Development of an ultra-thin fibroin membrane for RPE cell transplantation

Purpose: One of the challenges associated with cell-based therapies for repairing the retina is the development of suitable materials on which to grow and transplant retinal cells. Using the ARPE-19 cell line, we have previously demonstrated the feasibility of growing RPE-derived cells on membranes prepared from the silk protein fibroin. The present study was aimed at developing a porous, ultra-thin fibroin membrane that might better support development of apical-basal polarity in culture, and to extend this work to primary cultures of human RPE cells. Methods: Ultra-thin fibroin membranes were prepared using a highly polished casting table coated with Topas® (a cyclic olefin copolymer) and a 1:0.03 aqueous solution of fibroin and PEO (Mv 900 000 g/mol). Following drying, the membranes were water annealed to make them water-stable, washed in water to remove PEO, sterilised by treatment with 95% ethanol, and washed extensively in saline. Primary cultures containing human RPE cells were established from donor posterior eye cups and maintained in DMEM/F12 medium supplemented with 10% fetal bovine serum and antibiotics. First passage cultures were seeded onto fibroin membranes pre-coated with vitronectin and grown for 6 weeks in medium supplemented with 1% serum. Comparative cultures were established on porous 1.0 µm pore PET membrane (Millipore) and using ARPE-19 cells. Results: The fibroin membranes displayed an average thickness of 3 µm and contained numerous dimples/pore-like structures of up to 3-5 µm in diameter. The primary cultures predominantly contained pigmented epithelial cells, but mesenchymal cells (presumed fibroblasts) were also often present. Passaged cultures appeared to attach equally well to either fibroin or PET membranes. Over time cells on either material adopted a more cobblestoned morphology. Conclusions: Progress has been made towards developing a porous ultra-thin fibroin membrane that supports cultivation of RPE cells. Further studies are required to determine the degree of membrane permeability and RPE polarity.

Prognostic significance of platelet and microvessel counts in operable non-small cell lung cancer

Background: Microvessel density, an indirect measure of angiogenesis, has been shown to be an independent prognostic marker in many solid tumours including non-small cell lung cancer (NSCLC). Platelets transport and release angiogenic growth factors. Platelets are increasingly likely to adhere to tumour microvessels due to raised expression of platelet-binding proteins and stasis in blood-flow. Increased vascular permeability in tumour microvessels facilitates platelet extravasation into the extracellular matrix. Adherence and extravasation both lead to platelet activation and release of growth factors capable of instigating the angiogenic process. Methods: A total of 181 patients were identified who underwent resection of stage I-IIIa NSCLC with a post-operative survival >60 days. Patients were followed-up for a minimum of 24 months. Sections from the tumour periphery were stained for the endothelial marker CD34 (Novocastra NCL-END) using standard ABC immunohistochemistry. Chalkley counting was used to assess microvessel density. Results: A pre-operative platelet count greater than the median and above the normal range (>400) was associated with a poor outcome (P = 0.01 and P = 0.04, respectively). Tumours with an above median and high Chalkley count (upper tertile) had a worse prognosis (P = 0.007 and P = 0.0006, respectively). There was no association between platelet count and Chalkley count. Conclusions: Platelet and microvessel counts are both potential prognostic markers for NSCLC. The role of platelets in the angiogenic process needs to be further investigated. (C) 2000 Elsevier Science Ireland Ltd.

Phase III trial comparing paclitaxel poliglumex (CT-2103, PPX) in combination with carboplatin versus standard paclitaxel and carboplatin in the treatment of PS 2 patients with chemotherapy-naïve advanced non-small cell lung cancer

INTRODUCTION: Performance status (PS) 2 patients with non-small cell lung cancer (NSCLC) experience more toxicity, lower response rates, and shorter survival times than healthier patients treated with standard chemotherapy. Paclitaxel poliglumex (PPX), a macromolecule drug conjugate of paclitaxel and polyglutamic acid, reduces systemic exposure to peak concentrations of free paclitaxel and may lead to increased concentrations in tumors due to enhanced vascular permeability. METHODS: Chemotherapy-naive PS 2 patients with advanced NSCLC were randomized to receive carboplatin (area under the curve = 6) and either PPX (210 mg/m/10 min without routine steroid premedication) or paclitaxel (225 mg/m/3 h with standard premedication) every 3 weeks. The primary end point was overall survival. RESULTS: A total of 400 patients were enrolled. Alopecia, arthralgias/myalgias, and cardiac events were significantly less frequent with PPX/carboplatin, whereas grade ≥3 neutropenia and grade 3 neuropathy showed a trend of worsening. There was no significant difference in the incidence of hypersensitivity reactions despite the absence of routine premedication in the PPX arm. Overall survival was similar between treatment arms (hazard ratio, 0.97; log rank p = 0.769). Median and 1-year survival rates were 7.9 months and 31%, for PPX versus 8 months and 31% for paclitaxel. Disease control rates were 64% and 69% for PPX and paclitaxel, respectively. Time to progression was similar: 3.9 months for PPX/carboplatin versus 4.6 months for paclitaxel/carboplatin (p = 0.210). CONCLUSION: PPX/carboplatin failed to provide superior survival compared with paclitaxel/carboplatin in the first-line treatment of PS 2 patients with NSCLC, but the results with respect to progression-free survival and overall survival were comparable and the PPX regimen was more convenient. © 2008International Association for the Study of Lung Cancer.

Dynamic contrast-enhanced magnetic resonance imaging as a biomarker for the pharmacological response of PTK787/ZK 222584, an inhibitor of the vascular endothelial growth factor receptor tyrosine kinases, in patients with advanced colorectal cancer and liver metastases: Results from two phase I studies

Purpose: PTK787/ZK 222584 (PTK/ZK), an orally active inhibitor of vascular endothelial growth factor (VEGF) receptor tyrosine kinases, inhibits VEGF-mediated angiogenesis. The pharmacodynamic effects of PTK/ZK were evaluated by assessing changes in contrast-enhancement parameters of metastatic liver lesions using dynamic contrast-enhanced magnetic resonance imaging (DCE-MRI) in patients with advanced colorectal cancer treated in two ongoing, dose-escalating phase I studies. Patients and Methods: Twenty-six patients had DCE-MRI performed at baseline, day 2, and at the end of each 28-day cycle. Doses of oral PTK/ZK ranged from 50 to 2000 mg once daily. Tumor permeability and vascularity were assessed by calculating the bidirectional transfer constant (Ki). The percentage of baseline Ki (% of baseline Ki) at each time point was compared with pharmacokinetic and clinical end points. Results: A significant negative correlation exists between the % of baseline Ki and increase in PTK/ZK oral dose and plasma levels (P = .01 for oral dose; P = .0001 for area under the plasma concentration curve at day 2). Patients with a best response of stable disease had a significantly greater reduction in Ki at both day 2 and at the end of cycle 1 compared with progressors (mean difference in % of baseline Ki, 47%, P = .004%; and 51%, P = .006; respectively). The difference in % of baseline Ki remained statistically significant after adjusting for baseline WHO performance status. Conclusion: These findings should help to define a biologically active dose of PTK/ZK. These results suggest that DCE-MRI may be a useful biomarker for defining the pharmacological response and dose of angiogenesis inhibitiors, such as PTK/ZK, for further clinical development. © 2003 by American Society of Clinical Oncology.

Local stress-strain distribution and load transfer across cartilage matrix at micro-scale using combined microscopy-based finite element method

Articular cartilage is the load-bearing tissue that consists of proteoglycan macromolecules entrapped between collagen fibrils in a three-dimensional architecture. To date, the drudgery of searching for mathematical models to represent the biomechanics of such a system continues without providing a fitting description of its functional response to load at micro-scale level. We believe that the major complication arose when cartilage was first envisaged as a multiphasic model with distinguishable components and that quantifying those and searching for the laws that govern their interaction is inadequate. To the thesis of this paper, cartilage as a bulk is as much continuum as is the response of its components to the external stimuli. For this reason, we framed the fundamental question as to what would be the mechano-structural functionality of such a system in the total absence of one of its key constituents-proteoglycans. To answer this, hydrated normal and proteoglycan depleted samples were tested under confined compression while finite element models were reproduced, for the first time, based on the structural microarchitecture of the cross-sectional profile of the matrices. These micro-porous in silico models served as virtual transducers to produce an internal noninvasive probing mechanism beyond experimental capabilities to render the matrices micromechanics and several others properties like permeability, orientation etc. The results demonstrated that load transfer was closely related to the microarchitecture of the hyperelastic models that represent solid skeleton stress and fluid response based on the state of the collagen network with and without the swollen proteoglycans. In other words, the stress gradient during deformation was a function of the structural pattern of the network and acted in concert with the position-dependent compositional state of the matrix. This reveals that the interaction between indistinguishable components in real cartilage is superimposed by its microarchitectural state which directly influences macromechanical behavior.

Effects of scaffold architecture on mechanical characteristics and osteoblast response to static and perfusion bioreactor cultures

Tissue engineering focuses on the repair and regeneration of tissues through the use of biodegradable scaffold systems that structurally support regions of injury whilst recruiting and/or stimulating cell populations to rebuild the target tissue. Within bone tissue engineering, the effects of scaffold architecture on cellular response have not been conclusively characterized in a controlled-density environment. We present a theoretical and practical assessment of the effects of polycaprolactone (PCL) scaffold architectural modifications on mechanical and flow characteristics as well as MC3T3-E1 preosteoblast cellular response in an in vitro static plate and custom-designed perfusion bioreactor model. Four scaffold architectures were contrasted, which varied in inter-layer lay-down angle and offset between layers, whilst maintaining a structural porosity of 60 ± 5%. We established that as layer angle was decreased (90° vs. 60°) and offset was introduced (0 vs. 0.5 between layers), structural stiffness, yield stress, strength, pore size and permeability decreased, whilst computational fluid dynamics-modeled wall shear stress was increased. Most significant effects were noted with layer offset. Seeding efficiencies in static culture were also dramatically increased due to offset (~45% to ~86%), with static culture exhibiting a much higher seeding efficiency than perfusion culture. Scaffold architecture had minimal effect on cell response in static culture. However, architecture influenced osteogenic differentiation in perfusion culture, likely by modifying the microfluidic environment.

Assessment of freestanding membranes prepared from Antheraea pernyi silk fibroin as a potential vehicle for corneal epithelial cell transplantation

Freestanding membranes created from Bombyx mori silk fibroin (BMSF) offer a potential vehicle for corneal cell transplantation since they are transparent and support the growth of human corneal epithelial cells (HCE). Fibroin derived from the wild silkworm Antheraea pernyi (APSF) might provide a superior material by virtue of containing putative cell- attachment sites that are absent from BMSF. Thus we have investigated the feasibility of producing transparent, freestanding membranes from APSF and have analysed the behaviour of HCE cells on this material. No significant differences in cell numbers or phenotype were observed in short term HCE cell cultures established on either fibroin. Production of transparent freestanding APSF membranes, however, proved to be problematic as cast solutions of APSF were more prone to becoming opaque, displayed significantly lower permeability and were more brittle than BMSF-membranes. Cultures of HCE cells established on either membrane developed a normal stratified morphology with cytokeratin pair 3/12 being immuno-localized to the superficial layers. We conclude that while it is feasible to produce transparent freestanding membranes from APSF, the technical difficulties associated with this biomaterial, along with an absence of enhanced cell growth, currently favours the continued development of BMSF as a preferred vehicle for corneal cell transplantation. Nevertheless, it remains possible that refinement of techniques for processing APSF might yet lead to improvements in the handling properties and performance of this material.

International survey of rigid contact lens fitting

Purpose To determine the extent of rigid contact lens fitting worldwide and to characterize the associated demographics and fitting patterns. Methods Survey forms were sent to contact lens fitters in up to 40 countries between January and March every year for five consecutive years (2007 to 2011). Practitioners were asked to record data relating to the first 10 contact lens fits or refits performed after receiving the survey form. Survey data collected between 1996 and 2011 were also analyzed to assess rigid lens fitting trends in seven nations during this period. Results Data were obtained for 12,230 rigid and 100,670 soft lens fits between 2007 and 2011. Overall, rigid lenses represented 10.8% of all contact lens fits, ranging from 0.2% in Lithuania to 37% in Malaysia. Compared with soft lens fits, rigid lens fits can be characterized as follows: older age (rigid, 37.3 ± 15.0 years; soft, 29.8 ± 12.4 years); fewer spherical and toric fits; more bifocal/multifocal fits; less frequent replacement (rigid, 7%; soft, 85%); and less part-time wear (rigid, 4%; soft, 10%). High-Dk (contact lens oxygen permeability) (36%) and mid-Dk (42%) materials are predominantly used for rigid lens fitting. Orthokeratology represents 11.5% of rigid contact lens fits. There has been a steady decline in rigid lens fitting between 1996 and 2011. Conclusions Rigid contact lens prescribing is in decline but still represents approximately 10% of all contact lenses fitted worldwide. It is likely that rigid lenses will remain as a viable, albeit increasingly specialized, form of vision correction.

Development of Contact Lenses from a Biomaterial Point of View – Materials, Manufacture, and Clinical Application

Rigid lenses, which were originally made from glass (between 1888 and 1940) and later from polymethyl methacrylate or silicone acrylate materials, are uncomfortable to wear and are now seldom fitted to new patients. Contact lenses became a popular mode of ophthalmic refractive error correction following the discovery of the first hydrogel material – hydroxyethyl methacrylate – by Czech chemist Otto Wichterle in 1960. To satisfy the requirements for ocular biocompatibility, contact lenses must be transparent and optically stable (for clear vision), have a low elastic modulus (for good comfort), have a hydrophilic surface (for good wettability), and be permeable to certain metabolites, especially oxygen, to allow for normal corneal metabolism and respiration during lens wear. A major breakthrough in respect of the last of these requirements was the development of silicone hydrogel soft lenses in 1999 and techniques for making the surface hydrophilic. The vast majority of contact lenses distributed worldwide are mass-produced using cast molding, although spin casting is also used. These advanced mass-production techniques have facilitated the frequent disposal of contact lenses, leading to improvements in ocular health and fewer complications. More than one-third of all soft contact lenses sold today are designed to be discarded daily (i.e., ‘daily disposable’ lenses).

Not just another ball game : young adult and adult football fictions are different

Current media attention on the crossover novel highlights the increasing permeability of the boundaries between young adult and adult fiction. This paper will focus upon some of the difficulties around definitions of young adult fiction before considering the fiction of football, or soccer as it is more commonly known in Australia. The football genre exhibits a number of discrete and identifiable differences between young adult and adult readerships including, for example, the role of the protagonist, and the narrative’s distance from the game. This paper will use Franco Moretti’s Mapping as Distant Reading model of abstraction to highlight and unpack these and other characteristic differences in the narratological and stylistic techniques employed across adult and young adult texts. Close reading analysis of the adult football fiction Striker (1992) by Hunter Davies and young adult football fiction Lucy Zeezou’s Goal (2008) by Liz Deep-Jones’ will further illustrate the range of tensions and divergences as they are reflected across those readerships. The texts have been selected because they speak to themes of fear and safety; Joe Swift (Striker) is driven by a need to move away from childhood poverty and insecurity, while Lucy Zeezou shelters a homeless friend. With both protagonists being kidnapped for ransom for example, the texts have also been selected for their striking similarities in form and content.

Proteases and proteomics : cutting to the core of human skin pathologies

Preserving the integrity of the skin's outermost layer (the epidermis) is vital for humans to thrive in hostile surroundings. Covering the entire body, the epidermis forms a thin but impenetrable cellular cordon that repels external assaults and blocks escape of water and electrolytes from within. This structure exists in a perpetual state of regeneration where the production of new cellular subunits at the base of the epidermis is offset by the release of terminally differentiated corneocytes from the surface. It is becoming increasingly clear that proteases hold vital roles in assembling and maintaining the epidermal barrier. More than 30 proteases are expressed by keratinocytes or infiltrating immune cells and the activity of each must be maintained within narrow limits and confined to the correct time and place. Accordingly, over- or under-exertion of proteolytic activity is a common factor in a multitude of skin disorders that range in severity from relatively mild to life-threatening. This review explores the current state of knowledge on the involvement of proteases in skin diseases and the latest findings from proteomic and transcriptomic studies focused on uncovering novel (patho)physiological roles for these enzymes.

Differences in membrane acyl phospholipid composition between an endothermic mammal and an ectothermic reptile are not limited to any phospholipid class

This study examined questions concerning differences in the acyl composition of membrane phospholipids that have been linked to the faster rates of metabolic processes in endotherms versus ectotherms. In liver, kidney, heart and brain of the ectothermic reptile, Trachydosaurus rugosus, and the endothermic mammal, Rattus norvegicus, previous findings of fewer unsaturates but a greater unsaturation index (UI) in membranes of the mammal versus those of the reptile were confirmed. Moreover, the study showed that the distribution of phospholipid head-group classes was similar in the same tissues of the reptile and mammal and that the differences in acyl composition were present in all phospholipid classes analysed, suggesting a role for the physical over the chemical properties of membranes in determining the faster rates of metabolic processes in endotherms. The most common phosphatidylcholine (PC) molecules present in all tissues (except brain) of the reptile were 16:0/18:1, 16:0/18:2, 18:0/18:2, 18:1/18:1 and 18:1/18:2, whereas arachidonic acid (20:4), containing PCs 16:0/ 20: 4, 18: 0/ 20: 4, were the common molecules in the mammal. The most abundant phosphatidylethanolamines ( PE) used in the tissue of the reptile were 18:0/18:2, 18:0/20:4, 18:1/18:1, 18:1/18:2 and 18:1/20:4, compared to 16: 0/ 18: 2, 16: 0/ 20: 4, 16: 0/ 22: 6, 18: 0/ 20: 4, 18: 0/ 22: 6 and 18:1/20: 4 in the mammal. UI differences were primarily due to arachidonic acid found in both PC and PEs, whereas docosahexaenoic acid (22:6) was a lesser contributor mainly within PEs and essentially absent in the kidney. The phospholipid composition of brain was more similar in the reptile and mammal compared to those of other tissues.

Multivariate statistical analysis of hydrochemical data to assess alluvial aquifer–stream connectivity during drought and flood : Cressbrook Creek, southeast Queensland, Australia

A catchment-scale multivariate statistical analysis of hydrochemistry enabled assessment of interactions between alluvial groundwater and Cressbrook Creek, an intermittent drainage system in southeast Queensland, Australia. Hierarchical cluster analyses and principal component analysis were applied to time-series data to evaluate the hydrochemical evolution of groundwater during periods of extreme drought and severe flooding. A simple three-dimensional geological model was developed to conceptualise the catchment morphology and the stratigraphic framework of the alluvium. The alluvium forms a two-layer system with a basal coarse-grained layer overlain by a clay-rich low-permeability unit. In the upper and middle catchment, alluvial groundwater is chemically similar to streamwater, particularly near the creek (reflected by high HCO3/Cl and K/Na ratios and low salinities), indicating a high degree of connectivity. In the lower catchment, groundwater is more saline with lower HCO3/Cl and K/Na ratios, notably during dry periods. Groundwater salinity substantially decreased following severe flooding in 2011, notably in the lower catchment, confirming that flooding is an important mechanism for both recharge and maintaining groundwater quality. The integrated approach used in this study enabled effective interpretation of hydrological processes and can be applied to a variety of hydrological settings to synthesise and evaluate large hydrochemical datasets.

Microstructure and electromagnetic characteristics of Ni nanoparticle film coated carbon microcoils

Carbon microcoils (CMCs) have been coated with a Ni nanoparticle film using an electroless plating process. The morphology, the elemental composition and the phases in the coating layer, complex permittivity and permeability of the CMCs and Ni-coated CMCs were, respectively, investigated by X-ray diffraction (XRD), scanning electron microscopy (SEM), energy dispersive spectroscopy (EDS) and microwave vector network analysis at room temperature. A homogeneous dispersion of Ni nanoparticles on the outer surface of the CMCs was obtained, with a mean particle size of ∼34.4 nm and the phosphorus content of about 8.5 wt%. When comparing the coated and uncoated CMC samples, the real (ε′) and imaginary (ε″) part of the complex permittivity as well as dielectric dissipation factor (tgδε = ε″/ε′) of the Ni-coated CMCs were much smaller, while the real (μ′) and imaginary (μ″) part of the complex permeability and the magnetic dissipation factor (t g σμ = μ″ / μ′) were larger. The enhanced microwave absorption of Ni-coated CMCs resulted from stronger dielectric and magnetic losses. In contrast, the microwave absorption of uncoated CMCs was mainly attributed to the dielectric rather than magnetic losses.

Towards a multi-element simulation of heavily over-consolidated bagasse using a material subroutine

A better understanding of the behaviour of prepared cane and bagasse, and the ability to model the mechanical behaviour of bagasse as it is squeezed in a milling unit to extract juice, would help identify how to improve the current process. There are opportunities to decrease bagasse moisture from a milling unit. The behaviour of bagasse in chutes is poorly understood. Previous investigations have shown that juice flow through bagasse obeys Darcy’s permeability law, that the grip of the rough surface of the grooves on the bagasse can be represented by the Mohr-Coulomb failure criterion for soils, and that the internal mechanical behaviour of the bagasse is critical state behaviour similar to that for sand and clay. Progress has been made in the last 11 years towards implementing a mechanical model for bagasse in finite element software. The objective is to be able to correctly simulate various simple mechanical loading conditions measured in the laboratory. Combining these behaviours together is thought to have a high probability of reproducing the complicated stress conditions in a milling unit. This paper reports on progress made towards modelling the fifth and final (and most challenging) of the simple loading conditions: the shearing of heavily over-consolidated bagasse, using a specific model for bagasse in a multi-element simulation.