Comparative effects of alendronate and alfacalcidol on cancellous and cortical bone mass and bone mechanical properties in ovariectomized rats.

The purpose of the present study was to compare the effects of alendronate and alfacalcidol on cancellous and cortical bone mass and bone mechanical properties in ovariectomized rats. Twenty-six female Sprague-Dawley rats, 7 months of age, were randomized by the stratified weight method into four groups: the sham-operated control (Sham) group and the three ovariectomy (OVX) groups, namely, OVX + vehicle, OVX + alendronate (2.5 mg/kg, p.o., daily), and OVX + alfacalcidol (0.5 mug/kg, p.o., daily). At the end of the 8-week experimental period, bone histomorphometric analyses of cancellous bone at the proximal tibial metaphysis and cortical bone at the tibial diaphysis were performed, and the mechanical properties of the femoral distal metaphysis and femoral diaphysis were evaluated. OVX decreased cancellous bone volume per total tissue volume (BV/TV), and the maximum load of the femoral distal metaphysis, as a result of increases in serum osteocalcin (OC) levels, and also the number of osteoclasts (N.Oc), osteoclast surface (OcS) and bone formation rate (BFR) per bone surface (BS), and BFR/BV, without any effect on cortical area (Ct Ar), or maximum load of the femoral diaphysis. Alendronate prevented this decrease in cancellous BV/TV by suppressing increases in N.Oc/BS, OcS/BS, BFR/BS, and BFR/BV, without any apparent effect on Ct Ar, or maximum load of the femoral distal metaphysis and femoral diaphysis. On the other hand, alfacalcidol increased cancellous BV/TV, Ct Ar, and the maximum load of the femoral distal metaphysis and femoral diaphysis, by mildly decreasing trabecular BFR/BV, maintaining trabecular mineral apposition rate and osteoblast surface per BS, increasing periosteal and endocortical BFR/BS, and preventing an increase in endocortical eroded surface per BS. The present study clearly showed the differential skeletal effects of alendronate and alfacalcidol in ovariectomized rats. Alendronate prevented OVX-induced cancellous bone loss by suppressing bone turnover, while alfacalcidol improved cancellous and cortical bone mass and bone strength by suppressing bone resorption and maintaining or even increasing bone formation.     

Alfacalcidol prevents age-related bone loss and causes an atypical pattern of bone formation in aged male rats

The current study was designed to investigate the skeletal effects of alfacalcidol in aged rats. Eighteen-month-old male rats were treated with 0, 0.1, or 0.2 microg/kg/d of alfacalcidol by daily oral gavage, 5 days/week for 12 weeks. At the beginning of the treatments, one group of rats was euthanized to serve as a baseline control. At the end of the study, the second lumbar vertebrae and the right tibial diaphysess were processed for bone histomorphometric analysis. The fourth lumbar vertebrae were subjected to strength testing. The control group of rats at 21 months of age had decreased serum testosterone levels and decreased cancellous bone mass associated with increased bone turnover on the trabecular surface. The older rats had increased bone turnover on the endocortical surface and decreased bone formation on the periosteal surface compared with the 18-month group. In contrast, alfacalcidol treatment increased cancellous and cortical bone mass in aged male rats. Trabecular bone resorption was decreased whereas bone formation was maintained or increased in the rats treated with alfacalcidol. In addition, endocortical bone formation was decreased whereas periosteal bone formation was increased in the rats treated with alfacalcidol compared with vehicle-treated rats. Marrow trabecular bone area was increased by alfacalcidol treatment in tibial diaphyses. Furthermore, bone strength of the lumbar vertebral body was increased after alfacalcidol treatment. An atypical pattern of bone formation on endosteal bone surfaces was seen in the rats treated with alfacalcidol. The atypical bone formation is characterized by small, focal packets of newly formed bone on trabecular and endocortical bone surfaces. This gave the appearance of the formation of "bone buds" emanating from trabecular surfaces. These bony outgrowths were mineralized and demonstrated significant fluorochrome label indicating recent mineralization. Also, lamellae of the bony buds did not run parallel to those of the trabecular plate to which they are attached. Arrest lines presented in most of the "bone buds". In summary, alfacalcidol treatment increased cancellous and cortical bone mass and improved bone strength, resulting in the prevention of age-related bone loss in aged male rats. An atypical pattern of bone formation observed in this study may be a result of minimodeling based bone formation stimulated by alfacalcidol treatment.     

Alfacalcidol restores cancellous bone in ovariectomized rats

Active vitamin D metabolites have been demonstrated to reduce vertebral and hip fractures in elderly patients. A number of in vitro and in vivo pre-clinical studies have suggested that vitamin D may effectively stimulate osteoblastic activity and exert an anabolic effect on bone. The current study was designed to further explore the ability of an active vitamin D analog to restore bone in a skeletal site with established osteopenia in ovariectomized (OVX) rats. Female Sprague Dawley rats at five months of age and 8 weeks after sham ovariectomy or ovariectomy were randomly divided into 7 groups with 10 per group. At the beginning of the treatments, one group of sham-operated rats and one group of OVX rats were sacrificed to serve as baseline controls. Another group of sham-operated rats and one group of OVX rats were treated with vehicle for 4 weeks. The OVX rats in the remaining groups were treated with alfacalcidol at 0.05, 0.1 or 0.2 microg/kg/d by daily oral gavage, 5 days/week for 4 weeks. As expected, estrogen depletion caused high bone turnover and cancellous bone loss in lumbar vertebra of OVX rats. Alfacalcidol treatment at 0.1 or 0.2 but not 0.05 microg/kg/d increased serum calcium and phosphorus in OVX rats as compared with vehicle treatment. In addition, serum parathyroid hormone was suppressed, whereas serum osteocalcin was increased by alfacalcidol at all dose levels. Furthermore, histomorphometric data of 2nd lumbar vertebral body revealed that cancellous bone volume in OVX rats treated with alfacalcidol at 0.1 or 0.2 microg/kg/d was increased to the level of sham-operated rats treated with vehicle. This increment in cancellous bone mass was accompanied by increases in trabecular number and thickness and a decrease in trabecular separation. Moreover, osteoclast surface and number were significantly decreased, whereas bone formation variables such as mineralizing surface and bone formation rate were significantly increased in alfacalcidol- treated OVX rats compared with those of vehicle-treated OVX rats. Finally, a linear regression analysis showed that alfacalcidol treatment dose-dependently altered most of the variables measured in the current study. In conclusion, alfacalcidol completely restores cancellous bone by stimulating bone formation and suppressing bone resorption in lumbar vertebra of OVX rats when the treatment is started at an early phase of osteopenia. The evidence of increased bone formation by alfacalcidol treatments further supports the notion that active vitamin D metabolites or their analogs may exert anabolic effects on bone.     

Preventive effects of risedronate and calcitriol on cancellous osteopenia in rats treated with high-dose glucocorticoid.

We compared the effects of risedronate (Ris) and calcitriol (Cal) on cancellous osteopenia in rats treated with high-dose glucocorticoid (GC). Forty female Sprague-Dawley rats, 4 months of age, were randomized by the stratified weight method into four groups of 10 rats each according to the following treatment schedule: intact control, and GC administration with vehicle, Ris, or Cal. The GC (methylprednisolone sodium succinate, 5.0 mg/kg, s.c.), Ris (10 microg/kg, s.c.), and Cal (0.1 microg/kg, p.o.) were administered 3 times a week. At the end of the 4-week treatment period, bone histomorphometric analysis was performed for cancellous bone of the proximal tibial metaphysis. The GC administration decreased cancellous bone volume (BV/total tissue volume [TV]), trabecular number (Tb N), and trabecular thickness (Tb Th), as a result of increased bone resorption and decreased bone formation. Ris treatment markedly increased cancellous BV/TV and Tb N above the control level as a result of suppressed bone turnover. On the other hand, Cal treatment attenuated the GC-induced decrease in cancellous BV/TV and Tb Th as a result of suppressed bone resorption and maintained bone formation. This study showed the differential effects of Ris and Cal on cancellous osteopenia in rats treated with high-dose GC.     

The role of estrogen receptor-beta, in the early age-related bone gain and later age-related bone loss in female mice

The molecular and cellular mechanism of estrogen action in skeletal tissue remains unclear. The purpose of this study was to understand the role of estrogen receptor-beta, (ERbeta) on cortical and cancellous bone during growth and aging by comparing the bone phenotype of 6- and 13-month-old female mice with or without ERbeta. Groups of 11-14 wild-type (WT) controls and ERbeta knockout (BERKO) female mice were necropsied at 6 and 13 months of age. At both ages, BERKO mice did not differ significantly from WT controls in uterine weight and uterine epithelial thickness, indicating that ERbeta does not regulate the growth of uterine tissue. Femoral length increased significantly by 5.5% at 6 months of age in BERKO mice compared with WT controls. At 6 months of age, peripheral quantitative computerized tomography (pQCT) analysis of the distal femoral metaphysis (DFM) and femoral shafts showed that BERKO mice had significantly higher cortical bone content and periosteal circumference as compared with WT controls at both sites. In contrast to the findings in cortical bone, at 6 months of age, there was no difference between BERKO and WT mice in trabecular density, trabecular bone volume (TBV), or formation and resorption indices at the DFM. In 13-month-old WT mice, TBV (-41%), trabecular density (-27%) and cortical thickness decreased significantly. while marrow cavity and endocortical circumference increased significantly compared with 6-month-old WT mice. These age-related decreases in cancellous and endocortical bone did not occur in BERKO mice. At 13 months of age, BERKO mice had significantly higher total, trabecular and cortical bone, while having significantly lower bone resorption, bone formation and bone turnover in DFM compared with WT mice. These results indicate that deleting ERbeta protected against age-related bone loss in both the cancellous and endocortical compartments by decreasing bone resorption and bone turnover in aged female mice. These data demonstrate that in female mice, ERbeta plays a role in inhibiting periosteal bone formation, longitudinal and radial bone growth during the growth period, while it plays a role in stimulating bone resorption, bone turnover and bone loss on cancellous and endocortical bone surfaces during the aging process.     

Therapeutic effect of risedronate on cancellous and cortical bone in ovariectomized osteopenic rats: a comparison with the effects of alfacalcidol.

The purpose of the present study was to compare the therapeutic effects of risedronate (RIS) and alfacalcidol (ALF) on cancellous and cortical bone in ovariectomized osteopenic rats. Forty-two female Sprague-Dawley rats, 7 months of age, were randomized by the stratified weight method into six groups: the sham-operated control (Sham) group, and five ovariectomized groups: treated with vehicle, RIS (0.1, 1.0, or 2.5 mg/kg, p.o., daily), and ALF (0.5 microg/kg, p.o., daily). Treatment was started 6 weeks after surgery and continued for 6 weeks. Evaluation at 12 weeks after surgery revealed that ovariectomy (OVX) decreased the cancellous bone volume/total tissue volume (BV/TV) of the proximal tibial metaphysis as a result of an increase of the bone formation rate/bone surface (BFR/BS), BFR/BV, and eroded surface (ES/BS), while having no effect on the cortical area (Ct Ar) of the tibial diaphysis. OVX also decreased the maximum load of the femoral distal metaphysis, while having no effect on any mechanical property parameters of the femoral diaphysis. RIS (at all the doses) increased the BV/TV relative to the value in the OVX-Vehicle group, but the value was not restored to that observed in the Sham group. The effects of RIS (1.0 mg/kg and 2.5 mg/kg) were similar, and greater than those of RIS (0.1 mg/kg). ALF also increased the BV/TV relative to the OVX-Vehicle group, but the value was not restored to that observed in the Sham group, similar to the results of RIS (1.0 mg/kg and 2.5 mg/kg) treatment. The alterations of the structural parameters induced by RIS (at the doses) were attributable to suppression of the increase of ES/BS, BFR/BS, and BFR/BV. The alterations of the structural parameters induced by ALF were attributable to suppression of the increase of ES/BS and attenuation of the increase of BFR/BV, while the BFR/BS was maintained. ALF also increased the Ct Ar to beyond the value observed in the Sham group. RIS (at all the doses) had no effect on the mechanical properties of the femoral distal metaphysis, whereas ALF prevented the loss of the maximum load of the femoral distal metaphysis. Thus, the results of the present study show differential effects of RIS and ALF on cancellous and cortical bone in ovariectomized osteopenic rats.     

Effects of combined administration of alfacalcidol and risedronate on cancellous and cortical bone mass of the tibia in glucocorticoid-treated young rats

The purpose of the present study was to examine the effects of combined administration of alfacalcidol (ALF) and risedronate (RIS) on cancellous and cortical bone mass of the tibia in glucocorticoid (GC)-treated young rats. One hundred and nine female Sprague-Dawley rats, 3 months of age, were randomly divided by the stratified weight method into eleven groups according to the following treatment schedule: baseline control, a 4-week age-matched control, and a 4-week GC administration with a 4-week concomitant administration of vehicle, ALF, RIS, or ALF+RIS, and an 8-week age-matched control and 8-week GC administration with a 4-week administration of vehicle, ALF, RIS, or ALF+RIS that was initiated after a 4-week administration of GC. The GC (methylprednisolone sodium succinate, 5.0 mg/kg) and RIS (10 microg/kg) were administered subcutaneously 3 times a week. ALF (0.08 microg/kg) was administered orally 5 times a week. At the end of the experiment, static and dynamic bone histomorphometric analyses were performed on cancellous bone of the proximal tibial metaphysis and cortical bone of the tibial diaphysis. A 4-week GC administration induced a loss of cancellous bone volume/total tissue volume (BV/TV) compared with the age-matched control as a result of decreased bone formation and increased bone erosion, while an 8-week GC administration induced both losses of cancellous BV/TV and of percent cortical area (Ct Ar) compared with the age-matched control as a result of decreased trabecular bone formation and increased trabecular and endocortical bone erosion. ALF prevented the loss of cancellous BV/TV at the 4th week by mildly suppressing bone erosion without reducing bone formation, and it restored cancellous BV/TV and increased percentage of Ct Ar at the 8th week by preventing a reduction in trabecular bone formation and mildly suppressing trabecular bone erosion and by strongly suppressing endocortical bone erosion, respectively. RIS restored only cancellous BV/TV at 8 weeks by strongly suppressing bone formation and bone erosion. Combined administration of ALF and RIS increased total tissue area of cortical bone at the 4th and 8th weeks by markedly increasing periosteal bone formation. The present study showed the efficacy of combined administration of ALF and RIS for the geometry of cortical bone in GC-treated rats.     

A comparison of the anabolic effects of rat and bovine parathyroid hormone (1-34) in ovariectomized rats

The current study was designed to compare the skeletal effects of comparable doses of rat parathyroid hormone 1-34 (rPTH) and bovine parathyroid hormone 1-34 (bPTH) in ovariectomized (OVX) rats. Female Sprague-Dawley rats were OVX or sham-operated at 6 months of age and maintained untreated for 28 days after surgery. Baseline control and OVX rats were sacrificed at the beginning of treatment. Beginning 28 days post-OVX, the remaining rats were subcutaneously injected daily with rPTH or bPTH at 0, 5, 25, or 50 microg/kg/d for 28 days. Bone area, bone mineral content (BMC), and bone mineral density (BMD) of the distal femoral metaphyses were determined ex vivo using dual energy X-ray absorptiometry. Quantitative bone histomorphometry was performed on undecalcified longitudinal sections of the proximal tibia from each rat. Baseline OVX rats exhibited osteopenia as demonstrated by their significantly reduced femoral BMD and proximal tibia cancellous bone volume compared with those of baseline sham controls. Both rPTH and bPTH restored bone in OVX rats by markedly stimulating bone formation in a dose-dependent manner. However, a difference in potency between the two forms of PTH was evident. The percentage increases of BMC, BMD, cancellous bone volume, trabecular thickness, mineralizing surface, and bone formation rate in the OVX rats treated with bPTH at 5 microg/kg/d were the same as or above those treated with rPTH at the 25 microg/kg/d dose level. A relative potency analysis showed that bPTH was approximately 4- to 6-fold relatively more potent than rPTH in increasing distal femoral BMD as well as cancellous bone volume, mineralizing surface, and bone formation rate of proximal tibial metaphyses at comparable dose levels and a given time. These results may serve as a reference for in vivo study design when rPTH or bPTH are to be the agents for studies on bone anabolism.     

Protective actions of green tea polyphenols and alfacalcidol on bone microstructure in female rats with chronic inflammation

This study investigated the effects of green tea polyphenols (GTP) and alfacalcidol on bone microstructure and strength along with possible mechanisms in rats with chronic inflammation. A 12-week study using a 2 (no GTP vs. 0.5%, w/v GTP in drinking water)×2 (no alfacalcidol vs. 0.05 μg/kg alfacalcidol orally, 5×/week) factorial design was employed in lipopolysaccharide (LPS)-administered female rats. A group receiving placebo administration was used to compare with a group receiving LPS administration only to evaluate the effect of LPS. Changes in tibial and femoral microarchitecture and strength of femur were evaluated. Difference in expression of tumor necrosis factor-α (TNF-α) in proximal tibia using immunohistochemistry was examined. Compared to the placebo group, the LPS-administered-only group had significantly lower femoral mass, trabecular volume, thickness and number in proximal tibia and femur, and lower periosteal bone formation rate in tibial shafts but had significantly higher trabecular separation and osteoclast number in proximal tibia and eroded surface in endocortical tibial shafts. Both GTP and alfacalcidol reversed these LPS-induced detrimental changes in femur, proximal tibia and endocortical tibial shaft. Both GTP and alfacalcidol also significantly improved femoral strength, while significantly suppressed TNF-α expression in proximal tibia. There were significant interactions in femoral mass and strength, trabecular separation, osteoclast number and TNF-α expression in proximal tibia. A combination of both showed to sustain bone microarchitecture and strength. We conclude that a protective impact of GTP and alfacalcidol in bone microarchitecture during chronic inflammation may be due to a suppression of TNF-α.     

Disuse in adult male rats attenuates the bone anabolic response to a therapeutic dose of parathyroid hormone.

Intermittent treatment with parathyroid hormone (PTH) increases bone formation and prevents bone loss in hindlimb-unloaded (HLU) rats. However, the mechanisms of action of PTH are incompletely known. To explore possible interactions between weight bearing and PTH, we treated 6-mo-old weight-bearing and HLU rats with a human therapeutic dose (1 microg.kg(-1).day(-1)) of human PTH(1-34) (hPTH). Cortical and cancellous bone formation was measured in tibia at the diaphysis proximal to the tibia-fibula synostosis and at the proximal metaphysis, respectively. Two weeks of hindlimb unloading resulted in a dramatic decrease in the rate of bone formation at both skeletal sites, which was prevented by PTH treatment at the cancellous site only. In contrast, PTH treatment increased cortical as well as cancellous bone formation in weight-bearing rats. Two-way ANOVA revealed that hPTH and HLU had independent and opposite effects on all histomorphometric indexes of bone formation [mineral apposition rate (MAR), double-labeled perimeter (dLPm), and bone formation rate (BFR)] at both skeletal sites. The bone anabolic effects of weight bearing and hPTH on dLPm and BFR at the cortical site were additive, as were the effects on MAR at the cancellous site. In contrast, weight bearing and hPTH resulted in synergistic increases in cortical bone MAR and cancellous bone dLPm and BFR. We conclude that weight bearing and PTH act cooperatively to increase bone formation by resulting in site-specific additive and synergistic increases in indexes of osteoblast number and activity, suggesting that weight-bearing exercise targeted to osteopenic skeletal sites may improve the efficacy of PTH therapy for osteoporosis.     

Effects of disrupted beta1-integrin function on the skeletal response to short-term hindlimb unloading in mice.

The study was designed to determine whether beta1-integrin plays a role in mediating the acute skeletal response to mechanical unloading. Transgenic (TG) mice were generated to express a dominant negative form of beta1-integrin under the control of the osteocalcin promoter, which targets expression of the transgene to mature osteoblasts. At 63 days of age, wild-type (WT) and TG mice were subjected to hindlimb unloading by tail suspension for 1 wk. Pair-fed, normally loaded WT and TG mice served as age-matched controls. Bone samples from each mouse were processed for quantitative bone histomorphometry and biomechanical testing. The skeletal phenotype of TG mice was characterized by lower cancellous bone mass in the distal femoral metaphysis (-52%) and lumbar vertebral body (-20%), reduced curvature of the proximal tibia (-20%), and decreased bone strength (-20%) and stiffness (-23%) of the femoral diaphysis with relatively normal indexes of cancellous bone turnover. Hindlimb unloading for only 1 wk induced a 10% decline in tibial curvature and a 30% loss of cancellous bone in the distal femur due to a combination of increased bone resorption and decreased bone formation in both WT and TG mice. However, the strength and stiffness of the femoral diaphysis were unaffected by short-term hindlimb unloading in both genotypes. The observed increase in osteoclast surface was greater in unloaded TG mice (92%) than in unloaded WT mice (52%). Cancellous bone formation rate was decreased in unloaded WT (-29%) and TG (-15%) mice, but, in contrast to osteoclast surface, the genotype by loading interaction was not statistically significant. The results indicate that altered integrin function in mature osteoblasts may enhance the osteoclastic response to mechanical unloading but that it does not have a major effect on the development of cancellous osteopenia in mice during the early stages of hindlimb unloading.     

Continuous infusion of PGE2 is catabolic with a negative bone balance on both cancellous and cortical bone in rats

It is well documented that intermittent PGE(2) treatment increases both trabecular and cortical bone mass. However, the effects of continuous PGE(2) administration remain undocumented. The aim of the study was to investigate the effects of continuous prostaglandin E(2) (PGE(2)) on different bone sites in skeletally mature rats. Six-month-old Sprague Dawley rats were treated with PGE(2) at 1 or 3 mg/kg/d continuously via infusion pump for 21 days. Two other groups of rats received PGE(2) at the same doses by intermittent (daily) subcutaneous injections and served as positive controls. Histomorphometry was performed on cancellous bone of the proximal tibial metaphysis and cortical bone of the tibial shaft. As expected, intermittent PGE(2) treatment increased both cancellous and cortical bone mass by stimulating bone formation at the cancellous, periosteal and endocortical bone surfaces. In contrast, continuous PGE(2) treatment decreased cancellous bone mass with bone resorption exceeding bone formation. In addition, continuous PGE(2) treatment increased endocortical and intracortical bone remodeling, inducing bone loss which was partially offset by stimulating periosteal expansion. We conclude that continuous PGE(2) treatment induces overall catabolic effects on both cancellous and cortical bone envelopes, which differs from intermittent PGE(2) treatment that is anabolic. Lastly, we speculate that superior bone mass may be achieved by co-treatment of continuous PGE(2) in combination with an anti-catabolic agent.     

Skeletal effects of zinc deficiency in growing rats.

There is ample evidence that zinc plays an important role in bone metabolism and zinc deficiency has been implicated as a risk factor in the development of osteoporosis. It was the aim of the present study to investigate the skeletal effects of alimentary zinc deficiency in growing rats using quantitative bone histomorphometry. Twenty-four male Sprague Dawley rats with a mean initial body weight of 101 +/- 2 g were allocated in two groups of 12 rats each and had free access to a semi-synthetic, casein-based, zinc-deficient diet (0.76 mg zinc/kg) or to the same diet supplemented with 60 mg zinc per kg. All rats were sacrificed 42 days after the start of the experiment and the right distal femur was removed for bone histomorphometry. Relative to controls (+Zn), the zinc-deficient rats (-Zn) had a significantly lower body weight and about an 80% reduction in plasma and femur zinc concentration. The histomorphometric evaluation of the distal femoral metaphysis showed that zinc deficiency led to a 45% reduction (p < 0.01) in cancellous bone mass and to a deterioration of trabecular bone architecture, with fewer and thinner trabeculae. The osteopenia in -Zn rats was accompanied by significant reductions in osteoid perimeter (-31%, p < 0.05), osteoblast perimeter (-30%, p < 0.05), and osteoclast number (-38%, p < 0.01) relative to +Zn controls. We conclude that zinc deficiency induced low turnover osteopenia in femoral cancellous bone of growing rats. These results support the hypothesis that zinc deficiency during growth may impair the accumulation of maximal bone mass in humans; additionally, they suggest that zinc deficiency may play a role as a risk factor in the pathogenesis of osteoporosis.     

Estrogen-induced osteogenesis in intact female mice lacking ERbeta

We recently found that estrogen receptor (ER) antagonists prevent high-dose estrogen from inducing the formation of new cancellous bone within the medullary cavity of mouse long bones. In the present investigation, we studied the role of specific ER subtypes in this response by examining whether this is impaired in female ERbeta(-/-) mice previously generated by targeted gene deletion. Vehicle or 17beta-estradiol (E(2)) (range 4-4,000 microg. kg(-1). day(-1)) was administered to intact female ERbeta(-/-) mice and wild-type littermates by subcutaneous injection for 28 days. The osteogenic response was subsequently assessed by histomorphometry performed on longitudinal and cross sections of the tibia. E(2) was found to cause an equivalent increase in cancellous bone formation in ERbeta(-/-) mice and littermate controls, as assessed at the proximal and distal regions of the proximal tibial metaphysis. E(2) also resulted in a similar increase in endosteal mineral apposition rate in these two genotypes, as assessed at the tibial diaphysis. In contrast, cortical area in ERbeta(-/-) mice was found to be greater than that in wild types irrespective of E(2) treatment, as was tibial bone mineral density as measured by dual-energy X-ray absorptiometry, consistent with previous reports of increased cortical bone mass in these animals. We conclude that, although ERbeta acts as a negative modulator of cortical modeling, this isoform does not appear to contribute to high-dose estrogen's ability to induce new cancellous bone formation in mouse long bones.     

Local distribution and dosimetry of 226Ra in the trabecular skeleton of the beagle.

Young adult beagle dogs received a single injection of 38.1 kBq/kg body wt 226Ra and were serially sacrificed at 4 to 2955 days postinjection. Samples of sites of trabecular bone in the lumbar vertebral body, proximal ulna, and distal femoral metaphysis and epiphysis were analyzed autoradiographically. The time-dependent changes in the average 226Ra concentrations in the four regions were analyzed in terms of a compartmental model. The clearance rate from the lumbar vertebral body was about four times more rapid than for the proximal ulna and distal femoral epiphysis. Ratios of hotspot to diffuse label concentrations varied from about 10 to 23. The dose rate to the endosteum ranged between 8.7 and 39.5 mGy/day initially and 4 and 10.5 mGy/day toward the end of the observation period. Mean marrow dose rates were lower by a factor of 3 to 9.5. During their residence time the nuclei of bone lining cells receive a maximum dose of 8 Gy in the proximal ulna (2955 days after injection) and a minimum dose of 0.63 Gy in the lumbar vertebra (2955 days after injection). This corresponds on the average to 17 and 1.4 alpha-particle hits to the cell nuclei, respectively.     

Effect of gender on bone turnover in adult rats during simulated weightlessness.

Prologned spaceflight results in bone loss in astronauts, but there is considerable individual variation. The goal of this rat study was to determine whether gender influences bone loss during simulated weightlessness. Six-month-old Fisher 344 rats were hindlimb unweighted for 2 wk, after which the proximal tibiae were evaluated by histomorphometry. There were gender differences in tibia length, bone area, cancellous bone architecture, and bone formation. Compared with female rats, male rats had an 11.6% longer tibiae, a 27.8% greater cortical bone area, and a 37.6% greater trabecular separation. Conversely, female rats had greater cortical (316%) and cancellous (145%) bone formation rates, 28.6% more cancellous bone, and 30% greater trabecular number. Hindlimb unweighting resulted in large reductions in periosteal bone formation and mineral apposition rate in both genders. Unweighting also caused cancellous bone loss in both genders; trabecular number was decreased, and trabecular separation was increased. There was, however, no change in trabecular thickness in either gender. These architectural changes in cancellous bone were associated with decreases in bone formation and steady-state mRNA levels for bone matrix proteins and cancellous bone resorption. In conclusion, there are major gender-related differences in bone mass and turnover; however, the bone loss in hindlimb unweighted adult male and female rats appears to be due to similar mechanisms.     

The relationship of bone marrow type and microvasculature to the microdistribution and local dosimetry of plutonium in the adult skeleton

Most plutonium-induced bone tumors in beagles arise in trabecular bone sites surrounded by hematopoietic tissue. The microvasculature and the relative incorporation of 241Pu (a low energy beta emitter) on trabecular bone surfaces from red and fatty marrow sites were studied in the adult beagle. The vascular volume, as determined by India ink perfusion, was about 16% in the red marrow and about 2% in the fatty marrow. The red marrow has large vascular sinusoids with a discontinuous endothelial lining whereas fatty marrow has a closed capillary bed. Plutonium concentrations on bone surfaces of red marrow sites are about 0.43 pCi/cm2/nCi/kg of injected dosage, a factor of eight greater than at fatty marrow sites (0.053 pCi/cm2/nCi/kg). The differences in the bone marrow microvasculature between red and fatty marrow help to explain differences in plutonium incorporation and local bone physiology in these skeletal sites. These observations may relate to the nonuniform distribution of plutonium-induced osteosarcomas observed in adult, long-lived mammals.     

Effect of circular motion exercise on bone modeling and bone mass in young rats: an animal model of isometric exercise

The aims of the study are to develop a non-invasive animal model of circular motion exercise and to evaluate the effect of this type of exercise on bone turnover in young rats. The circular motion exercise simulates isometric exercise using an orbital shaker that oscillates at a frequency of 50 Hz and is capable of speeds from 0-400 rpm. A cage is fixed on top of the shaker and the animals are placed inside. When the shaker is turned on, the oscillatory movement should encourage the animals to hold on to the cage and use various muscle forces to stabilize themselves. Rats at 8 weeks of age were trained on the shaker for 6 weeks and static and dynamic histomorphometric analyses were performed for the proximal tibial metaphysis and the tibial shaft. The exercise resulted in no significant effect on animal body weight, gastrocnemius muscle weight and femoral weight. Although the bone formation rate of cancellous and cortical periosteum was increased by the exercise, trabecular bone volume was decreased. The exercise increased periosteal and marrow perimeters and the cross-sectional diameter of cortical bone from medial to lateral without a significant increase in the cortical bone area. These results suggest that circular motion exercise under force without movement or additional weight loading will cause bone-modeling drift with an increase in bone turnover to reconstruct bone shape in adaptation to the demand in strength. Since there is no additional weight loading during circular motion exercise, the net mass of bone is not increased. The bone mass lost in trabecular bone could possibly be due to a re-distribution of mineral to the cortical bone.     

Simvastatin did not prevent nor restore ovariectomy-induced bone loss in adult rats

Current published results on whether statins have beneficial effects on bone metabolism have been conflicting so far. In order to further investigate if statins were promising candidates for the treatment for osteoporosis, we conducted a study in which rats were ovariectomized (OVX) at 6 months of age, allowed to lose bone for 60 days and followed by oral administration of simvastatin at the dose levels of 0.3-10 mg/kg/d for 60 days. PGE2 (6 mg/kg) was used as a positive control. Study endpoints included bone histomorphometry on the proximal tibial metaphysis (PTM) and the tibial diaphysis (TX), dual-energy X-ray absorptiometry on the right femur and micro computed tomography (ICT) on the 5th lumbar vertebra (LV). After 120 days of OVX, cancellous bone lost by 80% in the PTM and 18% in the LV accompanied by increased bone formation and resorption. Simvastatin at all dose levels did not affect bone volume, bone formation rate and bone erosion surface when compared to 120 day ovariectomized animals at all bone sites studied. By contrast, PGE2 restored cancellous and cortical bone area to sham control levels. In conclusion, this study demonstrated that unlike PGE2, oral administration of simvastatin did not have effects on cancellous or cortical bone formation and resorption; and consequently was not able to prevent further bone loss or restore bone mass in the osteopenic, OVX rats.     

Rolipram, a phosphodiesterase 4 inhibitor, prevented cancellous and cortical bone loss by inhibiting endosteal bone resorption and maintaining the elevated periosteal bone formation in adult ovariectomized rats

Cyclic AMP (cAMP) is a continually produced nucleotide inactivated by hydrolysis to 5'AMP via phosphodiesterase (PDE) enzymes. Rolipram is a selective PDE4 inhibitor reported to have anti-inflammatory effects and used in the treatment of asthma and chronic obstructive pulmonary disease (COPD). The current study was designed to determine whether Rolipram could prevent and restore bone loss in ovariectomized (OVX) rats. Six-month-old Sprague Dawley rats underwent either sham-operated or bilateral ovariectomy, and were left untreated for 60 days to develop osteopenia. Then they were treated with vehicle, 6 mg/kg PGE(2), 3 microg/kg Alendronate or 0.1-1.0 mg/kg Rolipram for 60 days. At sacrifice, the right tibiae were processed for quantitative bone histomorphometric measurements. The right femurs were measured by dual energy A-ray absorptiometry and the 5th lumbar vertebrae were subjected to micro-computed tomography to access bone mass and architecture changes. Our results indicated that OVX induced negative bone balance in all five bone sites we tested, with bone resorption exceeding bone formation. Rolipram at 0.1-0.6 mg/kg dose levels prevented while at 1 mg/kg restored ovariectomy-induced cancellous and cortical bone loss in the tibia, femur and lumbar vertebra. Dynamic bone histomorphometry suggested that these beneficial effects were achieved by partially maintaining the elevated bone formation at the trabecular bone surface and increasing bone formation at the periosteal bone surface of the cortex. Furthermore, it reduced bone turnover at the trabecular and the endocortical bone surfaces. The prevention of further bone loss effects were comparable to those of an anti-resorption agent (Alendronate) but were not as great as those of an anabolic agent (PGE(2)). In addition, Rolipram treatment increased body and muscle weights compared to the vehicle-treated OVX rats. In conclusion, our study in an osteopenic rat model suggested that a selective PDE4 inhibitor may be used for the treatment of established osteoporosis.