Showing papers in "Calcified Tissue International in 2000"

Bioglass 45S5 stimulates osteoblast turnover and enhances bone formation In vitro: implications and applications for bone tissue engineering.

Abstract: We investigated the concept of using bioactive substrates as templates for in vitro synthesis of bone tissue for transplantation by assessing the osteogenic potential of a melt-derived bioactive glass ceramic (Bioglass® 45S5) in vitro. Bioactive glass ceramic and bioinert (plastic) substrates were seeded with human primary osteoblasts and evaluated after 2, 6, and 12 days. Flow cytometric analysis of the cell cycle suggested that the bioactive glass-ceramic substrate induced osteoblast proliferation, as indicated by increased cell populations in both S (DNA synthesis) and G2/M (mitosis) phases of the cell cycle. Biochemical analysis of the osteoblast differentiation markers alkaline phosphatase (ALP) and osteocalcin indicated that the bioactive glass-ceramic substrate augmented osteoblast commitment and selection of a mature osteoblastic phenotype. Scanning electron microscopic observations of discrete bone nodules over the surface of the bioactive material, from day 6 onward, further supported this notion. A combination of fluorescence, confocal, transmission electron microscopy, and X-ray microprobe (SEM-EDAX) examinations revealed that the nodules were made of cell aggregates which produced mineralized collagenous matrix. Control substrates did not exhibit mineralized nodule formation at any point studied up to 12 days. In conclusion, this study shows that Bioglass 45S5 has the ability to stimulate the growth and osteogenic differentiation of human primary osteoblasts. These findings have potential applications for tissue engineering where this bioactive glass substrate could be used as a template for the formation of bioengineered bone tissue.

Hypovitaminosis D myopathy without biochemical signs of osteomalacic bone involvement.

Abstract: The aims of this study were to investigate myopathy in relation to vitamin D status, and to study the muscular effects of vitamin D treatment on vitamin D-deficient individuals. Further, hypovitaminosis D myopathy was investigated in relation to alkaline phosphatase (ALP), the most commonly used marker for hypovitaminosis D osteopathy. Eight patients with osteomalacia had an isokinetic dynamometer test of all major muscle groups before and after 3 months of vitamin D treatment. The most pronounced improvements in muscle power were seen in the weight-bearing antigravity muscles of the lower limbs. A cross-sectional study was performed among 55 vitamin D-deficient veiled Arab women living in Denmark and 22 Danish controls. An isometric dynamometer model was used for determination of quadriceps muscle power. Both maximal voluntary contraction (MVC) and electrically stimulated values (single twitch, maximal production rate (MPR), and maximal relaxation rate (MRR)) were determined. The women underwent high-dose vitamin D treatment and were retested after 3 and 6 months. Prior to vitamin D treatment all parameters of muscle function in the group of vitamin D-deficient Arab women were significantly reduced compared with Danish controls. MVC: 259.4 +/- 11.0 N (Newton) versus 392.6 +/- 11. 4 N (P < 10(-6)), single twitch: 47.0 +/- 1.8 N versus 74.6 +/- 2.2 N (P < 10(-5)), MPR 8.9 +/- 0.3 N/10 ms versus 14.3 +/- 0.4 N/10 ms (P < 10(-6)), MRR 4.5 +/- 0.2 N/10 ms versus 6.2 +/- 0.2 N/10 ms (P < 10(-6)). Muscle function was affected to a similar degree in women with and without bone involvement (as indicated by elevated ALP). After 3 months of vitamin D treatment all muscle-related parameters improved significantly. After 6 months only MVC was reduced compared with Danish controls (320.7 +/- 14.3 N (P < 0.02)), whereas all other measurements were normalized. Hypovitaminosis D myopathy is a prominent symptom of vitamin D deficiency, and severely impaired muscle function may be present even before biochemical signs of bone disease develop. Full normalization of hypovitaminosis D myopathy demands high-dose vitamin D treatment for 6 months or more. Our findings indicate that serum levels of ALP cannot be used in the screening for hypovitaminosis D myopathy. Assessment of s-25OHD is the only reliable test.

Effects of Risedronate Treatment on Bone Density and Vertebral Fracture in Patients on Corticosteroid Therapy

Abstract: Men and women (n = 518) receiving moderate-to-high doses of corticosteroids were enrolled in two studies with similar protocols and randomly assigned to receive either placebo or risedronate (2.5 or 5 mg) for 1 year. All patients received daily calcium supplementation (500–1000 mg), and most also received supplemental vitamin D (400 IU). The primary endpoint was the difference between the placebo and active groups in lumbar spine bone mineral density (BMD) at 1 year; changes in BMD at other sites, biochemical markers of bone turnover, and the incidence of vertebral fractures were also assessed. In the overall population, the mean (SE) lumbar spine BMD increased 1.9 ± 0.38% from baseline in the risedronate 5 mg group (P < 0.001) and decreased 1.0 ± 0.4% in the placebo group (P= 0.005). BMD at the femoral neck, trochanter, and distal radius increased or was maintained with risedronate 5 mg treatment, but decreased in the placebo group. Midshaft radius BMD did not change significantly in either treatment group. The difference in BMD between the risedronate 5 mg and placebo groups was significant at all skeletal sites (P < 0.05) except the midshaft radius at 1 year. The 2.5 mg dose also had a positive effect on BMD, although of a lesser magnitude than that seen with risedronate 5 mg. A significant reduction of 70% in vertebral fracture risk was observed in the risedronate 5 mg group compared with the placebo group (P= 0.01). Risedronate was efficacious in both men and women, irrespective of underlying disease and duration of corticosteroid therapy, and had a favorable safety profile, with a similar incidence of upper gastrointestinal adverse events in the placebo and active treatment groups. Daily treatment with risedronate 5 mg significantly increases BMD and decreases vertebral fracture risk in patients receiving moderate-to-high doses of corticosteroid therapy.

Systematic Review of Randomized Trials of the Effect of Exercise on Bone Mass in Pre- and Postmenopausal Women

Abstract: Studies of the effect of exercise programs on bone mass appear inconsistent. Our objective was to systematically review and meta-analyze randomized trials of the effect of exercise on bone mass in pre- and postmenopausal women. A computerized MEDLINE search was conducted for the years 1966-1997. Thirty-five randomized trials were identified. Meta-analytic methods were used to statistically pool results of studies of the effect of impact (e.g., aerobics) and non-impact (e.g., weight training) exercise on the lumbar spine and femoral neck. The most studied bone site was the lumbar spine in postmenopausal women (15 studies), where both impact [1.6% bone loss prevented, 95% confidence intervals (CI): 1.0%-2.2%] and non-impact (1.0%, 95% CI: 0.4%-1.6%) exercise programs had a positive effect. Results for the lumbar spine in premenopausal women (eight studies) were similar: 1.5% (95% CI: 0.6%-2.4%) less bone loss (or net gain) after impact exercise and 1.2% (95% CI: 0.7%-1.7%) after non-impact exercise. Impact exercise programs appeared to have a positive effect at the femoral neck in postmenopausal women (five studies), 1.0% (95% CI: 0.4%-1.6%) bone loss prevented, and possibly in premenopausal women, 0.9% (95% CI: -0.2%-2.0%) bone loss prevented. There were too few trials to draw conclusions from meta-analyses of the effect of non-impact exercise on the neck of femur. This systematic review of randomized trials shows that both impact and non-impact exercise have a positive effect at the lumbar spine in pre- and postmenopausal women. Impact exercise probably has a positive effect at the femoral neck. More studies are required to determine the optimal intensity and type of exercise.

Serum CTX: a new marker of bone resorption that shows treatment effect more often than other markers because of low coefficient of variability and large changes with bisphosphonate therapy.

Abstract: Serum CrossLaps is a new assay for measuring carboxy-terminal collagen crosslinks (CTX) in serum. This measurement is reported to be more specific to bone resorption than other measurements. However, the utility of this and other markers in monitoring patients on antiresorptive therapy depends on how often changes anticipated with therapy exceed changes attributable to random variability. In a study where subjects received either placebo or pamidronate, we calculated the minimum significant change (MSC), that is, the change that was sufficiently large that it was unlikely to be due to spontaneous variability. We also examined the changes in markers of bone turnover in subjects treated with pamidronate (APD) (30 mg I.V. in 500 ml D5W over 4 hours) to see how often observed changes in turnover after treatment exceeded the MSC. The MSC for serum CTX was 30.2%, and was significantly (P < 0.05) lower than the MSC for urinary NTX (54.0%), and not significantly different from the MSC of urinary DPD (20.6%). Ninety percent of subjects treated with APD had a decline in serum CTX that exceeded the MSC, compared with 74% for bone-specific alkaline phophatase (BSAP), 57% for urinary N-telopeptide cross-links (NTX), and 48% for free deoxypyridinoline. Changes in serum CTX correlated reasonably well with changes in spine BMD after 2 years (r = 0.47), but this correlation did not quite reach statistical significance because of the small number of subjects. In conclusion, the serum CTX assay shows greater utility for assessing efficacy of antiresorptive treatment than some previously described markers.

Odontoblast differentiation of human dental pulp cells in explant cultures.

Abstract: In order to elucidate the mechanisms involved in human dentin formation, we developed a cell culture system to promote differentiation of dental pulp cells into odontoblasts. Explants from human teeth were cultured in Eagle's basal medium supplemented with 10% or 15% fetal calf serum, with or without β-glycerophosphate (βGP). Addition of βGP to the culture medium induced odontoblast features in the cultured pulp cells. Cells polarized and some of them exhibited a typical cellular extension. In some cases, cells aligned with their processes oriented in the same direction and developed junctional complexes similar to the terminal web linking odontoblasts in vivo. Fine structural analyses showed the presence of typical intracellular organelles of the odontoblast body, whereas the process contained only cytoskeleton elements and secretory vesicles. Polarized cells deposited onto the plastic dishes an abundant and organized type I collagen-rich matrix with areas of mineralization appearing thereafter. X-ray microanalysis showed the presence of calcium and phosphorus and the electron diffraction pattern confirmed the apatitic crystal structure of the mineral. High expression of α1(l) collagen mRNAs was detected in all polarized cells whereas dentin sialoprotein gene was mainly expressed in mineralizing areas. This cell culture system allowed for the differentiation of pulp cells into odontoblasts, at both the morphological and functional level. Moreover, these cells presented a spatial organization similar to the odontoblastic layer.

High intensity resistance training: effects on bone in older men and women.

Abstract: There is evidence that high intensity resistance training promotes bone maintenance in older women, however, the effect of high intensity free weight training has not been investigated in older men or women. Furthermore, little is known about the chronic effect of weight training on serum insulin growth factor-I (IGF-I) in this population. We compared the effects of a moderate intensity seated resistance-training program with a high intensity standing free weight exercise program on bone mass and serum levels of IGF-I and IGFBP3 in healthy older men and women. Twenty-eight men (54.6 ± 3.2 years) and 26 nonestrogen-replaced women (52.8 ± 3.3 years) served as their own controls for 12 weeks, then were randomly assigned to a moderate or high intensity training group and trained three times/week for 24 weeks. Prior to and after the control period and at the end of training, bone mass and body composition were assessed by dual energy X-ray absorptiometry (DXA), muscle strength by isokinetic dynamometry, muscular power by Wingate Anaerobic Power Test, and IGF-I by radioimmunoassay (RIA). A repeated measures analysis of covariance (ANCOVA) revealed that high intensity training resulted in a gain in spine BMD in men (1.9%), P < 0.05, but not in women, whereas moderate intensity training produced no changes in either gender at this site. Increases were observed at the greater trochanter, P < 0.03, in men regardless of training intensity, but not in women at any hip site. However, when compared with zero, both men and women in the high intensity group demonstrated significant increases in trochanteric BMD (1.3% and 2.0%, respectively) and a decrease in femoral BMD (−1.8%). Neither circulating serum IGF-I nor IGFBP3 were altered by either training regimen, but both training programs resulted in improvements in total body strength (37.62%) and lean mass (males 4.1%, females 3.1%). We conclude that although resistance training of moderate to high intensity produced similar muscle changes in older adults, a higher magnitude is necessary to stimulate osteogenesis at the spine. However, at the spine, intensity was not sufficient to offset low levels of estrogen in early postmenopausal women. Furthermore, bone changes were not accompanied by changes in circulating serum levels of IGF-I or IGFBP3.

Exercise and mechanical loading increase periosteal bone formation and whole bone strength in C57BL/6J mice but not in C3H/Hej mice.

Abstract: To identify the genes, and the mechanisms that account for the 53% higher peak bone density in C3H/HeJ (C3H) mice compared with C57BL/6J (B6) mice, we are performing quantitative trait locus and phenotypic analyses. The phenotypic studies revealed differences in bone formation and resorption, and showed that hindlimb immobilization (by sciatic neurectomy) caused a greater increase in endosteal resorption in the tibiae of B6 compared with C3H mice. The current studies were intended to examine the hypothesis that the bones of C3H mice are less sensitive to mechanical loading than the bones of B6 mice. To increase mechanical loading, 9-week-old female B6 and C3H mice (n = 10–13 mice/group) were subjected to a jumping exercise (20 jumps/day, 5 days/week, to heights of 20–30 cm) for a total of 4 weeks. Control mice did not jump. Osteocalcin, alkaline phosphatase (ALP) activity, and IGF-I were measured in serum. The left tibiae were used for histomorphometry (ground cross-sections prepared at the tibio-fibular junction) and the right tibiae and femora were used for determinations of bone breaking strength (3-point bending). The results of these studies revealed (1) significant effects of both mouse strain (B6 and C3H) and the jumping exercise on tibial strength; (2) an exercise-dependent increase in serum IGF-I in C3H, but not B6 mice; and (3) no effects on serum ALP or osteocalcin. The histomorphometric analyses showed no effect of exercise on C3H tibiae, but significant exercise-dependent increases in total bone area, periosteal perimeter, periosteal mineral apposition rate (MAR), and periosteal bone formation (P < 0.02 for each) in B6 tibiae. There were no effects of exercise on periosteal resorption or any endosteal measurement in either C3H or B6 mice. Since the jumping exercise was designed to cause a two–three fold increase in muscular-skeletal loading at the tibio-fibular junction, and the calculated stress (g/mm2) at this sampling site was only 16% greater for B6 compared with C3H mice, we had anticipated that both strains of mice would show exercise-dependent increases in periosteal bone formation, with a greater response in the B6 mice. The lack of a response in the C3H tibiae demonstrates that the bones of C3H mice are less sensitive to mechanical loading (and unloading) than the bones of B6 mice.

Efficacy of a 24-week aerobic exercise program for osteopenic postmenopausal women.

Abstract: Osteoporosis is one of the most common skeletal disorders affecting postmenopausal women. The purpose of this study was to investigate whether a 24-week program of aerobic high-impact loading exercise was beneficial for enhancing physical fitness and bone mineral density (BMD) in osteopenic postmenopausal women. Forty-three postmenopausal women aged 48-65 years participated in this study. The BMD of the spine (L2-L4) and right femoral neck of each woman was below 1 SD of the mean of premenopausal women, as examined by dual X-ray absorptiometry. The assignment of subjects into exercise or control group was not randomized but based on each subject's anticipated compliance to the 6-month long exercise program. Twenty-two subjects joined the exercise group and attended the training programs and 21 served as the control group. Exercise programs included treadmill walking at an intensity above 70% of maximal oxygen consumption (VO2max) for 30 minutes, followed by 10 minutes of stepping exercise using a 20-cm-high bench. The program was conducted three times per week for 24 weeks. Physical fitness measurements included testing of flexibility, muscular strength and endurance, body composition, and cardiopulmonary fitness. The results showed that the quadriceps strength, muscular endurance, and VO2max in the exercise group had significant improvements, whereas no improvement was found in any of the physical fitness parameters in the control group. The BMD of the L2-L4 and the femoral neck in the exercise group increased 2.0% (P > 0.05) and 6.8% (P 0.05), respectively. In conclusion, aerobics combined with high-impact exercise at a moderate intensity was effective in offsetting the decline in BMD in osteopenic postmenopausal women.

Association of methylenetetrahydrofolate reductase (MTHFR) polymorphism with bone mineral density in postmenopausal Japanese women.

Abstract: The pathogenesis of osteoporosis is controlled by genetic and environmental factors. Considering the high prevalence of osteoporosis in homocystinuria, abnormal homocysteine metabolism would contribute to the pathogenesis of osteoporosis. It is known that the polymorphism of methylenetetrahydrofolate reductase (MTHFR), the enzyme catalyzing the reduction of 5, 10-methylenetetrahydrofolate to 5-methyltetrahydrofolate, correlates with hyperhomocysteinemia. In this study, we examined the association of this polymorphism with bone mineral density (BMD). BMD was measured by dual-energy X-ray absorptiometry (DXA) in 307 postmenopausal women. MTHFR A/V polymorphism was analyzed using polymerase chain reaction restriction fragment length polymorphism (PCR-RFLP). We compared BMD, clinical characteristics, and bone metabolic markers among MTHFR groups (AA, AV, VV). The groups did not differ in terms of baseline data. The values of lumbar spine BMD and total body BMD were as follows: lumbar spine: AA, 0.91 +/- 0.18, AV, 0.88 +/- 0.16, VV, 0.84 +/- 0.14 g/cm(2); total body: AA, 0.97 +/- 0.11, AV, 0.96 +/- 0.11, VV, 0.93 +/- 0.09 g/cm(2). In the VV genotype, lumbar spine BMD values were significantly lower than those of the women with the AA genotype (P = 0.016) and total body BMD was significantly lower than those of the women with AA genotype (P = 0.03) and AV genotype (P = 0.04). This is the first report that suggests that the VV genotype of MTHFR is one of the genetic risk factors for low BMD.

Vitamin D deficiency and susceptibility to tuberculosis.

Abstract: Vitamin D, a modulator of macrophage function, can activate human anti-mycobacterial activity. Vitamin D deficiency is therefore associated with a higher risk of tuberculosis (TB) infection, as indicated by several observations. First, TB tends to occur during the colder seasons when cutaneous synthesis of vitamin D from sun exposure is reduced and serum vitamin D levels are lower. Second, patients with untreated TB, particularly those from a temperate climate, have lower serum vitamin D levels than healthy subjects. Third, the incidence of TB is higher among subjects with relatively low serum vitamin D levels, such as the elderly, uremic patients, and Asian immigrants in the U.K.

Effect of High Impact Activity on Bone Mass and Size in Adolescent Females: A Comparative Study Between Two Different Types of Sports

Abstract: The purpose of this cross-sectional study was to investigate the influence of two different types of weight-bearing activity, muscle strength, and body composition on bone mineral density (BMD), bone mineral content (BMC), and bone area in three different groups of late adolescent girls. The first group consisted of 10 females participating in competitive rope-skipping (age 17.8 +/- 0.8 years) training for 6.7 +/- 3.1 hours/week; the second group consisted of 15 soccer players (age 17.4 +/- 0.8 years) training for 6.1 +/- 2.0 hours/week; and the third group consisted of 25 controls (age 17.6 +/- 0.8 years) with physical activity of 0.9 +/- 1.1 hours/week. The groups were matched for age, height, and weight. BMD (g/cm(2)), BMC (g), and bone area (cm(2)) of the total body, lumbar spine, hip, total femur, distal femur, diaphyses of femur and tibia, proximal tibia, and humerus were measured using dual-energy X-ray absorptiometry (DXA). Bone density was also assessed in the radial forearm site of the dominant limb in the rope skippers and in 10 matched controls. The rope skippers had 22% higher BMD at the ultradistal site (P < 0.01). Both high-activity groups had significantly higher BMD (P < 0.05) at most loaded sites compared with the control group. When adjusting for differences in lean mass and starting age of sport-specific training between the activity groups, the rope-skipping group had a higher BMD of the total body, lumbar spine, and right humerus compared with the soccer group. They also had a significantly higher bone area of the total body, total femur, and the proximal femur than both other groups, and a significantly higher bone area of the tibia diaphysis, compared with the soccer group. In a multivariate analysis among all subjects (n = 50), all BMD sites, except the femur diaphysis, distal femur, and proximal tibia, were significantly related to type of physical activity (beta = 0.25-0.43, P < 0.05). The bone area values at different sites were strongly related to muscle strength and parameters related to body size [height, weight, lean mass, fat mass, and body mass index (BMI)]. In conclusion, it appears that in late adolescent women, weight-bearing activities are an important determinant for bone density, and high impact activities such as jumping also seem to be associated with a modification of the bone geometry (hence, the bone width) at the loaded sites.

Chondrocyte differentiation in human osteoarthritis: expression of osteocalcin in normal and osteoarthritic cartilage and bone.

Abstract: Osteocalcin (OC), which is a marker of the mature osteoblasts, can also be found in posthypertrophic chondrocytes of the epiphyseal growth plate, but not in chondrocytes of the resting zone or in adult cartilage. In human osteoarthritis (OA), chondrocytes can differentiate to a hypertrophic phenotype characterized by type X collagen. The protein- and mRNA-expression pattern of OC was systematically analyzed in decalcified cartilage and bone sections and nondecalcified cartilage sections of human osteoarthritic knee joints with different stages of OA to investigate the differentiation of chondrocytes in OA. In severe OA, we found an enhanced expression of the OC mRNA in the subchondral bone plate, demonstrating an increased osteoblast activity. Interestingly, the OC protein and OC mRNA were also detected in osteoarthritic chondrocytes, whereas in chondrocytes of normal adult cartilage, both the protein staining and the specific mRNA signal were negative. The OC mRNA signal increased with the severity of OA and chondrocytes from the deep cartilage layer, and proliferating chondrocytes from clusters showed the strongest signal for OC mRNA. In this late stage of OA, chondrocytes also stained for alkaline phosphatase and type X collagen. Our results clearly show that the expression of OC in chondrocytes correlates with chondrocyte hypertrophy in OA. Although the factors including this phenotypic shift in OA are still unknown, it can be assumed that the altered microenvironment around osteoarthritic chondrocytes and systemic mediators could be potential inducers of this differentiation.

Genetic variations in bone density, histomorphometry, and strength in mice

Abstract: The purpose of this study was to assess breed-related differences in bone histomorphometry, bone biomechanics, and serum biochemistry in three mouse breeds shown to differ in bone mineral density (BMD) (as measured by DXA) and bone mineral content (BMC). Femurs, tibiae, and sera were collected from 16-week-old C3H/HeJ {C3H}, C57BL/6J {BL6}, and DBA/2J {DBA}mice (n = 12/breed). Data collected included BMC and BMD (femora), histomorphometry of cancellous (distal femur) and cortical bone (diaphyseal tibiae and femora), bone strength (femora), and serum alkaline phosphatase (ALP). Consistent with previous reports, BMC and BMD were higher in C3H than in BL6 or DBA mice. The higher BMD in the C3H breed was associated with greater cancellous bone volume, cortical bone area, periosteal bone formation rate, biomechanical strength, and serum ALP. However, mid-diaphyseal total femoral and tibial cross-sectional area and moment of inertia were greatest in BL6, intermediate in C3H, and lowest in DBA mice. The specific distribution of cortical bone in C3H, BL6, DBA mice represents a difference in adaptive response to similar mechanical loads in these breeds. This difference in adaptive response may be intrinsic to the adaptive mechanism, or may be intrinsic to the bone tissue material properties. In either case, the bone-adaptive response to ordinary mechanical loads in the BL6 mice yields bones of lower mechanical efficiency (less stiffness per unit mass of bone tissue) and does not adapt as well as that of the C3H mice where the final product is a bone with greater resistance to bending under load. We suggest that the size, shape, and BMD of the bone are a result of breed-specific genetically regulated cellular mechanisms. Compared with the C3H mice, the lower BMD in BL6 mice is associated with long bones that are weaker because the larger cross-sectional area fails to compensate completely for their lower BMD and BMC.

Bone mineral density and muscle strength in young men with mental retardation (with and without Down syndrome).

Abstract: The objective of this study was to compare the bone mineral density (BMD) of men with Down syndrome (DS) to otherwise mentally retarded (MR) men and to investigate whether leg muscle strength of these patients is related to BMD. Two groups with MR (with and without DS) participated in the study, having met the following criteria: similar age, moderate to mild mental retardation, Tanner stage V of sexual development, similar age of beginning to walk, and equal motor activities. The DS group consisted of 8 men 23.9 +/- 4.2 years, and the MR group without DS consisted of 8 men 23.5 +/- 3.6 years. The two groups were compared with 10 sedentary students of the same age range (25.9 +/- 2.9 years) attending our University. The BMD of the 2(nd) to 4(th) lumbar vertebrae was measured in the PA projection and the mean density was expressed as g/cm(2). The isokinetic muscle strength of the right quadriceps femoris and hamstrings muscles was measured on a Cybex II isokinetic dynamometer. The value measured was peak torque at angular velocities at 60, 120, and 300 degrees.sec(-1). The results showed that BMD in DS individuals versus young adults (reference group of the scanner) was lower at the 26% level (T-score - 2.66 +/- 0.29) and significantly lower (P = 0.002) than that of the MR group. Significantly different muscle strength was observed between the DS and non-DS MR group (in quadriceps at 300 degrees.s(-1): P < 0.01, at 120 and 60 degrees. s(-1): P < 0.05; in hamstrings at 300 degrees.s(-1): P < 0.05). Higher differences in muscle strength were found between MR and control men, but no significant difference existed in BMD between them. Bivariate correlation showed that quadriceps strength significantly predicted the BMD in the DS patients. Active lifestyle and increased physical exercise to improve muscular strength should be instituted to avoid the development of osteoporosis in DS patients.

Time pattern of exercise-induced changes in type I collagen turnover after prolonged endurance exercise in humans.

Abstract: Type I collagen is known to adapt to physical activity, and biomarkers of collagen turnover indicate that synthesis can be influenced by a single intense exercise bout, but the exact time pattern of these latter changes are largely undescribed. In the present study, 17 healthy young males had their plasma concentrations of the carboxyterminal propeptide of type I procollagen (PICP), a marker of collagen formation, and the immunoactive carboxyterminal cross-linked telopeptide (ICTP), a marker of collagen resorption, measured before and immediately postexercise, as well as 1, 2, 3, 4, 5, and 6 days after completion of a marathon run (42 km). Serum concentrations of creatine kinase (S-CK) were measured as an indicator of muscular breakdown in response to the exercise bout. After a transient decrease in collagen formation immediately after exercise (plasma PICP concentration: 176 +/- 17 microg/liter to 156 +/- 9 microg/liter)(P 0.05). Muscle breakdown was elevated during the days following the exercise and peaked 24 hours after the exercise (S-CK concentration: 3,133 +/- 579 U/liter). The findings in the present study indicate that type I collagen synthesis is accelerated in response to prolonged strenuous exercise, reaching a peak after 3 days and returning to preexercising levels 5 days after the completion of a marathon run.

Low Dietary Protein and Low Bone Density

Abstract: Relatively little is known about the effects of low dietary protein intake on bone health. We have reported that in healthy young women, 4 days of a low-protein diet decreased urinary calcium excretion and was accompanied by secondary hyperparathyroidism (2 0 HPT) with twothreefold increases in serum PTH, 1,25(OH) 2 vitamin D, and nephrogenous cyclic adenosine monophosphate excretion (a bio-index of PTH action). The low-protein diet contained 45 g of protein (0.7 g/kg), 800 mg calcium, and was otherwise well balanced. The 2 0 HPT and reduced urinary calcium persisted for at least 2 weeks [1]. The etiology of the 20 HPT is due, in part, to a significant reduction in intestinal calcium absorption as measured by dual stable calcium isotopes [2]. We have observed that young women absorbed an average of 19% of dietary calcium after consuming the reduced protein diet for 4 days [2]. The longterm implications of these findings are uncertain, however, there is a clear potential to affect bone health. Data from the recently completed third National Health and Nutrition Examination Survey (NHANES III) [3] included information on protein intake and bone mineral density (BMD), and we therefore examined the relationship between these two parameters. Figure 1 shows mean total hip BMD, determined by dual energy X-ray absorptiometry (DXA), in 1822 non-Hispanic white women ages 50 years and older from NHANES III. Mean protein intakes (g/day ± SD) in each quartile were 31 ± 8, 50 ± 4, 65 ± 5, and 96 ± 22, respectively. After adjusting for age and body weight, there was a significant overall effect of protein intake ( P 4 0.037). A dietary protein intake in the lowest quartile (0–43 g/day) was associated with a significantly reduced BMD in the femur as compared with the quartile with the highest intake (>75 g/day); P 4 0.003. Women with intakes in the second quartile (44–58 g/day) also had significantly reduced BMD values compared with the highest quartile ( P 4 0.028). The same pattern occurred when the sample was restricted to women with calcium intakes $800 mg/day, which suggests that the relationship observed between dietary protein and BMD is probably not due to the effects of a concurrently low calcium intake. The mechanisms that underlie the apparent association of low protein intakes with low BMD are unknown. Our experimental data suggest one possibility which is that persistent 2 0 HPT and diminished calcium absorption at low levels of dietary protein intake cause a reduction in BMD due to the combined effects of calcium insufficiency and increased skeletal effects of PTH.

Changes in geometry and cortical porosity in adult, ovary-intact rabbits after 5 months treatment with LY333334 (hPTH 1-34).

Abstract: The purpose of this study was to determine if the increased cortical bone porosity induced by intermittently administered parathyroid hormone (PTH) reduces bone strength significantly. Mature ovary-intact New Zealand white rabbits were treated with once daily injections of vehicle, or PTH(1-34), LY333334, at 10 or 40 μg/kg/day for 140 days. Geometry of the femoral midshaft was measured to evaluate changes in the cross-sectional moment of inertia (CSMI). Cortical porosity was measured in the midshaft of the tibia by dividing cortical area into three zones based on equal divisions of cortical diameter: near endocortical (Zone I), near intermediate (Zone II), and near periosteal (Zone III) regions. Total cortical porosity significantly increased after PTH treatment from 1.4% in the controls to 6.3% in the higher dose group, but the location of the new porosities was not randomly distributed. In the controls, porosity of Zones I and II (both 1.7%) was almost twice as much as that of Zone III (0.9%). In the lower dose group, cortical porosity of Zone I (5.5%) and II (1.8%) was greater than in Zone III (0.9%), but these differences were not statistically significant. In the higher dose group, cortical porosity of Zone I (11.5%) and II (6.1%) significantly increased compared with Zone III (1.4%) (P < 0.0005). Histomorphometric measurements showed that bone formation rate on both periosteal and endocortical surfaces increased, resulting in increased bone area and cortical area in the higher dose group. A model was developed to evaluate the effect of the changes in geometry and porosity on CSMI in the different zones. This simulation model indicated that CSMI in the higher dose group was significantly greater than in the other two groups, despite the increased porosity. We speculate the reason to be that porosity increased near the endocortical surface, where its mechanical effect is small. This increase was more than offset by apposition of new bone on the periosteal surface. These data suggest that (1) PTH increases cortical porosity in a dose-dependent manner, primarily near endocortical surfaces; (2) because of this nonhomogeneous distribution, the mechanical effect of increased porosity is small; (3) the increased cortical porosity associated with PTH treatment is more than offset by periosteal apposition of new bone, causing an overall increase in the bending rigidity of cortical bone; and (4) these changes cannot be accurately evaluated using noninvasive methods of bone densitometry, which cannot account for the location of bone gain and bone loss.

Dentin sialoprotein (DSP) has limited effects on in vitro apatite formation and growth.

Abstract: Sialoproteins such as bone sialoprotein (BSP) and dentin sialoprotein (DSP) accumulate at the mineralization fronts in bone and dentin, respectively, suggesting they have some function in the mineralization process. BSP, a highly phosphorylated protein rich in polyglutamate repeats, is an effective nucleator of hydroxyapatite (HA) formation in vitro. The present study examines the effect of DSP, a low phosphorylated but related sialoprotein, on the formation and growth of HA. In vitro, in a gelatin gel diffusion system, DSP at low concentrations (<25 μg/ml) slightly increased the yield of HA formed at 3.5 and 5 days, while at higher concentrations (50–100 μg/ml) it slightly inhibited accumulation. Fewer mineral crystals were formed in the presence of high concentrations of DSP but they tended to aggregate (making them appear larger by electron microscopic analysis) than those formed in DSP-free gels. X-ray diffraction line broadening analysis failed to show significant changes in c-axis crystal dimensions with increasing DSP concentration. When HA-seed crystals were coated with DSP before inclusion in the gelatin gel there was a reduction in mineral accumulation relative to HA-seeds which had not been coated with DSP, but the extent of inhibition was significantly less than that seen in this system with other mineralized tissue matrix sialoproteins, such as osteopontin or BSP. The low affinity of DSP for well-characterized seed crystals and the limited effect of this protein on HA formation and growth suggest that the role of DSP in dentin is not primarily that of a mineralization regulator.

Establishment and characterization of a culture system for enzymatically released rat dental pulp cells.

Abstract: To establish a cell culture system that reflects the dentin formation in dental pulp tissue, we used dental pulp cells enzymatically isolated from rat incisor teeth. During the 20-day culture period, the cells exhibited various phenotypes of the odontoblast differentiation process, from the immature stage to the terminal mineralization stage. The cells began to form the mineralized nodules from day 10, and the nodules became larger by day 20. Alkaline phosphatase (ALP)-positive cells surrounded the mineralized nodules. The ALP activity in the cell layers was maximal on day 5, and gradually decreased to day 20. The calcium content in the cell layers was very low by day 10, and significantly increased from day 15. Sulfated glycosaminoglycans (GAGs) contained in the cell layers increased by day 15, but the content then decreased by day 20. The dental pulp cells produced a small amount of osteocalcin that was released into the culture medium by day 10, and the amount secreted increased markedly from day 15. The expression of osteocalcin and parathyroid hormone/parathyroid hormone-related peptide (PTH/PTHrP) receptor mRNA was evident as early as day 5, and the expression of each gradually increased with the number of days in culture. Dentin matrix protein (Dmp1) mRNA gene transcripts were identified by use of the reverse transcription polymerase chain reaction (RT-PCR) in the cells throughout the culture period. The present results demonstrate that this culture system is useful for studying the differentiation process of the odontoblast-like cells.

Expression of cathepsins B, H, K, L, and S and matrix metalloproteinases 9 and 13 during chondrocyte hypertrophy and endochondral ossification in mouse fracture callus.

Abstract: Fracture repair provides an interesting model for chondrogenesis and osteogenesis as it recapitulates in an adult organism the same steps encountered during embryonic skeletal development and growth. The fracture callus is not only a site of rapid production of cartilage and bone, but also a site of extensive degradation of their extracellular matrices. The present study was initiated to increase our understanding of the roles of different proteolytic enzymes, cysteine cathepsins B, H, K, L, and S, and matrix metalloproteinases (MMPs) 9 and 13, during fracture repair, as this aspect of bone repair has previously received little attention. Northern analysis revealed marked upregulation of cathepsin K, MMP-9, and MMP-13 mRNAs during the first and second weeks of healing. The expression profiles of these mRNAs were similar with that of osteoclastic marker enzyme tartrate-resistant alkaline phosphatate (TRAP). The changes in the mRNA levels of cathepsins B, H, L, and S were smaller when compared with those of the other enzymes studied. Immunohistochemistry and in situ hybridization confirmed the predominant localization of cathepsin K and MMP-9 and their mRNA in osteoclasts and chondroclasts at the osteochondral junction. MMP-13 was present in osteoblasts and individual hypertrophic chondrocytes near the cartilage-bone interphase. In cartilaginous callus, the expression of cathepsins B, H, L, and S was mainly related to chondrocyte hypertrophy. During bone remodeling both osteoblasts and osteoclasts contained these cathepsins. The present data demonstrate that degradation and remodeling of extracellular matrices during fracture healing involves activation of MMP-13 production in hypertrophic chondrocytes and osteoblasts, and cathepsin K and MMP-9 production in osteoclasts and chondroclasts.

Mechanical loading stimulates differentiation of periodontal osteoblasts in a mouse osteoinduction model: effect on type I collagen and alkaline phosphatase genes.

Abstract: The effects of mechanical loading on the osteoblast phenotype remain unclear because of many variables inherent to the current experimental models. This study reports on utilization of a mouse tooth movement model and a semiquantitative video image analysis of in situ hybridization to determine the effect of mechanical loading on cell-specific expression of type I collagen (collagen I) and alkaline phosphatase (ALP) genes in periodontal osteoblasts, using nonosseous cells as an internal standard. The histomorphometric analysis showed intense osteoid deposition after 3 days of treatment, confirming the osteoinductive nature of the mechanical signal. The results of in situ hybridization showed that in control periodontal sites both collagen I and ALP mRNAs were expressed uniformly across the periodontium. Treatment for 24 hours enhanced the ALP mRNA level about twofold over controls and maintained that level of stimulation after 6 days. In contrast, collagen I mRNA level was not affected after 24 hours of treatment, but it was stimulated 2.8-fold at day 6. This increase reflected enhanced gene expression in individual osteoblasts, since the increase in osteoblast number was small. These results indicate that (1) the mouse model and a semiquantitative video image analysis are suitable for detecting osteoblast-specific gene regulation by mechanical loading; (2) osteogenic mechanical stress induces deposition of bone matrix primarily by stimulating differentiation of osteoblasts, and, to a lesser extent, by an increase in number of these cells; (3) ALP is an early marker of mechanically-induced differentiation of osteoblasts. (4) osteogenic mechanical stimulation in vivo produces a cell-specific 2.8-fold increase in collagen gene expression in mature, matrix-depositing osteoblasts located on the bone surface and within the osteoid layer.

Bone sialoprotein (BSP) is a crucial factor for the expression of osteoblastic phenotypes of bone marrow cells cultured on type I collagen matrix.

Abstract: In this study, we demonstrated that type I collagen matrix induced the expression of osteoblastic phenotypes of bone marrow cells, and that antibone sialoprotein (BSP) monoclonal antibody suppressed the expression of these phenotypes. On the other hand, BSP accelerated the expression of osteoblastic phenotypes of bone marrow cells. The adherent bone marrow cells were harvested from rat femur and cultured on type I collagen matrix gels in medium containing 15% fetal calf serum, neither β-glycerophosphate nor glucocorticoid. Cells showed osteoblastic phenotypes (high alkaline phosphatase activity, osteocalcin synthesis, and responsiveness against parathyroid hormone) on collagen matrix gels at week 3 after the inoculation, and simultaneously, BSP was detected in the conditioned medium by Western blotting using an anti-BSP monoclonal antibody. However, cells in the conventional culture dishes did not show osteoblastic phenotypes during the experimental period. To investigate the physiological function of BSP in osteoblastic differentiation, bone marrow cells were cultured on collagen matrix with an anti-BSP monoclonal antibody for 3 weeks. This treatment suppressed the expression of the osteoblastic phenotypes, and the effect of the antibody was abolished by the addition of bovine bone BSP. Furthermore, bovine bone BSP stimulated the expression of osteoblastic phenotypes of bone marrow cells. Our results indicate that BSP plays a crucial role in the expression of osteoblastic phenotypes of bone marrow cells.

Acceleration of fresh fracture repair using the sonic accelerated fracture healing system (SAFHS): a review.

Abstract: The Sonic Accelerated Fracture Healing System (SAFHS) is a relatively new fracture management tool which incorporates the application of a specifically modified diagnostic ultrasound unit to healing fractures with the intention of accelerating repair. In an animal fracture model, this device has been shown to accelerate the rate of biomechanical healing by a factor of 1.4–1.6. In two randomized, controlled trials in humans, the same unit has been shown to reduce the time frame of clinical and radiographic healing by 38%. In the two fracture regions investigated, tibial diaphysis and distal radius, this represented a 58 day and 37 day reduction in healing time, respectively. Despite its effect on the entire process of fresh fracture repair, the effect of the SAFHS on the individual stages and processes involved has not been established. This paper reviews these stages and processes, and discusses the clinical and practical implications of the effect of the SAFHS on fracture repair and the need for further research into this modality.

Effect of silicon supplement on osteopenia induced by ovariectomy in rats.

Abstract: The effect of silicon (Si) supplement on preventing bone mass loss induced by ovariectomy (OVX) in rats was investigated. Three groups of 15, 100-day-old female Wistar rats each, with a mean initial weight of ∼260 g per animal, were selected for the present study. One of the experimental group consisting of 15 OVX rats was fed a diet supplemented with 500 mg of Si per kg of feed (Si + OVX). The other two groups consisting of 15 OVX and 15 sham-OVX rats did not receive these supplements. Morphometric (weight and length) and densitometric studies with dual-energy X-ray absorptiometry were performed on the whole femur and 5th lumbar vertebra of each animal 30 days after the experiment. The Si + OVX rats did not show a loss of bone mass induced by OVX at axial level (5th lumbar vertebra) or periphery (femur). Nonetheless, a significant increase (ANOVA with Bonferroni/Dunn post hocs test) of longitudinal development of the femur (P < 0.0001) was patent. These results, obtained through the measurements of axial and peripheral bones, warrant closer scrutiny in connection with the Si inhibitory effect on bone mass loss as well as the stimulatory effect on bone formation. Both actions, namely, inhibition of resorption and stimulation of formation, infer that Si may have a potential therapeutic application in the treatment of involutive osteoporosis.

Bone mass and muscle strength in young female soccer players.

Abstract: 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 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.

Semi-quantitative fluorescence analysis of calcein binding as a measurement of in vitro mineralization.

Abstract: We describe a new, highly sensitive semiquantitative method for rapid measurement of in vitro mineralization using calcein. Fluorescence analysis of the calcein bound to the calcium phosphate (hydroxyapatite) allows direct quantitation of extracellular matrix mineral content in monolayer cultures of bone-forming cells such as primary osteoblasts or osteosarcoma cells. Osteosarcoma cell lines UMR 106 and SaOS-2 were used to demonstrate that qualitatively, calcein was bound to the same regions of the mineralized cell monolayer as seen by conventional histological staining with von Kossa or Alizarin Red S. Moreover, total bound calcein could be quantitated by direct fluorescence analysis using a Cytofluor II plate reader. Changes in cell monolayer calcein fluorescence were shown to correlate well with direct colorimetric measurement of acid-solubilized Ca+2 from parallel cultures. Relative mineral quantitation by calcein fluorescence is rapid and more sensitive than colorimetric Ca+2 assays, can be performed directly on unfixed or fixed cell monolayers, and does not require the use of radioisotopes. The cell monolayer remains intact and potentially available for further analysis.

Prostaglandin E2 Directly Inhibits Bone-Resorbing Activity of Isolated Mature Osteoclasts Mainly Through the EP4 Receptor

Abstract: Prostaglandins (PGs) are well known to be important local factors in regulating bone formation and resorption. PGE2 is a potent stimulator of bone resorption because of enhancing osteoclast formation by its indirect action through stromal cells. However, the direct action of PGE2 on functionally mature osteoclasts is still controversial. In this study using highly purified rabbit mature osteoclasts, we examined the direct effect of PGE2 on osteoclastic bone-resorbing activity and its mechanism. PGE2 inhibited resorption pit formation on a dentine slice by the purified osteoclasts in a dose- and time-dependent manner. The inhibitory effect appeared as early as 4 hours after the PGE2 addition. Forskolin and 12-0-tetradecanoyl phorbol-13-acetate (TPA), respective activators of adenylate cyclase and protein kinase C, also decreased the osteoclastic bone-resorbing activity. PGE2 increased the content of intracellular cAMP in a dose range effective for the inhibition of bone resorption, whereas the prostanoid did not alter the intracellular level of inositol triphosphate. The inhibition of osteoclastic bone resorption by PGE2 was amplified and diminished by a cAMP phosphodiesterase inhibitor (isobutyl methylxanthine) and a protein kinase A inhibitor (Rp-cAMP), respectively. Of four different subtypes of PGE2 receptors (EPs), EP4 mRNA was predominantly expressed in isolated osteoclasts, whereas the other types of EP mRNA were detected in only small amounts. These results suggest that the PGE2 inhibitory effect was mediated by an adenylate cyclase system coupled with EP4. This possible association of PGE2 with EP4 in mature osteoclasts was supported by the finding that a specific agonist of EP4 (AE-604) inhibited the bone-resorbing activity and elevated the intracellular cAMP content. However, butaprost, a selective EP2 agonist, also mimicked the PGE2 effects on isolated osteoclasts although EP2 mRNA expression was minimal. In conclusion, PGE2 directly inhibits bone-resorbing activity of functionally mature osteoclasts by activation of the adenylate cyclase system, perhaps mainly through EP4.

Comparison of quantitative calcaneal ultrasound and dual energy X-ray absorptiometry in the evaluation of osteoporotic risk in children with chronic rheumatic diseases.

Abstract: Osteoporosis is a common complication in children with chronic rheumatic diseases (CRD). Although dual energy X-ray absorptiometry (DXA) is increasingly being used to determine bone mineral density (BMD) in children, it exposes the subject to ionizing radiation and does not provide a measure of true bone density; in fact, in growing bones the increase in BMD is mainly caused by the increase in bone size. In recent years, quantitative ultrasound techniques (QUS) have been used in radiation-free assessment of bone density and ``bone quality'' by measurement of the ultrasound waves attenuation by bone (BUA). In the present study we made a direct comparison of BUA in the calcaneum, determined by the pediatric contact ultrasound bone analyzer (CUBA) with lumbar BMD measured by DXA, in a group of 6–18-year-old patients with CRD. The study group consisted of 53 patients affected with juvenile rheumatoid arthritis (n = 29), systemic lupus erythematosus (n = 13), and juvenile dermatomyositis (n = 11). Mean age was 13.02 ± 2.69 years. In 22 patients (19 girls, 3 boys) both DXA and CUBA were repeated after 1 year in order to assess the mean percentage rate of BMD and BUA change over this time. Both lumbar spine BMD and calcaneal BUA measurements were lower in the CRD patients compared with a control group (P < 0.001). Calcaneal BUA was significantly correlated (r = 0.83, P < 0.001) with lumbar spine BMD. Age and sex correction (Z-score) did not change the relationship between BUA and BMD (r = 0.80, P < 0.001). A significant correlation between the mean percentage of variation (Δ%) of BMD and BUA (r = 0.76, P < 0.001) was also demonstrated in the 22 patients who were evaluated prospectively. Portability, ease of use, lower cost, and absence of radiation make CUBA a promising means of evaluating BMD in children.

Influence of Physical Activity on Ultrasound and Dual-Energy X-ray Absorptiometry Bone Measurements in Peripubertal Girls: A Cross-Sectional Study

Abstract: The aim of this cross-sectional study was to investigate whether two types of physical exercise affect the growing skeleton differently. We used calcaneal quantitative ultrasound measurements (QUS) and dual-energy X-ray absorptiometry (DXA) for measurement of bone mineral density (BMD), and to test how QUS values reflect the axial DXA values in these various study groups. A total of 184 peripubertal Caucasian girls aged 11-17 years (65 gymnasts, 63 runners, and 56 nonathletic controls) were studied. Weight, height, stage of puberty, years of training, and the amount of leisure-time physical activity were recorded. Broadband ultrasound attenuation (BUA) and sound of speed (SOS) through the calcaneus were measured. The BMD of the femoral neck and the lumbar spine were measured by DXA. The differences in mean values of bone measurements among each exercise group were more evident in pubertal than prepubertal girls. The mean BUA and SOS values of the pubertal gymnasts were 13.7% (77.8 dB/MHz versus 68.4 dB/MHz, P < 0.05) and 2.2% (1607.7 m/s versus 1572.4 m/s, P < 0.001) higher than of the controls, respectively. The mean BMD of the femoral neck in the pubertal gymnasts and runners was 20% (0.989 g/cm2 versus 0.824 g/cm2, P < 0.001) and 9.0% (0.901 g/cm2 versus 0.824 g/cm2, P < 0.05) higher than in the controls, respectively. The amount of physical activity correlated weakly but statistically significantly with all measured BMD and ultrasonographic values in the pubertal group (r = 0.19-0.35). The correlation between ultrasonographic parameters and BMD were weak, but significant among pubertal runners (r = 0.47-0.55) and controls (r = 0.39-0.42), whereas the DXA values of the femoral neck and the ultrasonographic parameters of the calcaneus did not correlate among highly physically active gymnasts. By stepwise regression analysis, physical activity accounted for much more of the variation in the DXA values than the ultrasonographic values. We conclude that the beneficial influence of exercise on bone status as measured by ultrasound and DXA was evident in these peripubertal girls. In highly active gymnasts the increase of the calcaneal ultrasonographic values did not reflect statistically significantly the BMD values of the femoral neck.