The esthetic nature of dental structures is significantly influenced by the materials used, the final restorative filling, and the overall health, hygiene, and condition of soft and hard tissues.^[1,2] Coronal discoloration is a common complication associated with calcium silicate-based cement used in vital pulp therapy (VPT), regenerative endodontics, and perforation repair, posing a challenge for dental clinicians. An ideal endodontic cement should offer excellent esthetic outcomes alongside optimal biological and mechanical properties.^[3,4]

Since the introduction of gray mineral trioxide aggregate (MTA) by Dr. Torabinejad et al. in 1991, white MTA was developed to address discoloration concerns. However, white MTA also presented limitations in clinical application.^[5,6] In response, alternative calcium silicate-based cement has been developed to address these challenges.^[7]

OrthoMTA is a novel orthograde root canal material that maintains its properties in the presence of moisture and blood. It exhibits excellent sealing ability, biocompatibility, radiopacity, and antibacterial properties. In addition, it contains no heavy metals, does not expand, is easy to handle and remove, and sets within 3 min.^[8]

RetroMTA, designed for VPT, comprises fine hydrophilic particles and achieves a compressive strength of 105 MPa. Calcium zirconia is used as its radiopacifier. This material is resistant to washing, has a rapid setting time of 150 s (extendable to 10 min by the dentist), and offers minimal discoloration and superior sealing ability.^[9]

Cold ceramic is another calcium silicate-based cement widely used for root-end filling, apical plug formation in teeth with open apices, root perforation repairs, and pulp capping procedures.^[10,11] It demonstrates comparable biocompatibility to MTA and superior sealing ability in the presence of blood compared to glass ionomer cement, calcium hydroxide, and MTA. Its initial setting time is approximately 15 min.^[12]

Despite the availability of multiple materials for perforation repair, VPT, and regenerative endodontics, none currently provides minimal discoloration suitable for use in the esthetic zone. This study aims to determine the discoloration caused by three calcium silicate-based cement – Cold Ceramic, RetroMTA, and OrthoMTA – in the presence of blood and normal saline for 1–2 months.

This study was approved by the local Ethics Committee (IR.MUI.RESEARCH.REC.1400.361) and adheres to the PRILE 2021 checklist [Supplementary Table 1].

Using the formula n = (z₁ + z₂)² × S²/d² and based on a 95% confidence level with a Type I error, the values are set as follows: z₁ = 1.96, z₂ = 0.84, P = 0.5, d = 0.98, and S = 0.98.

Accordingly, the minimum number of teeth required for each group in the study is 8. This experimental study included 48 extracted human maxillary anterior teeth, extracted due to periodontal disease and free of cracks and attrition. Exclusion criteria included teeth with discoloration (due to smoking, alcohol, or aging), crowns shorter than 8 mm, visible cracks, anatomical variations, or internal resorption. Debris pigments and periodontal ligament remnants

were removed using an ultrasonic scaler (NSK, Japan). The teeth were then polished with prophylaxis paste and disinfected in a 5.25% sodium hypochlorite solution (Chlora, Cerkamed, Poland) for 1 h before being stored in normal saline until further preparation and testing.

To standardize the samples, tooth roots were sectioned perpendicularly to the axial axis below the cementoenamel junction (CEJ) using a diamond fissure bur (Teeskavan, Iran), leaving 5 mm of root length. Standard access cavities were prepared, and the canals were shaped using Gates Glidden drills (#1 to #6, Dentsply Maillefer, Switzerland) with 2.5% sodium hypochlorite irrigation between each step. A final rinse with normal saline was performed following the completion of debridement.

Open root apices were sealed with adhesive wax, and the teeth were mounted and numbered by placing them in floral foam sponge cylinders, extending from the root to the CEJ. Glass ionomer restorative cement (GC Fuji II, Tokyo, Japan) was used to fill the root canal up to the CEJ as an apical restoration. On the labial surface of each tooth, a 2 mm × 2 mm window was created at the mid-cervical area using a thin taper bur (Teeskavan, Iran) to ensure consistent color measurement during the process.

The specimens were divided into six groups using simple randomization (n = 8 per group), based on the type of calcium silicate-based cement and the environment:

Cotton saturated with blood and OrthoMTA

Fresh venous blood was collected from the researcher by a trained professional. A 1-mm thick cotton pellet was placed on the apical restoration and saturated with either blood or normal saline using an insulin syringe. Each group was further subdivided for application of one of the three calcium silicate-based materials: OrthoMTA (BioMTA, Seoul, Korea), Cold Ceramic (SGM, Iran), or RetroMTA (BioMTA, Seoul, Korea). Each material was prepared according to the manufacturer’s instructions and applied in a 3-mm thick layer onto cotton pellets saturated with either blood or normal saline, using an MTA carrier (Juya, Tehran, Iran). The compatibility of the cement with the cotton pellet was ensured by gently contacting it with a 35# plugger (Densply, Mani, USA). A 1-mm thick cotton pellet moistened with distilled water was then placed on the material to facilitate setting, and the access cavity was sealed with a temporary restoration.

After placing the cement, the color at the mid-cervical labial surface of each specimen was measured three times with a spectrophotometer (Shade Pilot, DeguDent GmbH) to determine the initial enamel color. Specimens were incubated at 37°C and 100% humidity. After 24 h, temporary restorations were removed, and the setting of the cement was confirmed with a probe. Cavities were then etched, bonded, and sealed with an A3-colored resin composite (3M™ ESPE™, USA).

Color measurements were repeated at 30 and 60 days. Discoloration (ΔE) was calculated using the formula:

In this formula: L = Light level, a = Green-to-red axis, and b = Blue-to-yellow axis.

The experiment was conducted by a researcher who was blinded to the experimental conditions.

A ΔE value of <3.3 was considered clinically acceptable discoloration.^[13]

Data were analyzed using SPSS version 22 (IBM Corp., Armonk, NY). The Kruskal–Wallis test was used to compare the mean discoloration among the three materials across different environments and time points, and the Wilcoxon test was applied to evaluate changes over time within each group. A significance level of 0.05 was used.

According to the results in Table 1, the highest discoloration was observed in OrthoMTA at both 30 and 60 days. However, there were no significant differences in discoloration at either time point (P > 0.05, Wilcoxon test). In addition, the mean (standard deviation [SD]) discoloration did not change significantly over time (30 days vs. 60 days) for all calcium silicate-based cement, except for RetroMTA, where a significant difference was observed (P = 0.025, Wilcoxon test).

According to the results in Table 2, when in contact with saline, the highest discoloration was observed in Cold Ceramic at 30 days and OrthoMTA at 60 days. However, no significant differences were found at either time point (P > 0.05, Kruskal–Wallis test). Similarly, the mean (SD) discoloration did not differ significantly over time (30 vs. 60 days) for all calcium silicate-based cement (P > 0.05, Wilcoxon test).

Tooth discoloration is a significant disadvantage, particularly in VPT for anterior teeth.^[5] Clinicians should consider the potential impact of calcium silicate-based cement on tooth discoloration.^[14] This study showed that within 30 and 60 days, the mean discoloration of all three cement in a blood-contaminated environment exceeded the clinically detectable threshold (ΔE >3.3). Although OrthoMTA exhibited the highest average discoloration, this difference was not statistically significant among the three materials, indicating comparable discoloration.

According to the manufacturer, OrthoMTA has a composition similar to ProRoot MTA but contains lower levels of heavy metals. RetroMTA, on the other hand, uses zirconium oxide as a radiopacifier.^[15] Zirconium oxide is used as a radiopacifier in RetroMTA and Biodentine.^[15-18] In the present study, RetroMTA demonstrated minimal potential for staining.

Our results align with Kang et al., who reported the highest discoloration in materials containing bismuth oxide and minimal discoloration in those using zirconium oxide.^[9] Similarly, Możyńska et al.’s systematic review categorized OrthoMTA as having a high discoloration potential, while RetroMTA was among the materials with the lowest potential.^[5] Metlerska et al. further observed that RetroMTA and MTA repair HP caused less discoloration than ProRoot MTA, Biodentine, and Ortho MTA, supporting their suitability for esthetic zones. Based on clinical findings, Biodentine can be considered for use due to its absence of gray discoloration.^[14]

The presence of blood significantly exacerbates discoloration, as supported by previous research. This is related to material porosity and the presence or absence of the smear layer, both of which influence dentin permeability. Materials with longer setting times, such as MTA, remain porous for extended periods, absorbing more blood and undergoing hemolysis, leading to greater discoloration than faster-setting alternatives such as Biodentine.^[19] Within 24 h, blood can discolor teeth by more than 15%, depending on the type of material and the duration of exposure.^[5,20]

Shokouhinejad et al. reported similar findings, stating that blood-contaminated samples exhibited the highest discoloration, while samples without blood contamination showed significantly less discoloration.^[21]

Adl et al. concluded that time significantly impacts discoloration regardless of the material. While our study observed discoloration over 1 and 2 months, the study by Adl et al. included 11-week, 11-month, and 3-month intervals. These extended time intervals and other factors, such as a larger sample size in their study, likely account for differences in findings.^[22]

Moazzami et al. also noted that discoloration increases over time. Differences in experimental durations (1 and 2 months in our study vs. 1 and 3 months in theirs) and the materials used (RetroMTA, OrthoMTA, and Cold Ceramic in our study vs. ProRoot MTA and Nano Fast Cement in theirs) could explain variations in results.^[23]

Shokouhinejad et al. highlighted that both the presence of blood and the passage of time significantly affect discoloration, regardless of the type of material. Differences in experimental durations (1 and 2 months in our study vs. 1 and 6 months in theirs), materials tested (RetroMTA, OrthoMTA, and Cold Ceramic in our study vs. ProRoot MTA, Endosequence Root Repair Material, and Biodentine in theirs), and sample size (8 per group in our study vs. 12 per group in theirs) may contribute to discrepancies between the findings.^[21]

This study faced various limitations, including challenges in sourcing anterior teeth without cracks and crowns without wear, due to the stringent and time-consuming selection criteria, which reduced the sample size. Future studies should explore additional calcium silicate-based cement with varied compositions and radiopacifiers. Moreover, experiments with longer durations should be designed to more accurately investigate the impact of time on discoloration.

Discoloration among RetroMTA, OrthoMTA, and Cold Ceramic showed no significant differences in blood-contaminated and saline environments. However, over time, all materials exhibited clinically detectable discoloration (ΔE > 3.3).