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Role of the P38 Pathway in Calcium Silicate Cement–induced Cell Viability and Angiogenesis-related Proteins of Human Dental Pulp Cell In Vitro
Basic Research
Role of the P38 Pathway in Calcium Silicate Cement–induced Cell Viability and Angiogenesis-related Proteins of Human Dental Pulp Cell In Vitro Ming-Yung Chou, DDS, MD, PhD,*† Chia-Tze Kao, DDS, MD, PhD,*† Chi-Jr Hung, DDS, MD,*† Tsui-Hsien Huang, DDS, MD, PhD,*† Shu-Ching Huang, DDS, MD,*† Ming-You Shie, PhD,‡* and Buor-Chang Wu, DDS, MD*† Abstract Introduction: This study investigated that calcium silicate (CS) cement may influence the behavior of human dental pulp cells (hDPCs) via mitogen-activated protein kinase pathway, in particular p38. We have addressed that Si ion released from CS cement can influence osmolarity in the medium, which may stimulate hDPC viability and induce angiogenesis-related proteins through stimulation of the nitric oxide synthase and nitric oxide secretion. Methods: The hDPCs was cultured with CS cement to angiogenesis. Then, cell viability, ion concentration, osmolality, nitric oxide secretion, the von Willebrand factor, and angiopoietin-1 protein expression were examined. Results: CS cement elicited a significant (P < .05) increase of 15%, 20%, and 19% in viability compared with control on days 1, 3, and 5 of cell seeding, respectively. The CS cement consumed calcium and phosphate ions and released more Si ions in medium. The CS significantly (P < .05) increased the osmolality to 303.52 3.07, 315.03 5.80, and 319.95 4.68 mOsm/kg for 1, 3, and 5 days, respectively. P38 was activated through phosphorylation; the phosphorylation kinase was investigated in our cell system after culture with CS cement. Moreover, expression levels for angiopoietin-1 and von Willebrand factor in hDPCs on CS cement were higher than those of the CS + p38 inhibitor (SB203580) group (P < .05) at all of the analyzed time points. Conclusions: This study showed that CS cement was able to activate the p38 pathway in hDPCs cultured in vitro. Moreover, Si was shown to increase 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 was decreased. These findings verified that the p38 pathway plays a key role in regulating the angiogenic
behavior of hDPCs cultured on CS cement. (J Endod 2013;-:1–7)
Key Words Angiogenesis, calcium silicate cement, human dental pulp cell, osmolality, p38/MAPK
M
ineral trioxide aggregate (MTA) has been widely and successfully used with several clinical applications in endodontics (1). This material, which received approval in 1998, is basically a mixture of 75% Portland cement, 20% Bi2O3, and 5% gypsum (1). Not only does MTA have good biocompatibility (2), it has also been proven to enhance hard-tissue formation (3, 4). In dentistry, calcium silicate (CS)– based cements have been formulated into dentin replacement restorative materials (5). In a previous study, a quick-setting CS (Ca2SiO4) cement showed an advantageously shortened setting time, excellent bioactivity, and good biocompatibility (6–8). Moreover, we recently showed that CS cement stimulates the proliferation and differentiation of MG63 (7) and human dental pulp cells (hDPCs) in vitro (9). The inorganic ions released from silicate-based materials have been found to stimulate bone cell proliferation and osteogenic protein secretion (10, 11). 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 (11–13). However, the mechanism by which Si promotes cell behavior, including cell differentiation, remains unclear. Mitogen-activated protein kinase (MAPK) cascades play an important role in the modulation of gene expression and cytoplasmic signaling. In mammalian cells, there are 3 subfamilies of the MAPK family, namely extracellular signal-regulated kinase 1/2 (ERK1/2), c-jun N-terminal kinase (JNK), and p38. ERK1/2 preferentially regulates cell growth and differentiation (14, 15). In this regard, MTA has been shown to enhance proliferation of a human osteosarcoma cell line (16) and hDPCs via ERK1/2 activation (17). JNK signaling is mainly activated by cytokines and stress, and the p38 MAPK pathway can be activated by environmental stress, such as osmotic stress (18). 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 (19) and induces cell apoptosis.
From the *School of Dentistry and ‡Institute of Oral Science, Chung Shan Medical University; and †Department of Dentistry, Chung Shan Medical University Hospital, Taichung City, Taiwan. Ming-You Shie, and Buor-Chang Wu contributed equally to this work. Supported by a grant from the Chung Shan Medical University Hospital under the project CSH-2010-C-025 and the National Science Council grants (NSC 01-2314-B040-011-MY3) of Taiwan. Address requests for reprints to Dr Ming-You Shie, Institute of Oral Science, Chung Shan Medical University, Taichung City, Taiwan. E-mail address: eviltacasi@gmail. com 0099-2399/$ - see front matter Copyright ª 2013 American Association of Endodontists. http://dx.doi.org/10.1016/j.joen.2013.09.041
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P38 in CS-induced Pulp Cell Angiogenesis
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Figure 1. (A) PrestoBlue assay for hDPCs viability cultured on various specimens at various time points. CS cement elicited a significant (P < .05) increase in OD compared with Ctl for all time points. (B) Osmolality of cell culture medium at various time points. (C) Ca, Si, and P ion concentration of DMEM after culturing for different times. Data represent means standard deviation (n = 6). *Statistically significant difference from Ctl.
Taking these factors together, we hypothesized that CS cement may influence the behavior of hDPCs via the MAPK pathway, in particular the p38 pathway. The aim of this study was to verify the effect of CS cement contact on cell viability and endothelial-related protein expression through MAPK/p38 stimulation. In addition, this study was to investigate the specific role of the von Willebrand factor (vWF) and angiopoietin-1 (Ang-1) protein expression on Si ioninduced angiogenesis. Thus, the roles of MAPK/p38 involved in the regulation of angiogenic differentiation of hDPCs were also investigated.
Materials and Methods Specimen Preparation The CS cement was made according to our previously reported laboratory procedures (11). Appropriate amounts of CaO (65%; Showa, Tokyo, Japan), SiO2 (25%; High Pure Chemicals, Saitama, Japan), and Al2O3 (5%; Sigma-Aldrich, St Louis, MO) powders were mixed by a conditioning mixer (ARE-250; Thinky, Tokyo, Japan). After sintering at 1400 C for 2 hours, the granules were ball milled in EtOH by using a centrifugal ball mill (Retsch S 100, Hann, Germany) and then dried in an oven. CS cement was mixed according to the same liquid/ powder ratio of 0.3 mL/g. After mixing, the cement fully covered each well of the 24-well plate (GeneDireX, Las Vegas, NV) to a thickness of 2
Chou et al.
2 mm, and the samples were stored in an incubator at 100% relative humidity and 37 C for 1 day. Before cell experiments, all specimens were sterilized by immersion in 75% ethanol, followed by exposure to ultraviolet light for 1 hour.
HDPC Isolation and Culture The hDPCs were freshly derived from caries-free, intact premolars that were extracted for orthodontic treatment purposes, as described previously (20). The cells were freshly derived from a caries-free intact premolar that was extracted for orthodontic treatment. 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 phosphatebuffered saline (Caisson, North Logan, UT) solution and digested in 0.1% collagenase type I (Sigma-Aldrich) for 30 minutes. After being transferred to a new plate, the cell suspension was cultured in Dulbecco modified Eagle medium (DMEM) (Caisson), supplemented with 20% fetal bovine serum (Caisson) and 1% penicillin (10,000 U/mL)/streptomycin (10,000 mg/mL) (Caisson) in a humidified atmosphere with 5% CO2 at 37 C, and the medium was changed every 3 days. The cells were subcultured through successive passaging at a 1:3 ratio until they were used for experiments (passages 3–8). The JOE — Volume -, Number -, - 2013
Basic Research
Figure 2. (A) NO and (B) nitric oxide synthase (NOS3, eNOS) secretion by hDPCs in the presence of CS cement were higher than Ctl (P < .05). Data represent means standard deviation (n = 6). *Statistically significant difference from Ctl.
angiogenic induction reagent (2% fetal bovine serum, 1% penicillin [10,000 U/mL]/streptomycin [10,000 mg/mL], and 50 ng/mL vascular endothelial growth factor [Prospec, East Brunswick, NJ]) was mixed with DMEM.
Cell Viability Before the in vitro cell experiments, all cement specimens were sterilized by soaking in 75% ethanol, followed by exposure to ultraviolet light for 1 hour. After cell direct seeding on different substrates for various time periods, cell viability was evaluated by using the PrestoBlue assay (Invitrogen, Grand Island, NY), which is based on the detection of mitochondrial activity. Thirty microliters PrestoBlue solution and 300 mL DMEM were added to each well, followed by 30 minutes of incubation. After incubation, 100 mL of the solution in each well was transferred to a 96-well enzyme-linked immunosorbent assay (ELISA) plate. The plates were read in a Sunrise microtiter plate reader (Tecan Austria Gesellschaft, Salzburg, Austria) at 570 nm, with a reference wavelength of 600 nm. The results were obtained in triplicate from 3 separate experiments in terms of optical density (OD). Cells cultured on the tissue culture plate without the cement were used as a control (Ctl). Osmolality Measurement After 1, 3, and 5 days of culture, medium was collected for osmolality measurement. The results were obtained in triplicate from 3 separate experiments. The precise osmolality of the various culture media was determined directly on 20 mL medium by using a Model 3300 advanced micro-osmometer (Advanced Scientific Instruments, Norwood, MA). Ion Concentration The Ca, Si, and P ion concentration on DMEM was analyzed by using an inductively coupled plasma-atomic emission spectrometer (PerkinElmer OPT 1MA 3000DV, Shelton, CT) after culture for 1, 3, and 5 days. Three samples were measured for each data point. The results were obtained in triplicate from 3 separate samples for each test. Detection of Endothelial Nitric Oxide Synthase After 1, 3, and 5 days of culture, the hDPCs were processed for measurement of endothelial nitric oxide synthase (eNOS) by using a commercially available ELISA kit (Abcam, Cambridge, MA). Following the manufacturer’s instructions, we used the 2-hour assay, which has JOE — Volume -, Number -, - 2013
higher sensitivity. The reaction was terminated by the addition of stop solution and read at 450 nm by using a Sunrise microtiter reader. This experiment was repeated independently 3 times.
Nitric Oxide Colorimetric Assay After 1, 3, and 5 days of culture, culture medium was collected for NO colorimetric assay. The concentrations of NO in the culture medium were measured with the NO Detection Kit (BioVision, San Francisco, CA) in accordance with the manufacturer’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 by using Sunrise microtiter plate reader (21). Western Blotting Western blotting was performed on cells cultured on different cement specimens for a predetermined time to evaluate p38 and phospho-p38 levels. Cells were lysed in NP-40 lysis buffer (Invitrogen) at 4 C for 30 minutes, and the lysates were centrifuged at 13,000g. The protein concentrations of the lysates were measured by using a Bio-Rad DC Protein Assay kit (Richmond, CA), and proteins (30 mg) were resolved by using standard sodium dodecylsulfate–polyacrylamide gel electrophoresis and transferred to polyvinylidene difluoride membranes. After blocking with 5% bovine serum albumin (Sigma-Aldrich) in TBS-T (Tris-buffer saline containing 0.05% Tween-20) for 1 hour, the membranes were incubated with primary antibodies against b-actin, p38, and phospho-p38 (GeneTex, San Antonio, TX). The signal inhibitor used for the p38 was SB203580 (50 mmol/L; Invitrogen). A horseradish peroxidase–conjugated secondary antibody was subsequently added, and the proteins were visualized with enhancement by using enhanced chemiluminescent detection kits (Invitrogen). The stained bands were scanned and quantified by using a densitometer (Syngene Bioimaging System, Frederick, MD) and Scion Image software (Frederick, MD). Protein expression levels were normalized to the actin band for each sample. Intracellular Ang-1 and vWF Measurement The production of Ang-1 and vWF was quantified by using ELISA kits (Abcam; catalog no. ab99970 and ab108918) according to the manufacturer’s instructions. Briefly, hDPCs were cultured on substrates for 1, 3, and 5 days, and proteins from whole cell lysates were collected and quantified by using the ELISA kit. P38 in CS-induced Pulp Cell Angiogenesis
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Figure 3. Results of Western blot. (A) P38 activity in hDPCs after they were cultured on different substrates with or without pretreatment with SB203580 (50 mm). P38 activity is presented as ratio of phosphorylated p38 normalized to total p38. Data represent means standard deviation (n = 3). (B) Pp38/p38 ratio of untreated groups was used as 100% reference level. *Statistically significant difference from Ctl. pp38, phospho-38.
Statistical Analysis A one-way analysis of variance statistical analysis was used to evaluate the significance of the differences between the groups in each experiment. A Scheffe multiple comparison test was used to determine the significance of the deviations in the data for each specimen. In all cases, the results were considered statistically significant with P values < .05.
Results Cell Viability The cell viability of hDPCs grown on CS cement and Ctl is shown in Figure 1A. PrestoBlue showed that the number of cells cultured on CS cement surfaces had a significantly (P < .05) higher value amount at all culture times when compared with Ctl. CS elicited a significant (P < .05) increase of 15%, 20%, and 19% in OD compared with Ctl on days 1, 3 and 5 of cell seeding, respectively. 4
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Osmolality Variations of DMEM osmolality after the specified cultured time periods are shown in Figure 1B. The osmolality of the Ctl was similar for all time points. The CS was significant (P < .05); it increased the osmolality to 303.52 3.07, 315.03 5.80, and 319.95 4.68 mOsm/kg for 1, 3, and 5 days, respectively. Ion Concentration Variations of DMEM Ca, Si, and P ion concentrations after different cultured time periods are shown in Figure 1C. In CS cement, Ca ion concentration of the medium increased after 1 day of culture and then decreased to approximately 1.1 mmol/L after 3 days, which was lower than the baseline Ca concentration of DMEM (1.8 mmol/L) (P < .05). No significant difference was found between Ctl in Ca ion concentration for all time points. Si concentrations increased with increasing incubation time points. Si ion concentration was in the range of 1.3, 2.0, and 2.7 mmol/L at 1, 3, and 5 days, respectively. As for P ion concentration of the medium, it decreased after 1 day of culture and then decreased to JOE — Volume -, Number -, - 2013
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Figure 4. (A) Ang-1 and (B) vWF expression of hDPCs cultured on CS cement without and with SB203580 (50 mm) for different days. Protein secretion of untreated groups was used as 100% reference level. *Statistically significant difference from Ctl.
approximately 0.5 mmol/L after 5 days, and there was no difference in P ion concentration of Ctl for all time points.
eNOS and NO Detection The expression of eNOS and NO was also detected, and the results are shown in Figure 2. The eNOS protein expression in hDPCs cultured with CS substrates was nearly 1.4 times higher than in hDPCs cultured with Ctl (Fig. 2A). In addition, CS cement elicited a significant (P < .05) increase of 21% and 19% in NO synthesis compared with Ctl on days 3 and 5, respectively (Fig. 2B). These results indicate that the CS cement up-regulated the expression of eNOS and subsequently stimulated NO synthesis. P38 Pathway MAPK family was activated as a response of cells against several types of stress stimulation. Because p38 was activated through phosphorylation, the phosphorylation kinase was investigated in our cell sysJOE — Volume -, Number -, - 2013
tem after culture with CS cement (Fig. 3). P38 is strongly connected to hyperosmotic stress and has been found to be acutely activated in inorganic salt-treated cells (22); incubation of hDPCs in CS cement resulted in a time-dependent increase in pp38. Phosphorylation started to increase 13%, 21%, and 27% in hDPCs on CS compared with Ctl on days 1, 3, and 5, respectively (P < .05). Addition of the p38 inhibitor SB203580 to the hDPC culture resulted in inhibition of CS cement– induced p38 phosphorylation. After pretreatment with the p38 inhibitor, the activated p38 expression levels were reduced notably in all groups. The p38 synthesis was decreased 56% in hDPCs cultured on CS, which was more than Ctl (40%) on day 1 (Fig. 3B).
Effects of P38 Inhibitor on Angiogenesis Relative expression levels of Ang-1 and vWF protein secretion in hDPCs cultured on CS with the addition of the p38 inhibitor SB203580 (50 mmol/L) were evaluated at all time points. The Ang-1 expression in CS on days 1, 3, and 5 was enhanced 1.22, 1.26, and 1.31 times, respectively, as compared with that of Ctl (Fig. 4A). The P38 in CS-induced Pulp Cell Angiogenesis
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Basic Research vWF results shown in Figure 4B can be seen to be similar to Ang-1, both showing a time-dependent up-regulation in the CS group, which was significantly higher than that of controls for all time points (P < .05). Moreover, expression levels for Ang-1 and vWF in hDPCs on CS were higher than those of the CS + SB203580 group (P < .05) at all of the analyzed time points. Taken together, these results support a CSmediated up-regulation of angiogenesis-dependent protein occurring via the p38 pathway.
Discussion This study was designed to demonstrate the molecular effects of CS, a component of some novel capping agents used to preserve the viability of dental pulp tissue during reparative dentin formation. CS-based materials have been found to foster hDPC and hMSC adhesion, growth, and differentiation and have been used as implant materials for bone repair and regeneration (20). ERK/MAPK and p38/MAPK signaling pathways go through phosphorylation in cells cultured on CS substrates, and their inhibitors significantly reduce cell adhesion, proliferation, and differentiation as assessed according to total DNA and alkaline phosphatase activity (20). However, little information is known about the mechanisms by which CS identity regulates cell behavior and protein secretion in general, and much less is known about how they regulate the angiogenesis differentiation of hDPCs specifically. In this study, PrestoBlue analysis revealed differences between CS and Ctl. In all cases, absorbance values were higher for hDPCs cultured on CS in comparison with Ctl, and these differences were almost always significant (P < .05). Previous studies in our laboratory have demonstrated MG63 cell viability on 20 wt% Bi2O3-containing dicalcium silicate cement to be higher than that on white MTA and Ctl at all culture times (1, 7). Zhang et al (23) found that soluble factors from CaSiO3 substrates may be more important for proliferation and osteogenic differentiation in a growth medium (1). The hydrolysis of CS cement on immersion, and Si ion was dissolved during incubation (2, 9). Taking cell functions into account, the appropriate Si released from silicate-based materials may support cell behaviors (3, 4, 24). The current CS offers the ability of controlling the rate of soluble Si and Ca ions, which can promote cell adhesion and proliferation (5, 9). The bioactivity of silicate-based materials indicates 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 (6–8, 25). In addition, dissolution ions from the cement may change the osmolality of a culture medium. The osmolality of the CS group examined during the culturing periods in this study was in the range of the physiological osmolality for cells (280–320 mOsm/kg) (7, 10). In an earlier study, the relationship between the Si ion concentration and osmolality was demonstrated (9, 10). Angiogenesis is a critical physiology event in bone formation. Several studies have verified that biomaterials can promote the cell behavior of human umbilical vein endothelial cells (10, 11, 13, 26, 27). In angiogenesis, complex and diverse cellular actions are related to cell proliferation and migration. NO is the major regulator of cell migration and angiogenesis, which serves many important functions in the cardiovascular system such as vasodilation, inhibition of vasoconstrictor influences and platelet adhesion to the vascular endothelium (anti-thrombotic), and scavenging of superoxide anions (anti-inflammatory) (11–13, 28). We found that eNOS protein expression and NO secretion were significantly upregulated in HDPCs cultured on CS substrate for 3 and 5 days compared with those in hDPCs cultured with Ctl. The secretion of NO through eNOS has been shown to play an important role in angiogenesis and vasculogenesis, which are indispensable processes for tissue growth (14, 15, 6
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29). It has been reported that knockout mice for eNOS show impaired angiogenesis in response to ischemia (16, 30). Therefore, the stimulation of the angiogenesis of hDPCs by CS cement is possibly NO-dependent, ie, the CS cement enhanced the expression of certain proangiogenic factors, such as vWF and Ang-1, and initiated downstream NO production. We examined the phosphorylation p38/MAPK known to respond to various exogenous stresses by hyperosmolality. High osmolality acutely and fleetingly activated p38 and dephosphorylated JNK and ERK (17, 31). P38 has been shown to underlie cellular responses toward hyperosmotic stress (18, 22). A previous study suggested a common mechanism of p38/MAPK activation by high osmolality, possibly originating from cytoskeletal alterations due to cell shrinkage, as proposed previously in a report involving the Rac protein (19, 32). Our findings demonstrated Si ions released from CS cement and excited of osmolality to activate p38 hDPCs. Treatment of primary cells with SB203580 reduced the phosphorylation of p38. vWF is an important protein involved in coagulation and thrombus formation. After synthesis, it is found in secretory granules called Weibel-Palade bodies, and in vessels, it is released both constitutively and in a regulated manner (21, 33). Ang-1 is another family of growth factors that plays an important role in vascular development (22, 34). Moreover, NO may generally mediate proangiogenic activities of angiopoietin family members (20, 34). In this study, we tried to consider the possible angiogenesis induction mechanism by CS cement. Our data showed that CS cement had a higher potential to induce vWF and Ang-1 expression than the Ctl during angiogenesis. Furthermore, vWF and Ang-1 expression results indicated that SB203580 inhibitor significantly decreased the expression level when hDPCs were cultured with CS cement for all time points. This observation was in agreement with previous descriptions suggesting MAPK to be associated with CS-induced proliferation in hDPCs (20), particularly in the case of p38 in angiogenic differentiation. In conclusion, this study showed that CS cement was able to activate the p38 pathway in hDPCs cultured in vitro. Moreover, Si was shown to increase 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 was decreased. These findings verified that the p38 pathway plays a key role in regulating the angiogenic behavior of hDPCs cultured on CS cement. More specifically, we conclude that CS cement stimulates both proliferation and angiogenesis of hDPCs, at least partially via activation of the p38 pathway.
Acknowledgments The authors deny any conflicts of interest related to this study.
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Basic Research 7. Chiang TY, Ding SJ. Comparative physicochemical and biocompatible properties of radiopaque dicalcium silicate cement and mineral trioxide aggregate. J Endod 2010;36:1683–7. 8. Hung CJ, Kao CT, Shie MY, Huang TH. Comparison of host inflammatory responses between calcium-silicate base material and intermediate restorative material. J Dent Sci http://dx.doi.org/10.1016/j.jds.2013.08.002. 9. Shie MY, Chang HC, Ding SJ. Effects of altering the Si/Ca molar ratio of a calcium silicate cement on in vitro cell attachment. Int Endod J 2012;45:337–45. 10. Shie MY, Ding SJ, Chang HC. The role of silicon in osteoblast-like cell proliferation and apoptosis. Acta Biomater 2011;7:2604–14. 11. Zhai W, Lu H, Wu C, et al. Stimulatory effects of the ionic products from Ca-Mg-Si bioceramics on both osteogenesis and angiogenesis in vitro. Acta Biomater 2013;9: 8004–14. 12. Zhai W, Lu HX, Chen L, et al. Silicate bioceramics induce angiogenesis during bone regeneration. Acta Biomater 2012;8:341–9. 13. Li H, Chang J. Stimulation of proangiogenesis by calcium silicate bioactive ceramic. Acta Biomater 2013;9:5379–89. 14. Lai CF, Chaudhary L, Fausto A, et al. Erk is essential for growth, differentiation, integrin expression, and cell function in human osteoblastic cells. J Biol Chem 2001; 276:14443–50. 15. Du R, Wu T, Liu W, et al. 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. J Endod 2013;39:1023–9. 16. Huang TH, Ding SJ, Hsu TC, Kao CT. Effects of mineral trioxide aggregate (MTA) extracts on mitogen-activated protein kinase activity in human osteosarcoma cell line (U2OS). Biomaterials 2003;24:3909–13. 17. Chen CL, Kao CT, Ding SJ, et al. Expression of the inflammatory marker cyclooxygenase-2 in dental pulp cells cultured with mineral trioxide aggregate or calcium silicate cements. J Endod 2010;36:465–8. 18. Junttila MR, Li S-P, Westermarck J. Phosphatase-mediated crosstalk between MAPK signaling pathways in the regulation of cell survival. FASEB J 2008;22:954–65. 19. Brigotti M, Petronini PG, Carnicelli D, et al. Effects of osmolarity, ions and compatible osmolytes on cell-free protein synthesis. Biochem J 2003;369:369–74. 20. Shie MY, Ding SJ. Integrin binding and MAPK signal pathways in primary cell responses to surface chemistry of calcium silicate cements. Biomaterials 2013;34: 6589–606.
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21. Bi CWC, Xu L, Tian XY, et al. Fo Shou San, an ancient chinese herbal decoction, protects endothelial function through increasing endothelial nitric oxide synthase activity. PLoS ONE 2012;7:e51670. 22. Mavrogonatou E, Kletsas D. Differential response of nucleus pulposus intervertebral disc cells to high salt, sorbitol, and urea. J Cell Physiol 2011;227: 1179–87. 23. Zhang N, Molenda J, Fournelle J, et al. Effects of pseudowollastonite (CaSiO3) bioceramic on in vitro activity of human mesenchymal stem cells. Biomaterials 2010; 31:7653–65. 24. Schr€oder HC, Wang XH, Wiens M, et al. Silicate modulates the cross-talk between osteoblasts (SaOS-2) and osteoclasts (RAW 264.7 cells): inhibition of osteoclast growth and differentiation. J Cell Biochem 2012;113:3197–206. 25. Ding SJ, Shie MY, Wei CK. In vitro physicochemical properties, osteogenic activity, and immunocompatibility of calcium silicate–gelatin bone grafts for load-bearing applications. ACS Appl Mater Interfaces 2011;3:4142–53. 26. Leu A, Stieger SM, Dayton P, et al. Angiogenic response to bioactive glass promotes bone healing in an irradiated calvarial defect. Tissue Eng Part A 2009; 15:877–85. 27. Hoppe A, G€uldal NS, Boccaccini AR. A review of the biological response to ionic dissolution products from bioactive glasses and glass-ceramics. Biomaterials 2011;32:2757–74. 28. Fukumura D, Kashiwagi S, Jain RK. The role of nitric oxide in tumour progression. Nat Rev Cancer 2006;6:521–34. 29. Duda DG, Fukumura D, Jain RK. Role of eNOS in neovascularization: NO for endothelial progenitor cells. Trends Mol Med 2004;10:143–5. 30. Cooke JP, Losordo DW. Nitric oxide and angiogenesis. Circulation 2002;105:2133–5. 31. Sheikh-Hamad D, Gustin MC. MAP kinases and the adaptive response to hypertonicity: functional preservation from yeast to mammals. Am J Physiol Renal Physiol 2004;287:F1102–10. 32. Uhlik MT, Abell AN, Johnson NL, et al. Rac–MEKK3–MKK3 scaffolding for p38 MAPK activation during hyperosmotic shock. Nat Cell Biol 2003;5:1104–10. 33. Williamson MR, Black R, Kielty C. PCL-PU composite vascular scaffold production for vascular tissue engineering: attachment, proliferation and bioactivity of human vascular endothelial cells. Biomaterials 2006;27:3608–16. 34. Miller TW, Isenberg JS, Roberts DD. Molecular regulation of tumor angiogenesis and perfusion via redox signaling. Chem Rev 2009;109:3099–124.
P38 in CS-induced Pulp Cell Angiogenesis
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Role of the P38 Pathway in Calcium Silicate Cement–induced Cell Viability and Angiogenesis-related Proteins of Human Dental Pulp Cell In Vitro Ming-Yung Chou, DDS, MD, PhD,*† Chia-Tze Kao, DDS, MD, PhD,*† Chi-Jr Hung, DDS, MD,*† Tsui-Hsien Huang, DDS, MD, PhD,*† Shu-Ching Huang, DDS, MD,*† Ming-You Shie, PhD,‡* and Buor-Chang Wu, DDS, MD*† Abstract Introduction: This study investigated that calcium silicate (CS) cement may influence the behavior of human dental pulp cells (hDPCs) via mitogen-activated protein kinase pathway, in particular p38. We have addressed that Si ion released from CS cement can influence osmolarity in the medium, which may stimulate hDPC viability and induce angiogenesis-related proteins through stimulation of the nitric oxide synthase and nitric oxide secretion. Methods: The hDPCs was cultured with CS cement to angiogenesis. Then, cell viability, ion concentration, osmolality, nitric oxide secretion, the von Willebrand factor, and angiopoietin-1 protein expression were examined. Results: CS cement elicited a significant (P < .05) increase of 15%, 20%, and 19% in viability compared with control on days 1, 3, and 5 of cell seeding, respectively. The CS cement consumed calcium and phosphate ions and released more Si ions in medium. The CS significantly (P < .05) increased the osmolality to 303.52 3.07, 315.03 5.80, and 319.95 4.68 mOsm/kg for 1, 3, and 5 days, respectively. P38 was activated through phosphorylation; the phosphorylation kinase was investigated in our cell system after culture with CS cement. Moreover, expression levels for angiopoietin-1 and von Willebrand factor in hDPCs on CS cement were higher than those of the CS + p38 inhibitor (SB203580) group (P < .05) at all of the analyzed time points. Conclusions: This study showed that CS cement was able to activate the p38 pathway in hDPCs cultured in vitro. Moreover, Si was shown to increase 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 was decreased. These findings verified that the p38 pathway plays a key role in regulating the angiogenic
behavior of hDPCs cultured on CS cement. (J Endod 2013;-:1–7)
Key Words Angiogenesis, calcium silicate cement, human dental pulp cell, osmolality, p38/MAPK
M
ineral trioxide aggregate (MTA) has been widely and successfully used with several clinical applications in endodontics (1). This material, which received approval in 1998, is basically a mixture of 75% Portland cement, 20% Bi2O3, and 5% gypsum (1). Not only does MTA have good biocompatibility (2), it has also been proven to enhance hard-tissue formation (3, 4). In dentistry, calcium silicate (CS)– based cements have been formulated into dentin replacement restorative materials (5). In a previous study, a quick-setting CS (Ca2SiO4) cement showed an advantageously shortened setting time, excellent bioactivity, and good biocompatibility (6–8). Moreover, we recently showed that CS cement stimulates the proliferation and differentiation of MG63 (7) and human dental pulp cells (hDPCs) in vitro (9). The inorganic ions released from silicate-based materials have been found to stimulate bone cell proliferation and osteogenic protein secretion (10, 11). 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 (11–13). However, the mechanism by which Si promotes cell behavior, including cell differentiation, remains unclear. Mitogen-activated protein kinase (MAPK) cascades play an important role in the modulation of gene expression and cytoplasmic signaling. In mammalian cells, there are 3 subfamilies of the MAPK family, namely extracellular signal-regulated kinase 1/2 (ERK1/2), c-jun N-terminal kinase (JNK), and p38. ERK1/2 preferentially regulates cell growth and differentiation (14, 15). In this regard, MTA has been shown to enhance proliferation of a human osteosarcoma cell line (16) and hDPCs via ERK1/2 activation (17). JNK signaling is mainly activated by cytokines and stress, and the p38 MAPK pathway can be activated by environmental stress, such as osmotic stress (18). 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 (19) and induces cell apoptosis.
From the *School of Dentistry and ‡Institute of Oral Science, Chung Shan Medical University; and †Department of Dentistry, Chung Shan Medical University Hospital, Taichung City, Taiwan. Ming-You Shie, and Buor-Chang Wu contributed equally to this work. Supported by a grant from the Chung Shan Medical University Hospital under the project CSH-2010-C-025 and the National Science Council grants (NSC 01-2314-B040-011-MY3) of Taiwan. Address requests for reprints to Dr Ming-You Shie, Institute of Oral Science, Chung Shan Medical University, Taichung City, Taiwan. E-mail address: eviltacasi@gmail. com 0099-2399/$ - see front matter Copyright ª 2013 American Association of Endodontists. http://dx.doi.org/10.1016/j.joen.2013.09.041
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P38 in CS-induced Pulp Cell Angiogenesis
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Figure 1. (A) PrestoBlue assay for hDPCs viability cultured on various specimens at various time points. CS cement elicited a significant (P < .05) increase in OD compared with Ctl for all time points. (B) Osmolality of cell culture medium at various time points. (C) Ca, Si, and P ion concentration of DMEM after culturing for different times. Data represent means standard deviation (n = 6). *Statistically significant difference from Ctl.
Taking these factors together, we hypothesized that CS cement may influence the behavior of hDPCs via the MAPK pathway, in particular the p38 pathway. The aim of this study was to verify the effect of CS cement contact on cell viability and endothelial-related protein expression through MAPK/p38 stimulation. In addition, this study was to investigate the specific role of the von Willebrand factor (vWF) and angiopoietin-1 (Ang-1) protein expression on Si ioninduced angiogenesis. Thus, the roles of MAPK/p38 involved in the regulation of angiogenic differentiation of hDPCs were also investigated.
Materials and Methods Specimen Preparation The CS cement was made according to our previously reported laboratory procedures (11). Appropriate amounts of CaO (65%; Showa, Tokyo, Japan), SiO2 (25%; High Pure Chemicals, Saitama, Japan), and Al2O3 (5%; Sigma-Aldrich, St Louis, MO) powders were mixed by a conditioning mixer (ARE-250; Thinky, Tokyo, Japan). After sintering at 1400 C for 2 hours, the granules were ball milled in EtOH by using a centrifugal ball mill (Retsch S 100, Hann, Germany) and then dried in an oven. CS cement was mixed according to the same liquid/ powder ratio of 0.3 mL/g. After mixing, the cement fully covered each well of the 24-well plate (GeneDireX, Las Vegas, NV) to a thickness of 2
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2 mm, and the samples were stored in an incubator at 100% relative humidity and 37 C for 1 day. Before cell experiments, all specimens were sterilized by immersion in 75% ethanol, followed by exposure to ultraviolet light for 1 hour.
HDPC Isolation and Culture The hDPCs were freshly derived from caries-free, intact premolars that were extracted for orthodontic treatment purposes, as described previously (20). The cells were freshly derived from a caries-free intact premolar that was extracted for orthodontic treatment. 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 phosphatebuffered saline (Caisson, North Logan, UT) solution and digested in 0.1% collagenase type I (Sigma-Aldrich) for 30 minutes. After being transferred to a new plate, the cell suspension was cultured in Dulbecco modified Eagle medium (DMEM) (Caisson), supplemented with 20% fetal bovine serum (Caisson) and 1% penicillin (10,000 U/mL)/streptomycin (10,000 mg/mL) (Caisson) in a humidified atmosphere with 5% CO2 at 37 C, and the medium was changed every 3 days. The cells were subcultured through successive passaging at a 1:3 ratio until they were used for experiments (passages 3–8). The JOE — Volume -, Number -, - 2013
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Figure 2. (A) NO and (B) nitric oxide synthase (NOS3, eNOS) secretion by hDPCs in the presence of CS cement were higher than Ctl (P < .05). Data represent means standard deviation (n = 6). *Statistically significant difference from Ctl.
angiogenic induction reagent (2% fetal bovine serum, 1% penicillin [10,000 U/mL]/streptomycin [10,000 mg/mL], and 50 ng/mL vascular endothelial growth factor [Prospec, East Brunswick, NJ]) was mixed with DMEM.
Cell Viability Before the in vitro cell experiments, all cement specimens were sterilized by soaking in 75% ethanol, followed by exposure to ultraviolet light for 1 hour. After cell direct seeding on different substrates for various time periods, cell viability was evaluated by using the PrestoBlue assay (Invitrogen, Grand Island, NY), which is based on the detection of mitochondrial activity. Thirty microliters PrestoBlue solution and 300 mL DMEM were added to each well, followed by 30 minutes of incubation. After incubation, 100 mL of the solution in each well was transferred to a 96-well enzyme-linked immunosorbent assay (ELISA) plate. The plates were read in a Sunrise microtiter plate reader (Tecan Austria Gesellschaft, Salzburg, Austria) at 570 nm, with a reference wavelength of 600 nm. The results were obtained in triplicate from 3 separate experiments in terms of optical density (OD). Cells cultured on the tissue culture plate without the cement were used as a control (Ctl). Osmolality Measurement After 1, 3, and 5 days of culture, medium was collected for osmolality measurement. The results were obtained in triplicate from 3 separate experiments. The precise osmolality of the various culture media was determined directly on 20 mL medium by using a Model 3300 advanced micro-osmometer (Advanced Scientific Instruments, Norwood, MA). Ion Concentration The Ca, Si, and P ion concentration on DMEM was analyzed by using an inductively coupled plasma-atomic emission spectrometer (PerkinElmer OPT 1MA 3000DV, Shelton, CT) after culture for 1, 3, and 5 days. Three samples were measured for each data point. The results were obtained in triplicate from 3 separate samples for each test. Detection of Endothelial Nitric Oxide Synthase After 1, 3, and 5 days of culture, the hDPCs were processed for measurement of endothelial nitric oxide synthase (eNOS) by using a commercially available ELISA kit (Abcam, Cambridge, MA). Following the manufacturer’s instructions, we used the 2-hour assay, which has JOE — Volume -, Number -, - 2013
higher sensitivity. The reaction was terminated by the addition of stop solution and read at 450 nm by using a Sunrise microtiter reader. This experiment was repeated independently 3 times.
Nitric Oxide Colorimetric Assay After 1, 3, and 5 days of culture, culture medium was collected for NO colorimetric assay. The concentrations of NO in the culture medium were measured with the NO Detection Kit (BioVision, San Francisco, CA) in accordance with the manufacturer’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 by using Sunrise microtiter plate reader (21). Western Blotting Western blotting was performed on cells cultured on different cement specimens for a predetermined time to evaluate p38 and phospho-p38 levels. Cells were lysed in NP-40 lysis buffer (Invitrogen) at 4 C for 30 minutes, and the lysates were centrifuged at 13,000g. The protein concentrations of the lysates were measured by using a Bio-Rad DC Protein Assay kit (Richmond, CA), and proteins (30 mg) were resolved by using standard sodium dodecylsulfate–polyacrylamide gel electrophoresis and transferred to polyvinylidene difluoride membranes. After blocking with 5% bovine serum albumin (Sigma-Aldrich) in TBS-T (Tris-buffer saline containing 0.05% Tween-20) for 1 hour, the membranes were incubated with primary antibodies against b-actin, p38, and phospho-p38 (GeneTex, San Antonio, TX). The signal inhibitor used for the p38 was SB203580 (50 mmol/L; Invitrogen). A horseradish peroxidase–conjugated secondary antibody was subsequently added, and the proteins were visualized with enhancement by using enhanced chemiluminescent detection kits (Invitrogen). The stained bands were scanned and quantified by using a densitometer (Syngene Bioimaging System, Frederick, MD) and Scion Image software (Frederick, MD). Protein expression levels were normalized to the actin band for each sample. Intracellular Ang-1 and vWF Measurement The production of Ang-1 and vWF was quantified by using ELISA kits (Abcam; catalog no. ab99970 and ab108918) according to the manufacturer’s instructions. Briefly, hDPCs were cultured on substrates for 1, 3, and 5 days, and proteins from whole cell lysates were collected and quantified by using the ELISA kit. P38 in CS-induced Pulp Cell Angiogenesis
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Figure 3. Results of Western blot. (A) P38 activity in hDPCs after they were cultured on different substrates with or without pretreatment with SB203580 (50 mm). P38 activity is presented as ratio of phosphorylated p38 normalized to total p38. Data represent means standard deviation (n = 3). (B) Pp38/p38 ratio of untreated groups was used as 100% reference level. *Statistically significant difference from Ctl. pp38, phospho-38.
Statistical Analysis A one-way analysis of variance statistical analysis was used to evaluate the significance of the differences between the groups in each experiment. A Scheffe multiple comparison test was used to determine the significance of the deviations in the data for each specimen. In all cases, the results were considered statistically significant with P values < .05.
Results Cell Viability The cell viability of hDPCs grown on CS cement and Ctl is shown in Figure 1A. PrestoBlue showed that the number of cells cultured on CS cement surfaces had a significantly (P < .05) higher value amount at all culture times when compared with Ctl. CS elicited a significant (P < .05) increase of 15%, 20%, and 19% in OD compared with Ctl on days 1, 3 and 5 of cell seeding, respectively. 4
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Osmolality Variations of DMEM osmolality after the specified cultured time periods are shown in Figure 1B. The osmolality of the Ctl was similar for all time points. The CS was significant (P < .05); it increased the osmolality to 303.52 3.07, 315.03 5.80, and 319.95 4.68 mOsm/kg for 1, 3, and 5 days, respectively. Ion Concentration Variations of DMEM Ca, Si, and P ion concentrations after different cultured time periods are shown in Figure 1C. In CS cement, Ca ion concentration of the medium increased after 1 day of culture and then decreased to approximately 1.1 mmol/L after 3 days, which was lower than the baseline Ca concentration of DMEM (1.8 mmol/L) (P < .05). No significant difference was found between Ctl in Ca ion concentration for all time points. Si concentrations increased with increasing incubation time points. Si ion concentration was in the range of 1.3, 2.0, and 2.7 mmol/L at 1, 3, and 5 days, respectively. As for P ion concentration of the medium, it decreased after 1 day of culture and then decreased to JOE — Volume -, Number -, - 2013
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Figure 4. (A) Ang-1 and (B) vWF expression of hDPCs cultured on CS cement without and with SB203580 (50 mm) for different days. Protein secretion of untreated groups was used as 100% reference level. *Statistically significant difference from Ctl.
approximately 0.5 mmol/L after 5 days, and there was no difference in P ion concentration of Ctl for all time points.
eNOS and NO Detection The expression of eNOS and NO was also detected, and the results are shown in Figure 2. The eNOS protein expression in hDPCs cultured with CS substrates was nearly 1.4 times higher than in hDPCs cultured with Ctl (Fig. 2A). In addition, CS cement elicited a significant (P < .05) increase of 21% and 19% in NO synthesis compared with Ctl on days 3 and 5, respectively (Fig. 2B). These results indicate that the CS cement up-regulated the expression of eNOS and subsequently stimulated NO synthesis. P38 Pathway MAPK family was activated as a response of cells against several types of stress stimulation. Because p38 was activated through phosphorylation, the phosphorylation kinase was investigated in our cell sysJOE — Volume -, Number -, - 2013
tem after culture with CS cement (Fig. 3). P38 is strongly connected to hyperosmotic stress and has been found to be acutely activated in inorganic salt-treated cells (22); incubation of hDPCs in CS cement resulted in a time-dependent increase in pp38. Phosphorylation started to increase 13%, 21%, and 27% in hDPCs on CS compared with Ctl on days 1, 3, and 5, respectively (P < .05). Addition of the p38 inhibitor SB203580 to the hDPC culture resulted in inhibition of CS cement– induced p38 phosphorylation. After pretreatment with the p38 inhibitor, the activated p38 expression levels were reduced notably in all groups. The p38 synthesis was decreased 56% in hDPCs cultured on CS, which was more than Ctl (40%) on day 1 (Fig. 3B).
Effects of P38 Inhibitor on Angiogenesis Relative expression levels of Ang-1 and vWF protein secretion in hDPCs cultured on CS with the addition of the p38 inhibitor SB203580 (50 mmol/L) were evaluated at all time points. The Ang-1 expression in CS on days 1, 3, and 5 was enhanced 1.22, 1.26, and 1.31 times, respectively, as compared with that of Ctl (Fig. 4A). The P38 in CS-induced Pulp Cell Angiogenesis
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Basic Research vWF results shown in Figure 4B can be seen to be similar to Ang-1, both showing a time-dependent up-regulation in the CS group, which was significantly higher than that of controls for all time points (P < .05). Moreover, expression levels for Ang-1 and vWF in hDPCs on CS were higher than those of the CS + SB203580 group (P < .05) at all of the analyzed time points. Taken together, these results support a CSmediated up-regulation of angiogenesis-dependent protein occurring via the p38 pathway.
Discussion This study was designed to demonstrate the molecular effects of CS, a component of some novel capping agents used to preserve the viability of dental pulp tissue during reparative dentin formation. CS-based materials have been found to foster hDPC and hMSC adhesion, growth, and differentiation and have been used as implant materials for bone repair and regeneration (20). ERK/MAPK and p38/MAPK signaling pathways go through phosphorylation in cells cultured on CS substrates, and their inhibitors significantly reduce cell adhesion, proliferation, and differentiation as assessed according to total DNA and alkaline phosphatase activity (20). However, little information is known about the mechanisms by which CS identity regulates cell behavior and protein secretion in general, and much less is known about how they regulate the angiogenesis differentiation of hDPCs specifically. In this study, PrestoBlue analysis revealed differences between CS and Ctl. In all cases, absorbance values were higher for hDPCs cultured on CS in comparison with Ctl, and these differences were almost always significant (P < .05). Previous studies in our laboratory have demonstrated MG63 cell viability on 20 wt% Bi2O3-containing dicalcium silicate cement to be higher than that on white MTA and Ctl at all culture times (1, 7). Zhang et al (23) found that soluble factors from CaSiO3 substrates may be more important for proliferation and osteogenic differentiation in a growth medium (1). The hydrolysis of CS cement on immersion, and Si ion was dissolved during incubation (2, 9). Taking cell functions into account, the appropriate Si released from silicate-based materials may support cell behaviors (3, 4, 24). The current CS offers the ability of controlling the rate of soluble Si and Ca ions, which can promote cell adhesion and proliferation (5, 9). The bioactivity of silicate-based materials indicates 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 (6–8, 25). In addition, dissolution ions from the cement may change the osmolality of a culture medium. The osmolality of the CS group examined during the culturing periods in this study was in the range of the physiological osmolality for cells (280–320 mOsm/kg) (7, 10). In an earlier study, the relationship between the Si ion concentration and osmolality was demonstrated (9, 10). Angiogenesis is a critical physiology event in bone formation. Several studies have verified that biomaterials can promote the cell behavior of human umbilical vein endothelial cells (10, 11, 13, 26, 27). In angiogenesis, complex and diverse cellular actions are related to cell proliferation and migration. NO is the major regulator of cell migration and angiogenesis, which serves many important functions in the cardiovascular system such as vasodilation, inhibition of vasoconstrictor influences and platelet adhesion to the vascular endothelium (anti-thrombotic), and scavenging of superoxide anions (anti-inflammatory) (11–13, 28). We found that eNOS protein expression and NO secretion were significantly upregulated in HDPCs cultured on CS substrate for 3 and 5 days compared with those in hDPCs cultured with Ctl. The secretion of NO through eNOS has been shown to play an important role in angiogenesis and vasculogenesis, which are indispensable processes for tissue growth (14, 15, 6
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29). It has been reported that knockout mice for eNOS show impaired angiogenesis in response to ischemia (16, 30). Therefore, the stimulation of the angiogenesis of hDPCs by CS cement is possibly NO-dependent, ie, the CS cement enhanced the expression of certain proangiogenic factors, such as vWF and Ang-1, and initiated downstream NO production. We examined the phosphorylation p38/MAPK known to respond to various exogenous stresses by hyperosmolality. High osmolality acutely and fleetingly activated p38 and dephosphorylated JNK and ERK (17, 31). P38 has been shown to underlie cellular responses toward hyperosmotic stress (18, 22). A previous study suggested a common mechanism of p38/MAPK activation by high osmolality, possibly originating from cytoskeletal alterations due to cell shrinkage, as proposed previously in a report involving the Rac protein (19, 32). Our findings demonstrated Si ions released from CS cement and excited of osmolality to activate p38 hDPCs. Treatment of primary cells with SB203580 reduced the phosphorylation of p38. vWF is an important protein involved in coagulation and thrombus formation. After synthesis, it is found in secretory granules called Weibel-Palade bodies, and in vessels, it is released both constitutively and in a regulated manner (21, 33). Ang-1 is another family of growth factors that plays an important role in vascular development (22, 34). Moreover, NO may generally mediate proangiogenic activities of angiopoietin family members (20, 34). In this study, we tried to consider the possible angiogenesis induction mechanism by CS cement. Our data showed that CS cement had a higher potential to induce vWF and Ang-1 expression than the Ctl during angiogenesis. Furthermore, vWF and Ang-1 expression results indicated that SB203580 inhibitor significantly decreased the expression level when hDPCs were cultured with CS cement for all time points. This observation was in agreement with previous descriptions suggesting MAPK to be associated with CS-induced proliferation in hDPCs (20), particularly in the case of p38 in angiogenic differentiation. In conclusion, this study showed that CS cement was able to activate the p38 pathway in hDPCs cultured in vitro. Moreover, Si was shown to increase 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 was decreased. These findings verified that the p38 pathway plays a key role in regulating the angiogenic behavior of hDPCs cultured on CS cement. More specifically, we conclude that CS cement stimulates both proliferation and angiogenesis of hDPCs, at least partially via activation of the p38 pathway.
Acknowledgments The authors deny any conflicts of interest related to this study.
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21. Bi CWC, Xu L, Tian XY, et al. Fo Shou San, an ancient chinese herbal decoction, protects endothelial function through increasing endothelial nitric oxide synthase activity. PLoS ONE 2012;7:e51670. 22. Mavrogonatou E, Kletsas D. Differential response of nucleus pulposus intervertebral disc cells to high salt, sorbitol, and urea. J Cell Physiol 2011;227: 1179–87. 23. Zhang N, Molenda J, Fournelle J, et al. Effects of pseudowollastonite (CaSiO3) bioceramic on in vitro activity of human mesenchymal stem cells. Biomaterials 2010; 31:7653–65. 24. Schr€oder HC, Wang XH, Wiens M, et al. Silicate modulates the cross-talk between osteoblasts (SaOS-2) and osteoclasts (RAW 264.7 cells): inhibition of osteoclast growth and differentiation. J Cell Biochem 2012;113:3197–206. 25. Ding SJ, Shie MY, Wei CK. In vitro physicochemical properties, osteogenic activity, and immunocompatibility of calcium silicate–gelatin bone grafts for load-bearing applications. ACS Appl Mater Interfaces 2011;3:4142–53. 26. Leu A, Stieger SM, Dayton P, et al. Angiogenic response to bioactive glass promotes bone healing in an irradiated calvarial defect. Tissue Eng Part A 2009; 15:877–85. 27. Hoppe A, G€uldal NS, Boccaccini AR. A review of the biological response to ionic dissolution products from bioactive glasses and glass-ceramics. Biomaterials 2011;32:2757–74. 28. Fukumura D, Kashiwagi S, Jain RK. The role of nitric oxide in tumour progression. Nat Rev Cancer 2006;6:521–34. 29. Duda DG, Fukumura D, Jain RK. Role of eNOS in neovascularization: NO for endothelial progenitor cells. Trends Mol Med 2004;10:143–5. 30. Cooke JP, Losordo DW. Nitric oxide and angiogenesis. Circulation 2002;105:2133–5. 31. Sheikh-Hamad D, Gustin MC. MAP kinases and the adaptive response to hypertonicity: functional preservation from yeast to mammals. Am J Physiol Renal Physiol 2004;287:F1102–10. 32. Uhlik MT, Abell AN, Johnson NL, et al. Rac–MEKK3–MKK3 scaffolding for p38 MAPK activation during hyperosmotic shock. Nat Cell Biol 2003;5:1104–10. 33. Williamson MR, Black R, Kielty C. PCL-PU composite vascular scaffold production for vascular tissue engineering: attachment, proliferation and bioactivity of human vascular endothelial cells. Biomaterials 2006;27:3608–16. 34. Miller TW, Isenberg JS, Roberts DD. Molecular regulation of tumor angiogenesis and perfusion via redox signaling. Chem Rev 2009;109:3099–124.
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