Evaluation of fibroin-based scaffolds for ocular tissue reconstruction

The epithelium of the corneolimbus contains stem cells for regenerating the corneal epithelium. Diseases and injuries affecting the limbus can lead to a condition known as limbal stem cell deficiency (LSCD), which results in loss of the corneal epithelium, and subsequent chronic inflammation and scarring of the ocular surface. Advances in the treatment of LSCD have been achieved through use of cultured human limbal epithelial (HLE) grafts to restore epithelial stem cells of the ocular surface. These epithelial grafts are usually produced by the ex vivo expansion of HLE cells on human donor amniotic membrane (AM), but this is not without limitations. Although AM is the most widely accepted substratum for HLE transplantation, donor variation, risk of disease transfer, and rising costs have led to the search for alternative biomaterials to improve the surgical outcome of LSCD. Recent studies have demonstrated that Bombyx mori silk fibroin (hereafter referred to as fibroin) membranes support the growth of primary HLE cells, and thus this thesis aims to explore the possibility of using fibroin as a biomaterial for ocular surface reconstruction. Optimistically, the grafted sheets of cultured epithelium would provide a replenishing source of epithelial progenitor cells for maintaining the corneal epithelium, however, the HLE cells lose their progenitor cell characteristics once removed from their niche. More severe ocular surface injuries, which result in stromal scarring, damage the epithelial stem cell niche, which subsequently leads to poor corneal re-epithelialisation post-grafting. An ideal solution to repairing the corneal limbus would therefore be to grow and transplant HLE cells on a biomaterial that also provides a means for replacing underlying stromal cells required to better simulate the normal stem cell niche. The recent discovery of limbal mesenchymal stromal cells (L-MSC) provides a possibility for stromal repair and regeneration, and therefore, this thesis presents the use of fibroin as a possible biomaterial to support a three dimensional tissue engineered corneolimbus with both an HLE and underlying L-MSC layer. Investigation into optimal scaffold design is necessary, including adequate separation of epithelial and stromal layers, as well as direct cell-cell contact. Firstly, the attachment, morphology and phenotype of HLE cells grown on fibroin were directly compared to that observed on donor AM, the current clinical standard substrate for HLE transplantation. The production, transparency, and permeability of fibroin membranes were also evaluated in this part of the study. Results revealed that fibroin membranes could be routinely produced using a custom-made film casting table and were found to be transparent and permeable. Attachment of HLE cells to fibroin after 4 hours in serum-free medium was similar to that supported by tissue culture plastic but approximately 6-fold less than that observed on AM. While HLE cultured on AM displayed superior stratification, epithelia constructed from HLE on fibroin maintained evidence of corneal phenotype (cytokeratin pair 3/12 expression; CK3/12) and displayed a comparable number and distribution of ÄNp63+ progenitor cells to that seen in cultures grown on AM. These results confirm the suitability of membranes constructed from silk fibroin as a possible substrate for HLE cultivation. One of the most important aspects in corneolimbal tissue engineering is to consider the reconstruction of the limbal stem cell niche to help form the natural limbus in situ. MSC with similar properties to bone marrow derived-MSC (BM-MSC) have recently been grown from the limbus of the human cornea. This thesis evaluated methods for culturing L-MSC and limbal keratocytes using various serum-free media. The phenotype of resulting cultures was examined using photography, flow cytometry for CD34 (keratocyte marker), CD45 (bone marrow-derived cell marker), CD73, CD90, CD105 (collectively MSC markers), CD141 (epithelial/vascular endothelial marker), and CD271 (neuronal marker), immunocytochemistry (alpha-smooth muscle actin; á-sma), differentiation assays (osteogenesis, adipogenesis and chrondrogenesis), and co-culture experiments with HLE cells. While all techniques supported to varying degrees establishment of keratocyte and L-MSC cultures, sustained growth and serial propagation was only achieved in serum-supplemented medium or the MesenCult-XF„¥ culture system (Stem Cell Technologies). Cultures established in MesenCult-XF„¥ grew faster than those grown in serum-supplemented medium and retained a more optimal MSC phenotype. L-MSC cultivated in MesenCult-XFR were also positive for CD141, rarely expressed £\-sma, and displayed multi-potency. L-MSC supported growth of HLE cells, with the largest epithelial islands being observed in the presence of L-MSC established in MesenCult-XF„¥ medium. All HLE cultures supported by L-MSC widely expressed the progenitor cell marker £GNp63, along with the corneal differentiation marker CK3/12. Our findings conclude that MesenCult-XFR is a superior culture system for L-MSC, but further studies are required to explore the significance of CD141 expression in these cells. Following on from the findings of the previous two parts, silk fibroin was tested as a novel dual-layer construct containing both an epithelium and underlying stroma for corneolimbal reconstruction. In this section, the growth and phenotype of HLE cells on non-porous versus porous fibroin membranes was compared. Furthermore, the growth of L-MSC in either serum-supplemented medium or the MesenCult-XFR culture system within fibroin fibrous mats was investigated. Lastly, the co-culture of HLE and L-MSC in serum-supplemented medium on and within fibroin dual-layer constructs was also examined. HLE on porous membranes displayed a flattened and squamous monolayer; in contrast, HLE on non-porous fibroin appeared cuboidal and stratified closer in appearance to a normal corneal epithelium. Both constructs maintained CK3/12 expression and distribution of £GNp63+ progenitor cells. Dual-layer fibroin scaffolds consisting of HLE cells and L-MSC maintained a similar phenotype as on the single layers alone. Overall, the present study proposed to create a three dimensional limbal tissue substitute of HLE cells and L-MSC together, ultimately for safe and beneficial transplantation back into the human eye. The results show that HLE and L-MSC can be cultivated separately and together whilst maintaining a clinically feasible phenotype containing a majority of progenitor cells. In addition, L-MSC were able to be cultivated routinely in the MesenCult-XF® culture system while maintaining a high purity for the MSC characteristic phenotype. However, as a serum-free culture medium was not found to sustain growth of both HLE and L-MSC, the combination scaffold was created in serum-supplemented medium, indicating that further refinement of this cultured limbal scaffold is required. This thesis has also demonstrated a potential novel marker for L-MSC, and has generated knowledge which may impact on the understanding of stromal-epithelial interactions. These results support the feasibility of a dual-layer tissue engineered corneolimbus constructed from silk fibroin, and warrant further studies into the potential benefits it offers to corneolimbal tissue regeneration. Further refinement of this technology should explore the potential benefits of using epithelial-stromal co-cultures with MesenCult-XF® derived L-MSC. Subsequent investigations into the effects of long-term culture on the phenotype and behaviour of the cells in the dual-layer scaffolds are also required. While this project demonstrated the feasibility in vitro for the production of a dual-layer tissue engineered corneolimbus, further studies are required to test the efficacy of the limbal scaffold in vivo. Future in vivo studies are essential to fully understand the integration and degradation of silk fibroin biomaterials in the cornea over time. Subsequent experiments should also investigate the use of both AM and silk fibroin with epithelial and stromal cell co-cultures in an animal model of LSCD. The outcomes of this project have provided a foundation for research into corneolimbal reconstruction using biomaterials and offer a stepping stone for future studies into corneolimbal tissue engineering.

A tissue engineering solution for segmental defect regeneration in load-bearing long bones

The reconstruction of large defects (>10 mm) in humans usually relies on bone graft transplantation. Limiting factors include availability of graft material, comorbidity, and insufficient integration into the damaged bone. We compare the gold standard autograft with biodegradable composite scaffolds consisting of medical-grade polycaprolactone and tricalcium phosphate combined with autologous bone marrow-derived mesenchymal stem cells (MSCs) or recombinant human bone morphogenetic protein 7 (rhBMP-7). Critical-sized defects in sheep - a model closely resembling human bone formation and structure - were treated with autograft, rhBMP-7, or MSCs. Bridging was observed within 3 months for both the autograft and the rhBMP-7 treatment. After 12 months, biomechanical analysis and microcomputed tomography imaging showed significantly greater bone formation and superior strength for the biomaterial scaffolds loaded with rhBMP-7 compared to the autograft. Axial bone distribution was greater at the interfaces. With rhBMP-7, at 3 months, the radial bone distribution within the scaffolds was homogeneous. At 12 months, however, significantly more bone was found in the scaffold architecture, indicating bone remodeling. Scaffolds alone or with MSC inclusion did not induce levels of bone formation comparable to those of the autograft and rhBMP-7 groups. Applied clinically, this approach using rhBMP-7 could overcome autograft-associated limitations.

Bone tissue engineering : reconstruction of critical sized segmental bone defects in the ovine tibia

Well-established therapies for bone defects are restricted to bone grafts which face significant disadvantages (limited availability, donor site morbidity, insufficient integration). Therefore, the objective was to develop an alternative approach investigating the regenerative potential of medical grade polycaprolactone-tricalcium phosphate (mPCL-TCP) and silk-hydroxyapatite (silk-HA) scaffolds. Critical sized ovine tibial defects were created and stabilized. Defects were left untreated, reconstructed with autologous bone grafts (ABG) and mPCL-TCP or silk-HA scaffolds. Animals were observed for 12 weeks. X-ray analysis, torsion testing and quantitative computed tomography (CT) analyses were performed. Radiological analysis confirmed the critical nature of the defects. Full defect bridging occurred in the autograft and partial bridging in the mPCL-TCP group. Only little bone formation was observed with silk-HA scaffolds. Biomechanical testing revealed a higher torsional moment/stiffness (p < 0.05) and CT analysis a significantly higher amount of bone formation for the ABG group when compared to the silk-HA group. No significant difference was determined between the ABG and mPCL-TCP groups. The results of this study suggest that mPCL-TCP scaffolds combined can serve as an alternative to autologous bone grafting in long bone defect regeneration. The combination of mPCL-TCP with osteogenic cells or growth factors represents an attractive means to further enhance bone formation.

Vastu Purusha Mandala : A human ecological framework for designing living environments

This paper aims to present ‘vastu purusha mandala’(VPM), a symbolic diagram used in the indigenous system of Indian architecture as a human ecologic frame work for designing living environments. The article begins with an attempt to provide a working definition for the ‘living environment’ based on the theories developed by Rapoport (2005) and Lawrence (2001). It then discusses the symbolism and the human ecologic significance of VPM. This is substantiated through the works of Kramrisch (1976), Moore (1989), Shukla (1996) and Chakrabarthi (1998). Some recent papers on Vastu Shastra are also examined. Furthermore, VPM is compared with the livability guidelines developed for high-rise living by the Centre for Subtropical Design, Queensland University of Technology, Brisbane, Australia. A meaningful interpretation of vastushastra which is free from mysticism and symbolism is proffered through this paper.

Application of high voltage, high frequency pulsed electromagnetic field on cortical bone tissue

Over the last few decades, electric and electromagnetic fields have achieved important role as stimulator and therapeutic facility in biology and medicine. In particular, low magnitude, low frequency, pulsed electromagnetic field has shown significant positive effect on bone fracture healing and some bone diseases treatment. Nevertheless, to date, little attention has been paid to investigate the possible effect of high frequency, high magnitude pulsed electromagnetic field (pulse power) on functional behaviour and biomechanical properties of bone tissue. Bone is a dynamic, complex organ, which is made of bone materials (consisting of organic components, inorganic mineral and water) known as extracellular matrix, and bone cells (live part). The cells give the bone the capability of self-repairing by adapting itself to its mechanical environment. The specific bone material composite comprising of collagen matrix reinforced with mineral apatite provides the bone with particular biomechanical properties in an anisotropic, inhomogeneous structure. This project hypothesized to investigate the possible effect of pulse power signals on cortical bone characteristics through evaluating the fundamental mechanical properties of bone material. A positive buck-boost converter was applied to generate adjustable high voltage, high frequency pulses up to 500 V and 10 kHz. Bone shows distinctive characteristics in different loading mode. Thus, functional behaviour of bone in response to pulse power excitation were elucidated by using three different conventional mechanical tests applying three-point bending load in elastic region, tensile and compressive loading until failure. Flexural stiffness, tensile and compressive strength, hysteresis and total fracture energy were determined as measure of main bone characteristics. To assess bone structure variation due to pulse power excitation in deeper aspect, a supplementary fractographic study was also conducted using scanning electron micrograph from tensile fracture surfaces. Furthermore, a non-destructive ultrasonic technique was applied for determination and comparison of bone elasticity before and after pulse power stimulation. This method provided the ability to evaluate the stiffness of millimetre-sized bone samples in three orthogonal directions. According to the results of non-destructive bending test, the flexural elasticity of cortical bone samples appeared to remain unchanged due to pulse power excitation. Similar results were observed in the bone stiffness for all three orthogonal directions obtained from ultrasonic technique and in the bone stiffness from the compression test. From tensile tests, no significant changes were found in tensile strength and total strain energy absorption of the bone samples exposed to pulse power compared with those of the control samples. Also, the apparent microstructure of the fracture surfaces of PP-exposed samples (including porosity and microcracks diffusion) showed no significant variation due to pulse power stimulation. Nevertheless, the compressive strength and toughness of millimetre-sized samples appeared to increase when the samples were exposed to 66 hours high power pulsed electromagnetic field through screws with small contact cross-section (increasing the pulsed electric field intensity) compare to the control samples. This can show the different load-bearing characteristics of cortical bone tissue in response to pulse power excitation and effectiveness of this type of stimulation on smaller-sized samples. These overall results may address that although, the pulse power stimulation can influence the arrangement or the quality of the collagen network causing the bone strength and toughness augmentation, it apparently did not affect the mineral phase of the cortical bone material. The results also confirmed that the indirect application of high power pulsed electromagnetic field at 500 V and 10 kHz through capacitive coupling method, was athermal and did not damage the bone tissue construction.

Regulating virtual environments

Online gaming environments feature a number of challenging regulatory issues; a diverse player base, uneven power relationship, and lack of real dispute resolution mechanisms. By conducting an ethnographic study of the online environment Eve Online, and using as a comparative the offshore gaming industry, I consider how we might look to regulate, and resolve disputes within, online gaming environments. In doing so, I adopted a novel approach to the study of online gaming environments - that of norms - which gave significance not only to the terms of service dictated by platform providers and their legal advisors, but also to the social and ludic limitations and affordances players constructed themselves. Finally, through an account of the evolution of regulatory mechanisms and dispute resolution in the offshore gambling industry, I consider how an environment which features much in common with online gaming environments overcame a number of these challenges within the last 10-15 years, and what lessons might be taken from those experiences and applied to contemporary online gaming environments.

Selective essays in competitive environments

This thesis is a collection of essays that utilises descriptive and empirical tools to examine competitive environments such as in academia, superrich and sport. The essays capture different aspects of the winner-take-all phenomenon by looking at citation and publication inequality in a top tier economics journal namely the American Economic Review. How globalisation and corruption influence the accumulation of extraordinary wealth and finally, how in a fairly equal competition, that is in the National Rugby League in Australia, wearing red shirts could lead to a comparative advantage and hence, tip the balance between winning and losing. The results within academia indicate that a highly unequal distribution exist, in which only a few top authors or institutions produce the majority of output. Furthermore, the results obtained in the superrich environment indicate that corruption and globalisation enhances the accumulation of extraordinary wealth. Finally, the results in the sport environment are mixed. While we find support for a positive effect of wearing red jerseys in our descriptive analysis, we find a negative effect when we control at the team level. However, when we investigate the relative difference in the degree of redness between home and away team, we find a quite strong positive effect of wearing red shirts even after controlling at the team level.

Nonlinear H_infinity control of UAVs for collision avoidance in gusty environments

This paper proposes a nonlinear H_infinity controller for stabilization of velocities, attitudes and angular rates of a fixed-wing unmanned aerial vehicle (UAV) in a windy environment. The suggested controller aims to achieve a steady-state flight condition in the presence of wind gusts such that the host UAV can be maneuvered to avoid collision with other UAVs during cruise flight with safety guarantees. This paper begins with building a proper model capturing flight aerodynamics of UAVs. Then a nonlinear controller is developed with gust attenuation and rapid response properties. Simulations are conducted for the Shadow UAV to verify performance of the proposed con- troller. Comparative studies with the proportional-integral-derivative (PID) controllers demonstrate that the proposed controller exhibits great performance improvement in a gusty environment, making it suitable for integration into the design of flight control systems for cruise flight of UAVs.

Managing distraction in 21st century learning environments

First year students attend face-to-face classes armed with an arsenal of internet enabled digital devices. The conundrum is that while these devices offer scope for enhancing opportunities for engagement in face-to-face learning, they may simultaneously distract students away from learning and compound isolation issues. This paper considers how to best to use these devices for maximum engagement in first year face-to-face learning so as to assist students in connecting with other learners and instructors within the learning environment

Current status and future prospects for cultured limbal tissue transplants in Australia and New Zealand

Cultured limbal tissue transplants have become widely used over the last decade as a treatment for limbal stem cell deficiency (LSCD). While the number of patients afflicted with LSCD in Australia and New Zealand is considered to be relatively low, the impact of this disease on quality of life is so severe that the potential efficacy of cultured transplants has necessitated investigation. We presently review the basic biology and experimental strategies associated with the use of cultured limbal tissue transplants in Australia and New Zealand. In doing so, we aim to encourage informed discussion on the issues required to advance the use of cultured limbal transplants in Australia and New Zealand. Moreover, we propose that a collaborative network could be established to maintain access to the technology in conjunction with a number of other existing and emerging treatments for eye diseases.

Experimental study on mechanical properties of pumpkin tissue

Purpose: The purpose of this study was to calculate mechanical properties of tough skinned vegetables as a part of Finite Element Modelling (FEM) and simulation of tissue damage during mechanical peeling of tough skinned vegetables. Design/methodology: There are some previous studies on mechanical properties of fruits and vegetables however, behaviour of tissue under different processing operations will be different. In this study indentation test was performed on Peel, Flesh and Unpeeled samples of pumpkin as a tough skinned vegetable. Additionally, the test performed in three different loading rates for peel: 1.25, 10, 20 mm/min and 20 mm/min for flesh and unpeeled samples respectively. The spherical end indenter with 8mm diameter used for the experimental tests. Samples prepare from defect free and ripped pumpkin purchased from local shops in Brisbane, Australia. Humidity and temperature were 20-55% and 20-250C respectively. Findings: Consequently, force deformation and stress and strain of samples were calculated and shown in presented figures. Relative contribution (%) of skin to different mechanical properties is computed and compared with data available from literature. According the results, peel samples had the highest value of rupture force (291N) and as well as highest value of firmness (1411Nm-1). Research limitations/implications: The proposed study focused on one type of tough skinned vegetables and one variety of pumpkin however, more tests will give better understandings of behaviours of tissue. Additionally, the behaviours of peel, unpeeled and flesh samples in different speed of loading will provide more details of tissue damages during mechanical loading. Originality/value: Mechanical properties of pumpkin tissue calculated using the results of indentation test, specifically the behaviours of peel, flesh and unpeeled samples were explored which is a new approach in Finite Element Modelling (FEM) of food processes. Keywords: Finite Element Modelling (FEM), relative contribution, firmness, toughness and rupture force.

Towards visualising people’s ecology of hybrid personal learning environments

Ambient media architecture can provide place-based collaborative learning experiences and pathways for social interactions that would not be otherwise possible. This paper is concerned with ways of enhancing peer-to-peer learning affordances in library spaces; how can the library facilitate the community of library users to learn from each other? We report on the findings of a study that employed a participatory design method where participants were asked to reflect and draw places, social networks, and activities that they use to work (be creative, productive), play (have fun, socialize, be entertained), and learn (acquire new information, knowledge, or skills). The results illustrate how informal learning – learning outside the formal education system – is facilitated by a personal selection of physical and socio-cultural environments, as well as online tools, platforms, and networks. This paper sheds light on participants’ individually curated ecologies of their work, play, and learning related networks and the hybrid (physical and digital) nature of these places. These insights reveal opportunities for ambient media architecture to increase awareness of and connections between people’s hybrid personal learning environments.

Detection of tissue plasminogen activator (t-PA) and plasminogen activator inhibitor 2(PAI-2) in gingival crevicular fluid from healthy, gingivitis and periodontitis patients

Background: The regulation of plasminogen activation is a key element in controlling proteolytic events in the extracellular matrix. Our previous studies had demonstrated that in inflamed gingival tissues, tissue-type plasminogen activator (t-PA) is significantly increased in the extracellular matrix of the connective tissue and that interleukin 1β (IL-1β) can up regulate the level of t-PA and plasminogen activator inhibitor-2 (PAI-2) synthesis by human gingival fibroblasts. Method: In the present study, the levels of t-PA and PAI-2 in gingival crevicular fluid (GCF) were measured from healthy, gingivitis and periodontitis sites and compared before and after periodontal treatment. Crevicular fluid from106 periodontal sites in 33 patients were collected. 24 sites from 11 periodontitis patients received periodontal treatment after the first sample collection and post-treatment samples were collected 14 days after treatment. All samples were analyzed by enzyme-linked immunosorbent assay (ELISA) for t-PA and PAI-2. Results: The results showed that significantly high levels of t-PA and PAI-2 in GCF were found in the gingivitis and periodontitis sites. Periodontal treatment led to significant decreases of PAI-2, but not t-PA, after 14 days. A significant positive linear correlation was found between t-PA and PAI-2 in GCF (r=0.80, p<0.01). In the healthy group, different sites from within the same subject showed little variation of t-PA and PAI-2 in GCF. However, the gingivitis and periodontitis sites showed large variation. These results suggest a good correlation between t-PA and PAI-2 with the severity of periodontal conditions. Conclusion: This study indicates that t-PA and PAI-2 may play a significant rôle in the periodontal tissue destruction and tissue remodeling and that t-PA and PAI-2 in GCF may be used as clinical markers to evaluate the periodontal diseases and assess treatment.

Nitric oxide synthase type-II is synthesized by human gingival tissue and cultured human gingival fibroblasts

Nitric oxide is known to be an important inflammatory mediator, and is implicated in the pathophysiology of a range of inflammatory disorders. The aim of this study was to determine the localization and distribution of endothelial NOS (NOS-II) in human gingival tissue, and to ascertain if human gingival fibroblasts express NOS-II when stimulated with interferon gamma (IFN-gamma) and bacterial lipopolysaccharide (LPS). The distribution of NOS-II in inflamed and non-inflamed specimens of human gingivae was studied using a monoclonal antibody against nitric oxide synthase II. Cultures of fibroblasts derived from healthy human gingivae were used for the cell culture experiments. The results from immunohistochemical staining of the tissues indicated an upregulation of NOS-II expression in inflamed compared to non-inflamed gingival tissue. Fibroblasts and inflammatory cells within the inflamed connective tissue were positively stained for NOS-II. In addition, basal keratinocytes also stained strongly for NOS-II, in both healthy and inflamed tissue sections. When cultured human gingival fibroblasts were stimulated by INF-gamma and Porphyromonas gingivalis LPS, NOS-II was more strongly expressed than when the cells were exposed to LPS or IFN-gamma alone. These data suggest that, as for other inflammatory diseases, NO plays a role in the pathophysiology of periodontitis.

Effect of lipopolysaccharide from periodontal pathogens on the production of tissue plasminogen activator and plasminogen activator inhibitor 2 by human gingival fibroblasts.

Both tissue plasminogen activator (t-PA) and plasminogen activator inhibitor 2 (PAI-2) are important proteolysis factors present in inflamed human periodontal tissues. The aim of the present study was to investigate the effect of lipopolysaccharide (LPS) on the synthesis of t-PA and PAI-2 by human gingival fibroblasts (HGF). LPS from different periodontal pathogens including Actinobacillus actinomycetemcomitans, Porphyromonas gingivalis and Fusobacterium nucleatum were extracted by the hot phenol water method. The levels of t-PA and PAI-2 secreted into the cell culture media were measured by enzyme-linked immunosorbent assays (ELISA). The mRNA for t-PA and PAI-2 were measured by RT-PCR. The results showed t-PA synthesis was increased in response to all types of LPS studied and PAI-2 level was increased by LPS from A. actinomycetemcomitans and F. nucleatum, but not P. gingivalis. When comparing the effects of LPS from non-periodontal bacteria (Escherichia coli and Salmonella enteritidis) with the LPS from periodontal pathogens, we found that the ratio of t-PA to PAI-2 was greater following exposure of the cells to LPS from periodontal pathogens. The highest ratio of t-PA to PAI-2 was found in those cells exposed to LPS from P. gingivalis. These results indicate that LPS derived from periodontal pathogens may cause unbalanced regulation of plasminogen activator and plasminogen activator inhibitor by HGF and such an effect may, in part, contribute to the destruction of periodontal connective tissue through dysregulated pericellular proteolysis.