2-Methoxyestradiol Inhibits Longitudinal Bone Growth in Normal Female Rats

2-Methoxyestradiol (2-MeO-E₂), a major metabolite of 17β-estradiol, may function as a physiological tumor suppressor and is being investigated for clinical applications. It has been reported to target rapidly dividing cells. We investigated the effects of 2-MeO-E₂ on the growth plate of young rats because normal longitudinal bone growth requires rapid proliferation of cartilage and endothelial cells. Sexually mature (3-month-old) normal female rats were treated with 2-MeO-E₂ (100 mg/kg/day) for 13 days and it was found to have no effect on uterine weight but reduced serum cholesterol. The estrogen metabolite had no effect on either cortical or cancellous bone. In contrast, 2-MeO-E₂ dramatically reduced longitudinal bone growth rate at the proximal tibia from 55 ± 2 to 20 ± 2 μm/day (P < 0.001) and growth plate thickness from 153 ± 14 to 70 ± 6 μm (P < 0.001). The latter decrease was due to significant reductions in the height of both the proliferative (P < 0.001) and the hypertrophic (P < 0.001) zones. These results in normal female rats demonstrate that 2-MeO-E₂ inhibited longitudinal bone growth but had no effect on either radial bone growth or cancellous bone turnover. 2-MeO-E₂ was shown by these studies to have the ability to discriminate between bone and cartilage, as well as between reproductive and nonreproductive estrogen-target tissues. Thus, 2-MeO-E₂ is a naturally produced estrogen metabolite that demonstrates unique tissue selectivity. Received: 21 July 1999 / Accepted: 12 January 2000     

Effect of Lead on Bone Development and Bone Mass: A Morphometric,Densitometric, and Histomorphometric Study in Growing Rats

The effect of exposure to lead on the longitudinal development of bone and on bone mass was studied in rats. A group of 35, 50-day-old female Wistar rats was divided into a control group of 15 rats and an experimental group of 20 rats fed a diet supplemented with 17 mg of lead acetate per kg feed for 50 days. Total body bone densitometry (TBBMC) was performed the day before ending the 50-day experiment. On day 50, all rats were killed and their right femur and 5th lumbar vertebra were dissected. The bones were cleaned of soft tissue and femoral length and vertebral length were measured with a caliper and all bones were weighed on a precision scale. Final body weight (P < 0.05), TBBMC (P < 0.005), and femur weight (P < 0.005) were significantly lower in the control group. Femur length did not differ between groups, but the length of the 5th lumbar vertebra was greater in the control group (P < 0.05). Histomorphometry of the femur showed that Cn-BV/TV, Tb-N, Tb-Th were lower (P < 0.05 in all) and Tb-Sp was higher (P < 0.05) in the group given the lead-supplemented diet. These findings suggested lead-induced inhibition of axial bone development and a histomorphometric decrease in bone mass, produced mainly by enhanced resorption, and a densitometric increase in bone mass, produced by lead accumulation in bone. Received: 12 February 1996 / Accepted: 15 July 1996     

Effects of Indomethacin Administration on Bone Turnover and Bone Mass in Adjuvant-Induced Arthritis in Rats

We examined the bone turnover and bone mass in adjuvant-induced arthritis in rats and assessed the effects of indomethacin in this model. One hundred ten SD rats, 6 weeks of age, were assigned to 11 groups and injected with adjuvant or solvent in the right foot. Adjuvant-injected rats were orally administered indomethacin at doses of 0 (vehicle), 0.1 (low), 0.5 (medium), and 1.5 (high) mg/kg body weight from the start (day 0). Animals were sacrificed on days 0, 14 (acute phase), and 28 (chronic phase). In the arthritic-control group, serum osteocalcin level and bone mineral content of the fourth lumbar body (L4) and the femur were significantly reduced on day 14. Serum alkaline-phosphatase was increased on day 28. Trabecular bone volume of L4 was decreased on day 14, and the value was further decreased on day 28. Bone formation rate (BFR/BS) was significantly reduced on day 14, and then osteoclast number (Oc.N/BS) increased on day 28. Indomethacin treatment dose-dependently prevented increases in paw volume and osteoclast number. In the high dose group, these indices were maintained at the same level with those in the normal group. However, indomethacin treatments were not able to maintain the parameters of bone formation such as serum osteocalcin and BFR/BS values, and the trabecular bone mass decrease was only partially prevented. These data clearly indicated both reduced bone formation and increased bone resorption as the causes of bone loss in adjuvant-induced arthritis in rats. Increased bone resorption seemed to be due to the increased activity of prostaglandins, but bone formation defect would be related to other factors in this animal model. Received: 13 January 1996 / Accepted: 3 May 1996     

Effect of Lifelong Nicotine Inhalation on Bone Mass and Mechanical Properties in Female Rat Femurs

As tobacco smoking has been identified as a risk factor in the development of osteoporosis, possible deleterious effects of nicotine inhalation on bone mineral density (BMD) and mechanical properties of the femur in female rats were studied. Female Sprague Dawley rats were exposed to nicotine vapour 20 hours a day 5 days a week for 2 years. The nicotine concentration in the inhaled air was kept at a level, giving a plasma nicotine concentration exceeding that of heavy smokers. Throughout the study, the nicotine-exposed rats weighed approximately 10% less than the control rats. At the end of the study the rats were anesthesized and blood was collected by heart puncture for determination of nicotine in plasma. Both femurs were resected and scanned by dual X-ray absorptiometry (DXA). There was no difference in BMD between control rats (n = 7) and nicotine-exposed rats (n = 23) (mean 0.216 ± 0.021 g/cm² and 0.210 ± 0.014 g/cm², respectively (P= 0.19)). The left femur was used for mechanical testing of the shaft and the neck. No significant difference could be demonstrated in ultimate bending moment, ultimate energy absorbtion, stiffness, or deflection between the two groups. In conclusion, no negative effects of nicotine inhalation on the femurs of female rats were found. Received: 26 December 1997 / Accepted: 28 January 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.     

Prepubertal Oophorectomy Limits the Accumulation of Cancellous Bone in the Femur of Growing Rats with Long-Term Effects on Metaphyseal Bone Architecture

Bone loss after oophorectomy of adult rats is more rapid and complete in the metaphysis than in the epiphysis of the femur, particularly in the proximal region of the metaphysis distant from the growth plate. This study was undertaken to determine the effects of prepubertal oophorectomy on femoral cancellous bone acquisition during growth. Rats were oophorectomized (OVX) or sham operated at 3 weeks of age and killed at intervals up to 78 weeks for scanning electron microscopy and histomorphometry of the distal femur. Differences in cancellous bone architecture between the two groups was evident after 6 weeks of age. Relatively minor differences were found in the part of the metaphysis near the growth plate and in the epiphysis, with less trabeculae in the primary spongiosa and 1 to 2 less trabeculae/mm in the secondary spongiosa. However, as metaphyseal growth proceeded, trabeculae were present for a greater distance up the femoral shaft in controls than in OVX rats, with mean BV/TV in the proximal part of the metaphysis increasing from 1.4% at 6 weeks to 13.4% at 20 weeks in controls, with no increase in the OVX rats. We find that the lack of ovarian hormones increases the rate of destruction of trabeculae near the metaphyseal-diaphyseal junction. Received: 25 February 1997 / Accepted: 17 June 1997     

Effects of Pregnancy and Lactation on Trabecular Bone and Marrow Adipocytes in Rats

Changes in the structure and metabolism of trabecular bone and marrow adipocytes in rats during pregnancy and the early stage of postpartum were evaluated by investigating bone mineral density (BMD) and bone and fat histomorphometry. Forty-nine female virgin Sprague-Dawley rats aged 200 days were mated and divided into seven groups: (1) beginning controls; (2) antepartum-on-day-7; (3) antepartum-on-day-21; (4) nonlactating on the fourth postpartum day; (5) nonlactating on the sixth postpartum day; (6) nonlactating on the eighth postpartum day; (7) lactating on the eight postpartum day. The significant decreases occurred in the trabecular bone at the end of pregnancy and lactation, and the bone formation increases and the bone structure is almost recovered in nonlactating rats within 6 days of postpartum. The percent adipocyte volume, adipocyte number, and unit adipocyte volume significantly decreased during postpartum whether lactating or nonlactating, and they significantly showed negative correlation with the osteoid volume values. The serum triglyceride value and body weight of the seven groups correlated significantly with the unit adipocyte volume value (r = 0.49, P= 0.004; r = 0.58, P= 0.0005, respectively). We concluded that bone resorption and formation are regulated separately during late pregnancy and lactation and that the recovery of BMD from lactation appears to rely on an acceleration of bone formation. Furthermore, the metabolism of the marrow adipocyte may be correlated with bone formation rates, serum triglyceride value, and body weight during pregnancy and early stage of puerperium. Received: 5 October 1999 / Accepted: 27 April 2000 / Online publication: 27 July 2000     

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     

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     

Effects of Alprazolam Supplementation on Vertebral and Femoral Bone Mass in Rats on Strenuous Treadmill Training Exercise

The ability of alprazolam to diminish cortisol response and favor ovarian function could make it useful in the prevention of osteopenia in athletes in selected cases. A sample of 45 female Wistar rats, all 93 days old and with a mean initial weight of 267 ± 17 g, were studied. Rats were exposed to a high-performance level of exercise and were divided into two groups—one group received an alprazolam supplement and one did not—and compared with controls to determine the effect of alprazolam on bone mass as measured by dual-energy X-ray absorptiometry (DKA). Exercise consisted of treadmill running on 5 out of 7 days during a period of 11 weeks. A steep grade treadmill inclination was used to stimulate high-intensity muscle activity. Final inclination was 17.5° and treadmill speed was 45 cm/second. Upon completion of the experiment, all the rats were killed and the femur and 5th lumbar vertebra were dissected and cleaned. Length, weight, bone mineral content (BMC), and density (BMD) of the whole right femur and 5th lumbar vertebra were measured. In the exercise only group (no alprazolam), the length, weight, BMC, BMD, and femur BMC/final rat weight ratio of the femur, and the vertebral weight, vertebral BMD and BMC, and vertebral BMC/final rat weight ratio were lower than in the control and the exercise-alprazolam groups (P < 0.0167 – < 0.0001). Alprazolam preserves bone mass in rats exposed to intense exercise. Received: 1 June 1998 / Accepted: 15 December 1998     

Bone Mass in Female Volleyball Players: A Comparison of Total and Regional Bone Mass in Female Volleyball Players and Nonactive Females

The purpose of this cross-sectional study was to evaluate bone mass in female athletes participating in an impact loading sport (volleyball), and especially to investigate whether any changes in bone mass might be related to the type and magnitude of weightbearing loading and muscle strength. The volleyball group consisted of 13 first division players (age 20.9 ± 3.7 years) training for about 8 hours/week, and the reference group consisted of 13 nonactive females (age 25.0 ± 2.4 years) not participating in any kind of regular or organized sport activity. The groups were matched according to weight and height. Areal bone mineral density (BMD) was measured in total body, head, lumbar spine, femoral neck, Ward's triangle, trochanter, the whole femur, and humerus using dual-energy-X-ray absorptiometry. Isokinetic concentric peak torque of the quadricep and hamstring muscles was measured using an isokinetic dynamometer. Compared with the controls, the volleyball players had a significantly (P < 0.05–0.01) higher BMD of the total body (6.1%), lumbar spine (13.2%), femoral neck (15.8%), Ward's triangle (17.9%), trochanter (18.8%), nondominant femur (8.2%), and humerus (dominant 9.5%, nondominant 10.0%), but not of the head and the dominant whole femur. The dominant humerus showed significantly higher BMD than the nondominant humerus in both the volleyball and nonactive group (P < 0.05). There was no significant difference in muscle strength of the thigh between the two groups. In the nonactive group, muscle strength in the quadriceps, and especially hamstrings, was correlated to BMD of the adjacent bones (whole femur, hip sites) and also to distant sites (humerus). However, in the volleyball group there were no correlations between muscle strength and BMD of the adjacent bones, but quadricep strength correlated to BMD of the humerus. These results clearly show that young female volleyball players have a high bone mass. The demonstrated high bone mass seems to be related to the type of loading subjected to each BMD site. Muscle strength of the thigh seems to have little impact on BMD in female volleyball players. Received: 18 June 1996 / Accepted: 31 October 1996     

Low Bone Density with Normal Bone Turnover in Ovariectomized and Streptozotocin-Induced Diabetic Rats

Diabetes and estrogen deficit are known causes of osteopenia, diabetes being associated with a low bone turnover and estrogen deficit with a high bone turnover. In the present work, we studied the effect of combined ovariectomy and diabetes on bone mineral content (BMC) and bone mineral density (BMD) and several bone markers in the rat. Four groups of rats were studied: control (C), ovariectomized (O), diabetic (D), and ovariectomized and diabetic (DO). Twelve weeks after starting the experiments, BMC and BMD of the first six lumbar vertebrae were measured; a bone formation marker (BGP) and a bone resorption marker (free collagen cross-links, PYD) were also analyzed. Diabetic rats showed diminished gain in bone mass, BMC (D: 0.417 ± 0.028 g, DO: 0.422 ± 0.020 g) and BMDs (D: 0.171 ± 0.006 g/cm², DO: 0.174 ± 0.006 g/cm²) both being significantly (P < 0.001) lower than those of control (C: BMC 0.727 ± 0.024 g and BMD 0.258 ± 0.004 g/cm²) and ovariectomized (O: BMC 0.640 ± 0.044 g and BMD 0.240 ± 0.009 g/cm²) groups. Moreover, the BMC and BMD of the C group were significantly (P < 0.05) higher than that of the O group. BGP and PYD levels were significantly (P < 0.01) higher in the O group (BGP: 138.2 ± 16.8 ng/ml, PYD: 270.2 ± 17.8 nM/mM) than those found in the control rats (BGP: 44.7 ± 4.8 ng/ml, PYD: 165.6 ± 12.5 nM/mM); the D group showed significantly (P < 0.01) lower values (BGP: 27.4 ± 14.6 ng/ml, PYD: 55.0 ± 7.4 nM/mM) than those of the control group. The DO group showed similar levels (BGP: 43.4 ± 5.1 ng/ml, PYD: 146.7 ± 14.6 nM/mM) to those found in the C group. Although bone marker levels in the O and D groups were in accordance with those expected in these situations, in the DO group the corresponding levels are apparently ``normal.' Also, the decrease of gain in bone mass observed after combining estrogen deficit and diabetes (DO group) did not seem to be more marked than that caused by diabetes alone. Received: 7 January 1997 / Accepted: 7 August 1997     

Longitudinal Overgrowth of Bone After Osteotomy in Young Rats: Influence of Bone Stability

One hundred and five 5-week-old male rats were used to study the influence of the stability of an osteotomy on longitudinal overgrowth of the bone. In 45 rats (Group 1) a transverse diaphyseal osteotomy of the right tibia was made; the left tibia was left untreated. In the second 45 rats (Group 2) the right tibia was osteotomized after applying an external fixator; the left tibia underwent a sham operation without osteotomy. Fifteen further rats (Group 3) were used as a nonoperated control group. After being measured at different periods up to 20 weeks, the osteotomized tibiae of the Group 1 (unstable) were 16–25% longer than those of their nontreated limbs (P < 0.001). The osteotomized tibiae of the Group 2 (stable) were 6–11% longer than those of the sham-operated opposite limbs (P < 0.001). The osteotomized and unstable tibiae were 9–17% longer than the osteotomized and stable tibiae (P < 0.001) throughout the whole study. Although several factors have been considered to be responsible for longitudinal bone overgrowth after fracture in young animals, this work suggests that bone's stability plays a decisive role in the final outcome. Received: 20 February 1996 / Accepted: 24 September 1996     

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     

Bone Mass and Muscle Strength in Young Female Soccer Players

In this cross-sectional study, bone mass and muscle strength of the thigh were investigated in 51 female soccer players, age 16.3 ± 0.3 years, who had been playing soccer for 8.1 ± 2.1 years and were at the time of the study in soccer training for 5.0 ± 1.7 hours/week. They were compared with 41 nonactive females, age 16.2 ± 1.3 years. The groups were matched according to age, weight, and height. Areal bone mineral density (BMD) was measured of the total body, head, lumbar spine, femoral neck, Ward's triangle, and the greater trochanter using dual energy X-ray absorptiometry (DXA). Isokinetic muscle strength of the quadriceps and hamstrings muscles was measured using an isokinetic dynamometer. Compared with the nonactives, the soccer players had significantly higher BMD of the total body (2.7%), lumbar spine (6.1%), the dominant and nondominant hip (all sites). The largest differences were found in the greater trochanter on both sides (dominant, 16.5%, nondominant, 14.8%). The soccer players had significantly higher concentric and eccentric peak torque of the thigh muscles. In the soccer group, there was only a positive association between thigh muscle strength and BMD of the adjacent hip, and in the nonactive group there were several positive associations between muscle strength and BMD. However, when adjusting for the variation in weight and height all these associations became nonsignificant. Using multiple linear regression, the type of activity (soccer player, nonactive) independently predicted BMD of all dominant hip sites (β= 0.32–0.48, P < 0.01). No other variable was found to independently predict BMD of any site. In the younger subjects (≤16 years) only BMD of the greater trochanter was significantly higher in the soccer players. In the older subjects (>16 years) the soccer players had significantly higher BMD in all measured sites except for the nonweight-bearing head. The differences in muscle strength between soccer players and nonactives were already seen in the young age group. In conclusion, girls who train and play soccer in adolescence have a higher bone mass in the hip and lumbar spine, and a higher muscle strength of the thigh compared with nonactive controls, indicating a site-specific skeletal response of weight-bearing and impact-loadng acting on the skeleton. The differences in bone mass were already apparent in early adolescence, but became more pronounced in late adolescence, probably explained by a longer exposure to soccer training with time. Our results also indicate that muscle strength in itself might not be of decisive importance for bone mass in the hip of adolescent females. Received: 18 December 1998 / Accepted: 14 April 2000     

Effects of Magnesium Deficiency on Magnesium and Calcium Content in Bone and Cartilage in Developing Rats in Correlation to Chondrotoxicity

Quinolone-induced arthropathy has been described in juvenile rats between 3 and 6 weeks of age, but not in adult rats. The mechanism of this chondrotoxic effect is probably related to the Mg²⁺-chelating properties of the drugs, since identical cartilage lesions were observed in magnesium-deficient juvenile rats without quinolone treatment. However, the reasons for the phase-specificity of the effect are unknown. In the present study, we fed a magnesium-deficient diet to Wistar rats at different postnatal developmental stages. Cartilage lesions were only observed in magnesium-deficient rats between 3 and 5 weeks of age, but not in rats receiving the magnesium-deficient diet during weeks 5 to 8, weeks 8 to 11, or months 15 to 16. The formation of cartilage lesions was not related to the magnesium concentration in plasma, since magnesium concentrations in plasma were similarly reduced in rats with and without cartilage lesions. However, chondrotoxicity correlated with magnesium content in articular cartilage. In articular cartilage (articular and epiphyseal cartilage in immature rats) and bone, magnesium content was more reduced in rats receiving the magnesium-deficient diet between 3 and 5 weeks of age as compared with rats receiving the magnesium-deficient diet during weeks 8 to 11 postnatally. It was not possible to reduce the magnesium content in bone tissue of 15-month-old Wistar rats, which suggests a lower magnesium turnover in aged rats. Magnesium content in epiphyseal cartilage of 2-week-old rats (total femoral head) was 41.9 ± 16.9 mmol/kg dry weight. The magnesium content in joint hyaline cartilage was significantly lower in 4-week-old rats (19.5 ± 3.6 mmol/kg dry weight) and increased subsequently again to 48.5 ± 9.2 mmol/kg dry weight (mean ± SD; n= 8 to 16). Increase of the magnesium content in femoral bone between weeks 4 and 6 postnatally was less pronounced (139 ± 10 and 175 ± 15 mmol/kg dry weight, respectively). Taken together, these data show that in 4-week-old rats, magnesium concentration in joint hyaline cartilage is significantly lower than at other times during postnatal development. Only at this developmental stage can cartilage lesions be induced by feeding rats a magnesium-deficient diet. This period correlates well with the sensitive phase of immature rats toward the chondrotoxic action of quinolones. Received: 30 September 1996 / Accepted: 31 December 1996     

Differences in Bone Turnover and Skeletal Response to Thyroid Hormone Treatment Between Estrogen-Depleted and Repleted Rats

This study was undertaken to compare the effect of supraphysiological doses of thyroxine (T4) on bone metabolism in SHAM and OVX young adult rats. Female Sprague Dawley rats (220 ± 2 g, approx. 5 months of age) were divided into four groups of eight animals each. The animals were intraperitoneally injected 6 days per week with vehicle (Vh): 0.001 N NaOH/0.9% NaCl (SHAM+Vh and OVX+Vh) or 250 μg of thyroxine/kg/day (SHAM+T4 and OVX+T4) during a 5-week period. Serum T4 and osteocalcin (BGP), urinary pyridinolines (Pyr), and creatinine (creat) were determined. At the beginning and at end of the experiment, skeletal bone mineral content (BMC), bone mineral density (BMD), and area (A) of the total skeleton, femur, spine, and whole tibia, as well as proximal, middle, and distal areas of the tibia were assessed by dual X-ray absorptiometry (DXA) in an ultra-high-resolution mode. T4 treatment of the SHAM rats did not induce significant changes in BGP level or Pyr/creat excretion compared with the SHAM+Vh control group. However, these two biochemical bone markers significantly increased due to T4 treatment in OVX rats compared with both OVX+Vh and SHAM+T4 groups (P < 0.05 and P < 0.001, respectively). The OVX+T4 group had a significantly lower ΔBMD than SHAM+T4 rats in all studied regions (P < 0.05) except for the middle tibia region. OVX+T4 groups presented a significantly lower ΔBMC and ΔA compared with SHAM+T4 animals (P < 0.001). OVX+T4 rats significantly impaired the ΔBMD in the femur (P < 0.01), spine (P < 0.05), whole (P < 0.05) and middle (P < 0.05) tibia whereas T4 treatment of SHAM rats only affected, significantly, the whole (P < 0.05) and the proximal tibia region (P < 0.01). T4 treatment affects bone growth in young adult rats. The effect is significantly greater in the estrogen-depleted than in the estrogen-repleted state. The bone site most adversely affected by T4 treatment depends on the estrogen status. The proximal tibia (principally trabecular bone) was the most affected area in estrogen-repleted rats. Conversely, in OVX rats, the middle tibia (principally cortical bone) presented the greatest decrease in bone density. Received: 20 May 1999 / Accepted: 4 February 2000     

Improved Bone Biomechanical Properties in Rats after Oral Xylitol Administration

The effects of 5, 10, and 20% dietary xylitol supplementations on the biomechanical properties, histological architecture, and the contents of collagen, pyridinoline, and deoxypyridinoline in long bones of rats were studied. Tibiae were used for the three-point bending test, and femurs were used for the torsion and loading test of the femoral neck. The 10 and 20% oral xylitol administrations caused a significant increase of tibial stress, femoral shear stress, and stress of the femoral neck as compared with the controls. Parallel, but not significant, effects were also seen in the 5% xylitol supplementation group. No significant differences in strain or Young's modulus of the tibiae were detected between the groups. An increased shear modulus of elasticity in femurs was detected in the 20% supplementation group as compared with the controls. The histomorphometrical data for the secondary spongiosa of the proximal tibia revealed that trabecular bone volume was significantly greater in all dietary xylitol supplementation groups as compared with the controls. The bone volume increased along with increasing xylitol content. No significant differences between the groups were detected concerning the amount of collagen per dry weight of organic matrix, the concentrations of pyridinoline or deoxypyridinoline in collagen, or the ratio of these crosslinks. This suggests no xylitol-dependent selective changes in these structures of bone collagen. In conclusion, dietary xylitol supplementation in rats improves the biomechanical properties of bone and increases the trabecular bone volume dose dependently. Received: 30 January 1997 / Accepted: 1 October 1998     

Dietary Xylitol Retards the Ovariectomy-Induced Increase of Bone Turnover in Rats

The effects of 10% dietary xylitol supplementation in ovariectomized rats were studied on the degradation of bone organic and inorganic structures. The osseal concentrations of hydroxyproline, pyridinoline, and deoxypyridinoline were analyzed by high-performance liquid chromatography. Bone resorption was measured in [³H]tetracycline-prelabeled rats by urinary excretion of ³H, and by the amount of ³H preserved in bone. Bone trabeculation was measured by a computer image analyzer from sections stained by the method of von Kossa. The amount of collagen in bone organic fraction was lower in ovariectomized rats as compared with the sham-operated controls. This most likely is partly a consequence of an increased resorption, and partly a consequence of a higher proportion of immature periosteal bone in the ovariectomized animals, leading to a higher ratio of noncollagenous protein to collagen. The number of pyridinium crosslinks was lower in proportion, indicating no selective changes in the structure of collagen. Dietary xylitol significantly retarded the ovariectomy-associated decrease in the relative amount of collagen and the number of its mature crosslinks. Ovariectomy doubled the excretion of ³H and caused a significant decrease in the amount of ³H preserved in bone; both these changes were significantly retarded by the 10% dietary xylitol supplementation. Ovariectomy significantly decreased the volume of bone trabeculae, but this effect was also significantly inhibited by the xylitol supplementation in the diet. In conclusion, these findings suggest a dietary xylitol-induced normalizing effect on the rate of bone turnover in ovariectomized rats. Received: 12 February 1996 / Accepted: 20 August 1996     

A 12-Month Prospective Study of the Relationship Between Stress Fractures and Bone Turnover in Athletes

Bone remodeling may be involved in the pathogenesis of stress fractures in athletes. We conducted a 12-month prospective study to evaluate bone turnover in 46 female and 49 male track and field athletes aged 17–26 years (mean age 20.3; SD 2.0) 20 of whom developed a stress fracture. Baseline levels of bone turnover were evaluated in all athletes and monthly bone turnover levels were evaluated in a subset consisting of the 20 athletes who sustained a stress fracture and a matched comparison group who did not sustain a stress fracture. Bone formation was assessed using serum osteocalcin (OC) measured by human immunoradiometric assay and bone resorption by urinary excretion of pyridinium cross-links (Pyr and D-Pyr); high performance liquid chromatography and N-telopeptides of type 1 collagen (NTx) using ELISA assay. Athletes who developed stress fractures had similar baseline levels of bone turnover compared with their nonstress fracture counterparts (P > 0.10). Results of serial measurements showed no differences in average levels of Pyr, D-Pyr, or OC in those who developed stress fractures (P= 0.10) compared with the control group. In the athletes with stress fractures, there was also no difference in bone turnover levels prior to or following the onset of bony pain. Our results show that single and multiple measurements of bone turnover are not clinically useful in predicting the likelihood of stress fractures in athletes. Furthermore, there were no consistent temporal changes in bone turnover associated with stress fracture development. However, our results do not negate the possible pathogenetic role of local changes in bone remodeling at stress fracture sites, given the high biological variability of bone turnover markers and the fact that levels of bone turnover reflect the integration of all bone remodeling throughout the skeleton. Received: 12 August 1997 / Accepted: 8 January 1998