GDF-5 deficiency in mice alters the ultrastructure, mechanical properties and composition of the Achilles tendon.

Acromesomelic dysplasia of the Hunter-Thompson and Grebe types are rare human disorders based on growth/differentiation factor (GDF)-5/CDMP-1 genetic mutations. Numerous skeletal abnormalities are present in these individuals, including shortened limb bones and severe dislocations of the knee. In the GDF-5 deficient brachypodism mouse, similar, although less severe, phenotypes are observed. It is unknown whether the joint dislocations observed in these disorders are due to a defect in the original formation of joints such as the knee, or to abnormalities in the tendons and ligaments themselves. We hypothesized that tendons from GDF-5 deficient mice would exhibit altered composition, mechanical properties, and ultrastructure when compared with heterozygous control littermates. GDF-5 deficient Achilles tendons were structurally weaker than controls, and structural strength differences appeared to be caused by compromised material properties: after normalizing by collagen per unit length, mutant tendons were still 50% weaker (P < 0.0001) and 50% more compliant (P < 0.001) than controls. Despite comparable levels of skeletal maturity in the two cohorts, the majority of mutant tendon failures occurred in the mid-substance of the tendon (64% of all failures), whereas the majority of control failures occurred via avulsion (92% of all failures). Mutant Achilles tendons contained 40% less collagen per microgram of DNA when compared to controls (P = 0.004). No significant difference in glycosaminoglycan (GAG)/DNA was detected. Ultrastructural analyses indicated a slight trend toward increased frequency of small diameter (30-100 nm) collagen fibrils in the mutant Achilles. Our findings suggest that increased tendon and ligament laxity may be the cause of the joint dislocations seen in patients with Hunter-Thompson and Grebe type dysplasia, rather than developmental abnormalities in the joints themselves.     

The effects of growth and differentiation factor 5 on bone marrow stromal cell transplants in an in vitro tendon healing model

The effects of growth differentiation factor-5 (GDF-5) and bone marrow stromal cells (BMSCs) on tendon healing were investigated under in?vitro tissue culture conditions. BMSCs and GDF-5 placed in a collagen gel were interpositioned between the cut ends of dog flexor digitorum profundus tendons. The tendons were randomly assigned into four groups: 1) repaired tendon without gel; 2) repaired tendon with BMSC-seeded gel; 3) repaired tendon with GDF-5 gel without cells; and 4) repaired tendon with GDF-5 treated BMSC-seeded gel. At 2 and 4 weeks, the maximal strength of repaired tendons with GDF-5 treated BMSCs-seeded gel was significantly higher than in tendons without gel interposition. However, neither BMSCs nor GDF-5 alone significantly increased the maximal strength of healing tendons at 2 or 4 weeks. These results suggest that the combination of BMSCs and GDF-5 accelerates tendon healing, but either BMSCs or GDF-5 alone are not effective in this model.     

GDF-5 deficiency in mice delays Achilles tendon healing.

The aim of this study was to examine the role of one of the growth/differentiation factors, GDF-5, in the process of tendon healing. Specifically, we tested the hypothesis that GDF-5 deficiency in mice would result in delayed Achilles tendon repair. Using histologic, biochemical, and ultrastructural analyses, we demonstrate that Achilles tendons from 8-week-old male GDF-5 -/- mice exhibit a short-term delay of 1-2 weeks in the healing process compared to phenotypically normal control littermates. Mutant animals took longer to achieve peak cell density, glycosaminoglycan content, and collagen content in the repair tissue, and the time course of changes in collagen fibril size was also delayed. Revascularization was delayed in the mutant mice by 1 week. GDF-5 deficient Achilles tendons also contained significantly more fat within the repair tissue at all time points examined, and was significantly weaker than control tissue at 5 weeks after surgery, but strength differences were no longer detectable by 12-weeks. Together, these data support the hypothesis that GDF-5 may play an important role in modulating tendon repair, and are consistent with previously posited roles for GDF-5 in cell recruitment, migration/adhesion, differentiation, proliferation, and angiogenesis.     

Does body size account for gender differences in femur bone density and geometry?

The extent to which greater bone strength in men is caused by proportionately greater bone mass versus bigger bone size is not clear, primarily because the larger overall body size of men has made direct comparisons of skeletal measures difficult. We examined gender differences in femur neck (FN) areal bone mineral density (BMD) values collected from 5,623 non-Hispanic whites aged 20+ years in the third National Health and Nutrition Examination Survey (NHANES III, 1988-1994) before and after correction for measured height and weight. We supplemented the conventional areal BMD data (Hologic QDR 1000) with measurements of areal BMD and geometric properties (subperiosteal width, section modulus, and cortical thickness) made at narrow "cross-sectional" regions traversing the FN and the proximal shaft using a structural analysis program. Before body size adjustment, men had significantly higher values than women for all variables at the three measurement sites (p < 0.0001). Adjustment for body size reduced the differences between the sexes for all variables but had a greater effect on BMD (1-8% higher in men) than on geometry (5-17% higher in men). When examined by age, the sex discrepancy was significantly greater in the older group for all variables except subperiosteal widths. We conclude that although body size difference may account for most of the areal BMD difference between men and women, male bones are still bigger in ways that suggest greater bone strength. These differences may contribute importantly to lower fracture risk in men.     

Proximal femur bone geometry is appropriately adapted to lean mass in overweight children and adolescents

It is unclear if the bones of overweight children are appropriately adapted to increased loads. The objective of this study was to compare bone geometry in 40 overweight (body mass index [BMI] > 85th percentile) and 94 healthy weight (BMI < or = 85th percentile) subjects, ages 4-20 years. Dual energy X-ray absorptiometry (Hologic QDR 2000) scans were analyzed at the femoral shaft (FS) and narrow neck (NN) by the Hip Structure Analysis program. Subperiosteal width, cortical thickness and indices of bone axial and bending strength (bone cross-sectional area [CSA] and section modulus [Z]) were measured from bone mass profiles. Multivariate regression models were used to compare overweight and healthy weight subjects. Z was 11 (95% CI 5, 19) and 13 (7, 20) percent higher at the FS and NN, respectively, in overweight subjects (P < 0.001), adjusted for height, maturation and gender. At the NN, higher Z was due to greater subperiosteal width [4% (2, 7)] and bone CSA [10% (5, 16]) and at the FS, to higher bone CSA [10% (5, 16)] and thicker cortices [9% (3, 15)]. When lean mass was added to the models, bone variables did not differ between overweight and healthy weight subjects (P > 0.22), with the exception of NN subperiosteal width [3% (0, 6), P = 0.04]. Fat mass did not contribute significantly to any model. In summary, proximal femur bone geometric strength in overweight children was appropriately adapted to lean mass and height but greater weight in the form of fat mass did not have an independent effect on bone bending strength. These geometric adaptations are consistent with the mechanostat hypothesis that bone strength adapts primarily to muscle forces, not to static loads represented by body weight.     

The evaluation of a rat model for the analysis of densitometric and biomechanical properties of tumor-induced osteolysis.

Pathologic fractures from a reduction in bone mass and strength are a debilitating complication affecting the quality of life of individuals with metastatic lesions. There are a number of existing animal models for studying the effects of bone metastases experimentally, but these models are unsuitable for measuring structural changes in metastatic bone. Our goal was to present an in vivo model for directly investigating the densitometric and structural consequences of tumor-induced osteolysis in long bones. One femur from female Sprague Dawley rats was implanted with Walker Carcinosarcoma 256 malignant breast cancer cells or with a Sham implant. After 28 days, the animals were killed, and both femora of each animal evaluated using histomorphometry, densitometry, and mechanical testing. Compared to Sham-operated controls, we found an 11% decrease in bone mineral content, a 9% decrease in bone mineral density using dual energy X-ray absorptiometry, and a 16% decrease in bone density using peripheral quantitative computed tomography in the group with tumor cell implants. In addition, failure torque was decreased by 35% compared to the contralateral controls and by 41% compared to the Sham-operated controls. Torsional stiffness in the tumor cell-implanted femora was decreased by 35% compared to contralateral controls and by 39% compared to Sham-operated controls. Bone density was only weakly to moderately associated with bone strength in our model. By creating reproducible localized tumor-induced osteolytic lesions in a long bone, this model provides the most direct evaluation of the structural consequences of bone metastases. In the future, this model may provide a method for determining the effects of new therapeutic approaches on the preservation of bone mass and bone strength in the presence of metastatic bone disease.     

Type 1 Diabetes in Young Rats Leads to Progressive Trabecular Bone Loss,Cessation of Cortical Bone Growth,and Diminished Whole Bone Strength and Fatigue Life

People with diabetes have increased risk of fracture disproportionate to BMD, suggesting reduced material strength (quality). We quantified the skeletal effects of type 1 diabetes in the rat. Fischer 344 and Sprague‐Dawley rats (12 wk of age) were injected with either vehicle (Control) or streptozotocin (Diabetic). Forelimbs were scanned at 0, 4, 8, and 12 wk using pQCT. Rats were killed after 12 wk. We observed progressive osteopenia in diabetic rats. Trabecular osteopenia was caused by bone loss: volumetric BMD decreased progressively with time in diabetic rats but was constant in controls. Cortical osteopenia was caused by premature arrest of cortical expansion: cortical area did not increase after 4–8 wk in diabetic rats but continued to increase in controls. Postmortem μCT showed a 60% reduction in proximal tibial trabecular BV/TV in diabetic versus control rats, whereas moments of inertia of the ulnar and femoral diaphysis were reduced ～30%. Monotonic bending tests indicated that ulna and femora from diabetic animals were ～25% less stiff and strong versus controls. Estimates of material properties indicated no changes in elastic modulus or ultimate stress but modest (～10%) declines in yield stress for diabetic bone. These changes were associated with a ～50% increase in the nonenzymatic collagen cross‐link pentosidine. Last, cyclic testing showed diminished fatigue life in diabetic bones at the structural (force) level but not at the material (stress) level. In summary, type 1 diabetes, left untreated, causes trabecular bone loss and a reduction in diaphyseal growth. Diabetic bone has greatly increased nonenzymatic collagen cross‐links but only modestly reduced material properties. The loss of whole bone strength under both monotonic and fatigue loading is attributed mainly to reduced bone size.     

Mechanical and geometric changes in the growing femora of BMP-5 deficient mice

We examined the growth-related changes in femoral geometry and torsional strength in BMP-5 deficient short-ear mice over a 22-week time interval (“long-term” changes). Four groups of female mice (n = 6 per group) were examined: short-ear animals and their heterozygous control littermates at 4 and 26 weeks of age. In agreement with findings previously observed in a mixed-gender group of adult mice (26 weeks), the femora of short-ear animals were significantly smaller in length and cross section at both ages. The magnitudes of the differences between genotypes were comparable at each age, indicating that the overall rates of appositional and endochondral growth were similar for both genotypes over the 22-week period. In the adult animals, short-ear femora were 27 ± 7% weaker in torsional strength due to their smaller cross-sectional geometry. However, bone strength in adult short-ear mice appeared to be adequate for animal size: No significant difference was detected in maximum femoral torque when normalized by body mass. In 4-week old animals, BMP-5 deficiency was associated with a 27 ± 6% lower body mass, but the torsional strength of the femur was not significantly different from that of controls. Cross-sectional geometry was smaller in 4-week old short-ear mice, but the apparent bone material ultimate shear stress was elevated by 33 ± 10%, thereby resulting in a whole bone torsional strength equivalent to that of the larger control mice. While the data suggest a higher material strength in the 4-week-old short-ear animals, no significant difference in the level of bone mineralization was detectable between genotypes at either age.     

Decreased collagen organization and content are associated with reduced strength of demineralized and intact bone in the SAMP6 mouse.

To examine the link between bone material properties and skeletal fragility, we analyzed the mechanical, histological, biochemical, and spectroscopic properties of bones from a murine model of skeletal fragility (SAMP6). Intact bones from SAMP6 mice are weak and brittle compared with SAMR1 controls, a defect attributed to reduced strength of the bone matrix. The matrix weakness is attributed primarily to poorer organization of collagen fibers and reduced collagen content. INTRODUCTION: The contribution of age-related changes in tissue material properties to skeletal fragility is poorly understood. We previously reported that bones from SAMP6 mice are weak and brittle versus age-matched controls. Our present objectives were to use the SAMP6 mouse to assess bone material properties in a model of skeletal fragility and to relate defects in the mechanical properties of bone to the properties of demineralized bone and to the structure and organization of collagen and mineral. MATERIALS AND METHODS: Femora from 4- and 12-month-old SAMR1 (control) and SAMP6 mice were analyzed using bending and torsional mechanical testing of intact bones, tensile testing of demineralized bone, quantitative histology (including collagen fiber orientation), collagen cross-links biochemistry, and Raman spectroscopic analysis of mineral and collagen. RESULTS: Intact bones from SAMP6 mice have normal elastic properties but inferior failure properties, with 60% lower fracture energy versus SAMR1 controls. The strength defect in SAMP6 bones was associated with a 23% reduction in demineralized bone strength, which in turn was associated with poorer collagen fiber organization, lower collagen content, and higher hydroxylysine levels. However, SAMP6 have normal levels of collagen cross-links and normal apatite mineral structure. CONCLUSIONS: Bones from SAMP6 osteoporotic mice are weak and brittle because of a defect in the strength of the bone matrix. This defect is attributed primarily to poorer organization of collagen fibers and reduced collagen content. These findings highlight the role of the collagen component of the bone matrix in influencing skeletal fragility.     

Calcitonin Producing Tumour: Effects on Fracture Repair and Normal Bone in Rats

The mechanical properties and the collagen metabolism of healing fractures and intact bones have been studied in rats with a transplanted, calcitonin (CT) secreting, medullary thyroid carcinoma (MCT). Sham operated animals served as controls. the MCT was transplanted beneath the kidney capsule. Seven months later, when the rats with MCT had increased circulating levels of CT, a standardized femoral fracture was produced in all the animals.

The serum levels of CT were 3-40 times higher in tumour bearing rats than in controls in the period following the fracture. the fracture strength of rats with MCT was reduced by about 60 per cent compared to controls at 16 weeks after the fracture. the strength of intact femora (ultimate torsional moment) seemed to be progressively impaired by increasing levels of circulating CT. Also the strength of bone as a material (ultimate torsional stress) was reduced in the rats with MCT. the collagen synthesis was reduced in MCT rats, but the amounts of collagen in fractured or intact bones were not changed compared to controls.

We conclude that chronic hypercalcitoninaemia due to MCT seems to have a negative influence both on fracture healing and on bone metabolism.     

Calcitonin producing tumour. Effects on fracture repair and normal bone in rats

The mechanical properties and the collagen metabolism of healing fractures and intact bones have been studied in rats with a transplanted, calcitonin (CT) secreting, medullary thyroid carcinoma (MCT). Sham operated animals served as controls. The MCT was transplanted beneath the kidney capsule. Seven months later, when the rats with MCT had increased circulating levels of CT, a standardized femoral fracture was produced in all the animals. The serum levels of CT were 3-40 times higher in tumour bearing rats than in controls in the period following the fracture. The fracture strength of rats with MCT was reduced by about 60 per cent compared to controls at 16 weeks after the fracture. The strength of intact femora (ultimate torsional moment) seemed to be progressively impaired by increasing levels of circulating CT. Also the strength of bone as a material (ultimate torsional stress) was reduced in the rats with MCT. The collagen synthesis was reduced in MCT rats, but the amounts of collagen in fractured or intact bones were not changed compared to controls. We conclude that chronic hypercalcitoninaemia due to MCT seems to have a negative influence both on fracture healing and on bone metabolism.     

Calcitonin Producing Tumour: Effects on Fracture Repair and Normal Bone in Rats

The mechanical properties and the collagen metabolism of healing fractures and intact bones have been studied in rats with a transplanted, calcitonin (CT) secreting, medullary thyroid carcinoma (MCT). Sham operated animals served as controls. the MCT was transplanted beneath the kidney capsule. Seven months later, when the rats with MCT had increased circulating levels of CT, a standardized femoral fracture was produced in all the animals.

The serum levels of CT were 3-40 times higher in tumour bearing rats than in controls in the period following the fracture. the fracture strength of rats with MCT was reduced by about 60 per cent compared to controls at 16 weeks after the fracture. the strength of intact femora (ultimate torsional moment) seemed to be progressively impaired by increasing levels of circulating CT. Also the strength of bone as a material (ultimate torsional stress) was reduced in the rats with MCT. the collagen synthesis was reduced in MCT rats, but the amounts of collagen in fractured or intact bones were not changed compared to controls.

We conclude that chronic hypercalcitoninaemia due to MCT seems to have a negative influence both on fracture healing and on bone metabolism.     

Increased muscle mass with myostatin deficiency improves gains in bone strength with exercise.

We tested the hypothesis that increased muscle mass augments increases in bone strength normally observed with exercise. Myostatin-deficient mice, which show increased muscle mass, were exercised along with wildtype mice. Results indicate that increases in bone strength with exercise are greater in myostatin-deficient mice than in wildtype mice, suggesting that the combination of increased muscle mass and physical activity has a greater effect on bone strength than either increased muscle mass or intense exercise alone. INTRODUCTION: Muscle (lean) mass is known to be a significant predictor of peak BMD in young people, and exercise is also found to increase bone mass in growing humans and laboratory animals. We sought to determine if increased muscle mass resulting from myostatin deficiency would enhance gains in bone strength that usually accompany exercise. MATERIALS AND METHODS: Male mice lacking myostatin (GDF-8) were used as an animal model showing increased muscle mass. Wildtype and myostatin-deficient mice (n = 10-12 per genotype) were exercised on a treadmill for 30 minutes/day, 5 days/week, for 4 weeks starting at 12 weeks of age. Caged wildtype and myostatin-deficient mice (n = 10-12 per genotype) were included as sedentary controls. Structural and biomechanical parameters were measured from the radius. RESULTS: Ultimate force (F(u)), displacement (D(u)), toughness (energy-to-fracture; U), and ultimate strain (epsilon(u)) increased significantly with exercise in myostatin-deficient mice but not in normal mice. When F(u) is normalized by body mass, exercised myostatin-deficient mice show an increase in relative bone strength of 30% compared with caged controls, whereas exercised wildtype mice do not show a significant increase in ultimate force relative to caged controls. Relative to body weight, the radii of exercised myostatin-deficient mice are >25% stronger than those of exercised normal mice. CONCLUSIONS: Increased muscle mass resulting from inhibition of myostatin function improves the positive effects of exercise on bone strength.     

The mechanical phenotype of biglycan-deficient mice is bone- and gender-specific

Biglycan (bgn) is a small leucine-rich proteoglycan (SLRP) enriched in the extracellular matrix of skeletal tissues. While bgn is known to be involved in the growth and differentiation of osteoblast precursor cells and regulation of collagen fibril formation, it is unclear how these functions impact bone's geometric and mechanical properties, properties which are integral to the structural function of bone. Because the genetic control of bone structure and function is both local- and gender-specific and because there is evidence of gender-specific effects associated with genetic deficiencies, it was hypothesized that the engineered deletion of the gene encoding bgn would result in a cortical bone mechanical phenotype that was bone- and gender-specific. In 11-week-old C57BL6/129 mice, the cortical bone in the mid-diaphyses of the femora and tibiae of both genders was examined. Phenotypic changes in bgn-deficient mice relative to wild type controls were assayed by four-point bending tests to determine mechanical properties at the whole bone (structural) and tissue levels, as well as analyses of bone geometry and bone formation using histomorphometry. Of the bones examined, bgn deficiency most strongly affected the male tibiae, where enhanced cross-sectional geometric properties and bone mineral density were accompanied by decreased tissue-level yield strength and pre-yield structural deformation and energy dissipation. Because pre-yield properties alone were impacted, this implies that the gene deletion causes important alterations in mineral and/or the matrix/mineral ultrastructure and suggests a new understanding of the functional role of bgn in regulating bone mineralization in vivo.     

Altered hypertrophic chondrocyte kinetics in GDF-5 deficient murine tibial growth plates.

The growth/differentiation factors (GDFs) are a subgroup of the bone morphogenetic proteins best known for their role in joint formation and chondrogenesis. Mice deficient in one of these signaling proteins, GDF-5, exhibit numerous skeletal abnormalities, including shortened limb bones. The primary aim of this study was determine whether GDF-5 deficiency would alter the growth rate in growth plates from the long bones in mice and, if so, how this is achieved. Stereologic and cell kinetic parameters in proximal tibial growth plates from 5-week-old female GDF-5 -/- mice and control littermates were examined. GDF-5 deficiency resulted in a statistically significant reduction in growth rate (-14%, p=0.03). The effect of genotype on growth rate was associated with an altered hypertrophic phase duration, with hypertrophic cells from GDF-5 deficient mice exhibiting a significantly longer phase duration compared to control littermates (+25%, p=0.006). These data suggest that one way in which GDF-5 might modulate the rate of endochondral bone growth could be by affecting the duration of the hypertrophic phase in growth plate chondrocytes.     

Association of Body Composition and Physical Activity with Proximal Femur Geometry in Middle-Aged and Elderly Afro-Caribbean Men:

Osteoporotic fractures are less prevalent in African Americans than in caucasians, possibly because of differences in bone structural strength. Bone structural adaptation can be attributed to changes in load, crudely measured as lean and fat mass throughout life. The purpose of this analysis was to describe the associations of leg lean mass, total body fat mass, and hours walked per week with femoral bone mineral density (BMD) and bone geometry in a cross-sectional sample of 1,748 men of African descent between the ages of 40 and 79 years. BMD, section modulus (Z), cross-sectional area (CSA), and subperiosteal width were measured from dual energy X-ray absortiometry (DXA) scans using the hip structural analysis (HSA) program. Multiple linear regression models explained 35% to 48% of the variance in bending (Z) and axial (CSA) strength at the femoral neck and shaft. Independent of all covariates including total body fat mass, one standard deviation increase in leg lean mass was significantly associated with a 5% to 8% higher Z, CSA, and BMD (P < 0.010) at the neck and shaft. The number of hours walked per week was not a strong or consistent independent predictor of bone geometry or BMD. We have shown that weight is the strongest independent predictor of femur BMD and geometric strength although the effect appears to be mediated by lean mass since leg lean mass fraction and total body fat mass fraction had significant and opposing effects at the narrow neck and shaft in this group of middle aged and elderly men.     

Determinants of femoral geometry and structure during adolescent growth

Our goal was to understand developmental determinants of femoral structure during growth and sexual maturation by relating femoral measurements to gender and developmental factors (age, pubertal stage, height, and body mass). The bone mineral content of the femur was measured by dual energy x-ray absorptiometry in 101 healthy Caucasian adolescents and young adults, 9–26 years of age. After some simplifying assumptions had been made, cross-sectional geometric properties of the femoral midshaft were estimated. Two geometry-based structural indicators, the section modulus and whole bone strength index, were calculated to assess the structural characteristics of the femur. Femoral strength, as described by these structural indicators, increased dramatically from childhood through young adulthood. Regressions were performed between these femoral measurements and the developmental factors. Our data show that of age, pubertal stage, body mass, and height, body mass is the strongest predictor of femoral cross-sectional properties, and the correlation of body mass with femoral cross-sectional structure is independent of gender. A model including all four developmental factors and gender did not substantially increase the accuracy of predictions compared with the model with body mass alone. In light of previous research, we hypothesize that body mass is an indicator of in vivo loading and that this in vivo loading influences the cross-sectional growth of the long bones.     

Longitudinal analysis of mesenchymal progenitors and bone quality in the stem cell antigen-1-null osteoporotic mouse.

We performed a longitudinal analysis of bone quality in Sca-1-null mice. A tight temporal, site-specific association between Sca-1-deficient BMD deficiency and reduced mesenchymal progenitor frequency was observed. Defects in trabecular microarchitecture and mineralization were, at least partially, responsible for the age-related reduction in toughness of Sca-1(-/-) bones. INTRODUCTION: We previously showed that stem cell antigen 1 (Sca-1)-null mice undergo normal bone development but exhibit significantly decreased bone mass characteristic of age-dependent osteoporosis. The objective of this study was to characterize the initiation and progression of the Sca-1 mutant skeletal phenotype at the cellular, structural, material, and mechanical levels. MATERIALS AND METHODS: Sca-1-null and control mice were analyzed at 3, 5, 7, and 9 mo of age. In vitro osteoclastogenesis of bone marrow and spleen-derived progenitor populations was assessed. Bone marrow-derived mesenchymal progenitor frequency, along with osteogenic and adipogenic differentiation potential, was analyzed in vitro. Static histomorphometry of the sixth lumbar vertebrae was performed. Whole body, femoral, and vertebral BMD were assessed using DXA. Lumbar vertebrae were analyzed using microCT, back-scattered electron imaging, and compression tests. Three-point bending and femoral neck fracture tests were performed on excised femurs. RESULTS: Sca-1-null mice displayed an age-dependent, cell-autonomous osteoclast deficiency in vitro. From 7 mo of age onward, reduced Sca-1-null femoral BMD was observed alongside reduced mesenchymal progenitor frequency, and decreased in vitro osteogenic and adipogenic differentiation potential. Sca-1-deficient mice exhibited reduced whole body BMD compared with controls at all time-points analyzed. Although no differences in spinal BMD were observed, Sca-1(-/-) vertebrae exhibited decreased bone formation, with a maximal difference at 7 mo of age, inferior trabecular microarchitecture, and a greater degree of mineralization. At all sites tested, Sca-1-null bones exhibited reduced energy to failure from 5 mo onward. CONCLUSIONS: We showed a tight association within Sca-1-null mice between the initiation of stem cell defects and the exacerbation of deficiencies in bone quality at two sites clinically relevant to developing osteoporotic fractures. Sca-1-deficient mice, therefore, provide a novel and useful murine model of age-related osteoporosis, which with additional study, should further our understanding of the mechanisms underlying this increasingly prevalent disease.     

Diminished material properties and altered bone structure in rat femora during pregnancy

Pregnancy and lactation are known to cause structural and mechanical changes in bone, but the effects of pregnancy alone have not been evaluated thoroughly. This study used radiographic measurements, torsion testing, mineral analyses, and histological evaluation to determine whether there are changes in bone material and geometric properties during pregnancy in the growing rat, as implied by earlier biochemical and histological studies. The bones of pregnant 9 to 12-week-old rats and controls that were not pregnant and were matched by age (but not weight) were evaluated at times corresponding to 5, 10, 15 and 20 days of the 23-day gestation period to address the following questions: (a) How is the growth of whole bone affected by pregnancy in the growing rat (as determined by radiographic analyses)? (b) How are the mechanical properties (structural and material) of whole bone affected by pregnancy (as assessed by torsion testing)? (c) Are there changes in the characteristics of bone mineral during pregnancy (as determined by measurement of mineral content and x-ray diffraction analyses)? and (d) Are there detectable morphological or ultrastructural differences between the bones of pregnant and control rats (as assessed by analyses based on histology and back-scattered electron imaging)? The presence of statistically significant differences in this study was determined initially on the basis of a two-factor analysis of variance. In general, significant differences were noted only at late gestation (day 20), when the bones were longer and had a greater outer radius and cortical thickness; this indicates that more growth occurred during pregnancy. At day 20, biomechanical testing showed that the femora of pregnant rats had a 20% decrease in rotational angle to failure and a 30% increase in shear stiffness. No alterations in maximum torque or energy to failure were noted. Calculation of the properties of bone material revealed a 54% decrease in ultimate shear stress, a 50% decrease in shear modulus, and no alteration in shear strain when the bones from rats pregnant for 20 days were compared with controls. The bones of the pregnant rats at day 20 also differed from controls in terms of both mineral characteristics and morphology. X-ray diffraction showed larger mineral crystallites in the specimens from pregnant rats as compared with controls, whereas the mineral contents (ash weights) were similar. Scanning electron microscopy revealed qualitative differences, including increased periosteal vascularity and numerous large cortical cavities thought to be areas of resorption in the bones from rats pregnant for 20 days as contrasted with controls. These changes are consistent with the observed alteration in mechanical properties.