different loading frequencies on bone formation rate in vivo. The right tibiae of adult female rats were subjected to applied bending at frequencies of 0.05, 0.1 ...
RESEARCH COMMUNICATIONS
Mechanotransduction
in bone: do bone cells act as sensors
of
fluid flow? CHARLES
H. TURNER,1
Department
of Orthopaedic
Surgery,
Orthopaedic
Engineering
and Research
When
ABSTRACT
compact
ing loads, interstitial from regions of high
MARK
R. FORWOOD, Indiana Center,
bone is subjected
fluid in the bone matrix compressive stress. The
Helen
to bendflows away amount of
interstitial fluid flow is strongly influenced by the loading rate in a dose-dependent fashion. We hypothesize that interstitial fluid flow affects bone formation, and we tested this
hypothesis
indirectly
by
measuring
the
effect
of
different loading frequencies on bone formation rate in vivo. The right tibiae of adult female rats were subjected to applied bending at frequencies of 0.05, 0.1, 0.2, 0.5, 1.0, and 2.0 Hz for a 2-wk period. The rats were then killed and histomorphometric measurements of bone formation were made of the midshaft of the tibia, Bending of the tibia increased bone formation rate in the higherfrequency (0.5 to 2.0 Hz) loading groups as much as fourfold, yet no increase in bone formation rate was observed for loading frequencies below 0.5 Hz. In a separate experiment, we found stress-generated potentials the rat tibia to increase monotonically with
MARK
AND
University
(SGP) in increasing
loading frequency. The dose-response relationship between loading frequency and the bone formation response closely resembles the relationship between loading frequency and SGP within bone. The qualitative similarity between these two relationships suggests that increased bone formation is associated with increased SGP, which are caused by interstitial fluid flow. Bone cells are known to be sensitive to electric fields and may respond directly to SGP. Also, fluid shear forces have been shown to stimulate bone cells in culture, so it is possible that increased interstitial fluid flow directly affects bone formation.Turner, C. H., Forwood, M. R., Otter, M. W. Mechanotransduction in bone: do bone cells act as sensors of fluid flow? FASEBJ. 8: 875-878; 1994.
Medical
Hayes
Hospital,
. bone adaptation . histomorphometry
frequency
rats
not
19TH
CENTURY,
stress
was
ANATOMIST
responsible
Julius
Wolff
proposed
for determining important, that the
that the ar-
chitecture of bone and, more form of bone is related to mechanical stress by a mathematical law (1). For the past century, WolfFs law has become widely accepted if not scientifically proven (2). The prevailing view is that bone cells, namely osteoblasts and osteocytes, act as sensors of deformation in bone tissue that signal an anabolic response if a deformation threshold is surpassed (3, 4). In support of this view, experimental studies have shown that bone loss in weight-bearing limbs occurs during spaceflight or when a limb is placed in a cast (5-9), and bone hypertrophy occurs when bone is subjected to increased mechanical loads (10-14). However, there are many inconsistencies in the current paradigm. For instance, increased bone formation does not always occur near the tissue where deformation
0892-6638/94/0008-0875/$01.50.
© FASEB
Haverstraw,
46202, USA; and New York 10993, USA
Indiana
act
primarily
as sensors
of bone tissue deformation
but
as sensors of an indirect effect of tissue deformation interstitial fluid flow. Compact bone is similar to a stiff, water-soaked sponge. A compressive force on one side of the sponge will push water out the other side. The velocity at which the water moves through the sponge is related to the rate at which the force is applied. In bone that is loaded in bending, the stressgenerated electric potential, which is produced by the electrokinetic phenomenon known as streaming and therefore related to fluid flow, increases monotonically with increasing loading frequency (20-24). Theoretically the fluid shear forces generated on bone cells by stress-generated fluid flow are similar to those on vascular endothelial cells (25). Therefore, it is probable that extracellular fluid shear forces in
bone are of sufficient cause
new
goal
increasing formation. at frequencies
IN THE mechanical
Indianapolis, West
is the greatest (15). Simulated weightlessness experiments using 6#{176} head-down tilt bed rest with human subjects or tail suspension in rats have demonstrated increases in bone mass in the skull and mandible after a period of several weeks (16, 17). These changes cannot be explained by Wolff’s law because the bone tissue deformation in the skull should not have been increased significantly during head-down tilt. These results may possibly be caused by the fluid shifts that occur in head-down tilt experiments and the associated positive shift in interstitial pressure in the head leading to increased perfusion of the bone. Bone loss in the hindlimbs of rats subjected to tail suspension is associated with decreased fluid movement through the bone (18). It has been shown that bone cells respond to fluid shear forces much like endothelial cells (19), although the sensitivity to fluid shear is greater in bone cells. These results suggest that bone cells do
The Key Words:
W. OTTER
Center,
(Hz).
bone
magnitude
to activate
bone cells and
formation.
of the present
study
was to determine
whether
the rate of applied loading leads to increased bone Bending forces were applied to the tibiae of rats of 0.05,
Measurements
made during similar flow were compared
0.1, 0.2, 0.5,
1, and 2 cycles per second
of stress-generated
fluid
flow were
loading conditions and changes in fluid with bone formation measurements.
METHODS One hundred twenty-eight female, used for this study. All procedures
8-month-old throughout
Sprague-Dawley this experiment
rats were conformed
‘To whom correspondence should be addressed, at: Department of Orthopaedic Surgery, Indiana University Medical Center, 541 Clinical Dr., Rm 600, Indianapolis, IN 46202, USA. 2Abbreviations: SGP, stress-generated potential (or streaming potentials caused by mechanical stress); BFR, bone formation rate; BS, bone surface.
875
RESEARCH
COMMUNICATIONS
with the guidelines of the Animal Care and Use Committees from Indiana University and Helen Hayes Hospital, as set by the National Institutes of Health.
Streaming
potential
measurements
Streaming potential measurements were made cx vivo at Helen Hayes Hospital on the tibias of six rats, as an indirect measurement of bone fluid flow during cyclical bending. Within minutes after the rats were killed, whole limbs were excised at the hip and soft tissues were removed from the tibiae. Electrodes were attached to the medial and lateral surfaces of the mid-diaphyseal tibial shaft within 30 s of removal of soft tissue, and bones were immediately placed into a humidity chamber set to maintain relative humidity above 95%. Testing began within 4 mm of soft tissue removal and was completed after about 20 mm. Sintered Ag/AgCl pellet electrodes (205A In Vivo Metrics, Healdsburg, Calif.), protruding from the end of 4-40 nylon bolts, were held in place by a Delrin electrode holder that had a soft, closed-cell silicone rubber underpad, with circular cutout to provide an electrical seal around each electrode site. Electrical contact between the electrode and bone was made via a small cotton plug, saturated with buffered (to pH 7.0) saline. A materials test system (MTS 810, MTS Systems Corp., Minneapolis, Minn.) was used to load the tibiae in sinusoidal four-point bending in displacement control (displacement was 0.2 mm) at frequencies ranging from 0.1 to 30 Hz. Stressgenerated potential (SGP)’ data were preamplified and stored on a digital oscilloscope (Model 4094A, Nicolet, Burlington, Mass.). In-house software using Fast Fourier Transform routines was used to determine the magnitude of SOP per stroke at each frequency.
Bone
formation
measurements
RESULTS
Stress-generated potentials, generated by four-point bending, in the rat tibia increased monotonically with increased loading frequency (Fig. 1). As SGP are caused by bone fluid flow, this result indicates that bone fluid flow was progressively increased by increased loading frequency. Bone formation rates in the control (left) tibiae of the rats were significantly different among the experimental groups (P < 0.001, ANOVA), suggesting group differences in the baseline bone formation rates. Because of these differences, measurements in the loaded (right) tibiae were normalized by subtracting the corresponding measurement in the contralateral (control) tibia. Relative bone formation rate (BFR rt tibia - BFR lft tibia) was significantly higher with bending
Vol. 8
August
1994
V
6
N
4 0
z
2 C 0.1
0.05
0.2
0.5
1
2
5
LoadingFrequency
10
50
20
(Hz)
Figure
1. Stress-generated potential (SGP), generated by fourpoint bending of the rat tibia, increases monotonically with increased loading frequency. SGP is directly related to fluid flow in the bone. Similar results have been shown by others for human, bovine, and canine bone (22-24). SGP was normalized by the maximum bending displacement of the bone in millimeters. Error bars indicate standard deviation of the mean.
compared
Bone formation measurements were made at Indiana University on 122 rats. The rats were divided into 10 groups of 10 each and 2 groups of 11 each (average body wt was 308 ± 38 g). Of the 122 rats entering the study, specimens were analyzed from 114 animals. Three animals died from complications after anesthesia and five animals had missing bone labels. Rat weights did not change significantly over the course of the study (P - 0.5, paired test: data not shown). All rats were exposed to either bending or sham loading of their right tibiae. Bending loads were applied to the right tibia through a four-point loading apparatus (13), placing the lateral surface of the tibia in compression and the medial surface in tension. Sham loads were applied at the same magnitudes as bending forces but the loading pads were arranged so that they squeezed the leg without creating significant bending moments (13). Loading was applied as a sine wave at a magnitude of 52 N for 36 cycles per day. The frequency of applied load was either 0.05, 0.1, 0.2, 0.5, 1.0, or 2.0 Hz; therefore, the duration of loading varied from 12 mm/day (0.05 Hz) to 18 s/day (2.0 Hz). Loading was applied by an open-loop, stepper motordriven spring linkage. The loading apparatus incorporated a load cell so the applied load on each rat tibia could be monitored (applied loads varied by no more than ± 1 N). Before each loading session, rats were anesthetized by ether inhalation; between loading sessions they were allowed normal cage activity. Rats were given an i.p. injection of calcein green (7 mg/kg, Sigma Chemicals, St. Louis, Mo.) on days 5 and 12 and were killed on day 15. After death, the rats’ left and right tibiae were removed, embedded in plastic, sectioned transversely through the midshaft in the region of maximum bending moment (about 6 mm proximal to the tibiofibular junction), and analyzed microscopically using reflected UV light at x150 and x300 magnification. Bone formation rate (BFR/BS) was measured on the endocortical bone surface using a Bioquant semiautomatic digitizing system (R & M Biometrics, Nashville, Tenn.) attached to a Nikon Optiphot fluorescence microscope.
876
12 E > 10 E 0 8 C!, U)
groups
with sham (Fig.
significant However, relative
2).
The
loading 0.05,
in the 0.5,
0.1, and
0.2
1.0, and 2.0 Hz groups
showed
no
increase in relative bone formation with bending. sham and bending loading at 0.05 Hz decreased bone
formation.
DISCUSSION
The results show that bone formation is stimulated far more effectively by loading applied at frequencies of 0.5 Hz or greater.
Bone
formation
was decreased
by loading
applied
at
0.05 Hz, probably due to the long duration (12 mm) over which the loading was applied. Rats in the 0.05 Hz groups showed signs of decreased blood perfusion in the lower extremities, as evidenced by skin pallor and decreased skin temperature, immediately after the loading was applied. Blood flow apparently returned to normal within minutes after loading, but the 12 mm of decreased perfusion during loading may have caused local ischemia or possibly decreased the normal flow of extracellular fluid past bone cells, resulting in decreased bone formation. The association between increased loading duration and decreased bone formation raises the possibility that the lack of bone formation response for loading below 0.5 Hz may result
from
increased
loading
duration
and
not
decreased
loading frequency. We tested this possibility in another experiment (C. H. Turner and M. R. Forwood, unpublished results) by exposing rat tibiae to bending at 2 Hz for durations ranging from 36 to 360 s (the 18 s group from the current
study
was
included
in the analysis).
The
results
showed
that increasing the duration of loading did not decrease, but significantly increased, bone formation rate (Fig. 3). Furthermore, the association between increased loading duration and decreased bone formation was not seen in the sham loading groups when the 0.05 Hz group was removed (P = 0.27, ANOVA). Therefore, increased loading duration cannot be responsible for the lack of bone formation response below 0.5 Hz in Fig. 2. After exduding the effect of increased loading duration, there are two possible explanations for the relationship between loading frequency and an increase in bone formation rate. First, bone cells may be more sensitive to higher-
The FASEB journal
TURNER
ET AL.
RESEARCH COMMUNICATIONS frequency mechanical stimuli. Second, increasing loading frequency increases the extracellular fluid flow within the bone tissue, which increases fluid shear forces on bone cells and generates voltage gradients (SGP, ref 26) that may activate resting bone cells. Based on present evidence, the latter explanation seems most probable because culture experiments have shown no definitive evidence for loading frequency sensitivity of bone cell response. In culture, bone cells increase second-messenger production and increase cell proliferation in response to static mechanical loads (27, 28), yet static loads have not been shown to stimulate bone formation in vivo (29). Also, fluid flow producing static shear stresses on osteoblasts in culture increased cAMP production up to 12-fold and the response was proportional to the magnitude of shear stress (19). Furthermore, no loading frequency dependence could be shown for bone cells in culture loaded between 0.5 and 3.0 Hz (30), but loading frequencies lower than 0.5 Hz were not tested. These studies and others have led to the conclusion that bone cells are sensitive to the magnitude of deformation, not the frequency at which the deformation occurs (31). Therefore, one cannot explain the frequency dependence of bone formation in our experiment based on frequency sensitivity of bone cells. A more plausible explanation of our results inwlves the interaction between the solid and fluid compartments of the bone. As fluid flows through bone, a charge is convected with the fluid giving rise to electric potentials. When the fluid flow is caused by pressure gradients resulting from mechanical loading the resulting electric potentials are called SGP (21). Ex vivo experiments show SGP to increase monotonically within the same range of loading frequencies in which increases in bone formation were observed (Fig. 1). Although it has been hypothesized that the SGP have a direct effect on bone cells (26), SGP result from extracellular fluid flow within bone, which may play an even more important role in mechanotransduction. Recent studies suggest that it may be the fluid flow itself that triggers bone cells rather than the SOP. Fluid shear stress may directly stimulate secondmessenger production in osteoblasts independently of streaming electrical potentials (19). Furthermore, theoretical calculations of stress-generated fluid flow in the canaliculi of
E E a
U>
0
cc 18
36
72
180
360
Duration of Loading (sec) Figure
3. When loading is applied at 2 Hz, creases almost threefold if loading duration 360 s. BFR was significantly increased at a s (asterisk) compared with 18 and 36 s
bone formation
rate in-
is increased from 18 to loading duration of 360 of loading (P < 0.05,
Fisher’s protected least significant difference test, C. H. Turner and M. R. Forwood, unpublished data), which suggests that the decreased bone formation rate associated with lower loading frequencies was not caused by increased loading duration.
bone predict fluid shear stresses of a magnitude similar to that shown to cause bone cell response in vitro (25). We conclude that bone formation increases in a doseresponse manner in response to mechanical loading of increasing frequencies. This response may be caused by increased strain-generated, extracellular fluid flow within the bone that increases with increasing loading frequency. The qualitative similarities between measurements of stressgenerated potentials in the rat tibia (Fig. 1) and the frequency dependence of bone formation rate (Fig. 2) support our conclusions. This
work
was
(#AR40688). M. tralian National Fairley Fellowship. detail
in the
supported
by the
R. F.’s participation Health and Medical We thank Deanna
embedding
and
sectioning
National
Institutes
was supported
of Health
by an Aus-
Research Council N. Privette for her attention of bone
H. to
specimens.
REFERENCES
Loadng Frequency (Hz) Figure at lower
2. Bone formation rate (BFR) was not increased loading frequencies, but increased significantly
by loading at higher
frequencies (P < 0.001, ANOVA). Sham loading caused no increase in relative BFR, but bending increased relative BFR (BFR s-Stibia-BFR lft tibia) significantly compared with sham loading for loading frequencies of 0.5, 1.0, and 2.0 Hz. Asterisks signify significant increases in BFR for bending compared ing, P < 0.05, Fisher’s protected least significant Error bars indicate standard error of the mean.
MECHANOTRANSDUCTION
IN BONE
with sham difference
loadtest.
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mt.
21. Salzstein, R. A., Pollack, S. R., Malt, A. F. T, and Petrov, N. (1987) Electromechanical potentials in cortical bone - I. A continuum approach. j Biomech. 20, 261-270 22. Salzstein, R. A., and Pollack, S. R. (1987) Electromechanical potentials in cortical bone - II. Experimental analysis. j Biomec/z. 20, 271-280 23. Scott, G. C., and Korostoff, E. (1990) Oscillatory and step response electromechanical phenomena in human and bovine bone. j Biomech. 23, 127-143 24. Otter, M. W., Palmieri, V. R., Wu, D. D., Seiz, K. G., MacGinitie, L. A., and Cochran, G. V. B. (1992) A comparative analysis of streaming potentials in vivo and in vitro. j Orthop. R.es. 10, 710-719 25. Weinbaum, S., Cowin, S. C., and Zeng, Y. (1994) A model for the excitation of osteocytes by mechanical loading-induced fluid shear stresses. j Biomech. 27, 339-360 26. Eriksson, C. (1974) Streaming potentials and other water-dependent effects in mineralized tissues. Ann. N.Y Acad. &i. 238, 321-338 27. Somjen, D., Binderman, I., Berger, E., and Harell, A. (1980) Bone remodelling induced by physical stress is prostaglandin E2 mediated. Biochim. Biop/zys. Acta 627, 91-100 28. Binderman, I., Shimshoni, Z., and Somjen, D. (1984) Biochemical pathways involved in the translation of physical stimulus into biological message. Ca/cit Tissue mt. 36, S82-S85 29. Lanyon, L. E., and Rubin, C. T. (1984) Static vs. dynamic loads as an influence on bone remodelling. j Biomech. 17, 897-905 30. Jones, D. B., Nolte, H., Scholubbers, J-G., Turner, E., and Veltel, D. (1991) Biochemical signal transduction of mechanical strain in osteoblast-like cells. Biomaterials 12, 101-110 31. Burger, E. H., and Veldhuijzen, J. P. (1993) Influence of mechanical factors on bone formation, resorption and growth in vitro. In Bone Growth - B (Hall, B. K., ed) pp. 37-56, CRC Press, Boca Raton, Florida Received for publication February 11, 1994. Accepted for publication April 28, 1994.
ASPET Colloquium Structure
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