Browsing by Subject "Tissue engineering"
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Item Chemically modified hyaluronic acid biomaterials for cell culture and tissue engineering(2016-12) Joaquin, Alysa Marie; Zoldan, Janeta; Suggs, LauraThe fate and behavior of cells is strongly dependent on the cell microenvironment, and this knowledge has been applied to the design of biomaterials to influence cell growth, morphology, and differentiation. However, a dearth of research specifically focused on the effects of material hydrophobicity on cell behavior indicates that this easily controllable material property is being overlooked. The field of tissue engineering has a need for cost-efficient, scalable methods to both increase stem cell stocks and control cell behavior, and hydrophobic biomaterials may be a robust solution to these needs. To evaluate the utility of hydrophobicity in controlling cell behavior, hyaluronic acid was modified with amines representing a wide range of hydrophobicity, resulting in twelve new materials. Both mouse embryonic stem cells (mESCs) and fibroblasts were cultured on these materials to evaluate the differences in pluripotent and differentiated cell behavior in response to hydrophobic materials. The viability of cells cultured on these materials was tested as an indicator of biocompatibility, and cell morphology and spreading area was evaluated to relate cell behavior to biomaterial hydrophobicity. Eight of the twelve materials proved to be biocompatible, and hydrophobic materials inhibited cell spreading; fibroblasts cultured on modified HA hydrogels grew in populations of both compact cell clusters and elongated, multi-polar morphologies, and cell spreading area increased as hydrophobicity decreased. Similarly, mESC spreading area increased with decreasing hydrophobicity; mESCs grown on the least hydrophobic HA hydrogels also multi-polar spreading, while mESCs cultured on the more hydrophobic materials grew exclusively in compact cell clusters. As the morphology of cells is often indicative of cell fate, and as hydrophobic materials tended to inhibit cell spreading, we expected that mESCs cultured on hydrophobic materials would maintain pluripotency. To this end, hybrid scaffolds composed of modified HA and gelatin were developed as a platform for stem cell pluripotency maintenance. The mESCs seeded into these scaffolds had a higher expression of the pluripotency marker SSEA-1 compared to control mESCs grown in complete medium after 24 hours, indicating that hydrophobicity is an important material property to consider in stem cell culture.Item Clinically relevant adipose tissue engineering strategies and market potential(2010-12) Finkbiner, Jenny Jean; Ambler, Tony; Vail, Neal K.This thesis presents a foundation for developing a business case for companies interested in the reconstructive and cosmetic procedure markets. The focus is on reviewing adipose tissue engineering research and proposing technology opportunities that could be applied to challenging soft tissue reconstruction cases and adjacently applied to cosmetic applications. To establish the foundation for this type of program, this thesis includes an evaluation of the reconstructive and cosmetic procedure markets, current practices in these markets and their constraints, as well as a literature review of research in adipose tissue engineering and its potential clinical applications. Additionally it captures the competitive landscape of major players in the reconstructive market as well as up-and-coming players in the adipose tissue engineering field. Technology development opportunities with associated customer and business value are discussed with a recommendation for the development of a detailed business case to evaluate specific product development opportunities in these markets.Item Combined ultrasound, photoacoustic and elasticity microscope for tissue engineers(2006-08) Mallidi, Srivalleesha; Emelianov, Stanislav Y.Tissue engineering is an interdisciplinary field that combines various aspects of engineering and life sciences and aims to develop biological substitutes to restore, repair or maintain tissue function. Currently, the ability to have quantitative functional assays of engineered tissues is limited to existing invasive methods like biopsy. Hence, an imaging tool for non-invasive and simultaneous evaluation of the anatomical and functional properties of the engineered tissue is needed. In this thesis, we present an advanced in-vivo imaging technology - ultrasound biomicroscopy combined with complementary photoacoustic and elasticity imaging techniques, capable of accurate visualization of both structural and functional changes in engineered tissues, sequential monitoring of tissue adaptation and/or regeneration, and possible assistance of drug delivery and treatment planning. The combined imaging at microscopic resolution was evaluated on tissue mimicking phantoms imaged with 25 MHz single element focused transducer. The results of our study demonstrate that the ultrasonic, photoacoustic and elasticity images synergistically complement each other in detecting features otherwise imperceptible using the individual techniques. Finally, we illustrate the feasibility of the combined ultrasound, photoacoustic and elasticity imaging techniques in accurately assessing the morphological and functional changes occurring in engineered tissue.Item Creation of an optimized acellular scaffold for improved vascular engineering(2013-05) Nagao, Ryan Joseph; Suggs, Laura J.; Schmidt, Christine E.Engineering a complex tissue that exceeds 100-200 [mu]m requires a vascular connection. Methods to enhance vascularization include the delivery of angiogenic factors, and the use of scaffolds that encourage vascular ingrowth. However, these techniques rely on the host to vascularize the construct upon implantation, which is often too slow to provide nutrients to the entire construct. Hence, recent research has focused on creating de novo vascular networks prior to implantation. Such technologies would enable faster anastomosis with the host vascular system, as well as fully perfused constructs that can increase cell viability. Many techniques have been investigated to create de novo vascular networks with varying levels of success. Our approach was to utilize native vascular extracellular matrix (ECM) obtained from decellularizing highly vascularized tissue as a substrate for re-endothelialization and thus to create a three-dimensional vascular bed for ultimate use with various implant and tissue engineering applications. We have demonstrated a method of chemical decellularization that effectively removes cellular material while leaving behind an organized patent vascular network down to the capillary scale. Standard histological methods, DNA quantification, as well as vascular corrosion casting demonstrated this efficacy. Subsequent subcutaneous implantation then explantation of the scaffold at 7 and 28 days was used to assess the immunogenicity of the graft by analyzing the presence of immune cells. This scaffold was then re-endothelialized with human dermal microvascular endothelial cells (HDMECs) and conditioned with peristaltic flow for 60 hours to help improve vascular patency. Cellular distribution was determined qualitatively by first incubating the HDMECs with gold nanotracers, then imaging their presence upon implantation through ultrasound-guided photoacoustic (US/PA) imaging. Following the culture process, the scaffolds were analyzed for vascular patency through vascular corrosion casting, and cellular phenotype through histological methods---demonstrating a decrease in vascular damage. The re-endothelialized scaffolds were then assessed for functional vascular performance by perfusing whole blood through them. Results demonstrated better blood clearance in re-endothelialized scaffolds compared to scaffolds without cells. These results point to the ability of the optimized acellular (OA) scaffold to be used in future experiments focused on vascular and tissue engineering.Item Development of a bioreactor imaging system for characterizing embryonic stem cell-derived cardiomyocytes(2010-05) Abilez, Oscar John; Suggs, Laura J.; Roy, KrishnenduCardiovascular disease (CVD) affects more than 70 million Americans and is the number one cause of mortality in the United States. Because the regenerative capacity of adult tissues such as the heart is limited, human embryonic stem cells (hESC) have emerged as a source for potential cardiac therapies. However, despite the use of a variety of biochemical differentiation protocols, current yields of hESC-derived cardiomyocytes (CM) have been low. In the case of hESC-CM, which are inherently electromechanically active, additional forms of inducing a mature cardiac fate have not been fully explored. In order to non-invasively visualize and quantify biochemical, electrical, and mechanical stimulation on hESC-CM differentiation in future studies, a bioreactor imaging system has been developed and is described in this report.Item Growth factor presentation from PEGylated fibrin gels to enhance vasculogenesis(2010-05) Drinnan, Charles Thomas; Suggs, Laura J.; Farrar, Roger; Frey, Wolfgang; Roy, Krishnendu; Schmidt, ChristineI developed a system to release multiple growth factors from PEGylated fibrin gels with varying profiles to induce vasculogenesis from embedded human MSCs. Zero-order release can be obtained by conjugating a growth factor with a homobifunctional, amine-reactive, PEG derivative. Growth factors can be entrapped during thrombin-mediated crosslinking and released rapidly. Growth factors with physical affinity for fibrinogen or fibrin can be sequestered within the matrix and released via degradation and/or disassociation. PDGF-BB was loaded via entrapment while TGF-β1 was sequestered through a combination of physical affinity and conjugation. The affinity of TGF-β1 and fibrinogen had never been previously examined or quantified. I aimed to determine the Ka and Kd between TGF-β1 and fibrinogen through a variety of assays. Binding ELISAs were developed for TGF-β1 and fibronectin, a protein associated with fibrin gels, and TGF-β1 and fibrinogen. However, background was high due to insufficient blocking agents. Other assays explored included western blots, surface plasmon resonance, and radiolabeled TGF-β1 with limited success. The affect of TGF-β1 on human MSC differentiation towards vascular cell phenotypes was examined both in 2D and fibrin gels embedded with MSCs. With exposure to TGF-β1, MSC proliferation was significantly inhibited in both 2D and within fibrin gels indicating that loaded TGF-β1 maintained bioactivity for at least 7 days. Gene expression of MSCs exposed to TGF-β1 demonstrated inhibited endothelial cell differentiation and stimulated smooth muscle cell differentiation. However, confocal and light microscopy indicated that endothelial cell differentiation is maintained with TGF-β1 loaded PEGylated fibrin gels. The system developed is highly modular and can be applied to other tissue engineering systems. Furthermore, other growth factors could be incorporated to promote vascular cell differentiation.Item Hyaluronic acid hydrogel biomaterials for soft tissue engineering applications(2003) Baier, Jennie Melinda; Schmidt, Christine E.; Georgiou, GeorgeThe primary focus of this work was to develop new naturally derived hydrogel scaffolds for soft tissue engineering applications such as peripheral nerve repair. Ideal implants for soft tissues are degradable, porous, highly permeable, able to maintain a desired shape, and able to specifically modulate biological responses. Naturally derived materials are particularly suitable for these applications because they are degradable and intrinsically bioactive. As presented in this dissertation, we have made significant steps towards meeting these aims by creating new hydrogel scaffolds from crosslinked hyaluronic acid (HA). As a biomaterial candidate, HA presents a unique combination of advantages. Specifically, HA is a naturally derived, enzymatically degradable, non-immunogenic, non-adhesive, bioactive glycosaminoglycan that has been associated with several cellular processes, including angiogenesis, extracellular matrix homeostasis, and the regulation of inflammation. In our first studies, we developed a novel hydrogel tissue engineering scaffold from photocrosslinkable glycidyl methacrylate-modified HA (GMHA). The GMHA hydrogels were found to be enzymatically degradable, biocompatible, and the degradation products of GMHA retained a similar level of bioactivity as unmodified HA fragments. Further development of these GMHA hydrogels focused on methods to attach peptides (e.g., the fibronectin-derived cell adhesion sequence RGD or arg-gly-asp). Such sequences could provide a versatile method to control several adhesion-related cellular processes, including migration and proliferation. Finally, we explored GMHA-based hydrogels for controlled protein release. Localized delivery of bioactive proteins such as growth factors provides an additional level of versatility for modulating specific cellular behaviors involved in tissue repair. We believe that these novel GMHA-based biomaterials offer unique advantages over synthetic and non-degradable scaffolding biomaterials and provide a solid foundation for further modification and use in a variety of soft tissue engineering applications.Item Hybrid Polyethylene Glycol Hydrogels for Tissue Engineering Applications(2012-07-11) Munoz Pinto, Dany 1981-Currently, organ transplant procedures are insufficient to address the needs of the number of patients that suffer of organ failure related disease. In the United States alone, only around 19% of the patients are able to get an organ transplant surgery and 25% die while waiting for a suitable donor. Tissue engineering (TE) has emerged as an alternative to organ transplant; thus, the aim of the present study was to validate a poly(ethylene glycol) diacrylate (PEG-DA) hydrogel system as a model for material scaffolding in TE applications. This work explores the influence of scaffold material properties on cell behavior. Specifically, scaffold modulus, mesh size, and biochemical stimuli were characterized and their influence on cell response was analyzed at the biochemical, histological and microenvironmental levels. Three different TE targets were evaluated: vocal fold restoration, vascular grafts and osteochondral applications. Vocal fold fibroblast (VFF) phenotype and extracellular matrix (ECM) production were impacted by initial scaffold mesh size and modulus. The results showed increasing levels of SM-?-actin and collagen production with decreasing initial mesh size/increasing initial modulus, which indicated that VFFs were induced to take an undesirable myofibroblast-like phenotype. In addition, it was possible to preserve VFF phenotype in long-term cultured hydrogels containing high molecular weight hyaluronan (HAHMW). On the other hand, regarding vascular graft applications, smooth muscle cell (SMC) phenotype was enhanced by increasing scaffold mesh size and modulus. Finally, the effect of scaffold inorganic content (siloxane) on rat osteoblasts and mouse mesenchymal stem cells was evaluated. Interestingly, the impact of inorganic content on cell differentiation seemed to be highly dependent on the initial cell state. Specifically, mature osteoblasts underwent transdifferentiation into chondrocyte-like cells with increasing inorganic content. However, Mesenchymal stem cells appeared to be preferentially driven toward osteoblast-like cells with an associated increase in osteocalcin and collagen type I production.Item Manipulation of the embryoid body microenvironment to increase cardiomyogenesis(2014-08) Geuss, Laura Roslye; Suggs, Laura J.; Maynard, Jennifer; Stachowiak, Jeanne; Vokes, Steven; Zoldan, JanetaMyocardial Infarction (MI) is one of the most prevalent and deadliest diseases in the United States. Since the host myocardium becomes irreversibly damaged following MI, current research is focused on identification of novel, less invasive, and more effective treatment options for patients. Cellular cardiomyopathy, in which viable cells are transplanted into the necrotic tissue, has the potential to regenerate and integrate with the host myocardium. Stem cells, specifically pluripotent stem cells such as embryonic stem cells (ESCs) and induced pluripotent stem cells (iPSC), are ideal candidates for this procedure because they are pluripotent; however, ESCs must be predifferentiated to avoid teratoma formation in vivo. In this dissertation, our goal was improve upon current protocols to direct differentiation of ESCs into cardiomyocytes using an embryoid body (EB) model. We immobilized pro-cardiomyogenic proteins, specifically Sonic Hedgehog (SHH) and Bone Morphogenetic Protein 4 (BMP4) to paramagnetic beads and delivered them in the interior of the EB. While lineage commitment was indiscriminate, the presence of the beads alone appeared to guide differentiation into cardiomyocytes: there were significantly more contracting areas in EBs containing beads than in the presence of SHH or BMP4. To take advantage of this result, we immobilized Arginine-Glycine-Aspartic Acid (RGD) peptides to the beads and magnetized them following incorporation into the EB. Magnetically mediated strain increased the expression of mechanochemical markers, and in combination with BMP4 increased the percentage of cardiomyocytes. Finally, PEGylated fibrin gels were used to investigate the effect of seeding method and fibrinogen concentration on cardiomyocyte behavior and maturation. Cells seeded on top of compliant hydrogels had the most contracting regions compared to stiffer PEGylated fibrin gels, whereas cardiomyocytes seeded within the hydrogels could not remodel the matrix or maintain contractility. As an alternative to 3D culture, we seeded cardiomyocytes within gel layers, which maintained viability as well as contractile activity. We observed that PEGylated fibrin gels can maintain ESC-derived cardiomyocytes; however, the ratio of cardiomyocytes and non-cardiomyocytes should be optimized to maintain contractile phenotypes. Therefore, this dissertation presents novel methods to differentiate ESCs into cardiomyocytes, and subsequently promote their maturation in vitro, for the treatment of MI.Item Microfabrication of spatially-patterned, polymer scaffolds for applications in stem cell and tissue engineering(2007) Call, Mary Gazell Mapili, 1980-; Roy, KrishnenduTissue engineering is a recently developed field that combines material science, cell biology, and engineering to create or improve functional tissues/organs. The field of tissue engineering has progressed from a fledgling science to an emerging technology, in large part due to parallel advances in the application of biomaterials and understanding stem cell behavior. Current studies have evaluated certain types of natural and synthetic biomaterials for feasibility of replicating the physio-chemical microenvironments of stem cells. Furthermore, technologies derived from micro-machining and solid free-form fabrication industries have utilized these biomaterials to create scaffolds that resemble tissue-like structures. Recent scaffold fabrication methods have attempted to overcome certain challenges in engineering tissues and organs. One of the fundamental limitations in current tissue engineering efforts has been the inability to develop multiple tissue types (i.e. bone, cartilage, muscles, ligaments) within a single scaffold structure in a predesigned manner. The differentiation of multiple cells within a three-dimensional (3D) scaffold using a single stem cell population has yet to be developed due to challenges in integrating various biochemical factors in a spatially-patterned method. This dissertation discusses scaffold micro-fabrication techniques that use layerby-layer, ultraviolet-based (UV) stereolithography systems. These approaches in microfabricating scaffolds provide an optimal, biomimetic environment for the pre-patterned differentiation of mesenchymal stem cells into skeletal-type tissues. We demonstrated both laser-based and digital micromirror device-based stereolithography systems for creating intricate scaffold architectures with multiple bio-factors encapsulated in predetermined regions. We showed that micro-stereolithography has the powerful capability of building 3D complex scaffolds with specific pore sizes and shapes in a layer-by-layer fashion using photo-crosslinkable monomers. These polymer-based scaffolds were functionalized with specific signaling proteins to create a biomimetic niche in which stem cells can respond, attach, and differentiate. The ultimate goal of this project is to integrate novel concepts of micro-manufacturing along with polymer-controlled release kinetics and stem cell biology to attain pre-designed architectures of tissue structures.Item Nanoengineering of surfaces to modulate cell behavior : nanofabrication and the influence of nanopatterned features on the behavior of neurons and preadipocytes(2009-08) Fozdar, David Yash; Chen, ShaochenPromising strategies for treating diseases and conditions like cancer, tissue necrosis from injury, congenital abnormalities, etc., involve replacing pathologic tissue with healthy tissue. Strategies devoted to the development of tissue to restore, maintain, or improve function is called tissue engineering. Engineering tissue requires three components, cells that can proliferate to form tissue, a microenvironment that nourishes the cells, and a tissue scaffold that provides mechanical stability, controls tissue architecture, and aids in mimicking the cell’s natural extracellular matrix (ECM). Currently, there is much focus on designing scaffolds that recapitulate the topology of cells’ ECM, in vivo, which undoubtedly wields structures with nanoscale dimensions. Although it is widely thought that sub-microscale features in the ECM have the greatest vii impact on cell behavior relative to larger structures, interactions between cells and nanostructures surfaces is not well understood. There have been few comprehensive studies elucidating the effects of both feature dimension and geometry on the initial formation and growth of the axons of individual neurons. Reconnecting the axons of neurons in damaged nerves is vital in restoring function. Understanding how neurons react with nanopatterned surfaces will advance development of optimal biomaterials used for reconnecting neural networks Here, we investigated the effects of micro- and nanostructures of various sizes and shape on neurons at the single cell level. Compulsory to studying interactions between cells and sub-cellular structures is having nanofabrication technologies that enable biomaterials to be patterned at the nanoscale. We also present a novel nanofabrication process, coined Flash Imprint Lithography using a Mask Aligner (FILM), used to pattern nanofeatures in UV-curable biomaterials for tissue engineering applications. Using FILM, we were able to pattern 50 nm lines in polyethylene glycol (PEG). We later used FILM to pattern nanowells in PEG to study the effect of the nanowells on the behavior preadipocytes (PAs). Results of our cell experiments with neurons and PAs suggested that incorporating micro- and nanoscale topography on biomaterial surfaces may enhance biomaterials’ ability to constrain cell development. Moreover, we found the FILM process to be a useful fabrication tool for tissue engineering applications.Item Novel templating of three-dimensional hyaluronic acid soft tissue scaffolds(2013-12) Thomas, Richelle Czarina; Sanchez, Isaac C., 1941-; Schmidt, Christine E.Effective tissue engineering scaffolds should mimic the physical and chemical attributes of native tissue. Native tissues have intricate patterns, a multitude of porosities, and large water contents that are each directly associated with their ability to regulate and support life function. Therefore, the physical architecture of scaffolds intended to mimic these tissues for engineering applications plays an important role in scaffold performance both in vitro and in vivo. Self-assembling molecules organize into intricate patterns with a complexity that resembles that of native tissue. Hyaluronic acid (HA) hydrogels are widely used in tissue engineering for a variety of applications but fail to offer physical architecture beyond the inherent hydrogel porosity. To address this issue, a novel method to impose architecture within thin HA-based films using crystal nucleation was developed in the Schmidt lab [1]. The work described herein extends this method for use in three-dimensional matrices, with the main vii goal being the creation of hydrogels with a complex macroarchitecture. Four in situ self-assembling molecules were used: glycine, guanidine, urea and potassium dihydrogen phosphate. The crystallization of each molecule creates a specific porous network within the hydrogel that is the negative imprint of the crystalline geometry. The novel restriction of aqueous polymer into the molecule interstitial crystalline space allows hydrogels to embody complex geometric lumen architectures. The hydrogels were characterized in terms of their internal architectures, swelling, bulk moduli, biodegradability, cytotoxicity and in vitro cellular response. The unique structure-property relationships displayed by hydrogels templated by each of the crystallizing molecules were characterized in regards to mechanical properties. The need for complex microscopic architecture is conserved over many tissue engineering applications and templated scaffolds were evaluated for two unique applications. Crystal-templated hydrogels were investigated for use as an artificial stem cell niche environment to expand undifferentiated neural progenitor cells. Additionally, the templated hydrogels were evaluated for the in vitro study of myelin expression from Schwann cells. A hydrogel that combines the biocompatible properties of HA and the architectural complexity of native tissue may prove beneficial for biomedical applications.Item Photopolymerizable scaffolds of native extracellular matrix components for tissue engineering applications(2010-05) Suri, Shalu; Schmidt, Christine E.; Chen, Shaochen; Roy, Krishnendu; Suggs, Laura J.; Shear, Jason B.In recent years, significant success has been made in the field of regenerative medicine. Tissue engineering scaffolds have been developed to repair and replace different types of tissues. The overall goal of the current work was to develop scaffolds of native extracellular matrix components for soft tissue regeneration, more specifically, neural tissue engineering. To date, much research has been focused on developing a nerve guidance scaffold for its ability to fill and heal the gap between the damaged nerve ends. Such scaffolds are marked by several intrinsic properties including: (1) a biodegradable scaffold or conduit, consisting of native ECM components, with controlled internal microarchitecture; (2) support cells (such as Schwann cells) embedded in a soft support matrix; and (3) sustained release of bioactive factors. In the current dissertation, we have developed such scaffolds of native biomaterials including hyaluronic acid (HA) and collagen. HA is a nonsulphated, unbranched, high-molecular weight glycosaminoglycan which is ubiquitously secreted by cells in vivo and is a major component of extracellular matrix (ECM). High concentrations of HA are found in cartilage tissue, skin, vitreous humor, synovial fluid of joints and umbilical cord. HA is nonimmunogenic, enzymatically degradable, non-cell adhesive which makes HA an attractive material for biomedical research. Here we developed new photopolymerizable HA based materials for soft tissue repair application. First, we developed interpenetrating polymer networks (IPN) of HA and collagen with controlled structural and mechanical properties. The IPN hydrogels were enzymatically degradable, porous, viscoelastic and cytocompatible. These properties were dependent on the presence of crosslinked networks of collagen and GMHA and can be controlled by fine tuning the polymer ratio. We further developed these hydrogel constructs as three dimensional cellular constructs by encapsulating Schwann cells in IPN hydrogels. The hydrogel constructs supported cell viability, spreading, proliferation, and growth factor release from the encapsulated cells. Finally, we fabricated scaffolds of photopolymerizable HA with controlled microarchitecture and developed designer scaffolds for neural repair using layer-by-layer fabrication technique. Lastly, we developed HA hydrogels with unique anisotropic swelling behavior. We developed a dual-crosslinking technique in which a super-swelling chemically crosslinked hydrogel is patterned with low-swelling photocrosslinked regions. When this dual-crosslinked hydrogel is swelled it contorts into a new shape because of differential swelling among photopatterned regions.Item The impact of mechanical properties of poly(ethylene glycol) hydrogels on vocal fold fibroblasts' behavior(2009-05-15) Liao, HuiminVocal fold scarring, caused by injury and inflammation, presents significant treatment challenges. Tissue engineering might be a promising treatment for vocal fold restoration or regeneration. It is important to investigate how scaffold properties alter cell behavior instead of screening thousand of materials, which is fundamental knowledge for rational scaffold design. This work studies how tuning only one parameter, mechanical strength of the hydrogel scaffold, influences the extracellular matrix production of encapsulated porcine vocal fold fibroblast (PVFF). PVFF cells were encapsulated by photopolymerization in 10 wt%, 20 wt%, and 30 wt% poly(ethylene glycol) diacrylate (PEGDA) hydrogels (MW 10,000), with the similar biochemical environment and network structure but different mechanical properties. Cell adhesive peptide, RGDS, was grafted into each hydrogel network to mimic a cell adhesive environment. The glycosaminoglycans (GAGs) production per cell increased from 10 wt% to 20 wt%, 30 wt% gels, with an increase in hydrogel stiffness. The collagen production per cell increased from 10 wt% to 20 wt% gels but no further increase occurred with the increasing modulus from 20 wt% to 30 wt% gels. Interestingly, in hydrogels of intermediate modulus (20% PEGDA hydrogels), the highest elastin per cell was observed compared with gels with higher and lower storage modulus after day 30. Histological analysis showed GAGs, collagen and elastin were distributed pericellularly. However, the organization of collagen type I appeared to be influenced by gel mechanical properties, which was confirmed by immunohistological analysis. Furthermore, the immunohistological analysis showed that the phenotype of PVFF is regulated by the stiffness of the PEG hydrogel. This study demonstrates that different levels of VFF ECM formation may be achieved by varying the mechanical properties of PEG hydrogels and validates a systematic and controlled platform for further research of cell-biomaterials interaction.Item Tissue Engineering Approaches for Studying the Effect of Biochemical and Physiological Stimuli on Cell Behavior(2012-10-19) Jimenez Vergara, AndreaTissue engineering (TE) approaches have emerged as an alternative to traditional tissue and organ replacements. The aim of this work was to contribute to the understanding of the effects of cell-material and endothelial cell (EC) paracrine signaling on cell responses using poly(ethylene glycol) diacrylate (PEGDA) hydrogels as a material platform. Three TE applications were explored. First, the effect of glycosaminoglycan (GAG) identity was evaluated for vocal fold restoration. Second, the influence of GAG identity was explored and a novel approach for stable endothelialization was developed for vascular graft applications. Finally, EC paracrine signaling in the presence of cyclic stretch, and hydrophobicity and inorganic content were studied for osteogenic applications. In terms of vocal fold restoration, it was found that vocal fold fibroblast (VFF) phenotype and extracellular matrix (ECM) production were impacted by GAG identity. VFF phenotype was preserved in long-term cultured hydrogels containing high molecular weight hyaluronan (HAHMW). Furthermore, collagen I deposition, fibronectin production and smooth muscle alpha-actin (SM-alpha-actin) expression in PEG-HA, PEG-chondroitin sulfate C and PEG- heparan sulfate (HS) gels suggest that CSC and HS may be undesirable for vocal fold implants. Regarding vascular graft applications, the impact of GAG identity on smooth muscle cell (SMC) foam cell formation was explored. Results support the increasing body of literature that suggests a critical role for dermatan sulfate (DS)-bearing proteoglycans in early atherosclerosis. In addition, an approach for fabricating bi-layered tissue engineering vascular grafts (TEVGs) with stable endothelialization was validated using PEGDA as an intercellular "cementing" agent between adjacent endothelial cells (ECs). Finally, mesenchymal stem cell (MSC) differentiation toward osteogenic like cells was evaluated. ECM and cell phenotypic data showed that elevated scaffold inorganic content and hydrophobicity were indeed correlated with increased osteogenic differentiation. Moreover, the present results suggest that EC paracrine signaling enhances MSC osteogenesis in the presence of cyclic stretch.Item Ultrasound and photoacoustic imaging to monitor stem cells for tissue regeneration(2014-05) Nam, Seung Yun; Emelianov, Stanislav Y.; Suggs, Laura J; Pearce, John A; Dunn, Andrew K; Hall, Neal ARegenerative medicine is an interdisciplinary field which has advanced with the use of biotechnologies related to biomaterials, growth factors, and stem cells to replace or restore damaged cells, tissues, and organs. Among various therapeutic approaches, cell-based therapy is most challenging and exciting for both scientists and clinicians pursuing regenerative medicine. Specifically, stem cells, including mesenchymal stem cells and adipose-derived stem cells, are promising candidate cell types for cell-based therapy because they can differentiate into multiple cell types for tissue regeneration and stimulate other cells through neovascularization or paracrine signaling. Also, for effective treatment using stem cells, the tissue engineered constructs, such as bioactive degradable scaffolds, that provide the physical and chemical cues to guide their differentiation are incorporated with stem cells before implantation. Also, it was previously demonstrated that tissue-engineered matrices can promote tubulogenesis and differentiation of stem cells to vascular cell phenotypes. Hence, during tissue regeneration after stem cell therapy, there are numerous factors that need to be monitored. As a result, imaging-based stem cell tracking is essential to evaluate the distribution of stem cells as well as to monitor proliferation, differentiation, and interaction with the microenvironment. Therefore, there is a need for a stem cell imaging technique that is not only noninvasive, sensitive, and easy to operate, but also capable of quantitatively assessing stem cell behaviors in the long term with high spatial resolution. Therefore, the overall goal of this research is to demonstrate a novel imaging method capable of continuous in vitro assessment of stem cells as prepared with tissue engineered constructs and noninvasive longitudinal in vivo monitoring of stem cell behaviors and tissue regeneration after stem cell implantation. In order to accomplish this, gold nanoparticles are demonstrated as photoacoustic imaging contrasts to label stem cells. In addition, ultrasound and photoacoustic imaging was utilized to monitor stem cells and neovascularization in the injured rat tissue. Therefore, using these methods, tissue regeneration can be promoted and noninvasively monitored, resulting in a better understanding of the tissue repair mechanisms following tissue injury.Item Understanding mechanisms of stem cell tubulogenesis in PEGylated fibrin for improving neovascularization therapies(2013-12) Rytlewski, Julie Ann; Suggs, Laura J.Stem cell-based therapies are an important developing technology for treating cardiovascular ischemic disease, including subsequent co-morbidities such as ulcerative wounds. Mesenchymal stem cells (MSCs) have a proven ability to augment wound healing and neovascularization processes and have been more recently investigated for their endothelial-like behavior. This doctoral work aims to understand mechanisms underlying matrix-driven MSC tubulogenesis within PEGylated fibrin gels, specifically (1) why this behavior occurs and (2) if this behavior has clinical utility. Briefly, a three-dimensional morphological quantification pipeline was first developed for analyzing the maturity of vascular networks (Chapter 2). This method was applied in later studies that examined the full spectrum of MSC behavior in PEGylated fibrin gels, linking biomaterial properties with network development (Chapter 3). Mechanisms underlying the cell-matrix relationship were more fully clarified through gain-of-function cell studies. These studies indicated that PEGylated fibrin promotes endothelial-like MSC behavior through a combination of hypoxic stress and bioactive fibrin cues (Chapter 4). Notably, this endothelial-like MSC behavior closely mirrored vasculogenic mimicry, a process whereby tumors establish non-endothelialized vasculature in response to hypoxic stress. The functionality of these tumor vessels suggests that mature endothelial differentiation of MSCs may not be necessary to achieve therapeutically beneficial tissue perfusion. This hypothesis opens up new mechanisms for exploitation in vascular tissue engineering strategies.