Effect of Fluorohydroxyapatite on Biological and Physical Properties of MTA Angelus

Objectives This study aimed to assess the effect of addition of fluorohydroxyapatite (FHI) on biological and physical properties of mineral trioxide aggregate (MTA) Angelus. Materials and Methods In this in vitro, experimental study, nano-FHI powder was first synthesized, and the morphology and chemical structure of particles were evaluated by scanning electron microscopy (SEM), Fourier-transform infrared spectroscopy (FTIR), and X-ray diffraction (XRD). Three groups were evaluated in this study: MTA Angelus, MTA modified with 10% FHA, and MTA modified with 15% FHA. After mixing, the materials were applied to ring molds (10 mm diameter, 1 mm height), and the setting time of the three groups was evaluated according to ISO6876 and ASTMC266-03 with a Gillmore needle. The pH was measured using a pH meter at 24 and 48 hours and 7 days after mixing. The cytotoxicity of the materials was assessed in freshly mixed form and after 1 and 7 days using the methyl thiazolyl tetrazolium (MTT) assay according to ISO10993-5. Data were analyzed by one-way and repeated measures ANOVA and Tukey's test (alpha = 0.05). Results The addition of FHA to MTA significantly decreased the initial setting time (P < 0.05) and had no significant effect on cell viability (compared with pure MTA Angelus) at 1 and 7 days. However, modified MTA groups in freshly mixed form showed significantly lower cell viability (P < 0.05). The pH remained alkaline at all time points. Conclusion Addition of 15% FHA to MTA Angelus decreased its setting time with no adverse effect on cell viability (except for fresh form) or pH.


Introduction
Te ultimate goal of root canal therapy is to seal the communication between the root canal space and periapical tissue to prevent leakage of infective agents and microorganisms [1].Te success of endodontic treatment depends on several factors, particularly three-dimensional apical and coronal seals [2].Moreover, the materials used to create an optimal seal must be biocompatible, nontoxic, noncarcinogenic, and radiopaque, with optimal dimensional stability, antibacterial properties, and handling properties.Also, they must have favorable physical characteristics such as high compressive strength, bond strength to dentinal walls, and hardness [3].
Mineral trioxide aggregate (MTA) is currently one of the most commonly used dental materials.It is composed of tricalcium silicate, dicalcium silicate, tricalcium aluminate, tetracalcium aluminoferrite, calcium sulfate, and bismuth oxide [4].MTA was frst introduced for perforation repair [5] and as a root-end flling material [6].However, it currently has extensive applications for vital pulp therapy as a pulp capping agent [7], pulpotomy [8], root perforation repair [9], creation of an apical seal in nonvital, open-apex teeth [10], and regenerative endodontic treatments [11].Evidence shows that MTA has high biocompatibility [12] and low cytotoxicity [13] compared with other commonly used endodontic materials.It also elicits an optimal tissue response [14].Its application as a root-end flling material decreases periapical infammation and induces the formation of a new cementum layer in contact with the cement [15].MTA has high sealing ability [16], favorable marginal adaptation [17], and optimal antimicrobial activity [18].
Portland cement, which is a hydraulic cement, is the main constituent of MTA [5].Te hydration process of hydraulic cement is initiated after the contact of powder with water and leads to the formation of a porous gel.Te setting reaction of MTA takes several days.As the hydration process is accomplished, porosities decrease and resistance increases [19].
MTA has some shortcomings such as tooth discoloration, long setting time [20], and difcult handling due to its consistency after mixing the powder with water [21].High cost is another drawback of MTA, which limits its extensive application by dental clinicians [22].However, the manufacturer of MTA Angelus claims that it has a setting time of 15 minutes.
Attempts have been made to fx the drawbacks of MTA, and several materials were added to MTA powder to improve its physical, chemical, mechanical, and biological properties.Calcium chloride, K-Y gel, NaOCl gel, chlorhexidine gluconate, propylene glycol, aluminum fuoride, nanosilica, and methyl cellulose were added to MTA, and the physical, chemical, mechanical, and biological properties of the obtained product were studied [21,23,24].However, each material had a positive efect on one and a negative efect on another property of MTA, and thus, none of them appeared to be ideal for incorporation in the composition of MTA powder.
Hydroxyapatite (HA) is a calcium phosphate compound with a morphology and composition similar to human hard tissue.It has a hexagonal structure with chemical formula Ca 10 (PO 4 ) 6 (OH) 2 [25].HA is biocompatible, bioactive, osteoconductive, and nontoxic [26].It has been reported that addition of artifcially synthesized nano-HA and fuorapatite (FA) to glass ionomers improves their mechanical properties and bond strength to dentin.Another study showed that the addition of HA and fuorohydroxyapatite (FHA) to epoxy resin-based endodontic sealers increased their microhardness and bioactivity [27].Nano-FHA is a potential additive that can be used to improve the quality of dental materials [28].Shafaee et al. [29] added nano-FHA to Essix retainer to prevent the development of dental caries, and Wang et al. [30] demonstrated that the addition of nano-FHA to poly-ether ether ketone dental implants enhanced their biocompatibility, antimicrobial activity, and bone attachment.Another study used FHA to enhance the mechanical strength of a glass ionomer [28].Also, Lin et al. [31] indicated that the addition of nano-FA and nano-FHA to resin-modifed glass ionomer enhanced fuoride release with no adverse efect on bond strength.
To the best of the authors' knowledge, no previous study has assessed the efect of the addition of FHA to MTA.Tus, this study aimed to assess the efect of the addition of FHA on biological and physical properties of MTA Angelus.
Te null hypothesis of this study was that FHA could improve biological and physical properties of MTA Angelus.

Materials and Methods
Tis in vitro, experimental study evaluated the setting time, pH, and cytotoxicity of MTA Angelus (Angelus, Londrina, PR, Brazil) modifed with 10% and 15% FHA by weight.Te surface morphology and chemical composition of specimens were also evaluated by scanning electron microscopy (SEM), Fourier-transform infrared spectroscopy (FTIR), and X-ray difraction (XRD).
Te number of samples in each experiment was three.
2.1.Synthesis of FHA [32].Nano-FHA and MTA powders were weighed by a digital scale and divided into equal portions.Each portion of nano-FHA powder (10 wt% of the fnal powder in group 2 and 15 wt% in group 3) was manually mixed with MTA Angelus powder for 3 minutes.Subsequently, they were mixed in an amalgamator to achieve homogenous distribution of particles.
2.3.Cytotoxicity.Cytotoxicity of the three groups was evaluated according to ISO10993-5 in freshly mixed form and also at 1 and 7 days after setting.
Te powder and liquid were mixed according to the manufacturer's instructions in all groups, and the mixture was applied in molds measuring 5 mm in diameter and 2 mm in thickness to fabricate disc-shaped specimens for the three groups.At the respective time points, the specimens were removed from the incubator and also easily from the mold (since split molds were used).A serum-free culture medium was used for cytotoxicity testing.Te specimens were incubated in 650 µL of serum-free Dulbecco's modifed Eagle's medium at 37 °C, which yielded 1 mL/3 cm 2 2 Te Scientifc World Journal extraction ratio.Te specimens were then agitated at a rate of 100 rpm in an orbital shaker (Stuart Scientifc, Stone, UK).After 24 hours, the specimens were transferred to fresh culture medium and incubated for another 6 days.Accordingly, the cytotoxicity of the extract was evaluated at 1 and 7 days.Te cytotoxicity of the materials was evaluated by the methyl thiazolyl tetrazolium (MTT) assay.Fresh MTT was diluted in 0.5 mg/mL phosphate bufered saline.Te obtained solution was fltered by sterile syringe flters and used on the same day.
Dental pulp cells (291 w) were purchased from the Iranian Genetic Resources, seeded in 96-well plates, and incubated in culture medium for 24 hours to double in count and create a semiconfuent monolayer.Tey were then subjected to the test materials in 10% and 15% concentrations.After 24 hours, the treated cells were incubated with MTT solution (0.5 mg/mL in phosphate bufered saline) at 37 °C and 5% CO 2 with over 90% humidity.After 3-4 hours of incubation, 100 µL of dimethyl sulfoxide was added to dissolve the formed formazan crystals.Te optical density of the solution was read after 20 minutes at 570 nm wavelength with 630 nm reference wavelength using a multiwell plate reader and compared with control cultures to calculate the percentage of cell viability.All the experiments were performed in aseptic conditions under a laminar fow cabinet.Plain culture medium and distilled water were used as positive and negative controls, respectively.

Setting Time.
Te setting time of specimens in the three groups was measured according to ISO6876: 2012 and ASTM C266-03 under controlled temperature and moisture (37 ± 1 °C temperature and 95 ± 5% relative humidity).Te samples were mixed according to the manufacturer's instructions and applied in stainless steel ring molds with 10 mm diameter and 1 mm height.Excess material was removed to obtain a smooth surface.Prior to testing, all instruments and materials, glass slab, and spatula were incubated at 23 ± 1 °C temperature for one hour.Te mold was incubated at 37 ± 1 °C and 95% relative humidity for 24 hours.
To measure the initial setting time, a Gillmore needle with 100 ± 0.5 g weight and a cylindrical needle tip diameter of 2 mm with 5 mm length was used.According to the standards, 120 ± 10 seconds after completion of mixing in the mold, the mixture was incubated at 37 ± 1 °C with 95% relative humidity.Te Gillmore needle was gently pressed against the horizontal material surface for 5 seconds at 30second intervals according to ISO6876: 2012.Any indentation on the surface indicated that the material had yet to completely set.Tis process was repeated until no indentation was created on the surface.Tis time was recorded as the initial setting time.Te initial setting time was recorded for all three groups as explained above.
Te fnal setting time was evaluated using a Gillmore needle with 456 ± 0.5 g weight with a fat end with 1.0 ± 0.1 mm diameter.Te needle was gently pressed against the surface at 60-second intervals until a complete round-shaped indentation was no longer seen.Tis time was recorded as the fnal setting time.

Assessment of pH.
Te powder and liquid were mixed on a glass slab according to the manufacturer's instructions and applied in metal molds to create discs with 10 mm diameter.Tey were then incubated at 37 °C and 100% humidity.After setting, the cements were removed from the molds and placed in sealed plastic containers flled with 5 mL of distilled water and incubated at 37 °C and 100% humidity for 7 days.Te pH of the stored solution was measured using a pH meter (Metrohm AG, 781 pH/Ion Meter; Switzerland) at 24 and 48 hours and 7 days after mixing.Te pH of distilled water without cement was recorded as control.

XRD Analysis.
All synthesized powders underwent XRD analysis in an X-ray difractometer (X'Pert PRO MPD, Panalytical Company, Netherlands) at 40 kV voltage and 40 mA amperage using Cu-Ka (1.5406 Å).For qualitative analysis, XRD graphs were drawn in the range of 10 ≤ 2θ ≤ 70 with a scanning speed of 0.02 s −1 with 0.026 step size.

SEM.
Te specimens were gold sputter-coated (EMITECH K450X, England), and their surface microstructure was inspected under a scanning electron microscope (Sigma VP, ZEISS, Germany) at 10 kV.

Statistical Analysis.
Data were analyzed by one-way and repeated measures ANOVA.Pairwise comparisons were performed using Tukey's test at P < 0.05 level of signifcance. 1 presents the mean optical density of the three cements in freshly mixed form and 1 and 7 days after setting.As shown, the addition of FHA to MTA decreased cell viability in both groups at all time points except for day 1 in the 10% FHA group.According to one-way ANOVA, a signifcant diference was noted in cell viability of the three groups in fresh form (P � 0.00), but the diference was not signifcant at 1 or 7 days (P > 0.05).Pairwise comparisons of the cell viability of the three groups in fresh form were then performed using Tukey's test (Table 2).Te results showed that cell viability in the control and MTA groups was signifcantly higher than that in 10% FHA and 15% FHA groups (P < 0.05).No other signifcant diferences were noted (P > 0.05).Within-group comparison of cell viability over time revealed a similar trend in all groups.In all three groups, cell viability in fresh form was signifcantly lower than that after 1 and 7 days (P < 0.05), but the diference in cell viability between 1 and 7 days was not signifcant in any group (P > 0.05).

MTT Assay Results for Cytotoxicity. Table
Te Scientifc World Journal 3.2.Setting Time.Table 3 presents mean initial and fnal setting times in the three groups.One-way ANOVA revealed a signifcant diference in initial setting time among the three groups, such that the initial setting time in the 15% FHA group was signifcantly shorter than that in the 10% FHA group (P < 0.05).Also, the initial setting time of the 10% FHA group was signifcantly shorter than that of the MTA group (P < 0.05).
Te fnal setting time of the 15% FHA group was signifcantly shorter than that of 10% FHA and MTA groups (P < 0.05); the diference between 10% FHA and MTA was not signifcant (P > 0.05).4 presents the pH in the three groups at diferent time points.A signifcant diference was noted in pH among the three groups at each time point (P < 0.05).At 24 hours, the pH order was as follows: MTA > 10% FHA > 15% FHA.At 48 hours, the pH order was as follows: 10% FHA > MTA > 15% FHA.At 7 days, the following order was recorded: 15% FHA > MTA > 10% FHA.Te change in pH values at diferent time points within each group was not signifcant (P > 0.05).

XRD Results
. Figure 1 presents the XRD graph of the three tested cements.As shown, HA and FA were found in groups containing FHA. Tricalcium silicate, dicalcium silicate, tricalcium aluminate, and bismuth oxide were identifed in all groups.
XRD analysis of MTA Angelus showed a peak at 18 °2- theta for calcium hydroxide according to ICDD 01-076-0571.Considering the fact that bismuth oxide, tricalcium silicate, and tricalcium aluminate are present in the composition of MTA, the peaks related to these compounds were seen in all three groups as shown in Figure 1.Bismuth oxide and tricalcium silicate peaks were noted at 28 °.In groups 2 and 3 (FHA-modifed MTA), since no specifc pattern has been defned for FHA, assessment was performed according to FA (JCPDS card #15-0876) pattern.Te peaks at 26 °, 32 °, and 33 °belonged to the apatite compound.Such peaks were also noted in the MTA group due to its composition.However, in the apatite-containing groups, the apatite peaks merged with the peaks related to the MTA constituents, and thus, the intensity of the peaks and a shift to right were noted at 32 °and 33 °, indicating the presence of apatite in the compound.Te peak at 26 °confrmed the presence of apatite.No signifcant diference was noted between groups 2 and 3 due to diferent percentages of FHA. 2 presents the FTIR spectra of the three groups.As shown, in MTA Angelus, absorption bands were noted at 1410 cm −1 and 896 cm −1 related to the C-O group.Te band at 2900 cm −1 belonged to the -CH group, which becomes prominent in hydrated (set) materials [32].Also, a wide peak was seen at 3000-3600 cm −1 attributed to the water OH group present in the compound.Te bands at 420, 550, and 959 cm −1 were related to calcium silicate hydrate (C-S-H).A peak at 1410 cm −1 related to carbonate (CO 3 ) was also seen.

FTIR. Figure
In FHA groups, the peaks at 470, 870, and 1040 cm −1 were related to vibrations of ] 2 (P-O, v 1 (P − O) and v 3 (P − O).).Also, vibrations of v 2 (C − O 870 cm − 1 ) and v 3 (C − O 1419 − 1460 cm − 1 ) were seen in FHA groups.In FHA, the apatite structure was noted at 560-610 and 950-1100 cm −1 .Te peak at 1040 cm −1 was related to the phosphate bond in the structure of apatite, which was seen in both FHA groups.Te peaks at 1040 cm −1 and with a slight shift at 870 cm −1 in the 15% FHA group were longer and more distinct than those in the 10% FHA group.A new band (OH-F or OH-F-HO) was noted at 3540 cm −1 in FHA groups.3 presents the SEM micrographs of the completely set specimens in the three groups.SEM micrographs of all three groups showed apatite crystals in the form of plates with sharp margins along with spherical agglomerated particles.It appears that the addition of FHA slightly altered the morphology of crystals and increased their aspect ratio.Te addition of FHA increased the accumulation of plate-like particles, especially in the 15% FHA group.

Discussion
Tis study assessed the efect of the addition of 10% and 15% FHA on biological and physical properties of MTA Angelus.Te 10% and 15% values by weight for the addition of FHA were selected according to a previous study that showed lower concentrations did not add any advantage to MTA and higher concentrations complicated the manipulation and  [25].Te present results revealed that 15% FHA had the shortest initial and fnal setting time.Also, the initial setting time of the 10% FHA group was shorter than that of MTA, but its fnal setting time was comparable to that of MTA with no signifcant diference.Tis fnding was in line with the results of Antonijevic et al. [33] who showed that the addition of nano-HA to calcium silicate-based cement signifcantly decreased the initial setting time.However, Guerreiro-Tanomaru et al. [34] found that the addition of nano-HA to calcium silicate-based cement had no signifcant efect on initial setting time but decreased the fnal setting time.Eskandarinezhad et al. [35] used HA to shorten the setting time of MTA.Both HA and FHA nanoparticles are highly active due to their nanostructure and form bonds faster; thus, they shorten the setting time of MTA [35].
Te present results revealed that the pH of MTA Angelus at 24 hours was higher than that of the other groups.At 48 hours, the 10% FHA group showed a higher pH, while at 7 days, the pH of the 15% FHA group was higher.Although a signifcant diference existed in the pH of the three groups at each time point, the diference was not clinically important, and the pH values of all groups were in the alkaline range at diferent time points.Antonijevic et al. [33] and Guerreiro-Tanomaru et al. [34] reported that addition of nano-HA to calcium silicate-based cement did not change pH.Similar to the present study, all values were within the alkaline range.Although they added nano-HA instead of FHA, the alkaline pH of cements may biologically induce mineralization [36].Alkaline pH can cause infammatory reactions and formation of hydroxyapatite [37] and reparative dentin [38].Release of calcium ions from the MTA cements and increased alkalization of the surrounding environment are related to their ability to produce calcium hydroxide, which reacts with water and releases calcium and hydroxyl ions.Te released hydroxyl ions are responsible for the pH rise in the surrounding environment [39].
Te present results indicated that the addition of FHA to MTA did not change cell viability at 1 and 7 days, compared with MTA Angelus.However, in the fresh state, cell viability of FHA groups was lower than that of MTA Angelus.Tis result was in agreement with the fndings of Liu et al. [40] who showed that the addition of FHA adversely afected the cell viability.Tahriri and Moztarzadeh [41] and Montazeri and Shokrgozar [42] demonstrated that the addition of FHA did not increase cytotoxicity, which was diferent from the current results obtained in fresh form of cements.Since all studies used the MTT assay for cytotoxicity testing, variations in the results may be due to the use of diferent cell types.For instance, Liu et al. [40] used osteoclasts and Tahriri and Moztarzadeh [41] used G-292 cells and osteoblasts, while we used 291 w dental pulp cells, which are a type of mesenchymal stem cells.Montazeri and Shokrgozar [42] used osteoblasts and mesenchymal stem cells and showed that the presence of FHA increased the proliferation of osteoblasts but had no signifcant efect on mesenchymal  Te Scientifc World Journal stem cells.Te presence of fuoride the composition of FHA increases the release of alkaline phosphatase in osteoblasts; however, this is not the case for mesenchymal stem cells.Also, it appears that the fuoride ions in the structure of FHA afect the release of calcium and phosphate ions and the subsequent biological response.Tus, the selection of the most appropriate concentration for an optimal biological response is highly important [42].
In the present study, the XRD analysis showed that the XRD pattern of FHA was somewhere between that of HA and FA.Due to high similarity in the crystalline structure and the same size of network, all peaks of FA and HA could not be precisely diferentiated.Widening of the calcium hydroxide peak indicated a reduction of the crystalline phase and its conversion to an amorphous phase, which cannot be identifed by XRD [23].
Rocha et al. [43] disclosed the FTIR spectra of MTA Angelus and showed absorption bands at 660, 896, and 1410 cm −1 related to C-O.In the present study, absorption bands were noted at 896 and 1410 cm −1 for MTA Angelus.Abu Zeid et al. [44] showed bands at 2800-2900 cm −1 related to the -CH group, which were found at 2900 cm −1 for MTA Angelus in the present study.Te formation of CO 3 after setting of material results in the formation of peaks at 1440 and 1472 cm −1 , which were seen at 1410 cm −1 for MTA Angelus in the present study.Bands at 449, 524, and 996 cm −1 are related to calcium silicate hydrate in MTA Angelus, which were seen at 420, 550, and 959 cm −1 for the MTA Angelus used in the present study.An extensive peak at 3000-3600 cm −1 was attributed to the water OH group in completely set materials in the three groups, indicating the formation of hydrated phases (such as calcium hydroxide and calcium silicate hydrate) as reaction products.Even in case of presence of free calcium hydroxide as a reaction product, some additives can react with it and decrease its content.In the present study, the absence of calcium hydroxide in set MTA may indicate its reaction with other groups.
In FTIR of FHA groups, phosphate and carbonate functional groups were observed.Te presence of carbonate in all three study groups may be due to the absorption of carbon dioxide from the atmosphere, and the phosphate band was related to the apatite structure with a longer and more distinct peak in the 15% FHA group [45].A peak at 3540 cm −1 related to hydroxyl groups was noted as well.Te presence of hydroxyl groups was due to the hydrogen bond of fuorine ions.Tis peak confrmed the crystalline structure of FA with the hydroxyl group.Tese observations are due to the formation of hydrogen bonds between F and OH, which indicates that F ions have replaced OH ions in the structure of apatite.Nonetheless, the exact amount of F ions replaced with OH ions cannot be determined by FTIR [46].6 Te Scientifc World Journal microstructure of FHA is plate-like with diferent aspect ratios.Layered patterns have been seen in FHA specimens.Researchers claim that binding of F-ions afects the morphology of apatite crystals [47,48].In this study, the replacement of fuoride in the crystalline structure of apatite changed the morphology of crystals and increased their aspect ratio.Sharp margins of FHA crystals indicate their highly crystalline nature.
It has been reported that fuoride can cause epitaxial growth of crystals on the precursor of octa-calcium phosphate and change the morphology of apatite crystals as such [49].Fan et al. [50] reported that fuoride has a dosedependent efect on the morphology of calcium phosphate crystals [50].
Future studies are required on other percentages of FHA and their efects on other physical, mechanical, and biological properties of MTA.Further studies on the ionic and crystalline forms of the constituents of these cements are also recommended.In vivo studies are required to better simthe clinical setting.

Conclusion
Addition of 15% FHA to MTA Angelus decreased its setting time with no adverse efect on cell viability (except for fresh form) or pH.

Figure 2 :Figure 3 :
Figure 2: FTIR spectra of the three groups.

Table 2 :
Pairwise comparisons of cell viability of the three groups by Tukey's test.

Table 1 :
Mean optical density (indicative of cell viability) in the three cement groups in freshly mixed form and at 1 and 7 days after setting (n � 3).

Table 3 :
Mean initial and fnal setting time (in minutes) in the three groups (n � 3).

Table 4 :
pH in the three groups at diferent time points.