Browsing by Author "Grande-Allen, K. Jane"
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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 Multifaceted Approach to Enhance the Current Understanding and Treatment of Calcific Aortic Valve Disease(2013-11-27) Wiltz, Dena; Grande-Allen, K. Jane; Gustin, Michael C.; Mikos, Antonios G.Calcific aortic valve disease (CAVD) is a serious condition with unclear mechanisms driving this disease. This research focused on investigating the role of lysophosphatidylcholine (LPC) in CAVD and evaluating the efficacy of Raman spectroscopy (RS) to aid in current tissue engineering methods of heart valve replacements used to treat CAVD. Appropriate culture conditions for in vitro studies of CAVD were established. Specifically, the application of gentamicin in valvular interstitial cell (VIC) cultures was determined to significantly decrease mineralization of VICs in vitro in both normal and pre-calcified VIC culture conditions. Next, in vitro studies were conducted examining the role of LPC in a comparison of aortic and mitral VIC mineralization. Results indicated a higher percentage of LPC in calcified regions of tissue compared to non-calcified regions. In addition, 10000 nM LPC led to an increase in VIC mineralization, and aortic VICs displayed greater mineralization compared to mitral VICs. The role of the ryanodine receptor (RyR) in LPC-induced mineralization was evaluated. The presence of RyR isoforms 2 and 3 were confirmed in VICs. Next, in the presence of 10 µM LPC, the RyR was blocked and mineralization in VIC cultures significantly decreased compared to LPC treated cultures in which the RyR was not blocked. Several strategies exist for utilizing mesenchymal stem cells (MSCs) for tissue engineering of heart valves (TEHV) for valve replacement therapies. In this research, RS was able to detect distinct molecular characteristics of MSCs from different sources. This research has a significant impact on the study and understanding of CAVD. It suggests that gentamicin be used cautiously with in vitro studies of calcification, and suggest that mechanisms by which gentamicin acts in VICs may reverse calcification. In addition, these results showed that LPC has the capacity to promote VIC calcification, by interacting with the RyR, and that aortic VICs have a greater propensity for mineralization compared to mitral VICs. Also, RS may be used in future research to characterize MSCs prior to their use in TEHV. This research has highlighted the need for future investigations of LPC and the use of RS in understanding and treating CAVD.Item Amniotic Fluid-derived Stem Cell Isolation, Maintenance, and Differentiation for Cardiac Tissue Engineering(2014-12-05) Connell, Jennifer Petsche; Jacot, Jeffrey G; Fraser, Charles D; Lwigale, Peter; Grande-Allen, K. JaneCardiac tissue engineering is limited by the lack of a clinically relevant cell source. Amniotic fluid-derived stem cells (AFSC) are broadly multipotent and proliferate rapidly, making them a promising cell source for tissue engineering applications. AFSC can also be utilized autologously for congenital heart defects, the most severe of which are identified in utero, allowing for ample time to isolate and expand the cells to prepare a patch for implantation shortly after birth. This thesis focused on the characterization of AFSC and their potential to differentiate towards a cardiac lineage. For characterization studies, stem cells from amniotic fluid were sorted for c-kit protein expression at the first passage or left unfractionated and then expanded in 5 different media. Protein and gene expression of markers common to pluripotent stem cells were analyzed from passages 2 through 6, and differentiation capacity of the stem cells towards osteogenic, endothelial, and neurogenic lineages were compared at passages 5 and 6. The unfractionated AFSC maintained higher expression of stem cell markers but displayed a significant decrease in those markers at passage 6. Correspondingly, indicators of the lineages of interest were higher following differentiation at passage 5 compared to passage 6. To investigate the cardiac tissue engineering potential of AFSC, cells were differentiated in indirect co- cultures with neonatal rat ventricular myocytes (NRVM) and under a small molecule- based directed differentiation regime. NRVM induce AFSC to form functional gap junctions following indirect co-culture. AFSC undergoing directed differentiation also localized gap junctions to cell membranes and additionally demonstrated an up regulation in cardiac transcription factors and sarcomere proteins. In both co-culture and small molecule-based differentiation methods, however, no organized sarcomeres or spontaneously beating cells were observed. While AFSC hold great potential for regenerative medicine applications, particularly in congenital defect repair, functional cardiomyocytes have not yet been obtained, and it is likely that additional cues beyond chemical signaling and growth factors will be required. Overall, these studies led to a greater understanding of the cardiac potential of AFSC and the effect of sorting and culture conditions on maintenance of stem cell properties in AFSC.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 Assembly of a Three-Dimensional Multitype Bronchiole Coculture Model Using Magnetic Levitation(Liebert, 2013) Tseng, Hubert; Gage, Jacob A.; Raphael, Robert M.; Moore, Robert H.; Killian, Thomas C.; Grande-Allen, K. Jane; Souza, Glauco R.A longstanding goal in biomedical research has been to create organotypic cocultures that faithfully represent native tissue environments. There is presently great interest in representative culture models of the lung, which is a particularly challenging tissue to recreate in vitro. This study used magnetic levitation in conjunction with magnetic nanoparticles as a means of creating an organized three-dimensional (3D) coculture of the bronchiole that sequentially layers cells in a manner similar to native tissue architecture. The 3D coculture model was assembled from four human cell types in the bronchiole: endothelial cells, smooth muscle cells (SMCs), fibroblasts, and epithelial cells (EpiCs). This study represents the first effort to combine these particular cell types into an organized bronchiole coculture. These cell layers were first cultured in 3D by magnetic levitation, and then manipulated into contact with a custom-made magnetic pen, and again cultured for 48 h. Hematoxylin and eosin staining of the resulting coculture showed four distinct layers within the 3D coculture. Immunohistochemistry confirmed the phenotype of each of the four cell types and showed organized extracellular matrix formation, particularly, with collagen type I. Positive stains for CD31, von Willebrand factor, smooth muscle a-actin, vimentin, and fibronectin demonstrate the maintenance of the phenotype for endothelial cells, SMCs, and fibroblasts. Positive stains for mucin-5AC, cytokeratin, and E-cadherin after 7 days with and without 1% fetal bovine serum showed that EpiCs maintained the phenotype and function. This study validates magnetic levitation as a method for the rapid creation of organized 3D cocultures that maintain the phenotype and induce extracellular matrix formation.Item Assessment of spinal cord injury using ultrasound elastography in a rabbit model in vivo(Springer Nature, 2023) Tang, Songyuan; Weiner, Bradley; Taraballi, Francesca; Haase, Candice; Stetco, Eliana; Mehta, Shail Maharshi; Shajudeen, Peer; Hogan, Matthew; De Rosa, Enrica; Horner, Philip J.; Grande-Allen, K. Jane; Shi, Zhaoyue; Karmonik, Christof; Tasciotti, Ennio; Righetti, RaffaellaThe effect of the mechanical micro-environment on spinal cord injury (SCI) and treatment effectiveness remains unclear. Currently, there are limited imaging methods that can directly assess the localized mechanical behavior of spinal cords in vivo. In this study, we apply new ultrasound elastography (USE) techniques to assess SCI in vivo at the site of the injury and at the time of one week post injury, in a rabbit animal model. Eleven rabbits underwent laminectomy procedures. Among them, spinal cords of five rabbits were injured during the procedure. The other six rabbits were used as control. Two neurological statuses were achieved: non-paralysis and paralysis. Ultrasound data were collected one week post-surgery and processed to compute strain ratios. Histologic analysis, mechanical testing, magnetic resonance imaging (MRI), computerized tomography and MRI diffusion tensor imaging (DTI) were performed to validate USE results. Strain ratios computed via USE were found to be significantly different in paralyzed versus non-paralyzed rabbits. The myelomalacia histologic score and spinal cord Young’s modulus evaluated in selected animals were in good qualitative agreement with USE assessment. It is feasible to use USE to assess changes in the spinal cord of the presented animal model. In the future, with more experimental data available, USE may provide new quantitative tools for improving SCI diagnosis and prognosis.Item Bioinspired electrospun dECM scaffolds guide cell growth and control the formation of myotubes(AAAS, 2021) Smoak, Mollie M.; Hogan, Katie J.; Grande-Allen, K. Jane; Mikos, Antonios G.While skeletal muscle has a high capacity for endogenous repair in acute injuries, volumetric muscle loss can leave long-lasting or permanent structural and functional deficits to the injured muscle and surrounding tissues. With clinical treatments failing to repair lost tissue, there is a great need for a tissue-engineered therapy to promote skeletal muscle regeneration. In this study, we aim to assess the potential for electrospun decellularized skeletal muscle extracellular matrix (dECM) with tunable physicochemical properties to control mouse myoblast growth and myotube formation. The material properties as well as cell behavior – growth and differentiation – were assessed in response to modulation of crosslinking and scaffold architecture. The fabrication of a bioactive dECM-based system with tunable physicochemical properties that can control myotube formation has several applications in skeletal muscle engineering and may bring the field one step closer to developing a therapy to address these unmet clinical needs. Electrospun decellularized skeletal muscle with tunable physicochemical properties controls myoblast growth and myotube formation. Electrospun decellularized skeletal muscle with tunable physicochemical properties controls myoblast growth and myotube formation.Item Embargo Building a 3D genome atlas of human tissues down to base-pair resolutions(2023-03-21) Shamim, Muhammad Saad; Aiden, Aviva P; Grande-Allen, K. JaneThe three-dimensional organization of the genome affects cell function and can be interrogated in an unbiased manner via methods such as Hi-C, a genome-wide proximity ligation assay. Hi-C assays have led to a deeper understanding of the mechanisms underlying chromosome conformation, such as chromatin loops and compartments. Improvements to the Hi-C protocol have made it possible to generate meaningful contact maps down to base-pair-resolutions, comparable to the types of resolutions used to analyze most epigenetics assays, such as ChIP-Seq. Generating these ultra-high-resolution maps, however, requires terabases of DNA sequencing. We developed an ecosystem of open-source software tools to support the analysis and visualization of ultra-deep Hi-C datasets. These tools include the Juicer 2.0 pipeline for processing billions of DNA sequencing reads into contact maps at base-pair resolutions; the straw library for powering rapid programmatic access to Hi-C data from both local and remote files; a novel ensemble deep learning approach to annotate chromatin loops; and a novel algorithm that combines dimensionality reduction with unsupervised learning to reliably identify genomic subcompartments. Together, these tools enable the comprehensive processing and analysis of Hi-C data to unprecedented resolutions. We then applied these tools to generate and fully annotate 10-base-pair-resolution maps of nuclear architecture in over 100 primary samples and cell lines, spanning over 40 unique human tissues. Taken together, these datasets report over 100 terabases of raw sequence data, revealing hundreds of thousands of DNA loops localized down to 10-base-pair resolution, and help elucidate the function of genome architecture across human anatomy.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 Cellular and Extracellular Matrix Basis for Heterogeneity in Mitral Annular Contraction(Springer, 2015) Stephens, Elizabeth H.; Fahrenholtz, Monica M.; Connell, Patrick S.; Timek, Tomasz A.; Daughters, George T.; Kuo, Joyce J.; Patton, Aaron M.; Ingels, Neil B. Jr.; Miller, D. Craig; Grande-Allen, K. JaneRegional heterogeneity in mitral annular contraction, which is generally ascribed to the fibrous vs. muscular annular composition, ensures proper leaflet motion and timing of coaptation. It is unknown whether the fibroblast-like cells in the annulus modulate this heterogeneity, even though valvular interstitial cells (VICs) can be mechanically “activated.” Fourteen sheep underwent implantation of radiopaque markers around the mitral annulus defining four segments: septal (SEPT), lateral (LAT), and anterior (ANT-C) and posterior (POST-C) commissures. Segmental annular contraction was calculated using biplane videofluoroscopy. Immunohistochemistry of annular cross sections assessed regional matrix content, matrix turnover, and cell phenotype. Micropipette aspiration measured the effective modulus of the leaflets adjacent to the myocardial border. Whereas SEPT contained more collagen I and III, LAT demonstrated more collagen and elastin turnover as shown by greater decorin, lysyl oxidase, and matrix metalloprotease (MMP)-13 and smooth muscle alpha-actin (SMaA). This greater matrix turnover paralleled greater annular contraction in LAT vs. SEPT (22.5 vs. 4.1%). Similarly, POST-C had more SMaA and MMP13 than ANT-C, consistent with greater annular contraction in POST-C (18.8 vs. 11.1%). Interestingly, POST-C had the greatest effective modulus, significantly higher than LAT. These data suggest that matrix turnover by activated VICs relates to annular motion heterogeneity, maintains steady-state mechanical properties in the annulus, and could be a therapeutic target when annular motion is impaired. Conversely, alterations in this heterogeneous annular contraction, whether through disease or secondary to ring annuloplasty, could disrupt this normal pattern of cell-mediated matrix remodeling and further adversely impact mitral valve function.Item Characterization of hyaluronan in three-dimensional engineered tissues(2008) Allison, David D.; Grande-Allen, K. JaneHyaluronan is a fundamental component of the extracellular matrix (ECM), having essential roles in embryonic development, tissue hydration, and cell homeostasis. However, in many disease states hyaluronan can have a deleterious effect on tissue remodeling and function. To investigate the cell-mediated role of HA within the ECM, cells overexpressing the hyaluronan synthases (has-1, has-2, has-3) or the empty vector control (LXSN), were seeded within collagen gel scaffolds. In Specific Aim I, gels were grown under static tension for 6 weeks. The collagen gels containing has overexpressing cells had significantly increased retention and secretion of hyaluronan; reduced contraction; reduced collagen density; and significantly altered material properties vs. LXSN controls. These results indicate that the endogenous overproduction of hyaluronan within collagen gels alters their material, morphological and biochemical characteristics. In Specific Aim II, collagen gels containing has overexpressing cells and LXSN control cells were grown in a cyclic strain environment. Cyclic strain caused a significant elevation in the collagen fibril density, cell number, and hyaluronan content of the resulting collagen gels compared to those grown under a static strain regimen. The material behavior of collagen gels containing has overexpressing cells were weakened compared to controls. Transmission electron microscopy and immunohistochemistry showed that proteoglycan distribution was influenced by both strain and the specific has isozyme overexpressed. In Specific Aim III the effects of exogenous HA addition were examined in collagen gels containing either LXSN or has overexpressing cells. It was found that while exogenous high molecular weight HA reduced the diameter of collagen gels containing LXSN cells, the endogenous overproduction of HA (also of high molecular weight) caused the opposite effect. Furthermore, HA oligosaccharides caused an increase in the diameter of collagen gels containing LXSN cells, but had no effect in the has overexpressing groups. Similarly, the ultimate tensile strength was increased upon the addition of exogenous high molecular weight HA to collagen gels containing LXSN cells, but not has overexpressing cells. In this research collagen gels were used to simulate the impact of endogenously produced hyaluronan on overall extracellular matrix organization. This research provides an important step in understanding the role of hyaluronan within soft tissues, and offers insight into hyaluronan mediated pathological remodeling.Item Collagenous tissue engineered constructs to investigate the role of decorin on collagen fibrillogenesis and tissue mechanics(2008) Ferdous, Zannatul; Grande-Allen, K. JaneDecorin, a small leucine rich proteoglycan, has been reported to control collagen fibrillogenesis, thereby influencing the tensile properties of collagenous tissues. In this research work, decorin deficient cells (Dcn -/- ) were combined with tissue-engineered collagen gels to study the contribution of decorin to cell proliferation, collagen fibrillogenesis, and tensile strength, as well as the interaction between decorin and transforming growth factor (TGF)-beta. This project was driven by the hypothesis that decorin deficient cells grown in tissue-engineered constructs would show significant biomechanical influence over the tensile behavior of collagenous tissues by controlling collagen fibrillogenesis. Since decorin inhibits cell adhesion and cell migration on various matrix molecules, such as collagen and fibronectin, the adhesive characteristics of Dcn -/- cells were first investigated to understand how the absence of decorin would influence cell organization in the collagen gels. Since β 1 integrins play an integral role in cell-mediated adhesion, the contributions of α 2 and β 1 subunits of integrin were also investigated. In this study, Dcn -/- cells showed significantly higher adhesion to both collagen and fibronectin substrates. For both collagen and fibronectin substrates, blocking either α 2 or β 1 integrin subunits blocked cell adhesion differently for the wild-type and Dcn -/- cells, suggesting that distinct mechanisms of adhesion are utilized by these cell types. Finally, Dcn -/- cells showed greater migration on the collagen substrate compared to wild-type controls. To further determine how decorin participates in matrix organization, collagen gels containing the Dcn -/- cells and wild-type control cells were grown under 2 different mechanical conditions—static tension and dynamic tension. The static tension collagen gels seeded with Dcn -/- cells showed greater gel contraction, matrix organization, ultimate tensile strength and elastic modulus than those seeded with wild-type cells. Moreover, addition of TGF-beta to the wild-type cell-seeded gels made them similar to Dcn -/- cell-seeded gels. Conversely, when the collagen gels containing Dcn -/- cells were treated with a TGF-beta receptor kinase inhibitor, they demonstrated reduced contraction. These results indicate that the inhibitory interaction between decorin and TGF-beta significantly influenced the matrix organization and material behavior of these in vitro model tissues. The dynamic gels were grown with the above cell types in a Flexcell Tissue Train™ culture system under cyclic 5% uniaxial strain at 0.1 Hz, while the static gels were cultured under static tension. Interestingly, it was found that some measured outcomes such as collagen fibril density, PG density, maximum load and stiffness were altered with mechanical stimulation regardless of the cell type used. On the other hand, unique outcomes regarding cell density, collagen fibril diameter, and biglycan expression were observed in response to cyclic strain in the Dcn -/- cell-seeded gels only. These results led us to conclude that decorinmediated tissue organization is heavily dependent upon tissue type and the amount of strain imparted on the tissue.Item Comparing the Role of Mechanical Forces in Vascular and Valvular Calcification Progression(Frontiers, 2019) Gomel, Madeleine A.; Lee, Romi; Grande-Allen, K. Janeregulator of lysosomal function, or by inducing TFEB activation chemically. Because of its small format, rapidity, sensitivity and reproducibility, the NAG one-step cell assay is suitable for multiple procedures, including the high-throughput screening of chemical libraries to identify modulators of NAG expression, folding and activity, and the investigation of candidate molecules and constructs for applications in enzyme replacement therapy, gene therapy, and combination therapies.Item Composition, turnover, and mechanics of extracellular matrix in developing, aging, and pathological valves for application in the design of age-specific tissue engineered heart valves(2010) Stephens, Elizabeth Humes; Grande-Allen, K. JaneDebilitating valve disease necessitating valve replacement affects patients of all ages, all of whom would benefit from a tissue engineered heart valve with immunocompatibility, the ability of the valve to remodel in response to altered hemodynamics or patient growth, and physiologic mechanics. However, there may be age-specific requirements for such a valve. The overarching goal of this thesis was to characterize the extracellular matrix in developing, aging, and pathological mitral and aortic valves (MV, AV) in order to provide design criteria for an age-specific tissue engineered heart valve. The extracellular matrix plays a vital role in valve function; not only does it comprise the bulk of the valve tissue, but it determines the material properties of the valve, is integrally involved in biological signaling processes, and is altered in a number of valve pathologies. To this end, the composition, structure, and material properties of normal MV and AV were characterized with particular attention paid to valve heterogeneity and aging-related changes. Valves from disease states such as functional mitral regurgitation, dilated cardiomyopathy, iatrogenic valve wounds, calcific aortic valve disease, and myxomatous mitral valve disease were also analyzed to provide negative design criteria for a tissue engineered heart valve. Lastly, preliminary work was performed in developing a tissue engineered heart valve using poly(ethylene) glycol (PEG) hydrogels and valve cells of different ages. In sum, this body of work provides necessary design criteria for an age-specific tissue engineered heart valve, but in the process of analyzing various aspects of normal and diseased MV and AV, this thesis additionally provides insight into a variety of aspects of normal valve physiology, such as the relationship between valve composition and material properties and the mechanical environment, as well as insight into various valve diseases, such as the role of MV remodeling in functional mitral regurgitation and disease progression, with potential clinical implications for patients with these diseases.Item Culture Models for the Study of Hypoxia in Heart Valve Angiogenesis and Calcification(2016-11-29) Sapp, Matthew C; Grande-Allen, K. JaneCalcific aortic valve disease and mitral stenosis involve the thickening, fibrosis, and eventual calcification of the valve leaflets. The standard of treatment, repair or removal of the diseased leaflets, requires highly invasive surgery. Recent research into valve calcification has been focused on the development of pharmacological treatments for the reversal of valve disease. Limited understanding of the disease process, however, has hindered progress on the creation of noninvasive therapies. The goal of this research was to identify essential factors regulating the progression of valve disease with a focus on the role of hypoxia in pathological valve angiogenesis. A set of unique culture systems established through this research was used to induce hypoxia into 3D and whole tissue models of valve disease. A 3D paper-based gel culture system, adapted from a 3D cancer model, was customized for culturing valvular interstitial cells (VICs) in collagen gels. Using this system, thick paper-based cultures were used to generate large oxygen gradients. VICs in hypoxic regions of these cultures showed enhanced activation, a crucial transition step towards calcification. VIC responses to uniform oxygen levels were evaluated using a custom hypoxic chamber created from a glass desiccator paired with a gas cylinder. VICs in thin filter paper scaffolds were cultured under hypoxia and showed enhanced activation and expression of pro-angiogenic markers. These results provide the first evidence of elevated VIC angiogenic activity in response to hypoxic stimulation. Hypoxic whole leaflet cultures served as a highly integrative model for studying VICs in the native valve environment. Whole leaflets were cultured statically under hypoxia within the desiccator-based hypoxic incubator to study changes in VIC angiogenic and calcific behavior. Computational oxygen diffusion models, created using oxygen diffusion measurements from leaflets, were used to generate heatmaps of oxygen diffusion profiles throughout the valve tissue. Whole mitral leaflets showed a substantial response to hypoxia with a loss of half of the VIC population and elevated pro-angiogenic marker expression. Mechanical stimulation, an essential factor in the progression of valve disease, was incorporated into whole leaflet studies using a dynamic flow loop bioreactor. This bioreactor model of cyclic leaflet motion was used to culture whole aortic and mitral valve leaflets under hypoxic conditions in pulsatile flow. Similar to static cultures studies, whole aortic and mitral valve leaflets showed robust expression of hypoxic markers. These dynamic, hypoxic leaflet cultures also showed increased pro-angiogenic behavior in comparison to fresh tissue controls. The in vitro hypoxic leaflet cultures used in this research are the first experimental models demonstrating the contribution of hypoxia to enhanced pro-angiogenic activity in aortic and mitral valve leaflets. Future studies can use these models to avoid hypoxia-based degeneration in tissue engineered leaflets and to work towards an effective treatment for aortic and mitral valve diseases.Item Dantrolene inhibits lysophosphatidylcholine-induced valve interstitial cell calcific nodule formation via blockade of the ryanodine receptor(Frontiers Media S.A., 2023) Sylvester, Christopher B.; Amirkhosravi, Farshad; Bortoletto, Angelina S.; West, William J.; Connell, Jennifer P.; Grande-Allen, K. JaneCalcific aortic valve disease (CAVD), a fibrocalcific thickening of the aortic valve leaflets causing obstruction of the left ventricular outflow tract, affects nearly 10 million people worldwide. For those who reach end-stage CAVD, the only treatment is highly invasive valve replacement. The development of pharmaceutical treatments that can slow or reverse the progression in those affected by CAVD would greatly advance the treatment of this disease. The principal cell type responsible for the fibrocalcific thickening of the valve leaflets in CAVD is valvular interstitial cells (VICs). The cellular processes mediating this calcification are complex, but calcium second messenger signaling, regulated in part by the ryanodine receptor (RyR), has been shown to play a role in a number of other fibrocalcific diseases. We sought to determine if the blockade of calcium signaling in VICs could ameliorate calcification in an in vitro model. We previously found that VICs express RyR isotype 3 and that its modulation could prevent VIC calcific nodule formation in vitro. We sought to expand upon these results by further investigating the effects of calcium signaling blockade on VIC gene expression and behavior using dantrolene, an FDA-approved pan-RyR inhibitor. We found that dantrolene also prevented calcific nodule formation in VICs due to cholesterol-derived lysophosphatidylcholine (LPC). This protective effect corresponded with decreases in intracellular calcium flux, apoptosis, and ACTA2 expression but not reactive oxygen species formation caused by LPC. Interestingly, dantrolene increased the expression of the regulator genes RUNX2 and SOX9, indicating complex gene regulation changes. Further investigation via RNA sequencing revealed that dantrolene induced several cytoprotective genes that are likely also responsible for its attenuation of LPC-induced calcification. These results suggest that RyR3 is a viable therapeutic target for the treatment of CAVD. Further studies of the effects of RyR3 inhibition on CAVD are warranted.Item Demonstration of Piecewise Cubic Polynomial Fitting on Mesoscale Tester Data(Rice University, 2020) Mehta, Shail Maharshi; De Santos, Diego Ricardo; Sridhar, Shweta; Aguayo, Veronica Cristina; Meraz, Carlos Alberto; Mikos, Mary; Grande-Allen, K. Jane; BioengineeringAnimation of piecewise cubic polynomial fitting to data from a replicate of 1:15 PDMS performed in order to obtain stress values at exact increments of strainItem Development of 3D Printed Mitral Valve Constructs for Transcatheter Device Modeling of Tissue and Device Deformation(Springer Nature, 2022) Vukicevic, Marija; Mehta, Shail Maharshi; Grande-Allen, K. Jane; Little, Stephen H.Transcatheter mitral valve repair (TMVR) therapies offer a minimally invasive alternative to surgical mitral valve (MV) repair for patients with prohibitive surgical risks. Pre-procedural planning and associated medical device modeling is primarily performed in silico, which does not account for the physical interactions between the implanted TMVR device and surrounding tissue and may result in poor outcomes. We developed 3D printed tissue mimics for modeling TMVR therapies. Structural properties of the mitral annuli, leaflets, and chordae were replicated from multi-material blends. Uniaxial tensile testing was performed on the resulting composites and their mechanical properties were compared to those of their target native components. Mimics of the MV annulus printed in homogeneous strips approximated the tangent moduli of the native mitral annulus at 2% and 6% strain. Mimics of the valve leaflets printed in layers of different stiffnesses approximated the force–strain and stress–strain behavior of native MV leaflets. Finally, mimics of the chordae printed as reinforced cylinders approximated the force–strain and stress–strain behavior of native chordae. We demonstrated that multi-material 3D printing is a viable approach to the development of tissue phantoms, and that printed patient-specific geometries can approximate the local deformation force which may act upon devices used for TMVR therapies.Item Development of a Polymeric Gene Delivery Vector for Application in Osteochondral Tissue Engineering(2014-04-23) Needham, Clark James; Mikos, Antonios G.; Farach-Carson, Cindy; Grande-Allen, K. Jane; Kasper, KurtIn this work, the polymeric gene delivery vector poly(ethyenimine)-hyaluronic acid (bPEI-HA)was optimized for transfection efficiency, incorporated into microparticles for controlled release, and applied directly in an oligo[poly(ethylene glycol) fumarate] (OPF) composite scaffold for osteochondral tissue generation. First, the effect of bPEI-HA synthesis parameters, specifically primary amines concentration, ligand targeting, and overall charge on the effectiveness of the vectors were investigated by altering the type and amount of hyaluronic acid (HA) oligosaccharide in the polymer. It was found that the length of the HA oligosaccharide had the most significant effect on cytotoxicity and transfection efficiency with human mesenchymal stem cells, while molar incorporation of HA, as opposed to the saccharide length and HA mass incorporation, had the greatest effect on zeta potential, but a minor effect on both cytotoxicity and transfection efficiency. Next, bPEI-HA/DNA complexes were incorporated into poly(DL-lactic-co-glycolic acid) (PLGA) microparticles and compared to microparticles containing bPEI/DNA complexes at several incorporation concentrations. It was found that the addition of HA to the bPEI vector allowed for increased loading concentration within these systems and significantly altered release kinetics without changing the morphology of the particles. Furthermore, the incorporation of HA onto the bPEI backbone significantly increased the transfection efficiency of the complexes released from the corresponding microparticle formulation. Finally, bPEI-HA was complexed with DNA encoding for the transcription factors RUNX2 and the SOX trio and incorporated into a composite hydrogel scaffold which was implanted into a rat osteochondral defect for 6 weeks. The in vitro release of this system was characterized and found to have a significant burst release over the first week of exposure to water. The in vivo analysis showed that the incorporation of DNA encoding for RUNX2 in the bone layer of these scaffolds significantly increased bone growth. The results also indicate that a spatially loaded combination of RUNX2 and SOX trio DNA loading leads to significantly better healing than empty hydrogels or either factor alone. Finally, the results of this study suggest that subchondral bone is necessary for correct cartilage healing. This research demonstrates the potential for gene delivery, and specifically bPEI-HA combined with transcription factor DNA, to be applied to in vivo osteochondral situations and result in improved tissue growth and quality.