Tooth Discoloration after Regenerative Endodontic Procedures with Calcium Silicate-Based Cements—An Ex Vivo Study

applied sciences Article Tooth Discoloration after Regenerative Endodontic Procedures with Calcium Silicate-Based Cements—An Ex Vivo Study Paulo J. Palma 1,2,* , Joana A. Marques 1, Joana Santos 3, Rui I. Falacho 4, Diana Sequeira 1, Patrícia Diogo 1,2, Francisco Caramelo 5, João C. Ramos 2,6 and João Miguel Santos 1,2 1Institute of Endodontics, Faculty of Medicine, University of Coimbra, 3000-075 Coimbra, Portugal; [email protected] (J.A.M.); [email protected] (D.S.); [email protected] (P.D.); [email protected] (J.M.S.) 2Center for Innovation and Research in Oral Sciences (CIROS), Faculty of Medicine, University of Coimbra, 3000-075 Coimbra, Portugal; [email protected] 3Dentistry Department, Faculty of Medicine, University of Coimbra, 3000-075 Coimbra, Portugal; [email protected] 4Institute of Oral Implantology and Prosthodontics, Faculty of Medicine, University of Coimbra, 3000-075 Coimbra, Portugal; [email protected] 5Coimbra Institute for Clinical and Biomedical Research (iCBR) and Laboratory of Biostatistics and Medical Informatics (LBIM), Faculty of Medicine, University of Coimbra, 3000-548 Coimbra, Portugal; [email protected] 6Institute of Operative Dentistry, Faculty of Medicine, University of Coimbra, 3000-075 Coimbra, Portugal *Correspondence: [email protected]; Tel.: +351-239-249-151 Received: 13 July 2020; Accepted: 19 August 2020; Published: 21 August 2020   Abstract: The aim of the present ex vivo study was to assess and compare coronal discoloration induced by four endodontic biomaterials used in regenerative endodontic procedures (REPs). Root resection was executed horizontally, 2 mm apical to the cementoenamel junction, in all fifty-four teeth. After accessing the pulp chamber, specimens were randomly divided in groups and filled with either saline solution or blood, followed by calcium silicate-based cements (CSCs) placement: ProRoot mineral trioxide aggregate (MTA) (Dentsply Sirona), Biodentine (Septodont), TotalFill BC (FKG), or pulp capping material (PCM) (Colt è ne). Color change ( ∆ E) was assessed using the L* a* b* system at five different timepoints (before and immediately after biomaterial application, 72 h, 7 days, and 6 months). The significance level for statistical analysis was set at p<0.05. There are statistically significant differences regarding ∆ E over time (p<0.001). Statistical differences are found considering material (p<0.001), treatment (p=0.007), or both (p=0.002). If solely the material or treatment is considered, regardless of time, statistically significant differences are detected (p<0.001). After a six-month period of evaluation, blood exposure might be a critical factor in biomaterials’ color variation. Biodentine presents the lowest discoloration potential, followed by TotalFill and PCM, albeit without statistically significant differences. MTA exhibited the greatest color variation. The selection of biomaterial should consider the material’s discoloration potential. Keywords: calcium silicate-based cements; endodontic biomaterials; regenerative endodontic procedures; tooth discoloration; vital pulp therapy 1. Introduction Regenerative endodontic procedures (REPs) demonstrate excellent success rates for the resolution of periapical pathology and increase survival of the immature tooth [ 1 – 3 ]. However, tooth discoloration Appl. Sci. 2020,10, 5793; doi:10.3390/app10175793 www.mdpi.com/journal/applsci Appl. Sci. 2020,10, 5793 2 of 13 is a major esthetic concern reported in multiple studies as an undesirable result of regenerative endodontic procedures [ 4 – 6 ] or vital pulp therapy [ 7 ]. REPs are defined as “biologically based procedures designed to replace damaged tooth structures, including dentine and root structures, as well as cells of the pulp-dentine complex” [ 8 ]. Therefore, REPs aim is to regenerate the pulp-dentine complex damaged in immature permanent teeth with pulp necrosis [ 9 ]. The association between color variation and the use of different endodontic materials in REPs, namely calcium silicate-based cements (CSCs) such as mineral trioxide aggregate—MTA (ProRoot MTA, Dentsply Sirona, Johnson City, TN, USA)—and Biodentine (Septodont, Saint-Maur-des-Foss é s, France), has been described [ 10 , 11 ]. Thus, the selection of the biomaterial must consider functional, biological, and esthetic aspects, including its discoloration potential [12–14]. MTA was developed as a filling material, presenting a wide span of clinical applications including REPs, pulp capping procedures, apexification and apexogenesis, as well as root resorptions, furcation defects, and perforation repairs [ 15 , 16 ]. Although this bioceramic shows excellent biocompatibility and bioactivity, the original formulation (gray MTA) was associated with coronal discoloration [ 16 ]. This well-known drawback triggered the development of white MTA, by modifying the originally marketed composition. However, the latter formulation still results in dental staining [ 11 , 14 , 17 , 18 ]. Considering MTA’s main disadvantages, including its discoloration potential, a new generation of CSCs was introduced, such as Biodentine (with zirconium oxide as radiopacifier), pre-mixed TotalFill BC RRM Putty (FKG, La Chaux-de-Fonds, Switzerland) and an under development experimental material (pulp capping material (PCM), Coltène/Whaledent, Altstätten, Switzerland). Nowadays, multiple hypotheses have been outlined regarding the underlying mechanisms of color alteration following REPs, with the presence of bismuth oxide within the material composition as radiopacifier [ 19 – 21 ] and blood contamination [ 22 , 23 ] being suggested as key factors to trigger and exacerbate discoloration, respectively. Current literature states that Biodentine presents superior color stability over MTA, which might be associated with the faster setting time of the formerly mentioned biomaterial [ 11 , 18 , 24 ]. Therefore, bearing this potential explanation in mind, this study aims to analyze the discoloration potential of two more recently presented calcium silicate-based cements—TotalFill BC Putty and a new experimental cement (PCM)—which were selected because of their shorter setting times (approximately 2 h and 3 min, respectively) compared to MTA. Moreover, the present study belongs to an ongoing research line [ 3 , 11 ] that aims to investigate and compare the discoloration potential of several available bioactive cements used in regenerative therapy. Since the last study [ 24 ] of the mentioned research project was conducted on acrylic teeth and evaluated the role played by blood on color stability of both Biodentine and MTA, the present study intends to confirm the possible interaction between different biomaterials and blood, when in contact with the human tooth dental structure. Thus, the aim of the present ex vivo study was to assess and compare coronal discoloration induced by four endodontic biomaterials used in REPs—ProRoot MTA, Biodentine, TotalFill BC Putty, and an experimental pulp capping material (PCM)—in the presence of saline solution or blood. The null hypothesis states that there are no statistically significant differences between the experimental groups. 2. Experimental Section 2.1. Specimen Preparation Fifty-four premolars extracted for orthodontic purposes or periodontal reasons were included in the present study. The number of samples included in the present study was based on a previous sample size calculation, performed in G*Power 3.1 software. Only teeth clinically and radiographically free of caries, cracks, restorations, and pathologic or extrinsic discolorations were selected for the experimental procedures. External surfaces of each tooth were visually inspected and cleaned with ultrasonic scaler and periodontal scalers, and polished with pumice and water in order to remove any organic material, calculus, or extrinsic staining. Appl. Sci. 2020,10, 5793 3 of 13 Root resection was executed horizontally, 2 mm apical to the cementoenamel junction, in all teeth. The access cavity was prepared in all specimens through root-end preparation using a cylindrical diamond bur (2-mm diameter, Drendel+Zweiling Diamant GmbH, Kalletal, Germany) with high speed turbine, under copious irrigation. Cavities centered on the pulp chamber with 4 mm depth and 2 mm diameter were obtained, always ensuring a peripheral minimum of 1 mm of enamel and 1 mm of dentin. The access cavities were then irrigated with sodium hypochlorite (2.5% NaOCl) to remove pulp tissue remnants, followed by a 17% EDTA (CanalPro EDTA, Colt è ne/Whaledent, Altstätten, Switzerland) irrigation to eliminate the smear layer and expose tubular dentin, and then a final rinse with saline solution (0.9% NaCl). Specimens were stored in saline solution until experimental procedures started. 2.2. Blood Collection Informed consent was obtained from a participant, according to the approval of the Ethical Committee of IRB of the Faculty of Medicine—University of Coimbra (notification CE001/2013), and a blood (6 mL) sample was collected by venipuncture. The blood collection tubes were sterile and internally coated with spray-dried tripotassium ethylenediaminetetraacetic acid (K3EDTA) to prevent clotting, thus allowing both hematology analysis and handling during sample preparation. Hematologic parameters included hematocrit and hemoglobin determinations and an erythrocyte count. The obtained values were 37.2% for hematocrit, 4.22 × 10 12 /L for erythrocytes, and 11.9 g/dL for hemoglobin. Blood samples were sealed and stored at 4 ◦C until use. 2.3. Experimental Setup Samples were randomly divided into 10 groups by the stratified random sampling method: one negative control group (n=3), one positive control group (n=3), and 8 experimental groups (n=48), as shown in Figure 1. Appl. Sci. 2020, 10, x FOR PEER REVIEW 3 of 13 radiographically free of caries, cracks, restorations, and pathologic or extrinsic discolorations were selected for the experimental procedures. External surfaces of each tooth were visually inspected and cleaned with ultrasonic scaler and periodontal scalers, and polished with pumice and water in order to remove any organic material, calculus, or extrinsic staining. Root resection was executed horizontally, 2 mm apical to the cementoenamel junction, in all teeth. The access cavity was prepared in all specimens through root-end preparation using a cylindrical diamond bur (2-mm diameter, Drendel+Zweiling Diamant GmbH, Kalletal, Germany) with high speed turbine, under copious irrigation. Cavities centered on the pulp chamber with 4 mm depth and 2 mm diameter were obtained, always ensuring a peripheral minimum of 1 mm of enamel and 1 mm of dentin. The access cavities were then irrigated with sodium hypochlorite (2.5% NaOCl) to remove pulp tissue remnants, followed by a 17% EDTA (CanalPro EDTA, Coltène/Whaledent, Altstätten, Switzerland) irrigation to eliminate the smear layer and expose tubular dentin, and then a final rinse with saline solution (0.9% NaCl). Specimens were stored in saline solution until experimental procedures started. 2.2. Blood Collection Informed consent was obtained from a participant, according to the approval of the Ethical Committee of IRB of the Faculty of Medicine—University of Coimbra (notification CE001/2013), and a blood (6 mL) sample was collected by venipuncture. The blood collection tubes were sterile and internally coated with spray-dried tripotassium ethylenediaminetetraacetic acid (K3EDTA) to prevent clotting, thus allowing both hematology analysis and handling during sample preparation. Hematologic parameters included hematocrit and hemoglobin determinations and an erythrocyte count. The obtained values were 37.2% for hematocrit, 4.22 × 10 12 /L for erythrocytes, and 11.9 g/dL for hemoglobin. Blood samples were sealed and stored at 4 °C until use. 2.3. Experimental Setup Samples were randomly divided into 10 groups by the stratified random sampling method: one negative control group (n = 3), one positive control group (n = 3), and 8 experimental groups (n = 48), as shown in Figure 1. Experimental groups (N=54) Control Control / Saline (n=3) Control / Blood (n=3) MTA MTA / Saline (n=6) MTA / Blood (n=6) Biodentine Biodentine / Saline (n=6) Biodentine / Blood (n=6) TotalFill TotalFill / Saline (n=6) TotalFill / Blood (n=6) PCM PCM / Saline (n=6) PCM / Blood (n=6) Figure 1. Experimental groups description (Saline: saline solution; MTA: ProRoot MTA; TotalFill: TotalFill BC Putty; PCM: experimental pulp capping material). Appl. Sci. 2020,10, 5793 4 of 13 After preoperative color measurement of each specimen, cavities in the control group were filled with a sterile cotton pellet moistened with saline solution (negative control–Control/Saline) and blood (positive control-Control/Blood), respectively. The access cavities of the remaining experimental groups were filled with different combinations of liquid solution (saline solution or blood) and biomaterial (ProRoot MTA, Biodentine, TotalFill BC, and PCM) as follows: MTA/Saline; MTA/Blood; Biodentine/Saline; Biodentine/Blood; TotalFill/Saline; TotalFill/Blood; PCM/Saline and PCM/Blood. Cavities were firstly filled with either saline solution or blood, using a syringe and needle with lateral exit (Kendall, Monoject, New York, NY, USA). Subsequently, biomaterials were prepared according to the manufacturers’ instructions (Table 1) and inserted into the cavities directly over the liquid solutions. All cavities were then sealed with a resin-based flowable composite—SDR Flow+ Bulk Fill Flowable A2 (Dentsply DeTrey GmbH, Konstanz, Germany)—, and light-cured for 20 s with a polywave LED curing light source (Bluephase Style, Ivoclar Vivadent AG, Schaan, Liechtenstein). Table 1. Materials’ compositions, manufacturers, preparation procedures, lot numbers and expiration dates. Material Composition Manufacturer Preparation Procedure Lot Number Expiration Date ProRoot®MTA Tricalcium silicate, bismuth oxide, dicalcium silicate, tricalcium aluminate, calcium sulfate dehydrates or gypsum Dentsply Sirona, Johnson City, TN, USA Mix powder/liquid ratio 1:3 177918 08/2020 BiodentineTM Powder: tricalcium silicate, dicalcium silicate, calcium carbonate and oxide, iron oxide, zirconium oxide Liquid: calcium chloride, hydrosoluble polymer Septodont, Saint-Maur-des-Fossés, France Pour 5 drops of liquid into the capsule Place the capsule on a mixing device Mix for 30 s B21190 11/2019 TotalFill®BC RRMTM Putty Tricalcium silicate, tantalum oxide, zirconium oxide FKG, La Chaux-de-Fonds, Switzerland No mixing is required Remove the material and place it on glass slab. Place the material into canal with an instrument and compress it. 1702BPP 11/2019 PCM Silicates, polydimethylsiloxane, silicon oils, platinum catalyst, zinc oxide, zirconium dioxide, bioactive glass, pigment Coltène/Whaledent, Altstätten, Switzerland Ready to apply using auto-mixing tips 2018120-P3-RR 08/2019 SDR TM Bulk fill flowable composite Barium-alumino-fluoro-borosilicate glass, strontium-alumino-fluoro-silicate glass, modified urethanedimethacrylateresin, EBPADMA, TEGMA, CQ, photoaccelerator, BHT, UV stabilizer, titanium dioxide, iron oxide pigments, fluorescing agent Dentsply DeTrey GmbH, Konstanz, Germany Dispense SDRTM material Light-cure for at least 20 s 1803000656 02/2021 Tooth filling and photographic record were performed in the laboratory at 23.5 ◦ C in a 52% humidity environment. Immediate postoperative color measurements were recorded, and specimens were stored in a dark environment, in an incubator (Gallenkamp, London, UK) at 37 ◦ C and 100% humidity until subsequent color measurement evaluation periods. 2.4. Photographic Record Photographic register was performed with a Canon EOS 5DsR camera using a Canon EF 100 mm f/2.8 L Macro IS USM Lens and a Canon Macro Twin Lite MT-24EX with emitters positioned at a 45 ◦ angle and cross polarization filters (polar_eyes, Emulation Group). The following settings were used: F22 aperture, ISO 100, 1/125 shutter speed, Flash at Manual 1 2 power and custom white balance (with a 18% grey card–eLAB–Emulation Group). The photographs were taken from the buccal wall/side of the teeth and were saved in RAW file format. In order to assure standard positioning of the samples, a silicone device (Virtual Refill Putty Fast Set, Ivoclar Vivadent AG, Schaan, Liechtenstein) was prepared to hold the specimens and the grey card scale was used as a reference to properly center and place the samples, as seen in Figure 2. Appl. Sci. 2020,10, 5793 5 of 13 Appl. Sci. 2020, 10, x FOR PEER REVIEW 5 of 13 In post-production, before taking color measurements, all photographic registers were calibrated using the White Balance Selector and the Exposure slider of Adobe Photoshop Lightroom (Adobe Systems, San Jose, CA, USA). Figure 2. Positioning device for photographic record. The blue dashed line delimits the total area considered for shade analysis within the Commission International de l’Eclairage (CIE) L* a* b* color space for all timeframes. 2.5. Tooth Color Measurement In shade analysis there are five evaluation periods (Figure 3): T0: baseline (after cavity preparation, before biomaterial placement); TPO: immediately after biomaterial placement and provisional restoration; T72H: after 72 h of storage; T7D: after 7 days of storage; and T6M: after six months of storage. Color assessment was performed by a single operator using ImageJ (National Institutes of Health, NIH) software, considering a central circular area, focusing on the height and width of the tooth crown, with the total area of 256 × 256 pixels. The color space was measured with the rules of the Commission International de l’Eclairage (CIE) L * a * b * system, with L * values corresponding to the lightness or luminance (ranging from 0 [black] to 100 [white]), a * values matching the red-green axis (red—positive a *; green—negative a *) and the values of b* exhibiting the yellow-blue axis (yellow— positive b *; blue—negative b *). ∆E describes the color variation between baseline and each of the subsequent color measurement periods of evaluation (TPO, T72H, T7D, and T6M), determined by using the following formula: ∆E = [󰇛∆L󰇜+ 󰇛∆a󰇜+ 󰇛∆b󰇜] / (1) Perceptible chromatic alterations for the human eye were defined at the threshold value of ∆E ≥ 3.3 [25]. Figure 2. Positioning device for photographic record. The blue dashed line delimits the total area considered for shade analysis within the Commission International de l’Eclairage (CIE) L* a* b* color space for all timeframes. In post-production, before taking color measurements, all photographic registers were calibrated using the White Balance Selector and the Exposure slider of Adobe Photoshop Lightroom (Adobe Systems, San Jose, CA, USA). 2.5. Tooth Color Measurement In shade analysis there are five evaluation periods (Figure 3): T 0 : baseline (after cavity preparation, before biomaterial placement); T PO : immediately after biomaterial placement and provisional restoration; T 72H : after 72 h of storage; T 7D : after 7 days of storage; and T 6M : after six months of storage. Appl. Sci. 2020, 10, x FOR PEER REVIEW 6 of 13 Figure 3. Color assessment of randomly selected representative samples from each experimental group at the five evaluated timeframes (T0, TPO, T72H, T7D, and T6M). 2.6. Statistical Analysis Statistical analysis was performed using IBM SPSS Statistics version 24 software and the significance level was set at α = 0.05. The description of the results regarding each material (MTA, Biodentine, TotalFill, and PCM) and each treatment option (saline solution or blood) was attained using the mean and standard deviation. To evaluate color variation over time, considering both the biomaterial and the treatment, a repeated measures ANOVA with two independent factors was applied. For each material and condition, a graph was created depicting the chromatic coordinates (L*, a*, b*) and the variation of ΔE over time. A Friedman test allowed assessment of the differences over time. Kruskal–Wallis testing was executed to evaluate the differences between the groups at T6M. Kruskal–Wallis testing was repeated for each condition. 3. Results Table 2 shows the descriptive statistics obtained for L* a* b* chromatic coordinates mean values within the different study groups over time. The initial moment is represented by T0, which corresponds to the baseline. Also, color variation (ΔE) between baseline and each of the subsequent color measurement periods of evaluation is shown in Table 2. Figure 3. Color assessment of randomly selected representative samples from each experimental group at the five evaluated timeframes (T0, TPO, T72H, T7D, and T6M). Appl. Sci. 2020,10, 5793 6 of 13 Color assessment was performed by a single operator using ImageJ (National Institutes of Health, NIH) software, considering a central circular area, focusing on the height and width of the tooth crown, with the total area of 256 × 256 pixels. The color space was measured with the rules of the Commission International de l’Eclairage (CIE) L* a* b* system, with L* values corresponding to the lightness or luminance (ranging from 0 [black] to 100 [white]), a* values matching the red-green axis (red—positive a*; green—negative a*) and the values of b* exhibiting the yellow-blue axis (yellow—positive b*; blue—negative b*). ∆E describes the color variation between baseline and each of the subsequent color measurement periods of evaluation (T PO , T 72H , T 7D , and T 6M ), determined by using the following formula: ∆E=h(∆L)2+(∆a)2+(∆b)2i1/2(1) Perceptible chromatic alterations for the human eye were defined at the threshold value of ∆ E ≥ 3.3 [ 25 ]. 2.6. Statistical Analysis Statistical analysis was performed using IBM SPSS Statistics version 24 software and the significance level was set at α =0.05. The description of the results regarding each material (MTA, Biodentine, TotalFill, and PCM) and each treatment option (saline solution or blood) was attained using the mean and standard deviation. To evaluate color variation over time, considering both the biomaterial and the treatment, a repeated measures ANOVA with two independent factors was applied. For each material and condition, a graph was created depicting the chromatic coordinates (L*, a*, b*) and the variation of ∆ E over time. A Friedman test allowed assessment of the differences over time. Kruskal–Wallis testing was executed to evaluate the differences between the groups at T 6M . Kruskal–Wallis testing was repeated for each condition. 3. Results Table 2shows the descriptive statistics obtained for L* a* b* chromatic coordinates mean values within the different study groups over time. The initial moment is represented by T 0 , which corresponds to the baseline. Also, color variation ( ∆ E) between baseline and each of the subsequent color measurement periods of evaluation is shown in Table 2. Table 2. Mean and standard deviation values of each of the L* a* b* coordinates of each experimental group for all periods of evaluation. ∆ E values express color variation from baseline (T 0 ) to each evaluation timepoint (TPO, T72H, T7D, and T6M). Groups Coordinates T0TPO T72H T7D T6M p Control/Saline L* 58.6 ±6.6 61.6 ±6.0 58.6 ±6.5 60 ±6.6 59.5 ±7.1 =0.139 a* 1 ±5.2 2.8 ±5.2 3.5 ±6.5 3.2 ±5.9 3.4 ±5.3 =0.615 b* 18.1 ±12.6 17.6 ±11.7 19.1 ±12.5 19.3 ±12.3 19 ±12.3 =0.162 ∆E 3.8 ±2.1 2.0 ±0.7 2.2 ±0.9 1.6 ±0.6 =0.615 MTA/Saline L* 59.2 ±5.6 60.4 ±5.6 55.6 ±5.2 52.3 ±5.1 52.2 ±7.3 <0.001 † a* 2.0 ±2.4 2.0 ±2.5 1.1 ±1.6 0.8 ±1.5 0.7 ±1.1 =0.070 b* 15.6 ±6.5 14.7 ±6.0 12.0 ±5.3 11.1 ±4.9 11.3 ±5<0.001 † ∆E 1.8 ±0.8 5.5 ±1.1 8.6 ±1.2 8.5 ±2.6 =0.001 † Biodentine/Saline L* 55.6 ±4.9 56.0 ±4.0 55.6 ±4.4 52.5 ±5.0 55.5 ±4=0.012 † a* 2.8 ±3 3.2 ±2.8 3.7 ±2.6 3.3 ±2.7 4.8 ±2.6 <0.001 † b* 16.4 ±4.7 16.6 ±4.1 17.1 ±4.3 16.2 ±3.8 19 ±4.5 =0.003 † ∆E 1.5 ±0.6 1.4 ±0.7 3.4 ±0.9 3.4 ±0.9 =0.006 † TotalFill/Saline L* 60.1 ±5.4 61.9 ±4.7 61.2 ±5.2 60.3 ±5.4 60.1 ±4.7 =0.162 a* 0.8 ±2.7 0.9 ±3.1 1.1 ±2.9 1.2 ±3.0 1.9 ±3.0 =0.011 † b* 13.0 ±6.8 14.2 ±6.8 14.8 ±6.4 14.6 ±6.6 17.7 ±5.5 =0.001 † ∆E 2.3 ±0.9 2.4 ±0.7 2.2 ±0.5 4.9 ±2.4 =0.102 PCM/Saline L* 59.2 ±3.4 60.8 ±3.6 60.7 ±3.6 56.8 ±3.6 61.6 ±1.3 =0.003 † a* 1.4 ±3.1 1.5 ±2.7 1.2 ±2.9 1.2 ±2.7 0.1 ±2.5 =0.171 b* 15.1 ±6.5 15.4 ±5.1 15.7 ±6.3 14.6 ±6.0 14.4 ±5.3 =0.139 ∆E 2.5 ±0.5 1.7 ±0.8 2.6 ±1.5 5.1 ±3.9 =0.284 Appl. Sci. 2020,10, 5793 7 of 13 Table 2. Cont. Groups Coordinates T0TPO T72H T7D T6M p Control/Blood L* 58.4 ±9.2 58.0 ±8.9 53.5 ±12.0 53.6 ±10.3 51.3 ±7.9 =0.053 a* 1.0 ±3.2 4.9±3.7 3.9 ±4.0 3.4 ±3.6 3.3 ±2.8 =0.034 † b* 12.5 ±5.9 10.6 ±5.3 7.5 ±4.8 8.7 ±4.9 10.8 ±5.1 =0.022 † ∆E 4.4 ±1.4 7.7 ±4 6.7 ±2.8 8.0 ±1.7 =0.122 MTA/Blood L* 58.4 ±5.1 58.6 ±4.3 54.5 ±4.1 51.3 ±4.0 52.1 ±5.1 <0.001 † a* 1.0 ±2.0 1.5 ±1.6 0.0 ±2.0 -0.4 ±2.0 0.6 ±1.9 <0.001 † b* 16.0 ±3.2 13.5 ±4.3 11.2 ±4 9.7 ±3.8 12.2 ±3.1 <0.001 † ∆E 3.4 ±2.7 6.4 ±3.1 9.8 ±2.6 7.6 ±2.4 =0.004 † Biodentine/Blood L* 60.6 ±4.2 60.6 ±4.0 60.9 ±4.1 57.3 ±3.9 61.0 ±4.2 =0.013 † a* 0.8 ±2.4 0.9 ±2.2 1.4 ±2.5 1.0 ±1.9 1.9 ±2.3 =0.010 † b* 12.8 ±5.5 12.2 ±4.8 14.0 ±4.5 12.4 ±3.8 16.7 ±4.4 =0.004 † ∆E 1.6 ±1.4 2.1 ±1.0 3.8 ±1.2 4.6 ±1.6 =0.009 † TotalFill/Blood L* 59.1 ±7.8 59.4 ±7.0 57.5 ±7.1 54.6 ±6.5 56.7 ±6.7 =0.001 † a* 0.5 ±2.5 2.7 ±3.2 1.7 ±3.0 1.5 ±2.9 1.8 ±2.9 =0.015 † b* 11.0 ±4.3 10.3 ±4.5 11.4 ±3.5 10.2 ±3.3 15.5 ±3.0 =0.006 † ∆E 3.1 ±2.1 2.5 ±0.9 5.1 ±1.3 5.5 ±2.3 =0.086 PCM/Blood L* 58.6 ±6.9 58.6 ±4.8 55.0 ±5.9 51.0 ±5.6 55.9 ±5.6 =0.001 † a* 2.0 ±2.2 3.9 ±2.0 4.8 ±3.7 4.0 ±3.4 3.2 ±2.6 =0.004 † b* 15.9 ±6.8 14.3 ±7.1 15.8 ±4.9 14.5 ±4.6 18.6 ±5.6 =0.003 † ∆E 3.3 ±1.2 5.5 ±2.9 8.4 ±3.0 4.5 ±2.5 =0.006 † T 0 : baseline (after cavity preparation, before biomaterial placement); T PO : immediately after biomaterial placement and provisional restoration; T 72H : after 72 h of storage; T 7D : after 7 days of storage; and T 6M : after six months of storage. Mean ±standard deviation. †Statistically significant difference (p<0.05). There are statistically significant differences (F(2.11 92.90) =33.58; p<0.001) regarding color variation ( ∆ E) over time. All CSC groups showed a statistically significant variation (mainly decreasing) in L* values over time, except TotalFill/Saline group. MTA presented a noticeable reduction in L* values from T PO to T 7D , regardless of the treatment (saline or blood), as well as PCM contaminated by blood. However, at T 6M , group comparison regarding lightness revealed no statistical differences between the tested biomaterials either when in contact with saline solution (p=0.065) or blood (p=0.102), which also applies to a* and b* values in both conditions (saline: a* p=0.072; b* p=0.355; blood: a* p=0.148; b* p=0.060). When in contact with saline solution, both MTA and Biodentine exhibited statistically significant color alteration over time (p=0.001 and p=0.012, respectively). In the case of blood contamination, MTA kept presenting statistically significant color variation (p=0.001) alongside Biodentine (p=0.009), and PCM (p=0.006). No statistically significant differences were detected regarding ∆ E of TotalFill groups over time, either in contact with saline solution (p=0.102) or blood (p=0.086). At 6 months all CSC groups presented perceptible color changes (∆E>3.3). Statistical differences are found considering the factor material (F(8.45, 92.90) =5.635; p<0.001), the factor treatment (F(2.11, 92.90) =5.04; p=0.007) or both material and treatment (F(8.45, 92.90) =3.31; p=0.002). The graphs on Figure 4show the mean ∆ E over time, considering materials (Figure 4a) and treatment (Figure 4b) variation. Appl. Sci. 2020, 10, x FOR PEER REVIEW 8 of 13 Statistical differences are found considering the factor material (F(8.45, 92.90) = 5.635; p < 0.001), the factor treatment (F(2.11, 92.90) = 5.04; p = 0.007) or both material and treatment (F(8.45, 92.90) = 3.31; p = 0.002). The graphs on Figure 4 show the mean ΔE over time, considering materials (Figure 4a) and treatment (Figure 4b) variation. Figure 4. Color variation (ΔE) over time considering only the (a) materials (mineral trioxide aggregate (MTA), Biodentine, TotalFill, and PCM) and (b) treatment (Saline and Blood) variation. If solely the material is considered regardless the timing of evaluation, statistically significant differences are found (F(4, 44) = 13.05; p <0.001), which also applies to treatment (F(1, 44) = 23.11; p < 0.001). MTA is the material showing lowest color stability, with statistically significant differences (p < 0.001) being detected in regard to all other tested CSCs (Biodentine, TotalFill, and PCM). Furthermore, no statistically significant differences are found between Biodentine, TotalFill, and PCM. Additionally, there are statistically significant differences (F(4.44) = 2.71; p = 0.042) concerning the interaction between factor material and factor treatment. From all the CSC groups, MTA/Saline and MTA/Blood showed the highest color variation, with statistically significant differences. The total ∆E from T0 to each of the following assessment periods is presented in Figure 5. Figure 4. Color variation ( ∆ E) over time considering only the ( a ) materials (mineral trioxide aggregate (MTA), Biodentine, TotalFill, and PCM) and (b) treatment (Saline and Blood) variation. Appl. Sci. 2020,10, 5793 8 of 13 If solely the material is considered regardless the timing of evaluation, statistically significant differences are found (F(4, 44) =13.05; p<0.001), which also applies to treatment (F(1, 44) =23.11; p<0.001). MTA is the material showing lowest color stability, with statistically significant differences (p<0.001) being detected in regard to all other tested CSCs (Biodentine, TotalFill, and PCM). Furthermore, no statistically significant differences are found between Biodentine, TotalFill, and PCM. Additionally, there are statistically significant differences (F(4.44) =2.71; p=0.042) concerning the interaction between factor material and factor treatment. From all the CSC groups, MTA/Saline and MTA/Blood showed the highest color variation, with statistically significant differences. The total ∆ E from T0to each of the following assessment periods is presented in Figure 5. Appl. Sci. 2020, 10, x FOR PEER REVIEW 8 of 13 Statistical differences are found considering the factor material (F(8.45, 92.90) = 5.635; p < 0.001), the factor treatment (F(2.11, 92.90) = 5.04; p = 0.007) or both material and treatment (F(8.45, 92.90) = 3.31; p = 0.002). The graphs on Figure 4 show the mean ΔE over time, considering materials (Figure 4a) and treatment (Figure 4b) variation. Figure 4. Color variation (ΔE) over time considering only the (a) materials (mineral trioxide aggregate (MTA), Biodentine, TotalFill, and PCM) and (b) treatment (Saline and Blood) variation. If solely the material is considered regardless the timing of evaluation, statistically significant differences are found (F(4, 44) = 13.05; p <0.001), which also applies to treatment (F(1, 44) = 23.11; p < 0.001). MTA is the material showing lowest color stability, with statistically significant differences (p < 0.001) being detected in regard to all other tested CSCs (Biodentine, TotalFill, and PCM). Furthermore, no statistically significant differences are found between Biodentine, TotalFill, and PCM. Additionally, there are statistically significant differences (F(4.44) = 2.71; p = 0.042) concerning the interaction between factor material and factor treatment. From all the CSC groups, MTA/Saline and MTA/Blood showed the highest color variation, with statistically significant differences. The total ∆E from T0 to each of the following assessment periods is presented in Figure 5. Figure 5. ∆E of all experimental groups over time. Table 3is generated by performing group comparison according to individual evaluation of material/treatment interaction. PCM contaminated by blood led to a statistically higher color change when compared to Biodentine/Saline (p=0.012). Table 3. p-values obtained from group comparison regarding the interaction of factor material and factor treatment. MTA/Saline Biodentine/Saline TotalFill/Saline PCM/Saline Blood MTA/Blood Biodentine/Blood TotalFill/Blood PCM/Blood Saline 0.010 * 1.000 1.000 1.000 0.010 * 0.001 * 1.000 0.767 0.069 MTA/Saline 0.001 * 0.006 * 0.007 * 1.000 0.996 0.008 * 0.218 0.996 Biodentine/Saline 1.000 0.999 0.002 * <0.001 * 0.999 0.544 0.012 * TotalFill/Saline 1.000 0.010 * <0.001 * 1.000 0.918 0.069 PCM/Saline 0.010 * <0.001 * 1.000 0.921 0.071 Blood 1.000 0.012 * 0.174 0.943 MTA/Blood 0.001 * 0.028 * 0.750 Biodentine/Blood 0.939 0.082 TotalFill/Blood 0.739 * Statistically significant difference (p<0.05). 4. Discussion The aim of the present ex vivo study was to evaluate and compare the discoloration potential of four different CSCs used in regenerative endodontic procedures, as well as to assess the role played by blood contamination on color variation severity when CSCs are applied. Theoretically, recently introduced biomaterials (Biodentine, TotalFill, and PCM) with a faster setting time when Appl. Sci. 2020,10, 5793 9 of 13 compared to the gold standard MTA might present superior color stability. Moreover, it is crucial to unveil the possible role played by blood when in contact with CSCs since blood exposure is likely to occur in both REPs and vital pulp therapy. An insight into these matters would allow adequate selection of the biomaterial to use based on the clinical conditions, ultimately allowing the accomplishment of better esthetic results. In fact, it has been extensively reported that REPs lead to dental discoloration. This undesirable consequence of regenerative therapy is a major concern for both clinicians and patients, especially when an esthetic area is involved [ 4 – 7 ]. REPs protocol involves the creation of a cervical plug by placing a 3–4 mm CSC layer in the coronal portion of the root canal. Endodontic materials should present chromatic stability and optic properties similar to dental structures and not cause tooth discoloration over time [ 14 ]. However, as previously mentioned, the association of CSCs with color variation over time is well-described within the literature. The obtained results show that there are statistically significant differences in ∆ E over time (p<0.001). In fact, statistical differences were found considering the factor material (p<0.001), the factor treatment (p=0.007) or the interaction of both factors (p=0.001), over time. Therefore, the null hypothesis, stating that there are no statistically significant differences between the experimental groups, has been rejected. Several possible mechanisms have been outlined to explain color alteration following CSC placement, one being that the composition of the biomaterial is a major contributing factor to CSCs’ discoloration potential [ 26 ]. MTA formula contains bismuth oxide as a radiopacifier (Bi 2 O 3 ), which has been proven to lead to tooth discoloration through its (1) reduction or (2) oxidation when in contact with strong oxidizing agents, such as dentin collagen and sodium hypochlorite, used as an irrigation solution [ 26 ]. In fact, previous studies consistently demonstrate MTA to exhibit lower color stability when compared to alternative calcium silicate-based cements [ 10 , 27 ], hypothesizing that the obtained results could arise from the incorporation of different radiopacifiers such as zirconium oxide and tantalum oxide, instead of bismuth oxide, in the more recently introduced CSCs [26]. Concerning blood contamination, the overall available scientific evidence shows blood exposure to be a factor that significantly exacerbates CSC color alteration [ 2 ]. This discoloration severity increase by blood might be related to material porosity and presence or absence of smear layer, which can reduce or increase dentin permeability, respectively. MTA has a longer setting time (2 h and 45 min) compared with all the remaining three tested materials (Biodentine, 12 min; TotalFill, 2 h; PCM, 3 min). Therefore, it seems MTA remains porous for longer which results in increased blood absorption and subsequent hemolysis, with consequently greater discoloration. On the other hand, a recent in vitro study found that contact with blood after a follow-up period of 6 months does not modify color alterations suffered by the biomaterials [ 24 ]. Our findings do not line up with the results of the previously mentioned study since, after a 6-month period of evaluation, the present study found statistically significant differences regarding the factor material, as well as regarding the treatment (blood/saline solution), meaning that blood exposure might be a critical factor in biomaterials’ color variation. The contrasting results in respect to the role played by blood on color stability of CSCs, among studies with similar experimental protocols, might be related to the use of different experimental models. The study of Palma et al. [ 24 ] was conducted in an acrylic model, while the present study was made under ex vivo conditions. The use of natural human teeth may have led to a discoloration enhancement in the case of blood contamination due to the penetration of blood into the dentinal tubules [ 11 ]. For that reason the presence of blood has become a factor that significantly influences the chromatic result in the present study, which is in agreement with the overall literature [2]. It is, therefore, crucial to ensure complete hemostasis [ 26 ] and perform adequate blood clot stabilization, as well as to clean the blood present on dentin walls as meticulously as possible, to prevent blood influx into the tooth structure prior to CSC placement [ 24 ]. Moreover, the use of natural teeth as experimental model reproduces the clinical context more faithfully, thus allowing a better forecast of the impact of blood contamination on the esthetic outcome of the treatment.