ARSB IS REQUIRED FOR BONE REMODELING

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Entwicklung des CITRIC Stroboskop‐Kamerasystems für den Rotorversuchstand des DLR

Die Studienarbeit dient der Entwicklung des CITRIC Stroboskop‐Kamerasystems für den Rotorversuchstand des deutschen Zentrums für Luft‐ und Raumfahrt (DLR). Das Kamerasystem wird benötigt um Standbilder des sich drehenden Rotors aufzunehmen. Dabei soll das System über ein Bedienpanel verfügen, dass zur Steuerung des Objektivs an der Kamera bzw. des Schwenk‐/Neigekopfs, welcher die Kamera bewegt, dient. Ein besonderes Augenmerk bekommt die Steuerung der Blende (Autoiris) am Objektiv, da hierfür ein FBAS‐/BAS‐Signal erzeugt werden muss, welches mit einem Microcontroller nachgebildet wird. Desweiteren wird ein Mikrofonverstärker entwickelt, der auch bei erhöhtem Lärmpegel noch ausreichend gute Tonqualität gewährleistet

The impact of low-magnitude high-frequency vibration on fracture healing is profoundly influenced by the oestrogen status in mice

Fracture healing is impaired in aged and osteoporotic individuals. Because adequate mechanical stimuli are able to increase bone formation, one therapeutical approach to treat poorly healing fractures could be the application of whole-body vibration, including low-magnitude high-frequency vibration (LMHFV). We investigated the effects of LMHFV on fracture healing in aged osteoporotic mice. Female C57BL/6NCrl mice (n=96) were either ovariectomised (OVX) or sham operated (non-OVX) at age 41 weeks. When aged to 49 weeks, all mice received a femur osteotomy that was stabilised using an external fixator. The mice received whole-body vibrations (20 minutes/day) with 0.3 G: peak-to-peak acceleration and a frequency of 45 Hz. After 10 and 21 days, the osteotomised femurs and intact bones (contra-lateral femurs, lumbar spine) were evaluated using bending-testing, micro-computed tomography (μCT), histology and gene expression analyses. LMHFV disturbed fracture healing in aged non-OVX mice, with significantly reduced flexural rigidity (-81%) and bone formation (-80%) in the callus. Gene expression analyses demonstrated increased oestrogen receptor β (ERβ, encoded by Esr2) and Sost expression in the callus of the vibrated animals, but decreased β-catenin, suggesting that ERβ might mediate these negative effects through inhibition of osteoanabolic Wnt/β-catenin signalling. In contrast, in OVX mice, LMHFV significantly improved callus properties, with increased flexural rigidity (+1398%) and bone formation (+637%), which could be abolished by subcutaneous oestrogen application (0.025 mg oestrogen administered in a 90-day-release pellet). On a molecular level, we found an upregulation of ERα in the callus of the vibrated OVX mice, whereas ERβ was unaffected, indicating that ERα might mediate the osteoanabolic response. Our results indicate a major role for oestrogen in the mechanostimulation of fracture healing and imply that LMHFV might only be safe and effective in confined target populations

The Wnt serpentine receptor Frizzled-9 regulates new bone formation in fracture healing.

Wnt signaling is a key regulator of bone metabolism and fracture healing. The canonical Wnt/β-catenin pathway is regarded as the dominant mechanism, and targeting this pathway has emerged as a promising strategy for the treatment of osteoporosis and poorly healing fractures. In contrast, little is known about the role of non-canonical Wnt signaling in bone. Recently, it was demonstrated that the serpentine receptor Fzd9, a Wnt receptor of the Frizzled family, is essential for osteoblast function and positively regulates bone remodeling via the non-canonical Wnt pathway without involving β-catenin-dependent signaling. Here we investigated whether the Fzd9 receptor is essential for fracture healing using a femur osteotomy model in Fzd9(-/-) mice. After 10, 24 and 32 days the fracture calli were analyzed using biomechanical testing, histomorphometry, immunohistochemistry, and micro-computed tomography. Our results demonstrated significantly reduced amounts of newly formed bone at all investigated healing time points in the absence of Fzd9 and, accordingly, a decreased mechanical competence of the callus tissue in the late phase of fracture healing. In contrast, cartilage formation and numbers of osteoclasts degrading mineralized matrix were unaltered. β-Catenin immunolocalization showed that canonical Wnt-signaling was not affected in the absence of Fzd9 in osteoblasts as well as in proliferating and mature chondrocytes within the fracture callus. The expression of established differentiation markers was not altered in the absence of Fzd9, whereas chemokines Ccl2 and Cxcl5 seemed to be reduced. Collectively, our results suggest that non-canonical signaling via the Fzd9 receptor positively regulates intramembranous and endochondral bone formation during fracture healing, whereas it does not participate in the formation of cartilage or in the osteoclastic degradation of mineralized matrix. The finding that Fzd9, in addition to its role in physiological bone remodeling, regulates bone repair may have implications for the development of treatments for poorly or non-healing fractures

Gnathodiaphyseal dysplasia is not recapitulated in a respective mouse model carrying a mutation of the Ano5 gene

Mutations in the gene ANO5, encoding for the transmembrane protein Anoctamin 5 (Ano5), have been identified to cause gnathodiaphyseal dysplasia (GDD) in humans, a skeletal disorder characterized by sclerosis of tubular bones, increased fracture risk and fibro-osseous lesions of the jawbones. To better understand the pathomechanism of GDD we have generated via Crispr/CAS9 gene editing a mouse model harboring the murine equivalent (Ano5 p.T491F) of a GDD-causing ANO5 mutation identified in a previously reported patient. Skeletal phenotyping by contact radiography, μCT and undecalcified histomorphometry was performed in male mice, heterozygous and homozygous for the mutation, at the ages of 12 and 24 weeks. These mice did not display alterations of skeletal microarchitecture or mandible morphology. The results were confirmed in female mice and animals derived from a second, independent clone. Finally, no skeletal phenotype was observed in mice lacking ~40% of their Ano5 gene due to a frameshift mutation. Therefore, our results indicate that Ano5 is dispensable for bone homeostasis in mice, at least under unchallenged conditions, and that these animals may not present the most adequate model to study the physiological role of Anoctamin 5.ISSN:2352-187

Pharmacological estrogen administration causes a FSH-independent osteo-anabolic effect requiring ER alpha in osteoblasts.

Postmenopausal osteoporosis is characterized by declining estrogen levels, and estrogen replacement therapy has been proven beneficial for preventing bone loss in affected women. While the physiological functions of estrogen in bone, primarily the inhibition of bone resorption, have been studied extensively, the effects of pharmacological estrogen administration are still poorly characterized. Since elevated levels of follicle-stimulating hormone (FSH) have been suggested to be involved in postmenopausal bone loss, we investigated whether the skeletal response to pharmacological estrogen administration is mediated in a FSH-dependent manner. Therefore, we treated wildtype and FSHβ-deficicent (Fshb(-/-)) mice with estrogen for 4 weeks and subsequently analyzed their skeletal phenotype. Here we observed that estrogen treatment resulted in a significant increase of trabecular and cortical bone mass in both, wildtype and Fshb(-/-) mice. Unexpectedly, this FSH-independent pharmacological effect of estrogen was not caused by influencing bone resorption, but primarily by increasing bone formation. To understand the cellular and molecular nature of this osteo-anabolic effect we next administered estrogen to mouse models carrying cell specific mutant alleles of the estrogen receptor alpha (ERα). Here we found that the response to pharmacological estrogen administration was not affected by ERα inactivation in osteoclasts, while it was blunted in mice lacking the ERα in osteoblasts or in mice carrying a mutant ERα incapable of DNA binding. Taken together, our findings reveal a previously unknown osteo-anabolic effect of pharmacological estrogen administration, which is independent of FSH and requires DNA-binding of ERα in osteoblasts

Immunostaining of β-catenin at day 10 and histochemical TRAP staining indicating osteoclasts at day 24 in the fracture calli.

<p>WT: upper panel, <i>Fdz9</i>^−/−: lower panel. β-catenin was expressed in osteoblasts (OB) and proliferating chondroblasts (CB) but to a lesser extend in hypertrophic chondrocytes (HC). C: cortex. There were no differences between both genotypes. Only TRAP positive cells with ≥2 nuclei were identified as osteoclasts (OC). TRAP-staining either revealed no significant differences between both genotypes.</p

Immunostaining of chemokines Cxcl5 and Ccl2 at day 10.

<p>WT: upper panel, <i>Fdz9</i>^−/−: lower panel. C: cortex. Cxcl5 (<i>left</i>) and Ccl2 (<i>right</i>) were expressed by precursor cells, osteoblasts and chondrocytes in both genotypes, but staining was less intense in the absence of <i>Fzd9</i>. C: cortex, HC: hypertrophic chondrocytes, OB: osteoblasts, OT: osteocytes.</p

Histological evaluation of relative amounts of tissues in the fracture calli at different time points post fracture.

<p>WT: white columns, <i>Fzd9</i>^−/−: grey columns. All values are presented as median, interquartile ranges, minimum and maximum. n = 5–10; Mann-Whitney-U-test, *p<0.05. TOT: total osseous tissue, Cg: cartilage, FT: fibrous tissue. A: day 10, WT: n = 6, <i>Fzd9</i>^−/−: n = 5, B: day 24, WT: n = 7, <i>Fzd9</i>^−/−: n = 8, C: day 32, WT: n = 10, <i>Fzd</i>9^−/−: n = 8.</p

Immunostaining of osteoblast differentiation markers Runx2 and Osteocalcin at day 10.

<p>WT: upper panel, <i>Fdz9</i>^−/−: lower panel. Runx2 (<i>left</i>) was expressed mainly in the nucleus of preosteoblastic cells and osteoblasts (OB) located in the fracture callus. Osteocalcin (<i>right</i>) was mainly localized in the cytoplasma of osteoblasts near mineralized matrix and in the bone matrix. There were no differences in staining of Runx2 and Osteocalcin between both genotypes. C: cortex, OT: osteocytes.</p