MicroRaman Spectral Study of the PO4 and CO3 Vibrational Modes in Synthetic and Biological Apatites

The carbonate and phosphate vibrational modes of different synthetic and biological carbonated apatites were investigated by Raman microspectroscopy, and compared with those of hydroxyapatite. The ν₁ phosphate band at 960 cm⁻¹ shifts slightly due to carbonate substitution in both A and B sites. The spectrum of type A carbonated apatite exhibits two ν₁ PO₄ ³⁻ bands at 947 and 957 cm⁻¹. No significant change was observed in the ν₂ and ν₄ phosphate mode regions in any carbonated samples. The ν₃ PO₄ ³⁻ region seems to be more affected by carbonation: two main bands were observed, as in the hydroxyapatite spectrum, but at lower wave numbers. The phosphate spectra of all biominerals apatite were consistent with type AB carbonated apatite. In the enamel spectrum, bands were observed at 3513 and at 3573 cm⁻¹ presumably due to two different hydroxyl environments. Two different bands due to the carbonate ν₁ mode were identified depending on the carbonate substitution site A or B, at 1107 and 1070 cm⁻¹, respectively. Our results, compared with the infrared data already reported, suggest that even low levels of carbonate substitution induce modifications of the hydroxyapatite spectrum. Increasing substitution ratios, however, do not bring about any further alteration. The spectra of dentine and bone showed a strong similarity at a micrometric level. This study demonstrates the existence of acidic phosphate, observable by Raman microspectrometry, in mature biominerals. The HPO₄ ²⁻ and CO₃ ²⁻ contents increase from enamel to dentine and bone, however, these two phenomena do not seem to be correlated. Received: 5 January 1998 / Accepted: 12 May 1998     

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     

1α-Hydroxyvitamin D2 Partially Dissociates Between Preservation of Cancellous Bone Mass and Effects on Calcium Homeostasis in Ovariectomized Rats

Vitamin D metabolites can prevent estrogen depletion-induced bone loss in ovariectomized (OVX) rats. Our aim was to compare the bone-protective effects of 1α,25-dihydroxyvitamin D₃ (1,25(OH)₂D₃), 1α,25-dihydroxyvitamin D₂ (1,25(OH)₂D₂), 1α-hydroxyvitamin D₃ (1α(OH)D₃), and 1α-hydroxyvitamin D₂ (1α(OH)D₂) in OVX rats. 1α(OH)D₃ and 1α(OH)D₂ are thought to be activated in the liver to form 1,25(OH)₂D₃ and 1,25(OH)₂D₂, respectively. Forty-four 12-week-old female Fischer-344 rats were either OVX or sham-operated (SHAM). Groups of OVX rats (n = 7 each) received vehicle alone, 1,25(OH)₂D₃, 1,25(OH)₂D₂, 1α(OH)D₃, or 1α(OH)D₂, starting 2 weeks after surgery. All vitamin D metabolites were administered orally at a dose of 15 ng/day/rat. Urine and blood samples were collected 6, 9, 12, and 16 weeks after surgery. Serum samples were analyzed for total calcium and phosphate. Calcium, phosphate, creatinine, and free collagen cross-links (ELISA) were determined in urine. After tetracycline double labeling, the rats were sacrificed 16 weeks postsurgery, and the proximal tibiae and the first lumbar vertebrae were processed undecalcified for static and dynamic bone histomorphometry. 1,25(OH)₂D₃ and, to a slightly lesser extent, 1,25(OH)₂D₂ elevated vertebral cancellous bone mass in OVX rats to a level beyond that observed in SHAM animals, and both compounds increased serum calcium and urinary calcium excretion to similar extents. 1α(OH)D₃ and 1α(OH)D₂ resulted in a 64% and 84%, respectively, inhibition of ovariectomy-induced vertebral cancellous bone loss. In the proximal tibial metaphysis, all vitamin D metabolites tested could only partially prevent post-OVX trabecular bone loss, with a tendency for 1α(OH)D₃ to be the least active compound. The effects of 1α(OH)D₃ and 1α(OH)D₂ on calcium homeostasis differed markedly, however. The mean increase in urinary calcium excretion over the whole experiment was fivefold for 1α(OH)D₃, whereas the corresponding increase for 1α(OH)D₂ was only twofold. We conclude that, compared with 1α(OH)D₃, 1α(OH)D₂ combined at least equal or higher bone-protective activity in OVX rats with distinctly less pronounced effects on calcium homeostasis. This effect was not due to a differential action of the corresponding main activation products, 1,25(OH)₂D₃ and 1,25(OH)₂D₂. Received: 2 May 1996 / Accepted: 18 October 1996     

Fourier transform infrared microscopy of calcified turkey leg tendon

Fourier transform infrared microscopy (FT-IRMS) was used to monitor spatial variations in the quality and quantity of the mineral phase in calcified turkey tendon. Spectral maps were generated by analysis of 50 μm ×~ 50 μm areas within different regions of the tendon. Spectra of the transitional region, where nonmineralized matrix ends and mineralized matrix begins, revealed marked changes in the spectrally determined mineral-to-matrix ratio, whereas regions deeper into the mineralization front showed a relatively constant ratio. Since spectra of EDTA-demineralized matrix were similar to those of nonmineralized matrix, the nonmineralized regions of the tendon were used for spectral subtraction. The broad, relatively featureless contour of the mineral ν₁,ν₃ phosphate region (900–1200 cm⁻¹) showed only subtle changes at different stages of mineralization. Second derivatives of these spectra were calculated and compared with those of synthetic, poorly crystalline hydroxyapatite (HA). The peak positions seen in second-derivative spectra of the mineral near the transitional region were within ±2 cm⁻¹ of the least mature synthetic HAs whereas spectra of the mineral deeper into the mineralization front were within ±2 cm⁻¹ of the most mature synthetic HAs. Spectra from cross- and longitudinal sections at equivalent positions in the tendon, and polarized FT-IRMS data were analyzed to determine the effect of mineral orientation on the parameters used to characterize the mineral. Spectra of cross- and longitudinal sections of the tendon showed no major differences in either the ν₁,ν₃ phosphate region or the amide I, II, or III components (1200–1800 cm⁻¹). However, polarized FT-IR spectra revealed dramatic differences in both of these regions. Despite these differences, second-derivative analysis of the ν₁,ν₃ regions revealed no significant changes in the positions of the underlying bands used to characterize the environments of the phosphate ion in poorly crystalline HA. The results of this study demonstrate the power of FT-IRMS to monitor spatial variations of the mineral phase in calcified tissue. Also, the incorporation of polarized radiation provides a method capable of assessing the molecular orientation of the mineral phase relative to the collagen matrix. Received: 17 January 1995 / Accepted: 26 May 1995     

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.     

Prophylactic Effects of 1,24,25-Trihydroxyvitamin D3 on Ovariectomy-Induced Cancellous Bone Loss in the Rat

Vitamin D metabolites can prevent estrogen depletion-induced bone loss in ovariectomized (OVX) rats. In this study, we investigated the bone-sparing effects of oral 1α,24R,25-trihydroxyvitamin D₃ (1,24,25(OH)₃D₃) in a wide dose range in aged OVX rats. Fifty-three female Fischer-344 rats (6 months old, 170 g BW) were either ovariectomized or sham-operated (SHAM). Eight rats served as baseline controls. Groups of OVX rats (n= 7–8 each) received vehicle alone or graded oral doses of 1,24,25(OH)₃D₃ (0.05, 0.1, 0.2, and 0.3 μg/kg BW/day), starting five days after surgery. Urine and blood samples were collected one, two, three, and four months after surgery. Serum samples were analyzed for total calcium and alkaline phosphatase. Calcium, hydroxyproline, and collagen crosslinks (HPLC) were determined in urine. After fluorochrome double labeling, the rats were sacrificed four months postsurgery and the first lumbar vertebrae and the proximal tibiae were processed undecalcified for bone histomorphometry. Ovariectomy induced a 28% and a 69% reduction in vertebral and tibial cancellous bone area, respectively. Osteopenia in OVX rats was associated with increased histomorphometric and biochemical indices of bone turnover. The administration of 1,24,25(OH)₃D₃ to OVX rats dose-dependently increased vertebral and tibial cancellous bone mass, serum calcium, and urinary calcium excretion, and reduced histomorphometric and biochemical indices of bone resorption. 1,24,25(OH)₃D₃ at doses of 0.2 and 0.3 μg/kg/day produced strong anabolic effects, especially on vertebral cancellous bone in OVX rats, and increased mineral apposition rate and wall width of completed remodeling units relative to vehicle-treated OVX rats. Even at high doses, 1,24,25(OH)₃D₃ did not impair bone mineralization. We conclude that oral administration of 1,24,25(OH)₃D₃ can effectively prevent estrogen depletion-induced cancellous bone osteopenia in the aged OVX rat model. The therapeutic window for 1,24,25(OH)₃D₃ in OVX rats, however, is also narrow, comparable to that for calcitriol. Received: 29 March 1996 / Accepted: 23 September 1996     

Net Fluxes Over Working Thigh of Hormones,Growth Factors and Biomarkers of Bone Metabolism During Short Lasting Dynamic Exercise

The purpose of this study was to evaluate the responses of hormones, growth factors, and biomarkers involved in bone and muscle metabolism during exercise and in recovery. One leg knee-extension exercise and concomitant sampling from the artery and vein were performed. In 12 healthy individuals (6 men and 6 women; age 21–36 years) blood was drawn from the femoral artery and vein at rest, after 10 minutes warm-up, after 15 minutes work at 61% of peak one leg VO₂, and after 5 minutes work at peak one leg VO₂, as well as 5, 30, and 60 minutes in recovery. Blood flow in the femoral vein was measured using the thermodilution technique. Arteriovenous differences were measured over working thigh for growth hormone (GH), insulin-like growth factor I (IGF-I), insulin-like growth factor binding protein 3 (IGF BP3), parathyroid hormone (PTH) and bone biomarkers, i.e., the carboxyterminal propeptide of type I procollagen (PICP), the carboxyterminal cross-linked telopeptide of type I collagen (ICTP), osteocalcin, and bone-specific alkaline phosphatase (b-ALP). There was an uptake of GH (3.1 ± 1.2 mU · min⁻¹, P < 0.001; mean ± SE) over thigh during exercise and a release of IGF-I at the end of exercise (60 ± 36 μg · min⁻¹; P < 0.01). PICP was also released after the maximal exercise (23 ± 12 μg · min⁻¹; P < 0.01) as well as ICTP (0.5 ± 0.3 μg · min⁻¹; P < 0.05) and b-ALP (0.2 ± 0.1 μkat · min⁻¹; P < 0.05). Osteocalcin, IGF BP3, and PTH revealed no clearcut pattern. In the present study, exercise induces endocrine changes which point to anabolic effects on muscle and bone tissue. Received: 12 February 1996 / Accepted: 6 June 1996     

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     

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     

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     

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     

Transforming Growth Factor-β2 mRNA Level in Unloaded Bone Analyzed by Quantitative In Situ Hybridization

The effects of tail suspension hypokinesia on the gene expression for TGF-β₂ at different sites within bone were evaluated. TGF-β₂ mRNA signal levels were determined quantitatively by an image analysis system. The osteopenia induced by tail suspension was verified by histomorphometry. In the periosteum of nonsuspended control rats, TGF-β₂ mRNA was highly expressed in the preosteoblasts and osteoblast-rich cambial layers; very little signal was present within the middle and outer fibroblastic layers. Gene expression was significantly reduced in suspended rats, and this was evident both in terms of the number of silver grains in unit area or length of tissue and in each osteoblast and preosteoblast. Hypokinesia also reduced the expression of TGF-β₂ mRNA level in cortical and trabecular bone osteocytes, but did not adversely affect the mRNA level in chondrocytes in growth plate. The results affirm the site-specific response of TGF-β₂ gene expression in rats, and suggest that the cortical and trabecular bone osteopenia associated with hypokinesia in rats may be associated with a deficit in osteoblastic and osteocytic TGF-β₂ level. Received: 6 February 1998 / Accepted: 10 November 1998     

The Stimulation of Vertebral and Tibial Bone Growth by the Parathyroid Hormone Fragments, hPTH-(1-31)NH2, [Leu27]cyclo(Glu22-Lys26)hPTH-(1-31)NH2, and hPTH-(1-30)NH2

The native human parathyroid hormone, hPTH-(1-84), and certain carboxyl truncated analogs such as hPTH-(1-34) and even smaller fragments such as hPTH-(1-31)NH₂, [Leu²⁷]cyclo(Glu²²-Lys²⁶)hPTH-(1-31)NH₂, and hPTH-(1-30)NH₂ stimulate femoral trabecular and cortical bone growth in ovariectomized (OVX) rats. Here we show that when injected once daily for 6 weeks starting 2 weeks after OVX in doses of 1 or 2 nmol/100 g of body weight, hPTH-(1-31)NH₂, [Leu²⁷]cyclo(Glu²²-Lys²⁶)hPTH-(1-31)NH₂, and hPTH-(1-34)NH₂ prevented the loss of trabecular volume in the L5 vertebrae induced by OVX. In fact, by the end of the sixth week of injections (i.e., the eighth week after OVX) the fragments had increased the volume and trabecular thickness significantly above the values in vehicle-injected sham-operated rats. hPTH-(1-30)NH₂ can stimulate vertebral bone growth as much as the larger fragments, but 10–25 times more of it was needed to do so. The same daily doses of hPTH-(1-31)NH₂, [Leu²⁷]cyclo(Glu²²-Lys²⁶)hPTH-(1-31)NH₂, and hPTH-(1-34)NH₂ also raised the trabecular volume and thickness in the L5 vertebrae of rats well above the values in vehicle-treated animals when the injections were started 9 weeks after OVX. This restoration of trabecular bone in the L5 vertebrae in estrogen-deprived animals was accompanied by a significant increase in the bone mineral density (BMD) of the L1–L4 vertebrae and tibias. However, there was no significant drop in the pelvic BMD in the estrogen-deprived animals and the effects of hPTH-(1-31)NH₂, [Leu²⁷]cyclo(Glu²²-(Lys) hPTH-(1-31)NH₂, and hPTH-(1-34)NH₂ on the pelvic BMD were equivocal. Received: 11 May 1999 / Accepted: 2 November 1999     

Circulating β2 Microglobulin in Relation to Bone Metabolism: Implications for Bone Loss with Aging

The aim of this cross-sectional study was to evaluate the relationships between circulating β₂ microglobulin (β₂ m) and bone mineral density (BMD), parameters of bone remodeling, vitamin D metabolites, parathyroid hormone (PTH), estradiol levels, and age in a group of 165 clinically healthy or osteoporotic, but otherwise normal untreated women. In this group of women, systemic β₂ m correlated with BMD (g/cm²) levels for total hip and Ward's triangle (r =−0.298, P < 0.0001; and r =−0.299, P < 0.0001, respectively), but only at the borderline level with BMD at the spine (r =−0.145, P= 0.0604). Serum β₂ microglobulin markedly correlated with age (r = 0.512, P= 0.0001). β₂ m levels correlated with indices of bone remodeling, as well as with serum creatinine and estradiol levels. However, after stratification of all analyses by age, body mass index, and serum 25OHD₃, 1,25(OH)₂D₃, PTH, or estradiol levels (using standard multiple regression and stepwise forward regression models), only 25OHD₃ was found to be an independent predictor of BMD at the hip, including Ward's triangle, as estradiol of BMD at the spine. On the other hand, β₂ m was not associated with BMD at any of the measured regions. Also, no association was found between serum PTH and BMD values. Therefore, systemic β₂ m seems to be an indicator of bone remodeling in the course of natural skeletal aging rather than a variable independently predicting bone loss. Received: 21 July 1998 / Accepted: 10 June 1999     

Lifestyle Determinants of Bone Mineral: A Comparison Between Prepubertal Asian- and Caucasian-Canadian Boys and Girls

The purpose of this study was to examine the difference in lifestyle and morphometric factors that affect bone mineral and the attainment of peak bone mass in 168 healthy Asian (n = 58) and Caucasian (n = 110) Canadian, prepubertal girls and boys (mean age 8.9 ± 0.7) living in close geographical proximity. DXA (Hologic 4500) scans of the proximal femur (with regions), lumbar spine, and total body (TB) were acquired. We report areal bone mineral densities (aBMD g/cm²) at all sites and estimated volumetric density (νBMD, g/cm³) at the femoral neck. Dietary calcium, physical activity, and maturity were estimated by questionnaire. Of these prepubertal children, all of the boys and 89% of the girls were Tanner stage 1. A 2 × 2 ANOVA demonstrated no difference between ethnicities for height, weight, body fat, or bone mineral free lean mass. Asian children consumed significantly less dietary calcium (35%) on average and were significantly less active (15%) than their Caucasian counterparts (P < 0.001). There were significant ethnicity main effects for femoral neck bone mineral content (BMC) and αBMD (both P < 0.001) and significant sex by ethnicity interactions (P < 0.01). The Asian boys had significantly lower femoral neck BMC (11%), aBMD (8%), and νBMD (4.4%). At the femoral neck, BMFL mass, sex, and physical activity explained 37% of the total variance in aBMD (P < 0.05). In summary, this study demonstrated differences in modifiable lifestyle factors and femoral neck bone mineral between Asian and Caucasian boys. Received: 21 July 1998 / Accepted: 30 September 1999     

Effect of 17β-estradiol-bisphosphonate conjugates,potential bone-seeking estrogen pro-drugs,on 17β-estradiol serum kinetics and bone mass in rats

In order to target 17β-estradiol directly at bone we synthesized three 17β-estradiol-bisphosphonate conjugates (E₂-BPs) with different esterase-sensitive linkers between both molecular moieties. The systemic administration of these compounds should result primarily in local estrogenic effects on bone with no or negligible systemic hormonal effects. Only if a considerable margin exists between the doses required for inhibition of bone loss and those for systemic hormonal effects can such a pro-drug be considered acceptable for patients refusing systemic estrogen replacement therapy for several reasons. The conjugates were tested in vitro for their 17β-estradiol release in rat serum and in vivo for their local and systemic effects in rats: in vitro, the conjugates expressed cleavage resistance, low cleavage (4.8%), or high cleavage (33.1%) within 48 hours of incubation. The conjugate with the low-cleavage doubled 17β-estradiol serum half-life (3.78 hours) whereas the high-cleavage conjugate resulted in approximately four times higher serum half-life (8.36 hours) when compared with free 17β-estradiol. In ovariectomized rats, bone loss was optimally prevented by 50 nmol/kg/day of 17β-estradiol when administered S.C. over a period of 5 weeks, and protection against uterine atrophy was achieved at doses as low as 5 nmol/kg/day. The cleavage-resistant conjugate was ineffective in preserving bone and uterus in doses ranging from 5 to 150 nmol/kg/day. The other two E₂-BPs revealed a dose-dependent inhibition of bone loss which was paralleled by the respective uterus weight with a dose range of 1.5–150 nmol/kg/day being fully effective in a range similar to 17β-estradiol alone. The higher sensitivity of the uterus versus bone to protective estrogenic effects (1:10) was abolished by the conjugates. We conclude that E₂-BPs containing esterase-sensitive linkers failed to act as bone-seeking pro-drugs expressing primarily local effects on bone without systemic effects. Received: 1 September 1995 / Accepted: 23 February 1996     

Sustained Osteoblast Nuclear Receptor Binding of Converted 1α,25-Dihydroxyvitamin D3 after Administration of 3H-1α-Hydroxyvitamin D3: A Combined Receptor Autoradiography and Radioassay Time Course Study with Comparison to 3H-1α,25-Dihydroxyvitamin D3

The present study was undertaken to clarify the receptor distribution and the pharmacokinetics of ³H-1α(OH)D₃, and ³H-1α,25(OH)₂D₃ for comparison. Receptor autoradiography was used after intravenous injection to 3-day-old neonatal rats and radioassay-HPLC after oral application to young adult rats. Corresponding results were obtained from both receptor autoradiography and radioassay. After ³H-1α(OH)D₃ administration, uptake was delayed but sustained over a long period of time and the concentration of silver grains (autoradiography) or recovered ³H-1α,25(OH)₂D₃ (radioassay) peaked at a lower level. After ³H-1α,25(OH)₂D₃ administration, osteoblast nuclear, whole bone uptake and retention of radiolabeled compound were relatively rapid and short in duration. Nuclear uptake in osteoblasts after administration of ³H-1α(OH)D₃ was abolished in competition studies with 10-fold unlabeled 1α,25(OH)₂D₃. These results indicate that 1α(OH)D₃ continuously supplies osteoblasts with converted 1α,25(OH)₂D₃ and would not spread to the cells because of the low binding affinity of the receptor. Accordingly, 1α(OH)D₃ appears to have some therapeutic properties different from 1α,25(OH)₂D₃ because of a relatively slow and sustained accumulation of the receptor and less C_max (pharmacokinetics) compared with 1α,25(OH)₂D₃. Received: 26 August 1997 / Accepted: 20 February 1998     

High Bone Turnover is Associated with Low Bone Mass and Spinal Fracture in Postmenopausal Women

A group of 366 healthy, white postmenopausal women, aged 50–81 years, mean age 66 years, were selected from the screened population of Scandinavians who were part of a multicenter study of the efficacy of tiludronate, a new bisphosphonate, in established postmenopausal osteoporosis. Eighty-eight women had a lumbar spine bone mineral density (BMD) above 0.860 g/cm², and 278 women had a BMD below 0.860 g/cm². Spinal fracture was diagnosed from lateral spine X-ray studies and defined as at least 20% height reduction (wedge, compression, or endplate fracture) in at least one vertebra (T4–L4). Bone resorption was assessed by measurement of the urinary excretion of type I collagen degradation products by the CrossLaps™ enzyme-linked immunoassay (ELISA). Bone formation was assessed by ELISA measurement of the N-terminal-mid-fragment as well as the intact serum osteocalcin (OC_N-MID), thus omitting the influence of the instability of osteocalcin caused by the labile 6 amino acid C-terminal sequence. The women were divided into groups with high or low bone turnover according to the concentrations of urinary CrossLaps™ or OC_N-MID. Women in the quartiles with the highest concentrations of CrossLaps [519 ± 119 μg/mmol (SD)] or OC_N-MID [44.6 ± 7.5 ng/ml (SD)] had 10–16% lower spinal BMD compared with women in the lowest quartiles (CrossLaps 170 ± 48 μg/mmol (SD), and OC_N-MID [22.1 ± 3.0 ng/ml (SD)] (P < 0.0004). The prevalences of spinal fracture were 25 to 29% in the lowest quartiles, whereas the prevalences in the highest quartiles were almost double—53–54% (P < 0.006). If the women were subgrouped according to spinal BMD and prevalence of spinal fracture, corresponding results were found. Women with a BMD less than 0.860 g/cm², without or with spinal fracture (n = 136 and n = 142), had 36–43% higher concentration of CrossLaps (P= 0.0001) and 11–15% higher concentration of OC_N-MID (P < 0.02), as compared with women with a BMD above 0.860 g/cm² and no spinal fracture (n = 84). In conclusion, the results indicate a strong association among high bone turnover, low bone mass, and prevalence of spinal fracture, which supports the theory that high bone turnover is a risk factor for spinal fracture and osteoporosis. Received: 29 February 1996 / Accepted: 9 August 1996     

Treatment of Glucocorticoid-Induced Osteoporosis with Alfacalcidol/Calcium Versus Vitamin D/Calcium

Vitamin D/calcium substitution is generally regarded as an effective first step treatment for glucocorticoid-induced osteoporosis (GIOP). The aim of our study was to evaluate the efficacy of the active vitamin D metabolite alfacalcidol (1α) compared with the native vitamin D₃ in patients with established GIOP with or without vertebral fractures. Patients on long-term corticoid therapy were given either 1 μg alfacalcidol plus 500 mg calcium per day (group A, n = 43) or 1000 IU vitamin D₃ plus 500 mg calcium (group B, n = 42). The two groups were alike in age range, sex ratio, percentages of underlying diseases, average initial bone density values (lumbar spine: mean T-score −3.28 and −3.25, respectively), and rates of vertebral and nonvertebral fractures. During the 3-year study we found a small but significant increase of lumbar spine density in group 1α (+2.0%, P < 0.0001) and no significant changes at the femoral neck. In the D₃ group, there were no significant changes at both sites. At the end of the study, 12 new vertebral fractures had occurred in 10 patients of the group 1α and 21 in 17 patients of the D₃ group. In accordance with the observed fracture rates, the alfacalcidol group showed a significant decrease in back pain (P < 0.0001) whereas no change was seen in the vitamin D group. We conclude that with the doses used in this trial, alfacalcidol is superior to vitamin D in the treatment of established GIOP.     

In Vivo Assessment of Architecture and Micro-Finite Element Analysis Derived Indices of Mechanical Properties of Trabecular Bone in the Radius

Measurement of microstructural parameters of trabecular bone noninvasively in vivo is possible with high-resolution magnetic resonance (MR) imaging. These measurements may prove useful in the determination of bone strength and fracture risk, but must be related to other measures of bone properties. In this study in vivo MR imaging was used to derive trabecular bone structure measures and combined with micro-finite element analysis (μFE) to determine the effects of trabecular bone microarchitecture on bone mechanical properties in the distal radius. The subjects were studied in two groups: (I) postmenopausal women with normal bone mineral density (BMD) (n= 22, mean age 58 ± 7 years) and (II) postmenopausal women with spine or femur BMD −1 SD to −2.5 SD below young normal (n= 37, mean age 62 ± 11 years). MR images of the distal radius were obtained at 1.5 T, and measures such as apparent trabecular bone volume fraction (App BV/TV), spacing, number and thickness (App TbSp, TbN, TbTh) were derived in regions of interest extending from the joint line to the radial shaft. The high-resolution images were also used in a micro-finite element model to derive the directional Young’s moduli (E1, E2 and E3), shear moduli (G12, G23 and G13) and anisotropy ratios such as E1/E3. BMD at the distal radius, lumbar spine and hip were assessed using dual-energy X-ray absorptiometry (DXA). Bone formation was assessed by serum osteocalcin and bone resorption by serum type I collagen C-terminal telopeptide breakdown products (serum CTX) and urinary CTX biochemical markers. The trabecular architecture displayed considerable anisotropy. Measures of BMD such as the ultradistal radial BMD were lower in the osteopenic group (p<0.01). Biochemical markers between the two groups were comparable in value and showed no significant difference between the two groups. App BV/TV, TbTh and TbN were higher, and App TbSp lower, in the normal group than the osteopenic group. All three directional measures of elastic and shear moduli were lower in the osteopenic group compared with the normal group. Anisotropy of trabecular bone microarchitecture, as measured by the ratios of the mean intercept length (MIL) values (MIL1/MIL3, etc.), and the anisotropy in elastic modulus (E1/E3, etc.), were greater in the osteopenic group compared with the normal group. The correlations between the measures of architecture and moduli are higher than those between elastic moduli and BMD. Stepwise multiple regression analysis showed that while App BV/TV is highly correlated with the mechanical properties, additional structural measures do contribute to the improved prediction of the mechanical measures. This study demonstrates the feasibility and potential of using MR imaging with μFE modeling in vivo in the study of osteoporosis. Received: 13 December 2000 / Accepted: 30 May 2001