Alginate is a popular biomaterial in tissue engineering. When crosslinked with calcium ions (Ca2+), alginate forms a hydrogel which provides necessary mechanical support as a scaffold. The material properties as well as the biological properties of alginate scaffold are of great importance. In this thesis, the aim is to use traditional methods, such as scanning electron microscopy (SEM) and light microscopy, and emerging X-ray imaging techniques, such as micro-computed tomography (micro-CT) and synchrotron radiation (SR) X-ray imaging, to characterize the alginate scaffolds. Firstly, the material properties of freeze-dried alginate scaffolds were evaluated using micro-CT, as it is a non-destructive and non-invasive imaging method, and can provide three-dimensional information. Alginate scaffolds made with different sodium alginate concentrations and frozen to different temperatures were scanned and analyzed in micro-CT. Results indicated that lower freezing temperature and higher sodium alginate concentration lead to smaller pore size and porosity. Secondly, cell culture experiments were carried out to study the biological properties and the interactions of alginate hydrogel with cells. A Schwann cell line was either blended with alginate solution before crosslinking with calcium chloride (CaCl2) or put around alginate gel in the culture dish. Light microscopy of sectioned slices showed that cells surrounding the alginate gel could not grow into the gel, while cells blended with alginate solution before crosslinking could proliferate inside the hydrogel. Cells grown inside a thin slice of alginate gels appeared to be in better condition and were larger in size and also grew in clusters. Thirdly, in order to image soft tissue buried inside alginate gels, such as brain slices, novel imaging methods based on synchrotron radiation (SR) were applied, such as absorption and phase contrast imaging, diffraction-enhanced imaging (DEI) and also combined with computed tomography (CT). Synchrotron-based monochromatic X-ray imaging proved to be good at distinguish objects of similar density, especially biological soft tissue samples, even without any staining material, such as osmium tetroxide (OsO4). These three pieces of research work show the potential in applying the emerging X-ray imaging in soft tissue engineering.

Tissue-engineering (TE) is one of the most innovative approaches for tackling many diseases and body parts that need to be replaced, by developing artificial tissues and organs. For this, tissue scaffolds play an important role in various TE applications. A tissue scaffold is a 3D (3D) structure with interconnected pore networks and used to facilitate cell growth and transport of nutrients and wastes while degrading gradually itself. Many fabrication techniques have been developed recently for incorporating living cells into the scaffold fabrication process and among them; dispensing-based rapid prototyping techniques have been drawn considerable attention due to its fast and efficient material processing. This research is aimed at conducting a preliminary study on the dispensing-based biofabrication of 3D cell-encapsulated alginate hydrogel scaffolds. Dispensing-based polymer deposition system was used to fabricate 3D porous hydrogel scaffolds. Sodium alginate was chosen and used as a scaffolding biomaterial. The influences of fabrication process parameters were studied. With knowledge and information gained from this study, 3D hydrogel scaffolds were successfully fabricated. Calcium chloride was employed as crosslinker in order to form hydrogels from alginate solution. The mechanical properties of formed hydrogels were characterized and examined by means of compressive tests. The influences of reagent concentrations, gelation time, and gelation type were studied. A post-fabrication treatment was used and characterized in terms of strengthening the hydrogels formed. In addition, the influence of calcium ions used as crosslinker on cell viability and proliferation during and after the dispensing fabrication process was examined and so was the influence of concentration of calcium solutions and exposing time in both media and alginate hydrogel. The study also showed that the density of encapsulated cells could affect the viscosity of alginate solution. In summary, this thesis presents a preliminary study on the dispensing-based biofabrication of 3D cell-encapsulated alginate hydrogel scaffolds. The results obtained regarding the influence of various factors on the cell viability and scaffold fabrication would form the basis and rational to continue research on fabricating 3D cell-encapsulated scaffolds for specific applications.

Isolation of selected populations from heterogeneous cell mixtures and retrieval of the captured population of interest for regenerative medicine and diagnostics applications is one of the challenges that may be addressed by microfluidics. An affinity adhesion strategy was tested using the tetrapeptides RGDS (arg-gly-asp-ser), REDV (arg-glu-asp-val) and VAPG (val-ala-pro-gly) to modify an alginate hydrogel surface layer to selectively adhere fibroblast (FB), endothelial (EC) and smooth muscle cell (SMC) populations, respectively, of the non-myocyte cardiac cell fraction. Incorporation of peptides into sodium alginate gel surface coatings demonstrated a preferential, seeding density-dependent adhesion relationship on alginate-RGDS when tested with a cardiomyocyte-depleted cell suspension in both static culture and in microfluidic devices. Seeding density-dependent attachment was seen with close to 100% release of viable cells from coated surfaces upon application of ethylenediaminetetraacetic acid (EDTA). Further work will optimize the system with REDV and VAPG to capture ECs and SMCs.

Alginate is an exopolysaccharide secreted by Pseudomonas aeruginosa and is a major component of biofilms that infect the lungs of cystic fibrosis patients. Ten proteins have been implicated in alginate polymerization, modification and export, and are believed to assemble into a multi-protein complex that spans the cell envelope and coordinates the synthesis and secretion of alginate. AlgK is a protein encoded in the alginate biosynthetic operon, which is required for the secretion of high-molecular weight alginate. This study describes structural and functional studies of AlgK to improve our understanding of AlgK’s role in alginate biosynthesis. To shed light on the function of AlgK, C14-palmitic acid labeling and sucrose gradient fractionation studies confirmed that AlgK is an outer membrane lipoprotein. Cellular fractionation experiments also found that AlgK is involved in the proper localization of AlgE, the alginate secretion pore in the outer membrane. The structure of AlgK was determined to 2.5 Å resolution by X-ray crystallography and revealed that the protein folds into 22 alpha-helices that pack into a flexible right-handed solenoid. Closer examination of the amino acid sequence revealed that AlgK carries 9.5 tetratricopeptide repeat (TPR)-like elements. Given the role that TPR motifs generally play in protein-protein interaction and the assembly of multi-protein complexes, the presence of these motifs in AlgK suggests that it can bind to one or more proteins. Based on the results presented in this study, we propose that AlgK acts as a scaffold for the assembly of the alginate secretion complex. By mapping highly conserved residues onto the surface of our model, three putative sites of protein-protein interaction were identified. We hypothesize that the N-terminus of AlgK binds to AlgE in the outer membrane, and the C-terminus of AlgK binds to periplasmic and/or inner membrane Alg proteins, thereby acting as a linker between the inner and outer membrane components of the alginate biosynthetic complex. We further hypothesize that together AlgE and AlgK constitute a novel exopolysaccharide secretin. The alginate biosynthetic complex appears to be distinct from the canonical capsular polysaccharide systems currently described.

Alginate is a popular biomaterial in tissue engineering. When crosslinked with calcium ions (Ca2+), alginate forms a hydrogel which provides necessary mechanical support as a scaffold. The material properties as well as the biological properties of alginate scaffold are of great importance. In this thesis, the aim is to use traditional methods, such as scanning electron microscopy (SEM) and light microscopy, and emerging X-ray imaging techniques, such as micro-computed tomography (micro-CT) and synchrotron radiation (SR) X-ray imaging, to characterize the alginate scaffolds. Firstly, the material properties of freeze-dried alginate scaffolds were evaluated using micro-CT, as it is a non-destructive and non-invasive imaging method, and can provide three-dimensional information. Alginate scaffolds made with different sodium alginate concentrations and frozen to different temperatures were scanned and analyzed in micro-CT. Results indicated that lower freezing temperature and higher sodium alginate concentration lead to smaller pore size and porosity. Secondly, cell culture experiments were carried out to study the biological properties and the interactions of alginate hydrogel with cells. A Schwann cell line was either blended with alginate solution before crosslinking with calcium chloride (CaCl2) or put around alginate gel in the culture dish. Light microscopy of sectioned slices showed that cells surrounding the alginate gel could not grow into the gel, while cells blended with alginate solution before crosslinking could proliferate inside the hydrogel. Cells grown inside a thin slice of alginate gels appeared to be in better condition and were larger in size and also grew in clusters. Thirdly, in order to image soft tissue buried inside alginate gels, such as brain slices, novel imaging methods based on synchrotron radiation (SR) were applied, such as absorption and phase contrast imaging, diffraction-enhanced imaging (DEI) and also combined with computed tomography (CT). Synchrotron-based monochromatic X-ray imaging proved to be good at distinguish objects of similar density, especially biological soft tissue samples, even without any staining material, such as osmium tetroxide (OsO4). These three pieces of research work show the potential in applying the emerging X-ray imaging in soft tissue engineering.

Tissue-engineering (TE) is one of the most innovative approaches for tackling many diseases and body parts that need to be replaced, by developing artificial tissues and organs. For this, tissue scaffolds play an important role in various TE applications. A tissue scaffold is a 3D (3D) structure with interconnected pore networks and used to facilitate cell growth and transport of nutrients and wastes while degrading gradually itself. Many fabrication techniques have been developed recently for incorporating living cells into the scaffold fabrication process and among them; dispensing-based rapid prototyping techniques have been drawn considerable attention due to its fast and efficient material processing. This research is aimed at conducting a preliminary study on the dispensing-based biofabrication of 3D cell-encapsulated alginate hydrogel scaffolds. Dispensing-based polymer deposition system was used to fabricate 3D porous hydrogel scaffolds. Sodium alginate was chosen and used as a scaffolding biomaterial. The influences of fabrication process parameters were studied. With knowledge and information gained from this study, 3D hydrogel scaffolds were successfully fabricated. Calcium chloride was employed as crosslinker in order to form hydrogels from alginate solution. The mechanical properties of formed hydrogels were characterized and examined by means of compressive tests. The influences of reagent concentrations, gelation time, and gelation type were studied. A post-fabrication treatment was used and characterized in terms of strengthening the hydrogels formed. In addition, the influence of calcium ions used as crosslinker on cell viability and proliferation during and after the dispensing fabrication process was examined and so was the influence of concentration of calcium solutions and exposing time in both media and alginate hydrogel. The study also showed that the density of encapsulated cells could affect the viscosity of alginate solution. In summary, this thesis presents a preliminary study on the dispensing-based biofabrication of 3D cell-encapsulated alginate hydrogel scaffolds. The results obtained regarding the influence of various factors on the cell viability and scaffold fabrication would form the basis and rational to continue research on fabricating 3D cell-encapsulated scaffolds for specific applications.

Current treatments for spinal cord injury (SCI) are extremely limited due to the fact that the central nervous system lacks the intrinsic ability to regenerate, and constitutes a poor environment for regenerative axon growth. Nerve tissue engineering is an emerging field with the aim of repairing or creating new nerve tissues to promote functional recovery by using artificial tissue repair scaffolds. The design of a stable and consistent animal model of SCI is essential to study the effectiveness of scaffolds in promoting nervous system repair. In this study, a partial transection animal model was created with a three dimensional lesion at T8-T9 that disrupts axonal pathways unilaterally in the dorsal columns of the rat spinal cord. Alginate hydrogel scaffolds incorporating living Schwann cells were fabricated to evaluate the abilities of those scaffolds to foster axonal regeneration. The surgical technique was improved to provide better outcomes related to bleeding during surgery, weight control, neurological function and surgery duration. The survival rate of animals during the surgical procedure and post-surgery period was ultimately increased to 100%. Histology and immunohistochemistry results indicated that implanted alginate scaffolds may induce larger cavities and extenuate harmful inflammation responses, but that effect was ameliorated by inclusion of Schwann cells in the scaffold. However, neither plain alginate scaffolds nor scaffolds containing living Schwann cells were able to improve regeneration of identified axon tracts in the spinal dorsal columns. This research also employed a synchrotron based x-ray phase contrast imaging technique coupled with computed-tomography to visualize the low optical density structural features of scaffolds and spinal cord tissues in formaldehyde fixed specimens. The imaging results suggest that this is a promising method for analyzing the structure of tissue repair scaffolds within the spinal cord. This degree of structural characterization, potentially applicable to living tissue, is not afforded by other conventional image analysis techniques.

published_or_final_version / Botany / Master / Master of Philosophy

Currently, therapeutic proteins and peptides are delivered subcutaneously, as they are readily denatured in the acidic, protease rich environment of the stomach or gastrointestinal track and low bioavailability results from poor intestinal absorption through the paracellular route. Encapsulation of therapeutic peptides and proteins into polymeric micro- and nano- particle systems has been proposed as a possible strategy to overcome limitations to oral protein administration. Furthermore, it was shown that nanoparticles having diameters less than 5µm are able to be taken up by the M cells of Peyer’s patches found in intestinal mucosa . However, the current methodologies to produce particles within desired range involves organic solvents and several steps. In this study, spray drying was investigated as a microencapsulation alternative, as it offers the potential for single step operation, producing dry particles, with the potential for extending the microparticle size into the nano-range. The particles were produced by spray drying of alginate/protein solutions. The effect of spray drying operational parameters on particle properties such as recovery, residual activity and particle size was studied. Particle recovery depended on the inlet temperature of the drying air, whereas the particle size was affected by the feed rate and the alginate concentration of the feed solution. Increase in alginate:protein ratio increased protein stability during the process and shelf live experiments. Presence of 0.2 g trehalose/g particle increased the residual activity up to 90%. The resulting spherical micro and nanoparticles had smooth surfaces. Stable glycol-chitosan-ca-alginate particles were produced with single step operation. The resulting particles had mean diameter around 3.5μm and released 35% of the initial protein content to the simulated stomach environment within 2 hours. The protein distribution within the particle was studied by confocal laser scanning microscope with florescent labeled protein. The image showed protein deposition toward the surface of the particles. Total drying time and Peclet number was calculated for the particles and found to be 8.5 ms and 240, which indicates that particle formation was governed mainly by convection, which resulted in a hollow central region and protein distribution toward the particle surface. This study shows that stable alginate particles containing proteins can be produced in a single step by spray drying, where the particles had a mean size lower than the critical diameter necessary to be orally absorbed by M cell’s of the Peyer’s patches in the gastrointestinal tract and thus can be considered as a promising technology for oral peptide and protein delivery. / Thesis (Master, Chemical Engineering) -- Queen's University, 2007-10-30 12:20:47.728

A method was developed to produce and incorporate a network of discrete, genipin-crosslinked gelatin microfibers around a pancreatic islet within a barium alginate microcapsule. This technique allows for the encapsulation of a porous fibrous matrix without the geometrical restrictions required for cellular aggregate seeding. Microfibers were produced from a novel vortex-drawn extrusion system with an alginate support matrix. Optimization culminated in a hydrated fiber diameter of 22.3 ± 0.4 μm, a 98% reduction in cross sectional area, while making the process more reliable and less labour intensive. The optimized microfibers were encapsulated at 40 vol% within 294 ± 4 μm 1.6% barium alginate microparticles by an electrostatic-mediated dropwise extrusion system. Pancreatic islets extracted from Sprague Dawley rats were encapsulated within the microparticles, and analyzed over a 21-day preliminary in vitro study. Acridine orange and propidium iodide fluorescent viability staining and light microscopy indicated a significant increase in viability for the fiber-laden particles relative to fiber-free control particles at days 7, 14, and 21. The fiber-laden system also reduced the incidence of disrupted islet cohesion from 31% to 8% at day 21, and showed evidence of islet-fiber adhesion. Preliminary investigations into insulin secretion and metabolic activity showed no significant difference between test and control groups. Further investigation into benefits of islet encapsulation within an extracellular matrix fiber network will be the subject of future studies with this body of work serving as a foundation. The system developed in this investigation could be developed into a modular scaffold system for tissue engineering beyond the field of islet research. / Thesis (Master, Chemical Engineering) -- Queen's University, 2011-08-18 15:05:50.917