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


    ABSTRACT
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
APPENDIX
REFERENCES

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


    INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
APPENDIX
REFERENCES

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.


    MATERIALS AND METHODS
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
APPENDIX
REFERENCES

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.

                              
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Table 1.   Composition and physicochemical characteristics of the incubation media

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].


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Fig. 1.   Mechanical stimulator (see text for detailed description).

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).


    RESULTS
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
APPENDIX
REFERENCES

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.

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, tau  = 10.05 ± 0.9 min, xi  = 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.

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, tau  = 13.5 ± 0.92 min; 2 g, n = 4, tau  = 12.5 ± 0.7 min; 5 g, n = 9, tau  = 10.05 ± 0.9 min; 8 g, n = 4, tau  = 8.6 ± 0.7 min; 10 g, n = 4, tau  = 8.2. ± 0.85 min; and 12 g, n = 4, tau  = 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).

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, tau  = 13.9 ± 0.8 min; 1 Hz, n = 9, tau  = 10.05 ± 0.9 min; 1.5 Hz, n = 4, tau  = 8.8 ± 0.4 min; 1.75 Hz, n = 4, tau  = 8.3 ± 0.65 min; 2 Hz, n = 4, tau  = 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).

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
E<SUB>tot</SUB><IT>=E</IT><SUB>p</SUB><IT>+E</IT><SUB>c</SUB>
with
E<SUB>p</SUB><IT>=mgh=</IT>potential energy

E<SUB>c</SUB>½<IT>mv</IT><SUP>2</SUP><IT>=</IT>kinetic energy
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
E<SUB>transferred</SUB><IT>=n·E</IT><SUB>c</SUB>
with
n=f·t
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).

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.

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.

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.

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.


    DISCUSSION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
APPENDIX
REFERENCES

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 (Delta 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
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
APPENDIX
REFERENCES

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 (alpha ) linearly dependent upon n
for<IT> n=</IT>1<IT> ⇒ </IT>the system equation is not defined

for<IT> n≥</IT>2<IT> ⇒ &agr;=n−</IT>1
Each line of the tridiagonal system corresponds to a linear equation defined as reported below
y<SUB>i</SUB>=C<SUB>&agr;</SUB>·t<SUP>&agr;</SUP><SUB>i</SUB>+C<SUB>&agr;−1</SUB>·t<SUP>&agr;−1</SUP><SUB>i</SUB>+…+C<SUB>1</SUB>·t<SUB>i</SUB>+C<SUB>0</SUB>
where yi and ti are known data and Calpha , Calpha -1,...,C1,C0 are unknown variables.

The solution of this tridiagonal system is represented by a vector of alpha  + 1 coefficients
<B>C</B><IT>=</IT>[<IT>C<SUB>&agr;</SUB>, C</IT><SUB><IT>&agr;−</IT>1</SUB><IT>,…, C</IT><SUB>1</SUB><IT>, C</IT><SUB>0</SUB>]
Once having calculated C, the approximating polynomial function f(t) is defined as
f(t)=f(<B>C</B><IT>·</IT><B>t</B>)
with
<B>t</B><IT>=</IT>[<IT>t<SUB>&agr;</SUB>, t</IT><SUB><IT>&agr;−</IT>1</SUB><IT>,…, t</IT><SUB>1</SUB><IT>, </IT>1]<SUP><IT>T</IT></SUP>
where t is temperature. The equation reported above is satisfied by every experimental measurement
y<SUB>i</SUB>=f(t<SUB>i</SUB>)
and generates the most reliable value for each missing data [tmiss, ymiss] where
y<SUB>miss</SUB><IT>=f</IT>(<IT>t</IT><SUB>miss</SUB>)
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
g(t)=a·e<SUP>−t</SUP>+b (A1)
and representing the time progress of the postload current density, best nonlinear fit analysis was used to find its coefficients (a, tau , and b) that minimize the mean square error (xi )
&xgr;=<FR><NU><LIM><OP>∑</OP><LL>1</LL><UL>n</UL></LIM> <SUB>i</SUB>[g(t<SUB>i</SUB>)−y<SUB>i</SUB>]<SUP>2</SUP></NU><DE>n</DE></FR>=<FR><NU><LIM><OP>∑</OP><LL>1</LL><UL>n</UL></LIM> i<SUP>2</SUP></NU><DE>n</DE></FR> (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 xi 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 tau  defined in Eq. A1.

The algorithm is initialized by passing to it the temporary values atemp, btemp, tau temp of the g(t) coefficients that are calculated as a function of the disposal experimental measurements reported below
a<SUB>temp</SUB><IT>=y</IT><SUB>1</SUB><IT>−y<SUB>n</SUB></IT>

&tgr;<SUB>temp</SUB><IT>=k−t</IT><SUB>1</SUB>

b<SUB>temp</SUB><IT>=y<SUB>n</SUB></IT>
where
k=[y<SUB>n</SUB>−y<SUB>1</SUB>]·<FR><NU>&Dgr;<SUB>i</SUB></NU><DE>y<SUB>2</SUB>−y<SUB>1</SUB></DE></FR>+t<SUB>1</SUB>
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 Delta i is the time distance between y1 and y2 expressed in minutes.

The functions g(t) characterized by coefficients a, b, and tau  corresponding to an error xi  > 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 xi  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.


    REFERENCES
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
APPENDIX
REFERENCES

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Am J Physiol Endocrinol Metab 282(4):E851-E864
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