The skeleton fulfils its mechanical functions through structural organization and material properties of individual bones. It is stated that both cortical and trabecular morphology and mass can be (re)modelled in response to changes in mechanical strains engendered by load-bearing. To address this, animal models that enable the application of specific loads to individual bones have been developed. These are useful in defining how loading modulates (re)modeling and allow examination of the mechanisms that coordinate these events. This chapter describes how to apply mechanical loading to murine bones through points of articulation, which allows changes in endosteal, periosteal as well as trabecular bone to be revealed at multiple hierarchies, by a host of methodologies, including double fluorochrome labeling and computed tomography.

Low circulating IGF-I is associated with increased fracture risk. Conditional depletion of IGF-I produced in osteoblasts or osteocytes inhibits the bone anabolic effect of mechanical loading. Here, we determined the role of endocrine IGF-I for the osteogenic response to mechanical loading in young adult and old female mice with adult, liver-specific IGF-I inactivation (LI-IGF-I(-/-) mice, serum IGF-I reduced by ≈70%) and control mice. The right tibia was subjected to short periods of axial cyclic compressive loading three times/wk for 2 wk, and measurements were performed using microcomputed tomography and mechanical testing by three-point bending. In the nonloaded left tibia, the LI-IGF-I(-/-) mice had lower cortical bone area and increased cortical porosity, resulting in reduced bone mechanical strength compared with the controls. Mechanical loading induced a similar response in LI-IGF-I(-/-) and control mice in terms of cortical bone area and trabecular bone volume fraction. In fact, mechanical loading produced a more marked increase in cortical bone mechanical strength, which was associated with a less marked increase in cortical porosity, in the LI-IGF-I(-/-) mice compared with the control mice. In conclusion, liver-derived IGF-I regulates cortical bone mass, cortical porosity, and mechanical strength under normal (nonloaded) conditions. However, despite an ∼70% reduction in circulating IGF-I, the osteogenic response to mechanical loading was not attenuated in the LI-IGF-I(-/-) mice.

Estrogen receptor‐α (ERα) is crucial for the adaptive response of bone to loading but the role of endogenous estradiol (E2) for this response is unclear. To determine in vivo the ligand dependency and relative roles of different ERα domains for the osteogenic response to mechanical loading, gene‐targeted mouse models with (1) a complete ERα inactivation (ERα−/−), (2) specific inactivation of activation function 1 (AF‐1) in ERα (ERαAF‐10), or (3) specific inactivation of ERαAF‐2 (ERαAF‐20) were subjected to axial loading of tibia, in the presence or absence (ovariectomy [ovx]) of endogenous E2. Loading increased the cortical bone area in the tibia mainly as a result of an increased periosteal bone formation rate (BFR) and this osteogenic response was similar in gonadal intact and ovx mice, demonstrating that E2 (ligand) is not required for this response. Female ERα−/− mice displayed a severely reduced osteogenic response to loading with changes in cortical area (−78% ± 15%, p < 0.01) and periosteal BFR (−81% ± 9%, p < 0.01) being significantly lower than in wild‐type (WT) mice. ERαAF‐10 mice also displayed a reduced response to mechanical loading compared with WT mice (cortical area −40% ± 11%, p < 0.05 and periosteal BFR −41% ± 8%, p < 0.01), whereas the periosteal osteogenic response to loading was unaffected in ERαAF‐20 mice. Mechanical loading of transgenic estrogen response element (ERE)‐luciferase reporter mice did not increase luciferase expression in cortical bone, suggesting that the loading response does not involve classical genomic ERE‐mediated pathways. In conclusion, ERα is required for the osteogenic response to mechanical loading in a ligand‐independent manner involving AF‐1 but not AF‐2. © 2013 American Society for Bone and Mineral Research

The skeleton fulfils its mechanical functions through structural organisation and material properties of individual bones. Both cortical and trabecular morphology and mass can be (re)modelled in response to changes in mechanical strains engendered by load-bearing. To address this, animal models that enable the application of specific loads to individual bones have been developed. These are useful in defining how loading modulates (re)modelling and allow examination of the mechanisms that coordinate these events. This chapter describes how to apply mechanical loading to murine bones through points of articulation, which allows changes in endosteal, periosteal as well as trabecular bone to be revealed by double fluorochrome labelling and computed tomography, respectively.

Although Lrp5 is known to be an important contributor to the mechanisms regulating bone mass, its precise role remains unclear. The aim of this study was to establish whether mutations in Lrp5 are associated with differences in the growth and/or apoptosis of osteoblast-like cells and their proliferative response to mechanical strain in vitro. Primary osteoblast-like cells were derived from cortical bone of adult mice lacking functional Lrp5 (Lrp5(-/-)), those heterozygous for the human G171V High Bone Mass (HBM) mutation (LRP5(G171V)) and their WT littermates (WTLrp5, WTHBM). Osteoblast proliferation over time was significantly higher in cultures of cells from LRP5(G171V) mice compared to their WTHBM littermates, and lower in Lrp5(-/-) cells. Cells from female LRP5(G171V) mice grew more rapidly than those from males, whereas cells from female Lrp5(-/-) mice grew more slowly than those from males. Apoptosis induced by serum withdrawal was significantly higher in cultures from Lrp5(-/-) mice than in those from WTHBM or LRP5(G171V) mice. Exposure to a single short period of dynamic mechanical strain was associated with a significant increase in cell number but this response was unaffected by genotype which also did not change the 'threshold' at which cells responded to strain. In conclusion, the data presented here suggest that Lrp5 loss and gain of function mutations result in cell-autonomous alterations in osteoblast proliferation and apoptosis but do not alter the proliferative response of osteoblasts to mechanical strain in vitro.

Of the 1,328 genes revealed by microarray to be differentially regulated by disuse, or at 8 h following a single short period of osteogenic loading of the mouse tibia, analysis by predicting associated transcription factors from annotated affinities revealed the transcription factor EGR2/Krox-20 as being more closely associated with more pathways and functions than any other. Real time quantitative PCR confirmed up-regulation of Egr2 mRNA expression by loading of the tibia in vivo. In vitro studies where strain was applied to primary cultures of mouse tibia-derived osteoblastic cells and the osteoblast UMR106 cell line also showed up-regulation of Egr2 mRNA expression. In UMR106 cells, inhibition of β1/β3 integrin function had no effect on strain-related Egr2 expression, but it was inhibited by a COX2-selective antagonist and imitated by exogenous prostaglandin E2 (PGE2). This response to PGE(2) was mediated chiefly through the EP1 receptor and involved stimulation of PKC and attenuation by cAMP/PKA. Neither activators nor inhibitors of nitric oxide, estrogen signaling, or LiCl had any effect on Egr2 mRNA expression, but it was increased by both insulin-like growth factor-1 and high, but not low, dose parathyroid hormone and exogenous Wnt-3a. The increases by strain, PGE2, Wnt-3a, and phorbol 12-myristate 13-acetate were attenuated by inhibition of MEK-1. EGR2 appears to be involved in many of the signaling pathways that constitute early responses of bone cells to strain. These pathways all have multiple functions. Converting their strain-related responses into coherent "instructions" for adaptive (re)modeling is likely to depend upon their contextual activation, suppression, and interaction probably on more than one occasion.

Of the 1,328 genes revealed by microarray to be differentially regulated by disuse, or at 8 hours following a single short period of osteogenic loading of the mouse tibia, analysis by PASTAA (Predicting Associated Transcription Factors from Annotated Affinities) revealed the transcription factor EGR2/Krox-20 as being more closely associated with more pathways and functions than any other. Real time quantitative PCR confirmed up-regulation of Egr2 mRNA expression by loading of the tibia in vivo and in vitro by strain in primary cultures of mouse tibia-derived osteoblastic cells and the osteoblast UMR106 cell line. In UMR106 cells, inhibition of 1/ 3 integrin function had no effect on strain-related Egr2 expression but it was inhibited by a COX2 selective antagonist and imitated by exogenous PGE2. This response to PGE2 was mediated chiefly through the EP1 receptor and involved stimulation of PKC and attenuation by cAMP/PKA. Neither activators nor inhibitors of nitric oxide, estrogen signalling, or LiCl had any effect on Egr2 mRNA expression but it was increased by both IGF-1 and high, but not low, dose PTH and exogenous Wnt-3a. The increases by PGE2, Wnt-3a and PMA were attenuated by inhibition of MEK-1. EGR2 appears to be involved in many of the signalling pathways which constitute bone cells’ early responses to strain. These pathways all have multiple functions. Converting their strain-related responses into coherent “instructions” for adaptive (re)modelling is likely to depend upon their contextual activation, suppression and interaction probably on more than one occasion.

INTRODUCTION:To investigate the role of the low-density lipoprotein receptor-related protein 5 (Lrp5) in bones' responses to loading, we analysed changes in multiple measures of bone architecture in tibias subjected to loading or disuse in male and female mice with the Lrp5 loss of function mutation (Lrp5(-/-)) or heterozygous for the Lrp5 G171V High Bone Mass (HBM) mutation (Lrp5(HBM+)). MATERIALS AND METHODS:The right tibias of these 17week old male and female mice and their Wild Type (WT) littermates were subjected to short periods of loading three days a week for two weeks. Each tibia was loaded for 40 cycles, to produce peak strains at the midshaft within the low, medium or high physiological range (~1500, 2400 and 3000 microstrain, respectively). In similar groups of mice the right sciatic nerve was severed causing disuse of the right tibia for 3weeks. Data from microCT of loaded, neurectomised and contra-lateral control tibias were analysed to quantify changes in the cortical and cancellous regions of the bone in the absence of functional strains and in response to graded strains in addition to those derived from function. RESULTS AND CONCLUSION:Male WT(+/+) controls showed significant strain:response curves for cortical area and trabecular thickness, but Lrp5(-/-) mice showed no detectable strain:response in those same outcomes. Female mice of either WT(+/+) or Lrp5(-/-) genotype did not show significant strain:response curves for cortical or trabecular parameters, the one exception being Tb.Th in Lrp5(-/-) mice. Since female WT(+/+) mice did not respond to loading in a significant dose:responsive manner, the similar lack of responsiveness of the Lrp5(-/-) females could not be ascribed to their Lrp5 status. Cortical bone loss associated with disuse showed no differences between Lrp5(-/-) mice and WT(+/+) controls, but in cancellous bone of both male and females of these mice, there was a greater loss than in WT(+/+) controls. In contrast, the tibias of male and female mice heterozygous for the Lrp5 G171V HBM mutation showed greater osteogenic responsiveness to loading and less bone loss associated with disuse than their WT(HBM-) controls. These data indicate that the presence of the Lrp5 G171V HBM mutation is associated with an increased osteogenic response to loading but support only a marginal gender-related role for normal Lrp5 function in this loading-related response.

Pre- and early puberty may be the most opportune time to strengthen the female skeleton, but there are few longitudinal data to support this claim. Competitive female premenarcheal (pre/peri, n - 13) and postmenarcheal (post, n 32) tennis players aged 10 to 17 years were followed over 12 months. The osteogenic response to loading was studied by comparing the playing and nonplaying humeri for dual-energy X-ray absorptiometry (DXA) bone mineral content (BMC) and magnetic resonance imaging (MRI) total bone area (ToA), medullary area (MedA), cortical area (CoA), and muscle area (MCSA) at the humerus. Over 12 months, growth-induced gains (nonplaying arm) in BMC, ToA, and CoA were greater in pre/peri (10% to 19%, p<.001) than in post (3% to 5%, p<.05 to .001) players. At baseline, BMC, ToA, CoA, and MCSA were 8% to 18% greater in the playing versus nonplaying arms in pre/peri and post players (all p<.001); MedA was smaller in the playing versus nonplaying arms in post only players (p<.05). When comparing the annual gains in the playing arm relative to changes in the nonplaying arm, the increases in ToA and CoA were greater in pre/peri than post players (all p<.05). The smaller the side-to-side differences in BMC and CoA at baseline, the larger the exercise benefits at 12 months (r = -0.39 to =0.48, p<.01). The exercise-induced change in MCSA was predictive of the exercise benefits in BMC in pre/peri players only (p<.05). In conclusion, both pre/peri- and postmenarcheal tennis players showed significant exercise-induced skeletal benefits within a year, with greater benefits in cortical bone geometry in pre/perimenarcheal girls. (C) 2011 American Society for Bone and Mineral Research.

Bones' functionally adaptive responses to mechanical loading can usefully be studied in the tibia by the application of loads between the knee and ankle in normal and genetically modified mice. Such loading also deforms the fibula. Our present study was designed to ascertain whether the fibula adapts to loading in a similar way to the tibia and could thus provide an additional bone in which to study functional adaptation. The right tibiae/fibulae in C57BL/6 mice were subjected to a single period of axial loading (40 cycles at 10 Hz with 10-second intervals between each cycle; approximately 7 min/day, 3 alternate days/week, 2 weeks). The left tibiae/fibulae were used as non-loaded, internal controls. Both left and right fibulae and tibiae were analyzed by micro-computed tomography at the levels of the mid-shaft of the fibula and 25% from its proximal and distal ends. We also investigated the effects of intermittent parathyroid hormone (iPTH) on the (re)modelling response to 2-weeks of loading and the effect of 2-consecutive days of loading on osteocytes' sclerostin expression. These in vivo experiments confirmed that the fibula showed similar loading-related (re)modelling responses to those previously documented in the tibia and similar synergistic increases in osteogenesis between loading and iPTH. The numbers of sclerostin-positive osteocytes at the proximal and middle fibulae were markedly decreased by loading. Collectively, these data suggest that the mouse fibula, as well as the tibia and ulna, is a useful bone in which to assess bone cells' early responses to mechanical loading and the adaptive (re)modelling that this engenders.

Bone strength is, in part, dependent on a mechanical input that regulates the (re)modelling of skeletal elements to an appropriate size and architecture to resist fracture during habitual use. The rate of longitudinal bone growth in juveniles can also affect fracture incidence in adulthood, suggesting an influence of growth rate on later bone quality. We have compared the effects of fast and slow growth on bone strength and architecture in the tibiotarsi of embryonic and juvenile birds. The loading-related biochemical responses (intracellular G6PD activity and NO release) to mechanical load were also determined. Further, we have analysed the proliferation and differentiation characteristics of primary tibiotarsal osteoblasts from fast and slow-growing strains. We found that bones from chicks with divergent growth rates display equal resistance to applied loads, but weight-correction revealed that the bones from juvenile fast growth birds are weaker, with reduced stiffness and lower resistance to fracture. Primary osteoblasts from slow-growing juvenile birds proliferated more rapidly and had lower alkaline phosphatase activity. Bones from fast-growing embryonic chicks display rapid radial expansion and incomplete osteonal infilling but, importantly, lack mechanical responsiveness. These findings are further evidence that the ability to respond to mechanical inputs is crucial to adapt skeletal architecture to generate a functionally appropriate bone structure and that fast embryonic and juvenile growth rates may predispose bone to particular architectures with increased fragility in the adult.

Loading-related changes in gene expression in resident cells in the tibia of female mice in the contexts of normality (WT), estrogen deficiency (WT-OVX), absence of estrogen receptor alpha (ER alpha(-/-)) and disuse due to sciatic neurectomy (WT-SN) were established by microarray. Total RNA was extracted from loaded and contra-lateral non-loaded tibiae at selected time points after a single, short period of dynamic loading sufficient to engender an osteogenic response. There were marked changes in the expression of many genes according to context as well as in response to loading within those contexts. In WT mice at 3, 8, 12 and 24 h after loading the expression of 642, 341, 171 and 24 genes, respectively, were differentially regulated compared with contra-lateral bones which were not loaded. Only a few of the genes differentially regulated by loading in the tibiae of WT mice have recognized roles in bone metabolism or have been linked previously to osteogenesis (Opn, Sost, Esr1, Tgtb1, Lrp1, Ostn, Timp, Mmp, Ctgf Postn and Irs1, BMP and DLX5). The canonical pathways showing the greatest loading-related regulation were those involving pyruvate metabolism, mitochondrial dysfunction, calcium-induced apoptosis, glycolysis/gluconeogenesis, aryl hydrocarbon receptor and oxidative phosphorylation. In the tibiae from WT-OVX, ER alpha(-/-) and WT-SN mice, 440, 439 and 987 genes respectively were differentially regulated by context alone compared to WT. The early response to loading in tibiae of WT-OVX mice involved differential regulation compared to their contra-lateral non-loaded pair of fewer genes than in WT, more down-regulation than up-regulation and a later response. This was shared by WT-SN. In tibiae of ER alpha(-/-) mice, the number of genes differentially regulated by loading was markedly reduced at all time points.These data indicate that in resident bone cells. both basal and loading-related gene expression is substantially modified by context. Many of the genes differentially regulated by the earliest loading-related response were primarily involved in energy metabolism and were not specific to bone. (C) 2009 Elsevier Inc. All rights reserved.

The primary responsibility of the skeleton is to bear the loads involved in physical activity without sustaining damage. This capability involves a mechanism in which bone cells "assess" the suitability of the bones' existing architecture in relation to their prevailing loading environment and adapt or maintain it accordingly. It is widely assumed that the loading-related variable to which the bone cells respond is the strain engendered within the bone tissue. Strain-related adaptation is essential for normal bone development, regulation of strength in relation to exercise, healing of fractures and the success of orthopedic interference. The most widespread failure of this adaptive response is osteoporosis. Although strain-related bone adaptation can be investigated in these situations in humans in vivo, both the inputs and outputs are difficult to assess and control, and the tissues are unavailable for study. As a consequence much of our understanding of the mechanisms involved in strain-related adaptation have come from studies in animals where the strains within the bone are measured, the loads imposed on the bones can be controlled, and the adaptive changes to cells and architecture determined. Although many animals have been used in the past (1-3), now the most commonly used animal is the mouse. Normal and transgenic mice are available and inbred strains of mice are well characterized physiologically (4). Most importantly, the techniques for measuring strains in mouse bones, loading these bones in a controlled manner, and assessing changes in architecture are all available. This chapter outlines the techniques involved in these three phases of investigation in mouse bone.

The separate and combined effects of intermittent parathyroid hormone (iPTH) (1-34) and mechanical loading were assessed at trabecular and cortical sites of mouse long bones. Female C57BL/6 mice from 13 to 19 weeks of age were given daily injections of vehicle or PTH (1-34) at low (20 microg/kg/day), medium (40 microg/kg/day) or high (80 microg/kg/day) dose. For three alternate days per week during the last two weeks of this treatment, the tibiae and ulnae on one side were subjected to a single period of non-invasive, dynamic axial loading (40 cycles at 10 Hz with 10-second intervals between each cycle). Two levels of peak load were used; one sufficient to engender an osteogenic response, and the other insufficient to do so. The whole tibiae and ulnae were analyzed post-mortem by micro-computed tomography with a resolution of 5 microm. Treatment with iPTH (1-34) modified bone structure in a dose- and time-dependent manner, which was particularly evident in the trabecular region of the proximal tibia. In the tibia, loading at a level sufficient by itself to stimulate osteogenesis produced an osteogenic response in the low-dose iPTH (1-34)-treated trabecular bone and in the proximal and middle cortical bone treated with all doses of iPTH (1-34). In the ulna, loading at a level that did not by itself stimulate osteogenesis was osteogenic at the distal site when combined with high-dose iPTH (1-34). At both levels of loading, there were synergistic effects in cortical bone volume of the proximal tibia and distal ulna between loading and high-dose iPTH (1-34). Images of fluorescently labelled bones confirmed that such synergism resulted from increases in both endosteal and periosteal bone formation. No woven bone was induced by iPTH (1-34) or either level of loading alone, whereas the combination of iPTH (1-34) and the "sufficient" level of loading stimulated woven bone formation on endosteal and periosteal surfaces of the proximal cortex in the tibiae. Together, these data suggest that in female C57BL/6 mice, under some but not all circumstances, mechanical loading exerts an osteogenic response with iPTH (1-34) in trabecular and cortical bone.

The Wnt/beta-catenin pathway has been implicated in bone cell response to their mechanical environment. This response is the origin of the mechanism by which bone cells adjust bone architecture to maintain bone strength. Osteoporosis is the most widespread failure of this mechanism. The degree of osteoporotic bone loss in men and women is related to bio-available estrogen. Here we report that in osteoblastic ROS 17/2.8 cells and primary osteoblast cultures, a single short period of dynamic mechanical strain, as well as the glycogen synthase kinase-3 beta (GSK-3 beta) inhibitor LiCl, increased nuclear accumulation of activated beta-catenin and stimulated TCF/LEF reporter activity. This effect was blocked by the estrogen receptor ( ER) modulators ICI 182,780 and tamoxifen and was absent in primary osteoblast cultures from mice lacking ER alpha. Microarray expression data for 25,000 genes from total RNA extracted from tibiae of wild-type mice within 24 h of being loaded in vivo showed differential gene regulation between loaded and contralateral non-loaded bones of 10 genes established to be involved in the Wnt pathway. Only 2 genes were involved in loaded tibiae from mice lacking ER alpha (ER alpha(-/-)). Together these data suggest that Wnt/beta-catenin signaling contributes to bone cell early responses to mechanical strain and that its effectiveness requires ER alpha. Reduced effectiveness of bone cell responses to bone loading, associated with estrogen-related decline in ER alpha, may contribute to the failure to maintain structurally appropriate bone mass in osteoporosis in both men and women.

UNLABELLED:We examined the combined effects of exercise and calcium on BMC accrual in pre- and early-pubertal boys. Exercise and calcium together resulted in a 2% greater increase in femur BMC than either factor alone and a 3% greater increase in BMC at the tibia-fibula compared with the placebo group. Increasing dietary calcium seems to be important for optimizing the osteogenic effects of exercise. INTRODUCTION:Understanding the relationship between exercise and calcium during growth is important given that the greatest benefits derived from these factors are achieved during the first two decades of life. We conducted a blinded randomized-controlled exercise-calcium intervention in pre- and early-pubertal boys to test the following hypotheses. (1) At the loaded sites (femur and tibia-fibula), exercise and calcium will produce greater skeletal benefits than either exercise or calcium alone. (2) At nonloaded sites (humerus and radius-ulna), there will be an effect of calcium supplementation. MATERIALS AND METHODS:Eighty-eight pre- and early-pubertal boys were randomly assigned to one of four study groups: moderate impact exercise with or without calcium (Ca) (Ex+Ca and Ex+placebo, respectively) or low impact exercise with or without Ca (No-Ex+Ca and No-Ex+Placebo, respectively). The intervention involved 20 minutes of either moderate- or low-impact exercise performed three times a week and/or the addition of Ca-fortified foods using milk minerals (392+/-29 mg/day) or nonfortified foods over 8.5 months. Analysis of covariance was used to determine the main and combined effects of exercise and calcium on BMC after adjusting for baseline BMC. RESULTS:At baseline, no differences were reported between the groups for height, weight, BMC, or bone length. The increase in femur BMC in the Ex+Ca group was approximately 2% greater than the increase in the Ex+placebo, No-Ex+Ca, or No-Ex+Placebo groups (all p<0.03). At the tibia-fibula, the increase in BMC in the Ex+Ca group was approximately 3% greater than the No-Ex+placebo group (p<0.02) and 2% greater than the Ex+Placebo and the No-Ex+Ca groups (not significant). No effect of any group was detected at the humerus, ulna-radius, or lumbar spine for BMC, height, bone area, or volume. CONCLUSIONS:In this group of normally active boys with adequate calcium intakes, additional exercise and calcium supplementation resulted in a 2-3% greater increase in BMC than controls at the loaded sites. These findings strengthen the evidence base for public health campaigns to address both exercise and dietary changes in children for optimizing the attainment of peak BMC.