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Spatial patterning of cells is of great importance in tissue engrneenng and biotechnology, as it enables the creation of histoarchitectures of cells and cell aggregates for in vitro high throughput toxicological and therapeutic studies within 3-dimensional enviromnents. Using dielectrophoresis, homogeneous cells suspended in polyethylene glycol diacrylate polymer solution were patterned with repulsive dielectrophoretic forces, generating cell aggregates within a microarray formatted dielectrophoretic system. 2,2-Dimethoxy-2-phenylacetophenone photo initiator, at low concentrations, initiated PEG-diacrylate crosslinking through a uniform near-UV (peak = 350nm) irradiation of the micro-system within a compact, purpose built portable DV light-box for dielectrophoresis experimentation. The rate of induced cell aggregation over 5 minutes was observed to decrease with increasing PEG-diacrylate polymer concentration, while hydrogel water content remained high(>70%) at PEG- diacrylate concentrations up to 30%. These optimised conditions for rapid dielectrophoretic cell patteming low DV exposure times within the dedicated system and ease of hydrogel peelability were applied to yeast and human leukaemia and cervical cancer cell aggregates which showed 90% cell viability after one week which is significantly better than those published in previous studies. Drug testing study using vinblastine chemo-therapeutic agent showed that the optimised system of representing cells in 3D showed different IC50 graphs compared to results obtained from cells in 2D monolayer indicating the effect of cell-cell interactions. It was noted that the results obtained from mono layers and aggregates were widely apart for all I concentrations at all time points studied. Compared to constructing aggregates formed spontaneously or by culturing them on treated surfaces, our system represents a structure more like the original tissue in terms of having a polymer surrounding cells, which serves as a barrier that can represent blood (growth medium with dissolved drug) and extracellular matrix (hydrogel).This work demonstrates the use of this optimised system in cell, drug and tissue engineering studies; the system is easy to use, mimic cells in their natural tissue and it maintain high cell survival.

Introduction: Human umbilical cord (UC) may be a good source of mesenchymal stem cells (MSCs) for musculoskeletal tissue engineering, however their potential to form bone is incompletely understood. The aim of the present study was to evaluate the growth characteristics, the phenotype and the multipotentiality of UC MSCs in comparison to bone marrow (8M) MSCs, with a particular focus on the molecules involved in bone formation and vascular support. The phenotype of UC endothelial cells (ECs) was additionally investigated in order to isolate uncultured fractions of UC MSCs and ECs, and to compare their telomere status. Methodology: UC fragments were enzymatically digested and UC MSC and EC cultures were grown in specialised media. Quantitative in vitro assays were used to study osteogenic, chondrogenic, adipogenic and vessel formation capacities of UC MSCs in comparison to 8M MSCs. Phenotypic characterisation was performed using multiparameter flow cytometry with MSC-, EC-specific and haematopoietic lineage markers. Immunohistochemistry on UC tissue sections was undertaken to study cell topography and cell sorting was performed to purify putative native UC MSCs from whole UC digests. Relative telomere lengths were measured by qPCR in both cultured and purified UC MSCs. Gene expression was analysed using Taqman low density array for 48 transcripts chosen to reflect MSC osteogenenic, angiogenic and other lineage potentials. Results: Compared to BM MSCs, UC MSCs grew slightly faster and had greater telomere stability. Both BM and UC MSCs had a classic MSC phenotype (CD90•, CD73•, CD105•, CD146•, CD31-, CD34-, CD45-, CD235a'), UC MSCs could generate all three mesenchymal lineages but their differentiation levels were inferior to 8M MSCs. MSCs differentiated towards vasculogenesis on 3-D Matrigel scaffold but they did not differentiate towards mature C031+ ECs. Cells expressing MSC markers C090 and C0146 were mainly located in UC perivascular and Wharton's jelly areas. Putative native UC MSCs were electrostatically cell sorted based on the CD146+, CD45-, CD31' phenotype. Their telomere lengths were compared to electrostatically cell sorted CD146-, CD45-, CD31- cells and ECs (CD45-, CD31•) but contrary to the initial hypothesis, no differences in telomere lengths were found. In agreement with functional osteogenesis assays, UC MSCs expressed considerably lower levels (<5-200 fold) of osteogenesis-related transcripts compared to 8M MSCs. Although they were able to respond to osteogenic v stimulation by up-regulation of many osteogenesis-related molecules (up to 100- fold), the transcript expression levels of differentiated 8M MSCs were commonly not achieved. Conclusions: UC MSCs direct differentiation in standard osteogenic assays appeared inferior to BM MSCs, most likely due to their more immature status. However, UC digests could represent a potential source of regenerative cens (MSCs and ECs) for complex tissue engineering where functional and long-lasting vasculature is required. It is possible that bone-forming capacity of UC MSCs can be improved by the development of optimised expansion protocols andlor the use of purified uncultured MSCs.

Polypyrrole (PPy) is a conjugated polymer that displays special electronic properties including conductivity. It may be electrogenerated with the incorporation of any anionic species including negatively charged biological molecules such as proteins and polysaccharides. For this thesis, variously loaded-PPy films were prepared on gold sputter-coated coverslips. The growth and characteristics of epithelial cells, namely keratinocytes, were studied on these films by microscopy, biochemical assay, immunocytochemistry and electrochemical impedance spectroscopy. Keratinocyte viability was found to be PPy-load dependent. For chloride, polyvinyl sulphate, dermatan sulphate and collagen-loaded PPy films, polycarbonate and gold, keratinocyte viability, as assessed by the AlamarBlueTM assay, was respectively 47%, 60%, 88% and 23%, 75% and 61% of tissue culture polystyrene controls after 5 days. This was found to require a previously unreported polymer washing step prior to cell seeding due to the observed toxicity of untreated films. Keratinocytes stained positive for proliferation (PCNA), suprabasal differentiation (K10) and hyperproliferation (K16) markers although cell morphology was poor for organotypical cultures on dermatanloaded PPy compared with de-epidermalised dermis. Cell-induced impedance changes were detected in a three-electrode format over PPy modified electrodes. Results obtained showed the effects of cell density, cell type and monitoring frequencies. In particular, it was seen that lower cell densities could be detected on PPy compared to unmodified gold electrodes. Keratinocyte confluence as determined by impedimetric analysis was reached more rapidly on PPy than bare gold in agreement with AlamarBlueTM measurements. Electrical equivalent circuit analysis using parameters whose contributions may be directly mapped to intracellular and intercellular spaces, and membrane components suggested that the technique can be extended to cell morphology discrimination. This work shows that PPy biocomposites are attractive candidates for tissue engineering applications since they may incorporate biomolecules and are electrically addressable with the potential to both direct and report on cell activities.

In vitro modelling offers the potential of recapitulating human degenerate tissue for physiological studies and pharmacological screening. Yet, few systems have been developed to date, primarily due to the lack of vascularisation in engineered tissue. Here, the development of an in vitro pump is addressed. This will be the first component of a long term strategy to build internal circulatory systems for in vitro engineered tissue. Firstly, mechanical characterisation and surface biocompatibility of spin coated poly(dimethylsiloxane) (PDMS) elastomeric membranes was investigated to assess whether PDMS could be used as a structural constituent. Results showed that spin coating speed defines both film thickness and specific mechanical properties since tensile testing revealed that PDMS membranes exhibit thickness-dependent mechanical properties. Plasma polymerisation of allylamine was used to surface engineer the hydrophobic PDMS surface to promote cell adhesion and proliferation. Surface characterisation revealed that PDMS surfaces became hydrophilic and nitrogen enriched as a result of plasma polymerised allylamine (ppAAm) deposition. Additionally, a thick ppAAm deposition (92 nm) is required to minimise hydrophobic recovery of PDMS. Cell culture studies showed that cells readily attached to ppAAm (92 nm) deposited PDMS and that these surfaces were best suited to cultivating cells compared to other surface treatments. Secondly, a method was developed to control cell positioning on the PDMS surface, since cell alignment is required to generate directional and contractile force. The deposition of ppAAm (92 nm) and airbrushing of extracellular matrix (ECM) aerosols can be spatially restricted using a micro-stencil. Individual and multiple cell line patterns were achieved that remained faithful for ≤ 10 days. Importantly, this technique micropatterned cells at high resolution over macro scales. External mechanical stimulation was used to influence cell alignment and cytoskeletal organisation on ppAAm (92 nm)/ Fibronectln (Fn) micropatterned PDMS. Results showed that incorporating substrate strain with surface micropatterning can be used to control site- and alignment- specific cell attachment.

Récemment, l'utilisation de la technique d'assemblage couche par couche de polyélectrolytes naturels a été suggérée comme une nouvelle procédure de modification de surface. Le but de notre travail était de développer des films à base de CHI et de HA fonctionnalisés et réticulés. La biocompatibilité a été évaluée par MEB et visualisation des filaments d'actine. La différenciation endothéliale des CSMs a été suivie par Western Blot. Ensuite, la fonctionnalité des cellules a été évaluée par immunofluorescence du vWF et marquage au Dil-Ac-LDL. Le MEB a montré une morphologie fibroblastique des cellules sur CHI/HA et CHI-SH/HA-Thio. L'analyse par WB des cellules différenciées a montré une augmentation de l'expression de CD31, VEGF-R2 et VE-cadhérine sur CHI-SH/HA-Thio et CHI/HA. Les films CHI-SH/HA-Thio étaient biocompatibles et promouvaient le potentiel de différenciation endothéliale / Recently, the use L-b-L assembly of natural polyelectrolytes has been suggested as a new technique of surface modification. The aim of our work was to develop multilayered films based on CHI and HA polymers bearing chemical. WJ-MSCs were seeded on these substrates. Biocompatibility was assessed by SEM and actin visualization. WJ-MSCs differentiation into endothelial phenotype was followed by western blot. First of all, SEM showed a fibroblastic morphology of adherent cells on CHI/HA and CHI-SH/HA-Thio as for control. WB analysis showed a rise of CD31, VEGF-R2 and VE-cadherin expression on CHI-SH/HA-Thio and CHI/HA. Films CHI-SH/HA-Thio were biocompatible and promoted endothelial differentiation potential

L'ingénierie tissulaire a vu émerger dans la dernière décennie, la nanomédecine régénérative en combinant non seulement les cellules souches mais aussi les facteurs de croissance. Le but de ce travail a été d'utiliser les techniques de l'ingénierie osseuse et cartilagineuse pour améliorer ou créer des biomatériaux en les rendant bioactifs et vivants à une échelle nanométrique. Durant ce travail de thèse, nous nous sommes intéressés au développement de biomatériaux (naturels ou synthétiques), tridimensionnels (3D), implantables et utilisables pour le traitement de diverses pathologies de l'appareil loco-moteur, qui sont capables d'induire une meilleure différenciation cellulaire et une régénération tissulaire plus stable. Nous avons développé 3 types d'implants nanostructurés, à base de titane recouvert d'hydroxyapatite et de nanotubes de carbone, de capsules ou de membranes nanofibreuses nanofonctionnalisées par des facteurs de croissance, qui ont permis d'obtenir une régénération osseuse ou ostéo-articulaire, in vitro comme in vivo / During the last ten years, tissue engineering has merged with regenerative nanomedecine by combination, not only of stem cell technology but also of growth factors. The goal of this thesis was to use bone and cartilage engineering as a model, in order to improve and to develop active and living nanostructured implants. During this PhD studies, we were interested in the development of biomaterials (natural or synthetic), tridimentionnal (3D), transplantable for bone and cartilage diseases treatments, that are able to induce more cellular differentiation and improved tissue regeneration. In this thesis, we have developed 3 types of nanostructured implants, (i) titanium implants coated with hydroxyapatite and carbone nanotubes; (ii) active capsules functionalized by growth factors and stems cells for bone induction (in vitro/in vivo); (iii) electrospun nanofibrous membranes functionalized by growth factors and (Osteoblasts/Chondrocytes) for bone and cartilage regeneration, in vitro and in vivo