Fast and precise quantification of circulating tumour cells (CTC) in lung adenocarcinoma is a pivotal step in acceleration of diagnosis, selection of early therapy and estimation of treatment prognosis. Development of a new type of microfluidic device based on detection and quantification of epithelial- and mesenchymal-type CTC by high-affinity and cell-type specific protein binders anchored to a microfluidic chip surface represents a highly innovative approach. In this work, we used EpCAM membrane glycoprotein as a target for generation of epithelial cell- specific protein binders by a directed evolution of proteins selected from highly complex combinatorial libraries derived from albumin-binding domain scaffold (ABD) or human muscle protein domain-derived "Myomedin" scaffold. Collections of EpCAM-binding candidates from the both used libraries were generated and particular binding variants were further characterized by DNA sequencing, biochemically and by functional cell-surface binding assays. The best candidates might serve as robust anchor proteins of a microfludic chip. Key words: epithelial cell, EpCAM, protein binder, ribosome display, combinatorial library, protein scaffold

No description available.

This thesis presents a melt electrospinning technique to fabricate highly porous and controllable poly (ε-caprolactone) (PCL) microfibers for tissue engineering applications and rehabilitation applications. Electrospinning without solvents via melt methods may be an attractive approach to tissue engineering of cell constructs where solvent accumulation or toxicity is an issue. This method is also able to produce microfibers with controllable parameters. However, the fiber diameters resulting from melt electrospinning processes are relatively large when compared to the fibers from solution electrospinning. The typical microfiber diameter from melt electrospinning was reported to be approximately 0.1mm. In order to further develop the melt electrospinning technique, we focused on the design of a melt electrospinning setup based on numerical analysis using the Solidworks 2013 simulation package and practically established a melt electrospinning setup and thermal control system for accurate experiments. One of main purposes of this thesis is the build-up of mathematical modeling to control and predict the electrospun microfiber via a more intricate understanding of their parameters such as the nozzle diameter, applied voltage, distance between the nozzle and counter electrode, temperature, flow rate, linear transitional speed, among others. The model is composed of three parts: 1) melt electrospinning process modeling, 2) fibrous helix movement modeling, and 3) build-up of microfibers modeling. The melt electrospinning process model describes an electric field, the shape of jet’s continuously changing shape, and how the polymer melt is stretched into a Taylor cone and a straight jet. The fibrous helix movement model describes movement of electrospun microfibers influenced by Lorentz force, which moves along the helix pattern. Lastly, the build-up microfiber modeling describes the accumulation of the extruded microfibers on both flat and round counter electrodes based on the physical forces involved. These models are verified by experimental data from our own customized melt electrospinning setup. Moreover, the fabricated scaffolds are tested by seeding neural progenitors derived from murine R1 embryonic stem cell lines and it demonstrates the potential of scaffolds for tissue engineering applications. To increase cell attachment and proliferation, highly porous microfibers are fabricated by combination of melt electrospinning and particulate leaching technique. Finally, auxetic stretchable PCL force sensors are fabricated by melt electrospinning for hand rehabilitation. These stretchable sensors can be used to measure applied external loads or displacement and are also attachable to various substrates. We have attempted to apply the sensors to real human hand in order to prove their functionality. / Graduate / jko@me.uvic.ca

Defects in bone are a constant and serious problem. They occur as a result of high energy trauma, congenital conditions or are created surgically to treat bone tumours or infection. Currently the treatment for these conditions is awkward for the patient, takes a long time and has a high complication rate. An elegant solution would be to mend the bone defect using the patient own cells; osteoblasts or mesenchymal stem cells seeded onto a supportive material scaffold. For successful regeneration of bone structures, a scaffold production technique has to be adopted that can precisely control porosity, internal pore architecture and fibre thickness, as well as maximising media diffusion and optimising scaffold mechanical properties so that the scaffold can withstand bone bearing pressures. It would also be beneficial if the scaffold uniformly distributed surface strain along the fibres throughout the entire scaffold as this would encourage more even cell proliferation/differentiation in the structure. This was addressed by performing a series of finite element analyses on the computer aided design model where the mechanical properties of the natural or synthetic polymer used have been incorporated to yield an accurate strain profile of the entire scaffold. The process used here to generate the scaffolds is a Rapid Prototyping method that creates a three-dimensional object through the repetitive deposition of fibres in layers via extrusion. Due to the high accuracy and versatility of the extruder, the diameter of the pores can be precisely controlled to an accuracy of 10μm, in the manufactured scaffolds the pore size ranges from 100 to 300μm as that is what is found in trabecular bone. Natural and synthetic polymers were plotted which altered the biodegradability properties of the scaffold and the degrees of cell adhesion, proliferation and differentiation in the structure. Scaffolds were manufactured that demonstrated compatibility with cell adhesion, proliferation and osteogenic differentiation. On completion of the scaffolds, the latter were seeded with osteoblasts or marrow stromal cells and put into a mechanically stimulating bioreactor machine to induce a small strain in the scaffold; this was performed to encourage cell proliferation/differentiation. The structure was left until the osteoblasts or marrow stromal cells modified the scaffold through bone deposition. In-vivo experiments were then undertaken. Preliminary data indicated an effect of mechanical stimulation of the cell/scaffold construct on the degree of mineralization of cell matrix generated by human osteogenic cells.

<p>Burn injuries in the United States account for over one million hospital admissions per year, with treatment estimated at four billion dollars. Of severe burn patients, 30-90% will develop hypertrophic scars (HSc). Current burn therapies rely upon the use of bioengineered skin equivalents (BSEs), which assist in wound healing but do not prevent HSc. HSc contraction occurs of 6-18 months and results in the formation of a fixed, inelastic skin deformity, with 60% of cases occurring across a joint. HSc contraction is characterized by abnormally high presence of contractile myofibroblasts which normally apoptose at the completion of the proliferative phase of wound healing. Additionally, clinical observation suggests that the likelihood of HSc is increased in injuries with a prolonged immune response. Given the pathogenesis of HSc, we hypothesize that BSEs should be designed with two key anti-scarring characterizes: (1) 3D architecture and surface chemistry to mitigate the inflammatory microenvironment and decrease myofibroblast transition; and (2) using materials which persist in the wound bed throughout the remodeling phase of repair. We employed electrospinning and 3D printing to generate scaffolds with well-controlled degradation rate, surface coatings, and 3D architecture to explore our hypothesis through four aims.</p><p> In the first aim, we evaluate the impact of elastomeric, randomly-oriented biostable polyurethane (PU) scaffold on HSc-related outcomes. In unwounded skin, native collagen is arranged randomly, elastin fibers are abundant, and myofibroblasts are absent. Conversely, in scar contractures, collagen is arranged in linear arrays and elastin fibers are few, while myofibroblast density is high. Randomly oriented collagen fibers native to the uninjured dermis encourage random cell alignment through contact guidance and do not transmit as much force as aligned collagen fibers. However, the linear ECM serves as a system for mechanotransduction between cells in a feed-forward mechanism, which perpetuates ECM remodeling and myofibroblast contraction. The electrospinning process allowed us to create scaffolds with randomly-oriented fibers that promote random collagen deposition and decrease myofibroblast formation. Compared to an in vitro HSc contraction model, fibroblast-seeded PU scaffolds significantly decreased matrix and myofibroblast formation. In a murine HSc model, collagen coated PU (ccPU) scaffolds significantly reduced HSc contraction as compared to untreated control wounds and wounds treated with the clinical standard of care. The data from this study suggest that electrospun ccPU scaffolds meet the requirements to mitigate HSc contraction including: reduction of in vitro HSc related outcomes, diminished scar stiffness, and reduced scar contraction. While clinical dogma suggests treating severe burn patients with rapidly biodegrading skin equivalents, these data suggest that a more long-term scaffold may possess merit in reducing HSc.</p><p>In the second aim, we further investigate the impact of scaffold longevity on HSc contraction by studying a degradable, elastomeric, randomly oriented, electrospun micro-fibrous scaffold fabricated from the copolymer poly(l-lactide-co-ε-caprolactone) (PLCL). PLCL scaffolds displayed appropriate elastomeric and tensile characteristics for implantation beneath a human skin graft. In vitro analysis using normal human dermal fibroblasts (NHDF) demonstrated that PLCL scaffolds decreased myofibroblast formation as compared to an in vitro HSc contraction model. Using our murine HSc contraction model, we found that HSc contraction was significantly greater in animals treated with standard of care, Integra, as compared to those treated with collagen coated-PLCL (ccPLCL) scaffolds at d 56 following implantation. Finally, wounds treated with ccPLCL were significantly less stiff than control wounds at d 56 in vivo. Together, these data further solidify our hypothesis that scaffolds which persist throughout the remodeling phase of repair represent a clinically translatable method to prevent HSc contraction.</p><p>In the third aim, we attempt to optimize cell-scaffold interactions by employing an anti-inflammatory coating on electrospun PLCL scaffolds. The anti-inflammatory sub-epidermal glycosaminoglycan, hyaluronic acid (HA) was used as a coating material for PLCL scaffolds to encourage a regenerative healing phenotype. To minimize local inflammation, an anti-TNFα monoclonal antibody (mAB) was conjugated to the HA backbone prior to PLCL coating. ELISA analysis confirmed mAB activity following conjugation to HA (HA+mAB), and following adsorption of HA+mAB to the PLCL backbone [(HA+mAB)PLCL]. Alican blue staining demonstrated thorough HA coating of PLCL scaffolds using pressure-driven adsorption. In vitro studies demonstrated that treatment with (HA+mAB)PLCL prevented downstream inflammatory events in mouse macrophages treated with soluble TNFα. In vivo studies using our murine HSc contraction model suggested positive impact of HA coating, which was partiall impeded by the inclusion of the TNFα mAB. Further characterization of the inflammatory microenvironment of our murine model is required prior to conclusions regarding the potential for anti-TNFα therapeutics for HSc. Together, our data demonstrate the development of a complex anti-inflammatory coating for PLCL scaffolds, and the potential impact of altering the ECM coating material on HSc contraction.</p><p>In the fourth aim, we investigate how scaffold design, specifically pore dimensions, can influence myofibroblast interactions and subsequent formation of OB-cadherin positive adherens junctions in vitro. We collaborated with Wake Forest University to produce 3D printed (3DP) scaffolds with well-controlled pore sizes we hypothesized that decreasing pore size would mitigate intra-cellular communication via OB-cadherin-positive adherens junctions. PU was 3D printed via pressure extrusion in basket-weave design with feature diameter of ~70 µm and pore sizes of 50, 100, or 150 µm. Tensile elastic moduli of 3DP scaffolds were similar to Integra; however, flexural moduli of 3DP were significantly greater than Integra. 3DP scaffolds demonstrated ~50% porosity. 24 h and 5 d western blot data demonstrated significant increases in OB-cadherin expression in 100 µm pores relative to 50 µm pores, suggesting that pore size may play a role in regulating cell-cell communication. To analyze the impact of pore size in these scaffolds on scarring in vivo, scaffolds were implanted beneath skin graft in a murine HSc model. While flexural stiffness resulted in graft necrosis by d 14, cellular and blood vessel integration into scaffolds was evident, suggesting potential for this design if employed in a less stiff material. In this study, we demonstrate for the first time that pore size alone impacts OB-cadherin protein expression in vitro, suggesting that pore size may play a role on adherens junction formation affiliated with the fibroblast-to-myofibroblast transition. Overall, this work introduces a new bioengineered scaffold design to both study the mechanism behind HSc and prevent the clinical burden of this contractile disease.</p><p>Together, these studies inform the field of critical design parameters in scaffold design for the prevention of HSc contraction. We propose that scaffold 3D architectural design, surface chemistry, and longevity can be employed as key design parameters during the development of next generation, low-cost scaffolds to mitigate post-burn hypertrophic scar contraction. The lessening of post-burn scarring and scar contraction would improve clinical practice by reducing medical expenditures, increasing patient survival, and dramatically improving quality of life for millions of patients worldwide.</p> / Dissertation

This study is aimed to evaluate the degradation characteristics, cell viability and host tissue responses of PDLLA/PRGD/beta-TCP (PRT) composite nerve scaffold, which was fabricated by poly(D, L-lactic acid) (PDLLA), RGD peptide(Gly-Arg-Gly-Asp-Tyr, GRGDY, abbreviated as RGD) modified poly-{(lactic acid)-co-[(glycolic acid)-alt-(L-lysine)]}(PRGD) and beta-tricalcium phosphate (beta-TCP). The scaffolds' in vitro degradation behaviors were investigated in detail by analysing changes in weight loss, pH and morphology. Then, the 3-(4,5-dimethyl-2-thiazolyl) -2,5-diphenyl-2 -H-tetrazolium bromide (MTT) assay and cell live/dead assay were carried out to assess their cell viability. Moreover, in vivo degradation patterns and host inflammation responses were monitored by subcutaneous implantation of PRT scaffold in rats. Our data showed that, among the tested scaffolds, the PRT scaffold had the best buffering capacity (pH - 6.1-6.3) and fastest degradation rate (12.4%, 8 weeks) during in vitro study, which was contributed by the incorporation of beta-TCP nanoparticles. After in vitro and in vivo degradation, the high porosity structure of PRT could be observed using scanning electron microscopy. Meanwhile, the PRT scaffold could significantly promote cell survival. In the PRT scaffold implantation region, less inflammatory cells (especially for neutrophil and lymphocyte) could be detected. These results indicated that the PRT composite scaffold had a good biodegradable property; it could improve cells survival and reduced the adverse host tissue inflammation responses.

<p>To this day, cancer remains the leading cause of mortality worldwide1. A major contributor to cancer progression and metastasis is tumor angiogenesis. The formation of blood vessels around a tumor is facilitated by the complex interplay between cells in the tumor stroma and the surrounding microenvironment. Understanding this interplay and its dynamic interactions is crucial to identify promising targets for cancer therapy. Current methods in cancer research involve the use of two-dimensional (2D) monolayer cell culture. However, cell-cell and cell-ECM interactions that are important in vascularization and the three-dimensional (3D) tumor microenvironment cannot accurately be recapitulated in 2D. To obtain more biologically relevant information, it is essential to mimic the tumor microenvironment in a 3D culture system. To this end, we demonstrate the utility of poly(ethylene glycol) diacrylate (PEGDA) hydrogels modified for cell-mediated degradability and cell-adhesion to explore, in 3D, the effect of various tumor microenvironmental features such as cell-cell and cell-ECM interactions, and dimensionality on tumor vascularization and cancer cell phenotype. </p><p>In aim 1, PEG hydrogels were utilized to evaluate the effect of cells in the tumor stroma, specifically cancer associated fibroblasts (CAFs), on endothelial cells (ECs) and tumor vascularization. CAFs comprise a majority of the cells in the tumor stroma and secrete factors that may influence other cells in the vicinity such as ECs to promote the organization and formation of blood vessels. To investigate this theory, CAFs were isolated from tumors and co-cultured with HUVECs in PEG hydrogels. CAFs co-cultured with ECs organized into vessel-like structures as early as 7 days and were different in vessel morphology and density from co-cultures with normal lung fibroblasts. In contrast to normal lung fibroblasts (LF), CAFs and ECs organized into vessel-like networks that were structurally similar to vessels found in tumors. This work provides insight on the complex crosstalk between cells in the tumor stroma and their effect on tumor angiogenesis. Controlling this complex crosstalk can provide means for developing new therapies to treat cancer.</p><p>In aim 2, degradable PEG hydrogels were utilized to explore how extracellular matrix derived peptides modulate vessel formation and angiogenesis. Specifically, integrin-binding motifs derived from laminin such as IKVAV, a peptide derived from the α-chain of laminin and YIGSR, a peptide found in a cysteine-rich site of the laminin β chain, were examined along with RGDS. These peptides were conjugated to heterobifunctional PEG chains and covalently incorporated in hydrogels. The EC tubule formation in vitro and angiogenesis in vivo in response to the laminin-derived motifs were evaluated. </p><p>Based on these previous aims, in aim 3, PEG hydrogels were optimized to function as a 3D lung adenocarcinoma in vitro model with metastasis-prone lung tumor derived CAFs, HUVECs, and human lung adenocarcinoma derived A549 tumor cells. Similar to the complex tumor microenvironment consisting of interacting malignant and non-malignant cells, the PEG-based 3D lung adenocarcinoma model consists of both tumor and stromal cells that interact together to support vessel formation and tumor cell proliferation thereby more closely mimicking the functional properties of the tumor microenvironment. The utility of the PEG-based 3D lung adenocarcinoma model as a cancer drug screening platform is demonstrated with investigating the effects of doxorubicin, semaxanib, and cilengitide on cell apoptosis and proliferation. The results from drug screening studies using the PEG-based 3D in vitro lung adenocarcinoma model correlate with results reported from drug screening studies conducted in vivo. Thus, the PEG-based 3D in vitro lung adenocarcinoma model may serve as a better tool for identifying promising drug candidates than the conventional 2D monolayer culture method.</p> / Dissertation

Spinal cord injury (SCI) has presented itself as a multifaceted pathology that is largely inhibitory to regeneration, and therefore to functional recovery, even though spinal cord neurons have been found to be innately regenerative. Thus, having identified the key players in the inhibition of this innate regeneration, SCI researchers have focused on two major types of approaches: (1) blocking inhibitory cues and (2) promoting innate regeneration. Schwann cells (SCs) have long been shown to promote and enhance functional recovery after SCI through providing supplemental myelination and trophic and tropic factors to regenerating axons, though singular approaches rarely address the complex SCI pathology. Guidance channels and scaffolds have been shown to provide physical support and directional cues to regeneration axons. Therefore, a combinatorial approach in which SCs migrate into and throughout a guidance scaffold would be an ideal research focus for treating SCI. However, cell migration into guidance scaffolds has been shown to be problematic. This study attempts to assess and improve two- and three-dimensional SC migration on electrospun scaffolds. Additionally, we evaluate the ability of SCs, seeded on Matrigel-coated electrospun scaffolds, to improve functional recovery in rats with completely transected spinal cords.

Traumatic defects in articular cartilage can lead to joint disease and disability including osteoarthritis. Because cartilage is unable to regenerate when injured, the field of tissue engineering holds promise in restoring functional tissue. In this research, type II collagen was electrospun, cross-linked, and tested as scaffolds for supporting chondrocyte growth. The mechanical, biochemical, and histological characteristics of the engineered tissue were assessed as a function of the electrospinning solution concentration (i.e. scaffold fiber diameter and pore properties) and as a function of the time in culture (evaluated at 2, 4, and 6 weeks). Fiber diameter had a linear relationship with concentration: mean diameter increased from 107 to 446 nm and from 289 to 618 nm, measured with SEM and permeability meter, respectively, with increasing concentration, from 60 mg/mL to 120 mg/mL. Pore size revealed no relationship with concentration but mean values ranged in size from 1.76 to 3.17 μ2 or from 0.00055 to 0.0028 μ2, depending on the measurement technique. Average porosity ranged from 84.1 to 89.1%, and average permeability was between 6.82x10-4 and 35.0 x10-4 D. Histological analysis revealed a relatively high number of spherical cells, possibly indicating the expression of the chondrocyte phenotype. However, there were very little glycosaminoglycans and type II collagen synthesized by the cells despite an increase in the cell density over time for the 60, 80, and 100 mg/mL concentrations. The mean values for peak stress (between 0.17 and 0.35 MPa) and tangential modulus (between 0.32 and 0.64 MPa) for the mats are at least an order of magnitude less than that for native cartilage, while the mean values for strain at break (between 93 and 150%) for the mats are at least an order of magnitude greater than that for native cartilage. The equilibrium stiffness for all concentrations decreased from week 2 to week 6 of tissue culture (which may correlate with increasing cell density); the 100 mg/mL concentration had the highest mean value (0.084 MPa at week 2) and the lowest mean value (0.010 MPa at week 6). This research did not indicate any significant findings regarding the influence of concentration or culture time on chondrogenesis. Because the cells appeared to grow on the surface of the scaffold but there was a lack of cell migration into the scaffold, the scaffold material may be sufficient to support chondrocyte growth but the scaffold physical design must be reconsidered.

The goal of this thesis was to find a suitable method for evaluating structural changes of scaffolds as influenced by external mechanical pressure and to verify the validity of hypothesis which assumes a change of directionality of fibers and also thinning of fibers according to the stretching of a scaffold. Assumptions formulated in these three hypothesis were tested on a scaffold with a plasma surface treatment and without any plasma surface treatment. To examine structural changes an electrone microscope was used to observe the surface of scaffolds. Incurred photos were then processed with the help of automatic software picture analysis and observed data were statisticly evaluated. The result of this experiment is a description of used method which can be used in future for larger studies. It was found that by the effect of external mechanical pressure fibers of examinated scaffolds turn in the direction of the pull. It has also been found that the average thickness of fibers didn't change. Results of this work give insight into the evaluation of structural changes of scaffolds when pressured by an external mechanical power and open possibilities for deeper and more exact research in this field. Key words: scaffold, picture analysis, fiber directionality, fiber thickness.