Bone as an ion exchange system: evidence for a link between
mechanotransduction and metabolic needs
A.
Rubinacci1,
M.
Covini2,
C.
Bisogni2,
I.
Villa1,
M.
Galli2,
C.
Palumbo3,
M.
Ferretti3,
M. A.
Muglia3, and
G.
Marotti3
1 Bone Metabolic Unit, Scientific Institute H San Raffaele,
20132 Milano; 2 Department of Bioengineering, Politecnico of
Milano, 20133 Milano; and 3 Department of Morphological
Sciences Human Anatomy, University of Modena, 41100 Modena, Italy
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ABSTRACT |
To detect whether the
mutual interaction occurring between the osteocytes-bone lining cells
system (OBLCS) and the bone extracellular fluid (BECF) is affected by
load through a modification of the BECF-extracellular fluid (ECF;
systemic extracellular fluid) gradient, mice metatarsal bones
immersed in ECF were subjected ex vivo to a 2-min cyclic axial load of
different amplitudes and frequencies. The electric (ionic) currents at
the bone surface were measured by a vibrating probe after having
exposed BECF to ECF through a transcortical hole. The application of
different loads and different frequencies increased the ionic current
in a dose-dependent manner. The postload current density subsequently
decayed following an exponential pattern. Postload increment's
amplitude and decay were dependent on bone viability. Dummy and static
loads did not induce current density modifications. Because BECF is
perturbed by loading, it is conceivable that OBLCS tends to restore
BECF preload conditions by controlling ion fluxes at the bone-plasma interface to fulfill metabolic needs. Because the electric current reflects the integrated activity of OBLCS, its evaluation in transgenic mice engineered to possess genetic lesions in channels or matrix constituents could be helpful in the characterization of the mechanical and metabolic functions of bone.
osteocytes; bone lining cells; mineral homeostasis; mechanical
loading; fluid shear stress
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INTRODUCTION |
THE MUTUAL
INTERACTION occurring between the osteocyte-bone lining cell
system (OBLCS) and the surrounding bone extracellular fluid (BECF) has
been suggested to play a pivotal role in bone mechanotransduction
(3, 37). OBLCS is constituted by a network of stellate
cells buried within the bone matrix, the osteocytes, having an
asymmetrical arborization of dendrites polarized toward the bone
surfaces, where they come into contact with the bone lining cells or
the osteoblasts according to whether the bone is in a resting or a
growing phase, respectively. Because the cells forming such a
three-dimensional protoplasmic network are all joined by gap junctions,
OBLCS actually constitutes a functional syncytium (24, 27,
26). The lacuno and canalicular network of cavities, enclosing
the osteocyte cell bodies and dendrites, forms within the bone matrix a
complex microstructure of pores and channels filled by BECF that has a
different ionic composition from the systemic extracellular fluid (ECF)
of the perivascular loose connective tissue surrounding the bone surfaces.
This ionic difference is maintained by a pump-leak system that
selectively operates at the OBLCS level (32, 33, 34) as a
partition system (4) generating an ionic gradient between BECF and ECF (21, 22, 23, 30) with a subsequent electric potential difference at the bone membrane (36) that
appears to be under parathyroid hormone control (29). BECF
is forced to flow through the osteocyte lacunocanalicular network as a
result of bone loading, thus generating the following two phenomena: 1) a shear stress of the cell membrane that subsequently
activates specific cells functions (3) and 2) a
streaming potential as a result of the tangential motion of the
ion-carrying fluid at the interface of the charged bone matrix
(11). Both phenomena could imply a modification of BECF by
affecting ion pump-leak systems and ionic charge distribution. This
hypothesis is supported by the observations that the strain over the
plasma membrane of the cells lying on the bone surface activates ion
channels specific for potassium (6, 39), calcium
(31, 38), and sodium (18), which are known to
have a concentration gradient between BECF and ECF
(21-23).
Because the BECF-ECF gradient is the driving force for the
endogenous ionic current in bone, it is likely that the strain-induced modification of BECF could in turn modify such a signal. The ionic current in bone was demonstrated by Borgens (2) in 1984, subsequently characterized for its origin and ionic dependence by this
laboratory (32, 33, 34), and found to give reliable
information on the ionic exchange occurring at the bone-plasma interface.
To explore the hypothesis that strain modifies the BECF-ECF gradient
and associated electric signal, metatarsal bones of weanling mice were
subjected ex vivo and immersed in ECF medium to a cyclic axial load of
varying amplitudes and frequencies, and the electric (ionic) currents
at the bone-plasma interface were measured by a voltage-sensitive
two-dimensional vibrating probe system before and after load. Specific
aims of this study were to detect whether a cyclic load applied to a
viable bone induces a consistent change of the ionic fluxes at the
bone-medium (mimicking plasma) interface and whether the change is
dependent on the physical characteristics of the load and the viability
of the bone cells. To assess the presence and viability of OBLCS,
metatarsal bones were anatomically evaluated by light microscopy (LM),
transmission electron microscopy (TEM), and scanning electron
microscopy (SEM) analysis.
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MATERIALS AND METHODS |
Incubation Media
The living metatarsal bones were immersed in a medium
reproducing the physiological concentrations of the ionic species
present in plasma and defined ECF. The dead metatarsal bones were
immersed both in ECF medium and in ECF medium that was made bicarbonate free by substituting bicarbonate ions with isethionate. Experiments were also performed by immersing bones in defined external BECF (BECFext) incubation medium, according to Miller
(20) and Neuman (21). This medium could not
exactly reproduce BECF ionic concentrations; however, the small
residual sodium gradient was not considered sufficient to affect
current density (Table 1). Reagents were purchased from Sigma (St. Louis, MO). Osmolarity of solutions was measured by an Osmostat Os 6020 pressure osmometer (Damchi, Kyoto,
Japan). Resistivity was measured at 37°C with a multirange conductivity meter (HI 9033; PBI International, Milano, Italy). All
experiments in living bones were performed at a controlled temperature
(37°C), and experiments in dead bones were performed at room
temperature. Temperature was monitored by a T801 thermoprobe (Radiometer, Copenhagen, Denmark). The pH was 7.37 ± 0.04 at
37°C for living and 7.33 ± 0.04 at room temperature for dead
bones. Medium stability was monitored by a
pH-Po2-Pco2 automatic analyzer (Instrumentation
Laboratory, Lexington, MA) on aliquots taken at intervals during
readings.
Bone Samples
Weanling mice (Swiss; Charles River), 26 ± 3 days old and
weighing 16.7 ± 3.6 g, were killed with CO2 in a
gas chamber (Techniplast, Varese, Italy). The back limbs were
amputated at the distal tibia epiphysis and immersed in ECF. The
metatarsal bones (~7 mm long and 0.5 mm thick) were carefully
dissected to avoid damage to the bone surface. All manipulations were
carried out on samples immersed in the medium with an M3 surgical
microscope (Wild, Zurich, Switzerland). After the bone was freed of
soft tissue ensheathments, a transcortical hole of ~200 µm was
made with a thin stainless steel dental drill (Mani; Matsutani
Seisakusho, Ken, Japan). The animal use was approved by the local
Institutional Animal Care and Use Committee (protocol no. TS 9501, updated IACUC no. 135).
Experimental Setup and Data Acquisition
Experimental setup and data acquisition have been described
previously (32, 33, 34). The detailed procedure has been published previously (33). Data were recorded before and
after having applied a load to the bone (see Experimental
protocol).
Histology
LM.
Some tested metatarsal bones and controlateral bones were used as
controls, fixed in 4% buffered paraformaldehyde and embedded in
methylmethacrylate. By means of a Leitz saw microtome, the metatarsal
bones were serially (550 µm apart) cross-sectioned (200 µm thick).
The undecalcified sections were then ground to uniform thickness of 60 µm and examined under the light microscope (Zeiss Axiophot).
SEM.
To visualize tridimensionally the osteocyte lacunocanalicular casts,
some ground cross sections were decalcified in 0.1 N HCl for 1 min,
rinsed in fresh water for 30 min, macerated in 3% NaOCI, alcohol
dehydrated, embedded in methylmethacrylate, gold coated, and observed
under SEM (Philips SEM-515).
TEM.
Undecalcified cross sections 2 mm thick, taken with an indented blade
from the middiaphysial level of some metatarsal bones, were fixed with
4% paraformaldehyde (0.1 M cacodylate buffer, pH 7.4) for 2 h,
postfixed for 1% osmium tetroxide (0.1 M cacodylate buffer),
dehydrated in graded ethanol, and embedded in epoxy resin (Durcupan
ACM). To better visualize ultrastructural cellular details (for
instance, gap junctions), some specimens were decalcified in 2.5% EDTA
(0.1 cacodylate buffer). The specimens were sectioned with a diamond
knife mounted in an Ultracut-Reichert microtome. Ultrathin sections
(70-80 nm) were mounted on Formvar-coated and carbon-coated copper
grids, stained with 1% uranylacetate and lead citrate, and examined
under TEM (Zeiss EM 109).
Mechanical Stimulator
The mechanical stimulator was improved with respect to the
original one designed by Lozupone (14). This late friend
not only was the first to study osteocyte metabolism in ex vivo
conditions under pulsing compressive stresses but also provided the
seminal intuition for the present investigation. The improved
Lozupone's stimulator is a device enabling a direct, axial, and
compressive mechanical load on metatarsal bones of weanling mice (Fig.
1). It consists of 1) a
special holder to maintain bone in a vertical position; 2) a
Plexiglas chamber filled with ECF that contains the holder and the
metatarsal; 3) a piston linked together with a series of
shafts; and 4) an electric engine (engine no. 26569; PBI
International) driven by an electric generator of voltage and current
[input: voltage (V) = 230 V alternating current/50 Hz;
output: V = 0/30 V direct current and I = 0/2 A; Labornetzgerät/Regulated Power Supply Laboratory,
Munich, Germany].
The bone was inserted in the holder with one epiphysis lying on the
inferior base and the other rising from the superior plane to be hit by
the piston during the cyclic load. The Plexiglas chamber was filled
with prewarmed (37°C) ECF until the whole bone was covered. To assure
the stability of the ECF physicochemical characteristics (pH,
temperature, osmolarity, Po2, and Pco2)
during loading, a layer of light, white mineral oil (Sigma) was used to
cover the free surface of the medium. Before being loaded, the whole
stimulator was calibrated to equilibrate every external force and
torque working on the system, like weight of every shaft, weight of the
piston, Archimede's force, and tie reactions. The equilibrated
position, assessed by a horizontal air bubble level, is the one that
nullifies torque and both vertical and horizontal forces. Once
equilibrium was reached, electrical loading parameters (voltage and
current) were set to obtain the appropriate loading frequency (varying
from 0 to 2 Hz). This parameter was controlled by a digital multimeter
(601 Digital Multimeter; Hung-Chang, Seoul, Korea) and was assessed by
a photometer (Dt-2236 Digital Photometer; Elbro, Zurich, Switzerland).
Subsequently, the piston was charged with appropriate weight (varying
from 0 to 12 g), positioned along the axis of the piston to avoid
any torque generation. During loading, the rotating movement generated
by the electrical engine was first transferred on an eccentric axis
(cam), converted in a translator movement by two shafts linked
together, and finally transmitted to the piston. Cyclic loading could
be divided in two alternating loading and unloading phases. In the
loading phase, when the piston moved down and touched the free
epiphyses, the weight acting on the tip of the piston was transmitted
to the underlying bone; in the unloading phase, the piston moved up, lifting the weight from the bone. Piston diameter was smaller than the
chamber diameter to avoid friction between the piston and chamber and
the generation of a direct hydrostatic pressure on bone.
Experimental Protocol
Metatarsal bones of weanling mice were placed in the
experimental chamber under microscopic control. After the spatial
distribution of current density over the injury site was tested and the
expected geometry of the signal was assessed, the probe was located at the point of maximal density (generally found over the center of the
hole), and the current density was measured before loading. Once
a steady-state signal was obtained, the current was defined as
Jpreload ss and was recorded.
Next, the bones, immersed in ECF at 37°C, were subjected for 2 min to
cyclic axial loads of varying amplitudes and frequencies. After being
loaded, the bones were placed again in the experimental chamber under
microscopic control, and the electric (ionic) current was monitored for
30 min. Because the first reliable measurement was obtained in all bones with a time delay (because of the handling procedures) ranging from 8 to 12 min, the maximal current was measured at 12 min after having stopped the load and was defined as
Jpostload 12. Readings obtained with longer
delay were discarded. Jpostload t was monitored over time until a new steady state was reached; steady
current was defined as Jpostload ss.
To assure that the current measurements were taken at the same
location before and after loading, the maximal current was searched, and the correct positioning of the bone was assessed by
digital photographic control of the microscopic image.
To evaluate the role played by the cells in generating the postload
signal, dead bones, fixed by immersion in buffered formaldehyde (24 h)
and reequilibrated in ECF for 5 days according to Borgens (2), were submitted to the same procedure as living bones; to assure the stability of the physicochemical conditions resulting from the long reequilibration time, the experiment was performed in
bones immersed in bicarbonate-free ECF at room temperature, and the
effect of the bicarbonate removal was tested by comparing the data
obtained both in the presence and in the absence of the ion.
The viability of the OBLCS in living bone was tested by LM and TEM
analysis at the end of the experiment.
The following control experiments were performed: 1) to
verify whether the electric (ionic) current at the hole site was
strictly dependent on the ionic concentration gradient at the BECF-ECF interface, bone was immersed in an external medium with ionic concentrations equal to BECF (BECFext); and 2)
to evaluate the sources of errors resulting from repositioning, dead
and living bones were measured before and after a dummy load by keeping
all of the other procedures consistent with the protocol.
Statistics
Data were analyzed with the statistical package Prism version
3.02 (GraphPad Software, San Diego, CA). Significance among groups was
assessed by means of one-way ANOVA for nonparametric values
(Kruskal-Wallis test) and a multiple comparison test (Dunn's test).
The energy-response curve was characterized using nonlinear regression
analysis. All data are expressed as means ± SD. P < 0.05 was considered significant.
Mathematical Analysis of the Experimental Data
Because the ionic current was recorded with a time delay at
~12 min after loading because of the experimental handling time, the
time-dependent decay of the current was mathematically analyzed to
derive the missing data. Computing was based on spline functions and
nonlinear best fit analysis to minimize the mean square error on the
measured values, as reported below (see APPENDIX).
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RESULTS |
Basal Electric (Ionic) Current Density: Values and Distribution
All tested living bones (n = 87) showed the
expected spatial distribution of current vectors at the site of damage
in unloaded conditions. The maximal current density vector was normal
to the bone surface, whereas, moving along the bone, current density decreased and current direction became progressively parallel to the
bone longitudinal axis (Fig.
2A). The current appeared inward by the convention discussed previously (33). In
preload conditions, maximal current density becomes steady after an
initial slow decay. Jpreload ss in all tested
bones averaged 14.5 ± 4.16 µA/cm2, ranging from
6.37 to 27.19 µA/cm2.

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Fig. 2.
Distribution pattern of preload (A) and
postload (B) current density at the damage site of living
metatarsal bone of weanling mice immersed in control medium. Bone
damage is clearly visible as a round hole at the diaphysial cortex.
Vectors represent density (length), direction (angle), and sign
(inward) of the net current. By convention, direction of the current
flow is that of a cation flux. The arrowheads correspond to the point
of measurement of the current. Background value (<0.5
µA/cm2) is recognizable by the arrowhead far from the
bone surface. The scale of current density (10 µA/cm2) is
reported at top left.
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Postload Electric (Ionic) Current Density: Values, Distribution,
and Time Pattern
After having subjected the metatarsal bones to a cyclic (1 Hz) load of 5 g for 2 min, postload current density retained the same spatial distribution as the preload current (Fig.
2B); Jpreload ss of 15.58 ± 4.64 µA/cm2 increased significantly (P < 0.00001; n = 11) by a factor of 1.6 ± 0.25 but
decreased over time to a steady state without reaching the preload
value in all but two bones. The missing data between the first reliable
measurement and that at the end of loading were derived according to
the procedure described in the APPENDIX and reported in
Fig. 3. Derived data indicate that
Jpreload ss was significantly increased by a
factor of 3.06 ± 0.58 with a subsequent time-dependent
exponential decay to a plateau level higher than basal by a factor of
1.24 ± 0.2.

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Fig. 3.
Single exponential interpolations of the current density
measurements taken after a cyclic (1 Hz) load of 5 g and their
mean (n = 9, = 10.05 ± 0.9 min, = 0.178/0.35). Each symbol represents the single measurement at that
time for a single bone. Vertical continuous bars indicate the time at
which the loading cycle was started and stopped. See text for
definiions.
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Relationships Between Postload Current Density and Loading
Parameters (Weight and Frequency)
By varying the load from 0.7 to 12 g without changing either
the loading frequency (1 Hz) or the time (2 min), the increment in
postload current density was dependent on the applied loads, reaching a
plateau at 8 g (Fig.
4A).
Derived data indicate that postload current densities decreased after
different time-dependent exponential decays until different asymptotic
values were reached, depending on the applied load. The load amplitude
was found to affect the time-dependent exponential decay and the level
of the new steady state in current density after the load (Fig.
4B). For loads
2 g, the asymptotic level was equal to the
basal preload value, whereas for loads of 12 g, the asymptotic
level was significantly (P < 0.01) higher than the
basal preload value (Fig. 4C).

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Fig. 4.
A: current density increment as a
function of different loads (0.7 g, n = 6; 2 g,
n = 6; 5 g, n = 11; 8 g,
n = 6; 10 g, n = 6; and 12 g,
n = 6) at a fixed loading frequency (1 Hz). Dummy loads
(0 g, n = 4) did not induce any change in postload
current density with respect to the preload value. B:
time-dependent exponential decay of the mean increment of the derived
postload current density as a function of different loads (0.7 g,
n = 4, = 13.5 ± 0.92 min; 2 g,
n = 4, = 12.5 ± 0.7 min; 5 g,
n = 9, = 10.05 ± 0.9 min; 8 g,
n = 4, = 8.6 ± 0.7 min; 10 g,
n = 4, = 8.2. ± 0.85 min; and 12 g,
n = 4, = 7.8 ± 0.6 min) at a fixed
loading frequency (1 Hz). C: increment of the steady level
reached over time by the postload current density as a function of
different loads (0.7 g, n = 6; 2 g,
n = 8; 5 g, n = 11; 8 g,
n = 7; 10 g, n = 6; and 12 g,
n = 6) at a fixed loading frequency (1 Hz). Data are
expressed as means ± SD. Difference were considered significant
at P < 0.05. *P < 0.05, **P < 0.01, and ***P < 0.001 vs.
control (0 g).
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By varying the loading frequency from a static load to 2 Hz without
changing either the applied load (5 g) or the time (2 min), the
increment in current density over the basal preload value was dependent
on the applied frequencies and reached saturation at 1.5 Hz (Fig.
5A).
Static load did not induce any change in the current density, which
remained steady throughout the experimental time. Derived data indicate
that loading frequency affects both the time-dependent exponential
decay and the level of the new steady state in current density after
the load (Fig. 5B). For frequency
0.33 Hz, the asymptotic
level was equal to the basal preload value, whereas, for frequency of
1.5 Hz, the asymptotic level was significantly (P < 0.05) higher than the basal preload value (Fig. 5C).

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Fig. 5.
A: current density increment as a function
of different loading frequencies (0 Hz, n = 4; 0.33 Hz,
n = 6; 1 H z, n = 11; 1.5 Hz,
n = 6; 1.75 Hz, n = 6; and 2 Hz,
n = 6) at a fixed load (5 g). Static load did not
induce any change in current density that remained steady throughout
the experimental time. B: time-dependent mean exponential
decay of the increment of the derived postload current density as a
function of different loading frequencies (0.33 Hz, n = 4, = 13.9 ± 0.8 min; 1 Hz, n = 9, = 10.05 ± 0.9 min; 1.5 Hz, n = 4, = 8.8 ± 0.4 min; 1.75 Hz, n = 4, = 8.3 ± 0.65 min; 2 Hz, n = 4, = 7.8. ± 0.7 min) at a fixed load (5 g). C: increment of
the steady level reached over time by the postload current density as a
function of different loading frequencies (0 Hz, n = 4;
0.33 Hz, n = 6; 1 Hz, n = 11; 1.5 Hz,
n = 6; 1.75 Hz, n = 6; and 2 Hz,
n = 6) at a fixed load (5 g). Data are expressed as
means ± SD. Difference were considered significant at
P < 0.05. *P < 0.05, **P < 0.01, and ***P < 0.001 vs.
control (0 Hz).
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Relationships Between Postload Current Density and Maximal
Transferred Energy
By considering that the total energy associated to the system in
each loading phase is defined by
with
where m is related to the applied load, v is
the velocity associated to the applied frequency, h is the
position of the load, and g is the gravity force, and, by
considering that Ep is time after time nullified
by the tie reaction of the mechanical stimulator, the only kind of
energy acting on the underlying bone is Ec.
By assuming that the biological system does not dissipate the
transferred energy in each loading phase within the short loading time,
it is possible to evaluate the total energy transferred to the
underlying bone during the complete loading cycle
with
where n is the total number of the loading phase during
the loading cycle, f is the frequency of the applied load,
and t is the loading time expressed in seconds
(t = 2 min = 120 s).
By applying the relationships described above, the postload increment
in current density was significantly (r = 0.78;
P < 0.001) associated with the total energy
transferred to bone during the entire cycle (Fig.
6A). For energy = 5.75 µJ, Jpostload ss was significantly
(P < 0.05) higher than the basal preload value (Fig.
6B). The same response was therefore obtained by subjecting bones to either loads of small amplitude and high loading frequency, or
vice versa.

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Fig. 6.
A: relationship between the current density
increment and the maximal energy transferred to the bone during the
loading cycle. B: increment of the steady level reached over
time by the postload current density as a function of different values
of the maximal energy transferred to the bone during the loading cycle.
Data are expressed as means ± SD. Difference were considered
significant at P < 0.05. *P < 0.05, **P < 0.01, and ***P < 0.001 vs.
control (0 µJ).
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Relationship Between Postload Current Density and Viability of Bone
Cells
Jpreload ss in dead unloaded bones was not
significantly different from the background level (
0.5
µA/cm2). No spatial distribution of the current foci was
detectable (Fig. 7A). However,
after having submitted dead bones to the axial cyclic load (1 Hz,
5 g), a postload current density was measured (Jpostload 12 = 7.62 ± 2.65 µA/cm2; n = 9). Vectors were not
symmetrically orientated to the site of damage and did not exhibit the
same distribution pattern of postload current density as in viable bone
(Fig. 7B). Jpostload t linearly decayed, approaching the background level within 60 min in all
tested bones.

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Fig. 7.
Distribution pattern of preload (A) and
immediately postload (B) current density at the damage site
of dead metatarsal bone of weanling mice immersed in control medium.
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No relationship between postload current density and the applied load
was observed (Fig. 8). Any
inference resulting from the absence of bicarbonate from the medium in
dead bones was subsequently tested and found not relevant because no
changes in postload current density and subsequent time-dependent decay
were found between dead bones immersed in ECF or bicarbonate-free ECF
(Fig. 9).

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Fig. 8.
Time pattern of the mean derived current density before
and after a cyclic (1 Hz) load of 5 g (n = 9) and
10 g (n = 6) in dead bones. No relationship was
observed between the postload current density and the amplitude of the
applied load.
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Fig. 9.
Values of the current density measured before and after a
cyclic (1 Hz) load of 5 g in dead bones immersed in extracellular
fluid (ECF) medium (n = 4) and in ECF bicarbonate-free
medium (n = 9) and time-dependent linear decay of the
mean derived postload current density.
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Control Experiments
Experiment 1.
When external medium (n = 10) was changed with
BECFext, current density was at first nullified; then it
increased without reaching the basal value unless bone was
again exposed to ECF medium (Fig. 10).

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Fig. 10.
Values of the current density over time, entering or leaving the
site of damage of metatarsal bones incubated in different media (see
MATERIALS AND METHODS). When ECF was changed with external
bone ECF (BECFext) current density was nullified at first
and then increased without reaching the basal value unless bone was
exposed to ECF medium again. Each symbol represents the single
measurement at that time for a single bone.
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Experiment 2.
Errors resulting from repositioning and handling procedures were tested
and found to be not relevant. Dummy loads did not induce any change in
the current density before and after load either in viable
(Jpreload ss = 15.9 ± 3.16 µA/cm2; Jpostload 12 = 15.8 ± 2.66 µA/cm2; n = 4) or in
dead (Jpreload ss = 0.22 ± 0.3 µA/cm2 ~background value;
Jpostload 12 = 0.2 ± 0.29 µA/cm2; n = 3) bones.
Histology
LM analysis showed that the shaft of metatarsal bones in weanling
mice is made up of a very thin cortex (70-120 µm thick) of woven
bone containing vascular canals (Fig.
11). As shown by SEM observation of the
osteocyte lacunocanalicular cast, the cortex of metatarsal bones, at
the level where the holes were drilled, contains a continuous network
of lacunocanalicular microcavities (Fig.
12). TEM analysis demonstrated that the
osteocytes have the typical globose shape of those in woven bone and
are interconnected by a network of dendrites that, at the bone
envelopes, comes into contact with osteoblasts or bone lining cells,
depending on whether the surface is growing or resting. Several gap
junctions were observed among osteocytes and between osteocytes and
bone lining cells or osteoblasts. Osteocytes submitted to mechanical
loads show a normal ultrastructure as those in control unloaded bones (Fig. 13).

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Fig. 11.
Light microscopy micrograph under transmitted ordinary
light (×104) of an undecalcified cross section at the middiaphyseal
level of the metatarsal bone in a weanling mouse.
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Fig. 12.
Scanning electron microscopy micrograph (×570) of a
cross section at the midshaft level of the metatarsal bone in a
weanling mouse showing the methylmethacrylate casts of the osteocyte
lacunocanalicular microcavities. Arrows point to two vascular canals
filled with methylmethacrylate.
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Fig. 13.
Transmission electron microscopy (×14,000) micrographs of
osteocytes inside the cortex of a control unloaded (A) and a
loaded (B; 5 g, 1 Hz for 2 min) metatarsal bone. The
loading cycle did not affect the normal cell ultrastructure.
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DISCUSSION |
This study shows that cyclic axial load increases the steady
electric (ionic) inward current driven by living damaged bone at the
damage site. The increment in current density was significantly related
to the amplitude of the loading parameters and consequently to the
maximal transferred energy. The steady component of the current was
transiently lost after load as postload current density exponentially
decayed over time, but it was subsequently reestablished at levels
significantly higher than basal for specific loads, frequencies, and
maximal transferred energies. Dead bones that did not drive current
higher than background values when unstressed showed a transient inward
electric (ionic) current at the damaged site after loading that was
significantly lower than that in the living bones, decayed linearly to
background level, and was unrelated to the different loads applied.
This study confirms that living unstressed bone drives ionic currents
with a specific distribution pattern when the cortex is damaged by
drilling a hole through the medullar cavity. A detailed description of
the origin and meaning of this current, originally discovered by
Borgens (2), was previously reported (32, 33, 34). Briefly, the ionic current is measured at the hole site where the negative electrical potential difference of BECF with respect
to ECF (36) is shorted out. Ions are therefore free to
move along their electrochemical gradient through this low-resistance pathway (point sink). The activation of a pump-leak mechanism, devoted
to the maintenance of the ionic composition of BECF despite the leak,
generates the detectable inward electric current at the point sink
that, according to the model of ion fluxes in bone (33),
represents the return pathway of the current loop originated at the
intact portions of bone where cations flow out of BECF along their
concentration gradient through the paracellular spaces. The electrical
signal is sustained over time by a driving force provided by the OBLCS
(34) that, by compartmentalizing BECF from ECF (4,
10, 19, 20), generates the electrochemical gradient at the
bone-plasma interface. This view is confirmed by the observation that
basal preload maximal current density was reduced to the background
level when the electrochemical gradient was nullified by using BECF as
the external medium (BECFext). By considering that current
density tended to recover after having nullified the gradient, it is
conceivable that OBLCS could sense the modification of the
electrochemical gradient and activate the pump-leak mechanism that in
turn could restore it. It can be therefore assumed that the
injury-induced "short circuit" reflects the integrated activity of OBLCS.
The Model
The interpretative model of the ion fluxes at the BECF-ECF
interface after cyclic loading is based on the model of ionic currents in unstressed conditions published previously (33). The
present model implies load-related modifications of the composition of BECF (Fig. 14, A and
B) based on the following two different mechanisms: one cell
dependent while the other is of a physicochemical nature. According to
Duncan and Turner (8), the cell-dependent mechanism is
triggered by the Poisson's effect as follows: when a long bone is
subjected to an axial load, it expands in the radial direction, thus
causing a biaxial strain field on the OBLCS embedded in the bone
matrix. The strain gradient caused by the loading creates extracellular
fluid flow through the continuous network of lacunocanalicular cavities that in turn determines a shear stress over the osteocytes and
cell processes that could gate the opening of mechanosensitive channels. By considering that 1) mechanosensitive channels
are a large family of selective and nonselective cations; 2)
ionic current in bone is dependent on cations (2, 33) and
anions (2, 32) that are known to display a concentration
gradient between BECF and ECF as potassium, calcium, and sodium
(5, 9, 35) as well as chloride and bicarbonate (2,
30, 32); and 3) ionic current in bone is sensitive to
the blockers of cation channels (33), it can be
hypothesized that BECF is modified under the load by the opening of
mechanosensitive cation channels. This view is confirmed by the
following observations: 1) because of the distribution of
ion channels and transporters as well as the ion gradients across the
membrane of bone lining cells or osteoblasts, potassium could only
leave from the intracellular stores to the mineral-facing side and,
subsequently, out from BECF through the intercellular spaces along its
concentration gradient (35); 2) because
potassium outward flux increases after cell membrane physical
distortion (6, 17); and 3) because cyclic
stretch of the cell membrane induces an increase both in sodium
(17) and in calcium (12, 38) intracellular
concentration. Potassium outward flux to the ECF and calcium and sodium
shift into the intracellular compartment constitute the basis for a transient cation depletion of BECF that increase its electronegativity with respect to ECF, i.e., the electrochemical gradient at the BECF-ECF
interface and, subsequently, the returning pathway of the current loop
at the point sink. This mechanism is operative only in viable bones and
may justify the increment in current density measured after having
stopped the loading cycle.

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Fig. 14.
A and B: model of ion fluxes at
the BECF-ECF interface represents cells of osteogenic lineage,
distribution of channels and transporters, and source and direction of
measured extracellular net current. The model indicates both the
electrical potential of BECF and ECF with respect to the earth and the
electronegativity of BECF with respect to ECF. A: electrical
potential of BECF with respect to the earth, which is dependent on the
concentration in BECF of the different ion species, could be expressed
before the application of the load as a function of the number of
opened ionic membrane channels (OC) and the activity levels of
still-undefined membrane pumps (P). B: after a loading
compressive cycle, the electrical potential of BECF with respect to the
earth is also dependent upon the opening of stretch-activated channels,
streaming potentials ( Vstreaming), and bone
surface charge modification (m) induced by the application
of the load. The electrical potential of ECF (infinite bath) with
respect to the earth can be considered constant before and after the
load. The electrical potential difference between BECF and ECF is
shorted out (V = 0) at the point sink where the two
fluids are exposed to one another because of the damage of bone
cortex.
|
|
It is therefore conceivable that the relationships between postload
current density and loading parameters (weight and frequency) may be
the result of the characteristics of the mechanosensitive channels that
display an activation threshold, depending on the characteristics of
the applied loads. In fact, the response of osteoblasts in culture to
mechanical loading would indicate that the osteoblasts respond
differently to different magnitudes of strain, thus implying a
sensitive mechanism that adjusts cellular functions to the mechanical
environment (7). This view would imply that the load could
modulate the opening of the stretch-activated channels and the
subsequent cascade of events leading to the observed modifications of
the electric (ionic) current in bone.
The physicochemical mechanism might be explained by the streaming
potential effect. In fact, mechanical deformation of wet bone generates
long sustained electrical signals induced by the slow extracellular
fluid motion throughout the interconnected fine porous structure of the
canalicular network (3, 11). The electric signals are
driven by the potential difference generated by the tangential motion
of the ionic fluid far from the bone surface and the ionic phase of the
shear plane constituted by the ions next to the electrically charged
bone matrix. Obviously, this mechanism is operative in both viable and
dead bones. It is, in fact, likely that the generation of the
strain-related electric signal in bone amplifies the potential
difference between BECF and ECF in the viable bone and creates an
electrochemical gradient at the BECF-ECF interface in the dead bone
that drives the current density as measured after the loading cycle.
The existence of an electrochemical gradient at the BECF-ECF interface
in viable bone was shown to depend on OBLCS viability (2, 4, 29,
34, 36). In fact, structural and ultrastructural analyses
reported here clearly demonstrated that the osteocytes enclosed within
the lacunocanalicular network and the bone lining cells (or
osteoblasts) along the bone surfaces display a quite normal appearance
in both unloaded and loaded metatarsals, thus confirming that all
anatomical assumptions are correct.
Because the BECF-ECF electrochemical gradient at steady state is
maintained by a pump-leak system and is perturbed by a cyclic axial
load of physiological amplitude, it is likely that the latter modulates
the operational level of the former and thus the amplitude of the
associated electric (ionic) current. When we consider the external
medium as an infinite bath with constant ionic composition, the
variation of the measured current density at the point sink should be
associated with the postload-related modification of the BECF
composition according to the mechanisms outlined above.
Meaning of Postload Ionic Current Density Changes
The demonstration that the postload current density decayed over
time by two different patterns, depending on the bone viability, suggests that the restoration of the preload ionic composition of BECF
should depend on viable OBLCS. When bone is dead, only a
physicochemical equilibration phenomenon could take place that nullifies the electrochemical gradient generated by the strain. Because
in viable bone the higher the amplitude of the loading parameters the
faster the restoration of the preload steady-state conditions, it is
likely that OBLCS could participate in the short-term error correction
mechanism in BECF homeostasis that is proportional to the
strain-induced modification in the BECF composition. This short-term error correction mechanism implies that any perturbation of
the BECF-ECF electrochemical gradient, either by loading or by
selective modification of the ECF (2, 32, 33), activates a
pump-leak machinery that tends to restore its basal value and the
associated electric (ionic) current. By taking into account that
the plasma calcium homeostatic system could be located at the quiescent
bone surfaces, separated from bone remodeling and based on the
compartmentalizing role of bone lining cells (part of OBLCS; see Ref.
28), the corrective action of OBLCS after loading
perturbation may be integrated in a complex system that controls ion
balance at the bone-plasma interface. Considering that osteocytes are
likely inhibitory cells involved in the mechanism of osteocyte
recruitment from osteoblastic laminae (16) and in
maintaining the bone in a resting steady state (25), it
follows that the cells of the osteogenic system, particularly OBLCS,
occupy a central position in bone physiology and constitute a sort of "bone operations center" (15) capable of controlling
in a versatile manner both mineral and skeletal homeostases.
Perspectives
According to the view that a specific ionic milieu in bone is
pivotal for mineral homeostasis acid-base equilibrium and osteogenesis (1, 10, 13, 28, 29, 30, 33), this study showed that OBLCS
tends to restore the steady-state condition at the bone-plasma
interface by controlling ion fluxes after load perturbation. Because
OBLCS has been shown to operate as a partition system that selectively
controls the ionic composition of BECF for metabolic needs, OBLCS
itself seems to operate as a transducer and modulator of strain-related
signals to biochemical messages and as the link between mechanical
demands and metabolic needs. This study supported the concept that
osteocytes constitute a short-term error correction system that tends
to restore the load-related perturbation of the ionic endocanalicular
milieu. By confirming the view that bone is an ion exchange system
(1) and by extending this view to the fact that the bone
ion transporting system, still undefined, is perturbed by loading, this
study could allow the determination of a clinical model aimed to both
optimize the bone metabolic response to loading and understand when the
mechanism underlying this response is not operative, as in
osteoporosis. Because the ability of the OBLCS to detect the
load-induced perturbation of its ionic milieu was dependent on the
maximal transferred energy, the determination of a clinical model
appears to be feasible, because it will be possible to obtain the same
bone metabolic response without exposing the bones to the risk of
mechanical failure.
This study has established a model system for studying integrated
osseous responses to mechanical, pharmacological, and endocrine signals. Its application to transgenic mice engineered to possess genetic lesions in channels, gap junction constituents (connexin-43 and
-45), and/or noncollagen matrix proteins (osteopontin, osteocalcin) could contribute to the molecular characterization of the mechanisms underlying the interaction between mechanotransduction and metabolic needs.
 |
APPENDIX |
Spline
Spline functions were used both to mathematically analyze the
time-dependent decay of the postload current density since the 12th min
after having stopped the load until the end of the experimental time
and to reconstruct the experimental data missed because of the
periodical removal of the probe far from the bone surface to test the
background value.
Spline are smooth piecewise polynomial functions useful in local
approximation (interval of missing data) of the progress of a
mathematical function. Every single interval of missing data is
reconstructed using a polynomial function that is determined by
analyzing contemporaneously the temporal progress of the experimental measurements taken before and after the missing data, as reported below.
Starting from each couple of experimental measurements
([ti, yi], where
ti is time measurement expressed in min and
yi is the univocal value at
ti of the current density expressed in
µA/cm2) with i = 1,...,n
where n is the number of experimental data points, it is
possible to build a tridiagonal linear system of n different equations that has an order (
) linearly dependent upon
n
Each line of the tridiagonal system corresponds to a linear
equation defined as reported below
where yi and ti
are known data and C
,
C
1,...,C1,C0
are unknown variables.
The solution of this tridiagonal system is represented by a vector of
+ 1 coefficients
Once having calculated C, the approximating
polynomial function f(t) is defined as
with
where t is temperature. The equation reported
above is satisfied by every experimental measurement
and generates the most reliable value for each missing data
[tmiss, ymiss] where
The whole experiment is not analyzed by a unique curve but by
putting together a group of the most accurate polynomial functions approximating different intervals of missing data. The smaller the
interval of missing data and the greater the number of experimental measurements that precede and follow it, the better is the
corresponding polynomial reconstruction of the missing data obtained by
the spline algorithm. Spline appears to be very effective for data fitting because the linear systems to be solved for this are banded; hence, the work needed for their solution and their complexity grows
only linearly with the number of data points. Spline functions were
evaluated using Matlab (version 5.3.0 10183, R11; Matworks, Natick, MA).
Spline cannot be used to derive the missing data between the end of the
load and the minute at which the first reliable measurement is taken
because of the length of the time interval (~12 min) and the total
absence of the experimental measurement.
Best Nonlinear Fit Analysis
The best nonlinear fit analysis was used to determine the
time-dependent decay of the postload current density since the first minute after having stopped the load until the end of the experimental time. Given the generic negative exponential function
g(t), defined as reported below
|
(A1)
|
and representing the time progress of the postload current
density, best nonlinear fit analysis was used to find its coefficients (a,
, and b) that minimize the mean square
error (
)
|
(A2)
|
where n is the total number of disposal experimental
data, yi is the experimental measurement at time
ti expressed in µA/cm2,
g(ti) is the value assessed by
Eq. A1 at time ti expressed in µA/cm2, and
i is the error
calculated by Eq. A2 between yi and
g(ti).
An algorithm implemented in Matlab language (Matlab version 5.3.0 10183; Matworks) was used to determine by iteration the definitive
values of the coefficients a, b, and
defined
in Eq. A1.
The algorithm is initialized by passing to it the temporary values
atemp, btemp,
temp of the g(t)
coefficients that are calculated as a function of the disposal
experimental measurements reported below
where
where k is the elapsed time (expressed in minutes)
between the first minute after having stopped the load until the
beginning of the postload steady state, y1 is
the first reliable measurement since the end of the load,
y2 is the second reliable measurement since the
end of the load, yn is the last available
measurement at the end of the experimental time,
t1 is the minute at which the first reliable
measurement is taken, and
i is the time
distance between y1 and y2
expressed in minutes.
The functions g(t) characterized by coefficients
a, b, and
corresponding to an error
> 0.5 were discarded.
Any other best-fit analysis realized by using a linear or nonlinear
function instead of the one defined in Eq. A1 was found to
give an error
higher than that associated with the best exponential fit.
 |
ACKNOWLEDGEMENTS |
This work was supported in part by the Italian Ministry of
University and Scientific Research (MURST 40%).
 |
FOOTNOTES |
Address for reprint requests and other correspondence: A. Rubinacci, Bone Metabolic Unit, Scientific Institute H San Raffaele, Via Olgettina 60, 20132 Milano, Italy (E-mail:
alessandro.rubinacci{at}hsr.it).
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.
10.1152/ajpendo.00367.2001
Received 13 August 2001; accepted in final form 17 November 2001.
 |
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