On the relationship between streaming potential and strain in an in vivo bone preparation.

Transcortical streaming potentials were measured at each of two cortical-surface sites with respect to a reference electrode in the medullary canal, in the left ulnae of six live, adult (2 yr-old), male 18.2 +/- 1.4 kg domestic turkeys, under general anesthesia, for each of two loading conditions. We observed that the relationship among streaming potential magnitude, surface strain, and strain gradient is not as simple as anticipated. Under predominantly axial and bending load conditions, significantly different strain and strain gradients were generated at the two recording sites. However, no significant differences were detectable in transcortical streaming potentials for one of the loading conditions, and only a slight difference was detected in the other. Conversely, correlations of streaming potential magnitude to strain at both sites show robust relationships (r2 = 0.45, P - 0.02), albeit with different slopes for the two sites. These findings may have implications for the contribution of streaming potentials, or at least, fluid flow to the stimulation of bone cells.

Differential phosphorylation of paxillin in response to surface-bound serum proteins during early osteoblast adhesion.

An early signaling event during the adhesion and spreading of cells is integrin-mediated tyrosine phosphorylation of the cytoskeletal adaptor protein paxillin and the non-receptor tyrosine kinase pp125(FAK) at focal contacts. To determine the influence of surface-charge and -adsorbed adhesion proteins on this signaling pathway, paxillin phosphorylation was examined during attachment of MC3T3-E1 osteoblast-like cell onto charged and uncharged polystyrene, and on adsorbed layers of serum proteins, fibronectin (Fn), vitronectin (Vn), a mixture of Fn and Vn, and albumin. Paxillin phosphorylation was induced 2.4-fold (P < 0.05) on charged vs uncharged polystyrene only in the presence of serum proteins. Activation of paxillin via Fn or Vn alone, or in combination, resulted in significantly lower phosphorylation signals compared to whole serum (41 +/- 6.9%, P < 0.05, 45 +/- 5.9%, P < 0.05, and 76 +/- 9.8%, P < 0.075, respectively). Confocal laser microscopy confirmed increased co-localization of phosphotyrosine and paxillin at protruding lamellopodia of spreading osteoblasts on charged vs uncharged serum-pretreated polystyrene. Taken together, these data suggest that subtle differences in surface characteristics mediate effects on adhering cells via adsorbed serum proteins involving the cytoskeletal adaptor protein paxillin.

"And afterwards your body to be given for public dissection": a history of the murderers dissected in Glasgow and the west of Scotland.

Between 1752 and 1832, the bodies of hanged murderers were dissected or gibbeted. During this period, 38 murderers were executed in the West of Scotland. The bodies of at least 23 were dissected in Glasgow. The stories of these murders are recounted. Insight is also given into the attitudes of the public and the anatomists to dissection of executed murderers.

Integrin-mediated mechanotransduction in vascular smooth muscle cells: frequency and force response characteristics.

Blood vessels are continuously exposed to mechanical forces that lead to adaptive remodeling and atherosclerosis. Although there have been many studies characterizing the responses of vascular cells to mechanical stimuli, the precise mechanical characteristics of the forces applied to cells to elicit these responses are not clear. We designed a magnetic exposure system capable of producing a defined normal force on ferromagnetic beads that are specifically bound to cultured cells coated with extracellular matrix proteins or integrin-specific antibodies. Rat aortic smooth muscle cells were incubated with engineered fibronectin-coated ferromagnetic beads and then exposed to a magnetic field. With activation of extracellular signal-regulated mitogen-activated protein kinase 1/2 (ERK 1/2(MAPK)) used as a prototypical marker for cell responsiveness to mechanical forces, Western blot analysis demonstrated an increase in phosphorylated ERK 1/2(MAPK) expression reaching a maximal response of a 3.5-fold increase at a total force of approximately 2.5 pN per cell. The peak response occurred after 5 minutes of exposure and slowly decreased to baseline after 30 minutes. A cyclic, rather than static, force was required for this activation, and the frequency-response curve increased approximately 2-fold between 0.5 and 2.0 HZ: Vitronectin- and beta(3) antibody-coated beads showed a response nearly identical to those coated with engineered fibronectin, whereas forces applied to beads coated with alpha(2) and beta(1) antibodies did not significantly activate ERK 1/2(MAPK). Mechanical activation of the ERK 1/2(MAPK) system in rat aortic smooth muscle cells occurs through specific integrin receptors and requires a cyclic force with a magnitude estimated to be in the piconewton range.

Osteoblastic networks with deficient coupling: differential effects of magnetic and electric field exposure.

A gap junction-deficient cell line was utilized to test whether intercellular coupling plays a significant role in modulating the influence of biophysical stimuli such as extracellular electrical currents. ROS 17/2.8 cells, an osteosarcoma cell line, along with a control transfected cell line and a connexin 43-gap junction-deficient cell line, were exposed to a time-changing magnetic flux (30 Hz, 1.8 milliTesla) sufficient to induce an electric field in the cultures on the order of 2 mV/m. Field exposure inhibited cell growth independent of gap junctional coupling, while alkaline phosphatase activity was found to be dependent on gap junctional coupling. These findings can be interpreted to suggest that magnetic and electric field exposures have differential effects on cell cultures, with magnetic field exposure inhibiting cell growth through a mechanism independent of gap junctional coupling, while the alteration in enzyme activity appears to be stimulated by the induced electric field in a gap junction-dependent manner.

Suppression of a differentiation response in MC-3T3-E1 osteoblast-like cells by sustained, low-level, 30 Hz magnetic-field exposure.

Extremely low-frequency (ELF) magnetic fields have been reported to be capable of influencing both tissue remodeling and cell phenotypic expression in culture. However, whether the cells or tissues respond directly to the magnetic flux or to the electric field induced by the time-changing magnetic flux remains a controversial topic. To address this question, we developed an osteoblast cell assay based on the activity of alkaline phosphatase, an enzyme whose activity is up-regulated during the differentiation of bone cells. MC-3T3-E1 cells plated at a confluent density were allowed to proceed through the differentiation process for 3 days, after which they were exposed to a 30 Hz, 1.8-mT r.m.s. magnetic field inducing a spatially varying electric field with a maximum intensity of 0.9 mV/m r.m.s. In situ assays of alkaline phosphatase activity at 4, 8, 16 and 64 h demonstrated a progressive inhibition of enzyme activity, the pattern of which maps to the intensity of the induced electric field (R(2) = 0.5, P<0.001). We interpret these results to indicate that cells are capable of responding to ELF induced electric fields at intensities below 1 mV/m, and that the principal effect on cells is an inhibition of differentiation.

Morphologic responses of osteoblast-like cells in monolayer culture to ELF electromagnetic fields.

Osteoblast-like cells (MC 3T3-E1) were exposed for 24 h, immediately after plating, to a 60 Hz, 0.7 mT rms magnetic flux density, sufficient to induce an electric field of 0.5 mV/m rms, in order to investigate the influence of ELF field exposure on cell morphology. Using phase contrast images of the live cells, computerized image-analysis permitted rapid and objective quantification of cell length, width, area, perimeter, circularity and angular orientation. While the field-exposed cells were consistently smaller than sham treated cells, the morphologic alterations were not significantly different in the exposed cell population when cell orientation was not considered. When analyzed with respect to cell orientation, cells oriented parallel to the induced electric field (orthogonal to the applied magnetic field) demonstrated a significant decrease in cell length and an increase in roundness. These results confirm and extend previous studies on the morphologic adaptation of cells to low level ELF electromagnetic fields. The results suggest that the observed responses most likely depend on the induced electric field, with a field intensity threshold well below 1 mV/m. Further, these results provide important clues to the specific mechanism by which such low level fields may be capable of influencing cell behavior, and help to explain some of the difficulties in obtaining robust responses in in vitro EMF experiments.

Quantifying the strain history of bone: spatial uniformity and self-similarity of low-magnitude strains.

We hypothesize that when a broad spectrum of bone strain is considered, strain history is similar for different bones in different species. Using a data collection protocol with a fine resolution, mid-diaphyseal strains were measured in vivo for both weightbearing and non-weightbearing bones in three species: dog, sheep, and turkey, with strain information collected continuously while the animals performed their natural daily activities. The daily strain history was quantified by both counting cyclic strain events (to quantify the distribution of strains of different magnitudes) and by estimating the average spectral characteristics of the strain (to quantify the frequency content of the strain signals). Counting of the daily (12-24 h) strain events show that large strains (> 1000 microstrain) occur relatively few times a day, while very small strains (< 10 microstrain) occur thousands of times a day. The lower magnitude strains (< approximately 200 microstrain) are found to be more uniform around the bone cross-section than the higher magnitude, peak strains. Strain dynamics are found to be well described by a power-law relationship and exhibit self-similar characteristics. These data lead to the suggestion that the organization of bone tissue is driven by the continual barrage of activity spanning a wide but consistent range of frequency and amplitude, and until the mechanism of bone's mechanosensory system is fully understood, all portions of bone's strain history should be considered to possibly play a role in bone adaptation.

Changes in postural muscle dynamics as a function of age.

Histologic studies have demonstrated both a decrease in size and loss in number of type II muscle fibers with increasing age. Although these age-related histologic changes are believed to result in decreased strength and functional capacity, age-related changes in muscle force dynamics have not been clearly elucidated. Using vibromyographic (VMG) techniques, we recorded muscle activity of the soleus in 40 healthy adult volunteers spanning the age range of 20-82 years to test whether changes in postural muscle dynamics, in the frequency range of 0.1-50 Hz, were also associated with age. Although muscle dynamics below 15 Hz do not change with aging, the 30-50 Hz frequency components of the VMG were found to change significantly with advancing age (r = -.619, p = .0001). This was observed in both sexes independently. The observed age-related changes in muscle force dynamics demonstrate distinct physiologic alterations in muscle fiber activity. Further research will be required to fully elucidate the relationship between age-related changes in muscle fiber activity and other age-related conditions such as postural instability and osteoporosis.

Mechanically induced calcium waves in articular chondrocytes are inhibited by gadolinium and amiloride.

Chondrocytes in articular cartilage utilize mechanical signals from their environment to regulate their metabolic activity. However, the sequence of events involved in the transduction of mechanical signals to a biochemical signal is not fully understood. It has been proposed that an increase in the concentration of intracellular calcium ion ([Ca2+]i) is one of the earliest events in the process of cellular mechanical signal transduction. With use of fluorescent confocal microscopy, [Ca2+]i was monitored in isolated articular chondrocytes subjected to controlled deformation with the edge of a glass micropipette. Mechanical stimulation resulted in an immediate and transient increase in [Ca2+]i. The initiation of Ca2+ waves was abolished by removing Ca2+ from the extracellular media and was significantly inhibited by the presence of gadolinium ion (10 microM) or amiloride (1 mM), which have previously been reported to block mechanosensitive ion channels. Inhibitors of intracellular Ca2+ release (dantrolene and 8-diethylaminooctyl 3,4,5-trimethoxybenzoate hydrochloride) or cytoskeletal disrupting agents (cytochalasin D and colchicine) had no significant effect on the characteristics of the Ca2+ waves. These findings suggest that a possible mechanism of Ca2+ mobilization in this case is a self-reinforcing influx of Ca2+ from the extracellular media, initiated by a Ca2+-permeable mechanosensitive ion channel. Our results indicate that a transient increase in intracellular Ca2+ concentration may be one of the earliest events involved in the response of chondrocytes to mechanical stress and support the hypothesis that deformation-induced Ca2+ waves are initiated through mechanosensitive ion channels.

Does bone perfusion/reperfusion initiate bone remodeling and the stress fracture syndrome?

Stress fractures have been proposed to arise from repetitive activity of training inducing an accumulation of microfractures in locations of peak strain. However, stress fractures most often occur long before accumulation of material damage could occur; they occur in cortical locations of low, not high, strain; and intracortical osteopenia precedes any evidence of micro-cracks. We propose that this lesion arises from a focal remodeling response to site-specific changes in bone perfusion during redundant axial loading of appendicular bones. Intramedullary pressures significantly exceeding peak arterial pressure are generated by strenuous exercise and, if the exercise is maintained, the bone tissue can suffer from ischemia caused by reduced blood flow into the medullary canal and hence to the inner two-thirds of the cortex. Site specificity is caused by the lack, in certain regions of the cortex, of compensating matrix-consolidation-driven fluid flow which brings nutrients from the periosteal surface to portions of the cortex. Upon cessation of the exercise, re-flow of fresh blood into the vasculature leads to reperfusion injury, causing an extended no-flow or reduced flow to that portion of the bone most strongly denied perfusion during the exercise. This leads to a cell-stress-initiated remodeling which ultimately weakens the bone, predisposing it to fracture.

Skeletal cell stresses and bone adaptation.

There is no tissue in which mechanical stresses have been studied in more detail than the skeletal system, this focus arising primarily because bone plays a clear structural role in the body. However, the hypothesis that the skeleton represents an optimally designed structure has contributed remarkably little to our understanding of the development and adaptive capabilities of bone tissue. Recent investigations on the consequences of mechanical, hydrostatic, and electrical stresses on the cells of bone tissue have served to redirect the discussion of bone modeling and remodeling processes. These studies have refocused attention on the importance of chronic low-level dynamic stresses in mediating the physiologic response of bone tissue. Important recent observations suggest that an approach premised on the self-organizational properties of bone tissue may lead to significant improvements in our understanding and control of bone morphologic development, adaptation, and healing.

Nonlinear dependence of loading intensity and cycle number in the maintenance of bone mass and morphology.

The daily stress stimulus theory of bone adaptation was formulated to describe the loading conditions necessary to maintain bone mass. This theory identifies stress/strain magnitude and loading cycle number as sufficient to define an appropriate maintenance loading signal. Here, we extend the range over which loading cycle number has been evaluated to determine whether the daily stress stimulus theory can be applied to conditions of very high numbers of loading cycles at very low strain magnitudes. The ability of a relatively high-frequency (30-Hz) and moderate-duration (60-minute) loading regimen to maintain bone mass in a turkey ulna model of disuse osteopenia was evaluated by correlating the applied strain distributions to site-specific remodeling activity. Changes in morphology were investigated following 8 weeks of disuse compared with disuse plus daily exposure to 108,000 applied loading cycles sufficient to induce peak strains of approximately 100 microstrain. A strong correlation was observed between the preservation of bone mass and longitudinal normal strain (R = 0.91) (p < 0.01). The results confirm the strong antiresorptive influence of mechanical loading and identify a threshold near 70 microstrain for a daily loading cycle regimen of approximately 100,000 strain cycles. These results are not consistent with the daily stress stimulus theory and suggest that the frequency or strain rate associated with the loading stimulus must also play a critical role in the mechanism by which bone responds to mechanical strain.

Cloning of a novel cDNA expressed during the early stages of fracture healing.

Using differential mRNA display (DD-PCR), a novel cDNA, FxC1 (Fracture Callus 1) was isolated from the early stages of a healing fractured femur. Utilizing 5' RACE PCR, a 598-bp full-length cDNA was obtained for FxC1 that contains an open reading frame (ORF) of 243 bp, encoding for an 80 amino acid protein. Within this ORF, a leucine zipper motif was present. In vitro transcription/translation of the full-length cDNA generated the expected 9-kDa protein. Northern analysis reveals that this gene is expressed in calluses harvested from post-fracture day 5, 7 and 10, as well as in several other tissues and bone-derived cell lines. During the differentiation of MC3T3 cells along the osteoblast lineage, FxC1 expression increases 3- to 4-fold during the production and deposition of matrix proteins, suggesting a possible role for this protein in cell differentiation.

Patterns of strain in the macaque ulna during functional activity.

In vivo bone strain experiments were performed on the ulnae of three female rhesus macaques to test how the bone deforms during locomotion. The null hypothesis was that, in an animal moving its limbs predominantly in sagittal planes, the ulna experiences anteroposterior bending. Three rosette strain gauges were attached around the circumference of the bone slightly distal to midshaft. They permit a complete characterization of the ulna's loading environment. Strains were recorded during walking and galloping activities. Principal strains and strain directions relative to the long axis of the bone were calculated for each gauge site. In all three animals, the lateral cortex experienced higher tensile than compressive principal strains during the stance phase of walking. Compressive strains predominated at the medial cortex of two animals (the gauge on this cortex of the third animal did not function). The posterior cortex was subject to lower strains; the nature of the strain was highly dependent on precise gauge position. The greater principal strains were aligned closely with the long axis of the bone in two animals, whereas they deviated up to 45 degrees from the long axis in the third animal. A gait change from walk to gallop was recorded for one animal. It was not accompanied by an incremental change in strain magnitudes. Strains are at the low end of the range of strain magnitudes recorded for walking gaits of nonprimate mammals. The measured distribution of strains in the rhesus monkey ulna indicates that mediolateral bending, rather than anteroposterior bending, is the predominant loading regime, with the neutral axis of bending running from anterior and slightly medial to posterior and slightly lateral. A variable degree of torsion was superimposed over this bending regime. Ulnar mediolateral bending is apparently caused by a ground reaction force vector that passes medial to the forearm. The macaque ulna is not reinforced in the plane of bending. The lack of buttressing in the loaded plane and the somewhat counterintuitive bending direction recommend caution with regard to conventional interpretations of long bone cross-sectional geometry.

Effects of electromagnetic fields in experimental fracture repair.

The clinical benefits of electromagnetic fields have been claimed for 20 centuries, yet it still is not clear how they work or in what circumstances they should be used. There is a large body of evidence that steady direct current and time varying electric fields are generated in living bone by metabolic activity and mechanical deformation, respectively. Externally supplied direct currents have been used to treat nonunions, appearing to trigger mitosis and recruitment of osteogenic cells, possibly via electrochemical reactions at the electrode-tissue interface. Time varying electromagnetic fields also have been used to heal nonunions and to stabilize hip implants, fuse spines, and treat osteonecrosis and osteoarthritis. Recent research into the mechanism(s) of action of these time varying fields has concentrated on small, extremely low frequency sinusoidal electric fields. The osteogenic capacity of these fields does not appear to involve changes in the transmembrane electric potential, but instead requires coupling to the cell interior via transmembrane receptors or by mechanical coupling to the membrane itself.

Whole-body vibration in the skeleton: development of a resonance-based testing device.

Whole-body vibration (WBV) has been demonstrated to have a strong influence on physiological systems, ranging from severely destructive to potentially beneficial. Unfortunately, the study of WBV in a controlled manner is commonly constrained by space and budgetary factors, particularly where vibration in the low frequency range is considered. In the work presented here, a small, low-cost device for performing WBV of the human skeleton is developed to assist in studies of vertical acceleration in a clinical setting. The device design consists of a spring-supported plate driven by an 18 N peak-force electromagnetic actuator, and the associated driving and monitoring electronics. Animal and human lumped-mass models have been coupled with a model of the loading device to seek a resonance response in the vicinity of 30 Hz. This approach minimizes the loading requirements of such a device, and thus a major component of the cost, yet can provide peak accelerations of 0.15 g at a frequency of 30 Hz in a small, lightweight package capable of use in a clinical or laboratory setting.

Strain gradients correlate with sites of periosteal bone formation.

We examined the hypothesis that peak magnitude strain gradients are spatially correlated with sites of bone formation. Ten adult male turkeys underwent functional isolation of the right radius and a subsequent 4-week exogenous loading regimen. Full field solutions of the engendered strains were obtained for each animal using animal-specific, orthotropic finite element models. Circumferential, radial, and longitudinal gradients of normal strain were calculated from these solutions. Site-specific bone formation within 24 equal angle pie sectors was determined by automated image analysis of microradiographs taken from the mid-diaphysis of the experimental radii. The loading regimen increased mean cortical area (+/-SE) by 32.3 +/- 10.5% (p = 0.01). Across animals, some periosteal bone formation was observed in every sector. The amount of periosteal new bone area contained within each sector was not uniform. Circumferential strain gradients (r2 = 0.36) were most strongly correlated with the observed periosteal bone formation. SED (a scalar measure of stress/strain magnitude with minimal relation to fluid flow) was poorly correlated with periosteal bone formation (r2 = 0.01). The combination of circumferential, radial, and longitudinal strain gradients accounted for over 60% of the periosteal new bone area (r2 = 0.63). These data indicate that strain gradients, which are readily determined given a knowledge of the bone's strain environment and geometry, may be used to predict specific locations of new bone formation stimulated by mechanical loading.

Correlation of bony ingrowth to the distribution of stress and strain parameters surrounding a porous-coated implant.

The ability of shear strains to inhibit bony ingrowth was investigated by use of a transcortical porous-coated cylindrical plug implant in a functionally isolated turkey ulna model in which the mechanical loading environment could be accurately controlled and rigorously defined. The distribution of ingrowth at the bone-implant interface was quantified following 8 weeks of in vivo loading consisting of 100 seconds per day of a 20 Hz sinusoidal stimulus sufficient to cause a local peak strain of approximately 100 microstrain in the cortex at the bone-implant interface in four turkeys. A nonuniform but repeatable pattern of bony ingrowth, from 33 +/- 6 to 72 +/- 6% (mean +/- SE), was observed. The mechanical environment in the vicinity of the bone-implant interface was calculated using a three-dimensional elastic orthotropic finite element model. The general stress-strain state of the bone as predicted by the finite element model was validated in two additional turkeys using four three-element rosette strain gauges, while high resolution moiré interferometry was used to determine the mechanical state of the region immediately adjacent to the implant itself. Shear strains and stresses were evaluated at the interface and correlated to the pattern of bony ingrowth circumscribing the implant interface. Linear regressions between ingrowth and both shear strain and shear stress were negative, with the values of R = -0.75 and R = -0.78 (p < 0.001), respectively, indicating significant inhibition of ingrowth where shear components were maximal. These results suggest that the minimization of shear stress and strain components is a major determinant in achieving successful ingrowth of bone into a prosthesis.

Gap junctional intercellular communication contributes to hormonal responsiveness in osteoblastic networks.

To evaluate whether intercellular coupling via connexin43 gap junction channels modulates hormonal responsiveness of cells in contact, we have created osteoblastic cell lines deficient in connexin43. Osteoblastic ROS 17/2.8 cells were transfected with a plasmid containing an antisense cDNA construct to rat connexin43. Control transfection did not alter cell-to-cell coupling nor connexin43 mRNA or protein expression relative to nontransfected ROS 17/2.8 cells. In contrast, stable transfection with an antisense connexin43 cDNA resulted in two clones, RCx4 and RCx16, which displayed significant decreases in connexin43 mRNA and protein expression and were dramatically deficient in cell-to-cell coupling. Phenotypically, all transfectants retained osteoblastic characteristics. However, cells rendered connexin43-deficient through antisense transfection displayed a dramatic attenuation in the cAMP response to parathyroid hormone. Alterations in hormonal responses were not due to changes in parathyroid hormone receptor number or binding kinetics nor to alterations in adenylyl cyclase activity. These results indicate that gap junctions may be required for mediating hormonal signals. Furthermore, these experiments support a regulatory role for connexin43-mediated intercellular communication in the modulation of hormonal responses within elaborately networked bone cells.