Pharmacological treatment of osteopenia induced by gastrectomy or ovariectomy in young female rats

BACKGROUND: Both gastrectomy (GX) and ovariectomy (OVX) induce osteopenia in man and experimental animals. The present study addresses the question--can alendronate, estrogen or parathyroid hormone (PTH) be used to treat established GX- or OVX -evoked osteopenia? METHODS: Rats were GX-, OVX- or SHAM-operated 8 weeks before starting the treatment with drugs. Each group was then treated for 8 weeks with 50 microg/kg/day alendronate, 10 microg/kg/day estrogen or 75 microg/kg/day PTH(1-84); n = 8 rats/group. Peripheral Quantitative Computed Tomography (pQCT) was used to measure trabecular bone mineral density (BMD) and various cortical bone parameters. RESULTS: At killing, 16 weeks after surgery, GX and OVX rats had a greatly reduced trabecular BMD in the metaphysis of the distal femur (GX -44% and OVX -55%). Alendronate increased the trabecular BMD by 44% in GX rats and by 64% in OVX rats, while PTH increased it by 51% and 115%, respectively. However, estrogen increased the trabecular BMD in GX rats (35%), but not in OVX rats (15%, not significant). Cortical bone parameters were adversely (but moderately) affected by GX, but not by OVX or by treatment with the three drugs. INTERPRETATION: Alendronate, estrogen and PTH restored the trabecular bone loss in rats with an established GX-evoked osteopenia. In contrast, alendronate and PTH, but not estrogen, restored the trabecular bone loss after OVX. Hence, the mechanism underlying GX-evoked bone loss differs from that underlying OVX-evoked bone loss. The ability of alendronate, estrogen and PTH to reverse the GX-evoked osteopenia in the rat may be of clinical interest when dealing with bone loss in humans after GX.     

Bone tissue and its mineralization in aged estrogen-depleted rats after long-term intermittent treatment with parathyroid hormone (PTH) analog SDZ PTS 893 or human PTH(1-34)

Intermittently administered parathyroid hormone (PTH) is a potent bone anabolic agent. We aimed to determine the impact of long-term treatment with PTH on bone structure, dynamics, and mineralization. We ovariectomized (ovx) 1-year-old rats with the exception of a baseline and a sham-operated group. Twelve weeks later, a 36 week treatment with PTH analog SDZ PTS 893 (12.5, 25, 50, 100 microg/kg), human PTH(1-34) (25, 50, 100 microg/kg), or vehicle (ovx, sham) was initiated. Bone dynamics, structure, and mineralization were evaluated in the lumbar spine and in the femoral diaphysis. Cancellous bone turnover was elevated 12 weeks postovariectomy in estrogen-deficient, vehicle-treated animals, but returned to the level of the sham group by 48 weeks. The animals experienced substantial cancellous bone loss associated with a reduction of trabecular number and presented with a partly rod-like trabecular network. After 36 weeks of treatment with SDZ PTS 893 or human PTH(1-34), cancellous bone formation rates and turnover were raised in all treated groups compared with age-matched controls. The mineral apposition rate was increasing with dose. This amplified matrix synthesis led to trabecular thickening, but not to an increase in trabecular number, resulting in a crude, plate-like cancellous network with a high bone volume fraction. Fluorochrome label-based cortical bone dynamics demonstrated that a thick ring of new bone was formed at the endocortex by activation of modeling drifts during treatment. Treatment-induced cortical bone formation was increased with dose at the subperiosteal and endocortical envelopes, but substantially higher at the latter. Intracortical bone turnover was elevated near the endocortex. Bone mineralization was undisturbed in all compartments. The average degree of mineralization was lowered slightly, reflecting the increased portion of new bone formed during treatment. In summary, the main anabolic effect was mediated for both peptides by an increase in bone apposition with dose, persisting throughout treatment that lasted more than one third of the lifespan of the rats, and direct activation of bone-forming surfaces. As a result, a substantial amount of new bone, maintained at elevated turnover and adequate mineralization levels, formed predominantly at compartments exposed to bone marrow.     

Treatment of skeletally mature ovariectomized rhesus monkeys with PTH(1-84) for 16 months increases bone formation and density and improves trabecular architecture and biomechanical properties at the lumbar spine.

Histomorphometric studies of treatments for osteoporosis in humans are restricted to iliac crest biopsies. We studied the effects of PTH(1-84) treatment at the lumbar spine of skeletally mature ovariectomized rhesus monkeys. PTH increased bone turnover, rapidly normalized BMD, and increased vertebral compressive strength. PTH increased trabecular bone volume primarily by increasing trabecular number by markedly increasing intratrabecular tunneling. INTRODUCTION: Histomorphometric studies of the anabolic properties of PTH(1-84) (PTH) and related peptides in human bone are restricted to iliac crest biopsies. The ovariectomized (OVX) monkey is an accepted model of human postmenopausal bone loss and was used to study the effects of PTH treatment at clinically relevant skeletal sites. MATERIALS AND METHODS: Skeletally mature rhesus monkeys were OVX or sham-operated and, after a bone depletion period of 9 months, treated daily for 16 months with PTH (5, 10, or 25 microg/kg). Markers of bone formation (serum osteocalcin) and resorption (urine N-telopeptide [NTX]) and lumbar spine BMD were measured throughout the study. Trabecular architecture and vertebral biomechanical properties were quantified at 16 months. RESULTS: PTH treatment induced dose-dependent increases in bone turnover but did not increase serum calcium. Osteocalcin was significantly increased above OVX controls by 1 month. NTX was significantly elevated at 1 month with the highest dose, but not until 12 months with the 5 and 10 microg/kg doses. Lumbar spine BMD was 5% lower in OVX than in sham animals when treatment was started. All PTH doses increased BMD rapidly, with sham levels restored by 3-7 months with 10 and 25 microg/kg and by 16 months with 5 microg/kg. PTH treatment increased trabecular bone volume (BV/TV), primarily by increasing trabecular number, and dose-dependently increased bone formation rate (BFR) solely by increasing mineralizing surface. The largest effects on BV/TV and yield load occurred with the 10 microg/kg dose. The highest dose reduced trabecular thickness by markedly increasing intratrabecular tunneling. CONCLUSIONS: PTH treatment of OVX rhesus monkeys increased bone turnover and increased BV/TV, BMD, and strength at the lumbar spine. All PTH doses were safe, but the 10 microg/kg dose was generally optimal, possibly because the highest dose resulted in too marked a stimulation of bone remodeling.     

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.     

Long-term therapy of ovariectomy-induced osteopenia with parathyroid hormone analog SDZ PTS 893 and bone maintenance in retired breeder rats

The aim of the study was to assess the long-term anabolic effect of the parathyroid hormone (PTH) analog SDZ PTS 893 in a dose-response manner, and to determine the ability of the antiresorptive agents estradiol and alendronate to maintain bone mass after withdrawal of SDZ PTS 893. One hundred thirty retired breeder Wistar rats were distributed into 13 groups with 10 rats in each group: 1 baseline group, 2 sham groups, and 10 ovariectomized groups. Treatment was initiated 12 weeks after ovariectomy. SDZ PTS 893 treatment was administered daily subcutaneously (Monday to Friday) for 36 weeks. Treatment regimens were as follows: (1) baseline (-12 weeks); (2) ovariectomy (ovx) (0 weeks); (3) sham (36 weeks); (4) ovx (36 weeks); (5) SDZ PTS 893 12.5 microg/kg/day (36 weeks); (6) SDZ PTS 893 25 microg/kg/day (36 weeks); (7) SDZ PTS 893 50 microg/kg/day (36 weeks); (8) SDZ PTS 893 100 microg/kg/day (36 weeks); for the maintenance part of the study: (9) sham (48 weeks); ovx animals treated with SDZ PTS 893, 50 microg/kg/day for 36 weeks followed by 12 weeks of treatment regimens: (10) placebo; (11) SDZ PTS 893 50 microg/kg/day; (12) estradiol 10 microg/kg/day; or (13) alendronate 28 microg/kg (2 injections/week). The effects of ovx, SDZ PTS 893 treatment, and maintenance regimens were measured at four skeletal sites: lumbar vertebra; femoral diaphysis; distal femoral metaphysis; and proximal femoral metaphysis (femoral neck). At these sites, bone density and bone strength were measured as treatment endpoints. Furthermore, bone dimensions were measured at the midpoint of the femur. The results showed that SDZ PTS 893 increased bone strength in a dose-dependent manner at all skeletal sites tested. At the vertebral body and distal femoral metaphysis, apparent ash density increased in a similar way. There was a slight decrease in cortical density at the mid-diaphyseal site. Static histomorphometry showed increased bone area due to a decreased marrow area (endosteal net bone gain) but also due to increased tissue area (periosteal net bone gain). For maintenance, continuous SDZ PTS 893 therapy was most efficient, followed by alendronate and estradiol treatment with regard to preservation of bone mass and strength. It is concluded that the new PTH analog SDZ PTS 893 has a highly anabolic, dose- and time-dependent effect on all skeletal sites tested. Bone formation is induced at both endosteal and periosteal surfaces.     

Intermittent parathyroid hormone (1-34) treatment increases callus formation and mechanical strength of healing rat fractures.

The influence of intermittent parathyroid hormone (PTH(1-34)) administration on callus formation and mechanical strength of tibial fractures in rats was investigated after 20 and 40 days of healing. A dose of 60 microg of PTH(1-34)/kg/day and 200 microg of PTH(1-34)/kg/day, respectively, was administered during the entire periods of healing, and control animals with fractures were given vehicle. The dose of 200 microg of PTH(1-34)/kg/day increased the ultimate load and the external callus volume of the fractures by 75% and 99%, respectively, after 20 days of healing and by 175% and 72%, respectively, after 40 days of healing. The dose of 60 microg of PTH(1-34)/kg/day did not influence either ultimate load or external callus volume of the fractures after 20 days of healing, but the ultimate load was increased by 132% and the external callus volume was increased by 42% after 40 days of healing. During the healing period, the callus bone mineral content (BMC) increased in all groups. After 40 days of healing, the callus BMC was increased by 108% in the 200 microg of PTH(1-34)/kg/day group and by 76% in the 60 microg of PTH(1-34)/kg/day group. Both doses of PTH(1-34) steadily augmented the contralateral intact tibia BMC (20 days and 40 days: 60 microg of PTH (1-34)/kg/day 9% and 19%, respectively; 200 microg of PTH (1-34)/kg/day 12% and 27%, respectively) and bone mineral density (20 days and 40 days: 60 microg of PTH(1-34)/kg/day 11% and 12%, respectively; 200 microg of PTH(1-34)/kg/day 11% and 15%, respectively).     

Intermittently administered parathyroid hormone 1–34 reverses bone loss and structural impairment in orchiectomized adult rats

Male osteoporosis is emerging as a central theme in bone research. As in females, hypogonadism appears as a principal risk factor in men that leads to bone loss and increased fracture incidence. Intermittently administered parathyroid hormone (PTH) reverses bone loss in sex hormone-deprived women and female animals and increases bone mass in elderly men and normal male animals. This study was carried out to assess whether the PTH anabolic activity is also effective in adult castrated males and to gain insight into the underlying tissue processes. Bilateral orchiectomy (ORX) or sham-ORX was performed in 13-week old rats. Five weeks later, the ORX rats were treated intermittently with human PTH(1–34), 80 g/kg/day or vehicle for 6 weeks. Femora were evaluated by quantitative micro-computed tomography followed by dynamic histomorphometry. The trabecular bone volume density showed 40% and 56% ORX-induced loss in the distal metaphysis at 6 weeks and 12 weeks post-ORX, respectively. PTH(1–34) induced supraphysiologic recovery of this bone loss (155% recovery) consequent to a vast increase in trabecular thickness (174% over sham-ORX controls) and a partial reversal (62%) of the decrease in trabecular number. As compared with the results in 12-week, orchiectomized vehicle-administered rats, the PTH(1–34) treatment induced a significant decrease in osteoclast number (20%) and twofold increase in bone formation rate. While ORX did not affect the femoral diaphysis, PTH(1–34) induced marked cortical thickening via the stimulation of endosteal mineral appositional rate (154% over ORX rats). These data portray PTH(1–34) as a highly potent bone anabolic agent in adult ORX rats, mainly by increasing both the trabecular and cortical thicknesses through its effect on osteoblasts and osteoclasts. The adult ORX rat is useful for investigating the processes involved in bone anabolic activity in castrated osteoporotic males and for the development of bone anabolic agents for treating this condition.     

Effect of combined humanPTH(1-34) and calcitonin treatment in ovariectomized rats

We examined the combined effects of human parathyroid hormone 1-34 (hPTH) and elcatonin (ECT: a synthetic derivative of eel calcitonin) to prevent loss of bone mass, architecture and strength in ovariectomized (OVX) rats. Fifty-four female rats (aged 13 weeks) were assigned to one of nine groups: Sham (fake surgery performed), OVX, ECT (15 U/kg administered), PTH5, PTH10 and PTH20 (5, 10 or 20 microg/kg administered), and E+PTH5, E+PTH10 and E+PTH20 (15 U/kg of ECT and 5, 10 or 20 microg/kg of hPTH administered). The drug or vehicle was subcutaneously administered three times a week for 12 weeks. The femurs were removed at the completion of the experiment. The right distal femoral metaphysis was used for measuring bone mineral density (BMD), analyzing trabecular bone structure by micro-computed tomography (microCT), and conducting the bone strength test, and the left femur was used for histomorphometric analysis. Trabecular bone volume (BV/TV) and other bone mass parameters were greater in the ECT and PTH groups than in the OVX group. The number of nodes (N.Nd/TV) and trabecular number (Tb.N) were significantly greater in the ECT group, and trabecular thickness (Tb.Th) and trabecular bone pattern factor (TBPf) were significantly greater in the PTH group. These results indicate that these drugs preserve the bone architecture by different means. Analysis by means of microCT revealed that BV/TV, Tb.N, fractal D and N.Nd/TV were significantly greater in the E+PTH groups than in the PTH groups at each concentration. Trabecular separation (Tb.Sp) was significantly lower in the E+PTH5 and E+PTH10 groups than in the respective PTH5 and PTH10 groups. When the maximum load was applied in a compression test on the distal femur, the E+PTH groups had higher values than the PTH groups, however, the three point bending strength of the diaphysis of femur in the E+PTH10 and E+PTH20 groups tended to be low compared to those in the PTH10 and PTH20 groups. These results indicate that combination therapy using PTH and ECT preserves the trabecular microarchitecture better than single-drug therapy using ECT or PTH in OVX rats, however, it is necessary to optimize the calcitonin (CT) dosage and administration in order to achieve the optimal combined effect of PTH and CT.     

Bipedal stance exercise and prostaglandin E2 (PGE2) and its synergistic effect in increasing bone mass and in lowering the PGE2 dose required to prevent ovariectomized-induced cancellous bone loss in aged rats

Previous reports have shown that bone loss was partially prevented by bipedal stance "exercise" following ovariectomy (ovx), and it was well documented that prostaglandin E2 (PGE(2)) had an anabolic effect on the rat skeleton. The aim of this study was to determine whether lower doses of PGE(2) could prevent ovx-induced cancellous bone loss with the combination of bipedal stance exercise. Seventy-eight 10-month-old female Sprague-Dawley rats were either ovariectomized or sham-operated on day 0 and then treated with PGE(2) (0, 0.3, or 1 mg/kg per day) and/or housed in normal height cages (NC, 28 cm) or raised cages (RC, 33 cm) for 8 weeks. Bone histomorphometry was performed on the double-fluorescent-labeled proximal tibial metaphysis. In sham rats, 1 mg/kg PGE(2) + RC had synergistic effects in increasing trabecular bone area, width, and number by stimulating mineral apposition rate and bone formation rate. As expected, ovx induced cancellous bone loss, accompanied by elevated activation frequency. Without RC, PGE(2) monotherapy prevented ovx-induced bone loss at the 1 mg/kg per day dose, whereas this prevention effect was observed at the 0.3 mg/kg per day dose when combined with RC. Similar to their effects in sham rats, PGE(2) and RC had synergistic effects in augmenting cancellous bone mass and architecture and maintaining the elevated bone formation but depressing bone resorption and activation frequency. We conclude that bipedal stance exercise lowers the PGE(2) dose required to prevent ovx-induced cancellous bone loss in the proximal tibial metaphysis in aged rats.     

Treatment with human parathyroid hormone (1-34) for 18 months increases cancellous bone volume and improves trabecular architecture in ovariectomized cynomolgus monkeys (Macaca fascicularis)

A key feature of postmenopausal osteoporosis is the loss of trabecular bone mass and connectivity. The current study focuses on these parameters in the assessment of long-term (12 and 18 months) parathyroid hormone (PTH) therapy and its withdrawal (6 months) in the ovariectomized cynomolgus monkey (Macaca fascicularis), a well-characterized model for bone changes associated with postmenopausal osteoporosis. We used static and dynamic histomorphometric parameters to assess the amount and architecture of cancellous bone in four clinically important sites for osteoporotic fractures, including the lumbar vertebra, femoral neck, distal radius, and iliac crest. Recombinant human PTH(1-34) was administered daily to two groups for 18 months at 1.0 microg/kg per day (n = 19) and 5.0 microg/kg per day (n = 21). To study the effects of PTH withdrawal, two groups were administered PTH(1-34) daily for 12 months at 1.0 microg/kg per day (n = 20) and 5.0 microg/kg per day (n = 20), followed by daily administration of vehicle for 6 months. Sham-ovariectomized and ovariectomized (ovx) groups each received daily injections of vehicle for 18 months. Treatment with PTH had minimal effects on bone formation rates at the timepoints studied, but markedly increased cancellous bone volume relative to ovx monkeys in iliac crest biopsies at 6 and 15 months, as well as in terminal specimens of lumbar vertebrae, femoral neck, and distal radius after 18 months. At all sites, PTH significantly improved trabecular architecture, as evidenced by increased trabecular number (Tb.N) and decreased trabecular separation (Tb.Sp), with no significant change in trabecular thickness (Tb.Th). The mechanism of these structural changes is suggested by qualitative observations of trabecular tunneling observed in the iliac crest and vertebra. Longitudinal tunneling of thickened individual trabeculae is hypothesized to convert them into multiple trabeculae, resulting in a normalization of Tb.Th, but an increase in Tb.N. A significant positive effect on cancellous bone volume was still apparent after a 3-6 month withdrawal period following 12 months of PTH treatment in the iliac crest, vertebra, and femoral neck. Corresponding increases in Tb.N and decreases in Tb.Sp also remained significant after PTH withdrawal at these three sites. The distal radius was relatively insensitive to PTH treatment or its withdrawal, compared with the other bones. In summary, PTH therapy dramatically improved cancellous bone mass and architecture in both axial and appendicular sites.     

In vivo regulation of apoptosis in metaphyseal trabecular bone of young rats by synthetic human parathyroid hormone (1-34) fragment

Osteoblast differentiation and function can be studied in situ in the metaphysis of growing long bones. Proliferation and apoptosis dominate in the primary spongiosa subjacent to the growth plate, and differentiation and function dominate in the proximal metaphysis. Apoptosis of osteocytes dominates at the termination of the trabeculae in diaphyseal marrow. As parathyroid hormone regulates all phases of osteoblast development, we studied the in vivo regulation by human parathyroid hormone (1-34) (PTH) of apoptosis in bone cells of the distal metaphysis of young male rats. Rats were given PTH at 80 microg/kg per day, once daily, for 1-28 days. Bone cells were defined for flow cytometry as PTH1-receptor-positive (PTH1R(+)) and growth factor-receptor-positive (GFR(+)) cells. Apoptotic cells stained positive for either TdT-mediated dUTP-X nick end labeling (TUNEL) or annexin V (annV(+)) were detected by either flow cytometry or immunohistochemistry. Apoptosis was also assessed at the tissue level by RNAse protection and caspase enzyme activity assays. PTH increased apoptotic osteoblasts in the proliferating zone and apoptotic osteocytes in the terminal trabecular zone, by 40%-60% within 2-6 days of PTH treatment, but values became equivalent to controls after 21-28 days of treatment. This transient increase was confirmed in PTH1R(+), GFR(+) bone cells isolated by flow cytometry. There was no detectable change in the steady-state mRNA levels of selected apoptotic genes. Starting at 3 days, at the tissue level, PTH inhibited activity of caspases, which recognize the DEVD peptide substrate (caspases 2, 3, and/or 7), but not those caspases recognizing LEHD or YVAD peptide sequences. We speculate that the localized and tissue level effects of PTH on apoptosis can be explained on the basis of its anabolic effect on bone. The transient increase in apoptosis in the proliferating zone and terminal trabecular zone may be the result of the increased activation frequency and bone turnover seen with daily PTH treatment. As once-daily PTH increases the number of differentiated osteoblasts, and as these and hematopoietic marrow cells dominate metaphyseal tissue, inhibition of caspase activity may contribute to their prolonged survival, enabling extension of trabecular bone into the diaphyseal marrow to increase bone mass.     

Increased bone formation by intermittent parathyroid hormone administration is due to the stimulation of proliferation and differentiation of osteoprogenitor cells in bone marrow

In order to examine the mechanism of the anabolic effect of parathyroid hormone (PTH) on bone formation, human PTH(1-34) [hPTH(1-34)] (30 μg/kg) was injected subcutaneously to 9-week-old rats 5 times a week for 1 or 3 weeks. Trabecular bone volume (BV/TV) in the tibial metaphysis was not significantly different between the PTH- and vehicletreated groups, but the parameters related to bone formation, including osteoid surface (OS/BS), mineralizing surface (MS/BS), mineral apposition rate (MAR), and bone formation rate (BFR/BS), were significantly increased as early as 1 week after PTH treatment. And the parameters related to bone resorption including eroded surface (ES/BS) and osteoclast number (N.Oc/BS) were also significantly increased as early as 1 week after PTH treatment. Treatment with PTH for 1 week induced no significant increase in bone mineral density at the femoral metaphysis, whereas the same treatment for 3 weeks induced a significant increase. When bone marrow cells isolated from femora and tibiae of either PTH- or vehicle-treated rats were cultured at a high density (2 × 10⁷ cells/one well of 24-multiwell plate), cellular alkaline phosphatase (ALP) activity was significantly increased in the cells isolated from PTH-treated rats compared with vehicletreated rats. When bone marrow cells were cultured at a low density (4 × 10⁶ cells/a one well of 6-multiwell plate) to generate colonies (colony forming unit-fibroblastic, CFU-F), PTH induced apparent increases in both the total number of CFU-F and the number of ALP-positive CFU-F. The ratio of the latter to the former was significantly higher in the PTH-treated group than in the vehicle-treated group. These findings suggest that the anabolic effect of PTH is, at least in part, due to the stimulation of proliferation and differentiation of osteoprogenitor cells in bone marrow.     

Effects of separate and combined therapy with growth hormone and parathyroid hormone on lumbar vertebral bone in aged ovariectomized osteopenic rats

Previous studies have demonstrated that growth hormone (GH) has a marked anabolic effect on cortical bone, and parathyroid hormone (PTH) has been shown to increase cancellous bone markedly and cortical bone to some extent in ovariectomized (ovx) rats. Combined therapies mostly focused on combining a bone anabolic agent with an antiresorptive agent. The following study was carried out to examine the efficacy of combined therapy with GH and PTH, two bone anabolic agents in rebuilding bone after loss due to ovariectomy in lumbar vertebrae, which contain both cortical and cancellous bones. Twelve-month-old female F344 rats were divided into five groups: sham + solvent vehicle, ovx + solvent vehicle, ovx + GH (2.5 mg/kg/day), ovx + PTH (80 microg/kg/day), and ovx + GH (2.5 mg/kg/day) + PTH (80 microg/kg/day). After surgery, animals were left for 4 months to become osteopenic before the beginning of therapy. Hormone administrations were given 5 days per week for 2 months and the animals were killed. The L3 vertebra was removed and examined by pQCT densitometry and by histomorphometry. Compared with age-matched, sham-operated controls, there was a 21% decrease in total bone mineral content (BMC) (p < 0.0001), 17.0% decrease in total bone mineral density (BMD) (p < 0.0001), 25.4% decrease in cortical BMC (p < 0.001), 3.1% decrease in cortical BMD (p < 0.05), 50.5% decrease in cancellous BMC (p < 0.01), 47.3% decrease in cancellous BMD (p < 0.01), and 14.5% decrease in cancellous bone volume (BV/TV) (p < 0.05) in the vehicle-treated ovx rats. Compared with age-matched, vehicle-treated ovx controls, GH, PTH, and GH + PTH increased total BMC by 22.8% (p < 0.001), 32.4% (p < 0.0001), and 72.7% (p < 0.0001), respectively; total BMD by 9.7% (p > 0.05), 22.6% (p < 0.001), and 38.8% (p < 0.0001), respectively; cortical BMC by 28.8% (p < 0.01), 50.8% (p < 0.0001), and 98.4% (p < 0.0001), respectively; and cortical BMD by 4.5% (p < 0.01), 2.9% (p < 0.05), and 6.3% (p < 0.0001), respectively. PTH and GH + PTH significantly increased cancellous BMC by 95.3% (p < 0.01) and 255.8% (p < 0.0001), respectively; cancellous BMD by 77.6% (p < 0.05) and 181% (p < 0.0001), respectively; cancellous BV/TV by 38.6% (p < 0.0001) and 55.9% (p < 0.0001), respectively; and trabecular thickness by 48% (p < 0.0001) and 68.3% (p < 0.0001), respectively. Note that GH by itself had no significant effect on vertebral cancellous BMC, cancellous BMD, and cancellous BV/TV. In conclusion, the effect of PTH was mostly more marked than that of GH. GH acted mainly by increasing cortical bone with less effect on cancellous bone, while PTH acted by increasing both cortical and cancellous bones. Combined therapy with GH and PTH was more effective in rebuilding bone after ovariectomy than either therapy alone. The effects of combined therapy with GH and PTH were additive in vertebral bone in the aged osteopenic rats.     

Indomethacin inhibition of tenotomy-induced bone resorption in rats

Loss of biomechanical function results in rapid bone loss. This study assesses the role of arachidonic acid metabolites in immobilization-related osteopenia. A hind limb of the rat was immobilized by knee tenotomy and bone resorption and formation parameters were quantitated by histological methods in indomethacin-treated (0.5 mg/kg per day) and vehicle-treated animals. Control animals sacrificed 30, 72, and 240 hr post-tenotomy revealed a significant increase in osteoclast number (30 hr) and resorption surfaces (72 hr) and a decrease in trabecular bone volume (240 hr) in the tenotomized tibiae. In the indomethacin-treated tibial metaphysis, no significant differences were noted for these parameters by comparison to the nontenotomized leg. Bone formation parameters remained reduced in the tenotomized legs of both the indomethacin and vehicle-treated groups compared to the control legs. Indomethacin inhibited bone resorption, but did not prevent the decrease in bone formation produced by immobilization over the 10 days of these experiments.     

Bipedal stance exercise enhances antiresorption effects of estrogen and counteracts its inhibitory effect on bone formation in sham and ovariectomized rats

In this study we employed a raised cage model in combination with estrogen to observe their effects on the proximal tibial metaphysis (PTM) and tibial shaft (TX) in sham-operated or ovariectomized rats. A total of 105 6-month-old female Sprague-Dawley rats were used in the study. Bilateral sham ovariectomy or ovariectomy was performed at day 0 and the rats were housed in normal height or raised cages (RCs) and injected subcutaneously twice per week with 10 microg/kg of 17beta-estradiol (E2) or vehicle for 4 and 8 weeks. Because the time course of bone loss or bone gain distribution was not uniform in the metaphyses of the tibia, we subdivided the PTM into three zones (medial, central, and lateral) to observe the different bone loss or bone gain patterns after ovariectomy and/or raised cages. We found that: (1) E2 alone did not alter bone area or architecture in sham rats, whereas RC alone increased trabecular thickness and area of PTM, but had no effects on TX; (2) Ovx induced most bone loss from the central zone of the PTM and endocortical surface of TX, accompanied by decreased trabecular number and increased bone resorption; (3) E2 alone prevented ovx-induced bone loss by preserving trabecular number and depressing bone resorption; (4) RC alone partially compensated for bone loss following ovx by thickening the surviving trabeculae in lateral and medial zones, and tended to stimulate bone formation and decrease bone resorption; and (5) RC plus E2 increased trabecular bone area by having an additive effect on bone resorption and bone turnover. RCs helped to prevent the depressive effect of estrogen on periosteal bone formation. In conclusion, early and rapid bone loss occurred in the central zone of the metaphysis and endocortical surface after ovx. Estrogen replacement therapy prevented this loss. Raised cages partially compensated for bone loss following ovx by thickening the trabeculae in the lateral area of the metaphysis and decreased endocortical erosion. Combination treatment added bone to the PTM and prevented the decrease of periosteal bone formation after estrogen administration.     

A single intravenous administration of zoledronic acid prevents the bone loss and mechanical compromise induced by aromatase inhibition in rats

Recent evidence has demonstrated that long-term estrogen deprivation using aromatase inhibitor therapy in postmenopausal women with breast cancer results in bone loss and increased fracture risk. Bisphosphonates are potent inhibitors of bone resorption and have demonstrated efficacy in preventing bone loss in postmenopausal women with low bone mineral density (BMD) and in patients with breast cancer receiving estrogen deprivation therapy. Therefore, this study investigated the effects of the bisphosphonate zoledronic acid on BMD and bone strength in rats treated with the aromatase inhibitor, letrozole. Peripheral quantitative computed tomography demonstrated that treatment of rats with daily oral letrozole (1 mg/kg) induced significant bone loss and cortical thinning compared with control animals (P < 0.01). A single prior intravenous dose of zoledronic acid dose dependently protected against letrozole-induced bone loss and cortical thinning, with the highest evaluated dose (20 microg/kg) resulting in BMD values that were not significantly different from controls over the 24 weeks of letrozole treatment. Furthermore, biomechanical testing of the distal femoral metaphysis demonstrated that zoledronic acid (20 microg/kg) significantly prevented the decrease in stiffness and elastic modulus induced by letrozole treatment. Taken together, these data support the use of zoledronic acid for the prevention of bone loss in women with breast cancer receiving aromatase inhibitor therapy.     

Low dose parathyroid hormone maintains normal bone formation in adult male rats during rapid weight loss

A persistent negative energy balance results in bone loss. It is not clear whether the bone loss associated with chronic negative energy balance can be prevented. The objective of this study was to assess the efficacy of intermittent low dose parathyroid hormone (PTH) treatment in maintaining normal bone formation during severe energy restriction. Six-month-old male Fisher 344 rats were divided into 4 treatment groups: (1) baseline, (2) ad libitum (ad lib)-fed control, (3) energy-restricted (to consume 40% ad lib caloric intake), or (4) energy-restricted+low dose (1 μg/kg/d) PTH. Severe energy restriction for 14 d decreased body weight and serum leptin levels. Compared to ad lib-fed controls, energy-restricted rats had lower cancellous bone formation, higher osteoclast perimeter/bone perimeter and higher bone marrow adiposity in the proximal tibial metaphysis. Also, the energy-restricted rats had a lower periosteal bone formation rate at the tibia-fibula synostosis. Administration of PTH to energy-restricted rats had no effect on weight loss or osteoclast perimeter/bone perimeter. In contrast, energy-restricted rats treated with PTH had higher rates of cancellous and cortical bone formation compared to energy-restricted rats, and did not differ from the ad lib-fed control animals. Furthermore, PTH treatment maintained normal bone marrow adiposity. In conclusion, rapid weight loss in adult male rats was accompanied by decreased bone formation and increased bone marrow adiposity and these changes were prevented by low dose PTH treatment. Taken together, the results suggest that the energy cost of bone formation in adult rats is low and PTH therapy is effective in preventing the reduced bone formation associated with rapid weight loss.     

Effects of low-dose parathyroid hormone on bone mass, turnover, and ectopic osteoinduction in a rat model for chronic alcohol abuse

Parathyroid hormone (PTH) is used clinically in osteoporotic patients to increase bone mass by enhancing bone formation. PTH therapy is not uniformly effective at all skeletal sites and "life-style" factors may modulate the skeletal response to PTH. Alcohol may represent one of these factors. Chronic alcohol abuse is associated with osteoporosis and impaired fracture healing. Therefore, the present study investigated the effects of alcohol on the bone anabolic response to a dose of PTH similar to a human therapeutic dose 1) during normal cancellous and cortical bone growth and turnover, and 2) in a model of demineralized allogeneic bone matrix (DABM)-induced osteoinduction. Three-month-old male Sprague Dawley rats were fed a Lieber-DeCarli liquid diet with 35% of the calories derived from ethanol. The controls were pair-fed an alcohol-free isocaloric diet containing maltose-dextran. Following adaptation to the liquid diets, the rats were implanted subcutaneously with DABM cylinders prepared from cortical bone of rats fed normal chow. The rats were subsequently treated daily with PTH (1 microg/kg/d sc, 5 d/week) or vehicle and measurements on bone and DABM implants performed 6 weeks later. Total bone mass was evaluated on the day of necropsy using DXA. Tibiae were processed for histomorphometry. Bone mass and architecture in tibial diaphysis and DABM implants were evaluated by muCT. PTH treatment increased whole body bone mineral content (BMC) and bone mineral density (BMD). The hormone also increased bone formation and bone area/tissue area in the proximal tibial metaphysis. In contrast, PTH treatment had no effect on periosteal bone formation and minimal effects on DABM-induced osteoinduction. Alcohol consumption decreased whole body BMC. Alcohol also decreased cancellous as well as cortical bone formation and bone mass in tibia and impaired DABM-mediated osteoinduction. There was no interaction between PTH treatment and alcohol consumption for any of the endpoints evaluated. Our results indicate that the bone anabolic response to a therapeutic dose of PTH in the rat is largely confined to cancellous bone. In contrast, alcohol consumption inhibits bone formation at all sites. Furthermore, alcohol inhibits osteoinduction and reduces periosteal and cancellous bone formation, irrespective of therapeutic PTH administration. Based on the animal model, our findings suggest that alcohol consumption could impair the beneficial effects of PTH therapy in osteoporosis.     

Anabolic and catabolic bone effects of human parathyroid hormone (1-34) are predicted by duration of hormone exposure

Parathyroid hormone (PTH)(1-34), given once daily, increases bone mass in a variety of animal models and humans with osteoporosis. However, continuous PTH infusion has been shown to cause bone loss. To determine the pharmacokinetic profile of PTH(1-34) associated with anabolic and catabolic bone responses, PTH(1-34) pharmacokinetic and serum biochemical profiles were evaluated in young male rats using dosing regimens that resulted in either gain or loss of bone mass. Once-daily PTH(1-34) or 6 PTH(1-34) injections within 1 h, for a total daily dose of 80 microg/kg, induced equivalent increases in proximal tibia bone mass. In contrast, 6 PTH(1-34) injections/day over 6 h for a total dose of 80 microg/kg/day or 3 injections/day over 8 h for a total of 240 microg/kg/day decreased tibia bone mass. The PTH(1-34) pharmacokinetics of the different treatment regimens were distinctive. The magnitude of the maximum serum concentrations (Cmax) of PTH(1-34) and area under the curve (AUC) did not predict the catabolic bone outcome. Compared to the anabolic pharmacokinetic profile of a transient increase in PTH(1-34) with rapid decreases in serum calcium and phosphate, the catabolic regimen was associated with PTH(1-34) concentrations remaining above baseline values during the entire 6-h dosing period with a trend toward an increase in serum calcium and a prolonged decrease in phosphate. The pharmacokinetic profiles suggest that the anabolic or catabolic response of bone to PTH(1-34) is determined primarily by the length of time each day that serum concentrations of PTH(1-34) remain above baseline levels of endogenous PTH and only secondarily by the Cmax or AUC of PTH(1-34) achieved.     

Short-Term effects of high dose estrogen on tibiae of growing male rats

The short-term effects of estrogen at a single high dose (4 mg/kg body weight/day for 14 days) were determined on tibiae in the normal (noncastrate) growing male rat. In cortical periosteal bone, at a middiaphyseal site devoid of resorbing activity, estrogen suppressed periosteal bone formation and apposition rates, resulting in a smaller cross-sectional area. In middiaphyseal endocortical bone, estrogen had no effect on apposition and formation rates and, because medullary area was unchanged, probably had no effect on endocortical bone resorption. In the proximal tibial metaphysis, estrogen greatly suppressed longitudinal growth rate. In a site within the metaphysis adjusted for the effects of growth, cancellous mineral apposition was greatly reduced by the hormone. Estrogen-treated rats retained more of a fluorochrome label deposited in cancellous bone at the beginning of the study than vehicle-treated animals, indicating a reduced net bone loss. As a result of the lowered resorption induced by estrogen, cancellous bone mass (area and perimeter) were both significantly higher in estrogen-treated rats. No evidence was found for an anabolic action of the hormone in the male rat; indeed, estrogen reduced indices of bone formation. Received: 31 December 1995 / Accepted: 3 May 1996