Immobilization-related bone loss in the rat is increased by calcium deficiency

Summary The object of this study was to investigate whether a calcium-deficient diet increases the bone loss produced by mechanical hypofunction (disuse) in the rat. Male Sprague-Dawley rats of approximately 150 g were placed on either a normal diet or a calcium-deficient diet. After 7 days, all rats underwent unilateral hind-limb immobilization by sciatic neurectomy and were sacrificed 30 hours, 72 hours, or 10 days postsurgery. Femora were ashed and the total mineral content (ash weight) was determined. Tibiae were embedded, sectioned, and stained. The metaphyseal secondary spongiosa and the diaphyseal cortical bone were subjected to histomorphometric analysis. Femoral length and serum calcium were not affected by calcium intake or immobilization. Serum parathyroid hormone levels were elevated in rats on the calcium-deficient diet compared to those on the normal diet. Calcium deficiency caused a significant reduction in femoral ash weight (20–35%), tibial cortical thickness (16–20%), and trabecular bone volume (TBV) (33–39%) at 72 hours and 10 days postsurgery. Additional loss of bone mass occurred in the immobilized limb compared to the contralateral intact limb of both dietary groups. This loss occurred earlier (30 hours postsurgery versus 72 hours) in the animals on a calcium-deficient diet and was larger compared to animals on a normal diet (10.6% versus 4.8% at 72 hours and 17.9% versus 12.45% at 10 days). The total bone loss induced by the combination of a calcium-deficient diet and immobilization in this experiment was estimated to equal 46% of femoral ash weight and 79% of tibial TBV. Calcium deficiency increased the number of osteoclasts/mm bone surface in the secondary spongiosa by 25–45%. Immobilization caused a further increase of 50–60% by 72 hours postsurgery. Endosteal mineral apposition rate was significantly decreased (27–44%) by immobilization but was not altered by the low-calcium diet. These data suggest that dietary calcium deficiency augments immobilization-related osteopenia by increasing bone resorption.     

Calcium-deficient diet in ovariectomized dogs limits the effects of 17 beta-estradiol and nandrolone decanoate on bone.

Postmortem measurements by dual-photon absorptiometry of the femur and the second lumbar vertebra in adult dogs indicated bone loss after ovariectomy, which was more pronounced when calcium-deficient diet was given in ovariectomized dogs. This bone loss was nonhomogeneous throughout the femur. Ovariectomy resulted in trabecular and cortical bone loss, and additional calcium-deficient diet resulted in a further highly significant trabecular bone loss at the proximal epiphysis of the femur and in the vertebra. This bone loss was presumably the result of increased bone turnover, as reflected by the highly significant increase in serum alkaline phosphatase. Estrogens could only partially prevent the bone loss induced by calcium deficiency after ovariectomy, and nandrolone decanoate was not effective. We conclude that (1) ovariectomy results in bone loss in adult dogs, (2) this bone loss is more pronounced after calcium-deficient diet, (3) calcium deficiency could be a limiting factor for the preventive effect of estrogens and nandrolone decanoate, and (4) dual-photon absorptiometry allows the evaluation of nonhomogeneous bone loss throughout excised bones.     

Rabbit knee immobilization: bone remodeling precedes cartilage degradation.

This study analyzed processes underlying osteoporosis and osteoarthrosis after short-term immobilization of the right hind limb of postadolescent (2.8 kg) and mature (4.0 kg) rabbits. After 3 weeks, the lateral posterior aspect of the lateral tibial plateau and the lateral femoral condyle of the immobilized limb exhibited prominent subchondral vascular eruptions. Femoral metaphyseal bone density decreased 27 and 18% in the immobilized limbs of postadolescent and mature rabbits, respectively. Calcein green fluorescence increased 1.9-fold (p less than 0.001) in the metaphyseal trabeculae of immobilized femurs. With immobilization, sulfate incorporation into femoral cartilage glycosaminoglycan increased, although total cartilage glycosaminoglycan and hydroxyproline levels were unchanged. Thymidine incorporation into DNA increased four- to fivefold in tibial and femoral cartilage of the immobilized limb. In this study, bone loss and remodeling preceded erosive cartilage degradation.     

Dietary essential amino acid supplements increase bone strength by influencing bone mass and bone microarchitecture in ovariectomized adult rats fed an isocaloric low-protein diet.

This study was designed to investigate whether the administration of dietary essential amino acid supplements in adult rats made osteoporotic by estrogen deficiency and reduced protein intake could reverse the deleterious effects caused by these maneuvers. This animal model was selected to mimic the situation observed in elderly women in whom estrogen deficiency and/or low-protein intake (but also calcium and vitamin D deficiency) are known to contribute to the pathogenesis of osteoporosis. Six-month-old rats were ovariectomized (OVX) and fed an isocaloric 2.5% casein diet for 10 weeks or sham-operated (SHAM) and fed an isocaloric 15% casein diet. The animals fed the 2.5% casein diet were given isocaloric supplements of essential amino acids in similar relative proportion to that of casein at doses of 2.5% or 5% of total diet for an additional 16 weeks. Vertebrae, femur, and tibia bone mineral density (BMD); ultimate strength; and microtomographic histomorphometry were evaluated before and after dietary essential amino acid supplements. Essential amino acid supplements increased vertebrae, femur, and tibia bone strength in OVX rats fed a low-protein diet. The mechanical changes induced by this dietary isocaloric supplement were associated with the prevention of a further BMD decrease or even with some increases and changes in microarchitecture such as from a rod to a plate trabecular spacial configuration and increased cortical thickness. Higher insulin-like growth factor (IGF) I levels, as well as greater bone formation and reduced bone resorption as assessed by biochemical markers of bone remodeling, were found in rats receiving essential amino acid supplements. In conclusion, dietary essential amino acid supplements increased bone strength through modifications of BMD, trabecular architecture, and cortical thickness possibly by an IGF-I-mediated process.     

Calcium deficiency during lactation and in the first two weeks after weaning: decreased ash and increased magnesium in bone of rat pups

The mineral and skeletal status of offspring of calcium-deficient lactating dams was examined. At weaning pups of calcium deficient dams weigh less than controls and are hypocalcemic and hypermagnesemic, but have normal phosphorus levels. Bone ash expressed as percent dry weight is decreased, as is ash content of calcium and phosphorus, while magnesium is high. Histologically, except for thinner layers of lamellar bone, long bones and calvariae are unremarkable. Calcium-deficient pups subsequently fed a normal diet for two weeks gain weight rapidly, recover bone ash (increase of 62%) and normalize magnesium content of both blood and bone. At this point, bones from experimental and control animals are histologically indistinguishable. Weanlings of calcium-deficient mothers, themselves put onto the calcium-deficient diet for two weeks, show a further decline of blood calcium and a further decrease in bone ash. Blood and bone magnesium remain elevated. Long bone trabecular architecture and marrow cavity formation appear normal, but less compact bone is evident. These doubly deprived animals recover rapidly when placed on a normal diet. Within two weeks, mineral content of blood is in the control range, bone ash increases by 93%, and the slope of the weight gain curve parallels that of controls. However, in spite of the profound bone ash increase and linear weight gain, these animals remain deficient in both parameters when examined at 9 weeks of age. Similarly, bone mineral content, which also tended to normalize, fails to completely correct by this time point.     

Effect of exercise on tibial and lumbar vertebral bone mass in mature osteopenic rats: Bone histomorphometry study

The effect of moderate running exercise on tibial and lumbar vertebral bone mass was examined in mature osteopenic rats by bone histomorphometry. Ten 37-week-old female Wistar rats, with bone loss resulting from being fed a relatively low-calcium diet for 14 weeks after ovariectomy at the age of 23 weeks, were randomly divided into two groups of five animals each; control and exercise groups. The exercise consisted of treadmill running at 12 m/min for 1 h per day on 5 days per week for 12 weeks. During the exercise period, all animals were fed a standard calcium diet. After 12 weeks of exercise, bone histomorphometry was evaluated for cancellous bone (secondary spongiosa) of the proximal tibia and the fourth lumbar vertebra and for cortical bone of the tibial shaft. The findings suggested that in the mature osteopenic rat, there was a beneficial effect of moderate running exercise with adequate calcium intake on bone mass only in a weight-bearing long bone, the tibia. The mechanism for increased bone mass appeared to be both decreased bone resorption and increased bone formation in cancellous bone and increased bone formation in cortical bone. Received for publication on Dec. 18, 1997; accepted on April 2, 1998     

Effects of Clodronate on Cortical and Trabecular Bone in Ovariectomized Rats on a Low Calcium Diet

The aim of this study was to evaluate the contribution of a low calcium diet to the cortical and trabecular osteoporosis seen in ovariectomized rats after 7 weeks on a low calcium diet and to investigate the effects of the bisphosphonate clodronate on this development of osteoporosis. Thirty-six mature, female Wistar rats were randomized into four groups: Ovx−B (bisphosphonate) and Ovx−C (control) were ovariectomized, and Sham−Ca (low calcium) and Sham+Ca (normal calcium) were sham operated. The first three groups were fed a low calcium diet (0.01%) and Sham+Ca normal rat chow (Ca 1.1%). The Ovx−B received 10 mg/kg s.c. clodronate daily for nine weeks, and Ovx−C, Sham−Ca, and Sham+Ca received the same volumes of saline. Bone mineral turnover measured as ⁸⁵Sr-uptake was increased in all low calcium groups compared to Sham+Ca. The Sham+Ca femora had higher dry weight and ash weight than the other groups, and Ovx−C had higher dry weight compared with Ovx−B and Sham−Ca. Calcium content was lower in both Ovx groups compared to both Sham groups. Magnesium was lower in all groups compared to Sham+Ca and higher in Ovx−B compared with Ovx−C. In the femoral shaft, Sham+Ca had significantly higher ultimate bending moment, energy absorption, and deflection compared to the other three groups. Ultimate bending moment was higher in Sham−Ca than in Ovx−C. Stiffness was increased in both Sham+Ca and Ovx−B compared to Ovx−C. The maximum stress in the femoral midshaft was higher in Sham+Ca than in the other groups, and higher in Ovx−B than in Ovx−C. Histomorphometry showed increased medullary area in all low calcium groups compared to Sham+Ca and larger cortical area in Sham+Ca and Ovx−B compared to Ovx−C. Compared to Sham+Ca the trabecular bone volume was decreased to 30% in Sham−Ca and to 9% in Ovx−C, but was unchanged in Ovx−B. The low calcium diet generally increased bone mineral turnover and reduced the tibial bone volume. Femoral changes led to a reduction of cortical fracture strength and maximal stress. Ovariectomy in addition to a low calcium diet reduced femoral strength even more. Daily injections of clodronate to ovariectomized rats on a low calcium diet increased femoral shaft stiffness and maximum stress, and clodronate preserved both trabecular and cortical tibial bone volume completely. Received: 11 June 1996 / Accepted: 5 March 1997     

Soy moderately improves microstructural properties without affecting bone mass in an ovariectomized rat model of osteoporosis

Soy protein is reported to prevent bone loss in both women and rat models of osteoporosis. However, the role of soy isoflavones on the trabecular microarchitectural properties needs to be explored. In the present study, we examined whether soy protein with graded doses of isoflavones reverses loss of bone mineral density (BMD), bone mineral content (BMC), and trabecular microstructure in an ovariectomized (Ovx) osteopenic rat model. Seventy-eight 9-m old female Sprague-Dawley rats were either sham-operated (Sham; 1 group) or Ovx (5 groups) and fed a semi-purified casein-based diet. After 90 days, the occurrence of bone loss was confirmed using dual energy X-ray absorptiometry. Thereafter, rats were assigned to the following treatments: Sham, Ovx (control), Ovx + 17beta-estradiol (E(2); 10 microg/kg body wt. twice per week), Ovx + soy protein depleted of isoflavones (Soy-; 0.06 mg isoflavones/g protein), Ovx + soy protein with normal isoflavone content (Soy; 3.55 mg isoflavones/g protein), and Ovx + isoflavone-enriched soy protein (Soy+; 7.10 mg isoflavones/g protein). After 125 days of treatment, rats were euthanized, and tibia and lumbar bones were collected for the assessment of BMD, BMC, and trabecular microarchitectural properties using X-ray microcomputed tomography. None of the treatments had an effect on BMD or microarchitectural properties of the lumbar vertebra. However, Soy treatment significantly increased tibial BMC and BMD by 10% and 4.5% compared with Ovx control, but the increase in BMD was not enough to reach the BMD levels of the Sham control group. The Soy+ diet positively affected the tibial architectural properties including trabecular thickness, separation, and number. In summary, our findings suggest that soy protein does not restore bone loss in osteopenic rats; however, higher doses of isoflavones may be required to reverse the loss of tibial microstructural properties.     

Supplementation with Green Tea Polyphenols Improves Bone Microstructure and Quality in Aged,Orchidectomized Rats

Recent studies show that green tea polyphenols (GTPs) attenuate bone loss and microstructure deterioration in ovariectomized aged female rats, a model of postmenopausal osteoporosis. This study evaluated the efficacy of GTPs at mitigating bone loss and microstructure deterioration along with related mechanisms in androgen-deficient aged rats, a model of male osteoporosis. A 2 (sham vs. orchidectomy) × 2 (no GTP and 0.5% GTP in drinking water) factorial design was studied for 16 weeks using 40 aged male rats. An additional 10 rats (baseline group) were killed at the beginning of study to provide baseline parameters. There was no difference in femoral mineral density between baseline and the sham only group. Orchidectomy suppressed serum testosterone and tartrate-resistant acid phosphatase concentrations, liver glutathione peroxidase activity, bone mineral density, and bone strength. Orchidectomy also decreased trabecular bone volume, number, and thickness in the distal femur and proximal tibia and bone-formation rate in trabecular bone of proximal tibia but increased serum osteocalcin concentrations and bone-formation rates in the endocortical tibial shaft. GTP supplementation resulted in increased serum osteocalcin concentrations, bone mineral density, and trabecular volume, number, and strength of femur; increased trabecular volume and thickness and bone formation in both the proximal tibia and periosteal tibial shaft; decreased eroded surface in the proximal tibia and endocortical tibial shaft; and increased liver glutathione peroxidase activity. We conclude that GTP supplementation attenuates trabecular and cortical bone loss through increasing bone formation while suppressing bone resorption due to its antioxidant capacity.     

Trabecular bone turnover, bone marrow cell development, and gene expression of bone matrix proteins after low calcium feeding in rats

Low-calcium-fed animals have been accepted as one of the experimental models showing a reduction in bone mass. However, the effects of short-term low-calcium feeding on bone turnover, the development of osteoprogenitor cells, and gene expression of bone matrix proteins have not been reported. In this study, we examined the effect of a low-calcium diet on rat tibia and analyzed the changes in the bone by histomorphometry, bone marrow cell culture, and in situ and Northern hybridization of the bone matrix proteins. Rats were fed either a low-calcium diet (0.05% Ca) or a normal calcium diet (0.5% Ca) using the pair feeding technique. They were killed at day 0, 12 h, and days 1, 2, and 3. In the low-calcium group, the serum parathyroid hormone (PTH) level was temporarily increased in 12 h after feeding the low-calcium diet. Bone mineral density in the trabecular bone was significantly decreased from 1 day after the low-calcium feeding, but cortical bone did not show any changes during the experimental period. The bone volume per tissue volume in the proximal tibia also decreased from day 1 in the low-calcium group. The number of osteoclasts and osteoblasts on the trabecular bone surface was increased in the low-calcium group compared with the normal-calcium group. An ex vivo study showed that the number of progenitors of osteoclasts and osteoblasts in bone marrow was also increased in the low-calcium group of rats. The localization of type I collagen mRNA was observed in osteoblasts in the low-calcium group. The Northern hybridization study showed that the gene expression of type I collagen, osteopontin, and osteocalcin was increased at day 3 in the low-calcium group. These results indicated that the trabecular bone surface quickly responded to the low-calcium feeding and that bone remodeling activity was activated probably by PTH. The changes in bone marrow cell populations and the gene expression of bone matrix proteins are closely associated with increased bone turnover induced by the low-calcium diet, resulting in rapid bone loss of the trabecular bone.     

Assessment of effectiveness of oral administration of collagen peptide on bone metabolism in growing and mature rats

The purpose of this study was to examine the effectiveness of collagen peptide intake on bone metabolism in growing (G) and calcium-deficient mature (M) rats. As for the dosages used, they were amounts equal to the recommended supplements for humans (0.166g/kg body weight (BW) per day: Coll-1G and Coll-1M groups), 10-fold higher (1.66g/kg BW per day: Coll-10G and Coll-10M groups), and 100-fold higher (16.6g/kg BW per day: Coll-100G group). In growing male rats, bone mineral density (BMD) of the femur in the Coll-100G group was significantly higher than that in the other groups after the 4-week experimental period. On the other hand, kidneys in the rats from the Coll-100G group exhibited hypertrophy. To examine the effects of collagen peptide on bone metabolism in a calcium-deficient status, mature female rats were fed a 0.01% Ca diet for 9 weeks and then fed a diet with 0.2% calcium with or without collagen peptide (control, Coll-1M, and Coll-10M groups) or a 0.5% calcium diet (normal Ca) for 8 weeks. BMD of the whole femur in the Coll-10M group was significantly higher than that in the control and Coll-1M groups, and the level was similar to that in the normal Ca group. BMD of the lumbar spine in the Coll-10M group was significantly higher than their baseline value, as well as being significantly higher than that in the control and Coll-1M groups. These results suggest that orally administered collagen peptide may provide beneficial effects on bone metabolism, especially in the calcium-deficient condition, without obvious undesirable effects.     

High dietary phosphate intake reduces bone strength in the growing rat skeleton.

Nutrition influences peak bone mass development in early adulthood. The effect of high dietary phosphate intake on the growing skeleton of 1-month-old male rats (n = 30) was assessed in an 8-week intervention. High dietary phosphate intake increased bone remodeling and impaired bone material properties, diminishing bone mechanical strength. INTRODUCTION: High dietary phosphate intake is typical in the Western diet. Abundant phosphate intake enhances parathyroid secretion and bone metabolism. To study the influence of high dietary phosphate intake on growing bone homeostasis and structure, we submitted growing rats to experimental diets that varied in their phosphate content. MATERIALS AND METHODS: One-month-old intact male rats (n = 30) were fed a control diet (Ca:P 1:1) or an experimental diet of either Ca:P 1:2 or Ca:P 1:3 for 8 weeks. At the beginning and the end of the study period, the right femurs were measured using DXA. Double labeling with tetracycline injection was performed 12 and 2 days before death. After death, hind legs were cut loose. Left femurs were processed for histomorphometry. Right femurs were measured with pQCT. Mechanical testing was performed on the right femoral neck and tibial shaft. Six right tibias were analyzed with microCT. Serum PTH, calcium, and phosphate contents were analyzed. RESULTS: High-phosphate intake impaired growth of the animal, limited bone longitudinal growth, and restricted femur BMC and BMD build-up. Osteoclast number, osteoblast perimeter, and mineral apposition rate were increased, and trabecular area and width were decreased. Phosphate decreased femur midshaft total bone BMD, cortical bone BMD, and mean cortical thickness. High-phosphate diet reduced femoral neck and tibial shaft ultimate strength and tibia stiffness and toughness. In addition, serum PTH increased. CONCLUSIONS: High dietary phosphate intake reduced growth, skeletal material, and structural properties and decreased bone strength in growing male rats. Adequate calcium could not overcome this.     

Puerariae radix prevents bone loss in ovariectomized mice

Puerariae radix (PR), the root of Pueraria labata (Willd.) Ohwi, a wild creeper leguminous plant, is one of the earliest and most important crude herbs used in Chinese medicine for various medicinal purposes. PR contains a high amount of isoflavonoids such as daidzein and genistein, which are known to prevent bone loss induced by estrogen deficiency. We have demonstrated that soybean isoflavones prevent bone loss in an osteoporotic animal model. To examine the possible role of PR in bone metabolism, female mice were ovariectomized (OVX), and some OVX mice were fed a diet containing low, middle, and high doses (5%, 10%, and 20% of diet, respectively) of PR for 4 weeks. In OVX mice, the uterine weight declined, and intake of PR at any dose did not affect uterine weight. The total femoral bone mineral density (BMD) was significantly reduced by OVX, and the decrease in BMD caused by OVX was significantly inhibited by intake of the diet with the low dose of PR and completely prevented by the middle dose of PR. Histological analysis of the femoral metaphysis showed that intake of the diet with the middle dose of PR completely prevented decrease in trabecular bone volume (BV/TV) and trabecular thickness (Tb.Th) and restored the increase in trabecular separation (Tb.Sp) in OVX mice. In contrast, intake of the diet with the high dose of PR further increased BV/TV and Tb.Th and decreased Tb.Sp in OVX mice compared with that in the sham-operated mice. These results suggest that PR may represent a potential alternative medicine for hormone replacement therapy (HRT) in the prevention of osteoporosis in postmenopausal women.     

Inhibition of CaMKK2 reverses age-associated decline in bone mass

Decline in bone formation is a major contributing factor to the loss of bone mass associated with aging. We previously showed that the genetic ablation of the tissue-restricted and multifunctional Ca^2 +/calmodulin (CaM)-dependent protein kinase kinase 2 (CaMKK2) stimulates trabecular bone mass accrual, mainly by promoting anabolic pathways and inhibiting catabolic pathways of bone remodeling. In this study, we investigated whether inhibition of this kinase using its selective cell-permeable inhibitor STO-609 will stimulate bone formation in 32 week old male WT mice and reverse age-associated of decline in bone volume and strength. Tri-weekly intraperitoneal injections of saline or STO-609 (10 μM) were performed for six weeks followed by metabolic labeling with calcein and alizarin red. New bone formation was assessed by dynamic histomorphometry whereas micro-computed tomography was employed to measure trabecular bone volume, microarchitecture and femoral mid-shaft geometry. Cortical and trabecular bone biomechanical properties were assessed using three-point bending and punch compression methods respectively. Our results reveal that as they progress from 12 to 32 weeks of age, WT mice sustain a significant decline in trabecular bone volume, microarchitecture and strength as well as cortical bone strength. However, treatment of the 32 week old WT mice with STO-609 stimulated apposition of new bone and completely reversed the age-associated decrease in bone volume, quality, as well as trabecular and cortical bone strength. We also observed that regardless of age, male Camkk2^−/− mice possessed significantly elevated trabecular bone volume, microarchitecture and compressive strength as well as cortical bone strength compared to age-matched WT mice, implying that the chronic loss of this kinase attenuates age-associated decline in bone mass. Further, whereas STO-609 treatment and/or the absence of CaMKK2 significantly enhanced the femoral mid-shaft geometry, the mid-shaft cortical wall thickness and material bending stress remained similar among the cohorts, implying that regardless of treatment, the material properties of the bone remain similar. Thus, our cumulative results provide evidence for the pharmacological inhibition of CaMKK2 as a bone anabolic strategy in combating age-associated osteoporosis.     

Reductions in bone mass, structure, and strength in axial and appendicular skeletons associated with increased turnover after ovariectomy in mature cynomolgus monkeys and preventive effects of clodronate.

Over 16 months, we evaluated the effects of ovariectomy (OVX) and bisphosphonate clodronate (CLO) on bone in 48 cynomolgus monkeys (9-15 years old) fed a normal calcium diet. We established three OVX groups (oral CLO at 0 [OVX control], 12, or 60 mg/kg per day) and one sham-operated (SHAM) group. At 16 months, the bone mineral density (BMD) values (percentage of group baseline; OVX control vs. SHAM) for lumbar bone (L3-L5), proximal femur, midfemur, radius, and tibia were -2.6% versus 11.2%, -3.5% versus 8.9%, -3.0% versus 9.0%, -5.5% versus 15.7%, and -6.7% versus 13.9%, respectively. In OVX control (i) tibia showed significant loss of bone mineral content (BMC; vs. baseline), (ii) urinary deoxypyridinoline (DPD) and serum osteocalcin (OC) levels increased (peak = 182% and 168%, respectively, of SHAM), (iii) in lumbar bone and midfemur, ultimate load (UL) was reduced (vs. SHAM), (iv) in lumbar bone, trabecular bone-formation rates (BFRs) were not changed significantly, but tibial endocortical and intracortical bone formation rates were significantly raised (vs. SHAM), (v) the volumetric BMD (vBMD) and geometry of the tibial cortex (measured by peripheral quantitative computed tomography [pQCT]) were significantly reduced (vs. SHAM). CLO, 60 mg/kg per day but not 12 mg/kg per day, significantly inhibited OVX-induced changes, age-dependent increases in bone mass, and ability to maintain structure. Thus, in OVX mature cynomolgus monkeys (possibly, a unique model of the cortical bone loss secondary to estrogen deficiency), the post-OVX increases in systemic bone markers were slight, but stimulation of local turnover in the cortical envelope was enough to cause bone loss (more so in tibia than in lumbar trabecular bone). High-dose CLO prevented these changes.     

Moderate weight gain does not influence bone metabolism in skeletally mature female rats

Bone mass is correlated with body weight during growth. However, it is unclear how bone mass is influenced by weight gain following skeletal maturity. The purpose of this study was to determine the effects of weight maintenance and two rates of weight gain on bone metabolism using skeletally mature female rats. Eight-month-old female rats were fed one of 3 diets for 13 weeks: Lieber–DeCarli liquid diet ad lib (control diet), the same diet with caloric restriction to maintain initial body weight (calorie-restricted diet), and the same diet fed ad lib with the exception that appetite was enhanced (calorie-increased diet) by replacing a small quantity of maltose–dextran isolcalorically with ethanol (0.5% caloric intake). Compared to baseline, rats fed the calorie-restricted, control, and calorie-increased diets changed in weight by ?1 ± 2% (mean ± SE), 10 ± 3%, and 21 ± 2%, respectively. Weight gain was associated with a significant increase in serum leptin, a putative regulator of bone formation. In contrast, significant differences in tibial bone mineral content and density were not detected among treatments groups following dietary intervention or between treatment groups and the baseline group. Similarly, indices of cancellous bone architecture (area, trabecular number, thickness, and separation) and bone turnover (mineralizing perimeter, mineral apposition rate, and bone formation rate) did not differ among groups following dietary intervention. Our findings suggest that neither weight gain nor increased serum leptin levels, over the range evaluated, influence bone metabolism in skeletally mature female rats.     

Strontium ranelate does not stimulate bone formation in ovariectomized rats

INTRODUCTION: Strontium ranelate (SrR) is suggested to function as a dual-acting agent in the treatment of postmenopausal osteoporosis with anti-resorptive and anabolic skeletal benefits. We evaluated the effects of SrR on the skeleton in ovariectomized (OVX) rats and evaluated the influence of dietary calcium. METHODS: Three-month old virgin female rats underwent ovariectomy (OVX, n = 50) or SHAM surgery (SHAM, n = 10). Four weeks post-surgery, rats were treated daily by oral gavage with distilled water (10 ml/kg/day) or SrR (25 or 150 mg/kg/day) for 90 days. Separate groups of animals for each dose of SrR were fed a low (0.1%) or normal (1.19%) calcium (Ca) diet. Static and dynamic histomorphometry, DXA, mu-CT, mechanical testing, and serum and skeletal concentrations of strontium were assessed. RESULTS: SrR at doses of 25 and 150 mg/kg/day did not increase bone formation on trabecular or periosteal bone surfaces, and failed to inhibit bone resorption of trabecular bone regardless of Ca intake. There were no improvements in bone mass, volume or strength with either dose of SrR given normal Ca. CONCLUSION: These findings demonstrate that SrR at dosages of 25 and 150 mg/kg/day did not stimulate an anabolic bone response, and failed to improve the bone biomechanical properties of OVX rats.     

The effect of high or low dietary calcium on bone and calcium homeostasis in young male rats

Summary Young male rats (100 g body weight) were fed diets containing varying amounts of calcium. Body weight and bone development were studied together with various endocrine parameters, including blood levels of Ca²⁺, calcitonin, parathyroid hormone, vitamin D, and gastrin, and the enterochromaffin-like (ECL) cell-related parameters gastric mucosal histidine decarboxylase activity and histamine concentration. A diet containing 0.5% calcium resulted in ontimum body weight gain and bone development. A lower calcium intake impaired body weight gain and bone development. The impairment was manifested in reduced bone calcium content whereas the size of the bones was unaffected. The net absorption of calcium seemed to be proportional to the calcium intake. A low calcium diet (0.03%) raised the circulating levels of 1,25(OH)₂D and parathyroid hormone and lowered 25(OH)D₃ and Ca²⁺, whereas a high calcium diet (5.46%) raised calcitonin, Ca²⁺, 25(OH)D₃, and 1,25(OH)₂D. In addition, the low calcium diet lowered the circulating gastrin concentation and the histidine decarboxylase activity and histamine content of the ECL cells in the gastric mucosa. A high calcium diet raised the circulating gastrin concentration, but the rise was not associated with an increase in the histidine decarboxylase activity and histamine content.     

Osteoprotegerin abrogates chronic alcohol ingestion-induced bone loss in mice.

To investigate the role of osteoprotegerin (OPG) on alcohol (ethanol)-mediated osteoporosis, we measured a variety of bone remodeling parameters in mice that were either on a control diet, an ethanol (5%) diet, or an ethanol (5%) diet plus OPG administration. OPG diminished the ethanol-induced (1) decrease in bone mineral density (BMD) as determined by dual-energy densitometry, (2) decrease in cancellous bone volume and trabecular width and the increase of osteoclast surface as determined by histomorphometry of the femur, (3) increase in urinary deoxypyridinolines (Dpd's) as determined by ELISA, and (4) increase in colony-forming unit-granulocyte macrophage (CFU-GM) formation and osteoclastogenesis as determined by ex vivo bone marrow cell cultures. Additionally, OPG diminished the ethanol-induced decrease of several osteoblastic parameters including osteoblast formation and osteoblast culture calcium retention. These findings were supported by histomorphometric indices in the distal femur. Taken together, these data show that OPG diminishes ethanol-induced bone loss. Furthermore, they suggest that OPG achieves this through its ability to abrogate ethanol-induced promotion of osteoclastogenesis and promote osteoblast proliferation.     

Morphological changes in bone following intramedullary implantation of methyl methacrylate: A morphometrical study of calcium deficiency

Adult male Sprague-Dawley rats had a methyl methacrylate implant in their right femur. After 16 weeks a group of rats was given a calcium-deficient diet. The rats were followed for another 31 weeks. Due to calcium deficiency a loss of femoral bone mass occurred which was relatively greater in the non-operated femur, as compensatory periosteal bone apposition and remaining necrotic bone areas contributed to the bone mass in the operated femur. The calcium deficiency did not affect the interface between bone and implant, where a thin sleeve of new bone was formed. While the non-operated femur lost its bone through endosteal resorption, the loss of bone in the operated femur was due to intracortical resorption.