Browsing by Author "Jacot, Jeffrey G."
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Item A multilayered scaffold of a chitosan and gelatin hydrogel supported by a PCL core for cardiac tissue engineering(Elsevier, 2013) Pok, Seokwon; Myers, Jackson D.; Madihally, Sundararajan V.; Jacot, Jeffrey G.A three-dimensional scaffold composed of self-assembled polycaprolactone (PCL) sandwiched in a gelatin–chitosan hydrogel was developed for use as a biodegradable patch with a potential for surgical reconstruction of congenital heart defects. The PCL core provides surgical handling, suturability and high initial tensile strength, while the gelatin–chitosan scaffold allows for cell attachment, with pore size and mechanical properties conducive to cardiomyocyte migration and function. The ultimate tensile stress of the PCL core, made from blends of 10, 46 and 80 kDa (Mn) PCL, was controllable in the range of 2–4 MPa, with lower average molecular weight PCL blends correlating with lower tensile stress. Blends with lower molecular weight PCL also had faster degradation (controllable from 0% to 7% weight loss in saline over 30 days) and larger pores. PCL scaffolds supporting a gelatin–chitosan emulsion gel showed no significant alteration in tensile stress, strain or tensile modulus. However, the compressive modulus of the composite tissue was similar to that of native tissue (∼15 kPa for 50% gelatin and 50% chitosan). Electron microscopy revealed that the gelatin–chitosan gel had a three-dimensional porous structure, with a mean pore diameter of ∼80 μm, showed migration of neonatal rat ventricular myocytes (NRVM), maintained NRVM viability for over 7 days, and resulted in spontaneously beating scaffolds. This multi-layered scaffold has sufficient tensile strength and surgical handling for use as a cardiac patch, while allowing migration or pre-loading of cardiac cells in a biomimetic environment to allow for eventual degradation of the patch and incorporation into native tissue.Item A Phantom Study of In-beam PET Imaging for Proton Beam Range Verification(2013-12-17) Lou, Kai; Clark, John W., Jr.; Kelly, Kevin F.; Jacot, Jeffrey G.; Shao, YipingIn-beam PET imaging is an advanced image-based method to verify the proton beam range for proton therapy by measuring proton-induced positron activity distribution and activity range. This study investigates the feasibility, accuracy and precision of the activity range measurement with a high-performance compact PET prototype system for in-beam PET imaging. An experiment with a homogeneous PMMA phantom and several Monte Carlo simulation studies are conducted. The results have shown that the prototype PET can provide reasonably good images for the activity range measurement even with low count statistics; the accuracy of activity range measurement reaches sub-millimeter; 11C is the most dominating positron emission isotope contributing to the overall positron activity; the image quality and the precision of activity range measurement depend on the count statistics, and high count statistics leads to improved image quality and precision. Although the study is preliminary with simple system set-ups, it does provide interesting and important results which should lay the basis leading to future clinically relevant investigations.Item Amniotic fluid-derived stem cells demonstrate limited cardiac differentiation following small molecule-based modulation of Wnt signaling pathway(IOP Publishing, 2015) Connell, Jennifer Petsche; Ruano, Rodrigo; Jacot, Jeffrey G.Amniotic fluid-derived stem cells (AFSC) are a promising cell source for regenerative medicine and cardiac tissue engineering. However, a non-xenotropic differentiation protocol has not been established for cardiac differentiation of AFSC. We tested a small molecule-based modulation of Wnt signaling for directed cardiac differentiation of AFSC. Cells were treated with inhibitors of glycogen synthase kinase 3 and Wnt production and secretion in a time-dependent and sequential manner, as has been demonstrated successful for cardiac differentiation of embryonic and induced pluripotent stem cells. Cells were then analyzed for gene and protein expression of markers along the cardiac lineage at multiple days during the differentiation protocol. At the midpoint of the differentiation, an increase in the percentage of AFSC expressing Islet-1, a transcription factor found in cardiac progenitor cells, and Nkx-2.5, a cardiac transcription factor, was observed. After a 15 d differentiation, a subpopulation of AFSC upregulated protein expression of smooth muscle actin, myosin light chain-2, and troponin I, all indicative of progression down a cardiac lineage. AFSC at the end of the differentiation also demonstrated organization of connexin 43, a key component of gap junctions, to cell membranes. However, no organized sarcomeres or spontaneous contraction were observed. These results demonstrate that small molecule-based modulation of Wnt signaling alone is not sufficient to generate functional cardiomyocytes from AFSC, though an upregulation of genes and proteins common to cardiac lineage cells was observed.Item Amniotic Fluid-Derived Stem Cells Demonstrated Cardiogenic Potential in Indirect Co-culture with Human Cardiac Cells(Springer, 2014) Gao, Yang; Connell, Jennifer Petsche; Wadhwa, Lalita; Ruano, Rodrigo; Jacot, Jeffrey G.Amniotic fluid-derived stem cells (AFSC) have been shown to be broadly multipotent and non-tumorogenic. Previous studies of direct mixing of AFSC and neonatal rat ventricle myocytes indicated evidence of AFSC cardiogenesis. In this study, we examined human AFSC cardiogenic potential in indirect co-culture with human cardiac cells in conditions that eliminated the possibility of cell fusion. Human AFSC in contact with human cardiac cells showed expression of cardiac troponin T (cTnT) in immunohistochemistry, and no evidence of cell fusion were found through fluorescent in situ hybridization. When indirectly co-cultured with cardiac cells, human AFSC in contact with cardiac cells across a thin porous membrane showed a statistically significant increase in cTnT expression compared to non-contact conditions but lacked upregulation of calcium modulating proteins and did not have functional or morphological characteristics of mature cardiomyocytes. This suggests that contact is a necessary but not sufficient condition for AFSC cardiac differentiation in co-culture with cardiac cells.Item Amniotic fluid-derived stem cells for cardiovascular tissue engineering applications(Mary Ann Liebert, Inc., 2013) Petsche, Jennifer J.; Camci-Unal, Gulden; Khademhosseini, Ali; Jacot, Jeffrey G.Recent research has demonstrated that a population of stem cells can be isolated from amniotic fluid removed by amniocentesis that are broadly multipotent and non-tumorogenic. These amniotic fluid-derived stem cells (AFSC) could potentially provide an autologous cell source for treatment of congenital defects identified during gestation, particularly cardiovascular defects. In this review, the various methods of isolating, sorting and culturing AFSC are compared, along with techniques for inducing differentiation into cardiac myocytes and endothelial cells. Though research has not demonstrated complete and high yield cardiac differentiation, AFSC have been shown to effectively differentiate into endothelial cells and can effectively support cardiac tissue. Additionally, several tissue engineering and regenerative therapeutic approaches for the use of these cells in heart patches, injection after myocardial infarction, heart valves, vascularized scaffolds and blood vessels are summarized. These applications show great promise in the treatment of congenital cardiovascular defects, and further studies of isolation, culture, and differentiation of AFSC will help to develop their use for tissue engineering, regenerative medicine, and cardiovascular therapies.Item An Assessment of Gadonanotubes as Magnetic Nanolabels for Improved Stem Cell Detection and Retention in Cardiomyoplasty(2013-07-24) Tran, Lesa; Wilson, Lon J.; Matsuda, Seiichi P. T.; Jacot, Jeffrey G.; Perin, Emerson C.; Cabreira, Maria da GraçaIn this work, gadolinium-based carbon nanocapsules are developed as a novel nanotechnology that addresses the shortcomings of current diagnostic and therapeutic methods of stem cell-based cardiomyoplasty. With cardiovascular disease (CVD) responsible for approximately 30% of deaths worldwide, the growing need for improved cardiomyoplasty has spurred efforts in nanomedicine to develop innovative techniques to enhance the therapeutic retention and diagnostic tracking of transplanted cells. Having previously been demonstrated as a high-performance T1-weighted magnetic resonance imaging (MRI) contrast agent, Gadonanotubes (GNTs) are shown for the first time to intracellularly label pig bone marrow-derived mesenchymal stem cells (MSCs). Without the use of a transfection agent, micromolar concentrations of GNTs deliver up to 10^9 Gd(III) ions per cell, allowing for MSCs to be visualized in a 1.5 T clinical MRI scanner. The cellular response to the intracellular incorporation of GNTs is also assessed, revealing that GNTs do not compromise the viability, differentiation potential, or phenotype characteristics of the MSCs. However, it is also found that GNT-labeled MSCs exhibit a decreased response to select cell adhesion proteins and experience a non-apoptotic, non-proliferative cell cycle arrest, from which the cells recover 48 h after GNT internalization. In tandem with developing GNTs as a new stem cell diagnostic agent, this current work also explores for the first time the therapeutic application of the magnetically-active GNTs as a magnetic facilitator to increase the retention of transplanted stem cells during cardiomyoplasty. In vitro flow chamber assays, ex vivo perfusion experiments, and in vivo porcine injection procedures all demonstrate the increased magnetic-assisted retention of GNT-labeled MSCs in the presence of an external magnetic field. These studies prove that GNTs are a powerful ‘theranostic’ agent that provides a novel platform to simultaneously monitor and improve the therapeutic nature of stem cells for the treatment of CVD. It is expected that this new nanotechnology will further catalyze the development of cellular cardiomyoplasty and other stem cell-based therapies for the prevention, detection, and treatment of human diseases.Item Bioactive Poly(ethylene glycol)-based Hydrogels for Characterization of Matrix Influences on a Lung Cancer Metastasis Model(2013-09-16) Gill, Bj; West, Jennifer L.; Jacot, Jeffrey G.; Farach-Carson, CindyPathological changes to tumor extracellular matrix (ECM) composition, mechanics, and architecture promote cancer progression and metastasis. Exploration of tumor-ECM interactions using in vitro matrix-mimetic culture systems has largely been restricted to naturally-derived matrix materials that permit limited experimental control. Such study of a novel lung adenocarcinoma model in Matrigel™ (MG) has suggested key matrix cues that mediate epithelial-mesenchymal transition (EMT) and metastasis. In this thesis work, synthetic hydrogel scaffolds based on poly(ethylene glycol) (PEG) featuring high experimental control and modular bioactivity were used to study matrix influences on the EMT-prone model line 344SQ. Encapsulation of 344SQ cells in PEG hydrogels modified for cell adhesivity and cell-mediated enzymatic degradability induced formation of lumenized, polarized spheres mimicking the epithelial phenotype observed in three-dimensional MG. Tuning matrix stiffness, adhesive ligand concentration, and ligand spatial presentation altered epithelial morphogenesis. Exploration of the EMT phenotype of PEG-encapsulated 344SQ cells revealed TGFβ-initiated changes in morphology, polarity, expression levels of EMT marker genes and their epigenetic controller, and the organization of cell-secreted ECM. Notably, a potent role for adhesive ligand was illuminated as matrices with low PEG-RGDS concentration even in the absence of TGFβ induced formation of spheres with a post-EMT phenotype by several of these measures. A matrix-invasive phenotype was also revealed by altering matrix structural parameters and tuned with incorporation of an alternative protease-cleavable sequence. Finally, the influence of cell-cell contacts was explored by covalent incorporation of cadherin proteins into the matrix. Matrix-tethered E- and -N-cadherin affected 344SQ sphere development in otherwise non-cell-adhesive matrices and modulated polarity and the degree of TGFβ response. Further, in 344SQ with a knockdown of the essential polarity-determining protein Scribble, matrix-tethered cadherin influenced the formation of a phenotype with partially normalized epithelial polarity with corresponding differences in membrane localization of cell-expressed E-cadherin. Overall, this thesis demonstrates the utility of the more experimentally controllable PEG system in studying ECM influences on cancer progression with findings providing greater insight into stromal biomechanical, biochemical, and cell-cell factors that mediate lung adenocarcinoma epithelial morphogenesis and EMT. These contributions help advance the state of the field towards a goal of developing new metastasis-targeting cancer therapeutics.Item Biocompatible Carbon Nanotube–Chitosan Scaffold Matching the Electrical Conductivity of the Heart(American Chemical Society, 2014) Pok, Seokwon; Vitale, Flavia; Eichmann, Shannon L.; Benavides, Omar M.; Pasquali, Matteo; Jacot, Jeffrey G.; The Smalley Institute for Nanoscale Science & TechnologyThe major limitation of current engineered myocardial patches for the repair of heart defects is that insulating polymeric scaffold walls hinder the transfer of electrical signals between cardiomyocytes. This loss in signal transduction results in arrhythmias when the scaffolds are implanted. We report that small, subtoxic concentrations of single-walled carbon nanotubes, on the order of tens of parts per million, incorporated in a gelatin–chitosan hydrogel act as electrical nanobridges between cardiomyocytes, resulting in enhanced electrical coupling, synchronous beating, and cardiomyocyte function. These engineered tissues achieve excitation conduction velocities similar to native myocardial tissue (22 ± 9 cm/s) and could function as a full-thickness patch for several cardiovascular defect repair procedures, such as right ventricular outflow track repair for Tetralogy of Fallot, atrial and ventricular septal defect repair, and other cardiac defects, without the risk of inducing cardiac arrhythmias.Item Cardiac MRI: Improved Assessment of Left Ventricular Function, Wall Motion, and Viability(2013-09-16) Krishnamurthy, Ramkumar; Drezek, Rebekah A.; Muthupillai, Raja; Jacot, Jeffrey G.; Clark, John W., Jr.Heart failure is the clinical syndrome accompanying the inability of the heart to maintain a cardiac output required to meet the metabolic requirements and accommodate venous return, and is one of the leading causes of mortality in United States. Accurate imaging of the heart and its failure is important for successful patient management and treatment. Multiple cardiac imaging modalities provide complementary information about the heart – LV function and wall motion, anatomy, myocardial viability and ischemia. In many instances, it is necessary for a patient to undergo multiple imaging sessions to obtain diagnostic clinical information with confidence. It would be beneficial to the individual and the health care system if a single imaging modality could yield reliable clinical information about the heart, leading to a reduced cost, anxiety and an increased diagnostic confidence. This thesis proposes methods that would make cardiac MRI perform an improved assessment of LV function, wall motion, and viability, such that cardiac MRI is taken one step closer to being a single stop solution for imaging of heart. Conventional cardiac MR imaging is performed at a temporal resolution of around 40 ms per cardiac phase. While the global left ventricular (LV) function can be reliably established at this temporal resolution, functional metrics characterizing transient function like peak filling and ejection rates are not accurately assessed. A high temporal resolution is necessary to characterize such transient LV function and wall motion mechanics. This thesis proposes methods to acquire cine-images of the heart at a higher temporal resolution (~ 6 ms) and algorithms to acquire the LV volume across all cardiac phases that would yield functional metrics characterizing LV function and wall motion mechanics. The validation of these algorithms was performed on human subjects. Cardiac MR imaging is the current gold standard of myocardial viability imaging, in which scarred regions of the heart following myocardial infarction are visualized. However viability imaging faces image quality challenges in patients with severe arrhythmias and in cases where a higher spatial resolution, and hence a longer acquisition time, is desired. This thesis also proposes an arrhythmia insensitive inversion recovery (AIIR) algorithm that would significantly reduce artifacts that degrade image quality, thereby extending viability imaging to higher spatial resolution and in patients with severe arrhythmia. Simulations, experimental validation on phantoms and clinical verification on patients are performed. Results from high temporal resolution imaging reveal that obtaining cine cardiac MR images at a temporal resolution of 6 ms per cardiac phase is feasible. Appropriate validated algorithms yield LV time-volume curve from which LV functional metrics are reliably extracted. A dependence on temporal resolution is revealed, and a temporal resolution cut-off of 12 ms is proposed to reliably capture the temporal dynamics of the LV. Also, results from cardiac viability imaging show that the AIIR algorithm performs significantly better than conventional imaging methods in both phantoms and human subjects, as shown by the blinded expert scores, leading to a better image quality. In conclusion, this thesis proposes and implements methods that help cardiac MRI yield 1) a better function and wall motion assessment of the heart through high temporal resolution imaging and 2) a better assessment of myocardial viability through the AIIR algorithm.Item Clinical and Molecular Comparison of Pediatric and Adult Reverse Remodeling With Ventricular Assist Devices(Wiley, 2015) Weia, Benjamin C.; Adachi, Iki; Jacot, Jeffrey G.Ventricular assist device (VAD) support induces reverse remodeling of failing myocardium that leads to occasional functional recovery of the adult heart. While there have been numerous clinical reports in adult patients with end-stage cardiomyopathy, little is known about reverse remodeling in children, which has increasing clinical potential with the recent expansion of pediatric VADs in the setting of static organ supply for heart transplantation. Pediatric myocardium also promises theoretical advantages for recovery over adult myocardium due to its greater abundance of cardiac progenitor cells. To identify potential targets of future studies, we conducted a literature review with two aims: (i) to summarize clinical cases of pediatric patients who exhibited cardiac recovery following VAD support; and (ii) to analyze genetic changes in pediatric myocardium induced by VAD support compared with those observed in adult patients. Several clinical series of pediatric VAD cases report that small proportions of their cohorts were weaned off from device support, but a lack of information about the etiology and support duration of these patients limits the ability to determine whether they represent reverse remodeling of myocardial structure or just recovery from acute illness. A comparison of pediatric and adult gene expression changes with VAD support reveals approximately 40% of genes to be oppositely regulated, indicating that the pediatric genetic response is distinct. These observations highlight a necessity to better understand reverse remodeling specific to pediatric myocardium, which is crucial to improving clinical strategies for bridge-to-recovery in children.Item Development of a 3D Tissue Engineered Bone Tumor Model(2013-09-16) Burdett, Emily; Mikos, Antonios G.; Ludwig, Joseph A.; Kasper, Kurt; Jacot, Jeffrey G.; Zygourakis, Kyriacos3D ex vivo tumor models are required which better replicate the microenvironment encountered by tumor cells in vivo. In this study, we applied bone tissue engineering culture techniques to develop an ex vivo 3D bone tumor model. Ewing sarcoma cells were cultured on poly(ε-caprolactone) (PCL) microfiber scaffolds, and cellular growth kinetics, morphology, and infiltration were assessed. Cell/scaffold constructs were then exposed to anticancer drugs for up to 16 days and drug response was compared to 2D controls. Ewing sarcoma cells were capable of attachment and proliferation on PCL scaffolds and dense scaffold infiltration up to 200 micrometers. Constructs could be maintained in culture for up to 32 days, and high density 3D cell growth conferred an increased resistance to anticancer drugs over 2D controls. This 3D tumor model shows potential for use in future studies of bone tumor biology, especially as it pertains to the development of new anticancer drugs.Item Differentiation of spontaneously contracting cardiomyocytes from non-virally reprogrammed human amniotic fluid stem cells(Public Library of Science, 2017) Velasquez-Mao, Aaron J.; Tsao, Christopher J.M.; Monroe, Madeline N.; Legras, Xavier; Bissig-Choisat, Beatrice; Bissig, Karl-Dimiter; Ruano, Rodrigo; Jacot, Jeffrey G.Congenital heart defects are the most common birth defect. The limiting factor in tissue engineering repair strategies is an autologous source of functional cardiomyocytes. Amniotic fluid contains an ideal cell source for prenatal harvest and use in correction of congenital heart defects. This study aims to investigate the potential of amniotic fluid-derived stem cells (AFSC) to undergo non-viral reprogramming into induced pluripotent stem cells (iPSC) followed by growth-factor-free differentiation into functional cardiomyocytes. AFSC from human second trimester amniotic fluid were transfected by non-viral vesicle fusion with modified mRNA of OCT4, KLF4, SOX2, LIN28, cMYC and nuclear GFP over 18 days, then differentiated using inhibitors of GSK3 followed 48 hours later by inhibition of WNT. AFSC-derived iPSC had high expression of OCT4, NANOG, TRA-1-60, and TRA-1-81 after 18 days of mRNA transfection and formed teratomas containing mesodermal, ectodermal, and endodermal germ layers in immunodeficient mice. By Day 30 of cardiomyocyte differentiation, cells contracted spontaneously, expressed connexin 43 and β-myosin heavy chain organized in sarcomeric banding patterns, expressed cardiac troponin T and β-myosin heavy chain, showed upregulation of NKX2.5, ISL-1 and cardiac troponin T with downregulation of POU5F1, and displayed calcium and voltage transients similar to those in developing cardiomyocytes. These results demonstrate that cells from human amniotic fluid can be differentiated through a pluripotent state into functional cardiomyocytes.Item Evaluation of Endothelial Cells Differentiated from Amniotic Fluid-Derived Stem Cells(Mary Ann Liebert, Inc., 2012) Benavides, Omar M.; Petsche, Jennifer J.; Moise, Kenneth J. Jr.; Johnson, Anthony; Jacot, Jeffrey G.Amniotic fluid holds great promise as a stem cell source, especially in neonatal applications where autologous cells can be isolated and used. This study examined chemical-mediated differentiation of amniotic fluid-derived stem cells (AFSC) into endothelial cells and verified the function of AFSC-derived endothelial cells (AFSC-EC). AFSC were isolated from amniotic fluid obtained from second trimester amnioreduction as part of therapeutic intervention from pregnancies affected with twin-twin transfusion syndrome. Undifferentiated AFSC were of normal karyotype with a subpopulation of cells positive for the embryonic stem cell marker SSEA4, hematopoietic stem cell marker c-kit, and mesenchymal stem cell markers CD29, CD44, CD73, CD90, and CD105. Additionally, these cells were negative for the endothelial marker CD31 and hematopoietic differentiation marker CD45. AFSC were cultured in endothelial growth media with concentrations of vascular endothelial growth factor (VEGF) ranging from 1 to 100 ng/mL. After 2 weeks, AFSC-EC expressed von Willebrand factor, endothelial nitric oxide synthase, CD31, VE-cadherin, and VEGF receptor 2. Additionally, the percentage of cells expressing CD31 was positively correlated with VEGF concentration up to 50 ng/mL, with no increase at higher concentrations. AFSC-EC showed a decrease in stem cells markers c-kit and SSEA4 and were morphologically similar to human umbilical vein endothelial cells (HUVEC). In functional assays, AFSC-EC formed networks and metabolized acetylated low-density lipoprotein, also characteristic of HUVEC. Nitrate levels for AFSC-EC, an indirect measure of nitric oxide synthesis, were significantly higher than undifferentiated controls and significantly lower than HUVEC. These results indicate that AFSC can differentiate into functional endothelial-like cells and may have the potential to provide vascularization for constructs used in regenerative medicine strategies.Item Formation of functional gap junctions in amniotic fluid-derived stem cells induced by transmembrane co-culture with neonatal rat cardiomyocytes(Foundation for Cellular and Molecular Medicine/Blackwell Publishing Ltd, 2013) Connell, Jennifer Petsche; Augustini, Emily; Moise, Kenneth J. Jr.; Johnson, Anthony; Jacot, Jeffrey G.Amniotic fluid-derived stem cells (AFSC) have been reported to differentiate into cardiomyocyte-like cells and form gap junctions when directly mixed and cultured with neonatal rat ventricular myocytes (NRVM). This study investigated whether or not culture of AFSC on the opposite side of a Transwell membrane from NRVM, allowing for contact and communication without confounding factors such as cell fusion, could direct cardiac differentiation and enhance gap junction formation. Results were compared to shared media (Transwell), conditioned media and monoculture media controls. After a 2-week culture period, AFSC did not express cardiac myosin heavy chain or troponin T in any co-culture group. Protein expression of cardiac calsequestrin 2 was up-regulated in direct transmembrane co-cultures and media control cultures compared to the other experimental groups, but all groups were up-regulated compared with undifferentiated AFSC cultures. Gap junction communication, assessed with a scrape-loading dye transfer assay, was significantly increased in direct transmembrane co-cultures compared to all other conditions. Gap junction communication corresponded with increased connexin 43 gene expression and decreased phosphorylation of connexin 43. Our results suggest that direct transmembrane co-culture does not induce cardiomyocyte differentiation of AFSC, though calsequestrin expression is increased. However, direct transmembrane co-culture does enhance connexin-43-mediated gap junction communication between AFSC.Item In situ vascularization of injectable fibrin/poly(ethylene glycol) hydrogels by human amniotic fluid-derived stem cells(Wiley, 2015) Benavides, Omar M.; Brooks, Abigail R.; Cho, Sung Kyung; Connell, Jennifer Petsche; Ruano, Rodrigo; Jacot, Jeffrey G.One of the greatest challenges in regenerative medicine is generating clinically relevant engineered tissues with functional blood vessels. Vascularization is a key hurdle faced in designing tissue constructs larger than the in vivo limit of oxygen diffusion. In this study, we utilized fibrin-based hydrogels to serve as a foundation for vascular formation, poly(ethylene glycol) (PEG) to modify fibrinogen and increase scaffold longevity, and human amniotic fluid-derived stem cells (AFSC) as a source of vascular cell types (AFSC-EC). AFSC hold great potential for use in regenerative medicine strategies, especially those involving autologous congenital applications, and we have shown previously that AFSC-seeded fibrin-PEG hydrogels have the potential to form three-dimensional vascular-like networks in vitro. We hypothesized that subcutaneously injecting these hydrogels in immunodeficient mice would both induce a fibrin-driven angiogenic host response and promote in situ AFSC-derived neovascularization. Two weeks postinjection, hydrogels were sectioned, and the following was demonstrated: the average maximum invasion distance of host murine cells into the subcutaneous fibrin/PEG scaffold was 147 ± 90 µm after 1 week and 395 ± 138 µm after 2 weeks; the average number of cell-lined lumen per square millimeter was significantly higher in hydrogels seeded with stem cells or cocultures containing stem cells (MSC, 36.5 ± 11.4; AFSC, 47.0 ± 18.9; AFSC/AFSC-EC, 32.8 ± 11.6; and MSC/HUVEC, 43.1 ± 25.1) versus endothelial cell types alone (AFSC-EC, 9.7 ± 6.1; HUVEC, 14.2 ± 8.8); and a subset of these lumen were characterized by the presence of red blood cells. Select areas of cell-seeded hydrogels contained CD31+ lumen surrounded by α-smooth muscle cell support cells, whereas control hydrogels with no cells only showed infiltration of α-smooth muscle cell–positive host cells.Item Preparation of Monodomain Liquid Crystal Elastomers and Liquid Crystal Elastomer Nanocomposites(JoVE, 2016) Kim, Hojin; Zhu, Bohan; Chen, Huiying; Adetiba, Oluwatomiyin; Agrawal, Aditya; Ajayan, Pulickel; Jacot, Jeffrey G.; Verduzco, RafaelLCEs are shape-responsive materials with fully reversible shape change and potential applications in medicine, tissue engineering, artificial muscles, and as soft robots. Here, we demonstrate the preparation of shape-responsive liquid crystal elastomers (LCEs) and LCE nanocomposites along with characterization of their shape-responsiveness, mechanical properties, and microstructure. Two types of LCEs — polysiloxane-based and epoxy-based — are synthesized, aligned, and characterized. Polysiloxane-based LCEs are prepared through two crosslinking steps, the second under an applied load, resulting in monodomain LCEs. Polysiloxane LCE nanocomposites are prepared through the addition of conductive carbon black nanoparticles, both throughout the bulk of the LCE and to the LCE surface. Epoxy-based LCEs are prepared through a reversible esterification reaction. Epoxy-based LCEs are aligned through the application of a uniaxial load at elevated (160 °C) temperatures. Aligned LCEs and LCE nanocomposites are characterized with respect to reversible strain, mechanical stiffness, and liquid crystal ordering using a combination of imaging, two-dimensional X-ray diffraction measurements, differential scanning calorimetry, and dynamic mechanical analysis. LCEs and LCE nanocomposites can be stimulated with heat and/or electrical potential to controllably generate strains in cell culture media, and we demonstrate the application of LCEs as shape-responsive substrates for cell culture using a custom-made apparatus.Item Radiofrequency-induced Cellular Hyperthermia: Water-soluble Fullerene as a New Cancer Therapeutic Agent(2014-09-08) Cheney, Matthew Andrade; Wilson, Lon J.; Curley, Steven A.; Billups, W. Edward; Jacot, Jeffrey G.Due to the susceptibility of cancer to hyperthermia, abundant biomedical research is being conducted for the development of therapies using nanotechnology for noninvasive cancer hyperthermia. In this work, the water-soluble and neutrally-charged C60 fullerene, C60-ser, is presented as a new cancer therapeutic agent for the hyperthermia of liver cancer using radiofrequency (RF) energy. With liver cancer being the second leading cause of death in men and the sixth most in women worldwide, new and improved therapies are needed to combat the limitations associated with current cancer treatments. Using a RF generator operating at 13.56 MHz, aqueous heating of purified C60-ser within the RF field has been observed in a concentration- and aggregation-dependent manner. These findings now lay the foundation for a new cancer hyperthermia therapy using C60 fullerene. Though the exact mechanism of C60-ser heating within the RF field is still uncertain, evidence has shown that the six serinol malonate (ser) groups of C60-ser must be attached to C60 to produce any significant heating. C60 is known to play numerous roles in the biomedical field, including drug delivery, active cancer cell targeting, bone therapy, gene therapy, X-ray contrast enhancement, and now, through the present work, as a potential new cancer therapy by RF-induced hyperthermia. By attaching a fluorescent tag (PF) to C60-ser to form C60-serPF, passive and active internalization into Hep3B and Huh7 liver cancer cells has been observed, with evidence of internalization within the cytoplasm and, more surprisingly, the nucleus. Very few nanomaterials have been definitively proven to internalize within the nucleus of cells, raising the potential for both C60-ser and C60-serPF to be used for other biomedical purposes and not just solely for RF-induced hyperthermia effects on cellular DNA. In vivo biodistribution studies of C60-serPF also has showed its presence in all major organs, including the brain. Since C60-ser was shown to inhibit the internalization of C60-serPF in vitro, uptake by liver cancer cells of both nanomaterials proceed by similar pathways. Therefore, the ability of C60-serPF to traverse the blood brain barrier of mice suggests an array of opportunities for brain-related treatments and therapies using both C60-serPF and C60-ser. Taken together, the materials provide simultaneous diagnostic and therapeutic capabilities, making them a new class of theranostic agent. In vitro studies of C60-ser have shown that it is non-toxic at up to 1.0 mg/mL to liver cancer cells (Hep3B, SNU449, and HepG2), that it modulates cellular metabolic activity, that it negligibly influences the cell cycle, but that it does not promote radiofrequency-induced hyperthermia of individual liver cancer cells. However, when injected intratumorally in mice, C60-ser enhanced the RF heating of liver tumors by 89% and 26% when compared to PBS and water controls alone, respectively. This observed increase in heating led to significant necrosis for the C60-ser treated groups, while necrosis for the control groups was nonexistent. This work has shown in vivo efficacy of an RF/C60-ser combination therapy by hyperthermia for the treatment of liver cancer for the first time. Future directions must involve the incorporation of biological targeting groups conjugated to the C60 moiety to realize the full potential of a noninvasive and targeted approach to cancer cell hyperthermia for liver cancer and other types of cancer, as well.Item Remodeling of ECM patch into functional myocardium in an ovine model: A pilot study(Wiley, 2015) Scully, Brandi B.; Fan, Chris; Grigoryan, Bagrat; Jacot, Jeffrey G.; Vick, G. Wesley III; Kim, Jeffrey J.; Fraser, Charles D.; Grande-Allen, K. Jane; Morales, David L.S.Background: Previous studies have demonstrated that surgical patches comprised of small intestinal submucosa-derived extracellular matrix (ECM) have biological remodeling potential. This pilot study investigated histological, mechanical, and bioelectrical properties of an ECM patch implanted in the ovine right-ventricular outflow tract (RVOT). Materials and Methods: ECM patches (2 × 2 cm2) were implanted in four Western Range sheep (wether males, 37–49 kg, age <1 year) and explanted at 5 months (n = 2) and 8 months (n = 2). In vivo analysis included epicardial echocardiography and contact electrical mapping. Optical mapping was used to map electrical activity of two hearts on a Langendorff preparation. Mechanical testing quantified stiffness. Histological stains characterized structure, neovascularization, and calcification; immunohistochemistry (IHC) assessed cell phenotype. Results: In vivo analysis showed that ECM patch tissue was contractile by M-mode and two-dimensional echocardiographic evaluation. In vivo electrical mapping, and optical mapping confirmed that ECM conducted an organized electrical signal. Mechanical testing of native and ECM patched RVOT tissue showed an elastic modulus of the implanted patch comparable to native tissue stiffness. Conclusions: At 5 and 8 months, the ECM had undergone extracellular matrix remodeling and neovascularization without calcification. The ECM was populated with locally aligned muscle cells positive for sarcomeric alpha-actinin, CD45, and troponin I and T. In sheep, the ECM patch appears to have the potential of remodeling to resemble native, functional ventricular tissue as evidenced by histological, mechanical, and electrical properties.Item Responsive liquid crystal elastomers for enhanced cell sheet alignment(2017-04-18) Verduzco, Rafael; Jacot, Jeffrey G.; Adetiba, Oluwatomiyin; Agrawal, Aditya; Rice University; United States Patent and Trademark OfficeResponsive, biocompatible substrates are of interest for directing the maturation and function of cells in vitro during cell culture. This can potentially provide cells and tissues with desirable properties for regenerative therapies. The present disclosure provides a scalable approach to attach, align and dynamically load cells on responsive liquid crystal elastomer (LCE) substrates. Monodomain LCEs exhibit reversible shape changes in response to cyclic stimulus, and when immersed in an aqueous medium on top of, for example, resistive heaters, shape changes are fast, reversible and produce minimal temperature changes in the surroundings.Item Stem Cells and Progenitor Cells for Tissue-Engineered Solutions to Congenital Heart Defects(Libertas Academica Ltd, 2015) Gao, Yang; Jacot, Jeffrey G.Synthetic patches and fixed grafts currently used in the repair of congenital heart defects are nonliving, noncontractile, and not electrically responsive, leading to increased risk of complication, reoperation, and sudden cardiac death. Studies suggest that tissue-engineered patches made from living, functional cells could grow with the patient, facilitate healing, and help recover cardiac function. In this paper, we review the research into possible sources of cardiomyocytes and other cardiac cells, including embryonic stem cells, induced pluripotent stem cells, mesenchymal stem cells, adipose-derived stem cells, umbilical cord blood cells, amniotic fluid-derived stem cells, and cardiac progenitor cells. Each cell source has advantages, but also has technical hurdles to overcome, including heterogeneity, functional maturity, immunogenicity, and pathogenicity. Additionally, biomaterials used as patch materials will need to attract and support desired cells and induce minimal immune responses.