Fasciola hepatica Extracellular Vesicles isolated from excretory-secretory products using a gravity flow method modulate dendritic cell phenotype and activity.

Parasite-released extracellular vesicles (EVs) deliver signals to the host immune system that are critical to maintaining the long-term relationship between parasite and host. In the present study, total EVs (FhEVs) released in vitro by adults of the helminth parasite Fasciola hepatica were isolated using a recently described gravity flow method that protects their structural integrity. The FhEVs molecular cargo was defined using proteomic analysis and their surface topology characterised by glycan microarrays. The proteomic analysis identified 618 proteins, 121 of which contained putative N-linked glycosylation sites while 132 proteins contained putative O-linked glycosylation sites. Glycan arrays revealed surface-exposed glycans with a high affinity for mannose-binding lectins indicating the predominance of oligo mannose-rich glycoproteins, as well as other glycans with a high affinity for complex-type N-glycans. When added to bone-marrow derived dendritic cells isolated FhEV induced a novel phenotype that was categorised by the secretion of low levels of TNF, enhanced expression of cell surface markers (CD80, CD86, CD40, OX40L, and SIGNR1) and elevation of intracellular markers (SOCS1 and SOCS3). When FhEV-stimulated BMDCs were introduced into OT-II mice by adoptive transfer, IL-2 secretion from skin draining lymph nodes and spleen cells was inhibited in response to both specific and non-specific antigen stimulation. Immunisation of mice with a suspension of FhEV did not elicit significant immune responses; however, in the presence of alum, FhEVs induced a mixed Th1/Th2 immune response with high antigen specific antibody titres. Thus, we have demonstrated that FhEVs induce a unique phentotype in DC capable of suppressing IL-2 secretion from T-cells. Our studies add to the growing immuno-proteomic database that will be an important source for the discovery of future parasite vaccines and immunotherapeutic biologicals.

Laser Polishing of Additive Manufactured 316L Stainless Steel Synthesized by Selective Laser Melting.

One of the established limitations of metal additive manufacturing (AM) methods, such as selective laser melting (SLM), is the resulting rough surface finish. Laser polishing is one method that can be used to achieve an improved surface finish on AM printed parts. This study is focused on the laser surface polishing of AM parts using CO2 laser beam irradiation. Despite the fact that several researchers have investigated the traditional abrasive polishing method, there is still a lack of information reporting on the laser surface polishing of metal parts. In this study, AM 316L stainless steel cylindrical samples were polished using CO2 laser beam irradiation in continuous wave (CW) working mode. Two design of experiment models were developed for the optimization of the input processing parameters by statistical analysis of their effect on the resulting roughness. The processing parameters investigated were the laser beam power, the rotational speed of the sample, the number of laser scan passes, the laser beam focal position, and the percentage overlap of the laser tracks between consecutive passes. The characterization of the measured roughness and the modified layer microstructure was carried out using 3D optical and scanning electron microscopy (SEM). A maximum reduction of the roughness from 10.4 to 2.7 µm was achieved and no significant change in the microstructure phase type and micro-hardness was observed.

Synthesis and Characterization of Temperature-Sensitive and Chemically Cross-Linked Poly( N-isopropylacrylamide)/Photosensitizer Hydrogels for Applications in Photodynamic Therapy.

A novel poly( N-isopropylacrylamide) (PNIPAM) hydrogel containing different photosensitizers (protoporphyrin IX (PpIX), pheophorbide a (Pba), and protoporphyrin IX dimethyl ester (PpIX-DME)) has been synthesized with a significant improvement in water solubility and potential for PDT applications compared to the individual photosensitizers (PSs). Conjugation of PpIX, Pba, and PpIX-DME to the poly( N-isopropylacrylamide) chain was achieved using the dispersion polymerization method. This study describes how the use of nanohydrogel structures to deliver a photosensitizer with low water solubility and high aggregation tendencies in polar solvents overcomes these limitations. FT-IR spectroscopy, UV-vis spectroscopy, 1H NMR, fluorescence spectroscopy, SEM, and DLS analysis were used to characterize the PNIPAM-photosensitizer nanohydrogels. Spectroscopic studies indicate that the PpIX, Pba, and PpIX-DME photosensitizers are covalently conjugated to the polymer chains, which prevents aggregation and thus allows significant singlet oxygen production upon illumination. Likewise, the lower critical solution temperature was raised to ∼44 °C in the new PNIPAM-PS hydrogels. The PNIPAM hydrogels are biocompatible with >90% cell viability even at high concentrations of the photosensitizer in vitro. Furthermore, a very sharp onset of light-dependent toxicity for the PpIX-based nanohydrogel in the nanomolar range and a more modest, but significant, photocytotoxic response for Pba-PNIPAM and PpIX-DME-PNIPAM nanohydrogels suggest that the new hydrogels have potential for applications in photodynamic therapy.

Water-soluble, neutral 3,5-diformyl-BODIPY with extended fluorescence lifetime in a self-healable chitosan hydrogel.

3,5-Diformyl-4,4-difluoro-4-bora-3a,4a-diaza-s-indacene (3,5-diformyl-BODIPY) can be used as an efficient biofunctional cross-linker to generate a new class of chitosan-based hydrogels with fluorescence resonance energy transfer (FRET) dynamics and good solubility in water. The hydrogel was fully characterized by FT-IR, UV-vis, fluorescence, FE-SEM, AFM, rheology and picosecond time-resolved spectroscopic techniques. The self-healing ability was demonstrated by rheological recovery and macroscopic and microscopic observations. The fluorescence lifetime was found to increase in aqueous solution of the BODIPY-chitosan hydrogel compared to the 3,5-diformyl-BODIPY monomer. Calculations based on experimental results such as red-shift and decreased intensity of the emission spectrum of highly dye-concentrated hydrogel in comparison to dilute hydrogels, together with changes in the fluorescence lifetime of the hydrogel at different concentration of dyes, suggest that the BDP-CS hydrogels fluorescence dynamics obey the Förster resonance energy transfer (FRET). Improvements in mechanical and photochemical properties and the acceptable values of BODIPY fluorescence lifetime in the hydrogel matrix indicate the utility of the newly synthesized hydrogels for biomedical applications.

Improving smooth muscle cell exposure to drugs from drug-eluting stents at early time points: a variable compression approach.

The emergence of drug-eluting stents (DES) as a viable replacement for bare metal stenting has led to a significant decrease in the incidence of clinical restenosis. This is due to the transport of anti-restenotic drugs from within the polymer coating of a DES into the artery wall which arrests the cell cycle before restenosis can occur. The efficacy of DES is still under close scrutiny in the medical field as many issues regarding the effectiveness of DES drug transport in vivo still exist. One such issue, that has received less attention, is the limiting effect that stent strut compression has on the transport of drug species in the artery wall. Once the artery wall is compressed, the stents ability to transfer drug species into the arterial wall can be reduced. This leads to a reduction in the spatial therapeutic transfer of drug species to binding sites within the arterial wall. This paper investigates the concept of idealised variable compression as a means of demonstrating how such a stent design approach could improve the spatial delivery of drug species in the arterial wall. The study focused on assessing how the trends in concentration levels changed as a result of artery wall compression. Five idealised stent designs were created with a combination of thick struts that provide the necessary compression to restore luminal patency and thin uncompressive struts that improve the transport of drugs therein. By conducting numerical simulations of diffusive mass transport, this study found that the use of uncompressive struts results in a more uniform spatial distribution of drug species in the arterial wall.

Octavius 4D characterization for flattened and flattening filter free rotational deliveries.

PURPOSE: In this study the Octavius detector 729 ionization chamber (IC) array with the Octavius 4D phantom was characterized for flattening filter (FF) and flattening filter free (FFF) static and rotational beams. The device was assessed for verification with FF and FFF RapidArc treatment plans. METHODS: The response of the detectors to field size, dose linearity, and dose rate were assessed for 6 MV FF beams and also 6 and 10 MV FFF beams. Dosimetric and mechanical accuracy of the detector array within the Octavius 4D rotational phantom was evaluated against measurements made using semiflex and pinpoint ionization chambers, and radiochromic film. Verification FF and FFF RapidArc plans were assessed using a gamma function with 3%∕3 mm tolerances and 2%∕2 mm tolerances and further analysis of these plans was undertaken using film and a second detector array with higher spatial resolution. RESULTS: A warm-up dose of >6 Gy was required for detector stability. Dose-rate measurements were stable across a range from 0.26 to 15 Gy∕min and dose response was linear, although the device overestimated small doses compared with pinpoint ionization chamber measurements. Output factors agreed with ionization chamber measurements to within 0.6% for square fields of side between 3 and 25 cm and within 1.2% for 2 × 2 cm(2) fields. The Octavius 4D phantom was found to be consistent with measurements made with radiochromic film, where the gantry angle was found to be within 0.4° of that expected during rotational deliveries. RapidArc FF and FFF beams were found to have an accuracy of >97.9% and >90% of pixels passing 3%∕3 mm and 2%∕2 mm, respectively. Detector spatial resolution was observed to be a factor in determining the accurate delivery of each plan, particularly at steep dose gradients. This was confirmed using data from a second detector array with higher spatial resolution and with radiochromic film. CONCLUSIONS: The Octavius 4D phantom with associated Octavius detector 729 ionization chamber array is a dosimetrically and mechanically stable device for pretreatment verification of FF and FFF RapidArc treatments. Further improvements may be possible through use of a detector array with higher spatial resolution (detector size and∕or detector spacing).

An analysis of three dimensional diffusion in a representative arterial wall mass transport model.

The development and use of drug eluting stents has brought about significant improvements in reducing in-stent restenosis, however, their long term presence in the artery is still under examination due to restenosis reoccurring. Current studies focus mainly on stent design, coatings and deployment techniques but few studies address the issue of the physics of three dimensional mass transport in the artery wall. There is a dearth of adequate validated numerical mass transport models that simulate the physics of diffusion dominated drug transport in the artery wall whilst under compression. A novel experimental setup used in a previous study was adapted and an expansion of that research was carried out to validate the physics of three dimensional diffusive mass transport into a compressed porous media. This study developed a more sensitive method for measuring the concentration of the species of interest. It revalidated mass transport in the radial direction and presented results which highlight the need for an evaluation of the governing equation for transient diffusive mass transport in a porous media, in its current form, to be carried out.

Experimental determination of circumferential properties of fresh carotid artery plaques.

Carotid endarterectomy (CEA) is currently accepted as the gold standard for interventional revascularisation of diseased arteries belonging to the carotid bifurcation. Despite the proven efficacy of CEA, great interest has been generated in carotid angioplasty and stenting (CAS) as an alternative to open surgical therapy. CAS is less invasive compared with CEA, and has the potential to successfully treat lesions close to the aortic arch or distal internal carotid artery (ICA). Following promising results from two recent trials (CREST; Carotid revascularisation endarterectomy versus stenting trial, and ICSS; International carotid stenting study) it is envisaged that there will be a greater uptake in carotid stenting, especially amongst the group who do not qualify for open surgical repair, thus creating pressure to develop computational models that describe a multitude of plaque models in the carotid arteries and their reaction to the deployment of such interventional devices. Pertinent analyses will require fresh human atherosclerotic plaque material characteristics for different disease types. This study analysed atherosclerotic plaque characteristics from 18 patients tested on site, post-surgical revascularisation through endarterectomy, with 4 tissue samples being excluded from tensile testing based on large width-length ratios. According to their mechanical behaviour, atherosclerotic plaques were separated into 3 grades of stiffness. Individual and group material coefficients were then generated analytically using the Yeoh strain energy function. The ultimate tensile strength (UTS) of each sample was also recorded, showing large variation across the 14 atherosclerotic samples tested. Experimental Green strains at rupture varied from 0.299 to 0.588 and the Cauchy stress observed in the experiments was between 0.131 and 0.779 MPa. It is expected that this data may be used in future design optimisation of next generation interventional medical devices for the treatment and revascularisation of diseased arteries of the carotid bifurcation.

Factors that affect mass transport from drug eluting stents into the artery wall.

Coronary artery disease can be treated by implanting a stent into the blocked region of an artery, thus enabling blood perfusion to distal vessels. Minimally invasive procedures of this nature often result in damage to the arterial tissue culminating in the re-blocking of the vessel. In an effort to alleviate this phenomenon, known as restenosis, drug eluting stents were developed. They are similar in composition to a bare metal stent but encompass a coating with therapeutic agents designed to reduce the overly aggressive healing response that contributes to restenosis. There are many variables that can influence the effectiveness of these therapeutic drugs being transported from the stent coating to and within the artery wall, many of which have been analysed and documented by researchers. However, the physical deformation of the artery substructure due to stent expansion, and its influence on a drugs ability to diffuse evenly within the artery wall have been lacking in published work to date. The paper highlights previous approaches adopted by researchers and proposes the addition of porous artery wall deformation to increase model accuracy.

Demonstrating the influence of compression on artery wall mass transport.

The development of restenosis within the coronary arteries after a stenting procedure has been addressed with the development of the drug eluting stent device. However, in recent times the superiority of the drug eluting stent over bare metal stents has been brought into question. A lack of knowledge regarding the behavior of drug transport from the drug eluting devices contributes to this uncertainty. Questions arise as to whether drug eluting stents deliver sufficient amounts of therapeutic agents into the artery wall to suppress restenosis. Published investigations in this area have focused primarily on trends associated with how variations in stenting conditions affect mass transport behavior. However, experimentally validated numerical models that simulate mass transport within the artery wall are lacking. A novel experimental model was developed to validate computational predictions of species diffusion into a porous medium and an investigation into how stent strut compression influences mass transport was conducted. The study revealed that increased compressive forces on a porous media reduced the ability of species to diffuse through that media, and in relation to drug eluting stents will contribute to a reduction in therapeutic levels of drugs within the wall.

Variable temperature thin film indentation with a flat punch.

We present modifications to conventional nanoindentation that realize variable temperature, flat punch indentation of ultrathin films. The technique provides generation of large strain, thin film extrusion of precise geometries that idealize the essential flows of nanoimprint lithography, and approximate constant area squeeze flow rheometry performed on thin, macroscopic soft matter samples. Punch radii as small as 185 nm have been realized in ten-to-one confinement ratio testing of 36 nm thick polymer films controllably squeezed in the melt state to a gap width of a few nanometers. Self-consistent, compressive stress versus strain measurements of a wide variety of mechanical testing conditions are provided by using a single die-sample system with temperatures ranging from 20 to 125 degrees C and loading rates spanning two decades. Low roughness, well aligned flat punch dies with large contact areas provide precise detection of soft surfaces with standard nanoindenter stiffness sensitivity. Independent heating and thermometry with heaters and thermocouples attached to the die and sample allow introduction of a novel directional heat flux measurement method to ensure isothermal contact conditions. This is a crucial requirement for interpreting the mechanical response in temperature sensitive soft matter systems. Instrumented imprint is a new nanomechanics material testing platform that enables measurements of polymer and soft matter properties during large strains in confined, thin film geometries and extends materials testing capabilities of nanoindentation into low modulus, low strength glassy, and viscoelastic materials.

Room temperature mechanical thinning and imprinting of solid films.

The mechanical patterning of thin films has received recent attention due to significant potential for efficient nanostructure fabrication. For solid films, mechanically thinning wide areas remains particularly challenging. In this work, we introduce a new plastic ratchet mechanism involving small amplitude (<10 nm), oscillatory shear motion of the forging die. This isothermal mechanism significantly extends mass transport across surfaces, broadening the scope of nanoscale processing for a potentially wide class of solid ductile materials.