Evaluation of pH, calcium ion release and antimicrobial activity of a new calcium aluminate cement

Abstract

This study evaluated the pH, calcium ion release and antimicrobial activity of EndoBinder (EB), containing different radiopacifiers: bismuth oxide (Bi₂O₃), zinc oxide (ZnO) or zirconium oxide (ZrO₂), in comparison to MTA. For pH and calcium ion release tests, 5 specimens per group (n = 5) were immersed into 10 mL of distilled and deionized water at 37°C. After 2, 4, 12, 24, 48 h; 7, 14 and 28 days, the pH was measured and calcium ion release quantified in an atomic absorption spectrophotometer. For antimicrobial activity, the cements were tested against S. aureus, E. coli, E. faecalis and C. albicans, in triplicate. MTA presented higher values for pH and calcium ion release than the other groups, however, with no statistically significant difference after 28 days (p > 0.05); and the largest inhibition halos for all strains, with no significant difference (E. coli and E. faecalis) for pure EB and EB + Bi₂O₃ (p > 0.05). EB presented similar performance to that of MTA as regards pH and calcium ion release; however, when ZnO and ZrO₂ were used, EB did not present antimicrobial activity against some strains.

Endodontics; Root Canal Filling Materials; Disk Diffusion Antimicrobial Tests

Introduction

Among the desirable characteristics of sealing cements, reparative capacity and antimicrobial activity stand out in particular. Mineral Trioxide Aggregate (MTA) was originally developed for retrograde filling and treatment of root and furcal perforations,^{1 , 2} but its good clinical performance and biocompatibility^{1 , 2} led to its use in other situations, including pulp capping and pulpotomy.^{1 - 3}

Studies have reported the antimicrobial activity of MTA;^{4 , 5} however, its most interesting property is its ability to stimulate mineralized tissue formation,⁶ promoting less inflammatory reaction after direct application than calcium hydroxide cement.^{7 , 8}

During MTA hydration, calcium disilicate and trisilicate react to form calcium hydroxide and hydrated calcium silicate gel, thus making the pH of the medium alkaline.⁹ The calcium ions released during MTA setting are diffused through the dentinal tubules, increasing their concentration as the setting time increases, thus ensuring the good reparative capacity of the cement.^{6 - 8}

Nonetheless, MTA has some disadvantages that warrant the development of new materials with better properties, including long setting time,¹⁰ low flow capacity¹¹ and high incidence of dental structure staining.^{12 , 13}

With this in mind, a new calcium aluminate-based cement¹⁴ called EndoBinder (Binderware, São Carlos, Brazil) was developed. It preserves the properties and clinical applications of MTA, thus offering an alternative to compensate for MTA disadvantages. According to several studies, EndoBinder presents good biological, mechanical and anti-microbiological properties,^{15 - 17}and does not promote dental structure staining.¹⁸

Bismuth oxide, used as a radiopacifier in the MTA composition, increases MTA radiopacity, producing values higher than those equivalent to the Al scale.¹⁴ However it is not inert, and interferes in the hydration mechanism of MTA, altering its physicochemical properties.¹³

Thus, the aim of this study was to evaluate the pH, calcium ion release and antimicrobial activity of EndoBinder, composed with different radiopacifiers, as compared with MTA. The null hypothesis tested was that there would be no difference among the cements tested, in regard to the properties evaluated.

Methodology

The cements used in this study are described in Table 1.

Table 1
Cements used in the study.

Materials	Group	Manufacturer
EndoBinder pure	EBP	Binderware, São Carlos, Brazil
EndoBinder + 20% (weight) Bi2O3	EBBO
EndoBinder + 20% (weight) ZnO	EBZnO
EndoBinder + 20% (weight) ZrO2	EBZrO
MTA	MTA	Ângelus, Londrina, Brazil

Evaluation of pH and calcium ion release

In performing the calcium ion release and the pH tests, 1 mL of the tested cements (n = 5) were inserted into polyethylene tubes (10 mm long × 2 mm in diameter), using a plastic syringe. The cements were manipulated according to the manufacturers' recommendations, in the proportion of 1 g of powder to 0.21 mL of distilled water for EndoBinder; and 1 dose of powder to 1 drop of distilled water for MTA. After filling the tubes, their interiors were radiographed to evaluate whether they had been completely filled.

The specimens were immersed individually into Falcon tubes (BD Falcon, Franklin Lakes, USA), containing 10 mL of distilled and deionized water with a neutral pH (7.0), and stored at 37°C. The pH was assessed with a microprocessor controlled pH analyzer (pH 2000, Instrutherm, São Paulo, Brazil)at time intervals of 2, 4, 12, 24, 48 h and 7, 14 and 28 days of immersion. At each time interval, the polyethylene tube containing the cement was transferred to a new Falcon tube under the same conditions as the initial one, to assess pH and the ability of the cements to release calcium ions continuously. The resulting solution from each period of analysis was used to quantify the calcium ion release in the atomic absorption spectrophotometer (Perkin-Elmer Analyst 100, Perkin-Elmer Inc., Norwalk, USA).

Antimicrobial activity

The double-layer agar diffusion method was employed to evaluate the antimicrobial activity of the cements. Using a Teflon matrix (5 mm in internal diameter × 5 mm thick), 12 specimens of each cement (n = 3) were made in accordance with the manufacturers' instructions, as previously described.

S. aureus (ATCC 25923) and E. coli (ATCC 25922) were cultured aerobically on Muller Hinton Agar, E. faecalis (ATCC 29212), on Tryptic Soy Agar under microaerobic conditions in a candle jar with 3%-5% CO₂, and C. albicans (ATCC 10231), on Sabouraud Dextrose Agar under aerobic conditions.

Recent and pure bacterial cultures (37°C for 24 h) were placed on the appropriate culture media and transferred to test tubes with a 0.85% saline solution to obtain standard bacterial (~10⁸ CFU/mL) and fungal (~10⁶ CFU/mL) inocula, which were measured in a spectrophotometer (absorbance 0.08 to 0.1 and λ = 625 nm).

The culture media was poured into Petri plates to form the base layer, composed of 12 mL. After solidification, specimens of each cement type were applied to the base layer, and 8 mL of culture media, melted at 45°C with 1% standard microbial inoculum, was added to the base layer to form the seed layer. The plates were kept at room temperature for 2 h to allow pre-diffusion of the cements, and were then incubated at 37°C for 24 h.

The inhibition zones around the test specimens were measured by a millimeter ruler with a 0.5 mm accuracy. The tests were performed in triplicate, and the mean values of each test were submitted to statistical analysis.

Statistical analysis

The values obtained for the pH and the calcium ion release tests (2-way ANOVA, Bonferroni test, p < 0.05) and antimicrobial activity (1-way ANOVA, Tukey's test, p < 0.05) were statistically analyzed with Graphpad Prism 4.0 software (GraphPad Software, La Jolla, USA).

Results

pH and calcium ion release

The pH mean values are presented in Table 2.

Table 2
Mean and standard deviation of pH values at different time intervals.

	MTA	EBP	EBBO	EBZnO	EBZrO
2 h	8.0 (0.6) a,A	7.6 (0.5) ac,A	6.7 (0.1) b,A	6.7 (0.1) b,AB	7.1 (0.2) bc,A
4 h	8.1 (0.4) a,A	6.6 (0.2) b,B	6.8 (0.2) b,A	6.5 (0.1) b,A	6.7 (0.2) b,A
12 h	8.7 (0.8) a,AB	6.7 (0.2) b,B	6.7 (0.1) b,A	6.6 (0.0) b,AB	6.9 (0.6) b,A
24 h	8.2 (0.2) a,A	6.7 (0.1) b,B	6.9 (0.1) b,AB	6.6 (0.1) b,AB	6.8 (0.3) b,A
48 h	8.3 (0.2) a,A	7.6 (0.2) ab,A	7.6 (0.1) ab,B	7.3 (0.3) b,B	7.9 (0.3) ab,BC
7 days	9.4 (0.1) a,B	8.4 (0.2) b,C	9.1 (0.1) ab,C	8.6 (0.2) b,C	8.8 (0.2) ab,D
14 days	9.2 (0.4) a,B	8.6 (0.3) a,C	9.0 (0.1) a,C	8.6 (0.3) a,C	8.5 (0.3) a,CD
28 days	9.4 (0.4) a,B	8.9 (0.5) a,C	8.9 (0.4) a,C	9.0 (0.4) a,C	8.8 (0.5) a,D

• *Different lower case letters in the line and capital letters in the column indicate a statistically significant difference (2-way ANOVA, Bonferroni test, P < 0.05)

MTA presented the highest pH values, ranging from 8.0 to 9.4 at 28 days. In the initial period, there was no significant difference for EBP (p > 0.05); however, MTA differed from the other groups (p < 0.05). Only EBP presented a statistically significant difference for EBBO and EBZnO (p < 0.05). At 14 and 28 days, all cements presented similar results, with no statistically significant difference among them (p > 0.05).

Comparing the same material during the different periods (Figure 1), the pH of MTA increased progressively. EBP presented an increase in pH up until 28 days, but with no statistically significant difference as of the 7^th day (p > 0.05).

Figure 1
Graphic representation of the pH mean values at different time intervals.

The calcium ion release mean values may be seen in Table 3.

Table 3
Mean values and standard deviation for calcium ions (mg/dL) released at the different time intervals tested.

	MTA	EBP	EBBO	EBZnO	EBZrO
2 h	0.60 (0.01) a,A	1.54 (0.02) b,A	0.57 (0.01) a,A	0.68 (0.01) a,A	1.06 (0.02) ab,A
4 h	0.40 (0.01) a,A	0.24 (0.01) a,B	0.13 (0.01) a,A	0.19 (0.00) a,A	0.21 (0.01) a,B
12 h	0.54 (0.02) a,A	0.30 (0.01) a,B	0.24 (0.01) a,A	0.23 (0.00) a,A	0.23 (0.01) a,B
24 h	0.31(0.01) a,A	0.18 (0.00) a,B	0.19 (0.01) a,A	0.16 (0.00) a,A	0.17 (0.01) a,B
48 h	0.63(0.01) a,A	0.46 (0.01) a,B	0.52 (0.01) a,A	0.35 (0.01) a,A	0.36 (0.01) a,B
7 days	2.32 (0.04) a,B	1.88 (0.04) a,A	2.44 (0.05) a,B	1.94 (0.02) a,BC	2.02 (0.03) a,CD
14 days	2.00 (0.02) a,B	1.84 (0.05) a,A	2.00 (0.05) a,B	1.81 (0.03) a,B	1.76 (0.03) a,C
28 days	3.00 (0.07) a,C	2.66 (0.06) a,C	2.45 (0.07) a,B	2.50 (0.05) a,C	2.56 (0.06) a,D

• *Different lower case letters in the line and capital letters in the column indicate a statistically significant difference (2-way ANOVA, Bonferroni test, p < 0.05).

When the different materials were compared according to the same period (Figure 2), only EBP (2 h) presented a statistically significant difference in relation to the other groups (p < 0.05), which showed no significant difference among one another (p > 0.05). In the other periods of analysis, there was no statistically significant difference among the groups (p > 0.05).

Figure 2
Graphic representation of the calcium ion (mg/dL) release mean values at different time intervals.

Antimicrobial activity

The inhibition halo mean values may be seen in Table 4.

Table 4
Mean values of inhibition halo diameters (mm).

	MTA	EBP	EBBO	EBZrO	EBZnO
S. aureus	11.0 a	7.0 b	7.3 b	7.0 b	8.3 ab
E. coli	10.0 a	7.0 a	8.0 a	-	-
E. faecalis	10.0 a	7.3 a	8.0 a	-	-
C. albicans	13.7 a	7.0 b	8.3 b	-	-

• *Different letters in the lines indicate a statistically significant difference (1-way ANOVA, Tukey test, p < 0.05).

MTA presented an inhibition halo similar to that of EBZnO (p > 0.05) for S. aureus, but differing statistically from those of the other groups (p < 0.05). EBZrO and EBZnO presented no antimicrobial activity for E. coli and E. faecalis. The other cements presented activity against these strains, but with no statistically significant difference among one another (p > 0.05). EBZrO and EBZnO also presented no antimicrobial activity for C. albicans. MTA presented a statistically significant difference for EBP and EBBO (p < 0.05), which did not differ statistically between each other (p > 0.05).

Discussion

In the present study, the pH, the calcium ion release and the antimicrobial activity of EndoBinder with different radiopacifiers were evaluated in comparison with MTA. Thus, the null hypothesis tested was partially accepted, insofar as the cements showed a similar behavior for pH and calcium ion release. However, in terms of antimicrobial activity, the different radiopacifiers played a relevant role in the results for EB.

Unlike the results of this study, in which MTA presented a maximum pH value of 9.4, Cutajar et al. ¹⁹ found values higher than 12.0. This may be explained by the difference in the methodology used for pH measurement. Cutajar et al. ¹⁹ performed pH measurements directly in the cement after manipulation, and not in tubes containing distilled and deionized water, as in the present study. The method used in the present study has the advantage of allowing pH to be measured over longer periods of time, and reveals not only the pH of the material during setting, but also the medium alkalinization capacity of the material after setting.¹⁹

The initial pH value of MTA was higher than that of EndoBinder in its different forms, because of the calcium chloride in the MTA formula, added to accelerate its setting time.²⁰ This component significantly increases the pH after manipulation, thus keeping the medium alkaline, due to the additional calcium added to the formula.²⁰ However, some authors^{20 , 21} observed a reduction in pH when calcium chloride was added to MTA, because of the repulsion between chlorine and hydroxyl ions within the material.

During its hydration process, MTA yields hydrated calcium silicate, a by-product of calcium hydroxide.⁹ The reactions involving calcium silicate in the presence of water promote the hydrogenation of Ca(OH)₂ and CaO; therefore, MTA releases a high concentration of calcium ions into the medium⁹ produced from the calcium hydroxide, and from the decomposition of calcium silicate hydrate, which is released more slowly than calcium hydroxide.²² Thus, the dissociation of calcium hydroxide into calcium and hydroxyl ions results in a pH increase, and consequently, increased antimicrobial activity.²³

On the other hand, the hydration process of EndoBinder yields calcium aluminate and aluminum hydroxide hydrates.²⁴ The release of calcium and hydroxyl ions may be attributed to the decomposition of calcium aluminate hydrate, which occurs more slowly in EndoBinder than in MTA, and could explain the lower pH of EndoBinder in the initial periods, and the slower increase of pH in EndoBinder, in comparison with MTA.²⁵

In the particular case of EndoBinder, the water and cement (w/c) ratio, and also temperature, are determinants of the phases that will result from the hydration process.²⁵ Under the conditions in which the tests were performed (temperature of 37°C and w/c ratio = 0.21), the AH3 (Al₂O₃.3H₂O) and C3AH6 (3CaO.Al₂O₃.6H₂O) phases stood out particularly.²⁶ This considered, the results obtained for calcium ion release by EndoBinder are coherent with the above explanation. Because the dissolution and precipitation of the hydrated phases are quick for EBP, the concentration of calcium ions in solution is high in the first few hours.²⁶

Hydration process phases are more stable after 2 h, after which the calcium ion release rate is reduced.²⁵ This rate only begins to increase with the hydration of CaO, which occurs in the anhydrous composition of calcium aluminate cement, as a result of how the cement is obtained, i.e., from the calcination of aluminum (Al₂O₃) and calcium carbonate (CaCO₃).²⁵ The results demonstrated an increase in calcium ion release as of 48 h in an aqueous medium. The tendency is the same when distinct radiopacifiers are added to EBP. The same increased calcium ion release as of 48 hours applies to the pH results, although, in the first hours, these results are slightly influenced by the calcium chloride, which tends to act as a buffer of the solution in which the EndoBinder phases are hydrated.²⁰

Regarding antimicrobial activity, MTA inhibited all microorganisms effectively, as corroborated by the increase in the pH of the medium, due to calcium hydroxide formation.²³ Likewise, the antimicrobial activity of EndoBinder may be attributed to the decomposition of the calcium aluminate hydrate formed, which, in turn, releases calcium and hydroxyl ions.²⁴

It is known that the radiopacifiers present in cement formulation are not inert and interfere in the mechanism of cement hydration, decreasing the calcium ion release rates and affecting several physicochemical properties.^{13 , 22} This interference could be observed in the present study, insofar as the different radiopacifiers used influenced the antimicrobial activity of EB. Only EBZrO and EBZnO presented no antimicrobial activity against E. coli, E. faecalis and C. albicans; however, they were effective against S. aureus. The other forms of EndoBinder, namely, EBP and EBBO, also showed antimicrobial activity against E. faecalis, the microorganism most present in recurrent endodontic infections.^{27 , 28}

In another study, Jacobovitz et al. ¹⁵ evaluated the antimicrobial activity of MTA and EndoBinder + ZnO through apical microleakage tests, and found an absence of microbial growth after 30 days in both cements. The discrepant results could be explained by an important aspect of EndoBinder, namely, its good rheological properties (flowability and plasticity),²⁵ which offer an alternative to compensate for the negative features of MTA-based cements, such as sealing ability.

Conclusions

EndoBinder presented similar behavior to that of MTA for pH and calcium ion release, irrespective of the radiopacifier used. However, when ZnO and ZrO₂ were added, EndoBinder presented no antimicrobial activity against some microbial strains. In spite of the good performance of EndoBinder, further studies must be conducted before it can be validated as a sealing cement for root and furcal perforation treatment.

References

• ¹
  Al-kahtani A, Shostad S, Schifferle R, Bhambhani S. In vitro evaluation of microleakage of an orthograde apical plug of mineral trioxide aggregate in permanent teeth with simulated immature apices. J Endod. 2005 Feb;31(2):117-9.
• ²
  Andelin WE, Shabahang S, Wright K, Torabinejad M. Identification of hard tissue after experimental pulp capping using dentin sialoprotein (DSP) as a marker. J Endod. 2003 Oct;29(10):646-50.
• ³
  Accorinte ML, Holland R, Reis A, Bortoluzzi MC, Murata SS, Dezan Jr E, et al. Evaluation of mineral trioxide aggregate and calcium hydroxide cement as pulp-capping agent in human teeth. J Endod. 2008 Jan;34(1):1-6.
• ⁴
  Tanomaru JM, Tanomaru-Filho M, Hotta J, Watanabe E, Ito IY. Antimicrobial activity of endodontic sealers based on calcium hydroxide and MTA. Acta Odontol Latinoam. 2008;21(2):147-51.
• ⁵
  Odabas ME, Cinar C, Akça G, Araz I, Ulusu T, Yücel H. Short-term antimicrobial properties of mineral trioxide aggregate with incorporated silver-zeolite. Dent Traumatol. 2011 Jun;27(3):189-94.
• ⁶
  Cintra LT, Moraes IG, Estrada BP, Gomes Filho JE, Bramante CM, Garcia RB, et al. Evaluation of the tissue response to MTA and MBPC: microscopic analysis of implants in alveolar bone of rats. J Endod. 2006 Jun;32(6):556-9.
• ⁷
  Faraco Jr IM, Holland R. Response of the pulp of dogs to capping with mineral trioxide aggregate or a calcium hydroxide cement. Dent Traumatol. 2001 Aug;17(4):163-6.
• ⁸
  Min KS, Park HJ, Lee SK, Park SH, Hong CU, Kim HW, et al. Effect of mineral trioxide aggregate on dentin bridge formation and expression of dentin Sialoprotein and Heme Oxygenase-1 in human dental pulp. J Endod. 2008 Jun;34(6):666-70.
• ⁹
  Belío-Reyes IA, Bucio L, Cruz-Chavez E. Phase composition of ProRoot mineral trioxide aggregate by X-ray powder diffraction. J Endod. 2009 Jun;35(6):875-8.
• ¹⁰
  Torabinejad M, Hong CU, McDonald F, Pitt Ford TR. Physical and chemical properties of a new root-end filling material. J Endod. 1995 Jul;21(7):349-53.
• ¹¹
  Torabinejad M, Chivian N. Clinical applications of mineral trioxide aggregate. J Endod. 1999 Mar;25(3):197-205.
• ¹²
  Jacobovitz M, Lima RKP. The use of calcium hydroxide and mineral trioxide aggregate on apexification of a replanted tooth: a case report. Dent Traumatol. 2009 Jun;25(3):e32-6.
• ¹³
  Camilleri J. The physical properties of accelerated Portland cement for endodontic use. Int Endod J. 2008 Feb;41(2):151-7.
• ¹⁴
  Aguilar FG, Garcia LF, Rossetto HL, Pardini LC, Pires-de-Souza FC. Radiopacity evaluation of calcium aluminate cement containing different radiopacifying agents. J Endod. 2011 Jan;37(1):67-71.
• ¹⁵
  Jacobovitz M, Vianna ME, Pandolfelli VC, Oliveira IR, Rossetto HL, Gomes BP. Root canal filling with cements based on mineral aggregates: an in vitro analysis of bacterial microleakage. Oral Surg Oral Med Oral Pathol Oral Radiol Endod. 2009 Jul;108(1):140-4.
• ¹⁶
  Garcia LFR, Aguilar FG, Sabino MG, Rosseto HL, Pires-de-Souza FCP. Mechanical and microstructural characterization of new calcium aluminate cement (EndoBinder). Adv Appl Ceram. 2011 Nov;110(8),469-75.
• ¹⁷
  Aguilar FG, Garcia LFR, Pires-de-Souza FCP. Biocompatibility of new calcium aluminate cement (EndoBinder). J Endod. 2012 Mar;38(3):367-71.
• ¹⁸
  Garcia LD, Aguilar FG, Rossetto HL, Sabino MG, Pires-de-Souza FD. Staining susceptibility of new calcium aluminate cement (EndoBinder) in teeth: a 1-year in vitro study. Dent Traumatol. 2012 Oct 16. DOI: 10.1111/edt.12011.
  » https://doi.org/10.1111/edt.12011
• ¹⁹
  Cutajar A, Mallia B, Abela S, Camilleri J. Replacement of radiopacifier in mineral trioxide aggregate; characterization and determination of physical properties. Dent Mater. 2011 Sep;27(9):879-91.
• ²⁰
  Huang TH, Shie MY, Kao CT, Ding SJ. The effect of setting accelerator on properties of mineral trioxide aggregate. J Endod. 2008 May;34(5):590-3.
• ²¹
  Bortoluzzi EA, Juárez Broon N, Duarte MAH, Demarchi ACO, Bramante CM. The use of a setting accelerator and its effect on pH and calcium ion release of mineral trioxide aggregate and white Portland cement. J Endod. 2006 Dec;32(12):1194-7.
• ²²
  Camilleri J. Characterization of hydration products of mineral trioxide aggregate. Int Endod J. 2008 May;41(5):408-17.
• ²³
  Duarte MA, Demarchi ACCO, Yamashita JC, Kuga MC, Fraga SC. pH and calcium ion release of 2 root-end filling materials. Oral Surg Oral Med Oral Pathol Oral Radiol Endod. 2003 Mar;95(3):345-7.
• ²⁴
  Alt C, Wong L, Parr C. Measuring castable rheology by exothermic profile. Refractories Appl News. 2008 Mar;8(2):15-8.
• ²⁵
  Oliveira IR, Pandolfelli VC, Jacobovitz M. Chemical, physical and mechanical properties of a novel calcium aluminate endodontic cement. Int Endod J. 2010 Dec;43(12):1069-76.
• ²⁶
  Luz AP, Pandolfelli V. CaCO3 addition effect on the hydration and mechanical strength evolution of calcium aluminate cement for endodontic applications. Ceram Int. 2012 Mar;38(2):1417-25.
• ²⁷
  Ribeiro CS, Scelza MF, Hirata Júnior R, Oliveira LMB. The antimicrobial activity of gray-colored mineral trioxide aggregate (GMTA) and white-colored MTA (WMTA) under aerobic and anaerobic conditions. Oral Surg Oral Med Oral Pathol Oral Radiol Endod. 2010 Jun;109(6):e109-12.
• ²⁸
  Molander A, Reit C, Dahlén G, Kvist T. Microbiological status of root-filled teeth with apical periodontitis. Int Endod J. 1998 Jan;31(1):1-7.

Publication Dates

• Publication in this collection
  Jul-Aug 2013

History

• Received
  08 Nov 2012
• Accepted
  08 May 2013