Biomedical subjects

C T Rubin

Publications and source records attributed to C T Rubin.

At least 19 recordsLinked to original sources

PubMed1998

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.

Amino Acid Sequence↗

PubMed1998

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.

Animals↗

PubMed1998

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.

Animals↗

PubMed1998

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.

Adaptation, Physiological↗

PubMed1998

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.

Bone and Bones↗

PubMed1997

Testing the daily stress stimulus theory of bone adaptation with natural and experimentally controlled strain histories.

Theories of bone adaptation generally consider that a departure in some feature of the normal homeostatic mechanical stimulus governs mechanical adaptation. Specifically, the 'daily stress stimulus' theory commonly used in computational models of bone adaptation suggests that the mechanical stimulus arises from a synthesis of the peak magnitudes from each loading event during a day. In this study, the homeostatic daily strain history of the adult turkey ulna was established by categorizing and counting the natural wing activities of adult male turkeys over a full 24h period. Strain signals were recorded in vivo for each activity type at three mid-diaphysis sites using stacked rosette strain gages. Following surgical isolation and transverse metaphyseal pinning of the ulnae, additional strain signals were recorded during controlled axial and torsional loading regimens associated with documented maintenance, loss, or addition of bone mass. When the present data were incorporated into the daily stress stimulus formulation, the theory did not consistently discriminate maintenance versus formation regimens, i.e., some maintenance regimens were associated with a substantially higher daily stimulus than some regimens causing bone formation.

Adaptation, Physiological↗

PubMed1997

Experimental colitis impairs linear bone growth independent of nutritional factors.

BACKGROUND: Poor linear growth frequently complicates chronic inflammatory bowel disease in children. Circulating inflammatory mediators may play a role in this growth delay. We evaluated the effect of experimental colitis on bone growth in a nutritionally controlled rat model. METHODS: Experimental colitis was induced in male Sprague-Dawley rats (125-150 g) by enema with trinitrobenzene sulfonic acid in 50% ethanol on day 1 and 11 of a 14-day protocol. Control animals were pair-fed and all animals received a liquid rat diet (1 kcal/ml). Twenty-four-hour urine, collected on days 2 and 12 and serum samples, collected at death, were analyzed for calcium, zinc, and magnesium. Serum samples from a separate set of animals were studied for serial interleukin-6 levels. Right proximal tibias were processed for growth-plate histomorphometry, in which linear growth is proportional to the heights of the proliferative zone, and terminal hypertrophic chondrocyte, but inversely proportional to the height of the resting zone. RESULTS: Histology confirmed active inflammation in the animals given trinitrobenzene sulfonic acid. Weight gain and both urinary excretion and serum levels of zinc, calcium, and magnesium did not differ between treatment and nontreatment groups. Histologically, there was impaired linear bone growth. The resting zone was greater in the colitis group (94.5 +/- 32.6 microns versus 3.9 +/- 5.4 microns; p < 0.05); the proliferative zone was smaller in the colitis group (123.7 +/- 18.2 microns versus 78.9 +/- 11.2; p < 0.05 micron); the terminal hypertrophic chondrocyte was reduced in the colitis group (19.5 +/- 1.4 microns versus 28.8 +/- 3.6 microns; p < 0.05). At 6 and 24 hours after induction, the level of interleukin-6 was elevated in the colitis group. CONCLUSIONS: Experimental colitis results in a decreased linear bone growth, independent of nutritional intake. Circulating cytokines derived from intestinal inflammation may contribute to the suppression of bone growth.

Animal Nutritional Physiological Phenomena↗

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↗

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↗