Hydrogels are polymeric materials that have three-dimensional polymeric networks, which are able to absorb and retain a large amount of water within their structures without being dissolved. Among the synthetic hydrogel, poly(2-hydroxylethyl methacrylate) (poly(HEMA)) has been of great interest because of its excellent biocompatibility with the three-dimensional networks. Therefore, poly(HEMA) hydrogels have been widely used in many areas, especially in biomedical and pharmaceutical areas, for such applications as packing materials in chromatography, sorbents in controlled release and drug delivery, implanting materials in tissue engineering. However, the applications of poly(HEMA) are still limited because of its weak mechanical strength and network properties. Therefore, in recent decades, the challenge of how to modify and control the polymer properties and how to build highly porous structures in it has received considerable attention because these modifications could significantly improve the performance of poly(HEMA) hydrogels for more favorable applications. Although HEMA and its polymers have been studied for more than 40 years, few reports about the preparation of micro-/nano-porous poly(HEMA) hydrogel particles and the requirements of their applications have risen. Furthermore, how to control the porous structures and the properties of HEMA copolymers have not been well understood. Accordingly, the objectives of this research were to investigate the synthesis of the porous copolymeric particles of HEMA with various comonomers (MMA, St and NVP), to characterize the porous structures and particle morphology, to simulate the synthesis process and porous characteristics, to explore the effects of the polymer compositions and the porous structures on the swelling properties, and to apply the resultant polymeric particles in the controlled release of the hydrophilic model drug. In the present studies, HEMA was copolymerized with three different comonomers, methyl methacrylate (MMA), styrene (St) and N-vinyl-2-pyrrolidone (NVP), respectively, to prepare highly porous particles crosslinked using ethylene glycol dimethacrylate (EGDMA) in the presence of 1-octanol used as a porogen by means of suspension copolymerization in an aqueous phase initiated by 2,2-azobisisobutyronitrile (AIBN). Nano-pores were observed in the present studies. The pore size and the swelling properties of these particles can be successfully controlled by changing comonomers or adjusting the crosslinker and porogen concentration. The results indicate that lower crosslinker or porogen concentration favors generating smaller pores, whereas a higher concentration of a hydrophilic comonomer, higher crosslinker concentration and higher porogen volume ratio promote the generation of larger pores. In addition, the effects of the porous structures and the network properties on the swelling properties were explored. The swelling capacity of the porous particles is reduced with an increase in the EGDMA molar concentration. However, higher porosity in the particles and higher amount of hydrophilic comonomer result in a higher swelling capacity of the particles. The gel formation and the porous characteristics of HEMA/comonomer/EGDMA systems were simulated using the mathematical models combining the reaction kinetics and the thermodynamics. It was found that the model over-predicted the experimental results of the porosity because the pores and the networks are shrunk or collapsed during the porogen removal. Therefore, the model predicts the maximum porosity that the polymeric particles can reach. If the hydrophobic contents are higher, the model gives better prediction of the porosity. It is concluded that the microporous structures of HEMA related hydrogels could be controlled by a properly designed process based on the knowledge gained via this research. The output of this research helps with a better understanding for industrial production of micro-porous hydrogels and their applications.

The mature central nervous system has a very limited capacity for self-renewal and repair following injury. Neural stem cells (NSCs), however, provide a promising new therapeutic option and can be readily expanded in vitro . Towards the development of an effective therapy, greater understanding and control is needed over the mechanisms regulating the differentiation of these cells into function-restoring neurons. In vivo, the neural stem cell niche plays a critical role in directing stem cell self-renewal and differentiation. By understanding and harnessing the power of this niche, a tissue engineered system with encapsulated neural stem cells could be designed to encourage neuronal differentiation and ultimately regeneration of damaged neural tissue. Poly(ethylene glycol)-based hydrogels were used here as a platform for isolating and investigating the response of neural stem cells to various matrix, soluble, and cellular components of the niche. When covalently modified with a cyclic RGD peptide, the synthetic scaffold was demonstrated to support attachment and proliferation of a human NSC line under conditions permissive to cell growth. Under differentiating conditions, the scaffold maintained appropriate lineage potential of the cells by permitting the development of both neuronal and glial populations. Expansion and differentiation of NSCs was also observed in a more biomimetic, three dimensional environment following encapsulation within a degradable hydrogel material. To simulate the soluble signals in the niche, fibroblast growth factor and nerve growth factor were tethered to the hydrogel and shown to direct NSC proliferation and neuronal differentiation respectively. Finally, as an example of the cell-cell interactions in the niche, the pro-angiogenic capacity of encapsulated neural stem cells was evaluated both in vitro and in vivo. Ideally, the optimal scaffold design will be applied to guide NSCs in a therapeutic application. Toward this goal, a novel method was developed for encapsulation of the cells within injectable hydrogel microspheres. This technique was optimized for high cell viability and microsphere yield and was demonstrated with successful microencapsulation and delivery of neural stem cells in rodent model of ischemic stroke.

Hydrogels are polymeric materials that have three-dimensional polymeric networks, which are able to absorb and retain a large amount of water within their structures without being dissolved. Among the synthetic hydrogel, poly(2-hydroxylethyl methacrylate) (poly(HEMA)) has been of great interest because of its excellent biocompatibility with the three-dimensional networks. Therefore, poly(HEMA) hydrogels have been widely used in many areas, especially in biomedical and pharmaceutical areas, for such applications as packing materials in chromatography, sorbents in controlled release and drug delivery, implanting materials in tissue engineering. However, the applications of poly(HEMA) are still limited because of its weak mechanical strength and network properties. Therefore, in recent decades, the challenge of how to modify and control the polymer properties and how to build highly porous structures in it has received considerable attention because these modifications could significantly improve the performance of poly(HEMA) hydrogels for more favorable applications. Although HEMA and its polymers have been studied for more than 40 years, few reports about the preparation of micro-/nano-porous poly(HEMA) hydrogel particles and the requirements of their applications have risen. Furthermore, how to control the porous structures and the properties of HEMA copolymers have not been well understood. Accordingly, the objectives of this research were to investigate the synthesis of the porous copolymeric particles of HEMA with various comonomers (MMA, St and NVP), to characterize the porous structures and particle morphology, to simulate the synthesis process and porous characteristics, to explore the effects of the polymer compositions and the porous structures on the swelling properties, and to apply the resultant polymeric particles in the controlled release of the hydrophilic model drug. In the present studies, HEMA was copolymerized with three different comonomers, methyl methacrylate (MMA), styrene (St) and N-vinyl-2-pyrrolidone (NVP), respectively, to prepare highly porous particles crosslinked using ethylene glycol dimethacrylate (EGDMA) in the presence of 1-octanol used as a porogen by means of suspension copolymerization in an aqueous phase initiated by 2,2-azobisisobutyronitrile (AIBN). Nano-pores were observed in the present studies. The pore size and the swelling properties of these particles can be successfully controlled by changing comonomers or adjusting the crosslinker and porogen concentration. The results indicate that lower crosslinker or porogen concentration favors generating smaller pores, whereas a higher concentration of a hydrophilic comonomer, higher crosslinker concentration and higher porogen volume ratio promote the generation of larger pores. In addition, the effects of the porous structures and the network properties on the swelling properties were explored. The swelling capacity of the porous particles is reduced with an increase in the EGDMA molar concentration. However, higher porosity in the particles and higher amount of hydrophilic comonomer result in a higher swelling capacity of the particles. The gel formation and the porous characteristics of HEMA/comonomer/EGDMA systems were simulated using the mathematical models combining the reaction kinetics and the thermodynamics. It was found that the model over-predicted the experimental results of the porosity because the pores and the networks are shrunk or collapsed during the porogen removal. Therefore, the model predicts the maximum porosity that the polymeric particles can reach. If the hydrophobic contents are higher, the model gives better prediction of the porosity. It is concluded that the microporous structures of HEMA related hydrogels could be controlled by a properly designed process based on the knowledge gained via this research. The output of this research helps with a better understanding for industrial production of micro-porous hydrogels and their applications.

The potential application of a hydrogel-based strong anion-exchange (Q) membrane to purify plasmid DNAs was evaluated. The maximum binding capacity of plasmid DNA was estimated to be 12.4 mg/ml of membrane volume with a plasmid DNA recovery of ~ 90%, which is superior to other commercially available anion-exchange resins and membranes. The membrane was able to retain its structural integrity and performance after multiple cycles of usage (> 30 cycles). The inherent properties of plasmid DNA, membrane adsorbent, and the ionic environment on membrane performance were identified as the factors affecting membrane performance and their effects were systematically investigated. Plasmid DNAs with smaller tertiary structure have shorter dynamic radius and/or lowersurface charge densities, which tended to have a better adsorption and recovery than those with larger tertiary structure. Environmental Scanning Electron Microscopy (ESEM) revealed that the hydrogel structure is more porous on one side of membrane than the other, and higher plasmid DNA adsorption and recovery capacities were observed if the more porous side of the membrane was installed upward of flow in the chromatographic unit. ESEM also revealed improved pore distribution and increased membrane porosity if membrane was pre-equilibrated in the buffer solution for 16 hours. The development of better flow through channel in the hydrogel membrane upon extensive soaking further improved plasmid DNA adsorption and recovery capacities. The ionic environment affects the tertiary size of plasmid DNA; and the optimal operating pH of membrane chromatography was different for the plasmid DNAs investigated in this study. The relative contribution of these factors to improve membrane chromatography of plasmid DNAs was analyzed using statistical modeling. It was found that the adsorption of plasmid DNA was mainly affected by the available adsorptive area associated with membrane porosity, whereas the recovery of plasmid DNAs was mainly affected by the environmental pH. A novel, RNase-free, and potentially scalable bioprocess was synthesized using the hydrogel membrane as the technology platform for the manufacturing of pharmaceutical-grade plasmid DNA. High bioprocess recovery and product quality were primarily associated with the optimal integration of impurity removal by calcium chloride precipitation and anion-exchange membrane chromatography and the implementation of isopropanol precipitation as a coupling step between the two impurity-removing steps. Complete removal of total cellular RNA impurity was demonstrated without the use of animal-derived RNase. High-molecular-weight (HMW) RNA and genomic DNA (gDNA) were removed by selective precipitation using calcium chloride at an optimal concentration. Complete removal of the remaining low-molecular-weight (LMW) RNA was achieved by membrane chromatography using the high-capacity and high-productive hydrogel membrane. The simultaneous achievement of desalting, concentrating and buffer exchange by the coupling step of isopropanol precipitation and the high efficiency and resolution of DNA-RNA separation by anion-exchange membrane chromatography significantly reduced the operating complexity of the overall bioprocess, increased the overall recovery of plasmid DNA, and enhanced product quality by removing trace amounts of impurities of major concern for biomedical applications, such as gDNA, proteins, and endotoxin.

In this work, a gradient of interpenetrating polymer networks, consisting of anionic and cationic polymers, has been investigated with respect to protein resistant properties and swelling characteristics at different pH and ionic strength conditions. The swelling and protein adsorption have been studied using \emph{in situ} spectroscopic ellipsometry(SE) and imaging surface plasmon resonance(iSPR) respectively. It has been shown that, by altering the buffer pH, the region of lowest protein adsorption on the surface could be moved laterally. The swelling has similarly been shown to respond to both changes in pH and ionic strength. Additionally, the arise of surface charge and the polymer swelling in solution, both a consequence of the ionisation of fixed charges on the polymer, have been indicated to occur at different buffer pH. The studied polymer systems show promising properties for future applications in, for example, the biosensor area, where the surface chemistry can be tailor-made to work optimally in a given environment.

<p>Transplantation of a functional engineered skeletal muscle substitute is a promising therapeutic option to repair irreversible muscle damage, and, on the other hand, functional muscle tissue constructs can serve as in vitro 3D tissue models that complement the conventional 2D cell cultures and animal models to advance our limited understanding of intrinsic myogenesis and muscle regeneration process. However, the engineering of skeletal muscle constructs with comparable contractile function to the native muscle is hampered by the lack of 1) effective and reproducible methods to form relatively large muscle constructs composed of viable, dense, aligned and matured myofibers, and 2) beneficial microenvironmental cues as well as physiological stimulations that favor the growth, differentiation and maturation of myogenic cells. Thus, in this thesis, I have developed a mesoscopic hydrogel molding approach to fabricate relatively large engineered muscle tissue networks with controllable thickness, pore dimensions, overall myofiber alignment and regional myofiber orientation. I then investigated the effect of variation in pore length on the force generation and passive properties of engineered muscle networks and the potential to improve the contractile function of engineered muscle networks with the treatment of a soluble neurotrophic factor, agrin.</p><p>Specifically, high aspect-ratio soft lithography was utilized to precisely fabricate elastomeric molds containing an array of staggered hexagonal posts which created elliptical pores in muscle tissue sheets made from a mixture of primary skeletal myoblasts, fibrin and Matrigel. The improved oxygen and nutrient access through the pores increased the viability of the embedded muscle cells and prevented the formation of an acellular core. The differentiated myofibers were locally aligned in tissue bundles surrounding the elliptical pores. The length and direction of the microfabricate posts arbitrarily determined the length of elliptical pores and the mean orientation of myofibers formed around the pores, which enables the control of pore dimensions and regional myofiber orientation. Contractile force analysis revealed that engineered muscle networks with more elongated pores generated larger contractile force due to the increased myonuclear density and degree of overall myofiber alignment, despite the larger porosity and reduced tissue volume. Furthermore, the introduction of elliptical pores resulted in distinct deformational changes in tissue bundles and node regions that connect the ends of bundles with the applied unaxial macroscopic stretch, but such spatial alteration of local strain field resulted in no significant change in macroscopic length- tension relationships among engineered muscle networks with different pore length. </p><p>In addition, supplementing culture medium with soluble recombinant agrin significantly increased contractile force production of engineered muscle networks in the absence of nerve-muscle interaction, primarily or partially due to the agrin-induced upregulation of dystrophin. As expected, alteration in the levels endogenous ACh or ACh-like compound affected the agrin-induced AChR aggregation. Furthermore, increased autocrine AChR stimulation, a novel mechanism underlying survival and maturation of aneural myotubes, attenuated the agrin-induced force increase, while suppressed autocrine AChR stimulation severely comproised the overall force production of engineered muscle networks, of which the underlying mechanisms remains to be elucidated in the future studies. </p><p>In summary, a novel tissue engineering methodology that enables the fabrication of relative large muscle tissue constructs with controllable structure and function has been developed in this thesis. Future studies, such as optimizing cell-matrix interaction, incorporating beneficial regulatory proteins in the fibrin-based matrix, and applying specific patterns of electro-mechanical stimulations are expected to further augment the contractile function of engineered muscle networks. The potential application of this versatile tissue fabrication approach to engineer different types of soft tissue might further advance the development of tissue regeneration therapies as well as deepen our understanding of intrinsic tissue morphogenesis and regeneration process.</p> / Dissertation

Embryonic stem cells (ESCs) are pluripotent cells derived from the inner cell mass of the blastocyst that can give rise to cells of the ectoderm, endoderm and mesoderm lineages. Once isolated from the blastocyst, ESCs can be cultured indefinitely in vitro in an undifferentiated state or can be induced to differentiate. In the case of mouse ESCs (mESCs), the cytokine leukemia inhibitory factor (LIF) is added to culture media to maintain pluripotency and is removed to induce differentiation. Although it is known that extracellular matrix (ECM) components influence stem cell maintenance, proliferation and differentiation, the precise effects of ECM environments on embryonic stem cell behavior have not been systematically studied. The main purpose of this thesis project was to investigate the behavior of undifferentiated mESCs cultured in different 3D hydrogel matrices and to determine whether viscoelastic and biochemical variations in the matrices differentially affect the ability of stem cells to self-renew; that is, retain their pluripotency or undifferentiated phenotype. Their behavior in 3D environments was compared to mESC behavior in traditional 2D culture. In addition, a new method of casting hydrogels in polydimethylsiloxane (PDMS) molds was developed in order to efficiently cast multiple hydrogels of varying sizes and shapes. The findings of this thesis project will benefit both the scientific and engineering community as it encourages researchers to re-evaluate the quality of standard 2D embryonic stem cell culture methods versus potentially novel and advantageous 3D hydrogel culture methods.

Plasma polymerization was used to produce thermoresponsive, hydrogel films of N-Isopropylacrylamide (NIPAAm) in a single step. Through variation of reactor conditions such as deposition pressure and substrate temperature, physicochemical properties of the hydrogel films such as crosslink density and thus swelling could be controlled. Chemical bonding structures in plasma polymerized NIPAAm were studied using Fourier transform infrared spectroscopy (FTIR). Contact angle goniometry and quartz crystal microbalance with dissipation monitoring were used to confirm the existence of a hydrophilic-hydrophobic transition in plasma polymerized NIPAAm thin films, analogous to the lower critical solution temperature (LCST) transition in linear, uncrosslinked chains. Hydrogen bonding in NIPAAm thin films was found to control the moisture uptake capacity; films prepared at higher substrate temperatures and lower reactor pressures, and hence believed to possess greater crosslink density, showed the highest moisture uptake capacity in ambient humidity. Free volume characteristics of NIPAAm thin films were studied using Doppler broadening energy spectroscopy (DBES). Furthermore, a novel, electrophoretic procedure was conceived to incorporate biomolecules such as antibodies in plasma polymerized NIPAAm films for use as sensing layers in vapor phase, surface acoustic wave sensors.

There are more than 150,000 anterior cruciate ligament reconstructions each year with the goal of recovering the balance between knee stability and mobility. As many as 25 percent of these procedures will end in joint instability that can cause further damage. The risk of developing degenerative joint disease (DJD) increases in patients with previous knee injury, resulting in a higher instance of total knee arthroplasty (TKA). There are more than 400,000 TKA procedures each year, but the waiting lists for this surgery shows that many more patients are hoping to undergo this procedure. TKA provides improved knee function and pain relief for patients suffering from DJD. Although this procedure is considered successful, as younger patients undergo this treatment, the long-term performance must be improved. Major mechanisms of failure include component loosening from stress-shielding, poor integration of the implant with native tissue, and ion release from the implant. TiNb alloys are more biocompatible than currently used alloys, such as NiTi, and have mechanical properties closer to bone, so they would reduce the instance of stress shielding. TiNb can be made porous for better integration with the native bone and has superior corrosion resistance than NiTi. Engineered ligaments have generally failed to achieve mechanical properties sufficiently similar to their native counterparts, but also lack the osteochondral interface critical to the transfer of load between ligament and bone. The osteochondral interface could be incorporated through a gradient of inorganic content toward the bony insertion ends of the ligament graft, as we showed that in increase of inorganic content resulted in the transdifferentiation of osteoblasts toward chondrocyte-like cells (bone to cartilage-like). A composite scaffold composed of an electrospun mesh with either a hydrogel component or extracellular matrix (ECM) produced by the cells may be a suitable tissue engineered ligament graft. The non-linear stress-strain behavior seen in native ligament is exhibited by both of these systems, and the ECM produced by these systems is consistent with ligament tissue. The ECM-electrospun mesh composite exhibited higher elastic modulus than the fibrin-electrospun mesh composite, but required extensive pre culture while the fibrin-electrospun mesh composite could be fabricated in situ.

In this study, pH-sensitive poly(acrylamide-co-acrylic acid) hydrogels were synthesized as controlled release systems in the presence of N,N-methylene bisacrylamide as crosslinker and ammonium persulfate as initiator. A set of hydrogels were used in the form they were prepared. One set of hydrogels were prepared as porous networks by incorporating sodium chloride into the reaction medium and then leaching of it after the completion of polymerization reaction. Two sets of hydrogels were modified by argon-plasma at different discharge powers. Hydrogels were characterized by 13C-NMR, XPS, SEM, ATR-FTIR, ESR as well as equilibrium degree of swelling (EDS) and contact angle measurements. Prepared hydrogels were loaded with a model antibiotic, ciprofloxacin-HCl (CPFX), and in-vitro release of CPFX from hydrogel matrices were examined in buffer solutions of varying pH values. There are two factors determining the release rates of CPFX / one is the pH-dependent solubility of CPFX and the other is EDS of the hydrogel samples. For porous samples drug loading and release rates were higher when compared to the control samples and CPFX solubility dominated over release kinetics. Plasma treatment resulted in prolonged release rates in acidic medium.