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K J McLeod

Publications and source records attributed to K J McLeod.

At least 19 recordsLinked to original sources
PubMed1997

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.

Animals↗
PubMed1996

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.

Animals↗
PubMed1996

Formation of osteoclast-like cells is suppressed by low frequency, low intensity electric fields.

With use of a solenoid to generate uniform time-varying electric fields, the effect of extremely low frequency electric fields on osteoclast-like cell formation stimulated by 1,25(OH)2D3 was studied in primary murine marrow culture. Recruitment of osteoclast-like cells was assessed by counting multinuclear, tartrate-resistant acid phosphatase positive cells on day 8 of culture. A solenoid was used to impose uniform time-varying electric fields on cells; sham exposures were performed with an identical solenoid with a null net electric field. During the experiments, both solenoids heated interiorly to approximately 1.5 degrees C above ambient incubator temperature. As a result of the heating, cultures in the sham solenoid formed more osteoclast-like cells than those on the incubator shelf (132 +/- 12%). For this reason, cells exposed to the sham solenoid were used for comparison with cultures exposed to the active coil. Marrow cells were plated at 1.4 x 10(6)/cm2 in square chamber dishes and exposed to 60 Hz electric fields at 9.6 muV/cm from days 1 to 8. Field exposure inhibited osteoclast-like cell recruitment by 17 +/- 3% as compared with sham exposure (p < 0.0001). Several variables, including initial cell plating density, addition of prostaglandin E2 to enhance osteoclast-like cell recruitment, and field parameters, were also assessed. In this secondary series, extremely low frequency fields inhibited osteoclast-like cell formation by 24 +/- 4% (p < 0.0001), with their inhibitory effect consistent throughout all variations in protocol. These experiments demonstrate that extremely low intensity, low frequency sinusoidal electric fields suppress the formation of osteoclast-like cells in marrow culture. The in vitro results support in vivo findings that demonstrate that electric fields inhibit the onset of osteopenia and the progression of osteonecrosis; this suggests that extremely low frequency fields may inhibit osteoclast recruitment in vivo.

Animals↗
PubMed1996

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.

Animals↗
PubMed1995

Morphologic stages in lamellar bone formation stimulated by a potent mechanical stimulus.

The temporal stages of lamellar bone formation were studied using an animal model subject to up to 16 weeks of a controlled, externally applied load. The left ulnae of 15 adult male turkeys were functionally isolated via transverse metaphyseal osteotomies, while transcutaneous Steinmann pins permitted in vivo loading of the preparation via a servo-hydraulic actuator. For 5 days per week, the ulnae were exposed to 100 cycles per day of an applied load sufficient to cause a peak strain normal to the bone's longitudinal axis of 2000 microstrain (mu E). The contralateral limb was left surgically undisturbed and served as a baseline control. Following a loading period of 4, 8 or 16 weeks, ulnae were harvested and prepared for quantitative bone histomorphometry. Compared with each animal's contralateral ulna, the area of the experimental ulnae increased by 12.5% (+/- 5.6%) at 16 weeks. Periosteal mineral apposition rates in the loaded ulnae were significantly increased compared with control values, with a maximum rate of 6.0 +/- 3.4 microns/day at 5 weeks, slowing to 2.0 +/- 0.3 microns/day by 15 weeks. At 16 weeks, new bone was composed of primary and secondary osteons as well as circumferential lamellae, with osteocyte density and organization indistinguishable from that of the original cortex. Remnants of the initial woven bone response seen at 4 weeks remained clearly visible at both 8 and 16 weeks as diffusely labeled interstitial elements within the newly formed lamellar construct.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed1995

Cell-to-cell communication in osteoblastic networks: cell line-dependent hormonal regulation of gap junction function.

We have characterized the distribution, expression, and hormonal regulation of gap junctions in primary cultures of rat osteoblast-like cells (ROBs), and three osteosarcoma cell lines, ROS 17/2.8, UMR-106, and SAOS-2, and a continuous osteoblastic cell line, MC3T3-E1. All cell lines we examined were functionally coupled. ROS 17/2.8 were the more strongly coupled, while ROB and MC3T3-E1 were moderately coupled and UMR-106 and SAOS-2 were weakly coupled. Exposure to parathyroid hormone (PTH) for 1 h increased functional coupling in ROB cells in a concentration-dependent manner. Furthermore, PTH(3-34), an analog of PTH with binds to the PTH receptor and thus attenuates PTH-stimulated cAMP accumulation, also attenuated PTH-stimulated functional coupling in ROB. This suggests that PTH increases functional coupling partly through a cAMP-dependent mechanism. A 1 h exposure to PTH did not affect coupling in ROS 17/2.8, UMR-106, MC3T3-E1, or SAOS-2. To examine whether connexin43 (Cx43), a specific gap junction protein, is present in functionally coupled osteoblastic cells, we characterized Cx43 distribution and expression. Indirect immunofluorescence with antibodies to Cx43 revealed that ROS 17/2.8, ROB, and to a lesser extent MC3T3-E1 and UMR-106, expressed Cx43 immunoreactivity. SAOS-2 showed little if any Cx43 immunoreactivity. Cx43 mRNA and Cx43 protein were detected by Northern blot analysis and immunoblot analysis, respectively, in all cell lines examined, including SAOS-2. Our findings suggest that acute exposure to PTH regulates gap junction coupling, in a cell-line dependent manner, in osteoblastic cells.(ABSTRACT TRUNCATED AT 250 WORDS)

3T3 Cells↗
PubMed1995

Chondrocytes isolated from mature articular cartilage retain the capacity to form functional gap junctions.

The distribution, expression, and functionality of gap junctions was examined in bovine chondrocytes (BCs) isolated from mature articular cartilage. BC cells displayed immunoreactivity for connexin 43 (Cx43), a specific gap junction protein. Cx43 protein expression was confirmed by Western blot analysis, and Cx43 mRNA was detected by nuclease protection assay. Additionally, BCs were shown to be functionally coupled, as revealed by dye transfer studies, and octanol, a gap junction uncoupler, greatly attenuated coupling. Furthermore, confocal microscopy of fluo-3 loaded BC cells revealed that deformation-induced cytosolic Ca2+ ion (Ca2+) signals propagated from cell-to-cell via gap junctions. To our knowledge, this is the first evidence suggesting that chondrocytes isolated from adult articular cartilage express functional gap junctions.

Animals↗
PubMed1995

Mechanically induced periosteal bone formation is paralleled by the upregulation of collagen type one mRNA in osteocytes as measured by in situ reverse transcript-polymerase chain reaction.

Reverse transcript polymerase chain reaction (RT-PCR) was developed for use in situ to measure mechanically mediated changes in gene expression activity in osteocytes within dense cortical bone. Using the functionally isolated turkey ulna model of bone adaptation, the left ulna of 6 old adult (36-40 months) male turkeys were subject to 4 weeks of a mechanical regimen consisting either of (1) 3000 microstrain at 1 Hz for 5 minutes/day or (2) 500 microstrain at 30 Hz for 10 minutes/day. The right ulna of each bird remained intact and served as control. Only a small percentage of osteocytes in the intact control bones and the 3000 microstrain ulnae showed any evidence of mRNA for collagen (each 1.2% +/- 0.3%). However, mRNA for collagen type I was strongly evident in 92.4% (+/-2%) of the osteocytes within the ulnae subject to the high frequency, low magnitude load. Sense primer control sections from both experimental and intact animals were used to verify that only osteocytes of the loaded bone had elevated the level of collagen mRNA. This high frequency, low magnitude mechanical stimulus was also sufficient to stimulate substantial new bone formation (14% +/- 5% over intact controls), whereas the low frequency, high magnitude stimulus failed to elicit any bone formation (-3% +/- 7%). These experiments show that specific mechanical regimens can activate the osteocyte's expression of a message responsible for the synthesis of proteins remote from the site where the formation of bone is ultimately to occur, even under systemic distress such as aging. Further, these data suggest that osteocytes perceive the strain environment and that they play a role in orchestrating the modeling/remodeling response. By developing a technique as flexible and powerful as RT-PCR for use in dense cortical bone, determining the relative contribution of specific proteins to the transduction of regulatory signals to formative or resorptive responses is facilitated.

Adaptation, Physiological↗
PubMed1994

Promotion of bony ingrowth by frequency-specific, low-amplitude mechanical strain.

The ability of extremely low-amplitude mechanical strains to promote bony ingrowth was evaluated in an in vivo animal model, the functionally isolated turkey ulna. A cylindrical, porous-coated titanium implant was placed across the dorsal and ventral cortices of the left ulna diaphysis of 12 animals. Back scatter electron microscopy was used to quantify the relative bony ingrowth after eight weeks of: (1) disuse alone, (2) disuse plus 100 seconds per day of a 1-Hz, 150-microstrain (mu epsilon) mechanical stimulus, or (3) disuse plus 100 seconds per day of a 20-Hz stimulus of similar strain magnitude. Disuse alone caused a mean 8.3% (+/- 5.5%) less of bone away from the implant, with the area between implant and bone actively filling with a fibrous membrane. A daily 100-second regimen of low-magnitude, 1-Hz mechanical stimulation caused 28% (+/- 6.2%) of the implant area available for ingrowth to be filled with bone. At 20 Hz, the amount of bony ingrowth increased to 69% (+/- 3.0%). These data demonstrate that brief exposure to extremely low-amplitude mechanical strains can enhance the biologic fixation of cementless implants. Moreover, the degree of ingrowth is dependent on the frequency of the applied strain.

Animals↗
PubMed1993

Electric fields modulate bone cell function in a density-dependent manner.

The influence of an extremely low frequency (ELF) electric field stimulus (30 Hz at 6 microV/cm rms), known to promote bone formation in vivo, was evaluated for its ability to affect bone cell function in vitro. To accomplish this, we developed an apparatus for the exposure of monolayer cell systems to electric fields in a manner that provides relatively uniform electric field exposure of multiple cell samples as well as a rigorous sham exposure. We show that field exposure significantly limits the normal increase in osteoblastic cell number and enhances alkaline phosphatase activity compared to sham-exposed samples. Moreover, these alterations are shown to occur in a cell density-dependent manner. Samples plated at 6 x 10(3) cells/cm2 show no effect of field exposure. In samples plated at 30 x 10(3) cells/cm2, 72 h of field exposure resulted in 25% fewer cells in the exposed samples, and a doubling of alkaline phosphatase activity in those cells compared to sham exposure. Experiments using a 12 h exposure to preclude significant changes in cell number during the exposure show this density-dependent response to be biphasic. Sparse cultures (< 50 x 10(3) cells/cm2) were not found to be affected by the field exposure, but increases in alkaline phosphatase activity occurred in cultures at densities of 50-200 x 10(3) and 200-350 x 10(3) cells/cm2 and no effect on alkaline phosphatase activity was seen in confluent cell cultures of greater than 350 x 10(3) cells/cm2.(ABSTRACT TRUNCATED AT 250 WORDS)

Alkaline Phosphatase↗
PubMed1993

Optimization of electric field parameters for the control of bone remodeling: exploitation of an indigenous mechanism for the prevention of osteopenia.

The discovery of piezoelectric potentials in loaded bone was instrumental in developing a plausible mechanism by which functional activity could intrinsically influence the tissue's cellular environment and thus affect skeletal mass and morphology. Using an in vivo model of osteopenia, we have demonstrated that the bone resorption that normally parallels disuse can be prevented or even reversed by the exogenous induction of electric fields. Importantly, the manner of the response (i.e., formation, turnover, resorption) is exceedingly sensitive to subtle changes in electric field parameters. Fields below 10 microV/cm, when induced at frequencies between 50 and 150 Hz for 1 h/day, were sufficient to maintain bone mass even in the absence of function. Reducing the frequency to 15 Hz made the field extremely osteogenic. Indeed, this frequency-specific sinusoidal field initiated more new bone formation than a more complex pulsed electromagnetic field (PEMF), though inducing only 0.1% of the electrical energy of the PEMF. The frequencies and field intensities most effective in the exogenous stimulation of bone formation are similar to those produced by normal functional activity. This lends strong support to the hypothesis that endogenous electric fields serve as a critical regulatory factor in both bone modeling and remodeling processes. Delineation of the field parameters most effective in retaining or promoting bone mass will accelerate the development of electricity as a unique and site-specific prophylaxis for osteopenia. Because fields of these frequencies and intensities are indigenous to bone tissue, it further suggests that such exogenous treatment can promote bone quantity and quality with minimal risk or consequence.

Animals↗
PubMed1992

Microelectrode measurements of low frequency electric field effects in cells and tissues.

The average intensities of electric fields induced into tissue can be calculated if the morphology and conductivities of the tissue are known, and such values provide one estimate of dosage for a given field exposure level. However, the microanatomical structures of living tissue, which include gap junctions, tight junctions, highly charged cell coats, and extracellular matrices, as well as complex cell shapes, precludes a detailed characterization of the field and current distribution near the cells which are actually responding to the electric fields. This suggests that a more useful electric field dose metric may be one based on an induced physical effect on the cells. Electric fields have at least three distinct physical effects on cells: the normal plasma membrane potential will be altered; the ionic currents and ion distributions at the extracellular surface will be modified; and mechanical forces will be imposed at the cell surface. Each of these effects can, in principle, be measured through the application of specific microelectrode techniques. Here, the feasibility of using various intracellular and extracellular recording methods to obtain dosimetric values, as well as the contribution these measurements could make to our understanding of electric field interactions with biological tissue, are discussed.

Animals↗
PubMed1992

Suppression of the osteogenic response in the aging skeleton.

The ability of physical stimuli demonstrated as potently osteogenic in the young adult skeleton were evaluated for their capacity to stimulate new bone formation in the aging skeleton. Using the externally loadable, functionally isolated turkey ulna preparation, the ulnae of 1-year-old (n = 5), and 3-year-old (n = 3) turkeys were subjected to 300 cycles per day of a load regimen generating a high but physiologic level of normal strain (3,000 microstrain). Following 8 weeks of loading, areal properties and histomorphometry were performed on both the experimental and intact control ulnae. Bone cross-sectional areas in the 1-year-old animal increased by 30.2% (+/- 7.8%) as compared with the intact contralateral control ulnae, whereas the areal properties of the older skeleton remained essentially unchanged (-3.3 +/- 7.5%). Renewed bone formation in the experimental ulnae of the 1-year-old animals was characterized by the activation of periosteal bone apposition (4.0 +/- 0.4 microns/day). In comparison, periosteal bone formation in the 3-year-old males was activated in only 1 animal, and this at a significantly attenuated level (less than 0.8 micron/day). The histomorphometric evaluation of intracortical bone remodeling revealed no significant differences between the control and experimental ulnae in either age group. However, osteon mean wall thickness and bone formation sigma were significantly increased in the 3-year-old males (P less than 0.05). In conclusion, these data suggest that a physical signal that is clearly osteogenic in the young adult skeleton is hardly acknowledged in older bone tissue.(ABSTRACT TRUNCATED AT 250 WORDS)

Aging↗
PubMed1992

Characterizing bone strain distributions in vivo using three triple rosette strain gages.

Three triple-element rosette strain gages were attached to the equine third metacarpal midshaft to record site-specific strains engendered by locomotion. The distribution of strains acting upon the midshaft cross section were characterized using a combined beam theory and finite element model analysis that did not presume the manner by which the bone was inertially loaded. A medium-speed trot (3.6 ms-1) was chosen as a representative speed and gait, with normal and shear strains, and strain energy density (SED) distributions determined throughout the stance and subsequent swing phase. Importantly, the sites of maximum compression (-2400 mu epsilon), tension (810 mu epsilon), shear (1500 mu epsilon), and SED (54 kPa) were not located at any of the gage attachment sites, emphasizing that a minimum of three rosette gages are necessary to resolve the peaks and locations of functionally induced normal and shear strains. Considering the nonuniform strain distributions across the cortex, we conclude that the third metacarpal is subject to a complex loading milieu comprised of bending, axial compression, end shear, and torsion. As this complex manner of loading was consistent through the entire stance phase, it would appear that, at least during the trot, specific sites within the same cross section are subject to vastly different magnitudes of strain stimulus.

Animals↗
PubMed1992

The effect of low-frequency electrical fields on osteogenesis.

An in vivo animal model of disuse osteopenia was used to determine the osteogenic potential of specific components of electrical fields. The ability of a complex pulsed electrical field to inhibit loss of bone was compared with the remodeling response generated by extremely low-power, low-frequency (fifteen, seventy-five, and 150-hertz) sinusoidal electrical fields. The left ulnae of thirty adult male turkeys were functionally isolated by creation of distal and proximal epiphyseal osteotomies and then were exposed, for one hour each day, to an electrical field that had been induced exogenously by means of magnetic induction. After a fifty-six-day protocol, the remodeling response was quantified by a comparison of the cross-sectional area of the mid-part of the diaphysis of the functionally isolated ulna with that of the intact contralateral ulna. Disuse resulted in a 13 per cent mean loss of osseous tissue, which was not significantly different than the 10 per cent loss that was caused by disuse treated with inactive coils. Exposure to the pulsed electrical fields prevented this osteopenia and stimulated a 10 per cent mean increase in the bone area. The osteogenic influence of the sinusoidal electrical fields was strongly dependent on the frequency; the 150, seventy-five, and fifteen-hertz sinusoidal fields, respectively, generated a -3 per cent, + 5 per cent, and + 20 per cent mean change in the bone area. These results suggest a tissue sensitivity that is specific to very low-frequency sinusoidal electrical fields, and they imply that the induced electrical fields need not have complex waveforms to be osteogenic. Since the frequency and intensity range of the sinusoidal fields producing the greatest osteogenic response are similar to the levels produced intrinsically by normal functional activity, these results support the hypothesis that electricity plays a role in the retention of the normal remodeling balance within mature bone.

Animals↗
PubMed1990

Frequency specific modulation of bone adaptation by induced electric fields.

A frequency specificity for the response of bone tissue to a physical stimulus is proposed. This is obtained by comparing the spectral power of exogenously induced electric fields to the efficacy of those fields to inhibit immobilization induced bone loss in an in vivo model of skeletal adaptation. Analysis of a family of related waveforms shows that the effectiveness of the induced electric fields could be related to the induced spectral power below approximately 75 Hz. The analysis suggests that bone tissue may be extremely sensitive to induced power levels at or below this frequency, as amplitude variations of less than a factor of two within this range correspond to significantly different bone remodeling responses. The analysis also suggests that bone tissue may be strongly frequency selective, with bone capable of responding specifically to induced power in this osteogenic frequency band, even though the band includes less than 0.1% of the total induced power. As normal functional activity generates strain components encompassing this osteogenic frequency band, a distinct frequency selectivity may indicate that the tissue response is tuned to a specific endogenous stimulus. A detailed characterization of the frequency response of bone tissue could well point to the primary source for the control of the cells responsible for functional adaptation in the skeleton.

Adaptation, Physiological↗
PubMed1990

Functional strains and cortical bone adaptation: epigenetic assurance of skeletal integrity.

The skeleton's ability to withstand the extremes of physical activity is achieved in large part by its capacity to perceive and respond to small changes in its mechanical environment. Strains generated by functional activity would represent an efficient, epigenetic parameter by which the bone cell population could assess the skeleton's structural effectiveness, and subsequently use this information to influence its morphology. However, contrary to our normal interpretation of Wolff's Law, minimizing strain does not appear to be the paramount goal of adaptation, but rather skeletal morphology interacts with functional activity to generate a certain, perhaps cytologically beneficial, type of strain. Three sections are used to address the epigenetic impact on bone morphology: (a) at the level of the organ, the strains generated by functional activity; (b) at the level of the tissue, osteoregulatory parameters of the strain environment; and (c) at the level of the cell, the mechanisms by which physical information is translated to an adaptive response. Only when we understand the mechanisms and objectives of tissue adaptation in the normal skeleton can our perspective and treatment of functionally influenced skeletal pathologies (e.g. osteopenias, fractures) be enhanced.

Adaptation, Physiological↗