Characteristics of in vitro infection of human monocytes, by Rickettsia helvetica.

Eighteen species of rickettsiae are reported to cause infections in humans. One of these is Rickettsia helvetica, which is endemic in European and Asian countries and transmitted by the tick Ixodes ricinus. Besides fever, it has been demonstrated to cause meningitis and is also associated with perimyocarditis. One of the initial targets for rickettsiae after inoculation by ticks is the macrophage/monocyte. How rickettsiae remain in the macrophages/monocytes before establishing their infection in vascular endothelial cells remains poorly understood. The main aim of the present study was to investigate the impact on and survival of R. helvetica in a human leukemic monocytic cell line, THP-1. Our results show that R. helvetica survives and propagates in the THP-1 cells. The infection in monocytes was followed for seven days by qPCR and for 30 days by TEM, where invasion of the nucleus was also observed as well as double membrane vacuoles containing rickettsiae, a finding suggesting that R. helvetica might induce autophagy at the early stage of infection. Infected monocytes induced TNF-α which may be important in host defence against rickettsial infections and promote cell survival and inhibiting cell death by apoptosis. The present findings illustrate the importance of monocytes to the pathogenesis of rickettsial disease.

Titanium surface modification to enhance antibacterial and bioactive properties while retaining biocompatibility.

Bacterial infections associated with metal implants are severe problems affecting a considerable amount of people with dental or orthopedic implants. This study aims to examine the antibacterial effect of a Titanium-peroxy gel layer on the modified surface of commercially pure titanium grade 2. Variations in a multi-step surface modification procedure were tested to determine the best combination that provided an antibacterial effect while enhancing bioactivity without compromising biocompatibility. Soaking the surfaces in 30 wt% hydrogen peroxide held at 80 °C provided antibacterial activity while subsequent surface treatments in concentrated sodium and calcium hydroxide solutions were preformed to enhance bioactivity. Staphylococcus epidermidis was used to determine the antibacterial effect through both direct contact and biofilm inhibition tests while human dermal fibroblast cells and MC3T3 pre osteoblast cells were utilized to test biocompatibility. The greatest antibacterial effect was observed with only hydrogen peroxide treatment, but the resulting surface was neither bioactive nor biocompatible. It was found that subsequent surface treatments with sodium hydroxide followed by calcium hydroxide provided a bioactive surface that was also biocompatible. Additionally, a final treatment with autoclaving showed positive effects with regards to enhanced bioactivity. This multi-step surface modification procedure offers a promising, non-antibiotic, solution for combatting infections associated with biomedical implants.

The Influence of Hydroxyapatite Nanoparticle Morphology on Embryonic Development in a Zebrafish Exposure Model.

Nanomaterials are used in many different industries such as cosmetics, food, clothing, and electronics. There is increasing concern that exposure to nanoparticles (NPs) during pregnancy can adversely affect fetal development. It is well known that the size, charge, and chemistry of a nanoparticle can modulate embryological development. The role that particle morphology plays on early development, however, is still widely unknown. The present study aims to investigate the effect of hydroxyapatite nanoparticle (HANP) morphology on embryological development in a zebrafish exposure model. Four distinct HANP morphologies (dots, long rods, sheets, and fibers) were fabricated and characterized. Zebrafish embryos were exposed to HANPs (0-100 mg/L), and viability and developmental deformities were evaluated for up to 5 days post-fertilization (dpf). Malformations such as pericardial edema and axial curvature were apparent in embryos as early as 1 dpf, following exposure to the dot and fiber particles, and developed in embryos by 3 dpf in the sheet and long rod particle groups. Minimal death was observed in response to dot, long rod, and sheet particles (≤25%), while fiber particles induced overwhelming toxicity (≤60%) after 1 dpf, and complete toxicity during all subsequent time points. Collectively, these results suggest that nanoparticle morphology can significantly impact embryological development and should be a required consideration when designing nanomaterials for commercial use.

Photophysics of Coumarin and Carbostyril-Sensitized Luminescent Lanthanide Complexes: Implications for Complex Design in Multiplex Detection.

Luminescent lanthanide (Ln(III)) complexes with coumarin or carbostyril antennae were synthesized and their photophysical properties evaluated using steady-state and time-resolved UV-vis spectroscopy. Ligands bearing distant hydroxycoumarin-derived antennae attached through triazole linkers were modest sensitizers for Eu(III) and Tb(III), whereas ligands with 7-amidocarbostyrils directly linked to the coordination site could reach good quantum yields for multiple Ln(III), including the visible emitters Sm(III) and Dy(III), and the near-infrared emitters Nd(III) and Yb(III). The highest lanthanide-centered luminescence quantum yields were 35% (Tb), 7.9% (Eu), 0.67% (Dy), and 0.18% (Sm). Antennae providing similar luminescence intensities with 2-4 Ln-emitters were identified. Photoredox quenching of the carbostyril antenna excited states was observed for all Eu(III)-complexes and should be sensitizing in the case of Yb(III); the scope of the process extends to Ln(III) for which it has not been seen previously, specifically Dy(III) and Sm(III). The proposed process is supported by photophysical and electrochemical data. A FRET-type mechanism was identified in architectures with both distant and close antennae for all of the Lns. This mechanism seems to be the only sensitizing one at long distance and probably contributes to the sensitization at shorter distances along with the triplet pathway. The complexes were nontoxic to either bacterial or mammalian cells. Complexes of an ester-functionalized ligand were taken up by bacteria in a concentration-dependent manner. Our results suggest that the effects of FRET and photoredox quenching should be taken into consideration when designing luminescent Ln complexes. These results also establish these Ln(III)-complexes for multiplex detection beyond the available two-color systems.

Selenium- and Tellurium-Based Antioxidants for Modulating Inflammation and Effects on Osteoblastic Activity.

Increased oxidative stress plays a significant role in the etiology of bone diseases. Heightened levels of H2O2 disrupt bone homeostasis, leading to greater bone resorption than bone formation. Organochalcogen compounds could act as free radical trapping agents or glutathione peroxidase mimetics, reducing oxidative stress in inflammatory diseases. In this report, we synthesized and screened a library of organoselenium and organotellurium compounds for hydrogen peroxide scavenging activity, using macrophagic cell lines RAW264.7 and THP-1, as well as human mono- and poly-nuclear cells. These cells were stimulated to release H2O2, using phorbol 12-myristate 13-acetate, with and without organochalogens. Released H2O2 was then measured using a chemiluminescent assay over a period of 2 h. The screening identified an organoselenium compound which scavenged H2O2 more effectively than the vitamin E analog, Trolox. We also found that this organoselenium compound protected MC3T3 cells against H2O2-induced toxicity, whereas Trolox did not. The organoselenium compound exhibited no cytotoxicity to the cells and had no deleterious effects on cell proliferation, viability, or alkaline phosphatase activity. The rapidity of H2O2 scavenging and protection suggests that the mechanism of protection is due to the direct scavenging of extracellular H2O2. This compound is a promising modulators of inflammation and could potentially treat diseases involving high levels of oxidative stress.

Nitro-, Azo-, and Amino Derivatives of Ebselen: Synthesis, Structure, and Cytoprotective Effects.

Novel azo-bis-ebselen compounds 7 were prepared by reduction of 7-nitro-2-aryl-1,2-benzisoselenazol-3(2H)-ones 3 and 6 with sodium benzenetellurolate, NaTeC6H5, and by reaction of 2-bromo-3-nitrobenzamides with Na2Se2. The X-ray structure of 7b showed that the molecule, due to strong intramolecular secondary Se···N interactions, is completely planar. Azo-compounds 7 upon further reaction with NaTeC6H5 were reductively cleaved to provide 2 equiv of the corresponding aromatic amine. The weak Se-N bond was not stable enough to survive the reaction conditions, and diselenides 8 were isolated after workup. Whereas azo-bis-ebselens 7 were poor mimics of the glutathione peroxidase (GPx)-enzymes, nitroebselens 3, 6, and 11b and diselenides 8 were 3-6-fold more active than ebselen. Based on 77Se NMR spectroscopy, a catalytic cycle for diselenide 8b, involving aminoebselen 14, was proposed. As assessed by chemiluminescence measurements, the good GPx-mimics could reduce production of reactive oxygen species (ROS) in stimulated human mononuclear cells more efficiently than Trolox. No toxic effects of the compounds were seen in MC3T3-cells at 25 µM.

Pyrophosphate Stimulates Differentiation, Matrix Gene Expression and Alkaline Phosphatase Activity in Osteoblasts.

Pyrophosphate is a potent mitogen, capable of stimulating proliferation in multiple cell types, and a critical participant in bone mineralization. Pyrophosphate can also affect the resorption rate and bioactivity of orthopedic ceramics. The present study investigated whether calcium pyrophosphate affected proliferation, differentiation and gene expression in early (MC3T3 pre-osteoblast) and late stage (SAOS-2 osteosarcoma) osteoblasts. Pyrophosphate stimulated peak alkaline phosphatase activity by 50% and 150% at 100µM and 0.1µM in MC3T3, and by 40% in SAOS-2. The expression of differentiation markers collagen 1 (COL1), alkaline phosphatase (ALP), osteopontin (OPN), and osteocalcin (OCN) were increased by an average of 1.5, 2, 2 and 3 fold, by high concentrations of sodium pyrophosphate (100µM) after 7 days of exposure in MC3T3. COX-2 and ANK expression did not differ significantly from controls in either treatment group. Though both high and low concentrations of pyrophosphate stimulate ALP activity, only high concentrations (100µM) stimulated osteogenic gene expression. Pyrophosphate did not affect proliferation in either cell type. The results of this study confirm that chronic exposure to pyrophosphate exerts a physiological effect upon osteoblast differentiation and ALP activity, specifically by stimulating osteoblast differentiation markers and extracellular matrix gene expression.

In vivo and in vitro evaluation of hydroxyapatite nanoparticle morphology on the acute inflammatory response.

Biomedical implants have been widely used in bone repair applications. However, nanosized degradation products from these implants could elicit an inflammatory reaction, which may lead to implant failure. It is well known that the size, chemistry, and charge of these nanoparticles can modulate this response, but little is known regarding the role that the particle's morphology plays in inducing inflammation. The present study aims to investigate the effect of hydroxyapatite nanoparticle (HANPs) morphology on inflammation, in-vitro and in-vivo. Four distinct HANP morphologies were fabricated and characterized: long rods, dots, sheets, and fibers. Primary human polymorphonuclear cells (PMNCs), mononuclear cells (MNCs), and human dermal fibroblasts (hDFs) were exposed to HANPs and alterations in cell viability, morphology, apoptotic activity, and reactive oxygen species (ROS) production were evaluated, in vitro. PMNCs and hDFs experienced a 2-fold decrease in viability following exposure to fibers, while MNC viability decreased 5-fold after treatment with the dots. Additionally, the fibers stimulated an elevated ROS response in both PMNCs and MNCs, and the largest apoptotic behavior for all cell types. Furthermore, exposure to fibers and dots resulted in greater capsule thickness when implanted subcutaneously in mice. Collectively, these results suggest that nanoparticle morphology can significantly impact the inflammatory response.

Multifunctional Antioxidants: Regenerable Radical-Trapping and Hydroperoxide-Decomposing Ebselenols.

Regenerable, multifunctional ebselenol antioxidants were prepared that could quench peroxyl radicals more efficiently than α-tocopherol. These compounds act as better mimics of the glutathione peroxidase enzymes than ebselen. Production of reactive oxygen species (ROS) and reactive nitrogen species (RNS) in human mononuclear cells was considerably decreased upon exposure to the organoselenium compounds. At a concentration of 25 µm, the ebselenol derivatives showed minimal toxicity in pre-osteoblast MC3T3 cells.

The effect of oligo(trimethylene carbonate) addition on the stiffness of acrylic bone cement.

With the increasing elderly population an increase in the number of bony fractures associated to age-related diseases such as osteoporosis also follows. The relatively high stiffness of the acrylic bone cements used in these patients has been suggested to give raise to a suboptimal load distribution surrounding the cement in vivo, and hence contribute to clinical complications, such as additional fractures. The aim of this study was to develop a low-modulus bone cement, based on currently used, commercially available poly(methyl methacrylate) (PMMA) cements for vertebroplasty. To this end, acrylate end-functionalized oligo(trimethylene carbonate) (oTMC) was incorporated into the cements, and the resulting compressive mechanical properties were evaluated, as well as the cytotoxic and handling properties of selected formulations. Sixteen wt%oTMC was needed in the vertebroplastic cement Osteopal V to achieve an elastic modulus of 1063 MPa (SD 74), which gave a corresponding compressive strength of 46.1 MPa (SD 1.9). Cement extracts taken at 1 and 12 hours gave a reduced MG-63 cell viability in most cases, while extracts taken at 24 hours had no significant effect on cell behavior. The modification also gave an increase in setting time, from 14.7 min (SD 1.7) to 18.0 min (SD 0.9), and a decrease in maximum polymerization temperature, from 41.5°C (SD 3.4) to 30.7°C (SD 1.4). While further evaluation of other relevant properties, such as injectability and in vivo biocompatibility, remains to be done, the results presented herein are promising in terms of approaching clinically applicable bone cements with a lower stiffness.

Changes in the drug release pattern of fresh and set simvastatin-loaded brushite cement.

Calcium phosphate cements are synthetic bone graft substitutes able to set at physiological conditions. They can be applied by minimally invasive surgery and can also be used as drug delivery systems. Consequently, the drug release pattern from the cement paste (fresh cement) is of high clinical interest. However, previous studies have commonly evaluated the drug release using pre-set cements only. Therefore, the aim of this work was to determine if the time elapsed from cement preparation until immersion in the solution (3 min for fresh cements, and 1h and 15 h for pre-set cements) had an influence on its physical properties, and correlating these to the drug release profile. Simvastatin was selected as a model drug, while brushite cement was used as drug carrier. This study quantified how the setting of a material reduces the accessibility of the release media to the material, thus preventing drug release. A shift in the drug release pattern was observed, from a burst-release for fresh cements to a sustained release for pre-set cements.

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.

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.

Evaluation of Biocompatibility and Release of Reactive Oxygen Species of Aluminum Oxide-Coated Materials.

Surface properties of biomaterials can strongly influence biomaterial-host interactions. For this reason, coating processes open a wide range of possibilities to modulate the fate of a biomaterial in the body. This study evaluates the effect of a coating material intended for drug delivery capsules on biocompatibility and the release of reactive oxygen species (ROS), that is, respiratory burst in macrophages that indicates acute inflammation. In parallel with a new approach to develop drug-delivery capsules by directly coating solid-state drug particles, in this study, glass slides and silicon nanoparticles (NPs) were coated with aluminum oxide (Al2O3) using atomic layer deposition. Different sizes of NPs (20 and 310 nm) were suspended at different concentrations (10, 100, and 1000 µg/mL) and were evaluated. The homogeneous coating of slides was proved using X-ray photoelectron spectroscopy, and the coating on NP was observed using transmission electron microscopy. Human dermal fibroblasts and human osteoblasts were able to proliferate on the coated slides and in the presence of a suspension of coated NPs (20 and 310 nm) at a low concentration (10 µg/mL). The macrophages released ROS only when in contact with NPs at a concentration of 1000 µg/mL, where the 20 nm NPs caused a higher release of ROS than the 310 nm NPs. This study shows that Al2O3 coatings do not affect the cells negatively and that the cell viability was compromised only when in contact with a high concentration (1000 µg/mL) of smaller (20 nm) NPs.

In Vitro and In Vivo Response to Low-Modulus PMMA-Based Bone Cement.

The high stiffness of acrylic bone cements has been hypothesized to contribute to the increased number of fractures encountered after vertebroplasty, which has led to the development of low-modulus cements. However, there is no data available on the in vivo biocompatibility of any low-modulus cement. In this study, the in vitro cytotoxicity and in vivo biocompatibility of two types of low-modulus acrylic cements, one modified with castor oil and one with linoleic acid, were evaluated using human osteoblast-like cells and a rodent model, respectively. While the in vitro cytotoxicity appeared somewhat affected by the castor oil and linoleic acid additions, no difference could be found in the in vivo response to these cements in comparison to the base, commercially available cement, in terms of histology and flow cytometry analysis of the presence of immune cells. Furthermore, the in vivo radiopacity of the cements appeared unaltered. While these results are promising, the mechanical behavior of these cements in vivo remains to be investigated.

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.

The influence of Sr content in calcium phosphate coatings.

In this study calcium phosphate coatings with different amounts of strontium (Sr) were prepared using a biomineralization method. The incorporation of Sr changed the composition and morphology of coatings from plate-like to sphere-like morphology. Dissolution testing indicated that the solubility of the coatings increased with increased Sr concentration. Evaluation of extracts (with Sr concentrations ranging from 0 to 2.37 µg/mL) from the HA, 0.06Sr, 0.6Sr, and 1.2Sr coatings during in vitro cell cultures showed that Sr incorporation into coatings significantly enhanced the ALP activity in comparison to cells treated with control and HA eluted media. These findings show that calcium phosphate coatings could promote osteogenic differentiation even in a low amount of strontium.

Rebamipide delivered by brushite cement enhances osteoblast and macrophage proliferation.

Many of the bioactive agents capable of stimulating osseous regeneration, such as bone morphogenetic protein-2 (BMP-2) or prostaglandin E2 (PGE2), are limited by rapid degradation, a short bioactive half-life at the target site in vivo, or are prohibitively expensive to obtain in large quantities. Rebamipide, an amino acid modified hydroxylquinoline, can alter the expression of key mediators of bone anabolism, cyclo-oxygenase 2 (COX-2), BMP-2 and vascular endothelial growth factor (VEGF), in diverse cell types such as mucosal and endothelial cells or chondrocytes. The present study investigates whether Rebamipide enhances proliferation and differentiation of osteoblasts when delivered from brushite cement. The reactive oxygen species (ROS) quenching ability of Rebampide was tested in macrophages as a measure of bioactivity following drug release incubation times, up to 14 days. Rebamipide release from brushite occurs via non-fickian diffusion, with a rapid linear release of 9.70% ± 0.37% of drug per day for the first 5 days, and an average of 0.5%-1% per day thereafter for 30 days. Rebamipide slows the initial and final cement setting time by up to 3 and 1 minute, respectively, but does not significantly reduce the mechanical strength below 4% (weight percentage). Pre-osteoblast proliferation increases by 24% upon exposure to 0.4 uM Rebamipide, and by up to 73% when Rebamipide is delivered via brushite cement. Low doses of Rebamipide do not adversely affect peak alkaline phosphatase activity in differentiating pre-osteoblasts. Rebamipide weakly stimulates proliferation in macrophages at low concentrations (118 ± 7.4% at 1 uM), and quenches ROS by 40-60%. This is the first investigation of Rebamipide in osteoblasts.

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.

Scavenging effect of Trolox released from brushite cements.

In this study a brushite cement was doped with the chain-breaking antioxidant Trolox. The effect of the antioxidant on the physical properties of the cement was evaluated and the release of Trolox was monitored by UV spectroscopy. The ability of the Trolox set free to scavenge reactive oxygen species (ROS) released by macrophages was determined in vitro using a luminol-amplified chemiluminescence assay. Trolox did not modify the crystalline phases of the set cement, which mainly formed crystalline brushite after 7 days in humid conditions. The setting time, compressive strength and morphology of the cement also remained unaltered after the addition of the antioxidant. Trolox was slowly released from the cement following a non-Fickian transport mechanism and nearly 64% of the total amount was released after 3 days. Moreover, the capacity of Trolox to scavenge the ROS released by macrophages increased in a dose-dependent manner. Trolox-loaded cements are expected to reduce some of the first harmful effects of acute inflammation and can thus potentially protect the surrounding tissue during implantation of these as well as other materials used in conjunction.