Isolation of microvascular endothelial cells from cadaveric corneal limbus

Limbal microvascular endothelial cells (L-MVEC) contribute to formation of the corneal-limbal stem cell niche and to neovascularization of diseased and injuries corneas. Nevertheless, despite these important roles in corneal health and disease, few attempts have been made to isolate L-MVEC with the view to studying their biology in vitro. We therefore explored the feasibility of generating primary cultures of L-MVEC from cadaveric human tissue. We commenced our study by evaluating growth conditions (MesenCult-XF system) that have been previously found to be associated with expression of the endothelial cell surface marker thrombomodulin/CD141, in crude cultures established from collagenase-digests of limbal stroma. The potential presence of L-MVEC in these cultures was examined by flow cytometry using a more specific marker for vascular endothelial cells, CD31/PECAM-1. These studies demonstrated that the presence of CD141 in crude cultures established using the MesenCult-XF system is unrelated to L-MVEC. Thus we subsequently explored the use of magnetic assisted cell sorting (MACS) for CD31 as a tool for generating cultures of L-MVEC, in conjunction with more traditional endothelial cell growth conditions. These conditions consisted of gelatin-coated tissue culture plastic and MCDB-131 medium supplemented with fetal bovine serum (10% v/v), D-glucose (10 mg/mL), epidermal growth factor (10 ng/mL), heparin (50 μg/mL), hydrocortisone (1 μg/mL) and basic fibroblast growth factor (10 ng/mL). Our studies revealed that use of endothelial growth conditions are insufficient to generate significant numbers of L-MVEC in primary cultures established from cadaveric corneal stroma. Nevertheless, through use of positive-MACS selection for CD31 we were able to routinely observe L-MVEC in cultures derived from collagenase-digests of limbal stroma. The presence of L-MVEC in these cultures was confirmed by immunostaining for von Willebrand factor (vWF) and by ingestion of acetylated low-density lipoprotein. Moreover, the vWF+ cells formed aligned cell-to-cell ‘trains’ when grown on Geltrex™. The purity of L-MVEC cultures was found to be unrelated to tissue donor age (32 to 80 years) or duration in eye bank corneal preservation medium prior to use (3 to 10 days in Optisol) (using multiple regression test). Optimal purity of L-MVEC cultures was achieved through use of two rounds of positive-MACS selection for CD31 (mean ± s.e.m, 65.0 ± 20.8%; p<0.05). We propose that human L-MVEC cultures generated through these techniques, in conjunction with other cell types, will provide a useful tool for exploring the mechanisms of blood vessel cell growth in vitro.

Trashtopia by Maison Briz Vegas

Description of the Work Trashtopia was a fashion exhibition at Craft Queensland’s Artisan gallery showcasing outfits made entirely from rubbish materials. The exhibition was part of an on-going series by the Queensland Fashion Archives, called Remember or Revive. Maison Briz Vegas designers, Carla Binotto and Carla van Lunn created a dystopian beach holiday tableau referencing mid-century Californian and Gold Coast beach culture and style, and today’s plastic pollution of the world’s oceans. The display engaged a popular audience with ideas about environmental destruction and climate change while bringing twentieth and twenty-first century consumer and leisure culture into question. The medium of fashion was used as a means of amusement and provocation. The fashion objects and installation questioned current mores about the material value of rubbish and the installation was also a work of environmental activism. Statement of the Research Component The work was framed by critical reflections of contemporary consumer culture and research fields questioning value in waste materials and fashion objects. The work is situated in the context of conceptual and experimental fashion design practice and fashion presentation. The exhibited work transgressed the conventional production methods and material choice of designer fashion garments, for example, discarded plastic shopping bags were painstakingly shredded to mimic ostrich feathers. The viewer was prompted to reflect on the materiality of rubbish and its potential for transformation. The exhibition also sits in the context of culture jamming and contemporary activist practice. The work references and subverts twentieth century beach holiday culture, contrasting resort wear with a contemporary picture of plastic pollution of the oceans and climate change. Hawaiian style prints contained a playful and dark narrative of dying marine-life and the viewer was invited to take a “Greetings from Trashtopia” postcard depicting fashion models floating in oceans of plastic rubbish. This reflective creative practice sought to address the question of whether fashion made from recycled rubbish materials can critically and emotionally engage viewers with questions about contemporary consumer culture and material value. This work presents an innovative model of fashion design practice in which rubbish materials are transformed into designer garments and rubbish is placed centre stage in the public presentation of the designs. In overturning the traditional model of fashion presentation, the viewer is also given a deeper connection to the recycling process and complex ideas of waste and value. In 2015 two outfits from the exhibition were selected, along with works from three leading Australian fashion labels, and four leading New Zealand labels, for a commemorative ANZAC fashion collection shown at iD Dunedin Fashion Week. The show titled, “Together Alone, revisited” reprised an Australian and New Zealand fashion exhibition first held at the National Gallery of Victoria in 2009.

Design of stiffened plates with openings

The load-deflection and ultimate strength behaviour of longitudinally stiffened plates with openings was studied using a second-order elastic post-buckling analysis and a rigid-plastic analysis. The ultimate strength was predicted from the intersection point of elastic and rigid-plastic curves and the Perry strut formula. Comparison with experimental results shows that satisfactory prediction of ultimate strength can be obtained by this simple method. Effects of the size of opening, the initial geometrical imperfections and the plate slenderness ratio on the strength of perforated stiffened plates were also studied.

A micro-level investigation of the solid displacement method for porosity determination of dried food

Porosity is one of the key parameters of the macroscopic structure of porous media, generally defined as the ratio of the free spaces occupied (by the volume of air) within the material to the total volume of the material. Porosity is determined by measuring skeletal volume and the envelope volume. Solid displacement method is one of the inexpensive and easy methods to determine the envelope volume of a sample with an irregular shape. In this method, generally glass beads are used as a solid due to their uniform size, compactness and fluidity properties. The smaller size of the glass beads means that they enter into the open pores which have a larger diameter than the glass beads. Although extensive research has been carried out on porosity determination using displacement method, no study exists which adequately reports micro-level observation of the sample during measurement. This study set out with the aim of assessing the accuracy of solid displacement method of bulk density measurement of dried foods by micro-level observation. Solid displacement method of porosity determination was conducted using a cylindrical vial (cylindrical plastic container) and 57 µm glass beads in order to measure the bulk density of apple slices at different moisture contents. A scanning electron microscope (SEM), a profilometer and ImageJ software were used to investigate the penetration of glass beads into the surface pores during the determination of the porosity of dried food. A helium pycnometer was used to measure the particle density of the sample. Results show that a significant number of pores were large enough to allow the glass beads to enter into the pores, thereby causing some erroneous results. It was also found that coating the dried sample with appropriate coating material prior to measurement can resolve this problem.

Three dimensional in-vitro models for studying cancer angiogenesis

Introduction Hydrogels prepared from star-shaped poly(ethylene glycol) (PEG) and maleimide-functionalized heparin provide a potential matrix for use in developing three dimensional (3D) models. We have previously demonstrated that these hydrogels support the cultivation of human umbilical vein endothelial cells (HUVECs). We extend this body of work to study the ability to create an extracellular matrix (ECM)-like model to study breast and prostate cancer cell growth in 3D. Also, we investigate the ability to produce a tri-culture mimicking tumour angiogenesis with cancer spheroids, HUVECs and mesenchymal stem cells (MSCs). Materials and Methods The breast cancer cell lines, MCF-7 and MDA-MB-231, and prostate cancer cell lines, LNCaP and PC3, were seeded into starPEG-heparin hydrogels and grown for 14 Days to analyze the effects of varying hydrogel stiffness on spheroid development. Resulting hydrogel constructs were analyzed via proliferation assays, light microscopy, and immunostaining. Cancer cell lines were then seeded into starPEG-heparin hydrogels functionalized with growth factors as spheroids with HUVECs and MSCs and grown as a tri-culture. Cultures were analyzed via immunostaining and observed using confocal microscopy. Results Cultures prepared in MMP-cleavable starPEG-heparin hydrogels display spheroid formation in contrast to adherent growth on tissue culture plastic. Small differences were visualized in cancer spheroid growth between different gel stiffness across the range of cell lines. Cancer cell lines were able to be co-cultivated with HUVECs and MSC. Interaction was visualized between tumours and HUVECs via confocal microscopy. Further studies intend to further optimize and mimic the ECM environment of in-situ tumour angiogenesis. Discussion Our results confirm the suitability of hydrogels constructed from starPEG-heparin for HUVEC and MSC co-cultivation with cancer cell lines to study cell-cell and cell-matrix interactions in a 3D environment. This represents a step forward in the development of 3D culture models to study the pathomechanisms of breast and prostate cancer.

Developing a three-dimensional in-vitro cell culture model for studying breast and prostate cancer using starPEG-heparin hydrogels

Introduction Hydrogels prepared from poly(ethylene glycol) (PEG) and maleimide-functionalized heparin provide a potential matrix for use in developing three dimensional (3D) models. We have previously demonstrated that these hydrogels support the cultivation of human umbilical vein endothelial cells (HUVECs) (1). We extend this body of work to study the ability to create an extracellular matrix (ECM)-like model to study breast and prostate cancer cell growth in 3D. Also, we investigate the ability to produce a tri-culture mimicking tumour angiogenesis with cancer spheroids, HUVECs and mesenchymal stem cells (MSC). Materials and Methods The breast cancer cell lines, MCF-7 and MDA-MB-231, and prostate cancer cell lines, LNCaP and PC3, were seeded into starPEG-heparin hydrogels and grown for 14 Days to analyse the effects of varying hydrogel stiffness on spheroid development. Resulting hydrogel constructs were analyzed via Alamar Blue assays, light microscopy, and immunofluorescence staining for cytokeratin 8/18, Ki67 and E-Cadherin. Cancer cell lines were then pre-grown in hydrogels for 5-7 days and then re-seeded into starPEG-heparin hydrogels functionalised with RGD, SDF-1, bFGF and VEGF as spheroids with HUVECs and MSC and grown for 14 days as a tri-culture in Endothelial Cell Growth Medium (ECGM; Promocell). Cell lines were also seeded as a single cell suspension into the functionalised tri-culture system. Cultures were fixed in 4% paraformaldehyde and analysed via immunostaining for Von Willebrand Factor and CD31, as well as the above mentioned markers, and observed using confocal microscopy. Results Cultures prepared in MMP-cleavable starPEG-heparin hydrogels display spheroid formation in contrast to adherent growth on tissue culture plastic. Small differences were visualised in cancer spheroid growth between different gel stiffness across the range of cell lines. Cancer cell lines were able to be co-cultivated with HUVECs and MSC. HUVEC tube formation and cancer line spheroid formation occured after 3-4 days. Interaction was visualised between tumours and HUVECs via confocal microscopy. Slightly increased interaction was seen between cancer tumours and micro-vascular tubes when seeded as single cells compared with the pre-formed spheroid approach. Further studies intend to utilise cytokine gradients to further optimise the ECM environment of in situ tumour angiogenesis. Discussion and Conclusions Our results confirm the suitability of hydrogels constructed from starPEG-heparin for HUVECs and MSC co-cultivation with cancer cell lines to study cell-cell and cell-matrix interactions in a 3D environment. This represents a step forward in the development of 3D culture models to study the pathomechanisms of breast and prostate cancer. References 1. Tsurkan MV, Chwalek K, Prokoph S, Zieris A, Levental KR, Freudenberg U, Werner C. Advanced Materials. 25, 2606-10, 2013. Disclosures The authors declare no conflicts of interest

Numerical simulation of railway track supporting system using finite-infinite and thin layer elements under impulsive loads

The present study deals with two dimensional, numerical simulation of railway track supporting system subjected to dynamic excitation force. Under plane strain condition, the coupled finite-infinite elements to represent the near and far field stress distribution and thin layer interface element was employed to model the interfacial behavior between sleepers and ballast. To account for the relative debonding, slipping and crushing that could take place in the contact area between the sleepers and ballast, modified Mohr-Coulomb criterion was adopted. Furthermore an attempt has been made to consider the elasto-plastic material non-linearity of the railway track supporting media by employing different constitutive models to represent steel, concrete and supporting materials. Based on the proposed physical and constitutive modeling a code has been developed for dynamic loads. The applicability of the developed F.E code has been demonstrated by analyzing a real railway supporting structure.

Numerical modeling of railway track supporting system using finite–infinite and thin layer elements

The present contribution deals with the numerical modelling of railway track-supporting systems-using coupled finite-infinite elements-to represent the near and distant field stress distribution, and also employing a thin layer interface element to account for the interfacial behaviour between sleepers and ballast. To simulate the relative debonding, slipping and crushing at the contact area between sleepers and ballast, a modified Mohr-Coulomb criterion was adopted. Further more an attempt was made to consider the elasto plastic materials’ non-linearity of the railway track supporting media by employing different constitutive models to represent steel, concrete and other supporting materials. It is seen that during an incremental-iterative mode of load application, the yielding initially started from the edge of the sleepers and then flowed vertically downwards and spread towards the centre of the railway supporting system.

Effect of constitutive material models on seismic response of two-story reinforced concrete frame

This paper focuses on the finite element (FE) response sensitivity and reliability analyses considering smooth constitutive material models. A reinforced concrete frame is modeled for FE sensitivity analysis followed by direct differentiation method under both static and dynamic load cases. Later, the reliability analysis is performed to predict the seismic behavior of the frame. Displacement sensitivity discontinuities are observed along the pseudo-time axis using non-smooth concrete and reinforcing steel model under quasi-static loading. However, the smooth materials show continuity in response sensitivity at elastic to plastic transition points. The normalized sensitivity results are also used to measure the relative importance of the material parameters on the structural responses. In FE reliability analysis, the influence of smoothness behavior of reinforcing steel is carefully noticed. More efficient and reasonable reliability estimation can be achieved by using smooth material model compare with bilinear material constitutive model.

A finite element modelling method for thin layer mortared masonry systems

This paper deals with a finite element modelling method for thin layer mortared masonry systems. In this method, the mortar layers including the interfaces are represented using a zero thickness interface element and the masonry units are modelled using an elasto-plastic, damaging solid element. The interface element is formulated using two regimes; i) shear-tension and ii) shearcompression. In the shear-tension regime, the failure of joint is consiedered through an eliptical failure criteria and in shear-compression it is considered through Mohr Coulomb type failure criterion. An explicit integration scheme is used in an implicit finite element framework for the formulation of the interface element. The model is calibrated with an experimental dataset from thin layer mortared masonry prism subjected to uniaxial compression, a triplet subjected to shear loads a beam subjected to flexural loads and used to predict the response of thin layer mortared masonry wallettes under orthotropic loading. The model is found to simulate the behaviour of a thin layer mortated masonry shear wall tested under pre-compression and inplane shear quite adequately. The model is shown to reproduce the failure of masonry panels under uniform biaxial state of stresses.

Petrology and textural classification of the Jericho kimberlite, northern Slave Province, Nunavut, Canada

The paper presents data on petrology, bulk rock and mineral compositions, and textural classification of the Middle Jurassic Jericho kimberlite (Slave craton, Canada). The kimberlite was emplaced as three steep-sided pipes in granite that was overlain by limestones and minor soft sediments. The pipes are infilled with hypabyssal and pyroclastic kimberlites and connected to a satellite pipe by a dyke. The Jericho kimberlite is classified as a Group Ia, lacking groundmass tetraferriphlogopite and containing monticellite pseudomorphs. The kimberlite formed, during several consecutive emplacement events of compositionally different batches of kimberlite magma. Core-logging and thin-section observations identified at least two phases of hypabyssal kimberlites and three phases of pyroclastic kimberlites. Hypabyssal kimberlites intruded as a main dyke (HK1) and as late small-volume aphanitic and vesicular dykes. Massive pyroclastic kimberlite (MPK1) predominantly filled the northern and southern lobes of the pipe and formed from magma different from the HK1 magma. The MPK1 magma crystallized Ti-, Fe-, and Cr-rich phlogopite without rims of barian phlogopite, and clinopyroxene and spinel without atoll structures. MPK1 textures, superficially reminiscent of tuffisitic kimberlite, are caused by pervasive contamination by granite xenoliths. The next explosive events filled the central lobe with two varieties of pyroclastic kimberlite: (1) massive and (2) weakly bedded, normally graded pyroclastic kimberlite. The geology of the Jericho pipe differs from the geology of South African or the Prairie kimberlites, but may resemble Lac de Gras pipes, in which deeper erosion removed upper fades of resedimented kimberlites.

A new approach to kimberlite facies terminology using a revised general approach to the nomenclature of all volcanic rocks and deposits: Descriptive to genetic

Although kimberlite pipes/bodies are usually the remains of volcanic vents, in-vent deposits, and subvolcanic intrusions, the terminology used for kimberlite rocks has largely developed independently of that used in mainstream volcanology. Existing kimberlite terminology is not descriptive and includes terms that are rarely used, used differently, and even not used at all in mainstream volcanology. In addition, kimberlite bodies are altered to varying degrees, making application of genetic terminology difficult because original components and depositional textures are commonly masked by alteration. This paper recommends an approach to the terminology for kimberlite rocks that is consistent with usage for other volcanic successions. In modern terrains the eruption and emplacement origins of deposits can often be readily deduced, but this is often not the case for old, variably altered and deformed rock successions. A staged approach is required whereby descriptive terminology is developed first, followed by application of genetic terminology once all features, including the effects of alteration on original texture and depositional features, together with contact relationships and setting, have been evaluated. Because many volcanic successions consist of both primary volcanic deposits as well as volcanic sediments, terminology must account for both possibilities.