Effects of ageing on the biomechanical properties of rat articular cartilage.

Previous studies have demonstrated that male Sprague Dawley (SD) rats experience age-related bone loss with the same characteristics as that in ageing men. As articular cartilage, like bone, is a critical component of the health and function of the musculoskeletal system, the authors hypothesized that articular cartilage in the untreated male SD rats could be a suitable model for studying the age-related deterioration of articular cartilage in men. To test this hypothesis, male SD rats were killed at between 6 and 27 months. The right femur of each rat was removed. The effects of ageing on the structural integrity of the distal femoral articular cartilage were studied by biomechanical testing with a creep indentation apparatus. The aggregate modulus, Poisson's ratio, permeability, thickness, and percentage recovery of articular cartilage were determined using finite element/non-linear optimization modelling. No significant differences were observed in these biomechanical properties of the distal femoral articular cartilage as a function of age. Therefore, untreated male SD rats appear to be unsuitable for studying the age-related changes of articular cartilage as they occur in men. However, and more intriguingly, it is also possible that ageing does not affect the biomechanical properties of articular cartilage in the absence of cartilage pathology.

Effects of increased muscle mass on bone in male mice overexpressing IGF-I in skeletal muscles.

It has been hypothesized that increase in muscle mass increases the strain on bone resulting in increase in bone mass. The aim of the present study was to determine the effects of increased muscle mass on bone. A colony of transgenic mice that overexpress hIGF-I in muscle, resulting in larger muscles, was established. Six-month-old heterozygous and wild type males were used in this study. The tibial diaphysis, femoral diaphysis and distal femoral metaphysis were analyzed using pQCT densitometry. Heterozygous animals had significantly higher body weight, muscle weight and muscle area when compared with wild type animals. Tibia and femur of the heterozygous mice had significantly higher weights and lengths. The tibial and femoral diaphyses of heterozygous animals had significantly higher cortical bone area, cortical bone mineral content, cortical bone mineral density, cortical thickness and periosteal perimeter when compared with wild type animals. In the distal femoral metaphysis, the total bone area and the cancellous bone area of heterozygous mice were significantly higher than those of wild type animals. In conclusion, increased muscle mass was associated with bigger bones in animals overexpressing IGF-I. Only pure cortical bone increased in both area and mineral content in these animals; cancellous bone, however, increased only in area and not in mineral content and density.

Rodent model for investigating the effects of estrogen on bone and muscle relationship during growth.

It has been reported that in humans from about 11-12 years of age, bone mass begins to increase faster in girls than in boys with the same muscle mass, and by 14-15 years of age, bone mass per unit mass of muscle was found to be significantly higher in girls than in boys. Because around 15 years is the beginning of reproductive age in women, it was suggested that estrogen was involved in the higher bone mass in women during puberty. The present study was undertaken to determine if bone mass per unit muscle mass is higher in female than in male Sprague Dawley (SD) rats during growth, as has been reported in humans during growth and consequently, whether these SD rats are suitable for studying the musculoskeletal effects of estrogen, as may occur in humans during growth. L-4 vertebra of female and male SD rats aged 1-6 months were studied using peripheral quantitative computed tomography (pQCT). Muscle cross-sectional area was measured as a surrogate for muscle mass and bone mineral content (BMC) was measured as a surrogate for bone mass. From 1 to 6 months of age, total BMC, cortical BMC, and cancellous BMC increased faster in females than in males with similar muscle area, and at 3 and 6 months of age, the above vertebral indices of bone mass were significantly higher in female than in male rats. Since one of the main differences between female and male rats is the level of serum estrogen, the higher bone mass per unit muscle area seen at the L-4 vertebra in these female SD rats is similar to what has been reported in humans during puberty when serum estrogen level is high in females. The findings from this study indicate that female and male SD rats aged 1-6 months can be used as appropriate model for studying the effects of serum estrogen on the skeletal response of voluntary muscle forces, as has been reported in humans during growth.

Effects of cerivastatin and parathyroid hormone on the lumbar vertebra of aging male Sprague-Dawley rats.

Both men and women lose bone at a late age (aging bone loss). The aim of this study was to determine whether cerivastatin and parathyroid hormone (PTH) can prevent aging bone loss in men. Bone loss in aged male Sprague-Dawley (SD) rats was used as a model for age-related bone loss in men. Nine-month-old male SD rats were divided into six groups: (1) baseline controls (killed at the beginning of the study); (2) age-matched controls; (3) parathyroid hormone (PTH; 80 microg/kg body weight per day for 5 days/week) treated; (4) low-dose cerivastatin (0.2 mg/kg body weight per day) treated; and (5) medium-dose cerivastatin (0.4 mg/kg body weight per day) treated; and (6) high-dose cerivastatin (0.8 mg/kg body weight per day) treated. Groups 2-6 were treated for 23 weeks between the ages of 9 and 15 months and killed at the end of 23 weeks. The fourth lumbar vertebra was analyzed using peripheral quantitative computed tomography (pQCT). It is shown that age-matched controls had decreased cancellous bone mineral content (Cn. BMC) by 19% (p < 0.05) and cancellous bone mineral density Cn. BMD) by 22% (p < 0.01) when compared with baseline controls. All three doses of cerivastatin resulted in lower Cn. BMC and Cn. BMD when compared with age-matched controls, but this decrease was not statistically significant. In the PTH-treated group, Cn. BMC increased by 5% (p < 0.0001) and Cn. BMD increased by 37% (p < 0.0001) when compared with age-matched controls. In age-matched controls, cortical bone mineral content (Ct. BMC) and cortical bone mineral density (Ct. BMD) decreased slightly, but not significantly, when compared with baseline controls. Ct. BMD did not change significantly at any of the three doses in the cerivastatin-treated groups. In the PTH-treated group, Ct. BMC increased by 23% (p < 0.0001) when compared with age-matched controls. We confirmed that male SD rats lose bone with aging in the lumbar vertebra, and it is concluded that cerivastatin, at all doses administered, did not prevent this age-related bone loss. In contrast, PTH prevented age-related bone loss in the vertebra of male SD rats.

Age-related changes in bone mineral content and density in intact male F344 rats.

This study was undertaken to determine whether age-related bone loss occurs in intact male F344 rats. Bone loss was assessed in male F344 rats aged 3 to 27 months by scanning different bones using peripheral quantitative computed tomography (pQCT) densitometry. Cancellous and cortical bones were analyzed at the vertebra, proximal tibial metaphysis (PTM), and the neck of the femur. Cortical bone was also analyzed at the tibial and femoral diaphysis and at the tibio-fibula junction. In the vertebra, cancellous bone mineral content (Cn. BMC) did not change significantly with age. Cancellous bone mineral density (Cn. BMD) gradually decreased from 9 months onwards; and at 27 months of age, there was a 29% (p < 0.0001) decrease, when compared with 9-month-old animals. No significant change was observed in cortical bone mineral content (Ct. BMC) and cortical bone mineral density (Ct. BMD) with age. In the PTM, bone loss started to occur after 18 months of age. At 27 months of age, Cn. BMC decreased by 58% (p < 0.0001) and Cn. BMD also decreased by 58% (p < 0.0001). Ct. BMC decreased by 28% (p < 0.0001) in 27-month-old animals, whereas Ct. BMD was not affected by aging. At the tibio-fibula junction, Ct. BMC and Ct. BMD decreased after 18 months of age. At 27 months, Ct. BMC and Ct. BMD had decreased by 8% (p < 0.001) and 3% (p < 0.0001), respectively. Ct. BMC in the tibial diaphysis did not change significantly with age, whereas Ct. BMD decreased by 1% (p < 0.05) at 27 months. In the neck of the femur, Cn. BMC increased up to 24 months of age. Cn. BMD increased up to 18 months of age and decreased by 9% (p < 0.05) at 24 months and 11% (p < 0.001) at 27 months of age when compared with 18-month-old animals. Ct. BMC and Ct. BMD increased with age. In conclusion, although some components of the PTM decreased appreciably with age, in this study, most of the bone parameters analyzed either increased or did not change significantly with age. We conclude that unlike male Sprague Dawley rats, male F344 rats appear not to be a good model for studying age-related bone loss as occurs in aging men.

Separate and combined effects of growth hormone and parathyroid hormone on cortical bone osteopenia in ovariectomized aged rats.

The focus of this study is on whether cortical osteopenia occurs in ovariectomized aged female rats, and if so, whether growth hormone (GH) and parathyroid hormone (PTH) independently or together (GH+PTH) can rebuild the lost cortical bone. Tibio-fibula junction was analyzed by histomorphometry and peripheral quantitative computerized tomography (pQCT) densitometry. Significant loss of cortical bone area (Ct. BAr), cortical bone mineral content (Ct. BMC), cortical thickness (Ct. Th) and increase of endocortical perimeter occurred 4 months after ovariectomy. The rats were given GH, PTH, GH+PTH or vehicle for 2 months and sacrificed. GH, PTH and GH+PTH increased Ct. BAr, Ct. BMC, Ct. Th, periosteal perimeter, periosteal double-labeled perimeter, mineral apposition rate, and bone formation rate, but decreased marrow area. PTH and GH+PTH decreased endocortical perimeter, and increased endocortical double labeled perimeter and bone formation rate. In conclusion, ovariectomy induced cortical bone loss in aged rats by increasing endocortical bone resorption. Growth hormone increased periosteal bone formation, while PTH stimulated endocortical bone formation and in combination GH+PTH produced complementary effects thereby reversing osteopenia.

Male rodent model of age-related bone loss in men.

Osteoporosis is a common occurrence in aging men. There is currently no appropriate animal model for studying age-related bone loss in men. To determine whether male Sprague-Dawley (SD) rats experience bone loss with aging and whether this rodent model is appropriate for studying age-related bone loss in men, SD rats aged 1-27 months were examined at the L-4 vertebra, the left femoral neck, and the left proximal tibia using peripheral quantitative computed tomography (pQCT) densitometry. In the L-4 vertebra of the male SD rats, cortical bone mineral content (BMC), cortical bone mineral density (BMD), and cortical bone thickness (Ct.Th) increased to a maximum at about 4 months of age and then plateaued. Vertebral cortical BMC began to decrease after about 13 months and vertebral Ct.Th began to decrease after about 9 months. By 27 months of age, vertebral cortical BMC decreased by 26.1% (p < 0.0001) and vertebral Ct.Th decreased by 31% (p < 0.0001). Vertebral cancellous BMC and vertebral cancellous BMD increased to a maximum at about 3 months of age and then declined progressively with aging after a short plateau. From 3 to 27 months of age, vertebral cancellous BMC and vertebral cancellous BMD had decreased linearly by 35.4% (p < 0.0001) and 49.4% (p < 0.0001), respectively. Both vertebral periosteal and vertebral endocortical perimeters of the L-4 vertebra of the rats increased with aging. From 9 to 27 months of age, the percent increase of vertebral endocortical perimeter (19.8%, p < 0.0001) was higher than that of vertebral periosteal perimeter (7.4%, p < 0.0001). This process was associated with a decrease with aging in vertebral Ct.Th. In addition, cancellous bone in the femoral neck and the proximal tibia began to be lost at 9 months of age and, by 27 months of age, cancellous BMC and cancellous BMD decreased by 59.7% (p < 0.0001) and 58.4% (p < 0.0001), respectively, in the femoral neck and by 72.2% (p < 0.0001) and 71.4% (p < 0.0001), respectively, in the proximal tibia. To gain further insight into the effects of aging on cancellous bone in the L-4 vertebra, histomorphometry was done on the L-4 vertebral body of animals aged 3, 6, 9, 18, and 24 months after pQCT densitometry. From 3 months of age and thereafter, cancellous bone volume (BV/TV) decreased progressively and, by 24 months, there was a decrease of 35.7% (p < 0.0001). In the L-4 vertebra, single- and double-labeled surfaces, mineral apposition rate (MAR), and bone formation rate (BFR/BS) decreased with aging. In conclusion, age-related bone loss in male SD rats started mostly from 9 months of age when bone growth had been completed. Aging male SD rats experience bone loss comparable to that seen in men. Thus, male SD rats represent an appropriate animal model of age-related bone loss in men. We recommend using male SD rats that are 9 months old as the starting age for age-related bone loss. We also suggest using the L-4 vertebra and femoral neck as the clinically relevant bone sites for determining the cause of the loss of bone, and how and whether therapeutic agents could modulate age-related bone loss in men.

Analysis of the effects of growth hormone, exercise and food restriction on cancellous bone in different bone sites in middle-aged female rats.

The aim of this study is to determine the effects of growth hormone (GH), exercise (EX), GH+EX and food restriction on cancellous bone in middle-aged female rats. Female F344 rats aged 13 months were divided into (1) age-matched controls; (2) GH treated (2.5 mg/kg. 5 day/week); (3) EX (voluntary wheel running); (4) GH+EX; and (5) food restricted (FR) (fed 60% of the ad libitum food intake). The animals were treated for 18 weeks, at the end of which they were sacrificed. Cancellous bone and cortical bone in the fourth lumbar vertebra, proximal tibial metaphysis (PTM), distal femoral metaphysis (DFM) and femoral neck (NF) were analyzed using peripheral quantitative computerized tomography (pQCT) densitometry. Growth hormone increased cancellous bone area, cancellous bone mineral content, cortical bone area and cortical bone mineral content in the vertebra, PTM, DFM and NF. The tibial muscle wet weight was increased significantly after GH treatment. Exercise increased the cancellous bone area in the vertebra, PTM and DFM. Cortical bone area and cortical bone mineral content increased after EX in the vertebra, PTM, DFM and NF. No significant change was seen in the tibial muscle wet weight after EX. Growth hormone+EX increased cancellous bone area in the vertebra PTM and DFM but had no effect in neck of the femur. Cancellous bone mineral content, cortical bone area and cortical bone mineral content increased with GH+EX in the vertebra, PTM, DFM and NF. The tibial muscle wet weight was increased significantly with GH+EX. Food restriction decreased cancellous bone area and cancellous bone mineral content in all the bones studied. The decrease was statistically significant only at the distal femoral metaphysis. The tibial muscle wet weight decreased when compared with the age-matched control, but this decrease was not statistically significant. We conclude that the effect of the dose of GH used and the levels of voluntary wheel running EX used increased cancellous bone in intact rats; the effect of GH is much greater and different bones respond with varying intensities. The effects of combined treatment of GH and EX on cancellous bone are not always significantly higher than those of GH alone. FR at the level studied has a mostly negative effect on cancellous bone.

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.

Bone anabolic effects of separate and combined therapy with growth hormone and parathyroid hormone on femoral neck 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 and cortical bone markedly in ovariectomized (OVX) rats. Most previous combination therapies used the bone anabolic agent (PTH) and the anti-resorptive agents. In this study, two bone anabolic hormones, GH and PTH, were used in rebuilding bone following loss due to ovariectomy in the femoral neck, which contains 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). Following surgery, the animals were left for 4 months to become osteopenic before the beginning of hormone therapies. Hormone administrations were given 5 days per week for 2 months and the animals sacrificed. The right femurs were removed and the femoral necks were examined by pQCT densitometry and by histomorphometry. There was a 12.3% decrease in total bone mineral content (BMC) (P<0.01), a 6.2% decrease in total bone mineral density (BMD) (P<0.01), a 12.8% decrease in cortical BMC (P<0.05), a 25.9% decrease in cancellous BMC (P<0.0001), a 20.4% decrease in cancellous BMD (P<0.01), and a 34.2% decrease in cancellous bone volume (BV/TV) (P<0.0001) in vehicle-treated OVX rats. Growth hormone, PTH and GH+PTH treatment increased total BMC of the OVX rats by 14.4% (P<0.01), 23.5% (P<0.0001) and 30.6% (P<0.0001), respectively; increased total BMD by 7.0% (P<0.01), 9% (P<0.001) and 14.8% (P<0.0001), respectively; increased cortical BMC by 15.9% (P<0.05), 25.5% (P<0.001) and 29% (P<0.001), respectively; increased cancellous BMC by 40.9% (P<0.0001), 61.9% (P<0.0001) and 86.8% (P<0.0001), respectively; increased cancellous BMD by 31% (P<0.001), 41.8% (P<0.0001) and 61.8% (P<0.0001), respectively; increased cancellous BV/TV by 30.6% (P<0.05), 76.3% (P<0.0001) and 94.9% (P<0.0001), respectively; and increased trabecular thickness by 26.4% (P<0.05), 41.5% (P<0.001) and 43.2% (P<0.001), respectively, compared to the age-matched vehicle-treated OVX controls. In conclusion, both GH and PTH increased cortical and cancellous bone mass at the osteopenic femoral neck. Using two techniques, it was observed that the effects of PTH were mostly more marked than those of GH. Combined therapy with GH+PTH was more effective in rebuilding cortical bone and cancellous bone than either therapy alone in the aged ovariectomized osteopenic rats, which is in line with our hypothesis.

Parathyroid hormone and growth hormone have additive or synergetic effect when used as intervention treatment in ovariectomized rats with established osteopenia.

The severely osteoporotic human skeleton is characterized by thin cortices and a very fragile cancellous framework. To increase the biomechanical competence of such a skeleton, powerful anabolic agents are needed. The aim of the present study was to compare the effect of parathyroid hormone (PTH), growth hormone (GH) and combination treatment with PTH and GH in an aged, rat model with established osteopenia. Furthermore, envelope- and site-specific effects of the two agents are described. Twelve-month-old virgin F344 rats were divided into six groups with 11 animals per group: (1) baseline; (2) sham-operated + solvent vehicle (s.v.) (sham); (3) ovariectomized + s.v. (ovx); (4) ovx + GH 2.5 mg/kg body weight per day; (5) ovx + PTH 80 microg/kg body weight per day; and (6) ovx + GH and PTH treatment. Group 1 were killed to establish baseline values. Groups 2 (sham) and 3 (ovx) were killed after 24 weeks. Groups 4, 5, and 6 were allowed to develop osteopenia for 16 weeks before treatment was initiated. Treatment was given for a period of 8 weeks. The effects of GH, PTH, and GH + PTH cotherapy were measured by biomechanical testing at four different skeletal sites: lumbar vertebra; femoral diaphysis; femoral neck; and distal femoral metaphysis. In addition, static histomorphometry was performed at the middiaphyseal region. Ovx induced a loss of bone strength at all sites, but this was significant only at the femoral diaphysis and distal metaphysis. GH could reverse the loss of strength at the diaphysis, but not at the metaphysis. PTH, on the other hand, reversed the loss of strength to values significantly over ovx at all four sites. At the metaphysis, PTH monotherapy increased strength to above sham levels. However, GH + PTH cotherapy showed additive or synergistic effects at the four tested sites, leading to strength values significantly over sham at all these sites. Static histomorphometry showed that GH exerted its main effect on the periosteal envelope and PTH on the endocortical envelope; for this reason, the GH + PTH combination treatment had an additive or synergistic effect. We conclude that GH and PTH have a very pronounced anabolic effect when given in cotherapy. Therefore, this treatment regime seems promising in the clinical situation for management of patients with severe, established osteoporosis.

How cancellous and cortical bones adapt to loading and growth hormone.

There is great interest in the relationships between growth hormone (GH), muscle loading and bone, in part, because GH increases muscle mass which provides the largest signals that control bone modeling and remodeling. This study was designed to examine the effects of GH and muscle loading by exercise (EX) independently and in combination on bone and skeletal muscle. Thirteen-month-old female F344 rats were divided into 6 groups: Group 1, baseline controls (B); Group 2, agematched controls (C); Group 3, GH treated (2.5 mg rhGH/kg b. wt/day, 5 days per week); Group 4, voluntary wheel running exercise (EX); Group 5, GH+EX, and rats in Group 6 were food restricted (FR) to lower their body weight and examine the effects of decreased muscle load on bone. All animals, except the baseline controls, were sacrificed after 4.5 months. Growth hormone increased the body weight and tibial muscle mass of the rats markedly, while EX caused a slight decrease in body weight and partially inhibited the increase caused by GH in the GH+EX group. Food restriction greatly decreased body weight below that of age-matched controls but neither FR nor EX had a significant effect on the mass of the muscles around the tibia. Growth hormone and EX independently increased tibial diaphyseal cortical bone area (p<0.0001), cortical thickness (p<0.0001), cortical bone mineral content (p<0.0001), periosteal perimeter (p<0.0001) and bone strength-strain index (SSI) (p<0.0001). The effects of GH were more marked, and the combination of GH and EX produced additive effects on many of the tibial diaphyseal parameters including bone SSI. GH+EX, but not GH or EX alone caused a significant increase in endocortical perimeter (p<0.0001). In the FR rats, cortical bone area and cortical mineral content increased above the baseline level (p<0.0001) but were below the levels for age-matched controls (p<0.0001). In addition, marrow area, endocortical perimeter and endocortical bone formation rate increased significantly in the FR rats (p<0.01, p<0.0001, p<0.0001). Three-point bending test of right tibial diaphysis resulted in maximum force (Fmax) values that reflected the group differences in indices of tibial diaphyseal bone mass except that GH+EX did not produce additive effect on Fmax. The latter showed good correlation with left tibial diaphyseal SSI (r=0.857, p<0.0001) and both indices of bone strength correlated well with tibial muscle mass (r=0.771, Fmax; r=0.700, SSI; p<0.0001). We conclude that the bone anabolic effects of GH with or without EX may relate, in part, to increased load on bone from tibial muscles and body weight, which were increased by the hormone. The osteogenic effects of EX with or without GH may relate, in part, to increased frequency of muscle load on bone as EX decreased body weight (p<0.05) but had no significant effect on tibial muscle mass. The enhanced loss of endocortical bone by FR may relate, in part, to decreased load on bone due to low body weight (p<0.0001) as FR did not cause a significant decrease in tibial muscle mass (p=0.357). The roles of humoral and local factors in the bone changes observed remain to be established.

Strain differences in bone density and calcium metabolism between C3H/HeJ and C57BL/6J mice.

Previous reports indicate that peak bone density is significantly higher in C3H/HeJ (C3H) than in C57BL/6J (C57BL) mice, making these two inbred strains useful models for studying the genetic basis for peak bone density. The following study was undertaken to examine whether strain differences in the bone density of C3H and C57BL mice are associated with differences in intestinal calcium (Ca) absorption. Calcium absorption was measured by the balance technique and animals received two injections of fluorochromes 5 days apart before killing. Subsequently, the femurs were removed and, following measurement of volumetric density, the left femur was divided into three equal parts and the middle third served as the femoral cortical diaphysis. Femur diaphyseal volumetric bone density, ash, and Ca content were 10%, 29%, and 29% higher in C3H than in C57BL mice (p < 0.001), respectively. Bone length, periosteal mineral apposition rate, and periosteal bone formation rate of femoral diaphyseal cortical bone were not significantly different between the two strains of mice, but the marrow area of C57BL mice was almost twofold that of C3H mice (p < 0.0001). Intestinal Ca absorption and 1,25-dihydroxyvitamin D [1,25(OH)2D]-stimulated Ca2+ uptake by intestinal mucosal cells were 38% and 51% higher in C3H than in C57BL mice p < 0.001), respectively. Serum Ca and 1,25(OH)2D levels were 6% and 32% higher in C3H than in C57BL mice (p < 0.001), respectively, and the number of intestinal-occupied vitamin D receptors was 51% higher in C3H than in C57BL mice (p < 0.01). In a second experiment, three groups of C3H mice and three groups of C57BL mice were fed diets that contained 0.4%, 0.1%, or 0.02% Ca, and serum Ca, 1,25(OH)2D, parathyroid hormone (PTH), and intestinal Ca absorption measured. At all dietary Ca levels, C3H mice maintained positive Ca absorption and absorbed significantly more Ca than C57BL mice. In contrast, at low dietary Ca levels (0.1% and 0.02% Ca), C57BL mice maintained negative Ca absorption. Low dietary Ca increased serum PTH significantly in C57BL but not in C3H mice, and decreased serum 1,25(OH)2D and Ca levels in both strains of mice. Our findings indicate that the C57BL mice relied more on the mobilization of Ca from bone to maintain extracellular Ca homeostasis than the C3H mice. We conclude that strain differences in bone mass and density between C3H and C57BL mice is expressed, in part, through the vitamin D and PTH endocrine systems and their effects on the maintenance of extracellular Ca homeostasis.

Analysis of the effects of growth hormone, voluntary exercise, and food restriction on diaphyseal bone in female F344 rats.

The aim of this study is to examine the effects of growth hormone, exercise, and weight loss due to food restriction on tibial diaphyseal bone and on tibial muscle mass. Thirteen-month-old female F344 rats were divided into six groups: group 1, baseline controls (B); group 2, age-matched controls (C); group 3, GH treated (GH); group 4, voluntary wheel running exercise (EX); group 5, GH + EX; and group 6, food restricted (FR). The dose of GH was 2.5 mg recombinant human (rh) GH/kg body weight/day, 5 days per week, given in two divided doses of 1.25 mg at 9-10 A.M. and 4-5 P.M. Food-restricted rats were fed 60% of the mean food intake of the age-matched controls. All animals except the baseline controls were killed after 4.5 months. The baseline controls were killed at the beginning of the study. Growth hormone increased the body weight and tibial muscle mass of the rats markedly, while EX caused only a slight decrease in body weight and partially inhibited the increase caused by GH in the GH + EX group. Food restriction greatly decreased body weight below that of age-matched controls, but neither FR nor EX had a significant effect on the mass of the muscles around the tibia. Growth hormone and EX independently increased tibial diaphyseal cortical bone area (p < 0.0001, p < 0.0001), cortical thickness (p < 0.0001, p < 0.0001), cortical bone mineral content (p < 0.0001, p < 0.0001), periosteal perimeter (p < 0.0001, p < 0.0001), and bone strength-strain index (SSI) (p < 0.0001, p < 0.0001). The effects of GH were more marked and resulted in a greater increase in the weight of the mid tibial diaphysis (p < 0.0001). The combination of GH and EX produced additive effects on many of the tibial diaphyseal parameters, including bone SSI. GH + EX, but not GH or EX alone, caused a significant increase in endocortical perimeter (p < 0.0001). In the FR rats, cortical bone area and cortical mineral content increased above the baseline level (p < 0.001, p < 0.0001) but were below the levels for age-matched controls (p < 0.0001, p < 0.0001). In addition, marrow area, endocortical perimeter, and endocortical bone formation rate increased significantly in the FR rats (p < 0.01, p < 0.0001, p < 0.0001). Three-point bending test of right tibial diaphysis resulted in maximum force (Fmax) values that reflected the group differences in indices of tibial diaphyseal bone mass, except that GH + EX did not produce additive effect on Fmax. The latter showed good correlation with left tibial diaphyseal SSI (r = 0.857, p < 0.0001), and both indices of bone strength correlated well with tibial muscle mass (r = 0.771, Fmax; r = 0.700, SSI; p < 0.0001). GH increased serum IGF-I (p < 0.0001), and the increase was partially reduced by EX. Serum osteocalcin was increased by GH with or without EX (p < 0.01, p < 0.01), and FR or EX alone did not alter serum IGF-I and osteocalcin levels. The bone anabolic effects of GH with or without EX may relate, in part, to increased load on bone from tibial muscles and body weight, which were increased by the hormone. The osteogenic effect of EX with or without GH may relate, in part, to increased frequency of muscle load on bone as EX decreased body weight (p < 0.05), but had no significant effect on tibial muscle mass. The enhanced loss of endocortical bone by FR may relate, in part, to decreased load on bone due to low body weight (p < 0.0001), as FR did not cause a significant decrease in tibial muscle mass (p = 0.357). The roles of humoral and local factors in the bone changes observed remain to be established.

Calcium absorption and bone loss in ovariectomized rats fed varying levels of dietary calcium.

The following studies were undertaken to examine whether estrogen deficiency impairs calcium absorption in aged rats, and to determine whether impaired calcium absorption and the level of dietary calcium are related to the degree of bone loss due to estrogen deficiency. Sixty rats were sham operated (Sham) or ovariectomized (Ovx) to make them estrogen deficient and divided into three dietary groups of 10 rats per group: Group 1 (Sham) and Group 2 (Ovx) were maintained on a diet containing 0.5% calcium; Group 3 (Sham) and Group 4 (Ovx) were maintained on a diet containing 0.1% calcium; Group 5 (Sham) and Group 6 (Ovx) were maintained on a diet containing 0.02% calcium. Calcium absorption was measured in all animals at the beginning of the study and 2 weeks, 1 month, 2 months, and 3 months following surgery, then the animals were sacrificed. In Ovx rats fed 0.5% Ca diet, calcium absorption decreased progressively and the decrease became statistically significant 8 and 12 weeks following ovariectomy (P < 0.05). A similar ovariectomy-related impairment of calcium absorption was not observed in animals fed diets with lower calcium content, making the Ovx rat a tenuous model of intestinal calcium malabsorption. Low dietary calcium decreased cancellous bone mineral content and density at the proximal tibial metaphysis and the decrease was augmented by ovariectomy. The degree of osteopenia due to ovariectomy was not related to the level of dietary calcium or the efficiency of calcium absorption.

The effects of gonadectomy on bone size, mass, and volumetric density in growing rats are gender-, site-, and growth hormone-specific.

Peak volumetric bone mineral density (BMD) is determined by the growth in bone size relative to the mineral accrued within its periosteal envelope. Thus, reduced peak volumetric BMD may be the result of reduced mineral accrual relative to growth in bone size. Because sex steroids and growth hormone (GH) influence bone size and mass we asked: What are the effects of gonadectomy (Gx) on bone size, bone mineral content (BMC), areal and volumetric BMD in growing male and female rats? Does GH deficiency (GH-) reduce the amount of bone in the (smaller) bone, i.e., reduce volumetric BMD? Does GH- alter the effect of Gx on bone size and mineral accrual? Gx or sham surgery was performed at 6 weeks in GH- and GH replete (GH+) Fisher 344 male and female rats. Changes in bone size, volume, BMC, areal and volumetric BMD, measured using dual X-ray absorptiometry (DPX-L), were expressed as percentage of controls at 8 months (mean +/- SEM). All results shown were significant (p < 0.05 level) unless otherwise stated. In GH+ and GH- males, respectively, Gx was associated with: lower femur volume (24%, 25%), BMC (43%, 45%), areal BMD (21%, 14%), and volumetric BMD (30%, 28%); lower spine (L1-L3) volume (26%, 28%), BMC (26%, 30%), and areal BMD (28%, 12%), but not volumetric BMD. Following Gx, GH+ females had increased femur volume (11%), no effect on BMC, decreased areal BMD (6%) and decreased volumetric BMD (17%); GH- females had no change in femur volume, but decreased femur BMC (24%), areal BMD (10%), and volumetric BMD (25%). In GH+ and GH- females, respectively, Gx was associated with a decrease in spine (L1-L3) BMC (12%, 15%), areal BMD (16%, 15%), and volumetric BMD (10%, 16%) with no change in volume. Deficits in non-Gx GH- relative to non-Gx GH+ (males, females, respectively) were: femur BMC (49%, 37%), areal BMD (23%, 8%), volume (19%, 19%) and volumetric BMD (37%, 22%); spine (L1-L3) BMC (46%, 42%), areal BMD (37%, 43%), volume (10%, 15%), and volumetric BMD (40%, 33%). Testosterone and GH are growth promoting in growing male rats, producing independent effects on bone size and mass; deficiency produced smaller appendicular bones with reduced volumetric BMD because deficits in mass were greater than deficits in size. At the spine, the reduction in size and accrual were proportional, resulting in a smaller bone with normal volumetric BMD. In growing female rats, estrogen was growth limiting at appendicular sites; deficiency resulted in a GH-dependent increase in appendicular size, relatively reduced accrual, and so, reduced volumetric BMD in a bigger bone. At the spine, accrual was reduced while growth in size was normal, thus volumetric BMD was reduced in the normal sized bone. Understanding the pathogenesis of low volumetric BMD requires the study of the differing relative growth in size and mass of the axial and appendicular skeleton in the male and female and the regulators of the growth of these traits.

Ovariectomized murine model of postmenopausal calcium malabsorption.

Two experiments were carried out to examine whether the ovariectomized mouse is a potential in vivo model of intestinal calcium malabsorption as occurs in postmenopausal osteoporosis. In the first experiment, we compared the effects of ovariectomy and 17 beta-estradiol (E2) therapy on calcium absorption in C3H/HeJ (C3H) and C57BL/6J (C57BL) mice, which have high and low peak bone mass, respectively. For each strain of mice, three groups were studied: sham operated, ovariectomized, (OVX), and OVX + E2 (60 micrograms/kg of body weight [bw]/day). Therapy was continued for 35 days and calcium absorption measured. In the C3H mice, ovariectomy caused an increase in fecal calcium (17.6%), and a decrease in the amount (12.8%) and percentage (12.5%) of calcium absorbed. The decrease was prevented by E2 therapy, but the differences in the calcium absorption parameters among the groups were not statistically significant. In contrast, in the C57BL mice, ovariectomy caused a marked increase in fecal calcium (84.5%, p < 0.01), and a marked decrease in the amount (55%, p < 0.01) and percentage (34.8%, p < 0.001) of calcium absorbed, and the decrease in the percentage of calcium absorbed was partially prevented by E2 therapy (p < 0.05). In experiment 2, a dose-response study of the effects of E2 therapy on calcium absorption in OVX C57BL mice was carried out. Five groups of mice were studied: Group 1, sham operated; Group 2, OVX; Group 3, OVX + 60 micrograms of E2/kg of bw/day; Group 4, OVX + 120 micrograms of E2/kg of bw/day; Group 5, OVX + 240 micrograms of E2/kg of bw/day. Therapy was continued for 28 days and calcium absorption measured. Ovariectomy caused a marked increase in fecal calcium (50%, p < 0.0001) and urinary calcium (31%, p < 0.0001) and a marked decrease in calcium absorption (44.9%, p < 0.0001), and these changes were prevented by E2 therapy. The highest level of calcium absorption (109%, p < 0.0001, vs. OVX) was observed in the 120 micrograms of E2 group. Ovariectomy and E2 slightly increased plasma 1,25-dihydroxyvitamin D (1,25(OH)2D) levels but there was no significant correlation between 1,25(OH)2D levels and calcium absorption. The findings in the C57BL mice suggest that estrogen is a physiological regulator of calcium absorption in this strain of mice. Furthermore, these findings share many characteristics with intestinal calcium malabsorption and its reversal by estrogen therapy in hypoestrogenic women. We propose that the OVX C57BL mice warrants further characterization as a potential animal model for investigating issues related to calcium malabsorption in postmenopausal women.

Growth hormone and the expression of mRNAs for matrix proteins and oncogenes in bone.

To examine the effects of growth hormone (GH) on the expression of the mRNAs of bone matrix proteins, three experiments were carried out with 3-month-old female Sprague-Dawley rats. In the first experiment rats were given a single subcutaneous injection of recombinant human GH (8 mg rhGH/kg b. wt.), sacrificed 15 min, 1 h, 2 h, 4 h, 8 h, 16 h and 24 h later, and RNA isolated from cancellous bone from the distal femoral metaphysis. Growth hormone increased the level of type I collagen mRNA by 187, 417, and 509% over the control level at 15 min, 1 h and 2 h, respectively; the mRNA levels declined to 119 and 99% at 4 and 8 h, respectively, and then rose again to 351 and 423% over the control level at 16 and 24 h, respectively. Osteocalcin mRNA transcript increased by 89, 90, 325, 342, 361, and 407% over the control level at 15 min, 1 h, 2 h, 4 h, 8 h and 16 h, respectively, and fell to 66% at 24 h. The level of IGF-I mRNA increased by 45, 83, 120, 140, and 175% over the control level at 2, 4, 8, 16, and 24 h, respectively. In the second experiment, following the administration of rhGH (8 mg/kg b. wt.) bone osteocalcin mRNA increased by 127, 177, 361, and 413% over the control level at 30 min, 1 h, 2 h and 4 h, respectively; IGF-I mRNAs increased by 38, 33, 87, and 437 at 30 min, 1 h, 2 h and 4 h, respectively, but the levels did not become significant until 2 h; c-fos mRNA increased significantly at 30 min, and c-jun and c-myc mRNAs did not increase until 4 h. In the third experiment, animals were given a single injection of rhGH (8 mg/kg b. wt.) and the animals were bled at timed intervals and acid ethanol-extractable serum IGF-I determined. Serum IGF-I increased significantly only at 12 h following rhGH administration. Our data indicate that GH stimulates a rapid increase in the expression of mRNAs for the bone matrix proteins, type I collagen and osteocalcin, by a mechanism that appears to be independent of IGF-I, the early response oncogenes or an increase in osteoblast number.

Additive effect of voluntary exercise and growth hormone treatment on bone strength assessed at four different skeletal sites in an aged rat model.

The aim of the study was to assess the effect of growth hormone (GH), voluntary exercise (Ex), and the combination of GH and Ex on bone strength, mass, and dimensions in aged, intact female rats. In addition, the effect of food restriction (FR) was studied. Fourteen-month-old virgin F-344 rats were divided into 6 groups with 13 animals in each: (1) baseline (BSL); (2) control + solvent vehicle (CTRL); (3) GH 2.5 mg/kg/day (GH); (4) exercise, voluntary: 0.6-0.7 km/day (Ex); (5) GH treatment and voluntary exercise (GH + Ex); and (6) FR. Group 1 was killed at the beginning of the study and served as baseline. All the other groups were killed after 18 weeks' treatment. The effects of aging and treatment regimes were measured at four different skeletal sites: lumbar vertebrae, femoral cortical bone, femoral neck, and the distal femoral metaphysis. Aging in itself induced a decline in vertebral body strength and ash density. At the appendicular skeletal sites, bone mass and strength were unchanged or increased. Treatment with GH alone induced a significant increase in the biomechanical parameters at the vertebral body and the femoral diaphysis, but not at the femoral neck or the distal femoral metaphysis. Voluntary exercise on its own increased load values significantly over CTRL at the vertebral body site, but not at any of the appendicular skeletal sites. The combination of GH and voluntary exercise resulted in an additive effect at the vertebral site and at the femoral diaphysis, and a synergistic (potentiating) effect at the two femoral metaphyses. FR, on the other hand, had a negative effect on cortical bone area and strength at the femoral diaphysis, but no significant effect on the other sites tested. We conclude that GH treatment and voluntary exercise both have skeletal anabolic effects; however, these effects are exerted to differing degrees at different sites. Importantly, when dosed together, GH and Ex have either an additive or synergistic anabolic effect on all sites (axial and appendicular).

Aged-rodent models of long-term growth hormone therapy: lack of deleterious effect on longevity.

Studies were carried out to examine the effects of long-term recombinant human growth hormone (GH) therapy on longevity in rodents. In the first study, 150 18-month-old female F344 rats were divided into three groups of 50 rats per group: Group 1, solvent vehicle; Group 2, 10 microg GH/kg body weight three times per week; Group 3, 50 microg GH/kg body weight three times per week. GH and solvent vehicle therapies were started at 18 months of age and continued until all the animals died spontaneously. Serum insulin-like growth factor (IGF)-I was measured at 18 and 29 months of age and on 3-month-old rats. Serum IGF-I level decreased between 3 and 29 months of age. GH therapy reversed the decrease in a dose-dependent manner, with the 50 microg GH dose returning the serum IGF-I level to that of 3-month-old animals. However, statistical analysis revealed no significant effect of GH therapy on median life span, 10th percentile life span, or maximum life span. Similar observations on longevity were made on aged F344 male rats and on aged Balb/c mice, even when the dose of GH was increased to 1.0 mg/kg body weight two times per week. The main pathologic lesions in control animals were nephropathy, cardiomyopathy, leukemia, and testicular interstitial cell tumor; the prevalence of these lesions was not significantly altered by GH therapy. We conclude that long-term low-dose GH therapy that includes doses in the range that is given to humans in clinical trials in GH deficiency and to revert age-related physiologic declines has no overt deleterious effects on longevity and pathology in aged rodents.