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Biomedical subjects

C T Rubin

Publications and source records attributed to C T Rubin.

At least 19 recordsLinked to original sources
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

Effects of anisotropy and material axis registration on computed stress and strain distributions in the turkey ulna.

Finite element stress and strain distributions were studied parametrically for a curved long bone using several common material simplifications. A new technique is presented whereby local material axes conforming to local surface topology were automatically computed. Linearly elastic stress/strain solutions were evaluated as a function of the manner in which principal material directions are defined. The simplifications inherent in assumptions of local isotropy or globally registered transverse isotropy led to appreciably different solutions, particularly for some of the lesser-magnitude components of the strain tensor.

Algorithms↗
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

Three-dimensional geometric and structural symmetry of the turkey ulna.

Structural models of long-bone preparations usually assume left-right symmetry of contralateral bones under normal (baseline) conditions. To obtain insight on how this assumption affects the detection of subtle changes (as from functional adaptation), we formally examined the three-dimensional geometric and structural symmetry of paired long bones, using contemporary image reconstruction and stress analysis techniques. Nine pairs of ulnae from normal male turkeys were reconstructed computationally from serial transverse images obtained by either (a) mechanical sectioning and digital photographic imaging or (b) computed tomography. Computed tomography scans allowed greater precision in reconstruction than did digitally imaged photographs. Left-right comparisons of parameters of geometric symmetry (from computed tomography reconstructions) revealed average differences in whole bone volume and whole bone principal moments of inertia of 3.6 and 3.0%, respectively. Differences in bone curvature were indexed as noncolinearity of left compared with (mirrored) right centroidal axes, giving a disparity of 0.7 +/- 0.3 mm. Within the longitudinal central 20% of the diaphysis (the customary region for histomorphometry), average left-right differences in cross-sectional area and area principal moments of inertia for computed tomography images were 4.7 and 5.0%, respectively. The overlap of longitudinally paired cross sections of the mid-diaphysis, aligned at common centroids and oriented in the respective principal inertial directions, was greatest (as much as 95%) in the central 20% of the diaphysis. Paired three-dimensional finite element models demonstrated nearly identical left and right stress/strain fields throughout the ulnar diaphyses for both compressive and torsional loading. Our data suggest that the assumption of contralateral geometric symmetry in long bones should be judged in the context of the specific attribute of symmetry under consideration; however, we conclude that for purposes of finite element modeling the assumption of symmetry is reasonable.

Adaptation, Physiological↗
PubMed1995

Uniformity of resorptive bone loss induced by disuse.

Strains induced in the skeleton by functional activity are critical to the homeostasis of bone tissue. An in vivo model of disuse osteopenia was used to examine whether the removal of these regulatory stimuli induces a uniform loss of cortical bone through the whole organ or whether the loss of bone is focused at specific sites of the cortex. The right radii of five adult male turkeys were isolated from their normal functional loading for 8 weeks. The corresponding left radius from each animal served as an intact contralateral control. An additional group of five turkeys was used as time-zero controls to assess the initial areal symmetry of the left and right radii. Areal properties were assessed at three sites at equal intervals spanning the middle 3 cm of the diaphysis. Adaptation was determined for each cross section as a whole, as well as specifically by site by division of each cross section into 12 equal angle sectors. The average across all experimental sections after 8 weeks of disuse was 12.1 +/- 1.9% (+/- SE) loss of bone mass. The change in mean cross-sectional area varied little between the three diaphyseal sites (-10.2 +/- 3.3%, -13.5 +/- 3.8%, and -12.6 +/- 4.0%) and occurred primarily (84%) by uniform expansion of the endosteal envelope. However, elevated intracortical porosity following 8 weeks of disuse was highly nonuniform, with 58% of the increased porosity preferentially located in the ventral/caudal cortex (representing only 25% of the cortical area).(ABSTRACT TRUNCATED AT 250 WORDS)

Adaptation, Physiological↗
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

Regulation of cytoplasmic calcium concentration in tetracycline-treated osteoclasts.

The ability of low-dose tetracyclines to inhibit collagenase activity and inactivate osteoclasts suggests that these compounds have great potential as a prophylaxis for metabolic bone disease. However, the cellular mechanism by which tetracyclines interact with skeletal tissue is not yet clear. To better understand the effects of tetracyclines on bone metabolism, we examined their effect on osteoclast activity in vitro. Because tetracyclines can enter the cell and bind calcium and have been reported to directly interact with osteoclasts, we postulated that exposure to either of two tetracyclines, minocycline or doxycycline, would alter cytosolic Ca2+ regulation in rat osteoclasts. [Ca2+]i was measured in single rat osteoclasts utilizing fura-2. Addition of extracellular Ca2+ (5 mM CaCl2), a potent osteoclast inhibitor, increased [Ca2+]i in all osteoclasts, but 10(-6) M salmon calcitonin (sCT) did so only in a subpopulation of osteoclasts. Neither minocycline nor doxycycline (10 micrograms/ml) altered steady-state osteoclast [Ca2+]i. Further, neither minocycline nor doxycycline pretreatment affected the sCT-mediated increases in [Ca2+]i. However, tetracycline pretreatment significantly decreased the cytosolic Ca2+ response to extracellular CaCl2. Our results strongly suggest that tetracyclines have a specific effect on extracellular Ca(2+)-stimulated cytosolic Ca2+ mobilization in osteoclasts, which is not solely dependent on their ability to buffer Ca2+. Furthermore, these results point to the potential use of tetracyclines as probes to study cytosolic Ca2+ regulation. However, that tetracyclines attenuate a signal response associated with decreased osteoclastic resorption suggests that the reported antiresorptive attributes of tetracyclines must be achieved independently of an effect on osteoclastic cytosolic Ca2+.

Analysis of Variance↗
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↗