luedemann TNR, Ilie ADN, Hickel R, Kunzelmann KH. 2006. Micro-computed tomographic evaluation of a new enzyme solution for caries removal in deciduous teeth. Dent Mater J 25:675–683. Ericson D, Zimmerman M, Raber H, et al. 1999. Clinical evaluation of efficacy and safety of a new method for chemomechanical removal of caries—a multi-centre study. Caries Res 33:171–177. Gianini RJ, do Amaral FL, Florio FM, Basting RT. 2010. Microtensile bond strength of etch-and-rinse and selfetch adhesive systems to demineralized dentin after the use of a papain-based chemomechanical method. Am J Dent 23:23–28. Khattab NM, Omar OM. 2012. Papain-based gel for chemomechanical caries removal: influence on microleakage and microshear bond strength of esthetic restorative materials. J Am Sci 8:391–399. Kidd EA. 2010. Clinical threshold for carious tissue removal. Dent Clin North Am 54:541–549. Kinney JH, Marshall GW, Jr., Marshall SJ. 1994. Threedimensional mapping of mineral densities in carious dentin: theory and method. Scan Microsc 8:204–195. Kotb RM, Abdella AA, El Kateb MA, Ahmed AM. 2009. Clinical evaluation of Papacarie in primary teeth. J Clin Pediatr Dent 34:117–123. Kuboki Y, Ohgushi K, Fusayama T. 1977. Collagen biochemistry of the two layers of carious dentin. J Dent Res 56:1233–1237. Lopes MC, Mascarini RC, da Silva BM, Florio FM, Basting RT. 2007. Effect of a papain-based gel for chemomechanical caries removal on dentin shear bond strength. J Dent Child 74:93–97. Maltz M, Henz SL, de Oliveira EF, Jardim JJ. 2012. Conventional caries removal and sealed caries in permanent teeth: a microbiological evaluation. J Dent 40:776–782. Neves AA, Coutinho E, Vivan-Cardoso M, Jaecques S, Van Meerbeek B. 2010. Micro-CT based quantitative evaluation of caries-excavation. Dent Mater 26:579–588.
A. A. Neves et al.: Micro-CT Investigation of Papain Caries Removal Neves A, Coutinho E, De Munck J, Van Meerbeek B. 2011a. Caries-removal effectiveness and minimalinvasiveness potential of caries-excavation techniques: a micro-CT investigation. J Dent 39:154–162. Neves AA, Coutinho E, Cardoso MV, Lambrechts P, Van Meerbeek B. 2011b. Current concepts and techniques for caries excavation and adhesion to residual dentin. J Adhes Dent 13:7–22. Neves AA, Coutinho E, De Munck J, Lambrechts P, Van Meerbeek B. 2011c. Does DIAGNOdent provide a reliable caries-removal endpoint. J Dent 39:351–360. Ollion J, Cochennec J, Loll F, Escude C, Boudier T. 2013. TANGO: a generic tool for high-throughput 3D image analysis for studying nuclear organization. Bioinformatics 29:1840–1841. Pugach MK, Strother J, Darling CL, et al. 2009. Dentin caries zones: mineral, structure, and properties. J Dent Res 88:71–76. Schindelin J, Arganda-Carreras I, Frise E, et al. 2012. Fiji: an open-source platform for biological-image analysis. Nat Methods 9:676–682.
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Singh S, Singh DJ, Jaidka S, Somani R. 2011. Comparative clinical evaluation of chemomechanical caries removal agent Papacarie© with conventional method among rural population in India—in vivo study. Braz J Oral Sci 10: 193–198. Taylor AM, Satterthwaite JD, Ellwood RP, Pretty IA. 2010. An automated assessment algorithm for micro-CT images of occlusal caries. Surgeon 8:334–340. Yazici AR, Atilla P, Ozgunaltay G, Muftuoglu S. 2003. In vitro comparison of the efficacy of Carisolv (TM) and conventional rotary instrument in caries removal. J Oral Rehab 30:1177– 1182. Zhang X, Tu R, Yin W, et al. 2013. Micro-computerized tomography assessment of fluorescence aided caries excavation (FACE) technology: comparison with three other caries removal techniques. Aust Dent J 58:461–467. Zou W, Gao J, Jones AS, Hunter N, Swain MV. 2009. Characterization of a novel calibration method for mineral density determination of dentine by X-ray micro-tomography. Analyst 134:72–79.
Caries-Removal Effectiveness of a Papain-Based Chemo-Mechanical Agent: A Quantitative Micro-CT Study ALINE A. NEVES,1 ROSEANE A. LOURENSc O,2 HAIMON D. ALVES,3 RICARDO T. LOPES,3 AND LAURA G. PRIMO1 1
Departamento de Odontopediatria e Ortodontia, Universidade Federal do Rio de Janeiro, Rio de Janeiro, Brazil Escola de Ci^encias da Saude, Universidade do Grande Rio, Duque de Caxias, Rio de Janeiro, Brazil 3 Laboratorio de InstrumentaSc ~ao Nuclear – COPPE – Universidade Federal do Rio de Janeiro, Rio de Janeiro, Brazil 2
Summary: The aim of this study was to access the effectiveness and specificity of a papain-based chemomechanical caries-removal agent in providing minimum residual caries after cavity preparation. In order to do it, extracted carious molars were selected and scanned in a micro-CT before and after caries-removal procedures with the papain-based gel. Similar parameters for acquisition and reconstruction of the image stacks were used between the scans. After classification of the dentin substrate based on mineral density intervals and establishment of a carious tissue threshold, volumetric parameters related to effectiveness (mineral density of removed dentin volume and residual dentin tissue) and specificity (relation between carious dentin in removed volume and initial caries) of this caries-removal agent were obtained. In general, removed dentin volume was similar or higher than the initial carious volume, indicating that the method was able to effectively remove dentin tissue. Samples with an almost perfect accuracy in carious dentin removal also showed an increased removal of caries-affected tissue. On the contrary, less or no affected dentin was removed in samples where some carious tissue was left in residual dentin. Mineral density values in residual dentin were always higher or similar to the threshold for mineral density values in carious dentin. In conclusion, the papain-based gel was effective in removing carious dentin up to a conservative in vitro threshold. Lesion characteristics, such as activity and morphology of enamel lesion, may also influence caries-removal
Address for reprints: Aline de Almeida Neves, Universidade Federal do Rio de Janeiro, Departamento de Odontopediatria e Ortodontia, Rua Prof. Rodolpho Palo Rocco, 325, Cidade Universitaria, Rio de Janeiro 21941-617, Brazil E-mail: [email protected] Received 7 February 2015; revised 25 February 2015; Accepted with revision 10 March 2015 DOI: 10.1002/sca.21206 Published online XX Month Year in Wiley Online Library (wileyonlinelibrary.com).
properties of the method. SCANNING 9999:1–7, 2015. © 2015 Wiley Periodicals, Inc. Key words: residual caries, cariesremoving techniques, micro-CT, Papacarie
Introduction The concept of minimally invasive dentistry, among broader definitions, precludes a systematical and conservative removal of decayed tissue and its replacement by adhesive restorations to appropriately restore the integrity and aesthetics of the tooth (Banerjee, 2013). This approach means removing only infected and irreversibly destroyed dentin and leaving, as residual substrate for further adhesive procedures, a slightly demineralized caries-affected dentin (Kidd, 2010). Based on this philosophy, chemo-mechanical cariesremoving agents have been developed (Neves et al., 2011a). These products act on the degradation of the partially demineralized and altered dentin matrix which has been previously exposed to bacterial action (infected dentin), allowing its removal and preventing damage to the underlying remineralizable tissue (affected dentin) (Ericson et al., ’99). Among them, CarisolvTM (RLS Global AB, Gothenburg, Sweden), a sodium hypochloride-based gel added of an amino acid mixture became very popular among general dentists around the world. CarisolvTM has already proved its effectiveness and accuracy in the conservative removal of dentin carious tissue by different microscopy methods (Banerjee et al., 2000; Yazici et al., 2003; Neves et al., 2011b). More recently, a papain-based gel, also intended for chemomechanical caries removal has been developed and marketed (PapaCarieTM, Formula e ASc ~ao, S~ao Paulo, SP, Brazil) (Gianini et al., 2010; Khattab and Omar, 2012). This product contains the enzyme papain, a protease of broad proteolytic activity extracted from the latex of leaves and fruits of Carica papaya tree. It is
2
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suggested that this product also acts exclusively on breaking down the partially degraded collagen, without damaging intact collagen fibrils (Bussadori et al., 2005). The selective interaction of the papain enzyme with the affected components of the carious dentin is attributed to the lack of an antiprotease a-1-anti-trypsin, which inhibits protein digestion in sound collagenbased tissues, although this statement has recently been partially contested (Bertassoni and Marshall, 2009). In Brazil and other South American countries, PapaCarieTM has found a broad range of applications in dentistry, due to its clinical applicability, market availability, and relative low cost (Lopes et al., 2007), being nowadays one of the most used to conservatively remove carious dentin using a chemomechanical method in those regions. Its clinical effectiveness has also already been studied, and apparently, the use of the papain gel results in residual dentin with similar microbiological characteristics than that excavated by means of conventional burs (Kotb et al., 2009; Bussadori et al., 2011; Singh et al., 2011). In terms of lesion histopathology, however, the laboratorial micro-CT technique has a clear application in the study of caries-removing methods by allowing nondestructive analysis of samples (longitudinal evaluation), as recently demonstrated (Neves et al., 2010). Although some studies have been published on this respect for conventional and other improved cariesremoving methods (Neves et al., 2011; Ahmed et al., 2012; Zhang et al., 2013), caries-removal effectiveness of a papain-based caries-removing gel has not been accessed longitudinally in vitro. Thus, the aim of the present study was to access volumetric parameters related to effectiveness (mineral density of removed volume and residual dentin tissue) and specificity (relation between carious dentin in removed volume and initial caries) of dentin cariesremoval procedures performed by a papainbased chemo-mechanical caries-removal agent.
Materials and Methods Sample Selection and Preparation
Extracted human molar teeth presenting occlusal carious lesions were selected from a pool of extracted elements stored for less than 6 months in 1% aqueous chloramine solution under refrigeration. The teeth were collected in a faculty practice after written consent given by the patient. This studied complied with all ethical aspects of donation and use of biological human tissue and has been approved by the local university ethical committee. After ultrasonic cleaning of plaque, calculus, and other debris, a digital radiograph was obtained, so that
teeth without carious involvement in dentin and those in which the carious lesion was approaching the pulp chamber, were excluded. Ten teeth were initially selected and mounted by the roots in gypsum stubs to facilitate further manipulation. After that, the occlusal surface was submitted to a perpendicular flat cut with a diamond disk on a low speed cutting machine (Isomet, Buehler, Lake Bluff, IL) to expose the subjacent carious dentin and exclude any carious enamel. This procedure was performed to exclude possible areas of carious enamel, which by having similar micro-CT gray values than dentin, could impair some of the following proposed quantitative micro-CT analysis, as previously described (Neves et al., 2010).
Micro-CT Acquisition, Reconstruction, and Calibration
The teeth were subjected to a baseline scanning procedure using a Skyscan 1173 micro-CT system (Bruker micro-CT, Kontich, Belgium) (baseline stacks). The acquisition settings used were 70 kV, 114 mA, 14.6 mm pixel size, 260 ms exposure, frame averaging of 5 and rotation step of 0.5˚ through 360˚. A flatfield reference was obtained before the first scan and the random-movement amplitude was set to 20 lines to reduce ring artefacts. A 1-mm aluminium filter was also used to reduce the beam hardening effect. After cariesremoval procedures, the micro-CT scan was repeated for all studied samples using the same parameters described above (treated stacks). The acquired micro-CT projections were reconstructed into cross-section slices using a proprietary software interface (NRecon v.1.6.9, Bruker micro-CT). Specific reconstruction settings included a 50% beamhardening correction, ring artifact correction factor of 5 and input of optimal contrast limits (0–0.075) for all stack reconstructions. For calibration of micro-CT gray values into mineral density (MD) values of hydroxyapatite (HAp), three phantoms made of fine calcium hydroxyapatite powder embedded in epoxy resin, with different mineral densities (0.25, 0.75, and 3.14 g/cm3), were scanned and reconstructed using the same parameters described above. Gray values obtained by micro-CT were further converted into MD values of HA (g/cm3) after definition of a calibration curve using a linear equation fit. Caries-Removal Procedures
The teeth were treated with the papain-based cariesremoval gel (PapaCarie Duo1, Formula e ASc ~ao, S~ao Paulo, Brazil) following the manufacturer’s instructions. In short, the gel was dispensed over the carious lesion, and after 30 s the carious tissue was gently
A. A. Neves et al.: Micro-CT Investigation of Papain Caries Removal
scrapped with a blunt spoon excavator. When the gel turned grayish, the cavity was cleaned with a stream of water, air-dried, a new batch of gel was applied, and excavation procedure was started. The whole treatment was repeated until no more dentin tissue could be removed by the gel-excavation combination.
Image Registration of Baseline and Treated Image Stacks
After cross-section slices reconstruction, each treated stack was registered using its corresponding baseline stack as the template, by means of the Rigid Registration plugin implemented in the FIJI software interface (Schindelin et al., 2012). The rigid registration method defines six degrees of freedom for matching boundary surfaces in both images, and for this reason, this method was chosen since image boundaries were similar before and after caries-removal procedures. The similarity metric was based on the maximized mutual information algorithm, a registration method based on finding the alignment, or pairing of points, which match both histograms as closely as possible (Berry, 2008). The optimization steps of the registration algorithm were repeated until image stacks did not differ by more than 0.4 tolerance. In fact, after the stacks were placed at the same spatial position, further image operations allowed calculation of caries-removal parameters.
Quantitative Evaluation of Caries Removal
Pre-processing of digital images included a tridimensional median filter with a 2 2 2 kernel (8-voxel). The filtering algorithm used was part of the 3D ImageJ Suite built in the Process menu of the Fiji open-source software implementation (Ollion et al., 2013). After definition of cut-off points and arithmetic operations, the segmented
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TABLE I Classification of dentin substrate based on MD value intervals Type of dentin substrate Sound dentin (x)
Affected dentin Carious dentin
Range of MD values used to classify the dentin substrate Mean MD over a circular 100 pixel-radius ROI interpolated over 20 slices in unaffected dentin areas >1.11 g/cm3 HA x 0–1.11 g/cm3 HA
volumes were quantified by means of the 3D Object Counter plugin, after proper calibration of the resolution (Bolte and Cordelieres, 2006). For definition of the carious lesion volume, the cutoff point corresponding to a MD of 1.11 g/cm3 HA was defined in both baseline (initial carious volume) and treated stacks (residual dentin volume), based on previously published studies on microhardness (Kinney et al., ’94) and micro-CT (Neves et al., 2010; Neves et al., 2011c) of dentinal carious tissue. Tooth tissue presenting values that were lower or equal than the selected threshold were considered as carious tissue. Tooth tissue belonging to higher MD values than the threshold, but lower than sound dentin values obtained for the specific sample were considered as affected dentin (Table I). Removed (excavated) volume was obtained for each treated sample after an arithmetic XOR operation between baseline and treated stacks and volumetric quantification. From the removed tissue volumes, the carious and affected dentin contributions were calculated by defining specific MD thresholds, as described in Table I.
Results From 10 initial selected samples, four were excluded after the baseline scan due to an initial carious volume
Fig 1. Stereomicroscopic images of the studied samples 1–6 (a–f) before and after caries removal. Arrow in (d) indicates a dark pigmented arrested lesion, while arrow in (e) indicates residual enamel areas.
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lower than 0.5 mm3, which was considered too low to allow appropriate quantitative measurements, resulting in a final sample of six teeth included in the analysis. Figure 1 shows occlusal surface images of each studied sample before and after caries removal with the papain-based agent aided by a conventional spoon excavator. The initial carious dentin volume varied from 0.616 to 5.196 mm3, as seen in Figure 2(A) (dark gray bars). The respective excavated (removed) volumes (light gray bars on top) are also shown. Except for sample 5, in all other studied teeth, the removed dentin volume was similar or higher than the initial carious volume, indicating that the papain-based method was able to effectively remove dentin tissue. For the lesion in
sample 5, further removal of dentin could have been impaired by the remaining enamel area remaining over the lesion (red arrow, Fig. 1(E)). The specificity of carious tissue removal can be inferred by examining the volumetric difference between initial carious volume (dark gray bars, Fig. 2 (A)) and carious volume in excavated dentin (black dots, Fig. 2(A)). The difference between these two parameters results in the volume of carious tissue left at the cavity (Table II). It is possible to note that in samples in which an almost perfect accuracy in carious dentin removal was observed (samples 2, 4, and 6) were, in fact, followed by increased removal of caries-affected tissue (Fig. 2(B), dark gray bars). On the contrary, in those samples where apparently some carious tissue was left (samples 1, 3, and 5) less or no affected dentin was removed at the expense of some carious tissue in residual dentin. Figure 2(C) shows mean MD values in sound dentin for each studied sample (black lines). Mean MD values in residual dentin were always higher or, in case of sample 5, similar to the used threshold of 1.11 g/cm3 HA for carious dentin (dark gray line). Indeed, samples showing higher volumes of carious tissue left in the cavity (1, 3, and 5) showed also the higher differences between MD values in sound dentin and MD at residual dentin. Figure 3 shows volumetric renderings and selected micro-CT slices of baseline and treated stacks (sample 2) highlighting the effectiveness of carious dentin removal.
Discussion The present results show that excavation with the papain-based chemo-mechanical agent (PapaCarieTM) was able to reach an acceptable in vitro threshold, as seen by the MD in residual dentin (Fig. 2(C)). In all studied samples, carious tissue comprised most of the removed tissue after cavity preparation. In fact, mean MD in residual dentin and within excavated tissue were similar to those found in the literature (ClementinoLuedemann et al., 2006; Neves et al., 2011c; Ahmed et al., 2012).
TABLE II Carious dentin volume left after excavation with the papain-based gel Sample
Fig 2. Caries-removal parameters evaluated in each studied sample. (a) Initial caries volume, removed (excavated) tissue volume and carious tissue volume in removed tissue; (b) contributions of carious and affected dentin in removed volume; (c) mineral density values in sound and residual dentin and mean values in removed tissue volume.
1 2 3 4 5 6
Carious volume left at the cavity bottom 1.21 mm3 0.23 mm3 0.68 mm3 0.05 mm3 0.99 mm3 0.05 mm3
A. A. Neves et al.: Micro-CT Investigation of Papain Caries Removal
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Fig 3. Volume renderings and selected micro-CT slices of baseline and treated stacks (sample 2) calibrated for dentin mineral density values (g/cm3). (a) 3D volume rendering of baseline tooth; (b) 3D volume rendering of treated tooth; (c) micro-CT slice of baseline tooth; (d) micro-CT slice of treated (excavated) tooth. Arrow point to locations of carious dentin at the threshold for removal, suggesting a conservative excavation result.
Methodologically, the micro-CT technique in association with digital image analysis has a clear advantage over conventional microscopy methods for the study of caries-removing techniques as follows: it is nondestructive, allowing comparison of results obtained from the same sample before and after caries removal; its tridimensional nature allows obtaining volumetric results from the whole bulk of the specimen and also allows calibration against HA phantoms (g/cm3 of HA) into actual dentin mineral density data (Zou et al., 2009; Neves et al., 2010). To further increase the accuracy of the technique, in the present study, carious enamel areas were mechanically removed from the samples by a flat cut through the occlusal surface. This was performed to overcome the limitation to a fully quantitative volumetric approach on dentin caries removal related to the similarity in gray values between carious enamel and sound dentin (Neves et al., 2010; Taylor et al., 2010). However, this procedure also resulted in elimination of
some carious dentin areas, increasing the need to exclude some samples in the present study. Indeed, samples presenting less than 0.5 mm3 of initial carious volume were excluded since accurate results could not be achieved, as the micro-CT system used has limitations regarding spatial resolution. In fact, it has been already shown that chemomechanical methods resulted in the best agreement in conservative caries removal compared to conventional (burs) and laser-assisted methods, following similar mineral density parameters as used in the present study (Neves et al., 2011c). Even when a lower threshold for carious tissue is used, chemo-mechanical methods have proven to be more conservative than burs in establishing the endpoint of caries removal (Ahmed et al., 2012). The more specificity of chemo-mechanical methods is probably due to the specific chemical dissolution caused by the active component in these products, combined to the limited mechanical action of a hand instrument more conservatively used.
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Regarding the results showing some carious tissue at the cavity bottom for some samples (Table II), it is important to stress that the threshold for carious tissue used in the present study is originally obtained from in vitro measurements of mechanical properties of different zones of carious dentin (Pugach et al., 2009) and may not fully represent the more clinically relevant threshold of “infected dentin” (Kuboki et al., ’77). Moreover, some carious tissues eventually left at the bottom of a sealed cavity do not pose harm to longterm pulp vitality, as shown by recently controlled clinical trials (Alves et al., 2010; Maltz et al., 2012). Other specific discrepancies in caries removal, as seen for sample 5, where apparently not all carious tissue was removed (Fig. 2(A)) and, for sample 4, where MD in removed volume were slightly higher than the threshold for carious tissue (Fig. 2(C)) are related some factors as follows: in sample 5, some residual enamel originated from the fissures can still be identified, and this could have impaired further subjacent carious dentin removal (arrow, Fig. 1(E)). In sample 4, the higher mean MD value in removed tissue compared to the carious tissue threshold (1.37 g/cm3 against 1.11 g/cm3) can be explained by the presence of an arrested, dark colored lesion (arrow, Fig. 1(D)). These lesions normally present higher MD values (Bjorndal and Mjor, 2001; Neves et al., 2011c) due to formation of tertiary dentine, wearing off soft and necrotic tissue, and external pigmentation (Bjorndal and Darvann, ’99). These factors, however, can be promptly identified by the clinician during cavity preparation procedures and should not be considered to impair adequate caries removal by the papain-gel method.
Conclusion Dentin caries removal aided by a papain-based gel (PapaCarieTM) was effective in removing carious dentin up to a conservative in vitro threshold considered as endpoint of caries removal. Lesion characteristics, such as activity and morphology of enamel lesion, may also influence the caries-removal properties of the method.
Acknowledgements This study was supported by FAPERJ (APQ1 E-26/ 110.771/2012). Roseane de Araujo LourenSc o was supported by a research grant from Santander Universidades.
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