Tissue engineering is an interdisciplinary field that aims to restore, maintain, or improve diseased or damaged tissues. Electrospinning has become one of the most popular means to fabricate a scaffold for various tissue engineering applications as the process is extremely versatile and inexpensive. The ability for electrospinning to consistently create nanofibrous structures capable of mimicking the native extracellular matrix (ECM) is the basis behind why this technique is so successful in tissue engineering. Cardiovascular disease has been the leading cause of death in the United States for over 100 years, and because of this, the need for coronary artery replacements is in serious demand. More specifically, small diameter vessels (<6 mm I.D.) are most needed, due to the fact that they are most often affected and the current clinical replacements provide less than optimal long-term patency and regenerative ability. Tissue engineering of vascular grafts has been investigated for over 50 years, however, synthetic replacements made of Dacron® and expanded-poly(tetrafluoroethylene) (e-PTFE) still remain the clinical standard. This study examines a variety of different ways to alter different characteristics of electrospun constructs, to create scaffolds that would be favorable for use as a blood vessel replacement; the end goal being the creation of an acellular bioresorbable vascular graft that would provide sufficient mechanical support to withstand physiological forces, as well as ample biocompatibility to allow host cells to infiltrate and regenerate the graft as the structure degrades. As a way of tailoring the mechanical and thermal properties of a scaffold to be more conducive to that of a native artery, a novel co-polymer was created from the random copolymerization of two monomers; 1,4-Dioxan-2-one (DX) and DL-3-methyl-1,4-dioxan-2-one (DL-3-MeDX) were mixed at different ratios and electrospun, forming nanofibrous scaffolds that exhibited different mechanical and thermal properties. Next, scaffolds were electrospun from natural and synthetic polymers, and the potential for these materials to elicit the formation of an acute thrombotic occlusion was investigated by quantifying tissue factor expression from monocytes using a novel technique. Tissue factor expression by monocytes on the electrospun natural and synthetic polymer scaffolds was compared to that of e-PTFE to determine their potential for use as vascular graft materials. Platelet-rich plasma (PRP), a naturally occurring blood component which is comprised of supraphysiologic concentrations of autologous growth factors, was activated and lyophilized to form a preparation rich in growth factors (PRGF). PRGF was electrospun for the first time, to create a scaffold that would mimic the role of the native ECM in the wound healing cascade. Characterization of these scaffolds proved their bioactivity was enhanced, with cell infiltration occurring throughout the structures in as little as 3 days. Lastly, PRP/PRGF and/or heparin were incorporated into electrospun PCL scaffolds as a means of enhancing the regenerative potential and reducing the thrombogenic potential of the scaffolds, while supplying the constructs with mechanical stability. The release of several pro-regenerative growth factors and chemokines from the PRP incorporated scaffolds was analyzed and the effect of PRP and heparin on scaffold degradation characteristics was determined. Additionally, cell proliferation, migration, sprout formation, and chemokine release were evaluated, and results from these experiments proved the addition of PRP could enhance the regenerative potential of the electrospun scaffolds. The results from this study reveal the variety of ways in which a number of characteristics of an electrospun scaffold can be altered to create a more ideal bioresorbable vascular graft that has the potential to be regenerated within the body, while providing enough mechanical support for this to occur over time.

Tissue engineering scaffolds come in many shapes and sizes, however, due to difficulty manufacturing the microstructure architecture required in tissue engineering, most scaffolds are architecturally non-dynamic in nature. Because the microstructural architecture of all biological tissues is inherently complicated, non-dynamic tissue engineering scaffolds tend to be a poor platform for tissue regeneration. The current method for manufacturing dynamic tissue engineering scaffolds involves electrospinning successive layers of different fibers, an approach that exhibits no fiber transition between layers and subsequent delamination problems. In this study we aim to address the design challenges of tissue engineering scaffolds through our novel integrated fiber electrospinning technique. Developed in our lab, this electrospinning technique makes it possible to manufacture complex electrospun scaffolds tailorable to specific tissue engineering needs while minimizing delamination tendencies. Our goal is to enhance the capabilities of the tissue engineering field by increasing the manufacturable scaffold complexity and overall structural integrity of electrospun scaffolds.

Electrospinning is a technique used to generate scaffolds composed of nano- to micron-sized fibers for use in tissue engineering. This technology possesses several key weaknesses that prevent it from adoption into the clinical treatment regime. One major weakness is the lack of porosity exhibited in most electrospun scaffolds, preventing cellular infiltration and thus hosts tissue integration. Another weakness seen in the field is the inability to physically cut electrospun scaffolds in the frontal plane for subsequent microscopic analysis (current electrospun scaffold analysis is limited to sectioning in the cross-sectional plane). Given this it becomes extremely difficult to associate spatial scaffold dynamics with a specific cellular response. In an effort to address these issues the research presented here will discuss modifications to electrospinning technology, cryosectioning technology, and our understanding of cellular infiltration mechanisms into electrospun scaffolds. Of note, the hypothesis of a potentially significant passive phase of cellular infiltration will be discussed as well as modifications to cell culture protocols aimed at establishing multiple passive infiltration phases during prolonged culture to encourage deep cellular infiltration.

The need for tissue and organ replacements cannot be satisfied by autograft and allografts alone. The purpose of this study was to investigate the feasibility of electrospinning a blend of polydioxanone and fibrinogen to produce an engineered tissue scaffold. Fiber diameter and pore size of blends were characterized, as well as mechanical strength. Cell proliferation assays for 1 and 7 day cultures were preformed, and a histological evaluation was performed to determine how favorable the various blends were to cell infiltration and proliferation. Some ratios of blends were identified that contained both acceptable mechanical properties and properties that facilitated cell infiltration. These findings pave the way for future refinement and use of these scaffolds for a variety of tissue engineered targets.

Chronic respiratory diseases affects many people worldwide with little known about the mechanisms diving the pathology, making it difficult to find a cure. Improving the understanding of smooth muscle and extracellular matrix (ECM) interaction is key to developing a remedy to this leading cause of death. With currently no relevant or controllable in vivo or in vitro model to investigate diseased and normal interactions of small airway components, the development of a physiologically relevant in vitro model with comparable cell attachment, signaling, and organization is necessary to develop new treatments for airway disease. The goal of this study is to create a mechanically, biologically and structurally relevant in vitro model of small airway smooth muscle tissue. Synthetic Poly-L-Lactic Acid (PLLA) and decellularized pig lung ECM (DPLECM) were electrospun to form nanofibrous mats that can closely mimic natural bronchial tissue. The addition of DPLECM significantly changed the PLLA scaffold mechanically, biologically, and physically to bring it closer to the characteristics of the human lung. DPLECM scaffolds exhibited a significant decrease in the elastic modulus compared with PLLA alone. Histological staining and SDS-PAGE showed that after scaffold fabrication, essential proteins or protein fragments in natural ECM are still present after processing. Human bronchial smooth muscle cells (HBSMCs) seeded onto PLECM scaffolds formed multiple layers of cells compared to scaffolds composed solely of PLLA. Phenotype of smooth muscle is better maintained when DPLECM is incorporated into the scaffold shown by enhanced contractile protein expression and increased collagen production for normal smooth muscle remodeling of the scaffold. In summary, this research demonstrates that a PLLA/DPLECM composite electrospun mat is a promising tool to produce an in vitro model with the potential to uncover unknown characteristics of bronchiole smooth muscle behavior in diseased or normal states.

Three-dimensional collagen/chitosan scaffolds fabricated by freeze-drying technique in 96-well polystyrene and PDMS plates were optimized during this study. Surface tension is, by and large, one of the most limiting factors in fabricating freeze-dried scaffolds in small format well plates. Traditionally, bowl-shaped top surfaces of collagen/chitosan scaffolds were common in polystyrene 96-well plate; whereas for PDMS 96-well plate, dome-shaped surfaces were formed. These surface tension phenomena are not desirable in cell studies especially during initial cell seeding. A combination of surface treatment and change of freeze-drying regime were developed to mitigate the surface tension problem in PS and PDMS 96-well plates respectively. Collagen/chitosan scaffolds of varying concentration and composition were experimented in both polystyrene and PDMS 96-well plates. Thin water film treatment with UV cross-linking was successfully used to eliminate meniscus in PS well plates; pre-cooling, on the other hand, was utilised to treat scaffold solutions in PDMS well plates. The resultant matrices all had flat top surfaces and average thickness of 1 mm. As expected, scaffolds with lower overall polymer concentration or, from a compositional perspective, scaffolds with high chitosan content generally had larger pores. Microscopic observation by multi-photon microscope was performed and chemical analyses were conducted to characterize the surface-treated scaffolds. In addition, scaffolds were tested in vitro using DLD-1 cells, hMSCs and fibroblasts for their biological performance. The purpose of this study was to address the problem of using small format culture wells for the fabrication of freeze-dried collagen-based scaffolds for studies of cell growth in 3D culture and in microfluidic perfusion bioreactors.

Degenerative conditions of the spine are a major public health problem, leading to severe back pain, reduced quality of life and chronic disablement in a proportion of sufferers. For some of these patients, spinal fusion surgery is a treatment that can alleviate back pain and restore normal function. However, limitations in the availability of graft material mean that alternative grafts are needed and tissue-engineering approaches have been employed. Using a novel self-organising collagen scaffold combined with nano-hydroxyapatite and chondroitin sulphate and by employing the latest materials techniques, I have studied the osteogenic capability of a biomimetic graft for use in spinal fusion surgery. The mineralised collagen scaffold has compressive strength comparable to human cancellous bone and can support the proliferation of viable human mesenchymal stem cells. This porous scaffold can be combined with human mesenchymal stem cells to further promote bone growth, as evidenced by an upregulation in the levels of bone-forming genes and mineralisation of the scaffold. This scaffold can act as a carrier system for BMP-2, with wider application for other growth factors or drugs, providing sustained release when fabricated as a layer-by-layer scaffold. An alternative bone substitute for use in spinal surgery has been designed and characterised, with exciting potential for use in vivo.

Insulin maintains glucose homeostasis through its binding of the insulin receptor and activation of the insulin signalling cascade in insulin sensitive tissues. Skeletal muscle is a major endocrine organ, and is responsible for the majority of post-prandial glucose disposal. The maintenance of glucose homeostasis is a delicate balance and impairments in glucose disposal can have significant physiological effects, resulting in the onset of metabolic diseases such as diabetes mellitus. Insulin stimulated glucose uptake involves a number of signalling proteins to enable uptake to occur. In order to understand the complexities associated with the insulin signalling cascade, cell culture models have provided a controlled and easily manipulated environment in which to investigate insulin stimulated glucose uptake in skeletal muscle. While the majority of these experiments have been conducted in conventional monolayer cultures, the growing field of three-dimensional tissue engineering provides an alternative environment in which skeletal muscle cells can be grown to investigate their physiological function. The purpose of this thesis was to investigate the use of different cell culture models for investigating the effects of acute and chronic insulin exposure on skeletal muscle. Initial investigations aimed to establish glucose uptake in tissue engineering skeletal muscle constructs using tritium labelled (H3) 2-deoxy-d-glucose. Monolayer cultures were used to developed base line conditions. In these cultures, concentrations greater than 0.5 μCi for 15 minutes of insulin stimulation suggested an initial assay window for investigating insulin stimulated glucose uptake. However, the duration of insulin stimulation was not effective in measuring uptake in tissue engineered skeletal muscle constructs based upon western blot experiments of Akt phosphorylation, therefore insulin stimulation in skeletal muscle tissue engineered constructs was increased to 30 minutes. Glucose uptake is mediated via specific glucose transporter protein, GLUT1 and GLUT4. Therefore, the transcriptional profile of these transporters was elucidated in monolayer culture and tissue engineered skeletal muscle constructs. Time course experiments showed an increase in GLUT4 transcription in tissue engineered and monolayer culture systems which is associated with an increase in the transcription of skeletal muscle development and myogenic genes. In two dimensional culture, skeletal muscle cells were exposed to insulin during differentiation and in post-mitotic skeletal muscle myotubes to investigating the potential effects upon metabolic genes and proteins involved in insulin signalling. Chronic exposure to insulin during skeletal muscle differentiation reduced insulin signalling and resulted in an increase in basal glucose uptake and ablated insulin stimulated glucose uptake. In contrast, post-mitotic skeletal muscle myotubes did not shown similar changes and were not as responsive to acute insulin exposure. Therefore future experiments exposed skeletal muscle to insulin during differentiation. Using the previous findings as a basis for experimentation, the effects of chronic and acute insulin exposure upon three dimensional skeletal muscle constructs were investigated. Fibrin and collagen constructs were grown for a total period of 14 days. Constructs were exposed to insulin during differentiation and acutely stimulated for 30 minutes at day 14. Although there was a mean increase in Akt protein phosphorylation in both types of tissue-engineered constructs, these changes were not significant following acute insulin stimulation. In addition, glucose uptake in fibrin skeletal muscle constructs increased as a result of acute insulin stimulation however was not significantly difference to unstimulated constructs. The work presented in this thesis provides initial experimental data of the use of different skeletal muscle cell culture models for investigating insulin signalling and glucose uptake. Further research should further characterise these in vitro models for investigating skeletal muscle metabolism.

REGENERATIVE MEDICINE: VASCULARIZED SKELETAL MUSCLE Tissue engineering is a compelling strategy to create replacement tissues and in this study, skeletal muscle. One major hurdle in the field is how to vascularize large tissue-engineered constructs exceeding the nutrient delivery capability of diffusion. Endothelial colony forming cells and mesenchymal progenitor cells form blood vessels de novo and were co-injected with satellite cells in Matrigel, an extracellular matrix, or PuraMatrix, a synthetic hydrogel. Our approach focused on the ability of bioengineered vascular networks to induce murine and human satellite cells to differentiate and form organized skeletal muscle when injected. We found that perfused human blood vessels were formed in both Matrigel and PuraMatrix and that murine satellite cells differentiated and formed organized myotubes with striations, indicative of adult skeletal muscle. Mesenchymal progenitor cells also induced differentiation of satellite cells in vitro. Human Satellite cells, however, did not show signs of differentiation in either Matrigel or Puramatrix. These data have provided a proof of concept of engineering vascularized skeletal muscle using murine satellite cells. INDUCTION OF CARDIOMYOGENESIS The heart's regenerative capabilities are not robust enough to repair the amount of damaged tissue from myocardial infarction. A novel approach to relieve the ischemia is to deliver cells with vasculogenic ability, endothelial colony forming cells and mesenchymal progenitor cells, to assemble de novo blood vessels and support recovery of cardiomyocytes. In our study, we used an in vitro transwell system that prevent cell contact, but allow diffusion of soluble factors to investigate if endothelial colony forming cells or mesenchymal progenitor cells secrete factors that induce cardiomyogenesis. We found that neonatal rat cardiomyocyte proliferation is enhanced in the presence of endothelial colony forming cells and mesenchymal progenitor cells; however, presence of these cells without fetal bovine serum is not sufficient to initiate cardiomyogenesis. PERSONALIZED THERAPY FOR RENAL CELL CARCINOMA TESTING IN AN ENDOTHEIAL CELL MODEL Sunitinib and Pazopanib are both tyrosine kinase inhibitors with high specificity for vascular endothelial growth factor receptor 2 and are used in the treatment of Renal Cell Carcinoma to inhibit angiogenesis. Recent clinical findings suggest that a subset of the population with a single nucleotide polymorphism in vascular endothelial growth factor receptor 2 respond better to Pazopanib treatment. We used a standard in vitro angiogenesis assay, endothelial cell proliferation, to test the effects of the single nucleotide polymorphism on responsiveness to Sunitinib and Pazopanib. We found that cells containing the polymorphism are more sensitive to Pazopanib than Sunitinib, confirming the clinical finding. We also analyzed the inhibition of phosphorylated vascular endothelial growth factor receptor 2 and confirmed drug activity on the phosphorylated protein. These findings could have personalized clinical implications for the 3% of the population with the polymorphism.

The field of tissue engineering has grown immensely since its inception in the late 1980s. However, currently commercialized tissue engineered products are simple in structure. This is due to a pre-clinical bottleneck in which complex tissues are unable to be fabricated. 3D bioprinting has become a versatile tool in engineering complex tissues and offers a solution to this bottleneck. Characterizing the mechanical properties of engineered tissue constructs provides powerful insight into the viability of engineered tissues for their desired application. Current methods of mechanical characterization of soft hydrogel materials used in tissue engineering destroy the sample and ignore the effect of 3D bioprinting on the overall mechanical properties of a construct. Herein, this work reports on the novel use of a non-destructive method of viscoelastic analysis to demonstrate the influence of 3D bioprinting strategy on mechanical properties of hydrogel tissue scaffolds. 3D bioprinting is demonstrated as a versatile tool with the ability to control mechanical and physical properties. Structure-function relationships are developed for common 3D bioprinting parameters such as printed fiber size, printed scaffold pattern, and bioink formulation. Further studies include effective real-time monitoring of crosslinking, and mechanical characterization of multi-material scaffolds. We envision this method of characterization opening a new wave of understanding and strategy in tissue engineering. / Graduate