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The Biochemical Pathway Mediating the Proliferative Response of Bone Cells to a Mechanical Stimulus*

CARL T. BRIGHTON, M.D., PH.D., J. RUSH S. FISHER, JR., M.D., STUART E. LEVINE, M.D., JOHN R. CORSETTI, M.D., THOMAS REILLY, M.D., ADAM S. LANDSMAN, PH.D., JOHN L. WILLIAMS, PH.D. and LAWRENCE E. THIBAULT, PH.D., PHILADELPHIA, PENNSYLVANIA

Investigation performed at the Department of Orthopaedic Surgery, University of Pennsylvania School of Medicine, and the Department of Bioengineering, University of Pennsylvania School of Engineering and Applied Science, Philadelphia

	Abstract

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Calvarial bone cells of rats were subjected to either a cyclic biaxial strain of 0.17 per cent (1700 microstrain) or a hydrostatic pressure of 2.5, five, or ten pounds per square inch (17.2, 34.5, or sixty-nine kilopascals). The frequency was held constant at one hertz for both types of mechanical stimulation. When cultured bone cells that had been subjected to a cyclic biaxial strain for two hours were harvested twenty-two hours later, it was found that the level of prostaglandin E₂ had increased significantly (p < 0.01) as had cellular proliferation (p < 0.01), as indicated by the incorporation of [³H]-thymidine. The addition to the medium of indomethacin, an inhibitor of prostaglandin synthesis, at a ten-micromolar concentration significantly inhibited (p < 0.01) the increase in prostaglandin E₂ synthesis but had no effect on the strain-induced increase in cellular proliferation, as indicated by the incorporation of [³H]-thymidine. Twenty-four hours after exposure to the same cyclic biaxial strain for thirty seconds, other cultured bone cells showed a significant increase in the level of cytoskeletal calmodulin (p < 0.05) and in the DNA content (p < 0.05). N-(6-aminohexyl)-5-chloro-1-naphthalene-sulfonamide (W-7), a calmodulin antagonist, was added to the medium at a one-micromolar concentration, which had been shown to have no effect on the increase in the DNA content of control cells; W-7 completely blocked the increase in the level of cytoskeletal calmodulin and in the DNA content in the cells that were subjected to a cyclic biaxial strain. The bone cells subjected to a hydrostatic pressure showed a dose-dependent increase in the concentration of cytosolic Ca²⁺, as measured with Fura 2-AM, a fluorescent indicator of intracellular calcium. With a pressure of ten pounds per square inch (sixty-nine kilopascals), the increase in the concentration of cytosolic Ca²⁺ was nearly eight times greater than that at 2.5 pounds per square inch (17.2 kilopascals) (126 ± 15.2 compared with 16 ± 8.0 nanomolar, mean and standard deviation). The addition to the medium of neomycin, an inhibitor of the inositol phosphate cascade, at a ten-millimolar concentration completely blocked the increase in the concentration of cytosolic Ca²⁺ in these cells; this concentration of neomycin had been shown to have no effect on proliferation in control bone cells. There was also a dose-dependent relationship between the duration of the stimulus and the cellular proliferation. Remarkably, one cycle of pressure at ten pounds per square inch (sixty-nine kilopascals) and a frequency of approximately one hertz produced a 57 per cent increase in the incorporation of [³H]-thymidine at twenty-four hours (p < 0.001). From these findings, we hypothesized that the inositol phosphate cascade-cytosolic Ca²⁺-cytoskeletal calmodulin system plays a dominant role in the signal transduction of a mechanical stimulus into increased proliferation of bone cells, at least under the conditions reported here. CLINICAL RELEVANCE: Understanding the mechanisms by which bone cells convert a mechanical signal into a biological response is the beginning of an understanding of Wolff's law, which states that form follows function. A complete understanding of Wolff's law eventually should lead to an understanding of the cellular and molecular processes governing bone-remodeling and may allow therapeutic manipulation of bone-remodeling in clinical practice.

	Introduction

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Mechanical stimuli elicit the proliferation of bone cells^{4,8,10,26,30,31,51}. The conversion of an extracellular signal, such as a mechanical stimulus, into an intracellular response, such as cellular proliferation, is termed signal transduction. However, the biochemical mechanisms involved in the conversion of mechanical stimuli into biological responses by signal transduction in osteoblasts are poorly understood. In some forms of signal transduction, a ligand binds to a cell-surface receptor and produces a conformational change in the receptor protein that then converts the extracellular signal into an intracellular one³². There are several classes of cell-surface receptors^{11,23,32,41,56}; however, regardless of the type of receptor, once signal transduction has been initiated the conformational change in the receptor activates various biochemical pathways that bring about a physiological response of the cell to the extracellular signal. One well known biochemical pathway is the inositol phosphate cascade^3,49,58. The binding of ligand to receptor activates phospholipase C, a membrane-bound enzyme that hydrolyzes phosphatidyl inositol 4,5-bisphosphate, a phospholipid in the cell membrane, into two intracellular messengers, inositol 1,4,5-trisphosphate and diacylglycerol. Inositol 1,4,5-trisphosphate causes the rapid release of Ca²⁺ from intracellular stores of calcium, and the increased concentration of cytosolic Ca²⁺ triggers many intracellular processes^44,45. In turn, the inositol 1,4,5-trisphosphate is phosphorylated into inositol 1,3,4,5-tetrakisphosphate, which further increases the concentration of cytosolic Ca²⁺ by transiently opening Ca²⁺ channels in the cell membrane⁶². Diacylglycerol activates protein kinase C, which phosphorylates many proteins, activates many enzymes, and is important in controlling cellular division and proliferation². Protein kinase C also produces the synthesis of prostaglandins^3,49.

An increase in the concentration of cytosolic Ca²⁺ leads to an increase in the level of activated calmodulin, a ubiquitous intracellular calcium-binding protein that becomes reactive with a wide range of enzymes^40,55. Activated calmodulin interactions are responsible for many calcium-mediated processes, including contraction of muscles, chemotaxis, and proliferation of cells^12,13,15,55.

Mechanical perturbations serve as extracellular signals to a variety of cells, including bone cells. The concentration of cytosolic Ca²⁺ increases in response to shear stress or indentation of the cell membrane in endothelial cells^{1,20,48,50,56}, smooth muscle cells¹⁶, cardiac muscle cells⁵³, tracheal epithelial cells²⁴, and glial cells¹⁴. Several authors have found increased cellular proliferation^{4,10,26,30,31,54} and production of prostaglandin E₂^{4,5,25,42,47,54,64} after mechanical stimulation of bone cells by various means. Some investigators have found an increase in the level of cyclic adenosine monophosphate after mechanical stimulation of bone cells^4,5,54, but Harell et al.²⁵ found an initial increase in the level of cyclic adenosine monophosphate that was followed by a prolonged decrease. Sandy et al.⁴⁷ found a relatively slight increase in the level of cyclic adenosine monophosphate when bone cells were mechanically deformed and slightly but significantly (p < 0.01 to p < 0.05) increased levels of the inositol phosphates after thirty minutes of mechanical stimulation. Jones and Bingmann³⁰ detected increased levels of the inositol phosphates after ten seconds of mechanical strain. Increased concentrations of cytosolic Ca²⁺ have also been found in bone cells subjected to fluid shear stresses⁶¹ or to substrate stretching³¹.

We reported previously that isolated bone cells subjected to a cyclic biaxial mechanical strain showed significant increases (p < 0.01) in cellular proliferation and in the production of prostaglandin E₂ after stimulation for only fifteen and five minutes, respectively⁸. No significant change in the level of cyclic adenosine monophosphate was found at these times. Subsequent studies in our laboratory demonstrated that a cyclic biaxial mechanical strain of 0.17 per cent (1700 microstrain) applied to bone cells at one hertz produced highly significant (p < 0.0001 to p < 0.05) increases in the levels of phosphatidyl inositol 1,4-bisphosphate, inositol 1,4,5-trisphosphate, and inositol 1,3,4,5-tetrakisphosphate; peak concentrations were found after only twenty seconds of stimulation⁹. At a concentration that had no effect on the proliferation of normal bone cells, neomycin, which is an inhibitor of phospholipase C, a membrane-bound enzyme that hydrolyzes phosphatidyl inositol 4,5-bisphosphate to activate the inositol phosphate cascade, completely inhibited both the increased production of the various inositol phosphates during mechanical stimulation and the cellular proliferation seen after mechanical strain.

The objective of the present study was to examine the biochemical events, subsequent to activation of the inositol phosphate cascade, involved in signal transduction.

	Materials and Methods

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Synthesis of Prostaglandin E₂
Calvarial bone cells from neonatal rats were isolated by digestion with 0.2 per cent collagenase (United States Biochemical, Cleveland, Ohio), as previously described^8,31. The cells were grown in tissue-culture medium (Eagle minimum essential medium, NCTC-135, and 10 per cent fetal bovine serum; Gibco BRL Life Technologies, Grand Island, New York) in 5 per cent carbon dioxide and air at 37 degrees Celsius. Previous studies from this laboratory have demonstrated that, in culture, these cells exhibit an osteoblastic phenotype^6,7. The isolated bone cells were grown on membranes made of biocompatible polyether-based thermoplastic polyurethane (Pellethane 2363–80AE; Upjohn, Kalamazoo, Michigan) in specially constructed culture chambers incorporated into a mechanical testing device, which have been described previously^8,9,60.

The isolated bone cells were plated at a density of 35,000 cells per square centimeter in thirty-two wells. Two days after the cells had reached confluence, the wells were divided into four groups of eight wells each: (1) control cells, (2) control cells in medium containing indomethacin at a ten-micromolar concentration, (3) cells subjected to a cyclic biaxial strain of 0.17 per cent (1700 microstrain), and (4) cells in medium containing indomethacin at a ten-micromolar concentration that were subjected to the same cyclic biaxial strain. This concentration of indomethacin had been shown, in our laboratory, to have no effect on the proliferation of calvarial bone cells of rats. [³H]-thymidine (Amersham, Arlington Heights, Illinois) (five microcuries per milliliter), unlabeled thymidine (fourteen micrograms per milliliter), and 2'-deoxycytidine (2.4 micrograms per milliliter) were added to the medium in each well in each group. The experimental cells were subjected to the cyclic biaxial strain at a frequency of one hertz for two hours and then the cultures were incubated for twenty-two hours. Duplicate aliquots of the medium were taken from each well, lyophilized, reconstituted in an assay buffer from a commercially available prostaglandin E₂ radioimmunoassay kit (Dupont NEN Research Products, Boston, Massachusetts), and assayed on the basis of competitive protein-binding between labeled and unlabeled prostaglandin E₂, as previously described⁸. The cell layers in the culture wells were rinsed and then scraped into phosphate-buffered saline solution containing EDTA (two millimoles per liter). The cells were homogenized, and aliquots were taken from each cell homogenate for fluorometric DNA assay³⁴. The homogenates were then dialyzed against deionized water to remove unincorporated radioactivity. Duplicate 500-microliter samples were added to a 4.5-milliliter scintillation cocktail, and the cells were counted in a scintillation counter (Beckman Instruments, Fullerton, California). The mean incorporation of [³H]-thymidine and the concentration of prostaglandin E₂ were normalized to the total DNA content in each well.

All data were analyzed with use of one-way analysis of variance and the Scheffé multiple-comparisons test for significance between groups.

Cytosolic Ca²⁺
Experiments were conducted to determine the effect of mechanical strain on the concentration of cytosolic Ca²⁺ in bone cells. The apparatus to apply mechanical forces to isolated cells while they are observed under phase and fluorescent microscopy has been described previously³⁵. Cells were grown in a stainless-steel well with a quartz window in a chamber, the roof of which contained ports for the application and monitoring of pressure.

The bone cells were plated at a density of 50,000 cells per square centimeter in two milliliters of medium. Two days after the cells had reached confluence, they were loaded with 3.2-micromolar Fura 2-AM (Molecular Probes, Junction City, Oregon) in buffered saline solution containing calcium chloride (one millimole per liter), glucose (five millimoles per liter), and magnesium chloride (0.5 millimole per liter) at a pH of 7.35. The top portion of the chamber was threaded onto the bottom well and was sealed. Air was flushed from the well. Compressed nitrogen was directed through a pressure regulator and an electrically operated solenoid valve into a 150-cubic-centimeter reservoir fitted with a high-resistance evacuation valve to produce a more physiological cyclic ramp loading and unloading of the pressure wave. The pressure wave was applied through rigid tubing extending from the reservoir to the culture apparatus. The cells were exposed to light at wavelengths of 340 and 360 nanometers at intervals of sixty-six milliseconds for sixty seconds. The 340-nanometer wavelength excites Fura 2-AM in its Ca²⁺-bound form and the 360-nanometer wavelength, at its isosbestic point. The Fura 2-AM emissions at a 510-nanometer wavelength were measured and were used to calculate the concentration of cytosolic Ca²⁺. Images of fluorescing cells were captured on a silicon-intensified target camera (SIT-66; Dage-MTI, Michigan City, Indiana) and were stored on an optical memory disk with use of custom software (CASALS 2.21; Olympus, Lake Success, New York). Eight to twelve confluent cells were individually analyzed in each field at a magnification of 200 times.

The cells were then subjected to a cyclic hydrostatic pressure of 2.5, five, or ten pounds per square inch (17.2, 34.5, or sixty-nine kilopascals), applied at approximately one hertz, for ten cycles. The mean concentration of cytosolic Ca²⁺ was determined for eight to twelve responding cells at each load amplitude in each of two runs. Analysis for significance among the groups was performed with a one-way analysis of variance and the Scheffé multiple-comparisons test.

To determine whether an increase in the concentration of cytosolic Ca²⁺ was mediated by inositol 1,4,5-trisphosphate, two groups of cells in six wells each were subjected to cyclic hydrostatic pressure of ten pounds per square inch (sixty-nine kilopascals) at approximately one hertz for fifteen seconds. The cells in one of these groups were in medium containing neomycin at a ten-millimolar concentration. Neomycin is an inhibitor of the phospholipase C-mediated breakdown of phosphatidyl inositol 4,5-bisphosphate and, thus, is an inhibitor of the inositol phosphate cascade^43,58. In preliminary studies, a ten-millimolar concentration of neomycin had not affected the rate of proliferation in control cultures of bone cells.

To show that the same hydrostatic pressures that produced an increase in the concentration of cytosolic Ca²⁺ also produced an increase in cellular proliferation, bone cells were grown in twenty wells under the conditions already described, except that the cells were not loaded with Fura 2-AM. Two days after the cells had reached confluence, [³H]-thymidine (five microcuries per milliliter), unlabeled thymidine (fourteen micrograms per milliliter), and 2'-deoxycytidine (2.4 micrograms per milliliter) were added to the medium. The twenty wells were divided into five groups, four of which were subjected to a hydrostatic pressure of ten pounds per square inch (sixty-nine kilopascals) at approximately one hertz for one, ten, twenty-five, or fifty cycles (four wells each), and the fifth group of wells served as the control and was not subjected to hydrostatic pressure. After mechanical stimulation, the cultured cells were incubated in 5 per cent carbon dioxide and air at 37 degrees Celsius for twenty-four hours and then were analyzed for DNA content and incorporation of [³H]-thymidine. The results were expressed as the percentage increase of [³H]-thymidine incorporation per microgram of DNA in the stimulated wells compared with that in the control wells.

Calmodulin
Calvarial bone cells of rats were isolated and grown on Pellethane membranes in specially constructed culture chambers^8,9 until two days after they had reached confluence. The experiment was then divided into two parts: A and B.

Part A: Thirty-two wells were divided into two groups of sixteen wells each. One group, which served as the control, was not subjected to a mechanical strain, and the other group was subjected to a 0.17 per cent cyclic biaxial strain at one hertz for thirty seconds in 5 per cent carbon dioxide and air at 37 degrees Celsius. All of the cells in both groups were then maintained in culture for twenty-four hours under the conditions already described. The cells in eight wells from each group were analyzed fluorometrically for DNA content. The cells in the remaining eight wells from each group were analyzed for the levels of cytoskeletal and cytosolic calmodulin, with use of a modification of the technique of LeVine et al.³⁷. With this modified technique, immediately after the mechanical strain had been applied, the reaction was stopped by the addition of two milliliters of phosphate-buffered saline solution containing calcium chloride (0.1 millimole per liter) at 4 degrees Celsius. The solution was discarded, the cells were disrupted with 600 microliters of phosphate-buffered saline solution containing 0.1 per cent Triton X-100 (Fisher Scientific, Fair Lawn, New Jersey), and aliquots were taken from each well for fluorometric DNA assay. The disrupted cell suspension was then centrifuged at 40,000 times gravity for thirty minutes to separate the cytosolic (supernatant) from the cytoskeletal (pellet) fractions. The pellet was resuspended in phosphate-buffered saline solution containing calcium chloride (0.1 millimole per liter), and both fractions were boiled and then cooled in a mixture of dry ice and ethanol. Both fractions were centrifuged at 10,000 times gravity for thirty minutes, and the pellets were discarded. The calmodulin content in both remaining supernatants (one representing the cytosolic fraction and the other, the cytoskeletal fraction) was determined with use of competitive binding between unlabeled calmodulin as well as a fixed quantity of [¹²⁵I]-calmodulin (New England Nuclear, Boston, Massachusetts) for binding to a specific protein preparation, made from rat brain, containing a high concentration of calmodulin-dependent protein kinase³⁶. The results were expressed as micrograms of DNA per well, micrograms of total calmodulin per well, and the percentage of cytosolic and cytoskeletal fractions per total calmodulin.

Part B: Another thirty-two wells were divided into four groups of eight wells each: (1) control cells (not subjected to a mechanical strain); (2) control cells in medium containing N-(6-aminohexyl)-5-chloro-1-naphthalene-sulfonamide (W-7), a calmodulin antagonist³⁹, at a one-micromolar concentration, a concentration that had been shown in our laboratory to have no effect on the proliferation of calvarial bone cells of rats; (3) cells subjected to a 0.17 per cent cyclic biaxial strain at one hertz for thirty seconds; and (4) cells in medium containing W-7 at a one-micromolar concentration that were subjected to the same cyclic biaxial strain.

After the mechanical strain had been applied to groups 3 and 4, all of the cultures were incubated in 5 per cent carbon dioxide and air at 37 degrees Celsius. After twenty-four hours, the DNA content was analyzed. The results in all four groups were expressed as micrograms of DNA per well.

	Results

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Synthesis of Prostaglandin E₂
In the absence of indomethacin, the synthesis and release of prostaglandin E₂ increased significantly (p < 0.01) in response to a cyclic biaxial strain (Fig. 1). The levels of prostaglandin E₂ were significantly lower (p < 0.01) in the two groups of cells in medium with indomethacin than in the two groups in medium without indomethacin. In contrast, the presence of indomethacin had no effect on the cellular proliferation in response to the mechanical strain (Fig. 2). Specifically, the incorporation of [³H]-thymidine increased significantly (p < 0.01) after two hours of 0.17 per cent cyclic biaxial strain, regardless of the presence of indomethacin in the medium.

View larger version (41K): [in this window] [in a new window]   Aliquots taken from cultures of bone cells, two days after the cells had reached confluence, showed a significant increase (p < 0.01) in the synthesis and release of prostaglandin E2 (PGE2) in response to a cyclic biaxial strain of 0.17 per cent (1700 microstrain). This increase was significantly inhibited (p < 0.01) by the addition of indomethacin to the medium at a ten-micromolar concentration. The I-bars represent the standard error of the mean.

View larger version (53K): [in this window] [in a new window]   Twenty-two hours after cultured bone cells had been subjected to a cyclic biaxial strain of 0.17 per cent at one hertz for two hours, the incorporation of [3H]-thymidine increased significantly (p < 0.01). This increase in cellular proliferation was not inhibited by the presence of indomethacin in the medium, indicating that the proliferative response of bone cells to the mechanical strain was not mediated by prostaglandin E2. The I-bars represent the standard error of the mean. dpm = disintegrations per minute.

Cytosolic Ca²⁺
A mean of sixteen (27 per cent) of the sixty cells that were analyzed demonstrated a characteristic increase in the concentration of cytosolic Ca²⁺ in response to the mechanical strain. The latency period between the onset of hydrostatic pressure at ten pounds per square inch (sixty-nine kilopascals) and the onset of the increase in the concentration of cytosolic Ca²⁺ was a mean (and standard deviation) of 24.9 ± 1.09 seconds, and this increase lasted for a mean of 20.4 ± 4.16 seconds. All responding cells demonstrated a lower secondary peak, which occurred a mean of 7.6 ± 4.16 seconds after the primary peak (Fig. 3). Neomycin completely blocked the increase in the concentration of cytosolic Ca²⁺ (Fig. 4). The concentration of cytosolic Ca²⁺ increased in a dose-dependent manner: it increased a mean of 16 ± 8.0 nanomolar (range, eleven to thirty nanomolar) with 2.5 pounds per square inch (17.2 kilopascals) of applied hydrostatic pressure, 85 ± 27.2 nanomolar (range, forty-five to 120 nanomolar) with five pounds per square inch (34.5 kilopascals), and 126 ± 15.2 nanomolar (range, 106 to 200 nanomolar) with ten pounds per square inch (sixty-nine kilopascals) (Fig. 5).

View larger version (31K): [in this window] [in a new window]   The concentration of cytosolic Ca2+ increased in response to a hydrostatic pressure of ten pounds per square inch (sixty-nine kilopascals) at one hertz. The latency period between the onset of mechanical strain and the beginning of the increase in cytosolic Ca2+ was approximately twenty-five seconds, and the duration of the increase was approximately twenty seconds. The primary peak in the concentration of cytosolic Ca2+ was always followed by a lower secondary peak.

View larger version (29K): [in this window] [in a new window]   The increase in the concentration of cytosolic Ca2+ in response to a hydrostatic pressure was inhibited when the medium contained neomycin, an inhibitor of the inositol phosphate cascade, at a ten-millimolar concentration.

View larger version (39K): [in this window] [in a new window]   The concentration of cytosolic Ca2+ increased according to the amplitude of the hydrostatic pressure. The increases were significant in response to pressures of five and ten pounds per square inch (34.5 and sixty-nine kilopascals) applied at one hertz for ten cycles. The I-bars represent the standard deviation.

View larger version (42K): [in this window] [in a new window]   The incorporation of [3H]-thymidine increased in the cells subjected to a hydrostatic pressure of ten pounds per square inch (sixty-nine kilopascals) for a duration as short as one second. The I-bars represent the standard error of the mean. NS = not significant.

Calmodulin
The level of cytoskeletal calmodulin increased more than twofold (p < 0.05) and the DNA content increased 37 per cent (p < 0.05) in the cells that were subjected to mechanical strain, as compared with the control cells (Fig. 7). The fraction of cytosolic calmodulin decreased 13 per cent, the total calmodulin increased 3 per cent, and the percentage of cytoskeletal calmodulin per total calmodulin increased from 7 to 15 per cent. W-7 completely prevented the significant increase (p < 0.01) in cellular proliferation that was seen in the cells stimulated with a 0.17 per cent strain (Fig. 8).

View larger version (23K): [in this window] [in a new window]   Twenty-four hours after exposure to a cyclic biaxial strain of 0.17 per cent, the concentration of cytosolic calmodulin (CAM) increased more than twofold (p < 0.05) and the DNA content increased 37 per cent (p < 0.05). The I-bars represent the standard error of the mean.

View larger version (49K): [in this window] [in a new window]   The strain-induced proliferative response of bone cells was completely inhibited by the addition of N-(6-aminohexyl)-5-chloro- 1-naphthalene-sulfonamide (W-7), a calmodulin antagonist, at a one-micromolar concentration. The I-bars represent the standard error of the mean.

	Discussion

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The results of the experiments with cyclic biaxial strain suggest that, under the in vitro conditions described, the inositol phosphate cascade-activated calmodulin axis is the biochemical pathway involved in signal transduction of a mechanical strain to cellular proliferation in bone cells. When the inositol phosphate pathway was blocked with neomycin, cellular proliferation did not increase. The level of activated (cytoskeletal) calmodulin increased significantly (p < 0.05) after biaxial strain, but in the presence of W-7, an inhibitor of calmodulin, neither the concentration of cytoskeletal calmodulin nor the cellular proliferation increased. The concentration of cytosolic Ca²⁺ could not be measured during the experiments with cyclic biaxial strain because of optical limitations of the apparatus used to apply strain. However, several factors indicate that the concentration of cytosolic Ca²⁺ increased in the cells subjected to a biaxial strain. First, the level of inositol 1,4,5-trisphosphate had been shown to increase with use of the same apparatus and the same biaxial strain (0.17 per cent)⁹, and an increase in inositol 1,4,5-trisphosphate was inferred in this experiment as neomycin, an inhibitor of the inositol phosphate cascade, blocked the increase in cellular proliferation that otherwise occurred with biaxial strain. Second, inositol 1,4,5-trisphosphate brings about an increase in the concentration of cytosolic Ca²⁺ by releasing calcium from intracellular stores^44,45. Third, the level of activated (cytoskeletal) calmodulin increased after stimulation with a biaxial strain, and when this increase was inhibited with W-7 cellular proliferation was also inhibited. Fourth, it has been shown^40,55 that calmodulin is activated to cytoskeletal calmodulin by an increase in the concentration of cytosolic Ca²⁺.

The results of the experiments with hydrostatic pressure demonstrated that the concentration of cytosolic Ca²⁺ and the incorporation of [³H]-thymidine per microgram of DNA increased in a dose-dependent manner in response to hydrostatic pressure. The experiments with neomycin provided evidence that the increase in the concentration of cytosolic Ca²⁺ in response to an applied hydrostatic pressure is mediated by the inositol phosphate pathway. At a concentration that had no effect on the proliferation of control cells, neomycin completely blocked the increase in the concentration of cytosolic Ca²⁺ in response to ten pounds per square inch (sixty-nine kilopascals) of pressure at one hertz (Figs. 3 and 4). Moreover, the latency period between the onset of mechanical pressure and the beginning of the increase in the concentration of cytosolic Ca²⁺ (approximately twenty-five seconds) corresponds closely to the time to peak concentration of inositol 1,4,5-trisphosphate (approximately twenty seconds) in cultured bone cells subjected to mechanical strain. This finding suggests that, in the experiments with hydrostatic pressure, signal transduction was mediated by the inositol phosphate pathway. Thus, in both the experiments with biaxial strain and those with hydrostatic pressure, the inositol phosphate cascade appeared to be involved, the concentration of cytosolic Ca²⁺ increased, and the level of cytoskeletal calmodulin increased. On the basis of these observations, it is hypothesized that the inositol phosphate cascade-cytosolic Ca²⁺-cytoskeletal calmodulin axis plays a dominant role in the signal transduction of a mechanical stimulus into increased proliferation of bone cells under the conditions reported here (Fig. 9).

View larger version (18K): [in this window] [in a new window]   Biochemical pathway mediating the proliferative response of cultured bone cells to a mechanical stimulus, as determined in this study. PLC = phospholipase C, PIP2 = phosphatidyl inositol 4,5-bisphosphate, IP3 = inositol 1,4,5-triphosphate, DG = diacylglycerol, PLA2 = phospholipase A2, PGE2 = prostaglandin E2, and x = sites of inhibition of the inositol phosphate cascade (by neomycin), of the activation of calmodulin (by W-7), and of prostaglandin E2 synthesis (by indomethacin).

Several questions or concerns arise regarding these experiments. First, the cyclic biaxial strain applied to the bone cells was not uniform⁶⁰. The apparatus used in these and previous experiments^8,9 cyclically inflated a circular Pellethane membrane that was clamped at its periphery. At maximum deflection, the circumferential strain was 0.17 per cent at the center of the membrane and decreased linearly to 0 per cent at the periphery. The radial strain was 0.17 per cent at the center of the membrane and increased slightly to 0.22 per cent at the periphery⁶⁰. Thus, the bone cells were exposed to a heterogeneous strain. Obviously, studies involving dose-response relationships between strain characteristics and the kinetics of any given biological response cannot be performed with such an apparatus. However, the type of information sought in this experiment—namely, the identification of the biochemical pathway followed by bone cells stimulated by a mechanical strain of a maximum amplitude of 0.17 per cent—required only a consistent strain condition throughout those parts of the studies involving cyclic biaxial strain.

A second potential concern is the flexibility of the quartz window used in the experiments involving hydrostatic pressure and whether tension strain in the window was a co-stimulator. The circular quartz window bends as a linear thin plate, with maximum surface strains calculated to be 0.0006 per cent at a maximum pressure of ten pounds per square inch (sixty-nine kilopascals)¹⁹. These strains are far lower than the 0.04 per cent strain that we have found to be the lowest biaxial cyclic strain that elicited a significant increase in cellular proliferation⁸.

Also of concern are the actual waveforms (and their frequency content) that were used in the experiments with biaxial strain and hydrostatic pressure. With biaxial strain, the waveform was sinusoidal with a frequency of one hertz. To determine the frequency content of the waveform with hydrostatic pressure, a fast Fourier transformation power spectral analysis was performed, with use of MathCad software (MathSoft, Cambridge, Massachusetts), on a pressure tracing (Fig. 10). Eighty-six per cent of the curve was found to be described by a sine wave at 0.78 to 0.89 hertz.

View larger version (20K): [in this window] [in a new window]   Trace of the pressure used to deform bone cells with the hydrostatic pressure system used in these experiments.

A last potential concern with these and all similar studies is the lack of actual data on the type of mechanical signals a bone cell encounters in situ in living bone, as well as the amplitudes, frequencies, waveforms, and durations of those signals. It seems likely that the compressive, tensile, and shear forces to which the human skeleton is subjected lead to mechanical strain of bone cells in their lacunae and the cytoplasmic processes of bone cells in their canaliculi. With each step or muscle contraction, hydrostatic forces must also be generated in the haversian canals, lacunae, canaliculi, and other osseous pores, and bone cells are exposed to these pressures also. Ultimately, the type and parameters of the mechanical stimuli that bone cells encounter in situ must be determined, and similar mechanical signals must be applied to various bone-cell models both in vitro and in vivo, to understand the full nature and magnitude of signal transduction occurring when bone cells respond to mechanical stimuli.

NOTE: The authors thank Dr. Harry LeVine (Glaxo, Research Triangle Park, North Carolina) for his advice and assistance with the calmodulin assay, Krystyna Knight for performing the calmodulin assays, and Dr. Charles C. Clark for his critical reading of the manuscript.

	Footnotes

 
*No benefits in any form have been received or will be received from a commercial party related directly or indirectly to the subject of this article. Funds were received in total or partial support of the research or clinical study presented in this article. The funding sources were Grants AR18033 and AR39226 from the National Institutes of Health.

Department of Orthopaedic Surgery, University of Pennsylvania School of Medicine, 424 Edward J. Stemmler Hall, 36th and Hamilton Walk, Philadelphia, Pennsylvania 19104.

Department of Bioengineering, University of Pennsylvania School of Engineering and Applied Science, 220 South 33rd Street, Philadelphia, Pennsylvania 19104.

	References

Top Abstract Introduction Materials and Methods Results Discussion References

 

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