Search

Highlights • Hypoxia is crucial for regulating the angiogenic-osteogenic coupling process. • IL-6 and IL-8 secretion in osteoblastic cells could be increased by hypoxia stress via HIF-1α signaling. • Hypoxia can increase the binding ability of HIF-1α to the IL-6 and IL-8 promoters. • Autocrine levels of IL-6 and IL-8 may promote the angiogenic-osteogenic coupling process. Abstract Inappropriate angiogenesis and osteogenesis are considered as the crucial factors of osteoporotic fracture. Hypoxia is a primary driving force for regulating the angiogenic-osteogenic coupling process. Our recent results indicated that hypoxia could improve angiogenesis as well as differentiation and activity of osteoblastic cells via up-regulating VEGF through HIF-1α pathway. Here we demonstrated that in human osteoblastic MG-63, U2-OS and Saos-2 cells, besides VEGF, the other two pro-angiogenic factors IL-6 and IL-8 were also up-regulated by hypoxia and CoCl 2 (a mimic of hypoxia). Mechanism studies indicated overexpression of HIF-1α (generated from transfection with a plasmid encoding sense HIF-1α) markedly increased the levels of IL-6 and IL-8 in osteoblastic cells. Furthermore, a luciferase reporter assay was performed using the reporter vector containing the IL-6 or IL-8 promoter sequence to illustrate observably increased activity of hypoxia-induced IL-6 and IL-8 promoter caused by overexpression of HIF-1α. Additionally, chromatin immune-precipitation analysis showed hypoxia increased the DNA binding ability of HIF-1α to IL-6 or IL-8 promoter. Analysis in vitro by MTT test and Boyden chamber assay showed exogenous IL-6 and IL-8 (a relatively short period of treatment with recombinant IL-6 or IL-8 equivalent to the autocrine levels) could significantly promote the proliferation of human osteoblastic, endothelial and monocytic cells, as well as the migration of human endothelial cells. Taken together, these results indicate that IL-6 and IL-8 in osteoblastic cells may also contribute to the angiogenic-osteogenic coupling process via HIF-1α pathway. Besides VEGF, IL-6- or IL-8-targeted adjunctive therapy maybe a new strategy to improve the treatment of osteoporosis. [ABSTRACT FROM AUTHOR]

DMPC and DSPC liposomes were prepared via thin film hydration method followed by sonication. Propranolol solution was incorporated into liposomes at hydration stage. TEM images showed the sizes of DSPC and DMPC were around 88 and 137 nm, respectively. The highest encapsulation ratio of propranolol was approximately 70% using DSPC/CHO/OCT liposomes, which release the drug over 60% in 24 h and reached 100% in 48 h. Both propranolol (10-8-10-6 M) and DSCP liposomes-encapsulated propranolol showed over 1.5-fold increases in the proliferation of human osteoblastic cells hFOB1.19 while differentiation of the cells was approximately doubled by plain and liposomal propranolol, indicating that the stimulatory effects of liposomal propranolol are similar with those of propranolol on human osteoblastic hFOB1.19 cells. The phosphatidylcholine liposomes-encapsulated propranolol prepared in this study potentially possesses anabolic effects in vivo and is also a promising anti-osteoporotic agent in future. [ABSTRACT FROM AUTHOR]

Mechanical forces trigger biological responses in bone cells that ultimately control osteoblastogenesis and bone remodelling. Although several mechanosensors have been postulated, the precise mechanotransduction pathway remains obscure as the initial mechanosensing event has not yet been identified. Studies in kidney cells have shown that polycystin-1 (PC1), via its extracellular N-terminal part, may function as an “antenna-like” protein providing a linkage between environmental cues and their conversion into biochemical responses that regulate various cellular processes via the calcineurin/ NFAT pathway. Here we explored the involvement of PC1 in mechanical load (stretching)-induced signalling cascades that control osteoblastogenesis/bone formation. FACS and TransAM Transcription Factor ELISA analyses employing extracts from primary human osteoblast-like, PC1 expressing cells subjected to mechanical stretching (0-6 h) revealed that unphosphorylated/DNA-binding competent NFATc1 increased at 0.5-1 h and returned to normal at 6 h. In accordance with the activation mechanism of NFATc1, stretching of cultured cells pre-treated with cyclosporin A (CsA, a specific inhibitor of the calcineurin/NFAT pathway) abrogated the observed decrease in the abundance of the cytoplasmic pNFATc1 (phosphorylated/inactive) species. Furthermore, stretching of osteoblastic cells pre-treated with an antibody against the mechanosensing N-terminal part of PC1 also abrogated the observed decrease in the cytoplasmic levels of the inactive pNFATc1 species. Importantly, under similar conditions (pre-incubation of stretched cells with the inhibitory anti-PC1 antibody), the expression of the key osteoblastic, NFATc1-target gene runx2 decreased compared to untreated cells. Therefore PC1 acts as chief mechanosensing molecule that modulates osteoblastic gene transcription and hence bone-cell differentiation through the calcineurin/NFAT signalling cascade. [ABSTRACT FROM AUTHOR]

Objectives: The aim of this work was to analyse the behaviour of human bone marrow osteoblastic cells cultured on the surface of routinely used plasma-sprayed titanium implants in the presence of plasmatic and salivary nicotine levels reported in smokers. Material and methods: Human bone marrow cells (first subculture) were seeded on titanium implants and cultured for 35 days in α-minimal essential medium supplemented with 10% foetal bovine serum, 50 μg/ml ascorbic acid, 10 mM β-glycerophosphate and 10 nM dexamethasone. Seeded implants were exposed to nicotine, 10–1 mg/ml, from days 1 to 35, and characterized for cell morphology, viability/proliferation, alkaline phosphatase (ALP) activity and matrix mineralization. Results: Low levels of nicotine, 10 and 50 ng/ml, representative of the plasma concentrations reported in smokers, did not cause significant effects in the cell behaviour, although a small induction in cell growth and functional activity appeared to occur. Higher nicotine levels, 0.01–1 mg/ml, within those attained in saliva through tobacco use, caused evident dose-dependent effects in osteoblastic cell behaviour, i.e., a stimulatory effect in cell growth, ALP activity and matrix mineralization, at concentrations up to 0.2 mg/ml, and a deleterious effect at higher levels. Conclusions: Considering the high tissue diffusion potential of nicotine, the results suggest the possibility of a direct modulation of the osteoblast activity as a contributing factor to the overall effect of nicotine in the bone microenvironment around dental implants. [ABSTRACT FROM AUTHOR]

Abstract: Acanthamoeba spp. contains a group of free-living amoebae widespread in nature. These microorganisms may cause several diseases in humans including osteomyelitis. Here we characterize the cellular interaction between clinical and freshwater isolates of A. polyphaga with human osteoblasts. Amoeba cytoadherence was evaluated quantitatively and qualitatively. We observed that the clinical isolate readily adheres to human osteoblastic cells (HOB) in a saturable and time-dependent fashion. The cytoadhesion appears to be in part dependent on mannose-associated surface glycoconjugates, since prior incubation of the amoebae with α-mannose reduced cytoadhesion approximately 75%. Scanning electron microscopy revealed various amoebae exhibiting acanthapodia contacting the surface of osteoblasts. Some osteoblasts developed morphologies resembling apoptotic cells. The clinical isolate was highly toxic to HOB cells during 24h of cell–protozoan interaction. Cytotoxicity was also dependent on the amoeba-cell ratio. During the cytopathogenic process we observed amoebae in the apparent process of ingestion of target cells and also amoebae extending projections or digipodia into osteoblast targets. The results indicate that A. polyphaga trophozoites attach and destroy human osteoblasts. [Copyright &y& Elsevier]

In this work, the influence of thermal oxidation treatments of Ti6Al4V at 500°C and 700°C for 1 h on the in vitro corrosion behaviour and osteoblast response is studied. The potential of these treatments, aimed to improve the wear surface performance as biomaterial, relies in the formation of an outer “ceramic” layer of rutile. The corrosion behaviour was evaluated in simulated human fluids by electrochemical impedance spectroscopy and anodic polarisation tests. The effect of these thermal oxidation treatments on osteoblastic behaviour was studied in primary cultures of human osteoblastic cells. Results show that thermal oxidation treatments do not decrease the high in vitro corrosion resistance of the Ti6Al4V alloy. Osteoblast adhesion studies indicate that thermal oxidation treatments do not impair the material biocompatibility. Moreover, the thermal oxidation at 700°C enhances the in vitro osteoblastic cell attachment compared to the thermal oxidation at 500°C. [Copyright &y& Elsevier]

The effect of two biomaterials, polyethylene and α-alumina, on interleukin-6 (IL-6) secretion and expression has been studied in human osteoblasts in primary culture. Human osteoblastic cells were derived from fresh trabecular bone explants removed during total knee arthroplasty. On reaching confluence, cells were subcultured in 6 well plates; the resulting subcultures were incubated until confluence and polyethylene or α-alumina particles were added to some while the rest were left as controls. The IL-6 mRNA levels were assessed by reverse transcription (RT) followed by polymerase chain reaction (PCR). IL-6 secretion was measured in the conditioned medium. The IL-6 expression was higher in the presence of both biomaterials. Maximum expression occurred in response to a dose of 50 mg particles/well with both biomaterials and was greater after polyethylene particle addition than after α-alumina particle addition at this dose. The maximum IL-6 secretion elicited by α-alumina was produced at 10 mg particles/well while maximum response with polyethylene required 50 mg/well. At a dose of 10 mg/well, α-alumina particles induced more secretion than 10 mg of polyethylene particles. Nevertheless, at a dose of 50 mg/well maximum secretion was produced with polyethylene particles. In conclusion and in our experimental conditions, polyethylene as well as α-alumina increased both the expression and the secretion of IL-6 in human osteoblastic cells in primary culture and stimulation from polyethylene appears stronger than that from α-alumina at the same dose. [Copyright &y& Elsevier]

The physico-chemical surface design of implants influences the surrounding cells. Osteoblasts on sharp-edged micro-topographies revealed an impaired cell phenotype, function and Ca2+ mobilization. The influence of edges and ridges on the Wnt/β-catenin pathway in combination with the cells' stress response has not been clear. Therefore, MG-63 osteoblasts were studied on defined titanium-coated micro-pillars (5 × 5 × 5 µm) in vitro and in silico. MG-63s on micro-pillars indicated an activated state of the Wnt/β-catenin pathway. The β-catenin protein accumulated in the cytosol and translocated into the nucleus. Gene profiling indicated an antagonism mechanism of the transcriptional activity of β-catenin due to an increased expression of inhibitors like ICAT (inhibitor of β-catenin and transcription factor-4). Cells on pillars produced a significant reactive oxygen species (ROS) amount after 1 and 24 h. In silico analyses provided a detailed view on how transcriptional activity of Wnt signaling is coordinated in response to the oxidative stress induced by the micro-topography. Based on a coordinated expression of regulatory elements of the Wnt/β-catenin pathway, MG-63s are able to cope with an increased accumulation of β-catenin on micro-pillars and suppress an unintended target gene expression. Further, β-catenin may be diverted into other signaling pathways to support defense mechanisms against ROS. [ABSTRACT FROM AUTHOR]

Two series of segmented polyurethanes were obtained and their mechanical and thermal properties as well as their biodegradability and cytotoxicity were evaluated. The chemical nature of the polyurethanes was varied by using either 1,4 butanediol (poly-ester-urethanes, PEUs) or l-lysine ethyl ester dihydrochloride (poly-ester-urea-urethanes, PEUUs) as chain extenders. Results showed that varying the hard segment influenced the thermal and mechanical properties of the obtained polymers. PEUs showed strain and hardness values of about 10–20 MPa and 10–65 MPa, respectively. These values were higher than the obtained values for the PEUUs due to the phase segregation and the higher crystallinity observed for the polyester-urethanes (PEUs); phase segregation was also observed and analyzed by XRD and DSC. Moreover, both series of polymers showed hydrolytic degradation when they were submerged in PBS until 90 days with 20% of weight loss. In vitro tests using a Human Osteoblastic cell line (Hob) showed an average of 80% of cell viability and good adhesion for both series of polymers. [ABSTRACT FROM AUTHOR]