Chitosan is widely investigated for biomedical applications due to its excellent properties, such as biocompatibility, biodegradability, bioadhesivity, antibacterial, etc. In the field of neural engineering, it has been extensively studied in forms of film and hydrogel, and has been used as scaffolds for nerve regeneration in the peripheral nervous system and spinal cord. One of the main issues in neural engineering is the incapability of neuron to attach on biomaterials. The present study, from a new aspect, aims to take advantage of the bio-adhesive property of chitosan to develop chitosan-based materials for neural engineering, specifically in the fields of brain repair and neuroprosthetics. Neuronal responses to the developed biomaterials will also be investigated and discussed. In the first part of this study (Chapter II), chitosan was blended with a well-studied hydrogel material (agarose) to form a simply prepared hydrogel system. The stiffness of the agarose gel was maintained despite the inclusion of chitosan. The structure of the blended hydrogels was characterized by light microscopy and scanning electron microscopy. In vitro cell studies revealed the capability of chitosan to promote neuron adhesion. The concentration of chitosan in the hydrogel had great influence on neurite extension. An optimum range of chitosan concentration in agarose hydrogel, to enhance neuron attachment and neurite extension, was identified based on the results. A “steric hindrance” effect of chitosan was proposed, which explains the origin of the morphological differences of neurons in the blended gels as well as the influence of the physical environment on neuron adhesion and neurite outgrowth. This chitosan-agarose (C-A) hydrogel system and its multi-functionality allow for applications of simply prepared agarose-based hydrogels for brain tissue repair. In the second part of this study (Chapter III), chitosan was blended with graphene to form a series of graphene-chitosan (G-C) nanocomposites for potential neural interface applications. Both substrate-supported coatings and free standing films could be prepared by air evaporation of precursor solutions. The electrical conductivity of graphene was maintained after the addition of chitosan, which is non-conductive. The surface characteristic of the films was sensitively dependent on film composition, and in turn, influenced neuron adhesion and neurite extension. Biological studies showed good cytocompatibility of graphene for both fibroblast and neuron. Good cell-substrate interactions between neurons and G-C nanocomposites were found on samples with appropriate compositions. The results suggest this unique nanocomposite system may be a promising substrate material used for the fabrication of implantable neural electrodes. Overall, these studies confirmed the bio-adhesive property of chitosan. More importantly, the developed chitosan-based materials also have great potential in the fields of neural tissue engineering and neuroprosthetics.

In this thesis, chitosan, a biocompatible polysaccharide that has been approved as a food additive was selected as a platform for the development of safe, efficient non-viral gene delivery systems to mammalian cells. Previously, chitosan-based gene formulations had been generally associated with high molecular weight chitosans, which were poorly characterised in terms of molecular weight distribution and degree of acetylation. Therefore, in order to improve the properties of chitosan-based gene formulations, the research associated with this thesis focused on establishing the structure-property relationships of well-defined, low molecular weight chitosans (chitosan oligomers) as delivery systems for nucleic acids (pDNA and siRNA) in vitro and after lung administration in vivo. pDNA dissociated more easily from chitosan oligomers than from conventional high molecular weight chitosans, resulting in a faster onset and higher levels of in vivo gene expression, comparable to those mediated by polyethyleneimine (PEI), one of the most efficient non-viral delivery systems. Coupling of a trisaccharide branch to the chitosan oligomers so as to target extracellular lectins resulted in a significant improvement in transfection efficiency because of enhanced cellular uptake and colloidal stability. In contrast to pDNA, longer linear chitosan oligomers were required to form physically-stable nanoparticles with siRNA that mediated efficient, sustained gene silencing in vitro. Finally, the use of an optimised catheter device for the nebulisation of small volumes of pDNA formulations resulted in improved dose precision and lung distribution in vivo compared with conventional intratracheal instillation. In conclusion, chitosan oligomers are interesting and viable alternatives to other non-viral gene delivery systems.

Cell adhesion is the first step of cell response to materials and the extracellular matrix (ECM), and is essential to all cell behaviours such as cell proliferation, differentiation, migration and apoptosis for anchor-dependent cells. Therefore, studies of cell attachment have important implications to control and study cell behaviours. During many developed techniques for cell attachment, the manipulation of surface chemistry is a very important method to control initial cell attachment. To control cell adhesion on a two-dimensional surface is a simple model to study cell behaviours, and is a fundamental topic for cell biology, tissue engineering, and the development of biosensors. From the engineering point of view, the preparation of a material with controllable surface chemistry can help studies of cell behaviours and help scientists understand how surface features and chemistry influence cell behaviours. During the fabrication, the challenge is to create a surface with heterogeneous surface properties in the micro scale and subsequently to guide cell initial adhesion. In order to control cell adhesion in a spatial and temporal manner, a photochemical method to control surface chemistry was employed to control the surface property for cell adhesion in this project. Two photocleavable derivatives of the nitrobenzyl group were tried on two types of surfaces: a model self-assembled monolayer (SAM) with alkanethiol-gold surface and biodegradable chitosan. Reactive functional groups on two different surfaces can be inactivated by covalent binding with these photocleavable molecules, and light can be further introduced into the system as a stimulus to recover their reactivity. By simply applying a photomask with diffe

Spinal cord injury (SCI) is a devastating condition characterized by the loss of neuronal pathways responsible for coordinating motor and sensory information between the brain and the rest of the body. The mammalian spinal cord is limited in its ability to repair itself, so treatments devised to replace damaged tissue and promote regeneration are essential towards developing a cure. This work describes the development of a guidance channel strategy for spinal cord transection. Chitosan guidance channels were designed as a delivery vehicle for neural stem/progenitor cell (NSPC) transplants and drug-eluting poly(lactic-co-glycolic acid) (PLGA) microspheres. PLGA microspheres were embedded into chitosan channels by a spin-coating method. These microsphere-loaded channels demonstrated the ability for controlled short-term bioactive release of the small molecule drug dibutyryl cyclic-AMP (dbcAMP) and long-term bioactive release of the protein alkaline phosphatase. NSPCs were shown to be responsive to dbcAMP delivery, which results in greatly enhanced differentiation into neurons. The effect of directed neuronal differentiation was investigated after spinal cord transection in rat, resulting in a dramatic increase in NSPC transplant survival. Guidance channels containing NSPCs treated with dbcAMP resulted in robust tissue bridge formation after SCI, demonstrating extensive axonal regeneration and promoting functional recovery.

Spinal cord injury (SCI) is a devastating condition characterized by the loss of neuronal pathways responsible for coordinating motor and sensory information between the brain and the rest of the body. The mammalian spinal cord is limited in its ability to repair itself, so treatments devised to replace damaged tissue and promote regeneration are essential towards developing a cure. This work describes the development of a guidance channel strategy for spinal cord transection. Chitosan guidance channels were designed as a delivery vehicle for neural stem/progenitor cell (NSPC) transplants and drug-eluting poly(lactic-co-glycolic acid) (PLGA) microspheres. PLGA microspheres were embedded into chitosan channels by a spin-coating method. These microsphere-loaded channels demonstrated the ability for controlled short-term bioactive release of the small molecule drug dibutyryl cyclic-AMP (dbcAMP) and long-term bioactive release of the protein alkaline phosphatase. NSPCs were shown to be responsive to dbcAMP delivery, which results in greatly enhanced differentiation into neurons. The effect of directed neuronal differentiation was investigated after spinal cord transection in rat, resulting in a dramatic increase in NSPC transplant survival. Guidance channels containing NSPCs treated with dbcAMP resulted in robust tissue bridge formation after SCI, demonstrating extensive axonal regeneration and promoting functional recovery.

Naphthenic acids (NAs) are a group of carboxylic acids that are found in hydrocarbon deposits such as the oil sands bitumen. These compounds are a well-known corrosive agent and a toxic component in the oil sands process water (OSPW). Due to Alberta’s zero discharge policy, OSPW cannot be released and must be stored until toxic components like NAs are remediated. One technique that has shown potential is to physically adsorb NAs onto a copolymer generated from economical biomaterials. Therefore, the project can be divided into three sections: 1) Synthesis of β-cyclodextrin (β-CD) copolymer for the sorption of p-nitrophenol (PNP); 2) Synthesis of chitosan-based copolymers (Chi-Glu) for the sorption of PNP; 3) Sorption of carboxylates and NAs using Chi-Glu copolymers. PNP sorption was used as a probe to understand the physicochemical properties of the copolymers. In the first section, β-CD was reacted with sebacoyl chloride (SCl) and terephthaloyl chloride (TCl) at various mole ratios. Characterization was done using Fourier Transform Infrared Spectroscopy (FT-IR), thermogravimetric analysis (TGA), 1H NMR spectroscopy (1H NMR), elemental analysis (CHN), and nitrogen porosimetry. Copolymers synthesized at mole ratios of β-CD to SCl from 1:1 to 1:3 were hydrolyzed at acidic and basic conditions. Therefore, sorption studies were not done at these ratios. The same occurred for 1:1 to 1:3 TCl copolymers. Sorption studies with PNP at pH 4.6 demonstrated enhanced sorption capacity when comparing with a standard: granular activated carbon (GAC). The sorption capacity, Qm (mmol/g), ordered from largest to smallest is 1:9 SCl>1:9 TCl>1:6 SCl> GAC> 1:6 TCl. Chi-Glu copolymers were synthesized by cross-linking glutaraldehyde with pristine chitosan. A systematic study on the effects reaction conditions have on the sorption capacity of the materials was done. Three conditions were changed: pH, temperature, and mole ratios. Chi-Glu copolymers were synthesized at various chitosan to glutaraldehyde mole ratios (1:400, 1:700, 1:1000). Sufficient time was allowed for the aging process. Characterization was done using TGA, FT-IR, CHN, and nitrogen porosimetry. Sorption study with PNP were done at pH = 7.0 and 9.0. At pH = 7.0 sorption capacity appears to correlate to the quantity of homo-polymerized glutaraldehyde: 1:700>1:1000>1:400. While at pH = 9.0, the sorption capacity is inversely proportional to the degree of crosslinking: 1:400>1:700>1:1000. By increasing the pH at the shrinkage phase, PNP was weakly bound onto the Chi-Glu copolymer. Varying temperature before gelation caused a decrease in the sorption capacity with PNP. Sorption studies involving carboxylates and NAs were done at pH = 9.0 at ambient temperature using Chi-Glu copolymers (1:400, 1:700, and 1:1000) and chitosan. Three carboxylates were chosen to reflect the diverse components in NAs. Varying degrees of cyclization (Z = 0, -2, -4) and lipophilic surface area were the main criteria for carboxylates. The sorption capacity depended mainly on the lipophilic surface area (LSA) with sorption capacity highest for 2-hexyldecanoic acid (S1) which has the largest LSA and lowest for, trans-4-pentylcyclohexanecarboxylic acid (S2) and dicyclohexylacetic acid (S3). Unfortunately, cross-linking with glutaraldehyde does not enhance sorption as pristine chitosan retained a higher sorption capacity compared to Chi-Glu copolymers. Acros and Fluka NAs were chosen for sorption and no significant sorption was recorded for any copolymers. Problems involving the micellization process can explain the lack of sorption.

Chitosan membrane has found applications in biomedical, wastewater treatment, and petrochemical fields that involve the use of silver ions (Ag+). However, mobility of Ag+ in chitosan membranes has seldom been studied. In this study, transport properties of Ag+ in chitosan membranes are studied in-depth, to determine diffusivity coefficient, permeability coefficient, and sorption uptake of Ag+ in chitosan. All parameters are evaluated based on the influence of feed concentration, membrane thickness and operating temperature. The diffusivity is determined from the time lag obtained from transient diffusion experiments. The permeability is determined from the steady state of permeation experimentally. The diffusivity and corresponding permeability coefficients of Ag+ in chitosan range from to 2.0 10-7 (cm2/s) and from 6.6 10-8 to 2.0 10-7 {mol m/[m2 s (mol/L)]}, respectively, over the conditions tested. Temperature dependencies of these two parameters are found to follow the Arrhenius relationship. Sorption uptake of the silver salt in chitosan correlates well with the Langmuir isotherm. Also determined from the sorption tests are degree of membrane swelling at different concentrations. This information allows diffusivity coefficients to be determined from the steady state permeation rate. These values of diffusivity are compared with that obtained using the time lag method.

Cellulose is one of the most abundant biopolymers found in nature. Cellulose based derivatives have a number of advantages including recyclability, reproducibility, biocompatibility, biodegradability, cost effectiveness and availability in a wide variety of forms. Due to the benefits of cellulose based systems, this research study was aimed at developing novel cellulosic nanoparticles with potential pharmaceutical and personal care applications. Two different cellulosic systems were evaluated, each with its own benefits and proposed applications. The first project involves the synthesis and characterization of polyampholyte nanoparticles composed of chitosan and carboxymethyl cellulose (CMC), a cellulosic ether. EDC carbodiimide chemistry and inverse microemulsion technique was used to produce crosslinked nanoparticles. Chitosan and carboxymethyl cellulose provide amine and carboxylic acid functionality to the nanoparticles thereby making them pH responsive. Chitosan and carboxymethyl cellulose also make the nanoparticles biodegradable and biocompatible, making them suitable candidates for pharmaceutical applications. The synthesis was then extended to chitosan and modified methyl cellulose microgel system. The prime reason for using methyl cellulose was to introduce thermo-responsive characteristics to the microgel system. Methyl cellulose was modified by carboxymethylation to introduce carboxylic acid functionality, and the chitosan-modified methyl cellulose microgel system was found to be pH as well as temperature responsive. Several techniques were used to characterize the two microgel systems, for e.g. potentiometric and conductometric titrations, dynamic light scattering and zeta potential measurements. FTIR along with potentiometric and conductometric titration was used to confirm the carboxymethylation of methyl cellulose. For both systems, polyampholytic behaviour was observed in a pH range of 4-9. The microgels showed swelling at low and high pH values and deswelling at isoelectric point (IEP). Zeta potential values confirmed the presence of positive charges on the microgel at low pH, negative charges at high pH and neutral charge at the IEP. For chitosan-modified methyl cellulose microgel system, temperature dependent behaviour was observed with dynamic light scattering. The second research project involved the study of binding interaction between nanocrystalline cellulose (NCC) and an oppositely charged surfactant tetradecyl trimethyl ammonium bromide (TTAB). NCC is a crystalline form of cellulose obtained from natural sources like wood, cotton or animal sources. These rodlike nanocrystals prepared by acid hydrolysis of native cellulose possess negatively charged surface. The interaction between negatively charged NCC and cationic TTAB surfactant was examined and it was observed that in the presence of TTAB, aqueous suspensions of NCC became unstable and phase separated. A study of this kind is imperative since NCC suspensions are proposed to be used in personal care applications (such as shampoos and conditioners) which also consist of surfactant formulations. Therefore, NCC suspensions would not be useful for applications that employ an oppositely charged surfactant. In order to prevent destabilization, poly (ethylene glycol) methacrylate (PEGMA) chains were grafted on the NCC surface to prevent the phase separation in presence of a cationic surfactant. Grafting was carried out using the free radical approach. The NCC-TTAB polymer surfactant interactions were studied via isothermal titration calorimetry (ITC), surface tensiometry, conductivity measurements, phase separation and zeta potential measurements. The major forces involve in these systems are electrostatic and hydrophobic interactions. ITC and surface tension results confirmed two kinds of interactions: (i) electrostatically driven NCC-TTAB complexes formed in the bulk and at the interface and (ii) hydrophobically driven TTAB micellization on the NCC rods. Conductivity and surface tension results confirmed that the critical micelle concentration of TTAB (CMCTTAB) shifted to higher values in the presence of NCC. Phase separation measurements allowed us to identify the formation of large aggregates or hydrophobic flocs depending on the TTAB concentration. Formation of NCC-TTAB complexes in aqueous solutions was confirmed by a charge reversal from negative to positive charge on the NCC rods. The effect of electrolyte in shielding the negative charges on the NCC was observed from ITC, surface tensiometry and phase separation experiments. Several mechanisms have been proposed to explain the above results. Grafting of PEGMA on the NCC surface was confirmed using FTIR and ITC experiments. In phase separation experiments NCC-g-PEGMA samples showed greater stability in the presence of TTAB compared to unmodified NCC. By comparing ITC and phase separation results, an optimum grafting ratio (PEGMA : NCC) for steric stabilization was also proposed.

Tissue engineering has the potential to create new tissue and organs from cultured cells for transplantation. Biodegradable and biocompatible scaffolds play a vital role in the transfer of the cultured cells to a new tissue. Various scaffolds for soft tissue engineering have been developed, however there is not any structure totally mimicking the natural extracellular matrix (ECM), ready to use. In this study biodegradable and biocompatible scaffolds were developed from natural polymers by tissue engineering approach and tested in vitro. Scaffolds (SCAF) were prepared with freeze drying and composed of chitosan, gelatin and dermatan sulfate. Polymer solutions were treated with different stirring rates (500 rpm and 2000 rpm), freezing temperatures (-20 &deg / C and -80 &deg / C) and molding (cylindrical mold and petri dish) to achieve porous structure in order to provide sufficient space for cell growth and extracellular matrix production. Among the prepared scaffolds at different conditions, the scaffolds prepared at 500 rpm and frozen at -80 &deg / C, (SCAF-1), was chosen for further studies. These scaffolds achieved 0.512 MPa tensile strength, with 9.165 MPa tension modulus and 3.428 MPa compression modulus. Besides in lysozyme containing degradation medium they conserved their integrity and lost about 30 % of their initial weight in 30 days period. Mechanical and enzymatic degradation tests showed that scaffolds have physical integrity for the tissue engineering applications. To mimic the natural tissue and enhance cell growth, biologically active arginine &amp / #8211 / glycine - aspartic acid - serine (RGDS) peptides and platelet derived growth factor-BB (PDGF-BB) were immobilized on the SCAF-1. Fibroblast cells were seeded on the scaffolds containing RGDS, (SCAF-1-RGDS), and PDGF-BB, (SCAF-1-RGDS-PDGF), and incubated in media either free of serum or containing serum. Scaffolds immobilized with RGDS and PDGF-BB had the highest attached cell number by the day 15. Florescence microscopy studies also indicated that RGDS and RGDS-PDGF modified scaffolds were more suitable than controls, (SCAF-1), for cell growth and proliferation. According to scanning electron microscopy (SEM) results, modified scaffolds demonstrated better cell morphology and attachment of cells. Based on the obtained results, it can be concluded that RGDS-PDGF immobilized chitosan-gelatin-dermatan sulfate systems have a great potential to be used as a scaffold for soft tissue engineering applications.

Hard tissue engineering holds the promise of restoring the function of failed hard tissues and involves growing specific cells on extracellular matrix (ECM) to develop &bdquo / &bdquo / tissue-like&rdquo / structures or organoids. Chitosan is a linear amino polysaccharide that can provide a convenient physical and biological environment in tissue regeneration attempt. To improve chitosan&amp / #8223 / s mechanical and biological properties, it was blended with another polymer gelatin. 1-ethyl-3-(3-dimethylaminopropyl)-carbodiimide (EDC) and N-hydroxysuccinimide (NHS) were used to crosslink the chitosan-gelatin matrix to produce stable structures. These natural polymers are mechanically weak especially to serve as a bone substitude and therefore, an inorganic calcium phosphate ceramic, hydroxyapatite, was incorporated to improve this aspect. The objective of this study was to develop chitosan-gelatin/hydroxyapatite scaffolds for a successful hard tissue engineering approach. For this reason, two types of hydroxyapatite, as-precipitated non-sintered (nsHA) and highly crystalline sintered (sHA) were synthesized and blended into mixtures of chitosan (C) and gelatin (G) v to produce 2-D (film) and 3-D (sponge) structures. The physicochemical properties of the structures were evaluated by scanning electron microscopy, X-Ray Diffraction (XRD), Fourier Transform Infrared-Attenuated Total Reflectance spectrometer (FTIR-ATR), differential scanning calorimetry, contact angle and surface free energy measurements and swelling tests. Mechanical properties were determined through tensile and compression tests. In vitro cell affinity studies were carried out with SaOs-2 cells. MTS assays were carried out to study cell attachment and proliferation on the 2-D and 3-D scaffolds. Several methods such as confocal, fluorescence and scanning electron microscopy were used to examine the cell response towards the scaffolds. Cell affinities of the samples were observed to change with changing chitosan-gelatin ratio and hydroxyapatite addition into the matrices. XRD and FTIR results confirmed the purity of the hydroxyapatite synthesized. Mechanical test results showed that 2-D and 3-D chitosan-gelatin/hydroxyapatite constructs have similar properties as bones, and in vitro studies demonstrated that the prepared matrices have the potential to serve as scaffold materials in hard tissue engineering applications.