Browsing by Author "Benavides, Omar M."
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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 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 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 Use of Myocardial Matrix in a Chitosan-Based Full-Thickness Heart Patch(Mary Ann Liebert, Inc., 2014) Pok, Seokwon; Benavides, Omar M.; Hallal, Patrick; Jacot, Jeffrey G.A novel cardiac scaffold comprised of decellularized porcine heart matrix was investigated for use as a biodegradable patch with a potential for surgical reconstruction of the right ventricular outflow tract. Powdered heart matrix solution was blended with chitosan and lyophilized to form three-dimensional scaffolds. For this investigation, we examined the influence of different blending ratios of heart matrix to chitosan on porosity and mechanical properties, then gene expression and electrophysiological function of invading neonatal rat ventricular myocytes (NRVM) compared to type-A gelatin/chitosan composite scaffolds. Heart matrix/chitosanblended hydrogels (1.6 mg/mL heart matrix) had similar porosity (109 - 34 mm), and elastic modulus (13.2 - 4.0 kPa) as previously published gelatin/chitosan scaffolds. Heart matrix/chitosan hydrogels maintained > 80% viability and had higher NRVM retention (*1000 cells/mm2) than gelatin/chitosan scaffolds. There was a significant increase in a-myosin heavy chain and connexin-43 expression in NRVM cultured on heart matrix/chitosan scaffolds after 14 days compared with gelatin/chitosan scaffolds. Further, heart matrix/chitosan scaffolds had significantly higher conduction velocity (12.6 - 4.9 cm/s) and contractile stress (0.79 - 0.13 mN/mm2) than gelatin/chitosan scaffolds. In summary, NRVM cultured on heart matrix scaffold showed improvements in contractile and electrophysiological function.