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.

Combined VEGF and PDGF treatment reduces secondary degeneration after spinal cord injury

Trauma to the spinal cord creates an initial physical injury damaging neurons, glia, and blood vessels, which then induces a prolonged inflammatory response, leading to secondary degeneration of spinal cord tissue, and further loss of neurons and glia surrounding the initial site of injury. Angiogenesis is a critical step in tissue repair, but in the injured spinal cord angiogenesis fails; blood vessels formed initially later regress. Stabilizing the angiogenic response is therefore a potential target to improve recovery after spinal cord injury (SCI). Vascular endothelial growth factor (VEGF) can initiate angiogenesis, but cannot sustain blood vessel maturation. Platelet-derived growth factor (PDGF) can promote blood vessel stability and maturation. We therefore investigated a combined application of VEGF and PDGF as treatment for traumatic spinal cord injury, with the aim to reduce secondary degeneration by promotion of angiogenesis. Immediately after hemisection of the spinal cord in the rat we delivered VEGF and PDGF and to the injury site. One and 3 months later the size of the lesion was significantly smaller in the treated group compared to controls, and there was significantly reduced gliosis surrounding the lesion. There was no significant effect of the treatment on blood vessel density, although there was a significant reduction in the numbers of macrophages/microglia surrounding the lesion, and a shift in the distribution of morphological and immunological phenotypes of these inflammatory cells. VEGF and PDGF delivered singly exacerbated secondary degeneration, increasing the size of the lesion cavity. These results demonstrate a novel therapeutic intervention for SCI, and reveal an unanticipated synergy for these growth factors whereby they modulated inflammatory processes and created a microenvironment conducive to axon preservation/sprouting.

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.

The neglected dimension of community liveability : impact on social connectedness and active ageing in higher density accommodation

Purpose This thesis is about liveability, place and ageing in the high density urban landscape of Brisbane, Australia. As with other major developed cities around the globe, Brisbane has adopted policies to increase urban residential densities to meet the main liveability and sustainability aim of decreasing car dependence and therefore pollution, as well as to minimise the loss of greenfield areas and habitats to developers. This objective hinges on urban neighbourhoods/communities being liveable places, which residents do not have to leave for everyday living. Community/neighbourhood liveability is an essential ingredient in healthy ageing in place and has a substantial impact upon the safety, independence and well-being of older adults. It is generally accepted that ageing in place is optimal for both older people and the state. The optimality of ageing in place generally assumes that there is a particular quality to environments or standard of liveability in which people successfully age in place. The aim of this thesis was to examine if there are particular environmental qualities or aspects of liveability that test optimality and to better understand the key liveability factors that contribute to successful ageing in place. Method A strength of this thesis is that it draws on two separate studies to address the research question of what makes high density liveable for older people. In Chapter 3, the two methods are identified and differentiated as Method 1 (used in Paper 1) and Method 2 (used in Papers 2, 3, 4 and 5). Method 1 involved qualitative interviews with 24 inner city high density Brisbane residents. The major strength of this thesis is the innovative methodology outlined in the thesis as Method 2. Method 2 involved a case study approach employing qualitative and quantitative methods. Qualitative data was collected using semi-structured, in-depth interviews and time-use diaries completed by participants during the week of tracking. The quantitative data was gathered using Global Positioning Systems for tracking and Geographical Information Systems for mapping and analysis of participants’ activities. The combination of quantitative and qualitative analysis captured both participants’ subjective perceptions of their neighbourhoods and their patterns of movement. This enhanced understanding of how neighbourhoods and communities function and of the various liveability dimensions that contribute to active ageing and ageing in place for older people living in high density environments. Both studies’ participants were inner-city high density residents of Brisbane. The study based on Method 1 drew on a wider age demographic than the study based on Method 2. Findings The five papers presented in this thesis by publication indicate a complex inter-relationship of the factors that make a place liveable. The first three papers identify what is comparable and different between the physical and social factors of high density communities/neighbourhoods. The last two papers explore relationships between social engagement and broader community variables such as infrastructure and the physical built environments that are risk or protective factors relevant to community liveability, active ageing and ageing in place in high density. The research highlights the importance of creating and/or maintaining a barrier-free environment and liveable community for ageing adults. Together, the papers promote liveability, social engagement and active ageing in high density neighbourhoods by identifying factors that constitute liveability and strategies that foster active ageing and ageing in place, social connections and well-being. Recommendations There is a strong need to offer more support for active ageing and ageing in place. While the data analyses of this research provide insight into the lived experience of high density residents, further research is warranted. Further qualitative and quantitative research is needed to explore in more depth, the urban experience and opinions of older people living in urban environments. In particular, more empirical research and theory-building is needed in order to expand understanding of the particular environmental qualities that enable successful ageing in place in our cities and to guide efforts aimed at meeting this objective. The results suggest that encouraging the presence of more inner city retail outlets, particularly services that are utilised frequently in people’s daily lives such as supermarkets, medical services and pharmacies, would potentially help ensure residents fully engage in their local community. The connectivity of streets, footpaths and their role in facilitating the reaching of destinations are well understood as an important dimension of liveability. To encourage uptake of sustainable transport, the built environment must provide easy, accessible connections between buildings, walkways, cycle paths and public transport nodes. Wider streets, given that they take more time to cross than narrow streets, tend to .compromise safety - especially for older people. Similarly, the width of footpaths, the level of buffering, the presence of trees, lighting, seating and design of and distance between pedestrian crossings significantly affects the pedestrian experience for older people and impacts upon their choice of transportation. High density neighbourhoods also require greater levels of street fixtures and furniture for everyday life to make places more useable and comfortable for regular use. The importance of making the public realm useful and habitable for older people cannot be over-emphasised. Originality/value While older people are attracted to high density settings, there has been little empirical evidence linking liveability satisfaction with older people’s use of urban neighbourhoods. The current study examined the relationships between community/neighbourhood liveability, place and ageing to better understand the implications for those adults who age in place. The five papers presented in this thesis add to the understanding of what high density liveable age-friendly communities/ neighbourhoods are and what makes them so for older Australians. Neighbourhood liveability for older people is about being able to age in place and remain active. Issues of ageing in Australia and other areas of the developed world will become more critical in the coming decades. Creating livable communities for all ages calls for partnerships across all levels of government agencies and among different sectors within communities. The increasing percentage of older people in the community will have increasing political influence and it will be a foolish government who ignores the needs of an older society.

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.

The use of amenities in high density neighbourhoods by older urban Australians residents

Like other major cities, Brisbane (Australia) has adopted policies to increase residential densities to meet the liveability goal of decreasing car dependence. This objective hinges on urban neighbourhoods being amenity-rich spaces, reducing the need for residents to leave their neighbourhood for everyday living. While older people are attracted to urban settings, there has been little empirical evidence linking liveability satisfaction with older people's use of urban neighbourhoods. Using a case study approach employing qualitative (diaries, in-depth interviews) and quantitative (Global Positioning Systems and Geographical Information Systems mapping) methods,this paper explores the effect of the neighbourhood environment and its influence on liveability for older urban people. Reliance on motor vehicles and issues with availability and access to local amenities inhibit local participation for older people. Highlighting these issues furthers our understanding of the landscape planning and design factors that make urban neighbourhoods more liveable for older residents.

Experiences of neighbourhood walkability among older Australians living in high density inner-city areas

Walking as an out-of-home mobility activity is recognised for its contribution to healthy and active ageing. The environment can have a powerful effect on the amount of walking activity undertaken by older people, thereby influencing their capacity to maintain their wellbeing and independence. This paper reports the findings from research examining the experiences of neighbourhood walking for 12 older people from six different inner-city high density suburbs, through analysis of data derived from travel diaries, individual time/space activity maps (created via GPS tracking over a seven-day period and GIS technology), and in-depth interviews. Reliance on motor vehicles, the competing interests of pedestrians and cyclists on shared pathways and problems associated with transit systems, public transport, and pedestrian infrastructure emerged as key barriers to older people venturing out of home on foot. GPS and GIS technology provide new opportunities for furthering understanding of the out-of-home mobility of older populations.

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.