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Calcium silicate (CaSi) materials have been used for bone repair and generation due to their osteogenic properties. Tailoring the surface chemistry and structure of CaSi can enhance its clinical performance. There is no direct comparison between microscale and nanoscale CaSi particles. Therefore, this article aimed to compare and evaluate the surface chemistry, structure, and in vitro properties of microscale CaSi (μCaSi) and nanoscale CaSi (nCaSi) particles synthesized by the sol-gel method and precipitation method, respectively. As a result, the semi-crystalline μCaSi powders were assemblies of irregular microparticles containing a major β-dicalcium silicate phase, while the amorphous nCaSi powders consisted of spherical particles with a size of 100 nm. After soaking in a Tris-HCl solution, the amount of Si ions released from nCaSi was higher than that released from μCaSi, but there was no significant difference in Ca ion release between the two CaSi particles. Compared to microscale CaSi (μCaSi), nanoscale CaSi (nCaSi) significantly enhanced the growth and differentiation of human mesenchymal stem cells (hMSC) and inhibited the function of RAW 264.7 macrophages. In the case of antibacterial activity against Gram-negative Escherichia coli (E. coli) and Gram-positive Staphylococcus aureus (S. aureus), nanoscale nCaSi displayed a higher bacteriostatic ratio, a greater growth inhibition zone and more reactive oxygen species (ROS) production than microscale μCaSi. The conclusion is that nanoscale CaSi had greater antibacterial and osteogenic activity compared to microscale CaSi. Next generation CaSi-based materials with unique properties are emerging to meet specific clinical needs.

Calcium silicate cement has attracted much attention for bone defect repair and regeneration due to its osteogenic properties. Biomaterial-associated infections and washout have become a common clinical problem. In order to enhance the antibacterial and washout performance of calcium silicate cement to meet clinical needs, different types of chitosan, including chitosan polysaccharide (CTS), quaternary ammonium chitosan (QTS), and chitosan oligosaccharide (COS), as a liquid phase were added to the calcium silicate powder. The physicochemical properties, in vitro bioactivity, antibacterial efficacy, and osteogenic effects (MG63 cells) of the cement were evaluated. Antibacterial activity was conducted with Gram-negative Escherichia coli (E. coli) and a Gram-positive Staphylococcus aureus (S. aureus) bacteria. The amount of intracellular reactive oxygen species (ROS) produced in the bacteria cultured with the chitosan solution was also detected. The experimental results showed that the chitosan additive did not affect the crystalline phase of calcium silicate cement, but increased the setting time and strength of the cement in a concentration-dependent manner. Within the scope of this study, CTS and QTS solutions with a concentration of not <1 wt% improved the washout resistance of the control cement, while the COS solutions failed to strengthen the cement. When soaked in simulated body fluid (SBF) for 1 day, all cement samples formed apatite spherules. As the soaking time increased, the diametral tensile strength of all cements decreased and the porosity increased. The assays of MG63 cell function showed lower osteogenic activity of osteoblastic cells grown on the surfaces of the chitosan-incorporated cements in comparison with the control cement without chitosan. At the same 1% concentration, compared with QTS and COS cement, CTS cement had lower cell attachment, proliferation, differentiation, and mineralization. Conversely, the CTS cement resulted in the highest bacteriostasis ratio among the three hybrid cements against two bacteria. The ROS production followed the order of CTS > QTS > COS at the same 1% concentration. In conclusion, calcium silicate cement with 1% QTS may be a viable candidate for bone defect repair in view of anti-washout performance, setting time, antibacterial activity, and osteogenic activity shown in this study., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2020 Elsevier B.V. All rights reserved.)

Titanium and its alloys have been used as implant materials. Non-ideal osseointegration of the implant materials has facilitated the development of the bioactive coatings on the implant surfaces. In this work, the bioactive calcium silicate (CaSi) powder prepared in a green synthesis route was used to cover the surface of Ti implants by a facile electrospray deposition method. Post annealing in air was also applied to form the oxidation layer on the Ti surface with the aim of increasing the bond strength between the CaSi coating layer and Ti substrate. For the characterization of the coatings several analytical methods such as X-ray diffraction, scanning electron microscopy, secondary neutral mass spectrometry, and Raman-spectroscopy were used, in addition to the measurement of bond strength and corrosion resistance. The results indicated a uniform CaSi layer with a thickness of about 1 μm deposited on the Ti substrate. Annealing in the range of 700-900 °C in air resulted in the formation of rutile phase of TiO 2 ; more importantly, annealing at 800 °C did not significantly affect the composition of the CaSi layer consisting of β-Ca 2 SiO 4 . The bond strength between the coating layer and Ti substrate can be remarkably enhanced at an annealing temperature of 700 or 800 °C compared with the as-prepared coating without annealing. The annealed coatings had a better corrosion resistance than the as-prepared coating. It is concluded that the electrospray method associated with the post-annealing can be successfully used for the deposition of a CaSi layer with a defined structure and composition on titanium implants., (Copyright © 2019 Elsevier B.V. All rights reserved.)

Background: To enhance calcium silicate cements (CSCs) towards a specific clinical application of endodontics and vertebroplasty, the addition of oxide dopants (Bi2O3, SrO, ZnO, ZrO2) as radiopacifiers allows for tailoring of material properties., Objective: Effects of oxide dopants on the in vitro physicochemical properties and osetogenic activity of CSCs were investigated., Methods: The setting time, compressive strength, radiopacity, and osteogenic ability of the cements were evaluated. The ability of cement samples to support MG63 attachment, proliferation, differentiation, and mineralization was assessed., Results: The greater the oxide amount, the higher the setting time and radiopacity were in the cement. The effect of the oxide dopants on radiopacity followed the order Bi2O3 > ZrO2 > SrO > ZnO, which were greater than 3 mm of Al recommended by ISO 6876: 2001 standards. ZrO2 could reinforce compressive strength of the control cement, while ZnO remarkably reduced the strength. The adverse effect of Bi2O3 and ZrO2 was found on cell number, alkaline phosphatase (ALP) activity and mineralization of MG63 cells. SrO supported cell attachment, proliferation and differentiation, and significantly increased cellular mineralization compared to the control., Conclusions: The 20 wt% SrO-containing CSCs may be applied to endodontic treatment and vertebroplasty surgery.

To achieve the excellent mechanical properties of biodegradable materials used for cortical bone graft substitutes and fracture fixation devices remains a challenge. To this end, the biomimetic calcium silicate/gelatin/chitosan oligosaccharide composite implants were developed, with an aim of achieving high strength, controlled degradation, and superior osteogenic activity. The work focused on the effect of gelatin on mechanical properties of the composites under four different kinds of mechanical stresses including compression, tensile, bending, and impact. The evaluation of in vitro degradability and fatigue at two simulated body fluid (SBF) of pH 7.4 and 5.0 was also performed, in which the pH 5.0 condition simulated clinical conditions caused by bacterial induced local metabolic acidosis or tissue inflammation. In addition, human mesenchymal stem cells (hMSCs) were sued to examine osteogenic activity. Experimental results showed that the appropriate amount of gelatin positively contributed to failure enhancement in compressive and impact modes. The 10wt% gelatin-containing composite exhibits the maximum value of the compressive strength (166.1MPa), which is within the reported compressive strength for cortical bone. The stability of the bone implants was apparently affected by the in vitro fatigue, but not by the initial pH environments (7.4 or 5.0). The gelatin not only greatly enhanced the degradation of the composite when soaked in the dynamic SBF solution, but effectively promoted attachment, proliferation, differentiation, and formation of mineralization of hMSCs. The 10wt%-gelatin composite with high initial strength may be a potential implant candidate for cortical bone repair and fracture fixation applications., (Copyright © 2016 Elsevier Ltd. All rights reserved.)

The purpose of this study is to evaluate the physicochemical properties and in vitro osteogenic activity of radiopaque calcium silicate-gelatin cements. The radiopacity, setting time, working time, flow, diametral tensile strength, pH value, washout resistance and morphology of the cements with gelatin (0, 5 and 10% by weight) were measured, which compared to a popular endodontic material, ProRoot white-colored mineral trioxide aggregate (WMTA). The cell morphology, cell attachment and proliferation, alkaline phosphatase and osteocalcin levels on the cements were measured by culturing the specimens with dental pulp cells. The results indicated that the presence of gelatin significantly (P < 0.05) reduced radiopacity and diametral tensile strength and prolonged setting time. Nevertheless, the 5 wt% gelatin cement had a radiopacity (5.1 mm of Al thickness) higher than ISO 6876:2001 standards (3 mm of Al thickness). The setting time (33 min), working time (9 min) and flow value (17.4 mm) of the 5 wt% gelatin cement were significantly (P < 0.05) better than those of WMTA (corresponding 165, 6 min and 14.2 mm). The fresh WMTA completely degraded after soaking in a physiological solution for 1 h, while the gelatin cements resisted washout, showing no noticeable breakdown even after 1 day of soaking. The gelatin cement enhanced the higher expression of cell attachment, proliferation and differentiation as compared to WMTA. It was concluded that the 5 wt% gelatin-calcium silicate hybrid cement appears to be promising as a radiopaque biomaterial for medical applications such as endodontics and vertebroplasty.

The purpose of this study was to investigate the responses of the human osteosarcoma cell line MG63 to calcium silicate cements with different Si/Ca molar ratios and different surface roughness. In particular, the study evaluated integrin subunit levels, phosphor-focal adhesion kinase (pFAK) levels and protein production at the cell attachment stage. The results indicated that the surface roughness (variations within a factor of 10) of the cements did not play a prominent role in cell attachment and proliferation, but the effect of composition was highlighted. Increased pFAK and total integrin levels and promoted cell attachment and cell cycle progression were observed upon an increase in cement Si content. Cement with a higher Si content was beneficial for collagen Type I (COL I) adsorption, COL I secretion, and αlibβ3 subintegrin expression, whereas cement with a higher Ca content increased fibronectin (FN) adsorption, FN secretion, and enhanced αvβ1 subintegrin levels. These results establish composition-dependent differences in integrin binding as a mechanism regulating cellular responses to biomaterial surfaces., (Copyright © 2013 Wiley Periodicals, Inc.)

Cell attachment, proliferation and differentiation on different materials depend largely on the surface properties of the materials. This study sheds light on the mechanism by which the modulation of the chemical composition of calcium silicate cements with different Si/Ca molar ratios could produce different cell responses. Two primary cell types (human mesenchymal stem cells (hMSCs) and human dental pulp cells (hDPCs)) were used to elicit the changes in total DNA content, integrin subunit levels, phosphor-focal adhesion kinase (pFAK) levels, and mitogen-activated protein kinase (MAPK) signaling pathway activity at the cell attachment stage. The effect of small interfering RNA (siRNA) transfection targeting collagen type I (COL I) and fibronectin (FN) was also evaluated. The results indicated that increased total DNA content, pFAK and total integrin levels were observed upon an increase in cement Si content. Cements with different Si/Ca ratios did not cause the variations of interleukin 1β (IL-1β), epidermal growth factor (EGF) and tumor necrosis factor-α (TNF-α) ligands. The Si-rich cement facilitated COL I and α2β1 subintegrin expression, while Ca-rich cement promoted FN and αvβ3 subintegrin expression. Si component of the calcium silicates stimulated cell adhesion via activation of MAPK/extracellular signal-regulated kinase (ERK) and p38 signaling pathways more effectively than did by Ca component, but it did not affect c-Jun NH2-terminal kinase (JNK) activity. Inhibition of MAPK/ERK and MAPK/p38 signaling pathways in hMSCs and hDPCs significantly attenuated adhesion, proliferation and differentiation as assessed according to total DNA content and alkaline phosphatase activity. hMSCs and hDPCs from the three different donors exhibited a similar preference for cell behaviors. The results of the current study suggest that calcium silicate cements with a higher Si content have the potential to serve as excellent supports for primary cells. Unraveling the mechanism by which primary cells responded to calcium silicate materials will be beneficial for materials design in their eventual clinical use., (Crown Copyright © 2013. Published by Elsevier Ltd. All rights reserved.)

Calcium silicate cement (CSC) has favorable biocompatible properties that may support its clinical use as bone defect repair. A hybrid cement was developed consisting of a chitosan oligosaccharide (COS) solution in a liquid phase and gelatin (GLT)-containing calcium silicate powder in a solid phase. The combination of GLT and COS was chosen due to the benefits achieved from several synergistic effects and for their clinical applications. In this study, the in vitro physicochemical properties of CSC-GLT-COS hybrid cement were investigated. The results indicated that all cement specimens formed apatite spherulites in simulated body fluid (SBF) for as little as 1 h. After immersion in a Tris-HCl solution, a pore structure appeared on all specimen surfaces without any precipitation. The presence of GLT did not adversely affect the physiochemical properties of CSC, even after 30 days of immersion. In contrast, COS could promote the degradation of CSC, as evidenced in the results of mechanical strength, weight loss, and ion concentration. The in vitro physiochemical properties of the hybrid cement makes it an attractive choice for dental and orthopedic applications., (© 2010 Wiley Periodicals, Inc.)

Introduction: The purpose of this study was to comparatively examine physicochemical and biocompatible properties of 20 wt% bismuth oxide (Bi(2)O(3))-containing dicalcium silicate cement and white-colored mineral trioxide aggregate (WMTA)., Methods: The radiopacity, setting time, diametral tensile strength, pH value, morphology, and phase composition of the cements with and without Bi(2)O(3) were measured after mixing powders with water. Cement biocompatibility was evaluated by incubating the cement specimens with MG63 human osteoblast-like cells., Results: The addition of Bi(2)O(3) to the cement led to a significant increase (p < .05) in the setting time of 24 minutes. It was accompanied by a small decrease in the pH value but without adversely affecting diametral tensile strength. The radiopacity value of the Bi(2)O(3)-containing cement was equivalent to 7.3 mm of aluminum, which was significantly higher (p < 0.05) than pure cement without Bi(2)O(3) (1.1 mm of aluminum) but lower (p < .05) than WMTA (9.3 mm of aluminum). However, it was greater than 3 mm of aluminum, which is the value recommended by ISO 6876/2001 standards. MG63 cell viability cultured on Bi(2)O(3)-containing cement was higher than that cultured on WMTA at all culture times., Conclusions: The dicalcium silicate cement with 20 wt% Bi(2)O(3) showed shortened setting time and good biocompatibility and thus may have the potential to be a root-end filling material alternative to MTA., (Crown Copyright © 2010. Published by Elsevier Inc. All rights reserved.)

Calcium silicate cement (CSC) is biocompatible and possesses in vitro bioactivity. The aim of this study was to improve the handling and enhance osteogenic and immune properties of CSC by the addition of adjuvants to modify the cement. Human bone-marrow-derived mesenchymal stem cells were used to study the osteogenic behavior and immune response of cells on hybrid cements with added gelatin (GLT) and chitosan oligosaccharides (COS), which are analogs of the extracellular matrix components collagen and glycosaminoglycan, respectively. The addition of COS to the liquid phase slightly prolonged the setting time of CSC, whereas GLT in the solid phase significantly (p < 0.05) extended the hydration reaction. However, the addition of GLT appreciably improved the injectability of CSC, compared to COS. Cell viability was higher on CSC-COS than on the CSC control or on CSC-GLT at all culture times. The hybrid bone cements elicited less immune response than the CSC control. Additionally, COS inhibited expression of inducible nitric oxide synthase and interleukin-1 and activated interleukin-10 more effectively than GLT. Osteocalcin production and bone sialoprotein production were higher, and more calcium was detected in human bone-marrow-derived mesenchymal stem cells cultured on a CSC-GLT-COS surface than on CSC, CSC-GLT, or CSC-COS. These synergistic improvements in injectability, immune response, and osteogenesis suggest that the combination of bioactive calcium silicate, GLT, and COS has potential for use in clinical applications.

Introduction: Mineral trioxide aggregate (MTA) and calcium silicate (CS) cements exhibit acceptable levels of cytocompatibility. The aim of the present study was to evaluate MTA and CS cement genotoxicity in a human osteosarcoma cell line (MG63)., Methods: A mitochondrial colorimetric assay was used to evaluate the MG63 survival rate. A DNA precipitation assay was used to detect the MG63 DNA damage after contact with MTA or CS cement extracts., Results: The results showed that MTA and CS are cytocompatible with MG63 cells. There was no significant difference in the survival rate with MTA and CS materials (P > .05). Neither MTA nor CS cements causing MG63 cell DNA damage showed significant genotoxicity (P > .05)., Conclusions: Both MTA and CS cements are compatible with MG63 cells, and they are not cancer-causing agents., (Copyright 2010 American Association of Endodontists. Published by Elsevier Inc. All rights reserved.)

Novel washout-resistant bone substitute materials consisting of gelatin-containing calcium silicate cements (CSCs) were developed. The washout resistance, setting time, diametral tensile strength (DTS), morphology, and phase composition of the hybrid cements were evaluated. The results indicated that the dominant phase of beta-Ca(2)SiO(4) for the SiO(2)-CaO powders increased with an increase in the CaO content of the sols. After mixing with water, the setting times of the CSCs ranged from 10 to 29 min, increasing with a decrease in the amount of CaO in the sols. Addition of gelatin into the CSC significantly prolonged (P < 0.05) the setting time by about 2 and 8 times, respectively, for 5% and 10% gelatin. However, the presence of gelatin appreciably improved the anti-washout and brittle properties of the cements without adversely affecting mechanical strength. It was concluded that 5% gelatin-containing CSC may be useful as bioactive bone repair materials.

Introduction: Mineral trioxide aggregate (MTA) and calcium silicate (CS) cements exhibit acceptable physical and chemical properties. The aim of the present study was to evaluate the effects of MTA and CS cements on inflammatory reactions in primary cultured human dental pulp cells., Methods: The mitochondrial colorimetric assay was used to evaluate pulp cell survival rates. Fluorescent immunohistochemistry was used to observe focal adhesion kinase (FAK) and cyclooxygenase-2 (COX-2) distributions in the cells. Reverse transcription-polymerase chain reaction was used to assess COX-2 expression., Results: The results showed that MTA and CS are biocompatible with pulp cells (P>.05). FAK was well-distributed in pulp cells in contact with both cements. Both MTA and CS cements induced pulp cell inflammation as evidenced by increased COX-2 expression., Conclusions: The present study demonstrated that MTA and CS cements are biocompatible with primary cultured pulp cells. Both cements can induce inflammatory COX-2 expression in the pulp cells., (Copyright (c) 2010 American Association of Endodontists. Published by Elsevier Inc. All rights reserved.)

Introduction: The purpose of this study was to examine the in vitro bioactivity and biocompatibility of sol-gel-derived dicalcium silicate cements., Methods: The morphology, phase composition, and compressive strength of the novel cement were investigated after immersion in a simulated body fluid for different periods of time. Cement biocompatibility was evaluated by incubating the cement specimens with MG63 human osteoblast-like cells., Results: After immersion in a simulated body fluid as little as 1 hour, the cements were covered with clusters of bone-like apatite spherulites. The characteristic peaks of apatite at 2theta = 25.9 degrees and 31.8 - 32.9 degrees appeared. The compressive strength of the cement was increased from the initial strength value of 12.3 MPa to 1-day strength value of 20.2 MPa; these values were significantly different (P < .05). The MG63 cell viability increased 15% and 23% on the cement surfaces when compared with the control on hour 6 and day 7 of incubation, respectively. The cells appeared flat and exhibited intact, well-defined morphology on the cement surface., Conclusions: Both bioactivity and biocompatibility of the dicalcium silicate cement consistently make it a potential candidate for endodontic use.

Introduction: The purpose of this study was to examine the physicochemical properties of novel calcium silicate cements (CSCs) prepared by using a sol-gel method., Methods: The compressive strength, morphology, and phase composition of various cements were evaluated after mixing with water, in addition to setting time and pH value., Results: As solid phases, the sol-gel-derived powders mainly consisted of beta-dicalcium silicate. Setting times for cements mixed with water ranged from 12-42 minutes and were lower for cements with higher starting CaO content. The compressive strength of the CSCs ranged from 0.3-15.2 MPa; these values were significantly different (P < .05). Calcium silicate hydrate (C-S-H) was the principal phase that formed in the hydration process. The CSCs' pH values changed from an initial 11 to a high of 13., Conclusions: CSCs display advantageously shortened setting times and might have potential for endodontic use, although further tests are necessary to confirm this.

Bone cell (MG63) biocompatibility and bone marker expression were compared after calcium and silicate base cement (CS) and mineral trioxide aggregate (MTA) treatment. X-ray diffraction was used to identify material surface structure, and tetrazolium bromide colorimetric assay was used to evaluate the cell viability. The relative mitogen activation protein kinase expression was compared with Western blot, and bone marker expression was evaluated with reverse transcriptase polymerization chain reaction. The results showed that CS and MTA are similar chemical structures and biocompatible with MG63 cells. CS and MTA cements showed good MG63 cell proliferation by high phosphor extracellular signal-regulated kinase expression levels. CS and MTA cements showed the evident type I collagen, osteocalcin, alkaline phosphatase, bone sialoprotein, and osteopontin expression. Both MTA and CS cements are biocompatible and appear to have osetoconduction effects on bone cells.

The purpose of the study was to examine the physicochemical properties of mineral trioxide aggregate (MTA)-like cements prepared from SiO(2), CaO, and Al(2)O(3) as matrices, and ZnO, MgO, and Fe(2)O(3) as additives. The MTA-like cements showed a significantly (p < 0.05) faster setting time (<14 minutes) than MTA when mixed with water, although there was a significant decrease (p < 0.05) in diametral tensile strength, ranging from 0.9 to 3.1 MPa in comparison to MTA (4.4 MPa). The addition of ZnO, MgO, and Fe(2)O(3) to the MTA-like cement led to a significantly (p < 0.05) higher strength than MTA-like cement without additives. The pH values of the MTA-like cements were changed from an initial 11 to a high of 13, similar to the results of MTA. Future characterization of the examined MTA-like cement with shortened setting time is needed before it is considered a viable candidate for dental root-end-filling applications.

The purpose of this study was to investigate the effect of two solutions differing by pH (6.4 and 4.0) and the use of a setting accelerator (15% Na(2)HPO(4) solution) on the properties of white-colored mineral trioxide aggregate (WMTA). These studies indicated that pH 4.0 had a deleterious effect on the morphology of WMTA mixed with water, however, not for WMTA mixed with Na(2)HPO(4). When immersed in a pH 4.0 solution for 7 days, WMTA mixed with water or Na(2)HPO(4) achieved a diametral tensile strength of 7.9 and 9.0 MPa, respectively, which was significantly lower (p < 0.05) than those obtained at pH 6.4 (11.2 and 12.0 MPa) but significantly higher (p < 0.05) than day 0 samples (4.4 and 4.8 MPa). Mixing WMTA with the accelerator did not significantly affect the microstructure, solubility, or strength in an acidic environment.

The purpose of this study was to investigate the effect of a sodium phosphate dibasic (Na2HPO4) setting accelerator on the properties of white-colored mineral trioxide aggregate (WMTA). Setting times were measured by using a Gilmore needle. Changes in the pH value, diametral tensile strength, and phase composition of WMTA were evaluated. By using a 15% Na2HPO4 solution as a liquid phase mixed with WMTA, the setting time was significantly reduced to 26 minutes rather than the usual 3 hours. The 15% Na2HPO4 promoted WMTA to achieve a maximum diametral tensile strength of 4.9 MPa at an initial 6-hour aging time and 1 MPa for the cement mixed with water. The pH value of the 15% Na2HPO4-mixed cement was changed from an initial 11.0 to a high of 13.2, which was similar to the results using water. The results suggest that the Na2HPO4 solution may be an effective setting accelerator for WMTA.

Extracellular regulated kinases (ERKs)-1 and -2 are members of the MAPK family of protein kinases involved in the proliferation, differentiation and apoptosis of bone cells. The purpose of the present study investigated the biocompatibility role, and signaling pathways of components of mineral trioxide aggregate (MTA) by culturing human osteosarcoma cell line (U2OS) in the presence of materials. Biocompatibility effects were assessed using the MTT assay for mitochondrial enzyme activity. The statistical analysis of the survival rate was performed using one-way analysis of variance (ANOVA) with p<0.05 shown statistical difference. The signaling pathway of MTA-treated U2OS cells were assessed by the western blotting methods. Dose-dependent and time-dependent tests were conducted. The results showed that the survival rates of the MTA extract experimental groups were higher than that of the control group (p<0.05). ERKs activity was dose-dependent, decreasing as the concentrations of the MTA extract decreased, and was time-dependent, decreasing as the treatment time increased. Suppression of ERK pathway by PD98059 resulted in dose-dependent and time-dependent decreases. The findings suggest that MTA is a biocompatible material to U2OS cells, and the ERK kinase pathway plays a signal transduction role in the MTA treated U2OS cells.