Three-dimensional biofabrication of nanosecond laser micromachined nanofibre meshes for tissue engineered scaffolds.

There is a high demand for bespoke grafts to replace damaged or malformed bone and cartilage tissue. Three-dimensional (3D) printing offers a method of fabricating complex anatomical features of clinically relevant sizes. However, the construction of a scaffold to replicate the complex hierarchical structure of natural tissues remains challenging. This paper reports a novel biofabrication method that is capable of creating intricately designed structures of anatomically relevant dimensions. The beneficial properties of the electrospun fibre meshes can finally be realised in 3D rather than the current promising breakthroughs in two-dimensional (2D). The 3D model was created from commercially available computer-aided design software packages in order to slice the model down into many layers of slices, which were arrayed. These 2D slices with each layer of a defined pattern were laser cut, and then successfully assembled with varying thicknesses of 100 µm or 200 µm. It is demonstrated in this study that this new biofabrication technique can be used to reproduce very complex computer-aided design models into hierarchical constructs with micro and nano resolutions, where the clinically relevant sizes ranging from a simple cube of 20 mm dimension, to a more complex, 50 mm-tall human ears were created. In-vitro cell-contact studies were also carried out to investigate the biocompatibility of this hierarchal structure. The cell viability on a micromachined electrospun polylactic-co-glycolic acid fibre mesh slice, where a range of hole diameters from 200 µm to 500 µm were laser cut in an array where cell confluence values of at least 85% were found at three weeks. Cells were also seeded onto a simpler stacked construct, albeit made with micromachined poly fibre mesh, where cells can be found to migrate through the stack better with collagen as bioadhesives. This new method for biofabricating hierarchical constructs can be further developed for tissue repair applications such as maxillofacial bone injury or nose/ear cartilage replacement in the future.

Low-Cost Eye Phantom for Stereophotogrammetry-Based Optic Nerve Head Topographical 3D Imaging.

Glaucoma is the second leading cause of blindness globally. Stereophotogrammetry-based optic nerve head topographical imaging systems could potentially allow for objective glaucoma assessment in settings where technologies such as optical coherence tomography and the Heidelberg Retinal Tomograph are prohibitively expensive. In the development of such systems, eye phantoms are invaluable tools for both system calibration and performance evaluation. Eye phantoms developed for this purpose need to replicate the optical configuration of the eye, the related causes of measurement artefacts, and give the possibility to present to the imaging system the targets required for system calibration. The phantoms in the literature that show promise of meeting these requirements rely on custom lenses to be fabricated, making them very costly. Here, we propose a low-cost eye phantom comprising a vacuum formed cornea and commercially available stock bi-convex lens, that is optically similar to a gold-standard reference wide-angle schematic eye model and meets all the compliance and configurability requirements for use with stereo-photogrammetry-based ONH topographical imaging systems. Moreover, its modular design, being fabricated largely from 3D-printed components, lends itself to modification for other applications. The use of the phantom is successfully demonstrated in an ONH imager.

Probing fibronectin adsorption on chemically defined surfaces by means of single molecule force microscopy.

Atomic force microscope (AFM) based single molecule force spectroscopy (SMFS) and a quartz crystal microbalance (QCM) were respectively employed to probe interfacial characteristics of fibronectin fragment FNIII8-14 and full-length fibronectin (FN) on CH3-, OH-, COOH-, and NH2-terminated alkane-thiol self-assembled monolayers (SAMs). Force-distance curves acquired between hexahistidine-tagged FNIII8-14 immobilised on trisNTA-Ni2+ functionalized AFM cantilevers and the OH and COOH SAM surfaces were predominantly 'loop-like' (76% and 94% respectively), suggesting domain unfolding and preference for 'end-on' oriented binding, while those generated with NH2 and CH3 SAMs were largely 'mixed type' (81% and 86%, respectively) commensurate with unravelling and desorption, and 'side-on' binding. Time-dependent binding of FN to SAM-coated QCM crystals occurred in at least two phases: initial rapid coverage over the first 5 min; and variably diminishing adsorption thereafter (5-70 min). Loading profiles and the final hydrated surface concentrations reached (~ 950, ~ 1200, ~ 1400, ~ 1500 ng cm-2 for CH3, OH, COOH and NH2 SAMs) were consistent with: space-filling 'side-on' orientation and unfolding on CH3 SAM; greater numbers of FN molecules arranged 'end-on' on OH and especially COOH SAMs; and initial 'side-on' contact, followed by either (1) gradual tilting to a space-saving 'end-on' configuration, or (2) bi-/multi-layer adsorption on NH2 SAM.

3D Reconstruction of the Optic Nerve Head of a Phantom Eye from Images Obtained using a Slit Lamp Fitted with Low Cost Add-Ons.

Early detection and treatment are key in limiting vision loss from glaucoma, the second leading cause of blindness worldwide. Morphological alteration of the optic nerve head (ONH), detectable early in the condition, is a key clinical indicator. The mainstay for evaluation in clinics is the subjective assessment of stereoscopic ONH images. If quantitative diagnostic devices, which extract 3D information and use this to make an objective assessment, could be made affordable, it could mean greater diagnostic capability in primary/community care. A potentially cost-effective solution is to extract, using computer stereo vision, 3D information from stereo images obtained through a slit lamp, a mainstay of eye diagnostics, present in practically all ophthalmology and optometry practices. This work shows 3D ONH reconstruction in an eye phantom through a common slit lamp fitted with low cost cameras. Quantitative reconstructions, in close agreement with ground truths, were obtained.

Adsorption of Fibronectin Fragment on Surfaces Using Fully Atomistic Molecular Dynamics Simulations.

The effect of surface chemistry on the adsorption characteristics of a fibronectin fragment (FNIII8⁻10) was investigated using fully atomistic molecular dynamics simulations. Model surfaces were constructed to replicate self-assembled monolayers terminated with methyl, hydroxyl, amine, and carboxyl moieties. It was found that adsorption of FNIII8⁻10 on charged surfaces is rapid, specific, and driven by electrostatic interactions, and that the anchoring residues are either polar uncharged or of opposing charge to that of the targeted surfaces. On charged surfaces the presence of a strongly bound layer of water molecules and ions hinders FNIII8⁻10 adsorption. In contrast, adsorption kinetics on uncharged surfaces are slow and non-specific, as they are driven by van der Waals interactions, and the anchoring residues are polar uncharged. Due to existence of a positively charged area around its cell-binding region, FNIII8⁻10 is available for subsequent cell binding when adsorbed on a positively charged surface, but not when adsorbed on a negatively charged surface. On uncharged surfaces, the availability of the fibronectin fragment's cell-binding region is not clearly distinguished because adsorption is much less specific.

Electrospun collagen-based nanofibres: A sustainable material for improved antibiotic utilisation in tissue engineering applications.

For the creation of scaffolds in tissue engineering applications, it is essential to control the physical morphology of fibres and to choose compositions which do not disturb normal physiological function. Collagen, the most abundant protein in the human body, is a well-established biopolymer used in electrospinning compositions. It shows high in-vivo stability and is able to maintain a high biomechanical strength over time. In this study, the effects of collagen type I in polylactic acid-drug electrospun scaffolds for tissue engineering applications are examined. The samples produced were subsequently characterised using a range of techniques. Scanning electron microscopy analysis shows that the fibre morphologies varied across PLA-drug and PLA-collagen-drug samples - the addition of collagen caused a decrease in average fibre diameter by nearly half, and produced nanofibres. Atomic force microscopy imaging revealed collagen-banding patterns which show the successful integration of collagen with PLA. Solid-state characterisation suggested a chemical interaction between PLA and drug compounds, irgasan and levofloxacin, and the collagen increased the amorphous regions within the samples. Surface energy analysis of drug powders showed a higher dispersive surface energy of levofloxacin compared with irgasan, and contact angle goniometry showed an increase in hydrophobicity in PLA-collagen-drug samples. The antibacterial studies showed a high efficacy of resistance against the growth of both E. coli and S. Aureus, except with PLA-collagen-LEVO which showed a regrowth of bacteria after 48h. This can be attributed to the low drug release percentage incorporated into the nanofibre during the in vitro release study. However, the studies did show that collagen helped shift both drugs into sustained release behaviour. These ideal modifications to electrospun scaffolds may prove useful in further research regarding the acceptance of human tissue by inhibiting the potential for bacterial infection.

Fabrication and characterisation of drug-loaded electrospun polymeric nanofibers for controlled release in hernia repair.

The chemical distribution and mechanical effects of drug compounds in loaded electrospun scaffolds, a potential material for hernia repair mesh, were characterised and the efficacy of the material was evaluated. Polycaprolactone electrospun fibres were loaded with either the antibacterial agent, irgasan, or the broad-spectrum antibiotic, levofloxacin. The samples were subsequently characterised by rheological studies, scanning electron microscopy (SEM), atomic force microscopy (AFM), contact angle goniometry (CAG), in vitro drug release studies, antibacterial studies and time-of-flight secondary ion mass spectrometry (ToF-SIMS). Increased linear viscoelastic regions observed in the rheometry studies suggest that both irgasan and levofloxacin alter the internal structure of the native polymeric matrix. In vitro drug release studies from the loaded polymeric matrix showed significant differences in release rates for the two drug compounds under investigation. Irgasan showed sustained release, most likely driven by molecular diffusion through the scaffold. Conversely, levofloxacin exhibited a burst release profile indicative of phase separation at the edge of the fibres. Two scaffold types successfully inhibited bacterial growth when tested with strains of E. coli and S. aureus. Electrospinning drug-loaded polyester fibres is an alternative, feasible and effective method for fabricating non-woven fibrous meshes for controlled release in hernia repair.

Microparticle image velocimetry approach to flow measurements in isolated contracting lymphatic vessels.

We describe the development of an optical flow visualization method for resolving the flow velocity vector field in lymphatic vessels in vitro. The aim is to develop an experimental protocol for accurately estimating flow parameters, such as flow rate and shear stresses, with high spatial and temporal resolution. Previous studies in situ have relied on lymphocytes as tracers, but their low density resulted in a reduced spatial resolution whereas the assumption that the flow was fully developed in order to determine the flow parameters of interest may not be valid, especially in the vicinity of the valves, where the flow is undoubtedly more complex. To overcome these issues, we have applied the time-resolved microparticle image velocimetry (µ -PIV) technique, a well-established method that can provide increased spatial and temporal resolution that this transient flow demands. To that end, we have developed a custom light source, utilizing high-power light-emitting diodes, and associated control and image processing software. This paper reports the performance of the system and the results of a series of preliminary experiments performed on vessels isolated from rat mesenteries, demonstrating, for the first time, the successful application of the µ -PIV technique in these vessels.

The Genome of the "Great Speciator" Provides Insights into Bird Diversification.

Among birds, white-eyes (genus Zosterops) have diversified so extensively that Jared Diamond and Ernst Mayr referred to them as the "great speciator." The Zosterops lineage exhibits some of the fastest rates of species diversification among vertebrates, and its members are the most prolific passerine island colonizers. We present a high-quality genome assembly for the silvereye (Zosterops lateralis), a white-eye species consisting of several subspecies distributed across multiple islands. We investigate the genetic basis of rapid diversification in white-eyes by conducting genomic analyses at varying taxonomic levels. First, we compare the silvereye genome with those of birds from different families and searched for genomic features that may be unique to Zosterops. Second, we compare the genomes of different species of white-eyes from Lifou island (South Pacific), using whole genome resequencing and restriction site associated DNA. Third, we contrast the genomes of two subspecies of silvereye that differ in plumage color. In accordance with theory, we show that white-eyes have high rates of substitutions, gene duplication, and positive selection relative to other birds. Below genus level, we find that genomic differentiation accumulates rapidly and reveals contrasting demographic histories between sympatric species on Lifou, indicative of past interspecific interactions. Finally, we highlight genes possibly involved in color polymorphism between the subspecies of silvereye. By providing the first whole-genome sequence resources for white-eyes and by conducting analyses at different taxonomic levels, we provide genomic evidence underpinning this extraordinary bird radiation.

Electrospun polyurethane as an alternative ventricular catheter and in vitro model of shunt obstruction.

Intracranial pressure and volume vary considerably between hydrocephalic patients, and with age, health and haemodynamic status; if left untreated, intracranial pressure rises and the ventricular system expands to accommodate the excess cerebrospinal fluid, with significant morbidity and mortality. Cerebrospinal fluid shunts in use today have a high incidence of failure with shunt obstruction being the most serious. Conventional proximal shunt catheters are made from poly(dimethyl)siloxane, the walls of which are perforated with holes for the cerebrospinal fluid to pass through. The limited range of catheters, in terms of material selection and flow distribution, is responsible in large part for their poor performance. In this study, we present an alternative design of proximal catheter made of electrospun polyether urethane, and evaluate its performance in the presence of glial cells, which are responsible for shunt blockage. The viability and growth of cells on catheter materials such as poly(dimethyl)siloxane and polyurethane in the form of cast films, microfibrous mats and porous sponges were studied in the presence of proteins present in cerebrospinal fluid after 48 h and 96 h in culture. The numbers of viable cells on each substrate were comparable to untreated poly(dimethyl)siloxane, both in the presence and absence of serum proteins found in cerebrospinal fluid. A cell culture model of shunt obstruction was developed in which cells on electrospun polyether urethane catheters were subjected to flow during culture in vitro, and the degree of obstruction quantified in terms of hydraulic permeability after static and perfusion culture. The results indicate that a catheter made of electrospun polyether urethane would be able to maintain cerebrospinal fluid flow even with the presence of cells for the time period chosen for this study. These findings have implications for the design and deployment of microporous shunt catheter systems for the treatment of hydrocephalus.

Collagen-nanofiber hydrogel composites promote contact guidance of human lymphatic microvascular endothelial cells and directed capillary tube formation.

Collagen and fibronectin matrices are known to stimulate migration of microvascular endothelial cells and the process of tubulogenesis, but the physical, chemical, and topographical cues for directed vessel formation have yet to be determined. In this study, growth, migration, elongation, and tube formation of human lymphatic microvascular endothelial cells (LECs) were investigated on electrospun poly(D,L-lactic-co-glycolic acid) (PLGA) and poly(L-lactic-co-D-lactic acid) (PLDL) nanofiber-coated substrates, and correlated with fiber density and diameter. Directed migration of LECs was observed in the presence of aligned nanofibers, whereas random fiber alignment slowed down migration and growth of LECs. Cell guidance was significantly enhanced in the presence of more hydrophobic PLDL polymer nanofibers compared to PLGA (10:90). Subsequent experiments with tube-forming assays reveal the ability of resorbable hydrophobic nanofibers >300 nm in diameter to promote cell guidance in collagen gels without direct cell-fiber contact, in contrast to the previously reported contact-guidance phenomena. Our results show that endothelial cell guidance is possible within nanofiber/collagen-gel constructs that mimic the native extracellular matrix in terms of size and orientation of fibrillar components.