The limited treatment options available for heart disease patients has lead to increased interest in the development of embryonic stem cell (ESC) therapies to replace heart muscle. The challenges of developing usable ESC therapeutic strategies are associated with the limited ability to obtain a pure, defined population of differentiated cardiomyocytes, and the design of in vivo cell delivery platforms to minimize cardiomyocyte loss. These challenges were addressed in Chapter 2 by designing a cardiomyocyte selectable progenitor cell line that permitted evaluation of a collagen-based scaffold for its ability to sustain stem cell-derived cardiomyocyte function (“A P19 Cardiac Cell Line as a Model for Evaluating Cardiac Tissue Engineering Biomaterials”). P19 cells enriched for cardiomyocytes were viable on a transglutaminase cross-linked collagen scaffold, and maintained their cardiomyocyte contractile phenotype in vitro while growing on the scaffold. The potential for a novel cell-surface marker to purify cardiomyocytes within ESC cultures was evaluated in Chapter 3, “Dihydropyridine Receptor (DHP-R) Surface Marker Enrichment of ES-derived Cardiomyocytes”. DHP-R is demonstrated to be upregulated at the protein and RNA transcript level during cardiomyogenesis. DHP-R positive mouse ES cells were fluorescent activated cell sorted, and the DHP-R positive cultured cells were enriched for cardiomyocytes compared to the DHP-R negative population. Finally, in Chapter 4, mouse ESCs were characterized while growing on a clinically approved collagen I/III-based scaffold modified with the RGD integrin-binding motif, (“Collagen (+RGD and –RGD) scaffolds support cardiomyogenesis after aggregation of mouse embryonic stem cells”). The collagen I/III RGD+ and RGD- scaffolds sustained ESC-derived cardiomyocyte growth and function. Notably, no significant differences in cell survival, cardiac phenotype, and cardiomyocyte function were detected with the addition of the RGD domain to the collagen scaffold. Thus, in summary, these three studies have resulted in the identification of a potential cell surface marker for ESC-derived cardiomyocyte purification, and prove that collagen-based scaffolds can sustain ES-cardiomyocyte growth and function. This has set the framework for further studies that will move the field closer to obtaining a safe and effective delivery strategy for transplanting ESCs onto human hearts.

The limited treatment options available for heart disease patients has lead to increased interest in the development of embryonic stem cell (ESC) therapies to replace heart muscle. The challenges of developing usable ESC therapeutic strategies are associated with the limited ability to obtain a pure, defined population of differentiated cardiomyocytes, and the design of in vivo cell delivery platforms to minimize cardiomyocyte loss. These challenges were addressed in Chapter 2 by designing a cardiomyocyte selectable progenitor cell line that permitted evaluation of a collagen-based scaffold for its ability to sustain stem cell-derived cardiomyocyte function (“A P19 Cardiac Cell Line as a Model for Evaluating Cardiac Tissue Engineering Biomaterials”). P19 cells enriched for cardiomyocytes were viable on a transglutaminase cross-linked collagen scaffold, and maintained their cardiomyocyte contractile phenotype in vitro while growing on the scaffold. The potential for a novel cell-surface marker to purify cardiomyocytes within ESC cultures was evaluated in Chapter 3, “Dihydropyridine Receptor (DHP-R) Surface Marker Enrichment of ES-derived Cardiomyocytes”. DHP-R is demonstrated to be upregulated at the protein and RNA transcript level during cardiomyogenesis. DHP-R positive mouse ES cells were fluorescent activated cell sorted, and the DHP-R positive cultured cells were enriched for cardiomyocytes compared to the DHP-R negative population. Finally, in Chapter 4, mouse ESCs were characterized while growing on a clinically approved collagen I/III-based scaffold modified with the RGD integrin-binding motif, (“Collagen (+RGD and –RGD) scaffolds support cardiomyogenesis after aggregation of mouse embryonic stem cells”). The collagen I/III RGD+ and RGD- scaffolds sustained ESC-derived cardiomyocyte growth and function. Notably, no significant differences in cell survival, cardiac phenotype, and cardiomyocyte function were detected with the addition of the RGD domain to the collagen scaffold. Thus, in summary, these three studies have resulted in the identification of a potential cell surface marker for ESC-derived cardiomyocyte purification, and prove that collagen-based scaffolds can sustain ES-cardiomyocyte growth and function. This has set the framework for further studies that will move the field closer to obtaining a safe and effective delivery strategy for transplanting ESCs onto human hearts.

The limited treatment options available for heart disease patients has lead to increased interest in the development of embryonic stem cell (ESC) therapies to replace heart muscle. The challenges of developing usable ESC therapeutic strategies are associated with the limited ability to obtain a pure, defined population of differentiated cardiomyocytes, and the design of in vivo cell delivery platforms to minimize cardiomyocyte loss. These challenges were addressed in Chapter 2 by designing a cardiomyocyte selectable progenitor cell line that permitted evaluation of a collagen-based scaffold for its ability to sustain stem cell-derived cardiomyocyte function (“A P19 Cardiac Cell Line as a Model for Evaluating Cardiac Tissue Engineering Biomaterials”). P19 cells enriched for cardiomyocytes were viable on a transglutaminase cross-linked collagen scaffold, and maintained their cardiomyocyte contractile phenotype in vitro while growing on the scaffold. The potential for a novel cell-surface marker to purify cardiomyocytes within ESC cultures was evaluated in Chapter 3, “Dihydropyridine Receptor (DHP-R) Surface Marker Enrichment of ES-derived Cardiomyocytes”. DHP-R is demonstrated to be upregulated at the protein and RNA transcript level during cardiomyogenesis. DHP-R positive mouse ES cells were fluorescent activated cell sorted, and the DHP-R positive cultured cells were enriched for cardiomyocytes compared to the DHP-R negative population. Finally, in Chapter 4, mouse ESCs were characterized while growing on a clinically approved collagen I/III-based scaffold modified with the RGD integrin-binding motif, (“Collagen (+RGD and –RGD) scaffolds support cardiomyogenesis after aggregation of mouse embryonic stem cells”). The collagen I/III RGD+ and RGD- scaffolds sustained ESC-derived cardiomyocyte growth and function. Notably, no significant differences in cell survival, cardiac phenotype, and cardiomyocyte function were detected with the addition of the RGD domain to the collagen scaffold. Thus, in summary, these three studies have resulted in the identification of a potential cell surface marker for ESC-derived cardiomyocyte purification, and prove that collagen-based scaffolds can sustain ES-cardiomyocyte growth and function. This has set the framework for further studies that will move the field closer to obtaining a safe and effective delivery strategy for transplanting ESCs onto human hearts.

The limited treatment options available for heart disease patients has lead to increased interest in the development of embryonic stem cell (ESC) therapies to replace heart muscle. The challenges of developing usable ESC therapeutic strategies are associated with the limited ability to obtain a pure, defined population of differentiated cardiomyocytes, and the design of in vivo cell delivery platforms to minimize cardiomyocyte loss. These challenges were addressed in Chapter 2 by designing a cardiomyocyte selectable progenitor cell line that permitted evaluation of a collagen-based scaffold for its ability to sustain stem cell-derived cardiomyocyte function (“A P19 Cardiac Cell Line as a Model for Evaluating Cardiac Tissue Engineering Biomaterials”). P19 cells enriched for cardiomyocytes were viable on a transglutaminase cross-linked collagen scaffold, and maintained their cardiomyocyte contractile phenotype in vitro while growing on the scaffold. The potential for a novel cell-surface marker to purify cardiomyocytes within ESC cultures was evaluated in Chapter 3, “Dihydropyridine Receptor (DHP-R) Surface Marker Enrichment of ES-derived Cardiomyocytes”. DHP-R is demonstrated to be upregulated at the protein and RNA transcript level during cardiomyogenesis. DHP-R positive mouse ES cells were fluorescent activated cell sorted, and the DHP-R positive cultured cells were enriched for cardiomyocytes compared to the DHP-R negative population. Finally, in Chapter 4, mouse ESCs were characterized while growing on a clinically approved collagen I/III-based scaffold modified with the RGD integrin-binding motif, (“Collagen (+RGD and –RGD) scaffolds support cardiomyogenesis after aggregation of mouse embryonic stem cells”). The collagen I/III RGD+ and RGD- scaffolds sustained ESC-derived cardiomyocyte growth and function. Notably, no significant differences in cell survival, cardiac phenotype, and cardiomyocyte function were detected with the addition of the RGD domain to the collagen scaffold. Thus, in summary, these three studies have resulted in the identification of a potential cell surface marker for ESC-derived cardiomyocyte purification, and prove that collagen-based scaffolds can sustain ES-cardiomyocyte growth and function. This has set the framework for further studies that will move the field closer to obtaining a safe and effective delivery strategy for transplanting ESCs onto human hearts.

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Collagen scaffolds are porous structures which are used in bioreactors and in a wide range of tissue engineering applications. In these contexts, the scaffolds may be subjected to conditions in which fluid is forced through the structure and the scaffold is simultaneously compressed. It is clear that fluid transport within collagen scaffolds, and the inter-relationships between permeability, scaffold structure, fluid pressure and scaffold deformation are of key importance. However, these relationships remain poorly understood. In this thesis, a series of isotropic collagen structures were produced using a freeze-drying technique from aqueous slurry concentrations 0.5, 0.75 and 1 wt%, and fully characterised using X-ray micro-tomography and compression testing. It was found that collagen wt% influenced structural parameters such as pore size, porosity, relative density and mechanical properties. Percolation theory was used to investigate the pore interconnectivity of each scaffold. Structures with lower collagen fraction resulted in larger percolation diameters, but lower mechanical stiffness. Aligned collagen scaffolds were also produced by altering the freeze-drying protocol and using different types of mould materials and designs. It was found that a polycarbonate mould with stainless base resulted in vertically aligned structures with low angular variation. When compared with isotropic scaffolds from slurry of the same concentration, aligned scaffolds had a larger percolation diameter. Tortuosity was used as a mathematical tool to characterise the interconnected pathways within each porous structure. The effect of the size of the region of interest (ROI) chosen and the size of the virtual probe particle used in the analysis on the values of tortuosity calculated were determined and an optimised calculation methodology developed. Increasing the collagen fraction within isotropic scaffolds increased the tortuosity, and aligned structures had smaller tortuosity values than their isotropic counterparts. Permeability studies were conducted using two complementary experimental rigs designed to cover a range of pressure regimes and the results were compared with predictions from mathematical models and computational simulations. At low pressures, it was found that the lower collagen fraction structures, which had more open morphologies, had higher permeabilities. Alignment of the structure also enhanced permeability. The scaffolds all experienced deformation at high pressures resulting in a restriction of fluid flow. The lower collagen fraction scaffolds experienced a sharper decrease in permeability with increased pressure and aligned structures were more responsive to deformation than their isotropic counterparts. The inter-relationships between permeability, scaffold structure, fluid pressure and deformation of collagen scaffolds were explored. For isotropic samples, permeability followed a broad $(1- \epsilon)^2$ behaviour with strain as predicted by a tetrakaidecahedral structural model, with the constant of proportionality changing with collagen fraction. In contrast, the aligned structures did not follow this behaviour with the permeability dropping much more sharply in the early stages of compression. Open-cell polyurethane (PU) foams, sometimes used as dressings in wound healing applications, are often compared with collagen scaffolds in permeability models and were used in this thesis as a comparison structure. The foam had a higher permeability than the scaffolds due to its larger pore sizes and higher interconnectivity. In the light of the effects of compression on permeability, the changes in porous structure with compression were explored in isotropic and aligned 0.75 wt% scaffolds. Unlike the fluid flow experiments, these experiments were carried out in the dry state. Deformation in simple linear compression and in step-wise compression was studied, and the stress relaxation behaviour of the scaffolds characterised. A methodology was developed to characterise the structural changes accompanying compression using X-ray micro-tomography with an in situ compression stage. The methodology accounted for the need for samples to remain unchanged during the scan collection period for stable image reconstruction. The scaffolds were studied in uniaxial compression and biaxial compression and it was found that pore size and percolation diameter decreased with increasing compressive strain, while the tortuosity increased. The aligned structure was less affected than the isotropic at low compressions, in contrast to the results from the permeability study in which the aligned structure was more responsive to strain. This suggests that the degree of hydration may affect the structural changes observed. The insights gained in this study of the inter-relationships between microstructure, fluid dynamics and deformation in collagen scaffolds are of relevance to the informed design of porous structures for medical applications.

Pre-cellularised scaffolds are limited in volume due to the constraints of the time delay required for angiogenic cells ingrowth forming a vascular network and allowing for delivery of nutrients and waste exchange. Channels have the potential to improve the time taken for cellular penetration. The effectiveness of channels in improving angiogenic cells penetration was assessed in vitro and in vivo in porous 3-D collagen scaffolds. Initial studies conducted in vitro demonstrated that the scaffolds supported angiogenic cells ingrowth in culture and the channels improved the depth of penetration of cells into the scaffold. The cells reside mainly around the channels and migrate along the channels. In vivo, channels increased cell migration into the scaffolds and in particular angiogenic cells resulting in a clear branched vascular network of micro vessels in the channelled samples which was not apparent in the non-channelled samples. This correlated well with macrophage invasion into scaffolds since angiogenesis in vivo is usually accompanied by infiltration of macrophages which participate in organization of angiogenesis, and in regulation of tissue regeneration. Thus, macrophage-mediated biodegradation of collagen scaffolds in vitro was also assessed. Furthermore, pre-seeding channelled collagen scaffolds with endothelial cells implantation has potential of speeding up vascularisation of scaffolds compared to human bone marrow stromal cells.