For Western blot analysis, membranes were probed with the www.selleckchem.com/products/ganetespib-sta-9090.html following antibodies, phospho p42 44 9102 Cell Signaling, p44 4372 Cell Signaling. Serum deoxypyridinoline determination Serum deoxypyridinoline was measured with a METRA Serum PYD EIA Kit according to the supplied protocol. Results The process of bone repair was examined 7, 14 and 28 days post injury in transverse serial sections of decalcified as well as calcified tibia. In addition, serial longitudinal sections were prepared in order to ver ify the location of the injury site and to follow bone heal ing in another plane. The process of cortical and medullary defect healing was quantified with CT at day 14 after injury. Day 7 post injury In control mice the drill site was populated by connective tissue fibroblasts and mesenchymal progenitor cells at day 7 post injury.
Inhibitors,Modulators,Libraries In agreement with data reported previously, new bone formation was initiated at the periosteal surface and in Inhibitors,Modulators,Libraries the bone marrow cavity. The woven bone within the marrow cavity Inhibitors,Modulators,Libraries exhibited ini tial mineralisation as detected by VonKossa staining and small islands of mineralised tissue were detectable in the cortical region. Cartilage was also detected, indicating some degree of endochondral bone formation. In Nf1Prx1 mice connective tissue fibroblasts were present in the injury site but the deposition of extracellular matrix associated with osteoblast differentiation did not occur. Consequently, the mineralisation process was delayed in the marrow cavity and only marginally present in the cor tical region.
Quantifica tion by CT showed that the bone volume to total volume fraction in mutants was decreased by 40% in the bone Inhibitors,Modulators,Libraries marrow cavity and reduced five fold in the cortical regions when compared with con trols. Cartilage was formed excessively and fibro cartilaginous tissue accumulated on the margins. In addition, bone surrounding the drill site in Nf1Prx1 mice was generally unmineralised. Unmineralised bone was detectable in Inhibitors,Modulators,Libraries all tested animals on the 7th and 14th day post injury but it was consistently absent in the non injured Nf1Prx1 tibiae. Interestingly, this phenomenon was also observed at locations distant from the injury, suggesting the involvement of long range and or systemic signalling. In situ expression analysis showed a decreased level of Runx2 expression in the bone marrow cavity at day 7 post injury indicating impaired osteoblast formation and or recruitment of progenitor cells.
Day overnight delivery 14 post injury At day 14 post injury the drill site in control animals was filled with newly formed bone, osteoid and a few blood vessels. Mineralisation islands had developed to trabeculae by replacing cartilage and fibrous tissue. The newly formed trabeculae were thicker, and lined with a thin osteoid indicating timely minerali sation of the newly formed matrix.