Signal transduction in electrically stimulated bone cells.

BACKGROUND: Electrical stimulation is used to treat nonunions and to augment spinal fusions. We studied the biochemical pathways that are activated in signal transduction when various types of electrical stimulation are applied to bone cells. METHODS: Cultured MC3T3-E1 bone cells were exposed to capacitive coupling, inductive coupling, or combined electromagnetic fields at appropriate field strengths for thirty minutes and for two, six, and twenty-four hours. The DNA content of each dish was determined. Other cultures of MC3T3-E1 bone cells were exposed to capacitive coupling, inductive coupling, or combined electromagnetic fields for two hours in the presence of various inhibitors of signal transduction, with or without electrical stimulation, and the DNA content of each dish was determined. RESULTS: All three signals produced a significant increase in DNA content per dish compared with that in the controls at all time-points (p < 0.05), but only exposure to capacitive coupling resulted in a significant, ever-increasing DNA production at each time-period beyond thirty minutes. The use of specific metabolic inhibitors indicated that, with capacitive coupling, signal transduction was by means of influx of Ca(2+) through voltage-gated calcium channels leading to an increase in cytosolic Ca(2+) (blocked by verapamil), cytoskeletal calmodulin (blocked by W-7), and prostaglandin E2 (blocked by indomethacin). With inductive coupling and combined electromagnetic fields, signal transduction was by means of intracellular release of Ca(2+) leading to an increase in cytosolic Ca(2+) (blocked by TMB-8) and an increase in activated cytoskeletal calmodulin (blocked by W-7). CONCLUSIONS: The initial events in signal transduction were found to be different when capacitive coupling was compared with inductive coupling and with combined electromagnetic fields; the initial event with capacitive coupling is Ca(2+) ion translocation through cell-membrane voltage-gated calcium channels, whereas the initial event with inductive coupling and with combined electromagnetic fields is the release of Ca(2+) from intracellular stores. The final pathway, however, is the same for all three signals-that is, there is an increase in cytosolic Ca(2+) and an increase in activated cytoskeletal calmodulin.

Bone tissue engineering in a rotating bioreactor using a microcarrier matrix system.

A novel approach was utilized to grow in vitro mineralized bone tissue using lighter-than-water, polymeric scaffolds in a high aspect ratio rotating bioreactor. We have adapted polymer microencapsulation methods for the formation of hollow, lighter-than-water microcarriers of degradable poly(lactic-co-glycolic acid). Scaffolds were fabricated by sintering together lighter-than-water microcarriers from 500 to 860 microm in diameter to create a fully interconnected, three-dimensional network with an average pore size of 187 microm and aggregate density of 0.65 g/mL. Motion in the rotating bioreactor was characterized by numerical simulation and by direct measurement using an in situ particle tracking system. Scaffold constructs established a near circular trajectory in the fluid medium with a terminal velocity of 98 mm/s while avoiding collision with the bioreactor wall. Preliminary cell culture studies on these scaffolds show that osteoblast-like cells readily attached to microcarrier scaffolds using controlled seeding conditions with an average cell density of 6.5 x 10(4) cells/cm(2). The maximum shear stress imparted to attached cells was estimated to be 3.9 dynes/cm(2). In addition, cells cultured in vitro on these lighter-than-water scaffolds retained their osteoblastic phenotype and showed significant increases in alkaline phosphatase expression and alizarin red staining by day 7 as compared with statically cultured controls.

45S5 bioactive glass surface charge variations and the formation of a surface calcium phosphate layer in a solution containing fibronectin.

This study investigated the effect of fibronectin adsorption on surface charge variations and calcium phosphate (Ca-P) layer formation kinetics on the surface of 45S5 bioactive glass (BG). We hypothesize that the adsorption of fibronectin on BG changes the surface charge and alters the kinetics of Ca-P layer formation on the glass surface. The charge at a material's surface modulates surface chemistry, protein adsorption, and interactions with bone cells. The zeta potential of BG in a solution containing human plasma fibronectin (TE-FN) was measured as a function of time by particle electrophoresis, and Ca-P layer formation was characterized using SEM, EDXA, and FTIR. Si, Ca, and P solution concentrations also were determined. It was found that the adsorption of fibronectin reduced the initial electronegativity of the BG surface and delayed the formation of both the amorphous and the crystalline Ca-P layers. The delayed formation of these surface layers may be attributed to the competitive binding of Ca2+ ions by the fibronectin molecule. In addition, the formation of an amorphous Ca-P layer correlated with the reversal from a negatively to a positively charged surface, independent of the presence of fibronectin. The addition of a single protein (in this case fibronectin) can significantly alter material surface parameters, such as charge, and subsequently affect the formation of a surface Ca-P layer. Furthermore, the formation of an amorphous Ca-P layer is an important event in the reactions leading to bioactive behavior, and proteins such as FN are actively involved in the transformation of the surface into a Ca-P layer.

Numerical model and experimental validation of microcarrier motion in a rotating bioreactor.

The equations of motion for microcarriers in a rotating bioreactor have been formulated and trajectories obtained using numerical techniques. An imaging system was built to validate the results by direct observation of microcarrier trajectories in the rotating frame of reference. The microcarrier motion observed by this imaging system was in excellent agreement with the numerical predictions of that motion. In the rotating frame of reference, microcarriers with density greater than the surrounding fluid medium followed a circular motion relative to the culture medium combined with a persistent migration and eventual collision with the outer wall of the reactor. However, for microcarrier density less the fluid medium, their circular motion migrated toward the central region of the reactor. When multiple microcarrier beads that are lighter than water are inserted into the reactor, the centrally directed migration results in the formation of clusters that are stabilized by tissue bridges formed by osteoblasts seeded onto the microcarriers. This system offers unique opportunities to monitor tissue synthesis on microcarriers using real-time optical techniques and to optimize the bioreactor operating conditions for exploiting this technology to study early bone tissue synthesis in vitro.

Serum modulates the intracellular calcium response of primary cultured bone cells to shear flow.

We investigated the effect of newborn bovine serum on the intracellular calcium [Ca(2+)](i) response of primary cultured bone cells stimulated by fluid flow. As it has been previously established that these cells exhibit [Ca(2+)](i) responses to fluid flow shear stress in saline media without growth factors or other chemically stimulatory factors, we hypothesized that the addition of serum to the flow medium would enhance the mechanosensitivity of the cells. We examined the effect of a short-term (10-15min) exposure of the cells to 2 and 10% serum prior to flow stimulation (pretreated) compared to not exposing the cells prior to flow stimulation (unpretreated). The cells were subjected to a well-defined, 90-s flow stimulus with shear stress levels ranging from 0.02 to 3.5Pa in a laminar flow chamber using a saline medium supplemented with 2 or 10% serum. For pretreatment, the serum concentration was the same from pre-flow to flow exposure. We observed a differential effect in the magnitude of the peak [Ca(2+)](i) response modulated by the concentration of serum in the pre-flow medium. Additionally, ATP-supplemented flow was examined as a comparison to the serum-supplemented flow and exhibited a similar trend in the peak [Ca(2+)](i) flow response that was dependent on ATP concentration and pre-flow exposure conditions. These findings demonstrate that under the conditions of this study, chemical agonist exposure can modulate the [Ca(2+)](i) response in bone cells subjected to fluid flow-induced shear stress.

Temporal zeta potential variations of 45S5 bioactive glass immersed in an electrolyte solution.

45S5 bioactive glass (BG) is a bioactive material known to bond to bone in vivo through a surface calcium phosphate (Ca-P) layer. The goal of this study was to address the importance of BG surface charge in the bioactive response by examining the relationship between charge variations and the formation of the surface Ca-P layer. The zeta potential of BG in an electrolyte solution (TE) was measured by particle electrophoresis, and the formation of a Ca-P layer was characterized using SEM, EDXA, and FTIR. Si, Ca, and P solution concentrations also were determined. The initial BG surface was negatively charged, and two sign reversals were detected during 3 days of immersion. The first, from negative to positive after 1 day, is attributed to the adsorption of cations at the BG surface, and the second reversal was due to the precipitation of phosphate ions from solution. A strong correlation was found between the formation of a Ca-P layer and BG surface zeta potential variations. The dynamic shift in zeta potential from an initially negative surface to a positively charged surface directly corresponded with the formation of an amorphous Ca-P layer. In addition, when the glass surface matured into a crystalline Ca-P layer, it was associated with a reversal from a positive to a negative surface. Future work will focus on the effects of protein adsorption on BG surface charge and Ca-P layer formation kinetics as well as on cellular response to a changing BG surface.

Biochemical pathway mediating the response of bone cells to capacitive coupling.

Rat calvarial bone cells or mouse MC3T3-E1 bone cells subjected to a capacitively coupled electric field of 20 mV/cm consistently showed significant increases in cellular proliferation as determined by deoxyribonucleic acid content. Verapamil, a membrane calcium channel blocker; W-7, a calmodulin antagonist; indocin, a prostaglandin synthesis inhibitor; or bromophenacyl bromide, a phospholipase A2 inhibitor, each at a concentration that did not interfere with cell proliferation in control cultures, inhibited proliferation in those cultures subjected to the electric field. In contrast, neomycin, an inhibitor of the inositol phosphate cascade, did not inhibit this electrically induced cellular proliferation. Prostaglandin E2 production also was increased significantly with electrical stimulation, and this increase was inhibited by verapamil or indocin but not by neomycin. Thus, the data suggest that the signal transduction mediating the proliferative response of cultured bone cells to a capacitively coupled field involved transmembrane calcium translocation via voltage gated calcium channels, activation of phospholipase A2, and a subsequent increase in prostaglandin E2. Increases in cytosolic calcium and activated calmodulin are implied. The inositol phosphate pathway, unlike its dominant role in signal transduction in mechanically stimulated bone cells, does not appear to play a role in signal transduction in the proliferative response of bone cells to electrical stimulation.

Serum plays a critical role in modulating [Ca2+]c of primary culture bone cells exposed to weak ion-resonance magnetic fields.

Primary-culture bone cells were exposed to ion-resonance (IR) magnetic fields tuned to Ca2+. Cytosolic calcium concentration, [Ca2+]c, was measured by using fura-2 during field exposure. The fields investigated were 20 muT static + muT p-p at either 15.3 or 76.6 Hz, and 0.13 mT static + either 0.5 or 1.0 mT p-p at 100 Hz. Other parameters included field orientation, culture age (2 or 5 days after plating), and the presence of serum (0 or 2%) during exposure. Total experiment time was 29.5 min: The field was applied after 2 min, and bradykinin was added as an agonist control after 22 min. The data were quantified on a single-cell basis during the 2-22 min exposure period in terms of the magnitude of the largest occurring [Ca2+]c spike normalized to local baseline. Field-exposed and control groups were characterized in terms of the percent of cells exhibiting spike magnitudes above thresholds of 100 or 66% over baseline and were compared by using Fisher's exact test. Without serum, there was little evidence that IR magnetic fields altered [Ca2+]c. However, in the presence of 2% serum, 3 of the 16 experiments exhibited significant effects at the 100% threshold. Reducing this threshold to 66% resulted in five experiments exhibiting significant effects. Most strikingly, in all of these cases, the field acted to enhance [Ca2+]c activity as opposed to suppressing [Ca2+]c activity. These findings suggest a role for serum or for constituents within serum in mediating the effects of IR magnetic fields on cells and may provide a resolution pathway to the dilemma imposed by theoretical arguments regarding the possibility of such phenomena. Possible roles of serum and future studies are discussed.

Intracellular calcium response of ACL and MCL ligament fibroblasts to fluid-induced shear stress.

This study examines the real-time intracellular calcium concentration, [Ca2+]i, response of canine medial collateral ligament (MCL) and anterior cruciate ligament (ACL) fibroblasts subjected to a fluid-induced shear stress of 25 dynes/cm2. In experiments using a modified Hanks' Balanced Salt Solution (HBSS) perfusate, both cell types demonstrated a significant increase in peak [Ca2+]i compared to respective no-flow controls, the response of MCL fibroblasts being nearly 2-fold greater than that of ACL fibroblasts. In studies where the cells were bathed in a medium of HBSS supplemented with 2% newborn bovine serum (NBS) and then introduced to flow with the same medium, ACL fibroblasts responded nearly 3-fold greater than MCL fibroblasts. Neomycin (10 mM), thapsigarigin (1 microM) and Ca(2+)-free media supplemented with EGTA (1 mM) were able to inhibit significantly the [Ca2+]i response to flow with HBSS in both fibroblasts. Thapsigargin also blocked the NBS flow response in both cell types, while neomycin and Ca(2+)-free media significantly inhibited the ACL response. Our findings demonstrate that ACL and MCL cells are not the same. These differences may be related to the disparate healing capacity of the ACL and MCL observed clinically.

What is the role of the convective current density in the real-time calcium response of cultured bone cells to fluid flow?

Cultured cells subjected to fluid flow are exposed to mechanical forces and electrokinetic forces. The convective current establishes an electrokinetic force created by the flow-dependent transport of mobile ions in the media over the charged cell surfaces. This current can be expressed as a current density, the current normalized by the cross-sectional area in which it exists. In this study, we hypothesized that the convective current density has no role in the bone cell real-time intracellular calcium response to fluid flow. Our hypothesis was tested by incorporating electrokinetic measurements and classical electrokinetic double-layer theory to estimate the value of convective current density in a parallel-plate flow chamber and then to apply an external current during the presence of fluid flow that would alter convective current density. There was no difference between the mean peak calcium response of cells exposed to flow with an altered (canceled or doubled) convective current density versus flow with an unmodified convective current density, as was measured with fura-2 fluorescence microscopy. These results suggest that mechanical forces, such as fluid-induced shear stress, rather than concomitant electrokinetic forces are the primary stimuli in eliciting the observed calcium response of bone cells to fluid flow.

Intracellular Ca2+ stores and extracellular Ca2+ are required in the real-time Ca2+ response of bone cells experiencing fluid flow.

In this study, we sought to determine if there is a requirement for calcium entry from the extracellular space as well as calcium from intracellular stores to produce real-time intracellular calcium responses in cultured bone cells subjected to fluid flow. Understanding calcium cell signaling may help to elucidate the biophysical transduction mechanism(s) mediating the conversion of fluid flow to a cellular signal. An experimental design which utilized a scheme of pharmacological blockers was employed to distinguish between the biochemical pathways involved in this cell signaling. A parallel-plate flow chamber served as the cell stimulating apparatus and a fluorescence microscopy system using the calcium-sensitive dye fura-2 measured the intracellular calcium changes. In the present study, evidence for a role by the inositol-phospholipid biochemical pathway, specifically inositol trisphosphate (IP3) was obtained using neomycin which completely inhibited the calcium response to flow. Additionally, a concomitant role of extracellular calcium was demonstrated through experiments performed in calcium-free medium which also eliminated the flow response. Experiments conducted with gadolinium, a stretch-activated channel blocker, partially inhibited (approximately 30%) the flow response while verapamil, a type-L voltage sensitive channel blocker, had no effect on the flow response. These results suggest a requirement of extracellular calcium (or calcium influx) as well as IP3-induced calcium release from intracellular stores for generating the intracellular calcium response to flow in bone cells.

Effects of etidronate and oophorectomy on the zeta potential of rat bone.

Recent clinical trials in osteoporotic patients show that cyclical etidronate therapy can increase vertebral bone mass and reduce the incidence of vertebral fractures. Stress generated potentials, which are theorized to participate in a negative feedback arrangement regulating bone and which are electrokinetic in origin, can be characterized in vitro by the zeta potential, a measure of electrical surface charge. Thus, the possible effects of etidronate and oophorectomy on the zeta potential of bone were investigated with respect to bone remodeling, pathophysiology, and osteoporosis. Thirty-two oophorectomized and 48 sham oophorectomized rats received subcutaneous etidronate or saline. The zeta potential magnitude of the control group (sham oophorectomized, vehicle only) increased 23.6% from age 6 to 10 months, a period of bone growth. After 6 weeks of etidronate treatment, which decreased bone turnover, the zeta potential magnitude was decreased 14.9% (0.59 mV) compared with that of controls. Decreased zeta potential magnitudes with etidronate treatment also were observed in sham oophorectomized groups at 16 weeks and in oophorectomized groups at 6 and 16 weeks, but the differences were not statistically significant. A large bolus of etidronate had no effect. The zeta potential of bone is a dynamic quantity that may correlate with bone growth or bone turnover. Whether it exerts a causative effect or is a marker remains unknown.

Tibial nonunion treated with direct current, capacitive coupling, or bone graft.

Two hundred seventy-one tibial nonunions of average duration of 23.5 months (range, 9-69 months) were treated with direct current (167 patients), capacitive coupled electrical stimulation (56 patients), or bone graft surgery (48 patients). Logistic regression analysis was used to compare heal rates among the 3 treatment methods, to identify risk factors adversely affecting the heal rate, and to predict the probability of successful healing of a nonunion of any given risk profile treated with each of the 3 forms of therapy. Seven risk factors were identified: duration of nonunion, prior bone graft surgery, prior electrical treatment, open fracture, osteomyelitis, comminuted or oblique fracture, and atrophic nonunion. When no risk factors were present, there were no significant differences among the 3 treatment methods. As progressively more risk factors were present, the predicted heal rates decreased significantly regardless of the treatment method. Some differences among the treatment groups did appear in the heal rates: bone graft surgery yielded a worse heal rate when there was a previous bone graft failure, and capacitive coupling had a worse heal rate in the presence of an atrophic nonunion.

Real-time calcium response of cultured bone cells to fluid flow.

Using a parallel-plate flow chamber and fura-2 fluorescence microscopy, intracellular calcium was measured cell by cell in preconfluent primary culture rat calvarial bone cells to 18, 35, and 70 dynes/cm2 of fluid-induced shear stress. A heterogeneous response with respect to peak amplitude and latency was observed for the culture, with an overriding dose-dependent relationship between the mean peak amplitude of response and shear-stress magnitude. A dose dependence was observed between the number of responsive cells (responding > 50% over basal levels) and shear-stress magnitude. Not all cells could be restimulated by repeated exposure to flow. The observed cell response appears to be independent of whether cells are clustered together or isolated. Substratum stretch, hydrostatic pressure, and fluid shear stress have been shown in the literature to increase inositol phosphate (IP3) in bone cells, with IP3 causing the release of calcium from intracellular stores such as the endoplasmic reticulum. Therefore, a 6-fold inhibitory effect observed when calcium release from stores was blocked with 8-(n,N-diethylamino)octyl 1-3,4,5-atrimethoxybenzoate hydrochloride implicates an IP3 biochemical pathway mediating the fluid flow response in bone cells.

Zeta potential of bone from particle electrophoresis: solution composition and kinetic effects.

The morphology of bone may be influenced by many factors, including electromechanical ones such as electric potentials, electric fields, or zeta potentials. Stress-generated potential studies in bone and particle electrophoresis studies using calcium-deficient hydroxyapatite have shown that the zeta potential depends on the composition of the steeping fluid and steeping time. To better quantify and understand these in situ potential changes in bovine cortical bone, the effects of alterations in calcium, phosphate, and fluoride concentrations in Neuman's Fluid (NF), which simulates in vivo bone extracellular fluid, were investigated using particle electrophoresis. The zeta potential increased in magnitude with increased calcium concentration in NF in as little as 17 min. Increasing phosphate concentration in NF also increased the zeta potential magnitude. These results provide support for a structural model of the bone matrix surface-bone fluid interface, which incorporates the bone surface proper (composed of collagen, mineral, and boundary regions), stationary layer (in which ions, ionic complexes, and proteins may be adsorbed), and bone extracellular fluid. These results, coupled with those of previous studies, indicate that the protein phase probably has an important role in the determination of the physiologic zeta potential; the role of the mineral phase may also be important.

Low-power electromagnetic stimulation of osteotomized rabbit fibulae. A randomized, blinded study.

The purpose of this study was to determine whether low-power-consuming symmetrical-waveform electromagnetic stimuli could increase the stiffness of fracture sites in a rabbit fibular-osteotomy model. Both active and placebo devices were used in a blinded study protocol. Dose-response studies of pulse amplitude and pulse width were performed by continuous application (twenty-four hours a day) of repetitive (fifteen-hertz), bursted (five-millisecond-long) symmetrical, rectangular electromagnetic stimulus waveforms. The power consumed by these stimuli is approximately one-fifth that consumed by the pulsing electromagnetic field devices that are in current clinical use. Significant increase of callus bending stiffness was produced by pulse widths of five to seven microseconds and pulse amplitudes of fifty to 100 millivolts.

Stress-generated potentials in bone: effects of bone fluid composition and kinetics.

It is now generally accepted that stress-generated potentials (SGPs) at low frequencies are due to an electrokinetic phenomenon in the small interior surfaces of bone and are directly proportional to the zeta potential, a property of the poorly characterized bone surface-bone fluid interface. We hypothesized that this interface would be labile and might explain the controversy over whether or not the polarity of SGPs can invert under certain conditions. In this paper, the effects of alterations in the steeping fluid on SGPs for 87 samples from 15 animals were examined in four-point bending for steeping times of < or = 65 h. Calcium and fluoride in distilled-deionized water and constant ionic strength solutions produced concentration-dependent inversions in the SGP sign. A new steady state was reached in approximately 18 h. The effects of the fluoride anion (unlike the calcium cation) apparently were reversible. The results strongly suggest that the zeta potential at the labile bone surface-bone fluid interface can undergo dramatic changes, not only in magnitude but also in sign. The results further suggest that the preparation of bone samples is critical to the understanding of this interface in vivo, and they support the hypothesis that SGPs have a role in bone remodeling.

Particle microelectrophoresis of calcium-deficient hydroxyapatite: solution composition and kinetic effects.

Concurrent work demonstrates that the zeta potential of bone is multivalued and systematically alterable by changes in sample preparation, steeping fluid composition, and steeping time. Since bone mineral is a mixture of carbonated calcium-deficient hydroxyapatites, and since the zeta potential of calcium-deficient hydroxyapatite (CDHA) is altered by pH and time in HNO3-KOH solutions, the zeta potential of CDHA in physiologic Neuman's fluid (NF) compared with that seen in bone could reveal important information on the contribution of the mineral phase to the zeta potential of bone. In addition, such information may be valuable in designing and evaluating calcium-phosphate ceramics for increased bone ingrowth. Results demonstrate that the zeta potential of CDHA in NF is negative. With increasing calcium in NF, the zeta potential magnitude of CDHA decreases and inverts to positive values given sufficient calcium concentration and steeping time. This result is opposite to that seen in bone, suggesting that exposed CDHA is not the predominant bone microsurface and implicating a bone surface protein component. With increasing phosphate in NF, the zeta potential magnitude increases to more negative values. While low concentrations of fluoride showed no effect, the possibility of an effect with higher concentrations is still to be determined.

In vitro bone-cell response to a capacitively coupled electrical field. The role of field strength, pulse pattern, and duty cycle.

Newborn rat calvarial bone cells were grown to confluence and subjected to a matrix of sine wave 60-kHz capacitively coupled electrical signals of various field strengths, pulse-burst patterns, and duty cycles. Both [3H] thymidine incorporation into DNA and alkaline phosphatase activity were evaluated in field strengths ranging from 0.0001 to 20 mV/cm, with pulse-burst patterns ranging from continuous to 5 milliseconds ON/495 milliseconds OFF, with daily duty cycles ranging from 0.25% to 25%. A significant increase in proliferation occurred in field strengths of 0.1, 1, and 20 mV/cm when the signal was applied continuously for six hours. Significant proliferation also occurred when the 20-mV/cm field was pulsed for six hours at 5 milliseconds ON/495 milliseconds OFF and at 5 milliseconds ON/245 milliseconds OFF. No change in alkaline phosphatase activity occurred in the 20-mV/cm field with any signal. At 1 mV/cm, there was a significant decrease in alkaline phosphatase activity in the continuous signal and in the 5 milliseconds ON/62 milliseconds OFF signal; in the lower fields evaluated, there was an actual decrease in alkaline phosphatase activity with some of the signals. The field strength plays a dominant role in determining the bone-cell's proliferative response, and to a lesser extent the alkaline phosphatase activity response, to a capacitively coupled electric field. The pulse configuration and the duty cycle are also important, but only if the proper field strength is being applied to the cell.