by Keyword: Osteoblasts

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Canal, C., Fontelo, R., Hamouda, I., Guillem-Marti, J., Cvelbar, U., Ginebra, M. P., (2017). Plasma-induced selectivity in bone cancer cells death Free Radical Biology and Medicine 110, 72-80

Background: Current therapies for bone cancers - either primary or metastatic – are difficult to implement and unfortunately not completely effective. An alternative therapy could be found in cold plasmas generated at atmospheric pressure which have already demonstrated selective anti-tumor action in a number of carcinomas and in more relatively rare brain tumors. However, its effects on bone cancer are still unknown. Methods: Herein, we employed an atmospheric pressure plasma jet (APPJ) to validate its selectivity towards osteosarcoma cell line vs. osteoblasts & human mesenchymal stem cells. Results: Cytotoxicity following direct interaction of APPJ with cells is comparable to indirect interaction when only liquid medium is treated and subsequently added to the cells, especially on the long-term (72 h of cell culture). Moreover, following contact of the APPJ treated medium with cells, delayed effects are observed which lead to 100% bone cancer cell death through apoptosis (decreased cell viability with incubation time in contact with APPJ treated medium from 24 h to 72 h), while healthy cells remain fully viable and unaffected by the treatment. Conclusions: The high efficiency of the indirect treatment indicates that an important role is played by the reactive oxygen species (ROS) and reactive nitrogen species (RNS) in the gaseous plasma stage and then transmitted to the liquid phase, which overall lead to lethal and selective action towards osteosarcoma cells. These findings open new pathways for treatment of metastatic bone disease with a minimally invasive approach.

Keywords: Atmospheric pressure plasma jet, Bone cancer, hMSC, HOb, Liquids, Osteoblasts, Osteosarcoma, SaOS-2

Gustavsson, J., Planell, J., Engel, E., (2013). Ion-selective electrodes to monitor osteoblast-like cellular influence on the extracellular concentration of calcium Journal of Tissue Engineering and Regenerative Medicine 7, (8), 609-620

In bone tissue engineering, the composition of the ionic extracellular environment (IEE) can determine both cellular fate and a biomaterial's development and performance. Therefore, precise control of the IEE and a perfect understanding of the dynamic changes that it can be subject to due to cellular activity is highly desired. To achieve this, we initially monitored how two standard osteoblast-like cell models that expressed either high or low alkaline phosphatase activity - SAOS-2 and MG63 cells, respectively - affected the extracellular concentrations of calcium and phosphate during long-term cultures. It was observed that cellular influence on the IEE varied greatly between the two models and could be linked to the capacity of cells to deposit calcium in the extracellular matrix. Miniaturized ion-selective electrodes that could allow for real-time monitoring of calcium in a minimally invasive way were then constructed. The electrodes were characterized in standard in vitro cell culture environments, prior to being successfully applied for periods of 24h, to record the dynamics of cell-induced deposition of calcium in the extracellular matrix, while using osteogenic media of either high or low concentrations of phosphate. As a result, this study provides the background and technological means for the non-destructive evaluation of the IEE in vitro and allows for the optimization and development of better models of bone tissue construction.

Keywords: Extracellular ions, Ion-selective electrode, MG63, Mineralization, Osteoblasts, Saos-2, Sensor, Tissue engineering

Navarro, M., Pu, F., Hunt, J. A., (2012). The significance of the host inflammatory response on the therapeutic efficacy of cell therapies utilising human adult stem cells Experimental Cell Research 318, (4), 361-370

Controlling the fate of implanted hMSCs is one of the major drawbacks to be overcome to realize tissue engineering strategies. In particular, the effect of the inflammatory environment on hMSCs behaviour is poorly understood. Studying and mimicking the inflammatory process in vitro is a very complex and challenging task that involves multiple variables. This research addressed the questions using in vitro co-cultures of primary derived hMSCs together with human peripheral blood mononucleated cells (PBMCs); the latter are key agents in the inflammatory process. This work explored the in vitro phenotypic changes of hMSCs in co-culture direct contact with monocytes and lymphocytes isolated from blood using both basal and osteogenic medium. Our findings indicated that hMSCs maintained their undifferentiated phenotype and pluripotency despite the contact with PBMCs. Moreover, hMSCs demonstrated increased proliferation and were able to differentiate specifically down the osteogenic lineage pathway. Providing significant crucial evidence to support the hypothesis that inflammation and host defence mechanisms could be utilised rather than avoided and combated to provide for the successful therapeutic application of stem cell therapies.

Keywords: Co-culture, Inflammation, Mesenchymal stem cells, Monocytes, Osteoblasts

Byrne, Damien P., Lacroix, Damien, Prendergast, Patrick J., (2011). Simulation of fracture healing in the tibia: Mechanoregulation of cell activity using a lattice modeling approach Journal of Orthopaedic Research 29, (10), 1496-1503

In this study, a three-dimensional (3D) computational simulation of bone regeneration was performed in a human tibia under realistic muscle loading. The simulation was achieved using a discrete lattice modeling approach combined with a mechanoregulation algorithm to describe the cellular processes involved in the healing process namely proliferation, migration, apoptosis, and differentiation of cells. The main phases of fracture healing were predicted by the simulation, including the bone resorption phase, and there was a qualitative agreement between the temporal changes in interfragmentary strain and bending stiffness by comparison to experimental data and clinical results. Bone healing was simulated beyond the reparative phase by modeling the transition of woven bone into lamellar bone. Because the simulation has been shown to work with realistic anatomical 3D geometry and muscle loading, it demonstrates the potential of simulation tools for patient-specific pre-operative treatment planning.

Keywords: Tissue differentiation, Computational analysis, Mechanical conditions, Bone regeneration, Weight-bearing, Proliferation, Osteoblast, Stiffness, Ingrowth, Scaffold

Engel, E., Martinez, E., Mills, C. A., Funes, M., Planell, J. A., Samitier, J., (2009). Mesenchymal stem cell differentiation on microstructured poly (methyl methacrylate) substrates Annals of Anatomy-Anatomischer Anzeiger 191, (1), 136-144

Recent studies on 2D substrates have revealed the importance of surface properties in affecting cell behaviour. In particular, surface topography appears to influence and direct cell migration. The development of new technologies of hot embossing and micro-imprinting has made it possible to study cell interactions with controlled micro features and to determine how these features can affect cell behaviour. Several studies have been carried out on the effect of microstructures on cell adhesion, cell guidance and cell proliferation. However, there is still a lack of knowledge on how these features affect mesenchymal stem cell differentiation. This study was designed to evaluate whether highly controlled microstructures on PMMA could induce rMSC differentiation into an osteogenic lineage. Structured PMMA was seeded with rMSC and cell number; cell morphology and cell differentiation were evaluated. Results confirm that microstructures not only affect cell proliferation and alignment but also have a synergistic effect with osteogenic medium on rMSC differentiation into mature osteoblasts.

Keywords: Mesenchymal stem cells, Osteoblasts, Topography, Microstructures

Engel, E., Del Valle, S., Aparicio, C., Altankov, G., Asin, L., Planell, J. A., Ginebra, M. P., (2008). Discerning the role of topography and ion exchange in cell response of bioactive tissue engineering scaffolds Tissue Engineering Part A 14, (8), 1341-1351

Surface topography is known to have an influence on osteoblast activity. However, in the case of bioactive materials, topographical changes can affect also ion exchange properties. This makes the problem more complex, since it is often difficult to separate the strictly topographical effects from the effects of ionic fluctuations in the medium. The scope of this paper is to analyze the simultaneous effect of topography and topography-mediated ion exchange on the initial cellular behavior of osteoblastic-like cells cultured on bioactive tissue engineering substrates. Two apatitic substrates with identical chemical composition but different micro/nanostructural features were obtained by low-temperature setting of a calcium phosphate cement. MG63 osteoblastic-like cells were cultured either in direct contact with the substrates or with their extracts. A strong and permanent decrease of calcium concentration in the culture medium, dependent on substrate topography, was detected. A major effect of the substrate microstructure on cell proliferation was observed, explained in part by the topography-mediated ion exchange, but not specifically by the ionic Ca(2+) fluctuations. Cell differentiation was strongly enhanced when cells were cultured on the finer substrate. This effect was not explained by the chemical modification of the medium, but rather suggested a strictly topographical effect.

Keywords: Alkaline Phosphatase/metabolism, Bone Cements/pharmacology, Calcium/metabolism, Calcium Phosphates/pharmacology, Cell Adhesion/drug effects, Cell Differentiation/drug effects, Cell Proliferation/drug effects, Cell Shape/drug effects, Cells, Cultured, Culture Media, Durapatite/pharmacology, Humans, Interferometry, Ion Exchange, Materials Testing, Osteoblasts/ cytology/drug effects/enzymology/ultrastructure, Phosphorus/metabolism, Powders, Tissue Engineering, Tissue Scaffolds

Charles-Harris, M., Koch, M. A., Navarro, M., Lacroix, D., Engel, E., Planell, J. A., (2008). A PLA/calcium phosphate degradable composite material for bone tissue engineering: an in vitro study Journal of Materials Science-Materials in Medicine 19, (4), 1503-1513

Biodegradable polymers reinforced with an inorganic phase such as calcium phosphate glasses may be a promising approach to fulfil the challenging requirements presented by 3D porous scaffolds for tissue engineering. Scaffolds' success depends mainly on their biological behaviour. This work is aimed to the in vitro study of polylactic acid (PLA)/CaP glass 3D porous constructs for bone regeneration. The scaffolds were elaborated using two different techniques, namely solvent-casting and phase-separation. The effect of scaffolds' micro and macrostructure on the biological response of these scaffolds was assayed. Cell proliferation, differentiation and morphology within the scaffolds were studied. Furthermore, polymer/glass scaffolds were seeded under dynamic conditions in a custom-made perfusion bioreactor. Results indicate that the final architecture of the solvent-cast or phase separated scaffolds have a significant effect on cells' behaviour. Solvent-cast scaffolds seem to be the best candidates for bone tissue engineering. Besides, dynamic seeding yielded a higher seeding efficiency in comparison with the static method.

Keywords: Biocompatible Materials/ chemistry, Bone and Bones/ metabolism, Calcium Phosphates/ chemistry, Cell Differentiation, Cell Proliferation, Humans, Lactic Acid/ chemistry, Microscopy, Confocal, Microscopy, Electron, Scanning, Osteoblasts/metabolism, Permeability, Polymers/ chemistry, Porosity, Solvents/chemistry, Tissue Engineering/ methods

Gustavsson, J., Altankov, G., Errachid, A., Samitier, J., Planell, J. A., Engel, E., (2008). Surface modifications of silicon nitride for cellular biosensor applications Journal of Materials Science-Materials in Medicine 19, (4), 1839-1850

Thin films of silicon nitride (Si3N4) can be used in several kinds of micro-sized biosensors as a material to monitor fine environmental changes related to the process of bone formation in vitro. We found however that Si3N4 does not provide optimal conditions for osseointegration as osteoblast-like MG-63 cells tend to detach from the surface when cultured over confluence. Therefore Si3N4 was modified with self-assembled monolayers bearing functional end groups of primary amine (NH2) and carboxyl (COOH) respectively. Both these modifications enhanced the interaction with confluent cell layers and thus improve osseointegration over Si3N4. Furthermore it was observed that the NH2 functionality increased the adsorption of fibronectin (FN), promoted cell proliferation, but delayed the differentiation. We also studied the fate of pre-adsorbed and secreted FN from cells to learn more about the impact of above functionalities for the development of provisional extracellular matrix on materials interface. Taken together our data supports that Si3N4 has low tissue integration but good cellular biocompatibility and thus is appropriate in cellular biosensor applications such as the ion-sensitive field effect transistor (ISFET). COOH and NH2 chemistries generally improve the interfacial tissue interaction with the sensor and they are therefore suitable substrates for monitoring cellular growth or matrix deposition using electrical impedance spectroscopy.

Keywords: Adsorption, Amines/chemistry, Biocompatible Materials/ chemistry, Biosensing Techniques, Cell Differentiation, Cell Line, Cell Proliferation, Electric Impedance, Extracellular Matrix/metabolism, Fibronectins/chemistry, Humans, Materials Testing, Osteoblasts/ cytology, Silicon Compounds/ chemistry, Surface Properties

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