Abstract
Background
This study aimed to evaluate and compare the discoloration potential of four bioceramic pulp-capping materials in the presence of blood or 0.9% normal saline (NS) under in vitro conditions.
Methods
Extracted maxillary central incisors were prepared with standardized Class I cavities and allocated to blood or NS environments. Each medium was further subdivided according to the pulp-capping material used: Biodentine, NeoMTA, TotalFill Putty (TFP), and TheraCal PT (TPT). Following composite restoration, color measurements were obtained using a spectrophotometer at baseline, 1 week, 1 month, 3 months, and 6 months. All specimens were stored in phosphate-buffered saline at 37 °C throughout the experimental period. Color change (ΔE) was analyzed using nonparametric tests (α = 0.05).
Results
All materials demonstrated significant time-dependent increases in discoloration in both environments (p < 0.05). In NS, NeoMTA and TFP exhibited significantly lower ΔE values than Biodentine and TPT across all time points. In the presence of blood, Biodentine showed the greatest discoloration at each time point (p < 0.05), whereas NeoMTA and TFP maintained significantly lower values. TPT demonstrated comparable discoloration in blood and NS, with no significant differences between media (p > 0.05). Across all groups and evaluation periods, ΔE values ranged from 0.31 to 1.52.
Conclusions
Blood contamination appears to be an important factor influencing the esthetic performance of bioceramic pulp-capping materials. Therefore, esthetic behavior should be considered alongside biological properties when selecting materials for vital pulp therapy, particularly in clinically visible regions where discoloration may affect treatment outcomes.
Clinical Relevance
Blood contamination may increase discoloration in certain bioceramic materials; NeoMTA, TFP, and TPT may better preserve color in esthetically sensitive cases.
Keywords: Pulp capping materials, Bioceramics, Tooth discoloration, Color stability, Spectrophotometry, Vital pulp therapy
Introduction
Pulp capping is a vital therapeutic procedure in which a biocompatible material is placed over exposed or nearly exposed pulp to preserve vitality, maintain the pulp–dentin complex, and stimulate the formation of a dentin or dentin-like hard tissue bridge [1, 2]. The procedure is classified as direct—where the material is applied directly onto exposed pulp—or indirect, in which a residual dentin layer remains between the material and the pulp [3, 4].
Over time, several materials have been introduced for pulp capping. Calcium hydroxide, historically regarded as the gold standard, has been limited by high solubility, microleakage, and suboptimal long-term sealing despite its dentin bridge–forming capacity. As a result, bioceramic materials have become preferred alternatives. Mineral trioxide aggregate (MTA), initially developed as a root repair material, is now widely used in vital pulp therapy because of its excellent sealing ability, biocompatibility, and favorable mechanical behavior [3–6]. However, its long setting time, handling challenges, and propensity to cause tooth discoloration in esthetically sensitive areas remain significant drawbacks [6–9]. To address discoloration associated with bismuth oxide–containing formulations, newer calcium silicate–based cements—such as NeoMTA Plus and NeoMTA Putty, which incorporate tantalum oxide as a radiopacifier—were developed to improve color stability while maintaining biological properties [10, 11].
Another calcium silicate–based cement developed to overcome limitations of MTA is Biodentine, which offers shorter setting time, reduced solubility, high mechanical strength, and favorable sealing characteristics [12–15]. Both MTA and newer calcium silicates have been shown to induce thicker dentin bridges, reduce inflammation, and preserve pulp vitality more effectively than calcium hydroxide [16–18]. Despite its widespread clinical use, the long-term color stability of Biodentine—particularly under blood-contaminated conditions—remains insufficiently clarified [7–10, 19].
More recently, additional bioceramic alternatives have been introduced. TheraCal PT (TPT), a dual-cure resin-modified calcium silicate material, provides simplified handling, rapid polymerization, sustained calcium release, and an alkaline environment conducive to hydroxyapatite deposition and dentin bridge formation [20, 21]. TotalFill Putty (TFP), a premixed bioceramic material, has also emerged as a promising option in vital pulp therapy because of its consistent composition and ease of use [12, 22].
Recent clinical and systematic reviews have further reinforced the growing role of calcium silicate–based materials in vital pulp therapy, demonstrating high success rates, favorable biological outcomes, and increasing adoption in minimally invasive treatment approaches [23–26]. Nevertheless, esthetic outcomes remain an important consideration, particularly in anterior teeth and other highly visible regions.
The clinical success of pulp-capping materials depends not only on biological compatibility and dentinogenic potential but also on their esthetic behavior. Discoloration of anterior teeth after vital pulp therapy may compromise patient satisfaction and treatment acceptance. Although contemporary bioceramic materials have been developed to improve color stability, available evidence remains heterogeneous and direct comparisons among currently used materials are limited [27–29]. Although several studies have investigated discoloration associated with bioceramic materials, contemporary evidence highlights persistent uncertainty regarding their long-term esthetic performance under clinically relevant conditions, particularly in the presence of blood contamination [14, 24, 29].
Objective quantification of dental color change is typically performed using spectrophotometric analysis, with color expressed in the CIE Lab* color space system [4, 5, 30, 31]. Therefore, the aim of this in vitro study was to evaluate coronal discoloration induced by different bioceramic pulp-capping materials and to compare the effects of blood contamination (simulating direct pulp capping) with those of NS exposure (simulating indirect pulp capping).
Materials and methods
Informed consent and ethical approval
Informed consent was obtained from all patients prior to tooth extraction. The study protocol was approved by the Hamidiye Scientific Research Ethics Committee of the University of Health Sciences (Approval date: April 7, 2023; Document date and number: April 25, 2023 – 17431).
Sample size calculation
Sample size was calculated using G*Power 3.1 (Heinrich Heine University, Düsseldorf, Germany). Based on a 2 × 4 factorial design with five repeated measurements, an alpha level of 0.05, effect size f = 0.30 (medium effect according to Cohen), and statistical power (1 − β) = 0.95, a minimum of 160 specimens (20 teeth per subgroup) was required.
Tooth selection and preparation
A total of 160 maxillary central incisors extracted for periodontal reasons were included. Teeth were examined under a dental operating microscope (Labomed Magna; Labo America Inc., Fremont, CA, USA). Teeth presenting caries, cracks, fractures, resorption, erosion, abrasion, attrition, or previous restorations were excluded. To minimize anatomical variability that could influence discoloration outcomes, only maxillary central incisors with fully developed roots and intact coronal structures were selected.
All specimens were cleaned using an ultrasonic scaler and periodontal brush to remove calculus, soft tissue remnants, and extrinsic stains, and were subsequently stored in 1% chloramine-T solution until use.
Cavity preparation, group allocation, and restoration procedure
Crowns were sectioned 1 mm apical to the cementoenamel junction using a high-speed diamond bur under sterile saline irrigation. Only crown portions were used to isolate coronal discoloration relevant to esthetics. The pulp chamber was not externally exposed. Blood or normal saline (NS) was applied exclusively to the standardized cavity floor prior to placement of the test materials.
Standardized box-shaped cavities were prepared from the pulp chamber toward the incisal direction at the cementoenamel junction using a high-speed diamond bur under continuous saline irrigation. Each cavity had an internal depth of 5.0 mm toward the pulpal direction and a minimum remaining labial wall thickness of 3.0 mm between the outer surface and the cavity floor. Remaining wall thickness was verified at the coronal, middle, and cervical thirds using a digital caliper. Because discoloration is strongly influenced by dentin thickness, particular emphasis was placed on standardizing the remaining labial dentin wall among specimens. Following irrigation with 2.5% NaOCl and rinsing with NS, cavities were dried using sterile paper points.
Half of the specimens (n = 80) were assigned to the blood exposure group. Fresh, anticoagulant-free human blood obtained from a single donor was applied to the cavity floor in a standardized volume of 20 µL using a calibrated positive-displacement pipette. Excess blood was gently removed to leave a thin film. Ethical approval covered the use of donor blood.
The remaining specimens (n = 80) were exposed to NS using the same protocol. Each main group was subdivided according to the pulp-capping material used (Biodentine, NeoMTA, TotalFill Putty [TFP], and TheraCal PT [TPT]; n = 20 each), resulting in eight experimental groups.
Each bioceramic material was placed at a standardized thickness of 3.0 mm according to the manufacturers’ instructions. A self-etch adhesive (G-Premio BOND; GC Corp., Tokyo, Japan) was applied using a microbrush, gently air-thinned for 5 s, and light-cured for 20 s using an LED curing unit (1200 mW/cm²) (X-Cure; Woodpecker Medical Instrument Co. Ltd., Guangxi, China).
Cavities were restored using a single universal composite shade (A2 Body; Filtek Ultimate, 3 M ESPE, USA) to minimize shade-related variability. All specimens were stored in sterile phosphate-buffered saline at 37 °C and 100% humidity throughout the experimental period (Fig. 1).
Fig. 1.
Schematic illustration of the experimental model used to simulate direct and indirect pulp-capping conditions. Left: Blood-contaminated cavity simulating direct pulp-capping conditions. Right: Normal saline (NS)-exposed cavity simulating indirect pulp-capping conditions. In both models, a standardized cavity was filled with a bioceramic pulp-capping material and subsequently restored with composite resin to reproduce the experimental conditions used for color assessment
Color measurement
Baseline color measurements were performed using a VITA EasyShade spectrophotometer (VITA Zahnfabrik, Bad Säckingen, Germany). Baseline measurements were obtained after cavity preparation and standardization procedures but before application of blood, normal saline, pulp-capping materials, adhesive systems, or composite restorations. The device was calibrated prior to each measurement session using the manufacturer-provided built-in calibration block according to the manufacturer’s instructions.
Color was recorded from two regions of interest (ROIs): The central labial and central palatal thirds of each crown. A custom positioning jig indexed specimen position, ensuring orthogonal and reproducible probe placement at identical ROIs at each time point.
Three consecutive readings were obtained from each ROI and averaged to obtain a single-color value per specimen. Intra-operator reliability was assessed using a random subset of 20 teeth and demonstrated an intraclass correlation coefficient (ICC) ≥ 0.90.
Color measurements were repeated at 1 week (ΔE₁), 1 month (ΔE₂), 3 months (ΔE₃), and 6 months (ΔE₄). Measurements were performed under standardized laboratory lighting conditions inside a custom enclosure lined with white background paper. Reported values represent the mean of the averaged labial and palatal ROI values per specimen.
All measurements were performed by a single calibrated operator. Data were recorded in Microsoft Excel for statistical analysis.
Color parameters were expressed using the CIE Lab* color space system, where L represents lightness (0 = black, 100 = white), a represents the green–red chromatic axis, and b represents the blue–yellow chromatic axis. The overall color difference (ΔE) between time points was calculated using the CIE-recommended Eqs. (30,31):
 |
Statistical analysis and hypotheses
Statistical analyses were performed using IBM SPSS Statistics 22 (IBM Corp., Armonk, NY, USA). Normality was assessed using the Shapiro–Wilk test, which demonstrated a non-normal, right-skewed distribution of ΔE values. Therefore, nonparametric tests were applied.
Intergroup comparisons were performed using the Kruskal–Wallis test followed by Dunn’s post hoc test for pairwise comparisons. Two-group comparisons were performed using the Mann–Whitney U test. Intragroup differences across time were analyzed using the Friedman test, followed by Wilcoxon signed-rank tests for pairwise post hoc comparisons.
Holm–Bonferroni correction was applied to control for multiple pairwise testing. Effect sizes were calculated using Kendall’s W for Friedman tests and rank-biserial correlation (r) for pairwise comparisons. Results are presented as median (IQR). Statistical significance was set at p < 0.05.
The null hypothesis (H₀) stated that pulp-capping materials have no effect on tooth color over time. The alternative hypothesis (H₁) stated that pulp-capping materials influence tooth color, resulting in time-dependent discoloration.
Results
In the NS group (Table 1), all materials demonstrated a significant, time-dependent increase in discoloration (ΔE) (p < 0.05). Median (IQR) values for all materials and evaluation periods are presented in Tables 1, 2 and 3. At 1 week, NeoMTA exhibited significantly lower discoloration than Biodentine and TheraCal PT (TPT), with no significant difference compared with TotalFill Putty (TFP). At 1, 3, and 6 months, both NeoMTA and TFP consistently showed lower ΔE values than Biodentine and TPT (p < 0.05). Across all time points, Biodentine produced the highest ΔE values, whereas NeoMTA and TFP maintained the lowest and most stable color performance. TPT demonstrated moderate but progressively increasing discoloration.
Table 1.
Color change (ΔE) values of bioceramic pulp-capping materials exposed to normal saline (NS) at 1 week, 1 month, 3 months, and 6 months
| NS | ΔE (1 week) | ΔE (1 month) | ΔE (3 months) | ΔE (6 months) | P value† |
|---|---|---|---|---|---|
| Biodentine | 0.64 (0.49)Aa | 0.91 (0.48)Ab | 0.95 (0.44)Ab | 1.14 (0.42)Ac | 0.001* |
| NeoMTA | 0.31 (0.24)Ba | 0.43 (0.22)Bb | 0.48 (0.23)Bb | 0.55 (0.27)Bc | 0.001* |
| TFP | 0.41 (0.29)ABa | 0.46 (0.25)Ba | 0.69 (0.17)Bb | 0.73 (0.19)Bb | 0.001* |
| TPT | 0.64 (0.15)Aa | 0.76 (0.10)Ab | 0.98 (0.07)Ac | 1.11 (0.14)Ad | 0.001* |
| P value‡ | 0.004* | 0.001* | 0.001* | 0.001* |
Values are presented as median (IQR)
Different uppercase letters within the same column indicate statistically significant differences among materials
Different lowercase letters within the same row indicate statistically significant differences across time points
Abbreviations: NS Normal saline, TFP TotalFill Putty, TPT TheraCal PT, IQR Interquartile range
†Friedman test (within-material comparison across time)
‡Kruskal–Wallis test (between-material comparison at each time point)
*Statistically significant (P < 0.05)
Table 2.
Color change (ΔE) values of bioceramic pulp-capping materials exposed to blood at 1 week, 1 month, 3 months, and 6 months
| Blood | ΔE (1 week) | ΔE (1 month) | ΔE (3 months) | ΔE (6 months) | P value† |
|---|---|---|---|---|---|
| Biodentine | 0.70 (0.34)Aa | 1.04 (0.32)Ab | 1.39 (0.34)Ac | 1.52 (0.37)Ad | 0.001* |
| NeoMTA | 0.42 (0.34)Ba | 0.67 (0.28)Bb | 0.91 (0.27)Bc | 1.13 (0.28)Bd | 0.001* |
| TFP | 0.43 (0.22)Ba | 0.69 (0.17)Bb | 0.95 (0.17)Bc | 1.11 (0.20)Bd | 0.001* |
| TPT | 0.54 (0.19)ABa | 0.76 (0.13)Bb | 1.01 (0.11)Bc | 1.06 (0.09)Bd | 0.001* |
| P value‡ | 0.021* | 0.001* | 0.001* | 0.001* |
Values are presented as median (IQR)
Different uppercase letters within the same column indicate statistically significant differences among materials
Different lowercase letters within the same row indicate statistically significant differences across time points
Abbreviations: TFP TotalFill Putty, TPT TheraCal PT, IQR Interquartile range
†Friedman test (within-material comparison across time)
‡Kruskal–Wallis test (between-material comparison at each time point)
*Statistically significant (P < 0.05)
Table 3.
Comparison of color change (ΔE) values between blood- and normal saline (NS)-exposed groups for each bioceramic material at 1 week, 1 month, 3 months, and 6 months
| Material | Environment | ΔE (1 week) | ΔE (1 month) | ΔE (3 months) | ΔE (6 months) |
|---|---|---|---|---|---|
| Biodentine | Blood | 0.70 (0.34)a | 1.04 (0.32)a | 1.39 (0.34)a | 1.52 (0.37)a |
| NS | 0.64 (0.49)a | 0.91 (0.48)a | 0.95 (0.44)b | 1.14 (0.42)b | |
| P value† | 0.721 | 0.407 | 0.005* | 0.010* | |
| NeoMTA | Blood | 0.42 (0.34)a | 0.67 (0.28)a | 0.91 (0.27)a | 1.13 (0.28)a |
| NS | 0.31 (0.24)a | 0.43 (0.22)b | 0.48 (0.23)b | 0.55 (0.27)b | |
| P value† | 0.386 | 0.010* | 0.001* | 0.001* | |
| TFP | Blood | 0.43 (0.22)a | 0.69 (0.17)a | 0.95 (0.17)a | 1.11 (0.20)a |
| NS | 0.41 (0.29)a | 0.46 (0.25)b | 0.69 (0.17)b | 0.73 (0.19)b | |
| P value† | 0.935 | 0.006* | 0.001* | 0.001* | |
| TPT | Blood | 0.54 (0.19)a | 0.76 (0.13)a | 1.01 (0.11)a | 1.06 (0.09)a |
| NS | 0.64 (0.15)a | 0.76 (0.10)a | 0.98 (0.07)a | 1.11 (0.14)a | |
| P value† | 0.118 | 0.959 | 0.272 | 0.256 |
Values are presented as median (IQR)
Different lowercase letters within the same column for each material indicate statistically significant differences between blood and NS conditions
Abbreviations: NS Normal saline, TFP TotalFill Putty, TPT TheraCal PT, IQR Interquartile range
†Mann–Whitney U test
*Statistically significant (P < 0.05)
Within-material analyses confirmed significant time effects for all materials (p < 0.05). Biodentine showed minimal change until 3 months, followed by a marked increase at 6 months. NeoMTA demonstrated a gradual increase across all time points. TFP remained stable from 1 week to 1 month, increased at 3 months, and plateaued thereafter. TPT exhibited a steady, statistically significant increase throughout the experimental period.
In the blood-exposed group (Table 2), all materials also demonstrated significant time-dependent increases in discoloration (p < 0.05). At 1 week, Biodentine showed significantly greater ΔE values than NeoMTA and TFP (p < 0.05). At 1 month, Biodentine exhibited higher discoloration than all other materials. At 3 and 6 months, Biodentine consistently produced the highest ΔE values compared with NeoMTA, TFP, and TPT (p < 0.05).
Overall, ΔE values were higher under blood exposure than under NS for all materials. Biodentine exhibited the most pronounced increase, followed by TPT, whereas NeoMTA and TFP demonstrated lower but still blood-amplified discoloration. Longitudinally, each material (Biodentine, NeoMTA, TFP, and TPT) exhibited progressive and statistically significant increases in ΔE from 1 week through 6 months (p < 0.05), confirming cumulative discoloration under both environmental conditions.
Direct comparison of blood and NS groups (Table 3) demonstrated significantly greater discoloration under blood exposure for Biodentine at 3 and 6 months (p < 0.05), for NeoMTA beginning at 1 month (p < 0.05), and for TFP at 1, 3, and 6 months (p < 0.05). In contrast, TPT showed no significant differences between blood and NS conditions at any time point (p > 0.05). Blood exposure consistently amplified discoloration across materials, producing the greatest effect in Biodentine and the least effect in TPT (Fig. 2).
Fig. 2.
Comparison of color change (ΔE) values between normal saline (NS) and blood-exposed conditions for Biodentine, NeoMTA, TotalFill Putty (TFP), and TheraCal PT (TPT) at 1 week, 1 month, 3 months, and 6 months. Bars represent median ΔE values and error bars indicate interquartile ranges (IQRs). Each material is presented under both NS and blood-contaminated conditions to facilitate comparison of time-dependent color changes between environmental exposures
At the 6-month evaluation, Biodentine exhibited the highest overall ΔE values, particularly under blood exposure. NeoMTA and TFP demonstrated moderate discoloration, more pronounced in the presence of blood, whereas TPT showed similar ΔE values in both environments, indicating minimal influence of blood contamination (Fig. 3).
Fig. 3.
Comparison of six-month color change (ΔE) values of bioceramic pulp-capping materials under normal saline (NS) and blood-contaminated conditions. Bars represent median ΔE values and error bars indicate interquartile ranges (IQRs). Statistical comparisons between NS and blood conditions were performed separately for each material using the Mann–Whitney U test. Asterisks indicate statistically significant differences between environmental conditions (P < 0.05)
Discussion
This in vitro study evaluated the discoloration potential of four bioceramic pulp-capping materials under blood-contaminated and normal saline (NS) conditions over a six-month period. All materials demonstrated progressive increases in ΔE values, with blood contamination significantly intensifying discoloration for all materials except TheraCal PT (TPT), which showed comparable performance in both environments. Although several pairwise differences reached statistical significance, most six-month ΔE*ab values remained below the widely cited perceptibility (~ 1.2) and acceptability (~ 2.7) thresholds for ΔE*ab in dentistry [31, 32]. While the CIEDE2000 (ΔE00) formula better reflects human visual perception [31, 32], ΔE*ab was used to maintain comparability with prior endodontic literature, which predominantly reports color changes using the CIE Lab system. Consequently, ΔE00 was not calculated in the present study; however, future investigations should incorporate both metrics to enhance clinical interpretability and facilitate comparison between perceptual and instrumental assessments. Recent systematic reviews have similarly emphasized the need for standardized discoloration assessment protocols to improve interstudy comparability [26, 29]. Accordingly, the short-term clinical impact—particularly in anterior teeth—may be limited, and material selection should consider both biological performance and the relatively small magnitude of color change.
Spectrophotometry, widely regarded as the most objective method for in vitro and in vivo color assessment [33], enabled precise longitudinal evaluation of material-related color changes. Biodentine exhibited the greatest discoloration, particularly under blood-contaminated conditions, consistent with findings reported by Madani et al. [34]. In contrast, some studies have reported higher color stability for Biodentine under non-contaminated or short-term conditions [10, 34]. These discrepancies likely reflect methodological differences, including tooth substrate, contamination protocol, and observation duration. The present findings likely reflect enhanced penetration of hemoglobin and related pigments into dentinal tubules under controlled in vitro conditions.
NeoMTA demonstrated significantly lower discoloration, particularly relative to Biodentine and TPT in NS. This observation aligns with previous reports [10, 35] and can be attributed to replacement of bismuth oxide with tantalum oxide, a radiopacifier that does not undergo reduction or react with dentin collagen to form dark metallic by-products [7, 11, 27]. This intrinsic chemical difference appears to play a key role in color preservation. TFP, a pre-mixed calcium silicate material, exhibited color stability comparable to NeoMTA. Its homogeneous formulation and shorter setting time may reduce interaction with blood and limit pigment incorporation [27]. Although evidence regarding TFP-related discoloration remains limited, its performance in the present study suggests favorable esthetic potential.
TPT, a dual-cure resin-modified bioceramic, demonstrated minimal differences between blood and NS conditions. Because its initial set is driven by light polymerization, the material likely undergoes reduced direct interaction with blood [19, 28]. These findings are consistent with Türkoğlu Kayacı et al. [28], who reported comparable color stability for TPT and MTA over six months, supporting its potential use in esthetically demanding clinical scenarios where blood contamination is likely.
The pronounced discoloration observed under blood exposure is attributable to penetration of hemoglobin and other chromogenic blood components into dentinal tubules and subsequent interaction with bioceramic materials [6, 34]. Intrinsic material chemistry further modulates this effect. Calcium silicate–based cements generate an alkaline environment that may promote binding of hemoglobin degradation products to tooth structure. In contrast, tantalum-containing NeoMTA avoids the bismuth-associated darkening observed in conventional MTA systems [10, 35]. Additionally, shorter setting times (TFP) and resin modification (TPT) may reduce the exposure window for pigment incorporation.
The null hypothesis—that pulp-capping materials have no effect on tooth color over time—was rejected. A significant time-dependent increase in ΔE*ab was observed across all materials, confirming that discoloration is influenced by both environmental conditions (blood vs. NS) and material composition. Although ΔE*ab was used for consistency with previous literature, the superior perceptual fidelity of CIEDE2000 (ΔE00) supports its use in future studies to improve clinical relevance.
Most six-month ΔE*ab values remained below perceptibility (~ 1.2) and acceptability (~ 2.7) thresholds [31, 32], suggesting limited short-term esthetic impact. However, because blood contamination consistently amplified discoloration, longer observation periods may reveal greater divergence among materials. Therefore, in esthetically critical situations—particularly when blood exposure is likely, such as anterior vital pulp therapy—material selection should balance biological performance with the potential, even if modest, risk of color alteration. In such scenarios, NeoMTA, TFP, and TPT may be preferable to Biodentine due to their comparatively superior color stability [27], although Biodentine’s well-documented biological advantages should remain integral to clinical decision-making [23].
This study has several limitations. The in vitro design cannot fully replicate the oral environment. Thermocycling, pH cycling, and photoaging were not performed, which may underestimate long-term color instability beyond six months. Although a single composite resin and adhesive system were used to standardize restorations, composite-related discoloration effects cannot be completely excluded, and different restorative materials may produce variable results. Additionally, no untreated negative-control group or blood-only positive-control group was included, limiting the ability to isolate the independent contribution of blood contamination and its interaction with individual pulp-capping materials. Furthermore, remaining dentin thickness was standardized during specimen preparation and verified using digital caliper measurements; however, these measurements were not recorded for subsequent statistical analysis. Therefore, potential correlations between dentin thickness and discoloration outcomes could not be evaluated. Finally, only maxillary central incisors were evaluated; structural and optical differences in other tooth types may influence discoloration patterns. Future clinical studies with longer follow-up periods, broader material and tooth selection, inclusion of appropriate control groups, and assessment using both ΔE*ab and ΔE00 are warranted, in accordance with recent recommendations for vital pulp therapy research [24, 25].
Taken together, the present findings provide clinically relevant evidence that both environmental contamination and material composition influence esthetic outcomes in vital pulp therapy and should be considered during material selection in esthetically demanding cases. These findings should be interpreted within the context of controlled laboratory conditions and may vary under clinical intraoral conditions influenced by thermal cycling, biofilm formation, and long-term aging processes.
Conclusion
Within the limitations of this in vitro study, blood contamination was an important factor influencing the esthetic performance of several contemporary bioceramic pulp-capping materials. Although all tested materials demonstrated measurable color changes over time, the magnitude of discoloration varied according to material composition and environmental conditions.
These findings emphasize that esthetic behavior should be considered alongside biological properties when selecting materials for vital pulp therapy, particularly in anterior teeth and other esthetically demanding clinical situations. Further long-term clinical studies incorporating clinically relevant aging conditions are needed to confirm the translational significance of these findings.
Acknowledgements
Not applicable.
Abbreviations
- NS
Normal saline
- TFP
TotalFill Putty
- TPT
TheraCal PT
- ROI
Region of interest
- ICC
Intraclass correlation coefficient
- ΔE
Color difference
- CIE
Commission Internationale de l’Éclairage
Authors’ contributions
CT contributed to conceptualization, data curation, formal analysis, investigation, methodology, project administration, supervision, validation, visualization, writing of the original draft, and review and editing of the manuscript. EA contributed to data curation, investigation, methodology, project administration, and software. HNN contributed to funding acquisition, investigation, methodology, and writing of the original draft. HGG contributed to validation and review and editing of the manuscript. DAS contributed to supervision. All authors read and approved the final manuscript.
Funding
This research received no external funding.
Data availability
The datasets generated and/or analyzed during the current study are available from the corresponding author on reasonable request.
Declarations
Ethics approval and consent to participate
Informed consent was obtained from all patients prior to tooth extraction. The study protocol was approved by the Hamidiye Scientific Research Ethics Committee of the University of Health Sciences (Approval date: April 7, 2023; Document date and number: April 25, 2023–17431). All procedures were performed in accordance with relevant institutional guidelines and regulations.
Consent for publication
Not applicable.
Competing interests
The authors declare no competing interests.
Footnotes
Publisher’s note
Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.
References
- 1.Prati C, Gandolfi MG. Calcium silicate bioactive cements: biological perspectives and clinical applications. Dent Mater. 2015;31(4):351–70. [DOI] [PubMed] [Google Scholar]
- 2.Komabayashi T, Zhu Q, Eberhart R, Imai Y. Current status of direct pulp-capping materials for permanent teeth. Dent Mater J. 2016;35(1):1–12. [DOI] [PubMed] [Google Scholar]
- 3.Torabinejad M, Parirokh M, Dummer PMH. Mineral trioxide aggregate and other bioactive endodontic cements: an updated overview—part II: other clinical applications and complications. Int Endod J. 2018;51(3):284–317. [DOI] [PubMed] [Google Scholar]
- 4.O’Sullivan SM, Hartwell GR. Obturation of a retained primary mandibular second molar using mineral trioxide aggregate: a case report. J Endod. 2001;27(11):703–5. [DOI] [PubMed] [Google Scholar]
- 5.Hsien HC, Cheng YA, Lee YL, Lan WH, Lin CP. Repair of perforating internal resorption with mineral trioxide aggregate: a case report. J Endod. 2003;29(8):538–40. [DOI] [PubMed] [Google Scholar]
- 6.Adl A, Javanmardi S, Abbaszadegan A. Assessment of tooth discoloration induced by Biodentine and white mineral trioxide aggregate in the presence of blood. J Conserv Dent. 2019;22(2):164–8. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 7.Zafar K, Jamal S, Ghafoor R. Bioactive cements—mineral trioxide aggregate-based calcium silicate materials: a narrative review. J Pak Med Assoc. 2020;70(3):497–504. [DOI] [PubMed] [Google Scholar]
- 8.Camilleri J. Hydration characteristics of Biodentine and Theracal used as pulp-capping materials. Dent Mater. 2014;30(7):709–15. [DOI] [PubMed] [Google Scholar]
- 9.Raghavendra SS, Jadhav GR, Gathani KM, Kotadia P. Bioceramics in endodontics: a review. J Istanb Univ Fac Dent. 2017;51(Suppl 1):S128–37. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 10.Camilleri J. Staining potential of Neo MTA Plus, MTA Plus, and Biodentine used for pulpotomy procedures. J Endod. 2015;41(7):1139–45. 10.1016/j.joen.2015.02.032. [DOI] [PubMed] [Google Scholar]
- 11.Camilleri J, Sorrentino F, Damidot D. Investigation of the hydration and bioactivity of radiopacified tricalcium silicate cement, Biodentine and MTA Angelus. Dent Mater. 2013;29(5):580–93. 10.1016/j.dental.2013.03.007. [DOI] [PubMed] [Google Scholar]
- 12.Khalilak Z, Esnaashari E, Saati K, Bineshmarvasti D, Yousefshahi H, Nobakht M. An in vitro comparison of coronal discolouration caused by white mineral trioxide aggregate, Theracal, calcium-enriched mixture and Biodentine. Eur Endod J. 2022;7(1):47–51. 10.14744/eej.2020.83584. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 13.Kunert M, Lukomska-Szymanska M. Bio-inductive materials in direct and indirect pulp capping—a review. Mater (Basel). 2020;13(5):1204. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 14.Siboni F, Taddei P, Prati C, Gandolfi MG. Properties of NeoMTA Plus and MTA Plus cements for endodontics. Int Endod J. 2017;50:e83–94. [DOI] [PubMed] [Google Scholar]
- 15.Nagas E, Ertan A, Eymirli A, Uyanik O, Cehreli ZC. Tooth discoloration induced by different calcium silicate-based cements: a two-year spectrophotometric and photographic evaluation in vitro. J Clin Pediatr Dent. 2021;45(2):112–6. [DOI] [PubMed] [Google Scholar]
- 16.Rajasekharan S, Martens LC, Cauwels RGEC, Verbeeck RMH. Biodentine material characteristics and clinical applications: a review of the literature. Eur Arch Paediatr Dent. 2014;15:147–58. [DOI] [PubMed] [Google Scholar]
- 17.Grech L, Mallia B, Camilleri J. Investigation of the physical properties of tricalcium silicate cement-based root-end filling materials. Dent Mater. 2013;29:e20–8. [DOI] [PubMed] [Google Scholar]
- 18.Nowicka A, Lipski M, Parafiniuk M, Sporniak-Tutak K, Lichota D, Kosierkiewicz A, et al. Response of human dental pulp capped with Biodentine and mineral trioxide aggregate. J Endod. 2013;39(6):743–7. [DOI] [PubMed] [Google Scholar]
- 19.Miller AA, Takimoto K, Wealleans J, Diogenes A. Effect of 3 bioceramic materials on stem cells of the apical papilla proliferation and differentiation using a dentin disk model. J Endod. 2018;44(4):599–603. [DOI] [PubMed] [Google Scholar]
- 20.BISCO Inc. TheraCal PT product information/technical document (Archive Rev 4–19). Available at: https://www.bisco.com/content/bisco_inc-_b2b_/variants_files/TheraCal_PT_Archive_Rev_4-19_2024-10-09-08-35-52-2024-10-09-20-35-52-827.pdf. Accessed April 26, 2026.
- 21.Okabe T, Sakamoto M, Takeuchi H, Matsushima K. Effects of pH on mineralization ability of human dental pulp cells. J Endod. 2006;32(3):198–201. 10.1016/j.joen.2005.10.041. [DOI] [PubMed] [Google Scholar]
- 22.Chen Y, Chen X, Zhang Y, Zhou F, Deng J, Zou J, et al. Materials for pulpotomy in immature permanent teeth: a systematic review and meta-analysis. BMC Oral Health. 2019;19(1):227. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 23.Metiner AM, Aktören O. Çocuk diş hekimliğinde biyoseramik materyaller. Akd Dent J. 2023;2(3):151–6. [Google Scholar]
- 24.Wang X, Xiao Y, Song W, Ye L, Yang C, Xing Y, et al. Clinical application of calcium silicate-based bioceramics in endodontics. J Transl Med. 2023;21:853. 10.1186/s12967-023-04550-4. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 25.Xavier MT, Costa AL, Ramos JC, Caramês J, Marques D, Martins JNR. Calcium Silicate-Based Cements in Restorative Dentistry: Vital Pulp Therapy Clinical, Radiographic, and Histological Outcomes on Deciduous and Permanent Dentition—A Systematic Review and Meta-Analysis. Mater (Basel). 2024;17(17):4264. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 26.Cabrera-Fernández A, Dominguez-Dominguez L, Pérez-Pérez A, Santos JMM, Díaz-Cuenca A, Torres-Lagares D, Sequeira DB, Segura-Egea JJ, Martín-González J. Clinical and Radiographic Outcomes of Vital Pulp Therapy Using Resin-Modified Versus Conventional Calcium Silicate-Based Materials: A Systematic Review and Meta-Analysis. J Funct Biomater. 2026;17(1):32. 10.3390/jfb17010032. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 27.Palma PJ, Marques JA, Santos J, Falacho RI, Sequeira D, Diogo P, et al. Tooth discoloration after regenerative endodontic procedures with calcium silicate-based cements: an ex vivo study. Appl Sci (Basel). 2020;10:5793. [Google Scholar]
- 28.Türkoğlu Kayacı Ş, Solmazgül Yazıcı Z, Arslan H. Spectrophotometric analysis of color stability induced by various calcium silicate cements in full pulpotomy of permanent molars: Theracal PT, Biodentine, and ProRoot MTA. J Endod. 2024;50(2):229–34. 10.1016/j.joen.2023.11.008. [DOI] [PubMed] [Google Scholar]
- 29.Baranwal HC, Mittal N, Samad S, Ayubi A, Aggarwal H, Kharat SM. Comparative evaluation of dual-cure resin (TheraCal PT) and Biodentine in coronal pulpotomy of patients with symptoms indicative of irreversible pulpitis: A randomized clinical trial. J Conserv Dent Endod. 2024;27(8):822–7. 10.4103/JCDE.JCDE_280_24. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 30.Commission Internationale de l’Éclairage (CIE). Recommendations on Uniform Color Spaces, Color-Difference Equations, Psychometric Color Terms. CIE Publication No. 15 (E-1.3.1). Vienna, Austria: Bureau Central de la CIE; 1978. [Google Scholar]
- 31.Sharma G, Wu W, Dalal EN. The CIEDE2000 color-difference formula: implementation notes, supplementary test data, and mathematical observations. Color Res Appl. 2005;30(1):21–30. 10.1002/col.20070. [Google Scholar]
- 32.Paravina RD, Ghinea R, Herrera LJ, Bona AD, Igiel C, Linninger M, Sakai M, Takahashi H, Tashkandi E, Perez MM. Color difference thresholds in dentistry. J Esthet Restor Dent. 2015;27(Suppl 1):S1–9. 10.1111/jerd.12149. [DOI] [PubMed] [Google Scholar]
- 33.Dozić A, Kleverlaan CJ, El-Zohairy A, Feilzer AJ, Khashayar G. Performance of five commercially available tooth color-measuring devices. J Prosthodont. 2007;16(2):93–100. [DOI] [PubMed] [Google Scholar]
- 34.Madani Z, Alvandifar S, Bizhani A. Evaluation of tooth discoloration after treatment with mineral trioxide aggregate, calcium-enriched mixture, and Biodentine in the presence and absence of blood. Dent Res J (Isfahan). 2019;16:377–83. [PMC free article] [PubMed] [Google Scholar]
- 35.Tawfeek HA, El-Bardissy AA, Abou El-Yazeed M, Youssef R, Abd Alsamad AM. Clinical and radiographic evaluation of NeoMTA versus conventional white mineral trioxide aggregate in revascularization of non-vital immature permanent anterior teeth: a randomized controlled trial. BDJ Open. 2023;9(1):17. [DOI] [PMC free article] [PubMed] [Google Scholar]
Associated Data
This section collects any data citations, data availability statements, or supplementary materials included in this article.
Data Availability Statement
The datasets generated and/or analyzed during the current study are available from the corresponding author on reasonable request.