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.

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.

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.

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.

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.

Compressive mechanical properties and cytocompatibility of bone-compliant, linoleic acid-modified bone cement in a bovine model.

Adjacent vertebral fractures are a common complication experienced by osteoporosis patients shortly after vertebroplasty. Whether these fractures are due to the bone cement properties, the cement filling characteristics or to the natural course of the disease is still unclear. However, some data suggests that such fractures might occur because of an imbalance in the load distribution due to a mismatch between the elastic modulus (E) of the bone-cement composite, and that of the vertebral cancellous bone. In this study, the properties of bone-compliant linoleic acid-modified bone cements were assessed using a bovine vertebroplasty model. Two groups of specimens (cement-only and bone-cement composites), and four subgroups comprising bone cements with elastic moduli in the range of 870-3500MPa were tested to failure in uniaxial compression. In addition, monomer release as well as time and concentration-dependent cytocompatibility was assessed through the cement extracts using a Saos-2 cell model. Composites augmented with bone-compliant cements exhibited a reduction in E despite their relatively high bone volume fraction (BVF). Moreover, a significant positive correlation between the BVF and the E for the composites augmented with 870MPa modulus cements was found. This was attributed to the increased relative contribution of the bone to the mechanical properties of the composites with a decrease in E of the bone cement. The use of linoleic acid reduced monomer conversion resulting in six times more monomer released after 24h. However, the cytocompatibility of the bone-compliant cements was comparable to that of the unmodified cements after the extracts were diluted four times. This study represents an important step towards introducing viable bone-compliant bone cements into vertebroplasty practice.

Sustained release of simvastatin from premixed injectable calcium phosphate cement.

Locally applied simvastatin is known to promote bone regeneration; however, the lack of suitable delivery systems has restricted its clinical use. In this study we demonstrate for the first time the use of premixed acidic calcium phosphate cement (CPC) as a delivery system for water-solubilized simvastatin. Freeze-dried simvastatin ß-hydroxy acid (SVA) was added to the premixed cement paste in four different doses (1, 0.5, 0.25, and 0 mg SVA/g cement). The addition of the drug did not alter the cement setting time (38 min), compression strength (5.54 MPa), or diametral tensile strength (2.62 MPa). In a release study conducted in phosphate buffered saline at 37°C, a diffusion-controlled release was observed for over a week. Furthermore, the osteogenic effect of the released SVA was demonstrated in vitro. Cell proliferation, alkaline phosphatase activity, and mineralization were assayed after incubation with cement extracts. The lower doses of SVA (0.5 and 0.25 mg SVA/g cement) showed an approximately fourfold increase in mineralization as compared to the control. In conclusion, our findings suggest that premixed acidic CPC is a good option for local delivery of SVA, due to its ability of slowly releasing the drug, leading to a prolonged stimulation of osteogenesis.

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.

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.

Exploring a synthetic organoselenium compound for antioxidant pharmacotherapy--toxicity and effects on ROS-production.

The organoselenium antioxidant 1 was previously found to act as a catalytic antioxidant in a two-phase lipid peroxidation system. In aqueous environment, selenide 1 quenched ABTS-radicals more efficiently than Trolox and ascorbic acid. The selenide dose-dependently scavenged reactive oxygen and nitrogen species more efficiently than Trolox for neutrophils and PMA-stimulated macrophages, with 50% inhibitory concentrations in the low micromolar range. In addition no sign of toxicity or effect on cell viability was seen when culturing five human cell lines in concentrations up to 200 microM of selenide 1 for up to seven days. We therefore feel that the compound would be a good candidate for future drug development for prevention or treatment of disorders caused by or involving free radical-mediated or oxidative tissue damage.

Nanoporosity of alumina surfaces induces different patterns of activation in adhering monocytes/macrophages.

The present study shows that alumina nanotopography affects monocyte/macrophage behavior. Human mononuclear cells cultured on alumina membranes with pore diameters of 20 and 200 nm were evaluated in terms of cell adhesion, viability, morphology, and release of proinflammatory cytokines. After 24 hours, cell adhesion was assessed by means of light microscopy and cell viability by measuring LDH release. The inflammatory response was evaluated by quantifying interleukin-1ß and tumour necrosis factor-α. Finally, scanning electron microscopy was used to study cell morphology. Results showed pronounced differences in cell number, morphology, and cytokine release depending on the nanoporosity. Few but highly activated cells were found on the 200 nm porous alumina, while relatively larger number of cells were found on the 20 nm porous surface. However, despite their larger number, the cells adhering on the 20 nm surface exhibited reduced pro-inflammatory activity. The data of this paper implies that nanotopography could be exploited for controlling the inflammatory response to implants.