Higher Hydration Performance and Bioactive Response of the ... JCR/105-JBMR-B-Higher... · Key Words: bioactive endodontic cements, bioactive response, physico-chemical properties

Higher hydration performance and bioactive response of the new

endodontic bioactive cement MTA HP repair compared with ProRoot

MTA white and NeoMTA plus

María del Carmen Jiménez-Sánchez,1,2 Juan José Segura-Egea,1 Aránzazu Díaz-Cuenca2,3

1Department of Stomatology, Faculty of Dentistry, University of Sevilla, Sevilla, Spain2Materials Science Institute of Seville (ICMS), Joint CSIC-University of Seville Center, Sevilla, Spain3Networking Research Center on Bioengineering, Biomaterials and Nanomedicine (CIBER-BBN), Spain

Received 18 October 2018; accepted 19 December 2018

Published online 14 January 2019 in Wiley Online Library (wileyonlinelibrary.com). DOI: 10.1002/jbm.b.34304

Abstract: The aim of this study was to characterize the hydration

performance and the bioactive response of the new bioactive

endodontic cement MTA HP repair (HP), comparing its physico-

chemical parameters with those of ProRoot MTA White (Pro)

and NeoMTA Plus (Neo). Un-hydrated precursor materials were

characterized by X-ray fluorescence, laser diffraction, N2 physi-

sorption and field emission gun scanning electron microscopy

(FEG-SEM). Setting time was assessed according to ASTM speci-

fication C 266. Hydrated materials were analyzed by X-ray dif-

fraction, Fourier transform infrared spectroscopy (FT-IR) and

(FEG-SEM). Bioactivity evaluation in vitro was carried out, by

soaking processed cement disk in simulated body fluid (SBF)

during 168 h. The cements surface was studied by FT-IR, FEG-

SEM, and energy dispersive X-ray. Release to the SBF media of

ionic degradation products was monitored using inductively

coupled plasma atomic emission spectroscopy. HP showed

shorter initial setting time compared to Pro and Neo and pro-

duce a quick and effective bioactive response in vitro in terms of

phosphate phase surface coating formation. This higher bioac-

tive response for HP is correlated with increasing calcium alumi-

nate content, increasing surface area of un-hydrated powder

precursor and the increasing release capacity of Si ionic prod-

ucts of the final hydrated product. The higher bioactive response

of MTA HP repair highlights this material, as very interesting to

further investigate its performance to improve the outcome of

vital pulp therapy procedures. © 2019 Wiley Periodicals, Inc. J

BiomedMater Res Part B: Appl Biomater 107B:2109–2120, 2019.

Key Words: bioactive endodontic cements, bioactive response,

physico-chemical properties

How to cite this article: Jiménez-Sánchez MC, Segura-Egea JJ, Díaz-Cuenca A. 2019. Higher hydration performance and bioactive

response of the new endodontic bioactive cement MTA HP repair compared with ProRoot MTA white and NeoMTA plus.

J Biomed Mater Res Part B 2019:107B:2109–2120.

INTRODUCTION

Mineral trioxide aggregate (MTA) and related cements haveshown to stimulate the natural remineralization process at thematerial-tooth interface1 and then presently are considered bio-active endodontic cements (BECs).2 Since the first clinicalapproved formulation, ProRoot MTA,3 new products have beendeveloped and very intensive research is performed to findnew formulations with improved properties. In this respect,functional aspects of BECs, as the optimal hydration processand kinetics,4–6 biomechanical performance,7,8 and the roleplayed by the radiopacifying agents9,10 are being studied.Recently, research on BECs has focused on their applications asactive therapeutic agents to stimulate tissue regeneration.11–14

Bioactive endodontic cements are based on tricalcium sil-icate and include a radiopacifying material composed of bis-muth, zirconia, tantalum, or tungsten oxides, depending ofthe market product.2,15 Bismuth oxide, the radiopacifier

contained in the powder of MTA Plus,16 plays a main role incement hydration. However, it is responsible of toothdiscoloration,17 so new cements containing alternative radio-pacifiers elements to replace bismuth have been pre-pared.10,18 Moreover, other modifications have also beenintroduced seeking to overcome other problems such as longsetting time, high cost and difficult handling characteristics.These include, variations of cement formulation2,4 and theintroduction of additives.7,8,19 Compositional modificationsalter the material physico-chemical characteristics and thencorresponding functional properties, both biomechanically(setting) and biologically (bioactivity).

Among the new BECs that have come onto the market withnew radiopacifiers are NeoMTA Plus (Avalon Biomed Inc.,Bradenton, FL, USA) and MTA Repair HP (Angelus, Londrina,Brazil). NeoMTA Plus is a tricalcium silicate material, with tan-talum oxide (Ta2O5) as a radiopacifying agent.20 It releases

Correspondence to: Aránzazu Díaz-Cuenca; e-mail: [email protected] grant sponsor: Universidad de Sevilla; Fellowship PhD Program

© 2019 Wiley Periodicals, Inc. 2109

mailto:[email protected]

ions from compositional formulation and has calcium phos-phate forming capacity, which could enhance its bioactivityand biocompatibility for its use for vital pulp therapies, rootapexification, root repair, root-end filling, and sealing of rootcanals. 21 On the other hand, in MTA HP Repair bismuth oxidehas been replaced by calcium tungstate (CaWO4). This productis provided with liquid containing water and plasticizer andgreater handling in comparison with its predecessor AngelusWhite MTA has been reported.15

We argue the understanding of BEC physico-chemicalparameters–material properties relationships will help toachieve advanced BEC formulations. The aim of the work isto find ceramic powder parameters, which could representadvantages to produce materials with improved functional-ity. The chemical composition, textural properties and crystalphase formation of the new MTA HP Repair (HP) and twoother clinical approved, ProRoot MTA White (Pro) andNeoMTA Plus (Neo), are assessed. To eliminate the influenceof additional, and poorly specified, liquid additive agents, thebare ceramic powder formulation and water were used onlyto test the three products. BECs characterization is compara-tively analyzed and discussed in terms of hydration perfor-mance and bioactive response.

MATERIALS AND METHODS

ProRoot MTA White (DENTSPLY Tulsa Dental Specialties,DENTSPLY International, Inc. 5100 E. Skelly Drive, Suite300, Tulsa, OK – lot n. 0000163567), NeoMTA Plus (AvalonBiomed Inc., Bradenton, FL, – lot n. 2017021601) and MTAHP Repair (Angelus, Londrina, Brazil – lot n. 38585) wereused in this study.

Powder materials characterizationThe compositional analyses of un-hydrated BECs were per-formed by X-ray fluorescence (XRF) using the sequentialSpectrometer AXIOS WD-XRF (PANalytical, Malvern, UK). Theparticle size distributions of the three powders were mea-sured in a Malvern Sizer laser diffraction (LD) instrument(Southborough, MA), using an active beam length of 2.4 mmand a 300-RF lens. Likewise, textural parameters were

determined by N2 physisorption. Adsorption–desorption iso-therms were collected on a Micromeritics Tristar 3020 gasadsorption analyzer (Norcross, GA). The specific surface areawas determined by the BET (Brunauer–Emmett–Teller)method22 after degassing at 523 K for 2 h in a nitrogenstream. Total pore volume was obtained from the N2 amountadsorbed at 0.99 relative pressures.

Hydrated BECs process and analysesThe powders were mixed according to liquid volume amountmanufacture’s instructions but using Milli-Q water only.Although the three BECs used are supplied with a recom-mended liquid, Milli-Q water was chosen to avoid theinfluence of specific product manufacturer additives. Thisprocedure allows a standardization of the three differentBEC ceramic precursors. The manual mixing was performedadding the liquid to the powder on a glass slab, and thecement was blended using a metal spatula. A paste withhomogeneous consistency was obtained. The paste was com-pacted in a silicone mold of 10 mm in diameter and 4 mmhigh. Three silicone molds were filled with each material andstored in an incubator at 37�C and 95% relative humidity.

The setting time was determined according to AmericanSociety for Testing and Materials specification C 266.113.4 � 0.5 and 453.6 � 0.5 Gillmore needles were usedrespectively to determine the initial and the final settingtimes. This procedure was repeated at 60-s intervals, andthe time was measured using a digital chronometer. Thesetting times were measured from the start of mixing to thetime at which no indentations could be seen on the surfaceof the specimen. Measurements were performed three timesfor each material group.

X-ray diffraction (XRD) analysis of un-hydrated andhydrated materials was performed with a PANalytical X’PertPRO diffractometer (Almelo, Netherlands), using Cu-Kα radi-ation (0.154187 nm). The difractometer was operated at45 kV and 40 mA using a step size of 0.02 and a 500 s expo-sure time. Phase identification was accomplished by use ofsearch-match software utilizing ICDD database (2002,Pennsylvania, USA). Fourier transform infrared (FTIR)

TABLE I. BEC Studied Materials: Manufacturers and Chemical Composition Specifications

Materials Manufacturer Composition

ProRoot White Dentsply Tulsa Dental Specialties, Johnson City, TN, USA Tricalcium silicate (Ca3SiO5),

Dicalcium silicate (Ca2SiO4),

Bismuth oxide (Bi2O3),

Tricalcium aluminate (Ca3Al2O6),

Calcium sulfate dihydrate (Ca2(SO4)2H2O)

NeoMTA Plus Avalon Biomed Inc., Bradenton, FL, USA Tricalcium silicate (Ca3SiO5),


Tantalum oxide (Ta2O5),

Calcium sulphate (CaSO4),

Silica (SiO2)

HP Repair Angelus, Londrına, Parana, Brazil Tricalcium silicate (Ca3SiO5),


Calcium tungstate (CaWO4),

Tricalcium aluminate (3CaO.Al2O3),

Calcium oxide (CaO)

2110 JIMÉNEZ-SÁNCHEZ, SEGURA-EGEA AND DÍAZ-CUENCA MTA HP REPAIR PERFORMANCE & BIOACTIVITY

spectra were collected in transmission configuration in the4000–400 cm−1 range using 4 cm−1 interval in Nicolet IS50FT-IR of Thermo Scientific (Madison, WI).

The bioactivity evaluation was performed by soaking thecement disks in 13 mL simulated body fluid (SBF)23 during4, 24, 72, and 168 h at 36.5�C and 60 r.p.m. shaking. Previ-ously to the bioactivity assay, the samples were sterilizedunder UV light for 10 min period on each side. SBF solutionwas filtered using 0.2 mm bacteriostatic filter (Biofil). Con-centrations of Si, Ca, P, radiopacifying (Bi, Ta, and W) and Alions in the soaking media were monitored after 72 and168 h by inductively coupled plasma atomic emission spec-troscopy (ICP-AES) using the spectrometer Horiba JobinYvon (Ultima 2, Paris, France). Control solutions consistingof pure SBF was simultaneously prepared and stored underthe same conditions.

The microstructure of the un-hydrated, set materials andSBF treated samples was studied by field emission gunscanning electron microscopy (FEG-SEM) using a HITACHIS-4800 (Tokyo, Japan). Images were recorded at an acceler-ating voltage of 2 kV. Energy dispersive X-ray (EDX) analysiswas carried out at 10 kV with an EDX Bruker XFlash 4010detector. Fourier transform infrared (FTIR) spectra were col-lected in transmission configuration in the 4000–400 cm−1

range using 4 cm−1 interval in a Nicolet IS50 FT-IR (ThermoScientific, Madison, WI).

TABLEII.QuantitativeAnalysis

byX-rayFluorescence(X

RF)ofUn-hydratedBECPowderPrecursors

BECMaterial

Element(w

t%)

Ca

Si

Bi

Ta

WAl

SMg

Fe

Sr

Na

PTi

ProRootWhite

45.6

1,3�

0.0

8.5

1,3�

0.2

12.3

�0.0

––

0.9

1�

0.1

0.8

1�

0.1

0.3

�0.0

0.2

�0.0

0.1

�0.0

0.1

�0.0

0.1

�0.0

0.1

�0.0

NeoMTA

Plus

39.3

2,3�

0.4

9.6

2,3�

0.1

–16.3

�0.3

–0.8

2�

0.0

0.7

2�

0.0

0.3

�0.0

0.2

�0.0

0.1

�0.0

0.1

�0.0

0.1

�0.0

0.1

�0.0

HPRepair

42.4

1,2�

1.0

5.7

1,2�

0.1

––

22.0

�1.2

1.7

1,2�

0.0

0.0

1,2�

0.0

0.1

�0.0

0.0

�0.0

0.3

�0.0

0.1

�0.0

0.0

�0.0

0.0

�0.0

1p<0.01.

2p<0.01.

3p<=0.01.

Data

are

shownasmean�

standard

deviation.Sameletterin

acolumnindicatessignificantdifference(n

=3experiments

perform

edin

triplicate).

FIGURE 1. Bimodal particle size distributions of un-hydrated BEC

precursor materials. Each analysis was performed with three replicates.

Dashed line indicates maximum particle size volume corresponding to

the lower peak population distribution. (a.u.): arbitrary units.

TABLE III. Un-hydrated BECs Textural Parameters Obtained

by N2 Physisorption

BEC Material SBET (m2 g−1) VT (cm3 g−1)

ProRoot White 1.4 � 0.01 0.003 � 0.0001

NeoMTA Plus 1.3 � 0.12 0.003 � 0.0002

HP Repair 4.8 � 0.01,2 0.015 � 0.0011,2

1 p < 0.01.2 p < 0.01.

Specific surface area (SBET), calculated by the Brunauer–Elmet–Tellet

method, and total pore volume (VT) estimated from de nitrogen amount

absorbed at 0.99 relative pressure. Data are shown as mean � standard

deviation. Same letter in a column indicates significant difference (n = 3

experiments performed in triplicate).

JOURNAL OF BIOMEDICAL MATERIALS RESEARCH PART B: APPLIED BIOMATERIALS | AUG 2019 VOL 107B, ISSUE 6 2111

ORIGINAL RESEARCH REPORT

Statistical analysisData were analyzed using SPSS version 22.0 statistical soft-ware (SPSS, Inc., Chicago, IL). Each experiment was per-formed with three replicates and carried out at least threetimes. Quantitative data are presented as the mean � stan-dard deviation (SD). Statistical differences between the threeBECs were assessed using ANOVA and Student t-test. Ap < 0.05 was considered significant.

RESULTS

Un-hydrated BEC analysesThe studied BECs chemical compositions, supplied by themanufacturers, are listed in Table I. Tricalcium silicate,Ca3SiO5, and dicalcium silicate, Ca2SiO4, are major compo-nents of the three analyzed BECs, being the radiopacifyingsalt element the main distinctive difference between mate-rials. A quantitative analysis of the composition was carriedout using FRX as presented in Table II. As regards commonelements, Al content (1.7 � 0.0 wt %) (p < 0.01) was shownhighest for HP, while Si (5.7 � 0.1 wt %) (p < 0.01), and S

(0.0 � 0.0 wt %) (p < 0.01) were the lowest of the threestudied BECs.

Particle size measurements are displayed in Figure 1.Although bimodal distributions with two maxima at 0.3–0.4and 5–7 μm ranges are observed for the three studied mate-rials, somehow lower particle size can be inferred for the HP(dashed line). Likewise, specific surface area calculated byBET (Table III) confirms a significant higher surface(4.8 � 0.0 m2 g−1) (p < 0.01) and total pore volume(0.015 � 0.001 cm3 g−1) (p < 0.01) for HP. Besides, thestudy of the microstructure by FEG-SEM micrographs(Figure 2) indicates the presence of distinctive small needle-like morphologies of 50–100 nm size thickness decoratingall surface long for the HP material.

Hydrated BEC process and analysesOn addition of water BEC components (Table I) react to pro-duce calcium silicate hydrate (CSH) and calcium hydroxide(Ca(OH)2). Figure 3 displays XRD analysis, un-hydrated andhydrated (set), of the three studied BECs. For the un-hydrated

FIGURE 2. Field emission gun scanning electron microscopy (FEG-SEM) micrographs at two different magnifications of un-hydrated BEC showing

powder particles microstructure. (a, b) ProRoot White; (c, d) NeoMTA Plus; (e, f ) HP Repair exhibiting a distinctive finer microstructure consisting of

submicron elongated features of nanometric thickness (white arrows drawn perpendicular to larger axis). Scale bars: left (5 μm); right (1 μm).


BECs peaks corresponding to the radiopacifying elementoxides Bi2O3 (Powder diffraction file [PDF] 01-071-0465),Ta2O5 (PDF 00-019-1298), and CaWO4 (PDF 01-077-2233) ofPro, Neo and HP respectively are the more intense. For thethree materials, sharp peaks matching to Ca3SiO5 (PDF01-086-0402) and Ca2SiO4 (PDF 01-077-0409) are detected.Also, Ca3Al2O6 (PDF 00-032-0148) is detected for Pro and HPand minority sulfate phase such as Ca2(SO4)2H2O (PDF00-024-1067) in Pro and Neo. Upon hydration, the peak at

2θ = 18.0� is used to identify Ca(OH)2 (PDF 00-044-1481) for-mation for Pro and Neo. In case of HP, a shoulder signal atthis position merging to CaWO4 peak at 2θ = 18.6� isobserved. Distinctly a small angle peak at 2θ = 11.7� matchedwith Mg4Al2(OH)143H2O (PDF 00-035-0964) is revealed forthe hydrated HP material.

Figure 4 shows the results of infrared spectroscopy, usedto study both crystalline and amorphous phase formation.The major vibrational bands observed for un-hydrated BECs

FIGURE 3. X-ray diffraction (XRD) patterns of un-hydrated and hydrated BEC materials. The black arrow drawn in HP plot highlights detected shoul-

der signal assigned to Ca(OH)2.



spectra correspond to the Si–O asymmetric stretching (ν3), andSi–O bending (ν4 and ν2) of calcium silicate majority compo-nents and centered at 925, 522, and 452 cm−1, respec-tively.24,25 The triple bands defined in the 1155–1096 cm−1

range for Pro and Neo BECs are identified as S–O stretchingvibration (ν3) of SO4

2−.24 Also, bands at 3611 and 3557 cm−1

are adscribed to O–H stretching vibrations of sulfate com-pounds.26 Important changes in BECs spectra were observedupon hydration. Spectral intensity shifts of the Si–O bands areobserved from �900 cm−1 toward 1000–1100 cm−1 suggest-ing rearrangements in the silicate subsystem as result ofcalcium silicate dissolution and simultaneously polymerization

of silica to form CSH.25,27 The Si–O stretching band shiftingdue to polymerization obscures the SO4

2− bands24 resolved forun-hydrated Pro and Neo materials. Distinctly to the othertwo, un-hydrated HP BEC shows an intense Si–O symmetricvibration (ν1) at 815 cm−128 and a sharp Al–O vibration at860 cm−1.26 HP hydrated sharp peak observed at about420 cm−1 can be assigned to the lattice vibration (Ca–O) ofhydrated tricalcium aluminate.29

For the three materials, carbonate bands at 714, 875, and855 cm−1, 1490–1420 cm−1 and 2950–2500 cm−1 build upfrom the reactions of atmospheric CO2 with calcium hydrox-ide.24,26 The broad band centered at 3400 cm−1 is due tosymmetric and asymmetric stretching vibrations of O–Hadsorbed water molecules. The sharp band at 3640 cm−1 forNeo and HP corresponds to the OH band from Ca(OH)2.

25

Initial and final setting times for the three BECs are pre-sented in Table IV. HP showed shorter initial setting time(12 � 2 min) compared to Pro (p < 0.05) and Neo (p < 0.01).

Back-scatter electron images of final hydrated surfacesare displayed in Figure 5. The images show differencesbetween the materials. A porous irregular surface with longcracking features is observed for the Pro material. Brightparticles with sizes ≤1 μm corresponding to bismuth oxideappear homogeneously distributed over the surface. Aggre-gate particles formed as clusters over the surface are foundfor Neo and HP materials. A more flat background is noticedfor HP BEC although breaking off by big bumps where parti-cles with platelet morphology are visible. Bright featuresconsidered as the radiopacifying heavy element oxides Ta2O5

and CaWO4, respectively for Neo and HP are also markedwith arrows at the micrographs.

In vitro bioactivity assessmentFigure 6 shows the FT-IR absorbance spectra of BECs afteranalyzed SBF treatment times in comparison with the spectraof the hydrated (SBF un-treated) samples. An importantintensity increase with treatment time of CSH, broad bandwithin the 1000–1100 cm−1 range, is observed for the threematerials. Likewise, increasing formation of phosphate phasebands at 1097, 960, 607, and 570 cm−1,30 are clearlyobserved for the three BECs with prolonged SBF soaking. Cal-cium hydroxyapatite growing on the BEC surfaces after 72 htreatment can be inferred from the two bands at 607 and570 cm−1 characteristics of phosphate in a crystalline

FIGURE 4. Fourier transform infrared (FT-IR) analysis studying both

crystalline and amorphous phase formation of un-hydrated and

hydrated BEC materials.

TABLE IV. Initial and Final Setting Times of BECs

BEC Material Initial Time (min) Final Time (min)

ProRoot White 17 � 21,3 241 � 62,3

NeoMTA Plus 50 � 52,3 186 � 21,3

HP Repair 12 � 21,2 199 � 51,2

1 p < 0.05.2 p < 0.01.3 p < 0.01.

Data are shown as mean � standard deviation. Same letter in a col-

umn indicates significant difference (n = 3 experiments performed in

triplicate).


environment.31 Incipient signals at 607 and 570 cm−1 areparticularly observed for HP at only 24 h SBF treatment.

Secondary electron images of BECs after 24 h SBF treat-ment are presented in Figure 7(A,C, and E). New aggregateformations covering the surface are observed for the threematerials. Un-covered areas are more visible for Pro mate-rial. HP shows a distinctive coating formed with homoge-neous spheres with average diameter in the 0.5–1 μm range.Back-scatter electron images of BEC surfaces after 72 h SBFtreatment are also shown in Figure 7(B,D, and F ). BrightBi2O3 and Ta2O5 particles are observed for Pro and NeoBECs whilst HP does not show any bright contrast feature.In addition, a visible growing of characteristic sphericalphosphate aggregates is confirmed for the HP BEC surface(Figure 7F ).

Energy dispersive X-ray analysis comparing un-treated(hydrated), 24 h and 72 h SBF treated surfaces are displayedin Figure 8. Set BECs revealed phosphate deposition after 24 hfor the three materials. Particularly relevant is the high phos-phorous/silicon ratio intensity signal after 24 h measured forHP (4.46 � 1.80), in comparison with Pro (0.33 � 0.08)

(p < 0.05) and Neo (1.14 � 0.18) (p < 0.05), indicating higherphosphate phase deposition for the HP material.

Si, Ca, and P ionic product in solution after 72 and 168 hSBF soaking treatment are displayed in Figure 9. Notably, Sielution was significantly higher for HP both at 72 h(12.8 � 0.4 mg L−1) and at 168 h (12.2 � 0.1 mg L−1) treat-ment, compared to Pro (5.5 � 0.1 mg L−1) (p < 0.01) and(5.2 � 0.1 mg L−1) (p < 0.01), and Neo (3.2 � 0.0 mg L−1)(p < 0.01) and (3.8 � 0.0 mg L−1) (p < 0.01). On the contrary,HP showed the lowest Ca ionic product concentration at72 h (650.0 � 31.0 mg L−1) and at 168 h treatment(824.0� 14.3mgL−1), compared to Pro (1024.0� 19.0mg L−1)(p < 0.01) and (1205 � 30.5 mg L−1) (p < 0.01), and Neo(1299.0 � 18.6 mg L−1) (p < 0.01) and (1309 � 19.4 mg L−1)(p < 0.01) values. Al elution after 168 h is particularly high forthe HP material showing up to (0.401 � 0.004 mg L−1), fol-lowed by NEO eluting (0.034 � 0.001 mg L−1) (p < 0.01) andPro (0.008 � 0.000 mg L−1) (p < 0.01). As regards the radio-pacifying elements, bismuth and tungsten are detected respec-tively up to 0.183 � 0.007 mg L−1 and 0.106 � 0.002 mg L−1

(Table V).

FIGURE 5. Back-scatter electron images of hydrated BEC surfaces at two different magnifications. (A, B) ProRoot White; (C, D) NeoMTA Plus; (E, F )

HP Repair. White arrows indicate bright features corresponding to radiopacifying heavy element oxides. Scale bars: left (50 μm); right (10 μm).



DISCUSSION

Three commercial BECs used in endodontic therapy(ProRoot MTA White, NeoMTA Plus and MTA HP Repair),both in the form of powder precursor and final set processedproduct, are characterized in terms of chemical compositionand microstructure. After the measure of their physio-chemical properties, it can be concluded that MTA HP Repairshowed the lowest initial setting time, and produced a fasterand more effective bioactive response in terms of HA surfacecoating formation.

Biomineralization is the process by which a living organ-ism synthesizes mineral substance. One important challenge

facing endodontics today is precisely the correct formationof hard mineral tissue.32 To achieve this, it is essential thatthe materials used in endodontic treatments be bioactive.Bioactivity means the formation of calcium phosphatedeposits on the surface of materials placed in a simulatedbody fluid (SBF), that is, a buffer solution with an ion con-centration similar to that of human blood plasma.33 Theresults of the present investigation, showing a greater bioac-tive response of MTA HP repair, highlights this material asvery interesting candidate to further investigate its perfor-mance to improve the outcome of vital pulp therapy proce-dures in terms of increased biomineralization.

The results of quantitative XRF analysis reveals that, asexpected and with exception of the distinctive radiopacifyingelement, Bi, Ta, and W for Pro, Neo, and HP, respectively,major chemical composition consist of calcium silicates in theform of Ca3SiO5 and Ca2SiO4. Accordingly, major by-productphases produced during the hydration reaction, leading tothe setting and hardening of the cement, are CSH and calciumhydroxide Ca(OH)2. The peak at 2θ = 18� is used to identifythe production of Ca(OH)2

5 using XRD. However, the peakscorresponding to the major hydrated product, CSH, were notobserved by XRD analysis. This finding can be explained duenot only to poorly crystallized calcium silicate formation butalso to the nanoscale crystalline structure of SCH, causing toappear amorphous in XRD.5,34 Formation of CSH polymerizedsilica is confirmed for the three BECs by FTIR analysis show-ing bands within the 1011–1080 cm−1 range.25 Symmetricstretching ν1 of [SiO4]

4− tetrahedron at 815 cm−1 which isnot active for IR,28 increases upon hydration for Pro, alsoindicative of tetrahedron distortion modifications uponhydration. Interestingly, only HP powder precursor shows anintense Si-O (ν1) band before hydration which is indicative of[SiO4]

4− tetrahedron lower ordered structure. Besides, Alcontents of 1.7 wt % for HP double the 0.8–0.9 wt % mea-sured for Pro and Neo and a small angle peak matched withMg4Al2(OH)143H2O (PDF 00-035-0964) at 2θ = 11.7� is onlydetected for hydrated HP material. On the other side, XRFanalysis indicates S is only present for Pro and Neo as con-firmed by the S–O and O–H FTIR stretching vibrations of sul-fate compounds. Low amount of sulfate is correlated withshorter setting times as calcium sulfates are setting regula-tors to avoid a rapid desiccation of powder precursor paste.29

In this respect, our setting evaluation highlights the materialwithout sulfate, HP, with the lowest initial setting time. A toofast setting time could impede an effective and completehydration of the calcium silicate particles to form an hydratedgel which gradually fills in the spaces between the calciumsilicate grains. Therefore, in case of HP we argue that thequick setting time observed could well also be due to a signif-icant higher surface area of 4.7 m2 g−1 measured for thismaterial. Higher surface is correlated with smaller particlesizes which greatly faster the setting times.35

Bioactivity essay in vitro indicates that the three studiedBECs forms a robust crystallized calcium hydroxyapatite(HA) coating layer after 72 h treatment independently of theradiopacifying element Bi, Ta, or W. However, FEG-SEM obser-vations of BECs surfaces after 24 h SBF treatment shows HP

FIGURE 6. Fourier transform infrared (FT-IR) spectra of BECs after SBF

treatment of the different analyzed times plotted together with the spec-

tra of the hydrated, SBF un-treated, samples. (CSH = calcium silicate

hydrate).


material produce distinctively a phosphate phase coatingformed of homogeneous spheres with an average diameter inthe 0.5–1.0 μm range. Besides, a higher surface covering isobserved for HP at this short time in comparison with Pro andNeo materials. This higher bioactivity response of HP might beassociated to the higher calcium aluminate content of thismaterial29 but also to greater surface degradation as measuredby the Si ion product release detected by ICP. Accumulation ofsilica based materials dissolution products causes both thechemical composition and the pH of the solution to changeproviding surface sites and a pH conducive to HA nucleation.36

Particularly, the repolymerization of a porous silica-rich layerthrough silanol groups condensation from the soluble silica inthe form of Si(OH)4, followed by the migration of Ca2+ andPO4

3− groups to the surface forms a film rich in amorphousCaO–P2O5 on the silica-rich layer.

The clinical manifestation of bioactivity with the use ofBECs has been attributed to phosphate phase’s mineralizationinduction capacity30 and compared to that of calcium

hydroxide postulating that the mechanisms of action were sim-ilar.3 In case of BECs, when exposed to a phosphate-containingmedia such as SBF, a series of reactions take place on the sur-face between calcium from the cement and phosphate fromthe solution consisting of the absorption of Ca and P ions onthe silica-rich CSH surface and the precipitation of HPO4

2− con-taining phase which matures into a crystalline hydroxycarbo-nate apatite phase at increasing treatment times. The apatitelayer fills the superficial crack and voids providing a stablesealing at cement surface. Phosphate biological fluids andblood present at the external surface during surgical proce-dures provides phosphate ions able to promote apatite deposi-tion enhancing the formation of a mineralized biomimeticcement-tissue interphase. A recent investigation using MTAextract with concentration of Ca ions of 88.9 mg L−1 and Siions of 0.22 mg L−1 to the culture medium promotes the repairof injured pulp, potentially by accelerating proliferation andreducing the time required for hDPCs to enter into the odonto-blastic differentiation stage in the clinical setting.37

FIGURE 7. Field emission gun scanning electron microscopy (FEG-SEM) secondary electron micrographs after 24 h SBF treatment (left) and back-

scatter electron micrographs after 72 h SBF treatment of BEC surfaces. (A, B) ProRoot White; (C, D) NeoMTA Plus; (E, F ) HP Repair exhibiting a dis-

tinctive growing of homogeneous spherical aggregates. White arrows drawn in b and d indicate, respectively, bright Bi2O3 and Ta2O5 particles

observed for Pro and Neo BECs. Scale bars: (10 μm).



In comparison with Pro and Neo materials, our studyindicates that HP produces a quick and effective bioactiveresponse in vitro in terms of HA surface coating formation.Measurements of physico-chemical parameters for the threestudied BECs indicate that, the higher bioactivity responsefor HP correlates with increasing calcium aluminate content,increasing surface area of un-hydrated powder precursorand increasing release capacity of Si ionic products of the

final hydrated product. On the other side, our results indi-cate differences between materials for the release to themedia of unwanted ionic products, in terms of teeth discol-oration or toxicity. Hence, release up to 0.11 mg L−1 of Wand to 0.40 mg L−1 of Al to the SBF media were measuredfor HP. In comparison, for Pro the release of Bi reaches0.18 mg L−1. Ta was not detected for Neo material. AlthoughHP is a new product and then less reported than Pro andNeo materials, a recent study on human dental pulp stemcells biocompatibility of MTA Repair HP in comparisonwith NeoMTA Plus has shown a suitable degree of

FIGURE 8. Energy dispersive X-ray (EDX) analyses of un-treated

(hydrated) and after 24 h and 72 h SBF treatment of BEC surfaces. Each

analysis was performed with three replicates.

FIGURE 9. Si, Ca, and P ionic product concentration in the SBF soaking

media of BEC materials degradation monitored by inductively coupled

plasma atomic emission spectroscopy (ICP-AES). Values are presented

as mean � standard deviation (*p < 0.01). Horizontal lines indicate the

initial values of the ionic concentration of SBF as prepared media.


cytocompatibility.15 Further work will be needed to studydifferent physio-chemical parameters-material propertiesrelationships to optimize BECs functionality performance.Besides, supplementary agents as propylene glycol38 sodiumphosphate dibasic39 or methylcellulose and calcium chlo-ride8 could well improve significantly different properties offinal hydrated material although particular attention to allbiocompatibility aspects involved should be taken into con-sideration.38,40 Also, the presence of the plasticizer in MTARepair HP might increase its solubility and porosity.41

CONCLUSIONS

Physico-chemical characterization performed of three commer-cial BECs ProRoot MTA White, NeoMTA Plus and MTA HPRepair, containing different radiopacifying elements respec-tively, Bi, Ta, or W, has shown distinctive parameters, particu-larly for the HP material. Bioactivity article in vitro indicatesthat the three studied BECs forms a robust crystallized calciumhydroxyapatite coating layer after 72 h treatment. However, incomparison with Pro and Neo materials, HP produces a quickand effective bioactive response in vitro in terms of phosphatephase surface coating formation. This higher bioactiveresponse for HP is correlated with increasing calcium alumi-nate content, increasing surface area of un-hydrated powderprecursor and the increasing release capacity of Si ionic prod-ucts of the final hydrated product. The higher bioactiveresponse of MTA HP Repair highlights this material as veryinteresting candidate to further investigate its performance toimprove the outcome of pulp capping and other vital pulptherapy procedures in terms of increased biomineralization.

ACKNOWLEDGMENTS

MCJS acknowledges the financial support provided by theUniversity of Sevilla Fellowship PhD Program. Authors thankDr. J.J. Benítez (ICMS) to provide help and assistance withthe FT-IR spectroscopy measurements.

CONFLICT OF INTEREST

The authors deny any conflicts of interest related to thisstudy.

REFERENCES1. Torabinejad M, Chivian N. Clinical applications of mineral trioxide

aggregate. J Endod 1999;25:197–205.

2. Parirokh M, Torabinejad M, Dummer PMH. Mineral trioxide aggregate

and other bioactive endodontic cements: An updated overview – part

I: Vital pulp therapy. Int Endod J 2018;51:177–205.

3. Camilleri J, Pitt Ford TR. Mineral trioxide aggregate: A review of the

constituents and biological properties of the material. Int Endod J

2006;39:747–754.

4. Camilleri J, Sorrentino F, Damidot D. Investigation of the hydration

and bioactivity of radiopacified tricalcium silicate cement, bioden-

tine and MTA angelus. Dent Mater 2013;29:580–593.

5. Lee YL, Wang WH, Lin FH, Lin CP. Hydration behaviors of calcium

silicate-based biomaterials. J Formos Med Assoc 2017;116:424–431.

6. Duque JA, Fernandes SL, Bubola JP, Duarte MAH, Camilleri J,

Marciano MA. The effect of mixing method on tricalcium silicate-

based cement. Int Endod J 2018;51:69–78.

7. Kogan P, He J, Glickman GN, Watanabe I. The effects of various

additives on setting properties of MTA. J Endod 2006;32:569–572.

8. Ber BS, Hatton JF, Stewart GP. Chemical modification of ProRoot

MTA to improve handling characteristics and decrease setting time.

J Endod 2007;33:1231–1234.

9. Viapiana R, Guerreiro-Tanomaru JM, Hungaro-Duarte MA,

Tanomaru-Filho M, Camilleri J. Chemical characterization and bio-

activity of epoxy resin and Portland cement-based sealers with nio-

bium and zirconium oxide radiopacifiers. Dent Mater 2014;30:

1005–1020.

10. Camilleri J. Staining potential of neo MTA plus, MTA plus, and bio-

dentine used for pulpotomy procedures. J Endod 2015;41:

1139–1145.

11. Rodrigues EM, Gomes-Cornélio AL, Soares-Costa A, Salles LP,

Velayutham M, Rossa-Junior C, Guerreiro-Tanomaru JM,

Tanomaru-Filho M. An assessment of the overexpression of BMP-2

in transfected human osteoblast cells stimulated by mineral triox-

ide aggregate and biodentine. Int Endod J 2017;50:e9–e18.

12. Silva GF, Guerreiro-Tanomaru JM, da Fonseca TS, Bernardi MIB,

Sasso-Cerri E, Tanomaru-Filho M, Cerri PS. Zirconium oxide and

niobium oxide used as radiopacifiers in a calcium silicate-based

material stimulate fibroblast proliferation and collagen formation.

Int Endod J 2017;50:e95–e108.

13. Marí-Beffa M, Segura-Egea JJ, Díaz-Cuenca A. Regenerative end-

odontic procedures: A perspective from stem cell niche biology.

J Endod 2017;43:52–62.

14. Chang F, Kim JM, Choi Y, Park K. MTA promotes chemotaxis and

chemokinesis of immune cells through distinct calcium-sensing

receptor signaling pathways. Biomaterials 2018;150:14–24.

15. Tomás-Catalá CL, Collado-Gozález M, García-Bernal D, Oñate-

Sánchez FL, Llena C, Lozano A, Castelo-Baz P, Moraleda JM,

Rodríguez-Lozano FJ. Comparative analysis of the biological effects

of the endodontic bioactive cements MTA-angelus, MTA repair HP

and NeoMTA plus on human dental pulp stem cells. Int Endod J

2017;50:e63–e72.

TABLE V. Aluminum and Radiopacifying Elements Release Measured by Inductively coupled Plasma Atomic Emission

Spectroscopy (ICP-AES)

Element (mg L−1)

BEC material

ProRoot White NeoMTA Plus HP Repair

72 h 168 h 72 h 168 h 72 h 168 h

Aluminum 0.0031,3 � 0.000 0.0081,3 � 0.000 0.0092,3 � 0.000 0.0342,3 � 0.001 0.0161,2 � 0.001 0.4011,2 � 0.004

Bismuth 0.072 � 0.006 0.183 � 0.007 – – – –

Tantalum – – ≤0.004 ≤0.004 – –

Tungsten – – – – 0.106 � 0.002 0.070 � 0.005

1 p < 0.01.2 p < 0.01.3 p < 0.01.

Data are shown as mean � standard deviation. For equal treatment times, same letter in a row indicates significant difference (n = 3 experiments

performed in triplicate).



16. Gandolfi MG, Siboni F, Primus CM, Prati C. Ion release, porosity,

solubility, and bioactivity of MTA plus tricalcium silicate. J Endod

2014;40:1632–1637.

17. Camilleri J. Characterization of hydration products of mineral triox-

ide aggregate. Int Endod J 2008;41:408–417.

18. Vallés M, Mercadé M, Duran-Sindreu F, Bourdelande JL, Roig M.

Influence of light and oxygen on the color stability of five calcium

silcate-based materials. J Endod 2013;39:525–528.

19. Huang TH, Shie MY, Kao CT, Ding SJ. The effect of setting accelerator

on properties of mineral trioxide aggregate. J Endod 2008;34:590–593.

20. Avalon Biomed. NeoMTAPlus Directions for use. ftp://ftp.endoco.

com/links/AvalonMTAPlusDFU.pdf [accessed October 2018]

21. Siboni F, Taddei P, Prati C, Gandolfi MG. Properties of NeoMTA plus

and MTA plus cements for endodontics. Int Endod J 2017;50:e83–e94.

22. Sing KSW, Everett DH, Haul RAW, Moscou L, Pierotti RA. Reporting

physisorption data for gas/solid systems with special reference to

the determination of surface area and porosity. Pure Appl Chem

1985;57:603–619.

23. Kokubo T, Takadama H. How useful is SBF in predicting in vivo

bone bioactivity? Biomaterials 2006;27:2907–2915.

24. Mollah MYA, Yu W, Schennach R, Cocke DL. A Fourrier transform

infrared spectroscopic investigation of the early hydration of Port-

land cement and the influence of sodium lignosulfonate. Cement

Concrete Res 2000;30:267–273.

25. Ylmén R, Jäglid U, Steenari BM, Panas I. Early hydration and set-

ting of Portland cement monitored by IR, SEM and Vicat tech-

niques. Cement Concrete Res 2009;39:433–439.

26. Hughes TL, Methven CM, Jones TGJ, Pelham SE, Fletcher P, Hall C.

Determining cement composition by Fourier transform infrared

spectroscopy. Adv Cement Base Mater 1995;2:91–104.

27. Yu P, Kirkpatrick RJ, Poe B, McMillan PF, Cong X. Structure of cal-

cium silicate hydrate (C-S-H): Near-, mid-, and far-infrared spectros-

copy. J Am Ceram Soc 1999;82:742–748.

28. Ren X, Zhang W, Ye J. FTIR study on the polymorphic structure of

tricalcium silicate. Cement Concrete Res 2017;99:129–136.

29. Taddei P, Modena E, Tinti A, Siboni F, Patri C, Gandolfi MG. Effect

of the fluoride content on the bioactivity of calcium silicate-based

endodontic cements. Ceram Int 2014;40:4095–4107.

30. Tay FR, Pashley DH, Rueggeberg FA, Loushine RJ, Weller RN. Cal-

cium phosphate phase transformation produced by the interaction

of the Portland cement component of white mineral trioxide aggre-

gate with a phosphate-containing fluid. J Endod 2007;33:

1347–1351.

31. Salinas AJ, Vallet-Regí M. Bioactive ceramics: From bone grafts to

tissue engineering. RSC Adv 2013;3:11116–11131.

32. Proksch S, Brossart J, Vach K, Hellwig E, Altenburger MJ,

Karygianni L. Evaluation of the bioactivity of fluoride-enriched mineral

trioxide aggregate on osteoblasts. Int Endod J 2018;51:912–923.

33. Kokubo T. Bioactive glass ceramics: Properties and applications.

Biomaterials 1991;12:155–163.

34. Nonat A. The structure and stoichiometry of C-S-H. Cement Con-

crete Res 2004;34:1521–1528.

35. Ha WN, Bentz DP, Kahler B, Walsh LJ. D90: The strongest contribu-

tor to setting time in mineral trioxide aggregate and Portland

cement. J Endod 2015;41:1146–1150.

36. Hench LL. Bioceramics – From concept to clinic. J Am Ceram Soc

1991;74:1487–1510.

37. Qiu W, Sun B, He F, Zhang Y. MTA-induced notch activation

enhances the proliferation of human dental pulp cells by inhibiting

autophagic flux. Int Endod J 2017;50:e52–e62.

38. Marciano MA, Guimarães BM, Amoroso-Silva P, Camilleri J,

Hungaro Duarte MA. Physical and chemical properties and subcuta-

neous implantation of mineral trioxide aggregate mixed with pro-

pylene glycol. J Endod 2016;42:474–479.

39. Kulan P, Karabiyik O, Kose GT, Kargul B. Biocompatibility of accel-

erated mineral trioxide aggregate on stem cells derived from

human dental pulp. J Endod 2016;42:276–279.

40. Cavenago BC, Del Carpio-Perochena AE, Ordinola-Zapata R,

Estrela C, Garlet GP, Tanomaru-Filho M, Weckwerth PH, de

Andrade FB, Duarte MAH. Effect of using different vehicles on the

physicochemical, antimicrobial, and biological properties of white

mineral trioxide aggregate. J Endod 2017;43:779–786.

41. Guimarães BM, Prati C, Duarte MAH, Bramante CM, Gandolfi MG.

Physicochemical properties of calcium silicate-based formulations

MTA repair HP and MTA Vitalcem. J Appl Oral Sci 2018;26:

e2017115.


ftp://ftp.endoco.com/links/AvalonMTAPlusDFU.pdf

ftp://ftp.endoco.com/links/AvalonMTAPlusDFU.pdf

Documents

Higher Hydration Performance and Bioactive Response of the ... JCR/105-JBMR-B-Higher... · Key Words: bioactive endodontic cements, bioactive response, physico-chemical properties