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Mechanosensitivity of bone cells to oscillating fluid flow induced shear stress may be modulated by chemotransport
ARTICLE IN PRESS
Journal of Biomechanics 36 (2003) 1363–1371
Mechanosensitivity of bone cells to oscillating fluid flow induced shear stress may be modulated by chemotransport T.L. Haut Donahuea,b,*, T.R. Hauta, C.E. Yellowleya, H.J. Donahuea, C.R. Jacobsa,c a
Musculoskeletal Research Laboratory, Department of Orthopaedics and Rehabilitation, Pennsylvania State University, College of Medicine, Hershey, PA 17033, USA b Department of Mechanical Engineering—Engineering Mechanics, Michigan Technological University, 1400 Townsend Dr. Houghton, MI 49931, USA c Department of Mechanical Engineering, Palo Alto Veterans Affairs Medical Center, Stanford University, USA Accepted 12 March 2003
Abstract Fluid flow has been shown to be a potent physical stimulus in the regulation of bone cell metabolism. In addition to membrane shear stress, loading-induced fluid flow will enhance chemotransport due to convection or mass transport thereby affecting the biochemical environment surrounding the cell. This study investigated the role of oscillating fluid flow induced shear stress and chemotransport in cellular mechanotransduction mechanisms in bone. Intracellular calcium mobilization and prostaglandin E2 (PGE2) production were studied with varying levels of shear stress and chemotransport. In this study MC3T3-E1 cells responded to oscillating fluid flow with both an increase in intracellular calcium concentration ([Ca2+]i) and an increase in PGE2 production. These fluid flow induced responses were modulated by chemotransport. The percentage of cells responding with an [Ca2+]i oscillation increased with increasing flow rate, as did the production of PGE2. In addition, depriving the cells of nutrients during fluid flow resulted in an inhibition of both [Ca2+]i mobilization and PGE2 production. These data suggest that depriving the cells of a yet to be determined biochemical factor in media affects the responsiveness of bone cells even at a constant peak shear stress. Chemotransport alone will not elicit a response, but it appears that sufficient nutrient supply or waste removal is needed for the response to oscillating fluid flow induced shear stress. r 2003 Elsevier Science Ltd. All rights reserved. Keywords: Fluid flow; Bone; Mechanotransduction; Shear stress; Calcium
1. Introduction Bone cells are ideally situated to detect mechanical signals from the environment and transduce them into an appropriate biological response. The mechanism by which bone cells both sense and respond to changes in this mechanical environment is referred to as mechanotransduction. Several studies of mechanotransduction mechanisms (Hung et al., 1995; Jacobs et al., 1998; Turner and Pavalko, 1998) have identified a number of signals that bone cells respond to, including streaming potentials (Hung et al., 1996; Reich et al., 1990), chemotransport (Allen et al., 2000) and fluid flow (Allen *Corresponding author. Department of Mechanical Engineering, Michigan Technological University, 1400 Townsend Dr., Houghton, MI 49931, USA. Tel.: +1-906-487-2078; fax: +1-906-487-2822. E-mail address: [email protected] (T.L.H. Donahue).
et al., 2000; Frangos et al., 1985; Jacobs et al., 1998; McAllister and Frangos, 1999; Reich et al., 1990; You et al., 2000). Furthermore, previous studies suggest that mechanical loading-induced fluid flow is a more potent regulator of bone metabolism at typical loading magnitudes than mechanical strain (Smalt et al., 1997), substrate deformation (You et al., 2000), or streaming potentials (Reich et al., 1990). It is possible that bone cells directly sense loadinginduced flow of fluid through the lacunar–canalicular network (Burger and Klein-Nulend, 1999; Cowin et al., 1995; Kufahl and Saha, 1990). Weinbaum et al. (1994) predicted the membrane shear stress levels experienced by bone cells in vivo on the basis of theoretical models validated with respect to experimentally measured streaming potentials (Cowin et al., 1995; Weinbaum et al., 1994). They predicted magnitudes of fluid shear stress between 0.8 and 3 Pa on the cell membrane. Fluid
0021-9290/03/$ - see front matter r 2003 Elsevier Science Ltd. All rights reserved. doi:10.1016/S0021-9290(03)00118-0
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flow induced shear stress has been shown to stimulate the production of numerous intracellular and extracellular signaling molecules including intracellular calcium (Allen et al., 2000; Hung et al., 1995; Jacobs et al., 1998), cAMP (Reich et al., 1990), prostaglandin E2 (PGE2) (Ajubi et al., 1999; Klein-Nulend et al., 1997; Smalt et al., 1997; Sterck et al., 1998), and nitric oxide (McAllister and Frangos, 1999). These studies, with the exception of Jacobs et al. (1998), employed parallel plate flow chambers with either steady or pulsatile fluid flow. Jacobs et al. (1998) examined the [Ca2+]i response of bone cells subjected to both steady and oscillating fluid flow (OFF). When bone is loaded in vivo, the lacunar– canalicular network experiences a pressurization in response to the matrix deformation and this leads to flow along pressure gradients. When the loading is removed, the flows and pressure gradients are reversed and thus, the fluid motions are oscillatory in nature (Jacobs et al., 1998). Since the oscillatory component of the bone cell’s fluid flow environment has the potential to greatly exceed the steady component of flow driven by the arterial pressure head, Jacobs et al. (1998) suggested that OFF may be most representative of physiological loading. In addition to shear stress, loading-induced fluid flow will also induce chemotransport due to convection or mass transport thereby affecting the biochemical environment surrounding the cell. It has been demonstrated that the response of bone cells to fluid flow is modulated by the cell’s biochemical surroundings. For example, Allen et al. (2000) studied the effect of the addition of newborn bovine serum to the flow media on the [Ca2+]i response of primary cultured bone cells stimulated by fluid flow (Allen et al., 2000). They found that shear flow in serum-supplemented medium produces enhanced intracellular calcium responses in individual cells compared to serum-free flow stimulation. Cells exposed to serum-supplemented medium during flow exhibited a larger peak magnitude and longer duration calcium responses than those exposed to serum-free flow (Allen et al., 2000). This study suggests that chemical agonist exposure can modulate the intracellular calcium response in bone cells subjected to fluid flow induced shear stress. The hypothesis of the present study is that bone cells, specifically mouse osteoblastic MC3T3-E1 cells, respond to fluid flow induced shear stress with a biochemical response, and this response is modulated by chemotransport. Both intracellular calcium mobilization and PGE2 production were examined in this study. Intracellular calcium is an early second messenger that plays an important role in many downstream events, and is typically observed to increase dramatically within seconds of fluid flow stimulation. Therefore, [Ca2+]i was quantified as a measure of cell responsiveness (Hung et al., 1995; Jacobs et al., 1998). PGE2 was
studied because it has been implicated as an important mediator in the regulation of bone turnover (Imamura et al., 1990; Jee et al., 1985). Prostaglandins are produced by osteoblastic cells and are abundant in bone (Feyen et al., 1984; Nolan et al., 1983; Rodan et al., 1986). Prostaglandins have been shown to regulate bone metabolism by both stimulation and inhibition of bone formation (Jee et al., 1991; Raisz and Fall, 1990) and can also play a role in bone resorption (Dietrich et al., 1975; Fuller and Chambers, 1989). The goals of this study were to determine whether the effect of OFF can be ascribed to altered shear stress and/ or chemotransport in terms of its affect on (1) [Ca2+]i, and (2) production of PGE2. These results may lead to a better understanding of cellular mechanosensitivity in bone cells, and therefore, a better understanding of diseases involving altered mechanotransduction, such as osteoporosis.
2. Methods 2.1. Cell culture The immortalized mouse osteoblastic cell line MC3T3-E1 (mouse calveria) was employed in this study. MC3T3-E1 cells were cultured in minimum essential medium (MEM-a) (GIBCO BRL, Grand Island, NY) supplemented with 10% fetal bovine serum (FBS) (Hyclone, Logan, UT) and 1% penicillin/streptomycin (P/S) (GIBCO BRL, Grand Island, NY). Cells were maintained in an incubator at 37 C, 100% humidity, and 5% CO2. For intracellular calcium mobilization experiments, cells were plated on quartz slides (76 mm 26 mm 1.6 mm) at 0.65 105 cells/ slide and cultured for 48 h to 80% confluence. For PGE2 studies, cells were plated on glass slides (75 mm 38 mm 1 mm) at 3.5 105 cells/slide and cultured for 48 h to 85% confluence. 2.2. Oscillating fluid flow Cells were exposed to fluid flow as previously described (Jacobs et al., 1998; Kurokouchi et al., 2001; You et al., 2001; You et al., 2000). Briefly, two custom designed parallel plate flow chambers of similar construction, but varying capacity, were used to expose cells to fluid flow. [Ca2+]i imaging experiments utilized a vacuum-sealed flow chamber with a flow channel of 38 10 0.28 mm, whereas longer duration PGE2 studies were conducted with a larger chamber with a flow channel of 60 24 0.28 mm. The latter chamber utilizes a mechanical clamp to achieve a tight seal and can be assembled in a sterile environment and kept in a sterile incubator throughout the flow exposure period. Flow was delivered with Hamilton glass syringes
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custom mounted into a servo pneumatic loading frame (EnduraTec, Eden, Prairie, MN). To verify the output of the syringes an ultrasonic flow meter (Model 106, Transonic Systems, Ithaca, NY) was attached to the chamber inlet. This system generated sinusoidal OFF at 1 Hz. Various flow media were employed depending on the experiment as described below. Shear stress was kept constant by adding neutral dextran to the flow medium and decreasing the flow rate. To address the effect of nutrients on the cell’s response to OFF, Hanks’ buffered salt solution (HBSS) was also used as flow media during some experiments to create a flow media devoid of all nutrients and serum. 2.3. Viscosity measurements A capillary tube viscometer was used to quantify changes in viscosity with the addition of neutral dextran to the flow media. The accuracy of the viscometer was verified with ultrapure water. Both standard flow media containing 2% FBS and HBSS were tested and found to have a viscosity of 1.0 cP (1centiPoise (cP)=0.01 dyn s/ cm2). Neutral dextran (MW 500,000) (Sigma, St. Louis, MO) was added to the standard flow media in various quantities to modulate the viscosity; 0.5, 1, 1.5 or 2 g of dextran per 100 ml of media. These concentrations resulted in viscosity measurements of 1.2, 1.5, 2.0 and 2.4 cP, respectively. 2.4. Calcium imaging Pre-confluent cells were washed with MEM and 2% FBS solution at 37 C. Cells were then incubated with the dual excitation Ca2+-probe Fura-2 (acetoxymethyl (AM) ester 1 mM, Molecular Probes, Eugene, OR) for 30 min at 37 C. The cells were then washed with fresh media and 2% FBS solution and the slide was mounted in the parallel plate flow chamber and placed on an inverted fluorescence microscope (Nikon Diaphot 300) and left undisturbed for 30 min. The cells were illuminated as described previously (Jacobs et al., 1998; Kurokouchi et al., 2001; Yellowley et al., 1997, 1999; You et al., 2000, 2001). A Metafluor imaging system (Universal Imaging, West Chester, PA) was used to sample and record the emitted light from the cells in the field of view once every 2 s (emission wavelength 510 nm) and Metafluor imaging software was used to subtract the background fluorescence from each image and to outline and calculate the 340:380 ratio of light emitted in response to excitation at 340 and 380 nm for each cell in the field of view, as this ratio reflects [Ca2+]i. A calibration curve was constructed by acquiring 340:380 values (background subtracted) for a series of solutions of known free Ca2+ concentration (0– 39.8 mM, Molecular Probes) and 1 mM fura2 pentapotassium salt (Molecular Probes). This calibration curve
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Table 1 Intracellular calcium flow regimes Number of slides Standard media with a flow rate of 4.5 ml/min and a peak shear stress of 5 dyn/cm2 (0.5 Pa) 1 g of dextran per 100 ml of media with a flow rate of 4.5 ml/min and a peak shear stress of 8.7 dyn/cm2 (0.87 Pa) Standard media with a flow rate of 18 ml/min and a peak shear stress of 20 dyn/cm2 (2 Pa) 1 g of dextran per 100 ml of media with a flow rate of 11.5 ml/min and a peak shear stress of 20 dyn/cm2 (2 Pa) Hanks’ balance salt solution with a flow rate of 18 ml/min and a peak shear stress of 20 dyn/cm2 (2 Pa) Hanks’ balance salt solution with a flow rate of 36 ml/min and a peak shear stress of 40 dyn/cm2 (4 Pa)
3 4
6 7
4
4
was used to convert ratio values from individual cells into [Ca2+]i. To identify Ca2+ transients we used a numerical procedure adapted from mechanical fatigue analysis, known as Rainflow Cycle Counting (Downing and Socie, 1982). This simple algorithm reliably and automatically identifies and determines the amplitudes of spikes and transients in time history data even when superimposed over each other or in the presence of background noise (Jacobs et al., 2000). We have previously used this algorithm to identify transients in [Ca2+]i in chondrocytes and bone cells (Jacobs et al., 1998; Kurokouchi et al., 2001; Yellowley et al., 1997, 1999; You et al., 2000, 2001). We defined a response as a transient increase in [Ca2+]i of 50 nM or greater. Data were collected for 1 min at the start of each experiment prior to flow, and then for a period of 3 min during flow. Numerous flow regimes were applied while measuring [Ca2+]i (Table 1). Constant flow rate experiments were conducted at a lower shear stresses (5 and 8.7 dyn/cm2) compared to constant shear stress experiments, which were conducted at 20 dyn/cm2. This was necessary due to the fact that at 20 dyn/cm2 approximately 100% of the cells were responding, and so increasing the shear stress from 20 dyn/cm2 could not alter the percent of cells responding. 2.5. PGE2 experiments All experiments were performed with pre-confluent (85%) MC3T3-E1 cells. The cells were exposed to OFF for 1 h and control cells were placed in flow chambers, but were not exposed to fluid flow. Various flow regimes were employed (Table 2). Following the hour of flow, the slides were rinsed with phosphate buffered saline and placed in 10 ml of fresh standard flow media, incubated for an additional hour, and the media collected and
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Table 2 PGE2 fluid flow regimes
0.3
Standard media without dextran with flow rate of 43 ml/min and a peak shear stress of 20 dyn/cm2 (2 Pa) 1 g of dextran per 100 ml of media with flow rate of 28 ml/min and a peak shear stress of 20 dyn/cm2 (2 Pa) 2 g of dextran per 100 ml of media with flow rate of 18 ml/min and a peak shear stress of 20 dyn/cm2 (2 Pa) HBSS with flow rate of 43 ml/min and a peak shear stress of 20 dyn/cm2 (2 Pa)
Number of control slides
4
4
4
4
[Ca+2]i (µM)
0.25
Number of flow slides
0.2 0.15 0.1 0.05
4
4
0 0
4
50
100
150
200
250
Time (s)
4
Fig. 1. An example of [Ca2+]i response traces obtained for OFF in which 100% of individual MC3T3-E1 cells responded to flow. Note the arrow depicts the onset of flow.
2.6. Statistics For intracellular calcium mobilization experiments, the variability in the fraction of responding cells was characterized with the standard error of proportion (SEP) and statistical comparisons were made on the basis of the z-test statistic for comparison of proportions (Glantz, 1992; Jacobs et al., 1998). All data obtained from PGE2 experiments were expressed as mean7standard error (SEM). ANOVAs with Bonferroni/Dunn post hoc comparisons were completed using a commercially available software program (StatView, SAS Institute, San Francisco, CA).
3. Results 3.1. Calcium imaging MC3T3-E1 cells typically displayed a few small amplitude, intermittent Ca2+ response spikes during i the 1-min no flow period due to random system noise or an occasional spontaneous Ca2+ transient followed by a i more pronounced and coordinated response spike at the onset of flow (Fig. 1).
% of cells responding
100
stored at 80 C for PGE2 analysis while the cells were isolated and assayed for total protein. PGE2 accumulation was quantified using a commercially available, nonradioactive, competitive binding enzymeimmunoassay system (BioTrak, Amersham Pharmaceuticals, Piscataway, NJ), conducted at room temperature. Frozen media samples were thawed at room temperature and vortexed prior to use. The optical densities of the samples were read at 450 nm using a microplate reader (Dynex Technologies, Chantilly, VA). PGE2 accumulation was normalized to total cell protein determined by the Lowry method (Bio-Rad) for each slide.
80 60
*
40 20 0 4.5 ml/min 5 dynes/cm2 no dextran
4.5 ml/min 8.7 dynes/cm2 1 gram dextran
Fig. 2. The percentage of cells responding with a calcium transient for experiments using flow regimes at constant chemotransport (flow rate=4.5 ml/min) and varied peak shear stress. The number of cells for the 5 dyn/cm2, no dextran, 4.5 ml/min flow regime was n ¼ 111 (from 3 slides) and the number of cells for the 8.7 dyn/cm2, 1 g dextran, 4.5 ml/ min flow regime was n ¼ 190 (from 4 slides). The bars represent standard error of a proportion. *Significantly different from 5 dyn/ cm2, no dextran, 4.5 ml/min (po0:01).
The percentage of cells responding with an increase in [Ca2+]i was significantly dependent (po0:01) on the peak flow induced shear stress. At a constant flow rate of 4.5 ml/min, peak shear stresses of 5 (n ¼ 111) and 8.7 dyn/cm2 (n ¼ 190) resulted in 13.573.2% and 32.673.4% (7SEP) of the cells responding, respectively (Fig. 2). The mean amplitude of the cells responding was not significantly dependent on the peak shear stress. The amplitude was 38718 and 7277 nM, at 5 and 8.7 dyn/ cm2 (7SEM), respectively. The addition of neutral dextran did not change the percentage of cells responding in the no flow baseline period (data not shown). The percentage of cells responding with an increase in [Ca2+]i was also significantly dependent (po0:01) on the flow rate (chemotransport). At a peak shear stress of 20 dyn/cm2 and flow rate of 18 ml/min, 87.671.8% (7SEP) of individual cells responded to flow (n ¼ 347). This response was significantly reduced to 75.272.3%
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*
80 60 40 20 0
18 ml/min 20 dynes/cm2 no dextran
11.5 ml/min 20 dynes/cm2 1 gram dextran
Fig. 3. The percentage of cells responding with a calcium transient for experiments using flow regimes at a constant peak shear stress (20 dyn/ cm2) and varied chemotransport (flow rate). The number of cells for each of the regimes was n ¼ 347 (from 6 slides in standard media and 7 slides in media with dextran). The bars represent standard error of a proportion. *Significantly different from 11.5 ml/min, 1 g dextran, 20 dyn/cm2 flow regime (po0:01).
% of cells responding
100 80 60 40 20
*
*
0 18 ml/min 20 dynes/cm2 Standard Media
18 ml/min 20 dynes/cm2 HBSS
36 ml/min 40 dynes/cm2 HBSS
Fig. 4. The percentage of cells responding with a calcium transient for experiments using flow regimes with HBSS as the perfusing flow media. The number of cells for the 20 dyn/cm2, 18 ml/min, standard media flow regime was n ¼ 347 (from 6 slides). The number of cells for the 20 dyn/cm2, 18 ml/min, HBSS flow regime was n ¼ 214 (from 4 slides) and the number of cells for the 40 dyn/cm2, 36 ml/min, HBSS flow regime was n ¼ 189 (from 4 slides). The bars represent standard error of a proportion. *Significantly different from standard media, 20 dyn/cm2, 18 ml/min (po0:01).
of the cells responding (n ¼ 347) when the flow rate was reduced to 11.5 ml/min while maintaining a constant peak shear stress of 20 dyn/cm2 (Fig. 3). The average amplitude of the cells responding was not significantly affected by flow rate. The mean amplitude of the cells responding was 130728 and 82710 nM (7SEM) at 18 and 11.5 ml/min, respectively. To assess the role of nutrients in the [Ca2+]i response to OFF, experiments were conducted at a peak shear stress of 20 dyn/cm2 and a flow rate of 18 ml/min in standard flow media and nutrient free flow media. In standard media 87.671.8% (7SEP) of the cells responded. This response was significantly attenuated when HBSS (nutrient-free) was the flow media, with only 10.772.1% of the cells responding (n ¼ 214;
po0:01) (Fig. 4). Doubling the peak shear stress and flow rate to 40 dyn/cm2 and 36 ml/min, respectively, did not recover the attenuation seen with HBSS. Only 8.572.0% of individual cells responded to flow with an increase in [Ca2+]i (n ¼ 189) (Fig. 4). The mean amplitude of the cells responding was significantly attenuated for both conditions using HBSS; at 20 dyn/ cm2/18 ml/min and 40 dyn/cm2/36 ml/min the mean amplitudes were 7474 and 7078 nM, respectively (po0:01) compared to 130728 nM (7SEM) at 20 dyn/cm2/18 ml/min in standard media. 3.2. PGE2 production PGE2 production was significantly (po0:0001) increased with exposure to OFF. Total PGE2 production in cells exposed to a peak shear stress of 20 dyn/cm2 at a flow rate of 43 ml/min was 3473.1 pg/mg of total protein (7SEM) (n ¼ 4). This was significantly greater compared to PGE2 production in no flow, control cells, 14.870.85 pg/mg of total protein (n ¼ 4Þ (Fig. 5). This PGE2 production was modulated by flow rate (chemotransport). Reducing the flow rate from 43 ml/ min to either 28 ml/min or 18 ml/min and maintaining the peak shear stress at 20 dyn/cm2, reduced the total PGE2 production for the flow experiments to 25.876.8 and 9.171.8 pg/mg of total protein (n ¼ 4), respectively (Fig. 5), from 3473.1 pg/mg of total protein. At 18 ml/ min, the PGE2 production was not significantly different from no flow controls. The total PGE2 production was not affected by dextran concentration. For the no flow controls, the total PGE2 was not significantly different with various concentrations of dextran (Fig. 5). PGE2 production was also dependent on the level of nutrients in the flow media. At a peak shear stress of 20 dyn/cm2 and flow rate of 43 ml/min total PGE2 production was decreased from 18.374.3 pg/mg of total protein (7SEM) (n ¼ 4) in standard media, to 9.572.7 pg/mg of total protein (n ¼ 4) when the flow
Total PGE2 production (pg/µg of total protein)
% of cells responding
100
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40
^
flow no flow
30 20 **
10 0
43 ml/min 20 dynes/cm2
28 ml/min 20 dynes/cm2
18 ml/min 20 dynes/cm2
Fig. 5. PGE2 quantification in experiments at a constant peak shear stress (20 dyn/cm2) and varied chemotransport (flow rate). The numbers are representative of total PGE2 accumulation in the media normalized to total protein. All results are shown plotted as mean7SEM with each group containing four experiments. ^Significantly different from no flow control within group; **significantly different from flow at 43 ml/min and 28 ml/min (po0:0001).
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Total PGE2 production (pg/µg of total protein)
25 flow no flow
20 15 10 5 0 Standard Media 43 ml/min 20 dynes/cm2
HBSS 43 ml/min 20 dynes/cm2
Fig. 6. PGE2 quantification in experiments at a peak shear stress of 20 dyn/cm2 and flow rate of 43 ml/min, with varied flow media. The numbers are representative of total PGE2 accumulation in the media normalized to total protein. All results are shown plotted as mean7SEM with each group containing four experiments.
media was nutrient-free HBSS (Fig. 6). There was no difference in PGE2 production between the no flow, control cells, with HBSS.
4. Discussion This study investigated the role of OFF-induced shear stress and chemotransport in cellular mechanotransduction mechanisms in bone. These two were distinguished and independently studied in osteoblastic MC3T3-E1 cells. Studying these stimuli independently was accomplished via altering flow rate and/or flow media viscosity. To address the role of fluid flow induced shear stress and chemotransport, both intracellular calcium mobilization and PGE2 production were studied. The data from this study suggest that OFF induced intracellular calcium responses and PGE2 production are modulated by chemotransport. While bathed in standard media and exposed to a constant shear stress, the cells responded with both a robust intracellular calcium response and increase in PGE2 production. Both of these responses were decreased with decreasing flow rate while maintaining constant shear stress. In contrast, using HBSS as the flow media was not accompanied by significant changes in [Ca2+]i or PGE2 production with the onset of OFF. While many investigators have studied the effects of fluid flow induced shear stress on bone cells (Ajubi et al., 1996; Allen et al., 2000; Bakker et al., 2001; Hung et al., 1995; Klein-Nulend et al., 1995; Reich et al., 1990), few have studied oscillating fluid flow (Jacobs et al., 1998; You et al., 2001). It is important to make comparisons between these different types of flow; however, previous studies indicate that bone cells will respond differently to oscillating fluid flow induced shear stress versus
steady or pulsatile fluid flow induced shear stress (Jacobs et al., 1998; You et al., 2001). Allen and co-workers suggests only primary, not MC3T3-E1 cells respond with an increase in [Ca2+]i at 3.5 Pa of steady flow with serum-free HBSS flow media (Allen et al., 1996). However, at physiological levels of fluid flow (0.4–3.4 Pa), primary cells did not respond in the absence of serum (Allen et al., 2000). The authors suggest that the lack of response with MC3T3-E1 cells without serum may be an artifact of the cell line (Allen et al., 2000). Data from the current study agrees with the studies by Allen and co-workers showing no response of MC3T3-E1 cells in serum free flow media at 2 and 4 Pa. Jacobs et al. (1998) showed that steady flow is more stimulatory than oscillating flow (Jacobs et al., 1998). Hence, when comparing the current data with Allen and co-workers, it may be that in the current study oscillating fluid flow levels were not high enough to reach a threshold beyond which MC3T3-E1 cells will respond in the absence of serum. Perhaps the threshold in which osteoblastic MC3T3-E1 cells will show a response was not reached and is higher than the threshold for primary bone cells that may contain both osteoblasts and osteocytes. The current study did not go above physiological levels of fluid flow induced shear stress, which have been theorized to be 0.8–3.8 Pa (Cowin et al., 1995; Weinbaum et al., 1994). The current data does show a marked increase in the calcium response of MC3T3-E1 cells with serum-supplemented media. Reich and Frangos studied the effects of increasing shear on PGE2 production (Reich and Frangos, 1991). They showed a dose-dependent increase in the rate of PGE2 production in osteoblasts exposed to low (6 dyn/ cm2) and high (24 dyn/cm2) steady shear. While the current study did not test the effects of altered shear on PGE2 production, we would expect to see the same results as Reich and Frangos since we showed a dosedependent increase in the percentage of cells responding (with increase in intracellular calcium) with an increase in shear stress. The current study adds to the work of Reich and Frangos and investigated if the fluid flow induced shear stress response is modulated by chemotransport (flow rate). Our studies suggest that as flow rate increased, PGE2 production increased. PGE2 production decreased when cells were deprived of serum. While not statistically significant, PGE2 production was less in no flow control cells bathed in HBSS versus serum-supplemented media (Fig. 6). Perhaps if we designed the experiments to test this hypothesis the results may be significant with more experiments. Our findings demonstrate that although PGE2 production and intracellular calcium mobilization in response to OFF exhibit similar trends when chemotransport is altered, the magnitudes of these trends are
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dissimilar. That is, when HBSS was used as the flow media the effect of fluid flow on intracellular calcium mobilization was much greater than upon PGE2 production. Interestingly, these data suggest that PGE2 production in response to OFF may be occurring without intracellular calcium signaling. In support of this view, Saunders et al. (2001) has also found that there may be separate pathways involved in calcium wave propagation and PGE2 production in osteoblastic MC3T3-E1 cells exposed to fluid flow. In those studies, OFF in the presence of thapsigargin, a drug which empties the intracellular stores of calcium, induced a diminished calcium response, relative to fluid flow without thapsigargin. However, thapsigargin did not affect the PGE2 production. Therefore, these findings support our data suggesting there may be independent pathways involved in intracellular calcium mobilization and PGE2 production. A recent study by Bakker et al. (2001) addressed the nature of flow-derived cell stimulus by comparing variations in fluid transport with variations in shear stress, using nitric oxide and PGE2 production as parameters for bone cell activation (Bakker et al., 2001). These studies found that the production of NO and PGE2 is enhanced in a dose-dependent manner by pulsating fluid flow of increasing shear stress and flow rate. When these authors used neutral dextran to increase wall shear stress maintaining a constant flow rate, they showed an increase in both NO and PGE2 production. These findings are consistent with Reich and Frangos’s PGE2 results (Reich and Frangos, 1991). Similar to the current study, Bakker et al. (2001) maintained a constant shear and increased flow rate. In contrast to the current data, they showed no difference in PGE2 production with increasing flow rate. Our data shows an increase in PGE2 production with an increase in flow rate. This difference could be due to difference in fluid flow regimes. Bakker et al. (2001) applied steady flow while the current study used oscillating flow (Bakker et al., 2001). The current data suggests that chemotransport modulates the OFF induced shear stress response of osteoblastic cells. Studies by Allen et al. (2000) found that chemical agonist exposure (i.e. serum, and ATP) can modulate the [Ca2+]i response in bone cells subjected to fluid flow (Allen et al., 2000). Therefore, it is reasonable to hypothesize that a biochemical factor in the media is responsible for this modulation. ATP has previously been shown to be required for flow-induced [Ca2+]i changes in other cell types (Nollert and McIntire, 1992; Shen et al., 1992). These authors report that the convective mass transport of ATP from the bulk media is responsible for the flow-induced [Ca2+]i since the ATP concentration at the cell surface rises in shear flow. Additionally, ATP is also known to chemically activate certain cell receptors (Schofl et al., 1992) and
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thus, the observed ATP response in the previous studies provide supporting evidence that chemotransport is a likely explanation for the serum response (Allen et al., 2000). One limitation when interpreting our results is that there is no experimental quantification of lacunar– canalicular flow actually occurring in vivo. We have based our flow regime on theoretical predictions that have been validated with respect to experimental measurements of flow-induced streaming potentials (Cowin et al., 1995). However, the fact remains that the geometry of the flow chamber is different from that of a canalicula or lacuna and the flow rates used may differ from that which occurs in vivo through the lacunar–canalicular network. The current study suggests that oscillating fluid flow at peak shear stresses up to 4 Pa is not a sufficient enough stimulus to invoke a calcium response in serum free flow media. The percentage of cells responding in HBSS was not significantly different during flow compared to the no flow baseline. However, there may be a stimulus that is more powerful than oscillating fluid flow and would result in a greater calcium response in serum free media. Our data does not rule out the possibility that without serum the cells basal metabolism has changed and the cells are no longer capable of a calcium response. Further studies will need to be conducted to determine if the diminished response in HBSS is due to a decrease in basal metabolism or a decrease in mechanosensitivity. The HBSS data must be interpreted carefully due to confounding issues. The lack of response of cells in HBSS may be due to changes in either chemotransport or basal metabolism. In fact, without serum cells may be unable to respond to flow due to changes in their basal metabolism. While the HBSS data is consistent with the conclusion that chemotransport modulated the shear stress response in MC3T3-E1 cells, the data is not sufficient to rule out changes in basal metabolism as the reason for changes in the calcium response. The HBSS studies were performed in order to compare our data to Allen et al. (2000), which showed primary bone cells respond in serum free media, only when the steady fluid flow induced shear stress levels were beyond a certain threshold (3.5 Pa) (Allen et al., 2000). Our data shows no response of MC3T3-E1 cells to oscillating fluid flow induced shear stress levels as high as 4 Pa. In our lab and others, MC3T3-E1 cells have been used as a model for the study of mechanosensitivity in bone cells (Chen et al., 2000; You et al., 2001). These cells have previously been shown to respond to fluid flow with increases in intracellular calcium (You et al., 2001), gene expression (You et al., 2001), and reorganization of the actin cytoskeleton leading to increased expression of COX2 and cFOS (Pavalko et al., 1998). Additionally, in contrast to primary cells, the use of a cell line such as
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MC3T3-E1 cells ensures a homogeneous cell population. Primary bone cell culture often contains osteoblasts, osteocytes and fibroblasts in varying proportions, and thus observed difference may not always be due to difference in bone cell sensitivity. In this study, MC3T3-E1 cells responded to OFF with both a robust increase of [Ca2+]i and an increase in PGE2 production. These fluid flow induced responses were modulated by chemotransport. The percentage of cells responding with an [Ca2+]i oscillation increased with increasing flow rate, as did the production of PGE2. In addition, depriving the cells of nutrients during fluid flow resulted in a decreased [Ca2+]i and less PGE2 production. These data suggest that depriving the cells of a yet to be determined biochemical factor in media affects the responsiveness of bone cells even at a constant peak shear stress. Chemotransport alone will not elicit a response as shear stress will, but it appears that sufficient nutrient supply or waste removal is needed for the response to shear stress induced by OFF.
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ARTICLE IN PRESS T.L.H. Donahue et al. / Journal of Biomechanics 36 (2003) 1363–1371 mechanical signaling in MC3T3-E1 osteoblasts requires cytoskeleton–integrin interactions. American Journal of Physiology 275 (6 Pt 1), C1591–C1601. Raisz, L.G., Fall, P.M., 1990. Biphasic effects of prostaglandin E2 on bone formation in cultured fetal rat calvariae: interaction with cortisol. Endocrinology 126, 1654–1659. Reich, K.M., Frangos, J.A., 1991. Effect of flow on prostaglandin E2 and inositol trisphosphate levels in osteoblasts. American Journal of Physiology—Cell Physiology 261, C428–C432. Reich, K.M., Gay, C.V., Frangos, J.A., 1990. Fluid shear stress as a mediator of osteoblast cyclic adenosine monophosphate production. Journal of Cellular Physiology 143, 100–104. Rodan, S.B., Wesolowski, G., Rodan, G.A., 1986. Clonal differences in prostaglandin synthesis among osteosarcoma cell lines. Journal of Bone and Mineral Research 1 (2), 213–220. Saunders, M.M., You, J., Trosko, J.E., Yamasaki, H., Li, Z., Donahue, H.J., Jacobs, C.R., 2001. Gap junctions and fluid flow response in MC3T3-E1 cells. American Journal of Physiology— Cell Physiology 281, C1917–C1925. Schofl, C., Cuthbertson, K.S., Walsh, C.A., Mayne, C., Cobbold, P., von zur Muhlen, A., Hesch, R.D., Gallagher, J.A., 1992. Evidence for P2-purinoceptors on human osteoblast-like cells. Journal of Bone and Mineral Research 7 (5), 485–491. Shen, J., Luscinskas, F.W., Connolly, A., Dewey Jr., C.F., Gimbrone Jr., M.A., 1992. Fluid shear stress modulates cytosolic free calcium in vascular endothelial cells. American Journal of Physiology 262 (2 Pt 1), C384–C390. Smalt, R., Mitchell, T., Howard, R.L., Chambers, T.J., 1997. Induction of NO and prostaglandin E2 in osteoblasts by wall-
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shear stress but not mechanical strain. American Journal of Physiology 273 (Endocrinol. Metab. 36), E751–E758. Sterck, J.G., Klein-Nulend, J., Lips, P., Burger, E.H., 1998. Response of normal and osteoporotic human bone cells to mechanical stress in vitro. American Journal of Physiology 274 (6 Pt 1), E1113–1120. Turner, C.H., Pavalko, F.M., 1998. Mechanotransduction and function response of the skeleton to physical stress: the mechanisms and mechanics of bone adaptation. Journal of Orthopaedic Science 3, 346–355. Weinbaum, S., Cowin, S.C., Zeng, Y.A., 1994. A model for the excitation of osteocytes by mechanical loading induced bone fluid shear stresses. Journal of Biomechanics 27, 339–360. Yellowley, C.E., Jacobs, C.R., Li, Z., Zhou, Z., Donahue, H.J., 1997. Effects of fluid flow on intracellular calcium in bovine articular chondrocytes. American Journal of Physiology—Cell Physiology 273 (1 Pt 1), C30–C36. Yellowley, C.E., Jacobs, C.R., Donahue, H.J., 1999. Mechanisms contributing to fluid-flow-induced Ca2+ mobilizaion in articular chondrocytes. Journal of Cellular Physiology 180, 402–408. You, J., Yellowley, C.E., Donahue, H.J., Zhang, Y., Chen, Q., Jacobs, C.R., 2000. Substrate deformation levels associated with routine physical activity are less stimulatory to bone cells relative to loading-induced oscillatory fluid flow. Journal of Biomechanical Engineering 122 (4), 387–393. You, J., Reilly, G.C., Zhen, X., Yellowley, C.E., Chen, Q., Donahue, H.J., Jacobs, C.R., 2001. Osteopontin gene regulation by oscillatory fluid flow via intracellular calcium mobilization and activation of mitogen-activated protein kinase in MC3T3-E1 osteoblasts. Journal of Biological Chemistry 276 (16), 13365–13371.
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Mechanosensitivity of bone cells to oscillating fluid flow induced shear stress may be modulated by chemotransport T.L. Haut Donahuea,b,*, T.R. Hauta, C.E. Yellowleya, H.J. Donahuea, C.R. Jacobsa,c a
Musculoskeletal Research Laboratory, Department of Orthopaedics and Rehabilitation, Pennsylvania State University, College of Medicine, Hershey, PA 17033, USA b Department of Mechanical Engineering—Engineering Mechanics, Michigan Technological University, 1400 Townsend Dr. Houghton, MI 49931, USA c Department of Mechanical Engineering, Palo Alto Veterans Affairs Medical Center, Stanford University, USA Accepted 12 March 2003
Abstract Fluid flow has been shown to be a potent physical stimulus in the regulation of bone cell metabolism. In addition to membrane shear stress, loading-induced fluid flow will enhance chemotransport due to convection or mass transport thereby affecting the biochemical environment surrounding the cell. This study investigated the role of oscillating fluid flow induced shear stress and chemotransport in cellular mechanotransduction mechanisms in bone. Intracellular calcium mobilization and prostaglandin E2 (PGE2) production were studied with varying levels of shear stress and chemotransport. In this study MC3T3-E1 cells responded to oscillating fluid flow with both an increase in intracellular calcium concentration ([Ca2+]i) and an increase in PGE2 production. These fluid flow induced responses were modulated by chemotransport. The percentage of cells responding with an [Ca2+]i oscillation increased with increasing flow rate, as did the production of PGE2. In addition, depriving the cells of nutrients during fluid flow resulted in an inhibition of both [Ca2+]i mobilization and PGE2 production. These data suggest that depriving the cells of a yet to be determined biochemical factor in media affects the responsiveness of bone cells even at a constant peak shear stress. Chemotransport alone will not elicit a response, but it appears that sufficient nutrient supply or waste removal is needed for the response to oscillating fluid flow induced shear stress. r 2003 Elsevier Science Ltd. All rights reserved. Keywords: Fluid flow; Bone; Mechanotransduction; Shear stress; Calcium
1. Introduction Bone cells are ideally situated to detect mechanical signals from the environment and transduce them into an appropriate biological response. The mechanism by which bone cells both sense and respond to changes in this mechanical environment is referred to as mechanotransduction. Several studies of mechanotransduction mechanisms (Hung et al., 1995; Jacobs et al., 1998; Turner and Pavalko, 1998) have identified a number of signals that bone cells respond to, including streaming potentials (Hung et al., 1996; Reich et al., 1990), chemotransport (Allen et al., 2000) and fluid flow (Allen *Corresponding author. Department of Mechanical Engineering, Michigan Technological University, 1400 Townsend Dr., Houghton, MI 49931, USA. Tel.: +1-906-487-2078; fax: +1-906-487-2822. E-mail address: [email protected] (T.L.H. Donahue).
et al., 2000; Frangos et al., 1985; Jacobs et al., 1998; McAllister and Frangos, 1999; Reich et al., 1990; You et al., 2000). Furthermore, previous studies suggest that mechanical loading-induced fluid flow is a more potent regulator of bone metabolism at typical loading magnitudes than mechanical strain (Smalt et al., 1997), substrate deformation (You et al., 2000), or streaming potentials (Reich et al., 1990). It is possible that bone cells directly sense loadinginduced flow of fluid through the lacunar–canalicular network (Burger and Klein-Nulend, 1999; Cowin et al., 1995; Kufahl and Saha, 1990). Weinbaum et al. (1994) predicted the membrane shear stress levels experienced by bone cells in vivo on the basis of theoretical models validated with respect to experimentally measured streaming potentials (Cowin et al., 1995; Weinbaum et al., 1994). They predicted magnitudes of fluid shear stress between 0.8 and 3 Pa on the cell membrane. Fluid
0021-9290/03/$ - see front matter r 2003 Elsevier Science Ltd. All rights reserved. doi:10.1016/S0021-9290(03)00118-0
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flow induced shear stress has been shown to stimulate the production of numerous intracellular and extracellular signaling molecules including intracellular calcium (Allen et al., 2000; Hung et al., 1995; Jacobs et al., 1998), cAMP (Reich et al., 1990), prostaglandin E2 (PGE2) (Ajubi et al., 1999; Klein-Nulend et al., 1997; Smalt et al., 1997; Sterck et al., 1998), and nitric oxide (McAllister and Frangos, 1999). These studies, with the exception of Jacobs et al. (1998), employed parallel plate flow chambers with either steady or pulsatile fluid flow. Jacobs et al. (1998) examined the [Ca2+]i response of bone cells subjected to both steady and oscillating fluid flow (OFF). When bone is loaded in vivo, the lacunar– canalicular network experiences a pressurization in response to the matrix deformation and this leads to flow along pressure gradients. When the loading is removed, the flows and pressure gradients are reversed and thus, the fluid motions are oscillatory in nature (Jacobs et al., 1998). Since the oscillatory component of the bone cell’s fluid flow environment has the potential to greatly exceed the steady component of flow driven by the arterial pressure head, Jacobs et al. (1998) suggested that OFF may be most representative of physiological loading. In addition to shear stress, loading-induced fluid flow will also induce chemotransport due to convection or mass transport thereby affecting the biochemical environment surrounding the cell. It has been demonstrated that the response of bone cells to fluid flow is modulated by the cell’s biochemical surroundings. For example, Allen et al. (2000) studied the effect of the addition of newborn bovine serum to the flow media on the [Ca2+]i response of primary cultured bone cells stimulated by fluid flow (Allen et al., 2000). They found that shear flow in serum-supplemented medium produces enhanced intracellular calcium responses in individual cells compared to serum-free flow stimulation. Cells exposed to serum-supplemented medium during flow exhibited a larger peak magnitude and longer duration calcium responses than those exposed to serum-free flow (Allen et al., 2000). This study suggests that chemical agonist exposure can modulate the intracellular calcium response in bone cells subjected to fluid flow induced shear stress. The hypothesis of the present study is that bone cells, specifically mouse osteoblastic MC3T3-E1 cells, respond to fluid flow induced shear stress with a biochemical response, and this response is modulated by chemotransport. Both intracellular calcium mobilization and PGE2 production were examined in this study. Intracellular calcium is an early second messenger that plays an important role in many downstream events, and is typically observed to increase dramatically within seconds of fluid flow stimulation. Therefore, [Ca2+]i was quantified as a measure of cell responsiveness (Hung et al., 1995; Jacobs et al., 1998). PGE2 was
studied because it has been implicated as an important mediator in the regulation of bone turnover (Imamura et al., 1990; Jee et al., 1985). Prostaglandins are produced by osteoblastic cells and are abundant in bone (Feyen et al., 1984; Nolan et al., 1983; Rodan et al., 1986). Prostaglandins have been shown to regulate bone metabolism by both stimulation and inhibition of bone formation (Jee et al., 1991; Raisz and Fall, 1990) and can also play a role in bone resorption (Dietrich et al., 1975; Fuller and Chambers, 1989). The goals of this study were to determine whether the effect of OFF can be ascribed to altered shear stress and/ or chemotransport in terms of its affect on (1) [Ca2+]i, and (2) production of PGE2. These results may lead to a better understanding of cellular mechanosensitivity in bone cells, and therefore, a better understanding of diseases involving altered mechanotransduction, such as osteoporosis.
2. Methods 2.1. Cell culture The immortalized mouse osteoblastic cell line MC3T3-E1 (mouse calveria) was employed in this study. MC3T3-E1 cells were cultured in minimum essential medium (MEM-a) (GIBCO BRL, Grand Island, NY) supplemented with 10% fetal bovine serum (FBS) (Hyclone, Logan, UT) and 1% penicillin/streptomycin (P/S) (GIBCO BRL, Grand Island, NY). Cells were maintained in an incubator at 37 C, 100% humidity, and 5% CO2. For intracellular calcium mobilization experiments, cells were plated on quartz slides (76 mm 26 mm 1.6 mm) at 0.65 105 cells/ slide and cultured for 48 h to 80% confluence. For PGE2 studies, cells were plated on glass slides (75 mm 38 mm 1 mm) at 3.5 105 cells/slide and cultured for 48 h to 85% confluence. 2.2. Oscillating fluid flow Cells were exposed to fluid flow as previously described (Jacobs et al., 1998; Kurokouchi et al., 2001; You et al., 2001; You et al., 2000). Briefly, two custom designed parallel plate flow chambers of similar construction, but varying capacity, were used to expose cells to fluid flow. [Ca2+]i imaging experiments utilized a vacuum-sealed flow chamber with a flow channel of 38 10 0.28 mm, whereas longer duration PGE2 studies were conducted with a larger chamber with a flow channel of 60 24 0.28 mm. The latter chamber utilizes a mechanical clamp to achieve a tight seal and can be assembled in a sterile environment and kept in a sterile incubator throughout the flow exposure period. Flow was delivered with Hamilton glass syringes
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custom mounted into a servo pneumatic loading frame (EnduraTec, Eden, Prairie, MN). To verify the output of the syringes an ultrasonic flow meter (Model 106, Transonic Systems, Ithaca, NY) was attached to the chamber inlet. This system generated sinusoidal OFF at 1 Hz. Various flow media were employed depending on the experiment as described below. Shear stress was kept constant by adding neutral dextran to the flow medium and decreasing the flow rate. To address the effect of nutrients on the cell’s response to OFF, Hanks’ buffered salt solution (HBSS) was also used as flow media during some experiments to create a flow media devoid of all nutrients and serum. 2.3. Viscosity measurements A capillary tube viscometer was used to quantify changes in viscosity with the addition of neutral dextran to the flow media. The accuracy of the viscometer was verified with ultrapure water. Both standard flow media containing 2% FBS and HBSS were tested and found to have a viscosity of 1.0 cP (1centiPoise (cP)=0.01 dyn s/ cm2). Neutral dextran (MW 500,000) (Sigma, St. Louis, MO) was added to the standard flow media in various quantities to modulate the viscosity; 0.5, 1, 1.5 or 2 g of dextran per 100 ml of media. These concentrations resulted in viscosity measurements of 1.2, 1.5, 2.0 and 2.4 cP, respectively. 2.4. Calcium imaging Pre-confluent cells were washed with MEM and 2% FBS solution at 37 C. Cells were then incubated with the dual excitation Ca2+-probe Fura-2 (acetoxymethyl (AM) ester 1 mM, Molecular Probes, Eugene, OR) for 30 min at 37 C. The cells were then washed with fresh media and 2% FBS solution and the slide was mounted in the parallel plate flow chamber and placed on an inverted fluorescence microscope (Nikon Diaphot 300) and left undisturbed for 30 min. The cells were illuminated as described previously (Jacobs et al., 1998; Kurokouchi et al., 2001; Yellowley et al., 1997, 1999; You et al., 2000, 2001). A Metafluor imaging system (Universal Imaging, West Chester, PA) was used to sample and record the emitted light from the cells in the field of view once every 2 s (emission wavelength 510 nm) and Metafluor imaging software was used to subtract the background fluorescence from each image and to outline and calculate the 340:380 ratio of light emitted in response to excitation at 340 and 380 nm for each cell in the field of view, as this ratio reflects [Ca2+]i. A calibration curve was constructed by acquiring 340:380 values (background subtracted) for a series of solutions of known free Ca2+ concentration (0– 39.8 mM, Molecular Probes) and 1 mM fura2 pentapotassium salt (Molecular Probes). This calibration curve
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Table 1 Intracellular calcium flow regimes Number of slides Standard media with a flow rate of 4.5 ml/min and a peak shear stress of 5 dyn/cm2 (0.5 Pa) 1 g of dextran per 100 ml of media with a flow rate of 4.5 ml/min and a peak shear stress of 8.7 dyn/cm2 (0.87 Pa) Standard media with a flow rate of 18 ml/min and a peak shear stress of 20 dyn/cm2 (2 Pa) 1 g of dextran per 100 ml of media with a flow rate of 11.5 ml/min and a peak shear stress of 20 dyn/cm2 (2 Pa) Hanks’ balance salt solution with a flow rate of 18 ml/min and a peak shear stress of 20 dyn/cm2 (2 Pa) Hanks’ balance salt solution with a flow rate of 36 ml/min and a peak shear stress of 40 dyn/cm2 (4 Pa)
3 4
6 7
4
4
was used to convert ratio values from individual cells into [Ca2+]i. To identify Ca2+ transients we used a numerical procedure adapted from mechanical fatigue analysis, known as Rainflow Cycle Counting (Downing and Socie, 1982). This simple algorithm reliably and automatically identifies and determines the amplitudes of spikes and transients in time history data even when superimposed over each other or in the presence of background noise (Jacobs et al., 2000). We have previously used this algorithm to identify transients in [Ca2+]i in chondrocytes and bone cells (Jacobs et al., 1998; Kurokouchi et al., 2001; Yellowley et al., 1997, 1999; You et al., 2000, 2001). We defined a response as a transient increase in [Ca2+]i of 50 nM or greater. Data were collected for 1 min at the start of each experiment prior to flow, and then for a period of 3 min during flow. Numerous flow regimes were applied while measuring [Ca2+]i (Table 1). Constant flow rate experiments were conducted at a lower shear stresses (5 and 8.7 dyn/cm2) compared to constant shear stress experiments, which were conducted at 20 dyn/cm2. This was necessary due to the fact that at 20 dyn/cm2 approximately 100% of the cells were responding, and so increasing the shear stress from 20 dyn/cm2 could not alter the percent of cells responding. 2.5. PGE2 experiments All experiments were performed with pre-confluent (85%) MC3T3-E1 cells. The cells were exposed to OFF for 1 h and control cells were placed in flow chambers, but were not exposed to fluid flow. Various flow regimes were employed (Table 2). Following the hour of flow, the slides were rinsed with phosphate buffered saline and placed in 10 ml of fresh standard flow media, incubated for an additional hour, and the media collected and
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Table 2 PGE2 fluid flow regimes
0.3
Standard media without dextran with flow rate of 43 ml/min and a peak shear stress of 20 dyn/cm2 (2 Pa) 1 g of dextran per 100 ml of media with flow rate of 28 ml/min and a peak shear stress of 20 dyn/cm2 (2 Pa) 2 g of dextran per 100 ml of media with flow rate of 18 ml/min and a peak shear stress of 20 dyn/cm2 (2 Pa) HBSS with flow rate of 43 ml/min and a peak shear stress of 20 dyn/cm2 (2 Pa)
Number of control slides
4
4
4
4
[Ca+2]i (µM)
0.25
Number of flow slides
0.2 0.15 0.1 0.05
4
4
0 0
4
50
100
150
200
250
Time (s)
4
Fig. 1. An example of [Ca2+]i response traces obtained for OFF in which 100% of individual MC3T3-E1 cells responded to flow. Note the arrow depicts the onset of flow.
2.6. Statistics For intracellular calcium mobilization experiments, the variability in the fraction of responding cells was characterized with the standard error of proportion (SEP) and statistical comparisons were made on the basis of the z-test statistic for comparison of proportions (Glantz, 1992; Jacobs et al., 1998). All data obtained from PGE2 experiments were expressed as mean7standard error (SEM). ANOVAs with Bonferroni/Dunn post hoc comparisons were completed using a commercially available software program (StatView, SAS Institute, San Francisco, CA).
3. Results 3.1. Calcium imaging MC3T3-E1 cells typically displayed a few small amplitude, intermittent Ca2+ response spikes during i the 1-min no flow period due to random system noise or an occasional spontaneous Ca2+ transient followed by a i more pronounced and coordinated response spike at the onset of flow (Fig. 1).
% of cells responding
100
stored at 80 C for PGE2 analysis while the cells were isolated and assayed for total protein. PGE2 accumulation was quantified using a commercially available, nonradioactive, competitive binding enzymeimmunoassay system (BioTrak, Amersham Pharmaceuticals, Piscataway, NJ), conducted at room temperature. Frozen media samples were thawed at room temperature and vortexed prior to use. The optical densities of the samples were read at 450 nm using a microplate reader (Dynex Technologies, Chantilly, VA). PGE2 accumulation was normalized to total cell protein determined by the Lowry method (Bio-Rad) for each slide.
80 60
*
40 20 0 4.5 ml/min 5 dynes/cm2 no dextran
4.5 ml/min 8.7 dynes/cm2 1 gram dextran
Fig. 2. The percentage of cells responding with a calcium transient for experiments using flow regimes at constant chemotransport (flow rate=4.5 ml/min) and varied peak shear stress. The number of cells for the 5 dyn/cm2, no dextran, 4.5 ml/min flow regime was n ¼ 111 (from 3 slides) and the number of cells for the 8.7 dyn/cm2, 1 g dextran, 4.5 ml/ min flow regime was n ¼ 190 (from 4 slides). The bars represent standard error of a proportion. *Significantly different from 5 dyn/ cm2, no dextran, 4.5 ml/min (po0:01).
The percentage of cells responding with an increase in [Ca2+]i was significantly dependent (po0:01) on the peak flow induced shear stress. At a constant flow rate of 4.5 ml/min, peak shear stresses of 5 (n ¼ 111) and 8.7 dyn/cm2 (n ¼ 190) resulted in 13.573.2% and 32.673.4% (7SEP) of the cells responding, respectively (Fig. 2). The mean amplitude of the cells responding was not significantly dependent on the peak shear stress. The amplitude was 38718 and 7277 nM, at 5 and 8.7 dyn/ cm2 (7SEM), respectively. The addition of neutral dextran did not change the percentage of cells responding in the no flow baseline period (data not shown). The percentage of cells responding with an increase in [Ca2+]i was also significantly dependent (po0:01) on the flow rate (chemotransport). At a peak shear stress of 20 dyn/cm2 and flow rate of 18 ml/min, 87.671.8% (7SEP) of individual cells responded to flow (n ¼ 347). This response was significantly reduced to 75.272.3%
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*
80 60 40 20 0
18 ml/min 20 dynes/cm2 no dextran
11.5 ml/min 20 dynes/cm2 1 gram dextran
Fig. 3. The percentage of cells responding with a calcium transient for experiments using flow regimes at a constant peak shear stress (20 dyn/ cm2) and varied chemotransport (flow rate). The number of cells for each of the regimes was n ¼ 347 (from 6 slides in standard media and 7 slides in media with dextran). The bars represent standard error of a proportion. *Significantly different from 11.5 ml/min, 1 g dextran, 20 dyn/cm2 flow regime (po0:01).
% of cells responding
100 80 60 40 20
*
*
0 18 ml/min 20 dynes/cm2 Standard Media
18 ml/min 20 dynes/cm2 HBSS
36 ml/min 40 dynes/cm2 HBSS
Fig. 4. The percentage of cells responding with a calcium transient for experiments using flow regimes with HBSS as the perfusing flow media. The number of cells for the 20 dyn/cm2, 18 ml/min, standard media flow regime was n ¼ 347 (from 6 slides). The number of cells for the 20 dyn/cm2, 18 ml/min, HBSS flow regime was n ¼ 214 (from 4 slides) and the number of cells for the 40 dyn/cm2, 36 ml/min, HBSS flow regime was n ¼ 189 (from 4 slides). The bars represent standard error of a proportion. *Significantly different from standard media, 20 dyn/cm2, 18 ml/min (po0:01).
of the cells responding (n ¼ 347) when the flow rate was reduced to 11.5 ml/min while maintaining a constant peak shear stress of 20 dyn/cm2 (Fig. 3). The average amplitude of the cells responding was not significantly affected by flow rate. The mean amplitude of the cells responding was 130728 and 82710 nM (7SEM) at 18 and 11.5 ml/min, respectively. To assess the role of nutrients in the [Ca2+]i response to OFF, experiments were conducted at a peak shear stress of 20 dyn/cm2 and a flow rate of 18 ml/min in standard flow media and nutrient free flow media. In standard media 87.671.8% (7SEP) of the cells responded. This response was significantly attenuated when HBSS (nutrient-free) was the flow media, with only 10.772.1% of the cells responding (n ¼ 214;
po0:01) (Fig. 4). Doubling the peak shear stress and flow rate to 40 dyn/cm2 and 36 ml/min, respectively, did not recover the attenuation seen with HBSS. Only 8.572.0% of individual cells responded to flow with an increase in [Ca2+]i (n ¼ 189) (Fig. 4). The mean amplitude of the cells responding was significantly attenuated for both conditions using HBSS; at 20 dyn/ cm2/18 ml/min and 40 dyn/cm2/36 ml/min the mean amplitudes were 7474 and 7078 nM, respectively (po0:01) compared to 130728 nM (7SEM) at 20 dyn/cm2/18 ml/min in standard media. 3.2. PGE2 production PGE2 production was significantly (po0:0001) increased with exposure to OFF. Total PGE2 production in cells exposed to a peak shear stress of 20 dyn/cm2 at a flow rate of 43 ml/min was 3473.1 pg/mg of total protein (7SEM) (n ¼ 4). This was significantly greater compared to PGE2 production in no flow, control cells, 14.870.85 pg/mg of total protein (n ¼ 4Þ (Fig. 5). This PGE2 production was modulated by flow rate (chemotransport). Reducing the flow rate from 43 ml/ min to either 28 ml/min or 18 ml/min and maintaining the peak shear stress at 20 dyn/cm2, reduced the total PGE2 production for the flow experiments to 25.876.8 and 9.171.8 pg/mg of total protein (n ¼ 4), respectively (Fig. 5), from 3473.1 pg/mg of total protein. At 18 ml/ min, the PGE2 production was not significantly different from no flow controls. The total PGE2 production was not affected by dextran concentration. For the no flow controls, the total PGE2 was not significantly different with various concentrations of dextran (Fig. 5). PGE2 production was also dependent on the level of nutrients in the flow media. At a peak shear stress of 20 dyn/cm2 and flow rate of 43 ml/min total PGE2 production was decreased from 18.374.3 pg/mg of total protein (7SEM) (n ¼ 4) in standard media, to 9.572.7 pg/mg of total protein (n ¼ 4) when the flow
Total PGE2 production (pg/µg of total protein)
% of cells responding
100
1367
40
^
flow no flow
30 20 **
10 0
43 ml/min 20 dynes/cm2
28 ml/min 20 dynes/cm2
18 ml/min 20 dynes/cm2
Fig. 5. PGE2 quantification in experiments at a constant peak shear stress (20 dyn/cm2) and varied chemotransport (flow rate). The numbers are representative of total PGE2 accumulation in the media normalized to total protein. All results are shown plotted as mean7SEM with each group containing four experiments. ^Significantly different from no flow control within group; **significantly different from flow at 43 ml/min and 28 ml/min (po0:0001).
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Total PGE2 production (pg/µg of total protein)
25 flow no flow
20 15 10 5 0 Standard Media 43 ml/min 20 dynes/cm2
HBSS 43 ml/min 20 dynes/cm2
Fig. 6. PGE2 quantification in experiments at a peak shear stress of 20 dyn/cm2 and flow rate of 43 ml/min, with varied flow media. The numbers are representative of total PGE2 accumulation in the media normalized to total protein. All results are shown plotted as mean7SEM with each group containing four experiments.
media was nutrient-free HBSS (Fig. 6). There was no difference in PGE2 production between the no flow, control cells, with HBSS.
4. Discussion This study investigated the role of OFF-induced shear stress and chemotransport in cellular mechanotransduction mechanisms in bone. These two were distinguished and independently studied in osteoblastic MC3T3-E1 cells. Studying these stimuli independently was accomplished via altering flow rate and/or flow media viscosity. To address the role of fluid flow induced shear stress and chemotransport, both intracellular calcium mobilization and PGE2 production were studied. The data from this study suggest that OFF induced intracellular calcium responses and PGE2 production are modulated by chemotransport. While bathed in standard media and exposed to a constant shear stress, the cells responded with both a robust intracellular calcium response and increase in PGE2 production. Both of these responses were decreased with decreasing flow rate while maintaining constant shear stress. In contrast, using HBSS as the flow media was not accompanied by significant changes in [Ca2+]i or PGE2 production with the onset of OFF. While many investigators have studied the effects of fluid flow induced shear stress on bone cells (Ajubi et al., 1996; Allen et al., 2000; Bakker et al., 2001; Hung et al., 1995; Klein-Nulend et al., 1995; Reich et al., 1990), few have studied oscillating fluid flow (Jacobs et al., 1998; You et al., 2001). It is important to make comparisons between these different types of flow; however, previous studies indicate that bone cells will respond differently to oscillating fluid flow induced shear stress versus
steady or pulsatile fluid flow induced shear stress (Jacobs et al., 1998; You et al., 2001). Allen and co-workers suggests only primary, not MC3T3-E1 cells respond with an increase in [Ca2+]i at 3.5 Pa of steady flow with serum-free HBSS flow media (Allen et al., 1996). However, at physiological levels of fluid flow (0.4–3.4 Pa), primary cells did not respond in the absence of serum (Allen et al., 2000). The authors suggest that the lack of response with MC3T3-E1 cells without serum may be an artifact of the cell line (Allen et al., 2000). Data from the current study agrees with the studies by Allen and co-workers showing no response of MC3T3-E1 cells in serum free flow media at 2 and 4 Pa. Jacobs et al. (1998) showed that steady flow is more stimulatory than oscillating flow (Jacobs et al., 1998). Hence, when comparing the current data with Allen and co-workers, it may be that in the current study oscillating fluid flow levels were not high enough to reach a threshold beyond which MC3T3-E1 cells will respond in the absence of serum. Perhaps the threshold in which osteoblastic MC3T3-E1 cells will show a response was not reached and is higher than the threshold for primary bone cells that may contain both osteoblasts and osteocytes. The current study did not go above physiological levels of fluid flow induced shear stress, which have been theorized to be 0.8–3.8 Pa (Cowin et al., 1995; Weinbaum et al., 1994). The current data does show a marked increase in the calcium response of MC3T3-E1 cells with serum-supplemented media. Reich and Frangos studied the effects of increasing shear on PGE2 production (Reich and Frangos, 1991). They showed a dose-dependent increase in the rate of PGE2 production in osteoblasts exposed to low (6 dyn/ cm2) and high (24 dyn/cm2) steady shear. While the current study did not test the effects of altered shear on PGE2 production, we would expect to see the same results as Reich and Frangos since we showed a dosedependent increase in the percentage of cells responding (with increase in intracellular calcium) with an increase in shear stress. The current study adds to the work of Reich and Frangos and investigated if the fluid flow induced shear stress response is modulated by chemotransport (flow rate). Our studies suggest that as flow rate increased, PGE2 production increased. PGE2 production decreased when cells were deprived of serum. While not statistically significant, PGE2 production was less in no flow control cells bathed in HBSS versus serum-supplemented media (Fig. 6). Perhaps if we designed the experiments to test this hypothesis the results may be significant with more experiments. Our findings demonstrate that although PGE2 production and intracellular calcium mobilization in response to OFF exhibit similar trends when chemotransport is altered, the magnitudes of these trends are
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dissimilar. That is, when HBSS was used as the flow media the effect of fluid flow on intracellular calcium mobilization was much greater than upon PGE2 production. Interestingly, these data suggest that PGE2 production in response to OFF may be occurring without intracellular calcium signaling. In support of this view, Saunders et al. (2001) has also found that there may be separate pathways involved in calcium wave propagation and PGE2 production in osteoblastic MC3T3-E1 cells exposed to fluid flow. In those studies, OFF in the presence of thapsigargin, a drug which empties the intracellular stores of calcium, induced a diminished calcium response, relative to fluid flow without thapsigargin. However, thapsigargin did not affect the PGE2 production. Therefore, these findings support our data suggesting there may be independent pathways involved in intracellular calcium mobilization and PGE2 production. A recent study by Bakker et al. (2001) addressed the nature of flow-derived cell stimulus by comparing variations in fluid transport with variations in shear stress, using nitric oxide and PGE2 production as parameters for bone cell activation (Bakker et al., 2001). These studies found that the production of NO and PGE2 is enhanced in a dose-dependent manner by pulsating fluid flow of increasing shear stress and flow rate. When these authors used neutral dextran to increase wall shear stress maintaining a constant flow rate, they showed an increase in both NO and PGE2 production. These findings are consistent with Reich and Frangos’s PGE2 results (Reich and Frangos, 1991). Similar to the current study, Bakker et al. (2001) maintained a constant shear and increased flow rate. In contrast to the current data, they showed no difference in PGE2 production with increasing flow rate. Our data shows an increase in PGE2 production with an increase in flow rate. This difference could be due to difference in fluid flow regimes. Bakker et al. (2001) applied steady flow while the current study used oscillating flow (Bakker et al., 2001). The current data suggests that chemotransport modulates the OFF induced shear stress response of osteoblastic cells. Studies by Allen et al. (2000) found that chemical agonist exposure (i.e. serum, and ATP) can modulate the [Ca2+]i response in bone cells subjected to fluid flow (Allen et al., 2000). Therefore, it is reasonable to hypothesize that a biochemical factor in the media is responsible for this modulation. ATP has previously been shown to be required for flow-induced [Ca2+]i changes in other cell types (Nollert and McIntire, 1992; Shen et al., 1992). These authors report that the convective mass transport of ATP from the bulk media is responsible for the flow-induced [Ca2+]i since the ATP concentration at the cell surface rises in shear flow. Additionally, ATP is also known to chemically activate certain cell receptors (Schofl et al., 1992) and
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thus, the observed ATP response in the previous studies provide supporting evidence that chemotransport is a likely explanation for the serum response (Allen et al., 2000). One limitation when interpreting our results is that there is no experimental quantification of lacunar– canalicular flow actually occurring in vivo. We have based our flow regime on theoretical predictions that have been validated with respect to experimental measurements of flow-induced streaming potentials (Cowin et al., 1995). However, the fact remains that the geometry of the flow chamber is different from that of a canalicula or lacuna and the flow rates used may differ from that which occurs in vivo through the lacunar–canalicular network. The current study suggests that oscillating fluid flow at peak shear stresses up to 4 Pa is not a sufficient enough stimulus to invoke a calcium response in serum free flow media. The percentage of cells responding in HBSS was not significantly different during flow compared to the no flow baseline. However, there may be a stimulus that is more powerful than oscillating fluid flow and would result in a greater calcium response in serum free media. Our data does not rule out the possibility that without serum the cells basal metabolism has changed and the cells are no longer capable of a calcium response. Further studies will need to be conducted to determine if the diminished response in HBSS is due to a decrease in basal metabolism or a decrease in mechanosensitivity. The HBSS data must be interpreted carefully due to confounding issues. The lack of response of cells in HBSS may be due to changes in either chemotransport or basal metabolism. In fact, without serum cells may be unable to respond to flow due to changes in their basal metabolism. While the HBSS data is consistent with the conclusion that chemotransport modulated the shear stress response in MC3T3-E1 cells, the data is not sufficient to rule out changes in basal metabolism as the reason for changes in the calcium response. The HBSS studies were performed in order to compare our data to Allen et al. (2000), which showed primary bone cells respond in serum free media, only when the steady fluid flow induced shear stress levels were beyond a certain threshold (3.5 Pa) (Allen et al., 2000). Our data shows no response of MC3T3-E1 cells to oscillating fluid flow induced shear stress levels as high as 4 Pa. In our lab and others, MC3T3-E1 cells have been used as a model for the study of mechanosensitivity in bone cells (Chen et al., 2000; You et al., 2001). These cells have previously been shown to respond to fluid flow with increases in intracellular calcium (You et al., 2001), gene expression (You et al., 2001), and reorganization of the actin cytoskeleton leading to increased expression of COX2 and cFOS (Pavalko et al., 1998). Additionally, in contrast to primary cells, the use of a cell line such as
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MC3T3-E1 cells ensures a homogeneous cell population. Primary bone cell culture often contains osteoblasts, osteocytes and fibroblasts in varying proportions, and thus observed difference may not always be due to difference in bone cell sensitivity. In this study, MC3T3-E1 cells responded to OFF with both a robust increase of [Ca2+]i and an increase in PGE2 production. These fluid flow induced responses were modulated by chemotransport. The percentage of cells responding with an [Ca2+]i oscillation increased with increasing flow rate, as did the production of PGE2. In addition, depriving the cells of nutrients during fluid flow resulted in a decreased [Ca2+]i and less PGE2 production. These data suggest that depriving the cells of a yet to be determined biochemical factor in media affects the responsiveness of bone cells even at a constant peak shear stress. Chemotransport alone will not elicit a response as shear stress will, but it appears that sufficient nutrient supply or waste removal is needed for the response to shear stress induced by OFF.
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