Surface modification of ultrafine-grained titanium: Influence on mechanical properties, cytocompatibility, and osseointegration potential.

OBJECTIVE: The main objective of this study was to demonstrate that dental implants made from ultrafine-grain titanium (UFG-Ti) can be created that replicate state of the art surfaces of standard coarse-grain titanium (Ti), showing excellent cytocompatibility and osseointegration potential while also providing improved mechanical properties. MATERIAL AND METHODS: UFG-Ti was prepared by continuous equal channel angular processing (ECAP), and surfaces were treated by sandblasting and acid etching. Mechanical properties (tensile and fatigue strength), wettability, and roughness parameters were evaluated. Human trabecular bone-derived osteoblast precursor cells (HBCs) were cultured on all samples to examine cytocompatibility and mineralization after 4 and 28 days, respectively. Biomechanical pull-out measurements were performed in a rabbit in vivo model 4 weeks after implantation. RESULTS: Both yield and tensile strength as well as fatigue endurance were higher for UFG-Ti compared to Ti by 40%, 45%, and 34%, respectively. Fatigue endurance was slightly reduced following surface treatment. Existing surface treatment protocols could be applied to UFG-Ti and resulted in similar roughness and wettability as for standard Ti. Cell attachment and spreading were comparable on all samples, but mineralization was higher for the surfaces with hydrophilic treatment with no significant difference between UFG-Ti and Ti. Pull-out tests revealed that osseointegration of surface-treated UFG-Ti was found to be similar to that of surface-treated Ti. CONCLUSION: It could be demonstrated that existing surface treatments for Ti can be translated to UFG-Ti and, furthermore, that dental implants made from surface-treated UFG-Ti exhibit superior mechanical properties while maintaining cytocompatibility and osseointegration potential.

Novel antiviral properties of azithromycin in cystic fibrosis airway epithelial cells.

Virus-associated pulmonary exacerbations, often associated with rhinoviruses (RVs), contribute to cystic fibrosis (CF) morbidity. Currently, there are only a few therapeutic options to treat virus-induced CF pulmonary exacerbations. The macrolide antibiotic azithromycin has antiviral properties in human bronchial epithelial cells. We investigated the potential of azithromycin to induce antiviral mechanisms in CF bronchial epithelial cells. Primary bronchial epithelial cells from CF and control children were infected with RV after azithromycin pre-treatment. Viral RNA, interferon (IFN), IFN-stimulated gene and pattern recognition receptor expression were measured by real-time quantitative PCR. Live virus shedding was assessed by assaying the 50% tissue culture infective dose. Pro-inflammatory cytokine and IFN-ß production were evaluated by ELISA. Cell death was investigated by flow cytometry. RV replication was increased in CF compared with control cells. Azithromycin reduced RV replication seven-fold in CF cells without inducing cell death. Furthermore, azithromycin increased RV-induced pattern recognition receptor, IFN and IFN-stimulated gene mRNA levels. While stimulating antiviral responses, azithromycin did not prevent virus-induced pro-inflammatory responses. Azithromycin pre-treatment reduces RV replication in CF bronchial epithelial cells, possibly through the amplification of the antiviral response mediated by the IFN pathway. Clinical studies are needed to elucidate the potential of azithromycin in the management and prevention of RV-induced CF pulmonary exacerbations.

Comparison of three different brushing techniques to isolate and culture primary nasal epithelial cells from human subjects.

PURPOSE: Primary nasal epithelial cells are used for diagnostic purposes in clinical routine and have been shown to be good surrogate models for bronchial epithelial cells in studies of airway inflammation and remodeling. We aimed at comparing different instruments allowing isolation of nasal epithelial cells. METHODS: Primary airway epithelial cell cultures were established using cells acquired from the inferior surface of the middle turbinate of both nostrils. Three different instruments to isolate nasal cells were used: homemade cytology brush, nasal swab, and curette. Cell count, viability, time until a confluent cell layer was reached, and success rate in establishing cell cultures were evaluated. A standard numeric pain intensity scale was used to assess the acceptability of each instrument. RESULTS: Sixty healthy adults (median with interquartile range [IQR] age of 31 [26-37] years) participated in the study. Higher number of cells (×10(5) cells/ml) was obtained using brushes (9.8 [5.9-33.5]) compared to swabs (2.4 [1.5-3.9], p < 0.0001) and curettes (5.5 [4.4-6.9], p < 0.01). Cell viability was similar between groups. Cells obtained by brushes had the fastest growth rate, and the success rate in establishing primary cell cultures was highest with brushes (90% vs. 65% for swabs and 70% for curettes). Pain was highest with curettes (VAS score 4.0 [3.0-5.0] out of 10). The epithelial phenotype of the cultures was confirmed through cytokeratin and E-cadherin staining. CONCLUSIONS: All three types of instruments allow collection and growth of human nasal epithelial cells with good acceptability to study participants. The most efficient instrument is the nasal brush.

Impaired type I and type III interferon induction and rhinovirus control in human cystic fibrosis airway epithelial cells.

BACKGROUND: Rhinoviruses are important triggers of pulmonary exacerbations and possible contributors to long-term respiratory morbidity in cystic fibrosis (CF), but mechanisms leading to rhinovirus-induced CF exacerbations are poorly understood. It is hypothesised that there is a deficient innate immune response of the airway epithelium towards rhinovirus infection in CF. METHODS: Early innate immune responses towards rhinoviruses (RV-16, major-type and RV-1B, minor-type) in CF and non-CF bronchial epithelial cell lines and primary nasal and bronchial epithelial cells from patients with CF (n=13) and healthy controls (n=24) were studied. RESULTS: Rhinovirus RNA expression and virus release into supernatants was increased more than tenfold in CF cells compared with controls. CF cells expressed up to 1000 times less interferon (IFN) type I (ß) and type III (λ) mRNA and produced less than half of IFN-ß and IFN-λ protein compared with controls. In contrast, interleukin 8 production was not impaired, indicating a selective deficiency in the innate antiviral defence system. Deficient IFN production was paralleled by lower expression of IFN-stimulated genes including myxovirus resistance A, 2',5'-oligoadenylate synthetase, viperin and nitric oxide synthase 2. Addition of exogenous type I and III IFNs, particularly IFN-ß, restored antiviral pathways and virus control in CF cells, underscoring the crucial role of these molecules. CONCLUSIONS: This study describes a novel mechanism to explain the increased susceptibility of patients with CF to rhinovirus infections. A profound impairment of the antiviral early innate response in CF airway epithelial cells was identified, suggesting a potential use of IFNs in the treatment of rhinovirus-induced CF exacerbations.

Pharmacological, but not genetic, disruptions in 5-HT(2C) receptor function attenuate LPS anorexia in mice.

Peripheral administration of bacterial lipopolysaccharide (LPS) elicits anorexia in several species, including rats and mice. There is strong evidence that antagonism of serotonergic activity at 2C receptors (5-HT(2C)R) attenuates LPS anorexia in rats. Here we used pharmacological and genetic approaches to examine the role of the 5-HT(2C)R in LPS anorexia in mice. In Experiment 1, SB 242084, a potent and selective 5-HT(2C) antagonist (0.3 mg/kg) was injected intraperitoneally 15 min before intraperitoneal LPS (2 microg/kg) injections just prior to dark onset in c57BL/6 mice. Food intake was recorded 1, 2 and 4 h after LPS administration. In Experiment 2, we recorded 2, 4 and 24 h food intake following dark onset intraperitoneal LPS (0.125, 0.25, 0.5, 1 and 2 microg/kg) injections in mice with a genetic deletion of 5-HT(2C)R and their WT controls. Our pharmacological results suggest that at least part of the anorexia following peripheral LPS administration is mediated by an increase in 5-HT-ergic activity at the 5-HT(2C)R. Our genetic data, in contrast, suggest that 5-HT(2C)R is not a necessary part of LPS anorexia.

The role of nanostructures and hydrophilicity in osseointegration: In-vitro protein-adsorption and blood-interaction studies.

Protein adsorption and blood coagulation play important roles in the early stages of osseointegration and are strongly influenced by surface properties. We present a systematic investigation of the influence of different surface properties on the adsorption of the blood proteins fibrinogen and fibronectin and the degree of early blood coagulation. Experiments on custom-made and commercially available, microroughened hydrophobic titanium (Ti) surfaces (Ti SLA-Hphob ), hydrophilic (Hphil ) microroughened Ti surfaces with nanostructures (Ti SLActive-Hphil NS), and on bimetallic Ti zirconium alloy (TiZr, Roxolid®) samples were performed, to study the biological response in relation to the surface wettability and the presence of nanostructures (NS). Protein adsorption on the different substrates showed a highly significant effect of surface NS. Hydrophilicity alone did not significantly enhance protein adsorption. Overall, the combination of NS and hydrophilicity led to the highest adsorption levels; independent of whether Ti or TiZr were used. Hydrophilicity induced a strong effect on blood coagulation, whereas the effect of NS alone was weak. The combination of both surface characteristics led to early and most pronounced blood-coagulation. Therefore, nanostructured, hydrophilic Ti and TiZr surfaces may perform better in terms of osseointegration due to continuous protein adsorption and the formation of a layer of blood components on the implant surface.

Enhanced differentiation of human osteoblasts on Ti surfaces pre-treated with human whole blood.

Early and effective integration of a metal implant into bone tissue is of crucial importance for its long-term stability. While different material properties including surface roughness and wettability but also initial blood-implant surface interaction are known to influence this osseointegration, implications of the latter process are still poorly understood. In this study, early interaction between blood and the implant surface and how this affects the mechanism of osseointegration were investigated. For this, blood coagulation on a micro-roughened hydrophobic titanium (Ti) surface (SLA-H(phob)) and on a hydrophilic micro-roughened Ti surface with nanostructures (SLActive-H(phil)NS), as well as the effects of whole human blood pre-incubation of these two surfaces on the differentiation potential of primary human bone cells (HBC) was assessed. Interestingly, pre-incubation with blood resulted in a dense fibrin network over the entire surface on SLActive-H(phil)NS but only in single patches of fibrin and small isolated fibre complexes on SLA-H(phob). On SLActive-H(phil)NS, the number of HBCs attaching to the fibrin network was greatly increased and the cells displayed enhanced cell contact to the fibrin network. Notably, HBCs displayed increased expression of the osteogenic marker proteins alkaline phosphatase and collagen-I when cultivated on both surfaces upon blood pre-incubation. Additionally, blood pre-treatment promoted an earlier and enhanced mineralization of HBCs cultivated on SLActive-H(phil)NS compared to SLA-H(phob). The results presented in this study therefore suggest that blood pre-incubation of implant surfaces mimics a more physiological situation, eventually providing a more predictive in vitro model for the evaluation of novel bone implant surfaces.

Evidence that PGE2 in the dorsal and median raphe nuclei is involved in LPS-induced anorexia in rats.

Anorexia is an element of the acute-phase immune response. Its mechanisms remain poorly understood. Activation of inducible cyclooxygenase-2 (COX-2) in blood-brain-barrier endothelial cells and subsequent release of prostaglandins (e.g., prostaglandin E2, PGE2) may be involved. Therefore, we sought to relate the effects of prostaglandins on the anorexia following gram-negative bacterial lipopolysaccharide treatment (LPS) to neural activity in the dorsal and median raphe nuclei (DRN and MnR) in rats. COX-2 antagonist (NS-398, 10mg/kg; IP) administration prior to LPS (100µg/kg; IP) prevented anorexia and reduced c-Fos expression the DRN, MnR, nucleus tractus solitarii and several related forebrain areas. These data indicate that COX-2-mediated prostaglandin synthesis is necessary for LPS anorexia and much of the initial LPS-induced neural activation. Injection of NS-398 into the DRN and MnR (1ng/site) attenuated LPS-induced anorexia to nearly the same extent as IP NS-398, suggesting that prostaglandin signaling in these areas is necessary for LPS anorexia. Because the DRN and MnR are sources of major serotonergic projections to the forebrain, these data suggest that serotonergic neurons originating in the midbrain raphe play an important role in acute-phase response anorexia.

Serotonin 2C receptor signaling in a diffuse neuronal network is necessary for LPS anorexia.

LPS, a potent activator of the innate immune system, is commonly used to investigate the acute phase response to infection, including anorexia. Serotonin 2C-receptor signaling has been shown to be involved in the mediation of LPS anorexia. Here we used the selective, potent and brain-penetrant serotonin 2C-receptor antagonist SB 242084 to identify the brain sites involved in LPS anorexia. Male Long-Evans rats received 1 ml/kg intraperitoneal injections of 0 or 0.3 mg/kg SB 242084 and intraperitoneal injections of 0 or 100 microg/kg LPS 1 h later, at dark onset. Food intake was measured in one set of rats and c-Fos immunoreactivity in another, unfed, group 90 min after LPS injection. SB 242084 markedly attenuated the LPS-induced reduction in food intake, with no anorexia evident for the first 2 h after LPS. SB 242084 also completely blocked the LPS-induced increases in c-Fos expression in the paraventricular nucleus, central nucleus of the amygdala, nucleus tractus solitarii, median raphe nucleus and dorsal raphe nucleus and partially blocked it in the A1 noradrenergic area of the ventrolateral medulla and the raphe pallidus nucleus. SB 242084 did not significantly alter the c-Fos response in the arcuate nucleus or the raphe magnus nucleus. These data indicate that 2C receptor signaling activates a diffuse neural network, presumably mediating anorexia and other responses to LPS; they also suggest that the arcuate and the raphe magnus neurons that express c-Fos after LPS are not necessary for LPS anorexia.