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RESEARCH REPORT |
1 Departments of Orthodontics and Craniofacial Developmental Biology, Hiroshima University Graduate School of Biomedical Sciences, 1-2-3 Kasumi, Minami-Ku, Hiroshima 734-8553, Japan;
2 Department of Stomatology, University of California-San Francisco, San Francisco, CA, USA; and
3 Department of Orthodontics and Pediatric Dentistry, School of Dentistry, University of Michigan, Ann Arbor, USA;
* corresponding author, shigebon{at}hiroshima-u.ac.jp
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMATERIALS & METHODSRESULTSDISCUSSIONREFERENCES |
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KEY WORDS: mechanical strain • chondrocytes • PTHrP • ion channels
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMATERIALS & METHODSRESULTSDISCUSSIONREFERENCES |
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Given these findings, it is plausible that specific stress-mediated regulatory mechanisms for cartilage development are regulated via the mechano-induction of PTHrP. To begin assessing the potential link between mechano-modulation of PTHrP and subsequent events in chondrocytes, we conducted the present study to investigate the effects of cyclic mechanical strain on the expression of PTHrP in cultured growth plate chondrocytes. Specifically, we hypothesized that cyclic mechanical strain induces PTHrP expression in chondrocytes, and that the extent of this response is dependent on the status of chondrocytic differentiation and the magnitude of applied strain. Furthermore, based on the evidence that mechanical stress regulates chondrocyte proliferation and differentiation via some cell-surface ion channels (Wu and Chen, 2000), we also examined whether specific ion channels are involved in the cascade during the induction of PTHrP by mechanical strain.
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MATERIALS & METHODS |
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TOPABSTRACTINTRODUCTIONMATERIALS & METHODSRESULTSDISCUSSIONREFERENCES |
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-minimum essential medium (-MEM; Sanko Junyaku, Tokyo, Japan) containing 50 µg/mL ascorbic acid, 32 units/mL penicillin, 60 µg/mL kanamycin, and 250 ng/mL amphotericin B (Medium A). The cells (2 x 105) were seeded on Bioflex silicone-elastomer multiwell plates (35-mm diameter; Flexcell Co., McKeesport, PA, USA) or rigid-bottomed plates (control dish, 35-mm diameter; Corning Inc., Corning, NY, USA) and cultured in 2 mL Medium A containing 10% fetal bovine serum (FBS; Daiichi Kagaku, Tokyo, Japan). Before cells were seeded, experimental and control dishes were coated with 500 µL of collagen solution [10 µg/mL bovine type II collagen (acid-soluble, pepsin-resistant; Koken, Osaka, Japan) in phosphate-buffered saline containing 10 mM NaHCO3] to allow for the subsequent attachment of cells to the dishes. The cultures were maintained in an atmosphere of 5% CO2 at 37°C in a humidified incubator. Culture medium was changed every other day. Basic FGF (1 ng/mL) was added to the cultures to stimulate proliferation and maintain the chondrocytic phenotype until the cells became confluent. Once confluence was achieved, ascorbic acid (50 µg/mL) was added to the cultures to facilitate sequential differentiation of the cells to mature chondrocytes, as described previously (Yoshida et al., 2001).
Application of Cyclic Mechanical Strain
Cyclic mechanical strain was applied to the chondrocytes by means of a computer-driven vacuum-operated strain unit (Flexcell Co.). The strain conditions were 30 cycles/min (loading and relaxation on every alternate sec) at 7% or 12% elongation. These loading regimens were selected to be lower than the 23% elongation that has been shown to cause cartilage matrix catabolism (Honda et al., 2000). The experimental period was defined based on a protocol, previously described in the literature (Steers et al., 1998), in which PTHrP secretion was first observed at 8 hrs and was maintained at a high level for 24 hrs in cultured smooth-muscle cells in the same system. We used a 12-hour experimental period for examining gene expression analyses, and a 24-hour experimental period for protein analyses, to account for any potential temporal differences between transcriptional and post-transcriptional events. The cell appearance at proliferating and matrix-forming stages was observed after the application of 24-hour mechanical strain by phase-contrast microscopy, and photographed. To dissect the potential signaling cascade in the induction of PTHrP by mechanical strain at the matrix-forming stage, in some experiments, we added channel inhibitors—10 µM gadolinium, 10 µM nifedipine, 1 µM tetrodotoxin, or 1 mM 4-aminopyridine—to the cultures before and during strain application. Briefly, the cells were pre-incubated with Medium A containing each channel inhibitor for 1 hr, and then medium was changed to fresh Medium A containing each channel inhibitor immediately before 24-hour mechanical strain application.
Determination of the Rates of DNA, Proteoglycan, and Collagen Syntheses
For determining the rate of DNA synthesis, we exposed chondrocyte cultures at the proliferating stage (on day 4) to 5 µCi/mL[3H]thymidine in 2 mL of Medium A containing 0.5% FBS for the final 4 hrs of 24-hour mechanical stimulation. The radioactive counts of the cell layer were measured in a scintillation counter. To determine the rate of proteoglycan and collagen syntheses, we exposed chondrocyte cultures at the matrix-forming stage (on day 12) to 2.5 µCi/mL [35S]sulfate or 10 µCi/mL [2,3-3H]proline in 2 mL of Medium A containing 0.5% FBS for the final 4 hrs of 24-hour mechanical stimulation. We determined the rates of proteoglycan and collagen syntheses by measuring the incorporation of [35S]sulfate and [2,3-3H]proline into materials precipitated with cetyl pyridinium chloride (CPC; Nacalai Tesque Inc., Kyoto, Japan) in a scintillation counter.
Quantitative Real-time Polymerase Chain-reaction for PTHrP
Total RNA was isolated from the chondrocyte cultures from day 4 to day 20, with or without 12-hour mechanical strain, according to a guanidine thiocyanate method (Smale and Sasse, 1992). A single-stranded cDNA was synthesized from 1 µg of total RNA with the use of Oligo (dT)20 primer (Toyobo, Osaka, Japan) and a Rever Tra Ace- first-strand cDNA synthesis kit (Toyobo). The mRNA levels were estimated by quantitative real-time polymerase chain-reaction (PCR) analysis, with the use of the automated fluorometer (ABI Prism 7700 sequence detection system, Perkin-Elmer Biosystem, Foster, CA, USA). The sequences of the primers and probes for glyceraldehyde-3-phosphate dehydrogenase (GAPDH) and PTHrP are listed in the Table
. Quantitative results of real-time PCR were assessed with a cycle threshold (Ct) value, which identifies a cycle when the fluorescence of a given sample becomes significantly different from the base signal. We performed relative quantification of the PTHrP signals by normalizing the PTHrP signals to the GAPDH signals. Normalized PTHrP Ct values were expressed relative to the controls.
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RESULTS |
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TOPABSTRACTINTRODUCTIONMATERIALS & METHODSRESULTSDISCUSSIONREFERENCES |
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[a], inset), and synthesized abundant cartilagenous matrices from day 7 to day 14 (Fig. 1A [c,d], bright white outline in the pericellular region). Thus, we defined the cells on day 4 as proliferating, those on day 12 as matrix-forming, and those on day 20 as hypertrophic chondrocytes, according to previously published definitions (Yoshida et al., 2001). The cell appearance, including morphology and adherence, was unchanged between the cultures with and those without mechanical strain during the experimental period (Fig. 1A). Cyclic mechanical strains of both 7% and 12% elongation caused significant (p < 0.05) increases in cellular incorporation of [3H]thymidine in proliferating chondrocytes as compared with the control cells (Fig. 1B). Additionally, at the matrix-forming stage, both cyclic strains produced significant (p < 0.05) increases in proteoglycan and collagen syntheses, as measured by [35S]sulfate incorporation and [3H]proline incorporation, respectively (Figs. 1C, 1D).
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). The expression of PTHrP mRNA was significantly enhanced by about 4.4-fold (p < 0.01) and by about 2.3-fold (p < 0.05) at the proliferating and matrix-forming stages, respectively, in chondrocytes subjected to cyclic mechanical strain of 12% elongation relative to that in controls (Fig. 2B). Although chondrocytes at the hypertrophic stage showed a two-fold increase in PTHrP mRNA when subjected to strain of 12% elongation, this induction was statistically insignificant. Cyclic mechanical strain of 7% elongation also caused a significant increase in the expression of PTHrP mRNA by about 2.4-fold (p < 0.05) at the proliferating stage and by about 2.1-fold (p < 0.05) at the matrix-forming stage, but not at the hypertrophic stage (Fig. 2B). The effects of the extent of strain on the mechano-modulation of PTHrP were evident only in proliferating chondrocytes in which strain of 12% elongation induced significantly more (p < 0.05) PTHrP mRNA than that of 7% elongation.
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, 3B). Furthermore, both the Na+ channel blocker tetrodotoxin and the K+ channel blocker 4-aminopyridine did not produce a statistically significant decrease in the mechanical induction of PTHrP mRNA (Figs. 3C, 3D).
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMATERIALS & METHODSRESULTSDISCUSSIONREFERENCES |
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The induction of PTHrP was completely inhibited by the Ca2+ channel blocker nifedipine, but not by an SA channel blocker, gadolinium. The SA channel is known as a key channel for signal transduction induced by the deformation of the cell membrane (Sokabe et al., 1991). The cell shape was not changed by mechanical strain of 12% elongation at the proliferating and matrix-forming stages. This may be the reason why gadolinium did not affect the mechanical induction of PTHrP in this study. Alternatively, it is plausible that the effect of activation of the SA channel could not be obvious because the Ca2+ channel was primarily activated.
Yellowley et al.(1999) showed that the fluid flow caused mobilization of intracellular Ca2+ in articular chondrocytes by the activation of G-protein. Furthermore, Chang et al.(1999) reported that either intra- or extracellular Ca2+ mobilization was highly associated with the regulation of chondrocyte proliferation and maturation. Hence, when the cyclic mechanical forces are applied to the bottoms of the Flexercell dishes, the cells are exposed to fluid flow stress (Brown et al., 2000), which may consequently up-regulate the expression of PTHrP mRNA through a signal transduction pathway via the mobilization of Ca2+.
In conclusion, we have shown that the expression of PTHrP mRNA is enhanced by cyclic mechanical strain via signal transduction through the Ca2+ channel. We also demonstrate that the magnitude of mechano-induction of PTHrP is dependent on the stage of chondrocyte differentiation. These results suggest that mechanical induction of PTHrP allows chondrocytes for more replication and matrix-forming subsequently affects cartilage formation.
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ACKNOWLEDGMENTS |
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FOOTNOTES |
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These authors contributed equally to this work. 
Received September 29, 2003; Last revision September 23, 2004; Accepted September 28, 2004
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REFERENCES |
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| Journal of Dental Research ® | Critical Reviews (1990-2004) |
= negative stress; 
= positive stress. Mean ± SD, n = 3, *p < 0.05.
