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Effects of broad frequency vibration on cultured osteoblasts
Journal of Biomechanics 36 (2003) 73–80
Effects of broad frequency vibration on cultured osteoblasts Shigeo M. Tanakaa, Jiliang Lib, Randall L. Duncana, Hiroki Yokotab, David B. Burrb, Charles H. Turnera,* a
Department of Orthopedic Surgery, Biomaterials & Biomechanics Research Center, Indiana University School of Medicine, 541 Clinical Drive, Room 600, Indianapolis, IN46202-5111, USA b Department of Anatomy and Cell Biology, Indiana University School of Medicine, 635 Barnhill Drive, Indianapolis, IN, USA Accepted 17 July 2002
Abstract Bone is subjected in vivo to both high amplitude, low frequency strain, incurred by locomotion, and to low amplitude, broad frequency strain. The biological effects of low amplitude, broad frequency strain are poorly understood. To evaluate the effects of low amplitude strains ranging in frequency from 0 to 50 Hz on osteoblastic function, we seeded MC3T3-E1 cells into collagen gels and applied the following loading protocols for 3 min per day for either 3 or 7 days: (1) sinusoidal strain at 3 Hz, with 0–3000 mstrain peak-to-peak followed by 0.33 s resting time, (2) ‘‘broad frequency vibration’’ of low amplitude strain (standard deviation of 300 mstrain) including frequency components from 0 to 50 Hz, and (3) sinusoidal strain combined with broad frequency vibration (S þ V ). The cells were harvested on day 4 or 8. We found that the S þ V stimulation significantly repressed cell proliferation by day 8. Osteocalcin mRNA was up-regulated 2.6-fold after 7 days of S þ V stimulation, and MMP-9 mRNA was elevated 1.3-fold after 3 days of vibration alone. Sinusoidal stimulation alone did not affect the cell responses. No differences due to loading were observed in alkaline phosphatase activity and in mRNA levels of type I collagen, osteopontin, connexin 43, MMPs-1A, -3, -13. These results suggest that osteoblasts are more sensitive to low amplitude, broad frequency strain, and this kind of strain could sensitize osteoblasts to high amplitude, low frequency strain. This suggestion implies a potential contribution of stochastic resonance to the mechanical sensitivity of osteoblasts. r 2002 Elsevier Science Ltd. All rights reserved. Keywords: Bone; Osteoblasts; Biomechanics; Mechanical vibration; Osteoporosis
1. Introduction Bone cells of the osteoblastic lineage are known to sense and respond to mechanical stimuli. In other mechanosensory systems in the body, the mechanosensitivity of cells can be enhanced by stochastic resonance (Collins et al., 1996a; Cordo et al., 1996). This is a phenomenon in which mechanical noise enhances the response of a nonlinear system to a weak signal. Collins (Collins et al., 1996b) showed that the tactile sensation of the human fingertip was enhanced by incorporating mechanical noise onto a standard pressure pulse. However, the noise-enhancement of sensation occurred only for a narrow range of noise amplitudes. This technique might be used to improve cutaneous sensory *Corresponding author. Tel.: +317-274-3226; fax: +317-274-3702. E-mail address: [email protected] (C.H. Turner).
perception in individuals with peripheral neuropathies. Likewise, it may be possible to sensitize bone cells by mechanical noise-enhancement, thus improving the osteogenic response to mechanical loading. During normal locomotion, the frequency range of mechanical loading applied to bone is quite broad. In vivo bone strain measurements taken during locomotion demonstrate large amplitude strains at frequencies ranging from 0.5 to 2 Hz and low amplitude, but broad frequency strain vibration reaching to 30 Hz (Rubin et al., 1990; Turner et al., 1995). It is unclear whether the broad frequency vibration affects mechanical adaptation in bone. Previous studies of controlled skeletal loading in rats and turkeys showed that loading applied at higher frequencies (10–20 Hz) was more effective for stimulating new bone formation than loading at normal locomotor frequencies (1 Hz) (Hsieh and Turner, 2001; Rubin and McLeod, 1994). It was demonstrated that
0021-9290/02/$ - see front matter r 2002 Elsevier Science Ltd. All rights reserved. PII: S 0 0 2 1 - 9 2 9 0 ( 0 2 ) 0 0 2 4 5 - 2
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low amplitude, but higher frequency (50 Hz), mechanical vibration reduces early bone loss after ovariectomy in rats (Flieger et al., 1998). This observation suggests that high frequency vibration alters the balance between resorption and formation caused by estrogen deficiency either through inhibition of resorption or stimulation of formation. We hypothesized that osteoblasts might be sensitized to mechanical loading by low amplitude, broad frequency mechanical vibration. Others have shown that osteoblasts in culture respond to mechanical strain at frequency of 1 Hz (Kaspar et al, 2000; Meyer et al., 1997; Neidlinger-Wilke et al., 1994). Osteoblasts also respond to mechanical vibration (Tjandrawinata et al., 1997), yet we are not aware of a study that used vibration to sensitize osteoblasts to mechanical loading. In the present study, a newly developed mechanical stimulator was employed. The stimulator can generate low amplitude strain vibration with broad frequency components up to 50 Hz. Mechanical stimulation was given to MC3T3-E1 cells cultured in a collagen gel, and the effects of the broad frequency strain vibration on cellular proliferation, alkaline phosphatase activity, and mRNA expression of genes associated with the anabolic response in bone were investigated.
2. Materials and methods 2.1. Cell preparation MC3T3-E1 cells at passage 10 were pre-cultured in aMEM (Sigma, MO, USA) media supplemented with 10% FBS and antibiotics (100 U/ml penicillin and 0.1 mg/ml streptomycin). At passage 13, cells were seeded into collagen gels (1 mg/ml) consisting of rat-tail type I collagen (BD Biosciences, NJ, USA) and the media at a seeding density of 4.0 105 cells/ml. Mechanical stimulators were sterilized by ethylene oxide gas and 0.65 ml collagen gels with osteoblasts were placed into chambers of the stimulator with the media as previously described (Tanaka, 1999). The gels were cultured in the chambers for 24 h prior to initial mechanical stimulation. 2.2. Mechanical stimulator A mechanical stimulator using a piezoelectric actuator was described previously (Tanaka, 1999). In this study, we implemented a closed-loop (feedback) system to the piezo-driven stimulator. The stimulator has four chambers in which osteoblasts are cultured in a collagen gel with dimensions of 20 mm 13 mm 2 mm (Fig. 1A). Mechanical strain was applied by bimorphtype piezoelectric actuators (LPD30X, Megacera Inc.,
Fig. 1. Schematic diagram of mechanical stimulator with four chambers (A) and the motion of bimorph-type piezoelectric actuator (B). Bending of the actuator gives compression or tension to a collagen gel populated with MC3T3-E1 cells in a chamber: (1) collagen gel block with cells, (2) moving plunger, (3) fixed plunger, (4) bimorph-type piezoelectric actuator, (5) porous polyethylene strip, (6) gap sensor, and (7) medium.
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Saitama, Japan) to collagen gels, which were anchored by strips of porous polyethylene (Bel-Art Products, NJ, USA) fixed to plungers. To reduce hydrophobicity, the strips were soaked for 3 days in concentrated sulfuric acid and then washed 10 times over 2 days with distilled water (Kolodney and Wysolmerski, 1992). Bending of the actuator induced by voltage loading causes maximum displacement at the center of the actuator, and this displacement is transferred to the collagen gel via a moving plunger (Fig. 1B). Strain waveforms were prepared on a personal computer using programs based on LabVIEWR (National Instruments Co., TX, USA) and Visual Basic (Microsoft Co., WA, USA). A voltage-signal was sent to a high-speed amplifier with a frequency response of 1 kHz (HEOPS0.6B50, Matsusada Precision Inc., Siga, Japan), via an AD-DA board (aISA-A57, Adtek-system Science, Kanagawa, Japan). The displacement at the center of the actuator was measured by an eddy current-type gap sensor (AEC-55D15A, AEC, Kanagawa, Japan) with a frequency response of 0–20 kHz and resolution of 0.5 mm. The displacement signals from the gap sensor were collected by the AD-DA board, and errors between the desired and the measured waveforms were minimized by the feedback control at 250 ms interval. The strain waveforms were given to M3T3-E1 cells in collagen gels for 3 min per day for either 3 or 7 days, and cells were harvested on the following day (day 4 or 8). This stimulation protocol was selected based on in vivo studies reporting that 3 min of mechanical loading per day was sufficient to induce new bone formation on rat tibiae or ulnae (Forwood et al., 1996; Hsieh et al., 2001; Robling et al., 2000, 2002). Three waveforms employed in this study were sine (sinusoidal waves at 3 Hz with 0– 3000 mstrain peak-to-peak and 0.33 s of resting time
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between every wave); vibration (Gaussian quasi-white noise with a standard deviation of 300 mstrain and frequency components up to 50 Hz); and S þ V (in which the waveform was synthesized by superimposing the vibration on the sine waves using a computer, so that both waveforms were applied to the cells simultaneously (Fig. 2). 2.3. Cell counting Following the loading protocols, the collagen gels were dissolved in Hank’s balanced salt solution (HBSS) with collagenase. Cells were collected, and counted using a hematocytometer. The isolated cells were then washed and lysed in order to determine alkaline phosphatase activity and mRNA expression levels. 2.4. Alkaline phosphatase (ALP) activity Cells were rinsed twice with HBSS at 41C, solubilized in 50 mM Tris/HCl buffer, pH 7.6, containing 0.5% Triton X-100, and sonicated on ice for 30 s. The lysate was centrifuged for 10 min at 12,000 rpm at 41C. A protein concentration in the supernatant was determined by Bio-Rad DC protein assay using a standard curve for bovine serum albumin. The supernatant was mixed with 4 mM p-nitrophenyl-phosphate in a buffer consisting of 10 mM MgCl2 and 105 mM 2-amino-2methyl-2-propanol, pH 10.3, and incubated at 371C for 20 min. The reaction was terminated by adding 0.3 N NaOH solution. The absorbance of p-nitrophenol (pNP) at 410 nm was measured by spectrophotometry. A concentration of p-nitrophenol was determined using a standard curve and the measured data was converted to nmoles of p-NP per minute per cell or per mg of protein.
Fig. 2. Strain waveforms: (A) Control (no stimulation), (B) Sine (sinusoidal wave at 3 Hz with 0–3000 mstrain peak-to-peak followed by 0.33 s resting time), (C) Vibration (Gaussian quasi-white noise with standard deviation of 300 mstrain and frequency components from 0 to 50 Hz, (D) S þ V (sine (B) combined with vibration (C)).
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2.5. Semi-quantitative reverse-transcription polymerase chain reaction (RT-PCR) Semi-quantitative RT-PCR was performed to determine mRNA levels of osteocalcin (OC), pro-a-1 type I collagen (Collagen I), osteopontin (OPN), connexin 43 (Cx43), and four matrix metalloproteinases (MMPs-1A, -3, -9, -13) using a pair of primers specific to these genes (Table 1). Total RNA was extracted by using TRIzol reagent (GIBCO-BRL, Grand Island, NY, USA), and reverse-transcribed at 371C for 1 h using a cDNA synthesis kit (Amersham Pharmacia Biotech Inc., NJ, USA). Reverse-transcribed products were amplified by PCR in a reaction mixture consisting of 2.5 mM dNTPs, 20 pmol/ml primers, and 5 U/ml Taq poymerase. PCR was carried out using a thermal cycler (PTC-200, MJ Research, Inc., MA, USA) for 40 cycles (941C for 1 min, 551C for 1.5 min, and 721C for 2 min). Glyceraldehyde3-phosphate dehydrogenase (GAPDH) was used as an internal control. PCR products were electrophoresed on 1.2% agarose gel and visualized by ethidium bromide. To evaluate mRNA levels, an intensity of electrophoretic bands was quantified by using PhotoShop (Adobe Systems Inc., CA, USA), and normalized by GAPDH. However, this method for quantification of mRNA level is imprecise; therefore, this method was used only to screen genes that respond to the mechanical stimuli. For the responsive genes, mRNA levels were quantified using real time RT-PCR as described below. 2.6. Real-time RT-PCR Real-time RT-PCR was performed on an ABI PRISM 7700 Sequence Detection System (Applied Biosystems, CA, USA) using specific primers and probes for OC and
MMP-9 (Table 2). The primers and probe for GAPDH were provided from TaqMans Rodent GAPDH Control Reagents (Applied Biosystems, CA, USA). A reaction volume was 50 ml and the reaction mixture contained 5.5 mM MgCl2, 0.05% gelatin, 200 mM each of dATP, dCTP, dGTP, 400 mM dUTP, 0.05 U/ml AmpliTaq Gold, 0.01 U/ml uracil-N-glycosylase, 200 nM oligonucleotide primer, 100 nM probe, and 3.5 ml cDNA template. We used 40 PCR cycles with a 15-s melt at 951C and a 1-min annealing/extension at 601C. The level of OC mRNA and MMP-9 mRNA in the stimulated groups was normalized by the GAPDH mRNA level and compared to the mRNA level of control cells. 2.7. Statistical methods All experiments were performed three times. Analysis of variance (ANOVA) was used to examine statistically significant differences in cell proliferation, ALP activity, and gene expression of MC3T3-E1 cells. Fishers protected least significant difference tests were conducted for pairwise comparisons among groups, when po0:05 in ANOVA. Statistical significance was examined if the p-value was 0.05 or lower.
3. Results 3.1. Cell proliferation By day 8, the cell number in the S þ V group was reduced significantly (po0:05) compared to the control (Fig. 3). MC3T3-E1 cells proliferated over the first 4 days within the collagen gel, but then stopped. By day 4, control cell counts increased 2-fold, while sine,
Table 1 Oligonucleotides used in semi-quantitative RT-PCR mRNA Collagen I Cx43 OC OPN MMP-1A MMP-3 MMP-9 MMP-13 GAPDH
Sense Anti-sense Sense Anti-sense Sense Anti-sense Sense Anti-sense Sense Anti-sense Sense Anti-sense Sense Anti-sense Sense Anti-sense Sense Anti-sense
Primer sequences
PCR product size (bp)
50 -GGTGCCCCCGGTCTTCAG-30 50 -AGGGCCAGGGGGTCCAGCATTTC-30 50 -GTCAGCTTGGGGTGATGAACAG-30 50 -ATGGTTTTCTCCGTGGGACG-30 50 -GTCCTCCTGGTTCATTTCTTTGGGTAAC-30 50 -CACTACCTTATTGCCCTCCTGCTTGGAC-30 50 -CCAACGGCCGAGGTGATAG-30 50 -CAGGCTGGCTTTGGAACTTG-30 50 -ATACACAGTCTATGGATCCA-30 50 -GGTTCTTCAGTTAATAACTTC-30 50 -ACCGGATTTGCCAAGACAGAG-30 50 -AGCCTTGGCTGAGTGGTAGA-30 50 -AGTTTGGTGTCGCGGAGCAC-30 50 -TACATGAGCGCTTCCGGCAC-30 50 -CTGGTCTTCTGGCACACGCT-30 50 -GCAGCGCTCAGTCTCTTCAC-30 50 -GCCACCCAGAAGACTGTGGAT-30 50 -TGGTCCAGGGTTTCTTACTCC-30
529 499 294 333 271 332 755 611 477
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Table 2 Sequences of primers and probes used in real-time RT-PCR mRNA OC
MMP-9
Primer sequences Sense Anti-sense Probe Sense Anti-sense Probe
0
PCR product size (bp) 0
5 -GTGCTGCCCTAAAGCCAAAC-3 50 -GGAGGATCAAGTCCCGGAGA-30 50 -FAM-CTGGCAGCTCGGCTTTGGCTG-TAMRA-30 50 -CAGCTGGCAGAGGCATACTTG-30 50 -GCTTCTCTCCCATCATCTGGG-30 50 -FAM-CCGCTATGGTTACACCCGGGCC-TAMRA-30
63
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Fig. 3. Proliferation of MC3T3-E1 cells cultured in a collagen gel. Data represent the mean7SE (n ¼ 12). The dashed line indicates the mean cell number of samples on day 1 (n ¼ 19), which were incubated with no mechanical stimulation and harvested 24 h after seeding. The cell number in the S þ V group was reduced significantly by day 8 compared to the control group. Symbols: apo0:05; versus control on day 8. po0:01; po0:0001; versus no loading sample on day 1.
vibration, and S þ V stimulation only increased 1.8-, 1.6-, and 1.6-fold, respectively. 3.2. ALP activity ALP activity, an indicator of osteoblastic phenotype, increased significantly by day 4 (Fig. 4A and B). By day 8, the activity was reduced to the basal level of day 1. The results showed no significant differences among loading groups in ALP activities either per cell (Fig. 4A) or per protein (Fig. 4B). 3.3. Gene expression
Fig. 4. Alkaline phosphatase activity per cell (A) and per protein (B) of MC3T3-E1 cells in a collagen gel. Data are presented as the mean7SE (n ¼ 9). The dashed line indicates the average value for samples on day 1 (n ¼ 13), which were cultured for 1 day under no mechanical stimulation. No differences among groups within each time period. Symbols: po0:05; po0:01; po0:001; po0:0001; versus no loading sample on day 1.
An increase in OC mRNA expression was observed in the S þ V group. MMP-9 mRNA expression was also affected by mechanical stimuli. No detectable alteration was observed in the mRNA levels of collagen I, OPN, Cx43, MMPs-1A, -3, -13 by mechanical stimuli. No expression of MMP-1A mRNA was detected in any group.
The alterations in OC mRNA and MMP-9 mRNA levels were quantified using real-time RT-PCR (Fig. 5). Real-time RT-PCR revealed that the treatment of S þ V induced a 2.6-fold increase in OC mRNA level by day 8 over control (po0:01). Vibration treatment significantly increased MMP-9 mRNA level 1.3-fold over control by day 4 (po0:05).
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Fig. 5. Osteocalcin (A) and MMP-9 (B) mRNA expressions of MC3T3-E1 cultured in a collagen gel analyzed by real-time RTPCR. Levels of mRNA expression were normalized to GAPDH mRNA level, and expressed as a ratio to the level in controls. mRNA Level of Osteocalcin and MMP-9 increased significantly after 7 days of S þ V stimulation and 3 days of vibration alone, respectively. Data represent the mean7SE of triplicate experiments. po0:01 versus a,b control, csine. po0:05 versus dsine, evibration, fS þ V :
4. Discussion This study presents the effects of broad frequency vibration in osteoblasts on cellular proliferation and transcriptional regulation of genes involved in bone formation and remodeling. Our real time PCR results showed that S þ V stimulation up-regulated OC mRNA by 8 days and vibratory stimulation elevated MMP-9 mRNA expression by 4 days. Sinusoidal stimulation alone did not alter mRNA expression of either gene. These results suggest that osteoblasts are responsive to broad frequency vibration strain, and sensitized to sinusoidal stimulation by broad frequency vibration strain. It is possible that the broad frequency, low amplitude strains, such as vibration, used in this study
sensitize osteoblasts to mechanical strains through the phenomenon called stochastic resonance (Collins et al., 1996a, b; Cordo et al., 1996). Low amplitude strains commonly occur in bone. Over the course of a day, weight bearing bone tissue experiences large strains over 1000 mstrain only a few times, but small strains of less than 10 mstrain occur thousands of times (Fritton et al., 2000). The effect of mechanical vibration on OC expression suggests that low amplitude, broad high frequency bone strain makes osteoblasts more sensitive to the larger, but less frequent strains that occur during locomotor activities like walking. Our results demonstrate S þ V stimulation suppressed osteoblastic proliferation and promoted OC mRNA expression in 3D-collagen gel. This up-regulation of OC mRNA expression seems to be caused by a combined effect with a promotion of cell differentiation by 3D culture and S þ V stimulation. 3D cultures using a collagen gel promotes differentiation (Kinoshita et al., 1999) and mineralization (Rattner et al., 2000) of osteoblasts. MC3T3-E1 cells, an osteoblastic cell line derived from mouse calvaria cells (Kodama et al., 1981), also are capable of differentiating and initiating calcification in a collagen gel (Casser-Bette et al., 1990). The expression of OC is the terminal marker for differentiation in osteoblasts, and is associated with bone formation. Therefore, we interpret our results to mean that 3D-collagen culture promotes the onset of OC mRNA expression, and its expression is enhanced by S þ V stimulation. Our observations are consistent with previous findings on cell proliferation and differentiation of osteoblasts in 3D-collagen gels. Akhouayri (Akhouayri et al., 1999) reported reduced proliferation of osteoblasts in a floating gel exposed to shaking at 25 or 50 rpm after 8 days in culture. They also observed an elevated OC expression in the shaking gel on days 8–14. In 2D culture, a number of studies have reported enhanced osteoblastic proliferation (Kaspar et al., 2000; Meyer et al., 1997; Neidlinger-Wilke et al., 1994; Stanford et al., 1995) and collagen type I synthesis (Kaspar et al., 2000) in response to mechanical strain. However, mechanical strain in 2D culture was reported to suppress osteoblastic differentiation assessed by ALP activity or expression of OC (Kaspar et al., 2000; Stanford et al., 1995). These findings suggest that the responses of osteoblasts to mechanical stimuli are dependent upon culture environments. Cx43 is the major gap junction protein in osteoblasts. Our results showed no alteration of Cx43 mRNA expression after mechanical stimulation. However, there is a possibility that post-transcriptional processing of this gene is affected by mechanical stimuli. Ziambaras (Ziambaras et al., 1998) reported that mechanical stretch increased Cx43 protein synthesis without changing its mRNA expression in cultured osteoblasts. Therefore, investigation of post-transcriptional
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regulation of the genes we analyzed in this study is necessary to obtain further understanding about effects of broad frequency vibration on osteoblasts. The up-regulation of MMP-9 mRNA by vibratory stimulation in our study suggests a potential role of broad frequency vibration in remodeling of extracellular matrix. MMPs constitute a family of zincbinding proteolytic enzymes classified into 4 groups; collagenase, gelatinase, stomelysin, and a membrane type. MMP-9, gelatinase B, degrades components of extracellular matrix with a high specificity for denatured collagens (gelatin). In this study, mRNA levels of MMP-1 (collagenase 1), MMP-3 (stomelysin 1) and MMP-13 (collagenase 3) were not altered by the mechanical stimuli employed. Although MMP-9- null mice develop to term and survive normally after birth, a delay in vascularization and ossification of growth plates is reported (Engsig et al., 2000; Vu et al., 1998). Other studies have shown that the expression of MMP-9 is elevated in osteoblasts during osteogenesis (Filanti et al., 2000; Onyia et al., 1999). Further investigation is needed to understand the role of MMP-9 and other MMPs in response to broad frequency vibration on osteoblasts. In this study, we focused on gene expression that occurred 24 or more hours after a mechanical stimulus. The study did not address transient gene expression that might have occurred within minutes or a few hours after a mechanical stimulus. Further studies are needed to assess the effects of broad frequency vibration on early and intermediate response genes. Using a newly developed mechanical stimulator, we demonstrated that low amplitude, broad frequency vibration stimulated MMP-9 gene expression in MC3T3-E1 cells cultured in a collagen gel, and enhanced the expression of OC mRNA when mixed with high amplitude, low frequency sinusoidal strain. The sinusoidal strain alone had no effect on cell response. These results suggest that osteoblasts are sensitive to low amplitude, broad frequency strain vibration, and this strain vibration could sensitize them to high amplitude, low frequency strain. This suggestion implies a potential role for stochastic resonance in mechanical sensitivity of osteoblasts and modulating mechanical adaptation responses in bone.
Acknowledgements The technical assistance for RT-PCR by John W. Hawes and Hui Bin Sun is gratefully acknowledged. This study was supported by a grant from the Japan Science and Technology Corporation (JST Oversea Research Fellowship).
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References Akhouayri, O., Lafage-Proust, M.H., Rattner, A., Laroche, N., Caillot-Augusseau, A., Alexandre, C., Vico, L., 1999. Effects of static or dynamic mechanical stresses on osteoblast phenotype expression in three-dimensional contractile collagen gels. Journal of Cellular Biochemistry 76, 217–230. Casser-Bette, M., Murray, A.B., Closs, E.I., Erfle, V., Schmidt, J., 1990. Bone formation by osteoblast-like cells in a three-dimensional cell culture. Calcified Tissue International 46, 46–56. Collins, J.J., Imhoff, T.T., Grigg, P., 1996a. Noise-enhanced information transmission in rat sal cutaneous mechanoreceptors via aperiodic stochastic resonance. Journal of Neurophysiology 76, 642–645. Collins, J.J., Imhoff, T.T., Grigg, P., 1996b. Noise-enhanced tactile sensation. Nature 383, 770. Cordo, P., Inglis, J.T., Verschueren, S., Collins, J.J., Merfeld, D.M., Rosenblum, S., Buckley, S., Moss, F., 1996. Noise in human muscle spindles. Nature 383, 769–770. Engsig, M.T., Chen, Q.J., Vu, T.H., Pedersen, A.C., Therkidsen, B., Lund, L.R., Henriksen, K., Lenhard, T., Foged, N.T., Werb, Z., Delaisse, J.M., 2000. Matrix metalloproteinase 9 and vascular endothelial growth factor are essential for osteoclast recruitment into developing long bones. The Journal of Cell Biology 151, 879–890. Filanti, C., Dickson, G.R., Di Martino, D., Ulivi, V., Sanguineti, C., Romano, P., Palermo, C., Marduca, P., 2000. The expression of metalloproteinase –2, –9, and –14 and of tissue inhibitors –1 and –2 is developmentally modulated during osteogenesis in vitro, the mature osteoblastic phenotype expressing metalloproteinase –14. Journal of Bone and Mineral Research 15, 2154–2168. Flieger, J., Karachalios, T., Khaldi, L., Raptou, P., Lyritis, G., 1998. Mechanical stimulation in the form of vibration prevents postmenopausal bone loss in ovariectomized rats. Calcified Tissue International 63, 510–514. Forwood, M.R., Owan, I., Takano, Y., Turner, C.H., 1996. Increased bone formation in rat tibiae after a single short period of dynamic loading in vivo. American Journal of Physiology 270, E419–423. Fritton, S.P., McLeod, K.J., Rubin, C.T., 2000. Quantifying the strain history of bone: spatial uniformity and self-similarity of lowmagnitude strains. Journal of Biomechanics 33, 317–325. Hsieh, Y.F., Turner, C.H., 2001. Effects of loading frequency on mechanically induced bone formation. Journal of Bone and Mineral Research 16, 918–924. Hsieh, Y.F., Robling, A.G., Ambrosius, W.T., Burr, D.B., Turner, C.H., 2001. Mechanical loading of diaphyseal bone in vivo: the strain threshold for an osteogenic response varies with location. Journal of Bone and Mineral Research 16, 2291–2297. Kaspar, D., Seidl, W., Neidlinger-Wilke, C., Ignatius, A., Claes, L., 2000. Dynamic cell stretching increases human osteoblast proliferation and cicp synthesis but decreases osteocalcin synthesis and alkaline phosphatase activity. Journal of Biomechanics 33, 45–51. Kinoshita, S., Finnegan, M., Bucholz, R.W., Mizuno, K., 1999. Threedimensional collagen gel culture promotes osteoblastic phenotype in bone marrow derived cells. Kobe Journal of Medical Sciences 45, 201–211. Kodama, H., Amagai, Y., Sudo, H., Kasai, S., Yamamoto, S., 1981. Establishment of a clonal osteogenic cell line from newborn mouse calvaria. Japanese Journal of Oral Biology 23, 899–901. Kolodney, M.S., Wysolmerski, R.B., 1992. Isometric contraction by fibroblasts and endothelial cells in tissue culture: a quantitative study. The Journal of Cell Biology 117, 73–82. Meyer, U., Meyer, T., Jones, D.B., 1997. No mechanical role for vinculin in strain transduction in primary bovine osteoblasts. Biochemistry and Cell Biology 75, 81–87. Neidlinger-Wilke, C., Wilke, H.J., Claes, L., 1994. Cyclic stretching of human osteoblasts affects proliferation and metabolism: a new
80
S.M. Tanaka et al. / Journal of Biomechanics 36 (2003) 73–80
experimental method and its application. Journal of Orthopaedic Research 12, 70–78. Onyia, J.E., Hale, L.V., Miles, R.R., Cain, R.L., Tu, Y., Hwman, J.F., Hock, J.M., Santerre, R.F., 1999. Molecular characterization of gene expression charges in ros 17/2.8 cells cultured in diffusion chambers in vivo. Calcified Tissue International 65, 133–138. Rattner, A., Sabido, O., Le, J., Vico, L., Massoubre, C., Frey, J., Chamson, A., 2000. Mineralization and alkaline phosphatase activity in collagen lattices populated by human osteoblasts. Calcified Tissue International 66, 35–42. Robling, A.G., Burr, D.B., Turner, C.H., 2000. Partitioning a daily mechanical stimulus into discrete loading bouts improves the osteogenic response to loading. Journal of Bone and Mineral Research 15, 1596–1602. Robling, A.G., Hinant, F.M., Burr, D.B., Turner, C.H., 2002. Shorter, more frequent mechanical loading sessions enhance bone mass. Medicine and Science in Sports and Exercise 34, 196–202. Rubin, C.T., McLeod, K.J., 1994. Promotion of bony ingrowth by frequency-specific, low-amplitude mechanical strain. Clinical Orthopaedics and Related Research 298, 165–174. Rubin, C.T., McLeod, K.J., Bain, S.D., 1990. Functional strains and cortical bone adaptation: epigenetic assurance of skeletal integrity. Journal of Biomechanics 23, 43–54.
Stanford, C.M., Morcuende, J.A., Brand, R.A., 1995. Proliferative and phenotypic responses of bone-like cells to mechanical deformation. Journal of Orthopaedic Research 13, 664–670. Tanaka, S.M., 1999. A new mechanical stimulator for cultured bone cells using piezoelectric actuator. Journal of Biomechanics 32, 427–430. Tjandrawinata, R.R., Vincent, V.L., Hughes-Fulford, M., 1997. Vibrational force alters mRNA expression in osteoblasts. The FASEB Journal 11, 493–497. Turner, C.H., Yoshikawa, T., Forwood, M.R., Sun, T.C., Burr, D.B., 1995. High frequency components of bone strain in dogs measured during various activities. Journal of Biomechanics 28, 39–44. Vu, T.H., Shipley, J.M., Bergers, G., Berger, J.E., Helms, J.A., Hanahan, D., Shapiro, S.D., Senior, R.M., Werb, Z., 1998. Mmp-9/gelatinase b is a key regulator of growth plate angiogenesis and apoptosis of hypertrophic chondrocytes. Cell 93, 411–422. Ziambaras, K., Lecanda, F., Steinberg, T.H., Civitelli, R., 1998. C yclic stretch enhances gap junctional communication between osteoblastic cells. Journal of Bone and Mineral Research 13, 218–228.
Effects of broad frequency vibration on cultured osteoblasts Shigeo M. Tanakaa, Jiliang Lib, Randall L. Duncana, Hiroki Yokotab, David B. Burrb, Charles H. Turnera,* a
Department of Orthopedic Surgery, Biomaterials & Biomechanics Research Center, Indiana University School of Medicine, 541 Clinical Drive, Room 600, Indianapolis, IN46202-5111, USA b Department of Anatomy and Cell Biology, Indiana University School of Medicine, 635 Barnhill Drive, Indianapolis, IN, USA Accepted 17 July 2002
Abstract Bone is subjected in vivo to both high amplitude, low frequency strain, incurred by locomotion, and to low amplitude, broad frequency strain. The biological effects of low amplitude, broad frequency strain are poorly understood. To evaluate the effects of low amplitude strains ranging in frequency from 0 to 50 Hz on osteoblastic function, we seeded MC3T3-E1 cells into collagen gels and applied the following loading protocols for 3 min per day for either 3 or 7 days: (1) sinusoidal strain at 3 Hz, with 0–3000 mstrain peak-to-peak followed by 0.33 s resting time, (2) ‘‘broad frequency vibration’’ of low amplitude strain (standard deviation of 300 mstrain) including frequency components from 0 to 50 Hz, and (3) sinusoidal strain combined with broad frequency vibration (S þ V ). The cells were harvested on day 4 or 8. We found that the S þ V stimulation significantly repressed cell proliferation by day 8. Osteocalcin mRNA was up-regulated 2.6-fold after 7 days of S þ V stimulation, and MMP-9 mRNA was elevated 1.3-fold after 3 days of vibration alone. Sinusoidal stimulation alone did not affect the cell responses. No differences due to loading were observed in alkaline phosphatase activity and in mRNA levels of type I collagen, osteopontin, connexin 43, MMPs-1A, -3, -13. These results suggest that osteoblasts are more sensitive to low amplitude, broad frequency strain, and this kind of strain could sensitize osteoblasts to high amplitude, low frequency strain. This suggestion implies a potential contribution of stochastic resonance to the mechanical sensitivity of osteoblasts. r 2002 Elsevier Science Ltd. All rights reserved. Keywords: Bone; Osteoblasts; Biomechanics; Mechanical vibration; Osteoporosis
1. Introduction Bone cells of the osteoblastic lineage are known to sense and respond to mechanical stimuli. In other mechanosensory systems in the body, the mechanosensitivity of cells can be enhanced by stochastic resonance (Collins et al., 1996a; Cordo et al., 1996). This is a phenomenon in which mechanical noise enhances the response of a nonlinear system to a weak signal. Collins (Collins et al., 1996b) showed that the tactile sensation of the human fingertip was enhanced by incorporating mechanical noise onto a standard pressure pulse. However, the noise-enhancement of sensation occurred only for a narrow range of noise amplitudes. This technique might be used to improve cutaneous sensory *Corresponding author. Tel.: +317-274-3226; fax: +317-274-3702. E-mail address: [email protected] (C.H. Turner).
perception in individuals with peripheral neuropathies. Likewise, it may be possible to sensitize bone cells by mechanical noise-enhancement, thus improving the osteogenic response to mechanical loading. During normal locomotion, the frequency range of mechanical loading applied to bone is quite broad. In vivo bone strain measurements taken during locomotion demonstrate large amplitude strains at frequencies ranging from 0.5 to 2 Hz and low amplitude, but broad frequency strain vibration reaching to 30 Hz (Rubin et al., 1990; Turner et al., 1995). It is unclear whether the broad frequency vibration affects mechanical adaptation in bone. Previous studies of controlled skeletal loading in rats and turkeys showed that loading applied at higher frequencies (10–20 Hz) was more effective for stimulating new bone formation than loading at normal locomotor frequencies (1 Hz) (Hsieh and Turner, 2001; Rubin and McLeod, 1994). It was demonstrated that
0021-9290/02/$ - see front matter r 2002 Elsevier Science Ltd. All rights reserved. PII: S 0 0 2 1 - 9 2 9 0 ( 0 2 ) 0 0 2 4 5 - 2
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low amplitude, but higher frequency (50 Hz), mechanical vibration reduces early bone loss after ovariectomy in rats (Flieger et al., 1998). This observation suggests that high frequency vibration alters the balance between resorption and formation caused by estrogen deficiency either through inhibition of resorption or stimulation of formation. We hypothesized that osteoblasts might be sensitized to mechanical loading by low amplitude, broad frequency mechanical vibration. Others have shown that osteoblasts in culture respond to mechanical strain at frequency of 1 Hz (Kaspar et al, 2000; Meyer et al., 1997; Neidlinger-Wilke et al., 1994). Osteoblasts also respond to mechanical vibration (Tjandrawinata et al., 1997), yet we are not aware of a study that used vibration to sensitize osteoblasts to mechanical loading. In the present study, a newly developed mechanical stimulator was employed. The stimulator can generate low amplitude strain vibration with broad frequency components up to 50 Hz. Mechanical stimulation was given to MC3T3-E1 cells cultured in a collagen gel, and the effects of the broad frequency strain vibration on cellular proliferation, alkaline phosphatase activity, and mRNA expression of genes associated with the anabolic response in bone were investigated.
2. Materials and methods 2.1. Cell preparation MC3T3-E1 cells at passage 10 were pre-cultured in aMEM (Sigma, MO, USA) media supplemented with 10% FBS and antibiotics (100 U/ml penicillin and 0.1 mg/ml streptomycin). At passage 13, cells were seeded into collagen gels (1 mg/ml) consisting of rat-tail type I collagen (BD Biosciences, NJ, USA) and the media at a seeding density of 4.0 105 cells/ml. Mechanical stimulators were sterilized by ethylene oxide gas and 0.65 ml collagen gels with osteoblasts were placed into chambers of the stimulator with the media as previously described (Tanaka, 1999). The gels were cultured in the chambers for 24 h prior to initial mechanical stimulation. 2.2. Mechanical stimulator A mechanical stimulator using a piezoelectric actuator was described previously (Tanaka, 1999). In this study, we implemented a closed-loop (feedback) system to the piezo-driven stimulator. The stimulator has four chambers in which osteoblasts are cultured in a collagen gel with dimensions of 20 mm 13 mm 2 mm (Fig. 1A). Mechanical strain was applied by bimorphtype piezoelectric actuators (LPD30X, Megacera Inc.,
Fig. 1. Schematic diagram of mechanical stimulator with four chambers (A) and the motion of bimorph-type piezoelectric actuator (B). Bending of the actuator gives compression or tension to a collagen gel populated with MC3T3-E1 cells in a chamber: (1) collagen gel block with cells, (2) moving plunger, (3) fixed plunger, (4) bimorph-type piezoelectric actuator, (5) porous polyethylene strip, (6) gap sensor, and (7) medium.
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Saitama, Japan) to collagen gels, which were anchored by strips of porous polyethylene (Bel-Art Products, NJ, USA) fixed to plungers. To reduce hydrophobicity, the strips were soaked for 3 days in concentrated sulfuric acid and then washed 10 times over 2 days with distilled water (Kolodney and Wysolmerski, 1992). Bending of the actuator induced by voltage loading causes maximum displacement at the center of the actuator, and this displacement is transferred to the collagen gel via a moving plunger (Fig. 1B). Strain waveforms were prepared on a personal computer using programs based on LabVIEWR (National Instruments Co., TX, USA) and Visual Basic (Microsoft Co., WA, USA). A voltage-signal was sent to a high-speed amplifier with a frequency response of 1 kHz (HEOPS0.6B50, Matsusada Precision Inc., Siga, Japan), via an AD-DA board (aISA-A57, Adtek-system Science, Kanagawa, Japan). The displacement at the center of the actuator was measured by an eddy current-type gap sensor (AEC-55D15A, AEC, Kanagawa, Japan) with a frequency response of 0–20 kHz and resolution of 0.5 mm. The displacement signals from the gap sensor were collected by the AD-DA board, and errors between the desired and the measured waveforms were minimized by the feedback control at 250 ms interval. The strain waveforms were given to M3T3-E1 cells in collagen gels for 3 min per day for either 3 or 7 days, and cells were harvested on the following day (day 4 or 8). This stimulation protocol was selected based on in vivo studies reporting that 3 min of mechanical loading per day was sufficient to induce new bone formation on rat tibiae or ulnae (Forwood et al., 1996; Hsieh et al., 2001; Robling et al., 2000, 2002). Three waveforms employed in this study were sine (sinusoidal waves at 3 Hz with 0– 3000 mstrain peak-to-peak and 0.33 s of resting time
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between every wave); vibration (Gaussian quasi-white noise with a standard deviation of 300 mstrain and frequency components up to 50 Hz); and S þ V (in which the waveform was synthesized by superimposing the vibration on the sine waves using a computer, so that both waveforms were applied to the cells simultaneously (Fig. 2). 2.3. Cell counting Following the loading protocols, the collagen gels were dissolved in Hank’s balanced salt solution (HBSS) with collagenase. Cells were collected, and counted using a hematocytometer. The isolated cells were then washed and lysed in order to determine alkaline phosphatase activity and mRNA expression levels. 2.4. Alkaline phosphatase (ALP) activity Cells were rinsed twice with HBSS at 41C, solubilized in 50 mM Tris/HCl buffer, pH 7.6, containing 0.5% Triton X-100, and sonicated on ice for 30 s. The lysate was centrifuged for 10 min at 12,000 rpm at 41C. A protein concentration in the supernatant was determined by Bio-Rad DC protein assay using a standard curve for bovine serum albumin. The supernatant was mixed with 4 mM p-nitrophenyl-phosphate in a buffer consisting of 10 mM MgCl2 and 105 mM 2-amino-2methyl-2-propanol, pH 10.3, and incubated at 371C for 20 min. The reaction was terminated by adding 0.3 N NaOH solution. The absorbance of p-nitrophenol (pNP) at 410 nm was measured by spectrophotometry. A concentration of p-nitrophenol was determined using a standard curve and the measured data was converted to nmoles of p-NP per minute per cell or per mg of protein.
Fig. 2. Strain waveforms: (A) Control (no stimulation), (B) Sine (sinusoidal wave at 3 Hz with 0–3000 mstrain peak-to-peak followed by 0.33 s resting time), (C) Vibration (Gaussian quasi-white noise with standard deviation of 300 mstrain and frequency components from 0 to 50 Hz, (D) S þ V (sine (B) combined with vibration (C)).
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2.5. Semi-quantitative reverse-transcription polymerase chain reaction (RT-PCR) Semi-quantitative RT-PCR was performed to determine mRNA levels of osteocalcin (OC), pro-a-1 type I collagen (Collagen I), osteopontin (OPN), connexin 43 (Cx43), and four matrix metalloproteinases (MMPs-1A, -3, -9, -13) using a pair of primers specific to these genes (Table 1). Total RNA was extracted by using TRIzol reagent (GIBCO-BRL, Grand Island, NY, USA), and reverse-transcribed at 371C for 1 h using a cDNA synthesis kit (Amersham Pharmacia Biotech Inc., NJ, USA). Reverse-transcribed products were amplified by PCR in a reaction mixture consisting of 2.5 mM dNTPs, 20 pmol/ml primers, and 5 U/ml Taq poymerase. PCR was carried out using a thermal cycler (PTC-200, MJ Research, Inc., MA, USA) for 40 cycles (941C for 1 min, 551C for 1.5 min, and 721C for 2 min). Glyceraldehyde3-phosphate dehydrogenase (GAPDH) was used as an internal control. PCR products were electrophoresed on 1.2% agarose gel and visualized by ethidium bromide. To evaluate mRNA levels, an intensity of electrophoretic bands was quantified by using PhotoShop (Adobe Systems Inc., CA, USA), and normalized by GAPDH. However, this method for quantification of mRNA level is imprecise; therefore, this method was used only to screen genes that respond to the mechanical stimuli. For the responsive genes, mRNA levels were quantified using real time RT-PCR as described below. 2.6. Real-time RT-PCR Real-time RT-PCR was performed on an ABI PRISM 7700 Sequence Detection System (Applied Biosystems, CA, USA) using specific primers and probes for OC and
MMP-9 (Table 2). The primers and probe for GAPDH were provided from TaqMans Rodent GAPDH Control Reagents (Applied Biosystems, CA, USA). A reaction volume was 50 ml and the reaction mixture contained 5.5 mM MgCl2, 0.05% gelatin, 200 mM each of dATP, dCTP, dGTP, 400 mM dUTP, 0.05 U/ml AmpliTaq Gold, 0.01 U/ml uracil-N-glycosylase, 200 nM oligonucleotide primer, 100 nM probe, and 3.5 ml cDNA template. We used 40 PCR cycles with a 15-s melt at 951C and a 1-min annealing/extension at 601C. The level of OC mRNA and MMP-9 mRNA in the stimulated groups was normalized by the GAPDH mRNA level and compared to the mRNA level of control cells. 2.7. Statistical methods All experiments were performed three times. Analysis of variance (ANOVA) was used to examine statistically significant differences in cell proliferation, ALP activity, and gene expression of MC3T3-E1 cells. Fishers protected least significant difference tests were conducted for pairwise comparisons among groups, when po0:05 in ANOVA. Statistical significance was examined if the p-value was 0.05 or lower.
3. Results 3.1. Cell proliferation By day 8, the cell number in the S þ V group was reduced significantly (po0:05) compared to the control (Fig. 3). MC3T3-E1 cells proliferated over the first 4 days within the collagen gel, but then stopped. By day 4, control cell counts increased 2-fold, while sine,
Table 1 Oligonucleotides used in semi-quantitative RT-PCR mRNA Collagen I Cx43 OC OPN MMP-1A MMP-3 MMP-9 MMP-13 GAPDH
Sense Anti-sense Sense Anti-sense Sense Anti-sense Sense Anti-sense Sense Anti-sense Sense Anti-sense Sense Anti-sense Sense Anti-sense Sense Anti-sense
Primer sequences
PCR product size (bp)
50 -GGTGCCCCCGGTCTTCAG-30 50 -AGGGCCAGGGGGTCCAGCATTTC-30 50 -GTCAGCTTGGGGTGATGAACAG-30 50 -ATGGTTTTCTCCGTGGGACG-30 50 -GTCCTCCTGGTTCATTTCTTTGGGTAAC-30 50 -CACTACCTTATTGCCCTCCTGCTTGGAC-30 50 -CCAACGGCCGAGGTGATAG-30 50 -CAGGCTGGCTTTGGAACTTG-30 50 -ATACACAGTCTATGGATCCA-30 50 -GGTTCTTCAGTTAATAACTTC-30 50 -ACCGGATTTGCCAAGACAGAG-30 50 -AGCCTTGGCTGAGTGGTAGA-30 50 -AGTTTGGTGTCGCGGAGCAC-30 50 -TACATGAGCGCTTCCGGCAC-30 50 -CTGGTCTTCTGGCACACGCT-30 50 -GCAGCGCTCAGTCTCTTCAC-30 50 -GCCACCCAGAAGACTGTGGAT-30 50 -TGGTCCAGGGTTTCTTACTCC-30
529 499 294 333 271 332 755 611 477
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Table 2 Sequences of primers and probes used in real-time RT-PCR mRNA OC
MMP-9
Primer sequences Sense Anti-sense Probe Sense Anti-sense Probe
0
PCR product size (bp) 0
5 -GTGCTGCCCTAAAGCCAAAC-3 50 -GGAGGATCAAGTCCCGGAGA-30 50 -FAM-CTGGCAGCTCGGCTTTGGCTG-TAMRA-30 50 -CAGCTGGCAGAGGCATACTTG-30 50 -GCTTCTCTCCCATCATCTGGG-30 50 -FAM-CCGCTATGGTTACACCCGGGCC-TAMRA-30
63
67
Fig. 3. Proliferation of MC3T3-E1 cells cultured in a collagen gel. Data represent the mean7SE (n ¼ 12). The dashed line indicates the mean cell number of samples on day 1 (n ¼ 19), which were incubated with no mechanical stimulation and harvested 24 h after seeding. The cell number in the S þ V group was reduced significantly by day 8 compared to the control group. Symbols: apo0:05; versus control on day 8. po0:01; po0:0001; versus no loading sample on day 1.
vibration, and S þ V stimulation only increased 1.8-, 1.6-, and 1.6-fold, respectively. 3.2. ALP activity ALP activity, an indicator of osteoblastic phenotype, increased significantly by day 4 (Fig. 4A and B). By day 8, the activity was reduced to the basal level of day 1. The results showed no significant differences among loading groups in ALP activities either per cell (Fig. 4A) or per protein (Fig. 4B). 3.3. Gene expression
Fig. 4. Alkaline phosphatase activity per cell (A) and per protein (B) of MC3T3-E1 cells in a collagen gel. Data are presented as the mean7SE (n ¼ 9). The dashed line indicates the average value for samples on day 1 (n ¼ 13), which were cultured for 1 day under no mechanical stimulation. No differences among groups within each time period. Symbols: po0:05; po0:01; po0:001; po0:0001; versus no loading sample on day 1.
An increase in OC mRNA expression was observed in the S þ V group. MMP-9 mRNA expression was also affected by mechanical stimuli. No detectable alteration was observed in the mRNA levels of collagen I, OPN, Cx43, MMPs-1A, -3, -13 by mechanical stimuli. No expression of MMP-1A mRNA was detected in any group.
The alterations in OC mRNA and MMP-9 mRNA levels were quantified using real-time RT-PCR (Fig. 5). Real-time RT-PCR revealed that the treatment of S þ V induced a 2.6-fold increase in OC mRNA level by day 8 over control (po0:01). Vibration treatment significantly increased MMP-9 mRNA level 1.3-fold over control by day 4 (po0:05).
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Fig. 5. Osteocalcin (A) and MMP-9 (B) mRNA expressions of MC3T3-E1 cultured in a collagen gel analyzed by real-time RTPCR. Levels of mRNA expression were normalized to GAPDH mRNA level, and expressed as a ratio to the level in controls. mRNA Level of Osteocalcin and MMP-9 increased significantly after 7 days of S þ V stimulation and 3 days of vibration alone, respectively. Data represent the mean7SE of triplicate experiments. po0:01 versus a,b control, csine. po0:05 versus dsine, evibration, fS þ V :
4. Discussion This study presents the effects of broad frequency vibration in osteoblasts on cellular proliferation and transcriptional regulation of genes involved in bone formation and remodeling. Our real time PCR results showed that S þ V stimulation up-regulated OC mRNA by 8 days and vibratory stimulation elevated MMP-9 mRNA expression by 4 days. Sinusoidal stimulation alone did not alter mRNA expression of either gene. These results suggest that osteoblasts are responsive to broad frequency vibration strain, and sensitized to sinusoidal stimulation by broad frequency vibration strain. It is possible that the broad frequency, low amplitude strains, such as vibration, used in this study
sensitize osteoblasts to mechanical strains through the phenomenon called stochastic resonance (Collins et al., 1996a, b; Cordo et al., 1996). Low amplitude strains commonly occur in bone. Over the course of a day, weight bearing bone tissue experiences large strains over 1000 mstrain only a few times, but small strains of less than 10 mstrain occur thousands of times (Fritton et al., 2000). The effect of mechanical vibration on OC expression suggests that low amplitude, broad high frequency bone strain makes osteoblasts more sensitive to the larger, but less frequent strains that occur during locomotor activities like walking. Our results demonstrate S þ V stimulation suppressed osteoblastic proliferation and promoted OC mRNA expression in 3D-collagen gel. This up-regulation of OC mRNA expression seems to be caused by a combined effect with a promotion of cell differentiation by 3D culture and S þ V stimulation. 3D cultures using a collagen gel promotes differentiation (Kinoshita et al., 1999) and mineralization (Rattner et al., 2000) of osteoblasts. MC3T3-E1 cells, an osteoblastic cell line derived from mouse calvaria cells (Kodama et al., 1981), also are capable of differentiating and initiating calcification in a collagen gel (Casser-Bette et al., 1990). The expression of OC is the terminal marker for differentiation in osteoblasts, and is associated with bone formation. Therefore, we interpret our results to mean that 3D-collagen culture promotes the onset of OC mRNA expression, and its expression is enhanced by S þ V stimulation. Our observations are consistent with previous findings on cell proliferation and differentiation of osteoblasts in 3D-collagen gels. Akhouayri (Akhouayri et al., 1999) reported reduced proliferation of osteoblasts in a floating gel exposed to shaking at 25 or 50 rpm after 8 days in culture. They also observed an elevated OC expression in the shaking gel on days 8–14. In 2D culture, a number of studies have reported enhanced osteoblastic proliferation (Kaspar et al., 2000; Meyer et al., 1997; Neidlinger-Wilke et al., 1994; Stanford et al., 1995) and collagen type I synthesis (Kaspar et al., 2000) in response to mechanical strain. However, mechanical strain in 2D culture was reported to suppress osteoblastic differentiation assessed by ALP activity or expression of OC (Kaspar et al., 2000; Stanford et al., 1995). These findings suggest that the responses of osteoblasts to mechanical stimuli are dependent upon culture environments. Cx43 is the major gap junction protein in osteoblasts. Our results showed no alteration of Cx43 mRNA expression after mechanical stimulation. However, there is a possibility that post-transcriptional processing of this gene is affected by mechanical stimuli. Ziambaras (Ziambaras et al., 1998) reported that mechanical stretch increased Cx43 protein synthesis without changing its mRNA expression in cultured osteoblasts. Therefore, investigation of post-transcriptional
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regulation of the genes we analyzed in this study is necessary to obtain further understanding about effects of broad frequency vibration on osteoblasts. The up-regulation of MMP-9 mRNA by vibratory stimulation in our study suggests a potential role of broad frequency vibration in remodeling of extracellular matrix. MMPs constitute a family of zincbinding proteolytic enzymes classified into 4 groups; collagenase, gelatinase, stomelysin, and a membrane type. MMP-9, gelatinase B, degrades components of extracellular matrix with a high specificity for denatured collagens (gelatin). In this study, mRNA levels of MMP-1 (collagenase 1), MMP-3 (stomelysin 1) and MMP-13 (collagenase 3) were not altered by the mechanical stimuli employed. Although MMP-9- null mice develop to term and survive normally after birth, a delay in vascularization and ossification of growth plates is reported (Engsig et al., 2000; Vu et al., 1998). Other studies have shown that the expression of MMP-9 is elevated in osteoblasts during osteogenesis (Filanti et al., 2000; Onyia et al., 1999). Further investigation is needed to understand the role of MMP-9 and other MMPs in response to broad frequency vibration on osteoblasts. In this study, we focused on gene expression that occurred 24 or more hours after a mechanical stimulus. The study did not address transient gene expression that might have occurred within minutes or a few hours after a mechanical stimulus. Further studies are needed to assess the effects of broad frequency vibration on early and intermediate response genes. Using a newly developed mechanical stimulator, we demonstrated that low amplitude, broad frequency vibration stimulated MMP-9 gene expression in MC3T3-E1 cells cultured in a collagen gel, and enhanced the expression of OC mRNA when mixed with high amplitude, low frequency sinusoidal strain. The sinusoidal strain alone had no effect on cell response. These results suggest that osteoblasts are sensitive to low amplitude, broad frequency strain vibration, and this strain vibration could sensitize them to high amplitude, low frequency strain. This suggestion implies a potential role for stochastic resonance in mechanical sensitivity of osteoblasts and modulating mechanical adaptation responses in bone.
Acknowledgements The technical assistance for RT-PCR by John W. Hawes and Hui Bin Sun is gratefully acknowledged. This study was supported by a grant from the Japan Science and Technology Corporation (JST Oversea Research Fellowship).
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References Akhouayri, O., Lafage-Proust, M.H., Rattner, A., Laroche, N., Caillot-Augusseau, A., Alexandre, C., Vico, L., 1999. Effects of static or dynamic mechanical stresses on osteoblast phenotype expression in three-dimensional contractile collagen gels. Journal of Cellular Biochemistry 76, 217–230. Casser-Bette, M., Murray, A.B., Closs, E.I., Erfle, V., Schmidt, J., 1990. Bone formation by osteoblast-like cells in a three-dimensional cell culture. Calcified Tissue International 46, 46–56. Collins, J.J., Imhoff, T.T., Grigg, P., 1996a. Noise-enhanced information transmission in rat sal cutaneous mechanoreceptors via aperiodic stochastic resonance. Journal of Neurophysiology 76, 642–645. Collins, J.J., Imhoff, T.T., Grigg, P., 1996b. Noise-enhanced tactile sensation. Nature 383, 770. Cordo, P., Inglis, J.T., Verschueren, S., Collins, J.J., Merfeld, D.M., Rosenblum, S., Buckley, S., Moss, F., 1996. Noise in human muscle spindles. Nature 383, 769–770. Engsig, M.T., Chen, Q.J., Vu, T.H., Pedersen, A.C., Therkidsen, B., Lund, L.R., Henriksen, K., Lenhard, T., Foged, N.T., Werb, Z., Delaisse, J.M., 2000. Matrix metalloproteinase 9 and vascular endothelial growth factor are essential for osteoclast recruitment into developing long bones. The Journal of Cell Biology 151, 879–890. Filanti, C., Dickson, G.R., Di Martino, D., Ulivi, V., Sanguineti, C., Romano, P., Palermo, C., Marduca, P., 2000. The expression of metalloproteinase –2, –9, and –14 and of tissue inhibitors –1 and –2 is developmentally modulated during osteogenesis in vitro, the mature osteoblastic phenotype expressing metalloproteinase –14. Journal of Bone and Mineral Research 15, 2154–2168. Flieger, J., Karachalios, T., Khaldi, L., Raptou, P., Lyritis, G., 1998. Mechanical stimulation in the form of vibration prevents postmenopausal bone loss in ovariectomized rats. Calcified Tissue International 63, 510–514. Forwood, M.R., Owan, I., Takano, Y., Turner, C.H., 1996. Increased bone formation in rat tibiae after a single short period of dynamic loading in vivo. American Journal of Physiology 270, E419–423. Fritton, S.P., McLeod, K.J., Rubin, C.T., 2000. Quantifying the strain history of bone: spatial uniformity and self-similarity of lowmagnitude strains. Journal of Biomechanics 33, 317–325. Hsieh, Y.F., Turner, C.H., 2001. Effects of loading frequency on mechanically induced bone formation. Journal of Bone and Mineral Research 16, 918–924. Hsieh, Y.F., Robling, A.G., Ambrosius, W.T., Burr, D.B., Turner, C.H., 2001. Mechanical loading of diaphyseal bone in vivo: the strain threshold for an osteogenic response varies with location. Journal of Bone and Mineral Research 16, 2291–2297. Kaspar, D., Seidl, W., Neidlinger-Wilke, C., Ignatius, A., Claes, L., 2000. Dynamic cell stretching increases human osteoblast proliferation and cicp synthesis but decreases osteocalcin synthesis and alkaline phosphatase activity. Journal of Biomechanics 33, 45–51. Kinoshita, S., Finnegan, M., Bucholz, R.W., Mizuno, K., 1999. Threedimensional collagen gel culture promotes osteoblastic phenotype in bone marrow derived cells. Kobe Journal of Medical Sciences 45, 201–211. Kodama, H., Amagai, Y., Sudo, H., Kasai, S., Yamamoto, S., 1981. Establishment of a clonal osteogenic cell line from newborn mouse calvaria. Japanese Journal of Oral Biology 23, 899–901. Kolodney, M.S., Wysolmerski, R.B., 1992. Isometric contraction by fibroblasts and endothelial cells in tissue culture: a quantitative study. The Journal of Cell Biology 117, 73–82. Meyer, U., Meyer, T., Jones, D.B., 1997. No mechanical role for vinculin in strain transduction in primary bovine osteoblasts. Biochemistry and Cell Biology 75, 81–87. Neidlinger-Wilke, C., Wilke, H.J., Claes, L., 1994. Cyclic stretching of human osteoblasts affects proliferation and metabolism: a new
80
S.M. Tanaka et al. / Journal of Biomechanics 36 (2003) 73–80
experimental method and its application. Journal of Orthopaedic Research 12, 70–78. Onyia, J.E., Hale, L.V., Miles, R.R., Cain, R.L., Tu, Y., Hwman, J.F., Hock, J.M., Santerre, R.F., 1999. Molecular characterization of gene expression charges in ros 17/2.8 cells cultured in diffusion chambers in vivo. Calcified Tissue International 65, 133–138. Rattner, A., Sabido, O., Le, J., Vico, L., Massoubre, C., Frey, J., Chamson, A., 2000. Mineralization and alkaline phosphatase activity in collagen lattices populated by human osteoblasts. Calcified Tissue International 66, 35–42. Robling, A.G., Burr, D.B., Turner, C.H., 2000. Partitioning a daily mechanical stimulus into discrete loading bouts improves the osteogenic response to loading. Journal of Bone and Mineral Research 15, 1596–1602. Robling, A.G., Hinant, F.M., Burr, D.B., Turner, C.H., 2002. Shorter, more frequent mechanical loading sessions enhance bone mass. Medicine and Science in Sports and Exercise 34, 196–202. Rubin, C.T., McLeod, K.J., 1994. Promotion of bony ingrowth by frequency-specific, low-amplitude mechanical strain. Clinical Orthopaedics and Related Research 298, 165–174. Rubin, C.T., McLeod, K.J., Bain, S.D., 1990. Functional strains and cortical bone adaptation: epigenetic assurance of skeletal integrity. Journal of Biomechanics 23, 43–54.
Stanford, C.M., Morcuende, J.A., Brand, R.A., 1995. Proliferative and phenotypic responses of bone-like cells to mechanical deformation. Journal of Orthopaedic Research 13, 664–670. Tanaka, S.M., 1999. A new mechanical stimulator for cultured bone cells using piezoelectric actuator. Journal of Biomechanics 32, 427–430. Tjandrawinata, R.R., Vincent, V.L., Hughes-Fulford, M., 1997. Vibrational force alters mRNA expression in osteoblasts. The FASEB Journal 11, 493–497. Turner, C.H., Yoshikawa, T., Forwood, M.R., Sun, T.C., Burr, D.B., 1995. High frequency components of bone strain in dogs measured during various activities. Journal of Biomechanics 28, 39–44. Vu, T.H., Shipley, J.M., Bergers, G., Berger, J.E., Helms, J.A., Hanahan, D., Shapiro, S.D., Senior, R.M., Werb, Z., 1998. Mmp-9/gelatinase b is a key regulator of growth plate angiogenesis and apoptosis of hypertrophic chondrocytes. Cell 93, 411–422. Ziambaras, K., Lecanda, F., Steinberg, T.H., Civitelli, R., 1998. C yclic stretch enhances gap junctional communication between osteoblastic cells. Journal of Bone and Mineral Research 13, 218–228.