Aged mice have enhanced endocortical response and normal periosteal response compared with young‐adult mice following 1 week of axial tibial compression

With aging, the skeleton may lose its ability to respond to positive mechanical stimuli. We hypothesized that aged mice are less responsive to loading than young‐adult mice. We subjected aged (22 months) and young‐adult (7 months) BALB/c male mice to daily bouts of axial tibial compression for 1 week and evaluated cortical and trabecular responses using micro–computed tomography (µCT) and dynamic histomorphometry. The right legs of 95 mice were loaded for 60 rest‐inserted cycles per day to 8, 10, or 12 N peak force (generating mid‐diaphyseal strains of 900 to 1900 µε endocortically and 1400 to 3100 µε periosteally). At the mid‐diaphysis, mice from both age groups showed a strong anabolic response on the endocortex (Ec) and periosteum (Ps) [Ec.MS/BS and Ps.MS/BS: loaded (right) versus control (left), p < .05]. Generally, bone formation increased with increasing peak force. At the endocortical surface, contrary to our hypothesis, aged mice had a significantly greater response to loading than young‐adult mice (Ec.MS/BS and Ec.BFR/BS: 22 months versus 7 months, p < .001). Responses at the periosteal surface did not differ between age groups (p > .05). The loading‐induced increase in bone formation resulted in increased cortical area in both age groups (loaded versus control, p < .05). In contrast to the strong cortical response, loading only weakly stimulated trabecular bone formation. Serial (in vivo) µCT examinations at the proximal metaphysis revealed that loading caused a loss of trabecular bone in 7‐month‐old mice, whereas it appeared to prevent bone loss in 22‐month‐old mice. In summary, 1 week of daily tibial compression stimulated a robust endocortical and periosteal bone‐formation response at the mid‐diaphysis in both young‐adult and aged male BALB/c mice. We conclude that aging does not limit the short‐term anabolic response of cortical bone to mechanical stimulation in our animal model. © 2010 American Society for Bone and Mineral Research     

Tibial compression is anabolic in the adult mouse skeleton despite reduced responsiveness with aging

The ability of the skeleton to adapt to mechanical stimuli diminishes with age in diaphyseal cortical bone, making bone formation difficult for adults. However, the effect of aging on adaptation in cancellous bone, tissue which is preferentially lost with age, is not well characterized. To develop a model for early post-menopausal women and determine the effect of aging on cancellous bone adaptation in the adult mouse skeleton, in vivo tibial compression was applied to adult (26 week old) osteopenic female mice using loading parameters, peak applied load and peak diaphyseal strain magnitude, that were previously found to be osteogenic in young, growing (10 week old) mice. A Load-Matched group received the same peak applied loads (corresponding to +2100 με at the medial diaphysis of the tibia) and a Strain-Matched group received the same peak diaphyseal strains (+1200 με, requiring half the load) as the young mice. The effects of mechanical loading on bone mass and architecture in adult mice were assessed using micro-computed tomography and in vivo structural stiffness measures. Adaptation occurred only in the Load-Matched group in both the metaphyseal and diaphyseal compartments. Cancellous bone mass increased 54% through trabecular thickening, and cortical area increased 41% through medullary contraction and periosteal expansion. Adult mice were able to respond to an anabolic stimulus and recover bone mass to levels seen in growing mice; however, the adaptive response was reduced relative to that in 10 week old female mice for the same applied load. Using this osteogenic loading protocol, other factors affecting pathological bone loss can be addressed using an adult osteopenic mouse model.     

Connexin 43 Is Necessary for Murine Tendon Enthesis Formation and Response to Loading

The enthesis is a mineralized fibrocartilage transition that attaches tendon to bone and is vital for musculoskeletal function. Despite recent studies demonstrating the necessity of muscle loading for enthesis formation, the mechanisms that regulate enthesis formation and mechanoresponsiveness remain unclear. Therefore, the current study investigated the role of the gap junction protein connexin 43 in these processes by deleting Gja1 (the Cx43 gene) in the tendon and enthesis. Compared with their wild-type (WT) counterparts, mice lacking Cx43 showed disrupted entheseal cell alignment, reduced mineralized fibrocartilage, and impaired biomechanical properties of the supraspinatus tendon entheses during postnatal development. Cx43-deficient mice also exhibited reduced ability to complete a treadmill running protocol but no apparent deficits in daily activity, metabolic indexes, shoulder muscle size, grip strength, and major trabecular bone properties of the adjacent humeral head. To examine enthesis mechanoresponsiveness, young adult mice were subjected to modest treadmill exercise. Gja1 deficiency in the tendon and enthesis reduced entheseal anabolic responses to treadmill exercise: WT mice had increased expression of Sox9, Ihh, and Gli1 and increased Brdu incorporation, whereas Cx43-deficient mice showed no changes or decreased levels with exercise. Collectively, the results demonstrated an essential role for Cx43 in postnatal tendon enthesis formation, function, and response to loading; results further provided evidence implicating a link between Cx43 function and the hedgehog signaling pathway. © 2020 American Society for Bone and Mineral Research.     

Short-term and long-term site-specific effects of tennis playing on trabecular and cortical bone at the distal radius

Mechanical loading during growth magnifies the normal increase in bone diameter occurring in long bone shafts, but the response to loading in long bone ends remains unclear. The aim of the study was to investigate the effects of tennis playing during growth at the distal radius, comparing the bone response at trabecular and cortical skeletal sites. The influence of training duration was examined by studying bone response in short-term (children) and long-term (young adults) perspectives. Bone area, bone mineral content (BMC), and bone mineral density (BMD) of the radius were measured by DXA in 28 young (11.6 ± 1.4 years old) and 47 adult tennis players (22.3 ± 2.7 years old), and 70 age-matched controls (12 children, 58 adults) at three sites: the ultradistal region (trabecular), the mid-distal region, and the third-distal region (cortical). At the ultradistal radius, young and adult tennis players displayed similar side-to-side differences, the asymmetry in BMC reaching 16.3% and 13.8%, respectively (P < 0.0001). At the mid- and third-distal radius, the asymmetry was much greater in adults than in children (P < 0.0001) for all the bone parameters (mid-distal radius, +6.6% versus +15.6%; third-distal radius, +6.9% versus +13.3%, for BMC). Epiphyseal bone enduring longitudinal growth showed a great capacity to respond to mechanical loading in children. Prolonging tennis playing into adulthood was associated with further increase in bone mineralization at diaphyseal skeletal sites. These findings illustrate the benefits of practicing impact-loading sports during growth and maintaining physical activity into adulthood to enhance bone mass accrual and prevent fractures later in life.     

A Population‐Based Assessment of Rates of Bone Loss at Multiple Skeletal Sites: Evidence for Substantial Trabecular Bone Loss in Young Adult Women and Men

Using QCT, we made a longitudinal, population‐based assessment of rates of bone loss over life at the distal radius, distal tibia, and lumbar spine. Cortical bone loss began in perimenopause in women and later in life in men. In contrast, trabecular bone loss began in young adulthood in both sexes. Introduction: Although conventional wisdom holds that bone loss begins at menopause in women and later in life in men, this has not been examined longitudinally in population‐based studies using precise technology capable of distinguishing cortical and trabecular bone. Materials and Methods: In an age‐ and sex‐stratified population sample (n = 553), we measured volumetric BMD (vBMD) of trabecular and cortical bone by QCT annually for up to 3 yr at the distal radius (DR) and distal tibia (DT) (n = 552) and trabecular vBMD at baseline and 3 yr at the lumbar spine (LS) (n = 474). Results: Substantial cortical bone loss began in middle life in women but began mainly after age 75 in men. In contrast, substantial trabecular bone loss began in young adult women and men at all three skeletal sites and continued throughout life with acceleration during perimenopause in women. Women experienced 37% and men experienced 42% of their total lifetime trabecular bone loss before age 50 compared with 6% and 15%, respectively, for cortical bone. Median rates of change in trabecular bone (%/yr) were ?0.40, ?0.24, and ?1.61 in young adult women and ?0.38, ?0.40, and ?0.84 in young adult men at the DR, DT, and LS, respectively (all p < 0.001). The early trabecular bone loss did not consistently correlate with putative causal factors, except for a trend with IGF‐related variables at DT in women. However, in postmenopausal women and, to a lesser extent, in older men, higher rates of cortical and trabecular bone loss were associated with lower levels of biologically‐active sex steroids and with higher levels of follicle‐stimulating hormone and bone turnover markers. Conclusions: The late onset of cortical bone loss is temporally associated with sex steroid deficiency. However, the early‐onset, substantial trabecular bone loss in both sexes during sex steroid sufficiency is unexplained and indicates that current paradigms on the pathogenesis of osteoporosis are incomplete.     

Constrained tibial vibration does not produce an anabolic bone response in adult mice

Osteoporosis is characterized by low bone mass and increased fracture risk. High frequency, low-amplitude whole-body vibration (WBV) has been proposed as a treatment for osteoporosis because it can stimulate new bone formation and prevent trabecular bone loss. We developed constrained tibial vibration (CTV) as a method for controlled vibrational loading of the lower leg of a mouse. We first subjected mice to five weeks of daily CTV loading (0.5 G maximum acceleration) with loading parameters chosen to independently investigate the effects of strain magnitude, loading frequency, and cyclic acceleration on the adaptive response to vibration. We hypothesized that mice subjected to the highest magnitude of dynamic strain would have the largest bone formation response. We observed a slight, local benefit of CTV loading on trabecular bone, as BV/TV was 5.2% higher in the loaded vs. non-loaded tibia of mice loaded with the highest bone strain magnitude. However, despite these positive differences, we observed significantly lower measures of trabecular structure in both loaded and non-loaded tibias from CTV loaded mice compared to Sham and Baseline Control animals, indicating a negative systemic effect of CTV on trabecular bone. Based on this evidence, we conducted a follow-up study wherein mice were subjected to CTV or sham loading, and tibias were scanned at the beginning and end of the study period using in vivo microCT. Consistent with the findings of the first study, trabecular BV/TV in both tibias of CTV loaded and Sham mice was, on average, 36% and 31% lower on day 36 than day 0, respectively, compared to 20% lower in Age-Matched Controls over the same time period. Contrary to the first study, there were no differences between loaded and non-loaded tibias in CTV loaded mice, providing no evidence for a local benefit of CTV. In summary, 5 weeks of daily CTV loading of mice was, at best, weakly anabolic for trabecular bone in the proximal tibia, while daily handling and exposure to anesthesia was associated with significant loss of trabecular and cortical bone. We conclude that direct vibrational loading of bone in anesthetized, adult mice is not anabolic.     

Exercise does not enhance aged bone's impaired response to artificial loading in C57Bl/6 mice

Bones adapt their structure to their loading environment and so ensure that they become, and are maintained, sufficiently strong to withstand the loads to which they are habituated. The effectiveness of this process declines with age and bones become fragile fracturing with less force. This effect in humans also occurs in mice which experience age-related bone loss and reduced adaptation to loading. Exercise engenders many systemic and local muscular physiological responses as well as engendering local bone strain. To investigate whether these physiological responses influence bones' adaptive responses to mechanical strain we examined whether a period of treadmill exercise influenced the adaptive response to an associated period of artificial loading in young adult (17-week) and old (19-month) mice. After treadmill acclimatization, mice were exercised for 30 min three times per week for two weeks. Three hours after each exercise period, right tibiae were subjected to 40 cycles of non-invasive axial loading engendering peak strain of 2250 με. In both young and aged mice exercise increased cross-sectional muscle area and serum sclerostin concentration. In young mice it also increased serum IGF1. Exercise did not affect bone's adaptation to loading in any measured parameter in young or aged bone. These data demonstrate that a level of exercise sufficient to cause systemic changes in serum, and adaptive changes in local musculature, has no effect on bone's response to loading 3 h later. This study provides no support for the beneficial effects of exercise on bone in the elderly being mediated by systemic or local muscle-derived effects rather than local adaptation to altered mechanical strain.     

Blockade of beta-adrenergic signaling does not influence the bone mechano-adaptive response in mice

The involvement of the sympathetic nervous system (SNS) in the modulation of bone adaptation to its load-bearing demand remains controversial. This study tested the involvement of SNS in the adaptive response of trabecular and cortical bone to either external loading or disuse. External loading consisted of cyclic strain (40 cycles, peak 1500 microstrain) applied for 7 min, 3 days/week, while disuse was induced by unilateral sciatic neurectomy (SN). C57Bl/J6 mice, female, 9 weeks old, were subjected to loading or disuse for 2 weeks. Half of the loaded and SN mice were injected with the beta-adrenergic antagonist, propranolol (PRO, 20 mug/g) 1 week before the start of loading or disuse and during all the duration of the experiment. MicroCT analysis of the tibiae showed that the applied load induced significant changes on both trabecular architecture and cortical geometry compared to the contralateral controls, indicating increased bone mass. In contrast, disuse markedly reduced trabecular and cortical indexes. However, these adaptive responses were not altered by PRO treatment. We further tested whether the lack of protective effect of PRO against disuse-induced bone loss was due to the very short duration of treatment by blocking SNS signaling for 8 weeks with either PRO (0.5 mg/ml in drinking water) or guanethidine sulfate (GS, 40 mug/g, injected). At the end of fourth week of treatment, mice underwent SN surgery so that disuse was induced for the remaining 4 weeks. Again, neither PRO nor GS treatments altered the disuse-induced bone loss in the neurectomized tibia. In addition, blockade of SNS signaling for either 3 or 8 weeks did not affect the basal trabecular bone architecture in control tibiae and in L4 vertebrae. This study shows that the mechano-adaptive response occurring in trabecular and cortical bone upon loading or disuse is not altered by inactivation of beta-adrenergic signaling. Furthermore, sympathectomy had no effect on trabecular bone at different skeletal sites. This suggests that the osteo-regulatory action of beta-adrenergic signaling is not involved in the bone mechano-adaptive response and must therefore affect other bone regulatory pathways.     

Cortical and trabecular bone adaptation to incremental load magnitudes using the mouse tibial axial compression loading model

The mouse tibial axial compression loading model has recently been described to allow simultaneous exploration of cortical and trabecular bone adaptation within the same loaded element. However, the model frequently induces cortical woven bone formation and has produced inconsistent results with regards to trabecular bone adaptation. The aim of this study was to investigate bone adaptation to incremental load magnitudes using the mouse tibial axial compression loading model, with the ultimate goal of revealing a load that simultaneously induced lamellar cortical and trabecular bone adaptation. Adult (16 weeks old) female C57BL/6 mice were randomly divided into three load magnitude groups (5, 7 and 9 N), and had their right tibia axially loaded using a continuous 2-Hz haversine waveform for 360 cycles/day, 3 days/week for 4 consecutive weeks. In vivo peripheral quantitative computed tomography was used to longitudinally assess midshaft tibia cortical bone adaptation, while ex vivo micro-computed tomography and histomorphometry were used to assess both midshaft tibia cortical and proximal tibia trabecular bone adaptation. A dose response to loading magnitude was observed within cortical bone, with increasing load magnitude inducing increasing levels of lamellar cortical bone adaptation within the upper two thirds of the tibial diaphysis. Greatest cortical bone adaptation was observed at the midshaft where there was a 42% increase in estimated mechanical properties (polar moment of inertia) in the highest (9 N) load group. A dose response to load magnitude was not clearly evident within trabecular bone, with only the highest load (9 N) being able to induce measureable adaptation (31% increase in trabecular bone volume fraction at the proximal tibia). The ultimate finding was that a load of 9 N (engendering a tensile strain of 1833 με on medial surface of the midshaft tibia) was able to simultaneously induce measurable lamellar cortical and trabecular bone adaptation when using the mouse tibial axial compression loading model in 16 week old female C57BL/6 mice. This finding will help plan future studies aimed at exploring simultaneous lamellar cortical and trabecular bone adaptation within the same loaded element.     

Osteocyte Estrogen Receptor β (Ot-ERβ) Regulates Bone Turnover and Skeletal Adaptive Response to Mechanical Loading Differently in Male and Female Growing and Adult Mice

Age-related bone loss is a failure of balanced bone turnover and diminished skeletal mechanoadaptation. Estrogen receptors, ERα and ERβ, play critical roles in osteoprotective regulation activated by estrogen and mechanical signals. Previous studies mainly focused on ERα and showed that osteocyte-ERα (Ot-ERα) regulated trabecular, but not cortical bone, and played a minor role in load-induced cortical adaptation. However, the role of Ot-ERβ in bone mass regulation remains unrevealed. To address this issue, we characterized bone (re)modeling and gene expression in male and female mice with Ot-ERβ deletion (ERβ-dOT) and littermate control (LC) at 10 weeks (young) or 28 weeks (adult) of age, as well as their responses to in vivo tibial compressive loading. Increased cancellous bone mass appeared in the L₄ vertebral body of young male ERβ-dOT mice. At the same time, femoral cortical bone gene expression showed signs consistent with elevated osteoblast and osteoclast activities (type-I collagen, Cat K, RANKL). Upregulated androgen receptor (AR) expression was observed in young male ERβ-dOT mice relative to LC, suggesting a compensatory effect of testosterone on male bone protection. In contrast, bone mass in L₄ decreased in adult male ERβ-dOT mice, attributed to potentially increased bone resorption activity (Cat K) with no change in bone formation. There was no effect of ERβ-dOT on bone mass or gene expression in female mice. Sex-dependent regulation of Ot-ERβ also appeared in load-induced cortical responsiveness. Young female ERβ-dOT mice showed an enhanced tibial cortical anabolic adaptation compared with LC. In contrast, an attenuated cortical anabolic response presented at the proximal tibia in male ERβ-dOT mice at both ages. For the first time, our findings suggest that Ot-ERβ regulates bone (re)modeling and the response to mechanical signals through different mechanisms in males and females. © 2022 The Authors. Journal of Bone and Mineral Research published by Wiley Periodicals LLC on behalf of American Society for Bone and Mineral Research (ASBMR).     

Caloric restriction leads to high marrow adiposity and low bone mass in growing mice

The effects of caloric restriction (CR) on the skeleton are well studied in adult rodents and include lower cortical bone mass but higher trabecular bone volume. Much less is known about how CR affects bone mass in young, rapidly growing animals. This is an important problem because low caloric intake during skeletal acquisition in humans, as in anorexia nervosa, is associated with low bone mass, increased fracture risk, and osteoporosis in adulthood. To explore this question, we tested the effect of caloric restriction on bone mass and microarchitecture during rapid skeletal growth in young mice. At 3 weeks of age, we weaned male C57Bl/6J mice onto 30% caloric restriction (10% kcal/fat) or normal diet (10% kcal/fat). Outcomes at 6 (n = 4/group) and 12 weeks of age (n = 8/group) included body mass, femur length, serum leptin and insulin‐like growth factor 1 (IGF‐1) values, whole‐body bone mineral density (WBBMD, g/cm²), cortical and trabecular bone architecture at the midshaft and distal femur, bone formation and cellularity, and marrow fat measurement. Compared with the normal diet, CR mice had 52% and 88% lower serum leptin and 33% and 39% lower serum IGF‐1 at 6 and 12 weeks of age (p < .05 for all). CR mice were smaller, with lower bone mineral density, trabecular, and cortical bone properties. Bone‐formation indices were lower, whereas bone‐resorption indices were higher (p < .01 for all) in CR versus normal diet mice. Despite having lower percent of body fat, bone marrow adiposity was elevated dramatically in CR versus normal diet mice (p < .05). Thus we conclude that caloric restriction in young, growing mice is associated with impaired skeletal acquisition, low leptin and IGF‐1 levels, and high marrow adiposity. These results support the hypothesis that caloric restriction during rapid skeletal growth is deleterious to cortical and trabecular bone mass and architecture, in contrast to potential skeletal benefits of CR in aging animals. © 2010 American Society for Bone and Mineral Research.     

Periostin expression contributes to cortical bone loss during unloading

Periostin (a product of Postn gene) is a matricellular protein which is increased in periosteal osteoblasts and osteocytes upon mechanical stimulation. We previously reported that periostin-deficient mice (Postn^−/−) have low bone mass and a diminished response to physical activity due to a lack of sclerostin (a product of Sost gene) inhibition by mechanical loading. Here we hypothesized that periostin could play a central role in the control of bone loss during unloading induced by hindlimb suspension (HU).In Postn^+/+ mice (wildtype littermate), HU significantly decreased femur BMD, as well as trabecular BV/TV and thickness (Tb.Th). Cortical bone volume and thickness at the femoral midshaft, also significantly decreased. These changes were explained by an inhibition of endocortical and periosteal bone formation activity and correlated with a decrease of Postn expression and a consecutive increase in Sost early after HU. Whereas trabecular bone loss in Postn^−/− mice was comparable to Postn^+/+ mice, HU did not significantly alter cortical bone microstructure and strength in Postn^−/− mice. Bone formation remained unchanged in these mice, as Sost did not increase in the absence of periostin. In contrast, changes in Dkk1, Rankl and Opg expression in response to HU were similar to Postn^+/+ mice, indicating that changes in periostin expression were quite specifically related to changes in Sost. In conclusion, HU inhibits periostin expression, which in turn plays an important role in cortical bone loss through an increase in Sost. These results further indicate that periostin is an essential mediator of cortical bone response to mechanical forces (loading and unloading).     

Site-specific changes in bone microarchitecture, mineralization, and stiffness during lactation and after weaning in mice

Despite the dramatic bone loss that occurs during lactation, bone mineral density rapidly recovers after offspring are weaned and milk production stops. The goal of this study is to quantify site-specific changes in bone quantity and quality during and after lactation in a mouse model. We used micro computed tomography (μCT), individual trabecula segmentation (ITS), digital topological analysis (DTA)-based tissue mineral density (TMD) analysis, and micro finite element analysis (μFEA) to quantify the effects of lactation and weaning on bone microarchitecture, mineralization, and stiffness at the spine, tibia, and femur. We found a significant decrease in trabecular plate microarchitecture, tissue mineralization of the trabecular surface, trabecular central skeleton, and intervening envelopes, and whole bone stiffness in lactating versus nulliparous mice at all three sites. In recovered mice, all these different aspects of bone quality were comparable to nulliparous mice at the spine. In contrast, trabecular plate microarchitecture and whole bone stiffness at the tibia and femur in recovered mice were lower than nulliparous mice, as were central trabecular tissue mineralization and cortical structure at the femur. These findings are consistent with clinical observations of partial recovery of femoral bone mineral density BMD after lactation in humans. The observed differences in trabecular surface tissue mineralization in nulliparous, lactating, and recovered mice are consistent with prior observations that maternal bone turnover shifts from resorption to formation at the time of pup weaning. The significant differences in trabecular central tissue mineralization during these three states suggest that osteocytes may contribute to the reversible loss of mineral during and after lactation. Future studies are necessary to determine whether differing functions of various bone cells at individual skeletal sites cause site-specific skeletal changes during and after lactation.     

The cyclooxygenase-2 selective inhibitor NS-398 does not influence trabecular or cortical bone gain resulting from repeated mechanical loading in female mice

Summary

A single injection of the cyclooxygenase-2 (COX-2) selective inhibitor NS-398 reduces bone’s osteogenic response to a single period of mechanical loading in female rats, while women taking COX-2 selective inhibitors do not have lower bone mass. We show that daily NS-398 injection does not influence bone gain from repeated loading in female mice.

Introduction

Prostaglandins are mediators of bone cells’ early response to mechanical stimulation. COX-2 expression is up-regulated by exposure of these cells to mechanical strain or fluid flow, and the osteogenic response to a single loading period is reduced by COX-2 inhibition. This study determined, in female mice in vivo, the effect of longer term COX-2 inhibition on adaptive (re)modelling of cortical and trabecular bone in response to repeated loading.

Methods

Nineteen-week-old female C57BL/6 mice were injected with vehicle or NS-398 (5 mg/kg/day) 5 days a week for 2 weeks. On three alternate days each week, the right tibiae/fibulae were axially loaded [40 cycles (7 min)/day] three hours after injection. Left limbs acted as internal controls. Changes in three-dimensional bone architecture were analysed by high-resolution micro-computed tomography.

Results

In control limbs NS-398 was associated with reduced trabecular number but had no influence on cortical bone. In loaded limbs trabecular thickness and cortical periosteally enclosed volume increased. NS-398 showed no effect on this response.

Conclusion

Pharmacological inhibition of COX-2 by NS-398 does not affect trabecular or cortical bone’s response to repeated mechanical loading in female mice and thus would not be expected to impair the functional adaptation of bone to physical activity in women.     

Combined treatment with a transforming growth factor beta inhibitor (1D11) and bortezomib improves bone architecture in a mouse model of myeloma-induced bone disease

Multiple myeloma (MM) patients frequently develop tumor-induced bone destruction, yet no therapy completely eliminates the tumor or fully reverses bone loss. Transforming growth factor-β (TGF-β) activity often contributes to tumor-induced bone disease, and pre-clinical studies have indicated that TGF-β inhibition improves bone volume and reduces tumor growth in bone metastatic breast cancer. We hypothesized that inhibition of TGF-β signaling also reduces tumor growth, increases bone volume, and improves vertebral body strength in MM-bearing mice. We treated myeloma tumor-bearing (immunocompetent KaLwRij and immunocompromised Rag2 −/−) mice with a TGF-β inhibitory (1D11) or control (13C4) antibody, with or without the anti-myeloma drug bortezomib, for 4 weeks after inoculation of murine 5TGM1 MM cells. TGF-β inhibition increased trabecular bone volume, improved trabecular architecture, increased tissue mineral density of the trabeculae as assessed by ex vivo micro-computed tomography, and was associated with significantly greater vertebral body strength in biomechanical compression tests. Serum monoclonal paraprotein titers and spleen weights showed that 1D11 monotherapy did not reduce overall MM tumor burden. Combination therapy with 1D11 and bortezomib increased vertebral body strength, reduced tumor burden, and reduced cortical lesions in the femoral metaphysis, although it did not significantly improve cortical bone strength in three-point bending tests of the mid-shaft femur. Overall, our data provides rationale for evaluating inhibition of TGF-β signaling in combination with existing anti-myeloma agents as a potential therapeutic strategy to improve outcomes in patients with myeloma bone disease.     

Quantification of skeletal growth,modeling, and remodeling by in vivo micro computed tomography

In this study we established an image analysis scheme for the investigation of cortical and trabecular bone development during skeletal growth and tested this concept on in vivo μCT images of rats. To evaluate its efficacy, we applied the technique to young (1-month-old) and adult (3-month-old) rat tibiae with vehicle (Veh) or intermittent parathyroid hormone (PTH) treatment. By overlaying 2 sequential scans based on their distinct trabecular microarchitecture, we calculated the linear growth rate of young rats to be 0.31 mm/day at the proximal tibia. Due to rapid growth (3.7 mm in 12 days), the scanned bone region at day 12 had no overlap with the bone tissue scanned at day 0. Instead, the imaged bone region at day 12 represented newly generated bone tissue from the growth plate. The new bone of the PTH-treated rats had significantly greater trabecular bone volume fraction, number, and thickness than those of the Veh-treated rats, indicating PTH's anabolic effect on bone modeling. In contrast, the effect of PTH on adult rat trabecular bone was found to be caused by PTH's anabolic effect on bone remodeling. The cortical bone at the proximal tibia of young rats also thickened more in the PTH group (23%) than the Veh group (14%). This was primarily driven by endosteal bone formation and coalescence of trabecular bone into the cortex. This process can be visualized by aligning the local bone structural changes using image registration. As a result, the cortex after PTH treatment was 31% less porous, and had a 22% greater polar moment of inertia compared to the Veh group. Lastly, we monitored the longitudinal bone growth in adult rats by measuring the distance of bone flow away from the proximal tibial growth plate from 3 months to 19 months of age and discovered a total of 3.5 mm growth in 16 months. It was demonstrated that this image analysis scheme can efficiently evaluate bone growth, bone modeling, and bone remodeling, and is ready to be translated into a clinical imaging platform.     

Bone density, strength, and formation in adult cathepsin K (-/-) mice

Cathepsin K (CatK) is a cysteine protease expressed predominantly in osteoclasts, that plays a prominent role in degrading Type I collagen. Growing CatK null mice have osteopetrosis associated with a reduced ability to degrade bone matrix. Bone strength and histomorphometric endpoints in young adult CatK null mice aged more than 10 weeks have not been studied. The purpose of this paper is to describe bone mass, strength, resorption, and formation in young adult CatK null mice. In male and female wild-type (WT), heterozygous, and homozygous CatK null mice (total N=50) aged 19 weeks, in-life double fluorochrome labeling was performed. Right femurs and lumbar vertebral bodies 1-3 (LV) were evaluated by dual-energy X-ray absorptiometry (DXA) for bone mineral content (BMC) and bone mineral density (BMD). The trabecular region of the femur and the cortical region of the tibia were evaluated by histomorphometry. The left femur and sixth lumbar vertebral body were tested biomechanically. CatK (-/-) mice show higher BMD at the central and distal femur. Central femur ultimate load was positively influenced by genotype, and was positively correlated with both cortical area and BMC. Lumbar vertebral body ultimate load was also positively correlated to BMC. Genotype did not influence the relationship of ultimate load to BMC in either the central femur or vertebral body. CatK (-/-) mice had less lamellar cortical bone than WT mice. Higher bone volume, trabecular thickness, and trabecular number were observed at the distal femur in CatK (-/-) mice. Smaller marrow cavities were also present at the central femur of CatK (-/-) mice. CatK (-/-) mice exhibited greater trabecular mineralizing surface, associated with normal volume-based formation of trabecular bone. Adult CatK (-/-) mice have higher bone mass in both cortical and cancellous regions than WT mice. Though no direct measures of bone resorption rate were made, the higher cortical bone quantity is associated with a smaller marrow cavity and increased retention of non-lamellar bone, signs of decreased endocortical resorption. The relationship of bone strength to BMC does not differ with genotype, indicating the presence of bone tissue of normal quality in the absence of CatK.     

Low level irradiation in mice can lead to enhanced trabecular bone morphology

Charged particle radiation such as iron ions and their secondary fragmentation products are of particular concern to the skeleton due to their high charge and energy deposition. However, little is known about the long-term effects of these particles on trabecular and cortical bone morphology when applied at relatively low levels. We hypothesized that even a 4.4 cGy dose of a complex secondary iron ion radiation field will compromise skeletal quantity and architecture in adult mice. One year after radiation exposure and compared to age-matched controls, 4.4 cGy irradiated mice had 51 % more trabecular bone, 56 % greater trabecular bone volume fraction, 16 % greater trabecular number, and 17 % less trabecular separation in the distal metaphysis of the femur. Similar to the metaphysis, trabecular bone of the distal femoral epiphysis in 4.4 cGy mice had 33 % more trabecular bone, 31 % greater trabecular bone volume fraction, and a 33 % smaller structural model index. Cortical bone morphology, whole bone mechanical properties, and lower leg muscle mass were unaffected. When compared to two additional groups, irradiated at either 8.9 or 17.8 cGy, a (negative) dose response relationship was observed for trabecular bone in the metaphysis but not in the epiphysis. In contrast to our original hypothesis, these data indicated that a secondary field of low-level, high-linear energy transfer iron radiation may cause long-term augmentation, rather than deterioration, of trabecular bone in the femoral metaphysis and epiphysis of mice.     

Bone mass and microarchitecture of irradiated and bone marrow-transplanted mice: influences of the donor strain

Summary  Total body irradiation and bone marrow transplantation induced dramatic trabecular bone loss and cortical thickening in mice. Transplanted cells were engrafted in bone marrow, along trabeculae, and in periosteal and endosteal envelopes. None of the osteocytes were of donor origin. Bone microarchitecture of transplanted mice changed to tend toward the donor phenotype. Introduction  Osteopenia and osteoporosis are complications of bone marrow transplants (BMT) attributed to related chemotherapy. However, the specific influence of total body irradiation (TBI) is unknown. Methods  We investigated the effects of TBI and BMT on bone mass and microarchitecture by micro-CT. Eighteen C57Bl/6 (B6) mice receiving lethal TBI had a BMT with marrow cells from green fluorescent protein--transgenic-C57Bl/6 (GFP) mice. Transplanted (T^GFPB6), B6, and GFP mice were euthanized 1, 3, and 6 months after BMT or at a related age. Results  T^GFPB6 presented a dramatic bone loss compared with B6 and did not restore their trabecular bone mass over time, despite a cortical thickening 6 months after BMT. Serum testosterone levels were not significantly reduced after BMT. During aging, GFP mice have less trabeculae, thicker cortices, but a narrower femoral shaft than B6 mice. From 3 months after BMT, cortical characteristics of T^GFPB6 mice differed statistically from B6 mice and were identical to those of GFP mice. GFP⁺ cells were located along trabecular surfaces and in periosteal and endosteal envelopes, but none of the osteocytes expressed GFP. Conclusion  Our findings suggest that engrafted cells did not restore the irradiation-induced trabecular bone loss, but reconstituted a marrow microenvironment and bone remodeling similar to those of the donor. The effects of irradiation and graft on bone remodeling differed between cortical and trabecular bone.     

Administration of high-dose macrophage colony-stimulating factor increases bone turnover and trabecular volume fraction

Macrophage colony-stimulating factor (M-CSF) is a hematopoietic growth factor that plays a critical role in early osteoclastogenesis. To characterize the skeletal effects of M-CSF, we administered soluble M-CSF to mice. It was hypothesized that M-CSF would stimulate bone formation through coupled activity of osteoclasts and osteoblasts. Twenty-four male C57BL/6 J mice (n = 12/group, aged 7 weeks) received subcutaneous injections of human M-CSF [5 mg/(kg day)] or inert vehicle (VEH) for 21 days. M-CSF increased serum bone turnover markers (+57% TRAP-5b and +44% osteocalcin). Microcomputed tomography revealed an anabolic effect on tibial trabecular bone, with higher bone volume fraction (+35%), connectivity density (+79%), and number (+18%), as well as lower trabecular separation (−18%). M-CSF had no significant effect on cortical bone mineral content, geometry, or strength. There was no change in quantitative histomorphometry parameters of femoral cortical bone. These results reveal the complex, site-specific effects of M-CSF. In particular, we have demonstrated an anabolic effect of M-CSF on trabecular bone achieved through coupled activation of osteoblasts. However, in contrast to previous studies, M-CSF was found to have no effect on cortical bone. M-CSF was demonstrated to significantly influence both bone modeling and remodeling in relatively young animals.