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Involvement of p38 MAPK pathway in low intensity pulsed ultrasound induced osteogenic differentiation of human periodontal ligament cells
Ultrasonics 53 (2013) 686–690
Contents lists available at SciVerse ScienceDirect
Ultrasonics journal homepage: www.elsevier.com/locate/ultras
Involvement of p38 MAPK pathway in low intensity pulsed ultrasound induced osteogenic differentiation of human periodontal ligament cells Leixi Ren a, Zun Yang b, Jinlin Song b,⇑, Zhibiao Wang c, Feng Deng b, Wanshan Li a a
Department of Stomatology, Children’s Hospital of Chongqing Medical University, Chongqing, China Department of Orthodontics, The Affiliated Hospital of Stomatology, Chongqing Medical University, Chongqing, China c College of Biomedical Engineering, Chongqing Medical University, Chongqing, China b
a r t i c l e
i n f o
Article history: Received 25 November 2011 Received in revised form 12 October 2012 Accepted 21 October 2012 Available online 5 November 2012 Keywords: Human periodontal ligament cells Low intensity pulsed ultrasound p38 MAPK Osteogenesis
a b s t r a c t Objective: The purpose of this study was to clarify whether p38 MAPK is involved in the process of low intensity pulsed ultrasound (LIPUS) induced osteogenic differentiation of human periodontal ligament cells (HPDLCs). Methods: HPDLCs were isolated from premolars extracted for orthodontic reasons from healthy adolescences and used in the study at passage 5. They were pretreated with p38 specific inhibitor SB203580 and exposed daily to LIPUS with frequency of 1 MHz and intensity of 90 mW/cm2. Osteogenic differentiation was assayed by levels of alkaline phosphatase (ALP) and osteocalcin as well as calcium deposition. The levels of phosphorylated p38 (p-p38) and total p38 in HPDLCs in response to LIPUS for different times were detected by Western blot. Results: The enhanced ALP levels in media and cell lysate, osteocalcin level in media, as well as number of calcium nodules after LIPUS stimulation were decreased by SB203580 treatment. LIPUS stimulation did not change total p38 level, but time-dependently enhanced the level of p-p38; such enhancement was significantly blocked by preincubation with 10 lmol/L SB203580. Conclusion: The p38 MAPK is involved in the process of LIPUS-induced osteogenic differentiation of HPDLCs. Ó 2012 Elsevier B.V. All rights reserved.
1. Introduction HPDLCs are the main cellular component of periodontal tissues among different heterogeneous cells [1]. They have stem cell like properties and can protect and repair periodontal tissues by synthesizing collagen and producing cementum. In addition, HPDLC sheets created in cell-processing centers were safe products with the potential to regenerate periodontal tissues [2]. Thus, HPDLCs are considered a beneficial cell source for clinical periodontal regeneration [3–5]. LIPUS is an established therapy for fracture repair. It was approved by the Food and Drug Administration for use in fresh bone fracture healing in 1994 and in established nonunions in 2000 [6]. Studies have shown that LIPUS could increase calcium incorporation in cartilage and bone cell cultures, alter potassium flux across cell membrane, activate adenylyl cyclase in osteoblastic cells, modulate expression of numerous genes involved in fracture healing process, enhance angiogenesis, and so on [7]. In vitro studies suggested that when appropriately triggered, osteoblastic cells in hu⇑ Corresponding author. E-mail address: [email protected] (J. Song). 0041-624X/$ - see front matter Ó 2012 Elsevier B.V. All rights reserved. http://dx.doi.org/10.1016/j.ultras.2012.10.008
man periodontal ligament could differentiate into osteoblasts/ cementoblasts [8] and LIPUS could induce transient expression of the immediate-early response gene c-fos and elevate mRNA levels of insulin-like growth factor-I, osteocalcin and bone sialoprotein [9]. In addition, LIPUS has been demonstrated to have anabolic effects on cementoblasts, odontoblasts, and periodontal ligament cells [10]. In vitro studies, the frequency of 1 MHz and the intensity of 90 mW/cm2 for 20 min everyday was optimum when LIPUS induced osteogenic differentiation of HPDLCs [11]. Previous in vitro studies on cells including mesenchymal stem cells treated with mitogen-activated protein kinases (MAPKs) inhibitors showed that MAPKs p38 and ERK are important for early osteoblast differentiation [12]. LIPUS stimulation could induce mouse myoblast cells C2C12 differentiating into osteoblasts and/ or chondroblasts via ERK1/2 and p38 [13]. Moreover, LIPUS is able to initiate osteogenic differentiation and modulate ERK1/2 and p38 signaling pathways [14]. Exposure to LIPUS could accelerate softtissue healing by increasing connective tissue growth factors via MAPK signaling pathway [15]. Because PDLCs have similar effects with mesenchymal stem cells on osteogenic differentiation [16], we hypothesized that the p38 MAPK pathway is involved in LIPUS-induced osteogenic differentiation of HPDLCs.
L. Ren et al. / Ultrasonics 53 (2013) 686–690
2. Materials and methods 2.1. Cell isolation, culture and identification HPDLCs were prepared from premolars extracted for orthodontic reasons from healthy adolescences. In detail, the premolars as well as their surrounding gingivas were disinfected and washed three times with phosphate buffered saline (PBS) containing 100 lg/mL streptomycin and 100 U/mL penicillin. Their periodontal ligament tissues were scraped from the mid-third portion of premolar roots using No. 11 sharp surgical scalpels, cut into about 1 1 mm2 pieces, placed on the bottom of a 25 ml culture flask containing a-MEM (Hyclone, USA) supplemented with 20% fetal bovine serum (FBS, Hyclone, USA) and cultured at 37 °C in an incubator with 5% CO2 (Nuair, USA). The cells were passaged when reaching 60% confluency, and cells from passage 5 were used in all experiments. Informed consent was obtained from all patients and their parents. 2.2. ALP activity assay HPDLCs were inoculated into a 6-well plate containing a-MEM supplemented with 20% FBS at a density of 1 105 cells/cm2 and assigned into three groups. The cells in treatment group were treated with 10 lmol/L SB203580 (Santa Cruz, USA), a specific p38 MAPK inhibitor [17,18]. Cells in the control group were treated with 10 ll/L DMSO (Hyclone, USA), the solvent of SB203580. Cells in the blank group were treated with PBS. 2 h later, cells in the treatment and control groups were exposed to LIPUS with a frequency of 1 MHz and an intensity of 90 mW/cm2 for 20 min every 24 h. 11 d later, the media were collected and preserved at 80 °C, and cell lysates were obtained by digesting HPDLCs with trypsin, incubating in 0.2% Triton for 24 h at 4 °C and sonicating with a sonicator (Shanghai Bilon Instruction Co., Ltd., China). ALP activity in the media and cell lysates was measured by determining the formation of p-nitrophenol from p-nitrophenol phosphate using a kit (Nanjing Jiancheng Bioengineering Institute, China) according to the manufacturer’s instructions. 2.3. Quantitation of osteocalcin production Cells were similarly cultured and treated for 15 d instead of 11 d as mentioned above. Osteocalcin production in media was measured using radioimmunoassay kit (Beijing Atom High Tech Co., Ltd., China) following the manufacturer’s instructions. 2.4. Measurement of calcium deposition HPDLCs were inoculated into a 6-well plate containing a-MEM supplemented with 10% FBS, 50 lg/ml vitamin C and 10 mM bphosphoglycerol (Sigma, USA), at a density of 1 105 cells/cm2, to induce calcium phosphate deposition; the experiment was assigned into three groups with triplicate in each group. The cells were treated similarly as mentioned above for 21 d, washed with PBS for three times, and fixed with 0.05% glutaraldehyde for 10 min. After completely washed with demineralized water, cells were stained with alizarin red solution (Sigma, USA) for about 5 min. After washed 3 times again with demineralized water, cells were observed under an inverted microscope. The formation of one calcium nodule with diameter greater than 1 mm was considered as one counting unit [19]. 2.5. Protein extraction and western blot analysis To evaluate the involvement of p38 MAPK pathway in LIPUSinduced osteogenic differentiation of HPDLCs and its activity,
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HPDLCs (1 104 cells/cm2) were inoculated into a 6-well plate containing a-MEM supplemented with 10% FBS and cultured for 24 h at 37 °C in an incubator with 5% CO2. In the first set of experiments, HPDLCs were exposed to LIPUS under the frequency of 1 MHz and intensity of 90 mW/cm2 for 0 min, 15 min, 30 min, 60 min, 90 min, 120 min and 6 h, respectively. In the second set of experiments, cells were assigned into three groups and preincubated with 10 lmol/L SB203580 (treatment group), 10 lL DMSO (control group) and PBS (blank group), respectively, for 120 min and then exposed to LIPUS under the frequency of 1 MHz and intensity of 90 mW/cm2. The exposure time inducing the maximum ratio of phophorylated p38 (p-p38) to total p38 in the first set of experiments was chosen. HPDLCs were washed once with cold PBS, incubated on ice for 30 min, lysed with lysis buffer (Hyclone, USA) and centrifuged to obtain the whole cell lysates. Protein concentration was determined using Bradford assay (Applygen Technologies Inc., Beijing). Total 50 lg of each protein lysate were separated by electrophoresis on 5% linear gradient SDS polyacrylamide gel under reducing conditions (Bio-Rad, Hercules, CA). The separated proteins were electro-transferred onto polyvinylidine difluoride membranes. The membranes were washed once with Tris-buffered saline (TBS; pH 7.6) containing 0.1% Tween 20 (TBS-T) and blocked for 1 h in TBS-T containing 5% skimmed milk. After washed with TBS-T, the membranes were incubated overnight at 4 °C with antibodies against p38 MAPK and p-p38 MAPK (Sigma, USA) respectively at a dilution of 1:500 in TBS-T containing 5% BSA. Following three washes with BP solution, the membranes were incubated with HRP-conjugated secondary antibody (Sigma, USA) at 1:2000 dilution for 1 h at 37 °C. After another three washes with BP solution, the blots were developed with ECL and exposed to Kodak XAR-5 film. The intensity of each band was analyzed using Image tool V3.0 software. 2.6. Statistical analysis Statistical differences were evaluated by analysis of variance (ANOVA) and two-tailed Student’s t-test. P value less than 0.05 was considered as statistical significance. 3. Results 3.1. Effects of p38 MAPK inhibition on LIPUS-induced ALP, osteocalcin as well as calcium deposition ALP contents in the media and cell lysates of experimental, control and blank groups were significantly different (Flysate = 1207, Fsupernatant = 334, P < 0.05; Figs. 1 and 2). The ALP contents in the media and lysates of cells in experiment group were less than those in the control group (tlysate = 54.18, tsupernatant = 45.08, P < 0.05), while ALP contents in the media and cell lysates of control group were significantly higher than those in the blank group (tlysate = 24.47, tsupernatant = 22.98, P < 0.05). Osteocalcin contents in the media of experiment, control and blank groups were also significantly different (F = 28.2, P < 0.05). The osteocalcin content in the media of experiment group was the lowest (t = 7.48, P < 0.05), while the highest content was with the control group (t = 4.31, P < 0.05, Fig. 3). Many red calcium nodules in various size were found in the cells of control and blank groups (Figs. 4 and 5), but only few were in the cells of experimental group. In detail, the experiment group showed the least number of calcium nodules (Figs. 4c and 5), while the control group displayed the most nodules (Figs. 4b and 5), and the difference appeared statistically significant (F = 46.458, t = 3.214, P < 0.05).
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s). Fig. 1. ALP concentration in the cell lysates (v
s). Fig. 2. ALP concentration in the media (v
3.2. LIPUS stimulation enhances p38 MAPK phosphorylation The ratio of p-p38 to total p38 was enhanced in response to LIPUS stimulation. As shown in Fig. 6 and Table 1, this enhancement lasted for 6 h, reached its peak at 30 min, then gradually decreased (F = 170.01, P < 0.05). In addition, there was no significant difference in total p38 level in response to LIPUS stimulation (F = 0.748, P > 0.05). Moreover, while pretreatment of HPDLCs with SB203580 significantly attenuated the effect of LIPUS stimulation on p-p38 compared to control group (F = 2208.54, P < 0.05), it did not significantly affect the level of total p38 (F = 0.75, P > 0.05; Fig. 7 and Table 2). 4. Discussion Although the activation of p38 MAPK by LIPUS stimulation and the crucial role of p38 MAPK in osteoblast differentiation have
been reported previously [11,12,20], the role of p38 MAPK in LIPUS-induced osteogenic differentiation of HPDLCs remains unclear. In this study, we utilized a p38 specific inhibitor SB203580 to study the effects of blocking p38 MAPK signaling pathway on LIPUSinduced osteogenic differentiation of HPDLCs and found that blockade of p38 MAPK signaling pathway significantly attenuated LIPUS-induced ALP secretion, osteocalcin production and calcium deposition. The results demonstrated that p38 MAPK signaling pathway is involved in the LIPUS-induced osteogenic differentiation of HPDLCs. We also examined the effects of exposing HPDLCs to LIPUS for different time and found that although LIPUS exposure did not significantly affect the level of total p38, it significantly enhanced the ratio of p-p38 to total p38 in HPDLCs, which reached its peak at 30 min of exposure to LIPUS and declined gradually thereafter. However, the ratio of p-p38 to total p38 after 6 h exposure was still higher than the basal level, which might be because of the
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L. Ren et al. / Ultrasonics 53 (2013) 686–690
s). Fig. 3. Quantitation of the osteocalcin production (v
Blank group
Control group
Experiment group
Fig. 4. The calcium deposition (Alizarin red staining).
s). Fig. 5. Blocking the effect of LIPUS on the formation of calcium nodules by SB203580 pretreatment (v
tolerance of mechanical transmission channel receptor and the dephosphorylation effect of phosphoesterase on p-p38 [21]. More-
over, pretreatment of HPDLCs with SB203580 completely blunted the LIPUS-induced change in the ratio of p-p38 to total p38.
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L. Ren et al. / Ultrasonics 53 (2013) 686–690
ultrasound treatment instrument about periodontal disease’’ of High-tech Industry).
phospho-p38 total p38
0
5
15
30
60 120 360
Time after exposure to LIPUS (min) Fig. 6. LIPUS stimulation enhances phospho-p38, but not total p38 in a time dependent manner.
Table 1 s). The gray value of p38 and p-p38 at different time (v Different exposure time
p38
p-p38
0 min 5 min 15 min 30 min 60 min 120 min 6h
1.344 ± 0.017 1.350 ± 0.023 1.358 ± 0.015 1.346 ± 0.004 1.374 ± 0.047 1.343 ± 0.046 1.329 ± 0.017
2.107 ± 0.018 2.429 ± 0.078 2.532 ± 0.027 2.828 ± 0.039 2.325 ± 0.016 2.122 ± 0.031 1.463 ± 0.114
phospho-p38 total p38 SB203580 DMSO Fig. 7. Blocking the effect of LIPUS on phospho-p38 level by SB203580 pretreatment.
Table 2 The gray value of p38 and p-p38 after adding SB203580 (x ± s).
SB203580 groups DMSO groups
p38
p-p38
1.216 ± 0.012 1.225 ± 0.011
0.482 ± 0.023 1.099 ± 0.0009
Taken together, our results indicate that LIPUS stimulation can activate the p38 MAPK and strongly support that p38 is essential for LIPUS-induced osteogenic differentiation of HPDLCs. However, the target transcription factors of p38 pathway responsible for LIPUS-induced osteogenic differentiation remains poorly understood, which warrants further studies to fully reveal the underlying molecular mechanisms. Acknowledgments This work was supported by the College of Biomedical Engineering, Chongqing Medical University, China, the National Natural Science Foundation of China (Grant 30870754, 2009–2011), the Natural Science Foundation Project of CQ CSTC (Grant CSTC2010BB5355) and the National Development and Reform Commission of Chongqing (Grant [2007] 1110, 2008–2010, for the Technology Development Project ‘‘Study of the Low-intensity
References [1] H.W. Mi, M.C. Lee, E. Fu, L.P. Chow, C.P. Lin, Highly efficient multipotent differentiation of human periodontal ligament fibroblasts induced by combined BMP4 and hTERT gene transfer, Gene Ther. 18 (2011) 452–461. [2] D. Jönsson, D. Nebel, G. Bratthall, B.O. Nilsson, The human periodontal ligament cell: a fibroblast-like cell acting as an immune cell, J. Periodontal Res. 46 (2011) 153–157. [3] K. Washio, T. Iwata, M. Mizutani, T. Ando, M. Yamato, T. Okano, I. Ishikawa, Assessment of cell sheets derived from human periodontal ligament cells: a pre-clinical study, Cell Tissue Res. 341 (2010) 397–404. [4] S. Monnouchi, H. Maeda, S. Fujii, A. Tomokiyo, K. Kono, A. Akamine, The roles of angiotensin II in stretched periodontal ligament cells, J. Dent. Res. 90 (2011) 181–185. [5] L. Xia, Z. Zhang, L. Chen, W. Zhang, D. Zeng, X. Zhang, J. Chang, X. Jiang, Proliferation and osteogenic differentiation of human periodontal ligament cells on akermanite and b-TCP bioceramics, Eur. Cell Mater. 22 (2011) 68–82. [6] K. Sena, S.R. Angle, A. Kanaji, C. Aher, D.G. Karwo, D.R. Sumner, A.S. Virdi, Lowintensity pulsed ultrasound (LIPUS) and cell-to-cell communication in bone marrow stromal cells, Ultrasonics 51 (2011) 639–644. [7] K.N. Malizos, M.E. Hantes, V. Protopappas, A. Papachristos, Low-intensity pulsed ultrasound for bone healing: an overview, Injury 37 (2006) S56–62. [8] H.D. Choi, W.C. Noh, J.W. Park, J.M. Lee, J.Y. Suh, Analysis of gene expression during mineralization of cultured human periodontal ligament cells, J. Periodontal Implant Sci. 41 (2011) 30–43. [9] S.J. Warden, J.M. Favaloro, K.L. Bennell, J.M. McMeeken, K.W. Ng, J.D. Zajac, J.D. Wark, Low-intensity pulsed ultrasound stimulates a bone-forming response in UMR-106 cells, Biochem. Biophys. Res. Commun. 286 (2001) 443–450. [10] N.Z. Mostafa, H. Uludag˘, D.N. Dederich, M.R. Doschak, T.H. El-Bialy, Anabolic effects of low-intensity pulsed ultrasound on human gingival fibroblasts, Arch. Oral Biol. 54 (2009) 743–748. [11] J. Zhou, Effect of low intensity pulsed ultrasound on osteogenic differentiation of human periodontal ligament cells in vitro, Chongqing Medical University, 2010. [12] M.B. Greenblatt, J.H. Shim, W. Zou, D. Sitara, M. Schweitzer, D. Hu, S. Lotinun, Y. Sano, R. Baron, J.M. Park, S. Arthur, M. Xie, M.D. Schneider, B. Zhai, S. Gygi, R. Davis, L.H. Glimcher, The p38 MAPK pathway is essential for skeletogenesis and bone homeostasis in mice, J. Clin. Invest. 120 (2010) 2457–2473. [13] K. Ikeda, T. Takayama, N. Suzuki, K. Shimada, K. Otsuka, K. Ito, Effects of lowintensity pulsed ultrasound on the differentiation of C2C12 cells, Life Sci. 79 (2006) 1936–1943. [14] S.R. Angle, K. Sena, D.R. Sumner, A.S. Virdi, Osteogenic differentiation of rat bone marrow stromal cells by various intensities of low-intensity pulsed ultrasound, Ultrasonics 51 (2011) 281–288. [15] R. Shiraishi, C. Masaki, A. Toshinaga, T. Okinaga, T. Nishihara, N. Yamanaka, T. Nakamoto, R. Hosokawa, The effects of low-intensity pulsed ultrasound exposure on gingival cells, J. Periodontol. 82 (2011) 1498–1503. [16] J.H. Cho, S.K. Lee, J.W. Lee, E.C. Kim, The role of heme oxygenase-1 in mechanical stress-and lipopolysaccharide-induced osteogenic differentiation in human periodontal ligament cells, Angle Orthod. 80 (2010) 552–559. [17] C. Denkert, A. Siegert, A. Leclere, A. Turzynski, S. Hauptmann, An inhibitor of stress-activated MAP-kinases reduces invasion and MMP-2 expression of malignant melanoma cells, Clin. Exp. Metastasis 19 (2002) 79–85. [18] C. Chen, Y.H. Chen, W.W. Lin, Involvement of p38 mitogen-activated protein kinase in lipopolysaccharide-induced iNOS and COX-2 expression in J774 macrophages, Immunology 97 (1999) 124–129. [19] M.C. Castillo, M.J. Gimeno, B. Carro, J.L. Benito, E. Freile, J.M. Sainz, Diffuse pulmonary calcification in a patient with renal insufficiency, Arch. Bronconeumol 41 (2005) 587–589. [20] K.S. Lee, S.H. Hong, S.C. Bae, Both the SMAD and p38 MAPK pathways play a crucial role in Runx2 expression following induction by transforming growth factor-b and bone morphogenetic protein, Oncogene 21 (2002) 7156–7163. [21] P. Dang, S.G. Shi, Y.L. Song, Activation of p38 MAPK phosphorylation in human periodontal ligament cells induced by mechanical stress, Chin. J. Cinserv. Dent. 13 (2003) 202–204.
Contents lists available at SciVerse ScienceDirect
Ultrasonics journal homepage: www.elsevier.com/locate/ultras
Involvement of p38 MAPK pathway in low intensity pulsed ultrasound induced osteogenic differentiation of human periodontal ligament cells Leixi Ren a, Zun Yang b, Jinlin Song b,⇑, Zhibiao Wang c, Feng Deng b, Wanshan Li a a
Department of Stomatology, Children’s Hospital of Chongqing Medical University, Chongqing, China Department of Orthodontics, The Affiliated Hospital of Stomatology, Chongqing Medical University, Chongqing, China c College of Biomedical Engineering, Chongqing Medical University, Chongqing, China b
a r t i c l e
i n f o
Article history: Received 25 November 2011 Received in revised form 12 October 2012 Accepted 21 October 2012 Available online 5 November 2012 Keywords: Human periodontal ligament cells Low intensity pulsed ultrasound p38 MAPK Osteogenesis
a b s t r a c t Objective: The purpose of this study was to clarify whether p38 MAPK is involved in the process of low intensity pulsed ultrasound (LIPUS) induced osteogenic differentiation of human periodontal ligament cells (HPDLCs). Methods: HPDLCs were isolated from premolars extracted for orthodontic reasons from healthy adolescences and used in the study at passage 5. They were pretreated with p38 specific inhibitor SB203580 and exposed daily to LIPUS with frequency of 1 MHz and intensity of 90 mW/cm2. Osteogenic differentiation was assayed by levels of alkaline phosphatase (ALP) and osteocalcin as well as calcium deposition. The levels of phosphorylated p38 (p-p38) and total p38 in HPDLCs in response to LIPUS for different times were detected by Western blot. Results: The enhanced ALP levels in media and cell lysate, osteocalcin level in media, as well as number of calcium nodules after LIPUS stimulation were decreased by SB203580 treatment. LIPUS stimulation did not change total p38 level, but time-dependently enhanced the level of p-p38; such enhancement was significantly blocked by preincubation with 10 lmol/L SB203580. Conclusion: The p38 MAPK is involved in the process of LIPUS-induced osteogenic differentiation of HPDLCs. Ó 2012 Elsevier B.V. All rights reserved.
1. Introduction HPDLCs are the main cellular component of periodontal tissues among different heterogeneous cells [1]. They have stem cell like properties and can protect and repair periodontal tissues by synthesizing collagen and producing cementum. In addition, HPDLC sheets created in cell-processing centers were safe products with the potential to regenerate periodontal tissues [2]. Thus, HPDLCs are considered a beneficial cell source for clinical periodontal regeneration [3–5]. LIPUS is an established therapy for fracture repair. It was approved by the Food and Drug Administration for use in fresh bone fracture healing in 1994 and in established nonunions in 2000 [6]. Studies have shown that LIPUS could increase calcium incorporation in cartilage and bone cell cultures, alter potassium flux across cell membrane, activate adenylyl cyclase in osteoblastic cells, modulate expression of numerous genes involved in fracture healing process, enhance angiogenesis, and so on [7]. In vitro studies suggested that when appropriately triggered, osteoblastic cells in hu⇑ Corresponding author. E-mail address: [email protected] (J. Song). 0041-624X/$ - see front matter Ó 2012 Elsevier B.V. All rights reserved. http://dx.doi.org/10.1016/j.ultras.2012.10.008
man periodontal ligament could differentiate into osteoblasts/ cementoblasts [8] and LIPUS could induce transient expression of the immediate-early response gene c-fos and elevate mRNA levels of insulin-like growth factor-I, osteocalcin and bone sialoprotein [9]. In addition, LIPUS has been demonstrated to have anabolic effects on cementoblasts, odontoblasts, and periodontal ligament cells [10]. In vitro studies, the frequency of 1 MHz and the intensity of 90 mW/cm2 for 20 min everyday was optimum when LIPUS induced osteogenic differentiation of HPDLCs [11]. Previous in vitro studies on cells including mesenchymal stem cells treated with mitogen-activated protein kinases (MAPKs) inhibitors showed that MAPKs p38 and ERK are important for early osteoblast differentiation [12]. LIPUS stimulation could induce mouse myoblast cells C2C12 differentiating into osteoblasts and/ or chondroblasts via ERK1/2 and p38 [13]. Moreover, LIPUS is able to initiate osteogenic differentiation and modulate ERK1/2 and p38 signaling pathways [14]. Exposure to LIPUS could accelerate softtissue healing by increasing connective tissue growth factors via MAPK signaling pathway [15]. Because PDLCs have similar effects with mesenchymal stem cells on osteogenic differentiation [16], we hypothesized that the p38 MAPK pathway is involved in LIPUS-induced osteogenic differentiation of HPDLCs.
L. Ren et al. / Ultrasonics 53 (2013) 686–690
2. Materials and methods 2.1. Cell isolation, culture and identification HPDLCs were prepared from premolars extracted for orthodontic reasons from healthy adolescences. In detail, the premolars as well as their surrounding gingivas were disinfected and washed three times with phosphate buffered saline (PBS) containing 100 lg/mL streptomycin and 100 U/mL penicillin. Their periodontal ligament tissues were scraped from the mid-third portion of premolar roots using No. 11 sharp surgical scalpels, cut into about 1 1 mm2 pieces, placed on the bottom of a 25 ml culture flask containing a-MEM (Hyclone, USA) supplemented with 20% fetal bovine serum (FBS, Hyclone, USA) and cultured at 37 °C in an incubator with 5% CO2 (Nuair, USA). The cells were passaged when reaching 60% confluency, and cells from passage 5 were used in all experiments. Informed consent was obtained from all patients and their parents. 2.2. ALP activity assay HPDLCs were inoculated into a 6-well plate containing a-MEM supplemented with 20% FBS at a density of 1 105 cells/cm2 and assigned into three groups. The cells in treatment group were treated with 10 lmol/L SB203580 (Santa Cruz, USA), a specific p38 MAPK inhibitor [17,18]. Cells in the control group were treated with 10 ll/L DMSO (Hyclone, USA), the solvent of SB203580. Cells in the blank group were treated with PBS. 2 h later, cells in the treatment and control groups were exposed to LIPUS with a frequency of 1 MHz and an intensity of 90 mW/cm2 for 20 min every 24 h. 11 d later, the media were collected and preserved at 80 °C, and cell lysates were obtained by digesting HPDLCs with trypsin, incubating in 0.2% Triton for 24 h at 4 °C and sonicating with a sonicator (Shanghai Bilon Instruction Co., Ltd., China). ALP activity in the media and cell lysates was measured by determining the formation of p-nitrophenol from p-nitrophenol phosphate using a kit (Nanjing Jiancheng Bioengineering Institute, China) according to the manufacturer’s instructions. 2.3. Quantitation of osteocalcin production Cells were similarly cultured and treated for 15 d instead of 11 d as mentioned above. Osteocalcin production in media was measured using radioimmunoassay kit (Beijing Atom High Tech Co., Ltd., China) following the manufacturer’s instructions. 2.4. Measurement of calcium deposition HPDLCs were inoculated into a 6-well plate containing a-MEM supplemented with 10% FBS, 50 lg/ml vitamin C and 10 mM bphosphoglycerol (Sigma, USA), at a density of 1 105 cells/cm2, to induce calcium phosphate deposition; the experiment was assigned into three groups with triplicate in each group. The cells were treated similarly as mentioned above for 21 d, washed with PBS for three times, and fixed with 0.05% glutaraldehyde for 10 min. After completely washed with demineralized water, cells were stained with alizarin red solution (Sigma, USA) for about 5 min. After washed 3 times again with demineralized water, cells were observed under an inverted microscope. The formation of one calcium nodule with diameter greater than 1 mm was considered as one counting unit [19]. 2.5. Protein extraction and western blot analysis To evaluate the involvement of p38 MAPK pathway in LIPUSinduced osteogenic differentiation of HPDLCs and its activity,
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HPDLCs (1 104 cells/cm2) were inoculated into a 6-well plate containing a-MEM supplemented with 10% FBS and cultured for 24 h at 37 °C in an incubator with 5% CO2. In the first set of experiments, HPDLCs were exposed to LIPUS under the frequency of 1 MHz and intensity of 90 mW/cm2 for 0 min, 15 min, 30 min, 60 min, 90 min, 120 min and 6 h, respectively. In the second set of experiments, cells were assigned into three groups and preincubated with 10 lmol/L SB203580 (treatment group), 10 lL DMSO (control group) and PBS (blank group), respectively, for 120 min and then exposed to LIPUS under the frequency of 1 MHz and intensity of 90 mW/cm2. The exposure time inducing the maximum ratio of phophorylated p38 (p-p38) to total p38 in the first set of experiments was chosen. HPDLCs were washed once with cold PBS, incubated on ice for 30 min, lysed with lysis buffer (Hyclone, USA) and centrifuged to obtain the whole cell lysates. Protein concentration was determined using Bradford assay (Applygen Technologies Inc., Beijing). Total 50 lg of each protein lysate were separated by electrophoresis on 5% linear gradient SDS polyacrylamide gel under reducing conditions (Bio-Rad, Hercules, CA). The separated proteins were electro-transferred onto polyvinylidine difluoride membranes. The membranes were washed once with Tris-buffered saline (TBS; pH 7.6) containing 0.1% Tween 20 (TBS-T) and blocked for 1 h in TBS-T containing 5% skimmed milk. After washed with TBS-T, the membranes were incubated overnight at 4 °C with antibodies against p38 MAPK and p-p38 MAPK (Sigma, USA) respectively at a dilution of 1:500 in TBS-T containing 5% BSA. Following three washes with BP solution, the membranes were incubated with HRP-conjugated secondary antibody (Sigma, USA) at 1:2000 dilution for 1 h at 37 °C. After another three washes with BP solution, the blots were developed with ECL and exposed to Kodak XAR-5 film. The intensity of each band was analyzed using Image tool V3.0 software. 2.6. Statistical analysis Statistical differences were evaluated by analysis of variance (ANOVA) and two-tailed Student’s t-test. P value less than 0.05 was considered as statistical significance. 3. Results 3.1. Effects of p38 MAPK inhibition on LIPUS-induced ALP, osteocalcin as well as calcium deposition ALP contents in the media and cell lysates of experimental, control and blank groups were significantly different (Flysate = 1207, Fsupernatant = 334, P < 0.05; Figs. 1 and 2). The ALP contents in the media and lysates of cells in experiment group were less than those in the control group (tlysate = 54.18, tsupernatant = 45.08, P < 0.05), while ALP contents in the media and cell lysates of control group were significantly higher than those in the blank group (tlysate = 24.47, tsupernatant = 22.98, P < 0.05). Osteocalcin contents in the media of experiment, control and blank groups were also significantly different (F = 28.2, P < 0.05). The osteocalcin content in the media of experiment group was the lowest (t = 7.48, P < 0.05), while the highest content was with the control group (t = 4.31, P < 0.05, Fig. 3). Many red calcium nodules in various size were found in the cells of control and blank groups (Figs. 4 and 5), but only few were in the cells of experimental group. In detail, the experiment group showed the least number of calcium nodules (Figs. 4c and 5), while the control group displayed the most nodules (Figs. 4b and 5), and the difference appeared statistically significant (F = 46.458, t = 3.214, P < 0.05).
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s). Fig. 1. ALP concentration in the cell lysates (v
s). Fig. 2. ALP concentration in the media (v
3.2. LIPUS stimulation enhances p38 MAPK phosphorylation The ratio of p-p38 to total p38 was enhanced in response to LIPUS stimulation. As shown in Fig. 6 and Table 1, this enhancement lasted for 6 h, reached its peak at 30 min, then gradually decreased (F = 170.01, P < 0.05). In addition, there was no significant difference in total p38 level in response to LIPUS stimulation (F = 0.748, P > 0.05). Moreover, while pretreatment of HPDLCs with SB203580 significantly attenuated the effect of LIPUS stimulation on p-p38 compared to control group (F = 2208.54, P < 0.05), it did not significantly affect the level of total p38 (F = 0.75, P > 0.05; Fig. 7 and Table 2). 4. Discussion Although the activation of p38 MAPK by LIPUS stimulation and the crucial role of p38 MAPK in osteoblast differentiation have
been reported previously [11,12,20], the role of p38 MAPK in LIPUS-induced osteogenic differentiation of HPDLCs remains unclear. In this study, we utilized a p38 specific inhibitor SB203580 to study the effects of blocking p38 MAPK signaling pathway on LIPUSinduced osteogenic differentiation of HPDLCs and found that blockade of p38 MAPK signaling pathway significantly attenuated LIPUS-induced ALP secretion, osteocalcin production and calcium deposition. The results demonstrated that p38 MAPK signaling pathway is involved in the LIPUS-induced osteogenic differentiation of HPDLCs. We also examined the effects of exposing HPDLCs to LIPUS for different time and found that although LIPUS exposure did not significantly affect the level of total p38, it significantly enhanced the ratio of p-p38 to total p38 in HPDLCs, which reached its peak at 30 min of exposure to LIPUS and declined gradually thereafter. However, the ratio of p-p38 to total p38 after 6 h exposure was still higher than the basal level, which might be because of the
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s). Fig. 3. Quantitation of the osteocalcin production (v
Blank group
Control group
Experiment group
Fig. 4. The calcium deposition (Alizarin red staining).
s). Fig. 5. Blocking the effect of LIPUS on the formation of calcium nodules by SB203580 pretreatment (v
tolerance of mechanical transmission channel receptor and the dephosphorylation effect of phosphoesterase on p-p38 [21]. More-
over, pretreatment of HPDLCs with SB203580 completely blunted the LIPUS-induced change in the ratio of p-p38 to total p38.
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ultrasound treatment instrument about periodontal disease’’ of High-tech Industry).
phospho-p38 total p38
0
5
15
30
60 120 360
Time after exposure to LIPUS (min) Fig. 6. LIPUS stimulation enhances phospho-p38, but not total p38 in a time dependent manner.
Table 1 s). The gray value of p38 and p-p38 at different time (v Different exposure time
p38
p-p38
0 min 5 min 15 min 30 min 60 min 120 min 6h
1.344 ± 0.017 1.350 ± 0.023 1.358 ± 0.015 1.346 ± 0.004 1.374 ± 0.047 1.343 ± 0.046 1.329 ± 0.017
2.107 ± 0.018 2.429 ± 0.078 2.532 ± 0.027 2.828 ± 0.039 2.325 ± 0.016 2.122 ± 0.031 1.463 ± 0.114
phospho-p38 total p38 SB203580 DMSO Fig. 7. Blocking the effect of LIPUS on phospho-p38 level by SB203580 pretreatment.
Table 2 The gray value of p38 and p-p38 after adding SB203580 (x ± s).
SB203580 groups DMSO groups
p38
p-p38
1.216 ± 0.012 1.225 ± 0.011
0.482 ± 0.023 1.099 ± 0.0009
Taken together, our results indicate that LIPUS stimulation can activate the p38 MAPK and strongly support that p38 is essential for LIPUS-induced osteogenic differentiation of HPDLCs. However, the target transcription factors of p38 pathway responsible for LIPUS-induced osteogenic differentiation remains poorly understood, which warrants further studies to fully reveal the underlying molecular mechanisms. Acknowledgments This work was supported by the College of Biomedical Engineering, Chongqing Medical University, China, the National Natural Science Foundation of China (Grant 30870754, 2009–2011), the Natural Science Foundation Project of CQ CSTC (Grant CSTC2010BB5355) and the National Development and Reform Commission of Chongqing (Grant [2007] 1110, 2008–2010, for the Technology Development Project ‘‘Study of the Low-intensity
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