Effects of bisphosphate treatment and mechanical loading on bone modeling in the rat tibia.

Bisphosphonate treatment has been shown to decrease endosteal bone formation and increase periosteal bone apposition in the rat tibial diaphysis. This study tested the hypothesis that the increase in periosteal apposition is a compensatory attempt to maintain skeletal mass appropriate for the mechanical load. Twenty-four rats were divided into four groups. In two of the groups, one hindlimb in each rat was immobilized with a sling device to increase the mechanical load on the opposite limb. Daily injections of dichloromethylene bisphosphonate (Cl2MBP) were given at 10 mg/kg to one immobilized group and one mobile group. The other two groups were given daily injections of normal saline. Fluorescent bone labels were administered at two-week intervals. All rats were killed after ten weeks of treatment, and calcified tibial cross sections were prepared for fluorescence microscopy. Bone dimensions and periosteal and endosteal apposition rates were calculated. When compared with saline controls, Cl2MBP treatment decreased endosteal apposition rate in all tibias. Periosteal apposition rate was increased with Cl2MBP treatment in all tibias except the unloaded limb of immobilized rats. The Cl2MBP-induced increase in periosteal apposition rate was greatest in loaded limbs and was proportional to the relative amount of body weight supported.     

Effects of risedronate on trabecular microstructure and biomechanical properties in ovariectomized rat tibia

We determined the effect of risedronate on the trabecular microstructure of ovariectomized rat tibiae, using micro-computed tomography, in order to investigate how changes in microstructure contribute to biomechanical properties. Fifty 18-week-old rats underwent sham operation (n=10) or ovariectomy (OVX) (n=40). The OVX rats were further divided into four groups (n=10 for each group) and treated with risedronate at doses of 0, 0.1, 0.5 or 2.5 mg/kg for 9 months. OVX caused deterioration of three-dimensional trabecular microstructure, notably structure model index (SMI) and connectivity density, while treatment of OVX rats with risedronate at 0.5 and 2.5 mg/kg improved those deleterious microstructural changes. Biomechanical property, as assessed by finite element analysis (FEA), correlated significantly with trabecular bone volume fraction (BV/TV), and the correlation further increased substantially when microstructural parameters were added, especially SMI and connectivity density, with risedronate therapy. Thus, it is suggested that, in addition to increasing bone mass, risedronate improves biomechanical property by maintaining a plate-like structure as well as connectivity of trabeculae.     

Effects of different estrogen and progestin regimens on the mechanical properties of rat femur

The purpose of this study was to examine the effects of estrogen replacement, in concert with three different progestin regimens. On the mechanical properties of rat femoral cortical bone. Ninety-two 11-month-old female Sprague-Dawley rats were randomly divided into six groups and were treated for a duration of 6 months. Group-1 rats were intact controls. group-2 rats were ovariectomized controls, and groups 3-6 were Ovariectomized and given continuous doses of estrogen with 5% estradiol 17B silicone-rubber implants. Groups 4, 5, and 6 were also given different doses of progestin (norethindrone): group 4 received a continuous dose of 3 μg per animal per day, group 5 received a cyclic dose of 6 μ per animal per day for 14 days of a 28-day cycle, and group 6 received an interrupted dose of 3 μ per animal day for 3 days of a 6-day cycle. Femurs from each group were mechanically tested. Bending stiffness was measured by nondestructive three-point bending tests and maximum torque capacity, by destructive torsion tests. Geometrical properties and apparent density of cortical bone were also measured. The Significant differences were: the increases in elastic modulus (measured from the three-point bending stiffness) of group 5 (cyclic norethindrone) compared with those of group 2 (ovariectomized controls) and group 3 (estrogen only); the increases in the size represented by the moment of inertia, the moment of the area, and medial-lateral width of group 2 compared with those of group 5; and the increases in apparent density and decreases in moment of inertia of group 6 (interrupted norethindrone) compared with those of group2. Cyclic or interrupted treatment of progestin along with continuous treatment of estrogen after ovariectomy likely improves material properties of cortical bone, increases its density, and reduces the size of the bone compared with ovariectomized rats.     

Effects of age and sex on the regulation of plasma 1,25-(OH)2-D by phosphorus in the rat

Summary Dietary phosphate deprivation in women, but not men, is accompanied by a fall in plasma PO₄ and a rise in plasma 1,25-(OH)₂-vitamin D concentrations. In contrast, young male rats exhibit a fall in plasma PO₄ and a rise in plasma 1,25-(OH)₂-D concentrations in response to PO₄ deprivation. To evaluate whether age and sex influence basal plasma 1,25-(OH)₂-D levels and their regulation by PO₄ deprivation, plasma 1,25-(OH)₂-D, PO₄, and Ca levels were measured in male and female rats ranging in age from 6 weeks to 6 months while they were eating normal or low PO₄ diets for 1 to 16 days. Similar observations were also made in 6-week-old castrated male and female rats, males replaced with testosterone, and females replaced with estradiol. Basal plasma 1,25-(OH)₂-D levels were higher in 6-week-old males (228±76 pmol/l) than in 6-week-old females (148±62 pmol/l;P<0.01) and declined by age 11 weeks to stable levels averaging about 100 pmol/l without sex difference. Dietary PO₄ deprivation resulted in a three-to fourfold increase in plasma 1,25-(OH)₂-D concentrations regardless of age and sex, accompanied by a correlated rise in serum Ca concentrations. Castration of 6-week-old males and females eliminated the sex difference in basal plasma 1,25-(OH)₂-D levels and appeared to enhance the elevation of plasma 1,25-(OH)₂-D concentrations in response to PO₄ deprivation in females. Although gonadal hormones may modify basal plasma 1,25-(OH)₂-D levels, they are not required for the augmentation of plasma 1,25-(OH)₂-D levels in response to PO₄ deprivation.     

Effects of acetylsalicylic acid and naproxen on the mechanical properties of intact femora in rats

The influence of acetylsalicylic acid (ASA) and naproxen on growing bones was studied. Young male rats were used. The drugs were administered via gastric gavage twice a day for 9 or 18 days. Drug doses giving serum concentrations corresponding to ordinary anti-inflammatory steady-state levels in humans were used. There was a drug-related influence on the strength of the growing femur. After 9 days the ultimate bending moment of the distal femoral epiphyseal plate and ultimate torsional moment and stress of the femoral diaphysis increased by about 10% in the rats treated with 150 mg/kg/12 h of ASA as compared with controls. After 18 days there were no differences. The ultimate metaphyseal bending moment of the distal femur was not influenced after 9 days with this dose, but was reduced by about 10% compared with controls after 18 days. Doses of 100 mg/kg/12 h of ASA and 20 mg/kg/12 h of naproxen did not change the bone strength. The doses used were well tolerated and did not influence the bone growth or body weight of the rats. A naproxen dose of 40 mg/kg/12 h was lethal; rats that received this dose succumbed to jejunal perforations. The results indicate that ASA influences the remodeling of normally growing bones.     

Effects of acetylsalicylic acid and naproxen on the mechanical properties of intact femora in rats

Summary The influence of acetylsalicylic acid (ASA) and naproxen on growing bones was studied. Young male rats were used. The drugs were administered via gastric gavage twice a day for 9 or 18 days. Drug doses giving serum concentrations corresponding to ordinary anti-inflammatory steady-state levels in humans were used. There was a drug-related influence on the strength of the growing femur. After 9 days the ultimate bending moment of the distal femoral epiphyseal plate and ultimate torsional moment and stress of the femoral diaphysis increased by about 10% in the rats treated with 150 mg/kg/12 h of ASA as compared with controls. After 18 days there were no differences. The ultimate metaphyseal bending moment of the distal femur was not influenced after 9 days with this dose, but was reduced by about 10% compared with controls after 18 days. Doses of 100 mg/kg/12 h of ASA and 20 mg/kg/12 h of naproxen did not change the bone strength. The doses used were well tolerated and did not influence the bone growth or body weight of the rats. A naproxen dose of 40 mg/kg/12 h was lethal; rats that received this dose succumbed to jejunal perforations. The results indicate that ASA influences the remodeling of normally growing bones.

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Zusammenfassung Ziel dieser Studie war, die mechanischen Eigenschaften des Oberschenkelknochens junger männlicher Ratten nach 9- und 18tägi-ger Behandlung mit Azetylsalizylsäure (ASS) bzw. Naproxen zu untersuchen. Die Medikamente wurden per os als Suspension 2mal täglich verabreicht, so daß Serumkonzentrationen, vergleichbar mit den therapeutischen antiinflammatorischen Blutspiegeln in der Humanmedizin, erzielt wurden. Die Ratten, die mit der ASS in einer Dosierung von 150 mg/kg/12 Std behandelt wurden waren, zeigten nach 9 Tagen eine vorübergehend erhöhte Biegungsstabilität der Wachstumszone des distalen Femurendes sowie eine erhöhte Torsionsstabilität der Femurdiaphyse. Die Biegungsstabilität der Femurmetaphyse war dagegen nach 9 Tagen unbeeinflußt and nach 18 Tagen sogar herabgesetzt. ASS in einer Dosis von 100 mg/kg/12 Std und Naproxen in einer Dosis von 20 mg/kg/12 Std führten zu keiner Veränderung der Knochenstabilität. Die Ergebnisse deuten darauf hin, daß die Knochenumbauvorgänge des wachsenen Knochens durch ASS beeinflußt wurden.

     

Effects of exercise on bone morphology: Vascular channels studied in the rat tibia

14 male rats were divided into exercise and control groups to examine the effect of a 1-month exercise program on the vascular morphology of the tibial diaphysis. Following the exercise program the number of haversian canals per mm² increased in the middle third of the cortex, but not in the outer or inner zone. No change was observed in the number of non-haversian canals. The size of the nonhaversian canals increased following the training program, which was not evident in the haversian canals.     

The efficacy of the use of IR laser phototherapy (LPT) on bone defect grafted with biphasic ceramic on rats with iron deficiency anemia: Raman spectroscopy analysis

The aim of this study was to evaluate bone repair in anemic and non-anemic rats submitted or not to laser phototherapy and hydroxyapatite graft. Animals were divided in eight groups of five animals: Clot; Laser; Graft; Graft + Laser; iron deficiency anemia (IDA) + Clot; IDA + Laser; IDA + graft; IDA + graft + Laser. When appropriate irradiation with infrared laser was done during 15 days at a 48-h interval. Animals were killed at day 30; samples were analyzed by Raman spectroscopy. Three shifts were studied and statistically analyzed: ~960, ~1,070, and ~1,454 cm^?1. Graft + laser showed highest ~960 peak was statistically different from all other healthy groups. No statistical difference was found between Clot and IDA + Clot in any shift. The IDA + Graft and IDA + Graft + Laser groups had low mean peak values for shifts ~960, ~1,070, and ~1,454 cm^?1. The results in this study indicate that using hydroxyapatite (HA) and laser irradiation in healthy subjects is favorable to mineral deposition and bone maturation, this being of importance for some groups at risk, such as astronauts. In iron deficiency anemia cases, the use of graft, associated or not to laser irradiation, resulted in low collagen and low carbonate and phosphate HA.     

Effect of the laser and light-emitting diode (LED) phototherapy on midpalatal suture bone formation after rapid maxilla expansion: a Raman spectroscopy analysis

The aim of this study was to analyze the effect of laser or light-emitting diode (LED) phototherapy on the bone formation at the midpalatal suture after rapid maxilla expansion. Twenty young adult male rats were divided into four groups with 8 days of experimental time: group 1, no treatment; group 2, expansion; group 3, expansion and laser irradiation; and group 4, expansion and LED irradiation. In groups 3 and 4, light irradiation was in the first, third, and fifth experimental days. In all groups, the expansion was accomplished with a helicoid 0.020″ stainless steel orthodontic spring. A diode laser (λ780 nm, 70 mW, spot of 0.04 cm², t?=?257 s, spatial average energy fluence (SAEF) of 18 J/cm²) or a LED (λ850 nm, 150 mW?±?10 mW, spot of 0.5 cm², t?=?120 s, SAEF of 18 J/cm²) were used. The samples were analyzed by Raman spectroscopy carried out at midpalatal suture and at the cortical area close to the suture. Two Raman shifts were analyzed: ～960 (phosphate hydroxyapatite) and ～1,450 cm^?1 (lipids and protein). Data was submitted to statistical analysis. Significant statistical difference (p?≤?0.05) was found in the hydroxyapatite (CHA) peaks among the expansion group and the expansion and laser or LED groups. The LED group presented higher mean peak values of CHA. No statistical differences were found between the treated groups as for collagen deposition, although LED also presented higher mean peak values. The results of this study using Raman spectral analysis indicate that laser and LED light irradiation improves deposition of CHA in the midpalatal suture after orthopedic expansion.     

Characterization of a tartrate-resistant acid phosphatase (ATPase) from rat bone: hydrodynamic properties and N-terminal amino acid sequence

Certain physicochemical properties of rat bone tartrate-resistant acid ATPase (TrATPase), including the size and shape of the enzyme, potential subunit composition, and detergent binding, have been elucidated. SDS-polyacrylamide gel electrophoresis in combination with immunoblot analysis showed that the bone TrATPase has a molecular weight of 33,000 D and is composed of disulfide-linked polypeptides of 20,000 and 16,000 D. The enzyme contains 1.7 mol Fe per mol enzyme. Hydrodynamic studies allowed calculation of the Stokes radius (24 A), the sedimentation coefficient (3.19S), the partial specific volume (0.748 ml/g), the frictional ratio (0.995), and the axial ratio (1.0). The amount of detergent bound to the protein was determined to 4 mol of Triton X-100 per mol enzyme. The molecular weight of bone TrATPase derived from these parameters was 31,900 D. N-terminal amino acid sequence analysis of the Mr 20,000 subunit indicated a high degree of similarity with TRAP enzymes from spleen, uterus, placenta, hairy cell leukemia, and osteoclastoma. It is concluded that rat bone TrATPase belongs to the type 5 (tartrate-resistant and purple) acid phosphatase family. The similarities in the N-terminal amino acid sequences, iron content, and physicochemical properties of TRAP enzymes indicate a close structural relationship between type 5 acid phosphatases expressed in different tissues. The findings that TrATPase has a spherical shape and binds low amounts of detergent suggest that the enzyme is a soluble protein, compatible with the view that TrATPase is secreted by the osteoclast.     

Structural analysis of the human tibia in men with spinal cord injury by tomographic (pQCT) serial scans

Spinal cord injury (SCI), as a primarily neurological disorder that causes muscular atrophy, is well known to be associated with sub-lesional bone losses. These losses are more pronounced from epiphyseal than from diaphyseal regions. We hypothesized that this discrepancy may be explained by anatomical variation in endocortical circumference.Nine men who had attracted SCI 9 to 32 (mean 21.4) years prior to study inclusion were matched to able bodied control (Ctrl) people by age, height and weight. Serial scans by peripheral quantitative computed tomography were obtained from the tibia at steps corresponding to 5%-steps of the tibias length (s05 to s95, from distal to the proximal end of the tibia).As expected, SCI people had lower total bone mineral content (vBMC.tot) than able bodied control people (P < 0.001 at all sites). This group difference (ΔvBMC.tot) was more pronounced at the distal and proximal tibia than in the shaft (P < 0.001), and it amounted to 51% at s05, to 22% at s40, and to 47% at s95. Both endocortical and periosteal circumference were better predictors of ΔvBMC.tot (R² = 0.98 and R² = 0.97, respectively; P < 0.001 in both cases) than vBMC.tot (R² = 0.58, P < 0.001), suggesting that anatomical variation in geometry, rather than in bone mass can explain differential rates of bone loss after SCI. Moreover, the s04:s38 ratio in vBMC.tot was found to be 1.00 (95% confidence interval: 0.95–1.05) in the Ctrl group, and 0.63 in the SCI group (P < 0.001, 95% confidence interval: 0.54–0.68).These findings offer a rationale to account for the discrepancy between epiphyseal and diaphyseal bone losses following SCI. The suggestion is that the bone adaptive responses involved are limited in time, and that the reduced surface:volume ratio constitutes a limit within the available time window, in particular in the diaphysis. Finally, the drastically reduced s04:s38 vBMC.tot ratio observed in the SCI group in this study provides a rationale to scrutinize this Capozza index also in other studies as a general indicator of immobilisation-induced bone loss.     

Effects of parathyroid hormone (1-34) on tibia in an adult rat model for chronic alcohol abuse

Chronic alcohol abuse is a risk factor for osteoporosis in men. Human recombinant parathyroid hormone (1-34) (PTH) therapy increases bone mass in patients with osteoporosis. The purpose of the present study was to determine whether PTH is effective in increasing bone formation and bone mass in a rat model for established osteopenia caused by chronic alcohol abuse. Eight-month-old male Sprague Dawley rats were fed the Lieber-DeCarli liquid diet in which 35% of the calories were derived from either maltose-dextran or ethanol. Measurements were performed 16 weeks later to establish the magnitude of bone changes in the rats fed alcohol. High dose PTH (80 microg/kg/day) was administered 5 days/week for 6 weeks to establish the differential efficacy of hormone therapy on bone formation in alcohol consuming and alcohol withdrawn rats. The effects of alcohol and PTH on cancellous and cortical bone mass, architecture and turnover were determined by densitometry and histomorphometry. Rats fed alcohol had reduced bone mineral contents and densities, cancellous and cortical bone areas and cancellous bone formation rates compared to pair-fed controls. Following the withdrawal of alcohol, indices of bone formation increased compared to baseline values. PTH treatment increased bone mineral content and density, bone formation rates, cortical bone area, cancellous bone area and trabecular number and thickness, but several indices of bone formation were reduced in the presence of continued alcohol consumption. These results suggest that alcohol consumption, in addition to inducing bone loss, may reduce the efficacy of PTH therapy to reverse osteoporosis.     

Effects of irradiation on the appositional and longitudinal growth of the tibia and fibula of the rat with and without radioprotectant

The effect of therapeutic levels of irradiation on appositional bone growth was compared with its effect on longitudinal growth in the skeletally immature rat model. The widths and lengths of the tibiae and fibulae of young rats were studied at 2, 4, 6, and 12 weeks after exposure to 17.5 Gy x-irradiation to the knee region of the right leg, with and without the aminothiol radioprotectant amifostine 20 minutes before radiation. Irradiation retarded growth in the width of the tibia to a greater extent (19%-27%) than longitudinal growth (9%-21%). The appositional growth discrepancy decreased over time, whereas the length discrepancy increased. The proximal fibula, in contrast, undergoes a normal decrease in width over time, and irradiation retarded this contraction by 14%. Appositional growth does not appear to be spared from the damaging effects of irradiation, but a catch-up phenomenon is observed that is not seen in longitudinal growth. Amifostine reduced the radiation-induced loss in tibial width by 40% to 50% and in length by 12% to 30%.     

Assessment of the use of LED phototherapy on bone defects grafted with hydroxyapatite on rats with iron-deficiency anemia and nonanemic: a Raman spectroscopy analysis

This study aimed to assess bone repair in defects grafted or not with hydroxyapatite (HA) on healthy and iron-deficiency anemia (IDA) rats submitted or not to LED phototherapy (LED-PT) by Raman spectroscopy. The animals were divided in eight groups with five rats each: Clot; Clot?+?LED; IDA?+?Clot; IDA?+?LED; Graft; Graft?+?LED; IDA?+?Graft; and IDA?+?Graft?+?LED. When appropriated, irradiation with IR LED (λ850 ± 10 nm, 150 mW, CW, Φ?=?0.5 cm², 16 J/cm², 15 days) was carried out. Raman shifts: ～960 [symmetric PO₄ stretching (phosphate apatite)], ～1,070 [symmetric CO₃ stretching (B-type carbonate apatite)], and ～1,454 cm^?1 [CH₂/CH₃ bending in organics (protein)] were analyzed. The mean peak values for ～960, ～1,070, and ～1,454 cm^?1 were nonsignificantly different on healthy or anemic rats. The group IDA?+?Graft?+?LED showed the lowest mean values for the peak ～960 cm^?1 when compared with the irradiated IDA group or not (p?≤?0.001; p?≤?0.001). The association of LED-PT and HA-graft showed lowest mean peak at ～1,454 cm^?1 for the IDA rats. The results of this study indicated higher HA peaks as well as a decrease in the level of organic components on healthy animals when graft and LED phototherapy are associated. In the other hand, IDA condition interfered in the graft incorporation to the bone as LED phototherapy only improved bone repair when graft was not used.     

Ethane-1-hydroxy-1, 1-diphosphonate (EHDP) effects on growth and modeling of the rat tibia

One hundred gram male Sprague-Dawley rats were divided into groups which were injected daily for 10 or 30 days with vehicle (control group), 0.2, 0.4, 2.0, or 10.0 mg ethane-1-hydroxy-1,1-diphosphonate (EHDP)/kg/day. The proximal tibial metaphysis and epiphysis were assayed for changes in percentage of hard tissue and bone formation factors. Knowing these, information about hard tissue resorption was deduced. After ten or thirty days treatment with 2.0 or 10.0 mg EHDP/kg/day, there was an increase in percentage of hard tissue. This was due to a decrease in bone formation with a greater decrease in hard tissue resorption. Furthermore, after thirty days treatment with 0.2 and 0.4 mg EHDP/kg/day, there was an increase in percentage of hard tissue, which was due to a decrease in resorption with no change in formation. Ultrastructural studies on osteoclasts from EHDP-treated rats showed a general decrease in their vacuolization and amount of organelles as dosage of EHDP increased. Histologic findings suggest that EHDP is similar to fluoride in the way in which it depresses hard tissue resorption.     

Effect of short-term hypomagnesemia on the chemical and mechanical properties of rat bone.

Magnesium is known to have an essential role in determining the properties of bone, but the way in which Mg exerts its actions remains unclear. Although long-term Mg deficiency is known to produce osteopenia, the effects of short-term Mg deficiency have not been established. To test the hypothesis that Mg deficiency results in an altered pattern of initial mineralization and concomitant altered bone properties, the radiographic, histologic, chemical, and mechanical properties of the bones of rats given a Mg-deficient diet were compared to those of rats pair-fed the same diet supplemented with Mg. Short-term Mg-deficiency in the diet of growing rats produced a significant decrease in both the trabecular bone volume and the mineral content of the newly formed metaphysis, a significant increase in the Ca:P ratio, and a slight, but significant increase in hydroxyapatite crystallite size and/or perfection in the metaphysis. Comparable, but not significant, trends were found in the diaphyses. Metaphyseal bone osteocalcin levels were reduced in the Mg-deficient rats and lipid was more easily extracted from their bones. No detectable alterations in radiographic microstructure were noted. Mechanically, a significant decrease in the maximum three-point bend strength of the femurs of Mg-deficient rats was observed. These data support the hypothesis that short-term Mg deficiency affects the pattern of bone mineral formation.