Browsing by Author "West, Jennifer L."
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Item 3-Dimensional spatially organized PEG-based hydrogels for an aortic valve co-culture model(Elsevier, 2015) Puperi, Daniel S.; Balaoing, Liezl R.; O'Connell, Ronan W.; West, Jennifer L.; Grande-Allen, K. JanePhysiologically relevant inᅠvitro models are needed to study disease progression and to develop and screen potential therapeutic interventions for disease. Heart valve disease, in particular, has no early intervention or non-invasive treatment because there is a lack of understanding the cellular mechanisms which lead to disease. Here, we establish a novel, customizable synthetic hydrogel platform that can be used to study cell-cell interactions and the factors which contribute to valve disease. Spatially localized cell adhesive ligands bound in the scaffold promote cell growth and organization of valve interstitial cells and valve endothelial cells in 3D co-culture. Both cell types maintained phenotypes, homeostatic functions, and produced zonally localized extracellular matrix. This model extends the capabilities of inᅠvitro research by providing a platform to perform direct contact co-culture with cells in their physiologically relevant spatial arrangement.Item A gene therapy approach for tissue engineering applications(2007) Lau, Ying Ka Ingar; West, Jennifer L.; Cameron, Isabel C.; Grande-Allen, K. Jane; Gustin, Michael C.In this work, gene therapy was combined with cell therapy to tackle three tissue engineering applications. The goal of the first project was to promote endothelialization of tissue engineering vascular grafts (TEVGs). We developed a system called the collagen-based gene-activated matrix (GAM) which was able to retain plasmid DNA (pDNA) and allowed smooth muscle cells (SMCs) embedded to gradually take up and express the gene of interest, in this case, vascular endothelial growth factor (VEGF). To obtain better transfection efficiency, pDNA was complexed with polyethyleneimine (PEI) which dramatically improved transfection of SMCs in GAMs. Continual production of VEGF for approximately one month was observed. VEGF produced by SMCs in GAMs was bioactive and induced both enhanced migration and proliferation of endothelial cells (ECs) on collagen which is a common biomaterial for TEVGs. The goal of the second project was to potentiate angiogenesis through overexpression of VEGF in 10T1/2 cells for treatment of ischemic diseases and vascularization of tissue engineered constructs. 10T1/2 cells were transfected with the VEGF transgene successfully via retroviral transfection. VEGF-producing 10T1/2 cells were able to induce enhanced migration, proliferation, as well as invasion of underlying matrix in ECs. Potentiation of angiogenesis was further observed in 3D collagen models when ECs were co-cultured with VEGF-producing 10T1/2 cells. ECs formed extensive network of tubular structures and presence of a lumen in the vessels formed was confirmed by confocal microscopy. VEGF-producing 10TI/2 cells also rescued ECs from starvation and induced them to form organized tubular structures. The goal of the third project was to enhance mechanical strength in dermal wound through increased cross-linking of extracellular matrix (ECM) proteins via overexpression of lysyl oxidase (LO). Using the GAM system we developed and embedding transgene encoding LO with fibroblasts, we obtained enhanced mechanical strength in collagen constructs in vitro. We also demonstrated the same efficacy of these LO-producing GAMs in a dermal wound healing model in vivo.Item A Synthetic Matrix with Independently Tunable Biochemistry and Mechanical Properties to Study Epithelial Morphogenesis and EMT in a Lung Adenocarcinoma Model(American Association for Cancer Research, 2012) Gill, Bartley J.; Gibbons, Don L.; Roudsari, Laila C.; Saik, Jennifer E.; Rizvi, Zain H.; Roybal, Jonathon D.; Kurie, Jonathan M.; West, Jennifer L.Better understanding of the biophysical and biochemical cues of the tumor extracellular matrix environment that influence metastasis may have important implications for new cancer therapeutics. Initial exploration into this question has used naturally derived protein matrices that suffer from variability, poor control over matrix biochemistry, and inability to modify the matrix biochemistry and mechanics. Here, we report the use of a synthetic polymer-based scaffold composed primarily of poly(ethylene glycol), or PEG, modified with bioactive peptides to study murine models of lung adenocarcinoma. In this study, we focus on matrix-derived influences on epithelial morphogenesis of a metastatic cell line (344SQ) that harbors mutations in Kras and p53 (trp53) and is prone to a microRNA-200 (miR-200)–dependent epithelial–mesenchymal transition (EMT) and metastasis. The modified PEG hydrogels feature biospecific cell adhesion and cell-mediated proteolytic degradation with independently adjustable matrix stiffness. 344SQ encapsulated in bioactive peptide-modified, matrix metalloproteinase–degradable PEG hydrogels formed lumenized epithelial spheres comparable to that seen with three-dimensional culture in Matrigel. Altering both matrix stiffness and the concentration of cell-adhesive ligand significantly influenced epithelial morphogenesis as manifest by differences in the extent of lumenization, in patterns of intrasphere apoptosis and proliferation, and in expression of epithelial polarity markers. Regardless of matrix composition, exposure to TGF-β induced a loss of epithelial morphologic features, shift in expression of EMT marker genes, and decrease in mir-200 levels consistent with EMT. Our findings help illuminate matrix-derived cues that influence epithelial morphogenesis and highlight the potential utility that this synthetic matrix-mimetic tool has for cancer biology.Item A tunable hydrogel system and pulsatile flow bioreactor for the development of tissue engineered vascular grafts(2009) McHale, Melissa Knight; West, Jennifer L.The prevalence of coronary artery disease combined with a paucity of suitable vessel substitutes act as driving forces for cardiovascular tissue engineering research. In this thesis poly(ethylene glycol) diacrylate (PEGDA) hydrogels were investigated as a biomaterial for tissue engineered vascular grafts (TEVG). The global objectives for the work were two-fold. First, a thorough characterization of the hydrogels was warranted to determine material properties and cell interaction characteristics. The second objective was to develop a pulsatile flow culture system for TEVG that was capable of achieving physiologically relevant fluid flow parameters. Bulk properties of PEGDA hydrogels formed from a range of polymer molecular weights and solution concentrations were characterized. Resultant materials demonstrate tunable stiffness and strength, and network properties that are appropriate for supporting viability of encapsulated cells. Human coronary artery smooth muscle cells seeded on top of these PEGDA hydrogels exhibit changes in attachment, proliferation, and morphology that can be directly correlated to the rigidity of the substrate material. In general, stiffer materials encourage greater attachment, a higher rate of proliferation, and the development of a mature, spread morphology. Finally, these responses were shown to be independently modulated by changing either the hydrogel material properties or the peptide directed bio-adhesiveness of the substrate. The tissue bioreactor presented in this work is capable of imparting physiological fluid flow (120 mL/min), shear (5-10 dynes/cm2), pressure waveforms (120/80 mmHg), and pulse rates (60 or 120 bpm). Cell-laden hydrogel constructs cultured for up to 8 wk in this system responded to mechanical stimulation with increases in cell and extracellular matrix (ECM) content and positive modulations to material properties. Though the magnitude of ECM accumulation is quite low, changes in hydrogel stiffness and the presence of degrading enzymes indicate that the encapsulated vascular cells are working towards a more biologically appropriate surrounding. Since all parameters for appropriate TEVG culture are not yet understood, this device will serve as an important tool in the development of a small diameter vessel substitute.Item Application of Hydrogels in Heart Valve Tissue Engineering(Begell House, 2015) Zhang, Xing; Xu, Bin; Puperi, Daniel S.; Wu, Yan; West, Jennifer L.; Grande-Allen, K. JaneWith an increasing number of patients requiring valve replacements, there is heightened interest in advancing heart valve tissue engineering (HVTE) to provide solutions to the many limitations of current surgical treatments. A variety of materials have been developed as scaffolds for HVTE including natural polymers, synthetic polymers, and decellularized valvular matrices. Among them, biocompatible hydrogels are generating growing interest. Natural hydrogels, such as collagen and fibrin, generally show good bioactivity but poor mechanical durability. Synthetic hydrogels, on the other hand, have tunable mechanical properties; however, appropriate cell-matrix interactions are difficult to obtain. Moreover, hydrogels can be used as cell carriers when the cellular component is seeded into the polymer meshes or decellularized valve scaffolds. In this review, we discuss current research strategies for HVTE with an emphasis on hydrogel applications. The physicochemical properties and fabrication methods of these hydrogels, as well as their mechanical properties and bioactivities are described. Performance of some hydrogels including in vitro evaluation using bioreactors and in vivo tests in different animal models are also discussed. For future HVTE, it will be compelling to examine how hydrogels can be constructed from composite materials to replicate mechanical properties and mimic biological functions of the native heart valve.Item Applications of photothermally responsive composite materials(2002) Sershen, Scott Robert; West, Jennifer L.A composite material consisting of a thermally-sensitive hydrogel and optically active nanoparticles would possess the temperature sensitive characteristics of the NA copolymer as well as the absorption spectrum of the nanoshells. The combination of these two properties in a single material opens up several very interesting opportunities in a number of applications. Composite hydrogels consisting of either Au-Au2S or SiO2-Au nanoshells or gold colloid embedded in a N-isopropylacrylamide-co-acrylamide copolymer collapse upon exposure to light that matches the peak extinction wavelength of the nanoparticles. These composite materials have successfully delivered a wide range of molecular weight compounds, from methylene blue (MW 345) to bovine serum albumin (MW 66,000), in a pulsatile fashion in vitro and in vivo. Furthermore, insulin that has been released from the composite hydrogels in this manner retains its activity. Due to the ability of the nanoshell-composite to provide on-demand release of a therapeutic agent, such a system may prove to be beneficial in the treatment of diseases that require a flexible therapeutic regimen, such as insulin dependent diabetes mellitus. The collapse of the composite hydrogels has also been investigated for use as valves or gates in microfluidic devices. Hydrogels containing different nanoparticles exhibit independent optical addressibility, and were polymerized in situ within existing microfluidic chambers. Composite materials of optically active nanoparticles and thermally sensitive hydrogels should prove useful in a wide range of applications where remote optical activation of a mechanical device, such as a gate or switch, is desired, in particular where large relative displacements of such a structure are desirable.Item Bioactive Poly(ethylene glycol)-based Hydrogels for Angiogenesis in Tissue Engineering(2011) Saik, Jennifer Elaine; West, Jennifer L.Because engineered tissue constructs are inherently limited by their lack of microvascularization, which is essential to provide oxygen for cell survival, this thesis presents rationally designed materials and cell culture techniques capable of supporting functional tubule formation and stabilization. Combining a synthetic scaffold material with cells and their cell-secreted signals instigated tubule formation throughout the scaffold. Poly(ethylene glycol) (PEG) based hydrogels, biocompatible polymers which resist protein adsorption and subsequent nonspecific cellular adhesion, were modified to induce desired cell characteristics. Human umbilical vein endothelial cells were used as a reproducible and readily available cell type. Several tubule-stabilization signals, including platelet derived growth factor-BB (PDGF-BB) and ephrinA1, were covalently immobilized via conjugation to PEG to enable prolonged bioactive signaling and controlled local delivery. All hydrogels were further tested in a mouse cornea micropocket angiogenesis assay, a naturally avascular tissue for easy imaging in a reproducible and quantifiable assay. Hydrogels containing soluble growth factors induced vessel formation in the hydrogel, and the resulting vessel morphology was modulated using different growth factor concentrations. Immobilized PDGF-BB led to tubule formation in two dimensions, three dimensions, and in the mouse cornea while immobilized ephrinA1 stimulated secretion of extracellular matrix proteins laminin and collagen IV to stabilize the newly formed tubules. Finally, a co-culture of endothelial and pericyte cells encapsulated into hydrogels formed tubules that anastomosed to the host vasculature and contained red blood cells. PEG-based hydrogels represent a promising technique to induce microvascular formation in engineered constructs, leading to stable and functional vessel formation using covalently immobilized growth factors and encapsulated cells. These materials can be used for replacement of damaged or diseased tissues as the current supply of cadaveric donations cannot meet the demand of tissues for the 110,000 people awaiting an organ in the US.Item Bioactive Poly(ethylene glycol)-based Hydrogels for Characterization of Matrix Influences on a Lung Cancer Metastasis Model(2013-09-16) Gill, Bj; West, Jennifer L.; Jacot, Jeffrey G.; Farach-Carson, CindyPathological changes to tumor extracellular matrix (ECM) composition, mechanics, and architecture promote cancer progression and metastasis. Exploration of tumor-ECM interactions using in vitro matrix-mimetic culture systems has largely been restricted to naturally-derived matrix materials that permit limited experimental control. Such study of a novel lung adenocarcinoma model in Matrigel™ (MG) has suggested key matrix cues that mediate epithelial-mesenchymal transition (EMT) and metastasis. In this thesis work, synthetic hydrogel scaffolds based on poly(ethylene glycol) (PEG) featuring high experimental control and modular bioactivity were used to study matrix influences on the EMT-prone model line 344SQ. Encapsulation of 344SQ cells in PEG hydrogels modified for cell adhesivity and cell-mediated enzymatic degradability induced formation of lumenized, polarized spheres mimicking the epithelial phenotype observed in three-dimensional MG. Tuning matrix stiffness, adhesive ligand concentration, and ligand spatial presentation altered epithelial morphogenesis. Exploration of the EMT phenotype of PEG-encapsulated 344SQ cells revealed TGFβ-initiated changes in morphology, polarity, expression levels of EMT marker genes and their epigenetic controller, and the organization of cell-secreted ECM. Notably, a potent role for adhesive ligand was illuminated as matrices with low PEG-RGDS concentration even in the absence of TGFβ induced formation of spheres with a post-EMT phenotype by several of these measures. A matrix-invasive phenotype was also revealed by altering matrix structural parameters and tuned with incorporation of an alternative protease-cleavable sequence. Finally, the influence of cell-cell contacts was explored by covalent incorporation of cadherin proteins into the matrix. Matrix-tethered E- and -N-cadherin affected 344SQ sphere development in otherwise non-cell-adhesive matrices and modulated polarity and the degree of TGFβ response. Further, in 344SQ with a knockdown of the essential polarity-determining protein Scribble, matrix-tethered cadherin influenced the formation of a phenotype with partially normalized epithelial polarity with corresponding differences in membrane localization of cell-expressed E-cadherin. Overall, this thesis demonstrates the utility of the more experimentally controllable PEG system in studying ECM influences on cancer progression with findings providing greater insight into stromal biomechanical, biochemical, and cell-cell factors that mediate lung adenocarcinoma epithelial morphogenesis and EMT. These contributions help advance the state of the field towards a goal of developing new metastasis-targeting cancer therapeutics.Item Bioactive scaffolds for optimizing engineered tissue formation(2005) DeLong, Solitaire A.; West, Jennifer L.Tissue-engineering scaffolds were designed to mimic several features of the extracellular matrix using a combination of the synthetic polymer, PEG diacrylate, and bioactive factors. Scaffolds were formed by exposing aqueous solutions of PEG diacrylate and bioactive factors modified with PEG monoacrylate to ultraviolet or visible light in the presence of a suitable photoinitiator. Light exposure generated free radicals that targeted acrylate groups in the monomer and in PEG conjugated bioactive factors resulting in crosslinked hydrogel scaffolds with bioactive factors covalently incorporated. This study extended the capability for directing cell behavior using PEG-based hydrogels to include control over the spatial distribution of bioactive factors and the presentation of the growth factor, bFGF. Additionally, PEG hydrogels were modified with a degradable peptide sequence to enable cells to remodel the scaffold by secreting matrix metalloproteinases (MMPs). A continuous linear gradient was formed by simultaneously using a gradient maker to combine hydrogel precursor solutions with photopolymerization, which locks the gradient in place. Coomassie blue staining confirmed the formation of protein gradients. Fibroblast cells responded to covalently immobilized gradients of the adhesive peptide, RGD, by changing their morphology to align in the direction of increasing RGD concentration and by migrating differentially on RGD-gradient hydrogels compared to control hydrogels. Next, bFGF was covalently immobilized to hydrogels with retention of its mitogenic and chemotactic effects on smooth muscle cells (SMCs). A covalently immobilized bFGF gradient was also formed using the gradient maker and shown to increase linearly along the hydrogel's length by silver staining. SMCs responded to these bFGF-gradient hydrogels by aligning in the direction of increasing bFGF concentration and by migrating differentially, up the concentration gradient, compared to migration on control hydrogels. Finally, the MMP-sensitive peptide sequence, GPQGILGQ, was inserted into the main polymer chain's backbone to allow targeted degradation by cell-secreted proteases. Cells were observed to change their morphology and migrate when seeded within these degradable hydrogel scaffolds, but not in scaffolds lacking this degradable peptide sequence. This hydrogel system is expected to be useful for studying tissue formation leading eventually to an improved understanding of the factors needed to form engineered tissues.Item Biocompatible copolymers for localized cardiovascular drug delivery and tissue engineering(2005) Taite, Lakeshia J.; West, Jennifer L.The integration of bioactive and biomimetic signals into materials for drug delivery and tissue engineering serves to improve cellular responses and therefore healing by more closely resembling the natural cellular microenvironment. The materials developed in this thesis show promise in delivering therapeutic doses of nitric oxide (NO) to physiological systems and provide novel surfaces for the study of cell adhesion and spatial organization. NO has several biological functions that make it an ideal candidate therapeutic agent for the prevention of the occlusive scarring of blood vessels following treatment of coronary artery disease through procedures such as balloon angioplasty and bypass grafting. The present work incorporates NO donors into polymeric biomaterials, resulting in copolymers that release NO over controllable time frames depending on material design. These NO-generating polymers have proven effective in significantly reducing platelet adhesion and smooth muscle cell proliferation in vitro. Endothelial cells exposed to these materials displayed enhanced proliferation, which is essential in restoring vessel function. Local, sustained release of NO from perivascularly-applied hydrogels reduced unwanted neointimal formation by approximately 90% in an experimental balloon angioplasty model. Novel NO releasing dendrimers have been synthesized to establish the potential for injectible NO therapy and can be targeted to sites of active vascular disease. NO-releasing polyurethane has been synthesized as a candidate material for vascular grafts. The superior mechanical properties of polyurethane combined with the inhibition of platelet adhesion by NO promise increased patency in small diameter vascular prostheses. Bioactive poly(ethylene glycol) (PEG) hydrogels have also been synthesized with covalently bound cell adhesion moieties to elucidate the mechanisms of immune cell adhesion to the vascular wall under shear. Leukocytes perfused over the surfaces of these hydrogels in a parallel plate flow chamber display rolling and adhesion properties like those seen on vascular endothelium in vivo. This work also presents a system of patterning bioactive regions onto hydrogels using transparency masks. This system allows the formation of complex patterns of cell-adhesive regions that closely mimic in vivo cellular arrangement. The intrinsic biocompatibility of PEG and the decreased thrombogenicity that NO affords make these materials ideal for incorporation into blood contacting devices.Item Biomimetic PEG Hydrogels for ex vivo Hematopoietic Stem Cell Expansion(2012) Rowland, Maude Lucille; West, Jennifer L.Hematopoietic stem cells (HSCs) are commonly used in the treatment of blood cancers, like leukemia, and other cancers where radiation or chemotherapy damages the native HSC population. The development of a novel system to study and maintain HSCs ex vivo would give researchers and clinicians the ability to investigate the basic biological processes of HSCs, improve current treatment regimens, and explore their use in new therapies. The work in this thesis focuses on the development of a synthetic PEG hydrogel scaffold that accurately mimics aspects of the HSC microenvironment and can expand clinically relevant HSC populations. PEG hydrogel well surfaces were covalently functionalized with bioactive factors known to be critical in controlling HSC fate in vivo. In initial studies, 32D cells, a myeloid progenitor, were cultured in the wells for 6 days. On surfaces with the adhesive RGDS peptide sequence, 32D cell adhesion increased concurrently with RGDS surface concentrations. With the immobilization of two niche cytokines, SCF and SDF1α, onto hydrogel surfaces, 32D cells demonstrated significant increases in adhesion and spreading. These results confirmed that hematopoietic cell behavior could be controlled through the design of the bioactive PEG scaffold. In studies with a primary hematopoietic cell population (c-kit + , lin - ), the effects of bioactive molecules on cell expansion and differentiation were investigated after 2 weeks in culture. The adhesive peptides sequences, RGDS and CS1, and four niche proteins, SCF, SDF1α, JAG1, and IFNγ, were covalently tethered to hydrogel well surfaces. Primary cells proliferated significantly on gels containing SCF and IFNγ though only SCF was capable of preventing HSC differentiation. Cells cultured on surfaces functionalized with JAG1 and SDF1α did not proliferate extensively, but they were able to maintain primitive HSC populations. Primary c-kit + cells were also encapsulated within biodegradable PEG hydrogels and cultured for 2-5 weeks. Cells remained viable for 5 weeks in culture, and preliminary results indicated minimal cell differentiation. In this work, biomimetic PEG hydrogels were successfully employed to expand HSC populations in both two and three dimensions. The ability to generate large populations of primitive HSCs ex vivo has broad clinical and research implications.Item Cancer-Associated Fibroblasts Induce a Collagen Cross-link Switch in Tumor Stroma(American Association for Cancer Research, 2016) Pankova, Daniela; Chen, Yulong; Terajima, Masahiko; Schliekelman, Mark J.; Baird, Brandi N.; Fahrenholtz, Monica; Sun, Li; Gill, Bartley J.; Vadakkan, Tegy J.; Kim, Min P.; Ahn, Young-Ho; Roybal, Jonathon D.; Liu, Xin; Cuentas, Edwin Roger Parra; Rodriguez, Jaime; Wistuba, Ignacio I.; Creighton, Chad J.; Gibbons, Don L.; Hicks, John M.; Dickinson, Mary E.; West, Jennifer L.; Grande-Allen, K. Jane; Hanash, Samir M.; Yamauchi, Mitsuo; Kurie, Jonathan M.Intratumoral collagen cross-links heighten stromal stiffness and stimulate tumor cell invasion, but it is unclear how collagen cross-linking is regulated in epithelial tumors. To address this question, we used KrasLA1 mice, which develop lung adenocarcinomas from somatic activation of a KrasG12D allele. The lung tumors in KrasLA1 mice were highly fibrotic and contained cancer-associated fibroblasts (CAF) that produced collagen and generated stiffness in collagen gels. In xenograft tumors generated by injection of wild-type mice with lung adenocarcinoma cells alone or in combination with CAFs, the total concentration of collagen cross-links was the same in tumors generated with or without CAFs, but coinjected tumors had higher hydroxylysine aldehyde–derived collagen cross-links (HLCC) and lower lysine-aldehyde–derived collagen cross-links (LCCs). Therefore, we postulated that an LCC-to-HLCC switch induced by CAFs promotes the migratory and invasive properties of lung adenocarcinoma cells. To test this hypothesis, we created coculture models in which CAFs are positioned interstitially or peripherally in tumor cell aggregates, mimicking distinct spatial orientations of CAFs in human lung cancer. In both contexts, CAFs enhanced the invasive properties of tumor cells in three-dimensional (3D) collagen gels. Tumor cell aggregates that attached to CAF networks on a Matrigel surface dissociated and migrated on the networks. Lysyl hydroxylase 2 (PLOD2/LH2), which drives HLCC formation, was expressed in CAFs, and LH2 depletion abrogated the ability of CAFs to promote tumor cell invasion and migration.Item Cell migration through biomimetic hydrogel scaffolds(2003) Gobin, Andrea Samantha; West, Jennifer L.Cell migration is an essential step during processes such as embryonic development, wound healing, angiogenesis, and cancer metastasis. Migration is a complex integration of cellular adhesion to its substratum, reorganization of the cytoskeleton, proteolysis and remodeling of surrounding extracellular matrix (ECM), and activation and regulation of chemical signaling by growth factors and other mitogenic cues. These cooperative mechanisms enable a cell to move to its target to perform its function, whether to repair injured tissue, fight infections, or build new blood vessels. The main objective of this research is to study mechanisms of cell migration within a biomimetic hydrogel system. Because of the complexity of the ECM, studying cell migration in ECM derivatives or even single components of the ECM can make it difficult to decipher the importance of each factor involved. In addition, the ECM imposes a spatial barrier to cells. For migration, the cells must not only interact with matrix adhesive ligands for force generation, but also develop strategies to overcome biomechanical resistance imposed by the matrix. Thus, the biomimetic hydrogel system developed can provide the mechanical support, adhesion ligands, degradation sequences, and other signals, so that a cell can migrate. This system will provide tight control over many experimental parameters and minimize nonspecific cell-material interactions. Hence the aim of the hydrogel system is to stimulate an active interaction between the synthetic polymer and the biological environment. The biomimetic hydrogels are photopolymerizable hydrogels based on acrylated derivates of polyethylene glycol. This material contains proteolytically degradable peptide sequences, targeted for specific enzymes involved in cell migration, in the polymer backbone. Cell adhesion peptides are also grafted into the hydrogels during photopolymerization to promote interaction with specific cell surface receptors. Other bioactive signals, such as growth factors, can also be grafted into the network during photopolymerization. Thus a single hydrogel material can contain several different proteolytically sensitive segments, many cell adhesion ligands and various growth factors, allowing for one to mimic many properties of the ECM. This hydrogel system is used to assess cell migration mechanisms by controlling the identity and availability of bioactive signals presented to the cells and studying their affects.Item Development and characterization of a poly(ethylene glycol) hydrogel scaffold system for adipose tissue engineering(2005) Patel, Parul Natvar; West, Jennifer L.As the number of soft tissue correctional procedures increases yearly and the current methods being inadequate, alternative methods of rectifying defects has become a focus for tissue engineers. Our long-term goal is to engineer adipose tissue to correct soft tissue defects resulting from aging, trauma, congenital abnormalities, and tumor resection (including lumpectomies and mastectomies). The immediate objective is to develop and characterize a photopolymerizable synthetic hydrogel system to act as a bioactive scaffold that promotes preadipocyte (precursor cell to adipocyte) adhesion and proliferation for adipose tissue engineering. We have shown that poly(ethylene glycol) diacrylate derivatized with enzyme-sensitive degradation sites and cell-adhesion ligands promote preadipocyte adhesion, viability, proliferation and differentiation, and demonstrates mechanical properties able to withstand physiological strains and frequencies. The following specific aims address the scope of this thesis: (1) Determine the required polymer chemistry and fabricate a series of hydrogels with and without degradation peptides and cellular binding sites. (2) Quantitatively compare the physical properties (e.g., viscosity, elastic modulus, viscous modulus, complex modulus, and % recovery) of human adipose tissue with hydrogels fabricated. (3) Demonstrate that hydrogels containing adhesion ligands and degradation sites optimally promote preadipocyte adhesion, viability, and proliferation compared to other hydrogel configurations. (4) Demonstrate that preadipocyte-loaded hydrogels promote adipogenesis within a low-shear bioreactor system.Item Development of a Biomimetic Hydrogel Scaffold as an Artificial Niche to Investigate and Direct Neural Stem Cell Behavior(2012) Franco, Christy Lynn; West, Jennifer L.The mature central nervous system has a very limited capacity for self-renewal and repair following injury. Neural stem cells (NSCs), however, provide a promising new therapeutic option and can be readily expanded in vitro . Towards the development of an effective therapy, greater understanding and control is needed over the mechanisms regulating the differentiation of these cells into function-restoring neurons. In vivo, the neural stem cell niche plays a critical role in directing stem cell self-renewal and differentiation. By understanding and harnessing the power of this niche, a tissue engineered system with encapsulated neural stem cells could be designed to encourage neuronal differentiation and ultimately regeneration of damaged neural tissue. Poly(ethylene glycol)-based hydrogels were used here as a platform for isolating and investigating the response of neural stem cells to various matrix, soluble, and cellular components of the niche. When covalently modified with a cyclic RGD peptide, the synthetic scaffold was demonstrated to support attachment and proliferation of a human NSC line under conditions permissive to cell growth. Under differentiating conditions, the scaffold maintained appropriate lineage potential of the cells by permitting the development of both neuronal and glial populations. Expansion and differentiation of NSCs was also observed in a more biomimetic, three dimensional environment following encapsulation within a degradable hydrogel material. To simulate the soluble signals in the niche, fibroblast growth factor and nerve growth factor were tethered to the hydrogel and shown to direct NSC proliferation and neuronal differentiation respectively. Finally, as an example of the cell-cell interactions in the niche, the pro-angiogenic capacity of encapsulated neural stem cells was evaluated both in vitro and in vivo. Ideally, the optimal scaffold design will be applied to guide NSCs in a therapeutic application. Toward this goal, a novel method was developed for encapsulation of the cells within injectable hydrogel microspheres. This technique was optimized for high cell viability and microsphere yield and was demonstrated with successful microencapsulation and delivery of neural stem cells in rodent model of ischemic stroke.Item Development of bioactive polyurethaneureas to support endothelialization(2004) Jun, Ho-Wook; West, Jennifer L.Vascular diseases are responsible for the majority of deaths in the United States. Synthetic materials have been developed for blood vessel substitutes but not suitable for small diameter vascular applications such as coronary artery bypass grafting (CABG). Polyurethaneureas (PUU) have been widely used for biomedical applications due to their excellent mechanical properties and relatively good biocompatibility. However, like other synthetic materials, they are generally thrombogenic on exposure to blood. Endothelialization of synthetic grafts is a good strategy to improve graft patency. However, the graft patency is dependent on retention of endothelial cells on exposure to physiological shear stress. In this study, we developed bioactive polyurethaneureas to support endothelialization. First, we have demonstrated that endothelial cell behaviors could be altered by the surface YIGSR peptide concentrations. Bioactive polyurethanureas (PUUYIGSR) have been developed by incorporating YIGSR peptide sequences into polymer main chain, and improved endothelialization has been observed on the surface. In addition, PEG- and YIGSR-modified polyurethaneureas (PUUYIGSR-PEG) have been developed, and enhanced endothelialization and improved thromboresistance have been obtained simultaneously. Our bulk modification strategy allowed us to fabricate microporous scaffolds without interfering bioactivity of incorporated peptide sequences. Microporous scaffolds have been also used as a carrier of vascular endothelial growth factor (VEGF). The synergistic effects of peptide sequences, microporous structure, and incorporated VEGF on endothelialization have been observed. Additionally, nitric oxide (NO) releasing polyurethanes (PUBD-NO) have been developed by incorporating NO donor into the polymer main chain. NO was successfully released from the PUBD-NO in controlled manner and reduced platelet adhesion and smooth muscle cell proliferation but improved endothelialization proliferation.Item Development of hydrogel scaffolds and a bioreactor for vascular tissue engineering(2004) Schmedlen, Rachael Hope; West, Jennifer L.This dissertation determines the feasibility of photopolymerizable hydrogels as novel tissue engineering scaffolds and constructs a pulsatile flow bioreactor for the development of tissue engineered vascular grafts (TEVGs). The large number of small diameter bypass surgeries performed each year coupled with the shortage of suitable, patent vascular grafts has spurred the development of tissue engineered vascular substitutes. This investigation characterizes the mechanical properties of polyvinyl alcohol (PVA) and polyethylene glycol (PEG) hydrogels and evaluates their ability to support cell viability, proliferation, and extracellular matrix protein production for use as a tissue engineering scaffold. The elasticity and tensile strength of PVA and PEG hydrogels may be tailored by changing the polymer concentration, number of crosslinkable groups per PVA chain, PEG molecular weight, or using blends of high and low PEG molecular weights to transmit cyclic strain and still maintain structural integrity in a pulsatile flow bioreactor. At least 75% of cells cultured over two weeks inside PVA hydrogels and for four weeks in PEG hydrogels remained viable, with no differences in viability across the thickness of the hydrogel. Once seeded inside hydrogels, cells continue to function; following two weeks in culture, cells produced hydroxyproline in both PVA and PEG hydrogels. After determining that these hydrogels were suitable materials for scaffolds, a pulsatile flow bioreactor, mimicking transmural strain encountered in vivo, was constructed to culture tubular hydrogel-cell constructs. PEG hydrogels placed in the bioreactor exhibited strains at 2 Hz and between 5.9--15.9%, depending on the material elasticity, with pressures around 70/20 mmHg. Furthermore, smooth muscle cells seeded in PEG hydrogels and cultured in the bioreactor for one week showed similar DNA content to static gels, indicating that the bioreactor does not hinder cell viability. These results suggest that PVA and PEG hydrogels are appropriate materials for TEVG scaffolds and that the bioreactor generates conditions suitable for tissue formation and organization. In the future, this system will require optimization to incorporate the right combination of bioactive molecules, cell types, and bioreactor parameters to achieve a TEVG with composition, organization, and mechanical properties resembling those of native blood vessel.Item Diagnostic and therapeutic applications of metal nanoshells(2004) Hirsch, Leon Robert; West, Jennifer L.Metal nanoshells are a new class of optically tunable core/shell nanoparticles which are finding numerous applications in biomedicine due to their biocompatibility, chemical functionability, and optical tunability in the near infrared (a region of light where tissue is optically transmissive). The following work explores the design of near infrared resonant nanoshells for two new diagnostic and therapeutic biomedical applications. For diagnostic purposes, near infrared resonant nanoshells were found to be optically detectable in whole blood and proved capable of detecting sub-nanogram levels of analyte in whole blood in under 30 minutes. For therapeutic purposes, near infrared absorbing nanoshells were heated in tumors using higher laser powers. Excitation of near infrared resonant nanoshells promoted photothermal destruction of cancerous growths in vitro as well as in vivo. In vitro, nanoshell treated cells were photothermally ablated upon near infrared excitation with no detectable damage to cells receiving lasers only. Similar results were found in whole tissue. Using a live mouse model, a complete therapy system was demonstrated where systemically delivered nanoshells were found to preferentially accumulate into tumors. Near infrared excitation of these tumors provided complete recovery in 55% of the cases, while controls displayed a mean survival time of only 15--20 days.Item Endothelial cell and mural progenitor cell co-culture to promote angiogenesis in tissue engineered constructs(2008) Smith, April Ann; West, Jennifer L.The need for tissue engineered organs is great since only 15% of those on the transplant waiting list received organs in 2006. The ability to vascularize a tissue engineered construct is one of the major hurdles for tissue engineering because it can take 2 weeks for an implanted construct to become vascularized. To produce prevascularized tissues, the cellular interactions that occur during vascular formation need to be examined. In this work, human umbilical cord endothelial cells (HUVECs) and mural progenitor cells (10T1/2 cells) are cultured together for 6 days. Western blotting and immunofluorescence are performed to assess SM-specific marker expression. αSM-actin and calponin expression was observed in co-cultures seeded at 300,000 cells/well while caldesmon and SMMHC expression was observed in co-cultures seeded at 60,000 cells/well. HUVECs and 10T1/2 cells, seeded at 150,000 cells/gel, encapsulated in degradable PEG hydrogels expressed high levels of αSM-actin, calponin, and caldesmon.Item Engineering Vascularized Hepatic Tissue in Bioactive Poly(ethylene glycol)-based Hydrogels(2013-09-20) Higbee, Steven; West, Jennifer L.; Grande-Allen, K. Jane; Harrington, Daniel ATransport of oxygen and nutrients to cells within engineered tissues remains one of the most significant challenges in tissue engineering. This challenge has led researchers to seek new strategies to engineer vascularized tissues. Co-cultures of endothelial cells and pericytes can be used to form microvascular networks in bioactive scaffolds, and these networks have been shown to be perfusable and capable of anastomosis with host vasculature. These co-cultures are prevalent in the literature; however, little investigation has been done into the combination of cell-formed microvasculature with parenchymal cells. In our work, we used a co-culture approach to grow microvascular networks in a biomimetic poly(ethylene glycol) (PEG) hydrogel, in the presence of functional hepatocytes. Through the simultaneous encapsulation of three cell types – endothelial cells, pericyte precursors, and hepatocytes – in our biomimetic PEG system, we successfully engineered vascularized hepatic tissue. These vascularized tissues exhibited two distinct benefits when compared to non-vascularized controls. First, incorporation of the vasculogenic cells led to significant improvements in hallmark hepatocyte functions. Hepatocytes encapsulated alongside the vasculogenic cells demonstrated improved albumin synthesis and cytochrome P450 enzyme activity. These improvements result from physical and chemical cues from non-parenchymal cells, which regulate hepatocyte function in vivo and in vitro. Second, the cell-formed microvasculature led to improved mass transport within the hydrogel. In a microfluidic culture system designed to investigate the functionality of the cell-formed microvasculature, we demonstrated that the cell-formed networks are capable of anastomosis with prefabricated channels within the device. Further, transport through these networks significantly increased the distance from a media channel over which hepatocyte viability was supported. Our results suggest that a combination of prefabricated conduits and cell-formed microvasculature may be influential in the scaling up of engineered tissues.
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