22 research outputs found

    Effects of more natural housing conditions on the muscular and skeletal characteristics of female C57BL/6J mice

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    Background Enrichment of home cages in laboratory experiments offers clear advantages, but has been criticized in some respects. First, there is a lack of definition, which makes methodological uniformity difficult. Second, there is concern that the enrichment of home cages may increase the variance of results in experiments. Here, the influence of more natural housing conditions on physiological parameters of female C57BL/6J mice was investigated from an animal welfare point of view. For this purpose, the animals were kept in three different housing conditions: conventional cage housing, enriched housing and the semi naturalistic environment. The focus was on musculoskeletal changes after long-term environmental enrichment. Results The housing conditions had a long-term effect on the body weight of the test animals. The more complex and natural the home cage, the heavier the animals. This was associated with increased adipose deposits in the animals. There were no significant changes in muscle and bone characteristics except for single clues (femur diameter, bone resorption marker CTX-1). Additionally, the animals in the semi naturalistic environment (SNE) were found to have the fewest bone anomalies. Housing in the SNE appears to have the least effect on stress hormone concentrations. The lowest oxygen uptake was observed in enriched cage housing. Conclusions Despite increasing values, observed body weights were in the normal and strain-typical range. Overall, musculoskeletal parameters were slightly improved and age-related effects appear to have been attenuated. The variances in the results were not increased by more natural housing. This confirms the suitability of the applied housing conditions to ensure and increase animal welfare in laboratory experiments

    The role of collagen in bone apatite formation in the presence of hydroxyapatite nucleation inhibitors

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    Bone is a composite material in which collagen fibrils form a scaffold for a highly organized arrangement of uniaxially oriented apatite crystals. In the periodic 67¿nm cross-striated pattern of the collagen fibril, the less dense 40-nm-long gap zone has been implicated as the place where apatite crystals nucleate from an amorphous phase, and subsequently grow. This process is believed to be directed by highly acidic non-collagenous proteins, however, the role of the collagen matrix during bone apatite mineralization remains unknown. Here, combining nanometre-scale resolution cryogenic transmission electron microscopy and cryogenic electron tomography with molecular modelling, we show that collagen functions in synergy with inhibitors of hydroxyapatite nucleation to actively control mineralization. The positive net charge close to the C-terminal end of the collagen molecules promotes the infiltration of the fibrils with amorphous calcium phosphate (ACP). Furthermore, the clusters of charged amino acids, both in gap and overlap regions, form nucleation sites controlling the conversion of ACP into a parallel array of oriented apatite crystals. We developed a model describing the mechanisms through which the structure, supramolecular assembly and charge distribution of collagen can control mineralization in the presence of inhibitors of hydroxyapatite nucleatio

    Spine Metastases in Immunocompromised Mice after Intracardiac Injection of MDA-MB-231-SCP2 Breast Cancer Cells

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    Breast cancer cells frequently metastasize to bone, where their interaction with bone remodeling cell types enhances osteolytic bone destruction. Importantly, however, whereas skeletal analyses of xenograft models are usually restricted to hindlimb bones, human skeletal metastases are far more frequent in the spine, where trabecular bone mass is higher compared to femur or tibia. Here, we addressed whether breast cancer cells injected into immunocompromised mice metastasize to the spine and if this process is influenced by the amount of trabecular bone. We also took advantage of mice carrying the Col1a1-Krm2 transgene, which display severe osteoporosis. After crossing this transgene into the immunocompromised NSG background we injected MDA-MB-231-SCP2 breast cancer cells and analyzed their distribution three weeks thereafter. We identified more tumor cells and clusters of different size in spine sections than in femora, which allowed influences on bone remodeling cell types to be analyzed by comparing tumor-free to tumor-burdened areas. Unexpectedly, the Col1a1-Krm2 transgene did not affect spreading and metastatic outgrowth of MDA-MB-231-SCP2 cells, suggesting that bone tumor interactions are more relevant at later stages of metastatic progression

    The role of fetuin-a in endochondral ossification

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    The hepatic serum glycoprotein fetuin-A plays a role in mineralized matrix metabolism. It prevents unwanted ectopic calcification by stabilizing the amorphous phase of calcium phosphate and facilitating excess mineral clearance. Fetuin-A is one of the most highly abundant non-collagenous proteins in bone. As Ahsg-/- mice have shortened femoral bones, fetuin-A plays an important role in endochondral ossification. However, the underlying mechanisms are yet unknown. Earlier studies explained the shortened femora as a lack of TGF-β antagonization or as premature growth plate mineralization.In this study, I first hypothesized that fetuin-A regulates endochondral ossification through retinoic acid signaling, as retinoic acid intoxication leads to growth plate closure, especially in the femur. Using a postnatal bone culture system, the effect of fetuin-A and all-trans-retinoic acid (ATRA) on bone growth was investigated. Treatment of bones with ATRA alone led to growth plate cartilage degradation; a combination of ATRA and fetuin-A led to an increased degradation to the point of cartilage detachment. This indicated that fetuin-A might play a role in cartilage degradation, but it was not clear how, if at all, fetuin-A regulated retinoic acid signaling in cartilage.In histological sections from cultured bones, I discovered that bones from fetuin-A deficient mice had elongated hypertrophic zones in their growth plates. Histological sections of eight weeks old mice showed that growth plates of Ahsg-/- mice were grossly disordered and deformed. In order to gain three-dimensional information of growth plate morphology, bones were measured using μCT. Three-dimensional reconstructions of bones from eight weeks old mice revealed that the distal femora were displaced and deformed and the growth plates showed an angular deformation.Histological examination of bones from mice of different ages showed that, starting from three weeks of age, growth plates of both homo- and heterozygous fetuin-A deficient mice had lesions and were invaded by foreign cells. At eight weeks of age, lesions in the growth plates of Ahsg-/- mice had healed, partially by bone bridge formation, and growth plate architecture was distorted. Interestingly, in heterozygous Ahsg+/- mice, growth plates were either inconspicuous, or contained cavities which were lined with cellular infiltrate, although Ahsg+/- mice never developed the dysplasia.A microarray analysis of growth plates from 13 days old mice revealed a strong immune response in Ahsg-/- mice. A comparison to the Interferome database showed, that a huge amount of the upregulated genes were regulated through either a type I or a type II interferon response.I therefore hypothesize that fetuin-A plays an anti-inflammatory role in endochondral ossification and the dysplasia in Ahsg-/- mice is a consequence of an inflammatory response in the growth plate, which leads to cellular infiltration, mechanical weakening of growth plate cartilage and angular deformation of the distal femoral growth plate

    Deficiency of sphingosine-1-phosphate receptor 3 does not affect the skeletal phenotype of mice lacking sphingosine-1-phosphate lyase.

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    Albeit osteoporosis is one of the most prevalent disorders in the aged population, treatment options stimulating the activity of bone-forming osteoblasts are still limited. We and others have previously identified sphingosine-1-phosphate (S1P) as a bone remodeling coupling factor, which is released by bone-resorbing osteoclasts to stimulate bone formation. Moreover, S1pr3, encoding one of the five known S1P receptors (S1P3), was found differentially expressed in osteoblasts, and S1P3 deficiency corrected the moderate high bone mass phenotype of a mouse model (deficient for the calcitonin receptor) with increased S1P release from osteoclasts. In the present study we addressed the question, if S1P3 deficiency would also influence the skeletal phenotype of mice lacking S1P-lyase (encoded by Sgpl1), which display markedly increased S1P levels due to insufficient degradation. Consistent with previous reports, the majority of Sgpl1-deficient mice died before or shortly after weaning, and this lethality was not influenced by additional S1P3 deficiency. At 3 weeks of age, Sgpl1-deficient mice displayed increased trabecular bone mass, which was associated with enhanced osteoclastogenesis and bone resorption, but also with increased bone formation. Most importantly however, none of the skeletal parameters assessed by μCT, histomorphometry and serum analyses were significantly influenced by additional S1P3 deficiency. Taken together, our findings fully support the concept that S1P is a potent osteoanabolic molecule, although S1P3 is not the sole receptor mediating this influence. Since S1P receptors are considered excellent drug targets, it is now required to screen for the impact of other family members on bone formation

    Effects of more natural housing conditions on the muscular and skeletal characteristics of female C57BL/6J mice

    No full text
    Abstract Background Enrichment of home cages in laboratory experiments offers clear advantages, but has been criticized in some respects. First, there is a lack of definition, which makes methodological uniformity difficult. Second, there is concern that the enrichment of home cages may increase the variance of results in experiments. Here, the influence of more natural housing conditions on physiological parameters of female C57BL/6J mice was investigated from an animal welfare point of view. For this purpose, the animals were kept in three different housing conditions: conventional cage housing, enriched housing and the semi naturalistic environment. The focus was on musculoskeletal changes after long-term environmental enrichment. Results The housing conditions had a long-term effect on the body weight of the test animals. The more complex and natural the home cage, the heavier the animals. This was associated with increased adipose deposits in the animals. There were no significant changes in muscle and bone characteristics except for single clues (femur diameter, bone resorption marker CTX-1). Additionally, the animals in the semi naturalistic environment (SNE) were found to have the fewest bone anomalies. Housing in the SNE appears to have the least effect on stress hormone concentrations. The lowest oxygen uptake was observed in enriched cage housing. Conclusions Despite increasing values, observed body weights were in the normal and strain-typical range. Overall, musculoskeletal parameters were slightly improved and age-related effects appear to have been attenuated. The variances in the results were not increased by more natural housing. This confirms the suitability of the applied housing conditions to ensure and increase animal welfare in laboratory experiments

    Spine Metastases in Immunocompromised Mice after Intracardiac Injection of MDA-MB-231-SCP2 Breast Cancer Cells

    No full text
    Breast cancer cells frequently metastasize to bone, where their interaction with bone remodeling cell types enhances osteolytic bone destruction. Importantly, however, whereas skeletal analyses of xenograft models are usually restricted to hindlimb bones, human skeletal metastases are far more frequent in the spine, where trabecular bone mass is higher compared to femur or tibia. Here, we addressed whether breast cancer cells injected into immunocompromised mice metastasize to the spine and if this process is influenced by the amount of trabecular bone. We also took advantage of mice carrying the Col1a1-Krm2 transgene, which display severe osteoporosis. After crossing this transgene into the immunocompromised NSG background we injected MDA-MB-231-SCP2 breast cancer cells and analyzed their distribution three weeks thereafter. We identified more tumor cells and clusters of different size in spine sections than in femora, which allowed influences on bone remodeling cell types to be analyzed by comparing tumor-free to tumor-burdened areas. Unexpectedly, the Col1a1-Krm2 transgene did not affect spreading and metastatic outgrowth of MDA-MB-231-SCP2 cells, suggesting that bone tumor interactions are more relevant at later stages of metastatic progression

    Post-weaning epiphysiolysis causes distal femur dysplasia and foreshortened hindlimbs in fetuin-A-deficient mice

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    Fetuin-A / alpha(2)-Heremans-Schmid-glycoprotein (gene name Ahsg) is a systemic inhibitor of ectopic calcification. Due to its high affinity for calcium phosphate, fetuin-A is highly abundant in mineralized bone matrix. Foreshortened femora in fetuin-A-deficient Ahsg(-/-) mice indicated a role for fetuin-A in bone formation. We studied early postnatal bone development in fetuin-A-deficient mice and discovered that femora from Ahsg(-/-) mice exhibited severely displaced distal epiphyses and deformed growth plates, similar to the human disease slipped capital femoral epiphysis (SCFE). The growth plate slippage occurred in 70% of Ahsg(-/-) mice of both sexes around three weeks postnatal. At this time point, mice weaned and rapidly gained weight and mobility. Epiphysis slippage never occurred in wildtype and heterozygous Ahsg(+/-) mice. Homozygous fetuin-A-deficient Ahsg(-/-) mice and, to a lesser degree, heterozygous Ahsg(+/-) mice showed lesions separating the proliferative zone from the hypertrophic zone of the growth plate. The hypertrophic growth plate cartilage in long bones from Ahsg(-/-) mice was significantly elongated and V-shaped until three weeks of age and thus prior to the slippage. Genome-wide transcriptome analysis of laser-dissected distal femoral growth plates from 13-day-old Ahsg(-/-) mice revealed a JAK-STAT-mediated inflammatory response including a 550-fold induction of the chemokine Cxcl9. At this stage, vascularization of the elongated growth plates was impaired, which was visualized by immunofluorescence staining. Thus, fetuin-A-deficient mice may serve as a rodent model of growth plate pathologies including SCFE and inflammatory cartilage degradation
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