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The Role of Integrin αv in Proliferation and Differentiation of Human Dental Pulp Cell Response to Calcium Silicate Cement
Basic Research—Biology
The Role of Integrin av in Proliferation and Differentiation of Human Dental Pulp Cell Response to Calcium Silicate Cement Chi-Jr Hung, DDS, MD, PhD,*† Hsin-I. Hsu, MS,‡ Chi-Chang Lin, PhD,§ Tsui-Hsien Huang, DDS, MD, PhD,*† Buor-Chang Wu, DDS, MD, PhD,*† Chia-Tze Kao, DDS, MD, PhD,*† and Ming-You Shie, PhD* Abstract Introduction: It has been proved that integrin av activity is related to cell proliferation, differentiation, migration, and organ development. However, the biological functions of integrin av in human dental pulp cells (hDPCs) cultured on silicate-based materials have not been explored. The aim of this study was to investigate the role of integrin av in the proliferation and odontogenic differentiation of hDPCs cultured with the effect of calcium silicate (CS) cement and b-tricalcium phosphate (TCP) cement. Methods: In this study, hDPCs were cultured on CS and TCP materials, and we evaluated fibronectin (FN) secretion and integrin av expression during the cell attachment stage. After small interfering RNA transfection targeting integrin av, the proliferation and odontogenesis differentiation behavior of hDPCs were analyzed. Results: The results indicate that CS releases Si ion–increased FN secretion and adsorption, which promote cell attachment more effectively than TCP. The CS cement facilitates FN and av subintegrin expression. However, the FN adsorption and integrin expression of TCP are similar to that observed in the control dish. Integrin av small interfering RNA inhibited odontogenic differentiation of hDPCs with the decreased formation of mineralized nodules on CS. It also down-regulated the protein expression of multiple markers of odontogenesis and the expression of dentin sialophosphoprotein protein. Conclusions: These results establish compositiondependent differences in integrin binding and its effectiveness as a mechanism regulating cellular responses to biomaterial surface. (J Endod 2014;40:1802–1809)
From the *School of Dentistry and ‡Institute of Oral Science, Chung Shan Medical University; †Department of Stomatology, Chung Shan University Hospital; and §Department of Chemical and Materials Engineering, Tunghai University, Taichung City, Taiwan. Address requests for reprints to Dr Chia-Tze Kao, School of Dentistry, Chung Shan Medical University, Taichung City, Taiwan or Dr Ming-You Shie, Department of Chemical and Materials Engineering, Tunghai University, Taichung City, Taiwan. E-mail addresses: [email protected] or [email protected] 0099-2399/$ - see front matter Copyright ª 2014 American Association of Endodontists. http://dx.doi.org/10.1016/j.joen.2014.07.016
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Key Words Calcium silicate cement, fibronectin, human dental pulp cell, integrin av, odontogenesis
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ineral trioxide aggregate (MTA) has long been used as a root-end dental filling material and is widely used in several clinical applications of endodontics (1). MTA received approval for general medical use in 1998 and is comprised of a mixture of 75% Portland cement (CaO and SiO2), 20% Bi2O3, and 5% gypsum (1). Not only does MTA have good biocompatibility (2), it also has been proven to promote osteogenesis in human dental pulp cells (hDPCs) (3, 4). In dentistry, calcium silicate (CS)-based cements have been formulated into dentin replacement restorative materials (5), but there is reason to believe its performance can be made more effective by decreasing the setting time and improving the handling properties in the clinical setting (6). In our previous study, we produced a fast-setting CS cement that contains CaO, SiO2, and Al2O3, which were shown to have a reduced setting time (7). In addition, CS cement not only exhibits good osteoconduction effects (8, 9) but also reduces inflammation markers in primary hDPCs (10, 11) and in vivo (12). The release of Si concentrations from silicate-based materials influences the behavior of different cell types, such as inhibiting osteoclastogenesis in macrophages (13) and angiogenesis in hDPCs (14, 15). In addition, the amount of Si ions in CS-based materials can affect the adsorption of various types of extracellular matrices (ECMs) such as collagen I, fibronectin (FN), and vitronectin and promote the up-regulation of mitogen-activated protein kinase/ extracellular signal-regulated protein kinase 1/2 (MAPK/ERK 1/2) and MAPK/p38, signaling the pathway more effectively than Ca components (11, 16). Integrins are the major transmembrane receptors for cell adhesion to the ECM (17). All integrins are heterodimers composed of noncovalently linked alpha and beta subunits. Among the receptor systems, integrins bind to specific ECM components, such as collagen, vitronectin, and FN, influencing cell adhesion behavior (17, 18). Integrin-mediated interactions are vital to the maintenance of normal cell functions because they mediate the interaction between individual cells and the ECM and therefore have important implications for cell adhesion (19), proliferation (20), and differentiation (11). In addition, integrin protein expression changed accordingly, with higher levels of a2b1 and avb3 subintegrins in the cells on the Si- and Ca-rich substrates, respectively, which were ascribed to the collagen-binding and FN-binding subintegrin on primary cells, respectively (11). Integrin av is essential for various biological functions, and it binds to and competes for the arginine-glycine-aspartic site on FN (21, 22). Recent studies have shown that integrin av is not only required for osteoblast proliferation (23) but also for activation of the focal adhesion kinase/ ERK/MAPK and phosphoinositide 3-kinase signaling pathways (24). These findings indicate that integrin av might be affected by osteogenic differentiation of cells. The odontogenic differentiation of hDPCs is a biological process similar to osteogenic differentiation. There are several proteins involved in bone and dentin mineralization, and they are similar but distinct. In comparison with bone, dentin has been shown to contain less osteopontin, bone sialoprotein, and osteocalcin but more dentin sialoprotein (DSP) and dentin matrix protein 1 (DMP-1) (25). Thus, we suggest integrin av may be included in the odontogenic differentiation of hDPCs.
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Basic Research—Biology Based on these factors, we hypothesize that CS cement may influence FN adsorption and accelerate adhesion protein formation, in particular, integrin av. Thus, the roles of integrin av involved in the regulation of odontogenic differentiation of hDPCs are also investigated in the present study, which investigates the specific role of DMP-1 and DSP protein expression on CS-induced odontogenesis. Additionally, small interfering RNA (siRNA) transfection is used to explore the integrin av level in hDPCs during the proliferation and differentiation stages.
Materials and Methods Specimen Preparation The CS cement used in this research was made according to our previously reported laboratory procedures (13). Appropriate amounts of CaO (65%; Sigma-Aldrich, St Louis, MO), SiO2 (25%; High Pure Chemicals, Saitama, Japan), and Al2O3 (5%, SigmaAldrich) powders were mixed. After sintering at 1400 C for 2 hours, the granules were ball milled in 99.5% EtOH using a centrifugal ball mill (Retsch S 100, Hann, Germany) for 12 hours and then dried in an oven. In addition, beta-tricalcium phosphate (TCP, Sigma-Aldrich) was compared with CS in this study. Before the preparation of the cement specimens, 50 mg powdered zeta potential was placed in 1 mL phosphate-buffered saline (PBS; Gibco, Langley, OK) and after soaking for 3 hours was measured by particle electrophoresis using the Zetasizer Nano ZS (Malvern Instruments, Worcestershire, UK). Three samples were measured for each powder condition, and the average is reported in this analysis. The CS and TCP powders were mixed according to the liquid/powder ratio of 0.35 mL/g and placed in a 24-well plate (GeneDireX, Las Vegas, NV) to a thickness of 2 mm fully covering each well; the samples were then stored in an incubator at 100% relative humidity and 37 C for 1 day. Before performing the cell experiments, all the 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 had been extracted for orthodontic treatment purposes as described previously (11). 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 immersed in PBS (Caisson, North Logan, UT) solution and digested in 0.1% collagenase type I (Sigma-Aldrich) for 30 minutes. After being transferred to a new cultured dish, the cell suspension was cultured in Dulbecco modified Eagle medium (DMEM, Caisson) containing 20% fetal bovine serum (GeneDireX) and 1% penicillin (10,000 U/mL)/streptomycin (10,000 mg/mL) (Caisson) and was kept in a humidified atmosphere with 5% CO2 at 37 C. The medium was changed every 3 days. The hDPCs were subcultured through successive passaging at a 1:3 ratio until they were used for experiments (passages 3–8). Cell Adhesion Assays The suspended cells were kept at a density of 1.5 104/specimen and directly seeded over each sample. Cell cultures were incubated at 37 C in a 5% CO2 atmosphere. After being cultured for different lengths of time (ie, 1, 3, and 6 hours), cell adhesion ability was evaluated using the PrestoBlue assay (Invitrogen, Grand Island, NY). Briefly, each specimen was filled with medium with a 1:9 ratio of PrestoBlue in fresh DMEM and incubated at 37 C for 30 minutes. The solution in each well JOE — Volume 40, Number 11, November 2014
was then transferred to a new 96-well plate and read in a multiwell spectrophotometer (Hitachi, Tokyo, Japan) at 570 nm with a reference wavelength of 600 nm. The results were obtained in triplicate from 3 separate experiments for each test. Cells cultured on tissue culture plates without cement were used as a control (Ctl).
FN Secretion Cells were cultured on different substrates for 1, 3, and 6 hours, and the cell culture media were then collected and stored at room temperature. The enzyme-linked immunosorbent assay kits of human FN were obtained from Abcam (Cambridge, MA). Following the manufacturer’s instructions, we used a 3-hour assay, which has a high sensitivity. The reaction was terminated by the addition of stop solution and read at 450 nm using a multiwell spectrophotometer. FN Adsorption on Substrates After being cultured for different periods of time, the amount of FN secreted from cells onto the cement’s surface was analyzed using the enzyme-linked immunosorbent assay. The cells were detached using a trypsin-EDTA solution (Cassion) after being washed 3 times with cold PBS. Specimens were then washed 3 times with PBS containing 0.1% Tween 20 (PBS-T; Sigma-Aldrich, St Louis, MO) followed by blocking with 5% bovine serum albumin (Gibco) in PBS-T for 1 hour. Dilutions of primary antibodies were set at 1:500. After this procedure, samples were incubated with antihuman beta-actin or antihuman FN antibody (GeneTex, San Antonio, TX) for 3 hours at room temperature. Afterward, samples were washed 3 times with PBS-T for 5 minutes and incubated with horseradish peroxidase–conjugated secondary antibodies for 1 hour at room temperature with shaking. The samples were then washed 3 times with PBS-T for 10 minutes each, and then One-Step Ultra TMB substrate (Invitrogen) was added to the wells and developed for 30 minutes at room temperature in the dark; after this, an equal volume of 2M H2SO4 was added to stop and stabilize the oxidation reaction. The colored products were then transferred to new 96-well plates and read using a multiwell spectrophotometer at 450 nm with reference at 620 nm according to the manufacturer’s recommendations. All experiments were performed in triplicate. Additionally, beta-actin antibodies were used as a control. Western Blot Western blotting was performed on cells cultured on different cement specimens for a predetermined time to evaluate the differences in their protein 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 using a Bio-Rad DC Protein Assay kit (Richmond, CA), and the proteins (30 mg) were then resolved using standard sodium dodecyl sulfate–polyacrylamide gel electrophoresis and transferred to polyvinylidene difluoride membranes. After blocking with 5% bovine serum albumin (Sigma-Aldrich) in Tris-buffer saline containing 0.05% Tween 20 for 1 hour, the membranes were incubated with primary antibodies against beta-actin, integrin av (GeneTex), DMP-1, and DSP (Santa Cruz Biotechnology, Santa Cruz, CA). A horseradish peroxidase–conjugated secondary antibody was subsequently added, and the proteins were visualized with enhancement using enhanced chemiluminescent detection kits (Invitrogen). The stained bands were scanned and quantified using a densitometer (Syngene, Frederick, MD) and Scion Image software (Scion Corporation, Frederick, MD). Protein expression levels were normalized to the beta-actin band for each sample. The Role of Integrin av
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Figure 1. (A) Proliferation of hDPCs cultured on different substrates for various time points. (B) FN secretion and (C) adsorption from hDPCs after seeding on different substrates for 1, 3, and 6 hours. (D) Immunodetection of integrin protein expression in hDPCs cultured on various samples for 1, 3, and 6 hours. Integrin expression was quantified and normalized to beta-actin expression. *A significant difference (P < .05) compared with Ctl.
Ion Concentration The Ca and Si ion concentration on DMEM was analyzed using an inductively coupled plasma atomic emission spectrometer (OPT 1 MA 3000DV; Perkin-Elmer, Shelton, CT) after cell seeding for 1, 3, and 6 hours. Three samples were measured for each data point. The results were obtained in triplicate from 3 separate samples for each test. Preparation of Test Medium Containing Different Si Concentrations One gram of cement was immersed in 10 mL DMEM for 1 day, and the supernatants were passed through a 0.22-mm filter (Millipore, Billerica, MA) to obtain the cement extract. The Si ion concentration on extract was analyzed using an inductively coupled plasma atomic emission spectrometer. A detailed description of the dilution of the test medium with various Si ion concentrations is given elsewhere (13). The cement extract and DMEM were used to prepare 3 different media with various Si ion concentrations (0, 0.5, and 1 mmol/L). Then, FN secretion and cell adhesion of hDPCs after treatment with various Si concentrations were analyzed. siRNA Transfection For siRNA-mediated knockdown of integrin av, cells (wild type [WT]) were transfected with 50 nmol/L of either the targeting 1804
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or control siRNA (Genedirex) using Lipofectamine 2000 (Invitrogen) for 48 hours as recommended in the manufacturer’s instructions. The siRNAs targeting integrin av as well as the control nontargeting siRNAs were commercially available pools of 4 different siRNAs purchased from Santa Cruz Biotechnology. Cellular levels of the proteins specifically silenced were assessed using a Western blot test.
Proliferation Assay When cell proliferation was detected, it was then immediately measured through the incorporation of 5-ethynyl-20-deoxyuridine (EdU) with the Click-iT EdU Cell Proliferation Assay Kit (Invitrogen) and PrestoBlue assay. EdU solution with a final concentration of 105 mol/L was added into the culture medium and allowed to incubate overnight before the experiment. After being cultured for 1 day, the cells were fixed with 10% formalin and permeabilized using 0.5% Triton X-100. The EdU was stained according to the manufacturer’s protocol. Cells were also stained with 0.1 mg/mL 40 ,6-diamidino-2- phenylindole (Invitrogen) in 1 PBS for 30 minutes, which permitted the detection of all nuclei (blue). After washing 3 times with PBS-T, the cells were viewed under indirect immunofluorescence using a Zeiss Axioskop2 microscope (Carl Zeiss, Thornwood, NY). Images were analyzed with ImageJ software (National Institutes of Health, Bethesda, MD). JOE — Volume 40, Number 11, November 2014
Basic Research—Biology
Figure 2. (A) Ca and (B) Si ion concentration of DMEM after cell cultured for different times. *A significant difference (P < .05) compared with Ctl. (C) FN secretion and (D) hDPC adhesion after treatment with DMEM contained various concentrations. *A significant difference (P < .05) compared with 0 mmol/L.
Alkaline Phosphatase Assay The level of alkaline phosphatase (ALP) activity was determined on the third day after cell seeding. The process was as follows: the cells were lysed from discs using 0.2 % NP-40 and centrifuged for 10 minutes at 2000 rpm after washing with PBS. ALP activity was determined using p-nitrophenyl phosphate (SigmaAldrich) as the substrate. Each sample was mixed with p-nitrophenyl phosphate in 1 mol/L diethanolamine buffer for 15 minutes; after this, the reaction was stopped by the addition of 5 N NaOH and quantified by absorbance at 405 nm. All experiments were performed in triplicate. Alizarin Red S Stain The accumulated calcium deposition after 14 days was analyzed using alizarin red S staining as in a previous study (26). After the cells were washed with PBS, photographs were observed using an optical microscope (BH2-UMA; Olympus, Tokyo, Japan) equipped with a digital camera (Nikon, Tokyo, Japan) at 200 magnification. To quantify the stained calcified nodules after staining, samples were immersed with 1.5 mL 5% sodium dodecyl sulfate in 0.5 N HCl for 30 minutes at room temperature. After that, the tubes were centrifuged to 5000 rpm for 10 minutes, and the supernatant was transferred to the new 96-well plate (GeneDireX); absorbance was measured at 450 nm (Hitachi). JOE — Volume 40, Number 11, November 2014
Statistical Analysis One-way variance statistical analysis was used to evaluate the significance of the differences between the groups in each experiment. The 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 a P value <.05.
Results CS Enhances the Expression of FN and Integrin av in hDPCs The cell viability of hDPCs grown on CS and TCP is shown in Figure 1A. There were no significant differences (P > .05) between Ctl and TCP. Interestingly, more cells adhered to the CS surfaces than the Ctl surfaces and TCP for all of the times they were measured and compared (P < .05). After 6 hours, the optical density values of the cells in the presence of CS were 1.36 and 1.39 times higher (P < .05) than those obtained in Ctl and TCP, respectively. Figure 1B shows the amounts of FN protein in the culture medium secreted from cells cultured with different specimens. At 1 hour, FN secretion on CS was 3.41 and 2.56 times higher (P < .05) than on Ctl and TCP, respectively. There were no significant differences (P > .05) between Ctl and TCP. The effect of substrates on the adsorption of FN was also examined. As shown The Role of Integrin av
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Figure 3. (A) Western blotting of integrin av knockdown by pairs of integrin av–specific-specific siRNAs in the cells. The effects of siRNA integrin av transfection on (B) proliferation and (C) EdU expression by hDPCs cultured on various specimens. *A significant difference (P < .05) compared with Ctl.
in Figure 1C, there was a statistically significant difference (P < .05) in FN adsorption between means with respect to the TCP control. The value for the CS sample was significantly (P < .05) higher than Ctl and TCP. Western blot analyses of cell lysates revealed that integrin av expression was proportional in different specimens (Fig. 1D). CS was associated with significantly (P < .05) higher levels of integrin av compared with Ctl and TCP for all time points. Interestingly, integrin av expression was almost identical in Ctl and TCP.
Ion Concentration The variations of DMEM and Ca and Si ion concentrations as measured at different times during the period after cell culture are shown in Figure 2. In CS and TCP cement, the Ca ion concentration of the medium increased to approximately 1.9 mmol/L after being cultured for 1 hour and then went to levels higher than the baseline Ca concentration of DMEM (1.8 mmol/L) (P < .05). No significant difference in the Ca ion concentration levels was found between TCP and CS at any of the times they were measured (Fig. 2A). The Si concentration within the CS group rose in proportion with an increased incubation time (Fig. 2B). The Si ion concentration was approximately 0.2, 0.7, and 1.0 mmol/L at 1, 3, and 6 hours, respectively. Si ions were not detected in either the Ctl or the TCP group. The FN secretion (Fig. 2C) and the number of hDPCs (Fig. 2D) cultured in both 0.5 mmol/L and 1 mmol/L Si media progressively increased with an increased culture time. 1806
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Integrin av Modulation on Cell Adhesion Western blots showed effective integrin av knockdown by pairs of integrin av–specific siRNA in the cells compared with both WT and Ctl with siRNA (mock siRNA) transfection (Fig. 3A). Transfection with integrin av siRNA optimally reduced integrin av protein levels by 83% in hDPCs. The PrestoBlue assays (Fig. 3B) and EdU incorporation analysis (Fig. 3C) indicated cell proliferation on the various specimens after 1 day. Proliferation was remarkably reduced with the gene silencing of av, as can be seen in the siRNA experiments. One day after transfection of integrin av siRNA, no significant change in cell proliferation in the Ctl or TCP groups was observed. Indeed, integrin av depletion results in a >45% reduction in cell proliferation on CS. However, integrin av did not lead to cell adhesion defects on Ctl and TCP when depleted by siRNA knockdown. Integrin av Knockdown Inhibited Odontoblastic Behavior To investigate the role of integrin av–induced odontoblastic differentiation of hDPCs cultured on different substrates, hDPCs were cultured with different substrates both with and without transfection (Fig. 4A). DMP-1 expression of hDPCs with integrin av siRNA on Ctl, TCP, and CS was 1.02, 0.96, and 0.62 times more than that of WT, respectively. The DSP results are similar to DMP-1; the integrin av knockdown group decreased significantly compared with WT when cultured on CS (P < .05). The expressions of DMP-1 and DSP on hDPCs both with and without transfection are similar when cultured on Ctl and TCP. The ALP activity (Fig. 4B) and calcium deposition (Fig. 4C) levels were analyzed after the treatment of siRNA for integrin av on hDPCs. The JOE — Volume 40, Number 11, November 2014
Basic Research—Biology
Figure 4. (A) Immunodetection of DMP-1 and DSP expression levels in hDPCs without and with siRNA integrin av transfection, which was cultured on various specimens for 14 days. Values not sharing a common letter are significantly different at P < .05. (B) ALP activity and (C) calcium mineral deposits of hDPCs without and with siRNA integrin av transfection, which was cultured on various specimens for 14 days. White arrows point to calcium deposition, and the scale bar was 100 mm. Values not sharing a common letter are significantly different at P < .05.
ALP activity and calcium deposition of hDPCs with integrin av siRNA on CS showed a significant reduction (P < .05) when compared with WT and mock siRNA. In addition, siRNA for integrin av had no effect on the ALP activity in the Ctl and TCP substrates.
Discussion CS-based materials have been found to foster cell adhesion (11), growth (23), and differentiation (4, 14) and have been used as implant materials for bone repair and regeneration. However, little is known about the mechanisms by which material identity regulates cell behavior and protein expression in general, and even less is known about how they specifically regulate the differentiation of cells. The present study reveals that the Si ion plays an important role in the proliferation and differentiation of hDPCs into odontoblast cells. After an initial increase in Ca ion concentration corresponding to the dissolution of CaO (23), extracellular Ca ion is a potent regulator of cell migration, proliferation, and differentiation and has significant effects on the proliferation and differentiation of cells (26). In this study, Ca ion concentrations released from TCP and CS were found to be similar. Thus, we presumed Ca ions are not the major factor that affects cell adhesion and differentiation. However, we found that as the Si ions are released from CS, the concentration in DMEM increases, and this increase continues in proportion to the length of time the culture is maintained. The silicate cement undergoes hydrolysis when it is immersed, and the Si ions dissolve during incubation (23). It is evident that the cement containing Si is a more effective promoter of cell attachment than that without Si, emphasizing the importance of the material’s composition. Taking cell functions into account, the release of Si from silicate-based materials seems to support cell behaviors and functions (13, 24, 27). Valerio et al (28) have proven that the Si concentration JOE — Volume 40, Number 11, November 2014
(<2 mmol/L) from the dissolution of a bioglass not only promotes human mesenchymal stem cell (hMSC) proliferation but also stimulates ECM secretion compared with biphasic calcium phosphate and Ctl. Cell adhesion requires an appropriate proteinaceous substrate to which cell adhesion receptors, such as integrins, can attach and form cell-anchoring points. The dominating role of protein adsorption on substrates and the regulation of cell adhesion have been identified (16). FN is the main ECM molecule that is expressed and synthesized during the various stages of osteogenesis (29). In vivo, the adsorption to the biomaterial surface of bioactive proteins from the serum and bodily fluids at the surgical site is known to influence cellularmaterial interactions (30). The differences in FN adsorption between CS and TCP may be explained by the zeta potential, which represents the effective surface charge of a biomaterial in contact with solution. The charge of a material’s surface modulates its surface chemistry, protein adsorption, and interactions with bone cells. CS and TCP have zeta potential values of 9.2 0.5 and 4.7 0.3 mV, indicating there is a significant difference (P < .05). When compared with TCP, it can be seen that CS favors the increase of positively charged zeta potential. FN has an isoelectric point of 5.5–6.3 (31). Thus, FN has a net negative charge in the cultured medium within the pH range of the study (7.2–7.4). This may explain why FN is preferentially adsorbed on CS (zeta potential = 9.2 0.5 mV) as opposed to the less well-accepted TCP (zeta potential = 4.7 0.3 mV). These results verify that the cement containing Si provides cells with a more favorable microenvironment through enhanced adsorption of secretion proteins supporting cell adhesion during the initial culture period. An increase in the level of cell adhesion would be expected to result in increased cell proliferation. The initial adhesion of cells to a material’s surface can be affected by integrins, which are transmembrane receptors for specific amino acid sequences in ECM molecules. The receptor proteins are composed The Role of Integrin av
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Basic Research—Biology of alpha and beta transmembrane subunits. All osteoblastic cells express integrin av subunits (32). The receptor integrins not only allow binding to matrix proteins, such as FN (a target of avb3 [32]) but are also thought to be involved in the signaling events that lead to cell proliferation and differentiation. In the overexpression of integrin av, the proliferation rate of prostate cells can be induced (33), and, similarly, promotion, proliferation, and differentiation have been observed in stem cells (34). However, the mechanisms responsible for the opposing regulatory functions of integrin av in normal cell behavior on the biomaterials are poorly understood. The siRNA-mediated knockdown of integrin av shows a delay in the proliferation and DNA synthesis of hDPCs cultured on CS. Integrin av has been shown to be a potent cell cycle regulator and increases the expression of integrin av normal entry into the cell cycle (35). In addition, the expression of DSP and DMP-1 proteins is degraded in the siRNA-treated cells on CS over a 14-day period. Alizarin red S and Western blot analysis supply further evidence that integrin av positively regulates the odontogenic differentiation of hDPCs on CS but not on TCP. These results are the first to indicate that the knockdown of integrin av inhibits the odontogenic differentiation of hDPCs on silicate-based materials. Integrin av plays an important role in stimulating osteogenesis differentiation in hMSCs, and the overexpression of integrin av enhances both hMSCs osteogenesis differentiation in vitro and in vivo (36, 37). In summary, integrin av has the ability to regulate hDPC odontogenic differentiation between CS and TCP with different differentiation processes. We have shown that integrin av down-regulation inhibits the proliferative capacity and odontogenic differentiation of hDPCs on silicate-based materials. These findings suggest that integrin av has a positive influence on the odontogenesis differentiation of hDPCs and may help hDPCs stay in an adhesive and undifferentiated state. Unraveling the mechanisms causing cell attachment and proliferation enhancement as a response to the presence of CS-based materials is a crucial point for expanding the applications of silica-based materials. CS cements promote cell attachment and trigger greater integrin av expression than TCP cement. Integrin expression profiles change accordingly, with higher levels of av subintegrin in the cells on the CS cements, a difference ascribed to the FN-binding subintegrin on hDPCs. The hDPCs cultured on CS substrates promote cell adhesion, proliferation, and differentiation as assessed according to the EdU assay, PrestoBlue assay, and odontogenesis protein expression. This study provides new and important clues regarding the molecular mechanisms that may be involved in Si-induced cell behavior and provides insight into cell manipulation via material design.
Acknowledgments Chi-Jr Hung, Hsin-I Hsu, and Chia-Tze Kao contributed equally to this work. Supported by a grant from the National Science Council Taiwan (NSC 101-2314-B-040-011-MY3) of Taiwan and the THU project of Global Research and Education on Environment and Society (GREEnS 001). The authors deny any conflicts of interest related to this study.
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Antiosteoclastogenic activity of silicate-based materials antagonizing receptor activator for nuclear factor kappaB ligand–induced osteoclast differentiation of murine marcophages. J Endod 2013;39:1557–61. 14. Chou MY, Kao CT, Hung CJ, et al. Role of the p38 pathway in calcium silicate cementinduced cell viability and angiogenesis-related proteins of human dental pulp cell in vitro. J Endod 2014;40:818–24. 15. Huang SC, Wu BC, Kao CT, et al. Role of the p38 pathway in mineral trioxide aggregateinduced cell viability and angiogenesis-related proteins of dental pulp cell in vitro. Int Endod J 2014 Apr 28. http://dx.doi.org/10.1111/iej.12305. [Epub ahead of print]. 16. Shie MY, Chang HC, Ding SJ. Composition-dependent protein secretion and integrin level of osteoblastic cell on calcium silicate cements. J Biomed Mater Res Part A 2014;102:769–80. 17. Hynes RO. Integrins: bidirectional, allosteric signaling machines. Cell 2002;110: 673–87. 18. Schwartz MA, Schaller MD, Ginsberg MH. Integrins: emerging paradigms of signal transduction. Annu Rev Cell Dev Biol 1995;11:549–99. 19. Zurick KM, Qin C, Bernards MT. Adhesion of MC3T3-E1 cells bound to dentin phosphoprotein specifically bound to collagen type I. J Biomed Mater Res Part A 2012; 100:2492–8. 20. Yan YX, Gong YW, Guo Y, et al. Mechanical strain regulates osteoblast proliferation through integrin-mediated ERK activation. PLoS One 2012;7:e35709. 21. Siebers MC, Brugge PJ, Walboomers XF, Jasen JA. Integrins as linker proteins between osteoblasts and bone replacing materials. A critical review. Biomaterials 2005;26:137–46. 22. Keselowsky BG, Collard DM, Garcıa AJ. Integrin binding specificity regulates biomaterial surface chemistry effects on cell differentiation. Proc Natl Acad Sci U S A 2005; 102:5953–7. 23. 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. 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. Linde A, Goldberg M. Dentinogenesis. Crit Rev Oral Biol Med 1993;4:679–728. 26. Shie MY, Ding SJ, Chang HC. The role of silicon in osteoblast-like cell proliferation and apoptosis. Acta Biomater 2011;7:2604–14. 27. Wu BC, Kao CT, Huang TH, et al. Effect of a calcium channel blocker on the odontogenic activity of human dental pulp cells cultured with silicate-based materials. J Endod 2014;40:1105–11. 28. Valerio P, Pereira MM, Goes AM, Leite MF. The effect of ionic products from bioactive glass dissolution on osteoblast proliferation and collagen production. Biomaterials 2004;25:2941–8. 29. Kang Y, Kim SH, Bishop J, et al. The osteogenic differentiation of human bone marrow MSCs on HUVEC-derived ECM and b-TCP scaffold. Biomaterials 2012;33: 6998–7007. 30. Vohra S, Hennessy KM, Sawyer AA, et al. Comparison of mesenchymal stem cell and osteosarcoma cell adhesion to hydroxyapatite. J Mater Sci Mater Med 2008;19:3567–74. 31. Bouafsoun A, Helali S, Othmane A, et al. Evaluation of endothelial cell adhesion onto different protein/gold electrodes by EIS. Macromol Biosci 2007;7:599–610. 32. Anselme K. Osteoblast adhesion on biomaterials. Biomaterials 2000;21:667–81. 33. Cooper CR, Chay CH, Pienta KJ. The role of avb3 in prostate cancer progression. Neoplasia 2002;4:191–4.
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Basic Research—Biology 34. Goessler UR, Bugert P, Bieback K, et al. In vitro analysis of integrin expression in stem cells from bone marrow and cord blood during chondrogenic differentiation. J Cell Mol Med 2009;13:1175–84. 35. Shroff K, Pearce TR, Kokkoli E. Enhanced integrin mediated signaling and cell cycle progression on fibronectin mimetic peptide amphiphile monolayers. Langmuir 2012;28:1858–65.
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36. Hamidouche Z, Fromigue O. Priming integrin a5 promotes human mesenchymal stromal cell osteoblast differentiation and osteogenesis. Proc Natl Acad Sci U S A 2009;106:18587–91. 37. Fromigue O, Brun J, Marty C, et al. Peptide-based activation of alpha5 integrin for promoting osteogenesis. J Cell Biochem:3029–38, http://10.1111/iej.12305, 2012;113.
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The Role of Integrin av in Proliferation and Differentiation of Human Dental Pulp Cell Response to Calcium Silicate Cement Chi-Jr Hung, DDS, MD, PhD,*† Hsin-I. Hsu, MS,‡ Chi-Chang Lin, PhD,§ Tsui-Hsien Huang, DDS, MD, PhD,*† Buor-Chang Wu, DDS, MD, PhD,*† Chia-Tze Kao, DDS, MD, PhD,*† and Ming-You Shie, PhD* Abstract Introduction: It has been proved that integrin av activity is related to cell proliferation, differentiation, migration, and organ development. However, the biological functions of integrin av in human dental pulp cells (hDPCs) cultured on silicate-based materials have not been explored. The aim of this study was to investigate the role of integrin av in the proliferation and odontogenic differentiation of hDPCs cultured with the effect of calcium silicate (CS) cement and b-tricalcium phosphate (TCP) cement. Methods: In this study, hDPCs were cultured on CS and TCP materials, and we evaluated fibronectin (FN) secretion and integrin av expression during the cell attachment stage. After small interfering RNA transfection targeting integrin av, the proliferation and odontogenesis differentiation behavior of hDPCs were analyzed. Results: The results indicate that CS releases Si ion–increased FN secretion and adsorption, which promote cell attachment more effectively than TCP. The CS cement facilitates FN and av subintegrin expression. However, the FN adsorption and integrin expression of TCP are similar to that observed in the control dish. Integrin av small interfering RNA inhibited odontogenic differentiation of hDPCs with the decreased formation of mineralized nodules on CS. It also down-regulated the protein expression of multiple markers of odontogenesis and the expression of dentin sialophosphoprotein protein. Conclusions: These results establish compositiondependent differences in integrin binding and its effectiveness as a mechanism regulating cellular responses to biomaterial surface. (J Endod 2014;40:1802–1809)
From the *School of Dentistry and ‡Institute of Oral Science, Chung Shan Medical University; †Department of Stomatology, Chung Shan University Hospital; and §Department of Chemical and Materials Engineering, Tunghai University, Taichung City, Taiwan. Address requests for reprints to Dr Chia-Tze Kao, School of Dentistry, Chung Shan Medical University, Taichung City, Taiwan or Dr Ming-You Shie, Department of Chemical and Materials Engineering, Tunghai University, Taichung City, Taiwan. E-mail addresses: [email protected] or [email protected] 0099-2399/$ - see front matter Copyright ª 2014 American Association of Endodontists. http://dx.doi.org/10.1016/j.joen.2014.07.016
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Key Words Calcium silicate cement, fibronectin, human dental pulp cell, integrin av, odontogenesis
M
ineral trioxide aggregate (MTA) has long been used as a root-end dental filling material and is widely used in several clinical applications of endodontics (1). MTA received approval for general medical use in 1998 and is comprised of a mixture of 75% Portland cement (CaO and SiO2), 20% Bi2O3, and 5% gypsum (1). Not only does MTA have good biocompatibility (2), it also has been proven to promote osteogenesis in human dental pulp cells (hDPCs) (3, 4). In dentistry, calcium silicate (CS)-based cements have been formulated into dentin replacement restorative materials (5), but there is reason to believe its performance can be made more effective by decreasing the setting time and improving the handling properties in the clinical setting (6). In our previous study, we produced a fast-setting CS cement that contains CaO, SiO2, and Al2O3, which were shown to have a reduced setting time (7). In addition, CS cement not only exhibits good osteoconduction effects (8, 9) but also reduces inflammation markers in primary hDPCs (10, 11) and in vivo (12). The release of Si concentrations from silicate-based materials influences the behavior of different cell types, such as inhibiting osteoclastogenesis in macrophages (13) and angiogenesis in hDPCs (14, 15). In addition, the amount of Si ions in CS-based materials can affect the adsorption of various types of extracellular matrices (ECMs) such as collagen I, fibronectin (FN), and vitronectin and promote the up-regulation of mitogen-activated protein kinase/ extracellular signal-regulated protein kinase 1/2 (MAPK/ERK 1/2) and MAPK/p38, signaling the pathway more effectively than Ca components (11, 16). Integrins are the major transmembrane receptors for cell adhesion to the ECM (17). All integrins are heterodimers composed of noncovalently linked alpha and beta subunits. Among the receptor systems, integrins bind to specific ECM components, such as collagen, vitronectin, and FN, influencing cell adhesion behavior (17, 18). Integrin-mediated interactions are vital to the maintenance of normal cell functions because they mediate the interaction between individual cells and the ECM and therefore have important implications for cell adhesion (19), proliferation (20), and differentiation (11). In addition, integrin protein expression changed accordingly, with higher levels of a2b1 and avb3 subintegrins in the cells on the Si- and Ca-rich substrates, respectively, which were ascribed to the collagen-binding and FN-binding subintegrin on primary cells, respectively (11). Integrin av is essential for various biological functions, and it binds to and competes for the arginine-glycine-aspartic site on FN (21, 22). Recent studies have shown that integrin av is not only required for osteoblast proliferation (23) but also for activation of the focal adhesion kinase/ ERK/MAPK and phosphoinositide 3-kinase signaling pathways (24). These findings indicate that integrin av might be affected by osteogenic differentiation of cells. The odontogenic differentiation of hDPCs is a biological process similar to osteogenic differentiation. There are several proteins involved in bone and dentin mineralization, and they are similar but distinct. In comparison with bone, dentin has been shown to contain less osteopontin, bone sialoprotein, and osteocalcin but more dentin sialoprotein (DSP) and dentin matrix protein 1 (DMP-1) (25). Thus, we suggest integrin av may be included in the odontogenic differentiation of hDPCs.
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Basic Research—Biology Based on these factors, we hypothesize that CS cement may influence FN adsorption and accelerate adhesion protein formation, in particular, integrin av. Thus, the roles of integrin av involved in the regulation of odontogenic differentiation of hDPCs are also investigated in the present study, which investigates the specific role of DMP-1 and DSP protein expression on CS-induced odontogenesis. Additionally, small interfering RNA (siRNA) transfection is used to explore the integrin av level in hDPCs during the proliferation and differentiation stages.
Materials and Methods Specimen Preparation The CS cement used in this research was made according to our previously reported laboratory procedures (13). Appropriate amounts of CaO (65%; Sigma-Aldrich, St Louis, MO), SiO2 (25%; High Pure Chemicals, Saitama, Japan), and Al2O3 (5%, SigmaAldrich) powders were mixed. After sintering at 1400 C for 2 hours, the granules were ball milled in 99.5% EtOH using a centrifugal ball mill (Retsch S 100, Hann, Germany) for 12 hours and then dried in an oven. In addition, beta-tricalcium phosphate (TCP, Sigma-Aldrich) was compared with CS in this study. Before the preparation of the cement specimens, 50 mg powdered zeta potential was placed in 1 mL phosphate-buffered saline (PBS; Gibco, Langley, OK) and after soaking for 3 hours was measured by particle electrophoresis using the Zetasizer Nano ZS (Malvern Instruments, Worcestershire, UK). Three samples were measured for each powder condition, and the average is reported in this analysis. The CS and TCP powders were mixed according to the liquid/powder ratio of 0.35 mL/g and placed in a 24-well plate (GeneDireX, Las Vegas, NV) to a thickness of 2 mm fully covering each well; the samples were then stored in an incubator at 100% relative humidity and 37 C for 1 day. Before performing the cell experiments, all the 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 had been extracted for orthodontic treatment purposes as described previously (11). 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 immersed in PBS (Caisson, North Logan, UT) solution and digested in 0.1% collagenase type I (Sigma-Aldrich) for 30 minutes. After being transferred to a new cultured dish, the cell suspension was cultured in Dulbecco modified Eagle medium (DMEM, Caisson) containing 20% fetal bovine serum (GeneDireX) and 1% penicillin (10,000 U/mL)/streptomycin (10,000 mg/mL) (Caisson) and was kept in a humidified atmosphere with 5% CO2 at 37 C. The medium was changed every 3 days. The hDPCs were subcultured through successive passaging at a 1:3 ratio until they were used for experiments (passages 3–8). Cell Adhesion Assays The suspended cells were kept at a density of 1.5 104/specimen and directly seeded over each sample. Cell cultures were incubated at 37 C in a 5% CO2 atmosphere. After being cultured for different lengths of time (ie, 1, 3, and 6 hours), cell adhesion ability was evaluated using the PrestoBlue assay (Invitrogen, Grand Island, NY). Briefly, each specimen was filled with medium with a 1:9 ratio of PrestoBlue in fresh DMEM and incubated at 37 C for 30 minutes. The solution in each well JOE — Volume 40, Number 11, November 2014
was then transferred to a new 96-well plate and read in a multiwell spectrophotometer (Hitachi, Tokyo, Japan) at 570 nm with a reference wavelength of 600 nm. The results were obtained in triplicate from 3 separate experiments for each test. Cells cultured on tissue culture plates without cement were used as a control (Ctl).
FN Secretion Cells were cultured on different substrates for 1, 3, and 6 hours, and the cell culture media were then collected and stored at room temperature. The enzyme-linked immunosorbent assay kits of human FN were obtained from Abcam (Cambridge, MA). Following the manufacturer’s instructions, we used a 3-hour assay, which has a high sensitivity. The reaction was terminated by the addition of stop solution and read at 450 nm using a multiwell spectrophotometer. FN Adsorption on Substrates After being cultured for different periods of time, the amount of FN secreted from cells onto the cement’s surface was analyzed using the enzyme-linked immunosorbent assay. The cells were detached using a trypsin-EDTA solution (Cassion) after being washed 3 times with cold PBS. Specimens were then washed 3 times with PBS containing 0.1% Tween 20 (PBS-T; Sigma-Aldrich, St Louis, MO) followed by blocking with 5% bovine serum albumin (Gibco) in PBS-T for 1 hour. Dilutions of primary antibodies were set at 1:500. After this procedure, samples were incubated with antihuman beta-actin or antihuman FN antibody (GeneTex, San Antonio, TX) for 3 hours at room temperature. Afterward, samples were washed 3 times with PBS-T for 5 minutes and incubated with horseradish peroxidase–conjugated secondary antibodies for 1 hour at room temperature with shaking. The samples were then washed 3 times with PBS-T for 10 minutes each, and then One-Step Ultra TMB substrate (Invitrogen) was added to the wells and developed for 30 minutes at room temperature in the dark; after this, an equal volume of 2M H2SO4 was added to stop and stabilize the oxidation reaction. The colored products were then transferred to new 96-well plates and read using a multiwell spectrophotometer at 450 nm with reference at 620 nm according to the manufacturer’s recommendations. All experiments were performed in triplicate. Additionally, beta-actin antibodies were used as a control. Western Blot Western blotting was performed on cells cultured on different cement specimens for a predetermined time to evaluate the differences in their protein 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 using a Bio-Rad DC Protein Assay kit (Richmond, CA), and the proteins (30 mg) were then resolved using standard sodium dodecyl sulfate–polyacrylamide gel electrophoresis and transferred to polyvinylidene difluoride membranes. After blocking with 5% bovine serum albumin (Sigma-Aldrich) in Tris-buffer saline containing 0.05% Tween 20 for 1 hour, the membranes were incubated with primary antibodies against beta-actin, integrin av (GeneTex), DMP-1, and DSP (Santa Cruz Biotechnology, Santa Cruz, CA). A horseradish peroxidase–conjugated secondary antibody was subsequently added, and the proteins were visualized with enhancement using enhanced chemiluminescent detection kits (Invitrogen). The stained bands were scanned and quantified using a densitometer (Syngene, Frederick, MD) and Scion Image software (Scion Corporation, Frederick, MD). Protein expression levels were normalized to the beta-actin band for each sample. The Role of Integrin av
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Figure 1. (A) Proliferation of hDPCs cultured on different substrates for various time points. (B) FN secretion and (C) adsorption from hDPCs after seeding on different substrates for 1, 3, and 6 hours. (D) Immunodetection of integrin protein expression in hDPCs cultured on various samples for 1, 3, and 6 hours. Integrin expression was quantified and normalized to beta-actin expression. *A significant difference (P < .05) compared with Ctl.
Ion Concentration The Ca and Si ion concentration on DMEM was analyzed using an inductively coupled plasma atomic emission spectrometer (OPT 1 MA 3000DV; Perkin-Elmer, Shelton, CT) after cell seeding for 1, 3, and 6 hours. Three samples were measured for each data point. The results were obtained in triplicate from 3 separate samples for each test. Preparation of Test Medium Containing Different Si Concentrations One gram of cement was immersed in 10 mL DMEM for 1 day, and the supernatants were passed through a 0.22-mm filter (Millipore, Billerica, MA) to obtain the cement extract. The Si ion concentration on extract was analyzed using an inductively coupled plasma atomic emission spectrometer. A detailed description of the dilution of the test medium with various Si ion concentrations is given elsewhere (13). The cement extract and DMEM were used to prepare 3 different media with various Si ion concentrations (0, 0.5, and 1 mmol/L). Then, FN secretion and cell adhesion of hDPCs after treatment with various Si concentrations were analyzed. siRNA Transfection For siRNA-mediated knockdown of integrin av, cells (wild type [WT]) were transfected with 50 nmol/L of either the targeting 1804
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or control siRNA (Genedirex) using Lipofectamine 2000 (Invitrogen) for 48 hours as recommended in the manufacturer’s instructions. The siRNAs targeting integrin av as well as the control nontargeting siRNAs were commercially available pools of 4 different siRNAs purchased from Santa Cruz Biotechnology. Cellular levels of the proteins specifically silenced were assessed using a Western blot test.
Proliferation Assay When cell proliferation was detected, it was then immediately measured through the incorporation of 5-ethynyl-20-deoxyuridine (EdU) with the Click-iT EdU Cell Proliferation Assay Kit (Invitrogen) and PrestoBlue assay. EdU solution with a final concentration of 105 mol/L was added into the culture medium and allowed to incubate overnight before the experiment. After being cultured for 1 day, the cells were fixed with 10% formalin and permeabilized using 0.5% Triton X-100. The EdU was stained according to the manufacturer’s protocol. Cells were also stained with 0.1 mg/mL 40 ,6-diamidino-2- phenylindole (Invitrogen) in 1 PBS for 30 minutes, which permitted the detection of all nuclei (blue). After washing 3 times with PBS-T, the cells were viewed under indirect immunofluorescence using a Zeiss Axioskop2 microscope (Carl Zeiss, Thornwood, NY). Images were analyzed with ImageJ software (National Institutes of Health, Bethesda, MD). JOE — Volume 40, Number 11, November 2014
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Figure 2. (A) Ca and (B) Si ion concentration of DMEM after cell cultured for different times. *A significant difference (P < .05) compared with Ctl. (C) FN secretion and (D) hDPC adhesion after treatment with DMEM contained various concentrations. *A significant difference (P < .05) compared with 0 mmol/L.
Alkaline Phosphatase Assay The level of alkaline phosphatase (ALP) activity was determined on the third day after cell seeding. The process was as follows: the cells were lysed from discs using 0.2 % NP-40 and centrifuged for 10 minutes at 2000 rpm after washing with PBS. ALP activity was determined using p-nitrophenyl phosphate (SigmaAldrich) as the substrate. Each sample was mixed with p-nitrophenyl phosphate in 1 mol/L diethanolamine buffer for 15 minutes; after this, the reaction was stopped by the addition of 5 N NaOH and quantified by absorbance at 405 nm. All experiments were performed in triplicate. Alizarin Red S Stain The accumulated calcium deposition after 14 days was analyzed using alizarin red S staining as in a previous study (26). After the cells were washed with PBS, photographs were observed using an optical microscope (BH2-UMA; Olympus, Tokyo, Japan) equipped with a digital camera (Nikon, Tokyo, Japan) at 200 magnification. To quantify the stained calcified nodules after staining, samples were immersed with 1.5 mL 5% sodium dodecyl sulfate in 0.5 N HCl for 30 minutes at room temperature. After that, the tubes were centrifuged to 5000 rpm for 10 minutes, and the supernatant was transferred to the new 96-well plate (GeneDireX); absorbance was measured at 450 nm (Hitachi). JOE — Volume 40, Number 11, November 2014
Statistical Analysis One-way variance statistical analysis was used to evaluate the significance of the differences between the groups in each experiment. The 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 a P value <.05.
Results CS Enhances the Expression of FN and Integrin av in hDPCs The cell viability of hDPCs grown on CS and TCP is shown in Figure 1A. There were no significant differences (P > .05) between Ctl and TCP. Interestingly, more cells adhered to the CS surfaces than the Ctl surfaces and TCP for all of the times they were measured and compared (P < .05). After 6 hours, the optical density values of the cells in the presence of CS were 1.36 and 1.39 times higher (P < .05) than those obtained in Ctl and TCP, respectively. Figure 1B shows the amounts of FN protein in the culture medium secreted from cells cultured with different specimens. At 1 hour, FN secretion on CS was 3.41 and 2.56 times higher (P < .05) than on Ctl and TCP, respectively. There were no significant differences (P > .05) between Ctl and TCP. The effect of substrates on the adsorption of FN was also examined. As shown The Role of Integrin av
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Figure 3. (A) Western blotting of integrin av knockdown by pairs of integrin av–specific-specific siRNAs in the cells. The effects of siRNA integrin av transfection on (B) proliferation and (C) EdU expression by hDPCs cultured on various specimens. *A significant difference (P < .05) compared with Ctl.
in Figure 1C, there was a statistically significant difference (P < .05) in FN adsorption between means with respect to the TCP control. The value for the CS sample was significantly (P < .05) higher than Ctl and TCP. Western blot analyses of cell lysates revealed that integrin av expression was proportional in different specimens (Fig. 1D). CS was associated with significantly (P < .05) higher levels of integrin av compared with Ctl and TCP for all time points. Interestingly, integrin av expression was almost identical in Ctl and TCP.
Ion Concentration The variations of DMEM and Ca and Si ion concentrations as measured at different times during the period after cell culture are shown in Figure 2. In CS and TCP cement, the Ca ion concentration of the medium increased to approximately 1.9 mmol/L after being cultured for 1 hour and then went to levels higher than the baseline Ca concentration of DMEM (1.8 mmol/L) (P < .05). No significant difference in the Ca ion concentration levels was found between TCP and CS at any of the times they were measured (Fig. 2A). The Si concentration within the CS group rose in proportion with an increased incubation time (Fig. 2B). The Si ion concentration was approximately 0.2, 0.7, and 1.0 mmol/L at 1, 3, and 6 hours, respectively. Si ions were not detected in either the Ctl or the TCP group. The FN secretion (Fig. 2C) and the number of hDPCs (Fig. 2D) cultured in both 0.5 mmol/L and 1 mmol/L Si media progressively increased with an increased culture time. 1806
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Integrin av Modulation on Cell Adhesion Western blots showed effective integrin av knockdown by pairs of integrin av–specific siRNA in the cells compared with both WT and Ctl with siRNA (mock siRNA) transfection (Fig. 3A). Transfection with integrin av siRNA optimally reduced integrin av protein levels by 83% in hDPCs. The PrestoBlue assays (Fig. 3B) and EdU incorporation analysis (Fig. 3C) indicated cell proliferation on the various specimens after 1 day. Proliferation was remarkably reduced with the gene silencing of av, as can be seen in the siRNA experiments. One day after transfection of integrin av siRNA, no significant change in cell proliferation in the Ctl or TCP groups was observed. Indeed, integrin av depletion results in a >45% reduction in cell proliferation on CS. However, integrin av did not lead to cell adhesion defects on Ctl and TCP when depleted by siRNA knockdown. Integrin av Knockdown Inhibited Odontoblastic Behavior To investigate the role of integrin av–induced odontoblastic differentiation of hDPCs cultured on different substrates, hDPCs were cultured with different substrates both with and without transfection (Fig. 4A). DMP-1 expression of hDPCs with integrin av siRNA on Ctl, TCP, and CS was 1.02, 0.96, and 0.62 times more than that of WT, respectively. The DSP results are similar to DMP-1; the integrin av knockdown group decreased significantly compared with WT when cultured on CS (P < .05). The expressions of DMP-1 and DSP on hDPCs both with and without transfection are similar when cultured on Ctl and TCP. The ALP activity (Fig. 4B) and calcium deposition (Fig. 4C) levels were analyzed after the treatment of siRNA for integrin av on hDPCs. The JOE — Volume 40, Number 11, November 2014
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Figure 4. (A) Immunodetection of DMP-1 and DSP expression levels in hDPCs without and with siRNA integrin av transfection, which was cultured on various specimens for 14 days. Values not sharing a common letter are significantly different at P < .05. (B) ALP activity and (C) calcium mineral deposits of hDPCs without and with siRNA integrin av transfection, which was cultured on various specimens for 14 days. White arrows point to calcium deposition, and the scale bar was 100 mm. Values not sharing a common letter are significantly different at P < .05.
ALP activity and calcium deposition of hDPCs with integrin av siRNA on CS showed a significant reduction (P < .05) when compared with WT and mock siRNA. In addition, siRNA for integrin av had no effect on the ALP activity in the Ctl and TCP substrates.
Discussion CS-based materials have been found to foster cell adhesion (11), growth (23), and differentiation (4, 14) and have been used as implant materials for bone repair and regeneration. However, little is known about the mechanisms by which material identity regulates cell behavior and protein expression in general, and even less is known about how they specifically regulate the differentiation of cells. The present study reveals that the Si ion plays an important role in the proliferation and differentiation of hDPCs into odontoblast cells. After an initial increase in Ca ion concentration corresponding to the dissolution of CaO (23), extracellular Ca ion is a potent regulator of cell migration, proliferation, and differentiation and has significant effects on the proliferation and differentiation of cells (26). In this study, Ca ion concentrations released from TCP and CS were found to be similar. Thus, we presumed Ca ions are not the major factor that affects cell adhesion and differentiation. However, we found that as the Si ions are released from CS, the concentration in DMEM increases, and this increase continues in proportion to the length of time the culture is maintained. The silicate cement undergoes hydrolysis when it is immersed, and the Si ions dissolve during incubation (23). It is evident that the cement containing Si is a more effective promoter of cell attachment than that without Si, emphasizing the importance of the material’s composition. Taking cell functions into account, the release of Si from silicate-based materials seems to support cell behaviors and functions (13, 24, 27). Valerio et al (28) have proven that the Si concentration JOE — Volume 40, Number 11, November 2014
(<2 mmol/L) from the dissolution of a bioglass not only promotes human mesenchymal stem cell (hMSC) proliferation but also stimulates ECM secretion compared with biphasic calcium phosphate and Ctl. Cell adhesion requires an appropriate proteinaceous substrate to which cell adhesion receptors, such as integrins, can attach and form cell-anchoring points. The dominating role of protein adsorption on substrates and the regulation of cell adhesion have been identified (16). FN is the main ECM molecule that is expressed and synthesized during the various stages of osteogenesis (29). In vivo, the adsorption to the biomaterial surface of bioactive proteins from the serum and bodily fluids at the surgical site is known to influence cellularmaterial interactions (30). The differences in FN adsorption between CS and TCP may be explained by the zeta potential, which represents the effective surface charge of a biomaterial in contact with solution. The charge of a material’s surface modulates its surface chemistry, protein adsorption, and interactions with bone cells. CS and TCP have zeta potential values of 9.2 0.5 and 4.7 0.3 mV, indicating there is a significant difference (P < .05). When compared with TCP, it can be seen that CS favors the increase of positively charged zeta potential. FN has an isoelectric point of 5.5–6.3 (31). Thus, FN has a net negative charge in the cultured medium within the pH range of the study (7.2–7.4). This may explain why FN is preferentially adsorbed on CS (zeta potential = 9.2 0.5 mV) as opposed to the less well-accepted TCP (zeta potential = 4.7 0.3 mV). These results verify that the cement containing Si provides cells with a more favorable microenvironment through enhanced adsorption of secretion proteins supporting cell adhesion during the initial culture period. An increase in the level of cell adhesion would be expected to result in increased cell proliferation. The initial adhesion of cells to a material’s surface can be affected by integrins, which are transmembrane receptors for specific amino acid sequences in ECM molecules. The receptor proteins are composed The Role of Integrin av
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Basic Research—Biology of alpha and beta transmembrane subunits. All osteoblastic cells express integrin av subunits (32). The receptor integrins not only allow binding to matrix proteins, such as FN (a target of avb3 [32]) but are also thought to be involved in the signaling events that lead to cell proliferation and differentiation. In the overexpression of integrin av, the proliferation rate of prostate cells can be induced (33), and, similarly, promotion, proliferation, and differentiation have been observed in stem cells (34). However, the mechanisms responsible for the opposing regulatory functions of integrin av in normal cell behavior on the biomaterials are poorly understood. The siRNA-mediated knockdown of integrin av shows a delay in the proliferation and DNA synthesis of hDPCs cultured on CS. Integrin av has been shown to be a potent cell cycle regulator and increases the expression of integrin av normal entry into the cell cycle (35). In addition, the expression of DSP and DMP-1 proteins is degraded in the siRNA-treated cells on CS over a 14-day period. Alizarin red S and Western blot analysis supply further evidence that integrin av positively regulates the odontogenic differentiation of hDPCs on CS but not on TCP. These results are the first to indicate that the knockdown of integrin av inhibits the odontogenic differentiation of hDPCs on silicate-based materials. Integrin av plays an important role in stimulating osteogenesis differentiation in hMSCs, and the overexpression of integrin av enhances both hMSCs osteogenesis differentiation in vitro and in vivo (36, 37). In summary, integrin av has the ability to regulate hDPC odontogenic differentiation between CS and TCP with different differentiation processes. We have shown that integrin av down-regulation inhibits the proliferative capacity and odontogenic differentiation of hDPCs on silicate-based materials. These findings suggest that integrin av has a positive influence on the odontogenesis differentiation of hDPCs and may help hDPCs stay in an adhesive and undifferentiated state. Unraveling the mechanisms causing cell attachment and proliferation enhancement as a response to the presence of CS-based materials is a crucial point for expanding the applications of silica-based materials. CS cements promote cell attachment and trigger greater integrin av expression than TCP cement. Integrin expression profiles change accordingly, with higher levels of av subintegrin in the cells on the CS cements, a difference ascribed to the FN-binding subintegrin on hDPCs. The hDPCs cultured on CS substrates promote cell adhesion, proliferation, and differentiation as assessed according to the EdU assay, PrestoBlue assay, and odontogenesis protein expression. This study provides new and important clues regarding the molecular mechanisms that may be involved in Si-induced cell behavior and provides insight into cell manipulation via material design.
Acknowledgments Chi-Jr Hung, Hsin-I Hsu, and Chia-Tze Kao contributed equally to this work. Supported by a grant from the National Science Council Taiwan (NSC 101-2314-B-040-011-MY3) of Taiwan and the THU project of Global Research and Education on Environment and Society (GREEnS 001). The authors deny any conflicts of interest related to this study.
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