Reduced oxidative stress in primary human cells by antioxidant released from nanoporous alumina.

Nanoporous alumina elicits different inflammatory responses dependent on pore size, such as increased complement activation and reactive oxygen species (ROS) production, on 200 versus 20 nm pores. In this study, we attempt to further modulate inflammatory cell response by loading nanoporous alumina membranes (20, 100, and 200 nm pores), with an antioxidant, Trolox, for controlled drug release. For mononuclear cells (MNC) no difference in cell response, due to pore size, was seen when cultured on nonloaded membranes. However, when exposed to membranes loaded with Trolox, 100 uM was enough to quench ROS by more than 95% for all pore sizes. Polymorphonuclear cells (PMNC) produced significantly more ROS when exposed to 20 versus 100 nm pores. For Trolox loaded membranes, this trend reversed, due to slower release of antioxidant from the 20 nm pores. Furthermore, Trolox exhibited a unique effect on PMNCs that has not previously been reported: It delayed the production of ROS in a manner distinct from antioxidant activity. The present study confirms that nanoporous alumina is a suitable vehicle for drug delivery, and that Trolox can successfully modulate the inflammatory response of both MNC and PMNCs.

Simvastatin and zinc synergistically enhance osteoblasts activity and decrease the acute response of inflammatory cells.

Several ceramic biomaterials have been suggested as promising alternatives to autologous bone to replace or restore bone after trauma or disease. The osteoinductive potential of most scaffolds is often rather low by themselves and for this reason growth factors or drugs have been supplemented to these synthetic materials. Although some growth factors show good osteoinductive potential their drawback is their high cost and potential severe side effects. In this work the combination of the well-known drug simvastatin (SVA) and the inorganic element Zinc (Zn) is suggested as a potential additive to bone grafts in order to increase their bone regeneration/formation. MC3T3-E1 cells were cultured with Zn (10 and 25 µM) and SVA (0.25 and 0.4 µM) for 10 days to evaluate proliferation and differentiation, and for 22 days to evaluate secretion of calcium deposits. The combination of Zn (10 µM) and SVA (0.25 µM) significantly enhanced cell differentiation and mineralization in a synergetic manner. In addition, the release of reactive oxygen species (ROS) from primary human monocytes in contact with the same concentrations of Zn and SVA was evaluated by chemiluminescence. The combination of the additives decreased the release of ROS, although Zn and SVA separately caused opposite effects. This work shows that a new combination of additives can be used to increase the osteoinductive capacity of porous bioceramics.

Effect of a Bromo Substituent on the Glutathione Peroxidase Activity of a Pyridoxine-like Diselenide.

In search for better mimics of the glutathione peroxidase enzymes, pyridoxine-like diselenides 6 and 11, carrying a 6-bromo substituent, were prepared. Reaction of 2,6-dibromo-3-pyridinol 5 with sodium diselenide provided 6 via aromatic nucleophilic substitution of the 2-bromo substituent. LiAlH4 caused reduction of all four ester groups and returned 11 after acidic workup. The X-ray structure of 6 showed that the dipyridyl diselenide moiety was kept in an almost planar, transoid conformation. According to NBO-analysis, this was due to weak intramolecular Se···O (1.1 kcal/mol) and Se···N-interactions (2.5 kcal/mol). That the 6-bromo substituent increased the positive charge on selenium was confirmed by NPA-analysis and seen in calculated and observed (77)Se NMR-shifts. Diselenide 6 showed a more than 3-fold higher reactivity than the corresponding des-bromo compound 3a and ebselen when evaluated in the coupled reductase assay. Experiments followed for longer time (2 h) confirmed that diselenide 6 is a better GPx-catalyst than 11. On the basis of (77)Se-NMR experiments, a catalytic mechanism for diselenide 6 was proposed involving selenol, selenosulfide and seleninic acid intermediates. At low concentration (10 µM) where it showed only minimal toxicity, it could scavenge ROS produced by MNC- and PMNC-cells more efficiently than Trolox.

Inflammatory response to nano- and microstructured hydroxyapatite.

The proliferation and activation of leukocytes upon contact with a biomaterial play a crucial role in the degree of inflammatory response, which may then determine the clinical failure or success of an implanted biomaterial. The aim of this study was to evaluate whether nano- and microstructured biomimetic hydroxyapatite substrates can influence the growth and activation of macrophage-like cells. Hydroxyapatite substrates with different crystal morphologies consisting of an entangled network of plate-like and needle-like crystals were evaluated. Macrophage proliferation was evaluated on the material surface (direct contact) and also in extracts i.e. media modified by the material (indirect contact). Additionally, the effect of supplementing the extracts with calcium ions and/or proteins was investigated. Macrophage activation on the substrates was evaluated by quantifying the release of reactive oxygen species and by morphological observations. The results showed that differences in the substrate's microstructure play a major role in the activation of macrophages as there was a higher release of reactive oxygen species after culturing the macrophages on plate-like crystals substrates compared to the almost non-existent release on needle-like substrates. However, the difference in macrophage proliferation was ascribed to different ionic exchanges and protein adsorption/retention from the substrates rather than to the texture of materials.

Effects of nanoporous alumina on inflammatory cell response.

The present study focuses on the effects of nanoscale porosity on inflammatory response in vitro and in vivo. Nanoporous alumina membranes with different pore sizes, 20 and 200 nm in diameter, were used. We first evaluated cell/alumina interactions in vitro by observing adhesion, proliferation, and activation of a murine fibroblast and a macrophage cell line. To investigate the chronic inflammatory response, the membranes were implanted subcutaneously in mice for 2 weeks. Cell recruitment to the site of implantation was determined by histology and the production of cytokines was measured by protein array analysis. Both in vitro and in vivo studies showed that 200 nm pores induced a stronger inflammatory response as compared to the alumina with 20 nm pores. This was observed by an increase in macrophage activation in vitro as well as higher cell recruitment and generation of proinflammatory cytokines around the alumina with 200 nm pores, in vivo. Our results suggest that nanofeatures can be modulated in order to control the inflammatory response to implants.

Role of alumina nanoporosity in acute cell response.

This work studied the effect of nanoporous alumina in acute cellular response in an in vivo model. Nanoporous alumina membranes, with pore size diameters of 20 and 200 nm, were fabricated by anodic oxidation of aluminium. The membranes were thereafter characterized in terms of pore size distribution and chemical composition. To evaluate acute inflammatory response, the membranes were implanted in the peritoneal cavity of mice. Cell recruitment to the implant site was determined by fluorescence activated cell sorting (FACS) analysis. Cell adhesion to material surfaces was studied in terms of cell number, type, and morphology using scanning electron microscopy (SEM) and immunocytochemical staining followed by fluorescence microscopy. The fabricated nanoporous alumina membranes were found to have narrow pore size distribution. The in vivo study showed that 200 nm alumina membranes induced stronger inflammatory response than 20 nm membranes. This was reflected by the number of implant-associated phagocytes and the number of cells recruited to the implantation site. Since both pore-size membranes possess similar chemical composition, we believe that the observed difference in cell recruitment and adhesion is an effect of the material nanotopography. Our results suggest that nanotopography can be used to subtly control the recruitment and adherence of phagocytic cells during the acute inflammatory response to alumina membranes.

Low-modulus PMMA bone cement modified with castor oil.

Some of the current clinical and biomechanical data suggest that vertebroplasty causes the development of adjacent vertebral fractures shortly after augmentation. These findings have been attributed to high injection volumes as well as high Young's moduli of PMMA bone cements compared to that of the osteoporotic cancellous bone. The aim of this study was to evaluate the use of castor oil as a plasticizer for PMMA bone cements. The Young's modulus, yield strength, maximum polymerization temperature, doughing time, setting time and the complex viscosity curves during curing, were determined. The cytotoxicity of the materials extracts was assessed on cells of an osteoblast-like cell line. The addition of up to 12 wt% castor oil decreased yield strength from 88 to 15 MPa, Young's modulus from 1500 to 446 MPa and maximum polymerization temperature from 41.3 to 25.6°C, without affecting the setting time. However, castor oil seemed to interfere with the polymerization reaction, giving a negative effect on cell viability in a worst-case scenario.

Time sequence of blood activation by nanoporous alumina: Studies on platelets and complement system.

In the present work, the time sequence of blood activation by alumina membranes with different porosities (20 and 200 nm in diameter) was studied. The membranes were incubated with whole blood from 2 min to 4 h. Platelet adhesion and activation in addition to complement activation was monitored at different time points. Evaluation of platelet adhesion and activation was done by determining the change in platelet number and the levels of thrombospondin-1 (TSP-1) in the fluid phase. Scanning electron microscopy studies were done to further evaluate platelet adhesion and morphology. Immunocytochemical staining was used to evaluate the presence of CD41 and CD62P antigens on the material surface. Complement activation was monitored by measuring C3a and sC5b-9 in plasma samples by means of enzyme immunoassays. Both alumina membranes displayed similar complement activation time profiles, with levels of C3a and sC5b-9 increasing with incubation time. A statistically significant difference between the membranes was found after 60 min of incubation. Platelet activation characteristics and time profile were different between the two membranes. Platelet adhesion increased over time for the 20 nm surface, while the clusters of microparticles on the 200 nm surface did not appreciably change during the course of the experiment. The release of TSP-1 increased with time for both membranes; however, much later for the 200 nm alumina (240 min) as compared to the 20 nm membrane (60 min). The surface topography of the alumina most probably influence protein transition rate, which in turn affects material platelet activation kinetics.

Procoagulant behavior and platelet microparticle generation on nanoporous alumina.

In the present work, we have investigated platelet microparticle (PMP) generation in whole blood after contact with nanoporous alumina. Alumina membranes with pore sizes of 20 and 200 nm in diameter were incubated with whole blood and the number of PMP in the fluid phase was determined by flow cytometry. The role of the complement system in PMP generation was investigated using an analog of the potent complement inhibitor compstatin. Moreover, the procoagulant activity of the two pore size membranes were compared by measuring thrombin formation. Results indicated that PMP were not present in the fluid phase after whole blood contact with either of the alumina membranes. However, scanning electron microscope micrographs clearly showed the presence of PMP clusters on the 200 nm pore size alumina, while PMP were practically absent on the 20 nm membrane. We probed no influence of complement activation in PMP generation and adhesion and we hypothesize that other specific material-related protein-platelet interactions are taking place. A clear difference in procoagulant activity between the membranes could also be seen, 20 nm alumina showed 100% higher procoagulant activity than 200 nm membrane. By combining surface evaluation and flow cytometry analyses of the fluid phase, we are able to conclude that 200 nm pore size alumina promotes PMP generation and adhesion while the 20 nm membrane does not appreciably cause any release or adhesion of PMP, thus indicating a direct connection between PMP generation and nanoporosity.

Nanoporesize affects complement activation.

In the present study, we have shown the vast importance of biomaterial nanotexture when evaluating inflammatory response. For the first time in an in vitro whole blood system, we have proven that a small increase in nanoporesize, specifically 180 nm (from 20 to 200 nm), has a huge effect on the complement system. The study was done using nanoporous aluminiumoxide, a material that previously has been evaluated for potential implant use, showing good biocompatibility. This material can easily be manufactured with different pore sizes making it an excellent candidate to govern specific protein and cellular events at the tissue-material interface. We performed whole blood studies, looking at complement activation after blood contact with two pore size alumina membranes (pore diameters, 20 and 200 nm). The fluid phase was analyzed for complement soluble components, C3a and sC5b-9. In addition, surface adsorbed proteins were eluted and dot blots were performed to detect IgG, IgM, C1q, and C3. All results point to the fact that 200 nm pore size membranes are more complement activating. Significantly, higher values of complement soluble components were found after whole blood contact with 200 nm alumina and all studied proteins adsorbed more readily to this membrane than to the 20 nm pore size membrane. We hypothesize that the difference in complement activation between our two test materials is caused by the type and the amount of adsorbed proteins, as well as their conformation and orientation. The different protein patterns created on the two alumina membranes are most likely a consequence of the material topography.