In cardiovascular disease the repair response is insufficient to restore blood flow, leading to the death of muscle and loss of tissue function. Therefore, strategies to augment the endogenous cell response and its effects may help improve tissue recovery and function. In this study we explored the use of tissue-engineered collagen matrices for augmenting endogenous regenerative processes after myocardial infarction. Treatment with the sLeX-collagen matrix reduced inflammation and apoptosis and had a positive regenerative effect on the infarcted mouse heart, through improved vascular density and possibly enhanced cardiomyogenesis. Additionally, we investigated the effects of cryopreservation on generating circulating angiogenic cells (CACs) from peripheral blood mononuclear cells (PBMCs), as a potential source of stem cells that could be used in combination with our collagen scaffold. Our findings show that despite PBMCs experiencing phenotypic changes after cryopreservation, they may still be used to generate the same therapeutic CACs as freshly procured PBMCs.

A degradable/polar/hydrophobic/ionic (D-PHI) polyurethane with properties intended to promote tissue regeneration in a small diameter peripheral artery vascular graft was evaluated for cell biocompatibility and growth. Films were cast in polypropylene 96 well plates for monocyte/endothelial cell (EC) co-culture in vitro studies and porous scaffold discs were implanted in an in vivo subcutaneous mouse model. After 7 days in culture the co-culture demonstrated cell adhesion and growth, low esterase activity (a measure of degradative potential and cell activation), no detectable release of pro-inflammatory cytokine (tumour necrosis factor -α) but measurable anti-inflammatory interleukin (IL)-10. The EC and the co-culture expressed the EC biomarker CD31, whereas the monocyte monoculture did not. Cytokine array analysis of the in vivo characterization of D-PH supported an anti-inflammatory phenotype of cells at the site of the implant. Levels of IL-6 significantly decreased over time while IL-10 was significantly higher at 6 weeks post implant. TNF-α levels did not change significantly from 24 hours onwards, however the trend was towards lesser amounts following the initial time point. Histological analysis of the explanted scaffolds showed excellent tissue ingrowth and vascularization. A live/dead stain showed that the cells infiltrating the scaffolds were viable. Both the in vitro and in vivo results of this thesis indicate that D-PHI is a good candidate material for tissue engineering a peripheral artery vascular graft.

<p>Regenerative medicine holds the promise of providing relief for people suffering from diseases where treatment has been unattainable. The research is advancing rapidly; however, there are still many hurdles to overcome before the therapeutic potential of regenerative medicine and cell therapy can be realized. Low in frequency in all tissues, stem cell number is often a limiting factor. Approaches that can control the proliferation and direct the differentiation of stem cells would significantly impact the field. Developing an adequate environment that mimics in vivo conditions is an intensively studied topic for this purpose. Collaboratively, researchers have come close to incorporating nearly all biological cues representative of the human body. Arguably the most overlooked aspect is the influence of electrical stimulation. In this dissertation, we examined polyvinylidene fluoride (PVDF) as a new biomaterial and developed a 3D scaffold capable of providing mechanical and electrical stimuli to cells in vitro. </p><p>The fabrication of a 3D scaffold was performed using electrospinning. To obtain highly aligned fibers and scaffolds with controlled porosity, the set-up was modified by incorporating an auxiliary electrode to focus the electric field. Highly aligned fibers with diameters ranging from 500 nm to 15 µm were fabricated from colorless polyimide (CP2) and polyglycolic acid (PGA) and used to construct multilayer scaffolds. This experimental set-up was used to electrospin &#945;-phase PVDF into the polar &#946;-phase. We demonstrated the transition to the &#946;-phase by examining the crystalline structure using x-ray diffraction (XRD), differential scanning calorimetry (DSC), fourier transform infrared spectroscopy (FTIR) and polarized light optical microscopy (PLOM). We confirmed these results by observing a polarization peak at 80°C using the thermally stimulated current (TSC) method. Our results proved the electrospinning process used in our investigation poled the PVDF polymer in situ. </p><p>TThe influence of architecture and topographical cues was examined on 3D scaffolds and films of CP2 polyimide and PVDF. Culture of human mesenchymal stem cells (hMSCs) for 7 and 14 days demonstrated a significant difference in gene expression. The fibers upregulated the neuronal marker microtubule associated protein (MAP2), while downregulation of this protein was observed on films. Gap junction formation was observed by the expression of connexin-43 after 7 days on PVDF films attributed to its inherent pyroelectric properties. Connexin-43 expression on fibers showed cell-cell contact across the fibers indicating good communication in our 3D scaffold. </p><p>A scaffold platform was designed using PVDF fibers that allowed us to apply electrical stimulation to the cells through the fibers. The electrically stimulated PVDF fibers resulted in enhanced proliferation compared to TCPS as evidenced by a 10% increase in the uptake of EdU. Protein expression revealed upregulation of neuronal marker MAP2. Our findings indicate this new platform capable of delivering mechanical, electrical, topographical and biochemical stimuli during in vitro culture holds promise for the advancement of stem cell differentiation and tissue engineering.</p> / Dissertation

Tissue engineering has the potential to create new tissue and organs from cultured cells for transplantation. Biodegradable and biocompatible scaffolds play a vital role in the transfer of the cultured cells to a new tissue. Various scaffolds for soft tissue engineering have been developed, however there is not any structure totally mimicking the natural extracellular matrix (ECM), ready to use. In this study biodegradable and biocompatible scaffolds were developed from natural polymers by tissue engineering approach and tested in vitro. Scaffolds (SCAF) were prepared with freeze drying and composed of chitosan, gelatin and dermatan sulfate. Polymer solutions were treated with different stirring rates (500 rpm and 2000 rpm), freezing temperatures (-20 &deg / C and -80 &deg / C) and molding (cylindrical mold and petri dish) to achieve porous structure in order to provide sufficient space for cell growth and extracellular matrix production. Among the prepared scaffolds at different conditions, the scaffolds prepared at 500 rpm and frozen at -80 &deg / C, (SCAF-1), was chosen for further studies. These scaffolds achieved 0.512 MPa tensile strength, with 9.165 MPa tension modulus and 3.428 MPa compression modulus. Besides in lysozyme containing degradation medium they conserved their integrity and lost about 30 % of their initial weight in 30 days period. Mechanical and enzymatic degradation tests showed that scaffolds have physical integrity for the tissue engineering applications. To mimic the natural tissue and enhance cell growth, biologically active arginine &amp / #8211 / glycine - aspartic acid - serine (RGDS) peptides and platelet derived growth factor-BB (PDGF-BB) were immobilized on the SCAF-1. Fibroblast cells were seeded on the scaffolds containing RGDS, (SCAF-1-RGDS), and PDGF-BB, (SCAF-1-RGDS-PDGF), and incubated in media either free of serum or containing serum. Scaffolds immobilized with RGDS and PDGF-BB had the highest attached cell number by the day 15. Florescence microscopy studies also indicated that RGDS and RGDS-PDGF modified scaffolds were more suitable than controls, (SCAF-1), for cell growth and proliferation. According to scanning electron microscopy (SEM) results, modified scaffolds demonstrated better cell morphology and attachment of cells. Based on the obtained results, it can be concluded that RGDS-PDGF immobilized chitosan-gelatin-dermatan sulfate systems have a great potential to be used as a scaffold for soft tissue engineering applications.

Hard tissue engineering holds the promise of restoring the function of failed hard tissues and involves growing specific cells on extracellular matrix (ECM) to develop &bdquo / &bdquo / tissue-like&rdquo / structures or organoids. Chitosan is a linear amino polysaccharide that can provide a convenient physical and biological environment in tissue regeneration attempt. To improve chitosan&amp / #8223 / s mechanical and biological properties, it was blended with another polymer gelatin. 1-ethyl-3-(3-dimethylaminopropyl)-carbodiimide (EDC) and N-hydroxysuccinimide (NHS) were used to crosslink the chitosan-gelatin matrix to produce stable structures. These natural polymers are mechanically weak especially to serve as a bone substitude and therefore, an inorganic calcium phosphate ceramic, hydroxyapatite, was incorporated to improve this aspect. The objective of this study was to develop chitosan-gelatin/hydroxyapatite scaffolds for a successful hard tissue engineering approach. For this reason, two types of hydroxyapatite, as-precipitated non-sintered (nsHA) and highly crystalline sintered (sHA) were synthesized and blended into mixtures of chitosan (C) and gelatin (G) v to produce 2-D (film) and 3-D (sponge) structures. The physicochemical properties of the structures were evaluated by scanning electron microscopy, X-Ray Diffraction (XRD), Fourier Transform Infrared-Attenuated Total Reflectance spectrometer (FTIR-ATR), differential scanning calorimetry, contact angle and surface free energy measurements and swelling tests. Mechanical properties were determined through tensile and compression tests. In vitro cell affinity studies were carried out with SaOs-2 cells. MTS assays were carried out to study cell attachment and proliferation on the 2-D and 3-D scaffolds. Several methods such as confocal, fluorescence and scanning electron microscopy were used to examine the cell response towards the scaffolds. Cell affinities of the samples were observed to change with changing chitosan-gelatin ratio and hydroxyapatite addition into the matrices. XRD and FTIR results confirmed the purity of the hydroxyapatite synthesized. Mechanical test results showed that 2-D and 3-D chitosan-gelatin/hydroxyapatite constructs have similar properties as bones, and in vitro studies demonstrated that the prepared matrices have the potential to serve as scaffold materials in hard tissue engineering applications.

Meniscus is a wedge shaped structure, with a convex base attached to a flat tibial surface, and with a concave femoral surface, on which femur and tibia articulate. It has several functions including joint lubrication, shock absorption, load transmission and joint stability. Various methods were tried to treat meniscal tears but each has its own drawbacks. Tissue engineering seems to be a promising solution that avoids all the problems associated with the other approaches. In this study, a three dimensional (3D) collagen-based structure was prepared by tissue engineering to mimic the natural human meniscus. Three different foams prepared under different conditions were combined and nano/microfibrous layers were placed in between them. To mimic the properties of the natural tissue, the top layer was composed of collagen-chondroitin sulfate-hyaluronic acid (Coll-CS-HA) prepared by freezing at -20&ordm / C followed by lyophilization. The middle and bottom layers were made with just collagen after freezing at -20&ordm / C and -80&ordm / C, respectively and lyophilization. Aligned nano/microfibers were prepared using collagen-poly(L-lactic-co-glycolic acid (Coll-PLGA). Various crosslinking procedures such as 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide/N-hydroxysuccinimide (EDC/NHS), genipin (GP), glutaraldehyde (GLU) either alone or in combination with dehydrothermal treatment (DHT) were used and based on both compressive and tensile properties, the best crosslinker was chosen to be DHT+EDC/NHS. Mechanical properties (compressive, tensile and shear) of the dry foams and the final 3D construct were evaluated. The highest mechanical properties were obtained with the 3D construct. Then all these foams and the 3D construct were seeded with human fibrochondrocytes to study the cell behavior such as attachment, proliferation, and extracellular matrix (ECM) and glucosaminoglycan (GAG) production. Furthermore, the influence of cell seeding on the compressive properties of wet individual foams and the 3D construct was observed. As expected, the highest cell proliferation and compressive properties were obtained with the 3D construct. Finally, the 3D constructs, seeded with fibrochondrocytes, were implanted in New Zealand rabbits after meniscectomy. The initial microscopical examination show that the 3D construct has a significant potential as a meniscus substitute.

Due to their large internal surface area, microporous materials have been widely used in applications where high surface activity is desired. Example applications are extracellular scaffolds for tissue engineering, porous substrates for catalytic reaction, and permeable media for membrane filtration, etc. To realize these potential applications, various techniques such as TIPS (thermal induced phase separation), particle leaching, and SFF (solid freeform fabrication) were proposed and investigated. Despite of being able to generate microporous for specific applications, these available fabrication techniques have limitations on controlling the inner porous structure and the outer geometry in a cost-effective manner. To address these technical challenges, a systematic study focusing on the generation of microporous structures using co-continuous polymer blend was conducted. Under this topic, five subtopics were explored: 1) generation of gradient porous structures; 2) geometrical confining effect in compression molding of co-continuous polymer blend; 3) microporous composite with high nanoparticle loading; 4) micropatterning of porous structure; 5) simulation strategy for kinetics of co-continuous polymer blend phase coarsening process.

This dissertation presents the application of MEMS-based approaches for the construction of engineered tissue substitutes. MEMS technology can offer the physical scale, resolution, and organization necessary for mimicking native tissue architecture. Micromachined nozzles and templates were explored for the fabrication of acellular, biomimetic collagenous fibrous scaffolds, microvascular tissue structures, and the combination of these structures with cell-based therapeutics. The influence of the microstructure of the tissue constructs on their macro-scale characteristics was investigated.

Les protéines [Fe-S] sont des enzymes ubiquitaires, assurant des fonctions clés au sein des organismes vivants. La biosynthèse des centres [Fe-S], à savoir les processus permettant un assemblage correct des atomes de fer et de soufre au sein des protéines cibles, requièrent la participation de machineries protéiques complexes. Parmi elles se trouve la machinerie SUF qui intervient dans des conditions de stress oxydant et de carence en fer. Elle est composée de six gènes sufABCDSE. La protéine SufA est proposée comme étant une protéine scaffold ayant pour rôle de préassembler transitoirement des centres [Fe-S] et de les transférer à des protéines cibles. Elle possède trois résidus cystéines conservés proposés comme étant les ligands des centres [Fe-S].<br />SufA est obtenue principalement sous forme apo après purification. Le centre [Fe-S] peut être reconstitué chimiquement in vitro. Dans ces conditions, SufA contient un mélange de centres [2Fe-2S] et [4Fe-4S]. Nous avons alors isolé SufA native métallée après purification à partir de tout l'opéron suf en anaérobiose, et montré qu'elle contient un centre [Fe-S], plutôt de type [2Fe-2S], transférable efficacement à la ferrédoxine. Nous avons également étudié les mécanismes moléculaires de formation du cluster dans SufA. SufA est capable de fixer à la fois du soufre, au niveau de ses trois cystéines conservées, et du fer, majoritairement au niveau d'atomes d'azote et d'oxygène. Ces éléments sont mobilisables pour la formation d'un centre [Fe-S] en milieu réducteur. Enfin, des expériences préliminaires réalisées in vitro avec des mutants dirigés n'ont pas permis d'identifier la nature exacte des ligands du centre [Fe-S] dans SufA.

Data can be acquired from tissue's vascular structure and used for modeling and visualization. To acquire data from a vascular tree, we make its structure available for the gathering of data by separating it from the structures of surrounding tissues, which includes the capillary structure. The capillary structure contains important information, but, because of its size, is the most difficult to acquire data from. In this work, we look at methods for contrasting the vascular structure from surrounding tissues, and focus on the use of corrosion casting for this procedure. We collected image data using micro-computer tomography (micro-CT) and converted data into stereolithography models. Models were imported into computer aided design (CAD) software, which was used to further process the models in order to ensure that the necessary structures were in place for the recreation of the capillary structures' relationship to targeted cell systems. Recreating the cell system-capillary system relationship is the reason building this model is so important. It is this relationship that we seek to model so that, in the future, we can create designs that guide the fabrication of three-dimensional (3D) scaffolding, which mimic capillary patterns with supportive structure that serve as an extracellular matrix for 3D tissue engineering. This method had been designed to enhance a variety of therapeutic protocols including, but not limited to, organ and tissue repair, systemic disease mediation and cell/tissue transplantation therapy.