- Email: [email protected]
Coronal Tooth Discoloration and White Mineral Trioxide Aggregate
Basic Research—Biology
Coronal Tooth Discoloration and White Mineral Trioxide Aggregate Daniel Felman, BDSc, DCD, and Peter Parashos, MDSc, PhD Abstract Introduction: This study assessed and characterized discoloration when white MTA (wMTA) was placed in the coronal aspect of the root canal ex vivo and the influence of red blood cells on this discoloration. Methods: Canals were prepared from the apical aspect and restored with either wMTA + saline (n = 18), wMTA + blood (n = 18), or controls (n = 4 + 4) (blood or saline alone). Color was assessed according to the CIE L*a*b* color space using standardized digital photographs at 3 time points: baseline, day 1, and day 35. Statistical analysis was performed by using 1-way analysis of variance and a 2-sample t test with P < .05. Results: All teeth discolored when restored with wMTA, which was most prominent in the cervical third of the crown. The presence of blood within the canal adjacent to the setting wMTA exacerbated the discoloration (P = .03). Conclusions: wMTA induces the gray discoloration of the tooth crown, and the effect is compounded in the presence of blood. (J Endod 2013;39:484–487)
Key Words Discoloration, mineral trioxide aggregate, vital pulp therapy, white mineral trioxide aggregate
From Melbourne Dental School, University of Melbourne, Melbourne, Victoria, Australia. Supported by a grant from the Australian Society of Endodontology. Address requests for reprints to Dr Peter Parashos, Melbourne Dental School, University of Melbourne, 720 Swanston Street, Melbourne, Victoria, Australia 3010. E-mail address: [email protected] 0099-2399/$ - see front matter Copyright ª 2013 American Association of Endodontists. http://dx.doi.org/10.1016/j.joen.2012.11.053
T
he initial formulation of mineral trioxide aggregate (MTA) was a gray powder and was associated with coronal tooth discoloration (1, 2). Other studies have also reported discoloration after the placement of MTA but have not specified the type (3, 4). Because of the coronal discoloration caused by gray MTA, white MTA (wMTA) was developed and has been commercially available since 2002 (5). This formulation was thought to be more suitable for use as a pulp capping material in the esthetic region (6, 7) although slight discoloration may still result, necessitating a veneer or crown (7). Discoloration associated with wMTA was initially reported in in vitro and ex vivo studies, with the discoloration affecting the material both on its surface and internally (8, 9). Subsequent case reports have also described coronal tooth discoloration associated with wMTA (10, 11) although the discoloration is reversible with a simple walking bleach technique (11). To date, there are no studies that account for or quantify the discoloration of wMTA. Although the material itself may cause discoloration, another possible mechanism is an interaction between the red blood cells within the adjacent vital pulp and the setting wMTA. Red blood cells are a known tooth staining agent (12, 13). The aim of this study was to assess and quantify coronal tooth discoloration by wMTA.
Materials and Methods Tooth Preparation Forty-four single-rooted, unrestored premolar and incisor teeth extracted predominantly for orthodontic reasons were selected. The teeth were stored in chloramine T neutral (1%) followed by storage in buffered calcium phosphate solution until they were used in accordance with ethics approval guidelines (Ethics ID: 1035181, Human Research Ethics Committee, The University of Melbourne, Melbourne, Victoria, Australia). Extrinsic stain and calculus were removed with an ultrasonic scaler followed by polishing with pumice and water. The root tips were resected (2–3 mm) to expose the root canals and canal preparation standardized as follows. Canals were measured and enlarged from the apical aspect to the most coronal aspect of the pulp chamber to allow standardization of the thickness and volume of MTA and provide a closed system without the potential complication of coronal microleakage. Canal enlargement was initially performed with stainless steel K-files (VDW, Munich, Germany) followed by Gates-Glidden drills (#2–5). Finally, ParaPost drills (Coltene/Whaledent, Altst€atten, Switzerland) were used in sequence to produce a canal size equivalent to a ParaPost size 7 drill (Green, 1.75-mm diameter). Root canals were irrigated with sodium hypochlorite (6 mL, 4%; DentaLife, Croydon, Australia) to remove any pulp tissue remaining in pulp horns and not removed with the largest ParaPost drill followed by EDTA (4 mL, 15%, DentaLife) for 2 minutes to remove the smear layer and to expose the dentinal tubules. Canals were irrigated with a final rinse of sodium hypochlorite (1 mL, 4%). Blood Collection Whole blood (12 mL) was collected from a volunteer by venipuncture by a specialist medical hematologist. The blood collection tubes (3 4 mL) were sterile and spray coated with the anticoagulant K2EDTA to prevent clotting in order to facilitate the experiment. Hematologic testing (CELL-DYN Ruby; Abbott Laboratories, Abbott Park, IL) included a baseline hematocrit (percentage volume of erythrocytes).
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JOE — Volume 39, Number 4, April 2013
Basic Research—Biology TABLE 1. Teeth and Materials in Experimental and Control Groups Experimental groups
Control groups
wMTA + saline (wMTA and saline moistened sterile cotton pellets; n = 18)
Negative control (Saline moistened cotton pellets; n = 4)
wMTA + blood (wMTA and blood moistened sterile cotton pellets; n = 18)
Positive control (Blood moistened cotton pellets; n = 4)
To maximize the erythrocyte-mediated tooth discoloration in the experiment, the hematocrit was increased from 45% to 70% by volume. This involved fractionation via centrifugation (3000 rpm for 2 minutes), blood plasma removal, reconstitution, and reassessment. This cycle was repeated until a hematocrit level of 70% was achieved. The vials were then sealed and refrigerated (4 C) until they were used.
Experimental Setup Teeth were randomized into experimental or control groups (Table 1) using a random number generator (14). wMTA (ProRoot MTA; Dentsply Tulsa Dental, TN) was mixed according to the manufacturer’s directions and packed via the apical aspect to the most coronal aspect of the standardized preparation to a thickness of 3 mm with an endodontic plugger (size 9/11; Hu-Friedy, Chicago, IL). Indirect ultrasonic compaction (10 seconds) minimized voids within the material and ensured uniformity between the samples. Sterile cotton pellets (size 4; Richmond Dental, Charlotte, NC) were loosely placed within the prepared canals from the apical access. A pipette was used to transfer the blood or saline to saturate the cotton pellets. The apical openings were sealed with sticky wax (Dentsply Tulsa Dental, Johnson City, TN). Control teeth were identically prepared but without the placement of wMTA (Table 1). All samples were stored at 100% humidity in an incubator at 37 C for 35 days (ie, from T1 to T35) with 0.5 mL phosphate-buffered saline seeded in each specimen tube to act as the humidifying agent. Tooth Shade Assessment The CIE L*a*b* color space was used for tooth shade assessment (15). The nonlinear relations for L*, a*, and b* are designed to approximate the perceptual response of the human eye. Of particular interest in this study was the L* component that closely matches the human perception of lightness (ie, value) ranging from L* = 0 (black) to L* = 100 (white). A decrease in the L* value denotes increased grayness and therefore tooth discoloration. In addition, a* values represent color gradients spanning green to red, and b* values represent blue to yellow. Color measurements were recorded at 3 time points: (1) T0: baseline (after tooth preparation but before the placement of materials), (2) T1: 1 day after material placement, and (3) T35: 35 days upon retrieval.
Color measurement points (51 51 pixels) were identified and recorded in the cervical, middle, and incisal thirds. Images T0, T1, and T35 were overlaid within Adobe Photoshop CS4 (Adobe, San Jose, CA) to ensure that all color measurement positions were coincident between the samples. Digital photographic images were recorded under standardized lighting and desiccation conditions. Images were captured using a high-resolution digital camera (Canon EOS 450D, 12.2 MP; Canon Inc, Tokyo, Japan) in RAW file format with a 100-mm macrolens (Canon EF 100-mm F2.8 macro USM) under standardized conditions (exposure: 1/200 seconds, aperture: f/22; white balance flash: 5500 Ko) (16). Casmatch (Bear Medic Corp, Tokyo, Japan) shade reference cards were included in each image to provide a reference for shade control during analysis. Images were imported into Adobe Photoshop CS4 for shade analysis within the CIE L*a*b* color space. For reproducible positioning of each tooth within the photographic setup, composite resin was bonded onto each root (away from the crown) such that the tooth crown was parallel to the glass platform on which it was being photographed. All tooth samples were numerically coded before randomization with no additional identifying data present, and the researcher was blinded to the specific treatment history of any given sample.
Statistical Analysis Minitab statistical software (Minitab Inc, State College, PA) was used for the analysis with the level of significance set at P < .05. Two separate datasets were used to statistically analyze the relationships between the 4 study groups for the time points T0–T1, T0–T35, and T1–T35: (1) pooled (ie, cervical, middle, and incisal) and (2) cervical. Calculated from these datasets were !L*, !a*, !b*, !E, !Chroma, and !Hue values (15) for each of the 3 time intervals. One-way analysis of variance using the Fisher method for individual comparisons and 2-sample t tests were applied to analyze the data. The Pearson correlation coefficient (r) was used to assess the correlation between baseline L* values and !L* for both datasets over each of the previously described time periods.
Results The results relating to the gray discoloration (L*) data are summarized in Table 2. The percentage discoloration in the cervical region on days 1 and 35 is shown in Figure 1.
Control Groups The most significant discoloration was observed in positive control teeth, most intensely in the cervical third of the crown. One day after blood placement within the canal, the cervical region showed a diffuse pink-red background stain with a* values (red) increasing some
TABLE 2. Grayness Expressed as Mean Standard Deviation Values for Baseline (T0 L*) and Changes over the Time Periods T0–T1 and T1–T35 (DL*) DL* T0–T1
DL* T1–T35
DL* Total
% L* DTotal
Region
L* T0
wMTA + saline
Cervical Pooled
80.83 3.17 80.31 3.51
1.83 2.48 1.41 2.11
1.78 1.96 0.81 2.09
3.61 2.66 2.22 2.62
4.47 3.29 2.77 3.26
* A
wMTA + blood
Cervical Pooled
80.50 3.97 80.00 4.06
0.89 1.81 0.74 1.92
4.50 2.38 2.06 2.78
5.39 2.33 2.80 2.82
6.69 2.89 3.50 3.53
U A
Positive control
Cervical Pooled
77.75 4.35 79.25 4.41
12.50 3.11 10.08 3.58
11.50 7.59 6.58 7.06
24.00 5.35 16.67 6.77
30.87 6.89 21.03 8.55
# B
Negative control
Cervical Pooled
80.50 2.89 79.83 3.30
1.50 1.29 0.33 2.64
1.25 2.06 1.67 2.39
2.75 1.71 1.33 1.72
3.42 2.12 1.67 2.16
‡ C
The overall change in grayness values for each group and location is expressed as a percentage (% L* DTotal). Groups that do not share identical letters are significantly different (P < .05). Groups that do not share identical symbols are significantly different (P < .05).
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MTA Discoloration
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Basic Research—Biology
Figure 1. Percentage of discoloration in the cervical region at days 1 and 35.
206.7% 82.6 % above the baseline recording. Also at this time, the positive control teeth increased in grayness in the order of 16.12% 4.00 % in the cervical region. Over the following 34 days, 3 of the 4 positive control teeth showed a substantial reversal of the initial pink-red staining with a* values decreasing 151.1% 67.1%. Also in this time period, the positive control teeth continued to discolor gray (14.79% 9.77%). Although the positive control and the wMTA + blood groups contained the blood solution in the canal space, the discoloration shown by the positive control group was significantly greater in all tooth segments (P < .001). The negative control group showed a small increase in brightness with a mean increase of 3.42% at T35 (Fig. 1).
wMTA Groups One-way analysis of variance indicated that the teeth restored with wMTA discolored significantly in comparison with the negative control group (pooled P = .003, cervical P = .001). Both wMTA groups exhibited a discoloration gradient with the discoloration more prominent in the cervical third of the crown. In comparison with the baseline recordings, the cervical third discoloration shown by the wMTA + blood group was in the order of 6.69% 2.89% (P = .03), and for the wMTA + saline group it was 4.47% 3.29% (P = .03) (Table 2). This shade difference would be clearly discernible (17–19). When the cervical, middle, and incisal third data were combined, the wMTA + blood group continued to show greater discoloration, but this difference was not statistically significant (Table 2). The only statistically significant changes were related to the L* values. There were no significant changes in other color measures such as chroma or hue.
Discussion This study has shown that wMTA can discolor ex vivo teeth, and the presence of blood within the canal adjacent to the setting wMTA can exacerbate this discoloration. The factors responsible for the lightening of the negative control teeth are uncertain. Possibly, the sodium 486
Felman and Parashos
hypochlorite canal irrigation may have induced a mild bleaching effect, which would have affected all teeth in the study and thus been a constant for all the experimental groups. Discoloration was greatest in the wMTA + blood teeth, with mean gray value increases of 2.22% greater than the wMTA + saline teeth because of the presence of blood rather than saline within the canal. The fact that the wMTA + blood teeth did not discolor as severely as the blood-filled positive controls indicates that the wMTA material limited the diffusion of erythrocytes from the canal into the coronal dentin. The rapid development of a diffuse pink-red discoloration at day 1 in the positive control group affected the entire crown, with only minor loss of color intensity toward the incisal edge. This initial color change was likely caused by the transmission of the rich red-crimson hue of the freshly prepared erythrocytic concentrated blood solution through the tooth structure. Similarly, Marin et al (13) reported that dentin stained by blood initially stained red, whereas at a later time it showed a dark-brown hue. The authors hypothesized that this change may have resulted from the formaldehyde fixation process used in their experimental protocol. However, the present experiment used no fixation, and it is possible that this shade change reflects the interaction between light and the physiologic degradation of the internalized erythrocytes via hemolysis. Various studies have used whole blood to stain teeth in preparation for further experimentation, with centrifugation commonly used to encourage erythrocytic extravasation into the dentinal tubules to promote and accelerate tooth discoloration (12, 13, 20, 21). The present study did not use centrifugation, and the pattern of tooth discoloration in the positive control group may better reflect in vivo intrapulpal hemorrhage and its esthetic consequences. The mechanisms by which wMTA impacts on coronal tooth discoloration and those by which blood exacerbates this discoloration are currently unknown. One possible mechanism may relate to the oxidation and incorporation of the remaining iron content within the wMTA powder into the calcium aluminoferrite phase of the set wMTA cement. Although set wMTA contains only 9% of the iron oxide of gray MTA (22, 23), this quantity may be sufficient to result in the observed discoloration. Alternative materials to MTA have been developed to improve upon its handling (24), but the discoloration potential of these materials has not been assessed. Similarly, the release of heavy metal ions from MTA has been reported (25) although their effect on tooth discoloration is unknown but conceivably minimal because of the short-term release. Another mechanism may be the interaction between erythrocytes and the unset wMTA. Discoloration of traumatized teeth results from the hemolysis of erythrocytes and the accumulation of hemoglobin and hematin molecules within dentin tubules (20). The slow hydrating process of wMTA may permit the absorption and subsequent hemolysis of erythrocytes from the adjacent pulpal tissue, thus resulting in both material and subsequent tooth discoloration. Further research is required to determine the precise mechanisms responsible for this finding.
Acknowledgments The authors thank the following people: Dr Erik Magee for his support; Mr Chris Owen, Melbourne Dental School, for technical support; Dr Mark Levin for hematologic support; Ms Susan Evans of Graphic Partners for IT support; Dr Sandy Clarke, Statistical Consulting Center, The University of Melbourne. The authors deny any conflicts of interest related to this study. JOE — Volume 39, Number 4, April 2013
Basic Research—Biology References 1. Karabucak B, Li D, Lim J, et al. Vital pulp therapy with mineral trioxide aggregate. Dent Traumatol 2005;21:240–3. 2. Bogen G, Kim JS, Bakland LK. Direct pulp capping with mineral trioxide aggregate: an observational study. J Am Dent Assoc 2008;139:305–15. 3. Naik S, Hegde AM. Mineral trioxide aggregate as a pulpotomy agent in primary molars: an in vivo study. J Indian Soc Pedod Prev Dent 2005;23:13–6. 4. Jacobovitz M, de Pontes Lima RK. The use of calcium hydroxide and mineral trioxide aggregate on apexification of a replanted tooth: a case report. Dent Traumatol 2009; 25:e32–6. 5. Asgary S, Parirokh M, Eghbal MJ, et al. A comparative study of white mineral trioxide aggregate and white Portland cements using x-ray microanalysis. Aust Endod J 2004;30:89–92. 6. Bogen G, Kuttler S. Mineral trioxide aggregate obturation: a review and case series. J Endod 2009;35:777–90. 7. Bakland LK. Revisiting traumatic pulpal exposure: materials, management principles, and techniques. Dent Clin North Am 2009;53:661–73. 8. Watts JD, Holt DM, Beeson TJ, et al. Effects of pH and mixing agents on the temporal setting of tooth-colored and gray mineral trioxide aggregate. J Endod 2007;33: 970–3. 9. Boutsioukis C, Noula G, Lambrianidis T. Ex vivo study of the efficiency of two techniques for the removal of mineral trioxide aggregate used as a root canal filling material. J Endod 2008;34:1239–42. 10. Jacobovitz M, de Pontes Lima RK. Treatment of inflammatory internal root resorption with mineral trioxide aggregate: a case report. Int Endod J 2008; 41:905–12. 11. Belobrov I, Parashos P. Treatment of tooth discoloration after the use of white mineral trioxide aggregate. J Endod 2011;37:1017–20. 12. Freccia WF, Peters DD. A technique for staining extracted teeth: a research and teaching aid for bleaching. J Endod 1982;8:67–9.
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13. Marin PD, Bartold PM, Heithersay GS. Tooth discoloration by blood: an in vitro histochemical study. Endod Dent Traumatol 1997;13:132–8. 14. StatTrek. Available at: http://stattrek.com/Tables/Random.aspx. Accessed June 16, 2011. 15. Commission Internationale de l’Eclairage: Recommendations on uniform color spaces, color difference equations, psychometric color terms. CIE Publication No. 15 1978;(suppl 2). Available at: http://www.cie.co.at. Accessed March 20, 2011. 16. Wee AG, Lindsey DT, Kuo S, et al. Color accuracy of commercial digital cameras for use in dentistry. Dent Mater 2006;22:553–9. 17. Kuehni RG, Marcus RT. An experiment in visual scaling of small color differences. Col Res Appl 1979;4:83–91. 18. Seghi RR, Hewlett ER, Kim J. Visual and instrumental colorimetric assessments of small color differences on translucent dental porcelain. J Dent Res 1989;68: 1760–4. 19. Lindsey DT, Wee AG. Perceptibility and acceptability of CIELAB color differences in computer-simulated teeth. J Dent 2007;35:593–9. 20. Marin PD, Heithersay GS, Bridges TE. A quantitative comparison of traditional and non-peroxide bleaching agents. Endod Dent Traumatol 1998;14:64–7. 21. Maiolo K, Marin PD, Bridges TE, et al. Evaluation of a combined thiourea and hydrogen peroxide regimen to bleach bloodstained teeth. Aust Dent J 2007;52: 33–40. 22. Asgary S, Parirokh M, Eghbal MJ, et al. A qualitative x-ray analysis of white and gray mineral trioxide aggregate using compositional imaging. J Mater Sci Mater Med 2006;17:187–91. 23. Asgary S, Parirokh M, Eghbal MJ, et al. Chemical differences between white and gray mineral trioxide aggregate. J Endod 2005;31:101–3. 24. Porter ML, Berto A, Primus CM, et al. Physical and chemical properties of newgeneration endodontic materials. J Endod 2010;36:524–8. 25. Schembri M, Peplow G, Camilleri J. Analyses of heavy metals in mineral trioxide aggregate and Portland cement. J Endod 2010;36:1210–5.
MTA Discoloration
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Coronal Tooth Discoloration and White Mineral Trioxide Aggregate Daniel Felman, BDSc, DCD, and Peter Parashos, MDSc, PhD Abstract Introduction: This study assessed and characterized discoloration when white MTA (wMTA) was placed in the coronal aspect of the root canal ex vivo and the influence of red blood cells on this discoloration. Methods: Canals were prepared from the apical aspect and restored with either wMTA + saline (n = 18), wMTA + blood (n = 18), or controls (n = 4 + 4) (blood or saline alone). Color was assessed according to the CIE L*a*b* color space using standardized digital photographs at 3 time points: baseline, day 1, and day 35. Statistical analysis was performed by using 1-way analysis of variance and a 2-sample t test with P < .05. Results: All teeth discolored when restored with wMTA, which was most prominent in the cervical third of the crown. The presence of blood within the canal adjacent to the setting wMTA exacerbated the discoloration (P = .03). Conclusions: wMTA induces the gray discoloration of the tooth crown, and the effect is compounded in the presence of blood. (J Endod 2013;39:484–487)
Key Words Discoloration, mineral trioxide aggregate, vital pulp therapy, white mineral trioxide aggregate
From Melbourne Dental School, University of Melbourne, Melbourne, Victoria, Australia. Supported by a grant from the Australian Society of Endodontology. Address requests for reprints to Dr Peter Parashos, Melbourne Dental School, University of Melbourne, 720 Swanston Street, Melbourne, Victoria, Australia 3010. E-mail address: [email protected] 0099-2399/$ - see front matter Copyright ª 2013 American Association of Endodontists. http://dx.doi.org/10.1016/j.joen.2012.11.053
T
he initial formulation of mineral trioxide aggregate (MTA) was a gray powder and was associated with coronal tooth discoloration (1, 2). Other studies have also reported discoloration after the placement of MTA but have not specified the type (3, 4). Because of the coronal discoloration caused by gray MTA, white MTA (wMTA) was developed and has been commercially available since 2002 (5). This formulation was thought to be more suitable for use as a pulp capping material in the esthetic region (6, 7) although slight discoloration may still result, necessitating a veneer or crown (7). Discoloration associated with wMTA was initially reported in in vitro and ex vivo studies, with the discoloration affecting the material both on its surface and internally (8, 9). Subsequent case reports have also described coronal tooth discoloration associated with wMTA (10, 11) although the discoloration is reversible with a simple walking bleach technique (11). To date, there are no studies that account for or quantify the discoloration of wMTA. Although the material itself may cause discoloration, another possible mechanism is an interaction between the red blood cells within the adjacent vital pulp and the setting wMTA. Red blood cells are a known tooth staining agent (12, 13). The aim of this study was to assess and quantify coronal tooth discoloration by wMTA.
Materials and Methods Tooth Preparation Forty-four single-rooted, unrestored premolar and incisor teeth extracted predominantly for orthodontic reasons were selected. The teeth were stored in chloramine T neutral (1%) followed by storage in buffered calcium phosphate solution until they were used in accordance with ethics approval guidelines (Ethics ID: 1035181, Human Research Ethics Committee, The University of Melbourne, Melbourne, Victoria, Australia). Extrinsic stain and calculus were removed with an ultrasonic scaler followed by polishing with pumice and water. The root tips were resected (2–3 mm) to expose the root canals and canal preparation standardized as follows. Canals were measured and enlarged from the apical aspect to the most coronal aspect of the pulp chamber to allow standardization of the thickness and volume of MTA and provide a closed system without the potential complication of coronal microleakage. Canal enlargement was initially performed with stainless steel K-files (VDW, Munich, Germany) followed by Gates-Glidden drills (#2–5). Finally, ParaPost drills (Coltene/Whaledent, Altst€atten, Switzerland) were used in sequence to produce a canal size equivalent to a ParaPost size 7 drill (Green, 1.75-mm diameter). Root canals were irrigated with sodium hypochlorite (6 mL, 4%; DentaLife, Croydon, Australia) to remove any pulp tissue remaining in pulp horns and not removed with the largest ParaPost drill followed by EDTA (4 mL, 15%, DentaLife) for 2 minutes to remove the smear layer and to expose the dentinal tubules. Canals were irrigated with a final rinse of sodium hypochlorite (1 mL, 4%). Blood Collection Whole blood (12 mL) was collected from a volunteer by venipuncture by a specialist medical hematologist. The blood collection tubes (3 4 mL) were sterile and spray coated with the anticoagulant K2EDTA to prevent clotting in order to facilitate the experiment. Hematologic testing (CELL-DYN Ruby; Abbott Laboratories, Abbott Park, IL) included a baseline hematocrit (percentage volume of erythrocytes).
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JOE — Volume 39, Number 4, April 2013
Basic Research—Biology TABLE 1. Teeth and Materials in Experimental and Control Groups Experimental groups
Control groups
wMTA + saline (wMTA and saline moistened sterile cotton pellets; n = 18)
Negative control (Saline moistened cotton pellets; n = 4)
wMTA + blood (wMTA and blood moistened sterile cotton pellets; n = 18)
Positive control (Blood moistened cotton pellets; n = 4)
To maximize the erythrocyte-mediated tooth discoloration in the experiment, the hematocrit was increased from 45% to 70% by volume. This involved fractionation via centrifugation (3000 rpm for 2 minutes), blood plasma removal, reconstitution, and reassessment. This cycle was repeated until a hematocrit level of 70% was achieved. The vials were then sealed and refrigerated (4 C) until they were used.
Experimental Setup Teeth were randomized into experimental or control groups (Table 1) using a random number generator (14). wMTA (ProRoot MTA; Dentsply Tulsa Dental, TN) was mixed according to the manufacturer’s directions and packed via the apical aspect to the most coronal aspect of the standardized preparation to a thickness of 3 mm with an endodontic plugger (size 9/11; Hu-Friedy, Chicago, IL). Indirect ultrasonic compaction (10 seconds) minimized voids within the material and ensured uniformity between the samples. Sterile cotton pellets (size 4; Richmond Dental, Charlotte, NC) were loosely placed within the prepared canals from the apical access. A pipette was used to transfer the blood or saline to saturate the cotton pellets. The apical openings were sealed with sticky wax (Dentsply Tulsa Dental, Johnson City, TN). Control teeth were identically prepared but without the placement of wMTA (Table 1). All samples were stored at 100% humidity in an incubator at 37 C for 35 days (ie, from T1 to T35) with 0.5 mL phosphate-buffered saline seeded in each specimen tube to act as the humidifying agent. Tooth Shade Assessment The CIE L*a*b* color space was used for tooth shade assessment (15). The nonlinear relations for L*, a*, and b* are designed to approximate the perceptual response of the human eye. Of particular interest in this study was the L* component that closely matches the human perception of lightness (ie, value) ranging from L* = 0 (black) to L* = 100 (white). A decrease in the L* value denotes increased grayness and therefore tooth discoloration. In addition, a* values represent color gradients spanning green to red, and b* values represent blue to yellow. Color measurements were recorded at 3 time points: (1) T0: baseline (after tooth preparation but before the placement of materials), (2) T1: 1 day after material placement, and (3) T35: 35 days upon retrieval.
Color measurement points (51 51 pixels) were identified and recorded in the cervical, middle, and incisal thirds. Images T0, T1, and T35 were overlaid within Adobe Photoshop CS4 (Adobe, San Jose, CA) to ensure that all color measurement positions were coincident between the samples. Digital photographic images were recorded under standardized lighting and desiccation conditions. Images were captured using a high-resolution digital camera (Canon EOS 450D, 12.2 MP; Canon Inc, Tokyo, Japan) in RAW file format with a 100-mm macrolens (Canon EF 100-mm F2.8 macro USM) under standardized conditions (exposure: 1/200 seconds, aperture: f/22; white balance flash: 5500 Ko) (16). Casmatch (Bear Medic Corp, Tokyo, Japan) shade reference cards were included in each image to provide a reference for shade control during analysis. Images were imported into Adobe Photoshop CS4 for shade analysis within the CIE L*a*b* color space. For reproducible positioning of each tooth within the photographic setup, composite resin was bonded onto each root (away from the crown) such that the tooth crown was parallel to the glass platform on which it was being photographed. All tooth samples were numerically coded before randomization with no additional identifying data present, and the researcher was blinded to the specific treatment history of any given sample.
Statistical Analysis Minitab statistical software (Minitab Inc, State College, PA) was used for the analysis with the level of significance set at P < .05. Two separate datasets were used to statistically analyze the relationships between the 4 study groups for the time points T0–T1, T0–T35, and T1–T35: (1) pooled (ie, cervical, middle, and incisal) and (2) cervical. Calculated from these datasets were !L*, !a*, !b*, !E, !Chroma, and !Hue values (15) for each of the 3 time intervals. One-way analysis of variance using the Fisher method for individual comparisons and 2-sample t tests were applied to analyze the data. The Pearson correlation coefficient (r) was used to assess the correlation between baseline L* values and !L* for both datasets over each of the previously described time periods.
Results The results relating to the gray discoloration (L*) data are summarized in Table 2. The percentage discoloration in the cervical region on days 1 and 35 is shown in Figure 1.
Control Groups The most significant discoloration was observed in positive control teeth, most intensely in the cervical third of the crown. One day after blood placement within the canal, the cervical region showed a diffuse pink-red background stain with a* values (red) increasing some
TABLE 2. Grayness Expressed as Mean Standard Deviation Values for Baseline (T0 L*) and Changes over the Time Periods T0–T1 and T1–T35 (DL*) DL* T0–T1
DL* T1–T35
DL* Total
% L* DTotal
Region
L* T0
wMTA + saline
Cervical Pooled
80.83 3.17 80.31 3.51
1.83 2.48 1.41 2.11
1.78 1.96 0.81 2.09
3.61 2.66 2.22 2.62
4.47 3.29 2.77 3.26
* A
wMTA + blood
Cervical Pooled
80.50 3.97 80.00 4.06
0.89 1.81 0.74 1.92
4.50 2.38 2.06 2.78
5.39 2.33 2.80 2.82
6.69 2.89 3.50 3.53
U A
Positive control
Cervical Pooled
77.75 4.35 79.25 4.41
12.50 3.11 10.08 3.58
11.50 7.59 6.58 7.06
24.00 5.35 16.67 6.77
30.87 6.89 21.03 8.55
# B
Negative control
Cervical Pooled
80.50 2.89 79.83 3.30
1.50 1.29 0.33 2.64
1.25 2.06 1.67 2.39
2.75 1.71 1.33 1.72
3.42 2.12 1.67 2.16
‡ C
The overall change in grayness values for each group and location is expressed as a percentage (% L* DTotal). Groups that do not share identical letters are significantly different (P < .05). Groups that do not share identical symbols are significantly different (P < .05).
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Figure 1. Percentage of discoloration in the cervical region at days 1 and 35.
206.7% 82.6 % above the baseline recording. Also at this time, the positive control teeth increased in grayness in the order of 16.12% 4.00 % in the cervical region. Over the following 34 days, 3 of the 4 positive control teeth showed a substantial reversal of the initial pink-red staining with a* values decreasing 151.1% 67.1%. Also in this time period, the positive control teeth continued to discolor gray (14.79% 9.77%). Although the positive control and the wMTA + blood groups contained the blood solution in the canal space, the discoloration shown by the positive control group was significantly greater in all tooth segments (P < .001). The negative control group showed a small increase in brightness with a mean increase of 3.42% at T35 (Fig. 1).
wMTA Groups One-way analysis of variance indicated that the teeth restored with wMTA discolored significantly in comparison with the negative control group (pooled P = .003, cervical P = .001). Both wMTA groups exhibited a discoloration gradient with the discoloration more prominent in the cervical third of the crown. In comparison with the baseline recordings, the cervical third discoloration shown by the wMTA + blood group was in the order of 6.69% 2.89% (P = .03), and for the wMTA + saline group it was 4.47% 3.29% (P = .03) (Table 2). This shade difference would be clearly discernible (17–19). When the cervical, middle, and incisal third data were combined, the wMTA + blood group continued to show greater discoloration, but this difference was not statistically significant (Table 2). The only statistically significant changes were related to the L* values. There were no significant changes in other color measures such as chroma or hue.
Discussion This study has shown that wMTA can discolor ex vivo teeth, and the presence of blood within the canal adjacent to the setting wMTA can exacerbate this discoloration. The factors responsible for the lightening of the negative control teeth are uncertain. Possibly, the sodium 486
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hypochlorite canal irrigation may have induced a mild bleaching effect, which would have affected all teeth in the study and thus been a constant for all the experimental groups. Discoloration was greatest in the wMTA + blood teeth, with mean gray value increases of 2.22% greater than the wMTA + saline teeth because of the presence of blood rather than saline within the canal. The fact that the wMTA + blood teeth did not discolor as severely as the blood-filled positive controls indicates that the wMTA material limited the diffusion of erythrocytes from the canal into the coronal dentin. The rapid development of a diffuse pink-red discoloration at day 1 in the positive control group affected the entire crown, with only minor loss of color intensity toward the incisal edge. This initial color change was likely caused by the transmission of the rich red-crimson hue of the freshly prepared erythrocytic concentrated blood solution through the tooth structure. Similarly, Marin et al (13) reported that dentin stained by blood initially stained red, whereas at a later time it showed a dark-brown hue. The authors hypothesized that this change may have resulted from the formaldehyde fixation process used in their experimental protocol. However, the present experiment used no fixation, and it is possible that this shade change reflects the interaction between light and the physiologic degradation of the internalized erythrocytes via hemolysis. Various studies have used whole blood to stain teeth in preparation for further experimentation, with centrifugation commonly used to encourage erythrocytic extravasation into the dentinal tubules to promote and accelerate tooth discoloration (12, 13, 20, 21). The present study did not use centrifugation, and the pattern of tooth discoloration in the positive control group may better reflect in vivo intrapulpal hemorrhage and its esthetic consequences. The mechanisms by which wMTA impacts on coronal tooth discoloration and those by which blood exacerbates this discoloration are currently unknown. One possible mechanism may relate to the oxidation and incorporation of the remaining iron content within the wMTA powder into the calcium aluminoferrite phase of the set wMTA cement. Although set wMTA contains only 9% of the iron oxide of gray MTA (22, 23), this quantity may be sufficient to result in the observed discoloration. Alternative materials to MTA have been developed to improve upon its handling (24), but the discoloration potential of these materials has not been assessed. Similarly, the release of heavy metal ions from MTA has been reported (25) although their effect on tooth discoloration is unknown but conceivably minimal because of the short-term release. Another mechanism may be the interaction between erythrocytes and the unset wMTA. Discoloration of traumatized teeth results from the hemolysis of erythrocytes and the accumulation of hemoglobin and hematin molecules within dentin tubules (20). The slow hydrating process of wMTA may permit the absorption and subsequent hemolysis of erythrocytes from the adjacent pulpal tissue, thus resulting in both material and subsequent tooth discoloration. Further research is required to determine the precise mechanisms responsible for this finding.
Acknowledgments The authors thank the following people: Dr Erik Magee for his support; Mr Chris Owen, Melbourne Dental School, for technical support; Dr Mark Levin for hematologic support; Ms Susan Evans of Graphic Partners for IT support; Dr Sandy Clarke, Statistical Consulting Center, The University of Melbourne. The authors deny any conflicts of interest related to this study. JOE — Volume 39, Number 4, April 2013
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