The goal of this research was to develop a system of individualized medicine that could be applied to dermal wounds serving as a wound dressing and synthetic extracellular matrix while delivering stem cells to the wound bed. First, fabrication parameters for electrospinning polymer fibers were determined. This involved evaluating fiber morphology with respect to polymer selection and solution concentration. Next, construct fabrication was examined to produce an integrated void space, or cargo area, suitable to maintain stem cells. In vitro studies to ensure stem cell viability and phenotype were conducted, and results supported the notion that cells could be administered to the wound site through construct pre-seeding. Lastly, in vivostudies were conducted to evaluate the construct as an applied biomaterial and as a cellular delivery device. Wound closure and quality were assessed, and neo-vascularization quantified. This project will provide insight into the tissue engineering field regarding cell-based therapies and dermal wound healing.

This article describes the development of a component-based technology robot system that can be rapidly configured to perform a specific manufacturing task. The system is conceived with standard and inter-operable components including actuator modules, rigid link connectors and tools that can be assembled into robots with arbitrary geometry and degrees of freedom. The reconfigurable "plug-and-play" robot kinematic and dynamic modeling algorithms are developed. These algorithms are the basis for the control and simulation of reconfigurable robots. The concept of robot configuration optimization is introduced for the effective use of the rapidly reconfigurable robots. Control and communications of the workcell components are facilitated by a workcell-wide TCP/IP network and device level CAN-bus networks. An object-oriented simulation and visualization software for the reconfigurable robot is developed based on Windows NT. Prototypes of the robot systems configured to perform 3D contour following task and the positioning task are constructed and demonstrated. Applications of such systems for biomedical tissue scaffold fabrication are considered. / Singapore-MIT Alliance (SMA)

Despite the unfavorable pharmacokinetic properties associated with peptides, they are still of great interest in drug development due to a multitude of interesting biological functions. The development of peptidomimetics strives to maintain or improve the biological activity of a peptide concurrently with removing the unwanted properties. This thesis describes two synthetic approaches to peptidomimetics with particular emphasis on secondary structure mimetics. First the design, synthesis and evaluation of two beta-turn mimetics incorporated in the endorphin Leu-enkephalin is presented. The beta-turn mimetics were stabilized by replacement of the intramolecular hydrogen bond with an ethylene bridge, and the amide bond between Tyr and Gly was replaced with an ether linkage. Linear analogues of the two mimetics were also synthesized. The peptidomimetics and their linear analogues were evaluated in a competitive binding assay at two opiate receptors, my and delta. One of the cyclized beta-turn mimetics was found to be a delta receptor antagonist with an IC50 value of 160 nM. Second a synthetic strategy to a beta-strand mimetic using 2-fluoro-4-iodopyridine as scaffold is described. The synthesis involved a Grignard exchange reaction on the pyridine scaffold using an amino acid derivative as electrophile followed by an SNAr reaction using an amine as nucleophile. The synthesis of a tripeptidomimetic of Leu-Gly-Gly and attempts to introduce chiral building blocks at the C-terminal, as well as studies towards elongated mimetics are presented. Two additional studies deal with the synthesis of two classes of potential thrombin inhibitors based on the pyridine scaffold. The first class contain pyridine as central fragment (P2 residue) substituted with a para-amidinobenzylamine group as P1 residue and various benzoyl groups as P3 residues. Three potential thrombin inhibitors were synthesized and found to be microM inhibitors in an enzymatic assay. In the second class, the pyridine ring serves as P3 residue. This class also lacks a strongly basic group in the P1 position. A small library of eight compounds were synthesized and evaluated in the enzymatic assay. Unfortunately, these compounds lacked inhibitory activity.

The present work describes the preparation of hyaluronan derivatives and hydrogels with potential use in tissue engineering applications. A potentially injectable hydrogel consisting of hyaluronan and collagen was successfully used to grow neurons in vitro by encapsulation of neural stem and progenitor cells. Attempts were further made to establish a suitable modification strategy which could be used for the preparation of in vivo cross-linkable hyaluronan derivatives. The synthesis of a model substance consisting of a D-glucuronate derivative which could simplify the development of such a modification technique is described, although a new method to prepare hyaluronan derivatives was found without its use. The modification strategy involves the use of a triazine-reagent which enables the covalent attachment of hydrophilic and hydrophobic amines to hyaluronan carboxyl groups in a controlled fashion under mild conditions. Using triazine-activated amidation we synthesized an aldehyde-derivative of hyaluronan which was used to prepare gels by cross-linking with hydrazide-modified polyvinyl-alcohol. Gels were formed in less than 1 minute by mixing equal volumes of the polymer derivatives and they were subsequently used as a carrier for bone morphogenetic protein-2. An in vitro release study showed that approximately 88% of the growth factor is retained in the gel over a 4 week period. The ability to form new bone in vivo was further evaluated in an ectopic rat model by the injection of gels containing 30 µg BMP-2. Radiographic and histological examination 4 and 10 weeks after injection showed the formation of new bone without any signs of inflammation or foreign body response. Hydroxyapatite particles were further added to improve the mechanical properties of the gel, and a comparative study was conducted. This time the induced tissue consisted not only of bone, but also of interconnected cartilage and tendon, as confirmed by histology and immunohistochemistry.

The heart does not regenerate new functional tissue when myocardium dies following coronary artery occlusion, or is defective. Ventricular restoration involves excising the infarct and replacing it with a cardiac patch to restore the heart to a more efficient condition. The goal of this study was to design and develop a myocardial patch to replace myocardial infarctions. A basic design was developed that is composed of 3D microfibrous mats that house mesenchymal stem cells (MSCs) from umbilical cord matrix (Wharton&rsquo / s Jelly) aligned parallel to each other, and biodegradable macroporous tubings to supply growth media into the structure. Poly(glycerol sebacate) (PGS) prepolimer was synthesized and blended with P(L-D,L)LA and/or PHBV, to produce aligned microfiber (dia 1.16 - 1.37 &amp / #956 / m) mats and macroporous tubings. Hydrophilicity and softness of the polymer blends were found to be improved as a result of PGS introduction. The Wharton&rsquo / s Jelly (WJ) MSCs were characterized by determination of their cell surface antigens with flow cytometry and by differentiating them into cells of mesodermal lineage (osteoblasts, adipocytes, chondrocytes). Cardiomyogenic differentiation potential of WJ MSCs in presence of differentiation factors was studied with RT-PCR and immunocytochemistry. WJ MSCs expressed cardiomyogenic transcription factors even in their undifferentiated state. Expression of a ventricular sarcomeric protein was observed upon differentiation. The electrospun, aligned microfibrous mats of PHBV-P(L-D,L)LA-PGS blends allowed penetration of WJ MSCs and improved cell proliferation. To obtain the 3D myocardial graft, the WJ MSCs were seeded on the mats, which were then wrapped around macroporous tubings. The 3D construct (4 mm x 3.5 cm x 2 mm) was incubated in a bioreactor and maintained the uniform distribution of aligned cells for 2 weeks. The positive effect of nutrient flow within the 3D structure was significant. This study represents an important step towards obtaining a thick, autologous myocardial patch, with structure similar to native tissue and capability to grow, for ventricular restoration.

Tissue engineered human oral mucosa has the potential to fill tissue deficits caused by facial trauma or malignant lesion surgery. It can also help elucidate the biology of oral mucosa and serve as an alternative to in vivo testing of oral care products. The aim of this thesis was to construct a tissue engineered full-thickness human oral mucosa closely mimicking the native tissue. To this end, the feasibility of the concept was tested by co-culturing fibroblasts and epithelial cells isolated from normal human oral mucosa biopsies in a collagen-glycosaminoglycan-chitosan scaffold, developed in our laboratory to construct a skin equivalent. An oral mucosal equivalent closely mimicking the native one was obtained and characterized by histology, immunohistochemistry and transmission electron microscopy. Using the same model, the influence of mesenchymal cells on oral epithelial development was investigated by culturing epithelial cells on lamina propria, corneal stroma and dermal equivalents. They were found to significantly influence the thickness and the ultrastructure of the epithelium. Finally, in order to improve the adhesiveness of conventional scaffolds, an elastin-like recombinamer (ELR) containing the cell adhesion tripeptide, RGD, was used in the production of novel bilayer scaffolds employing lyophilization and electrospinning. These scaffolds were characterized by mercury porosimetry, scanning electron microscopy and mechanical testing. In vitro tests revealed positive contribution of ELR on the proliferation of both fibroblasts and epithelial cells. It was thus possible to construct a viable oral mucosa equivalent using the principles of tissue engineering.

In tissue engineering applications, use of biodegradable and biocompatible materials are essential. As the tissue regenerate itself on the material surface, the material degrades with enzymatic or hydrolytic reactions. After a certain time, natural tissue takes the place of the artificial support. Poly(&epsilon / -caprolactone) (PCL) is one of the preferable polymers used in the restoration of the bone defects due to its desirable mechanical properties and biocompatibility. Addition of inorganic calcium phosphate particles in PCL structures can improve the mechanical properties as well as osteoconductivity / and presence of an antibiotic can prevent infection that may occur at the defect site. In this study, three forms of biodegradable hard tissue supports which are bone fillers, bone regenerative membranes and 3D scaffolds were designed and prepared. As biodegradable bone fillers, composite microspheres containing gelatin and &beta / -tricalcium phosphate (&beta / -TCP) were prepared and characterized. Synthesized &beta / -TCP particles were coated with gelatin at different weight ratios and the effects of &beta / -TCP/Gelatin ratio on the morphology of the microspheres were evaluated. Also, a model antibiotic, gentamicin, was loaded to these microspheres and release behaviours of the drug and its antibacterial effect on E.Coli was determined. The selected composition of these microspherical bone fillers were used as additives in the preparation of bone regenerative membranes and scaffolds. For this purpose, microspheres were added into PCL solution and processed by either solvent casting or freeze-drying in order to prepare bone regenerative membranes or scaffolds, respectively. For every material, the ratio of constituents (microsphere and PCL) was altered in order to obtain optimum properties in the resulted hard tissue support structure. The effects of the ratio of the microspheres to PCL in terms of morphological, mechanical and degradation properties of composite films, as well as in vitro antibiotic release and antibacterial activities against E.Coli and S.Aureus were investigated. For scaffolds, the effects of the ratio of the microspheres to PCL on the morphological, mechanical, pore size distribution, degradation properties and in vitro antibiotic release were examined.

The focus of this dissertation is on the development of an additive manufacturing system and methodology for fabricating structures with functionally graded porous internal properties and complex three-dimensional external characteristics. For this purpose, a multi-scale three-dimensional printing system was developed, with capabilities and fabrication methodologies refined in the context of, but not limited to, manufacturing of porous bone substitutes. Porous bone implants are functionally graded structures, where internally, the design requires a gradient in porosity and mechanical properties matching the functional transition between cortical and cancellous bone regions. Geometrically, the three-dimensional shape of the design must adhere to the anatomical shape of the bone tissue being replaced. In this work, control over functionally graded porous properties was achieved by integrating specialized modules in a custom-made additive manufacturing system and studying their effect on fabricated constructs. Heterogeneous porous properties were controlled by: (i) using a micro-syringe deposition module capable of embedding sacrificial elements with a controlled feature size within the structure, (ii) controlling the amount of binder dispersed onto the powder substrate using a piezoelectric printhead, (iii) controlling the powder type or size in real-time, and/or (iv) selecting the print layer stacking orientation within the part. Characterization methods included differential scanning calorimetry (DSC)-thermo gravimetric analysis (TGA) to establish the thermal decomposition of sacrificial elements, X-ray diffraction (XRD) and dispersive X-ray spectroscopy (EDAX) to investigate the chemical composition and crystallinity, scanning electron microscopy (SEM) and optical microscopy to investigate the physical and structural properties, uniaxial mechanical loading to establish compressive strength characteristics, and porosity measurements to determine the bulk properties of the material. These studies showed that the developed system was successful in manufacturing embedded interconnected features in the range of 100-500 $ \mu m $, with a significant impact on structural properties resulting in bulk porosities in the range of 30-55% and compressive strength between 2-50 MPa. In this work, control over the the three-dimensional shape of the construct was established iteratively, by using a silhouette extraction image processing technique to determine the appropriate anisotropic compensation factors necessary to offset the effects of shrinkage in complex-shaped parts during thermal annealing. Overall shape deviations in the range of +/- 5-7 % were achieved in the second iteration for a femoral condyle implant in a sheep model. The newly developed multi-scale 3DP system and associated fabrication methodology was concluded to have great potential in manufacturing structures with functionally graded properties and complex shape characteristics.

Activity-induced immediate-early gene (IEG) transcription foci can be labelled with fluorescent probes, permitting high temporal and spatial resolution in mapping neuronal circuits. Previous quantification approaches have assumed a Boolean function of transcription foci, assuming that cells are either active or inactive. Due to multiple amplification steps in the in situ hybridization process, it was thought that information relating to magnitudes of firing rates was lost. However, the current data suggest that transcription foci actually exhibit non-Boolean intensity and size values which vary according to behavioural condition. Systematic characterization of transcription foci intensity and size revealed incremental variations such that: home-cage < one-environment exposure < five-environment exposure < maximal electroconvulsive shock. Visual differences in transcription foci may result from a quantifiable relationship between spiking patterns and transcription rates. The exact stoichiometry between neuronal spiking and transcription is not yet clear, but these results suggest that Boolean applications of IEG imaging may neglect accurate neuronal activation properties. / xvi, 125 leaves : ill. ; 29 cm

The current study characterizes the mechanical and biodegradation properties of a polycarbonate polyurethane (PU) electrospun nanofiber scaffold intended for use in the growth of a tissue engineered annulus fibrosus (AF) intervertebral disc component. Both the tensile strength and initial modulus of aligned scaffolds were higher than those of random scaffolds and remained unaffected during a 4 week biodegradation study, suggesting a surface-mediated degradation mechanism. The resulting degradation products were non-toxic. Confined compressive mechanical force of 1kPa, was applied at 1Hz to in vitro bovine AF tissue grown on the scaffolds to investigate the influence of mechanical force on AF tissue production, which was found to decrease significantly at 72 hours relative to 24 hours, independent of any effects from mechanical forces. Overall, the consistent rate of PU degradation, along with mechanical properties comparable to those of native AF tissue, and the absence of cytotoxic effects, make this polymer suitable for further investigation for use in tissue-engineering the AF.