Articular cartilage is a white connective tissue covering the ends of long bones to facilitate movement and articulation. Due to the avascular nature of the tissue, it has a limited capacity to repair once damaged. Human induced pluripotent stem cells are suggested as an ethical and ultimate source for cell based-therapies. Tissue engineering using these cells might present as a promising treatment for cartilage defects. Previous tissue engineering attempts using multipotent stem cells such as adult msenchymal stem cells or pluripotent stem cells such as embryonic stem cells were qualitative, variable and result in the formation of a tissue of a fibrocatilagenous phenotype. This study aims to identify the factors that control the differentiation of human induced pluripotent stem cells towards the chondrogenic lineage and to then assess their capacity to scale up and use in 3D cartilage tissue engineering. The hiPSC lines, C18 and C19, were shown to express pluripotent transcription factors OCT4 and Nanog by immunofluorescence and have the capacity to differentiate into the cells descended from the three germ layers. The pluripotent stem cells were directed to the chondrogenic lineage in a feeder-free and chemically defined system using a sequential addition of growth factors including Activin A, FGF-2 and BMP-4. The derived chondroprogenitors showed similar characteristics to genuine mesenchymal stem cells including adherence to plastic, fibroblastic morphology and expression of cell surface markers, except for the trilineage potential. The derived cells were seeded on 3D fibronectin coated PGA scaffolds and other biomaterials stimulated with TGF-p3 and BMP-7 resulting in a type IT collagen production reaching similar levels to previously reported levels with mesenchymal stem cells and a sGAG production of similar levels to native cartilage reaching 40% of dry weight.

No description available.

No description available.

The routine use of regenerative medicine products in patients requires cost-effective manufacturing processes and products that meet business and customer needs. The dermal skin substitute lCX-SKN, produced by lntercytex for the treatment of acute wounds, completed Phase I clinical trials in 2007. lCX-SKN is manufactured by seeding neonatal human dermal fibroblasts in a fibrin matrix and culturing for 49 days to form a collagen matrix synthesised by the cells. The results captured by this thesis demonstrate an integrated engineering and biological science approach to improve the current lCX-SKN process model and identify methods for process and product improvement. Measurement of the changes in the biochemical, mechanical and physical properties of lCX-SKN during the 49 day manufacturing period produced an improved four-phase process model describing cell proliferation, matrix compaction, fibrin degradation, collagen synthesis and matrix remodelling. Ultrasound was identified as a scalable form of mechanical stimulation for product improvement particularly as it does not require physical coupling to the constructs. A custom-built ultrasound device was used to investigate the effect of ultrasound on collagen synthesis and mechanical properties. A design of experiments showed that different combinations of ultrasound intensity (0.5-2.5W/cm2 ), duty cycle (5-80%) and duration (5-30minutes) affected the shear storage modulus (G') and collagen content. However, a significant effect on G' only resulted from combinations of duty cycle and duration. Further experiments to improve the properties of the construct, using 0.5W/cm2 intensity, 50% duty cycle and 14 minute duration resulted in a 73% increase in G' primarily through increased collagen deposition. The results showed that further work is required to minimise process variation through control of the input raw materials. Optimisation of the fibrin matrix and diffusion of the culture media were identified as key areas to improve manufacturing cost-effectiveness. Enhanced understanding of the physical and molecular mechanisms by which ultrasound elicits cell responses will enable further optimisation of the ultrasound process for product improvement.

This research characterized the cells used in the formation and growth of substitute arteries for use in treatments such as bypass surgery. Two cell types including immortalized rat smooth muscle cells (rSMCs) and adult human mesenchymal stem cells (hMSCs) were used to study this tissue engineered blood vessel system. Sodium alginate was used as the natural polymer to construct the tubular three-dimensional construct. Normally biologically inert, the alginate was modified to incorporate GRGDY peptides using carbodiiamide chemistry to provide cell adhesion sites for the cells. The growth characteristics of the rSMCs and hMSCs in tissue flasks were established. Each cell source was assessed according to the viability post-thaw and at each passage, the cell yield and the glucose concentration in the spent medium. Following these measurements, the population doubling per day, the specific growth rate and the glucose consumption rate of the cells were calculated. Concurrently, the alginate was formed into sheets, beads or hollow tubes and the interaction of the cells with the alginate was studied. The biological activity of the alginate matrices was assessed by observing the cells' interaction with the matrices. Measurements such as viability of the cells post-immobilization into the alginate, cell growth and glucose consumption were made and compare to data for cell growth in tissue flasks. The survival and metabolic activity of the cells within the alginate matrices were also measured using the MTT (3-(4,5-dimemylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) assay. Modifications were made to dissolve the cell/alginate matrix and incorporate a frozen storage step for analysis of time course samples. Both the rSMCs and hMSCs readily attached and elongated when seeded onto the surface of alginate-GRGDY hydrogel sheets. However, only the hMSCs attached and elongation when immobilized within the alginate-GRGDY tubes and beads. Differentiation studies were conducted on the hMSCs immobilized within alginate-GRGDY beads using conditioned-medium (microfiltrate) from rSMC co-cultures. Expression of the hMSC surface markers SH2 and SH4 were lost after 6 days in co-culture. Immunohistological preparations of the beads confirmed the hMSCs expressed a-smooth muscle actin (ASMA). Control cultures of hMSCs in alginate-GRGDY beads placed in unconditioned medium retained their expression of SH2 and SH4 and did not express ASMA. Using the MTT assay, the hMSCs were shown not to proliferate in unmodified alginate and alginate-GRGDY beads over the 2 week period examined.

Mixed treatment comparison (MTC) meta-analysis, or network meta-analysis, is a methodology developed in the last decade for synthesizing both direct and indirect evidence on multiple treatments from randomised controlled trials available in a therapeutic area, while respecting the randomisation of the included trials. As a natural extension of ordinary pairwise comparison meta-analysis, MTC has increasingly been used in medical research and health technology assessment. The eleven papers (PI-PI 1) on MTC meta-analysis included in this thesis were published in peer-viewed journals from 2004 to early 2013, which was a period of rapid growth of this research topic_ These papers have substantially contributed to the development of MTC meta-analysis in both theory and applications_ They are organised into four chapters (Chapters 3-6) according to their roles in the development of MTC rather than the chronological order of publishing, which are the body of the commentary part of this thesis. In each of these chapters, my comments will be focused on the basic idea behind each included paper and its role in developing the MTC methodology_ Treated as a system of methodology on MTC meta-analysis, these included publications are intrinsically related in a connection network (Chapter 7), where three pivotal papers (P 1, P4 and P 11) provided fundamental concepts and models, four extension papers (P7 -PlO) generalised the basic models to more complex situations, and the rest were concerned with practical applications (P2-3) and issues on assessing evidence inconsistency (P5-6). The significance of the included published work on the development of MTC is discussed in the conclusion chapter around several statistical and methodological aspects of evidence synthesis.

The research presented in this thesis investigates the potential for sputter deposited titanium and calcium phosphate thin films, singularly and in combination, to directly induce osteogenic differentiation in human bone marrow derived mesenchymal stem cells (MSCs). The nature and scale of these substrate driven effects have been compared with osteogenesis induced in MSCs by exposure to biochemical stimulants in the culture media. MSCs hold great promise for use in bone tissue engineering applications, therefore the provision of biomaterials that can direct the osteogenic differentiation of MSCs has significant implications for improving the repair or replacement of damaged or lost bone tissue. RF magnetron sputter deposition was used to create titanium and calcium phosphate thin films from titanium metal and hydroxyapatite powder targets respectively. The distinct chemical and topographical surface properties of each substrate were confirmed using XPS, FTIR, XRD, AFM and water contact angle analysis. The commercially obtained MSCs used for this work were characterised in detail to provide a benchmark data set for correlation with subsequent thin film substrate studies. To determine the previously unreported effects that these types of sputter deposited surfaces have on MSC behaviour, a range of relevant biological assays and detailed quantitative real time PCR gene expression studies were used. In this way, a comprehensive profile of MSC response to each type of surface condition was obtained over an in vitro culture period of up to 28 days. Early stage adhesion and morphology was also examined in order to attain a better understanding of the underlying mechanisms of the attendant MSC behaviour. All of the sputter deposited thin film surfaces directly promoted significant levels of osteogenic differentiation, to varying degrees, without the use of biochemical stimulants. This work shows for the first time, that the topography of the sputtered titanium coatings and the bioactive chemistry of the calcium phosphate coatings can individually direct the differentiation of MSCs towards an osteogenic lineage.

The aim of this thesis was to assess the effect of suboptimal handling on mouse pre-implantation embryo development, viability and gene expression of the HI9 and Igf2 imprinted genes and to investigate the effects on fctal and placental development following embryo transfers. Establishment of the expression pattern of H19 and Igf2 genes showed that H 19 was expressed only at the blastocyst stage whereas the Igf2 imprinted gene employed a gradual increase in expression from the l-cell to the blastocyst stage, with maximal tran script detection at the post-compaction stages (P< 0.05). Culture of pre-implantation embryos from 1-cell stage until the blastocyst stage of development under temperature variations as small as ± 0.5 °C impaired blastocyst development and significantly reduced blastocyst total cell numbers (P< 0.05). These blastocysts also showed effected levels of H19 and Igf2 expression. Daily exposure of pre- implantation embryos to ambient air at 37.0 •C for small amounts of time caused small fluctuations of the culture media pH between 7.20 and 7.30. This resulted in a statistically significant reduction in the first cleavage, blastocyst development and hatching rates, decreased trophectodenn and inner cell mass cell development and increased apoptotic cell index (P< 0.05). Expression of imprinted genes H 19 and Igf2 was perturbed in blastocysts (P< 0.05). Following embryo transfers, the implantation rates were effected and fetuses displayed reduced weight on day 18 of pregnancy (P< 0.05). Placentae displayed significantly reduced levels of H 19 and Jg£2 expression and in some cases the aberrant expression persisted from the blastocyst to post*implantation embryo development and was retained in placentae (P< 0.05). Vitrification of mouse 2-cell stage embryos, which are warmcd and grown to blastocysts, resulted in increased survival and blastocyst development rates. However, it was found that blastocyst vitrification at the 2-cell stage altered the expression of H 19 and Igf2 (P< 0.05). Artificially reduction of blastocoel cavities prior to vitrification using the ICSI needle or laser-pulse methods showed significantly increased survival and re-expansion rates when compared to blastocysts vitrified with intact blastocoel cavities (P< 0.05). Blastocoel reduction using these two methods prior to vitrification at the blastocyst stage did not compromise the expression of H 19 and Igf2. The results from this study clearly demonstrate that stress applied to the pre- implantation embryo during the in vitro culture and handling alters blastocyst homeostasis without compromising morphology. Even minimal micromanipulations of pre-implantation embryos during in vitro culture display a worrying domino effect. Morphologically good-looking embryos have the ability to mislead us as the negative effect mainly occurs at the global gene expression, imprinting and metabolism levels. Irreversible affects come at a cost of impaired post-implantation, post-natal development and disease in adult life. The current study underlines how suboptimal culturing and handling of embryos has long reaching effects far beyond blastocyst development and successful pregnancy.

Multifunctional tissue engineering scaffolds can be used to create a suitable niche for cells to grow and form neo-tissues. Microparticles with growth factorspecific release propelties and tissue-specific polymer degradation rates can serve as the building blocks that underlie the multifunctionality of these scaffolds. In this work, blending poly(DL-lactic acid-co-glycolic acid) - poly(ethylene glycol)poly( DL-lactic acid-co-glycolic acid) (PLGA-PEG-PLGA) triblock copolymers with poly(DL-lactic acid-co-glycolic acid) (POL LGA) as a foundation polymer is introduced as an approach to accelerate lysozyme release from large PLGA microparticles. Here, the reproducible synthesis of composition ally different triblock copolymers is reported. Their reproducibility is controlled by controlling the lactic acid /poly(ethylene glycol) (LA/PEG) ratio. It is shown that a difference in LA/GA ratio and molecular weight of PEG can result in structurally different triblock copolymers with different hydrophobicity and different sol-gel transition temperatures. Large micropalticles (~ 200 I-lm in diameter) were fabricated from PLGA foundation polymer blended with PLGA-PEG-PLGA with morphology and size distribution closely similar to the control group (PLGA foundation polymer (~300 I-lm in diameter)). Blending PLGA foundation polymer with PLGA-PEGPLGA triblock copolymers reduced the glass transition temperature (Tg) and the decrease in Tg was correlated inversely with the mass oftriblock copolymer present in the polymer formulation. A comparison of the effect of two different triblock copolymers, namely PLGA-PEG 1500-PLGA (LA/GA on feed ratio of 2.5) and PLGA-PEG 1 OOO-PLGA LA/GA on feed ratio of 3) on lysozyme release rate and profile from these microparticles was made. It was shown that blending these triblock copolymers with PLGA 85: 15 as foundation polymer prior to microparticle manufacture significantly accelerated lysozyme release from these microparticles. It is demonstrated that the lysozyme release rate and the total release was propOltionate to the mass of triblock copolymer blended with foundation polymer - in the way that the higher the mass oftriblock copolymer in the polymer formulation the higher the release rate. At 37°C, microparticles with :1 polymer formulation consisting of PLGA 85: 15 without triblock copolymer (the control group) were shown to have a biphasic release profile consisting of a slow release phase (first week) and a prolonged lag phase. The slow release phase was composed of a burst release during which totally ~5% of entrapped protein (in first week) was released. No release was detectable after this release phase. Microparticles with polymer formulation consisting of PLGA85: 15/PLGAPEG- PLGA 90: 1 0 showed a tri -phasic release curve. After a burst release of 14- 17% in the first 24 hours, a slow or not detectable level of release was observed for 20-30 days. After this lag phase, another 20-25% was released over 10-20 days and the total release achieved by day 60 was 40-50% (of entrapped protein). Microparticles with polymer formulation consisting of PLGA85: 15/PLGA-PEGPLGA 70:30 showed a continuous release profile and totally released 70% of entrapped protein in the first 30 days and the release was undetectable after 30 days to the end. These results demonstrated that blending PLGA-PEG-PLGA triblock in PLGA 85:15 accelerated lysozyme release from microparticles. It was 11 found that the effect of PLGA-PEG 1 OOO-PLGA and PLGA-PEG 1500-PLGA on release rate and profile was not different. The environment surrounding microparticles and in a controlled release context, the incubation medium, can affect the release behaviour. In this work, release behaviour of FITC labelled lysozyme from microparticles fabricated from two different polymer formulations into a fibrin clot (3.5 mg/mL fibrinogen; physiological concentration) was compared with the release to PBS and fibrin gel with 50 mg/mL fibrinogen. Fibrin clot was used as a model of blood clot. It was shown that the release profile ofFITC labelled lysozyme in PBS and in fibrin gels are similar for a celtain formulation. However, the release rates were different in each incubation media. Very low release (2 .7-4.5% depending on the polymer formulation) was detectable in fibrin gel (50 mg/mL fibrinogen). The release rate was higher (3 .7-7% depending on the formulation) in fibrin clot (3.5 mg/mL fibrinogen). The highest release rates were detected in PBS (11 and 13%). Finally, the bioactivity of bone morphogenetic protein-2 (BMP-2) released from two compositionally different microparticles laden in fibrin clot (3.5 mg/mL), was examined by measuring its effects on cells over a 20 day period. It was revealed that BMP-2 released from microparticles can escape the fibrin gel to the culture medium. These BMP-2 molecules triggered osteogenetic cellular pathways. This was shown by detection of increased levels of alkaline phosphatase compared to a negative control. I In conclusion, it was shown for the first time that PLGA-PEG-PLGA triblock copolymers blended with PLGA as a foundation polymer can accelerate protein release from large microparticles. Generally large microparticles have very slow release rate and assemble into highly porous scaffolds.

Tissue engineering of any tissue type requires the combination of a supporting scaffold, a range of biological factors and a suitable source of cells. This source of cells must satisfy a number of criteria:- • The ability to form all of the mature/specialised cell types found in the target tissue. • Readily obtainable. • Readily maintainable in the laboratory without requiring excessive resources or time. For intestinal tissue engineering there are a number of issues associated with the use of tissue derived stem cells particularly quantity of normal tissue available (from the patient) and maintenance and expansion of the cells when cultured in vitro. Using embryonic stem cells offers a potential alternative strategy but methods must be established to efficiently differentiate the cells towards the desired fate. Many strategies for differentiating embryonic stem cells are based upon treatment with growth factors in vitro. There is a (logistical) limit to the degree of complexity that these systems can achieve and therefore a limit to the number of differentiation signals that occur during in vivo development that can be mimicked. In recent years using embryonic tissue to provide signals to undifferentiated cells has proved a successful method of directing the differentiation of naïve cells towards a particular fate (with the choice of tissue determined by the desired target cell type). The aims of this thesis were to explore the potential of differentiating embryonic stem cells towards the intestinal progenitor fate using a combination of in vitro cell culture treatment with the growth factor Activin-A and ex vivo co-culture with embryonic chick gut tissue. Previous studies [Kubo et al 2004, Tada et al 2005, Yasunaga et al 2005, D’Amour et al 2005, MacClean et al 2007] have shown that Activin-A treatment will induce embryonic cells to more efficiently differentiate to definitive endoderm, the germ layer from which the intestines (and other visceral organs) arise. These techniques were applied to the Columnar Epithelial Epiblast murine embryonic stem cell line and cell differentiation was then evaluated at the molecular level using Reverse Transcription-Polymerase Chain Reaction, immunocytochemistry and Western blotting. Activin-A treatment produced an upregulation of definitive endoderm markers at both the mRNA and proteomic levels compared to the control conditions. However the cell population produced retained expression of pluripotent markers and showed some expression of markers of other cell lineages. Further studies [Sugie et al 2005, Fair et al 2003, Van Vranken et al 2005, Coleman et al 2007, Krassowska et al 2006] have shown that co-culture of embryonic stem cells with early stage embryonic tissue can induce the formation of particular tissue types; the tissue must be selected based on proximity to the target cell type during development. This exposes the embryonic stem cells to the signals that prompt differentiation towards the target tissue during normal development. With gut tissue much signalling occurs between the different tissue layers that make up the whole organ both during development and in adult tissue. Ex vivo co-culture of murine embryonic stem cells with embryonic chick gut tissue was used to direct their differentiation to the intestinal epithelial stem cell fate. Before the co-culture was carried out various experiments were carried out to establish if the proposed protocol was viable e.g. defining how long chick gut tissue explants could survive in culture. Once this had been established co-culture experiments were undertaken and cell differentiation was then evaluated at the molecular level using Reverse Transcription-Polymerase Chain Reaction, immunocytochemistry and Western blotting. The cells showed some expression of intestinal epithelial stem cell markers at both the mRNA and proteomic levels following co-culture. The cells were also assessed at a physiological/functional level by evaluating their ability to form a functional intestinal epithelial barrier. This was achieved using an in vitro co-culture model with intestinal subepithelial myofibroblasts by measuring transepithelial resistance, permeability to protein and morphology in a simple tissue model co-culture. The cells did not display the morphological or physiological characteristics associated with intestinal epithelial cells in the model system. Overall this work has shown that co-culturing pluripotent mES cells with embryonic chick gut tissue can induce differentiation towards the ISC fate. Pre-treating the cells with growth factors in vitro did not seem to enhance this differentiation but there was scope to refine these techniques. Following the differentiation protocols the cells did not display the desired physiological characteristics but again there was scope to refine the techniques particularly with regard to selecting cells positive for the expression of the chosen molecular markers. These techniques show promise but do require some further development.