Cortical Bone Volume Research Articles

Conditional Inactivation of the CXCR4 Receptor in Osteoprecursors Reduces Postnatal Bone Formation Due to Impaired Osteoblast Development

Cysteine (C)-X-C motif chemokine receptor 4 (CXCR4), the primary receptor for stromal cell-derived factor-1 (SDF-1), is involved in bone morphogenic protein 2 (BMP2)-induced osteogenic differentiation of mesenchymal progenitors. To target the in vivo function of CXCR4 in bone and explore the underlying mechanisms, we conditionally inactivated CXCR4 in osteoprecursors by crossing osterix (Osx)-Cre mice with floxed CXCR4 (CXCR4(fl/fl)) mice to generate knock-outs with CXCR4 deletion driven by the Osx promoter (Osx::CXCR4(fl/fl)). The Cre-mediated excision of CXCR4 occurred exclusively in bone of Osx::CXCR4(fl/fl) mice. When compared with littermate controls, Osx::CXCR4(fl/fl) mice developed smaller osteopenic skeletons as evidenced by reduced trabecular and cortical bone mass, lower bone mineral density, and a slower mineral apposition rate. In addition, Osx::CXCR4(fl/fl) mice displayed chondrocyte disorganization in the epiphyseal growth plate associated with decreased proliferation and collagen matrix syntheses. Moreover, mature osteoblast-related expression of type I collagen α1 and osteocalcin was reduced in bone of Osx::CXCR4(fl/fl) mice versus controls, suggesting that CXCR4 deficiency results in arrested osteoblast progression. Primary cultures for osteoblastic cells derived from Osx::CXCR4(fl/fl) mice also showed decreased proliferation and impaired osteoblast differentiation in response to BMP2 or BMP6 stimulation, and suppressed activation of intracellular BMP receptor-regulated Smads (R-Smads) and Erk1/2 was identified in CXCR4-deficient cells and bone tissues. These findings provide the first in vivo evidence that CXCR4 functions in postnatal bone development by regulating osteoblast development in cooperation with BMP signaling. Thus, CXCR4 acts as an endogenous signaling component necessary for bone formation.

Allometry in the distribution of material properties and geometry of the felid skull: Why larger species may need to change and how they may achieve it

Extant members of the cat family (Felidae) have been considered behaviourally and morphologically conservative, i.e., despite great differences in size, there is relatively little variation in either the shape of the felid skull and dentition across species, or in the way in which these structures are used to kill and dismember prey. Consequently felids have been considered an appropriate focus for a number of investigations into the influence of allometry on craniomandibular mechanics and morphology. However, although previous treatments have considered the role of shape, they have not investigated the influence of differences in the distribution of relatively stiff cortical and more compliant cancellous bone on performance. Here, using models that incorporate material properties for both cortical and cancellous bone, we apply three-dimensional (3D) finite element analysis (FEA) to models representing the skulls of seven extant felid species. Our objectives being to determine allometric trends regarding both overall geometry and the relative distributions of cortical and cancellous bone tissue. We also more comprehensively assess variation in the efficiency with which muscular force is converted to bite force and the capacity to resist associated stresses. Our results show that the cheetah ( Acinonyx jubatus) may be exceptional regarding both the efficiency with which muscular force is converted to bite force and the distribution of stress. We found a negative allometric trend between cortical bone volume and total skull bone volume, and positive allometry between the total skull bone volume and skull surface area. Results gained from mathematical modelling of beam analogies suggest that these trends reflect a need for larger species to respond to physical challenges associated with increased size, and, that changes in skull shape, bone composition, or a combination of both may be required to accommodate these challenges. With geometrical scaling stress increases by the same factor, and displacement by the same factor squared, but the ultimate failure stress of the material is invariant. We find that as species become larger, overall skull bone volume relative to surface area increases by adding a higher proportion of less dense and more compliant cancellous bone. This results in an increased cross-sectional area and second moment of inertia, which acts to reduce the overall stresses. An overall saving in mass is a likely additional consequence. Although we do find evidence that skull stiffness does diminish with size, we also argue that this is at least in part mitigated through the influence of these allometric trends. We further suggest that these trends and the explanations for them may be universal for vertebrates.

Fibroblast growth factor 23 overexpression impacts negatively on dentin mineralization and dentinogenesis in mice

1. Though previous studies have shown that fibroblast growth factor 23 (FGF23) mRNA expression localizes in ameloblasts and odontoblasts in teeth, it is unclear what effect FGF23 overexpression has on dentin mineralization and dentinogenesis. Toward this end, the phenotypes of mandibles and teeth were compared between 6-week-old FGF23 transgenic mice and their wild-type littermates by radiography, microcomputed tomography scanning, histology, histochemistry and immunohistochemistry. 2. The mineral density was reduced in all teeth, including molars and incisors, and in the mandible, and the mineralized tooth volume in incisor and molars, and the mineralized cortical and alveolar bone volume in mandibles were decreased in FGF23 transgenic mice compared with their wild-type littermates. The dental volume, reparative dentin area, the expression of dentin sialoprotein in dentin, and the deposition of type I collagen and osteocalcin in the dental matrix were significantly reduced. However, the predentin volume and the expression of biglycan in dentin were increased in FGF23 transgenic mice compared with their wild-type littermates. 3. The results of the present study show that FGF23 overexpression plays a negative regulatory role on dentin mineralization and dentinogenesis.

Deletion of Amp-activated Protein Kinase (AMPK)-Alpha 1 catalytic subunit in mice impairs the in vivo response of bones to hormonal challenges

Deletion of Amp-activated Protein Kinase (AMPK)-Alpha 1 catalytic subunit in mice impairs the in vivo response of bones to hormonal challenges

Oxytocin receptor nuclear translocation mediates upregulation of osteoblast specific genes

Oxytocin receptor nuclear translocation mediates upregulation of osteoblast specific genes

Risedronate does not reduce mechanical loading-related increases in cortical and trabecular bone mass in mice

To establish whether the combination of anti-resorptive therapy with mechanical loading has a negative, additive or synergistic effect on bone structure, we assessed the separate and combined effects of risedronate and non-invasive dynamic loading on trabecular and cortical bone. Seventeen-week-old female C57BL/6 mice were given daily subcutaneous injections of vehicle (n = 20) or risedronate at a dose of 0.15, 1.5, 15 or 150 μg/kg/day (n = 10 in each) for 17 days. From the fourth day of treatment, the right tibiae were subjected to a single period of axial loading (40 cycles/day) for three alternate days per week for two weeks. The left tibiae were used as internal controls. Trabecular and cortical sites in the tibiae were analyzed by high-resolution micro-computed tomography and imaging of fluorochrome labels. In the non-loaded tibiae, treatment with the higher doses of risedronate at 15 or 150 μg/kg/day resulted in higher trabecular bone volume and trabecular number than in vehicle-treated controls, whereas such treatment was associated with no differences in cortical bone volume at any dose. In the loaded tibiae, loading induced increases in trabecular and cortical bone volume compared with contra-lateral controls primarily through increased trabecular thickness and periosteal expansion, respectively, independently of risedronate treatment. In conclusion, the response to mechanical loading in both trabecular and cortical bone in mice is therefore not impaired by short-term treatment with risedronate, even over a 1000-fold dose range. In considering the optimization of treatments for osteoporosis, it is reassuring that anti-resorptive therapy and mechanical loading can exert independent beneficial effects.This article is part of a Special Issue entitled Bisphosphonates.

Experimental and finite element analysis of the mouse caudal vertebrae loading model: prediction of cortical and trabecular bone adaptation

In this study, we attempt to predict cortical and trabecular bone adaptation in the mouse caudal vertebrae loading model using knowledge of bone's local mechanical environment at the onset of loading. In a previous study, we demonstrated appreciable 25.9 and 11% increases in both trabecular and cortical bone volume density, respectively, when subjecting the fifth caudal vertebrae (C5) of C57BL/6 (B6) mice to an acute loading regime (amplitude of 8N, 3000 cycles, 10 Hz, 3 times a week for 4 weeks). We have also established a validated finite element (FE) model of the C5 vertebra using micro-computed tomography (micro-CT), which characterizes, in 3D, the micro-mechanical strains present in both cortical and trabecular compartments due to the applied loads. To investigate the relationship between load-induced bone adaptation and mechanical strains in-vivo and in-silico data sets were compared. Using data from the previous cross-sectional study, we divided cortical and trabecular compartments into 15 subregions and determined, for each region, a bone formation parameter ΔBV/BS (a cross-sectional measure of the bone volume added to cortical and trabecular surfaces following the described loading regime). Linear regression was then used to correlate mean regional values of ΔBV/BS with mean values of mechanical strains derived from the FE models which were similarly regionalized. The mechanical parameters investigated were strain energy density (SED), the orthogonal strains (e (x), e (y), e (z)) and the three shear strains (e (xy), e (yz), e (zx)). For cortical regions, regression analysis showed SED to correlate extremely well with ΔBV/BS (R (2) = 0.82) and e (z) (R (2) = 0.89). Furthermore, SED was found to predict expansion of the cortical shell correlating significantly with the regional percentage increases in cortical tissue volume (R (2) = 0.92), cortical marrow volume (R (2) = 0.91) and cortical thickness (R (2) = 0.56). For trabecular regions, FE parameters were found not to correlate with load-induced trabecular bone morphology. These results indicate that load-induced cortical morphology can be predicted from population data, whereas the prediction of trabecular morphology requires subject-specific micro- architecture.

Differences in leg bone geometry in young, old and very old women

Bone geometry is an important measure of bone strength and is known to be affected by weight-bearing and adult ageing. Engagement in weight-bearing activity decreases with age, thus in this study we compared bone geometry changes between weight-bearing (tibia) and non-weight-bearing (fibula) leg bones in three different age groups of women. Magnetic resonance images of the right leg were acquired in 9 young (20-27years), 7 old (61-69years) and 7 very old (71-80years) women. Total and cortical bone volumes and medullary cavity volumes (mm(3)) were calculated at proximal and distal sites for both bones. Tibial cortical bone volume was significantly less at the proximal site in old (17%) and very old (24%) groups versus young subjects. Cortical bone volume in the proximal fibula was also significantly reduced in the older groups (7 and 12%), but to a substantially lesser extent than in the tibia. In contrast, distal bone geometry appeared largely to be conserved in both tibia and fibula. Proximally, medullary cavity volume was greater in the older groups in the tibia but not the fibula. Distally, the only difference found in either bone was a significantly greater fibular medullary cavity in the very old group. These findings suggest weight-bearing bones in women are more susceptible than non-weight-bearing bones to age-related changes in bone geometry likely due to decreases in weight-bearing activities. Also, weight-bearing activity appears to provide a greater osteogenic stimulus at the distal portions of the leg bones.

An image-based skeletal dosimetry model for the ICRP reference adult male—internal electron sources

In this study, a comprehensive electron dosimetry model of the adult male skeletal tissues is presented. The model is constructed using the University of Florida adult male hybrid phantom of Lee et al (2010 Phys. Med. Biol. 55 339–63) and the EGSnrc-based Paired Image Radiation Transport code of Shah et al (2005 J. Nucl. Med. 46 344–53). Target tissues include the active bone marrow, associated with radiogenic leukemia, and total shallow marrow, associated with radiogenic bone cancer. Monoenergetic electron emissions are considered over the energy range 1 keV to 10 MeV for the following sources: bone marrow (active and inactive), trabecular bone (surfaces and volumes), and cortical bone (surfaces and volumes). Specific absorbed fractions are computed according to the MIRD schema, and are given as skeletal-averaged values in the paper with site-specific values reported in both tabular and graphical format in an electronic annex available from http://stacks.iop.org/0031-9155/56/2309/mmedia. The distribution of cortical bone and spongiosa at the macroscopic dimensions of the phantom, as well as the distribution of trabecular bone and marrow tissues at the microscopic dimensions of the phantom, is imposed through detailed analyses of whole-body ex vivo CT images (1 mm resolution) and spongiosa-specific ex vivo microCT images (30 µm resolution), respectively, taken from a 40 year male cadaver. The method utilized in this work includes: (1) explicit accounting for changes in marrow self-dose with variations in marrow cellularity, (2) explicit accounting for electron escape from spongiosa, (3) explicit consideration of spongiosa cross-fire from cortical bone, and (4) explicit consideration of the ICRP's change in the surrogate tissue region defining the location of the osteoprogenitor cells (from a 10 µm endosteal layer covering the trabecular and cortical surfaces to a 50 µm shallow marrow layer covering trabecular and medullary cavity surfaces). Skeletal-averaged values of absorbed fraction in the present model are noted to be very compatible with those weighted by the skeletal tissue distributions found in the ICRP Publication 110 adult male and female voxel phantoms, but are in many cases incompatible with values used in current and widely implemented internal dosimetry software.

Skeletal phenotype of the leptin receptor–deficient db/db mouse

Leptin, a major hormonal product of the adipocyte, regulates appetite and reproductive function through its hypothalamic receptors. The leptin receptor is present in osteoblasts and chondrocytes, and previously we have shown leptin to be an anabolic bone factor in vitro, stimulating osteoblast proliferation and inhibiting osteoclastogenesis. Leptin increases bone mass and reduces bone fragility when administered peripherally but also can indirectly reduce bone mass when administered into the central nervous system. However, data from animal models deficient in either leptin (ob/ob) or its receptor (db/db) remain contradictory. We compared the bone phenotype of leptin receptor-deficient (db/db) and wild-type mice using micro-computed tomographic (µCT) analysis of the proximal tibias and vertebrae. In the tibia, db/db mice had reduced percent trabecular bone volume (13.0 ± 1.62% in wild-type versus 6.01 ± 0.601% in db/db mice, p = .002) and cortical bone volume (411 ± 21.5 µm(3) versus 316 ± 3.53 µm(3), p = .0014), trabecular thickness (48.4 ± 001.07 µm versus 45.1 ± 0.929 µm, p = .041) and trabecular number (2.68 ± 0.319 mm(-1) versus 1.34 ± 0.148 mm(-1), p = .0034). In the fifth lumbar vertebral body, the trabecular thickness and cortical thickness were decreased in the db/db versus wild-type mice (0.053 ± 0.0011 mm versus 0.047 ± 0.0013 mm, p = .0002 and 0.062 ± 0.00054 mm versus 0.056 ± 0.0009 mm, p = .0001), respectively, whereas the trabecular and cortical percent bone volume and trabecular number did not reach significance. The total (endosteal and periosteal) cortical perimeter (12.2 ± 0.19 mm versus 13.2 ± 0.30 mm, p = .01) was increased. The serum osteocalcin levels were reduced in the db/db mice, suggesting that bone formation rates are decreased. The material properties of db/db femurs were determined by three-point bending and nanoindentation, showing decreased bone strength (13.3 ± 0.280 N versus 7.99 ± 0.984 N, p = .0074) and material stiffness (28.5 ± 0.280 GPa versus 25.8 ± 0.281 GPa, p < .0001). These results demonstrate that bone mass and strength are reduced in the absence of leptin signaling, indicating that leptin acts in vivo as an anabolic bone factor. This concurs with results of in vitro studies and of peripheral leptin administration in vivo and suggests that leptin's direct effects on bone cells are likely to override its actions via the central nervous system.

DLK1 is a novel regulator of bone mass that mediates estrogen deficiency–induced bone loss in mice

Delta-like 1/fetal antigen 1 (DLK1/FA-1) is a transmembrane protein belonging to the Notch/Delta family that acts as a membrane-associated or a soluble protein to regulate regeneration of a number of adult tissues. Here we examined the role of DLK1/FA-1 in bone biology using osteoblast-specific Dlk1-overexpressing mice (Col1-Dlk1). Col1-Dlk1 mice displayed growth retardation and significantly reduced total body weight and bone mineral density (BMD). Micro-computed tomographis (µCT) scanning revealed a reduced trabecular and cortical bone volume fraction. Tissue-level histomorphometric analysis demonstrated decreased bone-formation rate and enhanced bone resorption in Col1-Dlk1 mice compared with wild-type mice. At a cellular level, Dlk1 markedly reduced the total number of bone marrow (BM)-derived colony-forming units fibroblasts (CFU-Fs), as well as their osteogenic capacity. In a number of in vitro culture systems, Dlk1 stimulated osteoclastogenesis indirectly through osteoblast-dependent increased production of proinflammatory bone-resorbing cytokines (eg, Il7, Tnfa, and Ccl3). We found that ovariectomy (ovx)-induced bone loss was associated with increased production of Dlk1 in the bone marrow by activated T cells. Interestingly, Dlk1(-/-) mice were significantly protected from ovx-induced bone loss compared with wild-type mice. Thus we identified Dlk1 as a novel regulator of bone mass that functions to inhibit bone formation and to stimulate bone resorption. Increasing DLK1 production by T cells under estrogen deficiency suggests its possible use as a therapeutic target for preventing postmenopausal bone loss.

The atypical anti-psychotic clozapine decreases bone mass in rats in vivo

Background Fracture risk is increased in patients with schizophrenia, who often receive long-term therapy with anti-psychotic drugs. The mechanisms by which skeletal fragility is increased in patients with psychosis include increased risk of falling, but direct skeletal toxicity of anti-psychotic drugs is a possibility that has not been investigated. Methods We examined the skeletal effects, in vivo and in vitro, of a typical anti-psychotic drug, haloperidol, which primarily inhibits dopaminergic signaling, and an atypical anti-psychotic drug, clozapine, which predominantly inhibits serotonergic signaling. Results In growing rats, 42 days of clozapine treatment reduced whole body bone mineral density by 15% (P < 0.01 vs vehicle), and trabecular and cortical bone volume, as assessed by microcomputed tomography, by 29% and 15%, respectively (P < 0.05 vs vehicle for each). Treatment with haloperidol did not affect bone density. Clozapine, but not haloperidol, transiently increased levels of serum corticosterone, and decreased levels of serum testosterone. In vitro, clozapine dose-dependently decreased osteoblast mitogenesis, osteoblast differentiation and osteoclastogenesis, while haloperidol did not affect any of these parameters. Conclusions These data demonstrate that clozapine, but not haloperidol, exerts adverse skeletal effects in rodents, and that this effect may be attributable to direct actions to reduce osteoblast growth and function. Long-term administration of clozapine may therefore negatively affect bone health, and clinical studies to investigate this possibility are warranted.

Loss-of-function of SHARPIN causes an osteopenic phenotype in mice

SHARPIN is a novel protein thought to interact with SHANK family and is widely expressed in multiple tissues/cells, including osteoblasts and osteoclasts. Loss-of-function of Sharpin develops the chronic proliferative dermatitis mutation (CPDM) in mice as well as a severe inflammation in other organs. The actual function of SHARPIN is poorly understood. Our aim was to determine the functional roles of SHARPIN in bone metabolism by using CPDM mice. The skeletal phenotypes were determined by peripheral quantitative computed tomography, micro-computed tomography, and quantitative real-time RT-PCR, the cellular functions of osteoblasts and osteoclasts were investigated by ex vivo cell culture. Compared to wild-type controls, CPDM mice demonstrated significantly lower total and cortical bone mineral content and bone mineral density, trabecular and cortical bone volume, and trabecular number. The mRNA expression of Runx2, osterix, type I collagen, and osteocalcin was significantly lower in the bone from CPDM mice. Osteoclasts and osteoblasts from CPDM mice were functionally defective. Our result suggests that SHARPIN plays important regulating roles in bone metabolism. These functional roles may either come from systemic chronic inflammatory or directly signaling pathway within bone cells.

Low‐magnitude whole‐body vibration does not enhance the anabolic skeletal effects of intermittent PTH in adult mice

Whole-body vibration (WBV) is a low-magnitude mechanical stimulus that may be anabolic for bone, yet we recently found that WBV did not improve bone properties in adult mice. Because intermittent parathyroid hormone (PTH) enhances the anabolic effects of high-magnitude skeletal loading, we sought to determine the skeletal effects of WBV in combination with PTH. Seven-month-old male BALB/c mice were assigned to six groups (n = 13-14/group) based on magnitude of applied acceleration (0 or 0.3 G) and PTH dose (0, 10, or 40 µg/kg/day). Mice were exposed to WBV (0.3 G, 90 Hz, sine wave) or sham loading (0 G) for 15 min/day, 5 days/week for 8 weeks. Vehicle or hPTH (1-34) was administered prior to each WBV session. Whole-body bone mineral content increased by ~ 5% from 0 to 8 weeks in the 40 µg/kg PTH group only, independent of WBV loading. Similarly, PTH treatment increased tibial cortical bone volume by ~5% from 0 to 8 weeks, independent of WBV loading. Neither PTH nor WBV stimulated trabecular bone formation. Consistent with the cortical bone effect, tibias from the 40 µg/kg PTH group had significantly greater ultimate force and energy to failure than tibias in the 0 and 10 µg/kg PTH groups, independent of WBV treatment. In summary, 8 weeks of intermittent PTH treatment increased cortical bone volume and strength in adult male BALB/c mice. Daily exposure to low-magnitude WBV by itself did not improve skeletal properties and did not enhance the PTH effect. No WBV-PTH synergy was found in this preclinical study.

Effects of long-term supplementation with omega-3 fatty acids on longitudinal changes in bone mass and microstructure in mice

A diet rich in omega-3s has previously been suggested to prevent bone loss. However, evidence for this has been limited by short exposure to omega-3 fatty acids (FAs). We investigated whether a diet enriched in eicosapentaenoic acid (EPA) or docosahexaenoic acid (DHA) for the entire adult life of mice could improve bone microstructure and strength. Thirty female mice received a diet enriched in DHA or EPA or an isocaloric control diet from 3 to 17 months of age. Changes in bone microstructure were analyzed longitudinally and biomechanical properties were analysed by a three-point bending test. Bone remodelling was evaluated by markers of bone turnover and histomorphometry. Trabecular bone volume in caudal vertebrae was improved by EPA or DHA at 8 months (+26.6% and +17.2%, respectively, compared to +3.8% in controls, P=.01), but not thereafter. Trabecular bone loss in the tibia was not prevented by omega-3 FAs (BV/TV −94%, −93% and −97% in EPA, DHA and controls, respectively). EPA improved femur cortical bone volume (+8.1%, P<.05) and thickness (+4.4%, P<.05) compared to controls. EPA, but not DHA, reduced age-related decline of osteocalcin (−70% vs. −83% in controls, P<.05). EPA and DHA increased leptin levels (7.3±0.7 and 8.5±0.5 ng ml −1, respectively, compared to 4.5±0.9 ng ml −1 in controls, P=.001); however, only EPA further increased IGF-1 levels (739±108 ng ml −1, compared to 417±58 ng ml −1 in controls, P=.04). These data suggest that long-term intake of omega-3 FA, particularly EPA, may modestly improve the structural and mechanical properties of cortical bone by an increase in leptin and IGF-1 levels, without affecting trabecular bone loss.

Mechanical Loading-Related Bone Gain Is Enhanced by Tamoxifen but Unaffected by Fulvestrant in Female Mice

Accumulating evidence indicates that estrogen receptors (ERs) are involved in the mechano-adaptive mechanisms by which loading influences the mass and architecture of bones to establish and maintain their structural load-bearing competence. In the present study, we assessed the effects of the ER modulators tamoxifen and fulvestrant (ICI 182,780) on loading-related changes in the volume and structure of trabecular and cortical bone in the tibiae of female mice. Ten days after actual or sham ovariectomy, 17-wk-old female C57BL/6 mice were treated with vehicle (peanut oil), tamoxifen (0.02, 0.2, or 2 mg/kg · d), fulvestrant (4 mg/kg · d), or their combination and the right tibiae subjected to a short period of noninvasive axial loading (40 cycles/d) on 5 d during the subsequent 2 wk. In the left control tibiae, ovariectomy, tamoxifen, or fulvestrant did not have any significant effect on cortical bone volume, whereas trabecular bone volume was decreased by ovariectomy, increased by tamoxifen, and unaffected by fulvestrant. In the right tibiae, loading was associated with increases in both trabecular and cortical bone volume. Notably, the medium dose of tamoxifen synergistically enhanced loading-related gain in trabecular bone volume through an increase in trabecular thickness. Fulvestrant had no influence on the effects of loading but abrogated the enhancement of loading-related bone gain by tamoxifen. These data demonstrate that, at least in female mice, the adaptive response to mechanical loading of trabecular bone can be enhanced by ER modulators, in this case by tamoxifen.

Correlations between strength and quantitative computed tomography measurement of callus mineralization in experimental tibial fractures

Background The evaluation of fracture healing in the clinic has not changed significantly during the past few decades, despite the development of modern tissue-imaging tools. Recent publications have reported significant and interesting associations between biomechanical properties and quantitative computed tomography data of fractures and grafts. We therefore studied the correlations between the strength and segmented quantitative computed tomography data of tibial diaphyseal fractures. Methods Forty male rats received a tibial-shaft osteotomy that was initially stabilized with either intramedullary nailing or external fixation. Evaluation at 30 and 60 days post-osteotomy included X-ray, quantitative computed tomography and bending testing. Quantitative computed tomography data were segmented by voxel density into soft callus (171–539 mg/cm 3), hard callus (540–1199 mg/cm 3) and cortical bone (≥ 1200 mg/cm 3), and volumetric bone mineral density was calculated. Findings All fractures demonstrated pronounced formation of soft and hard callus tissues at 30 days post-osteotomy, and at 60 days the cortical bone volume was significantly increased with callus resorption. Bending strength correlated significantly and positively with fracture-site cortical bone volume and volumetric bone mineral density in the intramedullary nailed group in the early phase of healing. Interpretation Quantitative computed tomography was used to quantify characteristic secondary healing. The observed correlations indicate that biomechanically important mineralization can be measured by quantitative computed tomography in the early phase of healing in flexibly fixed fractures.

Loss-of-function of SHARPIN causes an osteopenic phenotype in mice

SHARPIN is a novel protein thought to interact with SHANK family and is widely expressed in multiple tissues/cells, including osteoblasts and osteoclasts. Loss-of-function of Sharpin develops the chronic proliferative dermatitis mutation (CPDM) in mice as well as a severe inflammation in other organs. The actual function of SHARPIN is poorly understood. Our aim was to determine the functional roles of SHARPIN in bone metabolism by using CPDM mice. The skeletal phenotypes were determined by peripheral quantitative computed tomography, micro-computed tomography, and quantitative real-time RT-PCR, the cellular functions of osteoblasts and osteoclasts were investigated by ex vivo cell culture. Compared to wild-type controls, CPDM mice demonstrated significantly lower total and cortical bone mineral content and bone mineral density, trabecular and cortical bone volume, and trabecular number. The mRNA expression of Runx2, osterix, type I collagen, and osteocalcin was significantly lower in the bone from CPDM mice. Osteoclasts and osteoblasts from CPDM mice were functionally defective. Our result suggests that SHARPIN plays important regulating roles in bone metabolism. These functional roles may either come from systemic chronic inflammatory or directly signaling pathway within bone cells.

CXC receptor knockout mice: Characterization of skeletal features and membranous bone healing in the adult mouse

The potential role of CXC chemokines bearing the glu-leu-arg (ELR) motif in bone repair was studied using a cranial defect (CD) model in mice lacking the CXC receptor (mCXCR −/− knockout mice), which is homologous to knockout of the human CXC receptor 2 (CXCR2) gene. During the inflammatory stage of bone repair, ELR CXC chemokines are released by inflammatory cells and serve as chemotactic and angiogenic factors. mCXCR −/− mice were smaller in weight and length from base of tail to nose tip, compared to WT littermates. DEXA analysis indicated that bone mineral density (BMD), bone mineral content (BMC), total area (TA), bone area (BA), and total tissue mass (TTM) were decreased in the mCXCR −/− mice at 6, 12, and 18 weeks of age. Trabecular bone characteristics in mCXCR −/− (% bone, connectivity, number, and thickness) were reduced, and trabecular spacing was increased as evidenced by μCT. There was no difference in bone formation or resorption indices measured by bone histomorphometry. Trabecular BMD was not altered. Cortical bone volume, BMD, and thickness were reduced; whereas, bone marrow volume was increased in mCXCR −/−. Decreased polar moment of inertia ( J) in the tibias/femurs suggested that the mCXCR −/− long bones are weaker. This was confirmed by three-point bending testing of the femurs. CDs created in 6-week-old male mCXCR −/− and WT littermates were not completely healed at 12 weeks; WT animals, however, had significantly more bone in-growth than mCXCR −/−. New bone sites were identified using polarized light and assessed for numbers of osteocyte (OCy) lacunae and blood vessels (BlV) around the original CD. In new bone, the number of BlV in WT was > 2× that seen in mCXCR −/−. Bone histomorphometry parameters in the cranial defect did not show any difference in bone formation or resorption markers. In summary, studies showed that mCXCR −/− mice have (1) reduced weight and size; (2) decreased BMD and BMC; (3) decreased amounts of trabecular and cortical long bone; (4) decreased femur bone strength; and (5) significantly reduced intramembranous bone formation and number of BlV in new calvarial bone during bone repair.

Chronic axial compression of the mouse tail segment induces MRI bone marrow edema changes that correlate with increased marrow vasculature and cellularity

Magnetic resonance imaging (MRI) of bone marrow edema (BME) has been found to be helpful in the diagnosis of back pain attributed to degenerative disk disease (DDD) and spondyloarthropathy (SA), but its interpretation is limited by a lack of knowledge of its nature and natural history. We assessed effects of compressive forces to mouse tail segments of WT and TNF-Tg mice with SA, via contrast enhanced-MRI and histology. Normalized marrow contrast enhancement (NMCE) of uninstrumented WT vertebrae significantly decrease, threefold (p < 0.01) from 8 to 12 weeks of age, while the NMCE of TNF-Tg vertebrae remained elevated. Compressive loading (6x body weight) increased NMCE twofold (p < 0.02) within 2 weeks in WT tails, which was equal to 6x loaded TNF-Tg tails within 4 weeks. Histology confirmed degenerative changes and that load-induced NMCE corresponded to increased vascular sinus tissue (35 +/- 3% vs. 19 +/- 3%; p < 0.01) and cellularity (4,235 +/- 886 vs.1,468 +/- 320 cells/mm(2); p < 0.01) for the loaded versus unloaded WT, respectively. However, micro-computed tomography (CT) analyses failed to detect significant load-induced changes to bone. While the bone marrow of loaded WT and TNF-Tg vertebrae were similar, histology demonstrated mild cellular infiltrate and increased osteoclastic resorption in the WT tails versus severe inflammatory-erosive arthritis in TNF-Tg joints. Significant (p < 0.05) decreases in cortical and trabecular bone volume in uninstrumented TNF-Tg versus WT vertebrae were confirmed by micro-CT. Thus, chronic load-induced DDD causes BME signals in vertebrae similar to those observed from SA, and both DDD and SA signals correlate with a conversion from yellow to red marrow, with increased vascularity.