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
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ABSTRACT |
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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
mechanotransduction; osteoblast; calcium signaling; bone adaptation
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INTRODUCTION |
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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
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METHODS |
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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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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.
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RESULTS |
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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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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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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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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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DISCUSSION |
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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
[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
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
Comparisons with in vivo data are further complicated when the
influence of frequency and shear stress on Ca
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
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
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ACKNOWLEDGEMENTS |
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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.
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FOOTNOTES |
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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.
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REFERENCES |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
1.
Ajubi, NE,
Klein-Nulend J,
Alblas MJ,
Burger EH,
and
Nijweide PJ.
Signal transduction pathways involved in fluid flow-induced PGE2 production by cultured osteocytes.
Am J Physiol Endocrinol Metab
276:
E171-E178,
1999
2.
Allen, FD,
Hung CT,
Pollack SR,
and
Brighton CT.
Serum modulates the intracellular calcium response of primary cultured bone cells to shear flow.
J Biomech
33:
1585-1591,
2000[ISI][Medline].
3.
Ashizawa, N,
Nonaka K,
Michikami S,
Mizuki T,
Amagai H,
Tokuyama K,
and
Suzuki M.
Tomographical description of tennis-loaded radius: reciprocal relation between bone size and volumetric BMD.
J Appl Physiol
86:
1347-1351,
1999
4.
Beit-Or, A,
Nevo Z,
Kalina M,
and
Eilam Y.
Decrease in the basal levels of cytosolic free calcium in chondrocytes during aging in culture: possible role as differentiation-signal.
J Cell Physiol
144:
197-203,
1990[ISI][Medline].
5.
Berridge, MJ,
Bootman MD,
and
Lipp P.
Calciuma life and death signal.
Nature
395:
645-648,
1998[ISI][Medline].
6.
Berridge, MJ,
and
Galione A.
Cytosolic calcium oscillators.
FASEB J
2:
3074-3082,
1988
7.
Burger, EH,
and
Klein-Nulend J.
Mechanotransduction in bonerole of the lacuno-canalicular network.
FASEB J
13, Suppl:
S101-S112,
1999
8.
Burger, EH,
Klein-Nulend J,
van der Plas A,
and
Nijweide PJ.
Function of osteocytes in bonetheir role in mechanotransduction.
J Nutr
125:
2020S-2023S,
1995[Medline].
9.
Chen, NX,
Ryder KD,
Pavalko FM,
Turner CH,
Burr DB,
Qiu J,
and
Duncan RL.
Ca2+ regulates fluid shear-induced cytoskeletal reorganization and gene expression in osteoblasts.
Am J Physiol Cell Physiol
278:
C989-C997,
2000
10.
Cowin, SC,
Weinbaum S,
and
Zeng Y.
A case for bone canaliculi as the anatomical site of strain generated potentials.
J Biomech
28:
1281-1297,
1995[ISI][Medline].
11.
Donahue, HJ.
Gap junctional intercellular communication in bone: a cellular basis for the mechanostat set point.
Calcif Tissue Int
62:
85-88,
1998[ISI][Medline].
12.
Donahue HJ, Li Z, and Zhou Z. Parathyroid hormone regulation of
gap junction expression and function in osteoblastic cells from young,
mature and old rats. 46th Annual Meeting of the Orthopaedic
Research Society, Orlando, FL, 2000, p. 689.
13.
Donahue, HJ,
McLeod KJ,
Rubin CT,
Andersen J,
Grine EA,
Hertzberg EL,
and
Brink PR.
Cell-to-cell communication in osteoblastic networks: cell line-dependent hormonal regulation of gap junction function.
J Bone Miner Res
10:
881-889,
1995[ISI][Medline].
14.
Donahue, HJ,
Zhou Z,
Li Z,
and
McCauley LK.
Age-related decreases in stimulatory G protein-coupled adenylate cyclase activity in osteoblastic cells.
Am J Physiol Endocrinol Metab
273:
E776-E781,
1997
15.
Duncan, RL,
and
Turner CH.
Mechanotransduction and the functional response of bone to mechanical strain.
Calcif Tissue Int
57:
344-358,
1995[ISI][Medline].
16.
Haapasalo, H,
Sievanen H,
Kannus P,
Heinonen A,
Oja P,
and
Vuori I.
Dimensions and estimated mechanical characteristics of the humerus after long-term tennis loading.
J Bone Miner Res
11:
864-872,
1996[ISI][Medline].
17.
Hung CT, Pollack SR, Reilly TM, and Brighton CT. Real-time calcium
response of cultured bone cells to fluid flow. Clin Orthopaed Rel
Res: 256-269, 1995.
18.
Jacobs, CR,
Yellowley CE,
Davis BR,
Zhou Z,
Cimbala JM,
and
Donahue HJ.
Differential effect of steady versus oscillating flow on bone cells.
J Biomech
31:
969-976,
1998[ISI][Medline].
19.
Jacobs, CR,
Yellowley CE,
Nelson DV,
and
Donahue HJ.
Analysis of time-varying biological data using rainflow cycle counting.
Comput Methods Biomech Biomed Eng
3:
31-40,
2000[Medline].
20.
Joldersma, M,
Burger EH,
Semeins CM,
and
Klein-Nulend J.
Mechanical stress induces COX-2 mRNA expression in bone cells from elderly women.
J Biomech
33:
53-61,
2000[ISI][Medline].
21.
Jorgensen, NR,
Geist ST,
Civitelli R,
and
Steinberg TH.
ATP- and gap junction-dependent intercellular calcium signaling in osteoblastic cells.
J Cell Biol
139:
497-506,
1997
22.
Jorgensen, NR,
Henriksen Z,
Brot C,
Eriksen EF,
Sorensen OH,
Civitelli R,
and
Steinberg TH.
Human osteoblastic cells propagate intercellular calcium signals by two different mechanisms.
J Bone Miner Res
15:
1024-1032,
2000[ISI][Medline].
23.
Kiebzak, GM,
Smith R,
Gundberg CC,
Howe JC,
and
Sacktor B.
Bone status of senescent male rats: chemical, morphometric, and mechanical analysis.
J Bone Miner Res
3:
37-45,
1988[ISI][Medline].
24.
Kirischuk, S,
Pronchuk N,
and
Verkhratsky A.
Measurements of intracellular calcium in sensory neurons of adult and old rats.
Neuroscience
50:
947-951,
1992[ISI][Medline].
25.
Klein-Nulend, J,
Semeins CM,
Ajubi NE,
Nijweide PJ,
and
Burger EH.
Pulsating fluid flow increases nitric oxide (NO) synthesis by osteocytes but not periosteal fibroblastscorrelation with prostaglandin upregulation.
Biochem Biophys Res Commun
217:
640-648,
1995[ISI][Medline].
26.
Knothe Tate, ML,
Knothe U,
and
Niederer P.
Experimental elucidation of mechanical load-induced fluid flow and its potential role in bone metabolism and functional adaptation.
Am J Med Sci
316:
189-195,
1998[ISI][Medline].
27.
Kufahl, RH,
and
Saha S.
A theoretical model for stress-generated fluid flow in the canaliculi-lacunae network in bone tissue.
J Biomech
23:
171-180,
1990[ISI][Medline].
28.
Leblanc, AD,
Schneider VS,
Evans HJ,
Engelbretson DA,
and
Krebs JM.
Bone mineral loss and recovery after 17 weeks of bed rest.
J Bone Miner Res
5:
843-850,
1990[ISI][Medline].
29.
Lewis, PL,
Brewster NT,
and
Graves SE.
The pathogenesis of bone loss following total knee arthroplasty.
Orthop Clin North Am
29:
187-197,
1998[ISI][Medline].
30.
Marchetti, ME,
Houde JP,
Steinberg GG,
Crane GK,
Goss TP,
and
Baran DT.
Humeral bone density losses after shoulder surgery and immobilization.
J Shoulder Elbow Surg
5:
471-476,
1996[Medline].
31.
Martin, RB,
and
Atkinson PJ.
Age and sex-related changes in the structure and strength of the human femoral shaft.
J Biomech
10:
223-231,
1977[ISI][Medline].
32.
McAllister, TN,
and
Frangos JA.
Steady and transient fluid shear stress stimulate NO release in osteoblasts through distinct biochemical pathways.
J Bone Miner Res
14:
930-936,
1999[ISI][Medline].
33.
Neter, J,
Kunter MH,
Nachtsheim CJ,
and
Wasserman W.
Applied Linear Statistical Models. Chicago: Irwin, 1996, p. 756-768.
34.
Parfitt, AM.
Age-related structural changes in trabecular and cortical bone: cellular mechanisms and biomechanical consequences.
Calcif Tissue Int
36, Suppl1:
S123-S128,
1984[ISI][Medline].
35.
Rawlinson, SC,
Pitsillides AA,
and
Lanyon LE.
Involvement of different ion channels in osteoblasts' and osteocytes' early responses to mechanical strain.
Bone
19:
609-614,
1996[ISI][Medline].
36.
Reich, KM,
and
Frangos JA.
Effect of flow on prostaglandin E2 and inositol trisphosphate levels in osteoblasts.
Am J Physiol Cell Physiol
261:
C428-C432,
1991
37.
Roholl, PJ,
Blauw E,
Zurcher C,
Dormans JA,
and
Theuns HM.
Evidence for a diminished maturation of preosteoblasts into osteoblasts during aging in rats: an ultrastructural analysis.
J Bone Miner Res
9:
355-366,
1994[ISI][Medline].
38.
Ryder, KD,
and
Duncan RL.
Parathyroid hormone modulates the response of osteoblast-like cells to mechanical stimulation.
Calcif Tissue Int
67:
241-246,
2000[ISI][Medline].
39.
Savineau, JP,
and
Marthan R.
Cytosolic calcium oscillations in smooth muscle cells.
News Physiol Sci
15:
50-55,
2000
40.
Smalt, R,
Mitchell FT,
Howard RL,
and
Chambers TJ.
Induction of NO and prostaglandin E2 in osteoblasts by wall-shear stress but not mechanical strain.
Am J Physiol Endocrinol Metab
273:
E751-E758,
1997
41.
Takahashi, A,
Camacho P,
Lechleiter JD,
and
Herman B.
Measurement of intracellular calcium.
Physiol Rev
79:
1089-1125,
1999
42.
Thomas, AP,
Bird GS,
Hajnaoczky G,
Robb-Gaspers LD,
and
Putney JW, Jr.
Spatial and temporal aspects of cellular calcium signaling.
FASEB J
10:
1505-1517,
1996
43.
Toescu, EC.
Temporal and spatial heterogeneities of Ca2+ signaling: mechanisms and physiological roles.
Am J Physiol Gastrointest Liver Physiol
269:
G173-G185,
1995
44.
Turner, CH,
and
Forwood MR.
Bone adaptation to mechanical forces in the rat tibia.
In: Bone Structure and Remodeling, edited by Odgard A,
and Weinans H.. River Edge, NJ: World Scientific, 1995, p. 65-77.
45.
Turner, CH,
Forwood MR,
Rho JY,
and
Yoshikawa T.
Mechanical loading thresholds for lamellar and woven bone formation.
J Bone Miner Res
9:
87-97,
1994[ISI][Medline].
46.
Turner, CH,
Takano Y,
and
Owan I.
Aging changes mechanical loading thresholds for bone formation in rats.
J Bone Miner Res
10:
1544-1549,
1995[ISI][Medline].
47.
Vander Molen, MA,
Donahue HJ,
Rubin CT,
and
McLeod KJ.
Osteoblastic networks with deficient coupling: differential effects of magnetic and electric field exposure.
Bone
27:
227-231,
2000[ISI][Medline].
48.
Vander Molen, MA,
Rubin CT,
McLeod KJ,
McCauley LK,
and
Donahue HJ.
Gap junctional intercellular communication contributes to hormonal responsiveness in osteoblastic networks.
J Biol Chem
271:
12165-12171,
1996
49.
Vico, L,
Collet P,
Guignandon A,
Lafage-Proust MH,
Thomas T,
Rehaillia M,
and
Alexandre C.
Effects of long-term microgravity exposure on cancellous and cortical weight-bearing bones of cosmonauts.
Lancet
355:
1607-1611,
2000[ISI][Medline].
50.
Weinbaum, S,
Cowin SC,
and
Zeng Y.
A model for the excitation of osteocytes by mechanical loading-induced bone fluid shear stresses.
J Biomech
27:
339-360,
1994[ISI][Medline].
51.
Yellowley, CE,
Jacobs CR,
and
Donahue HJ.
Mechanisms contributing to fluid-flow-induced Ca2+ mobilization in articular chondrocytes.
J Cell Physiol
180:
402-408,
1999[ISI][Medline].
52.
Yellowley, CE,
Jacobs CR,
Li Z,
Zhou Z,
and
Donahue HJ.
Effects of fluid flow on intracellular calcium in bovine articular chondrocytes.
Am J Physiol Cell Physiol
273:
C30-C36,
1997
53.
Yellowley, CE,
Li Z,
Zhou Z,
Jacobs CR,
and
Donahue HJ.
Functional gap junctions between osteocytic and osteoblastic cells.
J Bone Miner Res
15:
209-217,
2000[ISI][Medline].
54.
You, J,
Reilly GC,
Zhen X,
Yellowley CE,
Chen Q,
Donahue HJ,
and
Jacobs CR.
Osteopontin gene regulation by oscillatory fluid flow via intracellular calcium mobilization and activation of mitogen-activated protein kinase in MC3T3-E1 osteoblasts.
J Biol Chem
276:
13365-13371,
2001
55.
You, J,
Yellowley CE,
Donahue HJ,
Zhang Y,
Chen Q,
and
Jacobs CR.
Substrate deformation levels associated with routine physical activity are less stimulatory to bone cells relative to loading-induced oscillatory fluid flow.
J Biomech Eng
122:
387-393,
2000[ISI][Medline].