Mineral Trioxide Aggregate–based Endodontic Sealer Stimulates Hydroxyapatite Nucleation in Human Osteoblast-like Cell Culture

Mineral Trioxide Aggregate–based Endodontic Sealer Stimulates Hydroxyapatite Nucleation in Human Osteoblast-like Cell Culture

Basic Research—Technology Mineral Trioxide Aggregate–based Endodontic Sealer Stimulates Hydroxyapatite Nucleation in Human Osteoblast-like Cell Cultu...

1MB Sizes 6 Downloads 24 Views

Basic Research—Technology

Mineral Trioxide Aggregate–based Endodontic Sealer Stimulates Hydroxyapatite Nucleation in Human Osteoblast-like Cell Culture Loise Pedrosa Salles, MSc,*† Ana Lıvia Gomes-Cornelio, MSc,* Felipe Coutinho Guimar~ aes, MSc,† Bruno Schneider Herrera, PhD,‡ Sonia Nair Bao, PhD,† Carlos Rossa-Junior, PhD,‡ Juliane Maria Guerreiro-Tanomaru, PhD,* and Mario Tanomaru-Filho, PhD* Abstract Introduction: The main purpose of this study was to evaluate the biocompatibility and bioactivity of a new mineral trioxide aggregate (MTA)-based endodontic sealer, MTA Fillapex (MTA-F; Angelus, Londrina, Brazil), in human cell culture. Methods: Human osteoblast-like cells (Saos-2) were exposed for 1, 2, 3, and 7 days to MTA-F, Epiphany SE (EP-SE; SybronEndo, Orange, CA), and zinc oxide–eugenol sealer (ZOE). Unexposed cultures were the control group (CT). The viability of the cells was assessed by MTT assay and the morphology by scanning electron microscopy (SEM). The bioactivity of MTA-F was evaluated by alkaline phosphatase activity (ALP) and the detection of calcium deposits in the culture with alizarin red stain (ARS). Energy-dispersive X-ray spectroscopy (EDS) was used to chemically characterize the hydroxyapatite crystallites (HAP). Saos-2 cells were cultured for 21 days for ARS and SEM/EDS. ARS results were expressed as the number of stained nodules per area. Statistical analysis was performed with analysis of variance and Bonferroni tests (P < .01). Results: MTA-F exposure for 1, 2, and 3 days resulted in increased cytotoxicity. In contrast, viability increased after 7 days of exposure to MTA-F. Exposure to EP-SE and ZOE was cytotoxic at all time points. At day 7, ALP activity increase was significant in the MTA-F group. MTA-F presented the highest percentage of ARS-stained nodules (MTA-F > CT > EP-SE > ZOE). SEM/EDS analysis showed hydroxyapatite crystals only in the MTA-F and CT groups. In the MTA-F group, crystallite morphology and chemical composition were different from CT. Conclusions: After setting, the cytotoxicity of MTA-F decreases and the sealer presents suitable bioactivity to stimulate HAP crystal nucleation. (J Endod 2012;38:971–976)

Key Words Bioactivity, biocompatibility, hydroxyapatite, mineral trioxide aggregate sealer


ineral trioxide aggregate (MTA) emerged as the material of choice for root perforation repairs and root-end fillings in the 90s, a revolutionary period marked by many advances in endodontics (1). MTA was developed at Loma Linda University and received approval from the Food and Drug Administration for human use in 1998 (2, 3). Since then, MTA has shown excellent biological properties in several in vivo and in vitro studies (4–9). In cell culture systems, for example, MTA has been shown to enhance proliferation of periodontal ligament fibroblasts (6), to induce differentiation of osteoblasts (7, 8), and to stimulate mineralization of dental pulp cells (9). This biocompatibility and bioactive potential raised the interest of scientists worldwide to improve the handling characteristics and some physicochemical properties of MTA with the intention of expanding its applicability in endodontics. Consequently, new MTA-based root-end filling cements and root canal sealers have been proposed (10–12), such as MTA Fillapex (MTA-F; Angelus, Londrina, Brazil). The new MTA-based sealers reflect a current requirement to have materials for endodontic therapy that are able to stimulate the healing process of periapical tissues, instead of merely biocompatible or inert materials. As a result, MTA-F represents the effort in combining a material of excellent biological properties as MTA with resins and other components to improve diverse required properties of an endodontic sealer including adhesiveness, dimensional stability, working time, radiopacity, flow, and antibacterial effects. According to the manufacturer’s information, MTA-F is composed of salicylate resin, resin diluent, natural resin, bismuth oxide as radiopacifying agent, silica nanoparticles, MTA, and pigments. The MTA itself consists of fine hydrophilic particles of tricalcium silicate, tricalcium aluminum oxide, tricalcium oxide, gypsum (calcium sulfate dihydrate), and other mineral oxides (3). Gypsum is an important determinant of setting time. MTA cements generally contain less gypsum to allow more handling time. Unfortunately, MTA-F data sheet lacks details about the natural resin, pigments, and diluents composition. It is important to investigate if the combination of these resins and other constituents influence the bioactive potential of MTA in the new endodontic sealer. Therefore, the main purpose of this study was to evaluate the biocompatibility and the bioactivity of MTA-F in stimulating mineralization in Saos-2 cell culture compared with Epiphany SE

From the *Department of Restorative Dentistry, Dental School of S~ao Paulo State University, Araraquara, S~ao Paulo, Brazil; †Cellular Biology Department, Institute of Biological Sciences, University of Brasılia, Distrito Federal, Brazil; and ‡Department of Diagnosis and Surgery, Araraquara Dental School, S~ao Paulo State University, Araraquara, S~ao Paulo, Brazil. To CNPQ (Brazil) and CAPES (Brazil) for the fellowship grants (to L.P.S. and A.L.G.-C.). To CNPq, FAPDF (Brazil), FINEP (Brazil), and FAPESP (Brazil, grant 2010/10769-1) for supporting this study. Address requests for reprints to Dr Mario Tanomaru-Filho, Rua Humaita, 1680, Caixa Postal 331, Centro, 14801-903 Araraquara, SP, Brazil. E-mail address: [email protected] 0099-2399/$ - see front matter Copyright ª 2012 American Association of Endodontists. doi:10.1016/j.joen.2012.02.018

JOE — Volume 38, Number 7, July 2012

MTA-based Endodontic Sealer Stimulates Hydroxyapatite Nucleation


Basic Research—Technology (EP-SE; SybronEndo, Orange, CA) and zinc oxide–eugenol endodontic sealers. Saos-2 is a human osteoblast-like cell that provides a suitable model for studying late events of osteoblast differentiation (13). EP-SE is a self-etch, dual-cure, resin-based root canal filling system (14). Zinc oxide–eugenol (ZOE) is a commonly used endodontic sealer known to present cytotoxicity (15).

Materials and Methods Preparation of the Endodontic Sealers MTA-F, EP-SE, and EndoFill (prepared according to the manufacturer’s recommendations; Petropolis, RJ, Brazil) were inserted into sterile polythene molds measuring 6 mm in diameter and 2 mm in thickness (microcentrifuge tube caps). After 1 day of initial set at 37 C, 95% humidity, and 5% CO2, the sealer pellets were removed from the molds and placed on the bottom of transwell permeable supports (0.4-mm membranes; Corning, Union City, CA) on 12-well culture plates with culture medium for 1, 2, 3, and 7 days of cell exposure. Before performing the mineralization assays (alizarin red staining and scanning electron microscopy [SEM]), sealer samples were set for 7 days in the same conditions described previously before cell exposure. The 7 days setting time for mineralization assays was established in accordance with MTT results. Cell Culture Human osteoblast cells (Saos-2 line ATCC HTB-85) were grown as a monolayer culture in T-75 flasks (Corning, Union City, CA) containing Dulbecco modified Eagle medium (DMEM) supplemented with 10% fetal bovine serum (FBS), penicillin (100 IU/mL), and streptomycin (100 mg/mL) until confluent. The cells were subcultured twice a week at 37 C, 95% humidity, 5% CO2 (all culture supplies from Gibco-BRL, Gaithersburg, MD). Adherent cells in logarithmic growth phase were detached by a mixture of trypsin/EDTA (0.25%) at 37 C for 2 minutes. The collected cells were seeded on 12-well plates (Corning) at a density of 2  105 per well determined by hemocytometry. Then, cells were incubated in the same conditions described earlier for 24 hours to obtain exponential cell growth before exposure to the endodontic sealers. Unexposed cells were the positive control. The culture media was renewed every 3 days. To investigate the effect of endodontic sealers on the Saos-2 phenotype and analyze formation of mineralized nodules by scanning electron microscopy and energy dispersive X-ray spectroscopy (EDS), the cells were seeded over glass slides on 12-well plates (n = 3 slide samples/group for culture in osteogenic or nonosteogenic medium). After 1 day in culture, the cells were exposed to the endodontic sealers deposited on the transwell inserts for 21 days in osteogenic medium (DMEM, 10% FBS, 100 IU/mL penicillin, 100 mg/mL streptomycin, 0.0023 g/mL b-glycerophosphate, and 0.055 mg/mL L-ascorbate; Sigma Chemicals, St Louis, MO) or nonosteogenic medium (DMEM, 10% FBS, 100 IU/mL penicillin, and 100 mg/mL streptomycin). During the assays, the culture medium was renewed every 24 hours for 1 week and later on every 2 days. Cell Viability Cell proliferation was determined by MTT assay. This assay is based on the ability of the mitochondrial enzyme to convert the yellow water-soluble tetrazolium salt, 3-(4,5-dimethyl-thiazoyl)-2,5-diphenyltetrazolium bromide (MTT, Sigma Chemicals) into purple-colored formazan compounds. The absorbance measured is proportional to the amount of viable cells. After 0, 1, 2, 3, and 7 days of cell exposure to endodontic sealers (or nonexposure in the control group), the transwells with dental material samples were removed, and the culture medium was changed to DMEM containing 0.55 mg/mL MTT. The plates 972

Salles et al.

were incubated for an additional 4 hours in the same conditions described previously. Thereafter, each well was washed with 1 mL of phosphate-buffered saline (PBS 1x) and 500 mL acid-isopropyl alcohol (isopropyl alcohol, 0.04 N) were added to extract and solubilize the formazan. Aliquots of 150 mL of the formazan solution from each sample were transferred to a 96-well plate (Corning) and the optical density (OD = 570 nm) was measured using an automated microplate reader (ELx800; BioTek Instruments, Winooski, VT). Duplicate samples were prepared for each test and control groups at the different exposure times. The experiment was repeated three independent times (n = 6/group). Data were then exported to Excel spreadsheets (Office 2007; Microsoft Corporation, Redmond, WA) and subjected to statistical analysis.

Alkaline Phosphatase Activity Alkaline phosphatase (ALP) activity was determined using a commercial kit (Labtest; Lagoa Santa, MG, Brazil). Briefly, ALP hydrolyzes thymolphthalein monophosphate, releasing thymolphthalein in alkaline medium. After 1, 2, 3, and 7 days of exposure to the sealers, the attached Saos-2 cells were rinsed with PBS 1x and immersed in 1 mL sodium lauryl sulfate (1 mg/mL, Sigma Chemicals) for 30 minutes at room temperature. Aliquots of each sample solution (50 mL) were added to the kit contents according to the manufacturer’s instructions. Absorbance was spectrophotometrically measured at 590 nm, and ALP activity was calculated as mmol of thymolphthalein/min/L. Duplicate samples were prepared for each test and control group at the different exposure times. The experiment was repeated three times (n = 6/ group). Data were expressed as ALP activity normalized by the number of viable cells at the respective culture period (OD = 570 nm) (16). Mineralization and Alizarin Red Staining After 21 days of cell exposure to endodontic sealers, adherent Saos-2 was washed three times with PBS 1 and fixed in 10% (v/v) formaldehyde (Sigma) at room temperature for 15 minutes. The monolayers were then washed twice with dH2O before the addition of 1 mL ARS (2%, pH = 4.1) per well. The plates were incubated again at room temperature for 20 minutes. Then, the wells were washed five times with 2 mL dH2O. Stained monolayers were observed using an inverted microscope (Axiovert 100, Carl Zeiss, Jena, Germany) with 40 magnification. The wells were photographed (Canon EOS-1D, Canon Inc, Tokyo, Japan), and the digital images were processed using ImageJ 1.45 software (National Institutes of Health, Bethesda, MD). Two examiners individually counted the stained nodules in each macroscopic image (n = 6/group, including control). Morphological Analysis of Mineralized Nodules on Saos-2 by SEM and EDS After exposure to the endodontic sealers for 21 days in osteogenic or nonosteogenic medium, the cell slides were washed three times in PBS 1x and fixed in 2.5% glutaraldehyde for 2 hours. Specimens were then dehydrated in ethanol series (30%, 50%, 70%, 90%, and 100%) for 20 minutes at each concentration and dried in a criticalpoint dryer (LADD 28000; LADD, Williston, VT). Dried specimens were mounted on stubs, sputter coated with gold, and observed by SEM for morphological characterization of the Saos-2 cells (n = 3 slide samples/group). The crystallites on cells exposed or not (control) to endodontic sealers were screened by EDS for the presence of chemical elements (n = 30 nodules/group). The system was operated at an accelerating voltage of 15 kV (SEM-EDS JSM-7001F; JEOL, Tokyo, Japan). JOE — Volume 38, Number 7, July 2012

Basic Research—Technology

Figure 1. (A) Saos-2 viability and (B) ALP activity after cells exposure to different sealers: MTA-F, EP-SE, and ZOE. Unexposed cells were the control group. Mean  SEM (n = 6/group). *A significant difference between endodontic sealer treatment and control group. #A significant difference comparing to other groups of treatment and control. Analysis of variance, Bonferroni (P < .01).

Statistical Analysis The MTT results, ALP activity data, alizarin-stained nodules, and atom percentage of chemical elements (EDS of mineralized nodules) were evaluated by one-way analysis of variance. The mean differences between all cell treatment groups were compared by Bonferroni post hoc test and were considered to be significant at P < .01.

Results Cell Viability The MTT assay showed significant decrease in cell viability for all endodontic sealers after 1, 2, and 3 days of cell exposure when compared with the control group. At day 7, Saos-2 exposed to MTA-F revealed a recovery from cytotoxicity (Fig. 1A). ZOE and EP-SE presented cytotoxic effects at all time points. The groups of both dental materials showed a significant decline in cell viability. Exposure to EP-SE significantly reduced cell viability in a time-dependent manner. ALP Activity Saos-2 treated with all test materials had significantly lower metabolic activity and reduced levels of ALP activity compared with the control group after 1 and 2 days of exposure (Fig. 1B). After 7 days of exposure, only the MTA-F group showed enhancement of ALP activity (about two-fold compared with the control, P < .01). Mineralization and ARS After 21 days of cell exposure, only MTA-F had a significant stimulatory effect on the formation of a larger number of mineralized nodules than the control group. Moreover, the nodules in the MTA-F group appeared intensely stained in the microscopic analysis (Fig. 2A). Statistical analysis of ARS data showed significant differences among the sealers (Fig. 2B). ARS-stained nodules were observed in the EP-SE cell culture although in a significantly lower quantity than in the JOE — Volume 38, Number 7, July 2012

Figure 2. (A) Micrographs of calcium nodules on Saos-2 culture stained with ARS (2%). CT, control group; MTA-F, Saos-2 exposed to MTA Fillapex; EP-SE, Saos-2 exposed to Epiphany SE and ZOE, Saos-2 exposed to zinc eugenol endodontic sealer. The stain of MTA-F mineralized nodules was the most intense (bar = 100 mm). (B) Quantitative analysis of mineralized nodules after 21 days in culture. Data reported as mean  SEM (n = 6/group); different letter represents significant difference between groups. Analysis of variance, Bonferroni post test (P < .01).

MTA-F and control groups. Sparse mineralized nodules were observed in ZOE-treated cells at all time points (MTA-F > CT > EP-SE > ZOE).

Morphological Analysis of Saos-2 and Mineralized Nodules by SEM In EP-SE and ZOE groups, no difference was observed in cell morphology between cultures in nonosteogenic (Fig. 3A and B, respectively) or osteogenic medium (Fig. 3E and F). However, Saos-2 exposed to EP-SE and ZOE displayed morphology different from that of the control, and no mineralized nodules were observed at all. Briefly, we observed smaller cells with a loss of plasma membrane integrity, which suggested that cell necrosis was taking place (Fig. 3A, B, E, and F). In the MTA-F/nonosteogenic medium group, we observed a gel-like layer with nanoparticle precipitate (Fig. 3D). SEM cell morphology analysis showed plasmatic membrane integrity and adhesion to glass slides after MTA-based Endodontic Sealer Stimulates Hydroxyapatite Nucleation


Basic Research—Technology

Figure 3. Scanning electron micrographs of Saos-2 slides in (A-D) nonosteogenic medium and (E-H) osteogenic medium. (A and E) Saos-2 with disruption of plasma membrane after exposure to EP-SE. (B and F) Dead Saos-2 of ZOE group. (C and G) Saos-2 and HAP crystallites of the control group. (D and H) Micrographs of the gel-like layer and Saos-2 membrane with crystallites after exposure to MTA-F, respectively. (I) SEM of a MTA-F pellet sample surface, which was incubated in osteogenic medium at the same conditions of the cells for comparative purpose (bar = 1 mm [20,000]).

21 days of MTA-F treatment in osteogenic media (Fig. 3H) although we observed a lower number of cells in the MTA-F groups when compared with control cells. SEM analysis of MTA-F group confirmed the presence of numerous hydroxyapatite-like crystals (0.2-0.8 mm) deposited over the slide and attached to the cell membranes of Saos-2 in osteogenic medium, both unexposed and exposed to MTA-F (Fig. 3G and H, respectively). However, the crystals in MTA-F group had different morphological features from those observed in the control group. Mineralized nodules in the MTA-F group showed an irregularly smooth surface formed by compact spherical nanoparticles, whereas the nodules in the control cultures showed a rough surface formed by an aggregate of rod-shaped nanoparticles.

EDS Analysis of Mineralized Nodules EDS analysis of crystallites in the control and MTA-F groups of Saos-2 cells cultured in osteogenic media confirmed hydroxyapatite (HAP) formation and differences in chemical element composition (Fig. 4A and B). EDS on HAP crystallites from the MTA-F/osteogenic medium group displayed prominent peaks of Ca, P, O, and Si. Traces of K, Z, N, Na, and Mg were also detected. Cells exposed to MTA-F showed 974

Salles et al.

nanocrystallite deposits even in nonosteogenic media. However, the main minerals in this group were Ca and Si (Fig. 4C); no P was detected. Only EDS of the MTA-F pellet sample displayed a peak of Bi (Fig. 4D). The statistical analysis of the chemical elements showed significant differences in the percentages of Si, P, and Ca atoms in the MTA-F group crystals compared with the control (Fig. 5). The Ca/P atomic ratios of the control and MTA-F HAP crystallites in osteogenic medium were 2.01 and 1.5, respectively. The Si/Ca ratios were 0.18, 1.18, and 2.43 for the control, MTA-F/Saos-2 HAP crystals in osteogenic medium, and MTAF/Saos-2 nano-crystallites in nonosteogenic medium, respectively.

Discussion New MTA-based sealers and cements have been the focus of many studies in the endodontic field since the early investigations of Torabinejad et al (1, 2). The strong interest in developing MTA-based endodontic materials is because of the excellent biocompatibility, bioactivity, and osteoconductivity of MTA (17). In this study, we evaluated the bioactivity of a novel endodontic sealer, MTA Fillapex, to induce mineralization in human Saos-2 cell culture system. Saos-2 cell line has well-documented characterization, especially with regards to its high JOE — Volume 38, Number 7, July 2012

Basic Research—Technology

Figure 4. EDS of hydroxyapatite crystallites from Saos-2 culture in osteogenic media (A) unexposed and (B) exposed to MTA-F displaying high peaks of Ca, P, and O. (C) EDS of nanocrystallites from the MTA-F group in nonosteogenic media. (D) EDS of a MTA-F pellet sample for comparative purpose.

expression of bone ALP and its ability to deposit mineralizationcompetent extracellular matrix (13). The MTT results revealed that all materials, MTA-F, EP-SE, and ZOE, exhibited a cytotoxic effect. The cytotoxicity of MTA-F was likely related to the presence of resin in this material or the release of arsenic, a heavy metal that may be found as a contaminant in MTA (18). Arsenic reacts with protein thiols, and exposure to high concentrations of this element may induce genotoxicity. After 7 days of exposure to MTA-F, the cell culture showed evident recovery of viability. At this time point, the MTA-F sample was totally set. This suggests that the long setting time of MTA-F may play a role in its cytotoxicity because of the leakage of toxic compounds for a long drawn-out period. Interestingly, MTA-F maintained antibacterial activity during 7 days after mixture in a different study (19). Later, however, no antibacterial effect was detected. The antibacterial effect of MTA-F was related to its resin component and to the pH range of 10.14 to 10.5 that MTA-F promoted in suspension. The cytotoxicity observed for EP-SE and ZOE in the present study has been described (14, 20, 21). Two possible reasons for the high cytotoxicity of EP-SE include residual unreacted monomers such as

Figure 5. Atoms percentage in crystallites; * and # represent significant difference in P, Ca, and Si content between crystals of the different groups. Analysis of variance, Bonferroni post test (P < .01).

JOE — Volume 38, Number 7, July 2012

2-hydroxyethyl methacrylate (HEMA) release from the dual-cure composite and the filler particles leaching out (21). ZOE cements are known to cause inflammation and bone resorption in vivo. Furthermore, eugenol was shown to activate nuclear factor kappa B and to induce cyclooxygenase-2 expression, vacuolization, and toxicity in human osteosarcoma cells in vitro (15). ALP is a recognized marker of osteoblast differentiation and an essential enzyme in hydroxyapatite nucleation process. The initial cytotoxic effect of MTA-F possibly caused a decrease in ALP activity at the first days of exposure to the dental material. However, Saos-2 showed significant increase of ALP after 7 days of exposure to MTA-F, which supports the assumption of biocompatibility and bioactivity recovery once MTA-F is completely set. ALP activity in this study was similar to that of Saos-2 when exposed to MTA-based root-end filling materials after 3 days of treatment (22). The increase of ALP activity was consistent with the significantly high number of ARS-stained nodules observed in Saos-2 culture that had been exposed to MTA-F. In addition, the stain of mineralized nodules of MTA-F group appeared more intense under light microscopy. These findings suggested that MTA-F had the potential to induce formation of mineralized nodules. Furthermore, this possibility led to the characterization of mineralized nodules by SEM/EDS. Surprisingly, most of the mineralized nodules observed in SEM of the MTA- F group had morphology different from mineralized nodules in the control group. The diverse conditions of hydroxyapatite nucleation can explain this difference in nanocrystallites morphology. Nucleation is the first step in biomineralization and requires that a free energy barrier be overcome, which results in size and pattern formation of mineral crystallites through the mediating role of many organic and inorganic substrates (23). Super-saturation, foreign ions, cell membrane, and biomolecules, such as bone matrix proteins, may act as regulating agents in controlling the pattern of HAP crystallite assembly. In the control group, the confluence of Saos-2 associated with the massive presence of matrix biomolecules provided the template and site for a heterogeneous nucleation process. The presence of

MTA-based Endodontic Sealer Stimulates Hydroxyapatite Nucleation


Basic Research—Technology biomolecules lowers the energy barrier against nucleation and improves the structural match between crystalline phase and substrate, promoting structural synergy of the HAP crystals. On the other hand, it is possible that MTA-F promoted an environment highly saturated with foreign ions, a lower number of cells, and a Ca-Si hydrogel nucleation site. It was shown that relatively high super-saturation gives rise to a compact and random assembly of HAP crystallites (23). In addition, calcium silicate hydrate is the main byproduct of the reaction of MTA with calcium silicate cements (24). Therefore, the SEM characterization of the different morphology of HAP crystals in the MTA-F group, together with EDS analysis of the crystallites, showed that Ca and Si leached from the dental material participated in the nucleation process of HAP crystals, probably through formation of Ca-Si hydrogel nucleation sites. Previous studies have shown that Ca and Si ions readily leach out from MTA and calcium silicate cements in solution (24). Bioglass and tricalcium silicate were shown to release silicon to a level of 50 to 100 mg/L in cell culture medium (25). The strong peaks for Ca, P, and Si in EDS analysis of crystallites in the control and MTA-F groups are indicative of calcium phosphate deposition. The Ca/P ratio of 1.5 to 2.0 observed in the elemental analysis of crystallites from the MTA-F and control groups, respectively, is consistent with the apatite maturation stage in which carbonate ions may replace phosphate ions (type B carbonate apatite, type B CAp) or hydroxyl ions (type A carbonate apatite, type A CAp) in the apatite structure (26). Significant differences in the Si/Ca atomic ratios were observed between the control, MTA-F/osteogenic medium, and MTAF/nonosteogenic medium groups. Two distinct possibilities could explain the high Si/Ca ratio in the crystallites from the MTA-F/ osteogenic group: one is the occurrence of silicate coprecipitation with apatite. Metasilicate is soluble in an aqueous environment in which it reacts with calcium-forming polymerized gel-like insoluble silicate (25). The other possibility is the substitution of silicon for phosphorus in the apatite lattice of some crystallites (27) although there was no statistical difference in P content between crystallites of the control and MTA-F/osteogenic groups in this study. In the MTA-F/ nonosteogenic medium group, the 2.43 Si/Ca ratio reflects the presence of calcium-silicate hydrate gel, a fine-grained and highly disorganized hydrated silicate gel containing Si-OH groups that may provide nucleation sites for apatite formation (28, 29). Interestingly, the presence of silicon from the MTA-F sealer within the HAP crystallites enhances this sealer’s bioactive potential because silicon is an essential element for the normal growth of bone and connective tissues. Silicon was proven to stimulate DNA synthesis, ALP activity, osteocalcin expression, and fibroblast proliferation (30, 31). Despite the initial cytotoxic effect during setting, the endodontic sealer MTA Fillapex can be considered a promising material for root canal treatment, considering its bioactive potential. In this study, MTA-F clearly showed the ability to stimulate nucleation sites for the formation of apatite crystals in human osteoblast-like cell culture. Further analysis is needed to elucidate whether silicon and other mineral components of this material are incorporated into the apatite crystal lattice.

Acknowledgments The authors thank to the Brasilia University Microscopy Lab group for their cooperation with the micrograph images. The authors deny any conflicts of interest related to this study.

References 1. Torabinejad M, Hong CU, McDonald F, Pitt Ford TR. Physical and chemical properties of a new root-end filling material. J Endod 1995;21:349–53.


Salles et al.

2. Torabinejad M, White DJ. United States Patent 5, 415, 547 USPTO. Patent Full Text and Image Database 1995. 3. Parirokh M, Torabinejad M. Mineral trioxide aggregate: a comprehensive literature review—part I: chemical, physical, and antibacterial. J Endod 2010;36:16–27. 4. Holland R, Filho JA, de Souza V, Nery MJ, Bernabe PF, Junior ED. Mineral trioxide aggregate repair of lateral root perforations. J Endod 2001;27:281–4. 5. De Deus G, Petruccelli V, GurgelFilho E, Coutinho-Filho T. MTA versus Portland cement as repair material for furcal perforations: a laboratory study using a polymicrobial leakage model. Int Endod J 2006;39:293–6. 6. Bonson S, Jeansonne BG, Laillier TE. Root-end filling materials alter fibroblast differentiation. J Dent Res 2004;83:408–13. 7. Nakayama A, Ogiso B, Tanabe N, Takeichi O, Matsuzaka K, Inoue T. Behavior of bone marrow osteoblast-like cells on mineral trioxide aggregate: morphology and expression of type I collagen and bone-related protein mRNAs. Int Endod J 2005;38:203–10. 8. Gomes-Filho JE, de Faria MD, Barnabe PF, et al. Mineral trioxide aggregate but not lightcure mineral trioxide aggregate stimulated mineralization. J Endod 2008;34:62–5. 9. Yasuda Y, Ogawa M, Arakawa T, Kodowaki T, Takashi S. The effect of mineral trioxide aggregate on the mineralization ability of rat dental pulp cells: an in vitro study. J Endod 2008;34:1057–60. 10. Bortoluzzi EA, Guerreiro-Tanomaru JM, Tanomaru-Filho M, Duarte MAH. Radiographic effect of different radiopacifiers on a potential retrograde filling material. Oral Surg Oral Med Oral Pathol Oral Radiol Endod 2009;108:628–32. 11. Cornelio ALG, Salles LP, Campos da Paz M, Cirelli JA, Guerreiro-Tanomaru JM, Tanomaru-Filho M. Cytotoxicity of Portland cement with different radiopacifying agents: a cell death study. J Endod 2011;37:203–10. 12. Camilleri J. The physical properties of accelerated Portland cement for endodontic use. Int Endod J 2008;41:151–7. 13. McQuillan DJ, Richardson MD, Bateman JF. Matrix deposition by a calcifying human osteogenic sarcoma cell line (SAOS-2). Bone 1995;16:415–26. 14. Al-Hiyasat AS, Tayyar M, Darmani H. Cytotoxicity evaluation of various resin-based root canal sealers. Int Endod J 2010;43:148–53. 15. Lee Y, Yang S, Ho W, Lee YH, Hung S. Eugenol modulates cyclooxygenase-2 expression through the activation of nuclear factor kappa B in human osteoblasts. J Endod 2007;33:1177–82. 16. Westgard JO, Barry PL, Hunt MR, Groth T. A multi-rule Shewhart chart for quality control in clinical chemistry. Clin Chem 1981;27:493–501. 17. Torabinejad M, Parirokh M. Mineral trioxide aggregate: A comprehensive literature review—part II: leakage and biocompatibility investigations. J Endod 2010;36: 190–202. 18. Bramante CM, Demarchi AC, Moraes IG, et al. Presence of arsenic in different types of MTA and white and gray Portland cement. Oral Surg Oral Med Oral PatholOral Radiol Endod 2008;106:909–13. 19. Morgental RD, Vier-Pelisser FV, Oliveira SD, Antunes FC, Cogo DM, Kopper PM. Antibacterial activity of two MTA-based root canal sealers. Int Endod J 2011;44:1128–33. 20. Versiani MA, Carvalho-Junior JR, Padilha MI, Lacey S, Pascon EA, Sousa-Neto MD. A comparative study of physicochemical properties of AHPlus and Epiphany root canal sealants. Int Endod J 2006;39:464–71. 21. Yamanaka Y, Shigetani Y, Yoshiba K, Yoshiba N, Okiji T. Immunohistochemical analysis of subcutaneous tissue reactions to methacrylate resin-based root canal sealers. Int Endod J 2011;44:669–75. 22. Gandolfi MG, Perut F, Ciapetti G, Mongiorgi R, Prati C. New Portland cement-based materials for endodontics mixed with articaine solution: a study of cellular response. J Endod 2008;34:39–44. 23. Jiang H, Liu XY. Principles of mimicking and engineering the self-organized structure of hard tissues. J BiolChem 2004;279:41286–93. 24. Camilleri J. Hydration characteristics of calcium silicate cements with alternative radiopacifiers used as root-end filling materials. J Endod 2010;36:502–8. 25. Wang X, Ito A, Sogo Y, Li X, Oyane A. Silicate-apatite composite layers on external fixation rods and in vitro evaluation using fibroblast and osteoblast. J Biomed Mater Res 2010;92A:1181–9. 26. Gandolfi MG, Taddei P, Tinti A, Prati C. Apatite-forming ability (bioactivity) of ProRoot MTA. Int Endod J 2010;43:917–29. 27. Gibson IR, Best SM, Bonfield W. Chemical characterization of silicon-substituted hydroxyapatite. J Biomed Mater Res 1999;44:422–8. 28. Oliveira AL, Malafaya PB, Reis RL. Sodium silicate gel as a precursor for the in vitro nucleation and growth of a bone-like apatite coating in compact and porous polymeric structures. Biomaterials 2003;24:2575–84. 29. Carlisle EM. Silicon: a possible factor in bone calcification. Science 1970;167: 279–80. 30. Reffitt DM, Ogston N, Jugdaohsingh R, et al. Orthosilicic acid stimulates collagen type 1 synthesis and osteoblastic differentiation in human osteoblast-like cells in vitro. Bone 2003;32:127–35. 31. Hanasono MM, Lum J, Carroll LA, Mikulec AA, Koch RJ. The effect of silicone gel on basic fibroblast growth factor levels in fibroblast cell culture. Arch Facial PlastSurg 2004;6:88–93.

JOE — Volume 38, Number 7, July 2012