The cementogenic differentiation of periodontal ligament cells via the activation of Wnt/β-catenin signalling pathway by Li+ ions released from bioactive scaffolds

Lithium (Li) has been widely used as a long-term mood stabilizer in the treatment of bipolar and depressive disorders. Li+ ions are thought to enhance the remyelination of peripheral nerves and also stimulate the proliferation of neural progenitor cells and retinoblastoma cells via activation of the Wnt/β-catenin signalling pathway. Until now there have been no studies reporting the biological effects of released Li+ in bioactive scaffolds on cemetogenesis in periodontal tissue engineering applications. In this study, we incorporated parts of Li+ ions into the mesoporous bioactive glass (MBG) scaffolds and showed that this approach yielded scaffolds with a favourable composition, microstructure and mesopore properties for cell attachment, proliferation, and cementogenic differentiation of human periodontal ligament-derived cells (hPDLCs). We went on to investigate the biological effects of Li+ ions themselves on cell proliferation and cementogenic differentiation. The results showed that 5% Li+ ions incorporated into MBG scaffolds enhanced the proliferation and cementogenic differentiation of hPDLCs on scaffolds, most likely via activation of Wnt/β-catenin signalling pathway. Further study demonstrated that Li+ ions by themselves significantly enhanced the proliferation, differentiation and cementogenic gene expression of PDLCs. Our results indicate that incorporation of Li+ ions into bioactive scaffolds is a viable means of enhancing the Wnt canonical signalling pathway to stimulate cementogenic differentiation of PDLCs.

The microRNA expression signature on modified titanium implant surfaces influences genetic mechanisms leading to osteogenic differentiation

Topographically and chemically modified titanium implants are recognized to have improved osteogenic properties; however, the molecular regulation of this process remains unknown. This study aimed to determine the microRNA profile and the potential regulation of osteogenic differentiation following early exposure of osteoprogenitor cells to sand-blasted, large-grit acid-etched (SLA) and hydrophilic SLA (modSLA) surfaces. Firstly, the osteogenic characteristics of the primary osteoprogenitor cells were confirmed using ALP activity and Alizarin Red S staining. The effect of smooth (SMO), SLA and modSLA surfaces on the TGF-β/BMP (BMP2, BMP6, ACVR1) and non-canonical WNT/Ca2+ (WNT5A, FZD6) pathways, as well as the integrins ITGB1 and ITGA2, was determined. It was revealed that the modified titanium surfaces could induce the activation of TGF-β/BMP and non-canonical WNT/Ca2+ signaling genes. The expression pattern of microRNAs (miRNAs) related to cell differentiation was evaluated. Statistical analysis of the differentially regulated miRNAs indicated that 35 and 32 miRNAs were down-regulated on the modSLA and SLA surfaces respectively, when compared with the smooth surface (SMO). Thirty-one miRNAs that were down-regulated were common to both modSLA and SLA. There were 10 miRNAs up-regulated on modSLA and nine on SLA surfaces, amongst which eight were the same as observed on modSLA. TargetScan predictions for the down-regulated miRNAs revealed genes of the TGF-β/BMP and non-canonical Ca2+ pathways as targets. This study demonstrated that modified titanium implant surfaces induce differential regulation of miRNAs, which potentially regulate the TGF-β/BMP and WNT/Ca2+ pathways during osteogenic differentiation on modified titanium implant surfaces.

Development of a 3D culture system to study the skeletal metastasis of prostate cancer

In the cancer research field, most in vitro studies still rely on two-dimensional (2D) cultures. However, the trend is rapidly shifting towards using a three-dimensional (3D) culture system. This is because 3D models better recapitulate the microenvironment of cells, and therefore, yield cellular and molecular responses that more accurately describe the pathophysiology of cancer. By adopting technology platforms established by the tissue engineering discipline, it is now possible to grow cancer cells in extracellular matrix (ECM)-like environments and dictate the biophysical and biochemical properties of the matrix. In addition, 3D models can be modified to recapitulate different stages of cancer progression for instance from the initial development of tumor to metastasis. Inevitably, to recapitulate a heterotypic condition, comprising more than one cell type, it requires a more complex 3D model. To date, 3D models that are available for studying the prostate cancer (CaP)-bone interactions are still lacking. Therefore, the aim of this study is to establish a co-culture model that allows investigation of direct and indirect CaP-bone interactions. Prior to that, 3D polyethylene glycol (PEG)-based hydrogel cultures for CaP cells were first developed and growth conditions were optimised. Characterization of the 3D hydrogel cultures show that LNCaP cells form a multicellular mass that resembles avascular tumor. In comparison to 2D cultures, besides the difference in cell morphology, the response of LNCaP cells to the androgen analogue (R1881) stimulation is different compared to the cells in 2D cultures. This discrepancy between 2D and 3D cultures is likely associated with the cell-cell contact, density and ligand-receptor interactions. Following the 3D monoculture study, a 3D direct co-culture model of CaP cells and the human tissue engineered bone (hTEBC) construct was developed. Interactions between the CaP cells and human osteoblasts (hOBs) resulted in elevation of Matrix Metalloproteinase 9 (MMP9) for PC-3 cells and Prostate Specific Antigen (PSA) for LNCaP cells. To further investigate the paracrine interaction of CaP cells and (hOBs), a 3D indirect co-culture model was developed, where LNCaP cells embedded within PEG hydrogels were co-cultured with hTEBC. It was found that the cellular changes observed reflect the early event of CaP colonizing the bone site. In the absence of androgens, interestingly, up-regulation of PSA and other kallikreins is also detected in the co-culture compared to the LNCaP monoculture. This non androgenic stimulation could be triggered by the soluble factors secreted by the hOB such as Interleukin-6. There are also decrease in alkaline phosphatase (ALP) activity and down-regulation of genes of the hOB when co-cultured with LNCaP cells that have not been previously described. These genes include transforming growth factor β1 (TGFβ1), osteocalcin and Vimentin. However, no changes to epithelial markers (e.g E-cadherin, Cytokeratin 8) were observed in both cell types from the co-culture. Some of these intriguing changes observed in the co-cultures that had not been previously described have enriched the basic knowledge of the CaP cell-bone interaction. From this study, we have shown evidence of the feasibility and versatility of our established 3D models. These models can be adapted to test various hypotheses for studies pertaining to underlying mechanisms of bone metastasis and could provide a vehicle for anticancer drug screening purposes in the future.

Biological responses of human bone marrow mesenchymal stem cells to Sr-M-Si (M = Zn, Mg) silicate bioceramics

Strontium (Sr), Zinc (Zn), magnesium (Mg), and silicon (Si) are reported to be essential trace elements for the growth and mineralization of bone. We speculated that the combination of these bioactive elements in bioceramics may be effective to regulate the osteogenic property of boneforming cells. In this study, two Sr-containing silicate bioceramics, Sr2ZnSi2O7 (SZS) and Sr2MgSi2O7 (SMS), were prepared. The biological response of human bone marrow mesenchymal stem cells (BMSCs) to the two bioceramics (in the forms of powders and dense ceramic bulks) was systematically studied. In powder form, the effect of powder extracts on the viability and alkaline phosphatase (ALP) activity of BMSCs was investigated. In ceramic disc form, both direct and indirect coculture of BMSCs with ceramic discs were used to investigate their biological response, including attachment, proliferation, ALP activity, and bone-related genes expression. Beta-tricalcium phosphate (b-TCP) and akermanite (Ca2MgSi2O7, CMS) were used as control materials. The results showed that the Sr, Zn, and Si (or Sr, Mg, and Si)-containing ionic products from SZS and SMS powders enhanced ALP activity of BMSCs, compared to those from b-TCP. Both SZS and SMS ceramic discs supported the growth of BMSCs, and most importantly, significantly enhanced the ALP activity and bone-related genes expression of BMSCs as compared to b-TCP. The results suggest that the specific combination of bioactive ions (Sr, Zn, Si, e.g.) in bioceramics is a viable way to improve the biological performance of biomaterials, and the form of materials and surface properties were nonnegligible factors to influence cell response.

A biphasic scaffold design combined with cell sheet technology for simultaneous regeneration of alveolar bone/periodontal ligament complex

This study describes the design of a biphasic scaffold composed of a Fused Deposition Modeling scaffold (bone compartment) and an electrospun membrane (periodontal compartment) for periodontal regeneration. In order to achieve simultaneous alveolar bone and periodontal ligament regeneration a cell-based strategy was carried out by combining osteoblast culture in the bone compartment and placement of multiple periodontal ligament (PDL) cell sheets on the electrospun membrane. In vitro data showed that the osteoblasts formed mineralized matrix in the bone compartment after 21 days in culture and that the PDL cell sheet harvesting did not induce significant cell death. The cell-seeded biphasic scaffolds were placed onto a dentin block and implanted for 8 weeks in an athymic rat subcutaneous model. The scaffolds were analyzed by μCT, immunohistochemistry and histology. In the bone compartment, a more intense ALP staining was obtained following seeding with osteoblasts, confirming the μCT results which showed higher mineralization density for these scaffolds. A thin mineralized cementum-like tissue was deposited on the dentin surface for the scaffolds incorporating the multiple PDL cell sheets, as observed by H&E and Azan staining. These scaffolds also demonstrated better attachment onto the dentin surface compared to no attachment when no cell sheets were used. In addition, immunohistochemistry revealed the presence of CEMP1 protein at the interface with the dentine. These results demonstrated that the combination of multiple PDL cell sheets and a biphasic scaffold allows the simultaneous delivery of the cells necessary for in vivo regeneration of alveolar bone, periodontal ligament and cementum. © 2012 Elsevier Ltd.

Enterprise systems curricula for Asia-Pacific region : a vision for 2020

Enterprise Systems (ES) have emerged as possibly the most important and challenging development in the corporate use of information technology in the last decade. Organizations have invested heavily in these large, integrated application software suites expecting improvments in; business processes, management of expenditure, customer service, and more generally, competitiveness, improved access to better information/knowledge (i.e., business intelligence and analytics). Forrester survey data consistently shows that investment in ES and enterprise applications in general remains the top IT spending priority, with the ES market estimated at $38 billion and predicted to grow at a steady rate of 6.9%, reaching $50 billion by 2012 (Wang & Hamerman, 2008). Yet, organizations have failed to realize all the anticipated benefits. One of the key reasons is the inability of employees to properly utilize the capabilities of the enterprise systems to complete the work and extract information critical to decision making. In response, universities (tertiary institutes) have developed academic programs aimed at addressing the skill gaps. In parallel with the proliferation of ES, there has been growing recognition of the importance of Teaching Enterprise Systems at tertiary education institutes. Many academic papers have discused the important role of Enterprise System curricula at tertiary education institutes (Ask, 2008; Hawking, 2004; Stewart, 2001), where the teaching philosophises, teaching approaches and challenges in Enterprise Systems education were discussed. Following the global trends, tertiary institutes in the Pacific-Asian region commenced introducing Enterprise System curricula in late 1990s with a range of subjects (a subject represents a single unit, rather than a collection of units; which we refer to as a course) in faculties / schools / departments of Information Technology, Business and in some cases in Engineering. Many tertiary educations commenced their initial subject offers around four salient concepts of Enterprise Systems: (1) Enterprise Systems implementations, (2) Introductions to core modules of Enterprise Systems, (3) Application customization using a programming language (e.g. ABAP) and (4) Systems Administration. While universities have come a long way in developing curricula in the enterprise system area, many obstacles remain: high cost of technology, qualified faculty to teach, lack of teaching materials, etc.

Zoledronic acid preserves bone structure and increases survival but does not limit tumour incidence in a prostate cancer bone metastasis model

Background The bisphosphonate, zoledronic acid (ZOL), can inhibit osteoclasts leading to decreased osteoclastogenesis and osteoclast activity in bone. Here, we used a mixed osteolytic/osteoblastic murine model of bone-metastatic prostate cancer, RM1(BM), to determine how inhibiting osteolysis with ZOL affects the ability of these cells to establish metastases in bone, the integrity of the tumour-bearing bones and the survival of the tumour-bearing mice. Methods The model involves intracardiac injection for arterial dissemination of the RM1(BM) cells in C57BL/6 mice. ZOL treatment was given via subcutaneous injections on days 0, 4, 8 and 12, at 20 and 100 µg/kg doses. Bone integrity was assessed by micro-computed tomography and histology with comparison to untreated mice. The osteoclast and osteoblast activity was determined by measuring serum tartrate-resistant acid phosphatase 5b (TRAP 5b) and osteocalcin, respectively. Mice were euthanased according to predetermined criteria and survival was assessed using Kaplan Meier plots. Findings Micro-CT and histological analysis showed that treatment of mice with ZOL from the day of intracardiac injection of RM1(BM) cells inhibited tumour-induced bone lysis, maintained bone volume and reduced the calcification of tumour-induced endochondral osteoid material. ZOL treatment also led to a decreased serum osteocalcin and TRAP 5b levels. Additionally, treated mice showed increased survival compared to vehicle treated controls. However, ZOL treatment did not inhibit the cells ability to metastasise to bone as the number of bone-metastases was similar in both treated and untreated mice. Conclusions ZOL treatment provided significant benefits for maintaining the integrity of tumour-bearing bones and increased the survival of tumour bearing mice, though it did not prevent establishment of bone-metastases in this model. From the mechanistic view, these observations confirm that tumour-induced bone lysis is not a requirement for establishment of these bone tumours.

The roles of the preproghrelin-derived peptides - ghrelin, desacyl ghrelin and obestatin - in prostate cancer

Prostate cancer is the second most common cause of cancer related deaths in Western men. Despite the significant improvements in current treatment techniques, there is no cure for advanced metastatic, castrate-resistant disease. Early detection and prevention of progression to a castrate-resistant state may provide new strategies to improve survival. A number of growth factors have been shown to act in an autocrine/paracrine manner to modulate prostate cancer tumour growth. Our laboratory has previously shown that ghrelin and its receptors (the functional GHS-R1a and the non-functional GHS-R1b) are expressed in prostate cancer specimens and cell lines. We have shown that ghrelin increases cell proliferation in the PC3 and LNCaP prostate cancer cell lines through activation of ERK1/2, suggesting that ghrelin could regulate prostate cancer cell growth and play a role in the progression of the disease. Ghrelin is a 28 amino-acid peptide hormone, identified to be the natural ligand of the growth hormone secretagogue receptor (GHS-R1a). It is well characterised as a growth hormone releasing and as an orexigenic peptide that stimulates appetite and feeding and regulates energy expenditure and bodyweight. In addition to its orexigenic properties, ghrelin has been shown to play a regulatory role in a number of systems, including the reproductive, immune and cardiovascular systems and may play a role in a number of pathological conditions such as chronic heart failure, anorexia, cachexia, obesity, diabetes and cancer. In cancer, ghrelin and its receptor are expressed in a range of tumours and cancer cell lines and ghrelin has been demonstrated to modulate cell proliferation, apoptosis, migration and invasion in some cell types. The ghrelin gene (GHRL) encodes preproghrelin peptide, which is processed to produce three currently known functional peptides - ghrelin, desacyl ghrelin and obestatin. Prohormone convertases (PCs) have been shown to cleave the preproghrelin peptide into two primary products - the 28 amino acid peptide, ghrelin, and the remaining 117 amino acid C-terminal peptide, C-ghrelin. C-ghrelin can then be further processed to produce the 23 amino acid peptide, obestatin. Ghrelin circulates in two different forms - an octanoylated form (known as ghrelin) and a non-octanoylated form, desacyl ghrelin. The unique post-translational addition of octanoic acid to the serine 3 residue of the propeptide chain to form acylated ghrelin is catalysed by ghrelin O-acyltransferase (GOAT). This modification is necessary for binding of ghrelin to its only known functional receptor, the GHS-R1a. As desacyl ghrelin cannot bind and activate the GHS-R1a, it was initially thought to be an inactive peptide, despite the fact that it circulates at much higher levels than ghrelin. Further research has demonstrated that desacyl ghrelin is biologically active and shares some of the actions of ghrelin, as well as having some opposing and distinct roles. Interestingly, both ghrelin and desacyl ghrelin have been shown to modulate apoptosis, cell differentiation and proliferation in some cell types, and to stimulate cell proliferation through activation of ERK1/2 and PI3K/Akt pathways. The third known peptide product of the ghrelin preprohormone, obestatin, was initially thought to oppose the actions of ghrelin in appetite regulation and food intake and to mediate its effects through the G protein-coupled receptor 39 (GPR39). Subsequent research failed to reproduce the initial findings, however, and the possible anorexigenic effects of obestatin, as well as the identity of its receptor, remain unclear. Obestatin plays some important physiological roles, including roles in improving memory, the inhibition of thirst and anxiety, increased secretion of pancreatic juice, and regulation of cell proliferation, survival, apoptosis and differentiation. Preliminary studies have also shown that obestatin stimulates cell proliferation in some cell types through activation of ERK1/2, Akt and PKC pathways. Overall, however, at the commencement of this PhD project, relatively little was known regarding the functions and mechanisms of action of the preproghrelin-derived functional peptides in modulating prostate cancer cell proliferation. The roles of obestatin, and desacyl ghrelin as potential growth factors had not previously been investigated, and the potential expression and regulation of the preproghrelin processing enzymes, GOAT and prohormone convertases was unknown in prostate cancer cell lines. Therefore, the overall objectives of this study were to: 1. investigate the effects of obestatin on cell proliferation and signaling in prostate cancer cell lines 2. compare the effects of desacyl ghrelin and ghrelin on cell proliferation and signaling in prostate cancer cell lines 3. investigate whether prostate cancer cell lines possess the necessary enzymatic machinery to produce ghrelin and desacyl ghrelin and if these peptides can regulate GOAT expression Our laboratory has previously shown that ghrelin stimulates cell proliferation in the PC3 and LNCaP prostate cancer cell line through activation of the ERK1/2 pathway. In this study it has been demonstrated that treatments with either ghrelin, desacyl ghrelin or obestatin over 72 hours significantly increased cell proliferation in the PC3 prostate cancer cell line but had no significant effect in the RWPE-1 transformed normal prostate cell line. Ghrelin (1000nM) stimulated cell proliferation in the PC3 prostate cancer cell line by 31.66 6.68% (p<0.01) with the WST-1 method, and 13.55 5.68% (p<0.05) with the CyQUANT assay. Desacyl ghrelin (1000nM) increased cell proliferation in PC3 cells by 21.73 2.62% (p<0.01) (WST-1), and 15.46 7.05% (p<0.05) (CyQUANT) above untreated control. Obestatin (1000nM) induced a 28.37 7.47% (p<0.01) (WST-1) and 12.14 7.47% (p<0.05) (CyQUANT) significant increase in cell proliferation in the PC3 prostate cancer cell line. Ghrelin and desacyl ghrelin treatments stimulated Akt and ERK phosphorylation across a range of concentrations (p<0.01). Obestatin treatment significantly stimulated Akt, ERK and PKC phosphorylation (p<0.05). Through the use of specific inhibitors, the MAPK inhibitor U0126 and the Akt1/2 kinase inhibitor, it was demonstrated that ghrelin- and obestatin-induced cell proliferation in the PC3 prostate cancer cell line is mediated through activation of ERK1/2 and Akt pathways. Although desacyl ghrelin significantly stimulated Akt and ERK phosphorylation, U0126 failed to prevent desacyl ghrelin-induced cell proliferation suggesting ghrelin and desacyl ghrelin might act through different mechanisms to increase cell proliferation. Ghrelin and desacyl ghrelin have shown a proliferative effect in osteoblasts, pancreatic -cells and cardiomyocytes through activation of ERK1/2 and PI3K/Akt pathways. Here it has been shown that ghrelin and its non-acylated form exert the same function and stimulate cell proliferation in the PC3 prostate cancer cell line through activation of the Akt pathway. Ghrelin-induced proliferation was also mediated through activation of the ERK1/2 pathway, however, desacyl ghrelin seems to stimulate cell proliferation in an ERK1/2-independent manner. As desacyl ghrelin does not bind and activate GHSR1a, the only known functional ghrelin receptor, the finding that both ghrelin and desacyl ghrelin stimulate cell proliferation in the PC3 cell line suggests that these peptides could be acting through the yet unidentified alternative ghrelin receptor in this cell type. Obestatin treatment also stimulated PKC phosphorylation, however, a direct role for this pathway in stimulating cell proliferation could not be proven using available PKC pathway inhibitors, as they caused significant cell death over the extended timeframe of the cell proliferation assays. Obestatin has been shown to stimulate cell proliferation through activation of PKC isoforms in human retinal epithelial cells and in the human gastric cancer cell line KATO-III. We have demonstrated that all of the prostate-derived cell lines examined (PC3, LNCaP, DU145, 22Rv1, RWPE-1 and RWPE-2) expressed GOAT and at least one of the prohormone convertases, which are known to cleave the proghrelin peptide, PC1/3, PC2 and furin, at the mRNA level. These cells, therefore, are likely to possess the necessary machinery to cleave the preproghrelin protein and to produce the mature ghrelin and desacyl ghrelin peptides. In addition to prohormone convertases, the presence of octanoic acid is essential for acylated ghrelin production. In this study octanoic acid supplementation significantly increased cell proliferation in the PC3 prostate cancer cell line by over 20% compared to untreated controls (p<0.01), but surprisingly, not in the DU145, LNCaP or 22Rv1 prostate cancer cell lines or in the RWPE-1 and RWPE-2 prostate-derived cell lines. In addition, we demonstrated that exogenous ghrelin induced a statistically significant two-fold decrease in GOAT mRNA expression in the PC3 cell line (p<0.05), suggesting that ghrelin could pontentially downregulate its own acylation and, therefore, regulate the balance between ghrelin and desacyl ghrelin. This was not observed, however, in the DU145 and LNCaP prostate cancer cell lines. The GOAT-ghrelin system represents a direct link between ingested nutrients and regulation of ghrelin production and the ghrelin/desacyl ghrelin ratio. Regulation of ghrelin acylation is a potentially attractive and desirable tool for the development of better therapies for a number of pathological conditions where ghrelin has been shown to play a key role. The finding that desacyl ghrelin stimulates cell proliferation in the PC3 prostate cancer cell line, and responds to ghrelin in the same way, suggests that this cell line expresses an alternative ghrelin receptor. Although all the cell lines examined expressed both GHS-R1a and GHS-R1b mRNA, it remains uncertain whether these cell lines express the unidentified alternative ghrelin receptor. It is possible that the varied responses seen could be due to the expression of different ghrelin receptors in different cell lines. In addition to GOAT, prohormone convertases and octanoic acid availability may regulate the production of different peptides from the ghrelin preprohormone. The studies presented in this thesis provide significant new information regarding the roles and mechanisms of action of the preproghrelin-derived peptides, ghrelin, desacyl ghrelin and obestatin, in modulating prostate cancer cell line proliferation. A number of key questions remain to be resolved, however, including the identification of the alternative ghrelin/desacyl ghrelin receptor, the identification of the obestatin receptor, a clarification of the signaling mechanisms which mediate cell proliferation in response to obestatin treatment and a better understanding of the regulation at both the gene and post-translational levels of functional peptide generation. Further studies investigating the role of the ghrelin axis using in vivo prostate cancer models may be warranted. Until these issues are determined, the potential for the ghrelin axis, to be recognised as a novel useful target for therapy for cancer or other pathologies will be uncertain.

From slum to suburbia : examining a spectrum of planned and unplanned urban morphology in humid sub-tropical regions of the world

The current rapid urban growth throughout the world manifests in various ways and historically cities have grown, similarly, alternately or simultaneously between planned extensions and organic informal settlements (Mumford, 1989). Within cities different urban morphological regions can reveal different contexts of economic growth and/or periods of dramatic social/technological change (Whitehand, 2001, 105). Morpho-typological study of alternate contexts can present alternative models and contribute to the present discourse which questions traditional paradigms of urban planning and design (Todes et al, 2010). In this study a series of cities are examined as a preliminary exploration into the urban morphology of cities in ‘humid subtropical’ climates. From an initial set of twenty, six cities were selected: Sao Paulo, Brazil; Jacksonville, USA; Maputo, Mozambique; Kanpur, India; Hong Kong, China; and Brisbane, Australia. The urban form was analysed from satellite imagery at a constant scale. Urban morphological regions (types) were identified as those demonstrating particular consistant characteristics of form (density, typology and pattern) different to their surroundings when examined at a constant scale. This analysis was correlated against existing data and literature discussing the proliferation of two types of urban development, ‘informal settlement’ (defined here as self-organised communities identifiable but not always synonymous with ‘slums’) and ‘suburbia’ (defined here as master planned communities of generally detached houses prevalent in western society) - the extreme ends of a hypothetical spectrum from ‘planned’ to ‘spontaneous’ urban development. Preliminary results show some cities contain a wide variety of urban form ranging from the highly organic ‘self-organised’ type to the highly planned ‘master planned community’ (in the case of Sao Paulo) while others tend to fall at one end of the planning spectrum or the other (more planned in the cases of Brisbane and Jacksonville; and both highly planned and highly organic in the case of Maputo). Further research will examine the social, economical and political drivers and controls which lead to this diversity or homogeneity of urban form and speculates on the role of self-organisation as a process for the adaptation of urban form.

Preparation, characterization and in vitro angiogenic capacity of cobalt substituted β-tricalcium phosphate ceramics

Divalent cobalt ions (Co2+) have been shown to possess the capacity to induce angiogenesis by activating hypoxia inducible factor-1α (HIF-1α) and subsequently inducing the production of vascular endothelial growth factor (VEGF). However, there are few reports about Co-containing biomaterials for inducing in vitro angiogenesis. The aim of the present work was to prepare Co-containing β-tricalcium phosphate (Co-TCP) ceramics with different contents of calcium substituted by cobalt (0, 2, 5 mol%) and to investigate the effect of Co substitution on their physicochemical and biological properties. Co-TCP powders were synthesized by a chemistry precipitation method and Co-TCP ceramics were prepared by sintering the powder compacts. The effect of Co substitution on phase transition and the sintering property of the β-TCP ceramics was investigated. The proliferation and VEGF expression of human bone marrow mesenchymal stem cells (HBMSCs) cultured with both powder extracts and ceramic discs of Co-TCP was further evaluated. The in vitro angiogenesis was evaluated by the tube-like structure formation of human umbilical vein endothelial cells (HUVECs) cultured on ECMatrix™ in the presence of powder extracts. The results showed that Co substitution suppressed the phase transition from β- to α-TCP. Both the powder extracts and ceramic discs of Co-TCP had generally good cytocompatibility to support HBMSC growth. Importantly, the incorporation of Co into β-TCP greatly stimulated VEGF expression of HBMSCs and Co-TCP showed a significant enhancement of network structure formation of HUVECs compared with pure TCP. Our results suggested that the incorporation of Co into bioceramics is a potential viable way to enhance angiogenic properties of biomaterials. Co-TCP bioceramics may be used for bone tissue regeneration with improved angiogenic capacity.

Tactically-driven nonmonotone travelling waves

Models of cell invasion incorporating directed cell movement up a gradient of an external substance and carrying capacity-limited proliferation give rise to travelling wave solutions. Travelling wave profiles with various shapes, including smooth monotonically decreasing, shock-fronted monotonically decreasing and shock-fronted nonmonotone shapes, have been reported previously in the literature. The existence of tacticallydriven shock-fronted nonmonotone travelling wave solutions is analysed for the first time. We develop a necessary condition for nonmonotone shock-fronted solutions. This condition shows that some of the previously reported shock-fronted nonmonotone solutions are genuine while others are a consequence of numerical error. Our results demonstrate that, for certain conditions, travelling wave solutions can be either smooth and monotone, smooth and nonmonotone or discontinuous and nonmonotone. These different shapes correspond to different invasion speeds. A necessary and sufficient condition for the travelling wave with minimum wave speed to be nonmonotone is presented. Several common forms of the tactic sensitivity function have the potential to satisfy the newly developed condition for nonmonotone shock-fronted solutions developed in this work.

Introduction : creative industries and cities

The 2000s were marked by a resurgence of interest in creativity and cities. If the rapid global proliferation of the Internet and digital media technologies in the 1990s had set off enthusiasm for a post-industrial ‘new economy’, where the significance of location would be in decline, the 2000s saw an energetic search by artists, entrepreneurs, investors, policy-makers, journalists and many others to uncover the well-springs of creativity and its relationship to place (Flew 2012a). This chapter begins with a discussion of the discourses or ‘scripts’ that have emerged to try and conceptualise the relationship between creativity and cities, notably theories of creative clusters, creative cities and creative class theories. Such work can be seen as representing a growth in the field of cultural economic geography although – as is noted in the chapter – it possesses some significant gaps. Among the issues that are drawn out in this book, and discussed in this chapter, are: the need to move beyond ‘imagined geographies’ of creative inner cities and come to terms with empirical evidence that suggests significant concentrations of the creative workforce in suburbs and regional cities; the relevance of urban cultural policy as a variable in the rise of cities as creative hubs or, in a different model, media capitals; and the challenges of bringing together cultural research with economic discourses in ways that get beyond caricatured representations of the ‘other’, as found, for instance, in some of the most influential framings of the concept of neo-liberalism.

Leucocytes, cytokines and satellite cells : what role do they play in muscle damage and regeneration following eccentric exercise?

Exercise-induced muscle damage is an important topic in exercise physiology. However several aspects of our understanding of how muscles respond to highly stressful exercise remain unclear In the first section of this review we address the evidence that exercise can cause muscle damage and inflammation in otherwise healthy human skeletal muscles. We approach this concept by comparing changes in muscle function (i.e., the force-generating capacity) with the degree of leucocyte accumulation in muscle following exercise. In the second section, we explore the cytokine response to 'muscle-damaging exercise', primarily eccentric exercise. We review the evidence for the notion that the degree of muscle damage is related to the magnitude of the cytokine response. In the third and final section, we look at the satellite cell response to a single bout of eccentric exercise, as well as the role of the cyclooxygenase enzymes (COX1 and 2). In summary, we propose that muscle damage as evaluated by changes in muscle function is related to leucocyte accumulation in the exercised muscles. 'Extreme' exercise protocols, encompassing unaccustomed maximal eccentric exercise across a large range of motion, generally inflict severe muscle damage, inflammation and prolonged recovery (> 1 week). By contrast, exercise resembling regular athletic training (resistance exercise and downhill running) typically causes mild muscle damage (myofibrillar disruptions) and full recovery normally occurs within a few days. Large variation in individual responses to a given exercise should, however be expected. The link between cytokine and satellite cell responses and exercise-induced muscle damage is not so clear The systemic cytokine response may be linked more closely to the metabolic demands of exercise rather than muscle damage. With the exception of IL-6, the sources of systemic cytokines following exercise remain unclear The satellite cell response to severe muscle damage is related to regeneration, whereas the biological significance of satellite cell proliferation after mild damage or non-damaging exercise remains uncertain. The COX enzymes regulate satellite cell activity, as demonstrated in animal models; however the roles of the COX enzymes in human skeletal muscle need further investigation. We suggest using the term 'muscle damage' with care. Comparisons between studies and individuals must consider changes in and recovery of muscle force-generating capacity.

Vaccination of healthy and diseased koalas (Phascolarctos cinereus) with a Chlamydia pecorum multi-subunit vaccine : evaluation of immunity and pathology

Chlamydial infections represent a major threat to the long-term survival of the koala and a successful vaccine would provide a valuable management tool. Vaccination however has the potential to enhance inflammatory disease in animals exposed to a natural infection prior to vaccination, a finding in early human and primate trials of whole cell vaccines to prevent trachoma. In the present study, we vaccinated both healthy koalas as well as clinically diseased koalas with a multi-subunit vaccine consisting of Chlamydia pecorum MOMP and NrdB mixed with immune stimulating complex as adjuvant. Following vaccination, there was no increase in inflammatory pathological changes in animals previously infected with Chlamydia. Strong antibody (including neutralizing antibodies) and lymphocyte proliferation responses were recorded in all vaccinated koalas, both healthy and clinically diseased. Vaccine induced antibodies specific for both vaccine antigens were observed not only in plasma but also in ocular secretions. Our data shows that an experimental chlamydial vaccine is safe to use in previously infected koalas, in that it does not worsen infection-associated lesions. Furthermore, the prototype vaccine is effective, as demonstrated by strong levels of neutralizing antibody and lymphocyte proliferation responses in both healthy and clinically diseased koalas. Collectively, this work illustrates the feasibility of developing a safe and effective Chlamydia vaccine as a tool for management of disease in wild koalas.

Fibroblast growth factor receptor inhibition synergizes with paclitaxel and doxorubicin in endometrial cancer cells

OBJECTIVE: The fibroblast growth factor (FGF) family of signaling molecules has been associated with chemoresistance and poor prognosis in a number of cancer types, including lung, breast, ovarian, prostate, and head and neck carcinomas. Given the identification of activating mutations in the FGF receptor 2 (FGFR2) receptor tyrosine kinase in a subset of endometrial tumors, agents with activity against FGFRs are currently being tested in clinical trials for recurrent and progressive endometrial cancer. Here, we evaluated the effect of FGFR inhibition on the in vitro efficacy of chemotherapy in endometrial cancer cell lines. METHODS: Human endometrial cancer cell lines with wild-type or activating FGFR2 mutations were used to determine any synergism with concurrent use of the pan-FGFR inhibitor, PD173074, and the chemotherapeutics, doxorubicin and paclitaxel, on cell proliferation and apoptosis. RESULTS: FGFR2 mutation status did not alter sensitivity to either chemotherapeutic agent alone. The combination of PD173074 with paclitaxel or doxorubicin showed synergistic activity in the 3 FGFR2 mutant cell lines evaluated. In addition, although nonmutant cell lines were resistant to FGFR inhibition alone, the addition of PD173074 potentiated the cytostatic effect of paclitaxel and doxorubicin in a subset of FGFR2 wild-type endometrial cancer cell lines. CONCLUSIONS: Together these data suggest a potential therapeutic benefit to combining an FGFR inhibitor with standard chemotherapeutic agents in endometrial cancer therapy particularly in patients with FGFR2 mutation positive tumors.