Title: Methodological detection solution scaffolds response to mechanical stress, depending on the degree of hydration Objectives: Determining the extent of lateral deformation u scaffolds made of PVA polymer electrospinning technique. Identify the extent of differences in transverse deformation for different groups of nanofiber scaffolds made of PVA polymer electrospinning technique. Methods: Research scaffolds, we used a measuring device μ-tester, which has two jaws. For the measurement, we chose uniaxial tension test in -tester and record the fluorescence microscope was used with HD camera Olympus 320 for online video recording. Results: The results of this study showed that the ratio of the samples U: L and crosslinking time affects the degree of lateral deformation of the samples scaffolds. Samples scaffolds are compressible, some groups even reached the limits of incompressibility 0.5 Poisson's ratio. Keywords: Poisson, Poisson's ratio, scaffold, nanofiber scaffold, scaffold hydrated, electrospun scaffold, lateral deformation

Magnesium (Mg) and its alloys offer potential as a new class of degradable metallic orthopaedic biomaterials. In comparison with current metallic orthopaedic implant materials, Mg offers advantages such as, high specific strength, closer-to-bone stiffness and biodegradability, thereby eliminating the need for a second surgery to remove hardware. The use of porous metal foams as biomaterial scaffolds has been widely adopted, however, many of these porous structures are manufactured with pore architectures that are inherently random. This makes structural optimisation for a specific purpose challenging. Scaffolds containing ordered pore architectures can be fabricated to meet design criteria, such as porosity, stiffness, and volume fraction. Currently there are few methods described in the literature to manufacture ordered porous Mg. The main aim of this thesis was to determine the resolution of a novel indirect solid free-form fabrication (SFF) process for producing topologically-ordered porous Mg (TOPM) structures from pure Mg and commercial Mg alloys. The produced structures were examined for properties such as dimensional accuracy, microstructure, surface properties, mechanical properties and corrosion behaviour. The capability of the process was further examined in manufacturing structures with complex architecture for potential application as degradable metallic orthopaedic devices, namely a spinal fusion device (SFD) and screw. With the produced structures aimed at load-bearing applications in bone, the mechanical properties and behaviour of the TOPM and SFD made from Mg alloys were investigated using finite element analysis (FEA) and compression testing. The relationship between surface roughness and degradation behaviour in Mg biomaterials has received limited interest and is still a controversial issue. Therefore, it was necessary to accurately determine the effect of surface roughness on corrosion rate of Mg, especially samples manufactured from SFF and casting of molten Mg. Given the well-established need for improved corrosion resistance of Mg, two coating techniques, including biomimetic calcium phosphates and electrochemically-assisted deposition coating, were applied on Mg substrates cast via the SFF process. Corrosion testing was employed to investigate the effectiveness of the coating layers in improving corrosion resistance. In this thesis, the capability of the SFF manufacturing process and properties of the produced structures were thoroughly investigated. Results and findings contribute to the development of topology optimised, degradable Mg devices for biomedical applications.

The treatment of bone defects is facing the situation of lacking donations for autotransplantation. As a valid approach, scaffold-based tissue engineering combines the construction of well-defined porous scaffolds with advanced cell culturing technology to guide tissue regeneration. The role for the scaffold is to provide a suitable environment with a sufficient mechanical stiffness, supports for cell attachment, migration, nutrients and metabolite transport and space for cell remodeling and tissue regeneration. The random copolymers poly(L-lactide-co-ɛ-caprolactone) (poly(LLA-co-CL)) and poly(L-lactide-co-1,5-dioxepan-2-one) (poly(LLA-co-DXO)) have been successfully incorporated into 3D porous scaffolds to induce specific interactions with cells and direct osteogenic cell differentiation. In this thesis, these scaffolds have been modified in chemical and physical ways to map and understand requirements for bone regeneration. Scaffold functionalities and properties, such as hydrophilicity, stiffness, size/shape, and reproducibility, were studied. The hydrophilicity was varied by adding 3–20 % (w/w) Tween 80 to poly(LLA-co-CL) and poly(LLA-co-DXO) respectively, which resulted in contact angles from 35° to 15°. With 3 % Tween 80, the resultant mechanical and thermal properties were similar to pristine polymer materials. Tween 80 did not significantly influence cell attachment or proliferation but did stimulate the mRNA expression of osteogenetic markers. The surface functionality and mechanical properties were altered by introducing nanodiamond particles (n-DP) into poly(LLA-co-CL) scaffolds by means of surface physisorption or hybrid blending. Scaffold with n-DP physisorbed showed improved cell attachment, differentiation, and bone reformation. Hybrid n-DP/poly(LLA-co-CL) composites were obtained by direct blending of polylactide modified n-DP (n-DP-PLA) with poly(LLA-coCL). The n-DP-PLA was prepared by sodium hydride-mediated anionic polymerization using n-DP as the initiator. Prepared n-DP-PLA could be dispersed homogenously in organic solvents and blended with poly(LLA-coCL) solution. The n-DP-PLA particles were homogenously distributed in the composite material, which significantly improved mechanical properties. For comparison, the addition of benzoquinone-modified n-DP (n-DP-BQ) did not reinforce poly(LLA-co-CL). This indicated the importance of specific surface grafting, which determined different particle-polymer interactions. For the treatment of critical size defects, a large porous poly(LLA-co-CL) scaffold (12.5 mm diameter × 25 mm thickness) was developed and produced by molding and salt-leaching methods. The large porous scaffolds were evaluated in a scaffold-customized perfusion-based bioreactor system. It was obvious that the scaffold could support improved cell distribution and support the stimulation of human mesenchymal stem cell (hMSC) especially with dynamic flow in a bioreactor. To improve the scaffolding technique, a three-dimensional fiber deposition (3DF) technique was employed to build layer-based scaffolds. Poly(LLA-coCL) scaffolds produced by the 3DF method showed enhanced mechanical properties and a homogeneous distribution of human osteoblasts (hOBs) in the scaffolds. Although poly(LLA-co-CL) was thermally degraded, the degradation did not influence the scaffold mechanical properties. Based on the computerized design, a 3DF scaffold of amorphous copolymer poly(LLAco-CL) provides high-precision control and reproducibility. In summary, the design of porous scaffolds is one of the essential factors in tissue engineering as to mimicking the intrinsic extracellular environment. For bone tissue engineering, an optimized scaffold can maintain a contact angle greater than 35 degrees. Pristine or modified n-DP, introduced as an additive by surface physisorption or direct blending, can improve scaffold mechanical properties and cell response. Various sizes of scaffolds can be easily produced by a mold-mediated salt-leaching method. However, when 100 % reproducibility is required, the 3DF method can be used to create customizable scaffolds. / <p>QC 20140929</p>

Hydroxyapatite (HA) is the principal inorganic phase in bone. Synthetic hydroxyapatite particles, films, coatings, fibers and porous skeletons are used extensively in various biomedical applications. In this contribution, sol-gel processing and electrospinning have been used to develop a technique to produce fibrous structures. Poly(vinyl alcohol) (PVA) with an average molecular weight (MW) between 40,500 g/mol and 155,000 g/mol was electrospun with a calcium phosphate based sol. The sol was prepared by reacting triethyl phosphite and calcium nitrate and was directly added to an aqueous solution of PVA. This mixture was electrospun at a voltage of 20 - 30 kV. The results indicate that the sol particles were distributed uniformly within the PVA fibers. This electrospun structure was calcined at 600oC for 6 hr to obtain a residual inorganic, sub-micron fibrous network. The fibrous structure after electrospinning is retained after calcination. A variety of structures including solid fibers, micro-porous fibers and interconnected networks could be obtained after calcination. A bead-on-string structure was obtained after electrospinning for MW = 40,500 g/mol. X-Ray diffraction of this fibrous structure indicated that it consisted predominantly of hydroxyapatite with an average crystal size of almost 10-30 nm. The final morphologies of the ceramic fibers were found to depend on polymer molecular weight and sol volume fraction. Average fiber diameters were on the order of 200 nm and 800 nm for molecular weight of 67,500 g/mol and 155,000 g/mol, respectively. By judiciously controlling these material and process variables, non-woven mats of sub-micron fibers with varying degrees of interconnectivity and porosity have been produced. Such novel structures can be useful in drug delivery, tissue engineering and related biomedical applications.

Myocyte hypertrophy is the major compensatory response of the heart to chronic stress. It is induced by the activation of a network of interdependent, intracellular signaling pathways.1 An important pathway activated during the hypertrophic response is the calcineurin Abeta-NFATc transcription factor pathway.2 Our laboratory has recently discovered that calcineurin Abeta and NFATc transcription factors can associate with the scaffold protein mAKAPbeta.3 mAKAPbeta is a scaffold protein that forms a multimolecular signalosome located to the nuclear envelope of cardiac myocytes. Preliminary data demonstrate that calcineurin Abeta binds to a specific site on mAKAPbeta that lacks any of the consensus calcineurin binding sequences previously described. In this report, it is shown that a peptide, which contains the mAKAPbeta -calcineurin Abeta binding domain, associates with calcineurin Abeta in a calcium/calmodulin dependent manner. In addition, the binding of this mAKAPbeta peptide to calcineurin Abeta has no effect on calcineurin?s phosphatase activity. In fact, calcineurin Abeta bound to this mAKAPbeta peptide is catalytically active and capable of dephosphorylating NFAT. This is novel since other scaffold proteins that associate with calcineurin Abeta have been reported to inhibit its phosphatase activity. Furthermore, in our laboratory it has been shown that mAKAPbeta is required for both the nuclear translocation of NFATc and the induction of myocyte hypertrophy in vitro.4 In this report it is demonstrated that inhibition of calcineurin Abeta association to mAKAPbeta affects NFATc phosphorylation state and attenuates the norepinephrine induced hypertrophic response in primary neonatal cardiac myocytes. This study supports the hypothesis that the formation of multimolecular signaling complexes, like the mAKAPbeta signalosome, is necessary for the integration and fidelity of signal transduction involved in physiological processes like hypertrophy. Although hypertrophy is an adaptive response; it is often accompanied by maladaptive remodeling of the heart that can result in heart failure, a leading cause of death in the United States. Research in the signaling complexes involved in myocyte hypertrophy, like the mAKAPbeta signalosome, may lead to the development of novel treatments for pathologic hypertrophy and heart failure.

In coronary artery bypass surgery the autologous saphenous vein is the most commonly used vascular graft. However, in a growing number of patients this vein is not available due to disease or availability. To date, there are no commercially available vascular grafts to replace the autologous saphenous vein. Nevertheless, it is widely accepted that a successful small diameter blood vessel alternative will be found using a tissue engineering approach. A photo-cross-linked biodegradable elastomer of acrylated star-poly(ε-caprolactone-co-D,L-lactide) (ASCP) has recently been developed. The elastomer possesses many desirable properties, such as manufacturability and mechanical properties, making it an interesting scaffolding material candidate for this application. To test the feasibility of the ASCP elastomer as a scaffolding material, a porous scaffold with 90% porosity was constructed using paraffin microbeads combined with an emulsion of ASCP prepolymer and water. Native arterial mechanical properties were matched with an 1800 Da ASCP elastomeric scaffold (ELAS 1800) having 85% porosity. In vitro degradation of scaffolds prepared with two different ASCP Mn (1800 and 4500 Da) was investigated for 8 weeks. Bulk hydrolysis was the mode of degradation regardless of configuration, with the porous scaffold degrading slower than the nonporous control. In addition, the ELAS 4500 scaffold also degraded faster than the ELAS 1800 scaffold with the same porosity. In order to promote the cellular response to this potential vascular scaffold, the surface of the elastomer was modified to enhance bovine coronary artery smooth muscle cell (SMC) attachment and proliferation. Base etching the surface was not as effective as adding a small peptide sequence Gly-Arg-Gly-Asp-Ser (GRGDS) known to enhance cell adhesion. The surface modifications did not change SMC phenotype as all surfaces expressed the contractile marker proteins smooth muscle α-actin and h-caldesmon. The SMCs also expressed these marker proteins when seeded on porous scaffolds. Finally, it was possible to integrate the porous scaffold into a biomimetic blood vessel design. With this initial testing, it appears that the ASCP elastomer is a feasible scaffolding material for small diameter blood vessel tissue engineering. Nevertheless, more detailed testing of mechanical properties and cell behaviour must be conducted to ascertain that the ASCP elastomer and the proposed biomimetic blood vessel design can be appropriate replacements for the autologous saphenous vein. / Thesis (Master, Chemical Engineering) -- Queen's University, 2007-11-18 20:27:30.635

Development of novel oncology therapeutics is limited by a lack of accurate pre-clinical models for testing, specifically the inability of traditional 2D culture to accurately mimic in vivo tumors. Neuroblastoma (NB) is a heterogeneous tumor, that in high-risk patients exhibits a 5-year event free survival rate of less than 50%. As such, there is a clinical need for development of novel systems that can mimic the tumor microenvironment and allow for increased understanding of critical pathways as well as be used for preclinical therapeutic testing. In this thesis, lyophilized silk fibroin scaffolds were used to develop 3D neuroblastoma models (scaffolded NB) using multiple neuroblastoma cell lines. Cells grown on scaffolds in low (1%) and ambient (21%) oxygen were compared to traditional 2D (monolayer) cell culture using oxygen-controlled incubators. We hypothesized that scaffolded growth would promote changes in gene expression, cytokine secretion, and therapeutic efficacy both dependent and independent of hypoxia. Monolayer culturing in low oxygen exhibited increased expression of hypoxia related genes such as VEGF, CAIX, and GLUT1, while scaffolded NB exhibited increased expression of hypoxia related genes under both low and ambient oxygen conditions. Pimonidazole staining (hypoxia marker) confirmed the presence of hypoxic regions in the scaffolded NB. Cytokine secretion in monolayer and scaffolded NB suggested differential secretion of cytokines due to both oxygen concentrations (e.g. VEGF, CCL3, uPAR) and 3D culture (e.g. IL-8, GM-CSF, ITAC). Additionally, treatment with etoposide, a standard chemotherapeutic, demonstrated a reduced response in scaffolded culture as compared to monolayer culture regardless of oxygen concentration. However, use of a hypoxia activated therapeutic, tirapazamine exhibited response in low oxygen monolayer culture as well as scaffolded culture in both low and ambient oxygen. To further expand this model into a single culture system capable of generating cell driven oxygen gradients, a stacked culture system was developed. NB scaffolds were stacked using a holder designed based on COMSOL modeling of oxygen tension in the medium. Post-culture, the scaffolds can be separated for analysis on a layer-by-layer basis. Analysis of scaffolds demonstrated a decrease in dsDNA and an increase in hypoxia related genes (VEGF, CAIX, and GLUT1) at the interior of the stack, comparable to that of the scaffolded low oxygen culture. Scaffolds on the periphery of the stack retained gene expression levels similar to that of scaffolded ambient oxygen culture. COMSOL modeling of stacks suggests oxygen gradients present throughout the tumor model similar to that of an in vivo tumor. Gradients of oxygen were confirmed through positive pimonidazole staining. In summary, we developed a system capable of altering critical oxygen-dependent and independent pathways through controlled oxygen levels and 3D culturing. Further, we enhanced this system through the design of a culture system capable of controlling cell driven hypoxic microenvironments to mimic that of an in vivo tumor. This system has the potential to be applied to multiple cancer types, allowing for understanding of key pathway changes and better development of therapeutics.

Development of novel oncology therapeutics is limited by a lack of accurate pre-clinical models for testing, specifically the inability of traditional 2D culture to accurately mimic in vivo tumors. Neuroblastoma (NB) is a heterogeneous tumor, that in high-risk patients exhibits a 5-year event free survival rate of less than 50%. As such, there is a clinical need for development of novel systems that can mimic the tumor microenvironment and allow for increased understanding of critical pathways as well as be used for preclinical therapeutic testing. In this thesis, lyophilized silk fibroin scaffolds were used to develop 3D neuroblastoma models (scaffolded NB) using multiple neuroblastoma cell lines. Cells grown on scaffolds in low (1%) and ambient (21%) oxygen were compared to traditional 2D (monolayer) cell culture using oxygen-controlled incubators. We hypothesized that scaffolded growth would promote changes in gene expression, cytokine secretion, and therapeutic efficacy both dependent and independent of hypoxia. Monolayer culturing in low oxygen exhibited increased expression of hypoxia related genes such as VEGF, CAIX, and GLUT1, while scaffolded NB exhibited increased expression of hypoxia related genes under both low and ambient oxygen conditions. Pimonidazole staining (hypoxia marker) confirmed the presence of hypoxic regions in the scaffolded NB. Cytokine secretion in monolayer and scaffolded NB suggested differential secretion of cytokines due to both oxygen concentrations (e.g. VEGF, CCL3, uPAR) and 3D culture (e.g. IL-8, GM-CSF, ITAC). Additionally, treatment with etoposide, a standard chemotherapeutic, demonstrated a reduced response in scaffolded culture as compared to monolayer culture regardless of oxygen concentration. However, use of a hypoxia activated therapeutic, tirapazamine exhibited response in low oxygen monolayer culture as well as scaffolded culture in both low and ambient oxygen. To further expand this model into a single culture system capable of generating cell driven oxygen gradients, a stacked culture system was developed. NB scaffolds were stacked using a holder designed based on COMSOL modeling of oxygen tension in the medium. Post-culture, the scaffolds can be separated for analysis on a layer-by-layer basis. Analysis of scaffolds demonstrated a decrease in dsDNA and an increase in hypoxia related genes (VEGF, CAIX, and GLUT1) at the interior of the stack, comparable to that of the scaffolded low oxygen culture. Scaffolds on the periphery of the stack retained gene expression levels similar to that of scaffolded ambient oxygen culture. COMSOL modeling of stacks suggests oxygen gradients present throughout the tumor model similar to that of an in vivo tumor. Gradients of oxygen were confirmed through positive pimonidazole staining. In summary, we developed a system capable of altering critical oxygen-dependent and independent pathways through controlled oxygen levels and 3D culturing. Further, we enhanced this system through the design of a culture system capable of controlling cell driven hypoxic microenvironments to mimic that of an in vivo tumor. This system has the potential to be applied to multiple cancer types, allowing for understanding of key pathway changes and better development of therapeutics.

Enhancing productivity in bioprocesses, especially for biofuel production, is crucial for achieving an environmentally and economically sustainable biotechnology industry.Metabolic channelling occurs in nature when the intermediate between two consecutive enzymes in a pathway is directed from the first enzyme to the second avoiding diffusion in the cytosol. This would be very advantageous in bioprocesses as it would increase efficiency of a particular pathway, reducing side products and protecting the cells from potential toxic intermediates. In recent years different strategies for emulating channelling effect wereproposed and used with very promising results. Clustering of enzymes seems to be the simplest way to create metabolic channelling. In this master thesis, four different strategies to co-localize enzymes in clusters are compared. The metabolic pathway chosen as a model was ethanol production by pyruvate decarboxylase (Pdc) and alcohol dehydrogenase (Adh). Chimeric proteins were genetically engineered and transformed in E. coli creating different strains. Ethanol production by the different strains was measured to compare production efficiency. Cell growth and protein expression were used for further understanding of the results. Strengths and weaknesses of each strategy and proposals for further improvement were discussed.

Injuries to the meniscus are a common and important source of mobility issues in the knees of young active individuals, as well as elderly individuals. Conventional treatments for these injuries involve surgical resections of the damaged portions of tissue in order to relieve immediate clinical symptoms. However, with a decreased amount of meniscal tissue remaining, the load-bearing and load-distribution capacities remain compromised and inevitably lead to the development of osteoarthritis.1 In view of these deficiencies, tissue engineering has emerged as a promising alternative approach to meniscus repair. In this approach, biodegradable synthetic materials have been proposed as scaffolds to stimulate and support cell-mediated tissue remodeling. A wide range of synthetic materials have been developed to respond to the physical and chemical requirements of a scaffold, but many lack the necessary biological properties to respond to cellular stimuli. In addition, many of these materials are deficient in mechanical strength. The aim of this study was to develop a novel biomaterial that addresses these limitations. Poly(trimethylene carbonate) (PTMC) was selected as the main component of the scaffold due its highly suitable material properties. PTMC is a biocompatible, biodegradable polymer with excellent elastomeric properties and mechanical strength. It also offers the advantage of providing long-term mechanical support due to its low degradation rate. However, PTMC alone cannot stimulate tissue regeneration due to its bio-inert nature. In order to provide an ideal environment to support tissue repair, it must possess bioactive signals. PTMC was combined with a collagenase-sensitive peptide substrate to render the scaffold invasive by cells. The peptide also served to increase the slow degradation rate of PTMC by providing cleavage points throughout the network. The compressive strength of this material was significantly higher than previously used scaffold materials. Additionally, the material possessed enhanced toughness and elasticity, high equilibrium water content, and a tunable degradation profile. Unlike currently used scaffolding materials, this material satisfies all of the necessary requirements to function as an effective scaffold for meniscus regeneration. / Thesis (Master, Chemical Engineering) -- Queen's University, 2013-11-20 15:36:06.12