MICROSCOPY RESEARCH AND TECHNIQUE 77:52–56 (2014) Effects of Chemical Agents on Physical Properties and Structure of Primary Pulp Chamber Dentin FERNANDA MIORI PASCON,1 KAMILA ROSAMILIA KANTOVITZ,1 JULIANA FRANCIELE GASPAR,1 ANDREIA BOLZAN DE PAULA,2 AND REGINA MARIA PUPPIN-RONTANI1* 1 2 Pediatric Dentistry Department, Piracicaba Dental School, University of Campinas, Piracicaba, S~ ao Paulo, Brazil Operative Dentistry Department, Piracicaba Dental School, University of Campinas, Piracicaba, S~ ao Paulo, Brazil KEY WORDS dental pulp cavity; hardness; root canal irrigants; scanning electron microscopy ABSTRACT This study evaluated the effects of chemical agents on the physical properties and structure of primary pulp chamber dentin using surface roughness, microhardness tests, and scanning electron microscopy (SEM). Twenty-five primary teeth were sectioned exposing the pulp chamber and were divided into five groups (n 5 5): NT, no treatment; SH1, 1% sodium hypochlorite (NaOCl); SH1U, 1% NaOCl 1 Endo-PTCV; SH1E, 1% NaOCl 1 17% EDTA; and E, 17% EDTA. After dentin treatment, the specimens were submitted to roughness, microhardness testing, and SEM analysis. Roughness and microhardness data were submitted to one-way ANOVA and Tukey’s test (P < 0.05). The SH1E group showed the highest roughness, followed by the E group (P < 0.05) when compared with the NT, SH1, and SH1U groups. Microhardness values of SH1 and SH1U showed no significant difference as compared to the NT (control) group (P > 0.05). Microhardness values could not be obtained in the EDTA groups (SH1E and E). The presence of intertubular dentin with opened dentin tubules was observed in the NT, SH1, and SH1U groups. SH1E showed eroded and disorganized dentin with few opened tubules and the intertubular/peritubular dentin was partially removed. Considering the physical and structural approaches and the chemical agents studied, it can be concluded that NaOCl and NaOCl associated with Endo-PTCV were the agents that promoted the smallest changes in surface roughness, microhardness, and structure of the pulp chamber dentin of primary teeth. Microsc. Res. Tech. 77:52–56, 2014. V 2013 Wiley Periodicals, Inc. R R C INTRODUCTION Primary teeth with pulpal exposure or pathology must be treated by root canal treatment or extraction (Carrotte, 2005). The premature loss of primary teeth leads to undesirable tooth movements of primary and/ or permanent teeth, including the loss of arch length (Carrotte, 2005). Thus, maintaining arch length is important for good masticatory function and the future eruption of the permanent dentition with optimal occlusion development (Carrotte, 2005). Root canal treatment is a common dental procedure that is usually performed with mechanical debridement in the presence of chemical agents (McComb and Smith, 1975). Because irrigation with an inert solution cannot adequately reduce the microbial population in a root canal system, disinfection with other agents, such as NaOCl, is an important step in assuring the optimal bacterial decontamination of the canals (Zehnder, 2006). However, it has been reported that these agents are capable of causing alterations in the chemical composition of pulp chamber dentin, such as the inorganic content evaluated by Raman analysis (Borges et al., 2008) and the calcium/phosphorus ratio of the root canal dentin surface analyzed using inductively coupled plasma atomic emission spectrometry technique that measured the levels of five elements calcium, phosphorus, magnesium, potassium, and C V 2013 WILEY PERIODICALS, INC. sulfur (Ari and Erdemir, 2005). Despite the study of Borges et al. (2008), little is known at present regarding the mechanical and structural properties of primary pulp chamber dentin after using chemical agents. This subject can be important to suggest the performance of adhesion in this substrate (pulp chamber/restorative material) and can help the pediatric dentists to choose a chemical agent that present lower alterations in the substrate and to enhance the adhesion process. In addition, these alterations can be relevant for the long-term success of endodontics because they may affect the coronal bonding strength of dental materials (Kijsamanmith et al., 2002; Santos et al., 2006). Knowledge of the mechanical properties is important to predict the behavior of the dentin/restoration interface and for understanding how endodontic procedures alter dentin strength. As microhardness is dependent on the composition and surface structure (Panighi and G’Sell, 1992), attention has been focused *Correspondence to: R.M. Puppin-Rontani; Pediatric Dentistry Department, Piracicaba Dental School, University of Campinas, Av. Limeira, 901, 13414-903 Piracicaba, SP, Brazil. E-mail: rmpuppin@fop.unicamp.br Received 21 April 2013; accepted in revised form 24 October 2013 REVIEW EDITOR: Dr. Chuanbin Mao DOI 10.1002/jemt.22312 Published online 12 November 2013 in Wiley Online Library (wileyonlinelibrary.com). EFFECTS OF CHEMICAL AGENTS ON PULP CHAMBER on the relationship between dentin microhardness and the structural changes associated with root canal therapy and the application of materials within root canals. Thus, this issue is important in restorative dentistry and endodontics because determining microhardness can provide valuable evidence of mineral loss or gain in the dental hard tissue (Arends and ten Bosch, 1992). During endodontic treatment, enamel and coronal as well as radicular dentin are exposed to chemical agents deposited in the pulp chamber (Saleh and Ettman, 1999), which is submitted to agents with different wettability and surface tensions that tend to affect its mineral and organic contents and the surface energy (Ari and Erdemir, 2005). In addition, the chemical agents might change the radicular and coronal dentin wettability, which might affect the bacteria adhesion (Pringle and Fletcher, 1983) and the interaction between the dentin and restorative materials (Baier, 1992). Thus, the integrity of the dentin/resin bonding in the pulp chamber has important implications in improving the success of root canal treatment (Kijsamanmith et al., 2002). After the irrigation, the characteristics of dentinal tissues can be irreversibly altered. This may affect the interaction with the material used for coronal sealing (Santos et al., 2006). Besides, it is known that the prognosis of endodontically treated teeth does not rely on the apical sealing after the chemomechanical preparation of root canals; it is highly dependent on coronal sealing (Belli et al., 2001). The aim of this study was to evaluate the effects of chemical agents on the surface roughness, microhardness tests and scanning electron microscopy (SEM) of primary pulp chamber dentin. The tested hypothesis was that the agents studied would affect the physical properties and structure of the pulp chambers of primary teeth. MATERIALS AND METHODS Specimen Preparation Twenty-five sound primary human anterior teeth extracted for clinical or orthodontic reasons were used in this study. This research was conducted after obtaining approval from the Ethics Committee of Piracicaba Dental School, University of Campinas, and was undertaken according to their ethical principles (#041/2006). The teeth were cleaned and stored in 0.5% chloramine T solution at 4 C for up to 2 months after extraction. Debris and soft tissue remnants were removed and the roots were sectioned at the cementoenamel junction using a double-face diamond saw (KG Sorensen, S~ ao Paulo, SP, Brazil) and then discarded. The crowns were sectioned longitudinally in the mesiodistal direction to expose the pulp chamber. One side of the crown was randomly selected and embedded in polystyrene resin (Piraglass, Piracicaba, SP, Brazil), leaving the dentin pulp chamber exposed. The specimens were polished with 400-, 600-, and 1200-grit polishing papers (Arotec, S~ ao Paulo, SP, Brazil) under constant water irrigation and polished with 1.0-mm diamond paste (Buheler Metadi II, Buheler, Lake Buff, IL). The specimens were then immersed in deionized water and sonicated to clear the surface because during the endodontic treatment, a smear layer was not Microscopy Research and Technique 53 created on the pulp chamber. The specimens were randomly distributed into the following five groups (n 5 5): NT, no treatment (control group); SH1, 1% NaOCl (Proderma, Laboratory of Manipulation, Piracicaba, S~ ao Paulo, SP, Brazil); SH1U, 1% NaOCl 1 EndoPTCV gel (composed of 10% urea peroxide, 75% Carbowax 1500, and 15% tween 80) (Proderma, Laboratory of Manipulation, Piracicaba, S~ ao Paulo, SP, Brazil; Formula and Aç~ ao, Laboratory of Manipulation, S~ ao Paulo, SP, Brazil, Batch number 0028); SH1E, 1% NaOCl 1 17% EDTA (Proderma, Laboratory of Manip~o Paulo, SP, Brazil); and E, 17%, ulation, Piracicaba, Sa EDTA (Proderma, Laboratory of Manipulation, Piracicaba, S~ ao Paulo, SP, Brazil). R Experimental Procedure The specimens were individually immersed in 2 mL of the respective agent in polypropylene vials, which were agitated in an ultrasound bath at 37 C for 30 min. The treatment with chemical agents used in this study was validated in a previous study (Borges et al., 2008). The agents were changed every 5 min to prevent saturation of them. For treatments involving NaOCl and Endo-PTCV (SH1U), the gel was applied over the pulp chamber using a plastic spatula, and the solution was immediately dropped over the gel. In the SH1E group, a final flush of EDTA was used after using the NaOCl, as mentioned above. R Roughness Determination The surface roughness analyses were performed using a Surfcorder SE 1700 surface roughnessmeasuring instrument (Kosaka Corp, Tokyo, Japan). Three readings were recorded for each specimen at three different locations (parallel, perpendicular, and oblique) to scan the entire specimen area (pulp chamber). Then, a 0.25-mm cutoff was set and roughness measurements were recorded in Ra (mm). The Ra parameter describes the overall roughness of a surface and can be defined as the arithmetical value of all absolute distances of the roughness profile from the centerline within the measuring length. Ra values for each specimen were taken across the diameter over a standard length of 1.25 mm. The average of those three readings was used as the value for each specimen. Microhardness Determination The Vickers hardness values were measured using a Vickers diamond microhardness indenter under a 50-g load perpendicular to the indentation surface for 5 s (HMV 2000, Shimadzu, Tokyo, Japan). These parameters were chosen based on a pilot study, when it was observed that these were a minimum load/dwell time to visualize the pyramidal–indentation area. Three indentations spaced 200 mm apart were made on the pulp chamber for each specimen. The values were recorded as a Vickers hardness number (VHN) and the dentin microhardness of each specimen was obtained by averaging the mean VHN values for the three measurements. Scanning Electron Microscopy Impressions of all of the specimen surfaces were prepared using a low-viscosity polyvinyl siloxane material 54 F.M. PASCON ET AL. TABLE 1. Mean 6 standard deviations (SD) of the roughness numbers (Ra) and dentin microhardness numbers (VHN) for primary teeth Agents Roughness numbers (Ra) Microhardness numbers (VHN) NT SH1 SH1U SH1E E 0.254 6 0.015c 0.187 6 0.019c 0.151 6 0.016c 1.177 6 0.159a 0.612 6 0.335b 16.16 6 2.27A 11.46 6 2.26A 14.74 6 8.75A * * NT, no treatment; SH1, 1% sodium hypochlorite (NaOCl); SH1U, 1% V NaOCl 1 Endo-PTC ; SH1E, 1% NaOCl 1 17% EDTA; and E, 17% EDTA. Similar small letters in column (Ra) indicate no significant difference according to the ANOVA and Tukey’s test (P > 0.05), regarding groups. Similar capital letters in column (VHN) indicate no significant difference according to the ANOVA and Tukey’s tests (P > 0.05), regarding groups. *Microhardness could not be measured in the SH1E and E groups. R (Flexitime, Heraeus Kulzer, Hanau, Germany) to produce replicas. The replicas were poured with epoxy resin (Buehler, Lake, Buff, IL), gold-sputter coated (Balzers SCD 050/BAL-TEC, Schalksm€ uhle, Germany) and then observed in SEM (JEOL-JSM 5600LV, Tokyo, Japan) at an accelerating voltage of 15 kV at a working distance of 20 mm and a magnification of 1,0003. All micrographs were evaluated qualitatively regarding the dentin tubules and peritubular/intertubular dentin. Statistical Analysis The microhardness and roughness values were statistically analyzed with a one-way ANOVA and a means comparison was conducted using a post hoc Tukey’s multiple comparison test at the 95% confidence level. The software SAS system (version 9.2-SP2, SAS Institute Inc., Cary, NC, 2002) was used. RESULTS The mean and standard deviation of the roughness numbers (Ra) and dentin microhardness numbers (VHN) are shown in Table 1. The roughness values (Ra) indicated a statistically significant difference among the groups (P < 0.05). The SH1E group showed roughness significantly higher than the others groups (P < 0.05). The E group showed roughness significantly higher than NT, SH1, and SH1U (P < 0.05). Regarding microhardness, there was no significant difference among SH1, SH1U, and NT (control groups) (P > 0.05). Microhardness of the EDTA groups cannot be measured because of the erosive effect of EDTA on peritubular and intertubular dentin (Fig. 1). The representative SEM images are shown in Figure 1. SEM observations revealed different structural features for the groups. In the NT, SH1, and SH1U groups, the presence of intertubular dentin with open dentin tubules was observed. SH1E showed eroded and disorganized dentin with few opened tubules. In addition, the intertubular and peritubular dentin was partially removed. DISCUSSION Current concepts of canal chemomechanical preparation imply that chemical application is necessary to obtain a clean root canal system, which should be completely sealed. These procedures may induce considerable changes in the surface morphology of dentin, which may also exert changes in the mechanical and physical properties (Pascon et al., 2009). Thus, the tested hypothesis was accepted. EDTA-induced considerable changes to the surface roughness, microhardness and structural aspects in the pulp chamber dentin, which demonstrated that physical properties can be modified with chemical treatments (Eldeniz et al., 2005; Saleh and Ettman, 1999). NaOCl did not induced changes on roughness and microhardness. Thus, the tested hypothesis was partially accepted. Groups SH1 and SH1U showed similar roughness and microhardness than control group. Our results showed the highest surface roughness for dentin treated with 1% NaOCl associated with 17% EDTA. These results were similar to a published study that showed a roughness increase when associated solutions were used on root canal dentin of permanent teeth (Eldeniz et al., 2005). The roughness increase when EDTA is associated with irrigant agents can be related to its demineralizing effect on root canal dentin (Eldeniz et al., 2005). In addition, an increase in root dentin roughness was reported when 2.5% to 5.25% NaOCl was used alone (Ari et al., 2004; Hu et al., 2010). These results were quite different from ours, which showed that when 1% NaOCl was used alone or associated with Endo-PTCV there was no significant influence on the roughness. This could be explained by the NaOCl concentration used in the present study (1% NaOCl). This concentration is used frequently in endodontics of primary root canals since it has less irritant potential than 2.5% and 5.25%. The significant changes in roughness observed following the NaOCl treatment showed its potent direct effect on the organic and mineral content of dentin (Borges et al., 2007), and when this agent is associated with other agent, such as EDTA, the effects can be potentiated. As shown in the present study, the association between EDTA and NaOCl can increase surface roughness showing the combined effects of these agents. The intertubular dentin eroded can be related with higher surface roughness values as can be observed in Figure 1 (SH1E). A principal requirement for strong adhesive bonds is that the surface be clean and, therefore, in a high energy state (Marshall et al., 1993). In addition, wettability is enhanced for most practical dental situations by the presence of micro-surface roughness (Marshall et al., 2010). It is presumed that surface roughness promotes wettability by increasing the surface area and that the bond between the adhered surface and the adhesive will subsequently be stronger (Ayad et al., 2009). However, Tay et al. (2007) suggested that is difficult to simultaneously remove the smear layer and render dentinal tubules patent without demineralizing dentin with the commonly used smear layer-removing endodontic irrigants. Consequently, the presentation of a demineralized collagen matrix might be viewed as a consequence that accompanies the use of calciumdepleting agents as final rinses (Tay et al., 2007). Regarding the methodology adopted for the hardness measures, studies have shown the suitability and practicality of the Vickers microhardness test for evaluating the surface changes of dental hard tissues treated with chemical agents (Ballal et al., 2010; Unl€ u et al., 2004). When 1% NaOCl (used alone or associated with Endo-PTCV) was used, the results were similar to R R Microscopy Research and Technique EFFECTS OF CHEMICAL AGENTS ON PULP CHAMBER 55 Fig. 1. Representative SEM images of pulp chamber dentin surface replicas (1,0003), regarding the groups: NT, no treatment; SH1, R ; SH1E, 1% NaOCl; SH1U, 1% NaOCl associated with Endo-PTCV 1% NaOCl associated with 17% EDTA; and E, 17% EDTA. SEM observations revealed different structural features for the experimental groups. Major alterations were observed in the SH1E and E groups. SH1E showed eroded and disorganized dentin with few opened tubules. The intertubular and peritubular dentin was partially removed (asterisk). For the NT, SH1, and SH1U groups, the presence of intertubular dentin with open dentin tubules was observed. White arrows—opened dentin tubules; black arrows— intertubular dentin. the control group. However, the groups treated with EDTA yielded inconclusive results because these specimens could not be measured with the Vickers hardness test in the present study. Chelating agents are used for irrigation during mechanical instrumentation of the root canal system as adjuncts for root canal preparation and for the smear layer removal (McComb and Smith, 1975; Zehnder, 2006). Thus, the chelating action of EDTA solutions induced a softening potential on the calcified components of dentin, and a subsequent reduction in the microhardness was observed (Ari et al., 2004; Sayin et al., 2007). Previous studies have verified that 15% to 17% EDTA used on the root canal dentin also decreases microhardness (Ari et al., 2004; Sayin et al., 2007). Some studies have even detected erosion on the dentinal tubules caused by the dissolution of intertubular and peritubular dentin (Beltz et al., 2003; Niu et al., 2002; Saghiri et al., 2009). The explanation for this result is that 17% EDTA dissolves the mineral content of dentin (Beltz et al., 2003). In addition, studies have also confirmed the reduction of microhardness after irrigation with 17% EDTA as a result of its excessive demineralizing effect (Ari et al., 2004; Saghiri et al., 2009). This process could explain why, in the present study, it was not possible to Microscopy Research and Technique 56 F.M. PASCON ET AL. measure the pulp chamber hardness of the groups treated with EDTA. The chemical agents used in the present study are the most frequently employed during root canal therapy of primary teeth. All agents influenced the physical properties and structure of dentin surfaces. The SEM images confirmed the effect of these agents on dentin and their potential effect on roughness values. The erosive effect of EDTA on dentin has been established (Torabinejad et al., 2002), just as we detected the eroded and disorganized peritubular and intertubular dentin, in the SEM micrographs (Fig. 1) after using EDTA. From a clinical viewpoint, an ideal agent should be able to remove debris and the smear layer, to disinfect the root canal system, to allow penetration of antimicrobial agents into the dentinal tubules, and should have substantive antimicrobial activity. In addition, it should have no adverse effects on the sealing ability of filling materials (Torabinejad et al., 2002) and should not make changes in the dentin physical and chemical properties. Studies on the physical properties are important to provide clinicians with an understanding of how these tissues react under clinical conditions and for predicting the behavior of the tooth/restoration interface in a clinical setting. In this context, a better understanding of primary teeth is needed to improve dentin-bonding methods and to facilitate more effective and successful dental restorations. In conclusion, when considering the physical and structural approaches and the chemical agents studied, NaOCl and NaOCl associated with Endo-PTCV were the agents that promoted the smallest changes in the surface roughness, microhardness, and structure of pulp chamber dentin in primary teeth. R ACKNOWLEDGEMENTS This research was supported by the FAPESP – S~ ao Paulo Research Support Foundation (process #05/ 58561-1). The authors are grateful to the Pediatric Dentistry Department and Dental Materials, Piracicaba Dental School, University of Campinas for their cooperation. 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