48 research outputs found
Isolation and generation of osteoclasts
No full textThis chapter describes the isolation, culture, and staining of osteoclasts. The key advantages of this assay are
that it allows direct measurement of osteoclast number, bone resorption, as well as yielding good quantities
of osteoclasts at defined stages of formation for molecular analysis. An additional focus of this chapter will
be the generation of osteoclasts from less conventional animal species and cell lines
Effect of Shock Wave Treatment on Platelet-Rich Plasma Added to Osteoblast Cultures
No full text: The aim of this study was to verify the effects on osteoblast cultures of adding a platelet-rich plasma (PRP) concentrate pretreated with 500 shock wave (SW) at an energy flow density of 0.17 mJ/mm(2), emitted by an electromagnetic generator Minilith SL1 (STORZ, Germany), reproducing the conditions of our previous study in which we apply SW directly on osteoblasts. Real-time PCR showed that in osteoblast cultures with added PRP pretreated with SW, there was an increased expression at 48 h of insulin-like growth factor binding protein 3 (IGFBP-3) and runt-related transcription factor 2 (RUNX2) and at 72 h, of collagen type I, osteocalcin, insulin-like growth factor 1 (IGF-1) as well as IGFBP-3. Western blotting confirmed the increased protein synthesis of IGFBP-3. This experience suggests that extracorporeal shock wave treatment (ESWT) should stimulate osteogenesis also by indirect platelets-mediated network. It therefore seems possible that combining the two methods, ESWT and bioengineering procedures to infiltrate PRP and growth factors, could be a successful approach
Extracorporeal Shock Waves Stimulate Osteoblast Activities
No full textThe extracorporeal shock wave therapy (ESWT) is an extensively applied treatment for musculoskeletal disorders because it promotes bone repair. The aim of this study was to evaluate the direct effect of ESWT on murine osteoblasts to clarify the cellular mechanism that leads to the induction of osteogenesis. Osteoblasts in culture flasks were treated with ESWT pulses (500 impulses of 0.05 mJ/mm(2)) generated by an electromagnetic device. Using western blot analysis 3 h after ESWT, an increased expression of Bax was found, indicating a fast pro-apoptotic effect of treatment on some of the osteoblasts. Activation of the cyclin E2/CDK2 is the complex that regulates the G1-S transition and is essential for cell proliferation. It was evident 24 to 72 h after treatment, indicating a proliferative stimulus. A decreased expression of osteoprotegerin (OPG) and receptor activator NF kappa B ligand (RANKL) 24 and 48 h after ESW, followed by a later increase of OPG, paired with a much smaller increase of RANKL, was evident by real-time polymerase chain reaction (PCR). The decreased RANKL/OPG ratio suggests inhibition of osteoclastogenesis. We can conclude that ESWT induces bone repair through the proliferation and differentiation of osteoblasts and the reduction of their secretion of pro-osteoclastogenic factors. (E-mail: [email protected]) (C) 2009 World Federation for Ultrasound in Medicine & Biology
The V2 receptor antagonist tolvaptan raises cytosolic calcium and prevents AQP2 trafficking and function: an in vitro and in vivo assessment
No full textTolvaptan, a selective vasopressin V2 receptor antagonist, is a new generation diuretic. Its clinical efficacy is in principle due to impaired vasopressin-regulated water reabsorption via aquaporin-2 (AQP2). Nevertheless, no direct in vitro evidence that tolvaptan prevents AQP2-mediated water transport, nor that this pathway is targeted in vivo in patients with syndrome of inappropriate antidiuresis (SIAD) has been provided. The effects of tolvaptan on the vasopressin-cAMP/PKA signalling cascade were investigated in MDCK cells expressing endogenous V2R and in mouse kidney. In MDCK, tolvaptan prevented dDAVP-induced increase in ser256-AQP2 and osmotic water permeability. A similar effect on ser256-AQP2 was found in V1aR -/- mice, thus confirming the V2R selectively. Of note, calcium calibration in MDCK showed that tolvaptan per se caused calcium mobilization from the endoplasmic reticulum resulting in a significant increase in basal intracellular calcium. This effect was only observed in cells expressing the V2R, indicating that it requires the tolvaptan-V2R interaction. Consistent with this finding, tolvaptan partially reduced the increase in ser256-AQP2 and the water permeability in response to forskolin, a direct activator of adenylyl cyclase (AC), suggesting that the increase in intracellular calcium is associated with an inhibition of the calcium-inhibitable AC type VI. Furthermore, tolvaptan treatment reduced AQP2 excretion in two SIAD patients and normalized plasma sodium concentration. These data represent the first detailed demonstration of the central role of AQP2 blockade in the aquaretic effect of tolvaptan and underscore a novel effect in raising intracellular calcium that can be of significant clinical relevance
Microgravity during spaceflight directly affects in vitro Osteoclastogenesis and bone resorption
No full textFirst evaluation of the potential effectiveness in muscular dystrophy of a novel chimeric compound, BN 82270, acting as calpain-inhibitor and anti-oxidant
No full textBN 82270 is a membrane-permeable prodrug of a chimeric compound (BN 82204) dually acting as calpain inhibitor and anti-oxidant. Acute in vivo injection of dystrophic mdx mice (30 mg/kg, s.c.) fully counteracted calpain overactivity in diaphragm. A chronic 4-6 weeks administration significantly prevented in vivo the fore limb force drop occurring in mdx mice exercised on treadmill. Ex vivo electrophysiological recordings showed that BN 82270 treatment contrasted the decrease in chloride channel function (gCl) in diaphragm, an index of spontaneous degeneration, while it was less effective on both exercise-impaired gCl and calcium-dependent mechanical threshold of the hind limb extensor digitorum longus (EDL) muscle fibres. The BN 82270 treated mdx mice showed a marked reduction of plasma creatine kinase and of the pro-fibrotic cytokine TGF-β1 in both hind limb muscles and diaphragm; however, the histopathological profile of gastrocnemious muscle was poorly ameliorated. In hind limb muscles of treated mice, the active form was detected by HPLC in the low therapeutic concentration range. In vitro exposure to 100 μM BN 82270 led to higher active form in diaphragm than in EDL muscle. This is the first demonstration that this class of chimeric compounds, dually targeting pathology-related events, exerts beneficial effects in muscular dystrophy. The drug/prodrug system may require posology adjustment to produce wider beneficial effects on all muscle types. © 2006 Elsevier B.V. All rights reserved
