Abstract
Aim To investigate mineral trioxide aggregate (MTA) influence angiogenesis of primary human dental pulp cells (hDPCs) via the MAPK pathway, in particular p38.
Methodology hDPCs were cultured with MTA to angiogenesis, after which cell viability, ion concentration, osmolality, NO secretion, the von Willebrand factor (vWF) and angiopoietin-1 (Ang-1) protein expression were examined. PrestoBlue® was used for evaluating hDPCs proliferation. An enzyme-linked immunosorbent assay was employed to determine vWF and Ang-1 protein secretion in hDPCs cultured on MTA and the control. Cells cultured on the tissue culture plate without the cement were used as the control. The t-test was used to evaluate the significance of the differences between the mean values.
Results MTA elicited a significant (p < 0.05) increase viability compared with the control (15%, 16%, and 13% on days 1, 3 and 5 of cell seeding, respectively). MTA consumed calcium and phosphate ions, and released more Si ions in the medium.
MTA significantly (p < 0.05) increased the osmolality of the medium to 313, 328, and 341 mOsm/kg after 1 day, 3 days, and 5 days, respectively. P38 was activated through phosphorylation, and the phosphorylation kinase was investigated in the cell system after being cultured with MTA. Expression levels for Ang-1 and vWF in hDPCs on MTA were higher than those of the MTA + p38 inhibitor (SB203580) group (p <
0.05) at all of the time points.
Conclusions MTA was able to activate the p38 pathway in hDPCs cultured in vitro.
Moreover, Si increased the osmolality required to facilitate the angiogenic differentiation of hDPCs via the p38 signaling pathway. When the p38 pathway was blocked by SB203580, the angiogenic-dependent protein secretion decreasesd. These findings verify that the p38 pathway plays a key role in regulating the angiogenic behavior of hDPCs cultured on MTA.
Keywords: Human dental pulp cell, mineral trioxide aggregate, angiogenesis, p38/MAPK, osmolality.
Introduction
Mineral trioxide aggregate (MTA) has been widely and successfully used for several clinical applications in endodontics (Torabinejad et al. 1993). MTA has good biocompatibility (Silva et al. 2013), and on promote hard-tissue formation (Torabinejad & Parirokh 2010, Eid et al. 2013). In dentistry, silicate-based cements have been used on dentine replacement restorative materials (Wei et al. 2012), and recently our research team demonstrated that silicate-based substrate stimulates the proliferation and differentiation of MG63 (Shie et al. 2012), human dental pulp cells (hDPCs) (Shie & Ding 2013, Liu et al. 2013, Lai et al. 2014), and human periodontal ligament cells (hPDLs) (Huang et al. 2013) in vitro. The inorganic ions released from Si-based materials have been found to influence cell proliferation and osteogenesis maker protein secretion (Shie et al. 2011, Zhai et al. 2013). In addition, several studies have proven that angiogenic indicators can be promoted through indirect contact of relevant cells with Bioglass®, a silicate-based material, or with its dissolution products (Li & Chang 2013, Zhai et al. 2013, Chou et al. 2013). However, the mechanism by which Si effects cell behavior, including cell differentiation, remains unclear (Hung et al. 2013a).
Mitogen-activated protein kinase (MAPK) cascades are conserved signal transduction pathways, which translate external stimuli into cellular responses in all eukaryotes. In mammalian cells, there are three sub-families of the MAPK family:
extracellular signal-regulated kinase 1/2 (ERK1/2), c-jun N-terminal kinase (JNK), and p38. ERK1/2 signaling plays a central role in various biological processes, including cell growth and differentiation (Lai et al. 2001, Du et al. 2013). In this regard, MTA has been shown to promote proliferation of a human osteosarcoma cell line (Chen et al. 2009) and hDPCs via ERK1/2 activation (Chen et al. 2010). JNK
signaling is mainly activated by cytokines and stress in the environment. In fact, the p38 MAPK pathway can be activated by environmental stress, such as osmotic stress (Junttila et al. 2008). However, the presence of inorganic ions and osmolytes in cell protein synthesis is particularly important in a hypertonic medium because impairment of cell protein synthesis occurs immediately after cell shrinkage (Brigotti et al. 2003) and induces cell apoptosis (Shie et al. 2011).
Taking these factors altogether, it can be hypothesized that MTA may effect the behavior of hDPCs via the MAPK pathway, in particular, the p38 pathway. The aim of this study is to confirm the effect of MTA contact on cell viability and endothelial-related protein expression through MAPK/p38 stimulation. In addition, this study investigates the specific role of the von Willebrand factor (vWF) and angiopoietin-1 (Ang-1) protein expression on MTA-induced angiogenesis. To accomplish this, the roles of MAPK/p38 involved in the regulation of angiogenic differentiation of hDPCs were also investigated.
Materials and methods
Preparation of MTA specimens
MTA was prepared following the manufacturer’s instructions. MTA powder in a liquid/powder ratio of 0.3 mL/g was used to mix the cement. After mixing distilled H2O, the cement was used to fully cover each well of a 24-well plate (GeneDireX, Las Vegas, NV, USA) to a thickness of 2 mm; these specimens were then stored in an incubator at 37 °C and 100% relative humidity for 1 day to set.
HDPCs isolation and culture
The hDPCs used were freshly derived from caries-free, intact premolars that had been extracted for orthodontic treatment purposes, as described previously (Shie
& Ding 2013). The patient gave informed consent, and approval from the Ethics Committee of the Chung Shan Medicine University Hospital was obtained (CSMUH No. CS11187). The tooth was split sagittally with a chisel. The pulp tissue was then immersed in phosphate-buffered saline (PBS; Caisson, North Logan, UT, USA) solution and digested in 0.1% collagenase type I (Sigma-Aldrich) for 30 min. After being transferred to a new plate, the cell suspension was cultured in Dulbecco’s modified Eagle medium (DMEM; Caisson), supplemented with 20% fetal bovine serum (FBS; GeneDireX), 1% penicillin (10,000 U/mL)/streptomycin (10,000 mg/mL) (PS, Caisson) at 37°C in a humidified atmosphere with 5% CO2. The medium was changed every 3 days. The cells were sub-cultured through successive passaging at a 1:3 ratio until they were used for experiments (passages 3–8). The angiogenic induction reagent (2% fetal bovine serum, 1% PS, and 50 ng/mL VEGF (Prospec, East Brunswick, NJ, USA) was mixed with DMEM.
Fluorescent staining
The cell morphology of hDPCs after culturing on MTA using F-actin cytoskeleton stains. After incubation for 3 and 6 h, the cells were washed with PBS, fixed in 4% paraformaldehyde (Sigma-Aldrich) at room temperature for 20 min, and then permeabilized with PBS containing 0.1% Triton X-100 (Sigma-Aldrich). The F- actin filaments were stained with phalloidin conjugated to Alexa Fluor 594 (Invitrogen) for 1 h. The nuclei were then stained with 300 nM DAPI (Invitrogen) for 30 min. After washing, the morphology was obtained using a Zeiss Axioskop2 microscope (Carl Zeiss, Thornwood, NY, USA).
Cell viability
Before the in vitro cell experiments, MTA specimens were sterilized by soaking in 75% ethanol followed by exposure to ultraviolet (UV) light for 1 h. After cell direct seeding for various time periods, cell viability was evaluated using the PrestoBlue® assay (Invitrogen), which is based on the detection of mitochondrial activity. Thirty µL of PrestoBlue® solution and 300 µl of DMEM were added to each well followed by 30 min of incubation. After incubation, 100 µL of the solution in each well was transferred to a 96-well ELISA plate. The plates were read in a Sunrise microtiter plate reader (Hitachi, Tokyo, Japan) at 570 nm, with a reference wavelength of 600 nm. The results were obtained in triplicate from three separate experiments in terms of optical density. Cells cultured on the tissue culture plate without the cement were used as a control.
Osmolality measurement
After 1, 3, and 5 days of being cultured, the medium was collected for osmolality measurement. The results were obtained in triplicate from three separate experiments. The precise osmolality of the various culture media was determined directly on a 20 µL medium using a Model 3300 advanced micro-osmometer (Advanced Scientific Instruments, Norwood, MA, USA).
Ion concentration
The Ca, Si, and P ion concentration on DMEM was analyzed using an inductively coupled plasma-atomic emission spectrometer (ICP-AES; Perkin-Elmer OPT 1MA 3000DV, Shelton, CT, USA) after being cultured for 1, 3, and 5 days. Six
samples were measured for each data point. The results were obtained in triplicate from three separate samples for each test.
Detection of eNOS
After 1, 3, and 5 days of culturing, the hDPCs were processed for measurement of eNOS using a commercially available ELISA kit (Abcam, Cambridge, MA, USA). Following the manufacturer’s instructions, the 2-h assay was used which has higher sensitivity. The reaction was terminated by the addition of a stop solution in the ELISA kit and read at 450 nm using a multi-plate reader. This experiment was repeated independently 3 times.
Nitric oxide (NO) colorimetric assay
After 1, 3, and 5 days of culturing, the culture medium was collected for a nitric oxide colorimetric assay. The concentrations of NO in the culture medium were measured with a NO Detection Kit (BioVision, San Francisco, CA, USA) in accordance with the manufacture’s instructions. NO is quickly oxidized to nitrate and nitrite, which are the means by which to determine the NO concentration. The amount of nitrate was determined by converting it to nitrite, and the colorimetric determination of the total concentration of nitrite as a colored azo dye product at 540 nm using microtiter plate reader (Bi et al. 2012).
P38 and pp38 measurement
The production of p38 and pp38 were quantified using ELISA kits (Invitrogen, catalog no. KHO0061 and KHO0071) according to the manufacturer’s instructions.
To summarize the process briefly, hDPCs were cultured on substrates for 1, 3, and 5
days, and proteins from whole cell lysates were then collected and quantified using the ELISA kit.
Intracellular ang-1 and vWF measurement
The production of ang-1 and vWF were quantified using ELISA kits (Abcam, catalog no. ab99970 and ab108918) according to the manufacturer’s instructions.
Summarized briefly, the hDPCs were cultured on substrates for 1, 3, and 5 days, and proteins from the whole cell lysates were then collected and quantified using the ELISA kit.
Statistical analysis
To test for statistically significant differences, the t-test was used for pairwise comparisons. In all cases, the results were considered statistically significant with p values < 0.05.
Results
F-actin staining
Fluorescence staining shows that at hour 3, hDPCs cultured on MTA displayed rounded morphologies of cells. After 6 h of cell attachment, the hDPCs clearly expressed F-actin stress fibres.
Cell viability
Evidence of the cell viability of hDPCs grown on MTA and control is shown in Fig. 2A. PrestoBlue® revealed that the number of cells cultured on the MTA surfaces was significantly (p < 0.05) higher for all culture times when compared to
controls. MTA elicited a significant (p < 0.05) increase of 15%, 16% and 13% in the optical density compared with the control on days 1, 3 and 5 of cell seeding, respectively.
Osmolality
Variations of DMEM osmolality with and without MTA after the specified culturing time periods are shown in Fig. 2B. The osmolality of the control was similar for all time-points. MTA significantly (p < 0.05) increased the osmolality to 313, 328, and 341 mOsm/kg on days 1, 3, and 5, respectively.
Ion concentration
Variations in the DMEM Ca, Si, and P ion concentrations after being cultured for different time periods are shown in Fig. 3. For MTA, the Ca ion concentration of the medium increased after being cultured for 1 day but then decreased to approximately 1.61 mM by the third day, which is lower than the baseline Ca concentration of DMEM (1.8 mM) (p < 0.05). No significant difference was found between control in Ca ion concentration at any of the measured time points. Si concentrations increase steadily with time. Si ion concentrations are in the range of 1.87, 2.79, and 4.08 mM at 1, 3, and 5 days, respectively. As for the P ion concentration of the medium, it decreased after being cultured for 1 day and then decreased to approximately 0.71 mM after 5 days, and then stayed at that level for all time-points.
eNOS and NO detection
Expressions of eNOS and NO were also detected. The results are shown in Fig. 4. After 5 days, eNOS protein expression in hDPCs cultured with MTA substrates was nearly 1.4 times higher than in hDPCs cultured with the control (Fig.
4A). In addition, on days 1, 3, and 5, MTA elicited a significant (p < 0.05) increase of 17%, 28%, and 34%, respectively, in NO synthesis compared with the control (Fig.
4B). These results indicate that the MTA up-regulated the expression of eNOS and subsequently stimulated NO synthesis.
P38 Pathway
Cells in the MAPK family react against several types of stress stimulation.
Knowing that p38 is activated through phosphorylation, phosphorylation kinase in the cell system after culturing it with MTA cement (Fig. 4C). P38 is strongly connected to hyperosmotic stress and has been found to be acutely activated in inorganic salt- treated cells (Mavrogonatou & Kletsas 2011); the incubation of hDPCs in MTA results in a time-dependent increase in pp38. Phosphorylation increases to 15%, 22%, and 29% in hDPCs on MTA compared with the control on days 1, 3 and 5, respectively (p < 0.05). Adding the p38 inhibitor SB203580 to the hDPCs culture resulted in inhibition of MTA-induced p38 phosphorylation. After pretreatment with the p38 inhibitor, the activated p38 expression levels reduced notably in all groups.
Pp38 synthesis decreased 62% in hDPCs cultured on MTA, which was more than the control (50%) on the same day (Fig. 4D).
Effects of P38 Inhibitor on Angiogenesis
The relative expression levels of Ang-1 and vWF protein secretion in hDPCs cultured on MTA with the addition of the p38 inhibitor SB203580 (50 µM) were
evaluated at different time points. The Ang-1 expression in MTA on days 1, 3, and 5 were enhanced 1.25, 1.30, and 1.33 times, respectively, as compared to that of the control (Fig. 5A). It can be seen that the vWF results shown in Fig. 5B are similar to Ang-1, both meaning a time-dependent up-regulation in the MTA group, which is significantly higher than that of the control for all time-points (p < 0.05). Moreover, the expression levels for Ang-1 and vWF in hDPCs on MTA were higher than those of the MTA + SB203580 group (p < 0.05) at all of the analyzed time points.
Discussion
Calcium silicate-based materials have been found to promote the adhesion, growth and differentiation of hDPCs and hMSCs, and have been used as implant materials for tissue repair and formation (Shie & Ding 2013, Hung et al. 2013b).
MAPK/ERK and MAPK/p38 pathways go through phosphorylation in cells cultured on Si-based substrates, and their inhibitors significantly reduce cell attachment, proliferation and differentiation as assessed according to DNA and alkaline phosphatase activity (Shie & Ding 2013). However, little is known about the mechanisms by which silicate-based materials regulates cell behavior and protein secretion in general, and much less is known about how it regulates the angiogenic differentiation of hDPCs specifically.
In this study, PrestoBlue® analysis indicated differences between MTA and the control. In all cases, absorbance values were higher for hDPCs cultured on MTA and these differences were significant. Previous studies have demonstrated that the fibroblast and osteoblast viability on MTA is higher than the controls for all culture times (Ding et al. 2008, 2010). Zhang et al. (2010) found that the soluble factors from CaSiO3 substrates may be more important for proliferation and osteogenic
differentiation in a growth medium (Torabinejad et al. 1993, Zhang et al. 2010).
Taking cell behavior into account, the appropriate Si released from silicate-based materials may promote desired cell behavior (Schröder et al. 2012, Eid et al. 2013, Wu et al. 2013). MTA offers the ability of controlling the rate of soluble Ca and Si ions, which can enhance cell attachment and proliferation (Shie et al. 2012, Wei et al.
2012). The bioactivity of Si-based substrates shows that the presence of PO43- ions in the composition is not an essential requirement for the development of an apatite layer, which consumes calcium and phosphate ions (Kao et al. 2009, Hung et al.
2013b). In addition, the ions released from MTA may change the osmolality of a culture medium. The osmolality of the MTA group examined during the cultured periods was in the range of the physiological osmolality for cells (280–320 mOsm/kg) (Shie et al. 2011, Chou et al. 2013). In an earlier study, the relationship between the Si ion concentration and osmolality was demonstrated (Shie et al. 2011, 2012).
Angiogenic activity is a critical physiological event during bone regeneration and formation. Several studies have proven that biomaterials can enhance the desired cell behavior of HUVECs (Leu et al. 2009, Hoppe et al. 2011, Shie et al. 2011, Li &
Chang 2013, Zhai et al. 2013). NO is the major regulator of cell migration and angiogenesis. It is responsible for several important functions in the cardiovascular system, such as vasodilation, inhibition of vasoconstrictor influences and platelet adhesion to vascular endothelium (anti-thrombotic), as well as the scavenging of superoxide anions (anti-inflammatory) (Fukumura et al. 2006, Zhai et al. 2012, Li &
Chang 2013, Zhai et al. 2013). In the present research we find that eNOS protein expression and NO secretion were significantly up-regulated in hDPCs cultured on an MTA substrate for 3 and 5 days compared to those in hDPCs cultured on the control.
The secretion of NO through endothelial NO synthase (eNOS) has been shown to play
a key role in angiogenesis and vasculogenesis, which are indispensable processes for tissue growth (Lai et al. 2001, Duda et al. 2004, Du et al. 2013). It has been reported that knockout mouse for eNOS show impaired angiogenesis in response to ischemia (Cooke & Losordo 2002, Huang et al. 2003). Therefore, the stimulation of the angiogenesis of hDPCs by MTA is possibly NO-dependent, i.e. the MTA enhances the expression of certain proangiogenic factors, such as vWF and Ang-1, and initiate downstream NO production.
The phosphorylation MAPK/p38 known to respond to various exogenous stresses by hyper-osmolality was estimating. High osmolality acutely but fleetingly activates p38 and dephosphorylates JNK and ERK (Sheikh-Hamad & Gustin 2004, Chen et al. 2010). p38 has been shown to underlie cellular responses towards hyperosmotic stress (Junttila et al. 2008, Mavrogonatou & Kletsas 2011). A previous study suggests a common mechanism of MAPK/p38 activation by high osmolality, possibly originating from cytoskeletal alterations due to cell shrinkage, as proposed previously in a report involving the Rac protein (Brigotti et al. 2003; Uhlik et al.
2003). The previous findings demonstrate that Si ions are released from MTA when stimulated of osmolality to activate p38 hDPCs. Treating primary cells with SB203580 decreased the phosphorylation of p38.
vWF is an important protein involved in coagulation and thrombus formation.
Following synthesis, it is found in secretary granules called Weibel-Palade bodies and in vessels, and is released both constitutively and in a regulated manner (Williamson et al. 2006, Bi et al. 2012). Ang-1 is another family of growth factors that plays an important role in vascular development (Miller et al. 2009, Mavrogonatou & Kletsas 2011). NO generally mediates pro-angiogenic activities of angiopoietin family members (Miller et al. 2009, Shie & Ding 2013). In this study, we tried to determine
whether there may be a possible angiogenic induction mechanism triggered by MTA.
The data shows that MTA is more likely to promote vWF and ang-1 secretion than the control during angiogenesis. Furthermore, vWF and ang-1 expression results indicate that an SB203580 inhibitor significantly decreases the expression level when hDPCs are cultured with MTA for all culture times tested. This observation is in agreement with previous descriptions, suggesting that MAPK is associated with CS-induced proliferation in hDPCs (Shie & Ding 2013), particularly in the case of the p38 in angiogenic differentiation.
Conclusions
MTA was able to activate the p38 pathway in hDPCs cultured in vitro.
Moreover, Si ions increased the osmolality required to facilitate the angiogenesis differentiation of hDPCs via the p38 signaling pathway. When the p38 pathway was inhibited by SB203580, the angiogenic-dependent protein secretion was reduced.
These findings verify that the p38 pathway plays a important role in regulating the angiogenic behavior of hDPCs cultured on MTA. More specifically, it can be concluded that MTA stimulates both proliferation and angiogenesis of hDPCs, at least partially via activation of the p38 pathway.
Acknowledgements
The authors acknowledge receipt of a grant from the Chung Shan Medical University Hospital under the project CSH-2014-C-017 and the National Science Council grants (NSC 101-2314-B-040-011-MY3) of Taiwan. The authors declare that they have no conflicts of interest.
References
Bi CWC, Xu L, Tian XY et al. (2012) Fo Shou San, an ancient chinese herbal decoction, protects endothelial function through increasing endothelial nitric oxide synthase activity. PLoS ONE, 7, e51670.
Brigotti M, Petronini PG, Carnicelli D et al. (2003) Effects of osmolarity, ions and compatible osmolytes on cell-free protein synthesis. The Biochemical Journal 369, 369–74.
Chen CL, Huang TH, Ding SJ, Shie MY, Kao CT (2009) Comparison of calcium and silicate cement and mineral trioxide aggregate biologic effects and bone markers expression in MG63 cells. Journal of Endodontics 35, 682–5.
Chen CL, Kao CT, Ding SJ, Shie MY, Huang TH (2010) Expression of the inflammatory marker cyclooxygenase-2 in dental pulp cells cultured with mineral trioxide aggregate or calcium silicate cements. Journal of Endodontics 36, 465–8.
Chou MY, Kao CT, Hung CJ et al. (2013) Role of the p38 pathway in calcium silicate cement-induced cell viability and angiogenesis-related proteins of human dental
pulp cell in vitro. Journal of Endodontics
http://dx.doi.org/10.1016/j.joen.2013.09.041.
Cooke JP, Losordo DW (2002) Nitric oxide and angiogenesis. Circulation 105, 2133–
5.
Ding SJ, Kao CT, Chen CL, Shie MY, Huang TH (2010) Evaluation of human osteosarcoma cell line genotoxicity effects of mineral trixoide aggregate and calcium silicate cements. Journal of Endodontics 36, 1158–62.
Ding SJ, Kao CT, Shie MY, Hung CC, Huang TH (2008) The physical and cytological properties of white MTA mixed with Na2HPO4 as an accelerant.
Journal of Endodontics 34, 748–51.
Du R, Wu T, Liu W et al. (2013) Role of the extracellular signal-regulated kinase 1/2 pathway in driving tricalcium silicate-induced proliferation and biomineralization of human dental pulp cells In vitro. Journal of Endodontics 39, 1023–9.
Duda DG, Fukumura D, Jain RK (2004) Role of eNOS in neovascularization: NO for endothelial progenitor cells. Trends in Molecular Medicine 10, 143–5.
Eid AA, Niu L, Primus CM et al. (2013) In vitro osteogenic/dentinogenic potential of an experimental calcium aluminosilicate cement. Journal of Endodontics 39, 1161–6.
Fukumura D, Kashiwagi S, Jain RK (2006) The role of nitric oxide in tumour progression. Nature Reviews Cancer, 6, 521–534.
Hoppe A, Güldal NS, Boccaccini AR (2011) A review of the biological response to ionic dissolution products from bioactive glasses and glass-ceramics.
Biomaterials 32, 2757–74.
Huang TH, Ding SJ, Hsu TC, Kao CT (2003) Effects of mineral trioxide aggregate (MTA) extracts on mitogen-activated protein kinase activity in human osteosarcoma cell line (U2OS). Biomaterials 24, 3909–13.
Huang TH, Liu SL, Chen CL, Shie MY, Kao CT (2013) Low-level laser effects on simulated orthodontic tension side periodontal ligament cells. Photomedicine and Laser Surgery 31, 72–7.
Hung CJ, Kao CT, Chen YJ, Shie MY, Huang TH (2013a) Antiosteoclastogenic activity of silicate-based materials antagonizing receptor activator for nuclear factor kappaB ligand–induced osteoclast differentiation of murine marcophages.
Journal of Endodontics 39, 1557–61.
Hung CJ, Kao CT, Shie MY, Huang TH (2013b) Comparison of host inflammatory responses between calcium-silicate base material and intermediate restorative
material. Journal of Dental Sciences, http://dx.doi.org/10.1016/j.jds.2013.08.002.
Junttila MR, Li S-P, Westermarck J (2008) Phosphatase-mediated crosstalk between MAPK signaling pathways in the regulation of cell survival. The FASEB journal : official publication of the Federation of American Societies for Experimental Biology 22, 954–65.
Kao CT, Shie MY, Huang TH, Ding SJ (2009) Properties of an accelerated mineral trioxide aggregate-like root-end filling material. Journal of Endodontics 35, 239–
42.
Lai CF, Chaudhary L, Fausto A et al. (2001) Erk is essential for growth, differentiation, integrin expression, and cell function in human osteoblastic cells.
The Journal of Biological Chemistry 276, 14443–50.
Lai WY, Kao CT, Hung CJ et al. (2014) An evaluation of the inflammatory response of lipopolysaccharide-treated primary dental pulp cells with regard to calcium silicate-based cements. International Journal of Oral Science, http://dx.doi.org/10.1038/ijos.2014.5.
Leu A, Stieger SM, Dayton P, Ferrara KW, Leach JK (2009) Angiogenic response to bioactive glass promotes bone healing in an irradiated calvarial defect. Tissue engineering. Part A 15, 877–85.
Li H, Chang J (2013) Stimulation of proangiogenesis by calcium silicate bioactive ceramic. Acta Biomaterialia 9, 5379–89.
Liu CH, Huang TH, Hung CJ et al. (2013) The synergistic effects of fibroblast growth factor-2 and mineral trioxide aggregate on an osteogenic accelerator in vitro.
International Endodontic Journal, http://dx.doi.org/10.1111/iej.12227
Mavrogonatou E, Kletsas D (2011) Differential response of nucleus pulposus intervertebral disc cells to high salt, sorbitol, and urea. Journal of Cellular
Physiology 227, 1179–87.
Miller TW, Isenberg JS, Roberts DD (2009) Molecular regulation of tumor angiogenesis and perfusion via redox signaling. Chemical Reviews 109, 3099–
124.
Schröder HC, Wang XH, Wiens M et al. (2012) Silicate modulates the cross-talk between osteoblasts (SaOS-2) and osteoclasts (RAW 264.7 cells): Inhibition of osteoclast growth and differentiation. Journal of Cellular Biochemistry 113, 3197–206.
Sheikh-Hamad D, Gustin MC (2004) MAP kinases and the adaptive response to hypertonicity: functional preservation from yeast to mammals. American Journal of Physiology - Renal Physiology 287, F1102–10.
Shie MY, Ding SJ (2013) Integrin binding and MAPK signal pathways in primary cell responses to surface chemistry of calcium silicate cements. Biomaterials 34, 6589–606.
Shie MY, Chang HC, Ding SJ (2012) Effects of altering the Si/Ca molar ratio of a calcium silicate cement on in vitro cell attachment. International Endodontic Journal 45, 337–45.
Shie MY, Ding SJ, Chang HC (2011) The role of silicon in osteoblast-like cell proliferation and apoptosis. Acta Biomaterialia 7, 2604–14.
Silva EJNL, Rosa TP, Herrera DR et al. (2013) Evaluation of cytotoxicity and physicochemical properties of calcium silicate-based endodontic sealer MTA fillapex. Journal of Endodontics 39, 274–7.
Torabinejad M, Parirokh M (2010) Mineral trioxide aggregate: a comprehensive literature review—Part II: leakage and biocompatibility investigations. Journal of Endodontics 36, 190–202.
Torabinejad M, Watson T, Ford T (1993) Sealing ability of a mineral trioxide aggregate when used as a root end filling material. Journal of Endodontics 19, 591–5.
Uhlik MT, Abell AN, Johnson NL et al. (2003) Rac–MEKK3–MKK3 scaffolding for p38 MAPK activation during hyperosmotic shock. Nature Cell Biology 5, 1104–
10.
Wei W, Qi Y, Nikonov SY et al. (2012) Effects of an experimental calcium aluminosilicate cement on the viability of murine odontoblast-like cells. Journal of Endodontics 38, 936–42.
Williamson MR, Black R, Kielty C (2006) PCL-PU composite vascular scaffold production for vascular tissue engineering: attachment, proliferation and bioactivity of human vascular endothelial cells. Biomaterials 27, 3608–16.
Wu BC, Kao CT, Huang TH et al. (2013) Effect of a calcium channel blocker on the odontogenic activity of human dental pulp cells cultured with silicate-based materials. Journal of Endodontics, http://dx.doi.org/10.1016/j.joen.2013.12.019.
Zhai W, Lu HX, Chen L et al. (2012) Silicate bioceramics induce angiogenesis during bone regeneration. Acta Biomaterialia 8, 341–9.
Zhai W, Lu H, Wu C et al. (2013) Stimulatory effects of the ionic products from Ca- Mg-Si bioceramics on both osteogenesis and angiogenesis in vitro. Acta Biomaterialia 9, 8004–14.
Zhang N, Molenda J, Fournelle J, Murphy W, Sahai N (2010) Effects of pseudowollastonite (CaSiO3) bioceramic on in vitro activity of human mesenchymal stem cells. Biomaterials 31, 7653–65.
Figure Legends
Figure 1. Fluorescent staining of hDPCs cultured on MTA after 3 and 6 h of seeding.
Cells were stained for nuclei (blue) and F-actin (red).
Figure 2. (A) Cell proliferation assay for hDPCs cultured on MTA at various time- points. (B) Osmolality of cell culture medium at various time-points. “*”, statistically significant difference from control.
Figure 3. Ca, Si, and P ions concentration of DMEM after culturing for different times. Data represent means ± SD (n = 6). “*”, statistically significant difference from control.
Figure 4. (A) Nitric oxide synthase (NOS3, eNOS) and (B) nitric oxide secretion by hDPCs in the presence of MTA were higher than control (p < 0.05). P38 activity in hDPCs after cultured on MTA (C) without or (D) with pre-treatment with SB203580 (50 µm). P38 activity is presented as the ratio of phosphorylated p38 normalized to total p38.Data represent means ± SD (n = 6). “*”, statistically significant difference from control.
Figure 5. (A) Ang-1 and (B) vWF expression of hDPCs cultured on MTA cement without and with SB203580 (50 µm) for different days. Protein secretion of the untreated groups was used as the 100% reference level. “*”, statistically significant difference from control.
