Cerium oxide nanoparticle uptake kinetics from the gas-phase into lung cells in vitro is transport limited

Nowadays, aerosol processes are widely used for the manufacture of nanoparticles (NPs), creating an increased occupational exposure risk of workers, laboratory personnel and scientists to airborne particles. There is evidence that possible adverse effects are linked with the accumulation of NPs in target cells, pointing out the importance of understanding the kinetics of particle internalization. In this context, the uptake kinetics of representative airborne NPs over 30 min and their internalization after 24 h post-exposure were investigated by the use of a recently established exposure system. This system combines the production of aerosolized cerium oxide (CeO(2)) NPs by flame spray synthesis with its simultaneous particle deposition from the gas-phase onto A549 lung cells, cultivated at the air-liquid interface. Particle uptake was quantified by mass spectrometry after several exposure times (0, 5, 10, 20 and 30 min). Over 35% of the deposited mass was found internalized after 10 min exposure, a value that increased to 60% after 30 min exposure. Following an additional 24 h post-incubation, a time span, after which adverse biological effects were observed in previous experiments, over 80% of total CeO(2) could be detected intracellularly. On the ultrastructural level, focal cerium aggregates were present on the apical surface of A549 cells and could also be localized intracellularly in vesicular structures. The uptake behaviour of aerosolized CeO(2) is in line with observations on cerium suspensions, where particle mass transport was identified as the rate-limiting factor for NP internalization.

Direct combination of nanoparticle fabrication and exposure to lung cell cultures in a closed setup as a method to simulate accidental nanoparticle exposure of humans

The tremendous application potential of nanosized materials stays in sharp contrast to a growing number of critical reports of their potential toxicity. Applications of in vitro methods to assess nanoparticles are severely limited through difficulties in exposing cells of the respiratory tract directly to airborne engineered nanoparticles. We present a completely new approach to expose lung cells to particles generated in situ by flame spray synthesis. Cerium oxide nanoparticles from a single run were produced and simultaneously exposed to the surface of cultured lung cells inside a glovebox. Separately collected samples were used to measure hydrodynamic particle size distribution, shape, and agglomerate morphology. Cell viability was not impaired by the conditions of the glovebox exposure. The tightness of the lung cell monolayer, the mean total lamellar body volume, and the generation of oxidative DNA damage revealed a dose-dependent cellular response to the airborne engineered nanoparticles. The direct combination of production and exposure allows studying particle toxicity in a simple and reproducible way under environmental conditions.

Direct observation of liquid crystals using cryo-TEM: Specimen preparation and low-dose imaging

Liquid crystals (LCs) represent a challenging group of materials for direct transmission electron microscopy (TEM) studies due to the complications in specimen preparation and the severe radiation damage. In this paper, we summarize a series of specimen preparation methods, including thin film and cryo-sectioning approaches, as a comprehensive toolset enabling high-resolution direct cryo-TEM observation of a broad range of LCs. We also present comparative analysis using cryo-TEM and replica freeze-fracture TEM on both thermotropic and lyotropic LCs. In addition to the revisits of previous practices, some new concepts are introduced, e.g., suspended thermotropic LC thin films, combined high-pressure freezing and cryo-sectioning of lyotropic LCs, and the complementary applications of direct TEM and indirect replica TEM techniques. The significance of subnanometer resolution cryo-TEM observation is demonstrated in a few important issues in LC studies, including providing direct evidences for the existence of nanoscale smectic domains in nematic bent-core thermotropic LCs, comprehensive understanding of the twist-bend nematic phase, and probing the packing of columnar aggregates in lyotropic chromonic LCs. Direct TEM observation opens ways to a variety of TEM techniques, suggesting that TEM (replica, cryo, and in situ techniques), in general, may be a promising part of the solution to the lack of effective structural probe at the molecular scale in LC studies. Microsc. Res. Tech. 77:754-772, 2014. © 2014 Wiley Periodicals, Inc.

Airborne-particle Abrasion Is a Minimally Invasive Method to Pre-Treat Sclerotic, Non-Carious Cervical Dentin

Objectives: To investigate substance loss and bond strength capacity of sclerotic, non-carious cervical dentin after airborne-particle abrasion or diamond bur preparation. Methods: Fifteen non-sclerotic dentin specimens were made from crowns of extracted human incisors of which the labial surfaces had been ground with silicon carbide papers (non-sclerotic control; Group 1). Forty-five sclerotic dentin specimens (n=15/group) were made from the labial, non-carious cervical root part of extracted human incisors and underwent either no pre-treatment (sclerotic control; Group 2), pre-treatment with airborne-particle abrasion (CoJet Prep [3M ESPE] and 50 µm aluminium oxide; Group 3), or with diamond bur preparation (40 µm grit size; Group 4). Substance loss after pre-treatment was measured in Groups 3 and 4. Subsequently, Scotchbond Universal (3M ESPE) and resin composite (CeramX [DENTSPLY DeTrey]) were applied on the treated dentin surfaces. The specimens were stored at 37°C and 100% humidity for 24 h. After storage, shear bond strength (SBS) was measured and data analyzed with nonparametric ANOVA followed by Wilcoxon rank sum tests. Results: Substance loss (medians) was 19 µm in Group 3 and 113 µm in Group 4. SBS-values (MPa; medians) in Group 2 (9.24) were significantly lower than in Group 1 (13.15; p=0.0069), Group 3 (13.05; p=0.01), and Group 4 (13.02; p=0.0142). There were no significant differences in SBS between Groups 1, 3, and 4 (p≥0.8063). Conclusion: Airborne-particle abrasion and diamond bur preparation restored bond strength of Scotchbond Universal to sclerotic dentin to the level of non-sclerotic dentin, with airborne-particle abrasion being less invasive than diamond bur preparation.

Leukocyte- and Platelet-Rich Fibrin (L-PRF) for Long-Term Delivery of Growth Factor in Rotator Cuff Repair: Review, Preliminary Results and Future Directions

Surgical repair of the rotator cuff repair is one of the most common procedures in orthopedic surgery. Despite it being the focus of much research, the physiological tendon-bone insertion is not recreated following repair and there is an anatomic non-healing rate of up to 94%. During the healing phase, several growth factors are upregulated that induce cellular proliferation and matrix deposition. Subsequently, this provisional matrix is replaced by the definitive matrix. Leukocyte- and platelet-rich fibrin (L-PRF) contain growth factors and has a stable dense fibrin matrix. Therefore, use of LPRF in rotator cuff repair is theoretically attractive. The aim of the present study was to determine 1) the optimal protocol to achieve the highest leukocyte content; 2) whether L-PRF releases growth factors in a sustained manner over 28 days; 3) whether standard/gelatinous or dry/compressed matrix preparation methods result in higher growth factor concentrations. 1) The standard L-PRF centrifugation protocol with 400 x g showed the highest concentration of platelets and leukocytes. 2) The L-PRF clots cultured in medium showed a continuous slow release with an increase in the absolute release of growth factors TGF-β1, VEGF and MPO in the first 7 days, and for IGF1, PDGF-AB and platelet activity (PF4=CXCL4) in the first 8 hours, followed by a decrease to close to zero at 28 days. Significantly higher levels of growth factor were expressed relative to the control values of normal blood at each culture time point. 3) Except for MPO and the TGFβ-1, there was always a tendency towards higher release of growth factors (i.e., CXCL4, IGF-1, PDGF-AB, and VEGF) in the standard/gelatinous- compared to the dry/compressed group. L-PRF in its optimal standard/gelatinous-type matrix can store and deliver locally specific healing growth factors for up to 28 days and may be a useful adjunct in rotator cuff repair.

Osteo-odonto-keratoprosthesis (OOKP) and the testing of three different adhesives for bonding bovine teeth with optical poly-(methyl methacrylate) (PMMA) cylinder.

AIM Preparation of the lamina during osteo-odonto-keratoprosthesis (OOKP) design is complex, and its longevity and watertightness important. To date, only acrylic bone cements have been used for bonding the optical cylinder to the tooth dentine. Our aim was to evaluate different dental adhesives for OOKP preparation. METHODS Specimens of bovine teeth were produced by preparing 1.5-mm thick dentine slices with holes having a diameter of 3.5 mm. Each group (n=10 per group) was luted with either classic poly-(methyl methacrylate) (PMMA) bone cement, universal resin cement or glass ionomer cement. All specimens underwent force measurement using a uniaxial traction machine. RESULTS The highest mean force required to break the bond was measured for PMMA bone cement (128.2 N) followed by universal resin cement (127.9 N), with no statistically significant difference. Glass ionomer cement showed significantly lower force resistance (78.1 N). CONCLUSIONS Excellent bonding strength combined with easy application was found for universal resin cement, and thus, it is a potential alternative to acrylic bone cement in OOKP preparation.

Clinical results with two different methods of root-end preparation and filling in apical surgery: mineral trioxide aggregate and adhesive resin composite

INTRODUCTION: The aim of apical surgery is to hermetically seal the root canal system after root-end resection, thereby enabling periradicular healing. The objective of this nonrandomized prospective clinical study was to report results of 2 different root-end preparation and filling methods, ie, mineral trioxide aggregate (MTA) and an adhesive resin composite (Retroplast). METHODS: The study included 353 consecutive cases with endodontic lesions limited to the periapical area. Root-end cavities were prepared with sonic microtips and filled with MTA (n = 178), or alternatively, a shallow concavity was prepared in the cut root face, with subsequent placement of an adhesive resin composite (Retroplast) (n = 175). Patients were recalled after 1 year. Cases were defined as healed when no clinical signs or symptoms were present and radiographs demonstrated complete or incomplete (scar tissue) healing of previous radiolucencies. RESULTS: The overall rate of healed cases was 85.5%. MTA-treated teeth demonstrated a significantly (P = .003) higher rate of healed cases (91.3%) compared with Retroplast-treated teeth (79.5%). Within the MTA group, 89.5%-100% of cases were classified as healed, depending on the type of treated tooth. In contrast, more variable rates ranging from 66.7%-100% were found in the Retroplast group. In particular, mandibular premolars and molars demonstrated considerably lower rates of healed cases when treated with Retroplast. CONCLUSIONS: MTA can be recommended for root-end filling in apical surgery, irrespective of the type of treated tooth. Retroplast should be used with caution for root-end sealing in apical surgery of mandibular premolars and molars.

Comparison of different methods for thin section EM analysis of Mycobacterium smegmatis

Bacteria are generally difficult specimens to prepare for conventional resin section electron microscopy and mycobacteria, with their thick and complex cell envelope layers being especially prone to artefacts. Here we made a systematic comparison of different methods for preparing Mycobacterium smegmatis for thin section electron microscopy analysis. These methods were: (1) conventional preparation by fixatives and epoxy resins at ambient temperature. (2) Tokuyasu cryo-section of chemically fixed bacteria. (3) rapid freezing followed by freeze substitution and embedding in epoxy resin at room temperature or (4) combined with Lowicryl HM20 embedding and ultraviolet (UV) polymerization at low temperature and (5) CEMOVIS, or cryo electron microscopy of vitreous sections. The best preservation of bacteria was obtained with the cryo electron microscopy of vitreous sections method, as expected, especially with respect to the preservation of the cell envelope and lipid bodies. By comparison with cryo electron microscopy of vitreous sections both the conventional and Tokuyasu methods produced different, undesirable artefacts. The two different types of freeze-substitution protocols showed variable preservation of the cell envelope but gave acceptable preservation of the cytoplasm, but not lipid bodies, and bacterial DNA. In conclusion although cryo electron microscopy of vitreous sections must be considered the 'gold standard' among sectioning methods for electron microscopy, because it avoids solvents and stains, the use of optimally prepared freeze substitution also offers some advantages for ultrastructural analysis of bacteria.

Comparison of titanium-nitride-oxide-coated stents with zotarolimus-eluting stents for coronary revascularization a randomized controlled trial

Objectives This study sought to compare the efficacy of passive stent coating with titanium-nitride-oxide (TiNO) with drug-eluting stents releasing zotarolimus (ZES) (Endeavor, Medtronic, Minneapolis, Minnesota). Background Stent coating with TiNO has been shown to reduce restenosis compared with bare-metal stents in experimental and clinical studies. Methods In an assessor-blind noninferiority study, 302 patients undergoing percutaneous coronary intervention were randomized to treatment with TiNO or ZES. The primary endpoint was in-stent late loss at 6 to 8 months, and analysis was by intention to treat. Results Both groups were well balanced with respect to baseline clinical and angiographic characteristics. The TiNO group failed to reach the pre-specified noninferiority margin for the primary endpoint (in-stent late loss: 0.64 ± 0.61 mm vs. 0.47 ± 0.48 mm, difference: 0.16, upper 1-sided 95% confidence interval [CI]: 0.26; pnoninferiority = 0.54), and subsequent superiority testing was in favor of ZES (psuperiority = 0.02). In-segment binary restenosis was lower with ZES (11.1%) than with TiNO (20.5%; psuperiority = 0.04). A stratified analysis of the primary endpoint found particularly pronounced differences between stents among diabetic versus nondiabetic patients (0.90 ± 0.69 mm vs. 0.39 ± 0.38 mm; pinteraction = 0.04). Clinical outcomes showed a similar rate of death (0.7% vs. 0.7%; p = 1.00), myocardial infarction (5.3% vs. 6.7%; p = 0.60), and major adverse cardiac events (21.1% vs. 18.0%, hazard ratio: 1.19, 95% CI: 0.71 to 2.00; p = 0.50) at 1 year. There were no differences in rates of definite or probable stent thrombosis (0.7% vs. 0%; p = 0.51) at 1 year. Conclusions Compared with TiNO, ZES was superior with regard to late loss and binary restenosis. The concept of passive stent coating with TiNO remains inferior to drug-eluting stent technology in reducing restenosis. ([TIDE] Randomized Trial Comparing Titan Stent With Zotarolimus-Eluting Stent: NCT00492908)

Interaction Between Nitrous Oxide, Sevoflurane, and Opioids: A Response Surface Approach

BACKGROUND:: The interaction of sevoflurane and opioids can be described by response surface modeling using the hierarchical model. We expanded this for combined administration of sevoflurane, opioids, and 66 vol.% nitrous oxide (N2O), using historical data on the motor and hemodynamic responsiveness to incision, the minimal alveolar concentration, and minimal alveolar concentration to block autonomic reflexes to nociceptive stimuli, respectively. METHODS:: Four potential actions of 66 vol.% N2O were postulated: (1) N2O is equivalent to A ng/ml of fentanyl (additive); (2) N2O reduces C50 of fentanyl by factor B; (3) N2O is equivalent to X vol.% of sevoflurane (additive); (4) N2O reduces C50 of sevoflurane by factor Y. These four actions, and all combinations, were fitted on the data using NONMEM (version VI, Icon Development Solutions, Ellicott City, MD), assuming identical interaction parameters (A, B, X, Y) for movement and sympathetic responses. RESULTS:: Sixty-six volume percentage nitrous oxide evokes an additive effect corresponding to 0.27 ng/ml fentanyl (A) with an additive effect corresponding to 0.54 vol.% sevoflurane (X). Parameters B and Y did not improve the fit. CONCLUSION:: The effect of nitrous oxide can be incorporated into the hierarchical interaction model with a simple extension. The model can be used to predict the probability of movement and sympathetic responses during sevoflurane anesthesia taking into account interactions with opioids and 66 vol.% N2O.

A new microbiopsy system enables rapid preparation of tissue for high-pressure freezing

A microbiopsy system was developed to overcome long sampling times for tissues before they are cryo-fixed by high-pressure freezing. A commercially available biopsy gun was adapted to the needs of small-organ excisions, and biopsy needles were modified to allow small samples (0.6 mm x 1.2 mm x 0.3 mm) to be taken. Specimen platelets with a central slot of the same dimensions as the biopsy are used. A self-made transfer device (in the meantime optimized by Leica-Microsystems [Vienna, Austria]) coordinates the transfer of the excised sample from the biopsy needle into the platelet slot and the subsequent loading in a specimen holder, which is then introduced into a high-pressure freezer (Leica EM PACT; Leica Microsystems, Vienna, Austria). Thirty seconds preparation time is needed from excision until high-pressure freezing. Brain, liver, kidney and muscle excisions of anesthetised rats are shown to be well frozen.

Elevated exhaled nitric oxide in newborns of atopic mothers precedes respiratory symptoms

RATIONALE: Exhaled nitric oxide (NO) is a well-known marker of established airway inflammation in asthma. Its role in the disease process before the onset of respiratory symptoms remains unclear. Objectives: To examine whether elevated NO in newborns with clinically naive airways is associated with subsequent respiratory symptoms in infancy. METHODS: We measured exhaled NO concentration and output after birth and prospectively assessed respiratory symptoms during infancy in a birth cohort of 164 unselected healthy neonates. We examined a possible association between NO and respiratory symptoms using Poisson regression analysis. RESULTS: In infants of atopic mothers, elevated NO levels after birth were associated with increased risk of subsequent respiratory symptoms (risk ratio [RR], 7.5; 95% confidence interval [CI], 1.7-32.4 for each nl/s increase in NO output; p = 0.007). Similarly, a positive association between NO and symptoms was seen in infants of smoking mothers (RR, 6.6; 95% CI, 2.3-19.3; p = 0.001), with the strongest association in infants whose mothers had both risk factors (RR, 21.8; 95% CI, 5.8-81.3; p < 0.001). CONCLUSIONS: The interaction of NO with maternal atopy and smoking on subsequent respiratory symptoms is present early in life. Clinically, noninvasive NO measurements in newborns may prove useful as a new means to identify high-risk infants. Future confirmation of a role for NO metabolism in the evolution of respiratory disease may provide an avenue for preventative strategies.

Performance of four dentine excavation methods in deciduous teeth

This in vitro study aimed to assess the speed and caries removal effectiveness of four different new and conventional dentine excavation methods. Eighty deciduous molars were assigned to four groups. Teeth were sectioned longitudinally through the lesion centre. Images of one half per tooth were captured by light microscope and confocal laser scanning microscopy (CLSM) to assess the caries extension. The halves were then reassembled and caries removed using round carbide bur (group 1), Er:YAG laser (group 2), hand excavator (group 3) and a polymer bur (group 4). The time needed for the whole excavation in each tooth was registered. After excavation, the halves were photographed by light microscope. Caries extension obtained from CLSM images were superimposed on the post-excavation images, allowing comparison between caries extension and removal. The regions where caries and preparation limits coincided, as well as the areas of over- and underpreparation, were measured. Steel bur was the fastest method, followed by the polymer bur, hand excavator and laser. Steel bur exhibited also the largest overpreparation area, followed by laser, hand excavator and polymer bur. The largest underpreparation area was found using polymer bur, followed by laser, hand excavator and steel bur. Hand excavator presented the longest coincidence line, followed by polymer and steel burs and laser. Overall, hand excavator seemed to be the most suitable method for carious dentine excavation in deciduous teeth, combining good excavation time with effective caries removal.