Tissue engineering is an emerging biomedical innovation surrounded by potentiality and risk. Based on documentary analysis and expert interviews, this study discusses different constructions of risk according to main constituencies (scientists, clinicians and manufacturers), the way they prioritise and balance these risks, and how issues are framed as problematic or not. Complexity and uncertainty are the main drivers in this exercise, interpreted in terms of boundary drawing around contested risk domains. This is followed by a discussion of the translation of risk into regulatory policy, by focusing on two recent legislative initiatives by the European Commission: one to control the quality and safety aspects of human tissues and cells (DG SANCO Directive) and the other to facilitate the marketing of tissue engineered products in the EU (DG Enterprise Regulation). These two legislative initiatives aim to overcome the current regulatory lag in Europe, where tissue engineered applications are either unregulated or subject to a broad variety in national controls. This situation is problematic for manufacturers wanting to market their products in Europe, for regulators in evaluating the risks of these technologies and defining an appropriate approval route, and for patients in terms of unequal access to potentially beneficial therapies across the continent. Firmly rooted in ambitions to make the EU a techno-scientific and bio-economic powerhouse, regulation of this domain is troubled by competing agendas of promoting trade versus protecting public health. Social and ethical considerations about the impact of tissue engineering technology allow a reconsideration of the bio-society as alternative model, taking into account the technological as well as social character of innovation.

Stem cells are unspecialised cells found in the body which possess the ability to self renew and can be induced to proliferate and differentiate into more specialised cells. MSCs are adult stem cells that are capable of leaving the bone marrow and travelling in the bloodstream to a different site, where they may perform repair or regeneration processes of various mesenchymal tissues such as cartilage, bone and fat. Due to these properties of MSCs, it proves to be a useful source for the repair and regeneration of cartilage. The aims of this study were to: 1) characterise primary bovine MSCs by assessing the gene expression of hyaline cartilage-specific genes (SOX-9, Aggrecan and Collagen type II) and non-specific genes (Collagen type I and type X) 2) investigate the effects of culture medium supplemented with FGF-2 or with the addition of an extra growth factor, TGF-p2, on bovine MSCs in a two dimensional culture system 3) characterise the phenotypes of MSCs tissue grafts produced using MSCs pre-cultured in culture medium supplemented with FGF-2 or with the addition of TGF- p2 in a three dimension culture system (Transwell) seeded at high (6xl06 cells) and low (0.5 &times; 10<super>6</super> cells) cell density. Our study showed that different bone chamber size and thickness influenced the amount of marrow and cells harvested without interfering with the fibroblastic-shaped cell morphology and their adherence ability. The presence of stem/progenitor cell features is present in undifferentiated P0 BMSCs and PI and P2 BMSCs cultured in FGF-2 or with the addition of TGF-p2. Gene expression analyses on undifferentiated P0 BMSCs and PI and P2 BMSCs cultured in FGF-2 or with the addition of TGF-p2 suggested that the MSCs contain both fibroblastic and chondrogenic features. This implies that the MSCs are not fully committed towards a chondrogenic lineage. We have demonstrated that it is possible to generate a tissue graft using passaged BMSCs in a Transwell culture system but seeding density is important. The ideal seeding cell density is 0.5x106 cells/well using P2 and P3 BMSCs pre-cultured in either FGF-2 or with the addition of TGF-P2. The tissue graft produced has a high expression level of aggrecan, collagens type I and II, which suggested that it has a fibro-cartilage phenotype.

Impaired wound healing affects 3% of the population over 60 and costs over &pound;1 billion annually in the UK. Chronic wounds are characterised by prolonged inflammation, impaired re-epithelialisation and defective extracellular matrix (ECM) remodelling. Fibroblasts play an important role in the closure of skin wounds, they replace and remodel the lost tissue and also influence both re-epithelialisation and angiogenesis. Numerous <italic>in vivo</italic> chronic wound animal models have been reported however, they fail to accurately model human chronic wounds. This investigation aims to develop an <italic> in vitro</italic> chronic wound bioassay. Chronic wound fibroblast (CWF) and patient matched normal fibroblast (NF) cell strains (n=3) were retrovirally infected with a hTERT/puromycin construct. Population doubling levels (PDLs) were determined over an extended time in culture. RNA at defined time points was extracted and analysed using Asymetrix U133A microarrays. A cohort of gene expression changes were confirmed by QRT-PCR and the promoter regions of these genes used to construct fluorescent reporter human NF and CWF cell lines. Population doubling levels indicated that, compared to primary (non-immortalised) CWF and NF, the hTERT infected fibroblasts had escaped replicative senescence and formed an immortalised cell line. Affymetrix microarray analysis of the CWF-hTERT with the NF-hTERT identified 247 genes that were significantly up- or down-regulated in CWF (validated by QRT-PCR). Upstream promoter regions of 13 genes of interest and a housekeeping gene have been identified by database searches/sequence analysis, amplified and cloned into the promoterless reporter vector pZsGreen1-DR. Reporter constructs have been transiently transfected into human NF and CWF cell lines and are currently being tested for efficacy. CWF and NF cell lines have been created which demonstrated distinct gene expression profiles. The disease marker genes will form the basis of a low cost, highly reproducible fluorescent reporter cell-based bioassay which will be utilised in the discovery of novel therapeutics which have the potential to alter the chronic wound healing and, in turn, reduce unnecessary animal experimentation.

The mechanisms involved in osteoblast and adipocyte differentiation from the common progenitor, mesenchymal stem cell (MSC) are not fully understood. The nucleoside, adenosine, exists in all cells and is known to be involved in cell growth, proliferation and apoptosis by interacting with four distinct receptors (Ai, A2A, A2b and A3). The aims of this study was to investigate the expression and function of adenosine receptors in 1) MSCs and as they differentiated into osteoblasts and adipocytes and in 2) mouse 7F2 and human osteoblasts and during their transdifferentiation to adipocytes. Rat MSCs and 7F2 osteoblasts expressed all four adenosine receptors. Osteoblast differentiation was associated with increases in A2A and A2B receptor expression and their activation stimulated the expression of alkaline phosphatase, core binding factor a1 and mineralisation. Adenosine also stimulated adipogenesis (lipid accumulation, peroxisome proliferator-activated receptor, CCAAT/enhancer binding protein a and lipoprotein lipase expression) of MSCs which was accompanied by increased A1 and A2A receptor expression. Transdifferentiation of 7F2 cells to adipocytes was associated with increased Ai, but decreased A2A and A2b receptor expression. Loss of A2 receptors in adipocytes was supported by reduced cAMP and extracellular signal regulated kinase responses to adenosine. Adenosine also stimulated transdifferentiation of human osteoblasts to adipocytes by inducing lipoprotein lipase and inhibiting alkaline phosphatase and osteocalcin expression. Overexpression of Ai receptors in 7F2 cells stimulated adipogenesis (lipid accumulation and lipoprotein lipase expression) whereas overexpression of A2b receptors stimulated alkaline phophatase expression and inhibited adipogenesis. These results show that adenosine receptor expression and function is involved in lineage specific differentiation or transdifferentiation A2B receptors are associated with MSCs and osteoblasts and Ai receptors with adipocytes. Targeting adenosine signal pathways may thus be useful as an adjunct therapy for the prevention or treatment of conditions in which there is insufficient bone or excessive adipocyte formation.

The generation of neocartilage grafts <italic>ex vivo</italic> shows promise for the repair of articular cartilage defects. This thesis describes (1) the metabolism and macromolecular organisation of grafts produced utilising a Transwell culture system at biochemical and microscopic levels, (2) the effect of chondrocyte de-differentiation, induced by monolayer expansion, on resultant graft tissue architecture, and (3) how addition of exogenous hyaluronan of varying molecular weights, during and post graft generation effect chondrocyte metabolism, with a view to explaining effects of hyaluronan administration in viscosupplementation procedures. Grafts generated from primary chondrogenic sources were of hyaline nature with distinct zonal stratification as found in native articular cartilage evidenced by cell morphology, matrix organisation and immunohistochemical composition. Graft tissue produced from immature sources was more synthetically active than those produced from mature sources. Passage expansion of chondrocytes in monolayer culture caused progressive reduction in graft thickness, loss of zonal architecture, and a more fibrocartilaginous tissue histology, consistent with a de-differentiating chondrocyte phenotype. Grafts subjected to exogenous hyaluronan of smaller molecular weight than that typically found in native aggrecan aggregates (500kDa-1000kDa) had an increased release of proteoglycan from graft tissue. Addition of hyaluronan with significantly higher molecular weight than endogenous hyaluronan resulted in an increased retention of proteoglycan within graft tissue, suggesting that high molecular weight hyaluronan in viscosupplementation procedures may be facilitating the retention of newly synthesised 'repair aggrecan' in pathological cartilage, slowing down the rate of cartilage destruction through loss of proteoglycan from the tissue. Futhermore, the addition of 490kDa (similar molecular weight to endogenous hyaluronan) showed a marked increase in the amount of aggrecan synthesised. Collectively, this data suggests that viscosupplementation procedures should incorporate a mixture of hyaluronan ranging between 490kDa-3000+kDa in order to provide optimal benefits of aggrecan retention and biosynthesis to osteoarthritic patients receiving this treatment modality.

Parkinson's disease in reality arises as a result of a complex series of events, however it is strongly linked to the loss of a specific cellular population of midbrain dopaminergic neurons making it a candidate for stem cell based research. Stem cells can be cultured in vitro and via asymmetric cell division possess the capacity for both self renewal and the production of differentiated derivatives. The use of specific molecules and culture conditions can be applied to promote the differentiation of them towards particular cellular fates, in turn facilitating the possibility of producing enriched populations of cells displaying characteristics of a certain phenotype of interest. There has been much research focussed on the in vitro generation of dopaminergic neurons from various stem cell types. In this work the Tera2.cl.SP12 embryonal carcinoma stem cell line was the primary vehicle investigated for its ability to produce cells that were reflective of a dopaminergic phenotype. Retinoic acid was found to be able to up regulate the expression of a range of dopaminergic markers in the Tera2.cl.SP12 cell line over time. However it was clear that lowered oxygen culture, a method known to promote the production of neurons reflective of a dopaminergic phenotype in mesencephalic cultures, had no effect on the dopaminergic differentiation capacity of the embryonal carcinoma stem cells. The glycoprotein Wnt1 when applied to Tera2.cl.SP12 cultures in concert with retinoic acid was shown to increase the number and percentage of cells positive for the neuronal marker Beta III tubulin approximately 1.5 fold. This was accompanied by a concomitant rise in the mRNA expression of this marker, thus suggesting that the use of Wnt1 may be a means to produce cultures derived from embryonal carcinoma cells that are more neuronal, based on marker expression data. Other established methods to achieve dopaminergic differentiation such as suspension culture, stromal cell co-culture and the application of Sonic hedgehog and Fibroblast growth factor 8 are also able to induce a degree of neuronal and dopaminergic marker expression in Tera2.cl.SPI2 cultures. Overall these results suggest that the Tera2.cl.SP12 cell line might be one vehicle for the study of dopaminergic neurogenesis in vitro, in particular when Wnt1 and retinoic acid are used as a means to favourably enrich the population of cells displaying neuronal characteristics.

In Europe, diseases of the heart and circulatory system are the main cause of death; 48% of all deaths are from coronary heart disease, accounting for over 4.30 million deaths each year (European cardiovascular disease statistics 2008). Identifying stem cell types that can potentially contribute to the regeneration of cardiac tissue is one route to addressing this global problem. The follicular dermis has been proposed as a tissue containing cells with adult stem cell properties that could be used for regenerative medicine. This study showed that follicular dermal structures demonstrated spontaneous synchronised contractions in vitro, indicative of cardiomyocytic commitment. In contrast, isolated populations of follicular dermis, the dermal papilla and sheath, did not demonstrate spontaneous cardiomyocytic commitment in vitro. Co-culture of isolated follicular dermal populations with embryonic cardiomyocytes, a cell type previously reported to support cardiomyocytic commitment of mesenchymal stem cell types, induced expression of a panel of gene products indicative of cardiomyocytic commitment, but only when the inducing cell and the target cell were of the same species. The level of gene induction indicated that the number of cells affected were likely to be very modest. In order to better identify the stem cell types in the follicular dermis, this study used a novel mTert-GFP transgenic mouse to identify candidate stem cells on the basis of their expression of the stem cell phenotypic marker, telomerase. A small population of GFP-expressing non-haematopoietic cells present in the dermis (0.17- 0.49%) were identified and found associated with the follicular dermis and bulge region. This minor sub-population of GFP-expressing dermal cells contained all the detectable gene expression of four key markers of stem cells: Tert; Oct4; Nanog and Sox2, both at time of isolation and after 2 weeks in vitro culture. Intriguingly, non- haematopoietic, GFP-expressing cells were also detectable in the adult heart at low level (0.08%). After cryo-induced infarct, these non-haematopoietic GFP-expressing cells were associated with the epicardium, concentrated close to the infarct site, in a fashion consistent with other reports of candidate cardiac stem cells. This study indicates that the population of stem cells within the follicular dermis may be very modest. Further, it shows that phenotypic markers of stem cells from different organs may be a suitable approach for the investigation of the stem cell phenotype.

Islet purification prior to allo transplantation currently forms the clinical gold standard, despite the fact that purification protocols result in significant islet losses and remove potential islet precursors and tissue that may provide islets with bio-trophic support Conversely, non-purified islet preparations retain endocrine mass and within the sphere of islet auto transplantation have been associated with excellent long-term graft function. This study comprises of an analysis of peri-operative factors and the long-term graft function of patients’ auto transplanted with either purified (n=14) or nonpurified islets (n=23). Complementary in-vitro studies were carried out to assess the effect of non-islet tissue on islet viability, integrity and function (n=8), whilst a histology-based study (n=23) assessed whether transplantation of non-islet cells, particularly islet precursors, had a long-term effect on graft function in the clinical setting. Clinically, non-purified islets did not significantly increase peri-transplant venous pressures and perioperative factors including ITU stay, blood loss and liver function were comparable in both groups. Analysis of the 5 year post-transplant period demonstrated that although insulin release in response to glucose was initially superior following transplant of purified islets, non-purified islets were associated with stable long-term function. In-vitro studies reiterated these findings, revealing that islet viability and function were comparable in both groups, however, retention of intracellular insulin was found to be superior within non-purified preparations with some evidence that ductal tissue provided islets with bio-trophic support. Histology-based analysis of patient pancreata suggested a positive role for islet precursors demonstrating significantly superior blood glucose, HbA1c and C-peptide values associated with the transplantation of ductal cells and non-islet PDX-1 and glucagon positive cells. The results of this study indicate that transplantation of non-purified islets can be performed safely and with comparable long-term graft function as purified islets. Additionally, these studies potentially suggest that ductal tissue may help to preserve islet integrity, whilst certain precursors cells found within the acinar parenchyma and ductal epithelium may improve long-term islet graft function.

The aim of this project was to compare and contrast the results of three related methodologies which are of particular relevance or potential within our department -. a manually performed streptAvidin Biotin Complex (sABC/ HRP) method which for many years has served as our 'standard' immunocytochemical method; an integrated semi-automated systems from DAKO based on the TechMate 500 immunostainer and the ChemMate reagent package;. a manually performed tyramide amplification method regarded as amongst the most sensitive immunocytochemical method currently available. Antigen retrieval investigating differing durations of exposure to the proteolytic enzyme trypsin, and heat mediated antigen retrieval using citrate buffer pH6.0 or DAKO high pH antigen retrieval solution were also included as parameters for investigation. To this aim a panel of eleven specific antibodies were assessed, each of which had previously displayed individual qualities worthy of investigation -. PGP9.5, CD30, oestrogen receptor, CD3 polyclonal, CD2, CD3 monoclonal, CD4, cyclin D1, kappa immunoglobulin light chain, lambda immunoglobulin light chain and IgD immunoglobulin heavy chain. The results of this work demonstrate that each of the systems investigated has its individual merits -. The manual sABC/ HRP method is the least complicated and expensive of the methods to perform. There are significant disadvantages in its used however. Being a manual method there are many steps where individual variation in working practices within a laboratory can and do affect results. The TechMate/ ChemMate system is a more sensitive system than the manual sABC/ HRP method also providing a cleaner end result and allowing higher dilutions of primary antibody to be employed. Tyramide amplification is the most sensitive system evaluated, the antibodies in this work generally demonstrated immunostaining at higher dilutions than the other methods tested although some, in particular those directed against immunoglobulin light chains, operated better under the ChemMate/ TechMate system. On occasion specific positive results were obtained which none of the other methods tested could achieve with certain antibodies, CD3 monoclonal and to a lesser extent CD2.

No description available.