Structural Analysis of Bovine Root Dentin after Use of Different Endodontics Auxiliary Chemical Substances Danna Mota Moreira, DDS,* Jose´Fla ´vio Affonso Almeida, PhD,* Caio Cezar Randi Ferraz, PhD,* Brenda Paula Figueiredo de Almeida Gomes, PhD,* Se´rgio Roberto Peres Line, PhD, † and Alexandre Augusto Zaia, PhD* Abstract Introduction: The use of chemical substances in endodontics during chemical-mechanical preparation may alter the structure of dentin, mainly collagen, which contributes considerably to the mechanical properties of this structure. This study evaluated the effect of different auxiliary chemical substances during endodontic treat- ment in root canal walls by implementing polarized light microscopy (PLM) and scanning electron microscopy (SEM). Methods: Sixty bovine incisors were randomly divided into six groups (n = 10): G1, 5.25% NaOCl + 17% EDTA; G2, 2% chlorhexidine (CHX) gel; G3, 5.25% NaOCl; G4, 17% EDTA; G5, 2% CHX + 17% EDTA; and G6, 0.9% sodium chloride solution (control). Teeth were chemomechanically prepared and fractured into two fragments. One fragment was used for PLM, and the other was used for SEM analysis. Results: Groups treated with 5.25% NaOCl, whether associated or not associated to 17% EDTA, presented a significantly different birefringence pattern compared with the control group. The results revealed that groups irrigated with 2% CHX and EDTA, independently or combined, did not present any significant differences compared with the control group. SEM analysis showed structural alterations of the root canal inorganic matrix for all groups that used 17% EDTA. Conclusions: It is possible to conclude that 5.25% NaOCl, whether associated or not associated with 17% EDTA, causes alterations in the dentin collagen. The obtained SEM images revealed demineralization areas for groups that used 17% EDTA alone or associated with other substances. (J Endod 2009;35:1023–1027) Key Words Birefringence, chlorexidine, dentin, sodium hypochlorite T he use of chemical substances in endodontics during chemical-mechanical prepa- ration may alter the structure of dentin, mainly collagen, which contributes consid- erably to the mechanical properties of this structure. This may interfere with the penetration of monomers to within the demineralized dentin structure. The failed pene- tration compromises the bond strength between adhesive systems and the dental struc- ture and consequently puts the quality and durability of direct restorations and fiber post cementation at risk (1, 2). Among the auxiliary chemical substances used in endodontics during chemical- mechanical preparation, sodium hypochlorite, in different concentrations, has been the most commonly used. This endodontic irrigant has the ability to destroy a broad spectrum of microbes and to dissolve organic materials; however, it is known that sodium hypochlorite solutions has cytotoxic effects and that it alters the organic compo- nents of dentin, specially collagen (3, 4). Consequently, sodium hypochlorite may affect mechanical dentin properties via the degradation of organic dentin components. Chlorhexidine gluconate is another substance that has been used in endodontics. This substance boasts many properties such as wide antimicrobial spectrum, substan- tivity, and low toxicity (5, 6). The disadvantage of this substance is that it does not act as a tissue solvent (7). In the 1970s, chlorhexidine gluconate was suggested for use as a cavity cleanser for dentin surfaces before undertaking restorative procedures because of its antimicrobial properties (8). Tulunoglu et al (9) showed that chlorhexidine used as cavity cleanser affects the bonding between some adhesive systems and the dentin. On the other hand, Perdiga ˜o et al (10) did not find any influence from the use of chlorhexidine on bond strength between dentin surfaces and adhesive systems. The results of different studies suggest that the effect of chlorhexidine (CHX)-based cleansers on bond strength may be material specific regarding their interactions with various dentin bonding systems. Although CHX gel has been indicated as an auxiliary chemical substance for endodontic instrumentation (5–7), little information exists regarding the consequences of the use of the gel formulation and its effect on resin/root canal dentin adhesion (11). EDTA is often used with sodium hypochlorite to remove the smear layer. This de- mineralizing agent can expedite the destructive hypochlorite effect and facilitate hydroxyl ion diffusion through dentinal tubules (12, 13). However, EDTA alters the physicochemical properties of dentin and increases the adherence and adhesion force of Enterococcus faecalis to dentin, an important bacteria associated with posttreat- ment endodontic infections (14). Dentin collagen alteration after treatment with chemical substances can be observed by polarized light microscopy (PLM). This technique permits the determina- tion of optic anisotropies. Anisotropic substances exhibit double refraction (15, 16). Quantitative analysis of birefringence by PLM is a useful method to investigate the macromolecular orientation and organization of collagen fibers in connective tissues (17). Alterations of collagen’s birefringence pattern occur because of a disorganization in its micromolecular structure (15). Hence, it is possible to analyze dentin collagen structure alteration with birefringence after using the different chemical substances used in endodontics. It is possible to increase the collagen’s birefringence by dying the tissue with Sirius red stain. Sirius red molecules bind in parallel with collagen fiber molecules, increasing their birefringence under PLM (18, 19). From the Department of *Restorative Dentistry and † Morphology, Piracicaba Dental School, State University of Campinas, Piracicaba, SP, Brazil. Supported by the grants of the Brazilian agency FAPESP (04/11864-7 and 04/12474-8). Address requests for reprints to Dr Alexandre Augusto Zaia, Piracicaba Dental School, State University of Campinas, PO Box 52, 13414-903, Piracicaba, SP, Brazil E-mail address: zaia@fop. unicamp.br. 0099-2399/$0 - see front matter ª2009 American Association of Endodontists. doi:10.1016/j.joen.2009.04.002 Basic Research—Technology JOE — Volume 35, Number 7, July 2009 Bovine Root Dentin after Use of Different Endodontics Auxiliary Chemical Substances 1023