Flow-induced calcium oscillations in rat osteoblasts are age, loading frequency, and shear stress dependent

Seth W. Donahue1, Christopher R. Jacobs2,3, and Henry J. Donahue4

1 Department of Biomedical Engineering, Michigan Technological University, Houghton, Michigan 49931-1295; 2 Rehab Research and Development Center, Veterans Affairs Medical Center, Palo Alto 94304-1200; 3 Biomechanical Engineering Division, Stanford University, Stanford, California 94305-3030; and 4 Musculoskeletal Research Laboratory, Department of Orthopaedics and Rehabilitation and Department of Cellular and Molecular Physiology, The Pennsylvania State University, Hershey, Pennsylvania 17033


    ABSTRACT
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

Bone adaptation to mechanical loading is dependent on age and the frequency and magnitude of loading. It is believed that load-induced fluid flow in the porous spaces of bone is an important signal that influences bone cell metabolism and bone adaptation. We used fluid flow-induced shear stress as a mechanical stimulus to study intracellular calcium (Ca<UP><SUB>i</SUB><SUP>2+</SUP></UP>) signaling in rat osteoblastic cells (ROB) isolated from young, mature, and old animals. Fluid flow produced higher magnitude and more abundant [Ca2+]i oscillations than spontaneous oscillations, suggesting that flow-induced Ca<UP><SUB>i</SUB><SUP>2+</SUP></UP> signaling encodes a different cellular message than spontaneous oscillations. ROB from old rats showed less basal [Ca2+]i activity and were less responsive to fluid flow. Cells were more responsive to 0.2 Hz than to 1 or 2 Hz and to 2 Pa than to 1 Pa. These data suggest that the frequency and magnitude of mechanical loading may be encoded by the percentage of cells displaying [Ca2+]i oscillations but that the ability to transduce this information may be altered with age.

mechanotransduction; osteoblast; calcium signaling; bone adaptation


    INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

BONES ADAPT to mechanical loading. When normal mechanical loading is absent, bone mass is removed. For example, disuse osteopenia occurs in the tibias of astronauts who experience microgravity (49), in patients confined to prolonged bedrest (28), in immobilized bones following surgery (30), and in patients with total arthroplasty (29). When habitual bone loading is exceeded, bone mass is added. For instance, periosteal and endosteal bone areas have been found to be significantly higher in the dominant arm of tennis players (3). Bone mineral density and cross-sectional moment of inertia have been found to be significantly higher in the dominant humeri of tennis players, regardless of the age at which they started playing (16). However, the effect of mechanical loading on bone mass was more than twofold greater in young players than in players who began playing after reaching skeletal maturity. These data suggest that growing bones are more adaptable to mechanical loading than adult bones. Turner et al. (44-46) have demonstrated the ability of rat long bones to adapt to unaccustomed mechanical loading. They showed that new bone formation in tibias loaded in four-point bending was dependent on the frequency and magnitude of loading (44, 45). They also demonstrated that the ability of long bones to adapt to mechanical loading was diminished in 19-mo-old rats compared with 9-mo-old rats (46). At the highest bending load, the relative bone formation rate was more than 16-fold lower in the older rats. These findings parallel human studies, which suggest that cells in growing bones are more sensitive to mechanical signals than cells in adult bones.

It is believed that physical activities, which produce bending loads in bones, induce fluid flow in the porous spaces of bone (10, 15, 26, 27, 50). This fluid flow is believed to be an important physical signal that influences bone cell metabolism and bone adaptation to mechanical loading (7, 15, 27). Bone cells produce adaptations to mechanical loading: osteoblasts add bone mass when loading becomes excessive, and osteoclasts remove unneeded bone. However, the biochemical signaling pathways that mediate bone adaptation to mechanical loading are unknown. Understanding how individual bone cells respond to mechanical stimuli with biochemical responses and how these responses change with age may help elucidate our understanding of mechanically induced bone adaptations and the etiology of bone diseases such as osteoporosis.

In vitro, physical stimuli activate numerous signaling molecules in bone cells, including intracellular calcium (9, 17, 18, 51, 52, 55), prostaglandins (1, 36, 40), inositol trisphosphate (36), and nitric oxide (25, 32, 40). Mechanical stimuli also have been shown to upregulate gene expression in bone cells (9, 20, 54). Intracellular calcium oscillations are important signaling mechanisms for many cellular processes (e.g., differentiation, proliferation, and gene transcription) (5). Calcium signaling also is an early response in bone cell mechanotransduction and can influence downstream signaling events. For example, blocking mechanically induced intracellular calcium oscillations also blocks gene expression (9, 54), prostaglandin release (1), and cytoskeletal reorganization (9).

In vivo rat studies have demonstrated that long bone adaptation to mechanical loading is magnitude and frequency dependent and that the capacity for adaptation decreases with age (44-46). Therefore, it is likely that biochemical signaling in bone cells in response to mechanical stimuli is also dependent on age and on the magnitude and frequency of the physical signal. We hypothesized that fluid flow-induced oscillations in cytosolic calcium concentration ([Ca2+]i), in osteoblastic cells isolated from rat long bones, would be dependent on loading frequency, shear stress magnitude, and age of the rat from which the cells were isolated. Biochemical messages encoded by [Ca2+]i oscillations may be determined by the magnitude and/or frequency of the oscillation (6, 43). It also is thought that calcium signaling requires coordinated Ca<UP><SUB>i</SUB><SUP>2+</SUP></UP> signaling events in cell ensembles.(21) Therefore, we chose the percentage of cells displaying [Ca2+]i oscillations and the magnitude of the oscillations as the independent response variables to fluid flow-induced shear stress.


    METHODS
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

Bone cells. Rat osteoblastic cells (ROB) were isolated from the humeri, tibias, and femora of young (4 mo, n = 7), mature (12 mo, n = 7), and old (24 mo, n = 7) male Fisher 344 rats. All procedures were approved by the Institutional Animal Care and Use Committee at the M. S. Hershey Medical Center. The rats were anesthetized by an intraperitoneal injection of pentobarbital sodium (Nembutal; Abbott Laboratories, North Chicago, IL) with a dosage of 50 mg/kg of bodyweight and were euthanized by exsanguination. The bones were extracted from the animals, and subperiosteal ROB were obtained by removing all soft tissues, including cartilage and periosteum, from the bones and performing sequential collagenase (Worthington Biochemical, Lakewood, NJ) digestions at 37°C. Cells from the first digestion were collected by centrifugation and discarded to eliminate any residual non-bone cells that were not removed by dissection. Cells from the second digestion were collected by centrifugation and grown to confluency in Dulbecco's modified Eagle's medium (DMEM; GIBCO, Gaithersburg, MD), 20% fetal bovine serum (FBS), and 1% penicillin/streptomycin. Cells from all six bones were pooled and grown to confluency in the same culture flask. We have shown previously that ROB isolated by this technique display characteristics of the osteoblast phenotype (13). We also stained ROB for alkaline phosphatase activity and performed a dye transfer assay to demonstrate gap junctional intercellular communication (53).

Three days before experimentation, the cells were plated on quartz microscope slides (76 × 26 × 1.6 mm) at a density of 75,000 cells per slide; cells were ~70% confluent on the day of experimentation. The cells were incubated at 37°C with 10 µM fura 2-AM (Molecular Probes, Eugene, OR) for 30 min before mechanical stimulation.

Fluid flow system. After fura 2 loading, the cell-seeded microscope slides were mounted in a parallel-plate flow chamber that was fixed to the stage of a fluorescent microscope. A fresh bolus of flow medium was added to the chamber, and the cells were left undisturbed for 30 min. The flow medium consisted of DMEM and 2% FBS. We have described previously the fluid flow system in detail (18); it will be described briefly here. To generate fluid flow-induced shear stresses on the cells in the chamber, a material-testing machine was used to pump a syringe, which was in series with rigid wall tubing and a flowmeter (Transonic Systems, Ithaca, NY), driving fluid through the chamber. This system produces laminar fluid flow in the chamber with an oscillating flow profile. Shear stresses on the chamber walls are proportional to the chamber dimensions and the rate of fluid flow (17). Thus we were able to generate shear stresses on the cells with magnitudes that they are predicted to experience in vivo (50).

Oscillating fluid flow was used because it more closely simulates physiological bone loading than steady or pulsatile flow (18). During experimentation, the cells were exposed to 3 min of oscillating fluid flow that produced shear stresses of 1 or 2 Pa at frequencies of 0.2, 1, or 2 Hz. Six slides of cells from each rat were randomly assigned to one of the six shear stress/frequency combinations.

Calcium imaging. Real-time [Ca2+]i was quantified by using ratiometric dye methodology. When fura 2 binds Ca2+, its maximal absorption wavelength shifts from 363 nm for Ca2+-free fura 2 to 335 nm for Ca2+-bound fura 2 (41). In practice, wavelengths of 340 and 380 nm are used for ratiometric measurements. The emission peak is near 510 nm for both Ca2+-free and Ca2+-bound fura 2. ROB ensembles were illuminated at wavelengths of 340 and 380 nm, emitted light was passed through a 510-nm filter, and images were collected with a charge-coupled device camera. Images of fluorescence intensities were collected every 2 s for a 3-min no-flow period (baseline) and for 3 min of oscillating fluid flow. [Ca2+]i was determined from the ratio of the two emission intensities by using calibrated standards and image analysis software (Metaflour, West Chester, PA). Temporal [Ca2+]i profiles were determined for 25-35 individual cells for each slide (Fig. 1).


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Fig. 1.   Representative cytosolic calcium concentration ([Ca2+]i) profiles of 35 individual cells for a 3-min baseline period and 3 min of exposure to oscillating fluid flow [2 Pa, 1 Hz; rat osteoblastic cells (ROB) from a young rat]. Arrow indicates when flow was initiated. There were spontaneous [Ca2+]i oscillations in the baseline period, and with the onset of fluid flow there was a greater and more coordinated response.

Resting [Ca2+]i was typically <= 50 nM in ROB. We defined a responsive cell as one that displayed a transient increase in [Ca2+]i of at least 50 nM, because this represented at least a 100% increase over baseline. A numerical method known as Rainflow cycle counting was used to determine the magnitude of the calcium oscillations (19). We assessed the percentage of cells responding with a calcium oscillation and the magnitude of the responses.

Statistics. A factorial ANOVA was used to assess the influence of age, loading frequency, and shear stress on the percentage of cells responding to fluid flow with calcium oscillations and on the magnitude of the oscillations. ANOVAs were followed by Tukey's test for multiple mean comparisons. One-way ANOVAs were used to look for age-related differences for each shear stress/frequency combination. Use of ANOVA models requires the error terms to be normally distributed and requires constant variance for all factor levels (33). Studentized residuals were used to diagnose the validity of the model's assumptions. Frequency distributions of the residuals were used to check for outliers and normality of error terms. Plots of the residuals against fitted values were used to assess constancy of variance. Paired t-tests were used to compare the magnitudes of spontaneous and fluid flow-induced [Ca2+]i oscillations in cells that had responses in both periods. A significance level of 0.05 was used for all statistical analyses.


    RESULTS
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

ROB from all three age groups displayed abundant alkaline phosphatase staining in confluent cultures. Our laboratory has demonstrated previously (12) that confluent cultures of ROB from young, mature, and old animals display highly functional gap-junctional intercellular communication. We found comparable functional communication in subconfluent ROB, from all three age groups, that were seeded on quartz microscope slides (not shown). These data provide further verification that ROB display characteristics of the osteoblast phenotype.

Individual ROB demonstrated one of four [Ca2+]i profiles over the 6-min imaging period: 1) spontaneous oscillation in the baseline period and no oscillation in the flow period, 2) oscillations in both the baseline and flow periods, 3) no oscillation in the baseline period and an oscillation in the flow period, and 4) no oscillations in either period (Fig. 1). Rarely, cells displayed multiple oscillations in the baseline or flow periods. For the cells that did so, peak oscillations were used for statistical analyses. [Ca2+]i oscillations in both periods typically lasted 60 s and returned to near-baseline values.

There were spontaneous [Ca2+]i oscillations of at least 50 nM in ROB from all three age groups during the no-flow period. Of all the young ROB that were analyzed, 10% displayed spontaneous [Ca2+]i oscillations. Significantly (P = 0.04), fewer ROB from old rats displayed spontaneous [Ca2+]i oscillations during the no-flow period (Fig. 2A). However, there were no significant (P = 0.17) differences in the magnitude of the [Ca2+]i oscillations among the three age groups (Fig. 2B).


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Fig. 2.   A: influence of age on the percentage of cells displaying spontaneous [Ca2+]i oscillations during the baseline period. Values are means with SE bars (n = 42 slides for each age group). Groups with the same letter (A, B) were not significantly different from each other. Cells from young rats showed significantly more oscillations than cells from old rats. B: magnitudes of spontaneous [Ca2+]i oscillations in the baseline period were not significantly different among age groups.

With the onset of fluid flow, there were immediate and transient increases in [Ca2+]i that lasted ~60 s (Fig. 1). Peak values were reached ~15 s after the onset of fluid flow. Significantly (P < 0.0001), more cells displayed [Ca2+]i oscillations during the fluid-flow period than during the baseline period. Age (P = 0.008), loading frequency (P = 0.0001), and shear stress (P = 0.035) significantly influenced the percentage of cells responding to fluid flow. Mature ROB were more responsive than old ROB (Fig. 3). Cells were more responsive to 0.2 Hz than to 1 or 2 Hz (Fig. 4) and to 2 Pa than 1 Pa (Fig. 5). However, the magnitude of fluid flow-induced [Ca2+]i oscillations was not significantly (P = 0.367) affected by age, loading frequency, or shear stress magnitude. The magnitude (mean ± SD) of the fluid flow-induced [Ca2+]i oscillations, for all six loading regimes pooled, were 113 ± 60 nM for young ROB, 139 ± 102 nM for mature ROB, and 116 ± 85 nM for old ROB.


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Fig. 3.   Influence of age on the percentage of cells displaying [Ca2+]i oscillations during the fluid-flow period. Values are means of all 6 loading regimes with SE bars (n = 42 slides for each age group). Groups with the same letter (A, B) were not significantly different from each other. A significantly higher percentage of cells from mature rats responded to fluid flow than cells from old rats.



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Fig. 4.   Influence of loading frequency on the percentage of cells displaying [Ca2+]i oscillations during the fluid-flow period. Values are means of all age groups with SE bars (n = 42 slides for each frequency). A frequency of 0.2 Hz was significantly more stimulatory than a frequency of 1 or 2 Hz.



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Fig. 5.   Influence of shear stress on the percentage of cells displaying [Ca2+]i oscillations during the fluid-flow period. Values are means of all age groups with SE bars (n = 63 slides for each shear stress). Shear stress of 2 Pa was significantly more stimulatory than shear stress of 1 Pa.

When the subpopulation of cells that displayed [Ca2+]i oscillations in both the baseline and fluid-flow periods were considered, the magnitude of the fluid flow-induced [Ca2+]i oscillations were significantly (P < 0.0004) larger than the magnitude of the spontaneous [Ca2+]i oscillations for all three age groups. In this subpopulation, the magnitude of the fluid flow-induced [Ca2+]i oscillations were 58% greater than spontaneous [Ca2+]i oscillations in young ROB, 134% higher in mature ROB, and 81% higher in old ROB. Of the cells that displayed spontaneous [Ca2+]i oscillations in the baseline period, 80% of young ROB, 75% of mature ROB, and 76% of old ROB also exhibited flow-induced oscillations. Fluid flow was even able to potentiate the magnitude of the [Ca2+]i oscillation in cells that were displaying spontaneous oscillations at the onset of fluid flow (Fig. 6).


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Fig. 6.   [Ca2+]i profiles of 2 individual cells that displayed [Ca2+]i oscillations in both the baseline and flow periods. Fluid flow was able to induce larger magnitude [Ca2+]i oscillations in cells that displayed spontaneous oscillations and returned to basal levels before the onset of flow (dashed line) and in cells that had not returned to basal levels before the onset of flow (solid line).

The age of the rat from which cells were isolated significantly affected the percentage of cells responding to fluid flow in the factorial model. A significantly (P = 0.008) larger percentage of ROB from mature rats (71%) had [Ca2+]i oscillations than did ROB from old rats (49%) (Fig. 3). However, when each loading regime was considered separately, age did not significantly (P > 0.141) influence the percentage of cells responding to fluid flow at either 1 Pa (Fig. 7A) or 2 Pa (Fig. 7B). For each shear stress/frequency combination, however, old ROB were always the least responsive.


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Fig. 7.   Influence of age on the percentage of cells displaying [Ca2+]i oscillations for each flow regime. Values are means with SE bars (n = 7 slides for data point). When each loading regime was considered independently, there were no significant differences among age groups at either 1 (A) or 2 Pa (B).


    DISCUSSION
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

Bones adapt to mechanical loading in a frequency- and magnitude-dependent fashion (44, 45). However, the bones of mature rats adapt better to unaccustomed mechanical loading than do the bones of old rats (46). It is widely believed that bone cells mediate bone adaptations to mechanical loading by activating signaling pathways that regulate bone modeling and remodeling (7, 8, 11, 15). We found that Ca<UP><SUB>i</SUB><SUP>2+</SUP></UP> signaling in ensembles of osteoblastic cells was dependent on the frequency and magnitude of a mechanical stimulus. In addition, we found that ensembles of ROB from mature rats were more responsive to fluid flow than were ROB from old rats.

[Ca2+]i oscillations are involved in many normal cellular processes such as proliferation and gene expression (5). The spontaneous oscillations that occurred in ROB during the baseline period may have been manifestations of normal cell cycle processes. We found that the number of spontaneous [Ca2+]i oscillations declined with age in ROB, suggesting that bone cells from old animals are less metabolically active than cells from younger animals. To our knowledge, this is the first demonstration of an age-related decrease in basal Ca<UP><SUB>i</SUB><SUP>2+</SUP></UP> signaling activity in any cell type. Clearly, mechanical stimulation produced a much more abundant and synchronized pattern of Ca<UP><SUB>i</SUB><SUP>2+</SUP></UP> signaling than what occurred in the baseline period. Moreover, the magnitude of the flow-induced responses was significantly larger than the magnitude of the spontaneous oscillations. However, age, loading frequency, and shear stress influenced only the percentage of cells responding to fluid flow; they did not affect the magnitude of the [Ca2+]i oscillations. These findings suggest that the percentage of cells responding with [Ca2+]i oscillations may encode physical stimulus information. Indeed, the intercellular propagation of Ca<UP><SUB>i</SUB><SUP>2+</SUP></UP> waves is a mechanism for many cell types to coordinate their activities (21). It also is believed that a threshold of [Ca2+]i is required to activate a signaling cascade (43). In light of these views, it is reasonable to hypothesize that the abundant higher magnitude [Ca2+]i oscillations caused by fluid flow encode a different biochemical message than the sparse lower magnitude [Ca2+]i oscillations that occurred spontaneously during the baseline period.

Aging is known to impair osteoblast differentiation and activity, bone formation, and the material properties of bone (23, 31, 34, 37). Furthermore, aging impairs agonist-stimulated activity of second messengers such as cAMP and Ca2+ (14, 24), and Ca<UP><SUB>i</SUB><SUP>2+</SUP></UP> signaling has been linked to the differentiation of cells from the mesenchymal lineage (4). We found that the percentage of cells displaying spontaneous [Ca2+]i oscillations declined with age; significantly more ROB from young rats displayed spontaneous [Ca2+]i oscillations than did ROB from old rats. There were also age-related differences in fluid flow-induced [Ca2+]i oscillations, although in a more complex fashion. Significantly more ROB from mature rats displayed fluid flow-induced [Ca2+]i oscillations than did ROB from old rats, but there were no differences between young and old ROB. These differences in the age-related trends of basal and fluid flow-induced Ca<UP><SUB>i</SUB><SUP>2+</SUP></UP> signaling are difficult to interpret. Indeed, one would expect that ROB from rapidly growing young rats would display greater responses than ROB from old rats with slower growing bones. However, age-related differences in bone adaptation to unaccustomed mechanical loading has not been evaluated in the three age groups studied here. Although it has been demonstrated that 9-mo-old rats adapt better to mechanical loading than do 19-mo-old rats, it is unclear how the mechanical adaptations of young animals compare with those of mature and old animals (46).

Comparisons with in vivo data are further complicated when the influence of frequency and shear stress on Ca<UP><SUB>i</SUB><SUP>2+</SUP></UP> signaling is examined in ROB. In vivo, bone formation increases when the frequency and magnitude of the mechanical stimulus increases (44, 45). We found contradicting results with [Ca2+]i oscillations in ROB: the percentage of cells responding increased with increased shear stress but decreased with increased frequency. Clearly there is not a simple relationship between Ca<UP><SUB>i</SUB><SUP>2+</SUP></UP> signaling and bone adaptation. In fact, it is likely that bone adaptation to mechanical loading involves the complex interactions of several mechanotransduction signaling pathways (35). Mounting in vitro mechanotransduction data supports a role for Ca<UP><SUB>i</SUB><SUP>2+</SUP></UP> signaling in bone adaptation to mechanical loading (1, 54). For example, when mechanically induced calcium signaling in bone cells is inhibited by calcium channel blockers, mRNA expression of an abundant bone matrix protein (osteopontin) and the release of a potent stimulator of bone formation (prostaglandin E2) are also inhibited (1, 54). Moreover, calcium channel blockers prevent mechanical loading-induced prostaglandin release in bone organ culture (35). A well-defined role for Ca<UP><SUB>i</SUB><SUP>2+</SUP></UP> signaling in the bone adaptation, mechanotransduction signaling pathway has yet to be elucidated.

Primary bone cell cultures are necessary to study the effects of aging. However, a limitation of ROB cultures is that they likely contain a heterogeneous population of cells derived from bone. We cannot rule out the possibility that the cultures contain nonosteoblastic or preosteoblastic populations. However, the ROB display an osteoblastic morphology and express phenotypic markers of osteoblasts (i.e., alkaline phosphatase, type I collagen, osteopontin, and parathyroid hormone receptor) (14, 24). It is possible that age-related differences in the degree of cellular heterogeneity of our cell populations contributed to differences in responsiveness to fluid flow. Indeed, this also may be the case in vivo.

There were no differences in the percentage of ROB responding between loading frequencies of 1 and 2 Hz, but a significantly larger percentage of ROB responded to 0.2 Hz. One possible explanation for these findings is cellular viscoelasticity. Because cells are viscoelastic, they may be less stiff and more deformable at lower loading rates. Thus it is possible that the mechanotransducing "machinery" (e.g., stretch-activated ion channels, cell surface receptors, cytoskeleton, etc.) is more likely to be activated at lower loading rates. However, the possibility that the results can be explained, at least in part, by molecular transport phenomena cannot be overlooked. It is well known that serum constituents (e.g., ATP) can function as agonists for [Ca2+]i oscillations in a concentration-dependent fashion (39, 42, 43). Lower loading frequencies require the mechanical loading apparatus to pump larger volumes of medium, and thus more serum constituents, through the flow chamber to maintain shear stress levels. Therefore, the larger percentage of ROB responding to lower frequency loading may have resulted from larger agonist volumes flowing through the chamber. Similarly, this may explain why 2 Pa were significantly more stimulatory than 1 Pa; the development of higher shear stresses on the flow chamber walls, for a given frequency, requires larger volumes of fluid flow. However, it recently has been shown that shear stress in the absence of serum can stimulate a Ca<UP><SUB>i</SUB><SUP>2+</SUP></UP> signaling in bone cells, but the response is enhanced by mechanical stimulation in the presence of serum (2). Furthermore, it has been shown that parathyroid hormone modulates Ca<UP><SUB>i</SUB><SUP>2+</SUP></UP> signaling in bone cells, suggesting that bone cells may be sensitized to physical stimuli by biomolecules (38). These findings suggest that appropriate levels of both mechanical loading and biochemical constituents are required to mediate cellular mechanotransduction and bone adaptation to mechanical loading.

It has been postulated that gap junctions play a role in communicating mechanical signals in bone cell ensembles (11). In fact, a diverse array of extracellular stimuli (e.g., hormonal, electrical, and mechanical) has been shown to influence gap-junctional intercellular communication, which often involves the propagation of intercellular [Ca2+]i oscillations (12, 22, 47, 48). However, intercellular [Ca2+]i oscillations in bone cells also can be propagated by ATP activation of P2Y receptors (22). Because we found no age-related changes in gap-junctional communication in ROB, we postulate that the age-related decrease in the number of cells displaying mechanically induced [Ca2+]i oscillations involves a defect in the ATP/P2Y mechanism.

In summary, we found that fluid flow-induced Ca<UP><SUB>i</SUB><SUP>2+</SUP></UP> signaling in osteoblastic cells is age, frequency, and magnitude dependent. Cells from young rats showed more basal [Ca2+]i activity than did old ROB, and mature ROB were more responsive to fluid flow than were old ROB. Low frequency and high shear stress loading regimes were the most stimulatory. We also showed that fluid flow produced higher magnitude and more abundant [Ca2+]i oscillations than spontaneous oscillations. Ultimately, understanding mechanotransduction pathways in bone cells and how they are influenced by age and mechanical loading parameters may help elucidate the etiologies of bone diseases such as senile and disuse osteoporoses.


    ACKNOWLEDGEMENTS

This work was supported by National Institutes of Health Grants AR-45989, AG-13087, AG-00811, and AG-17021 and by United States Army Grant DAMD 17-98-1-8509.


    FOOTNOTES

Address for reprint requests and other correspondence: S. W. Donahue, Dept. of Biomedical Engineering, Michigan Technological Univ., 312 Chemical Sciences Bldg., 1400 Townsend Drive, Houghton, MI 49931-1295 (E-mail: swdonahu{at}mtu.edu).

The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

Received 20 February 2001; accepted in final form 10 July 2001.


    REFERENCES
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

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