Chemistry and Bioactivity of NeoMTA Plus TM versus MTA Angelus ® Root Repair Materials

Endodontic Department, Faculty of Dentistry, King Abdulaziz University, Jeddah, Saudi Arabia Endodontic Department, Faculty of Oral and Dental Medicine, Cairo University, Cairo, Egypt Department of Pediatric Dentistry, Faculty of Dentistry, King Abdulaziz University, Jeddah, Saudi Arabia Spectroscopy Department, Physics Division, National Research Center, Giza, Egypt Department of Operative Dentistry, Faculty of Dentistry, King Abdulaziz University, Jeddah, Saudi Arabia Biomaterials Division, Faculty of Oral and Dental Medicine, Tanta University, Tanta, Egypt Biomaterials and Tissue Engineering Division, UCL Eastman Dental Institute, 256 Gray’s Inn Road, London WC1X 8LD, UK


Introduction
Mineral trioxide aggregate (MTA) is a hydrophilic calcium silicate-based cement that is traditionally used as a root repair material, mainly developed from Portland cement [1,2].It consists of tricalcium and dicalcium silicate particles which harden in a wet environment forming calcium silicate hydrate [2][3][4][5][6][7].It proved to be an excellent material for pulp capping, pulpotomy, root perforation repair, root end filling, and pulp regeneration [8].Difficult manipulation and long setting time, however, limit the use of MTA.New calcium silicate-based, NeoMTA Plus, cement has been recently introduced as fast-set root and periapical tissue repair material.NeoMTA Plus is easily manipulated and remains in place without being washed out (due to its unique gel properties) and does not stain the tooth [9,10].Little information, however, is currently available on this material, and its hydration reaction is not yet well understood.
The aim of this study was to examine the chemical composition, surface structure, and bioactivity of the fast-set root repair material, NeoMTA Plus, in comparison with traditional White MTA (MTA Angelus) using Fourier transform infrared (FTIR) spectroscopy, X-ray diffraction (XRD), and scanning electron microscope with energy dispersive X-ray (SEM/EDX).The null hypothesis of this study is that there is no difference in their composition, surface structure, and bioactivity.

Method and Materials
The proposal of this study was accepted by the ethical committee of King Abdulaziz University.White MTA Angelus (Londrina, PR, Brazil) and NeoMTA Plus (Avalon Biomed Inc., Bradenton) were used in this study.The unhydrated dry powder of both materials was analysed with Fourier transform infrared (FTIR 6100, Jasco, Japan) spectroscopy and X-ray diffraction (XRD, Empyrean, Analytical 2010, Holland) to identify their original composition.According to the manufacturer's instructions, each material was mixed with its activator at a powder-to-liquid ratio of 3 : 1.The freshly mixed materials were analysed with FTIR to determine changes during the initial hydration (setting) reaction.Then, the samples were incubated at 37 °C and 100% humidity for 24 hours; the final setting was then analysed using the FTIR and XRD.
For bioactivity study, dentin discs of 8 × 8 mm dimension were trimmed from freshly extracted teeth.Using carbide round bur #2 and water coolant, small round holes of 2 mm diameter and 2 mm thickness were prepared.Fresh mix of either of the two investigated materials was packed in the dentin holes (three of each).They were incubated for 7 days at 37 °C and 100% humidity to ensure complete setting and then immersed in phosphate buffer solution (PBS) for 15 days.After the immersion period, the dentin discs were splashed with deionized water and air-dried for 24 hours before being analysed with FTIR spectroscopy and scanning electron microscope with energy dispersive X-ray (SEM/ EDX, field emission gun, Quanta 250, FEI, Czechoslovakia).From the FTIR spectra, the crystallinity index and carbonate/ phosphate (CO 3 /PO 4 ) ratio of the precipitate, formed on the surface of the incubated samples, have been calculated.The crystallinity index (also called infrared splitting factor (IRSF)) was calculated from the sum of heights of v 4 doublet orthophosphate bands at 601 and 557 cm −1 divided by the height of the line passing between them [11]; the base line was established between 515 and 630 cm −1 (Figure 1).The CO 3 /PO 4 ratio has been calculated from the deconvoluted FTIR spectra by dividing the integrated area under the carbonate (v 3 CO 3 ) band at 830-890 cm −1 by the integrated area under the phosphate (v 1 v 3 PO 4 ) band at 900-1200 cm −1 [12,13] (Figure 1).From the EDX data, the calcium/ phosphate (Ca/P) ratio of the precipitate formed on the surface of incubated samples was calculated.
For bioactivity, the data of crystallinity index and CO 3 / PO 4 ratio were analysed by Student's t-test; those of Ca/P ratio were analysed using one-way ANOVA and post hoc test.The statistical analysis was carried at 0.05 significance using SPSS WIN (16.0;SPSS, Munich, Germany).
FTIR spectra of hydrated (set) materials (Figures 1(b) and 1(c)) showed the presence of free -OH of calcium hydroxide at 3642 cm −1 ; this band decreased in initially set while disappeared in completely set materials.The associated -OH of water molecules at 3600-3000 cm −1 [5,6,21], however, decreased in initially set but become more prominent in completely set materials.The band at 2900-2800 cm −1 corresponding to -CH group becomes prominent for hydrated materials [14].The bands at 1668 cm −1 decreased and shifted to ≈1624 cm −1 and ≈1600 for NeoMTA Plus and MTA Angelus, respectively.The formation of CO 3 band in the initial set NeoMTA Plus and MTA Angelus has been detected at 1473 and 1498 cm −1 ; this was also confirmed by the appearance of band at 883 and 871 cm −1 , respectively [4,14,20,22,29].The former bands have been also shifted to lower frequency (1440 and 1472 cm −1 ) and become more intense for completely set NeoMTA Plus and MTA Angelus, respectively.The anhydrate calcium silicate of alite and belite phases, detected in initially set materials at ≈940, 515, and 456 cm −1 [22], decreased and shifted to 996, 524, and 449 cm −1 , respectively.These new bands have been assigned to calcium silicate hydrate (C-S-H) [4,15,21,30].Sulfate bands at 661 cm −1 and phosphate bands at 600 cm −1 were  3 Journal of Spectroscopy also shifted to 674 and 640 cm −1 , respectively.The calcium sulfate band at 1153 cm −1 has been shifted to 1113 cm −1 that was assigned to polymerized sulfate in ettringite (hydrated calcium aluminate sulfate) phase [31].
After incubation in PBS for 15 days after complete setting, the crystallinity index and CO 3 /PO 4 ratio of the precipitate formed on the surface of NeoMTA Plus samples (4.8 ± 0.37 and 0.12 ± 0.01, resp.) were significantly (P = 0 000) higher than those recorded for MTA Angelus (3.7 ± 0.52 and 0.52 ± 0.01, resp.).

Scanning Electron Microscopy/Energy Dispersive X-Ray.
In scanning electron microscopy images, the surface of hydrated MTA Angelus had spherical as well as needle-like particles (Figure 3(a)).The surface of hydrated NeoMTA Plus showed globular structure of spherical particles that vary from 1 to 3 μm in size (Figure 3(b)).The spherical particles of MTA Angelus were larger in diameter than those observed with NeoMTA Plus.MTA Angelus also showed the presence of more radiopaque phases as indicated by the presence of more contrast on SEM image.Generally, there was no evidence of unreacted powders present in both tested materials (i.e., both materials were completely set).5

Journal of Spectroscopy
The elemental analysis of hydrated mass of used root repair materials revealed that they were composed mainly of carbon (C), oxygen (O), calcium (Ca), and silicon (Si).The percentage of each element varied between them.Traces of aluminum (Al), sodium (Na), phosphorous (P), and sulfur (S) were also present.Tantalum (Ta) was only detected in NeoMTA Plus (Figures 3(c) and 3(d)).The ANOVA and post hoc tests revealed that there were statistical significant differences between both investigated materials regarding all their constituents (P = 0 000) except calcium (P = 0 53).
After 15 days of immersing in PBS, the crystals of MTA Angelus and NeoMTA Plus were covered with calcium phosphate precipitates (Figures 4(a) and 4(b)).From the elemental analysis (Figures 4(c) and 4(d)), a significant reduction in C and Si was detected for both materials; Ta was significantly reduced for NeoMTA Plus.P, however, increased.Ca/P ratio was higher in NeoMTA Plus (2.8) than that recorded in MTA Angelus (2.6).

Discussion
As seen from FTIR and XRD, both MTA Angelus and NeoMTA Plus are tricalcium silicate-based materials.The opacifier however varies between them (zirconium oxide in MTA Angelus but tantalum oxide in NeoMTA Plus) as detected in XRD.Recently, the biocompatible zirconium or tantalum is added to the root repair materials as radiopacifier instead of barium sulfate to eliminate the coronal tooth discoloration [33].Other elements as sulfur, indicating the presence of gypsum (calcium sulfate) phase of Portland cement [1], have also been detected from EDX.As indicated from EDX, NeoMTA Plus has higher amount (0.2 ± 0.02) of sulfur than MTA Angelus (0.14 ± 0.01).The presence of gypsum was also confirmed by FTIR; its presence has been detected in both powder and liquid (in particular).During setting reaction, gypsum plays an important role to harden the material as yielding ettringite [31].It is confirmed in the current study as the band of silicate oxide at 1120 cm −1 was dipped and shifted to 1110 cm −1 in the initial set spectra and to 996 cm −1 in the final set spectra.The changes were related to polymerization of calcium silicate hydrate during hydration reaction [15,34].
Aluminum was seen in FTIR of unhydrated powder of both materials as well as from EDX and FTIR of both hydrated root repair materials.NeoMTA Plus, however, has higher (0.54) aluminum content than MTA Angelus (0.48).Calcium aluminate oxide (mayenite) has been only detected in XRD of hydrated NeoMTA Plus.The absence of hydrated calcium aluminate from XRD of MTA Angelus could be related to the small size of its crystal or its amorphous nature.Aluminum has a strong effect on setting reaction of MTA; it rapidly reacts with the formed calcium hydroxide in the presence of water forming calcium aluminate hydrate (4CaO.Al 2 O 3 .13H 2 O) [1].Since gypsum and aluminum fasten the setting reaction [1], the presence of gypsum and aluminum in a relatively high amount in NeoMTA Plus could account for its rapid setting [1] as observed during mixing where it sets into a hard mass within few minutes, 6 Journal of Spectroscopy whereas the setting of MTA Angelus could take several hours.After incubation in PBS, aluminum disappeared from EDX analysis of both materials.This could be related to the presence of calcium phosphate precipitates on the surface of materials hiding the trace elements present in the materials' core.As indicated by FTIR, the investigated root repair materials showed the presence of free -OH (bands at 3642 cm −1 ) of calcium hydroxide that decreased in initially set materials and disappeared in completely set materials indicating that it has been consumed during the hydration reaction.The associated -OH of water molecules (band at 3600-3000 cm −1 ) becomes prominent in the spectra of completely set materials indicating the formation of hydrated phases (e.g., calcium hydroxide and calcium silicate hydrate) as the reaction products [5,6,21].Upon hydration, it is not necessary that all tricalcium silicate-based materials produce calcium hydroxide [35].Even with the presence of free calcium hydroxide as the reaction product, some additives could react with it and hence reduce its amount [36].In this study, the absence of calcium hydroxide in set MTA Angelus and NeoMTA Plus could indicate that it has been further reacted with the other groups such as phosphate, silicon oxide, or alumina-forming calcium hydroxide phosphate (monetite as in MTA Angelus), more calcium silicate hydrate (in both MTA Angelus and NeoMTA Plus), or calcium aluminate hydrate (as in NeoMTA Plus), respectively.The presence of calcium silicate hydrate was identified in FTIR for both MTA Angelus and NeoMTA Plus, but it is only seen from XRD of NeoMTA Plus.In XRD of MTA Angelus, calcium silicate (rankinite, Ca 3 Si 2 O 7 ) and tricalcium silicate oxide (Ca 3 (SiO 4 )O) have been detected.This could indicate the amorphous nature of calcium silicate hydrate seen in MTA Angelus.
After incubation in PBS for 15 days, calcium phosphate precipitates were observed on the surface of both MTA Angelus and NeoMTA Plus samples.The Ca/P ratio that was used as an indicator of bioactivity [37] of the precipitates was 2.6 and 2.8 for MTA Angelus and NeoMTA Plus, respectively.These values are close to those observed in a previous study [37].This high Ca/P ratio indicated that the formed precipitates could be a mixture of hydroxyapatite and may be calcium carbonate (calcite).This finding is also supported by FTIR that confirmed the presence of carbonated hydroxyapatite (β-type) at 1477 and 1441 cm −1 , respectively, for both MTA Angelus and NeoMTA Plus samples incubated in PBS for 15 days [7,22].The presence of these precipitates indicates that both materials are bioactive [37].
FTIR has been used as a reliable quantitative technique to measure the crystallinity index (a measure of crystal size and perfection) of biological as well as synthetic hydroxyapatite [11,38,39].For this purpose, the splitting of PO 4 vibration bending mode (515-630 cm −1 ) into doublet bands (v1v3 PO 4 ) at ≈601 and 557 cm −1 , corresponding to the transverse 7 Journal of Spectroscopy and longitudinal optical frequency, has been used as an indication of the hydroxyapatite's crystallinity [11,38,39].In amorphous calcium phosphate, due to lattice distortion, PO 4 vibration bending mode is usually seen as a single broad band [39].The finding of the present study indicated better crystallinity and bigger crystal size of NeoMTA Plus than of MTA Angelus [38].The crystallinity index of both NeoMTA Plus and MTA Angelus (4.8 ± 0.37 and 3.7 ± 0.52, resp.)falls in the range recorded for synthetic hydroxyapatite (3.6-6.07),but it is generally higher than that recorded for sound human tooth enamel (3.23) [38].Since the crystallinity increases with increasing Ca/P [39], the high crystallinity of NeoMTA Plus could be correlated with its high Ca/P ratio.The mineral composition of hydroxyapatite however has been measured by investigating the CO 3 /PO 4 ratio [11].The CO 3 /PO 4 ratio of NeoMTA Plus and MTA Angelus (0.12 ± 0.01 and 0.52 ± 0.01, resp.)falls in the range recorded for enamel (0.02-0.1) [40].Increasing carbonate content is associated with a distortion in lattice structure and hence reduces the crystallinity of hydroxyapatite [40].This could also explain the high crystallinity of NeoMTA Plus obtained in this study.

Conclusion
Both root repair materials used in this study, MTA Angelus and NeoMTA Plus, are tricalcium silicate-based materials, but they vary in the opacifier (zirconium oxide in MTA Angelus but tantalum oxide in NeoMTA Plus).NeoMTA Plus, however, has higher sulfur and aluminum content; this could account for its rapid setting compared to MTA Angelus.Upon hydration, tricalcium silicate hydrate has been observed in FTIR of both root repair materials but only in XRD of NeoMTA Plus.The absence of calcium hydroxide in both materials could indicate that it has been further reacted with the other groups such as phosphate, silicon oxide, or alumina-forming calcium hydroxide phosphate (monetite as in MTA Angelus), or more calcium silicate hydrate (in both MTA Angelus and NeoMTA Plus), or calcium aluminate hydrate (as in NeoMTA Plus), respectively.The precipitate formed after incubation in PBS for 15 days has higher crystallinity in NeoMTA Plus samples.This could be related to its high Ca/P but low CO 3 /PO 4 ratio.Finally, due to fast setting, higher crystallinity, and better bioactivity of NeoMTA PlusTM, it can be used as an alternative to MTA Angelus as pulp and root repair material.

Figure 1 :
Figure 1: FTIR spectra of (a) original components of MTA Angelus and NeoMTA Plus; completely versus initially set MTA Angelus (b) and NeoMTA Plus (c); deconvoluted spectra of completely set materials that were incubated for 15 days in PBS (d).Crystallinity was calculated by (a + b)/c (e).CO 3 /PO 4 ratio was calculated by dividing the area under v 3 CO 3 by area under v1v3PO 4 (f).

Figure 3 :
Figure 3: SEM micrographs of complete hydrated samples of MTA Angelus (a) and NeoMTA Plus (b) as well as EDX analysis of hydrated MTA Angelus (c) and NeoMTA Plus (d).

Figure 4 :
Figure 4: SEM micrographs of hydrated MTA Angelus (a) and NeoMTA Plus (c) after being immersed in PBS for 15 days as well as EDX analysis of hydrated MTA Angelus (c) and NeoMTA Plus (d) after being immersed in PBS for 15 days.