Tissue engineering is a multidisciplinary field that is rapidly emerging as a promising new approach in the restoration and reconstruction of tissues. In this approach, three dimensional (3D) scaffolds are of great importance. Scaffolds function both as supports for cell growth and depot for sustained release of required active agents (e.g. enzymes, genes, antibiotics, growth factors). Scaffolds should possess certain properties in accordance with usage conditions. Wet-spinning is a simple technique that has been widely used for the fabrication of porous scaffolds for tissue engineering applications. Natural polymers can effectively be used in scaffold fabrication due to their biocharacteristics. Among natural polymers, chitosan and alginate are two of the most studied ones in tissue engineering and drug delivery fields because of being biologically renewable, biodegradable, biocompatible, non-antigenic, non-toxic and biofunctional. In this study, two kinds of porous scaffolds were produced as chitosan and alginate coated chitosan fibrous scaffolds by wet-spinning technique In order to investigate the delivery characteristics of the scaffolds, loading of gentamicin as a model antibiotic and bovine serum albumin (BSA) as a model protein was carried out in different loading models. Resultant scaffolds were characterized in terms of their structural formation, biodegradation, biomineralization, water uptake and retention ability and mechanical properties. Additionally, release kinetics of gentamicin and BSA were examined. Efficiency of gentamicin on Escherichia coli (E.coli) was examined. Characterization of scaffolds revealed their adequacy to be used in bone tissue engineering applications and capability to be employed as bioactive agent delivery systems.

Tissue engineering applies principles of biology and engineering to the development of functional substitutes for damaged or lost tissues. Tools for the neo-generation of tissue in tissue engineering research include cells, biomaterials and soluble factors. One main obstacle in tissue engineering is the limited availability of autologous tissue specific progenitor cells. This has led to interest into using autologous cells with stem cell plasticity. Bone marrow derived stem cells were the first adult stem cells shown to have multilineage potential. Since, several reports have been published indicating that cells from other tissues; fat, muscle, connective tissue e.g., possess potential to differentiate into lineages distinct from their tissue of origin. The optimal cell type for use in tissue engineering applications should be easy to obtain, cultivate and store. The human dermal fibroblast is an easily accessible cell source, which after routine cell expansion gives a substantial cell yield from a small skin biopsy. Hence, the dermal fibroblast could be a suitable cell source for tissue engineering applications.The main aim of this thesis was to investigate the differentiation capacity of human dermal fibroblasts, and their possible applications in bone and cartilage tissue engineering applications. Human dermal fibroblasts were shown to differentiate towards adipogenic, chondrogenic, and osteogenic phenotypes upon subjection to specific induction media. Differentiation was seen both in unrefined primary cultures and in clonal populations (paper I). Fibroblasts could be used to create three-dimensional cartilage- and bone like tissue when grown in vitro on gelatin microcarriers in combination with platelet rich plasma (paper II). 4 weeks after in vivo implantation of osteogenic induced fibroblasts into a fracture model in athymic rats, dense cell clusters and viable human cells were found in the gaps, but no visible healing of defects as determined by CT-scanning (paper III). After the induction towards adipogenic, chondrogenic, endotheliogenic and osteogenic lineages, gene expression analysis by microarray and quantitative real-time-PCR found several master regulatory genes important for lineage commitment, as well as phenotypically relevant genes regulated as compared to reference cultures (paper IV). In conclusion, results obtained in this thesis suggest an inherent ability for controllable phenotype alteration of human dermal fibroblasts in vitro. We conclude that dermal fibroblasts could be induced towards adipogenic, chondrogenic, endotheliogenic or osteogenic novel phenotypes which suggest a genetic readiness of differentiated fibroblasts for lineage-specific biological functionality, indicating that human dermal fibroblasts might be a suitable cell source in tissue engineering applications.

This thesis demonstrated the effects of exogenous stimuli on engineered articular cartilage constructs and elucidated mechanisms underlying the responses to these agents. In particular, a series of studies detailed the effects of chondroitinase-ABC (C-ABC), hyaluronic acid (HA), and TGF-β1 on the biochemical and biomechanical properties of self-assembled articular cartilage. Work with C-ABC showed that this catabolic agent can be employed to improve the tensile properties of constructs. When constructs were cultured for 6 weeks, treating with C-ABC at 2 weeks enhanced the tensile stiffness. Furthermore, treating at 2 and 4 weeks synergistically increased tensile properties and allowed compressive stiffness to recover to control levels. Another study showed that combining C-ABC and TGF-β1 synergistically enhanced the biochemical and biomechanical properties of neotissue. Microarray analysis demonstrated that TGF-β1 increased MAPK signaling in self-assembled neocartilage whereas C-ABC had minimal effects on gene expression. SEM analysis showed that C-ABC increased collagen fibril diameter and fibril density, indicating that C-ABC potentially acts via a biophysical mechanism. Constructs treated with C-ABC and TGF-β1 also showed stability and maturation in vivo , exhibiting a tensile stiffness of 3.15±0.47 MPa compared to a pre-implantation stiffness of 1.95±0.62 MPa. To assess the response to HA application, studies were conducted to optimize HA administration and examine its effects in conjunction with TGF-β1. Applying HA increased the compressive stiffness 1-fold and increased GAG content by 35%, with these improvements depending on HA molecular weight, application commencement time, and concentration. Microarray and PCR analyses showed that HA also influenced genetic signaling, up-regulating multiple genes associated with the TGF-β1 pathway. In addition to genetic effects, the enhanced GAG retention due to HA treatment could increase the fixed charge density of the matrix and thereby increase resistance to compressive loading. Additive effects were observed when HA was applied in conjunction with TGF-β1, with the combined treatment increasing compressive stiffness and GAG content by 150% and 65%, respectively. In general, results demonstrated mechanisms underlying C-ABC, HA, and TGF-β1 treatments and showed how these agents can be applied to improve cartilage regeneration efforts.

Towards understanding regional meniscus characteristics important for tissue engineering efforts, meniscus cells were characterized biomechanically and an effective method for isolating these cells for tissue engineering was determined. It was found that the meniscus contains cells that are biomechanically distinct, with outer meniscus cells showing higher stiffness than inner cells. It was also determined that meniscus cells as a whole were more biomechanically similar to ligament cells than to articular chondrocytes, indicating that tissue properties may correlate with cellular mechanics. In addition to showing regionally distinct biomechanical properties, enzymatic isolation of meniscus cells was found to cause varying phenotypic changes in cells from the inner, middle, and outer regions. A comparison of isolation techniques also indicated that sequential digestion of meniscus tissue with pronase and collagenase was able to yield more cells with higher viability than other techniques tested, and those isolated cells created stiffer and more glycosaminoglycan (GAG) rich constructs when used in a tissue engineering modality than cells isolated using only collagenase. The identification of an effective mode of isolating meniscus cells is of great use to tissue engineering efforts, as they often require a large cell numbers. These findings illustrate that known regional variations in meniscus cell phenotype and biochemical composition are also evident in cellular mechanics, and phenotypic responses of these cells to isolation are varied and distinct. To be successful tissue replacements, tissue engineered meniscus constructs must not elicit an immune response and must have sufficient mechanical properties to survive when implanted. To determine if allogeneic or xenogeneic implantation of scaffold-free meniscus constructs could be feasible, the immunogenicity of bovine and leporine meniscus cells and articular chondrocytes were determined in an in vitro model system. It was found that neither bovine nor leporine meniscus cells or articular chondrocytes caused activation of leporine immune cells, suggesting that they may serve as allogeneic or xenogeneic cell sources for meniscus engineering. Additional analysis of the mechanical role of meniscus GAGs indicated that they are mechanically important in all regions of the meniscus, but especially in the inner region where the relatively high GAG content affects both compressive and tensile properties. Therefore, tissue engineering efforts should try to recapitulate GAG content and distribution to enhance the functionality of meniscus replacements. As a major obstacle for meniscus engineering is the identification of an abundant cell source, this thesis also investigated the use of skin cells as an alternative to primary cells for tissue engineering. Previously identified chondroinducible dermis cells were found to have multilineage differentiation capacity, and were subsequently termed dermis isolated adult stem cells (DIAS). DIAS cells were also able to be expanded in monolayer without losing chondroinductive capacity, and were able to create constructs with cartilaginous properties which could be varied with growth factor application. Given the ease of expansion and ability of DIAS cells to form fibrocartilaginous tissue, these cells present an abundant cell source for meniscus tissue engineering.

Fibrocartilages in the body, including the temporomandibular joint (TMJ) disc and knee meniscus, lack intrinsic healing capacity following trauma or disease. Current treatments only address the symptoms of fibrocartilage damage and do nothing to prevent further degradation of the joint. A tissue engineered replacement, with biochemical and biomechanical properties approaching those of native tissue, could provide a solution. This thesis investigates two components critical to the generation of a tissue engineered TMJ disc: 1) characterization of the native disc to identify a suitable animal model and create design parameters, and 2) development of approaches to use human embryonic stem cells (hESCs) in fibrocartilage tissue engineering. The first step to achieving this goal was to identify an animal model for the human TMJ disc based on quantitative biochemical and biomechanical properties. To this end, rabbit, goat, pig, cow, and human discs were analyzed, and the pig disc was shown to possess properties most similar to the human. The next step was to further characterize the pig TMJ, as many aspects of the joint were still poorly understood. Though the TMJ disc is anchored to the surrounding bony tissue on all sides by discal attachments, little was known about their properties. Biochemical and histological analysis was performed on these attachments and indicated that they are similar to the disc but possess distinct regional matrix content related to joint biomechanics. Finally, though the contribution of collagen to the mechanical properties of the TMJ disc was well characterized, the contribution of the glycosaminoglycans (GAGs) was unknown. By removing sulfated GAGs with chondroitinase ABC, it was found that these molecules contribute to the viscoelastic compressive properties of the disc, but only in regions with the highest native GAG content. The second aspect of this thesis involved producing fibrocartilage tissue from hESCs. The pluripotency and unlimited self-renewal of these cells makes them ideally suited for producing fibrocartilages that contain a spectrum of matrix components. This work began by investigating what factors are necessary for fibrochondrogenic differentiation of hESCs in embryoid bodies (EBs). Growth factors and co-cultures with primary fibrochondrocytes were both shown to be potent modulators of fibrochondrogenesis, although differentiation of hESCs consistently produced a heterogeneous cell population. To purify populations of fibrochondrocytes differentiated form hESCs, two inexpensive and novel techniques were investigated. First, density gradient separation was the first technique attempted. This technique was able to isolate distinct subpopulations of cells, some of which were mechanically similar to native chondrocytes. Second, a chondrogenic tuning technique was applied to differentiated hESCs. Following fibrochondrogenesis in EBs, cells were expanded in monolayer in chondrocyte specific media before being used for tissue engineering. Chondrogenic tuning produced several distinct cell populations during expansion, and, as a result, a spectrum of different cartilaginous tissues was achieved for tissue engineering. Three of the cell populations produced tissues similar to the native TMJ disc, outer meniscus, and inner meniscus. Overall, this thesis identified an animal model for TMJ characterization and in vivo studies, furthered understanding of structure-function relationships of the TMJ disc and its attachments, and developed a technique for producing a spectrum of engineered fibrocartilages from hESCs.

This study investigates the effect on: 1) the bulk surface; and 2) the three-dimensional non-woven microfabric scaffolds of poly(N-isopropylacylamide)-CNT-polyaniline on growth and viability of  mice fibroblast cells L929. The poly(N-isopropylacylamide)-CNT-polyaniline was prepared using coupling chemistry and electrospinning was then used for the fabrication of responsive, nonwoven microfabric scaffolds. The electrospun microfabrics were assembled in regular three-dimensional scaffolds with OD: 400-500 mm; L: 6-20 cm. Mice fibroblast cells L929 were seeded on the both poly(N-isopropylacylamide)-CNT-polyaniline bulk surface as well as non-woven microfabric scaffolds. Excellent cell proliferation and viability was observed on poly(N-isopropylacylamide)-CNT-polyaniline non-woven microfabric matrices in compare to poly(N-isopropylacylamide)-CNT-polyaniline bulk and commercially available Matrigel™ even with a range of cell lines up to 168 h. Temperature dependent cells detachment behaviour was observed on the poly(N-isopropylacylamide)-CNT-polyaniline scaffolds by varying incubation at below lower critical solution temperature (LCST) of poly(N-isopropylacylamide). The results suggest that poly(N-isopropylacylamide)-CNT-polyaniline non-woven microfabrics could be used as a smart matrices for applications in tissue engineering. / European Commission FP7 (PIIF-GA-2009-254955), JSPS, JST-CREST and MEXT

The growing incidences of coronary artery bypass graft surgeries have triggered a need to engineer a viable small diameter blood vessel substitute. An ideal tissue engineered vascular graft should mimic the microenvironment of a native blood vessel, while providing the adequate compliance post-implantation. Current vascular graft technologies lack the ability to promote vascular ECM deposition, leading to a compliance mismatch and ultimately, graft failure. Hence, in order to engineer suitable vascular grafts, this thesis describes the synthesis and characterization of novel elastin mimetic peptides, EM-19 and EM-23, capable of promoting vascular ECM deposition within a poly(ethylene glycol) diacrylate (PEG-DA) hydrogel. By combining the material properties of a synthetic and bio-inspired polymer, a suitable microenvironment for cell growth and ECM deposition can be engineered, leading to improved compliance. As such, characterization of EM-19 and EM-23 was conducted in human vascular smooth muscle cell (SMC) cultures, and the peptides self-assembled with a growing elastic matrix. After grafting the peptides onto the surface of PEG-DA hydrogels, EM-23 increased SMC adhesion by 6000% over PEG-RGDS hydrogels, which have been the gold standard of cell adhesive PEG scaffolds. Moreover, EM-23 grafted surfaces were able to promote elastin deposition that was comparable to tissue cultured polystyrene (TCPS) surface even though TCPS had roughly 4.5 times more SMCs adhered. Once translated to a 3D model, EM-23 also stimulated increased elastin deposition and improved the mechanical strength of the scaffold over time. Moreover, degradation studies suggested that EM-23 may serve as a template that not only promotes ECM deposition, but also allows ECM remodeling over time. The characterization studies in this thesis suggest that this peptide is an extremely promising candidate for improving vascular ECM deposition within a synthetic substrate, and that it may be beneficial to incorporate EM-23 within polymeric scaffolds to engineer compliant vascular grafts.

Three-dimensional (3D) tissue models have significantly improved our understanding of structure/function relationships and promise to lead to new advances in regenerative medicine. However, despite the expanding diversity of 3D tissue fabrication methods, in vitro approaches for functional assessments have been relatively limited. Herein, we describe the guidance of primary endothelial cells (ECs) in an agarose hydrogel scaffold that is chemically patterned with an immobilized concentration gradient of vascular endothelial growth factor 165 (VEGF165) using multiphoton laser patterning of VEGF165. This is the first demonstration of this patterning technology to immobilize proteins; and the first demonstration of immobilized VEGF165 to guide endothelial cell growth and differentiation in 3D environments. It is particularly compelling that this 3D hydrogels provide an excellent biomimetic environment for stem cell niche, thereby offering a new approach to study stem cell biology. In this thesis, we focused on the retinal stem cell niche, investigating cellular interactions between retinal stem and progenitor cells (RSPCs) and endothelial cells (ECs). By using this 3D in vitro model, we demonstrated the synergistic interactions between RSPCs and ECs wherein RSPCs migrated into 3D gels only in the presence of ECs and RSPCs stabilized EC tubular-like formations. Moreover, we characterized the contact-mediated effects of ECs on RSPC fate in terms of proliferation and differentiation.

Embryonic stem cells (ESC) constitute a promising source of cells for cardiac transplantation strategies. However, complexities associated with in vivo studies have made it difficult to develop a thorough understanding of cell integration. We have engineered an in vitro system that recapitulates the native cardiac environment using 300μm thick collagen scaffolds seeded with neonatal cardiomyocytes (CM) and electrical field stimulation. The injection of undifferentiated ESC served as a baseline to assess the validity of studying cell transplantation in this model. Yfp-ESC survived and proliferated over several days in model tissue. ESC were not observed to significantly differentiate into the cardiac lineage, and did not integrate with the cardiac cell population. While the injection of ESC improved cardiac cell number, tissue functional properties were hindered. The methods developed herein can be readily adapted to study ESC derived progenitor and differentiated cells, to elucidate the optimal cell state for ESC-mediated cell therapy.

This thesis presents a microfluidic strategy for the in-flow definition of a 3D soft material with a tunable and perfusable microstructure. The strategy was enabled by a microfluidic device containing up to fifteen layers that were individually patterned in polydimethylsiloxane (PDMS). Each layer contained an array of ten to thirty equidistantly spaced microchannels. Two miscible fluids (aqueous solutions of alginate and CaCl2) were used as working fluids and were introduced into the device via separate inlets and distributed on chip to form a complex fluid at the exit. The fluid microstructure was tuned by altering the flow rates of the working fluids. Upon solidification of alginate in the presence of calcium chloride, the created microstructure was retained and a soft material with a tunable microstructure was formed. The produced material was subsequently perfused using the same microfluidic architecture. The demonstrated strategy potentially offers applications in materials science and regenerative medicine.