Clodronate Prevents Bone Loss in Aged Ovariectomized Rats

The purpose of this study was to investigate the ability of clodronate to prevent ovariectomy (OVX)-induced osteopenia in aged rats. Fourteen-month-old female Sprague-Dawley rats (n = 166) were randomized into six groups. One group was sacrificed at the start of the study, four groups were ovariectomized, and one group was sham-operated (Sham). The OVX rats were given subcutaneously either vehicle (veh) or clodronate at doses of 3, 7, or 25 mg/kg once a week for 3 months, and the Sham rats were given the vehicle. At all dose levels clodronate inhibited trabecular bone loss in the distal femur and in the fourth lumbar vertebral body (L4), and decreased bone resorption as evidenced by urinary deoxypyridinoline excretion. The lowest dose of clodronate preserved serum osteocalcin and endosteal bone formation of secondary spongiosa in L4 at the level of the Sham/veh group. The OVX-induced increase in periosteal bone formation of femoral diaphysis was unaffected by two smaller doses of clodronate, but was decreased to the level of Sham rats after the highest dose. After 3 mg/kg clodronate, the percentage of femoral cortical bone area and the mean relative cortical thickness were higher compared with the OVX/veh group. There was a good positive correlation between the maximum load in three-point bending of the tibia and tibial ash weight. Normal lamellar pattern of newly formed cancellous and cortical bone was found after clodronate treatment. No signs of adverse accumulation of osteoid or any deleterious effect on mechanical strength of long bones and lumbar vertebrae were found. Received: 28 August 1996 / Accepted: 5 March 1997     

Ovariectomy-Induced Bone Loss Can be Affected by Different Intensities of Treadmill Running Exercise in Rats

Fifty-six Sprague-Dawley rats were either ovariectomized (OVX, n= 24), sham-operated (Sham, n= 24), or sacrificed (n= 8) at the beginning of the experiment to serve as a baseline group. The OVX and Sham groups were further randomly divided into control (CTRL), slow running (R10), and faster running (R18) groups. R10 and R18 groups ran for 2 × 30 min/day for 8 weeks at speeds of 10 m/min and 18 m/min, respectively. Exercise did not affect the mechanical or histomorphometric parameters of bone in the sham-operated rats. There was no effect of exercise on body weight gain in the OVX-R10 group, but in OVX-R18 it decreased the gain of body weight. In the OVX–CTRL group the maximal load and energy absorption of the femoral neck were 16.7% (P < 0.001) and 30.0% (P < 0.001) lower than in the Sham–CTRL group, respectively. In OVX animals, slow running had a positive effect on the maximal load of the femoral neck (86.5 N) when compared with OVX–CTRL rats (77.1 N, P < 0.07). 51.7% of the trabecular bone was lost in the distal femur as a result of OVX and exercise reduced this loss to 30.2% (R10) and 39.9% (R18). Ovariectomy increased the bone formation rate (BFR) and the mineral apposition rate (MAR) on the periosteum of the femoral shaft. Exercise decreased the periosteal BFR and MAR in OVX rats, but increased it at the endosteum. Osteoclast numbers in the femoral metaphysis were increased after OVX and running exercise inhibited this effect significantly. The maximal bending load of the humerus increased after OVX by 12.1% (P < 0.05). Exercise enhanced this effect, the slow running being more effective. These results suggest that bone in OVX rats is either more sensitive to exercise than in sham-operated rats or that the higher body weight with slow running induces optimal loading and strengthens the bones. Received: 2 May 1996 / Accepted: 15 October 1996     

Effect of Salmon Calcitonin on Femoral Bone Quality in Adult Ovariectomized Ewes

The aim of this study was to evaluate the effect of intermittent calcitonin on femoral bone quality in adult ewes from the time of ovariectomy. Six months after the start of the experiment, bone density measurements and mechanical testing (torsion and resonant frequency analysis of the diaphysis and compression of an excised trabecular bone cylinder from the femoral neck) were performed in sham-control and ovariectomized (OVX) ewes treated with placebo or salmon calcitonin (50 or 100 units, 3 times/week). Crystallinity of bone was evaluated by measuring X-ray diffraction line broadening. After OVX, a nonsignificant bone loss was found at all measured sites in the femur (−3 to −9%) together with a decreased biomechanical competence in the trabecular bone (compressive strain −28%, P < 0.05). Treatment with salmon calcitonin, 50 or 100 IU subcutaneously three times a week from the time of ovariectomy, resulted in a significant dose-dependent preservation of bone strength in the trabecular bone of the femoral neck compared with OVX. No adverse effects of calcitonin were observed on bone crystal composition as assessed by diffractiometry. We conclude that in adult ewes intermittent calcitonin treatment from the time of OVX was associated with a significant preservation of cancellous bone strength and strain in trabecular bone of the femoral neck, without affecting crystalline properties of bone. Received: 20 October 1995 / Accepted: 19 February 1996     

The Mechanical Properties of Fluoride-Treated Bone in the Ovariectomized Rat

The effect of fluoride therapy on the osteopenic bone of the ovariectomized rat was studied by comparing the densitometric and biomechanical data. Forty retired breeder female Sprague-Dawley rats aged 10 months were randomly divided into five groups. One group (Group A) was killed at the beginning of the study and was used as a baseline. Three groups were ovariectomized and one was sham-operated (Group B) and observed for the same period as a sham-aged group. A group of ovariectomized rats was used as a sham therapy control (Group C) and received only deionized drinking water; the other two groups (F1 and F2) received L-glutamine monofluorophosphate and calcium at a rate of 1:30 F/Ca at different doses by gavage (0.57 mg F/17 mg Ca per kg/day-Group F1; 0.21 mg F/6.30 mg Ca per kg/day-Group F2). Densitometric and biomechanical (compression and three-point bending test) assays, X-ray diffraction, and Fourier transformed infrared spectroscopy were performed on femoral specimens. Biomechanical data showed that the femoral heads of Group F2 required a significantly greater energy-to-failure than Group C (P < 0.05) as well as treated femoral diaphysis when compared with the others (P < 0.01). Significant increases in the elastic modules were observed in fluoride-treated groups (P < 0.001) when compared with other groups. Diffractometric and spectroscopic data showed the presence of fluorine-apatite in both treated groups with a high component of carbonates. Also, fluoride therapy causes an increase of bone stiffness due to the presence of fluoroapatite. It seems to produce two opposed properties in the osteopenic rat bone: a higher resistance to compression loading and a greater frailty to flexion loading. Received: 18 November 1997 / Accepted: 28 January 1999     

Effect of Estrogen Deficiency on Cancellous and Cortical Bone Structure and Strength of the Femoral Neck in Rats

Eighty mature Sprague-Dawley rats were weight matched before ovariectomy (Ovx) or Sham surgery (Sham). Sham rats had free access to food and water throughout the experiment, whereas Ovx rats were kept on the pair-fed diet. Rats were euthanized at 4, 8, and 12 weeks after surgery, and had received fluorochrome bone markers at 9 and 2 days prior to euthanasia. In addition 10 rats were euthanized at the time of surgery serving as baseline controls. All rats were also scanned for body composition and bone mineral parameters by DEXA before surgery and euthanasia. Left proximal femurs (femoral necks) were used for bone histomorphometry, whereas right femurs were used for in vitro DEXA measurements and mechanical testing. Despite pair-feeding, ovariectomized rats had increased body weights and fat body mass, whereas the percent lean body mass steadily declined throughout the experiment. Mineral density of the whole femur and femoral neck was significantly higher in the Sham rats relative to Ovx animals. Ovariectomy reduced trabecular number and thickness, and increased trabecular separation and bone marrow space at the femoral midneck location. The structure of the remaining trabeculae was dramatically changed toward simpler struts as revealed by nodal analyses. Cortical thickness in Ovx rats was reduced because of the high endocortical resorption, which, in addition to cancellous bone resorption, resulted in fewer endocortico-trabecular connections. Femoral necks obtained from ovariectomized rats had reduced strength and were less stiff relative to controls. Because of the enormous clinical significance of the proximal femur for osteoporosis in humans, and the opportunity for studying bone BMD, mass, structure, and strength at the same skeletal location, the femoral neck appears superior to other skeletal sites routinely used for bone histomorphometry or mechanical testing in the Ovx rat model. Received: 25 September 1996 / Accepted: 24 March 1997     

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. Received: 7 July 1998 / Accepted: 23 December 1998     

Collagen type I of rat cortical and trabecular bone differs in the extent of posttranslational modifications

This study sought to evaluate whether the architecture of the matrix of cortical and trabecular bone is exactly the same. For this purpose we analyzed the extent of some posttranslational modifications of type I collagen, which is the major component of bone matrix. Ten female and 10 male 100-day-old rats were sacrificed and the content of hydroxylysine, glycosylated hydroxylysine, and pyridinium cross-links of collagen from cortical and trabecular bone was determined. The amount of each compound was expressed as a molar ratio with hydroxyproline. The collagen posttranslational modification pattern appears to be the same in both sexes but with a higher extent of differences in females compared with males. Comparing cortical and trabecular bone, the former contains a higher amount of hydroxylysine residues whereas in the latter, glycosylation of hydroxylysine is higher and pyridinium cross-link concentration is lower. Moreover, an inverse linear relationship between glycosylated hydroxylysine and pyridinium cross-links concentration was established, both for female (r =−0.455, P= 0.04) and male rats (r =−0.426; P= 0.06). This paper discusses what these findings may mean in functional terms. Received: 14 March 1995 / Accepted: 9 August 1995     

Cyclical Etidronate Therapy for Prevention of Postmenopausal Bone Loss: A 1-Year Open-Label Follow-Up Study

The objective of this study was to evaluate whether the pharmacological activity of cyclical etidronate therapy is sustained beyond the dosing period. A group of 121 postmenopausal women who had completed a 2-year, double-blind, placebo-controlled parallel study with etidronate or placebo (400 mg/day for 14 days every 3 months) and calcium agreed to participate in a 1-year open-label follow-up study to evaluate the effect of discontinuing etidronate treatment. Fifty-nine subjects in the former etidronate group and 62 in the placebo group received 500 mg/day of elemental calcium; 54/59 and 58/62 subjects, respectively, completed the study. Outcomes of the study were bone mineral density (BMD), measured by dual energy X-ray absorptiometry (DXA), and biochemical markers of bone turnover (urinary deoxypyridinoline/creatinine and serum osteocalcin). To determine whether there was a residual effect of previous therapy we compared mean percentage changes from baseline (year 0) to year 3 for both spinal and femoral neck BMD and markers of bone turnover in the former cyclical etidronate and placebo groups. To evaluate the carryover effect of treatment we compared the percent change from year 2 to year 3 for the same variables. Mean percentage change (SEM) from year 2 to year 3 for spinal BMD in the former cyclical etidronate group was −2.87% (0.48%) versus −0.99% (0.36%) in the placebo group (P= 0.0022). In the femoral neck, the BMD changes were −0.86% (0.42%) versus −1.01% (0.41%) (NS). Biochemical markers increased within 6 months toward baseline levels. Mean percentage changes from baseline (year 0) in both spinal and femoral neck BMD were significantly different between groups 1 year after treatment discontinuation. No differences between groups were maintained in deoxypyridinoline and osteocalcin. It is concluded that following withdrawal of cyclical etidronate therapy bone loss resumes at a normal and moderately accelerated rate in the proximal femur and lumbar spine, respectively. A positive effect on BMD at both cortical and trabecular sites is maintained for 1 year after treatment withdrawal. Received: 8 May 1999 / Accepted: 10 December 1999     

Effect of Ovariectomy, Malnutrition and Glucocorticoid Application on Bone Properties in Sheep: A Pilot Study

The demographic changes in the human population continue to lead to an increasing incidence of osteoporosis. The main clinical symptom of osteoporosis is fracture. Fracture fixation in osteoporosis is frequently complicated by failure of fixation. There is a great need for a large-animal model of osteoporosis for controlled studies, which allows the investigation of fracture healing and fracture treatment in weak bone. Eight swiss mountain sheep, 7–9 years old, were divided into four treatment groups of two animals each. Group 1 was ovariectomized and fed a calcium/vitamin D-restricted diet (O+D). Group 2 was ovariectomized and given a daily intramuscular injection of 25 mg methylprednisolone (O+S). Group 3 was ovariectomized, fed a calcium/vitamin D-restricted diet and injected with 25 mg intramuscular methylprednisolone per day (O+D+S). Group 4 was used as an untreated, not sham operated control group. At the beginning of the study and every 2 months for 6 months the bone mineral density (BMD) was determined using quantitative computed tomography (pQCT) at the distal radius. Biopsies were taken after 6 months from vertebral bodies and femoral heads and the bone structure, i.e. trabecular thickness (Tb.Th), trabecular number (Tb.N), trabecular separation (Tb.Sp), bone surface fraction (BS/BV) and bone volume fraction (BV/TV), was determined by micro-CT. In vitro compression testing of the biopsies was performed to determine failure load and stiffness. The control group showed no changes in BMD. The greatest decrease in BMD was seen in group 3 (O+D+S), which had a decline of 58% in cancellous bone and 22% in cortical bone. In the vertebral body biopsies a prominent change in structural parameters was observed (Tb.N, –53%; Tb.Th, –63%, Tb.Sp, +150%). The changes were less pronounced in the femoral head biopsies. In the compression test the vertebral body biopsies of group 3 (O+D+S) had stiffness values 40% lower failure load 70% lower compared with the control group. The most effective method of inducing osteoporosis in sheep was found to be the combined treatment. These results need to be confirmed in a larger number of animals. Received: 4 May 2001 / Accepted: 13 December 2001     

Effects of Promethazine on Bone Mass and on Bone Remodeling in Ovariectomized Rats: A Morphometric, Densitometric, and Histomorphometric Experimental Study

The effect of promethazine on bone is debated. We studied the effect of promethazine on bone and the mechanism of action involved by densitometric and histomorphometric measurements in female Wistar rats (100 days old, mean weight 25 ± 20 g). A control group of 15 rats was not manipulated. An experimental group of 15 rats were ovariectomized (OVX) at 100 days of life and fed a diet supplemented with 4.8 mg/kg promethazine hydrochloride (OVX + Prom). The group that underwent OVX and a group of 15 rats that underwent sham ovariectomy (Sham-OVX) were not treated with promethazine. After 30 days, all the rats were killed. Their femur and 5th lumbar vertebra were dissected and cleaned of soft tissue. Femoral length and vertebral height were measured with a caliper and bones were weighed on a precision balance. The bone mineral content (BMC) and bone mineral density (BMD) of the whole right femurs and 5th lumbar vertebras were measured by dual-energy X-ray absorptiometry (DXA). Trabecular bone volume (Cn-BV-TV%), trabecular number (Tb-N mm⁻¹), trabecular thickness (Tb-Th μm), and trabecular separation (Tb-Sp μm) were measured in the femurs by histomorphometric study of nondecalcified bone. Our results showed that promethazine significantly inhibited postovariectomy loss of bone mass (P < 0.0001) by significantly reducing bone resorption, as shown by the smaller trabecular spaces observed in the treated OVX rats (P < 0.0001). Received: 1 June 1998 / Accepted: 17 February 1999     

Preventive Effects of Traditional Chinese (Kampo) Medicines on Experimental Osteoporosis Induced by Ovariectomy in Rats

Preventive effects by traditional Chinese (Kampo) medicines, Unkei-to, Hachimi-jio-gan, and Juzen-taiho-to, on the progress of bone loss induced by ovariectomy in rats were investigated for a period of 49 days. The bone mineral density (BMD) of tibia in ovariectomized (OVX) rats decreased by 20% from those in sham-operated (Sham) rats, with the decrease completely inhibited by the administration of any one of these Kampo medicines or 17β-estradiol. From scanning electron microscopic (SEM) analyses, the surface of a trabecular bone of tibia in OVX rats had a porous or erosive appearance, whereas that of the same bone in Sham rats was composed of fine particles. The administration of three Kampo medicines and 17β-estradiol to OVX rats preserved the fine particle surface of the trabecular bone. These results strongly suggest that any of these three gynecological Kampo medicines is as effective as 17β-estradiol in preventing the development of bone loss induced by ovariectomy in rats. Received: 18 December 1995 / Accepted: 24 September 1996     

骨质疏松绵羊模型松质骨及皮质骨的微观结构及力学性能变化的研究

目的观察去势手术对绵羊皮质骨和松质骨的骨密度、骨小梁结构及力学性能的影响。方法20只雌性成年绵羊（4±1.5）随机分为去势4个月组（OVX-4months）（4只）、去势12个月组（OVX-12months）（8只）和假手术（Sham）组（8只）。OVX组行双侧卵巢切除术,假手术组仅显露双侧卵巢,术中测定腰椎骨密度。分别与术后4、12个月处死动物,测定股骨颈、股骨干及股骨髁的骨密度,并行MicroCT分析及生物力学测试。结果去势12个月后（OVX-12months）组腰椎、股骨颈及股骨髁的骨密度较对照组显著降低,而皮质骨骨密度无明显降低。其松质骨的相对骨体积（BV/TV）、骨小梁厚度（Tb.Th）、骨小梁数目（Tb.N）较对照组显著降低,表面积体积比（BS/BV）、骨小梁间隙（Tb.Sp）则较对照显著增高。生物力学测试表明,去势12个月后,腰椎松质骨的最大压缩应力分别较Sham组和OVX-12months组下降82.5%和85.9%,力学强度显著下降,而皮质骨的力学强度无显著变化。结论去势12个月后,绵羊腰椎、股骨部的松质骨BMD及骨小梁空间结构参数明显降低,力学强度也显著下降,可以作为骨质疏松的大动物模型。而皮质骨的骨密度和力学强度下降不明显,需要更长的去势时间。     

Prevention of Bone Loss by Clodronate in Early Postmenopausal Women with Vertebral Osteopenia: A Dose-Finding Study

This double-masked, placebo-controlled study was undertaken to determine the efficacy and safety of oral clodronate in the prevention of bone loss in early postmenopausal women with vertebral osteopenia. Altogether 610 women with a mean age of 53 years were recruited for the study. They were 1–5 years postmenopausal and their lumbar spine bone mineral density (BMD) was at least 1 standard deviation below the mean of premenopausal women (T-score ≤−1). The subjects were randomized into five study groups to receive either placebo, clodronate 65 mg, 400 mg or 800 mg daily, or intermittent clodronate in 3 month cycles with 400 mg daily for 15 days followed with no treatment for 75 days for 3 years. One hundred and eighty-seven of 509 women who completed the primary study continued in the extension study of 2 years in which previous placebo users were switched to clodronate 800 mg daily, while previous users of 400 mg or 800 mg of clodronate used either placebo or 800 mg of clodronate daily. In the primary study clodronate was administered in the evening, and in the extension 1 h before breakfast on an empty stomach. In the primary study mean changes in lumbar spine BMD were −3.4% in the placebo group and +0.4% in 800 mg clodronate group [difference between groups at 3 years 3.8% (95% CI 2.7% to 4.9%, p<0.0001)], and in the trochanter area BMD −1.1% in the placebo group, and + 0.4% in the 800 mg clodronate group [difference between groups at 3 years 1.5% (95% CI 0.05% to 2.9%)]. During the extension study mean changes in lumbar spine BMD were +1.5% in the clodronate group and −0.2 % in the placebo group [difference between groups 1.7% (CI 0.4% to 3.0%, p = 0.010)] and in trochanter BMD were +2.5% in the clodronate group and no change in the placebo group [difference between groups 2.1% (CI 0.3% to 3.9%, p = 0.007)]. No statistically significant differences between the placebo and 800 mg clodronate groups were found in the femoral neck BMD. In the primary study the urinary excretion of type I collagen aminoterminal telopeptide (NTX) decreased by 44% (p<0.0001 compared with placebo) and that of deoxypyridinoline by 18% (p<0.0001) in the clodronate 800 mg group. In the extension study urinary NTX decreased by 51% (p<0.0001) in those who were switched to 800 mg of clodronate and increased by 67% (p<0.0001) in those who stopped using that dose. There was no difference in the frequency of gastrointestinal complaints between clodronate- and placebo-treated patients in the primary study, but they were more common among women who received clodronate in the extension phase. Clodronate in daily doses of 400–800 mg caused a slight elevation of aminotransferase levels, usually within the reference range. In bone biopsies no defect in mineralization was found. In conclusion, clodronate in a daily dose of 800 mg prevents early postmenopausal bone loss at the sites of the skeleton in which cancellous bone predominates. It effectively reduces bone resorption and bone turnover rate. Antifracture efficacy of clodronate remains to be established by prospective, placebo-controlled trials. Received: 4 March 2002 / Accepted: 9 July 2002     

Age-Associated Decline in Human Femoral Neck Cortical and Trabecular Content of Insulin-Like Growth Factor I: Potential Implications for Age-Related (Type II) Osteoporotic Fracture Occurrence

Recent evidence suggests that regulatory peptides such as insulin-like growth factor-I (IGF-I) are released locally from bone during resorption, and may then act in a sequential manner to regulate the cellular events required for the coupling of bone formation to resorption. Among other factors, a decrease in bone-associated IGF-I levels could therefore result in remodeling imbalance and contribute to the gradual loss of bone that occurs with age. As the femoral neck region is of primary concern for the clinical manifestations of osteoporosis, the current study was intended to assess the IGF-I contents in femoral neck cortical and trabecular bone from aging individuals. Bone samples from the neck region were obtained at postmortem from 39 females and 35 males, aged 23–92 years. Concentrations of IGF-I and osteocalcin were measured by radioimmunoassay in the supernatants obtained after EDTA and guanidine hydrochloride extraction. The total amount of protein present in the extracts was determined by spectrophotometry. IGF-I levels were significantly lower in trabecular compared with cortical bone. Though femoral neck total protein did not vary with donor age, both IGF-I and osteocalcin were found to decline markedly. Between the ages of 23 and 92 years, average yearly rates of loss of 0.30 and 0.21 ng IGF-I/mg protein were observed in cortical and trabecular bone, respectively, corresponding with net losses of nearly 35% of the cortical skeletal content of IGF-I and 41% of the trabecular skeletal content of IGF-I. These changes in bone-associated IGF-I paralleled those of osteocalcin, consistent with an overall decrease in osteoblast function with aging. In women, the rate of decline was significantly faster for trabecular than for cortical IGF-I, however in men, age-dependent changes in cortical and trabecular IGF-I were similar. These findings support the hypothesis that changes in the local IGF regulatory system over time could be a pathophysiologic component of the age-related (type II) femoral neck osteoporotic syndrome. Received: 12 December 1996 / Accepted: 23 April 1997     

Gonadal Steroids and Bone Metabolism in Young Castrated Male Rats

At 45 days of age, 40 male Wistar rats were castrated, then randomly divided into four groups, S.C. injected for 60 days after surgery either with 17β-estradiol (E) 10 μg/kg BW/48 hours, progesterone (P) 140 μg/kg BW/48 hours, dihydrotestosterone (D) 2 μg/kg BW/48 hours, E + P + D same doses, or solvent alone (CX). Ten other rats were sham-operated (SH) and used as controls. Animals were put in balance to determine Ca and phosphorus (Pi) intestinal apparent absorption (IA Ca, IA Pi) and urinary pyridinium crosslinks excretion. Plasma was collected for measurement of intact-parathyroid hormone (PTH), calcitonin (CT), insulin-like growth factor I (IGF-I), 1,25 dihydroxyvitamin D (1,25(OH)₂D), Ca, and Pi. Orchidectomy induced marked seminal vesicles atrophy and increased plasma CT, PTH, and Ca concentrations. IA Ca was significantly higher in P rats, however, neither castration nor any other treatment had significant effects. Orchidectomy decreased femoral length, dry weight, and Ca content, whereas E or D given alone or together with P improved endochondral growth and enhanced femoral Ca content. Again, bone mineral density was lowered by orchidectomy and reestablished by both E and EPD, even above SH values, this effect being more important at the metaphyseal levels. Urinary pyridinium cross-links excretion and plasma osteocalcin concentrations were higher in the CX animals than in the controls. Although E and D given alone did reduce both biochemical turnover markers, they showed additive effect when given together (EPD). In conclusion, in the young castrated male rat, E was more efficient than D for preventing bone loss, the most important effect being induced by a combination of E + P + D. Received: 28 June 1999 / Accepted: 12 January 2000     

Effects of drug treatment on bone strength and structural changes with aging: an experimental study in rats

 The purpose of the present study was to evaluate the profiles of the bone strength of rats treated from infancy with various drugs. Young female Sprague-Dawley rats were classified into five groups according to the composition of their diets. They underwent resection of their femurs for a three-point flexion test and an impaction test at 6, 8, 12, and 16 months of age. A microcomputed tomography unit was used to evaluate the microstructure of their femoral condyles at 16 months of age. The diet given to the rats in the control group contained 0.5% Ca. The rats in groups A, B, C, and D were placed on the following regimens, respectively: vitamin K mixed diet, vitamin D oral administration, 1.8% Ca-fortified diet, and 1.8% Ca-fortified diet plus vitamin K and vitamin D. In the impaction tests conducted on the rats at each age, the results from groups A and B were nearly the same as those from the control group. At 16 months of age, the rats in groups C and D had significantly higher test results than those in the control group. In the three-point flexion tests at 16 months of age, the results from groups A, B, and C were nearly the same as those from the control group. The results from group D, however, showed a significant increase. Examination of the microstructure of the femoral condyles at 16 months of age revealed controlled destruction of the trabecular structure in groups C and D. These findings suggest that supplementing the diet from infancy with Ca, vitamin K, and vitamin D might prevent bone fractures due to osteoporosis. Received: September 5, 2001 / Accepted: May 23, 2002     

Cytokine Production and Bone Mineral Density at the Lumbar Spine and Femoral Neck in Premenopausal Women

Cytokines such as interleukin-1 (IL-1β), interleukin-6 (IL-6) and tumor necrosis factor (TNF-α) can influence both bone resorption and bone formation. The objective of this cross-sectional study was to examine the relationship between cytokine production by peripheral blood mononuclear cells (PBMC) and bone mineral density (BMD); the annual rate of change in BMD was examined. Subjects participating in a randomized clinical trial entitled the Women's Healthy Lifestyle Project in Allegheny County, Pennsylvania were used. They included 50 healthy premenopausal women, aged 45–52 years, who had regular menses within the past 3 months and were not on replacement estrogens. Dual-energy X-ray absorptiometry measurements at the AP lumbar spine and femoral neck were made at baseline and at the first annual exam using a Hologic QDR 2000 densitometer. Cytokine production of IL-1β, IL-6, and TNF-α by PBMC was measured at the annual exam. The median values for stimulated cytokine production by PBMC were 3.92 ng/ml, 31.3 ng/ml, and 1.05 ng/ml, for IL-1β, IL-6, and TNF-α, respectively. There were modest correlations between cytokine production and cross-sectional BMD, ranging from r =−0.30 to r =−0.13. Trends of greater spinal bone loss were observed in women with ``high' (≥75th percentile) cytokine production of stimulated IL-1β and IL-6 (IL-1β: ``high' =−1.56% ± 0.70 versus ``low' (<75th percentile) =−0.56% ± 0.35, P= 0.21). In contrast, greater annual gains in femoral neck BMD were observed in those with high cytokine production of IL-1β and IL-6 (IL-1β: high = 3.39% ± 1.16 versus low =−0.85 ± 0.58, P= 0.002). There was no association between stimulated TNF production and annual change in BMD. In this population of healthy premenopausal women, the relationship between cytokine production by PBMC and the rate of change in BMD was significantly different for the lumbar spine and femoral neck, possibly reflecting differences in the proportion of trabecular and cortical bone at these sites. Received: 5 February 1997 / Accepted: 11 May 1998     

The synthetic phytoestrogen, ipriflavone, and estrogen prevent bone loss by different mechanisms

Ipriflavone (IP), a synthetic isoflavone has been reported to prevent bone loss in both postmenopausal women and ovariectomized (ovx) rats. The purpose of this study was to compare and contrast some of the bone protective mechanisms of IP to those of 17β-estradiol (E₂) in ovarian hormone deficiency. Forty-eight 95-day-old Sprague-Dawley rats were assigned to four groups: sham, ovx, ovx+IP, and ovx+E₂. The doses of IP and E₂ were 100 mg and 10 μg/kg body weight per day, respectively. Rats were fed a diet that contained 0.4% calcium, 0.3% phosphorus, and 0.195 nmol vitamin D₃/g diet. After sacrifice, left femoral bone densities were measured and bone histomorphometry was performed on the proximal tibial metaphysis. Ipriflavone as well as E₂ treatment completely prevented the ovx-induced femoral bone density loss. However, in contrast to E₂, IP did not lower the ovx-induced rise in serum alkaline phosphatase (ALP) activity or insulin-like growth factor (IGF)-I and IGF binding protein (IGFBP)-3 concentrations. On histomorphometry analysis, the ovariectomy-induced increase (P < 0.09) in bone formation rate (BFR) was significantly (P < 0.05) suppressed by E₂ treatment, whereas this higher BFR was maintained in IP-treated animals. These findings indicate that IP is effective in preventing the ovx-associated bone loss. The bone protective mechanisms of IP in ovarian hormone deficiency may be different from those of E₂ and may involve increased rates of bone formation. Received: 21 October 1998 / Acccepted: 26 July 1999     

Anabolic Effect of Prostaglandin E2 in Bone Is not Dependent on Pituitary Hormones in Rats

Prostaglandin E₂ (PGE₂) is an anabolic agent of bone in vivo but the mechanism of its action still remains unclear. The aim of this study was to determine whether the effect of PGE₂ on skeleton is mediated by pituitary hormones. Forty female, Sprague-Dawley rats were divided into four groups: baseline control (basal), age-matched intact control (CON), hypophysectomy (HX), and HX + PGE₂ (2 mg/kg/day) with 10 animals in each group. The basal group was sacrified at 2 months of age, and the remaining groups after 6 weeks of treatment. Cancellous and cortical bone histomorphometry was performed on double fluorescent-labeled 40 μm-thick sections of the proximal tibia and tibial shaft. Our results show that HX resulted in a cessation of bone growth, a decrease in cancellous bone volume, and cortical bone gain compared with the age-matched, intact CON rats. Compared with the HX group, the HX + PGE₂ group had a significantly greater tibial bone density (mean ± SE, HX + PGE₂:1.595 ± 0.007 versus HX:1.545 ± 0.013), percent cancellous bone volume (21.4 ± 2.0 versus 8.41 ± 1.70), percent cortical bone area (87.2 ± 0.85 versus 81.7 ± 0.7), and ratio of cortical area to marrow area (7.14 ± 0.56 versus 4.52 ± 0.21). Increased bone masses by PGE₂ in the HX animals were accompanied by an increase in the trabecular and endosteal-labeled surface and bone formation rate. The trabecular number and width were increased whereas trabecular separation was decreased in the HX + PGE₂ group compared with the HX group (P < 0.05). PGE₂ treatment also caused a decrease in the tibial endosteal eroded surface and medullar cavity of the HX animals. In conclusion, this study clearly demonstrates that PGE₂ (2 mg/kg/day) in the HX rats increases both cortical and cancellous bones and improves trabecular architecture in the tibia after 6 weeks of treatment. These skeletal alterations are due to a stimulation of bone formation and a suppression of bone resorption activity. These findings suggest that the anabolic effect of PGE₂ in bone is independent of pituitary hormones.     

Comparison of Three Bone Densitometry Methods in Osteoporotic Women

Three techniques of bone mass measurement were evaluated in the diagnosis of postmenopausal osteoporosis; the overlap in the measurements and the capacity for discriminating was determined among 51 postmenopausal normal (mean age 66.6 ± 8.4 years) and 42 postmenopausal osteoporotic women (mean age 68.5 ± 7.5 years). All bone mass was evaluated by total body bone mineral content (BMC_TB), density (BMD_TB), ultrasound bone velocity (UBV) in proximal phalanxes 2–5 of the nondominant hand (UBV = mean value of all ultrasound measurements), and peripheral quantitative computed tomography of the nondominant forearm (pQCT). BMC_TB was found to be significantly better (P < 0.0001) for diagnosing postmenopausal osteoporosis than the other methods; both cortical and trabecular pQCT measurements were more discriminating than the corresponding UBV measurements (P < 0.001). T-score values in normals, subjects versus osteoporotic ones were BMC_TB−1.15 ± 0.79 versus −3.17 ± 0.74; BMD_TB−1.01 ± 0.97 versus −3.28 ± 0.81; UBV −1.51 ± 1.02 versus −2.34 ± 1.21; trabecular-pQCT −0.40 ± 0.72 versus −1.57 ± 0.37; cortical-pQCT −1.00 ± 0.87 versus −2.67 ± 0.53; and total-pQCT −0.65 ± 1.01 versus −2.34 ± 0.27, respectively. The overlap in values between the postmenopausal normal and postmenopausal osteoporotic groups was 50% with UBV, 6% with BMC_TB, 9% with BMD_TB, 25% with cortical pQCT, and 42% with trabecular pQCT. BMC_TB, BMD_TB, UBV, and pQCT correlated well with each other as measurements of bone mass, but BMC_TB was more discriminating than the other measurements in the diagnosis of osteoporosis. Received: 7 June 1995 / Accepted: 21 May 1997