Influence of Thermal Treatment Conditions on the

2 downloads 0 Views 7MB Size Report
Feb 18, 2016 - (v = 150 rot/min), in order to obtain the MTA cements. The setting ... time of the MTA cement obtained by thermal treatment at 1400 ˝C/2 h (MTA1) was 55 min and ...... Tanomaru-Filho, M. Bioactivity of MTA Plus, Biodentine and ...
molecules Article

Influence of Thermal Treatment Conditions on the Properties of Dental Silicate Cements Georgeta Voicu 1 , Alexandru Mihai Popa 1 , Alina Ioana Badanoiu 1, * and Florin Iordache 2 1

2

*

Department of Science and Engineering of Oxide Materials and Nanomaterials, Faculty of Applied Chemistry and Material Science, Politehnica University of Bucharest, 1-7 Gh. Polizu Street, Bucharest RO-011061, Romania; [email protected] (G.V.); [email protected] (A.M.P.) Department of Fetal and Adult Stem Cell Therapy, Nicolae Simionescu Institute of Cellular Biology and Pathology of Romanian Academy, 8 B.P. Hasdeu Street, Bucharest RO-050568, Romania; [email protected] Correspondence: [email protected]; Tel.: +40-21-4023-884; Fax: +40-21-4023-815

Academic Editor: Alexandru Mihai Grumezescu Received: 25 January 2016 ; Accepted: 13 February 2016 ; Published: 18 February 2016

Abstract: In this study the sol-gel process was used to synthesize a precursor mixture for the preparation of silicate cement, also called mineral trioxide aggregate (MTA) cement. This mixture was thermally treated under two different conditions (1400 ˝ C/2 h and 1450 ˝ C/3 h) followed by rapid cooling in air. The resulted material (clinker) was ground for one hour in a laboratory planetary mill (v = 150 rot/min), in order to obtain the MTA cements. The setting time and mechanical properties, in vitro induction of apatite formation by soaking in simulated body fluid (SBF) and cytocompatibility of the MTA cements were assessed in this study. The hardening processes, nature of the reaction products and the microstructural characteristics were also investigated. The anhydrous and hydrated cements were characterized by different techniques e.g., X-ray diffraction (XRD), scanning electron microscopy (SEM), infrared spectroscopy (FT-IR) and thermal analysis (DTA-DTG-TG). The setting time of the MTA cement obtained by thermal treatment at 1400 ˝ C/2 h (MTA1) was 55 min and 15 min for the MTA cement obtained at 1450 ˝ C/3 h (MTA2). The compressive strength values were 18.5 MPa (MTA1) and 22.9 MPa (MTA2). Both MTA cements showed good bioactivity (assessed by an in vitro test), good cytocompatibility and stimulatory effect on the proliferation of cells. Keywords: mineral trioxide cement; composition; thermal treatment; hydration and hardening processes; properties; setting time; biocompatibility

1. Introduction Dental silicate cements, also known as mineral trioxide aggregate (MTA) cements, are currently used in endodontic procedures for root perforation repairs and root-canal sealing [1–11]. The mineral phases present in MTA cement are similar with those present in Portland cement, e.g., calcium silicates (3CaO¨SiO2 and 2CaO¨SiO2 ) and tricalcium aluminate (3CaO¨Al2 O3 ). For practical reasons MTA cement paste should be radioopaque therefore bismuth oxide can also be added to its formulations [1,2]. Our research group has reported the synthesis of mineralogical compounds specific for MTA cements e.g., 2CaO¨SiO2 [12] and 3CaO¨Al2 O3 [13] as well as MTA cements [14] using a non-conventional method—the sol-gel route. Compared with the solid state reaction route, the usual synthesis method for this type of materials, the sol-gel route presents some advantages like high purity of the resulted products and lower thermal treatment temperatures. Moreover, the MTA cements obtained by the sol-gel method have good biocompatibility and no recordable cytotoxicity [14]. Molecules 2016, 21, 233; doi:10.3390/molecules21020233

www.mdpi.com/journal/molecules

Molecules Molecules2016, 2016,21, 21,233 233

22ofof15 15

The setting time of MTA silicate cements, obtained by our group using this method, is still too The setting time of MTA silicate cements, obtained by our group using this method, is still too long [14,15], especially if dental applications are the aim; therefore, in this paper we present new MTA long [14,15], especially if dental applications are the aim; therefore, in this paper we present new MTA cement formulation, with a higher amount of 3CaO∙Al2O3, compound with high reactivity vs. water. cement formulation, with a higher amount of 3CaO¨Al2 O3 , compound with high reactivity vs. water. The influence of thermal treatment parameters (temperature and plateau) on the main properties of The influence of thermal treatment parameters (temperature and plateau) on the main properties of MTA cement, before and after hardening, is also presented in this paper. MTA cement, before and after hardening, is also presented in this paper. 2. Results 2. Results and and Discussion Discussion The powers powers obtained obtained by by the the grinding grinding of of MTA MTA clinkers clinkers for for 15 15 min min in in aa planetary planetary mill, mill, were were The analyzed by laser granulometry. The main granulometric characteristics of the two MTA cements analyzed by laser granulometry. The main granulometric characteristics of the two MTA cements are are presented in Table andgrains the grains size distributions curves are presented in Figure presented in Table 1 and1 the size distributions curves are presented in Figure 1. 1. Table 1. 1. Granulometric Granulometric characteristics characteristics of of MTA1 MTA1and andMTA2 MTA2cements. cements. Table

Cement Average Particle Size (μm) Cement Average Particle Size (µm) MTA1 15.03 MTA1 15.03 MTA2 10.77

d0.1 (μm) * d0.9 (μm) ** d0.9 (µm) ** 2.867 31.143 2.867 31.143 0.690 26.652

d0.1 (µm) *

MTA2 10.77 0.690 26.652 * The diameter where 10% of the population lies below this value; ** the diameter where 90% of the * The diameter where 10% of the population lies below this value; ** the diameter where 90% of the population population lies below this value. lies below this value.

The The data data presented presented in in Table Table11confirms confirmsthe the higher higher fineness fineness of of MTA2 MTA2powder powder as as compared compared with with MTA1. MTA1. The The amount amount of of grains grains with with particles particles sizes sizes below below 11 µm μm is is higher higher for for the the MTA MTA cement cement obtained obtained ˝ C/3 h (MTA2) as compared with the one obtained at 1400 ˝ C/2 h at at higher higher temperature temperature i.e., i.e., 1450 1450 °C/3 h (MTA2) as compared with the one obtained at 1400 °C/2 h (MTA1)—Figure (MTA1)—Figure1.1.

(a)

(b) Figure 1.1.Grain Grain size sizedistribution distributionof ofMTA MTAcements cementsobtained obtainedat: at:(a) (a)1400 1400˝ C/2 °C/2 hh (MTA1); (MTA1);(b) (b)1450 1450˝°C/ Figure C/ 3 h (MTA2). 3 h (MTA2).

Information regarding the crystalline compounds formed during the thermal treatment in MTA Information regarding the crystalline compounds formed during the thermal treatment in MTA cements were obtained by XRD (Figure 2). cements were obtained by XRD (Figure 2).

Molecules 2016, 21, 233

3 of 15

Molecules 2016, 21, 233

3 of 15 - C3S - C2S

2000

- C3A

- C3S - C2S

1000

- CaO

- C3A - CaO

1800 800

I (a.u.)

I (a.u.)

1600 1400 1200

600

1000 800 400

600 400

200

200 0 10

a

20

30

40

2 θ (deg.)

50

60

10

b

20

30

40

50

60

2 θ (deg.)

Figure 2. XRD patterns of MTA cements: (a) MTA1 and (b) MTA2.

Figure 2. XRD patterns of MTA cements: (a) MTA1 and (b) MTA2.

The main mineralogical crystalline compounds assessed by this technique in both MTA1 and MTA2main cements are: tricalciumcrystalline silicate—3CaO∙SiO 2 (C3S) (JCPDS 42-0551), silicate—2CaO∙SiO 2 The mineralogical compounds assessed by dicalcium this technique in both MTA1 (C 2 S) (JCPDS 76-0799), tricalcium aluminate—3CaO∙Al 2 O 3 (C 3 A) (JCPDS 38-1429) and free lime (JCPDS and MTA2 cements are: tricalcium silicate—3CaO¨SiO2 (C3 S) (JCPDS 42-0551), dicalcium 37-1497). The results obtained by Rietveld refinement of XRD patterns are presented in Table 2. silicate—2CaO¨SiO 2 (C2 S) (JCPDS 76-0799), tricalcium aluminate—3CaO¨Al2 O3 (C3 A) (JCPDS 38-1429) and free lime (JCPDS 37-1497). The results obtained by Rietveld refinement of XRD patterns are Table 2. Amount of main crystalline compounds present in MTA cements assessed by the Rietveld presented in Table 2. refinement technique. Specimen Table 2. Amount of main crystallineCompounds compounds present in MTA cements assessed by the Rietveld Mineralogic MTA1 MTA2 refinement technique. C3S (%) 68.60 71.40 C2S (%) 13.70 11.80 Specimen Mineralogic Compounds C3A (%) 13.40 15.70 MTA1 MTA2 CaO (%) 4.30 1.10 C3 S (%) 68.60 71.40 C2 S (%) 13.70 11.80 The first thermal treatment was performed at 1400 °C with a 2 h plateau based on previous C3 A (%) 13.40 15.70 results reported in other papers [14–16]. This thermal treatment ensured the formation of calcium CaO (%) 4.30 1.10 silicates and tricalcium aluminate, but the amount of free lime was high (4.3%), which implies that a higher amount of mineralogical compounds can be obtained if the thermal treatment temperature ˝ C with awas and first plateau increase. Therefore, second thermal performed at 1450 °C for 3 h. The thermal treatment wasthe performed at 1400treatment 2 h plateau based on previous results As expected, the increase of thermal treatment temperature (from 1400 °C to 1450 °C) and plateau reported in other papers [14–16]. This thermal treatment ensured the formation of calcium silicates and (from aluminate, 2 h at 3 h) determines a certainofincrease of the of C3S which and C3A, compounds a high tricalcium but the amount free lime wasamount high (4.3%), implies that awith higher amount reactivity vs. water [17,18]. Also, the amount of free lime decreases from 4.3% (MTA1) to 1.1% (MTA2); of mineralogical compounds can be obtained if the thermal treatment temperature and plateau increase. for this type of cements, free lime amount can also play an important role due to its antibacterial Therefore, the second thermal treatment was performed at 1450 ˝ C for 3 h. As expected, the increase of properties [2,7,19–21]. The setting time and compressive strength values of MTA cements are thermal treatment temperature (from 1400 ˝ C to 1450 ˝ C) and plateau (from 2 h at 3 h) determines presented in Table 3.

a certain increase of the amount of C3 S and C3 A, compounds with a high reactivity vs. water [17,18]. Also, the amount of free Table lime decreases from (MTA1) to 1.1%of(MTA2); for this type of cements, free 3. Setting time and4.3% compressive strengths MTA cements. lime amount can also play an important role due to its antibacterial properties [2,7,19–21]. The setting Compressive Strength Compressive Strength Cement strength Setting Time (min) time and compressive values of MTA cements presentedafter in Table 3. (MPa) after 7 Daysare (MPa) 28 Days MTA 1 55 9.2 18.2 MTA 2 Table 3. Setting 15 time and compressive 12.7 strengths of MTA cements. 22.9 Compressive Strength The Cement important decrease of the MTA2Compressive setting time Strength (as compared with MTA1) can be due to Setting Time (min) after 7 Days (MPa) after 28 Days (MPa) several factors e.g.,: MTA 1 55 9.2 18.2 MTA 2 15 12.7 22.9

Molecules 2016, 21, 233

4 of 15

The important decrease of the MTA2 setting time (as compared with MTA1) can be due to several factors e.g.,: (i) the higher amount of C3 S and C3 A formed in MTA2 ( as compared with MTA1)—see Table 2. Molecules 2016, 21, 233 4 of 15 Both tricalcium silicate and tricalcium aluminates are mineralogical compounds with high reactivity vs. 21, water to decrease of the with setting time; 2016, 233 [17]ofand 4 of 152. (i)Molecules the higher amount C3S actively and C3A contributes formed in MTA2 ( as compared MTA1)—see Table Both tricalcium silicate tricalcium aluminates compounds withTable high 1 and (ii) the higher amount of smalland cement grains in MTA2are as mineralogical compared with MTA1 (see (i) reactivity the higher amount of Cand 3S and C3A formed in MTA2 ( as compared with MTA1)—see Table 2. vs. water [17] actively contributes to decrease of the setting time; Figure 1) contributes also to the important decrease of the setting time noticed for the cement Both tricalcium silicate and tricalcium arecompared mineralogical with high (ii) the higher amount of small cement grainsaluminates in MTA2 as with compounds MTA1 (see Table 1 and thermally treated at higher temperature. reactivity vs. water [17] and actively contributes to decrease of the setting time; Figure 1) contributes also to the important decrease of the setting time noticed for the cement

the higher amount of smallstrengths, cement grains in MTA2 as7compared withof MTA1 (see Table and The(ii)values of the compressive assessed after and 28 days hardening, are1comparable thermally treated at higher temperature. Figure 1) contributes also to the important decrease of the setting time noticed for the cement for the two studied MTA cements (Table 3). Both setting time and compressive strengths have values The values treated of the atcompressive strengths, assessed after 7 and 28 days of hardening, are thermally higher temperature. similar with those reported in the literature for silicate dental cements [9,22]. comparable for the two studied MTA cements (Table 3). Both setting time and compressive strengths The values of the compressive strengths, assessed after 7 and depend 28 days both of hardening, are The MTA cements on the nature and havecompressive values similarstrengths with those values reportedofinhardened the literature for silicate dental cements [9,22]. comparable for the two studied MTA cements (Table 3). Both setting time and compressive strengths amount ofThe hydrates formed during values hardening processMTA as well as thedepend microstructure of hardened compressive strengths of hardened cements both on the nature andpastes. have values similar with those reported in the literature for silicate dental cements [9,22]. amount of hydrates formed during hardening process as cement well as the microstructure of hardened XRD patterns of anhydrous MTA cements and pastes hydrated for 1, 7 pastes. and 28 days, The compressive strengths values of hardened MTA cements depend both on the nature and XRD patterns of anhydrous MTA cements and cement pastes hydrated for 1, 7 and 28 days, presented in Figure 3b–d and Figure 4b–d,process provide information regarding the composition of amount of hydrates formed during hardening as well as the microstructure of hardened pastes. presented in Figures 3b–d and 4b–d, provide information regarding the composition of these materials. these materials. XRD patterns of anhydrous MTA cements and cement pastes hydrated for 1, 7 and 28 days,

I (a.u.) I (a.u.)

presented in Figures 3b–d and 4b–d, provide information regarding the composition of these materials. 3400 3200 3400 3000 3200 2800 3000 2600 2800 2400 2600 2200 2400 2000 2200 1800 2000 1600 1800 1400 1600 1200 1400 1000 1200 800 1000 600 800 400 600 200 400 20010 10

- C3S - C2S - C3S -CA - C23S

- C3A

- CaCO3 - CaO - CaCO3 - Ca(OH)2 - CaO

- Ca(OH)2

d

d c c

b b

20 20

30 40 30 2 θ (deg.) 40 2 θ (deg.)

50 50

a a

60 60

Figure 3. XRD patterns of MTA1: (a) anhydrous; (b) hydrated for 1 day; (c) hydrated for 7 days;

I (a.u.) I (a.u.)

Figure 3. XRD of MTA1: (a) anhydrous; (b) hydrated for 1 day; (c) hydrated for 7 days; 3. patterns XRD patterns (d)Figure hydrated for 28 days. of MTA1: (a) anhydrous; (b) hydrated for 1 day; (c) hydrated for 7 days; (d) hydrated for 28 days. (d) hydrated for 28 days. - C3S -- C C32SS -- C C2SA

2400 2400 2200 2200 2000 2000 1800 1800 1600 1600 1400 1400 1200 1200 1000 1000 800 800

3

- C3A

- CaCO3 --CaCO CaO 3 -- CaO Ca(OH)

- Ca(OH)2

2

d d c c

bb

600 600 400 400 aa

200 200 10 10

20 20

30 30

40 40

50 50

60 60

22 θθ (deg.)

4.4.XRD patterns (a) anhydrous; forfor day; (c) hydrated hydrated for7 7days; days; Figure XRD patterns ofMTA2: MTA2:(a) (a)anhydrous; anhydrous; (b) for 11 day; (c) for FigureFigure 4. XRD patterns of of MTA2: (b)hydrated hydrated 1 day; (c) hydrated for 7 days; hydratedfor for2828days. days. (d)(d) hydrated (d) hydrated for 28 days.

Molecules 2016, 21, 233

5 of 15

Molecules 2016, 21, 233 Molecules 21, 233 For 2016, the hydrated

5 of 15

5 of 15 cement pastes (Figures 3 and 4), a decrease of the intensity of peaks specific for anhydrous compounds (C S, C S and C A) due to their consumption in hydration processes it can be 3 2 3 For the hydrated cement pastes (Figures 3 and 4), a decrease of the intensity of peaks specific for ForThe the only hydrated cement pastes formed (Figuresduring 3 and 4), a decrease of the intensity of peaks specific for noticed. crystalline hydrate MTA cement hydration is portlandite—Ca(OH) 2; anhydrous compounds (C3S, C2S and C3A) due to their consumption in hydration processes it can be anhydrous compounds (C 3S, C2S and C3A) due to their consumption in hydration processes it can be the intensity of its specific peaks increases with the increase of the hydration time, a clear indication of noticed. The only crystalline hydrate formed during MTA cement hydration is portlandite—Ca(OH)2; noticed. The in onlyhigher crystalline hydratethe formed during MTA cement hydrationpastes is portlandite—Ca(OH) its amount. XRD patterns of both MTA area also the2; theformation intensity ofaits specific peaksOn increases with the increase of thecements hydration time, clearpresent indication the intensity of its specific peaks increases with the increase of the hydration time, a clear indication peaks for in calcium carbonate—this is most probably due to the carbonation of its specific formation a higher amount. Oncompound the XRD patterns of both formed MTA cements pastes are also ofportlandite its formation inatmospheric a higher amount. On the XRD patterns of both MTA cements pastes are also of with CO [18]. 2 present the peaks specific for calcium carbonate—this compound is most probably formed due to present the peaks specific for calcium carbonate—this compound most probably due to Thermal analysis can provide information about isthe hydrates (gelformed or crystalline the carbonation of portlandite with quantitative atmospheric CO 2 [18]. the carbonation of portlandite with atmospheric CO 2 [18]. compounds) Thecan endoeffectsquantitative present on the DTA curves (Figures 5 and 6) associated with the Thermal[17,18]. analysis provide information about the hydrates (gel or crystalline Thermal analysis can provide quantitative information about thefollowing hydrates processes (gel or crystalline weight loss assessed on the DTG and TG curves can be attributed to the compounds) [17,18]. The endo- effects present on the DTA curves (Figures 5 and 6) associated [17,18]: with the compounds) [17,18]. The endo- effects present on the DTA curves (Figures 5 and 6) associated with the weight loss assessed on theup DTG and˝TG curves be loss attributed to the following processes [17,18]: - endo-effects recorded to 100 C are due can to of moisture; weight loss assessed on the DTG and TG curves canthe be attributed to the following processes [17,18]: ˝ endo-effects recorded between 100 and 220 C are due to the dehydration of gel like calcium endo-effects up to 100 °C are due to the loss of moisture; endo-effects recorded up to 100 °C are due to the loss of moisture; silicates hydrates and calcium hydrates formed cement hydration; endo-effects recorded betweenaluminate 100 and 220 °C are due tobythe dehydration of gel like calcium endo-effects recorded between 100 and 220 are due to the dehydration of gel like calcium ˝ C°C silicates hydrates andbetween calcium 400 aluminate formed by by cement hydration; of portlandite; - endo-effects recorded and 500hydrates are determined the dehydration silicates hydrates and calcium aluminate hydrates formed by cement hydration; -- the endo-effects recorded between 400 and are˝ C determined by the of portlandite; endo-effects recorded between 600 500 and°C 850 are attributed to dehydration the decarbonation of CaCO endo-effects recorded between 400 and 500 °C are determined by the dehydration of portlandite; 3 the endo-effects recorded between 600asand 850 °C are attributed to compound the decarbonation CaCO3 with different crystallization degrees; previously presented, this is most of probably the endo-effects recorded between 600 and 850 °C are attributed to the decarbonation of CaCO3 with different crystallization degrees; as previously presented, this compound is most probably formed due to the carbonation of portlandite with atmospheric CO2 . with different crystallization degrees; as previously presented, this compound is most probably formed due to the carbonation of portlandite with atmospheric CO2. formed due to the carbonation of portlandite with atmospheric CO2.

1 day day 71 days 7 days 28 days 28 days

exo. exo. DTA DTA (a.u) (a.u) endo. endo.

150 150 133 133

83 83

127 127

DTG DTG (a.u) (a.u)

83 83

0 0

439 439

150 150 133 127133 127

100 100

200 200

461 461 451 451

692 692

461 461

665 665 700 700

451 439 451 439

300 300

400 400

500 500

600 600

700 700

800 800

900 900

1000 1100 1000 1100

Temperature (°C) Temperature (°C)

Figure 5. 5. DTA and and DTG curves curves of MTA1 MTA1 pastehydrated hydrated for1,1,77and and 28days. days. Figure Figure 5. DTA DTA and DTG DTG curvesof of MTA1paste paste hydratedfor for 1, 7 and28 28 days. 1 day day 71 days 7 28days days 28 days

exo. exo. DTA DTA (a.u) (a.u) endo. endo.

464 464

151 151 131 131 137 137

DTG DTG (a.u) (a.u)

446 446 457 457

151 151

0 0

100 100

137 137

200 200

300 300

400 400

464 464 447 447 457 457

500 500

687 687 684 684

600 600

700 700

800 800

900 900

1000 1100 1000 1100

Temperature (°C) Temperature (°C)

Figure 6. DTA and DTG curves of MTA2 paste hydrated for 1, 7 and 28 days. Figure 6. 6. DTA DTA and and DTG DTGcurves curvesof ofMTA2 MTA2paste pastehydrated hydratedfor for1,1,77and and28 28days. days. Figure

The weight losses recorded on TG curves were processed and are presented in Figure 7. The weight losses recorded on TG curves were processed and are presented in Figure 7.

Molecules 2016, 21, 233

6 of 15

Molecules 2016, 21, 233

6 of 15

The weight losses recorded on TG curves were processed and are presented in Figure 7. Molecules 2016, 3021, 233 30 25

20 Weight loss (%)

Weight loss (%)

25

6 of 15

MTA1 1 day

MTA1 28 days

MTA1 MTA211day day

MTA1 28 28 days MTA2 days

MTA2 1 day

MTA2 28 days

20

15 10

5 0

15 10 5 0

30 – 301000°C – 1000°C

30-400°C 30-400°C

400-480°C 400-480°C

Figure 7. Weight losses recorded TGcurves curves of MTA hydrated for 1for day and 28 days. Figure 7. Weight losses recorded ononTG MTAcement cementpastes pastes hydrated 1 day and 28 days. Figure 7. Weight losses recorded on TG curves of MTA cement pastes hydrated for 1 day and 28 days.

it can seen fromFigure Figure7,7, the the total total weight between 30 and 1000 1000 °C) °C) As itAscan be be seen from weightloss loss(recorded (recorded between 30 and ˝ C) increases increases from 1 day to Figure 28 days7,for both MTA cements, due to the progress of hydration processes. As it can be seen from the total weight loss (recorded between 30 and 1000 increases from 1 day to 28 days for both MTA cements, due to the progress of hydration processes. The weight corresponding to cements, the 30–400due °C interval provides of information the amount weight of from 1 day toloss 28 loss days for both MTA to the progress hydrationabout processes. The weight corresponding to the 30–400 °C interval provides information about theThe amount of ˝ calcium silicates hydrates (C-S-H) and calcium aluminate hydrates (C-A-H) formed in the hydrated loss corresponding to the 30–400 C interval provides information about the amount of calcium silicates calcium silicates hydrates (C-S-H) calcium aluminate hydrates (C-A-H) in the hydrated pastes; one can notice the higherand amount of C-S-H and C-A-H formed after 1 formed day of hydration in hydrates (C-S-H) and calcium aluminate hydrates (C-A-H) formed in the hydrated pastes; one can pastes; onepaste can notice the higher amount of C-S-H C-A-H formed 1 rate dayofofhydration hydration in MTA2 as compared with MTA1 paste—this canand be correlated with theafter higher notice the higher amount of C-S-H and C-A-H formed after 1 day of hydration in MTA2 paste as MTA2 paste as compared with MTA1 paste—this can be correlated with the higher rate of hydration in the first system, confirmed also by the lower values of the setting time (Table 3). The higher compared with paste—this can be with the of higher rate of °C) hydration in the in the first system, confirmed also the lower values theand setting time (Table 3).1first The higher amount of MTA1 portlandite (assessed byby thecorrelated weight loss between 400 480 formed after daysystem, of confirmed also by the lower values of the setting time (Table 3). The higher amount of portlandite hydration in MTA2 paste, as compared with MTA1 confirms also the hydration rate1 of amount of portlandite (assessed by the weight losspaste, between 400 and 480higher °C) formed after day of ˝ C) formed after 1 day of hydration in MTA2 paste, (assessed byin the weight loss 400with and 480 MTA2 cement, atpaste, early ages. hydration MTA2 asbetween compared MTA1 paste, confirms also the higher hydration rate of as compared paste, confirms also the higher hydration of MTA2 at early Thewith microstructure of MTA cement pastes, hydrated different rate periods of time,cement, are presented in ages. MTA2 cement, at MTA1 early ages. Figures 8 and 9. The of main hydrate phases assessed by XRD and DTA and TG, e.g., the calcium The microstructure MTA cement pastes, hydrated different periods of time, are presented The microstructure of MTA cement pastes, hydrated different periods of time, are presented in silicates and hydrates andmain portlandite, canphases be also assessed assessed inby theXRD SEMand images; calcium silicates hydrates in Figures The hydrate DTA and TG, Figures 8 8and 9.9.The main hydrate phases assessed by XRD and DTA and TG, e.g., e.g., the the calcium calcium have specific morphologies i.e., needle-like and plate microcrystals for calcium silicate hydrateshydrates and silicates hydrates and portlandite, can be also assessed in the SEM images; calcium silicates silicates hydrates and portlandite, can be also assessed in the SEM images; calcium silicates hydrates spongy agglomeration specific for carbonate phases [17,23]. In the microstructure of MTA1 paste can have specific morphologies i.e., and plate microcrystals for calcium silicate hydrates and havebe specific morphologies i.e.,needle-like needle-like and platefor microcrystals calcium silicate also assessed the hexagonal plate crystals specific of portlanditefor (see arrow in Figurehydrates 8c). The and spongy agglomeration specific for carbonate phases [17,23]. In the microstructure of MTA1 paste spongy agglomeration specific fortocarbonate phases [17,23]. In the microstructure of MTA1 paste can microstructure of MTA2 seems be also more compact as compared with the one assess for MTA1; can be also assessed the hexagonal plate crystals specific for of portlandite (see arrow in Figure 8c). be also thecorrelation hexagonalwith plate for of of portlandite (see arrow developed in Figure 8c). thisassessed is in good thecrystals slightly specific higher values compressive strength by The The microstructure of MTA2 seems also more compactasas compared withthe theone one assess assess for for MTA1; MTA1; MTA2 pastes 7 and 28 days ofbe hydration ascompact compared with MTA1. with microstructure ofafter MTA2 seems to to be also more compared this correlation with the the slightly higher values of compressive strength developed by MTA2 thisisisiningood good correlation with slightly higher values of compressive strength developed by pastes after 7 and 28 days of hydration as compared with MTA1. MTA2 pastes after 7 and 28 days of hydration as compared with MTA1.

(a) Figure 8. Cont.

(a) Figure8.8.Cont. Cont. Figure

Molecules 2016, 21, 233

7 of 15

Molecules 2016, 21, 233

7 of 15

Molecules 2016, 21, 233

7 of 15

(b) (b)

(c) (c) Figure 8. SEM images of MTA1 pastes hydrated for: (a)—1 day; (b)—7 days; (c)—28 days. Figure 8. SEM images of MTA1 pastes hydrated for: (a)—1 day; (b)—7 days; (c)—28 days. Figure 8. SEM images of MTA1 pastes hydrated for: (a)—1 day; (b)—7 days; (c)—28 days.

(a) (a)

(b) (b) Figure Figure9.9. 9.Cont. Cont. Figure Cont.

Molecules 2016, 21, 233

8 of 15

Molecules 2016, 21, 233 Molecules 2016, 21, 233

8 of 15 8 of 15

(c) (c) Figure 9. SEM images of MTA2 pastes hydrated for: (a)—1 day; (b)—7 days; (c)—28 days. Figure 9. SEM images of MTA2 pastes hydrated for: (a)—1 day; (b)—7 days; (c)—28 days. Figure 9. SEM images of MTA2 pastes hydrated for: (a)—1 day; (b)—7 days; (c)—28 days.

In vitro bioactivity tests were conducted on MTA pastes immersed in SBF at 37 °C for 14 days. In vitro bioactivity tests on MTA pastes immersed SBFatat3737°C˝ofC for days. XRD patterns of were these pastes (Figure show a decrease of the intensities diffraction InThe vitro bioactivity tests wereconducted conducted on 10) MTA pastes immersed ininSBF for 1414 days. TheThe XRD patterns of these pastes (Figure 10) show decrease of the intensities diffraction peaks peaks specific for anhydrous compounds (C310) S, Cshow 2S)a and portlandite (Ca(OH) 2) of confirming an XRD patterns of these pastes (Figure a decrease of the intensities of diffraction interaction of these phases withcompounds SBF; also, on(C the of MTA pastes immersed for 14 days specific for anhydrous compounds (C3 S, C2 S) and portlandite (Ca(OH) ) confirming an interaction peaks specific for anhydrous 3S,XRD C2S)spectra and portlandite (Ca(OH) 2 ) confirming an of 2 inphases SBF, new forthe hydroxyapatite (HAp) (JCPDS 84-1998) and calcium carbonate these with SBF;specific also, on XRDalso, spectra of MTA pastes immersed for 14immersed days in SBF, new peaks interaction ofpeaks these phases with SBF; on the XRD spectra of MTA pastes for(JCPDS 14 days are found. The (HAp) presence of HAp, compound with 84-1998) very good is a(JCPDS clear in03-0596) SBF, peaks specific for hydroxyapatite (HAp) (JCPDS andbiocompatibiliy, calcium specific fornew hydroxyapatite (JCPDS 84-1998) and calcium carbonate (JCPDScarbonate 03-0596) are found. indication of an adequate behavior of these materials if used as dental cements. areoffound. The presence of very HAp,good compound with very is good biocompatibiliy, is aadequate clear The03-0596) presence HAp, compound with biocompatibiliy, a clear indication of an

indication of anmaterials adequate behavior these materials as dental cements. behavior of these if500 used asofdental cements. if used -CS - C2S

- CaCO3 - CaO

C3S --HAp - C2S

CaCO32 - -Ca(OH) - CaO

- HAp

- Ca(OH)2

3

500 400

I (a.u.)I (a.u.)

400 after

300

after

300 200

before

200 100

before 10

100

20

30

20

30

40

50

2 θ (deg.) 10

40

(a)

2 θ (deg.)

500

(a) 500 400

50

60

- C3S - C2S

- CaCO3 - CaO

- HAp - C3S - C2S

- Ca(OH)2 - CaCO3 - CaO

- HAp

I (a.u.) I (a.u.)

60

- Ca(OH)2

400 300

after

after

300 200

before

200 100 before 10

20

30

40

50

60

2 θ (deg.)

100 10

20

30

(b)40

50

60

2 θ (deg.)

Figure 10. XRD patterns of hydrated MTA pastes before and after immersion in SBF for 14 days at 37 °C: (b) (a) MTA1; (b) MTA2.

Figure 10. XRD patterns of hydrated MTA pastes before and after immersion in SBF for 14 days at 37 °C:

Figure 10. XRD patterns of hydrated MTA pastes before and after immersion in SBF for 14 days at (a) MTA1; (b) MTA2. 37 ˝ C: (a) MTA1; (b) MTA2.

Molecules 2016, 21, 233

9 of 15

Molecules 2016, 21, 233

9 of 15

The FT-IR analysis (Figure 11) of MTA pastes immersed for 14 days in SBF shows the following Thebands FT-IR[24–26]: analysis (Figure 11) of MTA pastes immersed for 14 days in SBF shows the following specific specific bands [24–26]: bands at 1424 cm´1 and 872 cm´1 , specific for calcium carbonate, also assessed by XRD; −1 bands at2016, 1424 cm 872 cm−1 for calcium carbonate, also assessed by XRD; ´,1specific Molecules 233´1 and bands at 96221,cm and 518 cm , specific for the phosphate group (PO4 3´ ), from HAp;9 of 15 −1 −1, specific for the phosphate group (PO43−), bands at 962 cm and´518 cm from HAp; 1 is specific for carbonated HAp formed by the substitution of PO 3´ the band of 1411 cm−1 (Figure 4 The FT-IR analysis 11) of MTA pastes immersed for 14 days in SBF shows the of following the band of 1411 cm is specific for carbonated HAp formed by the substitution PO43− with 2´ ; with CO 3 specific bands [24–26]: CO 32−; ´1 is specific for hydroxyl groups (moisture). the band of 1639 cm−1 −1 is specific for −1 the band of 1639 cm hydroxyl groups (moisture). -

bands at 1424 cm and 872 cm , specific for calcium carbonate, also assessed by XRD;

bandscan at 962 cm on and 518FT-IR cm , specific for the phosphatefor group (PO4 ),phases—calcium from HAp; Also, silicates Also, one one can assess assess on −1the the FT-IR spectra spectra specific specific bands bands for hydrated hydrated phases—calcium silicates the band of 1411 cm is specific for carbonated HAp formed by the substitution of PO 43− with ´ 1 ´ 1 hydrates ), calcium or for hydrates (451, (451,2−962, 962, 1411 1411 and and1639 1639cm cm−1), calcium aluminate aluminate hydrate hydrate (424 (424 cm cm−1),), or for anhydrous anhydrous CO3 ; ´1 the bands between 400 and 150 cm´−11 can be also compounds (e.g., dicalcium silicate at cm compounds at 518 518hydroxyl cm−1);); the bands between 400 and 150 cm can be also the(e.g., band dicalcium of 1639 cm−1silicate is specific for groups (moisture). attributed attributed to to the the polymerized polymerized silicate silicate tetrahedra, tetrahedra, present present in in the the silicate silicate hydrate hydrate structure. structure. −1

−1

3−

Also, one can assess on the FT-IR spectra specific bands for hydrated phases—calcium silicates hydrates (451, 962, 1411 and 1639 cm−1), calcium aluminate hydrate (424 cm−1), or for anhydrous compounds (e.g., dicalcium silicate at 518 cm−1); the bands between 400 and 150 cm−1 can be also attributed to the polymerized silicate tetrahedra, present in the silicate hydrate structure.

Figure 11. FT-IR spectra of MTA pastes hydrated for 7 days and the immersed SBF for 14 days. Figure 11. FT-IR spectra of MTA pastes hydrated for 7 days and the immersed SBF for 14 days. Figure 11. FT-IR spectra of MTA pastes hydrated for 7 days and the immersed SBF for 14 days.

The SEM images of MTA pastes, immersed for 14 days in SBF (Figure 12), shows the presence of The SEMlayer images of MTA pastes, immersedoffor 14 days in SBF (Figure 12), shows theassociated presence of a thin friable formed agglomerations plate like crystals; this morphology The SEM images ofby MTA pastes, immersed for 14 days in SBF (Figure 12), shows theis presence of to aHAp thin[27–29]. friable layer formed by agglomerations of plate like crystals; this morphology is associated to a thin friable layer formed by agglomerations of plate like crystals; this morphology is associated to HAp [27–29]. HAp [27–29].

(a) (a)

(b) (b) Figure 12. Cont.

Figure 12. Cont.

Molecules 2016, 21, 233

10 of 15

Molecules 2016, 21, 233

10 of 15

Molecules 2016, 21, 233

10 of 15

(c)

(d)

Figure 12. SEM micrographs of MTA pastes hydrated for 7 days and the immersed SBF for 14 days at

Figure 12. SEM micrographs of MTA pastes hydrated for 7 days and the immersed SBF for 14 days at 37 °C: (a), (b)—MTA1; (c), (d)—MTA2. 37 ˝ C: (a), (b)—MTA1; (c), (d)—MTA2. (c) was verified by a MTT assay that is based (d) on biochemical reactions The cytotoxicity of MTA that cytotoxicity measure metabolic activity living cells. assay The MTT assay demonstrated the The of MTA was verified by ahydrated MTT thatand is the based on biochemical reactions Figure 12.the SEM micrographs of MTAofpastes for 7 days immersed SBF forthat 14 days athuman that endothelial cells presented metabolism growth the presence of MTA. measure the activity of(d)—MTA2. living cells. The and MTT assayin demonstrated that the human endothelial 37 metabolic °C: (a), (b)—MTA1; (c),normal The absorbance values measured at 570 nm showed a better proliferation of endothelial cells cells presented normal metabolism and growth in the presence of MTA. grown oncytotoxicity MTA2 (1450of°C/3 h) was compared to by those grown on MTA1 (1400 °C/2 h) (Figure 13); this can The MTA verified a MTT assay that is based on biochemical reactions The absorbance values measured at 570 nm showed a better proliferation of endothelial cells be to high in the MTA1 systemcells. (with high assay amount of ˝free lime—Table 2). The thatdue measure thebasicity metabolic activity of living Thea MTT demonstrated that the human ˝ grown on MTA2 (1450 C/3 h) compared to those grown on MTA1 (1400 C/2 h) (Figure 13); this can fluorescent microscopy images the biochemical test of results endothelial cells presented normalconfirm metabolism and growth inviability the presence MTA.showing that the be dueendothelial to high basicity in the MTA1 system (with a high amount of free lime—Table 2). The fluorescent cell viability is maintained 24nm h inshowed the presence of proliferation MTA hydrated for 7 and 28cells days. The absorbance values measured after at 570 a better of endothelial microscopy images confirm the biochemical viability test results showing that the endothelial grown on MTA2 cells (1450 retain °C/3 h)normal compared to those grown MTA1 (1400 h) a(Figure 13); this can cell The endothelial morphology, wereonadherent and°C/2 have relatively uniform viability is maintained after 24 h in the presence of MTA hydrated for 7 and 28 days. The endothelial be due to high in thesurfaces MTA1 (Figure system 14). (with a size highand amount free lime—Table 2).for The distribution on allbasicity investigated The shapeof of MTA are important the fluorescent microscopy images confirm the biochemical test granulometry results showing cells retain normal morphology, adherent and haveviability a relatively uniform distribution on all interaction with living cells. Thewere average particle size measured by laser wasthat 15.03the μm endothelial cell10.77 viability after 24 h inAt thethese presence of MTA hydrated forthe 7 and 28 days. for MTA1 and μm is formaintained MTA2, sizesare the proliferation endothelial cellswith investigated surfaces (Figure 14). The respectively. size and shape of MTA important forof interaction The endothelial cells particle retain normal morphology, wereMTA2 adherent and alter have a15.03 relatively uniform slightly decreased for MTA1—7 and 28 days. The did not the cellular livingwas cells. The average size measured by laser granulometry was µmmetabolism for MTA1 and distribution onthe all proliferation investigated surfaces (Figurecompared 14). The size and shapecells. of MTA are important for and moreover was increased with control The morphology wasthe not 10.77 µm for MTA2, respectively. At these sizes the proliferation of endothelial cells was slightly interactionthe with living cells.cells The retained average particle sizeshape measured by laser was 15.03 μm modified, endothelial a normal compared togranulometry control, adhered to culture decreased for MTA1—7 and 28 days. The MTA2 did not alter the cellular metabolism and moreover for MTA1 μm foruniform MTA2, respectively. these sizesup thetoproliferation endothelial cells 1 plates, and and had 10.77 a relatively distribution, At being viable 7 days in theofpresence of MTA the proliferation was increased compared with control cells. The morphology was not modified, was MTA2. slightlyThe decreased MTA1—7 andMTA 28 days. The MTA2 alter thebetter cellular metabolism and in situ for synthesis of the enables the celldid to not proliferate compared with the endothelial cells a the normal shape compared compared to of control, adhered to culturewas plates, and and moreover theretained proliferation was increased with control The morphology not control cells, demonstrating biocompatibility potential thesecells. cements and confirming their had apotential relatively uniform distribution, being viable up to 7 days in the presence of MTA 1 modified,application the endothelial cells retained normal compared control, adhered to culture in dentistry. These aresults areshape consistent with to those of other researchers that and plates, and had a relatively distribution, being viable up(80%–130% to 7 days in compared thecompared presence MTA 1 MTA2. The in situ synthesis ofuniform the MTA enables the cell to proliferate better control showed that different MTA cements have a good compatibility to of thewith control and MTA2. The in synthesiscells of the MTA enables the cellcements to proliferate better compared cells, group) demonstrating thesitu biocompatibility potential of these and confirming theirwith potential with human osteoblastic [30,31]. control cells, demonstrating the biocompatibility potential of these cements and confirming their application in dentistry. These results are consistent with those of other researchers that showed that potential application in dentistry. These results are consistent with those of other researchers that different MTA cements have a good compatibility (80%–130% compared to the control group) with showed that different MTA cements have a good compatibility (80%–130% compared to the control humangroup) osteoblastic cellsosteoblastic [30,31]. cells [30,31]. with human

Figure 13. Cont.

Molecules 2016, 21, 233

11 of 15

Molecules 2016, 21, 233 Molecules 2016, 21, 233

11 of11 15of 15

Figure 13. Endothelial cells proliferation profiles after growing on control (ctrl) and MTA for up to 48 h.

Figure 13. Endothelial cells proliferation profiles after growing on control (ctrl) and MTA for up to 48 h. Figure 13. Endothelial cells proliferation profiles after growing on control (ctrl) and MTA for up to 48 h.

(a)

(b)

(a)

(b)

(c)

(d)

(c)

(d)

(e) Figure 14. Fluorescence microscopic images of the endothelia cell monolayer in the presence of MTA (x20): (a) MTA1—7 days; (b) MTA1—28 days; (c) MTA2—7 days; (d) MTA2—28 days; (e) Ctrl.

(e) Figure 14. Fluorescence microscopic images of the endothelia cell monolayer in the presence of MTA Figure 14. Fluorescence microscopic images of the endothelia cell monolayer in the presence of MTA (x20): (a) MTA1—7 days; (b) MTA1—28 days; (c) MTA2—7 days; (d) MTA2—28 days; (e) Ctrl. (x20): (a) MTA1—7 days; (b) MTA1—28 days; (c) MTA2—7 days; (d) MTA2—28 days; (e) Ctrl.

Molecules 2016, 21, 233

12 of 15

3. Experimental Section The oxide composition for MTA cement was: CaO—69.4%, SiO2 —21.6%, Al2 O3 —5.8% and ZnO—3.2%. ZnO was added in this composition for two reasons: (i) Zn, in small quantities is generally harmless to human body and is a key element for bone development [16] and (ii) Zn can be incorporated in calcium silicates and calcium aluminates lattices increasing in this way the grindability of MTA cement [14]. In this study the MTA cement was obtained by a modified sol-gel method proposed by Voicu et al. [12–14]. As raw materials tetraethyl orthosilicate (C6 H16 O3 Si—TEOS), aluminium butoxide (C12 H27 O3 Al), zinc acetate (Zn(CH3 COO)2¨ 2H2 O) and calcium nitrate (CaNO3¨ 4H2 O) were used. The main steps of the MTA synthesis were: (a) (b)

(c)

aluminum butoxide and acetyl acetone (1:1 molar ratio) were magnetically stirred for 2 h; calcium nitrate was dissolved in distilled water and magnetically stirred until a clear solution was obtained; then zinc acetate was added and the solution was magnetically stirred for 2 h at 80 ˝ C; next, TEOS was added in this clear solution and the mixture was homogenized until a clear solution was obtained (molar ratio CaO:ZnO:SiO2 :H2 O was 1.24:0.04:0.36:10); the two solutions were mixed for 3 h at room temperature and then kept at 80 ˝ C until a gel was formed.

This gel was maturated for 24 h and then dried at 125 ˝ C for 24 h when a resin type precursor was obtained. The resin-type precursor was thermally treated in a platinum crucible at two different temperatures: 1400 ˝ C/2 h (MTA1) and 1450 ˝ C/3 h (MTA2), respectively. The heating was performed with 10 ˝ C /min and the cooling was performed rapidly in air. The MTA cements were obtained by the grinding of these clinkers (MTA1 and MTA2) for 15 min in a planetary ball mill (vrot = 150 rot/min). The resulted powder was analyzed by laser granulometry (by means of a Mastersizer 2000 laser granulometer, Malvern, U.K). The binding properties of MTA cements were assessed on pastes prepared with distilled water (cement to water weight ratio of 3:1). For the assessment of the setting time, 50 cm3 of MTA cement paste was filled in a metallic ring (φ = 10 mm, h = 5 mm) and kept on a glass plate in water bath (37 ˝ C and R.H. 80%). The setting time was assessed with a Vicat apparatus) and represents the time elapsed from the moment when the cement powder was mixed with distilled water until the Vicat needle do not leave any imprint on the surface of the paste. The compressive strength was assessed on paste specimens (cylinders φ = h = 10 mm) cured the first 24 h in the mold, placed on a glass plate in the water bath (37 ˝ C and R.H. 80%); after that the paste specimens were demoulded and stored in distilled water at 37 ˝ C up to 7 or 28 days. The compressive strength was assessed on a Cyber-Tronic testing machine (MATEST, Treviolo, Italy). The hydration and hardening processes of the MTA cements were assessed on paste specimens prepared and cured as presented above. After 1, 7 and 28 days, the pastes were ground until a fine powder was obtained. This powder was washed with ethyl alcohol and dried at 50 ˝ C for 24 h. The dried powder was analyzed by X-ray diffraction (XRD) and thermogravimentry (TG-DTG) coupled with differential thermal analysis (DTA). X-ray diffraction analysis was performed using an Empyrean diffractometer (Panalytical, Almelo, Netherland) with CuKα radiation (λ = 1.5418 Å), with scan step of 0.02˝ and counting time of 0.6 s/step; the diffractometer performs also Rietveld refinement of XRD patterns. The termogravimetry and differential thermal analysis were performed using a DTG-TA-60 derivatograph (Shimadzu, Kyoto, Japan); the analysis were performed in the 20–1000 ˝ C temperature range, with a heating rate of 10 ˝ C/min, in air. The microstructure of MTA cement pastes hydrated for 1, 7 and 28 days, was assessed by scanning electronic microcopy (SEM) using a S2600N instgrument (HITACHI, Kyoto, Japan). The specimens for SEM analysis were covered with a thin silver layer deposited by dc-sputtering.

Molecules 2016, 21, 233

13 of 15

The in vitro bioactivity of MTA cements was assessed on cement pastes hydrated for 7 days and soaked in simulated body fluid (SBF) (the specimen area to SBF volume ratio was 0.1 cm´1 ). The paste specimens were stored for 14 days in water bath at 37 ˝ C and then removed, gently rinsed with distilled water in order to remove all the soluble salts and dried at 60 ˝ C for 24 h. The specimen’s surface was then analyzed by XRD, Fourier Transformed Infrared Spectroscopy (FT-IR) and SEM. FT-IR measurements were performed using a Nicolet™ iS™50 spectrometer (Thermo Scientific, USA) equipped with an ATR module based on diamond crystal. The spectra were recorded over the wavenumber range of 150–1800 cm´1 with a resolution of 2 cm´1 . Also, in vitro biocompatibility tests were MTT assay and fluorescent microscopy for tracing of living cells: a) MTT assay The human endothelial cells line (EAhy923, American Type Culture Collection-ATCC, Manassas, VA, USA) was used to evaluate the biocompatibility of MTA. The cells were cultured in DMEM medium (Sigma-Aldrich, St. Louis, MO, USA) supplemented with 10% fetal bovine serum, 1% penicillin and 1% streptomycin antibiotics (Sigma-Aldrich). To maintain optimal culture conditions, medium was changed twice a week. The biocompatibility was assessed using a MTT assay (CellTiter 96® Non-Radioactive Cell Proliferation Assay, Promega, Fitchburg, WI, USA). This assay is a colorimetric method that allows quantitative assessment of proliferation, cell viability and cytotoxicity. The method is based on the reduction of yellow tetrazolium salt MTT (3-(4,5-dimetylthiazolyl)-2,5-diphenyltetrazolium bromide) to a dark blue formazan by the mitochondrial enzymes. Briefly, the human endothelial cells were grown in 96-well plates, with a seeding density of 3000 cells/well in the presence of MTA for 24–48 h. Then 15 mL Solution I was added and incubated at 37 ˝ C for 4 h. After that the Solution II was added and pipette vigorously to solubilise the formazan crystals. After 1 h the absorbance was read at 570 nm using a spectrophotometer (TECAN, Männedorf, Switzerland). b) Fluorescent microscopy for tracing of living cells A second method was additionally used for evaluation of the biocompatibility of MTA based on fluorescent microscopy using the RED CMTPX fluorophore (Life Technologies, Invitrogen, MA, USA, a cell tracker for long-term monitoring of living cells. The CMTPX was added after 7–28 days of cell culture in the presence of MTA for evaluating the viability and morphology of the endothelial. The CMTPX fluorophore was added in the culture medium at a final concentration of 5 µM, incubated for 30 min in order to allow the dye penetration into the cells. Next, the endothelial cells were washed with PBS and visualized by fluorescent microscopy. The photomicrographs were taken with a digital camera driven by Axio-Vision 4.6 software (Carl Zeiss, Oberkochen, Germany). The control cells were endothelial cells cultivated in the same medium, but without the MTA1 and MTA2 cements. 4. Conclusions The results presented in this paper show that MTA cements with short setting times can be obtained by a sol-gel synthesis route and an adequate thermal treatment. For the MTA cement obtained at a higher temperature e.g., 1450 ˝ C/3 h, the setting time was 15 min, an acceptable value for an endodontic bio-cement. The experimental results obtained by different analysis techniques (X ray diffraction, thermal analysis, scanning electronic microscopy and FT-IR spectroscopy) showed the presence of a high amount of hydrates (calcium silicate hydrates and calcium aluminate hydrates) in the hardened MTA cement pastes; the good compressive strengths assessed on hardened MTA cement pastes can be correlated with this high amount of hydrates.

Molecules 2016, 21, 233

14 of 15

The in vitro bioassays results demonstrate a high cell viability and good biocompatibility of MTA cements synthesized in this study. Acknowledgments: This research was financially supported by Sectoral Operational Programme Human Resources Development 2007–2013, financed by the European Social Fund and by the Romanian Government under the contract number POSDRU/156/1.2/G/135764 “Improvement and Implementation of University Master Programs in the Field of Applied Chemistry and Materials Science”—ChimMaster. Author Contributions: G.V. conceived the idea of this research; G.V., A.M.P. and A.I.B. designed the experiments and assessed the hydration and hardening processes and properties of MTA cements; F.I. performed the in vitro biocompatibility tests; G.V., A.I.B. and F.I. wrote the paper. Conflicts of Interest: The authors declare no conflict of interest.

References 1. 2.

3. 4. 5. 6.

7. 8. 9. 10. 11. 12. 13. 14. 15. 16. 17. 18. 19. 20.

Ribeiro da Silva, S.; Dias da Silva Neto, J.; Veiga, D.F.; Schnaider, T.B.; Ferreira, L.M. Portland cement vs. MTA as a root-end filling material. A pilot study 1. Acta Cir. Bras. 2015, 30, 160–164. [CrossRef] [PubMed] De-Deus, G.; Petruccelli, V.; Gurgel-Filho, E.; Coutinho-Filho, T. MTA vs. Portland cement as repair material for furcal perforations: A laboratory study using a polymicrobial leakage model. Int. Endod. J. 2006, 39, 293–298. [CrossRef] [PubMed] Roberts, H.W.; Toth, J.M.; Beryins, D.W.; Charlton, D.G. Mineral trioxide aggregate material use in endodontic treatment: A review of the literature. Dent. Mater. 2008, 24, 149–164. [CrossRef] [PubMed] Vasudev, S.K.; Goel, B.R.; Tyagi, S. Root end filling materials—A review. Endodontology 2003, 15, 12–18. Camilleri, J.; Ford, P.T.R. Mineral trioxide aggregate: A review of the constituents and biological properties of the material. Int. Endod. J. 2006, 39, 747–754. [CrossRef] [PubMed] Saidon, J.; He, J.; Zhu, Q.; Safavi, K.; Spångberg, L.S.W. Cell and tissue reactions to mineral trioxide aggregate and Portland cement. Oral Surg. Oral Med. Oral Pathol. Oral Radiol. Endod. 2003, 95, 483–489. [CrossRef] [PubMed] Casella, G.; Ferlito, S. The use of mineral trioxide aggregate in endodontics. Minerva Stomatol. 2006, 55, 123–143. [PubMed] Czarnecka, B.; Coleman, N.J.; Shaw, H.; Nicholson, J.W. The Use of Mineral Trioxide Aggregate in Endodontics—Status Report. Dent. Med. Probl. 2008, 45, 5–11. Viola, N.V.; Filho, M.T.; Cerri, P.S. MTA vs. Portland cement: Review of literature. South Braz. Dent. J. 2011, 8, 446–452. Gonçalves, J.L.; Viapiana, R.; Miranda, C.E.S.; Borges, A.H.; Cruz Filho, A.M. Evaluation of physico-chemical properties of Portland cements and MTA. Braz. Oral Res. 2010, 24, 277–283. [CrossRef] [PubMed] Steffen, R.; Van Waes, H. Understanding mineral trioxide aggregate/Portland-cement: A review of literature and background factors. Eur. Arch. Paediatr. Dent. 2009, 10, 93–97. [CrossRef] [PubMed] Voicu, G.; Ghi¸tulic˘a, C.D.; Dinu, E.; Andronescu, E. In-vitro behaviour of dicalcium silicate obtained through the sol-gel method. Rev. Romana Mater. 2011, 41, 229–233. Voicu, G.; Ghi¸tulic˘a, C.D.; Andronescu, E. Modified Pechini synthesis of tricalcium aluminate powder. Mater. Charact. 2012, 73, 89–95. [CrossRef] Voicu, G.; B˘ad˘anoiu, A.I.; Andronescu, E.; Chifiruc, C.M. Synthesis, characterization and bioevaluation of partially stabilized cements for medical applications. Cent. Eur. J. Chem. 2013, 11, 1657–1667. [CrossRef] Voicu, G.; B˘ad˘anoiu, A.I.; Andronescu, E.; Bleotu, C. Binding properties and biocompatibility of accelerated Portland cement for endodontic use. Rev. Chim. 2012, 63, 1031–1034. Lin, F.H.; Wang, W.H.; Lin, C.P. Transition element contained partial-stabilized cement (PSC) as a dental retrograde-filling material. Biomaterials 2003, 24, 219–233. [CrossRef] Taylor, H.F.W. Cement Chemistry; Academic Press: London, UK, 1997. B˘ad˘anoiu, A.; Paceagiu, J.; Voicu, G. Hydration and hardening processes of Portland cements obtained from clinkers mineralized with fluoride and oxides. J. Therm. Anal. Calorim. 2011, 103, 879–888. [CrossRef] Morrier, J.J.; Benay, G.; Hartmann, C.; Barsotti, O. Antimicrobial activity of Ca(OH)2 dental cements: An in vitro study. J. Endod. 2003, 29, 51–54. [CrossRef] [PubMed] Han, G.Y.; Park, S.H.; Yoon, T.C. Antimicrobial activity of Ca(OH)2 containing pastes with Enterococcus faecalis in vitro. J. Endod. 2001, 27, 328–332. [CrossRef] [PubMed]

Molecules 2016, 21, 233

21. 22.

23. 24. 25. 26.

27. 28. 29. 30.

31.

15 of 15

Mohammadi, Z.; Shalavi, S.; Yazdizadeh, M. Antimicrobial activity of calcium hydroxide in endodontics: A review. Chonnam Med. J. 2012, 48, 133–140. [CrossRef] [PubMed] Saghiri, M.A.; Garcia-Godoy, F.; Asatourian, A.; Lotfi, M.; Banava, S.; Khezri-Boukani, K. Effect of pH on compressive strength of some modification of mineral trioxide aggregate. Med. Oral Patol. Oral Cir. Bucal 2013, 18, e714–e720. [CrossRef] [PubMed] Campbell, D.H. Microscopical Examination and Interpretation of Portland Cement and Clinker, 2nd ed.; Portland Cement Association: Skokie, IL, USA, 1999. Ylmén, R.; Jäglid, U.; Steenari, B.M.; Panas, I. Early hydration and setting of Portland cement monitored by IR, SEM and Vicat techniques. Cem. Concr. Res. 2009, 39, 433–439. [CrossRef] Ylmén, R.; Wadsö, L.; Panas, I. Insights into early hydration of Portland limestone cement from infrared spectroscopy and isothermal calorimetry. Cem. Concr. Res. 2010, 40, 1541–1546. [CrossRef] Mollah, M.Y.A.; Kesmez, M.; Cocke, D.L. An X-ray diffraction (XRD) and Fourier transform infrared spectroscopic (FT-IR) investigation of the long-term effect on the solidification/stabilization (S/S) of arsenic(V) in Portland cement type-V. Sci. Total Environ. 2004, 325, 255–262. [CrossRef] [PubMed] Alves, N.M.; Leonor, I.B.; Azevedo, H.S.; Reis, R.L.; Mano, J.F. Designing biomaterials based on biomineralization of bone. J. Mater. Chem. 2010, 20, 2911–2921. [CrossRef] Caridade, S.G.; Merino, E.G.; Alves, N.M.; Mano, J.F. Bioactivity and viscoelastic characterization of chitosan/bioglass® composite membranes. Macromol. Biosci. 2012, 12, 1106–1113. [CrossRef] [PubMed] Voicu, G.; Jinga, S.I.; Tru¸sc˘a, R.; Iordache, F. Synthesis, characterization and bioevaluation of bioactive composites scaffolds based on collagen and glass ceramic. Dig. J. Nanomater. Biostr. 2014, 9, 99–108. Cornélio, A.L.; Rodrigues, E.M.; Salles, L.P.; Mestieri, L.B.; Faria, G.; Guerreiro-Tanomaru, J.M.; Tanomaru-Filho, M. Bioactivity of MTA Plus, Biodentine and experimental calcium silicate-based cements in human osteoblast-like cells. Int. Endod. J. 2016. [CrossRef] [PubMed] Rathinam, E.; Rajasekharan, S.; Chitturi, R.T.; Martens, L.; De Coster, P. Gene expression profiling and molecular signaling of dental pulp cells in response to tricalcium silicate cements: A systematic review. J. Endod. 2015, 41, 1805–1817. [CrossRef] [PubMed]

Sample Availability: Samples of the compounds presented in the manuscript are available from the authors. © 2016 by the authors; licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons by Attribution (CC-BY) license (http://creativecommons.org/licenses/by/4.0/).