In situ thin film growth stresses during chemical ordering.

The in situ growth stress and postgrowth stress relaxation during the L1(0) chemical ordering of Fe0.54Pt0.46 thin films have been characterized. The compressive stress is reduced with an increase in order parameter. The postgrowth stress relaxation rate increased with the order parameter and is rationalized in terms of an increase in the interfacial energy contribution at the grain boundaries because of chemical order. Density functional theory calculations were performed to quantify possible diffusion pathways and binding energies for Fe and Pt that may mitigate surface migration.

Skeletal phenotype of mice with a null mutation in Cav 1.3 L-type calcium channel.

This study aimed to understand the role of Cav1.3, one of the four L-type voltage sensitive calcium channels (VSCC) alpha(1) subunits, in the skeletal response to mechanical loading and intermittent PTH treatment. The Cav1.3 mRNA is expressed in osteoblasts. The Cav1.3 mRNA level in male wild type mice is higher than those in female. Loss of Cav1.3 resulted in a smaller skeleton in male mice as indicated by significantly lower body weight, less bone mineral content and smaller cross-sectional area of femoral midshaft. However, the osteogenic response to mechanical loading of the ulna was normal in Cav1.3(-/-) compared to the normal control mice. Male mice Cav1.3(-/-) were then treated daily with PTH at a dose of 40 microg/kg. A 6-week course of intermittent PTH treatment enhanced bone mineral content and mechanical strength equally in wild type control and Cav1.3 null mice. We also found that Cav1.2 subunit significantly increases in the absence of Cav1.3 gene. In conclusion, Cav1.3 is involved in bone metabolism, especially in male mice. Cav1.3 does not mediate osteoblast response to mechanical loading and PTH. Our data suggest that Cav1.1 and Cav1.2 subunits may substitute for Cav1.3 to maintain bone response to mechanical loading.

Heritability of lumbar trabecular bone mechanical properties in baboons.

Genetic effects on mechanical properties have been demonstrated in rodents, but not confirmed in primates. Our aim was to quantify the proportion of variation in vertebral trabecular bone mechanical properties that is due to the effects of genes. L3 vertebrae were collected from 110 females and 46 male baboons (6-32 years old) from a single extended pedigree. Cranio-caudally oriented trabecular bone cores were scanned with microCT then tested in monotonic compression to determine apparent ultimate stress, modulus, and toughness. Age and sex effects and heritability (h(2)) were assessed using maximum likelihood-based variance components methods. Additive effects of genes on residual trait variance were significant for ultimate stress (h(2)=0.58), toughness (h(2)=0.64), and BV/TV (h(2)=0.55). When BV/TV was accounted for, the residual variance in ultimate stress accounted for by the additive effects of genes was no longer significant. Toughness, however, showed evidence of a non-BV/TV-related genetic effect. Overall, maximum stress and modulus show strong genetic effects that are nearly entirely due to bone volume. Toughness shows strong genetic effects related to bone volume and shows additional genetic effects (accounting for 10% of the total trait variance) that are independent of bone volume. These results support continued use of bone volume as a focal trait to identify genes related to skeletal fragility, but also show that other focal traits related to toughness and variation in the organic component of bone matrix will enhance our ability to find additional genes that are particularly relevant to fatigue-related fractures.

Rapamycin impairs trabecular bone acquisition from high-dose but not low-dose intermittent parathyroid hormone treatment.

The osteo-anabolic effects of intermittent parathyroid hormone (PTH) treatment require insulin-like growth factor (IGF) signaling through the IGF-I receptor. A major downstream target of the IGF-I receptor (via Akt) is the mammalian target of rapamycin (mTOR), a kinase involved in protein synthesis. We investigated whether the bone-building effects of intermittent PTH require functional mTOR signaling. Mice were treated with daily PTH 1-34 (0, 10, 30, or 90 microg/kg) for 6 weeks in the presence or absence of rapamycin, a selective inhibitor of mTOR. We found that all PTH doses were effective in enhancing bone mass, whether rapamycin was present or not. Rapamycin had little to no effect on the anabolic response at low (10 microg) PTH doses, small effects in a minority of anabolic measures at moderate doses (30 microg), but the anabolic effects of high-dose PTH (90 microg) were consistently and significantly suppressed by rapamycin ( approximately 4-36% reduction). Serum levels of Trap5b, a marker of resorption, were significantly enhanced by rapamycin, but these effects were observed whether PTH was absent or present. Our data suggest that intermittent PTH, particularly at lower doses, is effective in building bone mass in the presence of rapamycin. However, the full anabolic effects of higher doses of PTH are significantly suppressed by rapamycin, suggesting that PTH might normally activate additional pathways (including mTOR) for its enhanced high-dose anabolic effects. Clinical doses of intermittent PTH could be an effective treatment for maintaining or increasing bone mass among patients taking rapamycin analogs for unrelated health issues.

Skeletal health: primate model of postmenopausal osteoporosis.

Currently, the nonhuman primate is the most widely used large animal model to evaluate the safety and efficacy of new drug entities to treat or prevent estrogen-deficiency-induced bone loss and osteoporosis. Surgical ovariectomy (OVX) induces a state of high bone turnover and rapid bone loss establishing a new steady-state bone mass within 8-9 months. Many systems in the monkey are similar to humans, including skeletal and reproductive physiology and the immune system, making this a plausible model suitable to evaluate the effects of new bone drugs. The long-term sequelae following OVX and withdrawal of monthly exposure to cyclic reproductive hormones in older female monkeys (cynomolgus and rhesus) mimics estrogen depletion and postmenopausal bone loss occurring in women. Characterization of the primate model revealed an apparent limitation to the extent of bone loss. Animals lose bone mass after OVX, but the extent of the bone loss cannot be described as osteoporotic. The small differences between OVX and sham-operated controls in many important bone measurements is overcome by including 15-20 animals per group to provide adequate statistical power. The long-term, at least 16 month, bone safety studies performed to satisfy regulatory guidelines provide an opportunity to study treatment effects for an extended period not covered in shorter-term safety studies. In vivo end-points such as densitometry and biochemical markers translate easily to clinical use, while biomechanical end-points that cannot be measured clinically can be used to predict fracture prevention. To date, the monkey OVX model has been used to support submissions for many new drugs including anabolics, bisphosphonates and selective estrogen receptor modulators. Despite its limitations, the OVX monkey model remains the best characterized of the large animal models of osteopenia and has become integral to osteoporosis drug development.

Bone parameters are improved with intermittent dosing of vitamin D3 and calcitonin.

Intermittent combination of an anabolic agent to promote bone formation and an antiresorptive agent that would prevent further bone loss is a theoretically attractive approach for restoring bone mass. We tested the potential of intermittently dosed calcitriol and calcitonin (CT) to restore bone properties in ovariectomized (Ovx) rats. Rats had Ovx or sham surgery at 8 weeks old and 4 weeks later were assigned to experimental groups: (1) sham vehicle, (2) Ovx vehicle, (3) Ovx + parathyroid hormone (PTH, 40 microg/kg), and (4) Ovx + calcitriol (2 microg/kg) + CT (2 microg/kg). Group 3 received PTH every week throughout the study, and group 4 received calcitriol at weeks 1, 3, 5, and 7 and CT at weeks 2, 4, 6, and 8. Dosing was carried out for 8 weeks with serum, and micro-computed tomographic analysis was done at 0, 4, and 8 weeks. Femurs and tibias were used for radiological analyses and for mechanical testing. Dosing with PTH improved bone mass and structure of cancellous bone at metaphyses of tibias and femurs as well as properties of cortical bone including geometry and strength. Intermittent dosing with calcitriol and CT was less potent in correcting loss of cancellous bone relative to treatment with PTH and had no effect on cortical bone parameters. However, intermittent dosing with calcitriol and CT was robust enough to improve cancellous bone mass and structure through bone formation without causing deleterious side effects. Our data provide additional evidence that therapies can be devised to ameliorate the skeletal defects associated with established osteoporosis.

Effects of treatment with parathyroid hormone 1-84 on quantity and biomechanical properties of thoracic vertebral trabecular bone in ovariectomized rhesus monkeys.

Osteoporosis is characterized by impaired bone quality leading to increased susceptibility to fracture, particularly of the thoracic spine. However, the lumbar spine is studied most commonly. We investigated the effects of 16 months of treatment with full-length parathyroid hormone (PTH) 1-84 (5, 10, or 25 microg/kg) on bone mineral density (BMD) and on architecture and biomechanical properties of trabecular bone at the thoracic spine of ovariectomized (OVX) adult rhesus monkeys and compared the results with those from the lumbar spine. At baseline, 9 months after surgery, dual-energy X-ray absorptiometric BMD at T9-T12 was 7% lower in OVX than in sham animals. All PTH(1-84) doses increased BMD to sham levels within 7 months. Micro-computed tomography of T10 vertebrae showed that trabecular bone volume and connectivity were higher in PTH(1-84)-treated animals than in sham controls, primarily through a significantly greater trabecular number. Peripheral quantitative computed tomography of trabecular bone cores from T11 and T12 confirmed that PTH(1-84) increased BMD. Compression testing of the cores showed that PTH(1-84) treatment increased stiffness, modulus, yield load, and yield stress to levels significantly greater than in sham animals, with the largest effect in the 10 microg/kg group (35-54% greater than in OVX controls). Thus, PTH(1-84) treatment increased BMD and the biomechanical properties of trabecular bone at the thoracic spine of OVX rhesus monkeys. The 10 microg/kg dose produced the greatest effect on trabecular strength, possibly because the highest dose stimulated bone remodeling excessively. Importantly, the changes observed were similar to those in lumbar vertebrae, thereby validating extrapolation of results from the lumbar to the thoracic spine.

High impact exercise is more beneficial than dietary calcium for building bone strength in the growing rat skeleton.

The benefits of impact exercise and dietary calcium on bone development are controversial. We used inbred rats under highly controlled conditions to test the independent and combined effects of impact exercise and physiological levels of calcium intakes on the growing skeleton. Forty growing F-344 female rats were fed diets containing either 100% (Ca+; 0.5% Ca) or 40% (Ca(-); 0.2% Ca) of their calcium requirements. Half of each dietary group was subjected to either 10 impacts per day from 45 cm freefall drops (Impact+), or no impact (Impact(-)). All rats received a free choice of physical activity period daily. After 8 weeks, the mechanical strength, volumetric density, geometry, and microarchitecture of their ulnae were measured. Body weight and bone length did not differ among groups. On both diets, freefall impact resulted in greater bone strength, cross-sectional moments of inertia, and endosteal and periosteal circumferences in the shaft. Only Ca+ resulted in greater shaft volumetric bone mineral density (vBMD) but that did not affect shaft breaking strength. In the bone ends, both Impact+ and Ca+ positively affected density and structure of both cortical and trabecular bone but the effects of Impact+ were more pervasive. In the proximal end, Impact+ resulted in greater bone volume fraction (BV/TV) in the trabecular bone due to greater trabecular thickness, and cortical thickness was greater due to a smaller endosteal circumference. Impact+ exerted a compensatory effect on vBMD and BV/TV in Ca(-) rats at the proximal site. In Impact(-) rats only, Ca+ resulted in greater total and cortical vBMD and BV/TV in the proximal ulna. Impact+ and Ca+ exerted additive effects on cortical bone area (BA) in the proximal ulna and on total BA, periosteal circumference, and trabecular vBMD in the distal ulna. In conclusion, impact exercise was more beneficial than adequate dietary calcium to growing bones, although sufficient dietary calcium was beneficial in rats not subjected to impact exercise.

Bone quality and bone strength in BXH recombinant inbred mice.

The relationship between bone quality and strength was studied in 11 BXH recombinant inbred (RI) strains of mice. The bone quality parameters studied were bone mineralization, microhardness, architecture, and connectivity. Previous studies have demonstrated considerable variability in bone density, biomechanical properties, and microstructure among inbred strains of mice. In particular, C3H/HeJ (C3H) mice exhibit thicker femoral and vertebral cortices and fewer trabeculae in the vertebral body compared with C57BL/6J (B6) mice, despite having similar vertebral bone strength. A set of RI mouse strains has been generated from B6 and C3H (denoted BXH) in an attempt to isolate genetic regulation of numerous traits, including bone. The objective of this study was to investigate relationships among bone quality and bone strength in femurs and vertebrae among BXH RI mice. The study involved 11 BXH RI strains of female mice (n = 5-7) as well as the B6 and C3H progenitor strains. Parameters contributing to bone quality were evaluated, including BMD, bone mineralization, microhardness, architecture, and connectivity. There was a strong correlation between femoral and vertebral BMD in all strains (P < 0.001) except in BXH-9 and -10 (P < 0.001). Within the vertebrae, cortical bone was more mineralized than trabecular bone, and a strong correlation existed between the two (P < 0.001). However, cortical microhardness did not differ from trabecular microhardness. Cortical bone was more mineralized in the femur than in the vertebrae and significantly harder, by 30%. There was a wide range in trabecular connectivity, architecture, and femur geometry among BXH RI strains. BMD explained 43% of vertebral bone strength but only 11% of femoral bone strength. Trabecular connectivity explained an additional 8% of vertebral strength, while mineralization and femur geometry explained 7% and 50% of femoral strength, respectively. Different bone quality parameters had varying influences on bone mechanical properties, depending on bone site. BMD may play a larger role in explaining bone strength in the vertebrae than in the femur. Moreover, cortical bone in the femur is harder than in vertebrae. The control of cortical bone material properties may be site-dependent.

Effects of treatment of ovariectomized adult rhesus monkeys with parathyroid hormone 1-84 for 16 months on trabecular and cortical bone structure and biomechanical properties of the proximal femur.

Treatment of monkeys and humans with parathyroid hormone (PTH) 1-84 stimulates skeletal remodeling, which increases trabecular (Tb) bone mineral density (BMD) but decreases cortical (Ct) BMD at locations where these bone types predominate. We report the effects of daily PTH treatment (5, 10, or 25 microg/kg) of ovariectomized (OVX) rhesus monkeys for 16 months on bone structure and biomechanical properties at the proximal femur, a mixed trabecular and cortical bone site. PTH reversed the OVX-induced decrease in BMD measured by dual-energy X-ray absorptiometry at the proximal femur, femoral neck, and distal femur. Peripheral quantitative computed tomography confirmed a significant decrease in Ct.BMD and an increase in Tb.BMD at the total proximal femur and at the proximal and distal femoral metaphyses. The decrease in Ct.BMD resulted primarily from increased area because cortical bone mineral content was unaffected by PTH. Histomorphometry revealed that PTH significantly increased the trabecular bone formation rate (BFR) as well as trabecular bone volume and number. PTH did not affect periosteal or haversian BFR at the femoral neck, but cortical porosity was increased slightly. PTH had no effects on stiffness or peak load measured using a shear test, whereas work-to-failure, the energy required to fracture, was increased significantly. Thus, PTH treatment induced changes in trabecular and cortical bone at the proximal femur that were similar to those occurring at sites where these bone types predominate. Together, the changes had no effect on stiffness or peak load but increased the energy required to break the proximal femur, thereby making it more resistant to fracture.

Effects of daily treatment with parathyroid hormone 1-84 for 16 months on density, architecture and biomechanical properties of cortical bone in adult ovariectomized rhesus monkeys.

Treatment with parathyroid hormone 1-84 (PTH) or teriparatide increases osteonal remodeling and decreases bone mineral density (BMD) at cortical (Ct) bone sites but may also increase bone size. Decreases in BMD and increases in size exert opposing effects on bone strength. In adult ovariectomized (OVX) rhesus monkeys, we assessed the effects of daily PTH treatment (5, 10 or 25 microg/kg) for 16 months on BMD at the radial, tibial and femoral diaphyses, and on biomechanical properties (3-point bending) of radial cortical bone and the femoral diaphysis. PTH treatment did not affect areal BMD measured by dual-energy X-ray absorptiometry at the tibial diaphysis but caused a rapid, dose-related decrease at the distal radial diaphysis. Peripheral quantitative computed tomography at the radial and femoral diaphyses confirmed a significant PTH dose-related decrease in volumetric Ct.BMD caused primarily by increased cortical area. Significant increases in cortical thickness were the result of nonsignificant increases in periosteal length and decreases in endocortical length. Histomorphometry revealed increased endocortical bone formation at the tibial diaphysis and rib, higher Haversian remodeling at the rib and increased cortical porosity at the rib and tibia. Biomechanical testing at the femoral diaphysis showed that PTH treatment had no effect on peak load, but significantly decreased stiffness and increased work-to-failure (the energy required to break the bone). Similar changes occurred in radial cortical beams but only stiffness was changed significantly. Thus, PTH treatment of OVX rhesus monkeys decreased BMD and stiffness of cortical bone but did not affect peak load, likely because of increased bone size. However, PTH treatment increased the energy required to break the femur making it more resistant to fracture.

The skeletal responsiveness to mechanical loading is enhanced in mice with a null mutation in estrogen receptor-beta.

Mechanical loading caused by physical activity can stimulate bone formation and strengthen the skeleton. Estrogen receptors (ERs) play some role in the signaling cascade that is initiated in bone cells after a mechanical load is applied. We hypothesized that one of the ERs, ER-beta, influences the responsiveness of bone to mechanical loads. To test our hypothesis, 16-wk-old male and female mice with null mutations in ER-beta (ER-beta(-/-)) had their right forelimbs subjected to short daily loading bouts. The loading technique used has been shown to increase bone formation in the ulna. Each loading bout consisted of 60 compressive loads within 30 s applied daily for 3 consecutive days. Bone formation was measured by first giving standard fluorochrome bone labels 1 and 6 days after loading and using quantitative histomorphometry to assess bone sections from the midshaft of the ulna. The left nonloaded ulna served as an internal control for the effects of loading. Mechanical loading increased bone formation rate at the periosteal bone surface of the mid-ulna in both ER-beta(-/-) and wild-type (WT) mice. The ulnar responsiveness to loading was similar in male ER-beta(-/-) vs. WT mice, but for female mice bone formation was stimulated more effectively in ER-beta(-/-) mice (P < 0.001). We conclude that estrogen signaling through ER-beta suppresses the mechanical loading response on the periosteal surface of long bones.

Daily treatment of aged ovariectomized rats with human parathyroid hormone (1-84) for 12 months reverses bone loss and enhances trabecular and cortical bone strength.

Most studies that have investigated the anabolic effects of parathyroid hormone (1-84) (PTH) or PTH fragments on the skeleton of ovariectomized (OVX) rats have evaluated the short-term effects of high-dose PTH(1-34) in young animals. This study used densitometry, histomorphometry, and biomechanical testing to evaluate the effects of 12-month daily treatment with low-dose PTH (15 or 30 microg/kg) in rats that were 10 months old at baseline, 4 months after OVX. Bone mineral density (BMD) and bone strength were reduced substantially in control OVX rats. The 15 microg/kg dose of PTH restored BMD to levels similar to those in sham animals within 6 months at the lumbar spine, distal and central femur, and whole body and maintained the BMD gain from 6 to 12 months. The 30 microg/kg dose produced greater effects. Both PTH doses normalized the trabecular bone volume-to-total volume ratio (BV/TV) at lumbar vertebra 3 but not at the proximal tibia (where baseline BV/TV was very low), solely by increasing trabecular thickness. PTH dose-dependently increased bone formation by increasing the mineralizing surface, but only the 30 microg/kg dose increased resorption. PTH increased cortical BMD, area, and thickness, primarily by increasing endocortical bone formation, and restored all measures of bone strength to levels similar to those in sham animals at all skeletal sites. PTH increased bone mass safely; there was no osteoid accumulation, mineralization defect, or marrow fibrosis and there were no abnormal cells. Thus, long-term PTH therapy normalized bone strength in the aged OVX rat, a model of postmenopausal osteoporosis, through increased bone turnover and enhanced formation of both trabecular and cortical bone.

Identification of a quantitative trait locus on rat chromosome 4 that is strongly linked to femoral neck structure and strength.

Risk factors for osteoporotic hip fracture include reduced bone mineral density and poor structure of the femoral neck, both of which are heritable traits. Previously, we showed that despite similar body size, Fischer 344 (F344) rats have significantly different skeletal traits compared with Lewis (LEW) rats. To identify a gene or genes regulating fracture risk at the femoral neck, we mapped quantitative trait loci (QTL) for femoral neck density and structure phenotypes using a 595 F2 progeny derived from the inbred F344 and LEW strains of rats. Femoral neck phenotypes included volumetric bone mineral density (vBMD), neck width, femoral neck cross-sectional area and polar moment of inertia (Ip). A 20-cM genome-wide scan was performed using 118 microsatellite markers and linkage analysis was conducted to identify chromosomal regions harbor QTL for femoral neck phenotypes. Strong evidence of linkage (P<0.01) to femoral neck vBMD was observed on chromosomes (Chrs) 1, 2, 4, 5, 7, 10 and 15. QTL affecting femoral neck structure and biomechanical properties were detected only on Chr 4 where the F344 alleles were shown to improve femoral neck structure, whereas these alleles had no effect on bone measurements at the lumbar spine and only modest effects at the femoral midshaft. In contrast, QTL on Chrs 1, 2 and 10 affected multiple skeletal sites. Several QTL regions in this study are homologous to human chromosomal regions, where linkage to femoral neck and related phenotypes has been reported previously. These findings represent an important first step in localizing and identifying genes that influence hip fragility.

Bone remodeling at the iliac crest can predict the changes in remodeling dynamics, microdamage accumulation, and mechanical properties in the lumbar vertebrae of dogs.

We previously demonstrated that suppression of bone remodeling allows microdamage to accumulate, thereby leading to reduced bone toughness in dog bone. In this study we evaluated the relationships between bone remodeling at the iliac crest and skeletal activation frequency, microdamage accumulation, or biomechanical properties of lumbar vertebrae using the same dogs to determine whether bone remodeling at the iliac crest can predict damage accumulation and mechanical parameters of the lumbar spine following treatment with antiresorptive agents. Thirty-six female beagles, 1 to 2 years old, were divided into three groups. The control group was treated daily for 12 months with saline vehicle. The remaining two groups were treated daily with oral risedronate at a dose of 0.5 mg/kg/day, or alendronate at 1.0 mg/kg/day orally. The doses of these bisphosphonates were 5 to 6 times the clinical doses approved for treatment of osteoporosis in humans. After sacrifice, the right ilium and L2 vertebra were assigned to histomorphometry. The left ilium and L3 vertebra were used for microdamage analysis. The L4 vertebra was mechanically tested to failure in compression, and bone toughness calculated from the stress-strain curve. There was a strong positive relationship for activation frequency (Ac.f) between ilium and lumbar vertebrae (r2 = 0.82; P < 0.0001). Iliac crest Ac.f underestimates Ac.f in L2, but L2 Ac.f reaches a minimum threshold and does not decline further when iliac crest Ac.f is below 0.10/yr. Microdamage (Cr.S.Dn) accumulation at the ilium was significantly associated with increased microdamage accumulation in the L3 lumbar vertebra (r2 = 0.43, P < 0.0001). The data also show that bisphosphonate treatment increased Cr.S.Dn at a faster rate in L3 than in the iliac crest. Although bisphosphonate treatment decreased bone toughness in L4, this decrease demonstrated no relationship to decreased Ac.f in the ilium. These results clearly indicate that bone remodeling data obtained from iliac crest biopsy could be used to estimate the activation frequency and microdamage burden in the vertebral column.