Crown discoloration promoted by materials used in regenerative endodontic procedures and effect of dental bleaching: spectrophotometric analysis

Abstract Regenerative endodontic procedure (REP) has been proposed as a new approach to treat immature permanent teeth. However, materials used in REP for root canal disinfection or cervical sealing may induce tooth discoloration. Objectives To assess tooth crown’s color after intracanal treatment with triple antibiotic paste (TAP) or calcium hydroxide (CH); cervical sealing with glass ionomer cement (GIC) or mineral trioxide aggregate (MTA); and bleaching with carbamide peroxide. Material and Methods After pulp removal and color spectrophotometer measurement, 50 bovine incisors were divided into 4 experimental groups and one control (untreated). Experiments were performed in phases (Ph). Ph1: TAP (ciprofloxacin, metronidazole, minocycline), TAPM (ciprofloxacin, metronidazole, amoxicillin), DAP (ciprofloxacin, metronidazole), or CH treatment groups. After 1 and 3 days (d); 1, 2, 3 weeks (w); and 1, 2, 3 and 4 months (m), color was measured and medications were removed. Ph2: GIC or MTA cervical sealing, each using half of the specimens from each group. Color was assessed after 1d, 3d; 1w, 2w, 3w; 1m and 2m. Ph3: Two bleaching sessions, each followed by color measurement. Data were analyzed with ANOVA and post-hoc Holm-Sidak method. Results Ph1: Specimens of TAP group presented higher color alteration (ΔE) mean than those of TAPM group. No significant difference was found among TAP or TAPM and CH, DAP or Control groups. Ph2: cervical sealing materials showed no influence on color alteration. Ph3: Different ΔE means (from different groups), prior to bleaching, became equivalent after one bleaching session. Conclusions TAP induces higher color alteration than TAPM; color alteration increases over time; cervical sealing material has no influence on color alteration; and, dental bleaching was able to recover, at least partially, the tooth crown’s color.


Introduction
Regenerative endodontic procedures (REP) have been proposed as an alternative to conventional endodontic treatment for immature permanent teeth, since it allows continuing root development in length and thickness 3,7,21,28 .
In Clinical Considerations for a Regenerative Procedure, American Association of Endodontists 2 (AAE) advises root canal disinfection, blood clot formation and capping, cervical sealing, and crown restoration. For root canal disinfection, AAE 2 recommends calcium hydroxide (CH) or triple antibiotic and minocycline 12 . Although TAP provides good antibacterial effect, case reports have shown tooth staining induced by minocycline 9,16,23 . Thus, alternative medications have been proposed, as double metronidazole 24 which minocycline is replaced by other antibiotics, as amoxicillin 14,24 or cefaclor 24,27 .
Subsequently to disinfection, AAE 2 advises a 3-4 mm layer of MTA, bioceramic, or glass ionomer cement (GIC) over the capped clot as cervical sealing. Since even white MTA could induce tooth discoloration 11,13 , alternatives to this material, such as bioceramics and GIC, have been strongly indicated in teeth where there is an esthetic concern 2 . Although REP provides satisfactory biological results, products used in this approach could impair dental esthetic, which may not be reverted by tooth bleaching 16,20 discoloration after each phase of REP, using different intracanal pastes and cervical sealing materials, followed by measuring color after bleaching. Therefore, investigation of alternatives to reduce tooth esthetic assess the effect of intracanal medications and sealing materials used in REP on tooth crown's color, as well as on tooth bleaching response. The null hypotheses are: (1) materials used as intracanal dressing or cervical sealing, in REP, are not able to induce tooth crown discoloration; and, (2) in case of tooth discoloration promoted by those materials, dental bleaching is not able to recover tooth color.

Material and methods
Tooth selection and specimen preparation Fifty bovine incisors with similar dentinal wall thickness were collected and disinfected by immersion in 1.5% sodium hypochlorite for 3 min. To simulate an immature tooth condition 6 and standardize specimen length (15 mm), each tooth was sectioned 5 mm coronally and 10 mm radicularly from the cementumenamel junction (CEJ) using a water-cooled diamond disc. After endodontic access, pulp tissue was removed and the root canal was enlarged with a 4103 diamond bur (KG Sorensen, Barueri, SP, Brazil) under irrigation with 3 mL of 1.5% NaOCl, to standardize the internal diameter in 1.6 mm and thickness of dentinal walls in 1.91±0.37 mm. The last radicular 5 mm were sealed with wax, and the root cervical third was irrigated with 3 mL of 1.5% NaOCl followed by 3 mL of 17% ethylenediaminetetraacetic acid (EDTA, Sigma-Aldrich, St. Louis, United States) to remove smear layer. Root canal was dried with absorbent paper points, a cotton pellet was placed inside the root canal and the crown was sealed with Citodur ® (DoriDent, Wien, Wien, Austria). During these procedures, teeth were wrapped up in gauzes embedded in distilled water to avoid dehydration; furthermore, after all preparations, they were stored submerged into 10 mL of distilled water.

Spectrophotometry
To standardize area and light conditions for color assessment, for each tooth, a custom silicone matrix was fabricated with impression material (Perfil ® , Vigodent S/A, Rio de Janeiro, RJ, Brazil), covering the entire buccal tooth surface. A perforation compatible with the size of the spectrophotometer tip (±6 mm diameter) was made with a cutting-edge cylinder at the crown area 5 (Figure 1a  Following coronal sealing, the specimens returned to the distilled water. After 1d and 3d; 1w, 2w, and 3w; and 1m and 2m, color shades were registered again.   Figure 2). Disregarding the period, week. On the other hand, the highest means were found at the last 3m (Table 1). It is worth pointing out that in CH/MTA group the became higher than at baseline ( Figure 4, Table 2).  Guide did not change in most samples (Supplemental Table 1).
Another explanation may be the interactions among the different materials used, which could produce formaldehyde as a byproduct of chemical reactions.
Bismuth oxide and calcium oxide could stain dental structure when in contact with formaldehyde 4 .
shades, according to VITA Classical A1-D4 ® Shade bleaching. It is possible since small color difference may not determine the change of a shade for another, as there is an interval in referential values of L*, a*, and, b* between shades 22 .
A digital spectrophotometer was used to register tooth shades along the three experimental phases.
From this device, it is possible to obtain values of "L", "a" and "b" of the dental structure and, then, became largely used in Dentistry due to their reliable method of reading tooth color shade. Here, Vita Easyshade ® was used as it has high reliability and accuracy 17 . Additionally to the numerical information of lightness and hue, this device provides shades according to VITA Classical A1-D4 ® Shade Guide, a standard and worldwide reference system in tooth shade determination. To further avoid external influences, silicone matrices were customized to ensure standardized area and light conditions along the experimental phases 5 . The selection of teeth with similar dentinal wall thickness is also important to improve standardized conditions 18 and, consequently, to improve reliability of the comparisons along the time.
Here, it was investigated the effect on dental crown discoloration of products used as intracanal medication or cervical sealing in regenerative endodontics procedures, as well as the impact of dental bleaching after regenerative approach. Under limitations of an ex vivo study, the methodology was designed to simulate procedures performed in clinics, applying sequentially products on the same tooth to assess, throughout the experimental phases, the impact of each of them on tooth color shade.

Conclusion
From the results of this experimental ex vivo study, we conclude that the evaluated intracanal medications, which are applied in regenerative endodontic being TAP the material that induces higher color alteration. On the other hand, cervical sealing material dental bleaching was able to recover, at least partially,