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.

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.

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 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 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.

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.

Estrogen receptor beta: the antimechanostat?

We have known for sometime that sex hormones influence the growth, preservation, and loss of bone tissue in the skeleton. However, we are only beginning to recognize how estrogen influences the responsiveness of the skeleton to exercise. Frost's mechanostat theory proposes that estrogen reduces the mechanical strain required to initiate an osteogenic response, but this may only occur at the endocortical and trabecular bone surfaces. The discovery of estrogen receptors alpha and beta may help us to understand the bone surface-specific effects of exercise. Findings from estrogen receptor knockout mice suggest that the activity of ERalpha may explain the positive interaction between estrogen and exercise on bone formation near marrow, that is, endocortical and trabecular bone surfaces. Estrogen inhibits the anabolic exercise response at the periosteal surface, and this we propose is due to the activation of ERbeta. Signaling through this receptor retards periosteal bone formation and suppresses gains in bone size and bone strength, and for these reasons it behaves as an antimechanostat.

Mechanosensitivity of the rat skeleton decreases after a long period of loading, but is improved with time off.

After the initial adaptation to large mechanical loads, it appears as though the skeleton's responsiveness to exercise begins to wane. To counteract the waning effects of long-term mechanical loading, "time off" may be needed to improve the responsiveness of bone cells to future mechanical signals and reinitiate bone formation. The aim of this study was to determine whether bone becomes less sensitive to long-term mechanical loading and whether time off is needed to improve mechanosensitivity. Fifty-seven female Sprague-Dawley rats (7-8 months of age) were randomized to one of following groups: Group 1 loading was applied for 5 weeks followed by 10 weeks of time off (1 x 5); Group 2 loading was applied for 5 weeks, followed by time off for 5 weeks and loading again for 5 weeks (2 x 5); Group 3 loading was applied continuously for 15 weeks (3 x 5); Group 4 age-matched control group; and Group 5 baseline control group. An axial load was applied to the right ulna for 360 cycles/day, at 2 Hz, 3 days/week at 15 N. At the end of the intervention, all three loaded groups showed similar increases in bone mass, cortical area, and I(MIN) in response to mechanical loading(.) Bone formation rate of the loaded ulna was increased in the first 5 weeks of loading for all three loaded groups; however, during the last 5 weeks, it was only significantly increased in the group that had time off (2 x 5) (P < 0.05). The group that had time off (2 x 5) also showed greater improvements in work to failure compared to the group loaded for 5 weeks (1 x 5) and the entire 15 weeks (3 x 5). A second experiment showed that the waning effect of long-term loading on the skeleton is not a result of aging. In conclusion, mechanical loading of the rat ulna results in large improvements in bone formation during the first 5 weeks of loading, but continual loading decreases the osteogenic response. Having time off increases bone formation and improves the resistance to fracture.

A "minimal epitope" anti-protein antibody that recognises a single modified amino acid.

Antibodies that recognise proteins bind to epitopes of varying size, but a grouping of the order of six amino acids, contiguous or not, is regarded as a typical number. By using as immunogen a highly abundant and universal eukaryotic nuclear protein (histone H4) modified in a manner not typical of secreted proteins (acetylation of lysine side chains), antiserum has been raised in rabbits having the single amino acid epsilon-N-acetyl lysine as the recognition epitope. The affinity-purified antibody should be useful for studying the functional role of this modification. The methodology has potential for raising antibodies to other types of post-translationally modified proteins.

Effects of loading frequency on mechanically induced bone formation.

The anabolic effect of mechanical loading on bone tissue is modulated by loading frequency. The objective of this study was to characterize the new bone formation on the periosteal and endocortical surfaces of the ulnar diaphysis in adult, female rats in response to controlled dynamic loading and to examine the interactions between strain magnitude, loading frequency, and bone formation rate (BFR/BS) for frequencies ranging from 1 to 10 Hz. Cyclic, compressive loading was applied to the ulnas of 60 adult, female rats divided into 12 loading groups. Loading was applied for 360 cycles/day with peak loads ranging from 4.3 to 18N at frequencies of 1, 5, and 10 Hz. After 2 weeks of loading, bone formation on the periosteal and endocortical surfaces of the ulna was quantified using double-label histomorphometry on transverse sections obtained at the middiaphysis. Periosteal bone formation increased in a dose-response manner with peak load at each of the three loading frequencies tested. Loading frequency significantly affected the x intercepts and slopes of the peak strain versus BFR/BS (p < 0.001) and peak strain versus mineralizing surface (MS/BS; p < 0.001) curves. Periosteal osteogenesis was best predicted by a mathematical model that assumed: (1) bone cells are activated by fluid shear stresses and (2) that stiffness of the bone cells and the extracellular matrix near the cells increases at higher loading frequencies because of viscoelasticity. Consequently, mechanotransduction appears to involve a complex interaction between extracellular fluid forces and cellular mechanics.

Aging changes mechanical loading thresholds for bone formation in rats.

The effect of aging on the mechanical loading thresholds for osteogenesis was investigated in rats. We applied mechanical loads varying from 30 to 64 N to the tibiae of 43 19-month-old rats using a four-point bending apparatus. Bone formation rates were measured on the periosteal and endocortical surfaces of the tibial midshaft using double-label histomorphometry. Bone formation rates from the old rats were compared with results from adult (9-month-old) rats that we reported earlier.(4) Bone formation on the periosteal surface of the old rats was predominantly woven-fibered. Periosteal bone formation was observed in a lower percentage of the old rats compared with the younger adult rats for applied loads of 40 N and greater (59% old, 100% adult). However, in the old rats that formed woven bone there were no significant differences in woven bone area (p=0.1) or surface (p=0.24) compared with younger adults. Therefore, the periosteum of old rats had a higher threshold for activation by mechanical loading, but after activation occurred, the cells had the same capacity to form woven bone as younger adult animals. On the endocortical surface, relative bone formation rates in old rats showed a marginal (p=0.06) increase in response to an applied load of 64 N but was not increased at lower loads. The relative bone formation rate in the old rats was over 16-fold less than that reported for the younger adult rats at an applied load of 64 N and the relative bone forming surface in old rats in this study was 5-fold less than it was younger rats under similar loading conditions. In the younger adult rats, a mechanical threshold for lamellar bone formation of 1050 microstrain was calculated for the endocortical bone surface. The old rats required over 1700 microstrain on the endocortical surface before bone formation was increased. The data suggest that both the periosteal and endocortical surfaces of the tibiae of older rats are less responsive to mechanical stimuli.

Mineral anisotropy in mineralized tissues is similar among species and mineral growth occurs independently of collagen orientation in rats: results from acoustic velocity measurements.

It has been reported that the mineral crystals in long bones have their c-axis aligned with the bone axis, presumably because collagen fibrils in bone also align with the bone axis. However, the predominant collagen orientation in bone often does not appear to be aligned with the mineral crystals, especially in rat primary bone. We hypothesized that mineral orientation in bone is not necessarily related to collagen orientation. An acoustic microscope was used to measure elastic constants of mineralized tissues from rat, cow, monkey, and human bone, and mineralized turkey leg tendon (MTLT). Measurements were made before and after demineralization with 10% ethylenediaminetetraacetic acid (EDTA) or decollagenization with 7% sodium hypochlorite. The elastic anisotropy ratio (AR) was defined as the ratio of the elastic coefficient in the longitudinal direction to the elastic coefficient in the transverse direction. Anisotropy ratios of mineralized tissues were not affected by formalin fixation or plastic embedding. An evaluation of tissues from the different species showed that the AR after decollagenization was not significantly different (p > 0.4, analysis of variance) among the groups, while AR after demineralization varied from 1.04 (rat bone) to 1.51 (MTLT). There was no correlation between AR after demineralization and AR after decollagenization (r = 0.13, p = 0.5). This showed that the elastic anisotropy of collagen is more variable than mineral anisotropy in bone and MTLT. Another experiment showed that mineralization of turkey leg tendon changes the elasticity of the collagen matrix, making it less anisotropic. A final, prospective experiment was performed in which tibiae of rats were subjected to mechanical loading for 16 weeks. After 12 days, new periosteal woven bone was observed on the tibiae and, after 16 weeks, this new bone was consolidated and mineralized. Mineral in the newly formed woven bone was virtually isotropic (AR = 1.07) after 12 days of loading, then became more anisotropic (AR = 1.52) after 16 weeks of mechanical loading, as the mineral density of the new bone increased. This increase in anisotropy of bone mineral occurred even though the collagen matrix was woven and had no measureable fibril orientation. We conclude that (1) collagen anisotropy and mineral anisotropy are not necessarily correlated in mineralized tissues, (2) mineralization can affect the collagen matrix elasticity of mineralized tissues, and (3) an organized mineral structure can form in the absence of an organized collagen matrix.

The biomechanical basis of vertebral body fragility in men and women.

The aim of this study was to quantify the biomechanical basis for vertebral fracture risk in elderly men and women. A bone is likely to fracture when the loads imposed are similar to or greater than its strength. To quantify this risk, we developed a fracture risk index (FRI) based on the ratio of the vertebral body compressive load and strength. Loads were determined by upper body weight, height, and the muscle moment arm, and strength was estimated from cross-sectional area (CSA) and volumetric bone mineral density (vBMD). With loads less than the strength of the bone, the FRI remains < 1. For any given load, once bone strength diminishes due to a falling vBMD, the FRI will increase. Should FRI approach or exceed unity, structural failure of the vertebra is likely. We measured vertebral body CSA vBMD of the middle zone of third lumbar vertebra by lateral and posteroanterior (PA) scanning using dual-energy X-ray absorptiometry (DXA) and calculated vertebral compressive stress (load per unit area) in 327 healthy men and 686 healthy women and 26 men and 55 postmenopausal women with vertebral fractures. Activities that require forward bending of the upper body caused approximately 10-fold more compressive stress on the vertebra compared with standing upright. Men and women had similar peak vBMD in young adulthood. Because men have greater stature than women, the loads imposed on the vertebral body are higher (3,754 +/- 65 N vs. 3,051 +/- 31 N; p < 0.001). However, because CSA also was higher in men than women, peak load per unit CSA (stress) did not differ by gender (317.4 +/- 4.7 N/cm2 vs. 321.9 +/- 3.3 N/cm2, NS). The FRI was similar in young men and women and well below unity (0.42 +/- 0.02 vs. 0.43 +/- 0.01; NS). Gender differences emerged during aging; CSA increased in both men and women but more so in men, so load per unit area (stress) diminished but more so in men than in women. vBMD decreased in both genders but less so in men. These changes were captured in the FRI, which increased by only 21% in men and by 102% in women so that only 9% of elderly men but 26% of elderly women had an FRI > or = 1. Men and women with vertebral fractures had an FRI that was greater than or equal to unity (1.03 +/- 0.13 vs. 1.35 +/- 0.13; p < 0.05) and was 2.04 SD and 2.26 SD higher than age-matched men and women, respectively. In summary and conclusion, young men and women have a similar vBMD, vertebral stress, and FRI. During aging, CSA increases more, and vBMD decreases less in men than in women. Thus, fewer men than women are at risk for fracture because fewer men than women have these structural determinants of bone strength below a level at which the loads exceed the bone's ability to tolerate them. Men and women with vertebral fractures have FRIs that are equal to or exceed unity. The results show that a fracture threshold for vertebrae can be defined using established biomechanical principles; whether this approach has greater sensitivity and specificity than the current BMD T score of -2.5 SD is unknown.

Partitioning a daily mechanical stimulus into discrete loading bouts improves the osteogenic response to loading.

A single 3-minute bout of mechanical loading increases bone formation in the rat tibia. We hypothesized that more frequent, shorter loading bouts would elicit a greater osteogenic response than a single 3-minute bout. The right tibias of 36 adult female Sprague-Dawley rats were subjected to 360 bending cycles per day of a 54 N force delivered in 1, 2, 4, or 6 bouts on each of the 3 loading days. Rats in the 6-bouts/day group received 60 bending cycles per bout (60 x 6); rats in the 4-bouts/day group received 90 bending cycles per bout (90 x 4); the 2- and 1-bouts/day groups received 180 and 360 bending cycles per bout, respectively (180 x 2 and 360 x 1). A nonloaded, age-matched control group (0 x 0) and two sham-bending groups (60 x 6 and 360 x 1) also were included. Fluorochrome labeling revealed a 10-fold increase in endocortical lamellar bone formation rate (BFR/bone surface [BS]) in the right tibia versus the left (nonloaded) side in the 60 x 6 bending group. Endocortical BFR/BS in the right tibia of the 4-, 2-, and 1-bout bending groups exhibited 8-, 4-, and 4-fold increases, respectively, over the control side. Relative (right minus left) values for endocortical BFR/BS, mineralizing surface (MS/BS), and mineral apposition rate (MAR) were 65-94% greater in the 90 x 4 and 60 x 6 bending groups compared to the 360 x 1 bending group. Sham-bending tibias exhibited relative endocortical bone formation values similar to those collected from the control (0 x 0) group. The data show that 360 daily loading cycles applied at intervals of 60 x 6 or 90 x 4 represent a more osteogenic stimulus than 360 cycles applied all at once, and that mechanical loading is more osteogenic when divided into discrete loading bouts. Presumably, bone cells become increasingly "deaf" to the mechanical stimulus as loading cycles persist uninterrupted, and by allowing a rest period between loading bouts, the osteogenic effectiveness of subsequent cycles can be increased.

Mechanical loading thresholds for lamellar and woven bone formation.

Bone formation was measured in rat tibiae after 12 days of applied loading. Bending forces were applied using a four-point loading apparatus. Sham loads were applied at the same magnitudes as bending forces but the loading pads were arranged so that bending was minimized. Bending and sham loading were applied to the right tibiae of rats and the left tibiae served as contralateral controls. Loading was applied as a sine wave with a frequency of 2 Hz for 18 s (36 cycles) per day. The peak magnitude of applied load was 27, 33, 40, 52, and 64 N. Woven bone was observed on the periosteal surface in all animals subjected to loads of 40 N or greater. Periosteal woven bone formation occurred in both bending and sham loading groups. Woven bone formation on the periosteal surface was either absent or responded at a maximal rate if the stimulus threshold was surpassed. The amount of new woven bone and the woven bone-forming surface were independent of the magnitude of applied strain. Bone formation on the endocortical surface was exclusively lamellar. Lamellar bone formation was stimulated by applied bending of the tibia but not by sham loading. Bending strains above a loading threshold of 40 N or about 1050 mu strain increased both bone-forming surface and the mineral apposition rate and subsequently increased the bone formation rate as much as sixfold. No evidence of increased bone formation was seen for applied strains below 1050 mu strain. Examination of bulk stained sections from animals exposed to the highest applied loads showed no evidence of microcracks.(ABSTRACT TRUNCATED AT 250 WORDS)

Sexual dimorphism in vertebral fragility is more the result of gender differences in age-related bone gain than bone loss.

Spine fractures usually occur less commonly in men than in women. To identify the structural basis for this gender difference in vertebral fragility, we studied 1013 healthy subjects (327 men and 686 women) and 76 patients with spine fractures (26 men and 50 women). Bone mineral content (BMC), cross-sectional area (CSA), and volumetric bone mineral density (vBMD) of the third lumbar vertebral body (L3) were measured by posteroanterior (PA) and lateral scanning using dual-energy X-ray absorptiometry (DXA). In this cross-sectional study, the diminution in peak vertebral body BMC from young adulthood to old age was less in men than in women (6% vs. 27%). This diminution was the net result of two opposing changes occurring concurrently throughout adult life: the removal of bone adjacent to marrow on the inner (endosteal) surface by bone resorption and the deposition of bone on the outer (periosteal) surface by bone formation. For L3, we estimated that men resorbed 3.7 g and deposited 3.1 g, producing a net loss of 0.6 g from young adulthood to old age and women resorbed 3.1 g and deposited only 1.2 g, producing a net loss of 1.9 g. Thus, based on our indirect estimates of periosteal gain and endosteal loss across life, the observed net diminution in BMC during aging was less in men than women because absolute periosteal bone formation was greater in men than women (3.1 g vs. 1.2 g) not because absolute bone resorption was less in men. On the contrary, the absolute amount of bone resorbed was greater in men than women (3.7 g vs. 3.1 g). Periosteal bone formation also increased vertebral body CSA 3-fold more in men than in women, distributing loads onto a larger CSA, so that the load imposed per unit CSA decreased twice as much in men than in women (13% vs. 5%). In men and women with spine fractures, CSA and vBMD were reduced relative to age-matched controls. However, vBMD was no different to the adjusted vBMD in age-matched controls derived assuming controls had no periosteal bone formation during aging. Thus, large amounts of bone are resorbed in men as well as in women, accounting for the age-related increase in spine fractures in both genders. Periosteal bone formation increases CSA and offsets bone loss in both genders but more greatly in men, accounting for the lower incidence of spine fractures in men than in women. We speculate that reduced periosteal bone formation, during growth or aging, may be in part responsible for both reduced vertebral size and reduced vBMD in men and women with spine fractures. Sexual dimorphism in vertebral fragility is more the result of gender differences in age-related bone gain than age-related bone loss.

Suppressed bone turnover by bisphosphonates increases microdamage accumulation and reduces some biomechanical properties in dog rib.

It has been hypothesized that suppression of bone remodeling allows microdamage to accumulate, leading to increased bone fragility. This study evaluated the effects of reduced bone turnover produced by bisphosphonates on microdamage accumulation and biomechanical properties of cortical bone in the dog rib. Thirty-six female beagles, 1-2 years old, were divided into three groups. The control group (CNT) was treated daily for 12 months with saline vehicle. The remaining two groups were treated daily with risedronate (RIS) at a dose of 0.5 mg/kg per day or alendronate (ALN) at 1.0 mg/kg per day orally. After sacrifice, the right ninth rib was assigned to cortical histomorphometry or microdamage analysis. The left ninth rib was tested to failure in three-point bending. Total cross-sectional bone area was significantly increased in both RIS and ALN compared with CNT, whereas cortical area did not differ significantly among groups. One-year treatment with RIS or ALN significantly suppressed intracortical remodeling (RIS, 53%; ALN, 68%) without impairment of mineralization and significantly increased microdamage accumulation in both RIS (155%) and ALN (322%) compared with CNT. Although bone strength and stiffness were not significantly affected by the treatments, bone toughness declined significantly in ALN (20%). Regression analysis showed a significant nonlinear relationship between suppressed intracortical bone remodeling and microdamage accumulation as well as a significant linear relationship between microdamage accumulation and reduced toughness. This study showed that suppression of bone turnover by high doses of bisphosphonates is associated with microdamage accumulation and reduced some mechanical properties of bone.

Anabolic effects of human biosynthetic parathyroid hormone fragment (1-34), LY333334, on remodeling and mechanical properties of cortical bone in rabbits.

Intermittent administration of parathyroid hormone (PTH) has an anabolic effect in cancellous bone of osteoporotic humans. However, the effect of PTH on cortical bone with Haversian remodeling remains controversial. The aim of this study was to determine the effects of biosynthetic human PTH(1-34) on the histology and mechanical properties of cortical bone in rabbits, which exhibit Haversian remodeling. Mature New Zealand white rabbits were treated with once daily injections of vehicle, or PTH(1-34), LY333334, at 10 micrograms/kg/day or 40 micrograms/kg/day for 140 days. Body weight in rabbits treated with PTH did not change significantly over the experimental period. Serum calcium and phosphate were within the normal range, but a 1 mg/ml increase in serum calcium was observed in rabbits given the higher dose of PTH. Histomorphometry of cortical bone in the midshaft of the tibia showed significant increases in periosteal and endocortical bone formation in these rabbits. Intracortical bone remodeling in the tibia was activated and cortical porosity increased by PTH. Cross-sectional bone area and bone mass of the midshaft of the femur increased significantly after PTH treatment. Ultimate force, stiffness, and work to failure of the midshaft of the femur of rabbits given the 40 micrograms dose of PTH were significantly greater than those in the control group, whereas elastic modulus was significantly lower than that in the rabbits given the 10 micrograms dose of PTH, but not different from controls. In the third lumbar vertebra, PTH increased both formation and resorption without increasing cancellous bone volume. The increases in bone turnover and cortical porosity were accompanied by concurrent increases in bone at the periosteal and endocortical surfaces. The combination of these phenomena resulted in an enhancement of the ultimate stress, stiffness, and work to failure of the femur.