| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
|
|
|
|||||||
|
|||||||||
RESEARCH REPORT |
Department of Orthodontics and Craniofacial Developmental Biology, Hiroshima University Graduate School of Biomedical Sciences, 1-2-3 Kasumi, Minami-ku, Hiroshima 734-8553, Japan;
* corresponding author, motumo{at}hiroshima-u.ac.jp
 |
ABSTRACT |
|---|
 TOP ABSTRACT INTRODUCTIONMATERIALS & METHODSRESULTSDISCUSSIONREFERENCES |
|---|
KEY WORDS: cyclic tensile forces • VEGF • M-CSF • MC3T3-E1 cells • S-A channel
|
INTRODUCTION |
|---|
|
TOPABSTRACTINTRODUCTIONMATERIALS & METHODSRESULTSDISCUSSIONREFERENCES |
|---|
There have been several reports about the effects of mechanical stimuli on bone cells (Neidlinger-Wilke et al., 1995). The effects of various mechanical stimuli—such as strain, compressive pressure (Ozawa et al., 1990), fluid flow (Kanno et al., 1999), and ultrasound (Reher et al., 1999)—have been examined in bone cells. A stretch-activated channel (S-A channel), which is a membrane stretch-activated ionic channel, was found in tissue-cultured embryonic chick skeletal muscle (Guharay and Sachs, 1984). Vascular endothelial cells have been reported to contain a cation-selective S-A channel permeable to calcium ions (Lansman et al., 1987). It was also demonstrated that intracellular Ca2+ increased in response to mechanical stretch via the S-A channel in human umbilical endothelial cells, and this response was blocked by gadolinium (Gd3+), a S-A channel blocker (Naruse and Sokabe, 1993). Recently, Kanzaki et al.(1999) identified a eukaryotic gene (Mid1) that encodes a mechanosensitive ion channel from yeast, and showed that Mid1 acts as a calcium-permeable cation-selective stretch-activated channel. Duncan and Misler (1989) reported that osteoblastic cells contain a S-A channel. Klein-Nulend et al.(1995) hypothesized that stress on bone causes fluid flow in the lacunar-canalicular system, which in turn stimulates the osteocytes to produce factors that regulate bone metabolism.
From these findings, it could be assumed that mechanical stimuli to osteoblasts are controlled through the S-A channel, and this results in the production of some bone remodeling factors.
In this study, we examined the effects of cyclic tensile forces on the expression of VEGF and M-CSF in osteoblastic MC3T3-E1 cells. We also investigated the influence of Gd3+, an S-A channel inhibitor, and the L-type calcium channel blocker nifedipine on VEGF and M-CSF expression.
|
MATERIALS & METHODS |
|---|
|
TOPABSTRACTINTRODUCTIONMATERIALS & METHODSRESULTSDISCUSSIONREFERENCES |
|---|
-modified MEM (Sigma, St. Louis, MO, USA) containing 10% FCS (Biological Industries, Kibbutz Beit-Haemek, Israel), 32 U/mL penicillin G (Meiji Seika, Tokyo, Japan), 250 µg/mL amphotericin B (Nacalai Tesque, Kyoto, Japan), and 60 µg/mL kanamycin (Meiji Seika, Tokyo, Japan) at 37°C in a humidified atmosphere of 5% CO2. The medium was changed twice a week. Cells were subcultured after treatment with 0.25% trypsin/EDTA and plated at 3 x 105 cells per 100-mm culture dish. For all experiments, cells between the 4th and the 10th passages were used.
Application of Cyclic Tensile Forces
A Flexcell strain unit FX-2000 (Flexcell International Co., Hillsborough, NC, USA) consists of a vacuum unit and a valve controlled by a computer program. MC3T3-E1 cells cultured on the flexible membrane base were subjected to cyclic tensile forces produced by the computer-controlled application of sinusoidal negative pressure. The flexible membranes supporting the cultured cells were deformed by negative pressure. Application of vacuum results in a maximum cell elongation of 20% at the well’s periphery, with the strain declining toward the center. The cells were placed in a humidified incubator in an atmosphere of 5% CO2 at 37°C. To examine the expression of VEGF and M-CSF mRNAs and their concentrations, we loaded various elongations of 7, 12, 17, and 24% at a frequency of 30 cycles/min for 12 hrs. Next, we investigated the influence of loading time changes (1, 3, 6, 12, and 24 hrs) upon the amounts of VEGF and M-CSF by 12% elongation.
Total RNA Extraction and cDNA Synthesis
Total RNA was isolated from the cell cultures with the use of a Quickprep Total RNA extraction kit (Amersham Biosciences Corp., Piscataway, NJ, USA). 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).
Primers
Primers for VEGF mRNA, which amplify VEGF188, VEGF164, and VEGF120, were purchased from R&D Systems (Minneapolis Inc., Minneapolis, MN, USA).
Primers for M-CSF mRNA were used as reported by Mitrasinovic et al.(2001): 5'-CCCATATT- GCGACACCGAA-3' (sense) and 5'-AAGCAGTAACTGAGCAACGGG-3' (antisense), with G3PDH primer (Rever Tra Ace- first-strand cDNA synthesis kit; Toyobo) as the control primer, and 5'-ACCACAGTCCATGCCATCAC-3' (sense) and 5'-TCCACCACCCTGTTGCTGTA-3' (antisense).
Real-time Quantitative PCR
Real-time PCR was performed with the SYBR Green I assay and the ABI Prism 7700 sequence detection system (Applied Biosystems, Foster City, CA, USA) from 1 µL of sample cDNA with three-stage program parameters as follows: 2 min at 50°C, 10 min at 95°C, and then 40 cycles of 45 sec at 94°C, 45 sec at 55°C, and 45 sec at 72°C. SYBER Green I dye intercalation into the minor groove of double-stranded DNA reaches maximum emission at 530 nm. PCR reactions for each sample were repeated three times for both target gene and the control.
Quantitative results of real-time fluorescence PCR were assessed by a cycle threshold (Ct) value, which identifies a cycle when the fluorescence of a given sample becomes significantly different from the baseline signal. Relative quantifications of the VEGF and M-CSF signals were normalized and expressed relative to the glyceraldehyde-3-phosphate dehydrogenase (G3PDH) signals.
Measurement of VEGF and M-CSF Concentrations
Conditioned medium from cultured MC3T3-E1 cells, with or without application of tensile force, was collected and centrifuged at 2000 rpm for 5 min. The amount of VEGF and M-CSF was measured by a quantitative sandwich enzyme immunoassay technique (Quantikine M mouse VEGF Immunoassay kit, Quantikine M mouse M-CSF Immunoassay kit; R&D Systems, Inc., Minneapolis, MN, USA), according to the manufacturer’s instructions. Standard curves were obtained, and the experiment was repeated 5 times.
Effects of Gd3+ and Nifedipine on the Expression of VEGF and M-CSF
The effects of Gd3+ and nifedipine on the expression of VEGF and M-CSF were examined in both force application and control groups. It has been reported that 1–100 µM Gd3+ inhibited S-A channels (Yang and Sachs, 1989). Cells were incubated with 10 and 100 µM Gd3+ chloridehexahydrate (Wako, Osaka, Japan) or 10 µM nifedipine (Sigma) for 30 min (Hung et al., 1996). After treatment of Gd3+ or nifedipine, 12% elongation was applied for 12 hrs.
Statistical Treatment
Statistical significance was evaluated by analysis of variance (ANOVA) and a multiple-comparison test (Scheffé’s test). A confidence level of p < 0.05 was defined as statistically significant.
|
RESULTS |
|---|
|
TOPABSTRACTINTRODUCTIONMATERIALS & METHODSRESULTSDISCUSSIONREFERENCES |
|---|
).
|
, 3).
|
|
, 3). VEGF protein production significantly increased in the experimental group compared with the control group at 3, 6, 12, and 24 hrs. M-CSF protein production also increased significantly in the experimental group in comparison with the control group at 6, 12, and 24 hrs.
Effect of Gd3+ and Nifedipine Treatment
Gd3+ treatment reduced the expression of VEGF mRNAs in a dose-dependent manner under tensile force, and a significant difference was shown after 10 µM treatment. Ten and 100 µM Gd3+ treatment reduced the expression of M-CSF mRNA. Control cells without tensile force were not influenced by 100 µM Gd3+ treatment. Nifedipine treatment did not inhibit the expression of either VEGF or M-CSF mRNA (Fig. 4A).
|
,4C). |
DISCUSSION |
|---|
|
TOPABSTRACTINTRODUCTIONMATERIALS & METHODSRESULTSDISCUSSIONREFERENCES |
|---|
A significant increase in VEGF and M-CSF mRNA and protein concentration was shown in the conditioned medium, under tensile force application over time. These results were similar to a previous study, which showed an enhancement of VEGF expression in human periodontal ligament fibroblasts treated in a Flexcell strain unit (Yoshino et al., 2003). The authors explained these findings by hypothesizing that mechanical stress activates cellular mechanotransducers, such as mechanosensitive ion channels, cytoskeleton, and integrins. A S-A channel is a stretch-activated ionic membrane channel, and the presence of such channels was first reported in tissue-cultured embryonic chick skeletal muscle (Guharay and Sachs, 1984) and osteoblast-like cells (Duncan and Misler, 1989). Naruse and Sokabe (1993) showed that stretching cellular membranes increased intracellular Ca2+ concentration in human umbilical endothelial cells cultured on silicon membranes. The Ca2+ response disappeared when extracellular Ca2+ was removed, or after Gd3+ treatment, which is a potent blocker of S-A channels. It was also demonstrated that orienting and elongating responses of cultured endothelial cells to cyclic stretch were inhibited by the removal of external Ca2+, or by the addition of Gd3+ (Naruse et al., 1998). These findings suggest that cell-orienting and -elongating responses are mediated by Ca2+-permeable S-A channels present on the cell membrane of endothelial cells. Recently, the homologue of the eukaryotic gene (Mid1) encoding the S-A channel was identified in yeast. It was revealed that Mid1 acts as a calcium-permeable, cation-selective stretch-activated channel (Kanzaki et al., 1999). Based on these findings, we hypothesized that mechanical stretch to osteoblastic MC3T3-E1 cells might cause them to express VEGF and M-CSF, and this may be mediated by S-A channel activation. Therefore, we investigated the effects of 10 and 100 µM Gd3+ on the expression of VEGF and M-CSF with tensile force application. Although Gd3+ is known as a potent S-A channel blocker (Hung et al., 1996), it also blocks voltage-dependent Ca2+ channels (Biagi and Enyeart, 1990). So, we examined the effect of 10 µM nifedipine, which is an L-type Ca2+ channel blocker. Gd3+ treatment inhibited the expression of VEGF and M-CSF genes and proteins, and the effect was dose-dependent. Nifedipine had no effect on VEGF and M-CSF genes or protein expression. Thus, it is clearly concluded that the expression of VEGF and M-CSF in cyclic tensile forces in osteoblast MC3T3-E1 cells is mediated by S-A channels.
VEGF, known as a vascular permeability factor (Senger et al., 1983), is a potent and specific mitogen for vascular endothelial cells and promotes neovascularization. Flt-1 and fetal liver kinase (Flk-1) (Terman et al., 1992) are the two main receptors for VEGF. It has been suggested that these receptors are involved in embryonic vascularization because of their localized expression during embryogenesis, and the impairment of blood vessel formation in Flt-1- and Flk-1-deficient mice (Fong et al., 1995). It is also well-documented that VEGF has various other biological functions, such as migration of human monocytes (Barleon et al., 1996) and osteoclast differentiation (Niida et al., 1999). Niida et al.(1999) showed that rhVEGF and receptor activator of nuclear factor 
B ligand (RANKL)-supported osteoclast generation in vitro, although the effect was significantly smaller than that generated in the presence of rhM-CSF and rhRANKL. No osteoclasts were detected in the presence of rhM-CSF or rhVEGF without rhRAKL. Also, in an in vivo study, rhVEGF was shown to induce significant osteoclast recruitment and stimulated bone resorption in op/op mice; however, the number was smaller than those induced by rhM-CSF (Niida et al., 1999). For this reason, it is likely that VEGF acts mainly as a neovascularization factor, and is less active in osteoclast recruitment than is M-CSF. It has also been shown that osteoclasts predominantly express Flt-1, but not Flk-1. PlGF, which shows a higher degree of homology to VEGF and binds only to Flt-1, could induce osteoclast recruitment to the same extent as VEGF. Thus, it was strongly suggested that VEGF and PlGF might induce osteclast recruitment via Flt-1 (Niida et al., 1999). These results revealed that VEGF functionally overlaps with M-CSF in osteoclast differentiation, and it is also clear that VEGF and M-CSF are intimately involved in bone remodeling through osteoclast bone resorption.
Orthodontic tooth movement is achieved with alveolar bone resorption and new bone formation. Osteoblasts communicate with osteocytes through gap junctions and constitute a complex level of cellular organization (Doty, 1981). The increase in intracellular Ca2+ propagates from cell to cell via gap junctions (Xia and Ferrier, 1992). Furthermore, osteoblasts also regulate osteoclast differentiation and activation through osteoclast differentiation factor (ODF)/osteoprotegerin ligand (OPGL)/TNF-related activation-induced cytokine (TRANCE)/RANKL (Lacey et al., 1998). Our recent studies have demonstrated that local administration of rhVEGF during experimental tooth movement enhanced the number of osteoclasts and the rate of tooth movement (Kaku et al., 2001; Kohno et al., 2003). Osteoblasts on the tension side of the alveolar bone expressed VEGF during tooth movement, suggesting that VEGF is an essential factor for bone remodeling (Kohno et al., 2003). It was also reported that periodontal cells, such as periodontal ligament fibroblasts, express VEGF and induce angiogenesis in response to mechanical stimuli (Yoshino et al., 2003). From these findings, it is concluded that osteoblasts promote expression of VEGF and M-CSF in response to mechanical stress such as orthodontic tooth movement, and this is mediated by the S-A channel. Thus, these factors (VEGF and M-CSF) may support angiogenesis and bone resorption. Furthermore, it is likely that a chain-reaction of angiogenesis and bone remodeling may be promoted by VEGF and M-CSF successively.
|
ACKNOWLEDGMENTS |
|---|
Received September 1, 2003; Last revision November 23, 2004; Accepted February 4, 2005
|
REFERENCES |
|---|
|
TOPABSTRACTINTRODUCTIONMATERIALS & METHODSRESULTSDISCUSSIONREFERENCES |
|---|
Biagi BA, Enyeart JJ (1990). Gadolinium blocks low- and high-threshold calcium currents in pituitary cells. Am J Physiol 259:C515–C520.
Doty SB (1981). Morphological evidence of gap junctions between bone cells. Calcif Tissue Int 33:509–512.[ISI][Medline]
Duncan R, Misler S (1989). Voltage-activated and stretch-activated Ba2+ conducting channels in an osteoblast-like cell line (UMR 106). FEBS Lett 251:17–21.[ISI][Medline]
Fong GH, Rossant J, Gertsenstein M, Breitman ML (1995). Role of the Flt-1 receptor tyrosine kinase in regulating the assembly of vascular endothelium. Nature 376:66–70.[Medline]
Guharay F, Sachs F (1984). Stretch-activated single ion channel currents in tissue-cultured embryonic chick skeletal muscle. J Physiol 352:685–701.
Hung CT, Allen FD, Pollack SR, Brighton CT (1996). Intracellular Ca2+ stores and extracellular Ca2+ are required in the real-time Ca2+ response of bone cells experiencing fluid flow. J Biomech 29:1411–1417.[ISI][Medline]
Iwasaki LR, Haack JE, Nickel JC, Morton J (2000). Human tooth movement in response to continuous stress of low magnitude. Am J Orthod Dentofacial Orthop 117:175–183.[ISI][Medline]
Kaku M, Kohno S, Kawata T, Fujita I, Tokimasa C, Tsutsui K, Tanne K (2001). Effects of vascular endothelial growth factor on osteoclast induction during tooth movement in mice. J Dent Res 80:1880–1883.
Kanno Z, Shibata T, Umezawa A, Kasugai S, Ohy K, Soma K (1999). Intracellular calcium response in cultured bone cells under mechanical stress. J Jpn Orthod Soc 58:389–395.
Kanzaki M, Nagasawa N, Kojima I, Sato C, Naruse K, Sokabe M, et al. (1999). Molecular identification of a eukaryotic, stretch-activated nonselective cation channel. Science 285:882–886; errata, Science 1999 Sep 3 285(5433):1493 and Science 2000 May 26 288 (5470):1347.
Klein-Nulend J, van der Plas A, Semeins CM, Ajubi NE, Frangos JA, Nijweide PJ, et al. (1995). Sensitivity of osteocytes to biomechanical stress in vitro. FASEB J 9:441–445.
Kohno S, Kaku M, Tsutsui K, Motokawa M, Ohtani J, Tenjo K, et al. (2003). Expression of vascular endothelial growth factor and the effects on bone remodeling during experimental tooth movement. J Dent Res 82:177–182.
Lacey DL, Timms E, Tan HL, Kelley MJ, Dunstan CR, Burgess T, et al. (1998). Osteoprotegerin ligand is a cytokine that regulates osteoclast differentiation and activation. Cell 93:165–176.[ISI][Medline]
Lansman JB, Hallam TJ, Rink TJ (1987). Single stretch-activated ion channels in vascular endothelial cells as mechanotransducers. Nature 325:811–813.[Medline]
Leung DW, Cachianes G, Kuang WJ, Goeddel DV, Ferrara N (1989). Vascular endothelial growth factor is a secreted angiogenic mitogen. Science 246:1306–1309.
Milgrom C, Finestone A, Simkin A, Ekenman I, Mendelson S, Millgram M, et al. (2000). In-vivo strain measurements to evaluate the strengthening potential of exercises on the tibial bone. J Bone Joint Surg Br 82:591–594.
Mitrasinovic OM, Perez GV, Zhao F, Lee YL, Poon C, Murphy GM Jr (2001). Overexpression of macrophage colony-stimulating factor receptor on microglial cells induces an inflammatory response. J Biol Chem 276:30142–30149.
Naruse K, Sokabe M (1993). Involvement of stretch-activated ion channels in Ca2+ mobilization to mechanical stretch in endothelial cells. Am J Physiol 264:C1037–C1044.
Naruse K, Yamada T, Sokabe M (1998). Involvement of SA channels in orienting response of cultured endothelial cells to cyclic stretch. Am J Physiol 274:H1532–H1538.
Neidlinger-Wilke C, Stalla I, Claes L, Brand R, Hoellen I, Rubenacker S, et al. (1995). Human osteoblasts from younger normal and osteoporotic donors show differences in proliferation and TGF beta-release in response to cyclic strain. J Biomech 28:1411–1418.[ISI][Medline]
Niida S, Kaku M, Amano H, Yoshida H, Kataoka H, Nishikawa S, et al. (1999). Vascular endothelial growth factor can substitute for macrophage colony-stimulating factor in the support of osteoclastic bone resorption. J Exp Med 190:293–298.
Ozawa H, Imamura K, Abe E, Takahashi N, Hiraide T, Shibasaki Y, et al. (1990). Effect of a continuously applied compressive pressure on mouse osteoblast-like cells (MC3T3-E1) in vitro. J Cell Physiol 142:177–185.[ISI][Medline]
Reher P, Doan N, Bradnock B, Meghji S, Harris M (1999). Effect of ultrasound on the production of IL-8, basic FGF and VEGF. Cytokine 11:416–423.[ISI][Medline]
Senger DR, Galli SJ, Dvorak AM, Perruzzi CA, Harvey VS, Dvorak HF (1983). Tumor cells secrete a vascular permeability factor that promotes accumulation of ascites fluid. Science 219:983–985.
Terman BI, Dougher-Vermazen M, Carrion ME, Dimitrov D, Armellino DC, Gospodarowicz D, et al. (1992). Identification of the KDR tyrosine kinase as a receptor for vascular endothelial cell growth factor. Biochem Biophys Res Commun 187:1579–1586.[ISI][Medline]
Xia SL, Ferrier J (1992). Propagation of a calcium pulse between osteoblastic cells. Biochem Biophys Res Commun 186:1212–1219.[ISI][Medline]
Yang X, Sachs F (1989). Block of stretch-activated ion channels in Xenopus oocytes by gadolinium and calcium ions. Science 243:1068–1071.
Yoshida H, Hayashi S, Kunisada T, Ogawa M, Nishikawa S, Okumura H, et al. (1990). The murine mutation osteopetrosis is in the coding region of the macrophage colony stimulating factor gene. Nature 345:442–444.[Medline]
Yoshino H, Morita I, Murota SI, Ishikawa I (2003). Mechanical stress induces production of angiogenic regulators in cultured human gingival and periodontal ligament fibroblasts. J Periodontal Res 38:405–410.[ISI][Medline]
This article has been cited by other articles:
|
|
 |
|
  Y. Khan and C. T. Laurencin Fracture Repair with Ultrasound: Clinical and Cell-Based Evaluation J. Bone Joint Surg. Am., February 1, 2008; 90(Supplement_1): 138 - 144. [Abstract] [Full Text] [PDF]
|
|
| ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
| IADR Journals | Advances in Dental Research ® |
| Journal of Dental Research ® | Critical Reviews (1990-2004) |
