Clcn7F318L/+ as a new mouse model of Albers-Schönberg disease.

Dominant negative mutations in CLCN7, which encodes a homodimeric chloride channel needed for matrix acidification by osteoclasts, cause Albers-Schönberg disease (also known as autosomal dominant osteopetrosis type 2). More than 25 different CLCN7 mutations have been identified in patients affected with Albers-Schönberg disease, but only one mutation (Clcn7G213R) has been introduced in mice to create an animal model of this disease. Here we describe a mouse with a different osteopetrosis-causing mutation (Clcn7F318L). Compared to Clcn7+/+ mice, 12-week-old Clcn7F318L/+ mice have significantly increased trabecular bone volume, consistent with Clcn7F318L acting as a dominant negative mutation. Clcn7F318L/F318L and Clcn7F318L/G213R mice die by 1month of age and resemble Clcn7 knockout mice, which indicate that p.F318L mutant protein is non-functional and p.F318L and p.G213R mutant proteins do not complement one another. Since it has been reported that treatment with interferon gamma (IFN-G) improves bone properties in Clcn7G213R/+ mice, we treated Clcn7F318L/+ mice with IFN-G and observed a decrease in osteoclast number and mineral apposition rate, but no overall improvement in bone properties. Our results suggest that the benefits of IFN-G therapy in patients with Albers-Schönberg disease may be mutation-specific.

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.

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.

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.

The relationship between muscle size and bone geometry during growth and in response to exercise.

As muscles become larger and stronger during growth and in response to increased loading, bones should adapt by adding mass, size, and strength. In this unilateral model, we tested the hypothesis that (1) the relationship between muscle size and bone mass and geometry (nonplaying arm) would not change during different stages of puberty and (2) exercise would not alter the relationship between muscle and bone, that is, additional loading would result in a similar unit increment in both muscle and bone mass, bone size, and bending strength during growth. We studied 47 competitive female tennis players aged 8-17 years. Total, cortical, and medullary cross-sectional areas, muscle area, and the polar second moment of area (I(p)) were calculated in the playing and nonplaying arms using magnetic resonance imaging (MRI); BMC was assessed by DXA. Growth effects: In the nonplaying arm in pre-, peri- and post-pubertal players, muscle area was linearly associated BMC, total and cortical area, and I(p) (r = 0.56-0.81, P < 0.09 to < 0.001), independent of age. No detectable differences were found between pubertal groups for the slope of the relationship between muscle and bone traits. Post-pubertal players, however, had a higher BMC and cortical area relative to muscle area (i.e., higher intercept) than pre- and peri-pubertal players (P < 0.05 to < 0.01), independent of age; pre- and peri-pubertal players had a greater medullary area relative to muscle area than post-pubertal players (P < 0.05 to < 0.01). Exercise effects: Comparison of the side-to-side differences revealed that muscle and bone traits were 6-13% greater in the playing arm in pre-pubertal players, and did not increase with advancing maturation. In all players, the percent (and absolute) side-to-side differences in muscle area were positively correlated with the percent (and absolute) differences in BMC, total and cortical area, and I(p) (r = 0.36-0.40, P < 0.05 to < 0.001). However, the side-to-side differences in muscle area only accounted for 11.8-15.9% of the variance of the differences in bone mass, bone size, and bending strength. This suggests that other factors associated with loading distinct from muscle size itself contributed to the bones adaptive response during growth. Therefore, the unifying hypothesis that larger muscles induced by exercise led to a proportional increase in bone mass, bone size, and bending strength appears to be simplistic and denies the influence of other factors in the development of bone mass and bone shape.

Mechanotransduction in bone: genetic effects on mechanosensitivity in mice.

Bone formation is enhanced by mechanical loading, but human exercise intervention studies have shown that the response to mechanical loading is variable, with some individuals exhibiting robust osteogenic responses while others respond modestly. Thus, mechanosensitivity - the ability of bone tissue to detect mechanical loads - could be under genetic control. We applied controlled mechanical loading to the ulnae of 20-week-old (adult) female mice derived from three different inbred strains (C3H/He, C57BL/6, and DBA/2), and measured the bone formation response with fluorochrome labels. Mechanical properties, including mechanical strain, second moments of area, and cortical bone material properties, were measured in a group of calibration animals not subjected to in vivo loading. The C3H/He mice were significantly less responsive to mechanical loading than the other two biological strains. Material properties (flexural elastic modulus, ultimate stress) were greatest in the C3H/He cortical tissue. Geometric and areal properties at the midshaft ulna were also greatest in the C3H/He mice. Based on the presumed role of osteocytes in strain detection, we measured osteocyte lacuna population densities in decalcified midshaft ulna sections. Osteocyte lacuna density was not related to mechanosensitivity. Our data suggest that bone mechanosensitivity has a significant genetic component. Identification of the genes that exert their influence on mechanosensitivity could ultimately lead to therapies that enhance bone mass and reduce fracture susceptibility.

The effect of mechanical loading on the size and shape of bone in pre-, peri-, and postpubertal girls: a study in tennis players.

Exercise during growth results in biologically important increases in bone mineral content (BMC). The aim of this study was to determine whether the effects of loading were site specific and depended on the maturational stage of the region. BMC and humeral dimensions were determined using DXA and magnetic resonance imaging (MRI) of the loaded and nonloaded arms in 47 competitive female tennis players aged 8-17 years. Periosteal (external) cross-sectional area (CSA), cortical area, medullary area, and the polar second moments of area (I(P), mm4) were calculated at the mid and distal sites in the loaded and nonloaded arms. BMC and I(P) of the humerus were 11-14% greater in the loaded arm than in the nonloaded arm in prepubertal players and did not increase further in peri- or postpubertal players despite longer duration of loading (both, p < 0.01). The higher BMC was the result of a 7-11% greater cortical area in the prepubertal players due to greater periosteal than medullary expansion at the midhumerus and a greater periosteal expansion alone at the distal humerus. Loading late in puberty resulted in medullary contraction. Growth and the effects of loading are region and surface specific, with periosteal apposition before puberty accounting for the increase in the bone's resistance to torsion and endocortical contraction contributing late in puberty conferring little increase in resistance to torsion. Increasing the bone's resistance to torsion is achieved by modifying bone shape and mass, not necessarily bone density.

Do bone cells behave like a neuronal network?

Bone cells are organized into an interconnected network, which extends from the osteocytes within bone to the osteoblasts and lining cells on the bone surfaces. There is experimental evidence suggesting that bone tissue exhibits basic properties of short- and long-term memory. An analogy might be made between the bone cell network and neuronal systems. For instance, recent studies suggest that the neurotransmitter glutamate may play a role in cell-to-cell communication among bone cells. Glutamate is a key neurotransmitter involved in learning and memory in reflex loops and the hippocampus. The simplest forms of memory include habituation (desensitization) and sensitization. It is argued that bone cells exhibit habituation to repeated mechanical stimuli and sensitization to mechanical loading by parathyroid hormone (PTH). Acquired long-term memory of a mechanical loading environment may influence the responsiveness of bone tissue to external stimuli. For instance, bone tissue from the skull shows markedly different responses to several stimuli, e.g., mechanical loading, disuse, and PTH, compared with long bones. We speculate that the history of weight bearing imparts long-term cellular memory to the bone cell network that modulates the cellular response to a wide variety of stimuli.

Effects of biomechanical stress on bones in animals.

The signals that allow bone to adapt to its mechanical environment most likely involve strain-mediated fluid flow through the canalicular channels. Fluid can only be moved through bone by cyclic loading, and the shear stresses generated on bone cells are proportional to the rate of loading. The proportional relation between fluid shear stresses on cells and loading rate predicts that the magnitude of bone's adaptive response to loading should be proportional to strain rate. For lower loading frequencies within the physiologic range, experimental evidence shows this is true. It is also true that the mechanical sensitivity of bone cells saturates quickly, and that a period of recovery either between loading cycles or between periods of exercise can optimize adaptive response. Together, these concepts suggest that short periods of exercise, with a 4-8 h rest period between them, are a more effective osteogenic stimulus than a single sustained session of exercise. The data also suggest that activities involving higher loading rates are more effective for increasing bone formation, even if the duration of the activity is short.

Recovery periods restore mechanosensitivity to dynamically loaded bone.

Bone cells are capable of sensing and responding to mechanical forces, but mechanosensitivity begins to decline soon after the stimulus is initiated. Under continued stimulation, bone is desensitized to mechanical stimuli. We sought to determine the amount of time required to restore mechanosensitivity to desensitized bone cells in vivo by manipulating the recovery time (0, 0.5, 1, 2, 4 or 8 h) allowed between four identical daily loading bouts. We also investigated the osteogenic effectiveness of shorter-term recovery periods, lasting several seconds (0.5, 3.5, 7 or 14 s), introduced between each of 36 identical daily loading cycles. Using the rat tibia four-point bending model, the right tibia of 144 adult female Sprague-Dawley rats was subjected to bending, sham bending or no loading. In the rats receiving recovery periods between loading bouts, histomorphometric measurements from the endocortical surface of the loaded and nonloaded control (left) tibiae revealed more than 100 % higher relative bone formation rates in the 8 h recovery group than in the 0 and 0.5 h recovery groups. Approximately 8 h of recovery was sufficient to restore full mechanosensitivity to the cells. In the rats allowed time to recover between load cycles, 14 s of recovery resulted in significantly higher (66-190 %) relative bone formation rates compared to any of the three shorter recovery periods. In both experiments, bone formation in the sham-bending animals was similar to that in the nonloaded control group. The results demonstrate the importance of recovery periods for (i) restoring mechanosensitivity to bone cells and (ii) maximizing the osteogenic effects of mechanical loading (exercise) regimens.

Modulation of appositional and longitudinal bone growth in the rat ulna by applied static and dynamic force.

Appositional and longitudinal growth of long bones are influenced by mechanical stimuli. Using the noninvasive rat ulna loading model, we tested the hypothesis that brief-duration (10 min/day) static loads have an inhibitory effect on appositional bone formation in the middiaphysis of growing rat ulnae. Several reports have shown that ulnar loading, when applied to growing rats, results in suppressed longitudinal growth. We tested a second hypothesis that load-induced longitudinal growth suppression in the growing rat ulna is proportional to time-averaged load, and that growth plate dimensions and chondrocyte populations are reduced in the loaded limbs. Growing male rats were divided into one of three groups receiving daily 10 min bouts of static loading at 17 N, static loading at 8.5 N, or dynamic loading at 17 N. Periosteal bone formation rates, measured 3 mm distal to the ulnar midshaft, were suppressed significantly (by 28-41%) by the brief static loading sessions despite normal (dynamic) limb use between the daily loading bouts. Static loading neither suppressed nor enhanced endocortical bone formation. Dynamic loading increased osteogenesis significantly on both surfaces. At the end of the 2 week loading experiment, loaded ulnae were approximately 4% shorter than the contralateral controls in the 17 N static and dynamic groups, and approximately 2% shorter than the control side in the 8.5 N static group, suggesting that growth suppression was proportional to peak load magnitude, regardless of whether the load was static or dynamic. The suppressed growth in loaded limbs was associated with thicker distal growth plates, particularly in the hypertrophic zone, and a concurrent retention of hypertrophic cell lacunae. Negligible effects were observed in the proximal growth plate. The results demonstrate that, in growing animals, even short periods of static loading can significantly suppress appositional growth; that dynamic loads trigger the adaptive response in bone; and that longitudinal growth suppression resulting from compressive end-loads is proportional to load magnitude and not average load.

Skeletal loading in animals.

A number of in vivo skeletal loading models have been developed to test specific hypotheses addressing the key mechanical and biochemical signals involved in bone's adaptive response to loading. Exercise protocols, osteotomy procedures, loading of surgically implanted pins, and force application through the soft tissues are common approaches to alter the mechanical environment of a bone. Although each animal overload model has a number of assets and limitations, models employing extrinsic forces allow greater control of the mechanical environment. Sham controls, for both surgical intervention (when performed) and loading, are required to unequivocally demonstrate that responses to loading are mechanically adaptive. Collectively, extrinsic loading models have fostered a greater understanding of the mechanical signals important for stimulating bone cells, and highlighted the roles of key signaling molecules in the adaptive response.

Mechanical loading of diaphyseal bone in vivo: the strain threshold for an osteogenic response varies with location.

Bone tissue responds to elevated mechanical loading with increased bone formation, which is triggered either directly or indirectly by the mechanical strain engendered in the bone tissue. Previous studies have shown that mechanical strain magnitude must surpass a threshold before bone formation is initiated. The objective of this study was to estimate the strain thresholds at three different locations along the ulna of adult rats. We hypothesized that the strain threshold would be greater in regions of the ulna habitually subjected to larger mechanical strains. New bone formation was measured on the periosteal and endocortical surfaces of the ulnar diaphysis in adult female rats exposed to controlled dynamic loading. Axial, compressive loading was applied daily at five different magnitudes for a period of 2 weeks. Bone formation rate (BFR) was measured, using double-label histomorphometry at the ulnar middiaphysis and at locations 3 mm proximal and 3 mm distal to the middiaphysis. Loading induced lamellar bone formation on the periosteal surface that was greater at the distal ulnar location and lower at the proximal location when compared with the middiaphysis. Likewise, peak strains on the periosteal surface were greatest distally and less proximally. There was a significant dose-response relationship between peak strain magnitude and periosteal new bone formation when the mechanically induced strain surpassed a threshold. The strain threshold varied from 1343 microstrain (mu strain) proximally to 2284 mu strain at the midshaft to 3074 mu strain distally. Unlike the periosteal response to mechanical loading, there was not a clear dose-response relationship between applied load and bone formation on the endocortical surface. Endocortical strains were estimated to be < 20% of periosteal strains and may not have been sufficient to initiate a bone formation response. Our results show that the osteogenic response on the periosteal surface of the ulna depends on peak strain level once a strain threshold is surpassed. The threshold strain is largest distally, where locomotor bone strains are typically higher and smallest proximally where locomotor bone strains are lower.

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.

Trends in chronic obstructive pulmonary disease mortality.

This study examines the mortality trends for chronic obstructive pulmonary disease (COPD) among whites and African Americans in Missouri from 1980-1996. Data from the Missouri Center for Health Information Management and Epidemiology were used to calculate mortality rates. Missouri's COPD deaths rose 40.6% from 1980-1996. Projections through the year 2006 predict continued escalation in rates. Much of the growth in COPD can be attributed to heavy tobacco use in the population.

Morphology of the drifting osteon.

Drifting osteons were followed longitudinally through the cortex of human and baboon long bones using serial sections. Direction of transverse drift was recorded at different cross-sectional levels of the same systems, and maximum angular change in drift direction was measured for each system. Most drifting osteons exhibit: (1) substantial ( approximately 90 degrees ) variation in the direction of transverse drift along their longitudinal axes, (2) intermittent regions of concentric (type I) morphology, and (3) change in drift direction over time, evident at single cross-sectional levels. Additionally, 3-dimensional reconstruction reveals that the basic multicellular units (BMUs) responsible for creating drifting osteons are morphologically distinct from the cutting-cone-closing-cone model BMUs that produce other types of osteons. The stimulus involved in the activation and guidance of drifting BMUs is unclear, but it is likely that the complex strain environment experienced by long bone cortices exerts a significant influence on their morphology.

Trends in cancer incidence and mortality in Missouri.

BACKGROUND: Cancer is the second leading cause of death in Missouri. Several factors influence public health planning and policy-making decisions, including assessment of the trends in disease and in related risk factors, of overall disease burden on the population, and of the effectiveness of intervention programs. To assess the influence of statewide cancer screening, education, and intervention programs on Missouri's cancer burden, recent trends in cancer incidence and mortality rates among Missourians were analyzed. METHODS: Age-standardized cancer incidence rates from 1985-1992 and cancer mortality rates from 1985-1994 and 1990-1996 were calculated by race, age group, and sex using data collected by the Missouri Cancer Registry and the Missouri Center for Health Statistics. Rates for each year examined were logged and regressed onto year. RESULTS: Between 1985 and 1992, the rate of cancer incidence increased by 16.1% (p < .05). From 1985-1994, cancer mortality rate increased by 3.5% (p < .05). Mortality among males, however, decreased between 1990 and 1996. This decrease was particularly pronounced among African-American males. Missouri males endured greater incidence of and mortality from cancer than Missouri females. Elderly males (both African American and white) exhibited the greatest increase in cancer incidence over the periods examined (+27.3% and +31.8% increase respectively, p < .05). Racial differences were nonexistent for cancer incidence rates, but the cancer mortality rate for African Americans was 45% greater than that for whites. CONCLUSIONS: Although Missouri cancer incidence and mortality rates are higher now compared to 16 years ago, data are presented that indicate a slight decline in mortality rates over the past six years. The recent decline (1990-96) in mortality was present only among men, particularly African-American men. The incidence increase was particularly important for women. Despite the implementation of some programs designed to target minorities and undeserved populations at greater risk, these data indicate that large demographic differences in cancer incidence and mortality still persist. A greater commitment to cancer screening, access to treatment, and cancer prevention programs are necessary to meet Missouri's year 2000 goals for cancer incidence and mortality.