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ORIGINAL RESEARCH
Low-Intensity Pulsed Ultrasound Induces Osteogenic Differentiation of Human Periodontal Ligament Cells Through Activation of Bone Morphogenetic Protein–Smad Signaling Zun Yang, MM, Leixi Ren, MM, Feng Deng, MD, Zhibiao Wang, MD, Jinlin Song, MD
Received April 1, 2013, from the Affiliated Hospital of Stomatology (Z.Y., F.D., J.S.) and College of Biomedical Engineering (Z.W.), Chongqing Medical University, Chongqing, China; Chongqing Key Laboratory for Oral Diseases and Biomedical Sciences, Chongqing, China (Z.Y., F.D., J.S.); and Department of Stomatology, Children’s Hospital of Chongqing Medical University, Chongqing, China (L.R.). Revision requested June 27, 2013. Revised manuscript accepted for publication August 30, 2013. We thank Jinyong Luo, PhD (Key Laboratory of Diagnostic Medicine, designated by the Chinese Ministry of Education, Chongqing Medical University). This work was supported by the College of Biomedical Engineering of Chongqing Medical University of China, the National Natural Science Foundation of China (grant 30870754, 2009–2011), the Natural Science Foundation Project of CQ CSTC (grant CSTC2010BB5355), a grant from the Affiliated Hospital of Stomatology of Chongqing Medical University, and the Chongqing Key Laboratory for Oral Diseases and Biomedical Sciences. Address correspondence to Jinlin Song, MD, Affiliated Hospital of Stomatology, Chongqing Medical University, 426 Songshi North St, Yubei District, 401147 Chongqing, China. E-mail: [email protected] Abbreviations
ALP, alkaline phosphatase; ANOVA, analysis of variance; BMP, bone morphogenetic protein; cDNA, complementary DNA; GAPDH, glyceraldehyde-3-phosphate dehydrogenase; mRNA, messenger RNA; PBS, phosphatebuffered saline; PCR, polymerase chain reaction; US, ultrasound doi:10.7863/ultra.33.5.865
Objectives—Low-intensity pulsed ultrasound (US) can accelerate fracture healing and osteogenic differentiation. The aim of this study was to investigate the osteogenic effect of low-intensity pulsed US on human periodontal ligament cells and to determine whether bone morphogenetic protein (BMP)-Smad signaling was involved. Methods—Human periodontal ligament cells were exposed to low-intensity pulsed US at a frequency of 1.5 MHz and intensity of 90 mW/cm2 for 20 min/d. Osteogenic differentiation was determined by assaying alkaline phosphatase (ALP) and calcium deposition. Expression of BMP-2, BMP-6, and BMP-9 was detected by real-time polymerase chain reaction analysis. Phosphorylated Smad was detected by western blotting; Smad in the cells was labeled by an immunofluorescent antibody and observed by laserscanning confocal microscopy. Results—The optical density of ALP stimulated by US at 1.5 MHz and 90 mW/cm2 for 20 min/d was significantly higher than in other groups (P < .01); therefore, this dosage was considered optimal for promoting osteogenic differentiation. After 13 days of US exposure, ALP increased gradually after 5 days, peaked at 11 days, and decreased at 13 days, with a significant difference compared with the control group (P < .05). Osteocalcin production increased from 9 to 13 days and peaked at 15 days, with a significant difference compared with the control group (P < .05). BMP-2 and BMP-6 increased dynamically after exposure for 13 days. BMP-2 increased 6.07-fold at 3 days, 6.39-fold at 11 days, and 5.97-fold at 13 days. BMP-6 expression increased 6.82-fold at 1 day and 51.5-fold at 3 days and decreased thereafter. BMP-9 was not expressed. Phospho-Smad1/5/8 expression was significantly increased after exposure (P< .05) and transferred from the cytoplasm into the nuclei. Conclusions—Low-intensity pulsed US effectively induced osteogenic differentiation of human periodontal ligament cells, and the BMP-Smad signaling pathway was involved in the mechanism. Key Words—basic science; bone morphogenetic protein–Smad; human periodontal ligament cells; low-intensity pulsed ultrasound; osteogenic differentiation; signal transduction
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uman periodontal ligament cells are among the main cellular components of the periodontal ligament connective tissue and play an important role in the maintenance, repair, and regeneration of periodontal tissue with the ability of osteogenic
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differentiation,1 which has shown the capacity to differentiate into osteoblast-like structures and contribute to periodontal tissue repair.1–4 It has been reported that periodontal ligament cells can express bone-associated molecules such as alkaline phosphatase (ALP), osteopontin, and osteocalcin.5,6 These cells have been considered a promising source for alveolar bone and periodontal ligament cell tissue regeneration and tissue engineering.7 Human periodontal ligament cells can undergo osteogenic differentiation by stimulation of cytokines and growth factors such as plateletderived growth factor,8 epidermal growth factor,9 transforming growth factor,10 and bone morphogenetic protein (BMP).11 Of those factors, BMP has been shown to be an important mediator for controlling osteoblast differentiation through the BMP-Smad signaling pathway.12 Gene responses to BMPs are mediated by Smad transcription factors; after phosphorylation on serine residues by the BMP receptor complex, Smad1, Smad5, and Smad8 move into the nucleus, where they regulate specific sets of target genes to mediate BMP-induced signals from the cell surface to the nucleus.13–16 Low-intensity pulsed ultrasound (US; intensity range, 30–100 mW/cm2) can be transmitted into the tissues as pressure waves to accelerate fracture healing in vivo.17–19 It has been proven to enhance ALP activity and accelerate calcium deposition20 in osteoblast cells. Low-intensity pulsed US exerts direct anabolic effects such as growth factor stimulation, osteogenic differentiation, and extracellular matrix production.21 The biophysical effects of low-intensity pulsed US are most likely caused by mechanical stress and microstreaming on the cellular plasma membrane and cytoskeletal structures, which induce intracellular signal transduction and gene transcription.22 However, to our knowledge, no studies have thus far evaluated the precise cellular molecular basis of osteogenic function in human periodontal ligament cells when stimulated by low-intensity pulsed US. This study investigated whether BMP-Smad signaling controlled the osteogenic regulation of human periodontal ligament cells under lowintensity pulsed US exposure. We assessed the differentiation effects of low-intensity pulsed US on the cells through ALP, osteocalcin, and BMP levels and explored the signal pathway by performing a phosphorylated Smad experiment and confocal laser-scanning microscopy.
Materials and Methods Cell Culture Human periodontal ligament cells were isolated from fresh premolars of healthy adolescents who were referred for
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orthodontic treatment to the Affiliated Hospital of Stomatology of Chongqing Medical University. The study was approved by the university’s Ethics Committee. The extracted teeth were immersed in Dulbecco’s modified Eagle’s medium (Hyclone, Logan, UT) supplemented with 10% fetal bovine serum (Hyclone), 125-U/mL penicillin, and 125-U/mL streptomycin (North China Pharmaceutical Group, Shijiangzhuang, China). The periodontal ligament tissue from the middle third of the root surface was scraped with a sharp No. 12 scalpel. The extracted tissues were immersed in 6-well plates in 1.5 mL of 20% fetal bovine serum and Dulbecco’s modified Eagle’s medium at 36.5°C and 5% carbon dioxide for primary culture, with medium replacement every other day.23 Cells at passage 4 were used for the experiments. Low-Intensity Pulsed US Exposure The low-intensity pulsed US exposure device was manufactured by the National Engineering Research Center of Ultrasound Medicine (Chongqing, China), which designed an array of 6 transducers fixed with a support device for using a 6-well culture plate. The plate was located above the array to touch the 6 transducers with dielectric coupling gel (Figure 1). Before the experiments, the US transducer was sterilized with 75% alcohol and ultraviolet light. The US exposure conditions were a frequency of 1.5 MHz, a 200-millisecond burst width sine wave, a pulse duty cycle of 1:4, a pulse repetition frequency of 1.0 kHz, intensity of 30, 60, or 90 mW/cm2, and an exposure time of 10, 20, or 30 minutes daily. The control group was treated in the same manner without US exposure. Optimizing the Dosage of Low-Intensity Pulsed US Human periodontal ligament cells (2.5 × 103) were seeded into a 6-well plate. The cells were exposed to low-intensity pulsed US for 0 seconds (control); 10, 20, or 30 min/d at 30 mW/cm2; 10, 20, or 30 min/d at 60 mW/cm2; or 10, 20, or 30 min/d at 90 mW/cm2 for 5 days, and then the US dosage was optimized by determining the ALP activity. Alkaline Phosphatase Activity Assay Human periodontal ligament cells at passage 4 were seeded into a 6-well plate at a density of 5 × 103 cells/cm2 (0.5 mL/well) with low-intensity pulsed US exposure at 90 mW/cm2 for 20 min/d for up to 13 days. The ALP activity in the lysate was assayed with a kit (Nanjing Jiancheng Bioengineering Institute, Nanjing, China) by measuring the formation of p-nitrophenol from p-nitrophenol phosphate at 3, 5, 7, 9, 11, and 13 days. The cells in each group were digested by trypsin, incubated in 0.2%
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Triton X-100 for 24 hours at 4°C, and split by ultrasonication (Shanghai Bilon Instruction Co, Ltd, Shanghai, China) to obtain the lysates. Osteocalcin Radioimmunoassay Human periodontal ligament cells at passage 4 were seeded into a 6-well plate at a density of 5 × 103 cells/cm2 (0.5 mL/well). Low-intensity pulsed US exposure at 90 mW/cm2 for 20 min/d was administered for up to 15 days. The culture medium of each group was collected at 5, 7, 9, 11, 13, and 15 days. The amount of osteocalcin in the supernatant was measured by radioimmunoassay (Department of Nuclear Medicine, First Affiliated Hospital of Chongqing Medical University). A mouse osteocalcin assay kit (Beijing Atom High Tech Co, Ltd, Beijing, China) was used according to the manufacturer’s instructions. RNA Isolation and Analysis Human periodontal ligament cells at passage 4 were cultured in a 6-well plate and exposed to low-intensity pulsed US at 90 mW/cm2 for 20 min/d and were harvested at 1, 3, 5, 7, 9, 11, and 13 days after US exposure. Total RNA was isolated with the RNAiso Plus reagent (TaKaRa
Biotechnology, Dalian, China). Aliquots that contained equal amounts of messenger RNA (mRNA) were subjected to real-time polymerase chain reaction (PCR) analysis. The mRNA was converted into complementary DNA (cDNA) by using a deoxynucleoside triphosphate mixture (TaKaRa Biotechnology), a random primer (TaKaRa Biotechnology), a ribonuclease inhibitor (TaKaRa Biotechnology), and Maloney murine leukemia virus reverse transcriptase (Promega, Madison, WI). The cDNA mixtures were diluted 5-fold in sterile distilled water, and 1 μL was subjected to real-time PCR using SYBR Premix Ex Taq (TaKaRa Biotechnology). Reactions were performed in 25-μL volumes of a solution containing 12.5 μL of SYBR Premix Ex Taq, 1 μL of primers (sense and antisense; TaKaRa Biotechnology) and 10.5 μL of distilled deionized water. The primers (Table 1) were designed using Primer3 software (Whitehead Institute for Biomedical Research, Cambridge, MA). Assays were performed on a RotorGene 6000 system (Corbett Life Science, Sydney, New South Wales, Australia) and analyzed with Rotor-Gene 6000 series software. Polymerase chain reaction conditions were 95°C for 10 seconds, 45 cycles at the annealing temperature (BMP-2 at 54°C and BMP-6 at 56°C) for
Figure 1. Experimental low-intensity pulsed US exposure setup.
Table 1. Real-time PCR Primers Used in the Experiments Target cDNA
Primer Sequence
Human BMP-2 CDS qPCR forward Human BMP-2 CDS qPCR reverse Human BMP-6 CDS qPCR forward Human BMP-6 CDS qPCR reverse Human GAPDH qRT-PCR forward, 18 mer Human GAPDH qRT-PCR reverse, 18 mer
5′-GGGCATCCTCTCCACAAA-3′ 5′-GTCATTCCACCCCACGTC-3′ 5′-TGCAGGAAGCATGAGCTG-3′ 5′-GTGCGTTGAGTGGGAAGG-3′ 5′-CAGCGACACCCACTCCTC-3′ 5′-TGAGGTCCACCACCCTGT-3′
CDS indicates coding sequence; q, quantitative; and RT, real-time.
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30 seconds, and 72°C for 30 seconds. All of the real-time PCR reactions were performed in triplicate, and the gene expression levels were calculated and normalized by dividing the calculated values for the mRNA samples by that of glyceraldehyde-3-phosphate dehydrogenase (GAPDH) mRNA at each time point. The 2(-Delta Delta C(T)) calculation method24 was used to analyze the results of amplification. Protein Extraction and Western Blot Analysis The involvement of BMP-Smad signal transduction in the osteogenic differentiation of human periodontal ligament cells was evaluated in this part of the experiment. The cells were exposed to low-intensity pulsed US at 90 mW/cm2 for 20 minutes after 5, 30, 60, and 120 minutes and 4 hours. At every time point, the samples were scraped and pelleted by centrifugation at 0°C at 1000g for 15 minutes. The pellets were washed twice in phosphate-buffered saline (PBS), homogenized in 0.2 N sulfuric acid, and centrifuged at 13,000g. Histones were pelleted from the supernatant by adding a 0.25 volume of 100% trichloroacetic acid. The pellets were suspended in 100% ethanol overnight and centrifuged again at 13,000g. The pellets were dissolved in ultrapure water and evaluated for protein concentration. An aliquot corresponding to 10 μg of protein was boiled twice in sodium dodecyl sulfate sample buffer (Tris-glycine– sodium dodecyl sulfate sample buffer; Invitrogen, Carlsbad, CA) and loaded onto a 4% to 20% Tris-glycine gel. The separated proteins were transferred to a polyvinylidene difluoride membrane. The membrane was blocked with TBST (10 mM Tris-Cl, pH 7.4, 200 mM sodium chloride, and 0.1% Tween 20) containing 5% nonfat milk for 60 minutes before incubation with 50-ng/mL antiphospho-Smad1/5/8 antibody (9511; Cell Signaling Technology, Beverly, MA) or anti-Smad1/5/8 antibody (sc-6031-R; Santa Cruz Biotechnology, Santa Cruz, CA) for 2 hours. The blots were washed in TBST and then incubated with a horseradish peroxidase–conjugated antimouse antibody (1:1000 dilution), and detection of the antigen-bound antibody was performed with an enhanced chemiluminescence western blotting analysis system (Amersham Life Science, Arlington Heights, IL).
(sc-6031-R; Santa Cruz Biotechnology) for 12 hours at 37°C. After the cells were washed 3 times with PBS, they were incubated with a goat antirabbit secondary antibody for an additional 1 hour. The cells were then washed with PBS, mounted, and observed on a laser-scanning confocal microscope (TCS SP2; Leica, Heidelberg, Germany) using a ×400 objective. Statistical Analysis The optimum dosage of low-intensity pulsed US was analyzed by 1-way analysis of variance (ANOVA). Alkaline phosphatase activity and the osteocalcin assay were analyzed by a paired t test, and P < .05 was considered statistically significant. Expression of BMP amplification was analyzed by the 2(-Delta Delta C(T)) calculation method.24 Expression of phospho-Smad1/5/8 was analyzed by 1-way ANOVA, and P < .05 was considered statistically significant.
Results Optimum Dosage of Low-Intensity Pulsed US Low-intensity pulsed US exposure increased the level of ALP in human periodontal ligament cells, with the highest optical density for US at 90 mW/cm2 for 20 min/d (Figure 2). The mean optical density of ALP ± SD in the cells was 0.0551 ± 0.0003, with significant differences (F = 0.59; P < .01) among the 9 exposure groups, as analyzed by 1-way ANOVA. This set of US parameters was therefore used for the following experiments. Figure 2. Alkaline phosphatase activity in human periodontal ligament cells after low-intensity pulsed US (LIPUS) exposure at different intensities and times after 5 days. *P < 01.
Confocal Microscopy Human periodontal ligament cells were exposed to lowintensity pulsed US at 90 mW/cm2 for 20 minutes. Cells were fixed after exposure for 1, 2, 4, and 6 hours in 0.4% paraformaldehyde for 20 minutes. Fixed cells were incubated with 10% normal goat serum for 40 minutes and then with the anti-Smad1/5/8 primary antibody
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Effect of Low-Intensity Pulsed US on ALP Activity and Osteocalcin as Well as Calcium Deposition As shown in Figure 3, the optical density of ALP in human periodontal ligament cells at 3, 5, 7, 9, 11, and 13 days was analyzed by a paired t test. Compared with the control group, ALP activity increased gradually up to 5 days, peaked at 11 days, and decreased at 13 days in the lowintensity pulsed US exposure groups. Osteocalcin production at 5, 7, 9, 11, 13, and 15 days was analyzed by a paired t test. Compared with the control group, osteocalcin increased at 9, 11, and 13 days and peaked at 15 days in the US exposure groups (P < .05). Real-time PCR Analysis of BMP-2, BMP-6, and BMP-9 As shown in Figure 4, after exposure to low-intensity pulsed US at 90 mW/cm2 for 20 min/d for 13 days, BMP2 mRNA expression increased over the 13 days in the US exposure groups. The BMP-2 mRNA level compared with Figure 3. A, Alkaline phosphatase activity in human periodontal ligament cells after low-intensity pulsed US (LIPUS) exposure at 90 mW/cm2 for 20min/d for 13 days. OD indicates optical density. B, Osteocalcin (OC) activity in the cells after low-intensity pulsed US exposure at the same dosage for 15 days.
the control group increased 6.07-fold at 3 days, 6.39-fold at 11 days, and 5.97-folds at 13 days. The BMP-6 level compared with the control group was 6.82-fold greater at 1 day, increased 51.5-fold at 3 days, and then decreased over the following days. No BMP-9 expression was observed in the cells in both the control and experimental groups. Smad1/5/8 Phosphorylation Compared with the control group, phospho-Smad1/5/8 protein expression was significantly increased in the western blot assay after treatment with low-intensity pulsed US at 90 mW/cm2 for 20 minutes. The phospho-Smad1/5/8 level gradually increased after US exposure at the 30minute point, reached its peak at the 60-minute point, maintained that level until the 120-minute point, and then decreased at the 4-hour point (Figure 5). The gray value of phospho-Smad1/5/8 was analyzed by 1-way ANOVA (P < .05). This result showed that the Smad1/5/8 underwent processes of phosphorylation and dephosphorylation right after US stimulation, indicating this pathway may be involved in the effect of low-intensity pulsed US on human periodontal ligament cells.
A Figure 4. Expression of BMP-2 and BMP-6 in human periodontal ligament cells over 13 days; con indicates control.
B
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Smad1/5/8 Transfer After Smad1/5/8 protein was stained by an immunofluorescent antibody, low-intensity pulsed US-stimulated human periodontal ligament cells were observed under confocal microscopy. The transfer activity is shown in Figure 6. The high green fluorescent label indicates Smad1/5/8. In the control group, the high green fluorescent label was mainly in the cytoplasm, and the nucleus was empty; 1 hour after US exposure, the label was in the nucleus, indicating that Smad1/5/8 started to shift into the nucleus; 2 hours after exposure, the label was still mainly in the nucleus; and 4 hours after exposure, the label was in the cell nucleus. However, 6 hours after exposure, the label was basically in the cytoplasm again.
Discussion It is well accepted that low-intensity pulsed US can induce cellular responses in cementoblasts, periodontal ligament cells, and bone cells. The positive influence of low-intensity pulsed US stimulation on tissue regeneration has been demonstrated in both animal and clinical trials.22 Human periodontal ligament cells, which are known to have a stem cell–like character, can differentiate into osteoblasts and cementoblasts, which act on periodontal regeneration.25 Among all of the research on periodontal regeneration, promoting human periodontal ligament cell proliferation and osteogenesis is the most important aspect. Osteogenesis proceeds through 3 overlapping phases: proliferation, differentiation, and calcification.26 During the differentiation phase, osteogenesis-related proteins are synthesized, and the extracellular matrix is formed and matures.27 This activity is characterized by expression of osteoblast phenotypes such as ALP activity and collagen 1 secretion in the early phase28 and extracellular matrix calcification induced by osteocalcin production in the later phase.29 This study indicated that ALP activity was increased in all low-intensity pulsed US exposure groups, although the highest activity occurred in the group with a dosage of 90 mW/cm2 for 20 min/d, which suggests that this dosage could be administered in subsequent research as the optimum dosage. Data indicated that low-intensity pulsed US might promote ALP synthesis at 5 days, peaking at 7 to 11 days, secreting out of the cells at 9 days, and decreasing at 13 days. Osteocalcin production in human periodontal ligament cells increased with time from 5 to 15 days in the US exposure groups. Significantly higher osteocalcin production was detected 11 and 13 days after exposure and peaked at 15 days. The time point of peak osteocalcin production was later than that of ALP, which might have
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been attributable to the fact that ALP production is an early osteogenic response in human periodontal ligament cells when stimulated by low-intensity pulsed US, whereas osteocalcin is a marker protein that is produced later in the osteogenic process. The mechanical and cavitation effects of low-intensity pulsed US are converted into stress30,31 on the cell membrane, which leads to surface structural changes.19,32,33 As a result, human periodontal ligament cells respond to stimulation by activating a signal transduction reaction to regulate osteogenic-related gene expression.34–37 Thus, we conducted an in-depth study of BMP-Smad signaling in human periodontal ligament cells stimulated by low-intensity pulsed US. Bone morphogenetic proteins constitute the largest group within the transforming growth factor β superfamily of hormonally active factors. Genetic studies have shown that BMPs and their related molecules are essential for normal skeletal development in vertebrates.38 In MC3T3-E1 osteoblastic cells, BMP-2 induced an increase in cellular ALP activity, and BMP-2 and BMP-3 slightly but significantly stimulated collagen synthesis.39 BMP-6 has been reported to display 2- to 2.5-fold more potent induction of osteoblast differentiation than BMP-2 or BMP-4 in a fetal rat secondary calvarial cell culture system, which induced the formation of more and larger bone nodules as well as increased osteocalcin secretion and might have acted at an earlier stage of cell differentiation.40 Figure 5. Expression of phospho-Smad1/5/8 (p-Smad) in human periodontal ligament cells by Western blotting; con indicates control.
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BMP-9 was proven to induce ALP in mesenchymal stem cells and mouse embryonic fibroblasts and to induce recruitment of both Runx2 and β-catenin to the osteocalcin promoter.41 Therefore, BMP-2, BMP-6, and BMP-9, which are the most potent factors of osteogenic differentiation attributable to low-intensity pulsed US, were chosen to assess in this study. The results showed that BMP-2 expression in human periodontal ligament cells exposed to low-intensity pulsed US at 90 mW/cm2 for 20 min/d for 13 days had a wavelike increase, peaking at 3 and 11 days. BMP-6 increased significantly at 3 days and then gradually decreased, which indicated that BMP-6 might act on the early response of human periodontal ligament cells stimulated by low-intensity pulsed US. However, BMP-9 was not expressed in both the control and experimental groups, indicating that BMP-9 did not exist in these cells. Gene responses to BMPs are mediated by Smad transcription factors after being phosphorylated on serine residues by the BMP receptor complex42 when stimulated
by the mechanical force of low-intensity pulsed US. Smads are divided into 3 classes: receptor-regulated Smads, common Smads, and inhibitory Smads,43 and each of them has a distinct function. Receptor-regulated Smads (Smad1/5/8) are phosphorylated by the type 1 receptor on the carboxylterminal SSXS motif. Once phosphorylated, receptorregulated Smads dissociate from the receptor, bind to common Smad (Smad4), and enter the nucleus.44 In the nucleus, heteromeric Smad complexes function as effectors of BMP signaling by regulating transcription of specific genes to increase various kinds of osteogenic differentiation indicators.38 In this study, when human periodontal ligament cells were stimulated by low-intensity pulsed US exposure at 90 mW/cm2 for 20 min/d, western blotting showed that Smad1/5/8 was phosphorylated from 30 minutes, peaked at 120 minutes, and then decreased at 4 hours. Immunofluorescence staining showed that Smad1/5/8 transferred from the cytoplasm to the nucleus in 4 hours and then retreated at 6 hours.
Figure 6. Nuclear accumulation of Smad1/5/8 in human periodontal ligament cells after low-intensity pulsed US exposure from 1 to 6 hours (original magnification ×400): A, control group; B, 1 hour after exposure; C, 2 hours after exposure; D, 4 hours after exposure; E, 6 hours after exposure.
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In conclusion, our results suggest that low-intensity pulsed US exposure induces human periodontal ligament cells into osteogenic differentiation, and the mechanism of osteogenic differentiation may be achieved through the BMP-Smad signaling pathway, which is involved in the early response of stimulation by low-intensity pulsed US. Although this study’s findings have merit for identifying the process of human periodontal ligament cell response to low-intensity pulsed US stimulation, an in vitro model cannot be extrapolated to in vivo conditions directly. Further experiments are needed to clarify this inference and to provide a basis for clinical applications.
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30. Perry MJ, Parry LK, Burton VJ, et al. Ultrasound mimics the effect of mechanical loading on bone formation in vivo on rat ulnae. Med Eng Phys 2009; 31:42–47. 31. Kasturi G, Adler RA. Mechanical means to improve bone strength: ultrasound and vibration. Curr Rheumatol Rep 2011; 13:251–256. 32. Appleford MR, Oh S, Cole JA, Protivínský J, Ong JL Ultrasound effect on osteoblast precursor cells in trabecular calcium phosphate scaffolds. Biomaterials 2007; 28:4788–4794. 33. Takeuchi R, Ryo A, Komitsu N, et al. Low-intensity pulsed ultrasound activates the phosphatidylinositol 3 kinase/Akt pathway and stimulates the growth of chondrocytes in three-dimensional cultures: a basic science study. Arthritis Res Ther 2008; 10:R77. 34. Chiquet M, Renedo AS, Huber F, Flück M. How do fibroblasts translate mechanical signals into changes in extracellular matrix production? Matrix Biol 2003; 22:73–80. 35. Ikeda K, Takayama T, Suzuki N, Shimada K, Otsuka K, Ito K. Effects of low-intensity pulsed ultrasound on the differentiation of C2C12 cells. Life Sci 2006; 79:1936–1943. 36. Sena K, Leven RM, Mazhar K, Sumner DR, Virdi AS Early gene response to low-intensity pulsed ultrasound in rat osteoblastic cells. Ultrasound Med Biol 2005; 31:703–708. 37. de Gusmão CV, Pauli JR, Saad MJ, Alves JM, Belangero WD. Low-intensity ultrasound increases FAK, ERK-1/2, and IRS-1 expression of intact rat bones in a noncumulative manner. Clin Orthop Relat Res 2010; 468:1149–1156. 38. Wan M, Cao X. BMP signaling in skeletal development. Biochem Biophys Res Commun 2005; 328:651–657. 39. Takuwa Y, Ohse C, Wang EA, Wozney JM, Yamashita K. Bone morphogenetic protein-2 stimulates alkaline phosphatase activity and collagen synthesis in cultured osteoblastic cells MC3T3-E1. Biochem Biophys Res Commun 1991; 174:96–101. 40. Boden SD, McCuaig K, Hair G, et al. Differential effects and glucocorticoid potentiation of bone morphogenetic protein action during rat osteoblast differentiation in vitro. Endocrinology 1996; 137:3401–3407. 41. Tang N, Song WX, Luo J, et al. BMP-9-induced osteogenic differentiation of mesenchymal progenitors requires functional canonical Wnt/ β-catenin signalling. J Cell Mol Med 2009; 13:2448–2464. 42. Shi Y, Massagué J. Mechanisms of TGF-β signaling from cell membrane to the nucleus. Cell 2003; 113:685–700. 43. Attisano L, Lee-Hoeflich ST. The Smads. Genome Biol 2001; 2:REVIEWS3010. 44. Taylor IW, Wrana JL. SnapShot: the TGFβ pathway interactome. Cell 2008; 133:378.e1.
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ORIGINAL RESEARCH
Low-Intensity Pulsed Ultrasound Induces Osteogenic Differentiation of Human Periodontal Ligament Cells Through Activation of Bone Morphogenetic Protein–Smad Signaling Zun Yang, MM, Leixi Ren, MM, Feng Deng, MD, Zhibiao Wang, MD, Jinlin Song, MD
Received April 1, 2013, from the Affiliated Hospital of Stomatology (Z.Y., F.D., J.S.) and College of Biomedical Engineering (Z.W.), Chongqing Medical University, Chongqing, China; Chongqing Key Laboratory for Oral Diseases and Biomedical Sciences, Chongqing, China (Z.Y., F.D., J.S.); and Department of Stomatology, Children’s Hospital of Chongqing Medical University, Chongqing, China (L.R.). Revision requested June 27, 2013. Revised manuscript accepted for publication August 30, 2013. We thank Jinyong Luo, PhD (Key Laboratory of Diagnostic Medicine, designated by the Chinese Ministry of Education, Chongqing Medical University). This work was supported by the College of Biomedical Engineering of Chongqing Medical University of China, the National Natural Science Foundation of China (grant 30870754, 2009–2011), the Natural Science Foundation Project of CQ CSTC (grant CSTC2010BB5355), a grant from the Affiliated Hospital of Stomatology of Chongqing Medical University, and the Chongqing Key Laboratory for Oral Diseases and Biomedical Sciences. Address correspondence to Jinlin Song, MD, Affiliated Hospital of Stomatology, Chongqing Medical University, 426 Songshi North St, Yubei District, 401147 Chongqing, China. E-mail: [email protected] Abbreviations
ALP, alkaline phosphatase; ANOVA, analysis of variance; BMP, bone morphogenetic protein; cDNA, complementary DNA; GAPDH, glyceraldehyde-3-phosphate dehydrogenase; mRNA, messenger RNA; PBS, phosphatebuffered saline; PCR, polymerase chain reaction; US, ultrasound doi:10.7863/ultra.33.5.865
Objectives—Low-intensity pulsed ultrasound (US) can accelerate fracture healing and osteogenic differentiation. The aim of this study was to investigate the osteogenic effect of low-intensity pulsed US on human periodontal ligament cells and to determine whether bone morphogenetic protein (BMP)-Smad signaling was involved. Methods—Human periodontal ligament cells were exposed to low-intensity pulsed US at a frequency of 1.5 MHz and intensity of 90 mW/cm2 for 20 min/d. Osteogenic differentiation was determined by assaying alkaline phosphatase (ALP) and calcium deposition. Expression of BMP-2, BMP-6, and BMP-9 was detected by real-time polymerase chain reaction analysis. Phosphorylated Smad was detected by western blotting; Smad in the cells was labeled by an immunofluorescent antibody and observed by laserscanning confocal microscopy. Results—The optical density of ALP stimulated by US at 1.5 MHz and 90 mW/cm2 for 20 min/d was significantly higher than in other groups (P < .01); therefore, this dosage was considered optimal for promoting osteogenic differentiation. After 13 days of US exposure, ALP increased gradually after 5 days, peaked at 11 days, and decreased at 13 days, with a significant difference compared with the control group (P < .05). Osteocalcin production increased from 9 to 13 days and peaked at 15 days, with a significant difference compared with the control group (P < .05). BMP-2 and BMP-6 increased dynamically after exposure for 13 days. BMP-2 increased 6.07-fold at 3 days, 6.39-fold at 11 days, and 5.97-fold at 13 days. BMP-6 expression increased 6.82-fold at 1 day and 51.5-fold at 3 days and decreased thereafter. BMP-9 was not expressed. Phospho-Smad1/5/8 expression was significantly increased after exposure (P< .05) and transferred from the cytoplasm into the nuclei. Conclusions—Low-intensity pulsed US effectively induced osteogenic differentiation of human periodontal ligament cells, and the BMP-Smad signaling pathway was involved in the mechanism. Key Words—basic science; bone morphogenetic protein–Smad; human periodontal ligament cells; low-intensity pulsed ultrasound; osteogenic differentiation; signal transduction
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uman periodontal ligament cells are among the main cellular components of the periodontal ligament connective tissue and play an important role in the maintenance, repair, and regeneration of periodontal tissue with the ability of osteogenic
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differentiation,1 which has shown the capacity to differentiate into osteoblast-like structures and contribute to periodontal tissue repair.1–4 It has been reported that periodontal ligament cells can express bone-associated molecules such as alkaline phosphatase (ALP), osteopontin, and osteocalcin.5,6 These cells have been considered a promising source for alveolar bone and periodontal ligament cell tissue regeneration and tissue engineering.7 Human periodontal ligament cells can undergo osteogenic differentiation by stimulation of cytokines and growth factors such as plateletderived growth factor,8 epidermal growth factor,9 transforming growth factor,10 and bone morphogenetic protein (BMP).11 Of those factors, BMP has been shown to be an important mediator for controlling osteoblast differentiation through the BMP-Smad signaling pathway.12 Gene responses to BMPs are mediated by Smad transcription factors; after phosphorylation on serine residues by the BMP receptor complex, Smad1, Smad5, and Smad8 move into the nucleus, where they regulate specific sets of target genes to mediate BMP-induced signals from the cell surface to the nucleus.13–16 Low-intensity pulsed ultrasound (US; intensity range, 30–100 mW/cm2) can be transmitted into the tissues as pressure waves to accelerate fracture healing in vivo.17–19 It has been proven to enhance ALP activity and accelerate calcium deposition20 in osteoblast cells. Low-intensity pulsed US exerts direct anabolic effects such as growth factor stimulation, osteogenic differentiation, and extracellular matrix production.21 The biophysical effects of low-intensity pulsed US are most likely caused by mechanical stress and microstreaming on the cellular plasma membrane and cytoskeletal structures, which induce intracellular signal transduction and gene transcription.22 However, to our knowledge, no studies have thus far evaluated the precise cellular molecular basis of osteogenic function in human periodontal ligament cells when stimulated by low-intensity pulsed US. This study investigated whether BMP-Smad signaling controlled the osteogenic regulation of human periodontal ligament cells under lowintensity pulsed US exposure. We assessed the differentiation effects of low-intensity pulsed US on the cells through ALP, osteocalcin, and BMP levels and explored the signal pathway by performing a phosphorylated Smad experiment and confocal laser-scanning microscopy.
Materials and Methods Cell Culture Human periodontal ligament cells were isolated from fresh premolars of healthy adolescents who were referred for
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orthodontic treatment to the Affiliated Hospital of Stomatology of Chongqing Medical University. The study was approved by the university’s Ethics Committee. The extracted teeth were immersed in Dulbecco’s modified Eagle’s medium (Hyclone, Logan, UT) supplemented with 10% fetal bovine serum (Hyclone), 125-U/mL penicillin, and 125-U/mL streptomycin (North China Pharmaceutical Group, Shijiangzhuang, China). The periodontal ligament tissue from the middle third of the root surface was scraped with a sharp No. 12 scalpel. The extracted tissues were immersed in 6-well plates in 1.5 mL of 20% fetal bovine serum and Dulbecco’s modified Eagle’s medium at 36.5°C and 5% carbon dioxide for primary culture, with medium replacement every other day.23 Cells at passage 4 were used for the experiments. Low-Intensity Pulsed US Exposure The low-intensity pulsed US exposure device was manufactured by the National Engineering Research Center of Ultrasound Medicine (Chongqing, China), which designed an array of 6 transducers fixed with a support device for using a 6-well culture plate. The plate was located above the array to touch the 6 transducers with dielectric coupling gel (Figure 1). Before the experiments, the US transducer was sterilized with 75% alcohol and ultraviolet light. The US exposure conditions were a frequency of 1.5 MHz, a 200-millisecond burst width sine wave, a pulse duty cycle of 1:4, a pulse repetition frequency of 1.0 kHz, intensity of 30, 60, or 90 mW/cm2, and an exposure time of 10, 20, or 30 minutes daily. The control group was treated in the same manner without US exposure. Optimizing the Dosage of Low-Intensity Pulsed US Human periodontal ligament cells (2.5 × 103) were seeded into a 6-well plate. The cells were exposed to low-intensity pulsed US for 0 seconds (control); 10, 20, or 30 min/d at 30 mW/cm2; 10, 20, or 30 min/d at 60 mW/cm2; or 10, 20, or 30 min/d at 90 mW/cm2 for 5 days, and then the US dosage was optimized by determining the ALP activity. Alkaline Phosphatase Activity Assay Human periodontal ligament cells at passage 4 were seeded into a 6-well plate at a density of 5 × 103 cells/cm2 (0.5 mL/well) with low-intensity pulsed US exposure at 90 mW/cm2 for 20 min/d for up to 13 days. The ALP activity in the lysate was assayed with a kit (Nanjing Jiancheng Bioengineering Institute, Nanjing, China) by measuring the formation of p-nitrophenol from p-nitrophenol phosphate at 3, 5, 7, 9, 11, and 13 days. The cells in each group were digested by trypsin, incubated in 0.2%
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Triton X-100 for 24 hours at 4°C, and split by ultrasonication (Shanghai Bilon Instruction Co, Ltd, Shanghai, China) to obtain the lysates. Osteocalcin Radioimmunoassay Human periodontal ligament cells at passage 4 were seeded into a 6-well plate at a density of 5 × 103 cells/cm2 (0.5 mL/well). Low-intensity pulsed US exposure at 90 mW/cm2 for 20 min/d was administered for up to 15 days. The culture medium of each group was collected at 5, 7, 9, 11, 13, and 15 days. The amount of osteocalcin in the supernatant was measured by radioimmunoassay (Department of Nuclear Medicine, First Affiliated Hospital of Chongqing Medical University). A mouse osteocalcin assay kit (Beijing Atom High Tech Co, Ltd, Beijing, China) was used according to the manufacturer’s instructions. RNA Isolation and Analysis Human periodontal ligament cells at passage 4 were cultured in a 6-well plate and exposed to low-intensity pulsed US at 90 mW/cm2 for 20 min/d and were harvested at 1, 3, 5, 7, 9, 11, and 13 days after US exposure. Total RNA was isolated with the RNAiso Plus reagent (TaKaRa
Biotechnology, Dalian, China). Aliquots that contained equal amounts of messenger RNA (mRNA) were subjected to real-time polymerase chain reaction (PCR) analysis. The mRNA was converted into complementary DNA (cDNA) by using a deoxynucleoside triphosphate mixture (TaKaRa Biotechnology), a random primer (TaKaRa Biotechnology), a ribonuclease inhibitor (TaKaRa Biotechnology), and Maloney murine leukemia virus reverse transcriptase (Promega, Madison, WI). The cDNA mixtures were diluted 5-fold in sterile distilled water, and 1 μL was subjected to real-time PCR using SYBR Premix Ex Taq (TaKaRa Biotechnology). Reactions were performed in 25-μL volumes of a solution containing 12.5 μL of SYBR Premix Ex Taq, 1 μL of primers (sense and antisense; TaKaRa Biotechnology) and 10.5 μL of distilled deionized water. The primers (Table 1) were designed using Primer3 software (Whitehead Institute for Biomedical Research, Cambridge, MA). Assays were performed on a RotorGene 6000 system (Corbett Life Science, Sydney, New South Wales, Australia) and analyzed with Rotor-Gene 6000 series software. Polymerase chain reaction conditions were 95°C for 10 seconds, 45 cycles at the annealing temperature (BMP-2 at 54°C and BMP-6 at 56°C) for
Figure 1. Experimental low-intensity pulsed US exposure setup.
Table 1. Real-time PCR Primers Used in the Experiments Target cDNA
Primer Sequence
Human BMP-2 CDS qPCR forward Human BMP-2 CDS qPCR reverse Human BMP-6 CDS qPCR forward Human BMP-6 CDS qPCR reverse Human GAPDH qRT-PCR forward, 18 mer Human GAPDH qRT-PCR reverse, 18 mer
5′-GGGCATCCTCTCCACAAA-3′ 5′-GTCATTCCACCCCACGTC-3′ 5′-TGCAGGAAGCATGAGCTG-3′ 5′-GTGCGTTGAGTGGGAAGG-3′ 5′-CAGCGACACCCACTCCTC-3′ 5′-TGAGGTCCACCACCCTGT-3′
CDS indicates coding sequence; q, quantitative; and RT, real-time.
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30 seconds, and 72°C for 30 seconds. All of the real-time PCR reactions were performed in triplicate, and the gene expression levels were calculated and normalized by dividing the calculated values for the mRNA samples by that of glyceraldehyde-3-phosphate dehydrogenase (GAPDH) mRNA at each time point. The 2(-Delta Delta C(T)) calculation method24 was used to analyze the results of amplification. Protein Extraction and Western Blot Analysis The involvement of BMP-Smad signal transduction in the osteogenic differentiation of human periodontal ligament cells was evaluated in this part of the experiment. The cells were exposed to low-intensity pulsed US at 90 mW/cm2 for 20 minutes after 5, 30, 60, and 120 minutes and 4 hours. At every time point, the samples were scraped and pelleted by centrifugation at 0°C at 1000g for 15 minutes. The pellets were washed twice in phosphate-buffered saline (PBS), homogenized in 0.2 N sulfuric acid, and centrifuged at 13,000g. Histones were pelleted from the supernatant by adding a 0.25 volume of 100% trichloroacetic acid. The pellets were suspended in 100% ethanol overnight and centrifuged again at 13,000g. The pellets were dissolved in ultrapure water and evaluated for protein concentration. An aliquot corresponding to 10 μg of protein was boiled twice in sodium dodecyl sulfate sample buffer (Tris-glycine– sodium dodecyl sulfate sample buffer; Invitrogen, Carlsbad, CA) and loaded onto a 4% to 20% Tris-glycine gel. The separated proteins were transferred to a polyvinylidene difluoride membrane. The membrane was blocked with TBST (10 mM Tris-Cl, pH 7.4, 200 mM sodium chloride, and 0.1% Tween 20) containing 5% nonfat milk for 60 minutes before incubation with 50-ng/mL antiphospho-Smad1/5/8 antibody (9511; Cell Signaling Technology, Beverly, MA) or anti-Smad1/5/8 antibody (sc-6031-R; Santa Cruz Biotechnology, Santa Cruz, CA) for 2 hours. The blots were washed in TBST and then incubated with a horseradish peroxidase–conjugated antimouse antibody (1:1000 dilution), and detection of the antigen-bound antibody was performed with an enhanced chemiluminescence western blotting analysis system (Amersham Life Science, Arlington Heights, IL).
(sc-6031-R; Santa Cruz Biotechnology) for 12 hours at 37°C. After the cells were washed 3 times with PBS, they were incubated with a goat antirabbit secondary antibody for an additional 1 hour. The cells were then washed with PBS, mounted, and observed on a laser-scanning confocal microscope (TCS SP2; Leica, Heidelberg, Germany) using a ×400 objective. Statistical Analysis The optimum dosage of low-intensity pulsed US was analyzed by 1-way analysis of variance (ANOVA). Alkaline phosphatase activity and the osteocalcin assay were analyzed by a paired t test, and P < .05 was considered statistically significant. Expression of BMP amplification was analyzed by the 2(-Delta Delta C(T)) calculation method.24 Expression of phospho-Smad1/5/8 was analyzed by 1-way ANOVA, and P < .05 was considered statistically significant.
Results Optimum Dosage of Low-Intensity Pulsed US Low-intensity pulsed US exposure increased the level of ALP in human periodontal ligament cells, with the highest optical density for US at 90 mW/cm2 for 20 min/d (Figure 2). The mean optical density of ALP ± SD in the cells was 0.0551 ± 0.0003, with significant differences (F = 0.59; P < .01) among the 9 exposure groups, as analyzed by 1-way ANOVA. This set of US parameters was therefore used for the following experiments. Figure 2. Alkaline phosphatase activity in human periodontal ligament cells after low-intensity pulsed US (LIPUS) exposure at different intensities and times after 5 days. *P < 01.
Confocal Microscopy Human periodontal ligament cells were exposed to lowintensity pulsed US at 90 mW/cm2 for 20 minutes. Cells were fixed after exposure for 1, 2, 4, and 6 hours in 0.4% paraformaldehyde for 20 minutes. Fixed cells were incubated with 10% normal goat serum for 40 minutes and then with the anti-Smad1/5/8 primary antibody
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Effect of Low-Intensity Pulsed US on ALP Activity and Osteocalcin as Well as Calcium Deposition As shown in Figure 3, the optical density of ALP in human periodontal ligament cells at 3, 5, 7, 9, 11, and 13 days was analyzed by a paired t test. Compared with the control group, ALP activity increased gradually up to 5 days, peaked at 11 days, and decreased at 13 days in the lowintensity pulsed US exposure groups. Osteocalcin production at 5, 7, 9, 11, 13, and 15 days was analyzed by a paired t test. Compared with the control group, osteocalcin increased at 9, 11, and 13 days and peaked at 15 days in the US exposure groups (P < .05). Real-time PCR Analysis of BMP-2, BMP-6, and BMP-9 As shown in Figure 4, after exposure to low-intensity pulsed US at 90 mW/cm2 for 20 min/d for 13 days, BMP2 mRNA expression increased over the 13 days in the US exposure groups. The BMP-2 mRNA level compared with Figure 3. A, Alkaline phosphatase activity in human periodontal ligament cells after low-intensity pulsed US (LIPUS) exposure at 90 mW/cm2 for 20min/d for 13 days. OD indicates optical density. B, Osteocalcin (OC) activity in the cells after low-intensity pulsed US exposure at the same dosage for 15 days.
the control group increased 6.07-fold at 3 days, 6.39-fold at 11 days, and 5.97-folds at 13 days. The BMP-6 level compared with the control group was 6.82-fold greater at 1 day, increased 51.5-fold at 3 days, and then decreased over the following days. No BMP-9 expression was observed in the cells in both the control and experimental groups. Smad1/5/8 Phosphorylation Compared with the control group, phospho-Smad1/5/8 protein expression was significantly increased in the western blot assay after treatment with low-intensity pulsed US at 90 mW/cm2 for 20 minutes. The phospho-Smad1/5/8 level gradually increased after US exposure at the 30minute point, reached its peak at the 60-minute point, maintained that level until the 120-minute point, and then decreased at the 4-hour point (Figure 5). The gray value of phospho-Smad1/5/8 was analyzed by 1-way ANOVA (P < .05). This result showed that the Smad1/5/8 underwent processes of phosphorylation and dephosphorylation right after US stimulation, indicating this pathway may be involved in the effect of low-intensity pulsed US on human periodontal ligament cells.
A Figure 4. Expression of BMP-2 and BMP-6 in human periodontal ligament cells over 13 days; con indicates control.
B
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Smad1/5/8 Transfer After Smad1/5/8 protein was stained by an immunofluorescent antibody, low-intensity pulsed US-stimulated human periodontal ligament cells were observed under confocal microscopy. The transfer activity is shown in Figure 6. The high green fluorescent label indicates Smad1/5/8. In the control group, the high green fluorescent label was mainly in the cytoplasm, and the nucleus was empty; 1 hour after US exposure, the label was in the nucleus, indicating that Smad1/5/8 started to shift into the nucleus; 2 hours after exposure, the label was still mainly in the nucleus; and 4 hours after exposure, the label was in the cell nucleus. However, 6 hours after exposure, the label was basically in the cytoplasm again.
Discussion It is well accepted that low-intensity pulsed US can induce cellular responses in cementoblasts, periodontal ligament cells, and bone cells. The positive influence of low-intensity pulsed US stimulation on tissue regeneration has been demonstrated in both animal and clinical trials.22 Human periodontal ligament cells, which are known to have a stem cell–like character, can differentiate into osteoblasts and cementoblasts, which act on periodontal regeneration.25 Among all of the research on periodontal regeneration, promoting human periodontal ligament cell proliferation and osteogenesis is the most important aspect. Osteogenesis proceeds through 3 overlapping phases: proliferation, differentiation, and calcification.26 During the differentiation phase, osteogenesis-related proteins are synthesized, and the extracellular matrix is formed and matures.27 This activity is characterized by expression of osteoblast phenotypes such as ALP activity and collagen 1 secretion in the early phase28 and extracellular matrix calcification induced by osteocalcin production in the later phase.29 This study indicated that ALP activity was increased in all low-intensity pulsed US exposure groups, although the highest activity occurred in the group with a dosage of 90 mW/cm2 for 20 min/d, which suggests that this dosage could be administered in subsequent research as the optimum dosage. Data indicated that low-intensity pulsed US might promote ALP synthesis at 5 days, peaking at 7 to 11 days, secreting out of the cells at 9 days, and decreasing at 13 days. Osteocalcin production in human periodontal ligament cells increased with time from 5 to 15 days in the US exposure groups. Significantly higher osteocalcin production was detected 11 and 13 days after exposure and peaked at 15 days. The time point of peak osteocalcin production was later than that of ALP, which might have
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been attributable to the fact that ALP production is an early osteogenic response in human periodontal ligament cells when stimulated by low-intensity pulsed US, whereas osteocalcin is a marker protein that is produced later in the osteogenic process. The mechanical and cavitation effects of low-intensity pulsed US are converted into stress30,31 on the cell membrane, which leads to surface structural changes.19,32,33 As a result, human periodontal ligament cells respond to stimulation by activating a signal transduction reaction to regulate osteogenic-related gene expression.34–37 Thus, we conducted an in-depth study of BMP-Smad signaling in human periodontal ligament cells stimulated by low-intensity pulsed US. Bone morphogenetic proteins constitute the largest group within the transforming growth factor β superfamily of hormonally active factors. Genetic studies have shown that BMPs and their related molecules are essential for normal skeletal development in vertebrates.38 In MC3T3-E1 osteoblastic cells, BMP-2 induced an increase in cellular ALP activity, and BMP-2 and BMP-3 slightly but significantly stimulated collagen synthesis.39 BMP-6 has been reported to display 2- to 2.5-fold more potent induction of osteoblast differentiation than BMP-2 or BMP-4 in a fetal rat secondary calvarial cell culture system, which induced the formation of more and larger bone nodules as well as increased osteocalcin secretion and might have acted at an earlier stage of cell differentiation.40 Figure 5. Expression of phospho-Smad1/5/8 (p-Smad) in human periodontal ligament cells by Western blotting; con indicates control.
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BMP-9 was proven to induce ALP in mesenchymal stem cells and mouse embryonic fibroblasts and to induce recruitment of both Runx2 and β-catenin to the osteocalcin promoter.41 Therefore, BMP-2, BMP-6, and BMP-9, which are the most potent factors of osteogenic differentiation attributable to low-intensity pulsed US, were chosen to assess in this study. The results showed that BMP-2 expression in human periodontal ligament cells exposed to low-intensity pulsed US at 90 mW/cm2 for 20 min/d for 13 days had a wavelike increase, peaking at 3 and 11 days. BMP-6 increased significantly at 3 days and then gradually decreased, which indicated that BMP-6 might act on the early response of human periodontal ligament cells stimulated by low-intensity pulsed US. However, BMP-9 was not expressed in both the control and experimental groups, indicating that BMP-9 did not exist in these cells. Gene responses to BMPs are mediated by Smad transcription factors after being phosphorylated on serine residues by the BMP receptor complex42 when stimulated
by the mechanical force of low-intensity pulsed US. Smads are divided into 3 classes: receptor-regulated Smads, common Smads, and inhibitory Smads,43 and each of them has a distinct function. Receptor-regulated Smads (Smad1/5/8) are phosphorylated by the type 1 receptor on the carboxylterminal SSXS motif. Once phosphorylated, receptorregulated Smads dissociate from the receptor, bind to common Smad (Smad4), and enter the nucleus.44 In the nucleus, heteromeric Smad complexes function as effectors of BMP signaling by regulating transcription of specific genes to increase various kinds of osteogenic differentiation indicators.38 In this study, when human periodontal ligament cells were stimulated by low-intensity pulsed US exposure at 90 mW/cm2 for 20 min/d, western blotting showed that Smad1/5/8 was phosphorylated from 30 minutes, peaked at 120 minutes, and then decreased at 4 hours. Immunofluorescence staining showed that Smad1/5/8 transferred from the cytoplasm to the nucleus in 4 hours and then retreated at 6 hours.
Figure 6. Nuclear accumulation of Smad1/5/8 in human periodontal ligament cells after low-intensity pulsed US exposure from 1 to 6 hours (original magnification ×400): A, control group; B, 1 hour after exposure; C, 2 hours after exposure; D, 4 hours after exposure; E, 6 hours after exposure.
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In conclusion, our results suggest that low-intensity pulsed US exposure induces human periodontal ligament cells into osteogenic differentiation, and the mechanism of osteogenic differentiation may be achieved through the BMP-Smad signaling pathway, which is involved in the early response of stimulation by low-intensity pulsed US. Although this study’s findings have merit for identifying the process of human periodontal ligament cell response to low-intensity pulsed US stimulation, an in vitro model cannot be extrapolated to in vivo conditions directly. Further experiments are needed to clarify this inference and to provide a basis for clinical applications.
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