Browsing by Subject "Tissue Engineering"

Now showing 1 - 7 of 7
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    Bioreactor Conditioning for Accelerated Remodeling of Fibrin-Based Tissue Engineered Heart Valves
    (2015-05) Schmidt, Jillian
    Fibrin is a promising scaffold material for tissue engineered heart valves, as it is completely biological, allows for engineered matrix alignment, and is able to be degraded and replaced with collagen by entrapped cells. However, the initial fibrin matrix is mechanically weak, and extensive in vitro culture is required to create valves with sufficient mechanical strength and stiffness for in vivo function. Culture in bioreactor systems, which provide cyclic stretching and enhance nutrient transport, has been shown to increase collagen production by cells entrapped in a fibrin scaffold, accelerating strengthening of the tissue and reducing the required culture time. In the present work, steps were taken to improve bioreactor culture conditions with the goal of accelerating collagen production in fibrin-based tissue engineered heart valves using two approaches: (i) optimizing the cyclic stretching protocol and (ii) developing a novel bioreactor system that permits transmural and lumenal flow of culture medium for improved nutrient transport. The results indicated that incrementally increasing strain amplitude cyclic stretching with small, frequent increments in strain amplitude was optimal for collagen production in our system. In addition, proof of concept studies were performed in the novel bioreactor system and increased cellularity and collagen deposition near the lumenal surface of the tissue were observed.
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    Creation of Perfusable Tissue Engineering Constructs through Biological Assembly
    (2015-08) Riesberg, Jeremiah
    One of the biggest barriers in creating large tissues is the lack of oxygen and nutrient transport required for cell growth and tissue development in the interior region. The post-implantation cell survival in large tissue engineering constructs can be assisted by prevascularization. In this work we used a bottom-up approach to prepare large, prevascularized tissue constructs through perfusion culture of porous, cell-laden hydrogel constructs biologically assembled from smaller gel modules. The small gel modules had a controlled shape and were laden with HUVECs and hMSCs. They were packed in a bioreactor for perfusion culture, during which capillary formation inside and between individual gel modules led to the assembly of the small modules into a nearly centimeter sized porous construct. Viable cells and hollow lumen-like structures were observed throughout the porous construct, while in a nonporous control construct with similar dimensions viable cells were only observed in a peripheral layer several hundred micron thick. This modular assembly approach allows for creation of prevascularized large tissue constructs through the biological assembly of gels, which was difficult previously.
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    Design of Silica-Collagen Nanocomposite for Corneal Replacement
    (2015-09) DiVito, Michael
    The cornea is the most commonly transplanted tissue in the United States. Globally, corneal diseases are the second leading cause of blindness. Due to strict FDA regulations, lack of eye banking facilities, and other factors which limit the supply of donor tissue, designing an artificial cornea made of readily available materials is of great interest. The synthetic constructs that are currently clinically available in the United States have had moderate success, but biocompatibility issues such as stromal melting and epithelial defects are still common. When considering a potential material for corneal replacement, it must meet the design criteria of the normal functioning cornea. The relevant design criteria can be broken down into three main groups: optical behavior, biomechanical properties, and biocompatibility. The presented work proposes silica-collagen nanocomposites as a viable candidate material to meet these design criteria. A bottom-up approach starting from the molecular level is utilized to modify the surface chemistry and physical properties of collagen fibrils. In doing so, methodologies are presented which allow for fine-tuning of optical, biomechanical, and biodegradation behavior. The first part of this work validates the theory that light scattering of collagen hydrogels is heavily dependent on the change in the material’s index of refraction over length scales comparable to the wavelength of incident light. This work shows that light scattering of collagen hydrogels can be minimized by a rapid neutralization technique, and by the addition of nanocrystalline cellulose. Additionally, collagen hydrogels with embedded magnetic nanowires can be polarized to form an aligned fibril microstructure and show an increase in light transmission. The second part of this thesis characterizes the mechanical and optical behavior, as well as the biocompatibility of silica-collagen nanocomposites. This work shows that a copolymerization method can be used to make implants which have improved biomechanical properties (when compared to pure collagen hydrogels) and can be re-epithelialized in an ex vivo rabbit model. Additionally, an improved two-step process for silica deposition onto collagen fibrils is presented. This new method shows that poly-L-lysine can be used to induce a uniform silica shell around collagen fibrils in the absence of large silica aggregates. This new method increases mechanical stiffness and enzymatic degradation resistance without producing any additional light scattering in the material. Silica-collagen nanocomposites show great potential in the context of corneal replacement. The methods developed and results presented here can be useful for improving any collagen-based corneal replacement, as well as in other applications such as drug delivery and silica nanoparticle templating.
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    Development of a completely biological tissue engineered heart valve.
    (2009-04) Syedain, Zeeshan Hayder
    In the United States alone, over 100,000 heart valve replacement procedures are performed each year, with approximately 45% of patients below age 65. While current mechanical and bioprosthetic heart valves are viable options, they have several limitations. The most significant limitation is for pediatric patients, since neither of these valve types grow and remodel with the patient. Tissue engineering provides a methodology to create functional heart valves that can grow and remodel similar to native tissue once implanted. Several tissue engineering approaches have been proposed using decellularized native scaffolds, synthetic biopolymers, and biological polymers seeded with cells. Fibrin provides a scaffold to create tissue-engineered heart valves (TEHV) that are completely biological with an environment permissive for extracellular matrix (ECM) deposition. Previous research in our lab has demonstrated the feasibility of creating a fibrin-based TEHV with neonatal human dermal fibroblast (nHDF) that yields valve leaflets with structural and mechanical anisotropy similar to native leaflets. However, the TEHV had sub-optimal tensile mechanical properties and was thus unable to withstand physiological forces. The development of tissue can be accelerated by both chemical and mechanical stimulus. Previously, fibrin based TEHV were cultured with chemical stimulus in the form of growth factors supplemented in the culture medium resulting in improved ECM deposition by the cells; however, no mechanical stimulation was applied. Prior research in both our lab and by other researchers has shown cyclic stretching with constant strain amplitude is a method to stimulate remodeling of biological scaffolds seeded with cells. Initial experiments were conducted to evaluate the effect of cyclic stretching on fibrin-based tubular constructs seeded with porcine valve interstitial cells (PVIC) and nHDF. Cyclic stretching with 10% constant strain amplitude applied for 3 weeks led to modest improvement in tensile properties of the tubular constructs. We hypothesized that long-term cyclic stretching, as was used in this study, could induce cellular adaptation, minimizing the benefits of cyclic stretching. This hypothesis was tested in subsequent experiments using tubular constructs cultured with incremental strain amplitude cyclic stretching, with an average strain of 10% for 3 weeks. Both PVIC and nHDF seeded constructs exhibited a 2-fold improvement in ultimate tensile strength (UTS) and collagen density over samples conditioned with constant strain amplitude strteching. To verify that this was the result of a cellular response, phosphorylation of extracellular signal-regulated kinase (ERK) was measured by western blot. At 5 weeks, the phosphorylated ERK was 255% higher in incremental cyclic strained samples compared to constant strain samples. nHDF-seeded tubular constructs were also used to optimize the use of transforming growth factor beta (TGF-β). Studies showed that under cyclic stretching conditions, TGF-β has detrimental effects on total collagen deposition and collagen maturation. Western blot analysis showed a decrease in p-ERK signaling in TGF-β treated samples. However, TGF-β use demonstrated a benefit by increasing the elastin content of the tissue constructs. In subsequent experiments, a sequence of cyclic stretching and TGF-β supplementation was used to optimize tensile mechanical properties and elastin content of the engineered tissue. Based on the results with tubular constructs, a novel bioreactor was designed to apply controlled cyclic stretching to the fibrin-based TEHV. Briefly, the valve was mounted on two plastic end-pieces with elastic latex tube placed around TEHV. Using a reciprocating syringe pump, culture medium was cyclically pumped into the bioreactor. The root distension, which was determined by the stiffer latex was used as a control parameter, and in turn stretched the leaflets. A separate flowloop (connected to the bioreactor end-pieces) was used to control nutrient transport to the TEHV. Using an incremental strain amplitude stretching regime, fibrin-based TEHV were conditioned in the bioreactor for 3 weeks. Cyclically stretched valves (CS valve) had improved tensile properties and collagen deposition compared to statically-cultured valves. The mechanical stiffness (modulus) and anisotropy (measured as ratio of leaflet modulus in circumferential to radial directions) in the leaflets was comparable to native sheep pulmonary valve leaflets. Collagen organization/ maturation also improved in CS valves over statically-cultured valves as observed by picrosirius red staining of tissue crosssections. In addition, the CS valve root could withstand pressures of up to 150 mmHg and its compliance was comparable to that of the sheep pulmonary artery at physiological pressures. To assess in vivo remodeling TEHV were implanted in the pulmonary artery of two sheep for 4.5 weeks with the pulmonary valve either left intact or rendered incompetent by leaflet excision. Echocardiography immediately after implantation showed functional coapting leaflets, with normal right heart function. It was also performed just prior to explantation, revealing functional leaflets although with moderate regurgitation in both cases and a partial detachment of one leaflet from the root in one case. The explanted leaflets had thickness and tensile properties comparable the implanted leaflets. There was endothelialization on the lumenal surface of the TEHV root. These preliminary results are unprecedented for a TEHV developed from a biological scaffold; however, many issues remain to be surmounted. In further development of the TEHV with a fibrin scaffold, photo-cross linking of the fibrin gel was utilized as a method to stiffen the matrix, thereby inhibiting excessive cell-induced compaction. Preliminary studies with tubular constructs demonstrated reduced compaction of cross-linked fibrin gel during cyclic stretching with no effect on nHDF proliferation or deposited collagen. In addition, a preliminary investigation using blood outgrowth endothelial cells (BOEC) has been conducted to assess their adhesion to the remodeled TEHV surface. Studies showed BOEC adhesion and proliferation on remodeled fibrin surface creating a confluent layer after 4 days of culture. Successful seeding of sheep BOEC on the TEHV surface prior to implantation would reduce the risk of clotting. Overall, the studies presented in this dissertation advance the development of a completely biological tissue-engineered heart valve. These studies improve our understanding of the role of cyclic stretching in tissue remodeling and have furthered the science of mechnotransduction and tissue remodeling.
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    Engineered Cardiac Tissues for Delivery of Cells to the Injured Myocardium
    (2015-07) Wendel, Jacqueline
    With the high incidence of heart failure in the developing world and the inherent risks and limited availability of donor hearts, cell-based solutions have become an attractive solution. However, current methods to deliver cells to the heart have resulted in limited long term cell retention and consequently minimal therapeutic efficacy. In this work, we aim to use engineered tissues as a means to deliver cells to the injured myocardium post- infarction with increased cell retention. The results detailed in this dissertation indicate that engineered tissues can be constructed from both primary rodent cardiomyocytes and human pluripotent stem cell derived cardiomyocytes, and that these tissues not only engraft post-infarction with high cell retention , but in some instances also result in improved cardiac function and limitation of left ventricular remodeling postinfarction.
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    Supporting data for 3D Printed Stem-Cell Derived Neural Progenitors Generate Spinal Cord Scaffolds
    (2020-05-15) Joung, Daeha; Truong, Vincent; Neitzke, Colin C; Guo, Shuang-Zhuang; Walsh, Patrick J; Monat, Joseph R; Meng, Fanben; Park, Sung Hyun; Dutton, James R; Parr, Ann M; McAlpine, Michael C; mcalpine@umn.edu; McAlpine, Michael C; McAlpine Research Group
    A bioengineered spinal cord is fabricated via extrusion-based multilateral 3D bioprinting, in which clusters of induced pluripotent stem cell (iPSC)-derived spinal neuronal progenitor cells (sNPCs) and oligodendrocyte progenitor cells (OPCs) are placed in precise positions within 3D printed biocompatible scaffolds during assembly. The location of a cluster of cells, of a single type or multiple types, is controlled using a point-dispensing printing method with a 200 μm center-to-center spacing within 150 μm wide channels. The bioprinted sNPCs differentiate and extend axons throughout microscale scaffold channels, and the activity of these neuronal networks is confirmed by physiological spontaneous calcium flux studies. Successful bioprinting of OPCs in combination with sNPCs demonstrates a multicellular neural tissue engineering approach, where the ability to direct the patterning and combination of transplanted neuronal and glial cells can be beneficial in rebuilding functional axonal connections across areas of central nervous system (CNS) tissue damage. This platform can be used to prepare novel biomimetic, hydrogel-based scaffolds modeling complex CNS tissue architecture in vitro and harnessed to develop new clinical approaches to treat neurological diseases, including spinal cord injury.
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    Utilization of A 3D Culture System of Collagen-Mimic Peptide Gfoger-Based Hydrogel to Model Osteosarcoma from Engineered Ipsc
    (2021-05) Thueson, Hanna
    Modeling the early stages of human osteosarcoma development remains a significant challenge. Most existing human models are derived from patient tumor tissue which is used to establish tumor cell lines or xenograft models in immunodeficient mice. These models are largely derived from late or end stage disease and do not allow the study of the early events of transformation. Further, 2D cell lines are largely homogenous and do not replicate the heterogeneity of primary tumors. Xenografted models more closely replicate the primary tumor but can have low engraftment rates and are logistically challenging to maintain. The immunocompromised nature of xenografted mice limits the potential for immunotherapy studies. The work presented here establishes a 3D culture system to model early-stage osteosarcoma development from engineered human iPSC. When cultured as aggregates in a GFOGER (integrin-specific glycine-phenylalanine-hydroxyproline-glycine-glutamate-arginine) based hydrogel known to promote osteoblastic differentiation, osteoblasts engineered with osteosarcoma-associated mutations readily form 3D organoids. Histological analysis supports that 3D culture of iPSC-derived osteoblasts promotes a more tissue-like phenotype with increased mineralization and ECM development within the tissue construct. In addition, preliminary functional studies suggest that 3D culture promotes transformative properties and an osteosarcoma phenotype. This novel approach has potential for future applications in disease modeling, in vivo studies, and drug discovery.