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A critical challenge in the design of biomaterials for tissue engineering relies on the development of tissue-specific biomimetic scaffolds capable of replacing cell-matrix interactions required for the repair of injured tissues. Further, such biomaterials with the additional capacity to prevent bacteria contamination can resolve issues surrounding surgical prosthesis infection. Fibrous micro- and nanostructures are extensively researched in tissue engineering due to their intrinsic similarities to decellularised human tissues. Among the several fibre-forming processes, electrospinning has drawn much attention due to its ability to produce scaffolds that morphologically resemble the native extracellular matrix (ECM) of human tissues. Electrospinning is a versatile method that uses electrohydrodynamic principles to produce fibres with diameters ranging from microns to tens of nanometres. By varying the chemistry and morphology of the fibres, it is feasible to attain different physiological and mechanical responses. The wide array of raw natural and synthetic materials - including polymers and complex molecules - that can be used to electrospin fibres can resolve well-documented problems associated with the inferiority of synthetic biomaterials and the limitations of biological tissues. In this thesis, electrospinning is utilised to contribute to the engineering of advanced ECM-mimicking biomaterials. The work will focus on (1) improving the physicochemical and mechanical responses of skin substitutes and (2) preventing mesh-associated surgical site infection. The initial study of this thesis presents the design and construction of a nozzle-free electrospinning device, which is an economically viable method of scaling-up fibre production output. The equipment is then used to fabricate elastic skin-like composite nanofibres consisting of poly(vinylpyrrolidone) (PVP) and poly(glycerol sebacate) (PGS). The findings indicate that the mechanical properties of the electrospun mats could be tuned by varying the concentration of PGS and the molecular weight of PVP within the blends. Photocrosslinking the fibres prevented the rapid degradation of the composite mats due to the hydrophilic nature of PVP, making it feasible to assess the biological responses of the construct in vitro, displaying good viability and proliferation of human dermal fibroblasts. This study provides a different approach towards the development of skin substitutes, based on the fact that mechanical stimuli influence the ability of dermal cells to adapt and reconstruct the ECM at an injured site; being able to adjust the mechanics to those of different anatomical sites of the body can have a positive effect on the overall outcome of a healing wound. Synthetic biomaterials tend to present suboptimal cell growth and proliferation, with many studies linking this phenomenon to the hydrophobicity of such surfaces. This thesis continues with the development of a protocol for silk fibroin extraction from Bombyx mori cocoons, which achieved significantly increased yields of the protein in a third of the time required by the conventional molecular cut-off extraction approach. The extracted silk fibroin was then used to produce electrospun membranes consisting of poly(caprolactone) (PCL) blended with variant forms of PGS. The main aim of this work was the development of fibre mats with tuneable hydrophobicity/hydrophilicity properties, depending on the esterification degree and concentration of PGS within each composite. By altering the surface properties of the electrospun membranes, the trinary composite biomaterial presented improved fibroblast attachment behaviour and optimal growth in comparison to PCL-only fibrous mats. The study continued with the development of an ultralight-weight nanostructured bicomponent antimicrobial construct with a similar microstructure to biologic meshes, which preserved the required mechanical integrity of synthetic mesh materials. A core/shell nanofibrous structure was developed, consisting of nylon-6 in the core and chitosan/polyethylene oxide in the shell. The bicomponent fibre structure comprised a binary antimicrobial system incorporating 5-chloro-8-quinolinol in the chitosan-shell, with the sustained release of polyhexamethylene biguanide from the nylon-6 core of the fibres. The antimicrobial nanofibres were found to elicit a robust bactericidal response, in vitro, against the two most commonly occurring pathogenic bacteria in deep incisional surgical site infections; Staphylococcus aureus and Pseudomonas aeruginosa. The results of this study advocate that the bicomponent nanofibres developed can be a promising alternative to biologic meshes, employed for hernia repair today, due to similar architecture and mechanics, but at the same time capable of actively protecting the patient from subsequent mesh-associated infections, thus tackling this life-threatening postoperative complication. Overall, the work in this thesis has expanded upon the fields of skin tissue engineering and drug-eluting antimicrobial biomaterials, potentially guiding new areas of research.

Osteoarthritis is the leading cause of pain and disability worldwide. Progressive pathological changes in osteoarthritis are attributed to the fact that articular cartilage has a lack of vasculature, limiting its regenerative properties. Despite the availability of current treatment options, the prevalence of osteoarthritis is on the rise. Fully functional and suitable long-term treatments for large cartilage defects are yet to be sought. Over the years, considerable effort has been directed towards developing scaffolds for cartilage tissue engineering which consider the structural and biological function of the extracellular matrix (ECM). Tissue engineering combines structure, biological factors and signals to provide mechanical support and regulate cellular activities. This PhD focused on making scaffolds which captured the complex architecture and biological components of the native cartilage. To achieve this, a new technique called cyro-printing was developed to fabricate highly porous scaffolds with reproducible macro and micro pore structures. Through the adjustment of polymer solution concentration and print-head temperature, scaffold morphology, pore diameter and mechanical properties were altered. These cryo-printed scaffolds possess similar columnar pore structure as the deep zone of the cartilage. Cryo-printing was then combined with electrospinning to produce multizone scaffolds which mimic the collagen fibre orientation in the various zones of the native cartilage. Multizone scaffolds provided a viable initial platform that captures the complex structure and compressive properties of the native cartilage. Moreover, they supported long-term chondrocyte and MSC attachment with expression of key chondrogenic genes and the production of the essential biomolecule, glycosaminoglycan. Investigation continued with the incorporation of bioactive molecules, collagen and antioxidants, into PCL scaffolds. Phase separated hybrid collagen and PCL scaffolds were produced that displayed an interconnected and porous structure with adequate mechanical properties. Interestingly, these properties could be altered with an increase in collagen concentration. The collagen was evenly distributed throughout the scaffold. These scaffolds successfully allowed chondrocyte attachment and viability. Vitamin E was then incorporated into PCL fibres with the aim to regulate oxidative stress. Electrospun vitamin E/PCL scaffolds were successfully produced which demonstrated antioxidant potential and the ability to support chondrocyte attachment and growth. Another antioxidant, glutathione was also combined with PCL and these scaffolds successfully allowed cell attachment and viability, as well as modulation of markers of oxidative stress. Overall, the findings of this thesis demonstrate preliminary success with using scaffolds in vitro, highlighting their potential as platforms in cartilage tissue engineering.

The first part of this project was inspired by the fascinating internal porosity of Polypore fungi, characterised by channel-like and micro-fibrous porosities, which create a highly efficient directional release system for their basidiospores. An initial analysis of their natural porous structures revealed the effect of their morphology on the sorption of liquids; sections of the channel-like regions of Ganoderma applanatum fungi were first dip-coated with octadecyltrichlorosilane to achieve superhydrophobicity (water contact angle higher than 150°) and selective sorption of oils and organic solvents. The treated fungi showed oil sorption capacity between 1.8 (rapeseed oil) and 3.1 g/g (chloroform), and the combination of channel-like porosity and hydrophobic properties suggested its use as naturally derived filtration element in micro fluidic networks, showing the possibility of efficiently separating water from chloroform. The second part of this research work replicated the Polypore fungi porosity through the fabrication of polymeric membranes by means of immersion precipitation technique, a specific variant of phase inversion methodologies, thus biomimicking the dual porosity of Polypore fungi. Poly(vinylidene fluoride) (PVDF) has been widely adopted to produce porous membranes due to its membrane forming capability and outstanding mechanical, chemical and thermal properties. N,N-dimethylformamide (DMF) (solvent) and water (coagulant) were then chosen as part of the PVDF membrane forming system to achieve the formation of a net separation between finger-like and sponge-like porosity regions within the membrane thickness. By precisely varying the coagulation bath temperature, the extension of the finger-like region was controlled; the use of 90 °C as temperature for the coagulant led to a predominance of sponge-like morphologies within the PVDF membranes, where only ≈2.5 % of the membrane thickness at the coagulation bath/casting solution interface was characterised by macrovoids; the macrovoids disappearance at high temperatures was addressed to the rapid polymer precipitation and skin layer formation at the early stage of the casting solution phase separation, which prevented the fast solvent/coagulant mass exchange within the inner casting solution layers. On the other hand, lowering the coagulation bath temperature led to a significant increase of the macrovoids length, which were present in more than 50% of the membrane thickness when the temperature was decreased below 20 °C. Also, the production of PVDF membranes at low coagulant temperatures affected the shape of the macrovoids, and finger-like porosity was obtained; this effect was addressed to the early gelation of the polymer-rich phase during the occurrence of phase separation, which generated a contraction force around the macrovoids which turned into narrow and long finger-like pores. Finally, the resulting membrane morphologies, which mimicked the Polypore fungi porosity, showed directional sorption and release of therapeutic essential oils to be applied as drug delivery systems. Novel research from the past years has shown the possibility of combining phase inversion techniques with more recent scaffolds production technologies, such as electrospinning, to achieve superior membrane properties. The last part of this work developed an innovative approach to improve the mechanical properties of polycaprolactone (PCL) electrospun membranes by first dip-coating them into PVDF casting solution and then inducing phase separation through water vapor. The slow phase separation of PVDF caused the solid-liquid demixing to occur and lead to the formation of PVDF spheres with diameters ranging from 4.8 to 10.3 m; the electrospun polycaprolactone nanofibers acted as nucleation sites, and the PVDF spherulites grew onto the porous surface of the fibers and physically bonded multiple fibers together. The fabricated composite scaffolds showed increased mechanical properties if compared to the polycaprolactone mats alone; the ultimate tensile strength, Young's modulus and elongation at break increased from 1.2 MPa, 62.9 kPa, 634.1 % for PCL electrospun mats to 2.1 MPa, 764.5 % and 136.7 kPa for the fabricated PCL-PVDF composite scaffolds, respectively. Finally, cytotoxicity studies using neuroblastoma cells showed the biocompatibility of the fabricated composite scaffolds and suggested their potential to be applied in neural tissue engineering.

Liver disease is one of the top five leading causes of premature death in the UK, with incidence rising sharply by 20% over the last decade, and mortality increasing over 400% since 1970. Liver disease incidence and mortality is rising in stark contrast to trends in the other top healthcare burdens, with stroke, cancer, heart disease and lung disease incidence and mortality rates plummeting and continuing to fall. Liver disease's hallmark pathology of late diagnosis and rapid acute disease progression leads to an urgent need for donor organs; the only curative treatment for end stage liver disease. However, a chronic and ongoing shortage of suitable organs for transplant means many die before a donor liver can be found, and countless others live with severe, debilitating symptoms at a high cost to both the patient and the healthcare system. As part of the push for a solution to this problem, tissue engineers are focussing on creating niche microenvironments for hepatocytes which support their survival and function in as close to an in vivo like state as possible; addressing the need for an ideal in vitro model of the human liver and for lab created 'organoids' which could be used to treat patients. Such an environment would allow for the study of new pharmaceuticals, disease biology and hepatocyte behaviour in the laboratory and lead to more effective treatments for patients. While research to date is making inroads into this dilemma, we are yet to see a lab created environment which accurately recapitulates the complex, finely tuned and responsive extracellular matrix (ECM) of the liver. In an effort to address this, researchers have been incorporating bioactivity into scaffold environments for hepatocytes. This thesis presents three methods of incorporating bioactivity into scaffolds for liver tissue engineering; drug induced ECM biodecoration, synthetically derived ECM biodecoration and decellularized human liver ECM incorporation. Scaffolds were seeded with hepatocyte cells and their response to their microenvironment analysed. Mechanical characterisation and immunohistochemical analyses demonstrated the differences between the scaffold and the ECM biodecoration, as well as retention of ECM proteins through the manufacturing process. Each method altered the protein production and gene expression of hepatocytes, indicating that these methods provide a viable, translatable platform for creating a niche microenvironment for hepatocytes, supporting and manipulating phenotype and function. These scaffolds offer great potential for tissue engineering and regenerative medicine strategies for liver and a translatable method for other whole organ tissue engineering.

There is a pressing need for further advancement in tissue engineering of functional organs with a view to providing a more clinically relevant model for drug development and reduce the dependence on organ donation. Polymer based scaffolds, such as polycaprolactone (PCL), have been highlighted as a potential avenue for tissue engineered kidneys, but there is little investigation down this stream. The focus within kidney tissue engineering has been on two-dimensional cell culture and decellularised tissue. The aim of this project is to utilise electrospun scaffolds within a three-dimensional printed bioreactor system to create an ex vivo environment, to be used as either a conditioning tool for kidney tissue engineering scaffolds or as a model for disease. Electrospun polymer scaffolds can be created with a variety of fibre diameters and the variation in morphology of tissue engineered scaffolds has been shown to affect the way cells behave and integrate. The first study of this thesis examined the cellular response of a kidney cell-line to scaffold architecture using novel electrospun scaffolds. Two fibre diameters were used and three distinct scaffold architectures: random, aligned and cryogenic. The results showed that architecture of the scaffold has a profound effect on kidney cells; whether that is effects of fibre diameter on the cell attachment and viability or the effect of fibre arrangement on the distribution of cells and their alignment with fibres, overall there was a preference for a larger fibre diameter of around 4 μm. Following this, electrospun scaffolds were investigated for their potential to host a multicell population. Rat primary kidney cells were used, and results showed that the scaffolds were capable of sustaining a multi-population of kidney cells, determined by the presence of: aquaporin-1 (proximal tubules), aquaporin-2 (collecting ducts), synaptopodin (glomerular epithelia) and von Willebrand factor (glomerular endothelia cells). Viability of cells appeared to be unaffected by fibre diameter. Overall, the ability of electrospun polymer scaffolds to act as conveyors for kidney cells is a promising strategy for kidney tissue engineering and one that should be explored further; the 'non-woven path' provides benefits over decellularised tissue by offering a high degree of morphological control with a scalable fabrication process and a tuneable rate of degradation. Investigation continued with the development of a 3D printed bioreactor. This was a proof of concept device aiming to represent the in vivo environment. The device was designed to be simple to use, delivering a finely controlled shear stress to cells adhered to the scaffold in a dual chamber system, using a modified cell culture media. Computational fluid dynamics was used to gain a better insight into the forces experienced by cells and a modified cell culture media was used to give a better shear distribution across the scaffold, whilst keeping the flow chamber height large. Our investigation demonstrated the ability of the lab scale system to sustain cell life whilst upregulating key transmembrane (AQP-2), cytoskeletal (KRT-8, KRT-18) and tight junction (E-CAD) proteins. Development continued, consolidating the positive characteristics of the bioreactor whilst providing a simplified redesign. In this second-generation bioreactor, the shear profile delivered to each side of the scaffold was mirrored with a flow regime intended to have a low Reynolds number, allowing for the use of two different media without mixing by convection. The device produced a shear stress of 18 to 21 mPa over 80% of the scaffold surface on both superior and inferior sides. The bioreactor maintained a co-culture of endothelial (HUVEC) and epithelial (RC-124) cells, showing the distinct cell types localised on opposite sides of the bioreactor, identified by aquaporin-2 and von Willebrand factor. This bioreactor is a useful tool for modelling of kidney tubules, but has applications in any area where a dual environment with a controlled shear stress is needed. Overall, this work has expanded the breadth of kidney tissue engineering potentially guiding new potential areas of research.

Macromolecular biomaterials often require covalent crosslinking to achieve adequate stability and mechanical strength for their given application. However, the use of auxiliary chemicals may be associated with long-term toxicity in the body. Oppositely-charged polyelectrolytes (PEs) have the advantage that they can self-crosslink electrostatically and those derived from marine organisms are an inexpensive alternative to glycosaminoglycans present in the extracellular matrix of human tissues. A range of different combinations of PEs and preparation conditions have been reported in the literature. However, although there has been some work on complex formation between chitosan (CS) and carrageenan (CRG), much of the work undertaken has ignored the effect of pH on the consequent physicochemical properties of self-crosslinked polyelectrolyte complex (PEC) gels, films and scaffolds. Chitosan is a positively-charged polysaccharide with NH3+ side groups derived from shrimp shells and, carrageenan is a negatively-charged polysaccharide with OSO3- side groups derived from red seaweed. These abundant polysaccharides possess advantageous properties such as biodegradability and low toxicity. However, at present, there is no clear consensus on the cell binding properties of CS and CRG or CS-CRG PEC materials. The aim of this study was to explore the properties of crosslinker-free PEC gels, solvent-cast PEC films and freeze-dried PEC scaffolds based on CS and CRG precursors for medical applications. The objective was to characterise the effect of pH of the production conditions on the physicochemical and biological properties of CS-CRG PECs. Experimental work focused on the interaction between PEs, the composition of PECs, the rheological properties of PEC gels and the mechanical properties of PEC films and scaffolds. In addition, cell and protein attachment to the PEC films was assessed to determine their interactions in a biological environment. For biomedical applications, these materials should ideally be stable when produced such that they can be processed to form either a film or a scaffold and have mechanical properties comparable to those of collagenous soft tissues. FTIR was used to confirm PEC formation. Zeta potential measurements indicated that the PECs produced at pH 2-6 had a high strength of electrostatic interaction with the highest occurring at pH 4-5. This resulted in stronger intra-crosslinking in the PEC gels which led to the formation of higher yield, solid content, viscosity and fibre content in PEC gels. The weaker interaction at pH 7-12 resulted in higher levels of CS incorporated into the complex and the formation of inter-crosslinking through entanglements between PEC units. This resulted in the production of strong and stiff PEC films and scaffolds appropriate for soft tissue implants. The PECs prepared at pH 7.4 and 9 also exhibited low swelling and mass loss, which was thought to be due to the high CS content and entanglements. From the range of samples tested, the PECs produced at pH 7.4 appeared to show the optimum combination of yield, stability and homogeneity for soft tissue implants. Biological studies were performed on CS, CRG and PECs prepared at pH 3, 5, 7.4 and 9. All of the PE and PEC films were found to be non-cytotoxic. When the response of three different cell types and a high binding affinity protein (tropoelastin) was evaluated; it was found that the CS-CRG PEC films displayed anti-adhesive properties. Based on these experimental observations and previous studies, a mechanistic model of the anti-adhesive behaviour of PEC surfaces was proposed. It was therefore concluded that the CS-CRG PECs produced might be suitable for non-biofouling applications.

Thermoplastic elastomers (TPE) are a set of materials with characteristics of elastomers and thermoplastics. There is an increasing demand for polymers to be processed into three dimensional porous constructs for tissue engineering. Aliphatic polyester-based, poly(butylene succinate-co-dilinoleic succinate) (PBS-DLS) and polyisobutylene-based, poly(alloocimene-b-isobutylene-b-alloocimene) thermoplastic elastomer copolymers and their development will be presented for end use as biomaterial-based therapies in this dissertation. Electrospun fibrous scaffolds are favored for tissue engineering for their micro-structured networks creating a high surface area to volume ratio and this high interconnected porosity. These properties help mimic natural tissue structure for better tissue integration and diffusion through the network. Applying thermoplastic elastomers as scaffolds offers materials whose material properties can be tailored for specific applications. This dissertation presents work to advance biodegradable aliphatic copolymers for tissue scaffolds, and polyisobutylene copolymers for drug delivery. Cardiac soft tissue regenerations strategies employ biodegradative copolymers for cell delivery. Completely bio-based and biodegradable PBS-DLS copolymers have shown great potential for coiled 3D scaffolds for cardiac applications. This dissertation presents the kinetics of a step enzymatic polycondensation of PBS-DLS copolymers with varying feed ratios. 1H NMR and SEC results found that hydrophobic soft segment DLS was incorporated into the hard segment PBS within the first 3 hours. After which, the pressure was increased during second stage and complete DLS incorporation and high Mn oligomers occurred between 24 and 48 hours. MALDI-ToF analysis showed that the lower molecular weight fractions cyclic formation of long PBS sequences are favored during early stages of reactions. Poly(styrene-b-isobutylene-b-styrene) is currently used as the coating on the Taxus coronary drug eluting stent. Sequential generations of polyisobutylne-based materials have been made and studied for drug release applications. Specific aim two of this dissertation presents the development of poly(ρ-methylstyrene-b-isobutylene-b-ρ-methylstyrene) (arb_SIBS) and poly(alloocimene-b-isobutylene-b-alloocimene) (AIBA) for drug delivery. Analysis foundmaterials to be non-toxic and able to release anti-inflammatory drugs. AIBA was loaded with ZAF and implanted in vivo. Analysis found this combination to illicit less inflammation thansilicone rubber and is as effective as orally administrated zafirlukast to mitigate fibrous capsule development for up to 90 days in vivo.

Three-dimensional collagen/chitosan scaffolds fabricated by freeze-drying technique in 96-well polystyrene and PDMS plates were optimized during this study. Surface tension is, by and large, one of the most limiting factors in fabricating freeze-dried scaffolds in small format well plates. Traditionally, bowl-shaped top surfaces of collagen/chitosan scaffolds were common in polystyrene 96-well plate; whereas for PDMS 96-well plate, dome-shaped surfaces were formed. These surface tension phenomena are not desirable in cell studies especially during initial cell seeding. A combination of surface treatment and change of freeze-drying regime were developed to mitigate the surface tension problem in PS and PDMS 96-well plates respectively. Collagen/chitosan scaffolds of varying concentration and composition were experimented in both polystyrene and PDMS 96-well plates. Thin water film treatment with UV cross-linking was successfully used to eliminate meniscus in PS well plates; pre-cooling, on the other hand, was utilised to treat scaffold solutions in PDMS well plates. The resultant matrices all had flat top surfaces and average thickness of 1 mm. As expected, scaffolds with lower overall polymer concentration or, from a compositional perspective, scaffolds with high chitosan content generally had larger pores. Microscopic observation by multi-photon microscope was performed and chemical analyses were conducted to characterize the surface-treated scaffolds. In addition, scaffolds were tested in vitro using DLD-1 cells, hMSCs and fibroblasts for their biological performance. The purpose of this study was to address the problem of using small format culture wells for the fabrication of freeze-dried collagen-based scaffolds for studies of cell growth in 3D culture and in microfluidic perfusion bioreactors.

Tendons serve to attach muscles to bones, and are dense structures composed offibers. Tendon injuries, as well as tendinopathies, in which a tendon is overused or hasbeen degenerated due to sport injuries or age, are a large problem for many adult patientsand account for about 30% of musculoskeletal diseases. Around 33 millionmusculoskeletal injuries have been reported per year in the United States alone, 50% ofwhich involve tendon and ligament injuries. In American healthcare, only the flexortendon lacerations sustain estimated costs of anywhere between $240.8 to $409.1 millionper year. Having a slow metabolism, tendon tissue needs a substantial period to redevelopenough strength after injury. In most of the cases, tendon tissue does not have fullfunctional recovery because of low regeneration capacity and scar tissue formation.Therefore, the healing of tendon injuries is a significant and clinically challengingproblem requiring an urgent need to find alternative and cost-effective treatments. Toaddress this problem, a combinatorial approach involving synergetic use of a 3-dimensional (3D) scaffold system with adult mesenchymal stem cells (MSCs) and locallyapplied electrical stimulation (ES) was used to produce bioactive extracellular vesicles(EVs) as novel therapeutic tools to enhance tendon regeneration. For this purpose, wheyprotein isolate (WPI)-based 3D scaffolds were developed to provide favorablemicroenvironment for MSCs attachment and growth. Conductive graphene andbiodegradable Polylactic Acid (PLA) based flexible electronic coil was integrated to the 3D WPI scaffold to provide wireless ES of MSCs and modulate EVs secretion. Theisolated EVs were characterized and applied to primary tenocyte cells to evaluate theregenerative activity. The results indicated that the isolated EVs, as well as applied ES,promoted the regeneration capacity of tenocyte cells in vitro and promoted the expressionof tendon markers. This research demonstrated the proof of concept for potential use ofEVs as therapeutic tools for tendon regeneration and can have important impacts inadvancing the tissue regeneration field by offering a less invasive and more effectivetreatment option for tendon injuries, eventually bringing relief to millions of patients.

The deposition of stem cells at sites of injury is a clinically relevant approach to facilitate tissue repair and angiogenesis. However, insufficient cell engraftment and survival require the engineering of novel scaffolds. Here, a regular network of microscopic poly(lactic-co-glycolic acid) (PLGA) filaments was investigated as a promising biodegradable scaffold for human Adipose-Derived Stem Cell (hADSC) tissue integration. Via soft lithography, three different microstructured fabrics were realized where 5 x 5 and 5 x 3 & mu;m PLGA 'warp' and 'weft' filaments crossed perpendicularly with pitch distances of 5, 10 and 20 & mu;m. After hADSC seeding, cell viability, actin cytoskeleton, spatial organization and the secretome were characterized and compared to conventional substrates, including collagen layers. On the PLGA fabric, hADSC re-assembled to form spheroidal-like structures, preserving cell viability and favoring a nonlinear actin organization. Moreover, the secretion of specific factors involved in angiogenesis, the remodeling of the extracellular matrix and stem cell homing was favored on the PLGA fabric as compared to that which occurred on conventional substrates. The paracrine activity of hADSC was microstructure-dependent, with 5 & mu;m PLGA fabric enhancing the expression of factors involved in all three processes. Although more studies are needed, the proposed PLGA fabric would represent a promising alternative to conventional collagen substrates for stem cell implantation and angiogenesis induction.

Myocardial ischaemia is one of the leading dead causes worldwide. Although animal experiments have histor-ically provided a wealth of information, animal models are time and money consuming, and they usually miss typical human patient's characteristics associated with ischemia prevalence, including aging and comorbidities. Generating reliable in vitro models that recapitulate the human cardiac microenvironment during an ischaemic event can boost the development of new drugs and therapeutic strategies, as well as our understanding of the underlying cellular and molecular events, helping the optimization of therapeutic approaches prior to animal and clinical testing. Although several culture systems have emerged for the recreation of cardiac physiology, mimicking the features of an ischaemic heart tissue in vitro is challenging and certain aspects of the disease process remain poorly addressed. Here, current in vitro cardiac culture systems used for modelling cardiac ischaemia, from self-aggregated organoids to scaffold-based constructs and heart-on-chip platforms are described. The advantages of these models to recreate ischaemic hallmarks such as oxygen gradients, patho-logical alterations of mechanical strength or fibrotic responses are highlighted. The new models represent a step forward to be considered, but unfortunately, we are far away from recapitulating all complexity of the clinical situations.

Mechanical environment plays a crucial role in regulating bone regeneration in bone defects. Assessing the mechanobiological behavior of patient-specific orthopedic scaffolds in-silico could help guide optimal scaffold designs, as well as intra- and post-operative strategies to enhance bone regeneration and improve implant longevity. Additively manufactured porous scaffolds, and specifically triply periodic minimal surfaces (TPMS), have shown promising structural properties to act as bone substitutes, yet their ability to induce mechanobiologially-driven bone regeneration has not been elucidated. The aim of this study is to i) explore the bone regeneration potential of TPMS scaffolds made of different stiffness biocompatible materials, to ii) analyze the influence of pre-seeding the scaffolds and increasing the post-operative resting period, and to iii) assess the influence of patient-specific parameters, such as age and mechanosensitivity, on outcomes. To perform this study, an in silico model of a goat tibia is used. The bone ingrowth within the scaffold pores was simulated with a mechano-driven model of bone regeneration. Results showed that the scaffold's architectural properties affect cellular diffusion and strain distribution, resulting in variations in the regenerated bone volume and distribution. The softer material improved the bone ingrowth. An initial resting period improved the bone ingrowth but not enough to reach the scaffold's core. However, this was achieved with the implantation of a pre-seeded scaffold. Physiological parameters like age and health of the patient also influence the bone regeneration outcome, though to a lesser extent than the scaffold design. This analysis demonstrates the importance of the scaffold's geometry and its material, and highlights the potential of using mechanobiological patient-specific models in the design process for bone substitutes.

To meet the requirements for full production in cellular agriculture, a multi-disciplinary approach is required. New questions and understandings between different concepts, such as texture and cooking, need to be identified and how they correlate with cellular behavior and taste. This work focuses on understanding and developing photolithographic systems for scalable cellular agriculture. There were two specific research directions: 1) a traditional photolithography printing system was improved upon for mass manufacturing purposes and 2) mechanical analysis and degradation of key potential photolithographic biomaterials were tested for a food product. A novel digital rotational ultraviolet manufacturing (DRUM) process was developed, and initial photolithographic printing tests were carried out. GelMA hydrogels were tested in uniaxial compression and compared to a range of meat samples at 20°C using constant displacement speed control. The results showed effective mimicry under the defined environment to that of raw scallops, but not of higher order constructs. A soy-based resin was also tested in degradation 37°C for 0 to 28 days in phosphate buffered solution and ethanol solutions. Degradation was successfully mapped via thermomechanical means in dynamic mechanical analysis. Stability of the material was verified upon addition of EBC-1 enzyme. Initial values for the mechanical behavior of pure soya scaffolds at 100% density were too stiff and unable to match desired findings in literature. The achieved results showed promise as a proof of concept for this methodology and emphasizes the importance of standardization of results, ultimately leading to the conclusion that the requirements for these products will likely require complex modular, multi-step, multi-material systems at scale. Advisor: Michael P. Sealy

Bacterial vaginosis (BV) is a condition in which healthy lactobacilli are replaced by an overabundance of pathogenic bacteria in the female reproductive tract. Current antibiotic treatments often fail to “cure” infection, resulting in recurrence in more than 50% of women, 6 months post-treatment. Recently, probiotics have demonstrated promise to restore vaginal health; however, as with other active agents, delivery requires once-to-twice daily administration. Recently, three-dimensional (3D)-bioprinting has enabled the fabrication of well-defined cell-laden architectures with tunable agent release, thereby presenting a novel approach with which to deliver probiotics. One promising bioink, gelatin alginate, was selected for study, due to its ability in other work to provide structural stability, host compatibility, viable probiotic incorporation, and nutrient diffusion. The focus of this study was to formulate and characterize 3D-bioprinted Lactobacillus crispatus (L.cr.)-containing gelatin alginate scaffolds for reproductive health applications. Different weight to volume (w/v) ratios of gelatin alginate were bioprinted to determine the formulation with the highest printing resolution, and different crosslinking reagents were evaluated for effect on scaffold integrity, via mass loss and swelling measurements. Additionally, post-print viability, sustained-release, and vaginal keratinocyte cytotoxicity assays were conducted. A 10:2 (w/v) gelatin alginate formulation was selected based on line continuity and resolution, while degradation and swelling experiments demonstrated the greatest structural stability with dual-crosslinking, showing minimal mass loss and swelling over 28 days. Last, 3D-bioprinted L.cr.-containing scaffolds demonstrated sustained-release of therapeutically-relevant levels of probiotics over 28 days, while maintaining the viability of vaginal epithelial cells. For the first time, this study shows that 3D-bioprinted scaffolds may provide a new alternative to sustain probiotic delivery with future goals to help maintain or restore female reproductive health after BV infection.

The co-administration of glial cell line-derived neurotrophic factor (GDNF) and mesenchymal stem cells (MSCs) in hydrogels (HGs) has emerged as a powerful strategy to enhance the efficient integration of transplanted cells in Parkinson's disease (PD). This strategy could be improved by controlling the cellular microenvironment and biomolecule release and better mimicking the complex properties of the brain tissue. Here, we develop and characterize a drug delivery system for brain repair where MSCs and GDNF are included in a nanoparticle-modified supramolecular guest-host HA HG. In this system, the nanoparticles act as both carriers for the GDNF and active physical crosslinkers of the HG. The multifunctional HG is mechanically compatible with brain tissue and easily injectable. It also protects GDNF from degradation and achieves its controlled release over time. The cytocompatibility studies show that the developed biomaterial provides a friendly environment for MSCs and presents good compatibility with PC12 cells. Finally, using RNA-sequencing (RNA-seq), we investigated how the three-dimensional (3D) environment, provided by the nanostructured HG, impacted the encapsulated cells. The transcriptome analysis supports the beneficial effect of including MSCs in the nanoreinforced HG. An enhancement in the anti-inflammatory effect of MSCs was observed, as well as a differentiation of the MSCs toward a neuron-like cell type. In summary, the suitable strength, excellent self healing properties, good biocompatibility, and ability to boost MSC regenerative potential make this nanoreinforced HG a good candidate for drug and cell administration to the brain.

Glaucoma is the leading cause of irreversible blindness worldwide and ischaracterized by the progressive degeneration of the optic nerve. Intraocularpressure (IOP), which is considered to be the main risk factor for glaucomadevelopment, builds up in response to the resistance (resistance to what?) providedby the trabecular meshwork (TM) to aqueous humor (AH) outflow. Although theTMand its relationship to AH outflow have remained at the forefront of scientificinterest, researchers remain uncertain regarding which mechanisms drive thedeterioration of the TM. Current tissue‐engineering fabrication techniques havecome up with promising approaches to successfully recreate the TM. Nonetheless,more accurate models are needed to understand the factors that make glaucomaarise. In this review, we provide a chronological evaluation of the technologicalmilestones that have taken place in the field of glaucoma research, and we conduct acomprehensive comparison of available TM fabrication technologies. Additionally,we also discuss AH perfusion platforms, since they are essential for the validation ofthese scaffolds, as well as pressure–outflow relationship studies and the discovery ofnew IOP‐reduction therapies.

Immunotherapies involving engineered T cells have shown remarkable clinical outcomes for the treatment of cancer, particularly hematological malignancies, such as leukemia and lymphoma. However, several barriers need to be overcome before these adoptive cellular therapies can be accessed by a broader patient population and be applied to other cancers. Currently, the lack of control over the quality of T-cell products limits the cells’ intrinsic ability to proliferate, persist, and effectively kill tumor cells in vivo. This can, in part, be attributed to the synthetic materials used to mediate T-cell activation, a key step in therapeutic T-cell production. Conventional materials fail to recapitulate key features of physiological T-cell activation, resulting in poor control over T-cell quality and yield. In this thesis, we address these challenges by developing material scaffolds that mimic physiological antigen presentation and provide precise control over T-cell activation to create and manipulate T cells ex vivo and in vivo. First, we design and characterize the material scaffolds, defining and optimizing key parameters for ex vivo human T-cell activation and expansion. The materials are used to improve T-cell immunotherapies in two approaches: (1) by engineering the materials to tune T cell-intrinsic attributes to enhance therapeutic T-cell products, and (2) by using the materials as an injectable scaffold niche for local T-cell stimulation, to boost systemic antitumor responses of prior-administered T-cell immunotherapies. Together, this thesis describes a materials-based approach to manipulate T-cell biology, enabling the controlled manufacture of high-quality T-cell products ex vivo and the enhancement of their therapeutic functions in vivo.

Abstract: The menisci are a pair of knee joint structures that are wedge-shaped in cross-section and semi-lunar in superior view. They play roles in force distribution that help maintain the health of the articular cartilage. In adults, the menisci can be divided into a fibrous, vascularized outer region and a fibrocartilaginous, avascular inner region. Being avascular, the inner regions cannot heal upon injury, which predisposes the knee to early osteoarthritis development. The principal biochemical constituents of meniscus fibrocartilage include type I collagen and hyaline cartilage-associated biochemicals type II collagen and aggrecan. Meniscus fibrocartilage is synthesized and maintained by cells known as meniscus fibrochondrocytes (MFCs), which exist natively in a hypoxic and mechanically-loaded environment. Meniscus tissue engineering (MTE) aims to generate cell-based meniscus replacements in vitro that slow or prevent osteoarthritis development after incidence of non-healing meniscus injuries. This is accomplished using a suitable combination of biomaterial scaffolds, cells, and growth signals. To this end, this thesis focuses on: i) development of biomimetic meniscus scaffolds; ii) investigating the MFC response to combined hypoxia and mechanical loading; and iii) identifying how this knowledge can be used to enhance fibrocartilage histogenesis in vitro. Chapters 1 and 2 introduced the research topic with a review of meniscus development, comparative anatomy, and hypoxia and mechanical loading in MTE. This review suggested that the fibrocartilaginous phenotype of the adult human inner meniscus is likely an adaptation to its hypoxic and mechanically-loaded environment. The first project (Chapter 3) described design and production of 3D printed biomimetic meniscus scaffolds. The second project (Chapter 4) presented an investigation of the simultaneous response of MFCs to combined hypoxia and supplementation of the growth factor TGF-β3 in high-density cell aggregates (pellets). These treatments had synergistic interactions that potently promoted fibrocartilage histogenesis. Chapter 5 extended this study to a larger-scale model with a porous type I collagen scaffold. Hypoxia had few effects in this scaffold, even when combined with mechanical loading (dynamic compression (DC)). Chapter 6 investigated the factorial effects of cell expansion, hypoxia, and culture time to identify more suitable static pre-culture conditions on the scaffold before treatment with hypoxia and mechanical loading. Cell expansion had few effects, but mechanical properties and the hypertrophic phenotype increased continually up to the latest measured time point (9 weeks). Hypoxia suppressed both the hypertrophic phenotype and mechanical property development. Based on these outcomes, Chapter 7 investigated an intermediate pre-culture period in normal oxygen levels prior to a short-term treatment with hypoxia and mechanical loading (DC) to identify their effects on transcriptome-wide gene expression. The immediate MFC response to the combined treatment supported fibrocartilage histogenesis in an additive rather than synergistic manner. Chapter 8 described a series of four follow-up studies that used a shortened pre-culture period of 3 weeks based on experiences in Chapter 7 prior to hypoxia and mechanical loading treatments. First, the treatments were extended to five days and to include additional types of daily mechanical loading: i) a novel DC regime combining displacement and load control, and ii) cyclic hydrostatic pressure. The original DC regime, used in Chapter 7, was most effective in gene regulation. RNA sequencing showed that the longer-term combined treatment also supported fibrocartilage histogenesis, especially in promoting the hyaline cartilage-like aspect of the inner meniscus phenotype. The second and third studies of Chapter 8 showed that the gene expression response to DC saturated after 5 minutes and increased in magnitude with higher applied strain. The last study considered a long-term treatment of mechano-hypoxia conditioning, applying continuous hypoxia with four daily incidents of DC. The outcomes included increased equilibrium dynamic modulus to levels that rival the native meniscus, although further experimentation is required. Chapter 9 summarized the work and suggested future research directions. This thesis has three main contributions: i) the design of biomimetic meniscus scaffolds that can be used for complete meniscus tissue engineering; ii) the knowledge that the gene expression response of MFCs to combined hypoxia and mechanical loading is, after up to five days, primarily additive; and iii) the early evidence that combined hypoxia and mechanical loading, “mechano-hypoxia conditioning,” can be a useful strategy to promote fibrocartilage histogenesis in vitro for MTE.

The ideal “off the shelf” tissue engineering, small-diameter (< 6 mm inner diameter (ID)) vascular graft hinges on designing a template that facilitates transmural ingrowth of capillaries to regenerate an endothelized neointimal surface. Previous traditionally electrospun (TES) approaches to create bioresorbable vascular grafts lack the pore sizes required to facilitate transmural capillary ingrowth required for successful in situ neovascular regeneration. Therefore, the ability to create scaffolds with program-specific architectures independent of fiber diameter via the relatively recent sub-technique of near-field electrospinning (NFES) represents a promising solution to create tissue engineering vascular grafts. These programmed large pore sizes are anticipated to promote in situ regeneration and improve the outcomes as well as the quality of life of patients with arterial disease. In this dissertation, we manufactured via NFES as well as characterized biodegradable polydioxanone (PDO) small-diameter vascular grafts. Chapter 1 introduces the need for off-the-shelf, small-diameter vascular grafts to facilitate in situ regeneration, the process and pore size limitations of TES vascular grafts, and the promising use of NFES to develop precisely tailored PDO vascular grafts. Chapter 2 describes the process of NFES and details the current progress in NFES of biomedical polymers as well as the major limitations that exist in the field. Chapters 3, 4, and 5 contain primary research exploring the creation of an NFES vascular graft scaffold and characterizing the mechanical as well as biological response of these scaffolds. Specifically, in Chapter 3 we demonstrate a NFES apparatus designed around a commercial 3D printer to write PDO microfibers. The processing parameters of air gap, polymer concentration, translational velocity, needle gauge, and applied voltage were characterized for their effects on PDO fiber diameter. The processing parameters of polymer concentration and translational fiber deposition velocity were further characterized for their effects on fiber crystallinity and individual fiber uniformity. The precision of fiber stacking via a 3D printer was qualitatively evaluated to inform the creation of 3D scaffolds to guide the alignment of human gingival fibroblasts. It was found that fiber diameters correlate positively with polymer concentration, applied voltage, and needle gauge and inversely correlate with translational velocity and air gap distance. Individual fiber diameter variability decreases, and crystallinity increases with increasing translational fiber deposition velocity. These data resulted in the creation of tailored PDO 3D scaffolds which guided the alignment of primary human fibroblast cells. Together, these results suggest that NFES of PDO can be scaled to create precise geometries with tailored fiber diameters for vascular graft scaffolds. In Chapter 4, we demonstrated a NFES device to semi-stably write PDO microfibers. The polymer spinneret was programmed to translate in a stacking grid pattern, which resulted in a scaffold with highly aligned grid fibers that were intercalated with low density, random fibers. As a consequence of this random switching process, increasing the grid dimensions resulted in both a lower density of fibers in the center of each grid in the scaffold as well as a lower density of “rebar-like” stacked fibers per unit area. These hybrid architecture scaffolds resulted in tailorable as well as greater surface pore sizes as given by scanning electron micrographs and effective object permeability as indicated by fluorescent microsphere filtration compared to TES scaffolds of the same fiber diameter. Furthermore, these programmable scaffolds resulted in tailorability in the characterized mechanical properties ultimate tensile strength, percent elongation, yield stress, yield elongation, and Young’s modulus independent of fiber diameter compared to the static TES scaffold characterization. Lastly, the innate immune response of neutrophil extracellular traps (NETs) was further attenuated on NFES scaffolds compared to TES scaffolds. These results suggest that this novel NFES scaffold architecture of PDO can be highly tailored as a function of programming for small diameter vascular graft scaffolds. In Chapter 5, we created two types of NFES PDO architectures, as small-diameter vascular graft scaffolds. The first architecture type consisted of a 200 x 200 µm and 500 x 500 µm grid geometry with random fiber infill produced from one set of processing parameters, while the second architecture consisted of aligned fibers written in a 45°/45° and 20°/70° offset from the long axis, both on a 4 mm diameter cylindrical mandrel. These vascular graft scaffolds were characterized for their effective object transit pore size, mechanical properties, and platelet-material interactions compared to TES scaffolds and Gore-Tex® vascular grafts. It was found that effective pore size, given by 9.9 and 97 µm microsphere filtration through the scaffold wall for NFES grafts, was significantly more permeable compared to TES grafts and Gore-Tex® vascular grafts. Furthermore, the characterized mechanical properties of ultimate tensile strength, percent elongation, suture retention, burst pressure, and Young’s modulus were all tailorable for NFES grafts, independent of fiber diameter, compared to TES graft characterization. Lastly, platelet adhesion was attenuated on large pore size NFES grafts compared to the TES grafts which approximated the low level of platelet adhesion measured on Gore-Tex® grafts, with all grafts showing minimal platelet activation given by P-selectin surface expression. Together, these results suggest a highly tailorable process for the creation of the next generation of small-diameter vascular grafts. Lastly, Chapter 6 expounds future considerations for continuing research in NFES technology, NFES for general tissue engineering, and NFES for vascular tissue engineering as well as gives final conclusions. Together, the finding of this dissertation indicated that NFES vascular grafts result in seamless, small diameter tubular scaffolds with programmable pore sizes on the magnitude anticipated to facilitate transmural endothelialization as well as programmable mechanical properties that approximate native values. Thus, this work represents the next step in developing bioinstructive designed scaffolds to facilitate in situ vascular regeneration to improve the outcomes as well as the quality of life of patients with arterial vascular disease.

In this dissertation, small and large NaCl particle-derived surfaces (Ra > 40 microns) were generated on 2D Zn materials, and the surfaces were carefully studied concerning topography, corrosion behavior, and bone cell compatibility. Increases in surface roughness accelerated the corrosion rate, and cell viability was maintained. This method was then extended to 3D porous scaffolds prepared by a hybrid AM/casting technique. The scaffolds displayed a near-net shape, an interconnected pore structure, increasing porosity paralleled to an increased corrosion rate, an ability to support cell growth, and powerful antibacterial properties. Lastly, nano/micro (Rz 0.02–1 microns) topographies were generated on 2D Zn materials, and the materials were comprehensively studied with special attention devoted to corrosion behavior, biocompatibility, osteogenic differentiation, immune cell response, hemocompatibility, and antibacterial performance. For the first time, the textured nonhemolytic surfaces on Zn were shown to direct cell fate, and the micro-textures promoted bone cell differentiation and directed immune cells away from an inflammatory phenotype.

Load-bearing soft tissue (LBSTs) injuries are multi-causal, resulting from physical activities, infection, and aging, affecting a wide array of patients. These injuries are expensive to treat and require long-term care, increasing the health care cost burden by billions of dollars annually in the United States. Commonly reported injuries of LBSTs, such as damage to knee menisci and nucleus pulposi, could be addressed by a tissue-engineered scaffold. This study investigated a novel PVA/alginate composite scaffold as a potential tissue-engineered construct that can be used to treat LBST injuries. PVA with embedded alginate beads was physically crosslinked by repeated freezing and thawing to produce composite scaffolds (PVA-ALG scaffolds). After crosslinking, bovine articular chondrocytes were seeded in these constructs. All the constructs maintained cell viability and had compressive properties similar to nucleus pulposi. Among the PVA concentrations used, 10% PVA produced constructs with a high compressive modulus (5.49 kPa) that could be handled easily during the fabrication process. Further characterization of 10% PVA-ALG showed that the scaffolds promoted cell growth. SEM micrographs and nitrogen adsorption analysis showed that the PVA-ALG scaffolds are mostly macroporous. These constructs also contained a significant amount of micropores and some mesopores. As the PVA concentration increased, the surface area of the scaffolds increased, and the porosity decreased in the scaffolds. In addition, the swelling decreased with the increase in the PVA concentration. However, the material properties in all the constructs were favorable for cell growth. After 11 weeks in PBS, 10% PVA-ALG scaffolds showed 68% degradation (reduction in weight in the swollen state) with neutral degradation products. Cell proliferation studies showed that the chondrocytes expanded well in both alginate beads and PVA regions, even though the cell growth was higher in alginate. While live/dead cell analysis helped determine the cell growth at 1 mm depth, histological analysis proved useful in evaluating cell growth in the inner regions of the sample. Gene expression studies and histological analysis indicated that the PVA-ALG scaffolds provided an environment favorable for the chondrocytes to maintain their cartilaginous phenotype. However, colorimetric analyses failed to evaluate the overall cell growth as the scaffolds interacted with MTT, resazurin, and WST-1 dyes. Optimizing the cell seeding method used in the scaffolds could yield a more homogenous cell proliferation. Also, higher cell seeding densities could help distribute more cells in the scaffold, thereby achieving faster cell growth.During the fabrication process, the compressive modulus of 10% PVA-ALG scaffolds dropped by 27% compared to non-sterile PVA hydrogels due to autoclave sterilization. Longer freeze times and higher freeze-thaw cycles yielded 10% PVA-ALG scaffolds with higher mechanical strength while encouraging good cell growth. The studies performed can be used as a framework to develop PVA-ALG scaffolds with more consistent cell seeding mechanisms and optimum crosslinking conditions to suit a variety of load-bearing soft tissues. In addition, this study presented several characterization methods that can be utilized in evaluating the viability of the PVA-ALG scaffold for LBST regeneration.

Periodontitis is a leading chronic oral inflammatory disease and primary cause of permanent tooth loss estimated to affect 47.2% of adults in the United States. Damage to the tooth-supporting apparatus, which includes periodontal ligament (PDL) fibers that anchor the tooth root to alveolar bone, subsequently initiates osseous tissue resorption. Multi-tissue morbidity is a significant challenge given lack of predictability in reconstructing tissues with physiologic functionality native to healthy periodontium. Tissue engineering strategies have potential to address existing deficiencies in clinically-induced regeneration through combinational approaches using biomaterials, growth factors, and cell-based therapy. The purpose of this work was to develop scaffolds incorporating micropatterned topography for guidance of cell growth and periodontal tissue formation, in conjunction with localized, spatiotemporally-controlled growth factor delivery via gene therapy vectors. Micropatterned polycaprolactone (PCL) films were designed to assess PDL cell orientation in vitro, with incorporation of the patterned film into a 3D-printed PCL scaffold for evaluation of varying topography on oriented tissue formation in an ectopic murine model. Specifically, pillars with varying groove depths (30um, 10um) and groove widths (15um, 60um) were used for the scaffold “PDL” region in combination with human PDL (hPDL) cell seeding, while the 3D-printed base served as a region for osseous tissue formation via delivery of human gingival fibroblasts (hGFs) transduced with adenoviral bone morphogenetic protein (Ad-BMP7). Micropatterned films with pillars containing deeper grooves (30um) provided greater control over hPDL cell orientation and subsequent alignment of soft collagenous tissue compared to non-grooved pillars or an amorphous PCL film, with significant (p

Abstract: Chicken feathers are an abundant by-product from poultry industry. Feathers are composed of around 90% keratin, a valuable protein bioresource that have great potential for non-food applications. Keratin is characterized by a high content of cysteine residues forming disulfide bonds. This feature makes keratin an interesting biopolymer for the fabrication of mechanically resistant materials, including thermoplastics and tissue engineered scaffolds. Therefore, the overall objectives of the thesis are 1) to fabricate and improve mechanical properties of chicken feather thermoplastics; 2) to study and characterize the self-assembly of feather keratin hydrogels and their physical and biological properties; 3) to compare hydrogel properties of hair, wool and feather keratins; and 4) to evaluate the feasibility of electrospinning of feather keratin for the fabrication of nanofibrous scaffolds. Chicken feathers were mixed with glycerol/propylene glycol plasticizers and thermally processed into plastic films. The tensile mechanical properties of films were evaluated. Incorporation of up to 2% graphite oxide nanoparticles resulted in enhanced tensile strength and Young modulus of plastic films, which were attributed to keratin intercalation into graphene oxide nanosheets and interaction between oxygen functionalities of graphite oxide with amino acid side groups of keratin, and plasticizers. Chicken feather keratin was also studied for developing scaffolds for potential application in tissue engineering. Keratin from chicken feathers was solubilized in a solution containing urea, thiourea, and sodium metabisulfite. Keratin hydrogels were spontaneously formed during controlled dialysis of extracted keratin solution. Keratin gelation and stability of gels was mainly controlled by the extent of disulfide bond re-formation. Gelation at neutral pH proceeded slowly towards the formation of transparent gels, whereas rapid gelation occurred at pHs of 3 and 9 due to isoelectric aggregation and increased rate of disulfide exchange reactions, respectively. Hydrogels had similar viscoelastic properties of adipose and dermal tissue. Increasing keratin concentration resulted in increased storage modulus of hydrogels; however, swelling capacity and porosity decreased with increased concentration. Hydrogel scaffolds supported the growth of human dermal fibroblasts (HDFa) over a period of 21 days. Cells infiltration was affected by the dense arrangement of keratin and lower porosity in hydrogels prepared at (10 and 12.5%), where the growth of cells was limited to the surface of scaffolds. Keratin from different sources was compared for their hydrogel properties. Keratin from hair, wool and feathers were characterized by their molecular weight, amino acid composition, and thermal and conformational properties. Hydrogels from feather keratin demonstrated substantially higher storage modulus (~10 times higher) than others. However, higher swelling capacity (>3000%) was determined in hair and wool over feather keratin (1500%) hydrogels. The smaller molecular weight and -sheet structure of feather keratin facilitated the self-assembly of rigid hydrogels. Whereas, higher molecular weight stretchable -helix keratins in hair and wool resulted in weaker hydrogels. Fibroblasts showed the highest proliferation rate on feather keratin hydrogel scaffolds, which was attributed to their superior viscoelastic properties. In the last part of the thesis, the potential of forming keratin nanofibers through electrospinning was evaluated. Electrospun keratin nanofiber were fabricated with non-toxic solvents and crosslinking reagents. Keratin was solubilized at room temperature in a 1 M NaOH solution. Poly(vinyl alcohol) (PVA)/citric acid aqueous solution was used as an aid for electrospinning. Solutions containing 10, 20, and 30% keratin/PVA mass ratio were successfully electrospun. The diameter of nanofibers decreased from 565 ± 154 nm in PVA to 274 ± 42nm in 20% keratin electrospun mats, which was attributed to a reduction in the viscosity of the solutions. Further decrease in viscosity with 30% keratin resulted in beads-on-fiber. The resultant nanofibrous mats were thermally cross-linked by esterification of PVA hydroxyl groups with carbonyl groups in citric acid. The incorporation of keratin in PVA nanofiber was confirmed by FTIR and XPS results. Proliferation of fibroblasts after 14 days was higher in scaffolds containing 20% keratin, which was attributed to the superior biological properties of keratin and higher surface area to volume ratio compared to 30% keratin. Feather keratin is a valuable bioresource for material applications. Feather plastics can be evaluated for applications including agricultural films. Keratin hydrogel and electrospun mat scaffolds support the proliferation of fibroblasts, making them potential materials for in vivo studies in skin tissue engineering. 

Mesenchymal stem cells (MSCs) have great potential to improve clinical outcomes for many inflammatory and degenerative diseases through delivery of exogenous MSCs via injection or cell-laden scaffolds and through mobilization and migration of endogenous MSCs to injury sites. MSC fate and function is determined by microenvironmental cues, specifically dimensionality, topography, and cell-cell interactions. MSC responses of migration and differentiation are the focus of this dissertation. Cell migration occurs in several physiological and pathological processes; migration mode and cell signaling are determined by the environment and type of confinement in three-dimensional (3D) models. Tendon injury is a common musculoskeletal disorder that occurs through cumulative damage to the extracellular matrix (ECM). Studies combining nanofibrous scaffolds and MSCs to determine an optimal topographical environment have promoted tenogenic differentiation under various conditions. We investigated cellular response of MSCs on specifically designed nanofiber matrices fabricated using a novel spinneret-based tunable engineered parameters production method (STEP). We designed suspended and aligned nanofiber scaffolds to study cellular morphology, tendon marker gene expression, and matrix deposition as determinants for tendon differentiation. The delivery and maintenance of MSCs at sites of inflammation or injury are major challenges in stem cell therapies. Enhancing stem cell homing could improve their therapeutic effects. Homing is a process that involves cell migration through the vasculature to target organs. This process is defined in leukocyte transendothelial migration (TEM); however, far less is known about MSC homing. We investigated two population subsets of MSCs in a Transwell system mimicking the vasculature; migrated cells that initiated transmigration on the endothelium and nonmigrated cells in the apical chamber that failed to transmigrate. Gene and protein expression changes were observed between these subsets and evidence suggests that multiple signaling pathways regulate TEM. The results of these experiments have demonstrated that microenvironmental cues are critical to understanding the cellular and molecular mechanisms of MSC response, specifically in homing and differentiation. This knowledge has identified scaffold parameters required to stimulate tenogenesis and signaling pathways controlling MSC homing. These findings will allow us to target key regulatory molecules and cell signaling pathways involved in MSC response towards development of regenerative therapies.

Signaling through Fas/FasL is critical to immune homeostasis and tolerance to self-antigens. SA-FasL is a chimeric protein of FasL and streptavidin. SA-FasL exists as oligomers with potent apoptotic function on Fas expressing immune cells and tightly binds to biotinylated surfaces. Islet grafts engineered to transiently display SA-FasL on their surface established tolerance in allogeneic recipients with a short course of rapamycin. We hypothesized that SA-FasL on the islet allograft will induce apoptosis in alloreactive T effector (Teff) cells and phagocytes clearing apoptotic bodies will produce tolerogenic molecules, such as TGF-β, that will lead to the generation and/or expansion of Treg cells at the induction phase. Treg cells will then home to allografts in response to inflammatory cues and will be maintained in the graft by alloantigens for long-term graft protection. In support of this hypothesis, we demonstrated reduced alloreactive T cells in the draining lymph nodes of SAFasL- engineered grafts as compared to controls. Depletion of phagocytes or blocking TGF-β peri- and immediate post-transplantation of allogeneic SA-FasLislet grafts abrogated tolerance as the grafts were rejected acutely. Systemic nature of the tolerance at the induction phase was shown by demonstrating that both un-engineered as well as SA-FasL-engineered islet grafts simultaneously transplanted under the contralateral kidney capsules survived indefinitely. SA-FasL-islets had characteristics of immune privilege as chemical destruction of the long-term graft followed by the transplantation of an un-engineered graft at the same location 4 days later resulted in the protection of the second set graft. We also tested whether tolerance to allografts requires physical presence of SA-FasL on the graft. To investigate this, we engineered biotinylated poly ethylene glycol (PEG) hydrogel or poly(lactic-co-glycolide) (PLGA) scaffolds with SA-FasL and co-transplanted with unmanipulated allogeneic islets under the kidney capsule or epididymal fat pad, respectively, resulted in indefinite ( > 200 days) islet survival. Flow cytometry revealed increased amounts of Tregs in the graft and draining lymph nodes in the PEG model. Mice rejected these grafts when Tregs were depleted. Taken together, the studies presented herein elucidate the mechanistic basis of SA-FasL-mediated tolerance and show that SA-FasL-engineered biomaterials are also effective in inducing tolerance.

ABSTRACT OF THE THESISFeasibility Studies on Using a Thermoplastic Polyurethane Scaffold for Hybrid Tissue-Engineered Heart ValvesBySamuel David ZukeMaster of Science in Biomedical EngineeringUniversity of California, Irvine, 2017Professor Arash Kheradvar, ChairHeart valve replacement procedures currently use either mechanical or bioprosthetic valves made from animal tissue. This thesis examined the use of polymers as the scaffold material for novel hybrid tissue-engineered heart valves and as the leaflet material for polymer heart valves. The concept of a polymeric heart valve was also examined when testing material durability and functionality with accelerated wear testing and a pulsatile flow simulator. Human aortic smooth muscle cells (HASMC), normal human lung fibroblasts (NHLF), and umbilical vein endothelial cells (HUVEC) were examined on a thermoplastic polyurethane, Aromatic Carbothane. Each cell type was seeded on individual pieces of both fibronectin-treated and untreated Carbothane to test biocompatibility. Samples were stained using Cell Tracker Red CMTPX and imaged using fluorescence microscopy. Fluorescence imaging confirmed HASMC and NHLF adhesion and viability on the polyurethane substrate. HUVECs initially attached on both fibronectin-treated and untreated substrate, but they began to detach and clump together within 24 hours. Trilayered tissue samples were successfully grown on a lasercut Carbothane mesh scaffold using the three cell types. A Carbothane heart valve was sewn and placed in a heart flow simulator to examine geometric orifice area. The valve opened to about 49% of the calculated orifice area, but it showed very symmetrical coaptation as it closed. The valve showed no signs of damage after 20 million cycles in the AWT. Carbothane exhibited excellent biocompatibility, and its durability, ease of manufacturing, and flexibility make it a promising candidate for future studies in the field of heart valve engineering.

The fate and behavior of cells is strongly dependent on the cell microenvironment, and this knowledge has been applied to the design of biomaterials to influence cell growth, morphology, and differentiation. However, a dearth of research specifically focused on the effects of material hydrophobicity on cell behavior indicates that this easily controllable material property is being overlooked. The field of tissue engineering has a need for cost-efficient, scalable methods to both increase stem cell stocks and control cell behavior, and hydrophobic biomaterials may be a robust solution to these needs. To evaluate the utility of hydrophobicity in controlling cell behavior, hyaluronic acid was modified with amines representing a wide range of hydrophobicity, resulting in twelve new materials. Both mouse embryonic stem cells (mESCs) and fibroblasts were cultured on these materials to evaluate the differences in pluripotent and differentiated cell behavior in response to hydrophobic materials. The viability of cells cultured on these materials was tested as an indicator of biocompatibility, and cell morphology and spreading area was evaluated to relate cell behavior to biomaterial hydrophobicity. Eight of the twelve materials proved to be biocompatible, and hydrophobic materials inhibited cell spreading; fibroblasts cultured on modified HA hydrogels grew in populations of both compact cell clusters and elongated, multi-polar morphologies, and cell spreading area increased as hydrophobicity decreased. Similarly, mESC spreading area increased with decreasing hydrophobicity; mESCs grown on the least hydrophobic HA hydrogels also multi-polar spreading, while mESCs cultured on the more hydrophobic materials grew exclusively in compact cell clusters. As the morphology of cells is often indicative of cell fate, and as hydrophobic materials tended to inhibit cell spreading, we expected that mESCs cultured on hydrophobic materials would maintain pluripotency. To this end, hybrid scaffolds composed of modified HA and gelatin were developed as a platform for stem cell pluripotency maintenance. The mESCs seeded into these scaffolds had a higher expression of the pluripotency marker SSEA-1 compared to control mESCs grown in complete medium after 24 hours, indicating that hydrophobicity is an important material property to consider in stem cell culture.

The objective of this study was to evaluate four methods to generate a decellularized synovium scaffold (SynECM) for use as a biologic transplant. Villous synovium was harvested and frozen (-80oC) from the femoropatellar and medial femorotibial joints of four adult normal horses 25,000 base pairs (bp). 2XPAA treatment also had retained matrix integrity, but low DNA content with short DNA fragments (< 300 bp). The Triton and the NaCl preparations damaged villous structure leaving little to no discernible synovial villi, no identifiable residual cells and short (

This self-study focused of an intervention specialist's decision-making process in designing instruction for students with special needs and those at risk in learning. Vygotsky's Sociocultural Theory and the Zone of Proximal Development (ZPD) provided the lens through which this research was conceptualized and viewed. The purpose of this research study was to utilize a reflective thinking practice in examining my part of the teaching/learning cycle discerning what information lead to decisions in creating scaffolds for students' zone of proximal development. The findings included: (a) the practitioner ignited an invitation to learn and be motivated, designed scaffolds, and created environments to encourage student empowerment; (b) personal attention was directed to those tensions that required change by igniting a problem solving sequence that offered possible strategies and solutions; and (c) outside factors including institutional influences, limitations, and interruptions distracted the practitioner from the primary purpose of teaching. The findings of this study offered the following implications for intervention specialists. First, a reflective thinking practice enables an intervention specialist to discern thoughts that transpire from school/societal deliberations and those that result from his/her own viewpoint. In grappling with the contradictory messages of these two forces a practitioner develops his/her voice and the self-knowledge needed to define his/her position within a given tension. Second, incorporating the element of play into daily lessons encourages children to acquire greater attention to task, thus, increasing cognitive development. Third, the development of individualized scaffolds employs a deconstructive / reconstructive nature to achieve independence in skill execution.

The goal of this research was to engineer a scaffold composed of a mechanical gradient and understand the effects of dynamic stimulation on the morphogenesis of the interfacial tissue via spatially controlled differentiation of a single stem cell source. Osteoarthritis is a degenerative joint disease affecting articular cartilage and the underlying subchondral bone resulting in severe pain and disability of the joints. While current clinical treatments provide limited efficacy in long-term success, tissue engineering strategies prove to be a viable option to address regeneration of the osteochondral gradient interface. To address this challenge spatial patterning of mechanical factors are essential for the development of a gradient tissue structure in three dimensions. Here, the overarching goal of this work was to examine (1) the preferential differentiation capacity of MSCs towards multi-phenotypic lineages when subjected to variations in compressive strain, (2) the control of micro- and macro-scale scaffold mechanics through utilizing a core-shell microfiber electrospinning technique, and finally (3) the engineering of a dynamic mechanical gradient that can spatially control the local strain that MSCs will sense and differentiate into multi-phenotypic lineages. My findings demonstrate that MSCs differentiate in a magnitude-dependent and phenotype-specific manner in response to different magnitudes of dynamic compressive strain suggesting that MSCs are mechano-responsive and their multi-phenotypic differentiation can be controlled by varying the strain regimens. Furthermore, it was demonstrated that the mechanical properties of core-shell electrospun fibers can be modulated by controlling the composition and the dimension of core, decoupled from the cell-interfacing surface chemistry. More importantly, it was shown that mechanical properties of such fibers/scaffolds at the micro- and macroscale can be independently regulated by modulating micro- and macro-structure. Finally, spatial control of the mechanical properties of individual core-shell fibers in a monolithic scaffold was able to modulate the preferential multi-phenotypic differentiation of MSCs when subjected to dynamic compressive strain. Thus, these findings provide an additional avenue for directing stem cell differentiation in lieu of biochemical cues commonly used for osteochondral tissue regeneration. Addressing the influence of mechanical stimulation on spatially controlled multi-phenotypic MSC differentiation is the basis behind the work presented in the following dissertation.

Many important biomaterials are composed of multiple layers of networked fibers. A prime example is in the field of tissue engineering, in which damaged or diseased native tissues are replaced by artificial tissues that are grown on fibrous polymer networks. For load bearing tissues, it is critical that the mechanical behavior of the engineered tissue be similar to the behavior of the native tissue that it will replace. In the case of soft tissues such as heart valves, the macroscale mechanical behavior is highly anisotropic and nonlinear. This behavior is a result of complex deformations of the collagen and elastin fibers that form the extracellular matrix (ECM). The microstructure of engineered tissues must be properly designed to reproduce this unique macroscopic behavior. While there is a growing interest in modeling and simulation of the mechanical response of this class of biomaterials, a theoretical foundation for such simulations has yet to be firmly established. This work introduces a method for modeling materials that have a layered, fibrous network microstructure. Methods for characterizing the complex network geometry are first established. Then an algorithm is developed for generating realistic network geometry that is a good representation of electrospun tissue scaffolds, which serve as the primary synthetic structure on which engineered tissues are grown. The level of fidelity to the real geometry is a significant improvement on previous representations. This improvement is important, since the scaffold geometry has a strong influence over the macroscopic mechanical behavior of the tissue, cell proliferation and attachment, nutrient and waste flows, and extracellular matrix (ECM) generation. Because of the importance of scaffolds in tissue formation and function, this work focuses on characterizing scaffold network geometry and elucidating the impact of geometry on macroscale mechanics. Simulation plays an important role in developing a detailed understanding of scaffold mechanics. In this work, Cosserat rod theory is used to model individual fibers, which are connected to form a network that is treated as a representative volume element (RVE) of the material. The continuum theory is the basis for a finite element discretization. The nonlinear equations are solved using Newton's method in a parallel implementation that is capable of accurately capturing the large, three-dimensional fiber rotations and large fiber stretches that result from the large macroscopic deformations experienced by these biomaterials in their natural environment. Comparisons of simulation results with existing analytical models of soft tissues show that these models can predict the behavior of scaffold networks with reasonable accuracy, despite the significant differences between soft tissue and scaffold network microstructural geometry. The simulations also reveal how macroscale loading is related to the microscale fiber deformations and the load distribution among the fibers. The effects of different characteristics of the microstructural geometry on macroscopic behavior are explored, and the implications for the design of scaffolds that produce the desired macroscopic behavior are discussed. Overall, the improved modeling of electrospun scaffolds presented in this work is an important step toward designing more functional engineered tissues.

Learners who are novice problem solvers often encounter difficulty when solving complex problems. One explanation for this difficulty is that the cognitive requirements of problem-solving are sufficiently high that learners easily become overwhelmed and frustrated, leading to a state known as cognitive overload in which learning is obstructed. Cognitive Load Theory is concerned with the design of instructional approaches intended to manage the cognitive load required for thinking and problem-solving tasks. Scaffolds are any kind of support that facilitates the accomplishment of a difficult task that a learner would not be able to accomplish on their own. They are potential mechanisms to support students in negotiating the potentially high cognitive load required by complex problem-solving. The purpose of this study was to examine the effects of technology-based scaffolds within a problem-based learning environment known as Alien Rescue. The study investigated the impact of scaffolds on cognitive load, problem-solving behaviors, science knowledge, and student perceptions of the learning environment. Participants for this study included sixth grade students from a suburban middle school in the southwestern United States. Student classes were assigned to one of three treatment conditions: (a) a problem constraint condition in which students were guided through a problem-solving process similar to that of an expert, (b) a prompt condition in which students were provided with guiding messages during problem-solving, and (c) a control condition with no scaffolding. All conditions participated in the use of Alien Rescue for three weeks. Measures including a self-report measure of mental effort, calculated instructional efficiency scores, problem solution scores, student activity logs, and science knowledge test performance were used to evaluate students' cognitive load, problem-solving performance, problem-solving strategies, and learning gains. An open-ended questionnaire and student interviews were used to gather data on students' perceptions of the program. Results of the study indicate statistically significant differences between treatment conditions with respect to problem-solving efficiency, student problem-solving behaviors, and scientific knowledge gain. Additionally, qualitative findings highlight problematic aspects of the highly structured condition as implemented within the classroom context while also identifying components of the learning environment that were perceived as helpful and useful to participants. Teacher interviews also provided insight into classroom implementation of the program and opportunities to further enhance scaffolds to support student learning. Implications of the study from research, instructional design, and technology perspectives are discussed along with a treatment of study limitations and opportunities for further research in this area.

Clinical translation of cardiovascular tissue engineering (CVTE) is rapidly shifting from concept to application, granting a myriad of opportunities for the treatment of cardiovascular disorder (CVD). There remains, however, a critical hurdle to overcome: the application of tissue engineering to a comprised patient - more specifically, a patient with diabetes mellitus (DM). The alarming prevalence of DM is of great concern due to its duel threat as both a risk factor for CVD and a predictor of biomedical device failure. Elevated levels of inflammation and impaired wound healing are hallmarks of DM contributing to cardiomyopathy, atherosclerosis, and valve disease. The primary focus of my research was two-fold: 1) to evaluate diabetes-related complications to scaffolds and stem cells used for cardiovascular tissue engineering; and 2) to attenuate these complications by addition of a non-toxic matrix-binding polyphenolic antioxidant, pentagalloyl glucose (PGG). Two types of extracellular matrix (ECM) scaffolds were investigated in this study: collagen-based and elastin based. In vivo biocompatibility studies revealed that the diabetic environment invoked detrimental alterations to the matrix scaffolds including crosslinking, advanced glycation end product (AGE) accumulation, and elevated inflammation. However, these complications could be mitigated by scaffold pre- treatment with PGG. By virtue of its antioxidant properties, PGG halted diabetes-related stiffening, AGE accumulation, inflammation, and calcification. The effect of seeded autologous adipose stem cells (ASCs) were also investigated in vivo. We observed immunomodulatory capabilities of ASCs to the implanted constructs by reducing the pro-inflammatory response, shifting the polarization of macrophages towards constructive remodeling, and preventing inflammation-driven calcification. The combination of ASCs with PGG formed a truly diabetic-resistant construct capable of combating glycoxidation, crosslinking, destructive inflammation, and calcification. The overall goal of this research was to establish the framework of clinical translation of tissue engineering. Tissue engineering is often heralded as a patient- tailored approach for disease treatment; however, our translational efforts are useless if we cannot address the comorbidities associated with the patient. This research takes a step towards the development of a deliverable and robust tissue engineered construct for use in treatment of cardiovascular disease.

Two lines of investigation have highlighted the importance of antibodies to the V1/V2 domain of gp120 in providing protection from HIV-1 infection. First, the recent RV144 HIV-1 vaccine trial documented a correlation between non-neutralizing antibodies to the V2 domain and protection. Second, multiple broadly neutralizing monoclonal antibodies to the V1/V2 domain (e.g. PG9) have been isolated from rare infected individuals, termed elite neutralizers. Interestingly, the binding of both types of antibodies appears to depend on the same cluster of amino acids (positions 167-171) adjacent to the junction of the B and C strands of the four-stranded V1/V2 domain Beta-sheet structure. However, the broadly neutralizing mAb, PG9, additionally depends on mannose-5 glycans at positions 156 and 160 for binding. Because the gp120 vaccine immunogens used in previous HIV-1 vaccine trials were enriched for complex sialic acid-containing glycans, and lacked the high mannose structures required for the binding of PG9-like mAbs, we wondered if these immunogens could be improved by limiting glycosylation to mannose-5 glycans. Here, we describe the PG9 binding activity of monomeric gp120s from multiple strains of HIV-1 produced with mannose-5 glycans. We also describe the properties of glycopeptide scaffolds from the V1/V2 domain also expressed with mannose-5 glycans. The V1/V2 scaffold from the A244 isolate was able to bind the PG9, CH01, and CH03 mAbs with high affinity provided that the proper glycans were present. We further show that immunization with A244 V1/V2 fragments alone, or in a prime/boost regimen with gp120, enhanced the antibody response to sequences in the V1/V2 domain associated with protection in the RV144 trial.

Bone tissue engineering is an emerging field that seeks to improve the treatment of bone defects by restoring the functions of bone using the body’s natural healing processes. Polymer scaffolds seeded with osteoblast and growth factors is one technique that has shown the potential to speed the healing process and decrease the rehabilitation time from bone defects. The goal of this study is to create viable polymer/ceramic scaffolds through melt processing of polycaprolactone and hydroxyapatite and using polyethylene oxide as porogen. The results of this study show that melt processing of these materials is an effective method for creating stable scaffolds. The properties of these scaffolds can be altered by changing several factors including polymer ratio, ceramic and salt content, and the pressure applied during the fabrication process. Biological analysis shows that the scaffolds seeded with MC3T3-E1 cells are capable of facilitating cell attachment and proliferation in vitro over time.

After skeletal muscle damage, satellite cells move towards the injured area to assist in regeneration. However, these cells are rare as their numbers depend on the age and composition of the injured muscle. This regeneration method often results in scar tissue formation along with loss of function. Although several treatment methods have been investigated, no muscle replacement treatment currently exists. Tissue engineering attempts to create, repair, and/or replace damaged tissue by combining cells, biomaterials, and tissue-inducing substances such as growth factors. Electrospinning produces a non-woven scaffold out of biomaterials with fiber diameters ranging from nanometers to microns to create an extracellular-like matrix on which cells attach and proliferate. Our focus is on synthetic polymers, specifically poly(D,L-lactide) (PDLA), poly(L-lactide) (PLLA), and poly(ε-caprolactone) (PCL). Skeletal muscle cells grown on electrospun scaffolds tend to elongate and fuse together thus, mimicking natural tissue. Electrical stimulation has been shown to increase the number of cells fused in culture and decreased the time needed in culture for cells to contract. Therefore, a conductive element was added to each scaffold, specifically polyaniline (PANi), gold nanoparticles (Au Nps), and multi-walled carbon nanotubes (MWCNT). Our project goal is to create a polymeric, conductive, and biocompatible scaffold for skeletal muscle regeneration. PANi and PDLA were mixed to form the following solutions 24% (83% PDLA/17% PANi), 24% (80% PDLA/20% PANi), 22% (75%PDLA/25% PANi), 29% (83% PDLA/17% PANi), and 29% (80% PDLA/20% PANi). Only the 75/25 electrospun scaffold was conductive and had a calculated conductivity of 0.0437 S/cm. Scaffolds with larger amounts of PANi were unable to be electrospun. PDLA/PANi scaffolds were biocompatible as primary rat skeletal muscle cells cultured in vitro did attach. However, the scaffolds shrunk, degraded easily, and became brittle. Although PDLA/PANi scaffolds were easily manufactured, our results indicate that this polymer mixture is not appropriate for skeletal muscle scaffolds. PLLA and Au Nps were electrospun together to form three composite scaffolds: 7% Au-PLLA, 13% Au-PLLA, and 21% Au-PLLA. These were compared to PLLA electrospun scaffolds. Measured scaffold conductivities were 0.008 ± 0.015 S/cm for PLLA, 0.053 ± 0.015 S/cm for 7% Au-PLLA, 0.076 ± 0.004 S/cm for 13% Au-PLLA, and 0.094 ± 0.037 S/cm for 21% Au-PLLA. It was determined via SEM with a Bruker energy dispersive x-ray spectrometer (EDS) that the Au Nps were not evenly distributed within the scaffolds as they had agglomerated. Rat primary muscle cells cultured on the three Au-PLLA scaffolds displayed low cellular activity. A second cell study was conducted to determine Au NPs toxicity. The results show that the Au Nps were not toxic to the cells and the low cellular activity may be a marker for myotube fusion. Elastic modulus and yield stress values for the three Au-PLLA scaffolds measured on days 0, 7, 14, 21, and 28 were much larger than skeletal muscle tissue. Due to the larger mechanical properties and Au Nps agglomeration, a third polymer and conductive element scaffold was investigated. PCL was chosen as the new synthetic polymer as it had a lower elastic modulus and high elongation. MWCNT were chosen as the conductive element as they disperse well within PCL when acid functionalized. A third component was added to the scaffold to help it move similar to skeletal muscle. Ionic polymer gels (IPG) are hydrogels that respond to an external stimulus such as temperature, pH, light, and electric field. A poly(acrylic acid)/poly(vinyl alcohol) (PAA/PVA) mixture is one type of IGP that responds to an electric field. The scaffolds were coaxially electrospun so that each fiber had a PCL-MWCNT interior with a PAA/PVA sheath. These scaffolds were compared to electrospun PCL and PCL-MWCNT ones. The addition of MWCNT to the PCL did increase scaffold conductivity. Actuation of the PCL-MWCNT-PAA/PVA scaffold occurred when 15V and 20V were applied. All three scaffolds had rat primary skeletal muscle cells attached but, more multinucleated cells with actin interaction were seen on PCL-MWCNT-PAA/PVA scaffolds. Once again the mechanical properties were greater than muscle, but because of its ability to actuate we believe the PCL-MWCNT-PAA/PVA scaffold has potential as a bioartificial muscle. Further characterization of the PCL-MWCNT-PAA/PVA included varying the ratios of PAA/PVA, smaller crosslinking times, and lower amounts of MWCNT. Four ratios, 83/17, 60/40, 50/50, and 40/60, were successfully coaxially electrospun with PCL and MWCNT. Overall, very few differences were seen between the four ratios in conductivity, cellular biocompatibility, actuation angular speed, and mechanical properties. The 83/17 and 40/60 ratios were chosen for additional investigation into mechanical properties and actuation. As the mechanical properties of the two types of scaffolds did not change significantly through degradation, lower PVA crosslinking times were tested. No significant effects were found and it was hypothesized that the evaporation of the solution played a role in the crosslinking process. The smaller MWCNT amount scaffolds also did not significantly affect the mechanical properties or the actuation angular speeds. More work into lowering the scaffold mechanical properties while increasing the actuation angular speed is necessary. Though the mechanical properties for the 83/17 and 40/60 scaffolds remained high compared to skeletal muscle, we also looked for differences in in vivo biocompatibility. Both scaffolds were implanted into the right vastus lateralis muscle of Sprague-Dawley rats. The left vastus lateralis muscle served as either the PBS injected sham surgery or an unoperated control. Biocompatibility was evaluated using enzymes, creatine kinase (CK) and lactate dehydrogenase (LDH), levels, fibrosis formation, inflammation, scaffold cellular infiltration, and neovascularization on days 7, 14, 21, and 28 post-implantation. Fibrotic tissue formation, inflammation, and elevated CK and LDH levels were observed initially but responses decreased during the four week study. Cells infiltrated the scaffolds and histological staining showed more fibroblasts than myogenic cells initially but over time, the fibroblasts decreased and myogenic cells increased. Neovascularization of both scaffolds was also recorded. PCL-MWCNT-PAA/PVA scaffolds were determined to be biocompatible, but some differences between the two types were noted. The 83/17 scaffolds caused less of a response from the body compared to the 40/60 scaffolds and had more myogenic cells attached. However, the 40/60 scaffolds had a larger number of blood vessels running through the scaffold. In conclusion, we have successfully fabricated a polymeric, conductive, and biocompatible scaffold that can actuate for skeletal muscle tissue engineering. Although our results are promising, more work is necessary to continue developing and refining the scaffold.

Scaffolds that mimic the structural features of natural extracellular matrix and can deliver biomolecules in a controlled fashion may provide cells with a favorable microenvironment to facilitate tissue regeneration. Biodegradable nanofibrous scaffolds with interconnected pore network have previously been developed in our laboratory to mimic collagen matrix and advantageously support both bone and cartilage regeneration. This dissertation project aims to expand both the structural complexity and the biomolecule delivery capacity of such biomimetic scaffolds for tissue engineering. We first developed a nanofibrous scaffold that can release an antibiotic (doxycycline) with a tunable release rate and a tunable dosage, which was demonstrated to be able to inhibit bacterial growth over a prolonged time period. We then developed a nanofibrous tissue-engineering scaffold that can release basic fibroblast growth factor (bFGF) in a spatially and temporally controlled fashion. In a mouse subcutaneous implantation model, the bFGF-releasing scaffold was shown to enhance cell penetration, tissue ingrowth and angiogenesis. It was also found that both the dose and the release rate of bFGF play roles in the biologic function of the scaffold. After that, we developed a nanofibrous PLLA scaffold that can release both bone morphogenetic protein 7 (BMP-7) and platelet-derived growth factor (PDGF) with distinct dosages and release kinetics. It was demonstrated that BMP-7 and PDGF could synergistically enhance bone regeneration using a mouse ectopic bone formation model and a rat periodontal fenestration defect regeneration model. The regeneration outcome was dependent on the dosage, the ratio and the release kinetics of the two growth factors. Last, we developed an anisotropic composite scaffold with an upper layer mimicking the superficial zone of cartilage and a lower layer mimicking the middle zone of cartilage. The thin superficial layer was fabricated using an electrospinning technique to support a more parallel ECM orientation to the cartilage surface. The lower layer was fabricated using a phase-separation technique to support a more isotropic ECM distribution. Human bone marrow-derived mesenchymal stem cells (hMSCs) were seeded on this complex scaffold and cultured under chondrogenic conditions. The results showed that the composite scaffold was indeed able to support anisotropic cartilage tissue structure formation.

Ever since the discovery of conducting polymers (CPs)in the late 1970s, organic conjugated materials and polymers is one of the most popular and fascinating research area among the scientists due to the remarkably unique properties and broad range of applications of (CPs), notably organic photovoltaic (OPVs), organic or polymer based light emitting devices (OLEDs / PLEDs), field effect transistors (FETs), nonlinear optical (NLO) devices and sensor for biologically relevant analytes, metal ions and anions. Optical properties of these materials depend on intra / interchromophoric interaction, geometry and relative orientation in space. Detailed study of such materials can also help us to understand various intriguing properties of conducting polymers such as charge carrier mobility, quantum yield, and how the excited states transfer energy. The primary focus of my research is to develop novel materials which can be employed as sensors for biological relevant molecules and ions and the synthesis of novel scaffolded chromophores to investigate the interchromophoric interactions and to tune their photophysical properties. The basic introduction of conducting polymers and its applications are described in chapter one followed by the detailed study of m-xylene and m-terphenyl based phosphate and pyrophosphate anions sensors in chapter two and three. Also polymer supported m-xylene based materials were studied for catalytic decomposition of phosphoesters in chapter two. Synthesis and photophysical characterization of π-conjugated cruciforms is reported in chapters four and five. Chapter four accounts for the synthesis of 1,3-bis(dimethylaminomethyl)phenyl based cruciform receptors as metal ion sensor whereas chapter five describes the synthesis and photophysical characterization of five phosphorous-containing cruciform derivatives. Synthesis of novel m-terphenyl based halogenated oxacyclophane scaffolds for covalent attachment of chromophores using C-C coupling reaction is reported in chapter six to study the controlled interchain interaction for both monomer and polymers.

Primary open angle glaucoma (POAG) is the second leading blind disease in the world. The pathological mechanism of glaucoma has been still debated. The general representative symptom of glaucoma is the abnormally increased intraocular pressure (IOP), which causes optic nerve damage and permanent vision loss. According to previous research, the breakdown of production and clearance of aqueous humor (AH) increases the IOP. The specific ocular tissue called as a “trabecular meshwork (TM)” in the AH outflow drainage system is believed to play an important role in the regulation of IOP in the normal range from 15mmHg to 22mmHg. Clinical treatments of glaucoma are to lower the IOP to the normal range. In general, the invasive surgical techniques such as “trabeculectomy”, and “laser trabeculoplasty” are performed with relevant medications. Although the coagulation of tissue by laser based surgeries is less invasive than surgical removal, patients are still exposed to the post-operative risks such as tissue coagulation, inflammation, cornea injury, and cataract. These may cause secondary visual loss. In this work, due to the complex morphology of TM tissue and interrelated parameters involved in the IOP regulation, the key factors governing the outflow facility are investigated using multi-disciplinary approaches. First, the perfusion test was conducted to examine the effect of low fluence diode laser (wavelength: 830nm) which generates the non-invasive level of energy on the hydraulic resistivity of in vitro cultured TM monolayer on the solid porous membrane. The expression of heat shock protein upon low fluence laser treatment was also assessed. The dose-dependent effect of glucocorticoid drug (dexamethasone) was investigated using the electrical impedance spectroscopy. To take into account the three dimensional (3D) porous morphology of TM, polymer based micro/nanofibrous membranes mimicking the in vivo-like environment of natural TM was constructed by combining an array of electrospinning and microfabrication technologies. The effect of topographical parameters on 3D TM growth was quantified. Finally, a 3D co-culturing strategy was developed, which allows positioning of TM cells and Schlemm’s canal (SC) endothelial cells within a single specimen to mimic the natural composite cellular structures in the outflow facility of aqueous humor. This work presents a set of enabling technology for systematic in vitro investigation of the outflow facility of AH. This is critical for exploring the key factor that governs the IOP and is expected to provide useful insights for therapeutics of POAG. The outcome can also be applicable for in vitro study of other tissues with morphological complexity.

The goal of this research is to investigate the effects of the microenvironment on the osteogenic differentiation of human embryonic stem cells (hESCs). Human embryonic stem cells present a potentially unlimited supply of cells that may be directed to differentiate into all cell types within the body and used in regenerative medicine for tissue and cell replacement therapies. Current techniques used for bone tissue repair employ the use of auto- and allografting methods, however, these methods have inherent limitations that restrict their universal application. The limitations of these reparative strategies suggest that an alternative approach is required, and hESCs may provide a repository of cells for such an approach. One of the major gaps in the knowledge regarding hESCs is the lack of understanding the biological cues from the microenvironment that control and direct differentiation. Therefore, we hypothesized that controlling the local in vitro and in vivo microenvironments can direct osteoblastic differentiation of hESCs. We showed the importance of cell culture conditions in the in vitro microenvironment, and the importance of implantation site and scaffold design in the in vivo microenvironment. First, we developed a transwell co-culture system consisting of hESCs with human bone marrow stromal cells (hBMSCs). This demonstrated that pro-osteogenic soluble signaling factors secreted by hBMSCs into the cellular microenvironment directed differentiation of hESCs into osteoblasts. Secondly, we reproducibly derived mesenchymal progenitors from hESCs (hES-MSCs) that possess the characteristic hBMSC immunophentoype, are capable of multilineage differentiation along, and genetically modified them with bone specific transgenes to track osteoblastic differentiation. Distinct osteoprogenitor cells were identified and when implanted in an orthotopic calvarial defect microenvironment, participated in the bone regeneration process. Lastly, we delivered osteoprogenitor hES-MSCs in vivo within hydroxyapatite/tri-calcium phosphate (HA/TCP) scaffolds to investigate the effects that porosity and permeability design have on cell differentiation and overall bone tissue formation. We demonstrated osteoprogenitors derived from hESCs survive and play a role in overall bone tissue formation within HA/TCP scaffolds with high porosity and permeability.

Polymers are widely used in bioengineering for a wide range of applications, including substrates for in vitro cell culture and scaffolds for in vivo tissue engineering. Because polymer surfaces are usually non-polar and exhibit low biocompatibility, surface chemical modification must be used to enhance biocompatibility. In this study, biopolymer surfaces were modified by various plasma treatments and the resulting surface properties were characterized in detail by various microanalysis techniques. Although surface chemistry modification of biopolymers is important, modification of the near-surface structure of biopolymers is also critical because it affects cell attachment, proliferation, and infiltration, which is of paramount importance in the fabrication of scaffolds for tissue engineering. Plasma polymerized fluorocarbon (FC) films grafted onto Ar plasma-treated low-density polyethylene surfaces were shown to increase the surface shear strength while maintaining low friction. These surface characteristics illustrate the potential of FC films as coating materials of bioinstruments, such as catheters used for the treatment of diseased arteries where blood flow is restricted by plaque deposits onto the inner wall of the vessel. In addition to FC film grafting, plasma polymerization with diethylene glycol dimethyl ether monomer was used to graft non-fouling polyethylene glycol (PEG)-like films on various substrates to prevent both protein adsorption and cell attachment, which is of great importance to the fabrication of non-clotting artificial grafts for bypass surgery. Non-fouling PEG-like films were used to chemically pattern substrate surfaces for single-cell culture. Polystyrene culture dishes coated with a PEG-like film were chemically patterned using a silicon shadow mask or a poly(dimethyl siloxane) (PDMS) membrane mask, fabricated by standard lithography methods, to locally remove the PEG film by Ar plasma etching through the mask windows. Another surface chemical patterning method for long-term single-cell culture was accomplished with polystyrene and parylene C surfaces by taking advantage of the change in surface hydrophilicity induced by plasma treatment. These surface chemical patterning methods were used to regulate the shape and size of smooth muscle cells (SMCs). A strong effect of the shape and size of SMCs on proliferation rate was observed, which was correlated to changes in nuclei shape and volume of the SMCs.In contrast to solid polymers, plasma surface treatment of fibrous polymer materials to improve biocompatibility has received relatively less attention. Thus, another objective of this dissertation was to explore how plasma surface modification with inert (e.g., Ar) and reactive (e.g., NH3) gas plasmas can be used to enhance cell attachment, growth and infiltration into fibrous polymer scaffolds. Poly(L-lactide) (PLLA) microfibrous scaffolds synthesized by electrospinning were plasma treated with Ar and NH3 gases to improve cell affinity and incorporate functional groups for biomolecule immobilization. Both Ar and NH3 plasma treatments were shown to improve the cell attachment and growth onto the fabricated microfibrous scaffolds, while surface functional groups produced by NH3 plasma treatment were also effective in immobilizing biomolecules. In addition to the surface chemistry, the structure of biopolymer materials also impacts the effectiveness of tissue engineering scaffolds. Using microfabrication technology to produce a patterned PDMS template for electrospinning, patterned PLLA microfibrous scaffolds with different structures were fabricated and their potential for tissue engineering was demonstrated by in vitro and in vivo cell culture experiments. The results of this thesis indicate that surface chemistry and structure modification of biopolymers by combining plasma treatment with microfabrication/micropatterning techniques is an effective method of engineering surfaces for single-cell culture and scaffold materials with tailored two- and three-dimensional structures that enhance cell growth and infiltration. The findings of this work have direct application in the development of patterned surfaces for controlled single-cell attachment, which is of particular value to studies of individual cell behavior, and scaffolds for tissue engineering and repair.

Rotator cuff tears are a source of debilitating pain that commonly affects more than 40% of our aging population. Despite advances in surgical treatment, the failure rate of rotator cuff repairs is as high as 20-90%. Extracellular matrix (ECM) derived scaffolds have recently been investigated as augmentation devices for rotator cuff repairs, but none has yet demonstrated both the appropriate biological and mechanical properties for mitigating re-tears and enhancing healing. This dissertation proposes to engineer the mechanical properties of allograft fascia lata in a manner that will allow its use as an augmentation device for rotator cuff repairs. This dissertation also aims to develop a simple quasi-linear spring-network model for rotator cuff repairs to elucidate the basic biomechanics of these repairs. The central hypothesis is that engineered fascia lata will have suture retention strength similar to that of human rotator cuff tendon (~250N), even after in vivo implantation. The specific aims are to engineer the mechanical properties of allograft fascia lata ECM and to subsequently evaluate the host response and concomitant mechanical properties of the engineered (reinforced) fascia in a rat model. Further, this dissertation will also develop and validate a spring-network model for simplified rotator cuff repairs. Studies presented in this dissertation demonstrate stitching as a technology to engineer the suture retention and stiffness of allograft (human derived) fascia lata ECM. Stitching fascia ECM with braided, resorbable, polymer fibers in a unique, controlled manner increased the suture retention load of reinforced fascia scaffolds by six fold over non-reinforced fascia. Additionally, the suture retention properties of reinforced fascia scaffolds were comparable to that of human rotator cuff tendon (~250N) at time zero and even after in vivo implantation for twelve weeks. Except for the increased presence of foreign body giant cells in areas concentrated around the polymer fibers, the host response of the reinforced fascia scaffolds were comparable to the non-reinforced fascia at the time points investigated. The spring-network model predicted that the scaffold component carries ~20-30% of the total load on the repair. Parametric sensitivity analysis predicted that greatest improvements in the force carrying capacity of the repair may be achieved by improving the properties of the tendon-to-bone repair. Parametric simulation studies suggested that in the clinical setting of a weak tendon-to-bone repair, scaffold augmentation could significantly off-load the repair and largely mitigate the poor construct properties. However, engineering a scaffold with supra-physiologic stiffness would not translate into stiffer or stronger repairs. The results of this dissertation show that reinforced fascia scaffolds may have and possibly maintain mechanical properties comparable to the suture retention properties of human rotator cuff tendon. This suggests that reinforced fascia scaffolds may be able to provide mechanical augmentation to rotator cuff repairs and also modulate tendon retraction in a manner that reduces the incidence of tendon re-tear. The spring-network model provides a starting point to develop more clinically relevant models for rotator cuff repairs.

In recent years, it has been shown that the nanofiber structures prepared using electrospinning can serve as near ideal substrates for engineering tissues. Various biodegradable polymers of natural and synthetic origins have been used to construct the nanofiber scaffolds. The use of natural polymers is important in that they contain specific cell recognition sites that are capable of binding cells. Synthetic biodegradable polymers, on the other hand, can provide the necessary mechanical properties and their degradation rate can be controlled positively. When used alone, however, neither can provide an ideal structure for long-term development of tissues. This is because the regenerated natural polymers, although greatly biocompatible, are weak and degrade rapidly and uncontrollably, while the synthetic polymers, although mechanically more stable, are not as biocompatible. The focus of the current investigation was, therefore, to combine natural and synthetic polymers and to produce materials that have novel hybrid properties at the nano level. An optimum structure proposed was a differentially biodegradable bicomponent nanofiber with the sheath of natural and the core of synthetic polymers. Co-axial electrospinning was used to prepare the proposed core-sheath nanofibers. A major objective of the current research was to develop and optimize the technology to produce uniform bicomponent nanofibers of predictable morphologies by understanding the effects of various material and process variables such as solution concentration, solvent type, solution flow rate, and applied voltage. Two natural polymers (collagen and gelatin) and one synthetic biodegradable polymer (PCL) were used to develop the proposed structures. The factors that affected the bicomponent fiber formation were: interfacial tension between sheath and core solutions, volatility of the solvent, and applied voltage. By minimizing the interfacial tension, selecting the solvents with low vapor pressure, and adjusting the voltage to a value lying within a particularly narrow range, uniform bicomponent fibers were obtained. Other factors such as polymer concentrations and flow rates were shown to directly affect the dimensions of the sheath and the core. It was hypothesized that the bicomponent structures produced would be differentially biodegradable in that the natural polymer sheath would degrade relatively faster, after initiating the cell activity, and the synthetic polymer core would degrade at a much slower rate and continue to support the cell growth over the required longer period of time. Collagen-PCL sheath-core bicomponent structure was visualized as the most suitable combination for tissue engineering applications and hence, the degradation behavior of this particular structure was investigated. The results showed that for the observed thirty day period of degradation, only collagen from the sheath degraded. The degradation rate of the polymer was highest within the first hour and decreased continuously with time. The degradation within the first hour was high enough to cause the polymer to lose about 50% of the weight. Degradation in PCL was too small to be effectively measured. The degradation behavior of collagen was modeled using a mathematical approach. The model suggested that the mass loss of the polymer due to degradation was proportional to the natural logarithm of time. Cell culture studies on the collagen-PCL bicomponent structures demonstrated, as hypothesized, the improvement in biocompatibility and hydrophilicity of the structure due to the presence of collagen on the surface of PCL. The superiority of the bicomponent structure (over that of the PCL control) was evidenced by the uniform cell seeding and the relatively much higher differentiation of the human adipose derived adult stem cells (hADASCs) into osteoblasts. It could, therefore, be concluded that the hybrid structures developed in this research had very high potential for use as scaffolds for engineering tissues.

Teachers face many challenges in enacting innovative curriculum materials and often must make modifications to fit local contexts and constraints. However, without an understanding of curriculum designers' intentions, modifications can lead to a loss of coherence with respect to the embedded reform ideas of the curriculum. As new curriculum materials are used more widely, it becomes increasingly important to understand how to support teacher decision making about curriculum implementation in ways that are coherent with respect to designers' intentions. This research posits that teachers need to understand underlying curriculum design ideas, such as the coherence of curriculum materials, which refer to the relationships between individual lessons and broader learning goals as well as connections between and across lessons that are intended to create effective learning experiences. I examined how the following three scaffolding strategies help teachers understand curricular coherence: (1) Providing visualizations to help teachers inspect multiple aspects of curricular coherence; (2) Demonstrating changes in the coverage rates of learning goals as a consequence of modification; and (3) Encouraging reflection about the modifications and their impact on coherence. The research questions addressed in this study are: (1) How does amount of teaching experience relate to teachers' understanding of coherence? (2) What are the roles of software scaffolds in helping teachers consider higher levels of coherence when modifying materials? (3) When teachers make changes in curriculum units with the assistance of software scaffolds, how do they reflect on their understanding of curriculum structure and modification strategies? To answer these research questions, I developed a software tool as a context to support and examine teachers' curriculum modification practices. I studied twenty teachers' modification practices with and without scaffolding support. Findings indicate that: (1) Teachers' level of understanding of curriculum coherence is not related to experience. (2) Software scaffolds helped teachers consider higher levels of curriculum coherence. (3) Software scaffolds encouraged teachers to examine their understanding of unit structures and modification strategies. This study informs the design of environments that aim to support more teachers in enacting curricula congruently with respect to design intent in the context of large-scale curriculum implementation.

Despite the demonstration of various engineered tissues, clinically useful bone tissue regeneration remains a challenge. Osteogenic cells, a three-dimensional (3D) osteoconductive scaffold, and osteoinductive factors are integral to successful bone regeneration. Pivotal in bone regeneration will be the ability to deliver appropriate growth factors in a controlled manner using 3D scaffolds and advanced release technologies within a tissue engineering strategy. The overall goal of this dissertation is to develop a growth factor delivering 3D scaffold platform for bone tissue regeneration. First, we have developed a phase separation/sugar sphere template leaching technique to fabricate 3D porous scaffolds which mimic natural bone extracellular matrix in structure, morphology and/or composition. The novel scaffolds possess high porosities, interconnected macropores, and collagen resembling nano fibers. The technique advantageously controls macropore shape and size by sugar spheres, interpore opening size by sugar template assembly conditions, and pore wall morphology by phase separation parameters. The interconnected macroporous and nano-fibrous scaffold is designed to provide an advantageous environment for cell attachment, proliferation and differentiation. In addition, the biomimetic growth of bone-like apatite crystals in scaffold was investigated in simulated body fluid and the apatite coated scaffolds demonstrated improved mechanical properties and bioactivity. Second, biodegradable micro/nanospheres are prepared for the controlled release of several biological molecules including parathyroid hormone (PTH), recombinant human platelet-derived growth factor-BB (rhPDGF-BB) and bone morphogenetic protein (rhBMP-7). The released proteins were biological active to stimulate corresponding cellular functions. Furthermore, a 3D scaffold delivery system has been developed by immobilizing growth factor (rhPDGF-BB or rhBMP-7) containing nanospheres onto a scaffold. In vitro, different biodegradable nanospheres have been utilized to individually control the release profiles of the growth factors from 3D scaffold. In vivo, enhanced ectopic bone formation has been demonstrated in rhBMP-7 delivering nanosphere-scaffold. The nanosphere-scaffold technology platform is further expanded to deliver multiple proteins from one single scaffold, each with individualized release profile. Multiple proteins released from a single scaffold over days, weeks, and months are demonstrated. In summary, this dissertation demonstrates the successful generation of a biomimetic scaffold capable of controlled growth factor delivery which indicates significant potentials in tissue engineering and regenerative medicine.

This is a study investigating how the presentation of scaffolds impacts what preservice teachers are able to learn and notice from video. A mixed method approach was utilized to study the change in teacher knowledge and ability to notice content from assigned treatment videos. The sample (n=41) was drawn from three randomly distributed sections of a science methods course designed to teach preservice teachers how to plan for and carry-out project-based science lessons by working with an inquiry-based unit on simple machines. The focus of this research was a lever investigation from that unit and a collection of videos that were produced showing a classroom enactment of that investigation. Subjects were placed into one of three conditions, the Available Scaffolds (AS) group, Integrated Scaffolds (IS) group and comparison group based on the course section in which they enrolled. During the treatment period members from the AS and IS groups viewed a set of videos designed to help prepare teachers to enact the lever investigation. The same classroom footage was used for each set of videos but the arrangement of scaffolds differed for each group. The AS group had access to scaffolds such as static text summaries and introductory teacher commentary while the IS group was presented with scaffolds such as hyperlinks to specific sections of the videos, onscreen text and titles and voice-over narration that were more explicitly integrated within the video. The comparison group did not watch any videos as part of their treatment experience. They participated in an enactment of the lever investigation with the methods course instructor in order to learn more about facilitating the lesson in their own classrooms. Findings from this research indicate that the impact of scaffolds is dependent on their presentation and alignment with the stated learning objectives of the video they are designed to support. Scaffolds such as onscreen text and teacher commentary can be used effectively to draw attention to specific content and prompt preservice teachers to notice pre-identified classroom interactions. The use and purpose of scaffolds is not always apparent to preservice teachers and therefore may require explicit instructions in order to be used effectively. As the use of video in teacher education has become more prevalent it is imperative that we have an empirically sound understanding of how scaffolds can be used effectively to support learning from videos. The main purpose of this research is to provide that empirical foundation and inform the design of video-based materials.

Organic molecules that mimic key protein structural motifs have been shown to mediate protein-protein interactions involved in a number of biological processes. An area where such chemical tools are particularly useful is gene regulation at the transcriptional level, a process mediated via many poorly understood protein-protein interactions. The correlation between a number of human disease states and the aberrant regulation of these events has led to a concentrated effort in developing peptidomimetics to elucidate these complex mechanisms and to serve as ligands for the artificial control of this process. Chapter I details the historical challenges and some advances in the development of such molecules. beta-Peptides, oligomers of beta-amino acids, have enormous potential in this context due to their capacity to adopt discrete, stable conformations in biological systems. However, the use of beta-peptides in this application is ultimately limited by the lack of efficient synthetic methods for the preparation of the highly substituted and stereoisomerically pure constituents. Chapter II details the development of a conceptually unique synthetic approach that uses chiral isoxazolidines as key intermediates to access densely functionalized beta-amino acids difficult to realize by conventional means. Chapter III details the expansion of this synthetic method to provide isoxazolidines that contain functional groups analogous to those found in natural transcriptional activators that serve as effective small molecule components in gene upregulation, presumably by mediating protein-protein interactions involved in this process. Chapter IV details the development of an iterative synthetic approach to synthesize extended versions of the active isoxazolidines that have enhanced biological activity, seemingly due to increased surface area and functionality available for protein surface recognition. An account of the structural details of this class of compounds is also discussed. By utilizing a synthetic strategy that provides control over both the stereochemistry and functionality present in the molecule, this novel foldamer motif is also an attractive target for future applications ranging from catalysis to hydrogen storage.

Surface chemistry of the scaffold material, the fiber diameter, pore size, structure and configuration and porosity can regulate tissue development. The overall objective was to study the effect of the three dimensional structure of tissue scaffolds on animal cell culture. To achieve this, the growth of human astrocyte cells, secreting Glial-cell-line Derived Neurotrophic Factor (GDNF), in non-woven polyethylene terephthalate (PET) matrices was specifically targeted. The ability of GDNF to selectively nourish and regenerate dopaminergic neurons makes it an ideal candidate for the treatment of Parkinson's disease. On the basis of cell morphology, density and GDNF secretion, compressed PET matrices (porosity 88.8%, mean pore diameter 64 microns), treated with boiling sodium hydroxide, were found to be very effective in supporting high density growth of astrocytes. Most scaffolds used thus far, are anisotropic in their architecture and have a wide pore size distribution, making the task of identifying optimum values of the parameters important for tissue development even more difficult. However, microfabricated poly(lactic co-glycolic acid) (PLGA) scaffolds with a uniform structure supported a higher cell density, longer cell proliferation times than in non-uniform fibrous matrices, and induced less apoptosis, implying that uniformity in scaffold microstructure was a more important parameter than the mean pore size of the scaffolds. Cytocompatibility studies on PLGA scaffolds also revealed that cells could span in three-dimensional space over distances greater than four times their average length. To develop a fast convenient cell based model to study cell culture in three dimensional (3D) scaffolds, cells were transfected with an Enhanced Green Fluorescent Protein (EGFP) based reporter system. This allowed fast, sensitive and noninvasive monitoring of cell growth in suspension and could be used to detect the spatial distribution of these cells in 3D scaffolds. The fluorescence of these cells was linked to the S-phase of the cell cycle and decreased in a dosage dependent manner when the cells were treated with chemotherapeutic drugs 5-fluorouracil and doxorubicin, making these cells an ideal and reliable model for reporting cellular proliferation and drug effects on cell growth.

Varied clinical problems have created a need to repair and regenerate bone. Tissue engineering is an alternative to conventional therapies that may allow the generation of functional tissue. The hypothesis of this work is that combining solid freeform fabricated scaffolds and localized gene therapy will result in the formation of biologically and mechanically functional bone throughout the scaffold. Initial experiments focused on the use of fibrin and collagen hydrogels to suspend and deliver adenoviral vectors. Results showed that the hydrogels maintained or extended the activity of adenoviral vectors in vitro. In vivo, gels containing adenovirus expressing bone morphogenetic protein-7 led to ectopic bone formation. In vivo experiments were undertaken to optimize the combination of scaffolds and gene therapy, focusing on the type of gene therapy (in vivo or ex vivo), the carrier (hydrogel or polymer), and the scaffold pore size. In vivo implantation triggered the generation of bone with marrow space and vasculature. The variables tested had various effects on bone generation: gene therapy method had the largest effect, with ex vivo gene therapy generating significantly more bone than in vivo gene therapy; carrier type also had a significant effect, with scaffolds seeded with fibrin gel showing more bone generation; pore size did not have a significant effect. The results demonstrated the conditions best suited to maximize the formation of bone and in the final phase of the thesis these conditions were combined with topology optimized scaffolds. These scaffolds were implanted in vivo and then evaluated mechanically and histologically. Over extended implantation times, large amounts of bone formed. Though the bone volume was relatively constant over time, it was increasingly localized on the scaffold contours as time increased. The compressive modulus of the bone increased as the modulus of the degrading scaffold declined. Thus, the overall modulus of the implant was nearly constant, an excellent indicator that such an implant could continuously function in a load bearing site. These results show designed solid freeform fabricated scaffolds combined with optimized gene therapy can be used to achieve rapid osteogenesis and maintain appropriate mechanical properties, thereby meeting the essential requirements for a bone tissue engineering implant.