This work examines the osteogenic potential and immune-privileged properties of human umbilical cord perivascular cells (HUCPVCs) in normal Wistar rats and athymic rnu/rnu rats for up to 60 days. HUCPVCs demonstrated a mesenchymal stromal cell phenotype, assayed through flow cytometry, and RT-PCR analysis detected their expression of osteogenic genes. A bone tissue engineering construct was developed through centrifugal seeding of HUCPVCs onto calcium phosphate-coated PLGA scaffolds. These cell-scaffold constructs were transplanted into bilateral femoral defects. HUCPVCs did not induce any systemic biological response in normal rats; however, they did not engraft and impaired bone healing up to 60 days. When transplanted into athymic rats, HUCPVCs were detected up to 30 days in the femoral defects, improved bone regeneration at 15 and 30 days, as measured by micro computed tomography, and expressed osteogenic proteins. These findings demonstrate that HUCPVCs are suitable for bone tissue engineering studies in larger animals.

Tissue engineering the annulus fibrosus (AF) for use in a functional intervertebral disc replacement is a promising alternative to current treatments of degenerative disc disease. Polycarbonate urethane (PU) scaffolds have demonstrated the ability to support AF cell attachment and matrix synthesis and are suitable for tissue engineering the AF. The present study investigates the effects of the physical and biochemical environment on AF cells grown on aligned nanofibrous PU scaffolds. First, the effect of dynamic spinner flask culture and fibronectin pre-coating on tissue formation was analyzed and then the role of scaffold fibre tension on annulus fibrosus cells was examined using a tailored culture system. The results of these studies demonstrated that AF cells are sensitive to differences in biochemical cues at the scaffold surface and their physical environment and respond by altering their cellular responses and, potentially by manipulating their microenvironments, including the physical characteristics of the PU-ADO scaffolds.

Tissue engineering is limited by inability to create early and adequate blood supply. In-vivo DCE-MRI has imaged angiogenesis in soft tissues, yet has not been considered in hard tissues. Bilateral critical defects created in parietal bones of eighteen adult rabbits were left void, treated with haluronic acid acellular matrix (HA-ACM), or HA-ACM impregnated with vascular endothelial growth factor (VegF). DCE- MRI was acquired at weeks 1,2,3,6, and 12. Histologic analysis of HA-ACM treated defects demonstrated quantitatively greater immature bone formation, increased quantity and larger blood vessels compared to void. Statistically significant greater angiogenesis evidenced by quantitative perfusion on MRI supported histologic findings. DCE MRI is a novel means of imaging angiogenesis in grafted bone defects. DCE-MRI discerns physiologically important phases of angiogenesis: Initial vasoactive response, vessel network initiation, establishment, and pruning. DCE-MRI is adaptable to non-invasive study of candidate tissue engineered constructs and in evaluating scaffolds and treatments on angiogenesis.

Degenerative disc disease is a common ailment with enormous medical, psychosocial and economic ramifications. This study was designed to investigate the utility of a thiol-modified hyaluronan(TMHA) and elastin-like polypeptide(EP) composite material as a potential tissue engineering scaffold to reconstitute the nucleus pulposus in early degenerative disc disease. TMHA and EP were combined in various concentrations and cross-linked using poly(ethylene glycol)diacrylate. Resulting materials were evaluated biomechanically and biologically. Confined compression testing revealed that the addition of EP to TMHA-based gels resulted in a stiffer construct, but remained an order of magnitude less stiff than native nucleus. The in vitro cell culture experiments with human intervertebral disc cells demonstrated 70% cell viability at three weeks with apparent maintenance of phenotype. The addition of EP did not have a significant biologic effect. An in vivo pilot study demonstrated biocompatibility of the TMHA-based hydrogels; additional power is required to adequately assess treatment effect.

This work examines the osteogenic potential and immune-privileged properties of human umbilical cord perivascular cells (HUCPVCs) in normal Wistar rats and athymic rnu/rnu rats for up to 60 days. HUCPVCs demonstrated a mesenchymal stromal cell phenotype, assayed through flow cytometry, and RT-PCR analysis detected their expression of osteogenic genes. A bone tissue engineering construct was developed through centrifugal seeding of HUCPVCs onto calcium phosphate-coated PLGA scaffolds. These cell-scaffold constructs were transplanted into bilateral femoral defects. HUCPVCs did not induce any systemic biological response in normal rats; however, they did not engraft and impaired bone healing up to 60 days. When transplanted into athymic rats, HUCPVCs were detected up to 30 days in the femoral defects, improved bone regeneration at 15 and 30 days, as measured by micro computed tomography, and expressed osteogenic proteins. These findings demonstrate that HUCPVCs are suitable for bone tissue engineering studies in larger animals.

Tissue engineering the annulus fibrosus (AF) for use in a functional intervertebral disc replacement is a promising alternative to current treatments of degenerative disc disease. Polycarbonate urethane (PU) scaffolds have demonstrated the ability to support AF cell attachment and matrix synthesis and are suitable for tissue engineering the AF. The present study investigates the effects of the physical and biochemical environment on AF cells grown on aligned nanofibrous PU scaffolds. First, the effect of dynamic spinner flask culture and fibronectin pre-coating on tissue formation was analyzed and then the role of scaffold fibre tension on annulus fibrosus cells was examined using a tailored culture system. The results of these studies demonstrated that AF cells are sensitive to differences in biochemical cues at the scaffold surface and their physical environment and respond by altering their cellular responses and, potentially by manipulating their microenvironments, including the physical characteristics of the PU-ADO scaffolds.

Tissue engineering is limited by inability to create early and adequate blood supply. In-vivo DCE-MRI has imaged angiogenesis in soft tissues, yet has not been considered in hard tissues. Bilateral critical defects created in parietal bones of eighteen adult rabbits were left void, treated with haluronic acid acellular matrix (HA-ACM), or HA-ACM impregnated with vascular endothelial growth factor (VegF). DCE- MRI was acquired at weeks 1,2,3,6, and 12. Histologic analysis of HA-ACM treated defects demonstrated quantitatively greater immature bone formation, increased quantity and larger blood vessels compared to void. Statistically significant greater angiogenesis evidenced by quantitative perfusion on MRI supported histologic findings. DCE MRI is a novel means of imaging angiogenesis in grafted bone defects. DCE-MRI discerns physiologically important phases of angiogenesis: Initial vasoactive response, vessel network initiation, establishment, and pruning. DCE-MRI is adaptable to non-invasive study of candidate tissue engineered constructs and in evaluating scaffolds and treatments on angiogenesis.

This study aimed at guiding osteoblast cells on biodegradable polymer carriers with well-defined surface microtopography and chemistry, and investigating the effect of cell alignment on osteoblast phenotype expression. A blend of two different polyesters, one being natural in origin (PHBV) and the other synthetic (P(L/DL)LA), was used to form a film with parallel macro- (250 &micro / m wide) or microgrooves (27 &micro / m wide) on its surface, by solvent casting on patterned templates. The micropatterned Si template was produced by photolithography, while the Teflo macropatterned template was lathe cut. Fibrinogen (Fb) was adsorbed or immobilized via epichlorohydrin spacer/crosslinker on the film surfaces to enhance cell attachment by increasing the surface hydrophilicity and by providing RGD amino acid sequence for integrin binding. Surface hydrophilicity was assessed by water contact angle measurements. Adsorption of Fb caused an increase in hydrophilicity, while the opposite was achieved with its covalent immobilization. Fb was homogeneously immobilized throughout the whole micropatterned film surface with amount of 153.1 &plusmn / 42.4 g Fb/cm2, determined with the Bradford assay, while it was adsorbed within the grooves of the micropattern. Surface characteristics of the films were studied with Scanning Electron (SEM) and Light microscopy. Osteoblast cells derived from rat bone marrow were seeded on the polymeric films with different surface topography and chemistry and were grown for one and three weeks. Osteoblast proliferation on the films was determined with Cell Titer 96 TM Non-Radioactive Cell Proliferation (MTS) test. Alkaline Phosphatase (ALP) assay and tetracycline labelling of mineralized matrix were carried out to determine osteoblast phenotype expression on different surfaces. SEM and fluorescence microscopy were used to evaluate the cell alignment. Osteoblasts on the micropatterned films with adsorbed Fb aligned along the groove axis with a mean deviation angle of 13.1o, while on the unpatterned films deviation from horizontal axis was 63.2o and cells were randomly distributed. Cell alignment did not affect cell proliferation. However, the highest ALP specific activity and the most homogeneous mineral distribution were obtained on the Fb adsorbed micropatterned films.

ABSTRACT CORNEA ENGINEERING ON BIODEGRADABLE POLYESTERS Zorlutuna, Pinar M. Sc., Department of Biotechnology Supervisor: Prof. Vasif Hasirci Co-Supervisor: Asst. Prof. AySen Tezcaner January 2005, 66 pages Cornea is the outermost layer of the eye and has an important role in vision. Damage of cornea due to injuries or infections could lead to blindness lowering the quality of life of the patient severely. In such cases, transplantation or artificial corneas have been used for treatment but both had drawbacks. The novel approach for corneal replacements is the tissue engineering of the cornea, a promising method which would be free of these drawbacks, if successful. In this study, carriers for tissue engineering of the cornea were designed and tested in vitro. Blends of biodegradable and biocompatible polyesters of natural (PHBV8) and synthetic (PLLA) origin were used to construct these carriers. For the epithelial layer of the cornea, PLLA-PHBV8 micropatterned films were prepared with solvent casting and seeded with D407 (retinal pigment epithelial) cells. In order to achieve proper cell growth, the films were coated with fibronectin. For the stromal layer of the cornea, highly porous foams of PLLA-PHBV8 were prepared by lyophilization and seeded with 3T3 cells (fibroblasts). A new approach was developed to create a combination of the film and the foam to obtain a surface patterned, 3 dimensional cell carrier. These carriers were seeded with Saos-2 cells (osteosarcoma cells) in the preliminary optimization studies and with D407 and 3T3 cells in further studies. The cell numbers on the carriers were quantified by using MTS assay (non-radioactive cell proliferation assay) and the cell proliferation on polymeric carriers was significantly higher than that of control (Tissue culture polystyrene) by the day 14. Characterization of these cells and the carrier was done using a variety of microscopic methods. The micrographs showed that the foam had a highly porous structure and the pores were interconnected. 3T3 cells were found to be distributed quite homogeneously at the seeding site, but due to the high thickness of the foam, the cells could not sufficiently populate the core (central parts of the foam) during the given incubation time. The micropatterned film allowed multilayer formation of D407 cells. The functionality of the cells seeded on the carriers was examined by immunohistochemistry. These analyses proved that the cells retained their phenotype during culturing. D407 cells formed tight junctions characteristic of epithelial cells, and 3T3 cells deposited collagen type I into the foams. Based on the results, it can be concluded that the 3-D PLLA-PHBV8 construct with surface patterns have a serious potential for use as a tissue engineering carrier for the reconstruction of the cornea. Key words: Tissue engineering, cornea, polymeric carrier, biodegradable, polyester.

Key to the success of natural and engineered tissues becoming clinically available until needed is their long-term storage at low temperatures. This can be implemented by means of freezing or vitrification. To this end, vitrification offers an attractive approach for tissue banking by forming an amorphous glass both intra- and extracellularly and thereby avoiding the harmful effects of ice formation. Generally, high concentrations of cryoprotectants (CPAs) are used in conjunction with high cooling and warming rates to achieve this. However, hurdles associated with applying this technique include the ability to adequately deliver and remove CPAs due to cellular osmotic and cytotoxic effects as well as achieving adequate cooling and warming rates throughout the tissue to avoid ice formation. The aim of this work was to account for these factors in designing cryopreservation protocols for native and engineered tissues that had intrinsically different characteristics, including tissue size and extracellular matrix properties. The tissues investigated were two types of three-dimensional, cell encapsulated systems consisting of murine insulinomas and murine embryonic stem cells, and native articular cartilage. A mathematical 3-D CPA transport model was developed to predict cell volume excursions and intracellular CPA equilibration and applied to cryopreserve an engineered tissue. This thesis established a systematic methodology to design cryopreservation protocols using experimental measurements and a mathematical model for tissues.