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.

Prevention and treatment of sciatic denervation disuse osteoporosis in the rat tibia with capacitively coupled electrical stimulation.

Osteoporosis in the sciatic-denervated rat tibia was both prevented and reversed with a capacitively coupled electrical field. In both the prevention of the development of osteoporosis and the reversal of a previously established osteoporosis, a statistically significant enhancement of wet weight, dry weight, ashed weight, ultimate strength, cortical area, cortical thickness, and a concomitant decrease in cortical porosity occurred in the stimulated, denervated tibiae of the experimental animals compared with the nonstimulated, denervated tibiae of the control animals. These effects exhibited dose-response characteristics. A 60 kHz symmetrical sinewave signal was effective in preventing osteoporosis at a range of 5-10 peak-to-peak, and it was effective in reversing osteoporosis at 10 V peak-to-peak. Reversal of a well-established osteoporosis in laboratory animals has not been reported previously. Continued investigation into the use of a capacitively coupled electrical field in the prevention and treatment of osteoporosis seems warranted from these studies.

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.

The origin of stress-generated potentials in fluid-saturated bone.

The objective of this study was to determine the origin of stress-generated potentials (SGPs) in fluid-saturated bone. Stress-generated potentials were studied as a function of the conductivity, NaCl concentration, and viscosity of the fluid contained within cortical human and bovine bone. Bone samples were soaked in solutions in which NaCl and sucrose concentrations were systematically varied. Macroscopic SGPs and their relaxation times were measured as a function of these properties. Stress-generated potentials were also measured as a function of conductivity and NaCl concentration by using a microelectrode. The results of this study confirmed that the properties of the fluid in bone have a great influence on the magnitude and time dependence of the SGP. Especially notable was the observation that solutions of high NaCl concentration consistently reversed the polarity of the SGP. These results are consistent with streaming potential theory. Although fluid-saturated bone may retain some piezoelectric properties, SGPs are predominantly caused by streaming potentials.

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.

Ion concentration effects on the zeta potential of bone.

Stressed bone generates an electrical potential, the sign of which reverses in the presence of specific concentrations of sodium, potassium, or calcium ions. A study was designed to test the hypotheses, drawn from stress-generated potential (SGP) studies, that the reversal in polarity of the zeta potential of bone is the cause of this SGP polarity reversal. The zeta potential of bone particles, prepared from fresh bovine metatarsals as homogeneous 5 microns dispersions, was measured by free-fluid electrophoresis in different concentrations of sodium chloride. The zeta potential of bone particles reversed sign at a sodium ion concentration that was the same as that found to reverse measured SGPs, namely 0.74 molar. This finding is consistent with the hypothesis that streaming potentials are the cause of SGPs in fluid-saturated bone. It was possible to obtain electrophoretic mobility histograms, and hence zeta-potential histograms, of bone particles by using a Pen Kem, Inc. (Bedford Hills, NY, U.S.A.) "3000" Automated Electrokinetic Analyzer. At sodium ion concentrations at which bone particles were nearly neutral, the zeta-potential histograms indicated a broad distribution of particle charge, with some particles being negative, some positive, and some neutral. In addition, it was found that particles prepared from Formalin-fixed bone produced the same electrokinetic results as those prepared from fresh bone, and that the addition of MOPS buffer caused the zeta potential to invert sign from negative to positive values at ion concentrations exceeding 3.0 molar.

Comparison of asymmetrical and symmetrical pulse waveforms in electromagnetic stimulation.

Pulsing electromagnetic field (PEMF) stimulation is a noninvasive therapeutic modality that has been successfully used to stimulate healing of surgically resistant human bone fracture nonunions. Asymmetry of the stimulus pulse waveform was thought to be necessary for therapeutic effectiveness, but asymmetrical pulses require significant electrical energy that constrains clinical delivery systems to suboptimal designs. Development of low-energy consuming stimuli will enable clinical device improvements and may provide additional information about the interaction of electromagnetic fields with tissues. The objectives of this study were (a) to determine if asymmetry of the stimulus pulse waveform is needed for efficacy and (b) to determine if symmetrical pulse waveform stimuli also can produce a beneficial therapeutic response. The rabbit fibular osteotomy model was used to answer these questions and to identify which components of the clinically used asymmetrical PEMF produce the therapeutic response. The results suggest that asymmetry is not necessary and that a narrow pulse width, symmetrical square wave signal can also stimulate stiffness increases in this model. The data also suggest that the high-amplitude, narrow-pulse portion of the asymmetrical PEMF is the principal component of the signal pulse that is responsible for the clinical therapeutic effect.

In vivo growth plate stimulation in various capacitively coupled electrical fields.

The right proximal tibial growth plates of adolescent New Zealand white rabbits were stimulated with various capacitively coupled electrical fields. Capacitor plates attached to plastic jigs placed over the proximal tibiae were connected to function generators which supplied sine wave signals of 60 kHz frequency and various voltages (2.5, 5, 10, and 20 V peak-to-peak). At 0 h and at 48 h, each animal was labeled with intravenously injected oxytetracycline. For the next 48 h, each right proximal tibial growth plate was stimulated with one of the above electrical signals. At the end of the 48 h of stimulation, the animals were sacrificed, and the tibiae were excised; histological sections of the proximal growth plate in each tibia were made, and the distance the labels moved away from the bone-cartilage junction down into the metaphysis was measured under fluorescent microscopy. Results indicate that the rabbit growth plate can be consistently stimulated to statistically significant accelerated growth in a capacitively coupled electrical field. A dose-response effect was noted, with 5 V peak-to-peak exhibiting maximum growth acceleration. Thus, the application of the proper capacitively coupled electrical field significantly stimulated the rabbit growth plate at voltage and current levels that are safe for human use.

Pulsing electromagnetic field stimulation of the in vitro growth plate.

Specific pulsing electromagnetic fields (PEMFs) have been used to stimulate growth and repair of osteogenic tissues; however, the basis for this specificity is unknown. Previously, we determined the relevant electromagnetic field parameters of the clinically used PEMF and independently verified the beneficial effects of PEMFs on the rabbit fibula fracture healing model. The goal of the present study was to develop an in vitro model that would permit the effectiveness of various electric and magnetic field components of the PEMF to be determined. The costochondral junction (CCJ) of the 21-day-old rat was exposed in vitro to PEMFs with various electric and magnetic field component amplitudes. Response of this model to PEMFs was determined by nondestructive macrophotographic measurement of CCJ growth. Preliminary data indicated that temperature effects were present in this in vitro system. Subsequent experiments designed to separate the effects of temperature and PEMFs on the growth of CCJs in tissue culture were performed. Results indicate that accurate and frequent temperature measurements must be made for in vitro models being used to study effects of PEMFs. Small temperature differences induced by the coils used to produce PEMFs in the CCJ experimental system can have significant stimulatory effects, and the combined effects of temperature and PEMFs are not linearly additive in this model. Furthermore, our results suggest that thermal and PEMF stimuli could affect macrophotographically measured growth of the CCJ by separate mechanisms or could have a synergistic effect. Therefore, PEMF stimulation experiments should be performed under strictly "athermal" conditions.

Proliferative and synthetic response of bovine growth plate chondrocytes to various capacitively coupled electrical fields.

In vitro monolayer cultures of growth plate chondrocytes isolated from newborn calf costochondral junctions were subjected to capacitively coupled electrical fields for 48 h. In part A, the electrical signal was a 60-kHz sine wave applied at different voltages so as to produce electrical fields at the pericellular level of 7, 20, 50, and 126 mV/cm. Incorporations of [3H]thymidine and [35S]sulfate were assayed to determine the effect of the above fields on cells proliferation and matrix synthesis, respectively. Proliferation was increased by 47% in the 20 mV/cm field whereas the same field decreased [35S]sulfate incorporation by 21%. These changes were significant at p less than 0.05 in both instances. In part B, the 20 mV/cm field was applied in a pulsed fashion to produce daily duty cycles of 100, 25, 2, and 0.25%. Incorporation of [3H]thymidine, [35S]sulfate, and [14C]proline per DNA were assayed. Results indicated that the 100, 25, and 0.25% percent duty cycles showed significantly (p less than 0.01-0.05) increased proliferation, whereas the 0.25% signal (5 ms on/495 ms off for 6 h/day) significantly decreased [14C]proline incorporation. We conclude that the biologic response of cells in vitro is signal specific, and that the total amount of electrical energy required to stimulate the growth plate chondrocyte to increased proliferation is very small since the total time the 0.25% duty cycle signal was only 3.6 min of a 24-h period.

Treatment of denervation/disuse osteoporosis in the rat with a capacitively coupled electrical signal: effects on bone formation and bone resorption.

Utilizing a sciatic neurectomy model of disuse osteoporosis, the effects on rates of bone formation and bone resorption were examined when a capacitively coupled electrical signal was applied to the denervated tibia in the rat. It was found that a low-voltage, symmetrical sine wave, 60-kHz, capacitively coupled signal had no significant effect on the amount of bone resorption occurring in denervated right tibiae in rats previously labeled with [3H]tetracycline. This was true whether the signal was applied while osteoporosis was developing (prevention of osteoporosis) or after it had been established (treatment of osteoporosis). If a similar capacitively coupled signal was applied to rats in which osteoporosis was well established, and the rats were labeled with [3H]tetracycline daily during a 12-day treatment period, it was found that there was statistically significant enhancement of the amount of new bone formation (increased [3H]tetracycline incorporation) in the tibiae that received the signal as compared with that of the controls. These results indicate that prevention or amelioration of disuse osteoporosis that occurs with a capacitively coupled electrical signal is due not to a change in the rate of bone resorption, but to an increase in the rate of bone formation.

Fracture healing in the rabbit fibula when subjected to various capacitively coupled electrical fields.

The effect of capacitively coupled electrical stimulation on the healing of midshaft transverse osteotomies of the rabbit fibula is assessed roentgenographically, mechanically, and histologically. The results show that a dose-response curve for capacitive coupling and fracture healing exists and that a 220 mV, 250 microA, 60 kHz applied electrical signal (0.33 V/cm internal electric field) is the most effective signal for fracture stimulation in this model.

The influence of functional use of endosseous dental implants on the tissue-implant interface. I. Histological aspects.

The tissue-implant interfaces of functional and non-functional endosseous dental implants were compared histologically for up to one year post-operatively. Nonmineralized connective tissue zones (a "fibrous capsule") existed in all functional interfaces. Direct, or nearly direct, bone apposition to implants occurred in non-functional interfaces. The origin of this result and its significance in dental implantology is discussed.

The influence of functional use of endosseous dental implants on the tissue-implant interface. II. Clinical aspects.

Functional and non-functional endosseous dental implants were clinically compared in beagle mandibles for up to one year post-operatively. Differing biomechanical conditions led to clinical differences between functional and non-functional implants. Typical clinical tests, however, did not always reveal detailed histological differences between implant-tissue interfaces of functional and non-funcional implants.

Treatment of recalcitrant non-union with a capacitively coupled electrical field. A preliminary report.

Twenty-two well established non-unions in twenty patients were treated with a capacitively coupled electrical signal (sine wave, sixty kilohertz, five volts peak to peak) that was applied non-invasively through stainless-steel capacitor plates placed on the skin surface overlying the approximate site of the non-union. The average age of the eleven female and nine male patients in this series was 38.4 years, and the average duration of the twenty-two non-unions was 3.3 years. Seventeen of the non-unions were labeled recalcitrant, meaning that they had failed to heal after either previous bone-grafting or another type of electrical stimulation, or both. Five of the non-unions had not been previously treated. Seventeen (77.3 per cent) of the non-unions achieved solid osseous union after an average of 22.5 weeks of treatment with capacitive coupling. The results in this small series were not affected by the non-union being recalcitrant, by the fact that one patient bore full weight on the extremity in a cast, by the presence of osteomyelitis, or by the presence of remaining metallic internal-fixation devices in the bone. Since capacitive coupling is non-invasive, involves portable equipment, allows full weight-bearing on the lower extremity in a cast, is easy to apply, and does not require precise localization of the capacitor plates, it has distinct advantages over other methods of treating non-union with electricity.

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.

Treatment of sciatic denervation disuse osteoporosis in the rat tibia with capacitively coupled electrical stimulation. Dose response and duty cycle.

We determined the dose-response characteristics of a low-voltage, high-frequency, capacitively coupled electrical signal that reverses an established osteoporosis in a rat tibial model with sciatic neurectomy. The electrical signals were delivered by means of stainless-steel gel-coated electrodes. In the first part of the study, the shape (sine wave) and frequency (sixty kilohertz) of the signal were kept constant while the voltage was varied from 0.01 to ten volts, peak to peak. Control osteoporotic and sham osteoporotic animals showed a mean loss of tibial ash weight of 19 and 17 per cent, respectively, twenty-eight days after sciatic neurectomy plus twelve days of no treatment using sham electrodes. Rats that were subjected to 0.25, 0.50, 1.0, 2.5, and 5.0 volts, peak to peak, for twelve days beginning on the twenty-eighth day after sciatic neurectomy all showed mean losses of tibial ash weight that were significantly less than those of the controls. The rats that had a 0.5-volt peak-to-peak signal showed the least mean loss of tibial ash weight (only 6 per cent). We concluded that a capacitively coupled electrical signal, delivered through gel-coated electrodes, can largely reverse an established disuse osteoporosis due to neurectomy in the rat tibia. In the second part of the study, the duty cycles of a sine-wave, sixty-kilohertz, 0.5-volt peak-to-peak signal were varied (12.5, 50, and 100 per cent on), and the wet, dry, and ash weights were determined and compared with those of unstimulated osteoporotic controls. Only the 100 per cent duty-cycle signal was effective in reversing the loss of bone mass in the neurectomized tibiae.

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.

Treatment of castration-induced osteoporosis by a capacitively coupled electrical signal in rat vertebrae.

Castrated male Sprague Dawley rats were subjected to various capacitively coupled electrical fields for six and eight weeks at two and 4.5 months after castration, respectively, with pairs of electrodes that were located paraspinally on the surface of the skin dorsally at the eleventh thoracic and fourth lumbar levels. When the animals were killed, dry and ash weights per unit of volume (apparent density), elastic modulus, ultimate stress, work to failure, trabecular area fraction, and mean trabecular width were determined for selected vertebrae. The results indicated that a sixty-kilohertz, 100-microampere signal (a calculated current density of five microamperes root-mean-square per square centimeter and a field of twelve millivolts root-mean-square per centimeter) significantly reversed the castration-induced osteoporosis in the lumbar vertebrae and restored bone mass per unit of volume in rats that had been stimulated for eight weeks after castration.

The proliferative and synthetic response of isolated calvarial bone cells of rats to cyclic biaxial mechanical strain.

Isolated bone cells from the calvaria of newborn rats were grown in monolayer on polyurethane membranes in specially constructed culture chambers. These were subjected to cyclic biaxial mechanical strains of 0.02 per cent (200 microstrain), 0.04 per cent (400 microstrain), and 0.1 per cent (1000 microstrain) at a frequency of one hertz for periods ranging from fifteen minutes to seventy-two hours. DNA content, an index of proliferation, was significantly increased at a strain of 0.04 per cent applied for fifteen minutes and for twenty-four and forty-eight hours. DNA content was not increased at the other amplitudes of strain that were evaluated, nor was it increased after prolonged mechanical stimulation for forty-eight hours or longer. Synthesis of collagen, non-collagenous protein, and proteoglycan, as well as activity of alkaline phosphatase, all indicators of macromolecular synthesis, were significantly decreased at a strain of 0.04 per cent applied for fifteen minutes and for twenty-four, forty-eight, and seventy-two hours. Macromolecular synthesis was not affected by the other amplitudes of strain that were evaluated in this study. At a strain of 0.04 per cent, prostaglandin E2 content was significantly increased after five, fifteen, and thirty minutes of mechanical stimulation, whereas net cAMP content did not change significantly. This suggests that the described cellular events (increased proliferation and decreased macromolecular synthesis) that occur secondary to mechanical strain are mediated, at least in part, by prostaglandin E2.