Effects of oxygen and culture system on in vitro propagation and redifferentiation of osteoarthritic human articular chondrocytes

Regenerative medicine-based approaches for the repair of damaged cartilage rely on the ability to propagate cells while promoting their chondrogenic potential. Thus, conditions for cell expansion should be optimized through careful environmental control. Appropriate oxygen tension and cell expansion substrates and controllable bioreactor systems are probably critical for expansion and subsequent tissue formation during chondrogenic differentiation. We therefore evaluated the effects of oxygen and microcarrier culture on the expansion and subsequent differentiation of human osteoarthritic chondrocytes. Freshly isolated chondrocytes were expanded on tissue culture plastic or CultiSpher-G microcarriers under hypoxic or normoxic conditions (5% or 20% oxygen partial pressure, respectively) followed by cell phenotype analysis with flow cytometry. Cells were redifferentiated in micromass pellet cultures over 4 weeks, under either hypoxia or normoxia. Chondrocytes cultured on tissue culture plastic proliferated faster, expressed higher levels of cell surface markers CD44 and CD105 and demonstrated stronger staining for proteoglycans and collagen type II in pellet cultures compared with microcarrier-cultivated cells. Pellet wet weight, glycosaminoglycan content and expression of chondrogenic genes were significantly increased in cells differentiated under hypoxia. Hypoxia-inducible factor-3alpha mRNA was up-regulated in these cultures in response to low oxygen tension. These data confirm the beneficial influence of reduced oxygen on ex vivo chondrogenesis. However, hypoxia during cell expansion and microcarrier bioreactor culture does not enhance intrinsic chondrogenic potential. Further improvements in cell culture conditions are therefore required before chondrocytes from osteoarthritic and aged patients can become a useful cell source for cartilage regeneration.

Navigating the muddy waters of the research into single sex classrooms in co-educational middle years settings

Establishing single sex classes within co-educational sites is an option that Australian schools are again exploring. To date Australia has experienced three ‘waves’ of interest in establishing single sex classes, the first focused on equitable education opportunities for girls (Alloway & Gilbert, 1997), the second centered on boys’ literacy and engagement (Gilbert & Gilbert, 1998) and this current wave focuses on perceived difference between the sexes in co-educational classrooms (Protheroe, 2009; Gurian, Stevens & Daniels, 2009). With the intersection of middle schooling movement, focusing on learner centered classrooms (Pendergast & Bahr, 2010) and current educational agendas aimed at improving student performance and measurable learning outcomes (Ministerial Council on Education, Employment, Training and Youth Affairs, 2008), it is understandable that schools are exploring such student grouping options. However, after thirty years of international research into the efficacy of single sex classes in co-educational settings, the results still remain unclear. This paper seeks to navigate the ‘muddy waters’ of this body of research and suggests a framework to help guide school communities through the decision-making process associated with considering single sex classes.

Stromal upregulation of lateral epithelial adhesions : gene expression analysis of signalling pathways in prostate epithelium

BACKGROUND: Stromal signalling increases the lateral cell adhesions of prostate epithelial cells grown in 3D culture. The aim of this study was to use microarray analysis to identify significant epithelial signalling pathways and genes in this process. METHODS: Microarray analysis was used to identify genes that were differentially expressed when epithelial cells were grown in 3D Matrigel culture with stromal co-culture compared to without stroma. Two culture models were employed: primary epithelial cells (ten samples) and an epithelial cell line (three experiments). A separate microarray analysis was performed on each model system and then compared to identify tissue-relevant genes in a cell line model. RESULTS: TGF beta signalling was significantly ranked for both model systems and in both models the TGF beta signalling gene SOX4 was significantly down regulated. Analysis of all differentially expressed genes to identify genes that were common to both models found several morphology related gene clusters; actin binding (DIAPH2, FHOD3, ABLIM1, TMOD4, MYH10), GTPase activator activity (BCR, MYH10), cytoskeleton (MAP2, MYH10, TMOD4, FHOD3), protein binding (ITGA6, CD44), proteinaceous extracellular matrix (NID2, CILP2), ion channel/ ion transporter activity (CACNA1C, CACNB2, KCNH2, SLC8A1, SLC39A9) and genes associated with developmental pathways (POFUT1, FZD2, HOXA5, IRX2, FGF11, SOX4, SMARCC1). CONCLUSIONS: In 3D prostate cultures, stromal cells increase lateral epithelial cell adhesions. We show that this morphological effect is associated with gene expression changes to TGF beta signalling, cytoskeleton and anion activity.

Stroma regulates increased epithelial lateral cell adhesion in 3D culture : a role for actin/cadherin dynamics

BACKGROUND: Cell shape and tissue architecture are controlled by changes to junctional proteins and the cytoskeleton. How tissues control the dynamics of adhesion and cytoskeletal tension is unclear. We have studied epithelial tissue architecture using 3D culture models and found that adult primary prostate epithelial cells grow into hollow acinus-like spheroids. Importantly, when co-cultured with stroma the epithelia show increased lateral cell adhesions. To investigate this mechanism further we aimed to: identify a cell line model to allow repeatable and robust experiments; determine whether or not epithelial adhesion molecules were affected by stromal culture; and determine which stromal signalling molecules may influence cell adhesion in 3D epithelial cell cultures. METHODOLOGY/PRINCIPAL FINDINGS: The prostate cell line, BPH-1, showed increased lateral cell adhesion in response to stroma, when grown as 3D spheroids. Electron microscopy showed that 9.4% of lateral membranes were within 20 nm of each other and that this increased to 54% in the presence of stroma, after 7 days in culture. Stromal signalling did not influence E-cadherin or desmosome RNA or protein expression, but increased E-cadherin/actin co-localisation on the basolateral membranes, and decreased paracellular permeability. Microarray analysis identified several growth factors and pathways that were differentially expressed in stroma in response to 3D epithelial culture. The upregulated growth factors TGFβ2, CXCL12 and FGF10 were selected for further analysis because of previous associations with morphology. Small molecule inhibition of TGFβ2 signalling but not of CXCL12 and FGF10 signalling led to a decrease in actin and E-cadherin co-localisation and increased paracellular permeability. CONCLUSIONS/SIGNIFICANCE: In 3D culture models, paracrine stromal signals increase epithelial cell adhesion via adhesion/cytoskeleton interactions and TGFβ2-dependent mechanisms may play a key role. These findings indicate a role for stroma in maintaining adult epithelial tissue morphology and integrity.

Krüppel-associated box (KRAB)-associated co-repressor (KAP-1) Ser-473 phosphorylation regulates heterochromatin protein 1 (HP1- ) mobilization and DNA repair in heterochromatin

The DNA damage response encompasses a complex series of signaling pathways that function to regulate and facilitate the repair of damaged DNA. Recent studies have shown that the repair of transcriptionally inactive chromatin, named heterochromatin, is dependent upon the phosphorylation of the co-repressor, Krüppel-associated box (KRAB) domain-associated protein (KAP-1), by the ataxia telangiectasia-mutated (ATM) kinase. Co-repressors, such as KAP-1, function to regulate the rigid structure of heterochromatin by recruiting histone-modifying enzymes, such HDAC1/2, SETDB1, and nucleosome-remodeling complexes such as CHD3. Here, we have characterized a phosphorylation site in the HP1-binding domain of KAP-1, Ser-473, which is phosphorylated by the cell cycle checkpoint kinase Chk2. Expression of a nonphosphorylatable S473A mutant conferred cellular sensitivity to DNA-damaging agents and led to defective repair of DNA double-strand breaks in heterochromatin. In addition, cells expressing S473A also displayed defective mobilization of the HP1-β chromodomain protein. The DNA repair defect observed in cells expressing S473A was alleviated by depletion of HP1-β, suggesting that phosphorylation of KAP-1 on Ser-473 promotes the mobilization of HP1-β from heterochromatin and subsequent DNA repair. These results suggest a novel mechanism of KAP-1-mediated chromatin restructuring via Chk2-regulated HP1-β exchange from heterochromatin, promoting DNA repair.

Engineering across cultures : new learning resources for intercultural competency in engineering

BACKGROUND The work described in this paper has emerged from an ALTC/OLT funded project, Exploring Intercultural Competency in Engineering. The project indentified many facets of culture and intercultural competence that go beyond a culture-as-nationality paradigm. It was clear from this work that resources were needed to help engineering educators introduce students to the complex issues of culture as they relate to engineering practice. A set of learning modules focussing on intercultural competence in engineering practice were developed early on in the project. Through the OLT project, these modules have been expanded into a range of resources covering various aspects of culture in engineering. Supporting the resources, an eBook detailing the ins and outs of intercultural competency has also been developed to assist engineering educators to embed opportunities for students to develop skills in unpacking and managing cross-cultural challenges in engineering practice. PURPOSE This paper describes the key principles behind the development of the learning modules, the areas they cover and the eBook developed to support the modules. The paper is intended as an introduction to the approaches and resources and extends an invitation to the community to draw from, and contribute to this initial work. DESIGN/METHOD A key aim of this project was to go beyond the culture-as-nationality approach adopted in much of the work around intercultural competency (Deardorff, 2011). The eBook explores different dimensions of culture such as workplace culture, culture’s influence on engineering design, and culture in the classroom. The authors describe how these connect to industry practice and explore what they mean for engineering education. The packaged learning modules described here have been developed as a matrix of approaches moving from familiar known methods through complicated activities relying to some extent on expert knowledge. Some modules draw on the concept of ‘complex un-order’ as described in the ‘Cynefin domains’ proposed by Kurtz and Snowden (2003). RESULTS Several of the modules included in the eBook have already been trialled at a variety of institutions. Feedback from staff has been reassuringly positive so far. Further trials are planned for second semester 2012, and version 1 of the eBook and learning modules, Engineering Across Cultures, is due to be released in late October 2012. CONCLUSIONS The Engineering Across Cultures eBook and learning modules provide a useful and ready to employ resource to help educators tackle the complex issue of intercultural competency in engineering education. The book is by no means exhaustive, and nor are the modules, they instead provide an accessible, engineering specific guide to bringing cultural issues into the engineering classroom.

Reconceptualising mega project success in Australian Defence : recognising the importance of value co-creation

Recent literature in project management has urged a re-conceptualisation of projects as a value co-creation process. Contrary to the traditional output-focused project methodology, the value creation perspective argues for the importance of creating new knowledge, processes, and systems for suppliers and customers. Stakeholder involvement is important in this new perspective, as the balancing of competing needs of stakeholders in mega projects becomes a major challenge in managing the value co-creation process. In this study we present interview data from three Australian defence mega projects to demonstrate that senior executives have a more complex understanding of project success than traditional iron triangle measures. In these mega defence projects, customers and other stakeholders actively engage in the value creation process, and over time both content and process value are created to increase defence and national capability. Value created and captured during and post projects are the key to true success.

Residence time investigation in a co-axial dielectric barrier discharge reactor

The residence time distribution (RTD) is a crucial parameter when treating engine exhaust emissions with a Dielectric Barrier Discharge (DBD) reactor. In this paper, the residence time of such a reactor is investigated using a finite element based software: COMSOL Multiphysics 4.3. Non-thermal plasma (NTP) discharge is being introduced as a promising method for pollutant emission reduction. DBD is one of the most advantageous of NTP technologies. In a two cylinder co-axial DBD reactor, tubes are placed between two electrodes and flow passes through the annuals between these barrier tubes. If the mean residence time increases in a DBD reactor, there will be a corresponding increase in reaction time and consequently, the pollutant removal efficiency can increase. However, pollutant formation can occur during increased mean residence time and so the proportion of fluid that may remain for periods significantly longer than the mean residence time is of great importance. In this study, first, the residence time distribution is calculated based on the standard reactor used by the authors for ultrafine particle (10-500 nm) removal. Then, different geometrics and various inlet velocities are considered. Finally, for selected cases, some roughness elements added inside the reactor and the residence time is calculated. These results will form the basis for a COMSOL plasma and CFD module investigation.

The Yonder project : literacy and social competency development in primary school students through co-creation of live performance

Since 2007 Kite Arts Education Program (KITE), based at Queensland Performing Arts Centre (QPAC), has been engaged in delivering a series of theatre-based experiences for children in low socio-economic primary schools in Queensland. The artist in residence (AIR) project titled Yonder includes performances developed by the children with the support and leadership of teacher artists from KITE for their community and parents/carers,supported by a peak community cultural institution. In 2009,Queensland Performing Arts Centre partnered with Queensland University of Technology (QUT) Creative Industries Faculty (Drama) to conduct a three-year evaluation of the Yonder project to understand the operational dynamics, artistic outputs and the educational benefits of the project. This paper outlines the research findings for children engaged in the Yonder project in the interrelated areas of literacy development and social competencies. Findings are drawn from six iterations of the project in suburban locations on the edge of Brisbane city and in regional Queensland.

A spectroscopic comparison of YBCO superconductors synthesised by solid-state and co-precipitation methods

Raman spectroscopy, X-ray diffraction (XRD), and scanning electron microscopy (SEM) have been used to compare samples of YBa2Cu3O7 (YBCO) synthesised by the solid-state method and a novel co-precipitation technique. XRD results indicate that YBCO prepared by these two methods are phase pure, however the Raman and SEM results show marked differences between these samples.

Fine-grained Y2Cu2O5 powder from a co-precipitated precursor

A co-precipitation process is utilized to manufacture Y2Cu2O5 precursor powders. Upon calcination at high temperatures, such as 800 degrees C, the co-precipitated powder transforms to Y2Cu2O5. By selective variation of calcination parameters, grain-growth can be controlled to yield different sized Y2Cu2O5 powder, including sub-micron average sizes. ICP analysis, X-ray diffraction, electron microscopy, a.c. magnetic susceptibility and FT Raman are used to characterize phase development, morphology and purity of the powders.

Manufacture of specific BSCCO powder compositions by co-precipitation

A co-precipitation process for large-scale manufacture of bismuth-based HTSC powders has been demonstrated. Powders manufactured by this process have a high phase purity and precisely reproducible stoichiometry. Controlled time and temperature variations are used to convert precursors to HTSC compounds and to obtain specific particle-size distributions. The process has been demonstrated for a variety of compositions in the BSCCO system. Electron microscopy X-ray diffraction, inductively coupled plasma spectroscopy and magnetic-susceptibility measurements are used to characterize the powders.

Using the Depression Anxiety Stress Scale 21 (DASS-21) across cultures

DASS-21 has been validated in a number of populations such as Hispanic adults, American, British and Australian. The findings show that the DASS-21 is psychometrically sound with good reliability and validity. It is clear from the literature that the DASS-21 is a well established instrument for measuring depression, anxiety and stress in the Western world. Nonetheless, the lack of appropriate validation amongst Asian populations continues to pose concerns over the use of DASS-21 in Asian samples. Cultural variation may influence the individual’s experience and emotional expression. Thus, when researchers and practitioners employ Western-based assessments with Asian populations by directly translating them without an appropriate validation, the process can be challenging. In summary, we have conducted a series of rigorous statistical tests and minimised any potential confounds from the demographic information. The advantages of this revised DASS-18 stress scale are twofold. First, the revised DASS-18 stress scale possessed fewer items, which resulted in a cleaner factorial structure. Second, it also had a smaller inter-factor correlation. With these justifications, the revised DASS-18 stress scale is potentially more suitable for the Asian populations.

Blood cultures for evaluation of early postoperative fever after femoral neck fracture surgery

PURPOSE. To evaluate the utility of blood cultures in the assessment of early postoperative fever in hip fracture patients with no other indicators of sepsis. METHODS. 101 blood cultures were drawn on postoperative days 0 to 5 to investigate 84 febrile episodes in 31 women and 30 men (mean age, 80 years) whose body temperature measured via the tympanic route was ≥38ºC. Culture results of these 61 patients were divided into culture-positive and culture-negative groups for comparison. RESULTS. Of the 101 blood cultures, only 2 were positive: one was obtained 5 days after dynamic hip screw fixation, and the other 4 days after hemiarthroplasty. Both blood cultures grew coagulase-negative staphylococcal species, which were deemed to be skin contaminants not requiring change of patient management. 44 of these patients were treated with oral or intravenous antibiotics for a period of time. CONCLUSION. The risk of bacteraemia in patients with postoperative fever but no other symptoms of infection is low. Routine procurement of blood cultures in such patients is ineffective and of limited utility.