Showing papers in "Journal of Bone and Mineral Research in 1997"
TL;DR: Dual‐energy X‐ray absorptiometry measurements of femoral bone mineral density (BMD) from the third National Health and Nutrition Examination Survey (NHANES III, 1988–1994) are used to estimate the overall scope of the disease in the older U.S. population and explore different approaches for defining low BMD in older men in that age range.
Abstract: Data on the number of U.S. women with low femoral bone mineral density (BMD) are currently available only from indirect estimates. We used dual-energy X-ray absorptiometry (DXA) measurements of femoral BMD from phase 1 of the third National Health and Nutrition Examination Survey (NHANES III, 1988-1991) to estimate prevalences of low femoral BMD in women ages 50 years and older using an approach proposed recently by an expert panel of the World Health Organization (WHO). Cutpoints for low BMD were derived from BMD data of 194 non-Hispanic white (NHW) women aged 20-29 years from the NHANES III dataset. The prevalence of older U.S. women with femoral osteopenia (BMD between 1 standard deviation [SD] and 2.5 SD below the mean of young NHW women) ranged from 34-50% in four different femur regions, which corresponds to approximately 12-17 million women. The prevalence with osteoporosis (BMD > 2.5 SD below the mean of young NHW women) ranged from 17-20%, or approximately 6-7 million women. Prevalences were 1.3-2.4 times higher in NHW women than non-Hispanic black women (NHB), and 0.8-1.2 times higher in NHW versus Mexican American (MA) women. The estimated numbers of NHW, NHB, and MA women with osteopenia were 10-15 million, 800,000-1.2 million, and 300,000-400,000, respectively; corresponding figures for osteoporosis were 5-6 million, 200,000-300,000, and 100,000 respectively. Thus, the first data on BMD from a nationally representative sample of older women show a substantial number with low femoral BMD.(ABSTRACT TRUNCATED AT 250 WORDS)
1,396 citations
TL;DR: Although the majority of U.S. health care expenditures for the treatment of osteoporotic fractures were for white women, one‐fourth of the total was borne by other population subgroups.
Abstract: Osteoporotic fractures are a significant public health problem, resulting in substantial morbidity and mortality. Previous estimates of the economic burden of osteoporosis, however, have not fully accounted for the costs associated with treatment of nonhip fractures, minority populations, or men. Accordingly, the 1995 total direct medical expenditures for the treatment of osteoporotic fractures were estimated for all persons aged 45 years or older in the United States by age group, sex, race, type of fracture, and site of service (inpatient hospital, nursing home, and outpatient). Osteoporosis attribution probabilities were used to estimate the proportion of health service utilization and expenditures for fractures that resulted from osteoporosis. Health care expenditures attributable to osteoporotic fractures in 1995 were estimated at $13.8 billion, of which $10.3 billion (75.1%) was for the treatment of white women, $2.5 billion (18.4%) for white men, $0.7 billion (5.3%) for nonwhite women, and $0.2 billion (1.3%) for nonwhite men. Although the majority of U.S. health care expenditures for the treatment of osteoporotic fractures were for white women, one-fourth of the total was borne by other population subgroups. By site-of-service, $8.6 billion (62.4%) was spent for inpatient care, $3.9 billion (28.2%) for nursing home care, and $1.3 billion (9.4%) for outpatient services. Importantly, fractures at skeletal sites other than the hip accounted for 36.9% of the total attributed health care expenditures nationally. The contribution of nonhip fractures to the substantial morbidity and expenditures associated with osteoporosis has been underestimated by previous researchers.
1,321 citations
TL;DR: Analysis of bone formation in vivo by single‐colony derived strains of human marrow stromal fibroblasts provides evidence that individual human CFU‐Fs have osteogenic potential and yet differ from each other with respect to their osteogenic capacity.
Abstract: Populations of marrow stromal fibroblasts (MSFs) can differentiate into functional osteoblasts and form bone in vivo. It is not known, however, what proportion of MSF precursor cells, colony forming units-fibroblast (CFU-Fs), have osteogenic potential. In the present study, analysis of bone formation in vivo by single-colony derived strains of human marrow stromal fibroblasts (HMSFs) has been performed for the first time. Each strain originated from an individual CFU-F and underwent four passages in vitro prior to subcutaneous implantation into immunodeficient mice within vehicles containing hydroxyapatite-tricalcium phosphate ceramic. Multicolony derived HMSF strains were also transplanted to serve as positive controls. After 8 weeks, abundant bone formation was found in the transplants of all multicolony derived HMSF strains, whereas 20 out of 34 (58.8%) single-colony derived strains from four donors formed bone. Immunostaining with antibody directed against human osteonectin and in situ hybridization for human-specific alu sequences demonstrated that cells forming new bone were of human origin and were vital for at least 45 weeks post-transplantation. Both the incidence of bone-forming colonies and the extent of bone formation by single-colony derived HMSF strains were increased by cultivation with dexamethasone and ascorbic acid phosphate. Other factors, including type of transplantation vehicle, morphology, size, and structure of the original HMSF colonies showed no obvious correlation with the incidence or extent of bone formation. Hematopoietic tissue within the newly formed bone was developed in the transplants exhibiting exuberant bone formation. These results provide evidence that individual human CFU-Fs have osteogenic potential and yet differ from each other with respect to their osteogenic capacity.
744 citations
TL;DR: It is proposed that osteoporotic fracture may be a consequence of a positive feedback between damage accumulation and the increased remodeling space associated with repair.
Abstract: The accumulation of bone microdamage has been proposed as one factor that contributes to increased skeletal fragility with age and that may increase the risk for fracture in older women. This paper reviews the current status and understanding of microdamage physiology and its importance to skeletal fragility. Several questions are addressed: Does microdamage exist in vivo in bone? If it does, does it impair bone quality? Does microdamage accumulate with age, and is the accumulation of damage with age sufficient to cause a fracture? The nature of the damage repair mechanism is reviewed, and it is proposed that osteoporotic fracture may be a consequence of a positive feedback between damage accumulation and the increased remodeling space associated with repair.
671 citations
TL;DR: The results suggest that the polymorphism at the translation start site of the VDR gene may modulate BMD in premenopausal Japanese women.
Abstract: The effect of a T-C transition polymorphism at the translation initiation codon of the human vitamin D receptor (VDR) gene on the biological function of the encoded protein was investigated. Of 239 Japanese women volunteers subjected to genotype analysis for this polymorphism, 32 (13%) were genotype MM (the M allele is ATG at the putative translation start site), 75 (31%) were genotype mm (the m allele is ACG at the putative translation start site), and 132 (55%) were genotype Mm. The bone mineral density (BMD) in the lumbar spine (L2–L4) was determined for 110 healthy premenopausal women from the volunteers and was shown to be 12.0% greater (p < 0.05) for mm homozygotes than for MM homozygotes. Synthesis of the proteins by the M and m alleles from the cloned cDNAs in vitro and in transfected COS-7 cells revealed them to have a size of 50 and 49.5 kD, respectively, as determined by sodium dodecyl sulfate polyacrylamide gel electrophoresis. This size difference is consistent with initiation of translation of the M allele-encoded protein from an ATG codon located at nucleotides +10 to +12 in the conventional open reading frame. The extent of vitamin D–dependent transcriptional activation of a reporter construct under the control of a vitamin D response element in transfected HeLa cells was ∼1.7-fold greater for the m type VDR than for the M type protein. These results suggest that the polymorphism at the translation start site of the VDR gene may modulate BMD in premenopausal Japanese women.
627 citations
TL;DR: It is concluded that endogenous sex steroids levels are significantly related to bone density in older women and men and individual variation in age‐related bone loss may be partially accounted for by alterations in sex steroid levels with aging.
Abstract: This study examines the associations between endogenous sex steroids and bone mineral density (BMD), using data from a geographically defined cohort in Rancho Bernardo, California. Participants were community-dwelling women and men aged 50-89 years who took part in a study of endogenous sex steroid measurement between 1984-1987 and who had BMD measured in 1988-1991. Those taking corticosteroids or estrogen at the time of sex steroid determination were excluded. The main study outcomes were BMD of the ultradistal radius, midshaft radius, lumbar spine, and total hip by sex steroid level, adjusted for age, body mass index, cigarette smoking, alcohol consumption, leisure exercise, use of thiazides, thyroid hormones, and former estrogen use (women only). At the time of the hormone measurements, the mean age of the 457 women was 72.1 years and that of the 534 men was 68.6 years. A statistically significant positive relation was seen between bioavailable estradiol and BMD at all sites in women and men. Total estradiol was significantly associated with BMD at all sites in women and at all but the ultradistal radius in men. Estrone had a global effect on BMD in women and was not measured in men. Higher bioavailable (but not total) testosterone levels were associated with higher BMD of the ultradistal radius, spine, and hip in men and the ultradistal radius in women. Dehydroepiandrosterone was positively associated with BMD of the midradius, spine, and hip in women and was not associated with BMD at any site in men. Of the sex steroids tested, bioavailable estrogen was most strongly associated with BMD in both women and men. We conclude that endogenous sex steroid levels are significantly related to bone density in older women and men. Individual variation in age-related bone loss may be partially accounted for by alterations in sex steroid levels with aging. Further study to elucidate safe environmental and medical methods to maintain optimal sex steroid levels in old age is needed.
553 citations
TL;DR: It was estimated that at least 90% of all hip and spine fractures among elderly white women should be attributed to osteoporosis, and regardless of fracture type, attribution probabilities were less for men than women and generally less for non‐whites than whites.
Abstract: To assess the cost-effectiveness of interventions to prevent osteoporosis, it is necessary to estimate total health care expenditures for the treatment of osteoporotic fractures. Resources utilized for the treatment of many diseases can be estimated from secondary databases using relevant diagnosis codes, but such codes do not indicate which fractures are osteoporotic in nature. Therefore, a panel of experts was convened to make judgments about the probabilities that fractures of different types might be related to osteoporosis according to patient age, gender, and race. A three-round Delphi process was applied to estimate the proportion of fractures related to osteoporosis (i.e., the osteoporosis attribution probabilities) in 72 categories comprised of four specific fracture types (hip, spine, forearm, all other sites combined) stratified by three age groups (45-64 years, 65-84 years, 85 years and older), three racial groups (white, black, all others), and both genders (female, male). It was estimated that at least 90% of all hip and spine fractures among elderly white women should be attributed to osteoporosis. Much smaller proportions of the other fractures were attributed to osteoporosis. Regardless of fracture type, attribution probabilities were less for men than women and generally less for non-whites than whites. These probabilities will be used to estimate the total direct medical costs associated with osteoporosis-related fractures in the United States.
539 citations
TL;DR: Based on the predictions of theoretical analyses and computational models, and based on supporting experimental data, it is well established that forces applied to bone are primarily the result of muscular contraction.
Abstract: THE EFFECTS OF DISUSE, or conversely, of elevated physical activity on muscle strength and bone mass are well known. Disuse causes muscle wasting and bone loss; physical activity increases muscle strength and bone mass. The association between muscle strength and bone mass is therefore clearly established, although neither the dominant effect of muscle on bone mass, nor the cause and effect relationship is established by these simple correlations alone. Frost proposes that “voluntary muscle forces . . . dominate a bone’s postnatal structural adaptations to mechanical usage, modified . . . by body weight and one’s voluntary physical activity.” Neither body weight nor physical activity is independent of muscle mass, but it is true that muscle forces place greater loads on bones than do gravitational forces associated with weight. This was shown theoretically nearly 50 years ago by Pauwels, and is easily deduced from a simple analysis of muscle pull and levers such as the one described by Frost. More recently, this has been demonstrated using experimental data from implanted hip prostheses. This analysis shows that .70% of the bending moments on a bone are transmitted by muscle force rather than body weight. Computational models employing finite elements together with the application of computer-aided optimization to predict femoral geometry during growth are unable to simulate the morphology of the femoral shaft without the addition of muscle forces. Application of various combinations of axial, bending, and torsional loading to a cylinder were not able to generate femoral diaphyseal morphology when compared with the cross-sectional geometry of real bones, but the introduction of adductor muscle forces in various combinations with these loadings did produce geometries comparable to observed femoral geometry. Therefore, based on the predictions of theoretical analyses and computational models, and based on supporting experimental data, it is well established that forces applied to bone are primarily the result of muscular contraction.
527 citations
TL;DR: Evidence supports the use of QUS techniques for the assessment of fracture risk in elderly women and best established for water‐based calcaneal QUS systems, while future studies should include the predictive validity of other Q US systems.
Abstract: Quantitative ultrasound (QUS) methods have been introduced in recent years for the assessment of skeletal status in osteoporosis. The performance of QUS techniques has been evaluated in a large number of studies. Reviewing existing knowledge, an international expert panel formulated the following consensus regarding the current status of this technology. To date, evidence supports the use of QUS techniques for the assessment of fracture risk in elderly women. This has been best established for water-based calcaneal QUS systems. Future studies should include the predictive validity of other QUS systems. Additional clinical applications of QUS, specifically the assessment of rates of change for monitoring disease progression or response to treatment, require further investigation. Its low cost and portability make QUS an attractive technology for assessing risk of fractures in larger populations than may be suitable or feasible for bone densitometry. Additional investigations that assess innovative QUS techniques in well defined research settings are important to determine and utilize the full potential of this technology for the benefit of early detection and monitoring of osteoporosis.
525 citations
TL;DR: High resolution magnetic resonance imaging, a potentially useful tool for quantifying trabecular structure in vivo, may have applications for understanding and evaluating skeletal changes related to age and osteoporosis.
Abstract: High resolution magnetic resonance (MR) images of the distal radius were obtained at 1.5 Tesla in premenopausal normal, postmenopausal normal, and postmenopausal osteoporotic women. The image resolution was 156 μm in plane and 700 μm in the slice direction; the total imaging time was ∼16 minutes. An intensity-based thresholding technique was used to segment the images into trabecular bone and marrow, respectively. Extensions of standard stereological techniques were used to derive measures of trabecular bone structure from these segmented images. The parameters calculated included apparent measures of trabecular bone volume fraction, trabecular thickness, trabecular spacing, and trabecular number. Fractal-based texture parameters, such as the box-counting dimension, were also derived. Trabecular bone mineral density (BMD) and cortical bone mineral content (BMC) were measured in the distal radius using peripheral quantitative computed tomography (pQCT). In a subset of patients, spinal trabecular BMD was measured using quantitative computed tomography (QCT). Correlations between the indices of trabecular bone structure measured from these high-resolution MR images, age, BMD, and osteoporotic fracture status were examined. Cortical BMC and trabecular BMD at the distal radius, spinal BMD, trabecular bone volume fraction, trabecular thickness, trabecular number, and fractal dimension all decreased with age. Trabecular spacing showed the greatest percentage change and increased with age. In addition, significant differences were evident in spinal BMD, radial trabecular BMD, trabecular bone volume fraction, trabecular spacing, and trabecular number between the postmenopausal nonfracture and the postmenopausal osteoporotic subjects. Trabecular spacing and trabecular number showed moderate correlation with radial trabecular BMD but correlated poorly with radial cortical BMC. High resolution MR imaging, a potentially useful tool for quantifying trabecular structure in vivo, may have applications for understanding and evaluating skeletal changes related to age and osteoporosis.
454 citations
TL;DR: The results support the concept that osteoporosis is a loss of normal bone and provide evidence for the hypothesis that osteoarthritis is, at least partly, a bone disease in which proliferation of defective bone results in an increase in bone stiffness.
Abstract: The material properties of cancellous bone from patients with osteoporosis (OP) or osteoarthritis (OA) were determined and compared with normal controls. Samples were selected from defined sites in human femoral heads which are subjected to different loads in vivo. Overall, OP bone had the lowest stiffness and OA the highest, and this same order was reflected in the apparent densities of the bone, with OA being the most dense and OP the least. Normal and OP bone were found to have very similar stiffness-density relationships and composition. However, OA bone differed significantly from normal. The stiffness of OA bone increased more slowly with apparent density and its material density was significantly reduced. These findings were due to an altered composition of the bone in which the mass fraction of mineral is 12% less than normal. There was also greater site variation of both apparent and material density, suggesting an altered sensitivity to applied load. These results support the concept that osteoporosis is a loss of normal bone. They also provide evidence for the hypothesis that osteoarthritis is, at least partly, a bone disease in which proliferation of defective bone results in an increase in bone stiffness.
TL;DR: Although a large proportion of bone mineral accrual in the premenarcheal skeleton was related to growth, an osteogenic effect was associated with exercise, suggesting that high‐impact, strength building exercise is beneficial for premenARcheal strength, lean mass gains, and bone mineral acquisition.
Abstract: Enhancement of bone mineral acquisition during growth may be a useful preventive strategy against osteoporosis. The aim of this study was to explore the lean mass, strength, and bone mineral response to a 10-month, high-impact, strength-building exercise program in 71 premenarcheal girls, aged 9–10 years. Lean body mass, total body (TB), lumbar spine (LS), proximal femur (PF), and femoral neck (FN) bone mineral were measured using the Hologic QDR 2000+ bone densitometer. Strength was assessed using a grip dynamometer and the Cybex isokinetic dynamometer (Cybex II). At baseline, no significant difference in body composition, pubertal development, calcium intake, physical activity, strength, or bone mineral existed between groups. At completion, there were again no differences in height, total body mass, pubertal development, calcium intake, or external physical activity. In contrast, the exercise group gained significantly more lean mass, less body fat content, greater shoulder, knee and grip strength, and greater TB, LS, PF, and FN BMD (exercise: TB 3.5%, LS 4.8%, PF 4.5%, and FN 12.0%) compared with the controls (controls: TB 1.2%, LS 1.2%, PF 1.3%, and FN 1.7%). TB bone mineral content (BMC), LS BMC, PF BMC, FN BMC, LS bone mineral apparent density (BMAD), and FN bone area also increased at a significantly greater rate in the exercise group compared with the controls. In multiple regression analysis, change in lean mass was the primary determinant of TB, FN, PF, and LS BMD accrual. Although a large proportion of bone mineral accrual in the premenarcheal skeleton was related to growth, an osteogenic effect was associated with exercise. These results suggest that high-impact, strength building exercise is beneficial for premenarcheal strength, lean mass gains, and bone mineral acquisition.
TL;DR: The present results indicate that a large number of strains per day is not necessary for bone hypertrophy to develop in rats.
Abstract: The effects of jump training on bone morphological and mechanical properties were investigated in immature bones of female Fischer 344 rats. Five-week-old rats were divided into control or five jump-trained groups comprised of 5-, 10-, 20-, 40-, and 100-jump groups, representing the number of jumps per day. The rats were jump-trained 5 days/week for 8 weeks, and the height of jump was increased to 40 cm progressively. The femur and tibia in the 5-jump group had significantly greater fat-free dry weights per body weight and maximum loads at the fracture tests than those in the control group. The tibia in the 5-jump group also had significantly larger cortical area at the cross-sectional analysis. Although a slight tendency toward increase according to the number of jumps per day was observed, there were few differences in bone morphological and mechanical parameters among the 10-, 20-, and 40-jump groups. The present results indicate that a large number of strains per day is not necessary for bone hypertrophy to develop in rats.
TL;DR: A new skeletal paradigm suggests the above mechanical influences would dominate control of the process in time and anatomical space.
Abstract: Bone strength and "mass" normally adapt to the largest voluntary loads on bones. The loads come from muscles, not body weight. Bone modeling can increase bone strength and "mass," bone remodeling can conserve or reduce them, and each can turn ON and OFF in response to its own threshold range of bone strains. During growth, the loads on bones from body weight and muscle forces increase, and modeling correspondingly increases bone strength and "mass." In young adults those loads usually plateau, so bone strength can "catch up" and modeling can turn OFF. Meanwhile remodeling keeps existing bone. After about 30 years of age, muscle strength usually decreases. In aging adults this would put bones that had adapted to stronger young-adult muscles into partial disuse and make remodeling begin to reduce their strength and "mass," as disuse regularly does in experimental situations in other mammals, both growing and adult. Those changes associate strongly with the size of the bone strains caused by the loads on bone. While nonmechanical effects associated with aging should contribute to that age-related bone loss too, a new skeletal paradigm suggests the above mechanical influences would dominate control of the process in time and anatomical space.
TL;DR: It is suggested that the three muscle variables have a modest genetic component, suggesting the potential for clinical intervention and lifestyle modifications and confirming the rationale for research into bone‐specific genes.
Abstract: Lean body mass and muscle strength are both associated with bone mineral density (BMD), which is known to be under strong genetic control. In this classical twin study, we examine the size of the genetic component of both muscle strength and lean body mass and to what degree they account for the genetic component of BMD. In all, 706 postmenopausal women were examined; 227 pairs of monozygous (MZ) twins and 126 pairs of dizygous (DZ) twins. Grip strength was measured using a hand-help grip bulb and leg strength using a dynamic leg extensor power rig. Lean body mass and BMD at multiple sites were measured by dual-energy X-ray absorptiometry. BMD correlated with both leg extensor strength (r = 0.16-0.26) and grip strength (r = 0.12-0.21). Lean mass was significantly correlated with BMD at all sites (r = 0.20-0.39). All three muscle variables have a moderate genetic component with heritability estimates of 0.52 for lean body mass, 0.46 for leg extensor strength, and 0.30 for grip strength (all p < 0.05). The genetic component of BMD was not significantly reduced after adjusting for lean mass and muscle strength, with less than 20% of the genetic variance of BMD explained by the muscle variables. In conclusion, these data suggest that the three muscle variables have a modest genetic component, suggesting the potential for clinical intervention and lifestyle modifications. The genetic component to muscle bulk and strength accounts for little of the genetic component to BMD, confirming the rationale for research into bone-specific genes.
TL;DR: The regulatory mechanisms of osteoclast function are reviewed mainly on the basis of the experimental results obtained in the murine culture system.
Abstract: OSTEOCLASTIC BONE RESORPTION consists of several complicated processes: osteoclast development, attachment of osteoclasts to calcified tissues, development of a ruffled border and clear zone, followed by the secretion of acids and lysosomal enzymes into the space beneath the ruffled border. During the past decade, several new approaches have been taken to investigate osteoclast function. Highly purified functionally active osteoclasts can be isolated, which allows osteoclast function to be studied biochemically. The mechanisms of reduced bone resorption have been elucidated in several types of congenital osteopetrotic mutant mice. For example, the important role of macrophage-colony stimulating factor (M-CSF, colony-stimulating factor 1) in osteoclast development has been established in the osteopetrotic (op/op) mutant mice. Another osteopetrotic mutant mouse model has been generated by targeted disruption of the proto-oncogene c-src. Studies using this model showed that tyrosine kinase–mediated signals are essentially involved in osteoclast function. Using a purified osteoclast preparation, we showed that osteoclast function was activated by osteoblasts through a mechanism involving cell-to-cell contact. On the basis of these new approaches and experimental findings, we examined several fundamental issues regarding osteoclastic bone resorption. We considered how osteoclasts form ruffled borders and clear zones, which signals are necessary for osteoclast polarization, which mechanisms regulate the life span of osteoclasts, how calcitonin and bisphosphonates inactivate osteoclast function, and how osteoblasts regulate osteoclast function. In this article, we review the regulatory mechanisms of osteoclast function mainly on the basis of the experimental results obtained in our murine culture system. DEVELOPMENT AND FUNCTION OF OSTEOCLASTS
TL;DR: The results demonstrate that the toxic effect of clodronate on J774 macrophages, and probably on osteoclasts, is due to the metabolism of cladronate to a nonhydrolyzable ATP analog.
Abstract: Clodronate, alendronate, and other bisphosphonates are widely used in the treatment of bone diseases characterized by excessive osteoclastic bone resorption. The exact mechanisms of action of bisphosphonates have not been identified but may involve a toxic effect on mature osteoclasts due to the induction of apoptosis. Clodronate encapsulated in liposomes is also toxic to macrophages in vivo and may therefore be of use in the treatment of inflammatory diseases. It is generally believed that bisphosphonates are not metabolized. However, we have found that mammalian cells in vitro (murine J774 macrophage-like cells and human MG63 osteosarcoma cells) can metabolize clodronate (dichloromethylenebisphosphonate) to a nonhydrolyzable adenosine triphosphate (ATP) analog, adenosine 5'-(β,γ-dichloromethylene) triphosphate, which could be detected in cell extracts by using fast protein liquid chromatography. J774 cells could also metabolize liposome-encapsulated clodronate to the same ATP analog. Liposome-encapsulated adenosine 5'-(β,γ-dichloromethylene) triphosphate was more potent than liposome-encapsulated clodronate at reducing the viability of cultures of J774 cells and caused both necrotic and apoptotic cell death. Neither alendronate nor liposome-encapsulated alendronate were metabolized. These results demonstrate that the toxic effect of clodronate on J774 macrophages, and probably on osteoclasts, is due to the metabolism of clodronate to a nonhydrolyzable ATP analog. Alendronate appears to act by a different mechanism.
TL;DR: Combined treatment with bFGF and BMP‐2 synergistically enhances the osteogenic potency of bF GF in rat marrow MSC culture, and porous calcium phosphate ceramic cubes implanted in vivo showed higher histologic score for bone formation than those with MSCs pre‐exposed to either BFGF or BMP-2 alone.
Abstract: Bone marrow stroma contains multipotential mesenchymal progenitor cells which can differentiate into osteoblastic cells; we refer to these cells as mesenchymal stem cells (MSCs). Basic fibroblast growth factor (bFGF) and bone morphogenetic protein-2 (BMP-2) have been implicated in the osteogenic regulatory process by virtue of their mitogenic and differentiation activities, respectively. This study examines and compares the effects of bFGF and BMP-2 on dexamethasone (Dex)-dependent in vitro osteogenic differentiation of rat marrow-derived MSCs. A 6-day exposure to bFGF markedly stimulated cell growth and induced osteoblastic differentiation as shown by osteocalcin mRNA expression (day 14), bone nodule formation (day 18), and calcium deposition (day 18). These results indicate that bFGF enhances both mitogenic activity and osteogenic development of Dex-treated marrow MSCs. In contrast, BMP-2 did not induce osteogenesis as strongly as bFGF. Thus, exposure to BMP-2 slightly increased bone nodule number and calcium content compared with the control. Exposure of MSCs to both BMP-2 and bFGF induced expression of osteocalcin mRNA and mineralizing bone-like nodules as early as day 11 and resulted in enhancement of bone formation more markedly than either factor alone. Consistent with these results, porous calcium phosphate ceramic cubes implanted in vivo, which were loaded with MSCs pre-exposed to both bFGF and BMP-2, showed higher histologic score for bone formation than those with MSCs pre-exposed to either bFGF or BMP-2 alone. These data indicate that combined treatment with bFGF and BMP-2 synergistically enhances the osteogenic potency of bFGF in rat marrow MSC culture.
TL;DR: This study examined the commonly available methods of noninvasively assessing bone mineral status across three defined female populations to examine their interrelationships, compare their respective abilities to reflect age‐ and menopause‐related bone loss, discriminate osteoporotic fractures, and classify patients diagnostically.
Abstract: The purpose of this study was to examine the commonly available methods of noninvasively assessing bone mineral status across three defined female populations to examine their interrelationships, compare their respective abilities to reflect age- and menopause-related bone loss, discriminate osteoporotic fractures, and classify patients diagnostically. A total of 47 healthy premenopausal (age 33 +/- 7 years), 41 healthy postmenopausal (age 64 +/- 9 years), and 36 osteoporotic postmenopausal (age 70 +/- 6 years) women were examined with the following techniques: (1) quantitative computed tomography of the L1-L4 lumbar spine for trabecular (QCT TRAB BMD) and integral (QCT INTG BMD) bone mineral density (BMD); (2) dual X-ray absorptiometry of the L1-L4 posterior-anterior (DXA PA BMD) and L2-L4 lateral (DXA LAT BMD) lumbar spine, of the femoral neck (DXA NECK BMD) and trochanter (DXA TROC BMD), and of the ultradistal radius (DXA UD BMD) for integral BMD; (3) peripheral QCT of the distal radius for trabecular BMD (pQCT TRAB BMD) and cortical bone mineral content (BMC) (pQCT CORT BMC); (4) two radiographic absorptiometric techniques of the metacarpal (RA METC BMD) and phalanges (RA PHAL BMD) for integral BMD; and (5) two quantitative ultrasound devices (QUS) of the calcaneus for speed of sound (SOS CALC) and broadband ultrasound attenuation (BUA CALC). In general, correlations ranged from (r = 0.10-0.93) among different sites and techniques. We found that pQCT TRAB BMD correlated poorly (r 0.05) using age-adjusted receiver operating characteristic (ROC) curve analysis. Nevertheless, kappa score analysis (using -2.0 T score as the cut-off value for osteopenia and -2.5 T score for osteoporosis) showed that in general the diagnostic agreement among these measurements in classifying women as osteopenic or osteoporotic was poor, with kappa scores averaging about 0.4 (exceptions were QCT TRAB/INTG BMD, DXA LAT BMD, and RA PHAL BMD, with kappa scores ranging from 0.63 to 0.89). Often different patients were estimated at risk by using different measurement sites or techniques.
TL;DR: A substantial subset of elderly women has elevated bone turnover, which appears to adversely influence BMD and fracture risk, and combined biochemical and BMD screening may provide better prediction of future fracture risk than BMD alone.
Abstract: To assess the influence of bone turnover on bone density and fracture risk, we measured serum levels of osteocalcin (OC), bone alkaline phosphatase (BAP), and carboxy-terminal propeptide of type I procollagen (PICP), as well as 24-h urine levels of cross-linked N-telopeptides of type I collagen (NTx) and the free pyridinium cross-links, pyridinoline (Pyd) and deoxypyridinoline (Dpd), among 351 subjects recruited from an age-stratified random sample of Rochester, Minnesota women. PICP, NTx, and Dpd were negatively associated with age among the 138 premenopausal women. All of the biochemical markers were positively associated with age among the 213 postmenopausal women, and the prevalence of elevated turnover (>1 standard deviation [SD] above the premenopausal mean) varied from 9% (PICP) to 42% (Pyd). After adjusting for age, most of the markers were negatively correlated with bone mineral density (BMD) of the hip, spine, or forearm as measured by dual-energy X-ray absorptiometry, and women with osteoporosis were more likely to have high bone turnover. A history of osteoporotic fractures of the hip, spine, or distal forearm was associated with reduced hip BMD and with elevated Pyd. After adjusting for lower BMD and increased bone resorption, reduced bone formation as assessed by OC was also associated with prior osteoporotic fractures. These data indicate that a substantial subset of elderly women has elevated bone turnover, which appears to adversely influence BMD and fracture risk. Combined biochemical and BMD screening may provide better prediction of future fracture risk than BMD alone.
TL;DR: The in vivo temporospatial expression patterns of two dentin NCP genes, dentin matrix protein 1 (Dmp1), and dentin sialophosphoprotein (DSPP) in developing molars are compared, implying that these molecules serve different biological functions in vivo.
Abstract: Although the precise mechanisms of the conversion of predentin to dentin are not well understood, several lines of evidence implicate the noncollagenous proteins (NCPs) as important regulators of dentin biomineralization. Here we compared the in vivo temporospatial expression patterns of two dentin NCP genes, dentin matrix protein 1 (Dmp1), and dentin sialophosphoprotein (DSPP) in developing molars. Reverse transcription-polymerase chain reaction was performed on embryonic day 13 to 1-day-old first molars using Dmp1- and DSPP-specific primer sets. Dmp1 transcripts appeared at the late bud stage, while DSPP mRNA was seen at the cap stage. Expression of both genes was sustained throughout odontogenesis. In situ hybridization analysis revealed interesting differences in the expression patterns of these genes. While Dmp1 and DSPP showed coexpression in young odontoblasts before the start of mineralization, the expression of these genes was notably distinct at later stages. Dmp1 expression decreased in secretory odontoblasts after the appearance of mineral, while high levels of DSPP were sustained in odontoblasts. In early secretory ameloblasts, DSPP expression was transient and down-regulated with the appearance of dentin matrix. Interestingly, Dmp1 expression became evident in ameloblasts during the maturative phase of amelogenesis. In contrast to Dspp expression that was tooth-specific, Dmp1 was expressed by osteoblasts throughout ossification in the skeleton. Probes directed to the “DSP” and “DPP” regions of the DSPP gene showed identical patterns of mRNA expression. These data show that the developmental expression patterns of Dmp1 and DSPP are distinct, implying that these molecules serve different biological functions in vivo.
TL;DR: The purpose of this review is to amplify this message by drawing attention to several misconceptions regarding the earlier gain and later loss of bone that have developed because of reliance on bone densitometry as the investigative method of choice or convenience in the study of skeletal health and disease, and by demonstrating that the behavior of each of the surfaces of the skeleton, during both growth and aging, differ from one ethnic group to another and in men and women.
Abstract: THE REASON FOR comparing ethnic and gender differences in skeletal mass, size, and architecture is to define the structural basis underlying the differences in fracture rates seen between these groups. Han et al. have examined histomorphometric and biochemical measurements of bone turnover in premenopausal and postmenopausal white and black women in this, and the December 1996, edition of the Journal. The authors take us on a journey across ethnic groups, across age, and across menopause, exploring the static and dynamic parameters of bone resorption and formation on the surfaces of the iliac crest—on the endosteal surface of its trabeculae, and on the endocortical, intracortical, and periosteal surfaces of its cortex. The studies are a contribution because they answer and raise questions concerning ethnic differences in bone fragility and show us a way of thinking about the skeleton that is a little different. The main observations presented in these two manuscripts are: (i) the higher trabecular bone density in blacks is due to greater trabecular thickness not trabecular numbers; (ii) the lower bone turnover in blacks is likely to be due to the lower bone surface-to-volume ratio of their cancellous bone; (iii) the increase in bone remodeling associated with aging is similar in blacks and whites; (iv) despite the thicker trabeculae and lower bone turnover, blacks are not spared the loss of bone and the architectural disruption that accompanies advancing age. The work of Han et al. is a contribution in the broader context of the study of bone remodeling: the surface phenomenon that forms the morphological basis of bone turnover and bone loss. These studies, and a great deal of research by the senior investigator of the group, Michael Parfitt, emphasize the need to study the surfaces of the skeleton. The purpose of this review is to amplify this message by (i) drawing attention to several misconceptions regarding the earlier gain and later loss of bone that have developed because of reliance on bone densitometry as the investigative method of choice or convenience in the study of skeletal health and disease, and by (ii) demonstrating that the behavior of each of the surfaces of the skeleton, during both growth and aging, differ from one ethnic group to another and in men and women. The comparison of the remodeling behavior of these surfaces during growth and aging in women of different ethnic groups, in men of different ethnic groups, and women and men of the same ethnic group may be a step toward identifying the structural components responsible for the differing fracture patterns seen in the groups.
TL;DR: The availability of a cloned sequence for 1α‐OHase generates novel tools for the study of the molecular etiology of PDDR, and will allow the investigation of other disturbances of vitamin D metabolism.
Abstract: Pseudovitamin D-deficiency rickets (PDDR) is an autosomal recessive disorder that may be due to impaired activity of 25-hydroxyvitamin D-1α-hydroxylase, a renal cytochrome P450 enzyme (P4501α) of the vitamin D pathway. The disease locus for PDDR has been mapped by linkage analysis to 12q13-q14, but the molecular defect underlying the enzyme dysfunction has remained elusive due to the lack of sequence information for the P4501α gene (hereafter referred to as 1α-OHase). We have used a probe derived from the rat 25-hydroxyvitamin D-24-hydroxylase (CYP24; 24-OHase) sequence to identify and clone the 1α-OHase cDNA. The full-length 1α-OHase clone of 2.4 kb codes for a protein of predicted Mr 55 kDa. Functional activity of the cloned sequence was assessed using transient transfection, and the production of authentic 1α,25-dihydroxyvitamin D3 [1α,25(OH)2D3] was confirmed using high performance liquid chromatography fractionation and time-of-flight mass spectrometry. The expression of the gene was analyzed in vitamin D–replete animals; treatment with 1α,25(OH)2D3 reduced 1α-OHase transcript levels by 70%, while administration of parathyroid hormone led to a 2-fold increase in the expression of the gene, thus confirming the hormonal regulation previously described using biochemical methods. The rat cDNA was used to obtain a human genomic clone. Interestingly, the human 1α-OHase gene mapped to 12q13.1-q13.3, providing strong evidence that a mutation in the 1α-OHase gene is responsible for the PDDR phenotype. The availability of a cloned sequence for 1α-OHase generates novel tools for the study of the molecular etiology of PDDR, and will allow the investigation of other disturbances of vitamin D metabolism.
TL;DR: There is a moderate apparent premenopausal bone loss that occurs only at cancellous bone sites and that apparent bone loss is accelerated at most skeletal sites after the age of 75 years, suggests this cross‐sectional but large study.
Abstract: We measured the bone mineral density (BMD) at various skeletal sites (total body, hip, anteroposterior [AP] and lateral [lat] spine, and forearm) in a large population-based cohort of women aged 31-89 years (the OFELY cohort), and results were analyzed according to age and postmenopausal years. A significant apparent bone loss was found before the menopause in cancellous bone, i.e., at the lat spine and Ward's triangle (-10%; p < 0.05-0.001). Cross-sectional analysis indicated that, after the menopause, apparent bone loss was accelerated within the 10 years following menopause, continued thereafter at all sites except the AP spine, and was again accelerated in elderly menopausal for more than 25 years. Between 30 and 80 years, BMD decreased by 15 to 44% (T score -1.6 to -3.4) according to the site. The amount of apparent bone loss was highest at the Ward's triangle when expressed in percentage (44%) and at the mid- and distal radius when expressed in number of standard deviations from the peak bone mass (-3.4). As a result, the percentage of women classified as osteoporotic according to the World Heath Organization, i.e., with a T score < or = -2.5, varied substantially from site to site and was highest at the radius (37% and 46%) and lateral spine (25-31%), intermediate at the Ward's triangle, AP spine, and whole body BMD, and lowest at the whole body bone mineral content, femoral neck, and trochanter (10-12%). In conclusion, this cross-sectional but large study suggests that there is a moderate apparent premenopausal bone loss that occurs only at cancellous bone sites and that apparent bone loss is accelerated at most skeletal sites after the age of 75 years. Because of the highly variable coefficient of variation of the peak bone mass at various skeletal sites, the percentage of postmenopausal women identified as being osteoporotic varies widely according to the site of measurement.
TL;DR: The results indicate that bone mineral at clinically relevant sites, the lumbar spine and femoral neck, can respond dramatically to mechanical loading characteristic of gymnastics training in college‐aged women.
Abstract: Maximizing peak bone mass, as well as reducing its loss after menopause, is important for the prevention of osteoporosis. One mode of activity, gymnastics training, invokes high impact loading strains on the skeleton which may have powerful osteogenic effects. To examine the role of athletic activity, specifically gymnastics, on bone mineral density (BMD) accretion, we monitored longitudinal changes in regional and whole body BMD in collegiate women gymnasts and competitive athletes whose skeletons are exposed to differential loading patterns: runners and swimmers. Two cohorts were studied. Cohort I = 26 gymnasts (19.7 ± 1.2 years), 36 runners (21.1 ± 2.7 years) and 14 nonathletic women (19.3 ± 1.7 years) followed over an 8-month period. Cohort II = 8 gymnasts (18.9 ± 1.1 years), 11 swimmers (20.0 ± 2.3 years) and 11 nonathletic women (19.0 ± 1.2 years) followed over a 12-month period. Lumbar spine (L2–4), femoral neck, and whole body BMD (g/cm2) were assessed by dual-energy X-ray absorptiometry. For cohort I, the percent change in lumbar spine BMD after 8 months was significantly greater (p = 0.0001) in the gymnasts (2.8 ± 2.4%) than in the runners (−0.2 ± 2.0%) or controls (0.7 ± 1.3%). An increase in femoral neck BMD of 1.6 ± 3.6% in gymnasts was also greater (p < 0.05) than runners (−1.2 ± 3.0%) and approached significance compared with controls (−0.9 ± 2.2%, p = 0.06). For cohort II, gymnasts gained 2.3 ± 1.6% at the lumbar spine which differed significantly (p < 0.01) from changes in swimmers (−0.3 ± 1.5%) and controls (−0.4 ± 1.7%). Similarly, the change at the femoral neck was greater (p < 0.001) in gymnasts (5.0 ± 3.4%) than swimmers (−0.6 ± 2.8%) or controls (2.0 ± 2.3%). The percent change in BMD at any site did not differ between eumenorrheic and irregularly menstruating athletes. These results indicate that bone mineral at clinically relevant sites, the lumbar spine and femoral neck, can respond dramatically to mechanical loading characteristic of gymnastics training in college-aged women. This occurred despite high initial BMD values and was independent of reproductive hormone status. The results provide evidence to support the view that high impact loading, rather than selection bias, underlies high BMD values characteristic of women gymnasts. Because all athletes underwent resistance training throughout the year of study, muscle strengthening activity did not appear to be a significant factor in the skeletal response observed in gymnasts. We conclude that activities resulting in high skeletal impacts may be particularly osteotropic for young women.
TL;DR: It is demonstrated that ATDC5 cells keep track of the multistep differentiation process encompassing the stages from mesenchymal condensation to calcification in vitro, as well as the molecular mechanism underlying regulation of cartilage differentiation during endochondral bone formation.
Abstract: During the process of endochondral bone formation, proliferating chondrocytes give rise to hypertrophic cells, which then deposit a mineralized matrix to form calcified cartilage prior to replacement by bone. Previously, we reported that a clonal cell line, ATDC5, undergoes efficient chondrogenic differentiation through a cellular condensation stage. Here we report that the differentiated ATDC5 cells became hypertrophic at the center of cartilage nodules, when the cells ceased to grow. Formation of hypertrophic chondrocytes took place in association with type X collagen gene expression and a dramatic elevation of alkaline phosphate (ALPase) activity. After 5 weeks of culture, mineralization of the culture could be discerned as Alizarin red-positive spots, which spread throughout the nodules even in the absence of beta-glycerophosphate. Electron microscopy and electron probe microanalysis revealed that calcification was first initiated at matrix vesicles in the territorial matrix and that it advanced progressively along the collagen fibers in a manner similar to that which occurs in vivo. The infrared spectrum of the mineralized nodules indicated two absorption doublets around 1030 cm-1 and 600 cm-1, which are characteristic of apatitic mineral. Calcifying cultures of ATDC5 cells retained responsiveness to parathyroid hormone (PTH): PTH markedly inhibited elevation of ALPase activity and calcification in the culture in a dose-dependent manner. Thus, we demonstrated that ATDC5 cells keep track of the multistep differentiation process encompassing the stages from mesenchymal condensation to calcification in vitro. ATDC5 cells provide an excellent model to study the molecular mechanism underlying regulation of cartilage differentiation during endochondral bone formation.
TL;DR: Prostaglandin up‐regulation is associated with an induction of PGHS‐2 enzyme mRNA, which may subsequently provide a means for amplifying the cellular response to mechanical stress.
Abstract: Bone tissue responds to mechanical stress with adaptive changes in mass and structure. Mechanical stress produces flow of fluid in the osteocyte lacunar-canalicular network, which is likely the physiological signal for bone cell adaptive responses. We examined the effects of 1 h pulsating fluid flow (PFF; 0.7 ± 0.02 Pa, 5 Hz) on prostaglandin (PG) E2, PGI2, and PGF2α production and on the expression of the constitutive and inducible prostaglandin G/H synthases, PGHS-1, and PGHS-2, the major enzymes in the conversion of arachidonic acid to prostaglandins, using mouse calvarial bone cell cultures. PFF treatment stimulated the release of all three prostaglandins under 2% serum conditions, but with a different time course and to a different extent. PGF2α was rapidly increased 5–10 minutes after the onset of PFF. PGE2 release increased somewhat more slowly (significant after 10 minutes), but continued throughout 60 minutes of treatment. The response of PGI2 was the slowest, and only significant after 30 and 60 minutes of treatment. In addition, PFF induced the expression of PGHS-2 but not PGHS-1. One hour of PFF treatment increased PGHS-2 mRNA expression about 2-fold relative to the induction by 2% fresh serum given at the start of PFF. When the addition of fresh serum was reduced to 0.1%, the induction of PGHS-2 was 8- to 9-fold in PFF-treated cells relative to controls. This up-regulation continued for at least 1 h after PFF removal. PFF also markedly increased PGHS activity, measured as the conversion of arachidonic acid into PGE2. One hour after PFF removal, the production of all three prostaglandins was still enhanced. These results suggest that prostaglandins are important early mediators of the response of bone cells to mechanical stress. Prostaglandin up-regulation is associated with an induction of PGHS-2 enzyme mRNA, which may subsequently provide a means for amplifying the cellular response to mechanical stress.
TL;DR: The SCP polymorphism, detected with the endonuclease FokI, appears to influence peak bone density, particularly at the femoral neck, particularly in premenopausal American women.
Abstract: This study examines the association between bone mineral density (BMD) and a start codon polymorphism (SCP) at the translation initiation site of the vitamin D receptor (VDR) gene. The thymine/cytosine (T/C) polymorphism in the first of two start (ATG) codons can be detected by a restriction fragment length polymorphism (RFLP) using the endonuclease FokI, which recognizes ATG as part of its restriction site. F indicates absence of the first ATG and a VDR that is shorter by three amino acids. The FokI genotype was determined in 154 premenopausal American women (72 black and 82 white) who were 20-40 years old. BMD of the total body, femoral neck, and lumbar spine were measured by dual-energy X-ray absorptiometry. The distribution of the SCP genotypes differed significantly by race (p < 0.001): 4% of blacks versus 18% of whites were ff homozygous and 65% of blacks versus 37% of whites were FF homozygous. There was no statistically significant interaction between race and SCP genotype in analyses of BMD at any skeletal site. In the group as a whole, the ff women had femoral neck BMD that was 7.4% lower than that of the FF women. The ff white women had total body BMD values that were 4.3% lower and femoral neck values that were 12.1% lower than FF white women. Total body and femoral neck BMD did not differ significantly by genotype in black women, and spine BMD did not differ by genotype in either race. Addition of the SCP genotype to analysis of covariance models comparing BMD of the black and white women reduced estimated differences in femoral neck BMD between the two groups by about 35%. In conclusion, the SCP polymorphism, detected with the endonuclease FokI, appears to influence peak bone density, particularly at the femoral neck. Racial differences in its distribution may explain some of the racial difference in femoral neck BMD.
TL;DR: It is concluded that a short course of high doses of intravenous alendronate is associated with a prolonged skeletal retention of the agent, and suggests that this regimen has a sustained effect on bone turnover persisting for at least 1 year.
Abstract: Postmenopausal women with established vertebral osteoporosis were studied for 2 years to determine the terminal elimination half-life and the duration of response to treatment with intravenous alendronate (30 mg) given over 4 days. The urinary excretion of alendronate followed a multiexponential decline. Approximately 50% of the total dose was excreted over the first 5 days, and a further 17% was excreted in the succeeding 6 months. Thereafter, there was a much slower elimination phase with an estimated mean terminal half-life of greater than 10 years (n = 11). Urinary excretion of hydroxyproline and calcium decreased significantly from pretreatment values by day 3, reaching a nadir by 1 week (40% and 67% decrease, respectively). Thereafter, hydroxyproline remained suppressed for the following 2 years. In contrast, urinary calcium excretion returned gradually toward pretreatment values over the first year and during the second year was comparable to pretreatment values. Serum activity of alkaline phosphatase activity decreased over 3 months (23% reduction), increased gradually thereafter, and returned to pretreatment values at month 24. Bone mineral density measured at the spine increased by approximately 5% during the first year and remained significantly higher than pretreatment values at 2 years. We conclude that a short course of high doses of intravenous alendronate is associated with a prolonged skeletal retention of the agent. This open study also suggests that this regimen has a sustained effect on bone turnover persisting for at least 1 year.
TL;DR: Although femoral neck BMD was responsive only to the GRF exercise program, some adaptations that were specific to the JRF exerciseprogram may be important in preventing osteoporotic fractures by reducing the risk for falls.
Abstract: This study compared the effects of two exercise training programs, 11 months in duration, on bone mineral density (BMD) in older, sedentary women. Thirty-nine women, aged 60-74 years, were assigned to the following groups: (a) a group that performed exercises that introduced stress to the skeleton through ground-reaction forces (GRF) (i.e., walking, jogging, stairs); (b) a group that performed exercises that introduced stress to the skeleton through joint-reaction forces (JRF) (i.e., weight lifting, rowing); or (c) a no-exercise control group. BMD of the whole body, lumbar spine, proximal femur, and distal forearm was assessed five times at approximately 3-month intervals. The GRF and JRF exercise programs resulted in significant and similar increases in BMD of the whole body (2.0 +/- 0.8% and 1.6 +/- 0.4%, respectively), lumbar spine (1.8 +/- 0.7% and 1.5 +/- 0.5%, respectively), and Ward's triangle region of the proximal femur (6.1 +/- 1.5% and 5.1 +/- 2.1%, respectively). There was a significant in BMD of the femoral neck only in response to the GRF exercise program (GRF, 3.5 +/- 0.8%; JRF, -0.2 +/- 0.7%). There were no significant changes in BMD in control subjects. Among all exercisers, there was a significant inverse (r = -0.52, p < 0.01) relationship between increases in whole body BMD and reductions in fat mass, suggesting a dose response effect of exercise on bone mass. Although femoral neck BMD was responsive only to the GRF exercise program, some adaptations (i.e., increase in lean body mass and strength) that were specific to the JRF exercise program may be important in preventing osteoporotic fractures by reducing the risk for falls. It remains to be determined whether all of these benefits can be gained through a training program that combines the different types of exercises employed in this study.