Evaluation of cellular interactions with functionalized scaffolds for cardiovascular tissue engineering

Introduction: Cardiovascular disease is a leading cause of death worldwide, with heart valve disease in particular becoming a rising problem in developing countries. Tissue engineering offers the next step in heart valve tissue replacement surgery. Synthetic scaffolds used in tissue engineering often do not have sufficient cell adhesion, thus the addition of biosignals is crucial. Surface modifications can be used to improve desired cell adhesion and proliferation. To covalently attach cell adhesion peptides/biosignals, synthetic hydrogel spacers are often used in an interim grafting step. Poly (acrylic acid) (PAA) is a non-toxic, FDA approved hydrogel that has been shown in preliminary studies (previous research and own experience) to improve cell adhesion even without the addition of specific cell-binding compounds. This project aims to show the effect of systematically increasing the concentration of PAA modified 2D and 3D polyurethanes (biodegradable and biostable) on cell adhesion, persistence, and proliferation. Experimental Methods: In Part 1, 2D nondegradable Pellethane® films were surface modified by varying the poly (acrylic acid-co-acrylamide) (P(AA-co-AM)) comonomer feed ratio from 0 to 100 % PAA in 20 % increments, using poly (acrylamide) (PAM) as a copolymer (unmodified and collagen coated controls). Surface properties were analysed using SEM imaging, staining, energy-dispersive X-ray spectrometry (SEM-EDS) and toluidine blue carboxyl assays (TBCA). Endothelial cells were isolated from human saphenous veins using an enzymatic digestion method, and identified by staining with DAPI and Cy3 against CD31. Isolated endothelial cells and human dermal fibroblasts (Cell bank: R039/2016) were seeded onto Pellethane® films (8 000 cells/film). Live/dead staining and XTT cell viability assays were performed over 24 and 72 hrs, respectively. Following this, XTT cell viability assays were performed at 7 and 24 hrs post-seeding on endothelial cells cultured under serum-free conditions (20 000 cells/film; unmodified and 80 % PAA). In Part 2, both Pellethane® and DegraPol® (degradable) 2D films and 3D electrospun scaffolds were used. The polymer samples were surface modified with 0, 40, and 80 % PAA. All samples were imaged using SEM prior to in vitro cell culture evaluation. Endothelial cells were seeded (8 000 cells/film) onto surface modified polymer samples, and XTT cell viability assays were performed over 72 hrs. Three-dimensional scaffolds seeded with endothelial cells (20 000 and 50 000 cells/film; unmodified and 80 % PAA) were immunocytochemically stained (Hoechst and CD31) at Day 1, 3, and 7 post-seeding. Results and discussion: In Part 1, SEM imaging and preliminary staining confirmed the addition of PAA to polymer surfaces. Systematically increasing [AA] in the P(AA-co-AM) comonomer ratio resulted in the expected increase in surface-COOH functional groups (TBCA and SEM-EDS). The number of COOH groups increased as [PAA] increased from 0-40 % (R 2=0.76; P 0.0001) before plateauing (TBCA). This was further confirmed by a decreasing N/O ratio with increasing [AA] monomer (R2=0.70; P< 0.001) (SEM-EDS). An increase in [PAA] resulted in a linear increase in endothelial cell adhesion and persistence (R 2=0.92 (live/dead staining) and 0.96 (XTT cell viability assays); P< 0.05). Endothelial cell viability on surfaces modified with 80 and 100 % PAA was comparable to that achieved on the collagen positive control. High concentrations of PAA also showed improved fibroblast adhesion (R2= 0.71 (live/dead staining) and 0.54 (XTT cell viability assays); P< 0.05) but did not display any persistence or viability close to that obtained on the collagen. Collagen coated surfaces displayed the highest cell adhesion and proliferation for both cell types (XTT cell viability assays and live/dead staining). Endothelial cell adhesion was improved by both the addition of PAA to the polymer surface, and FBS to the cell culture medium (P< 0.05) (cells cultured under serum-free conditions). In Part 2, the improvement of endothelial cell adhesion on PAA modified 2D Pellethane® films was confirmed and additionally shown on 2D DegraPol® (P ≤0.05) (XTT cell viability assays). However, the endothelial cell persistence seen in earlier assays was not observed. The positive effect of increasing [PAA] did not translate to 3D scaffolds, and cell behaviour was improved on unmodified surfaces in comparison to any of the PAA modified groups (XTT cell viability assays and immunocytochemical staining). This discrepancy is proposed to be a difference in grafting efficiency on the degradable materials and the 3D structure of the electrospun scaffolds. Conclusions: An increase in PAA surface modification on polyurethane can improve endothelial cell adhesion and persistence on nondegradable 2D polyurethane scaffolds. These results did not translate to electrospun scaffolds, probably due to the complex 3D cell environment. Further investigation is required for use in TEHV and other applications

Modeling an In-Register, Parallel “Iowa” Aβ Fibril Structure Using Solid-State NMR Data from Labeled Samples with Rosetta

SummaryDetermining the structures of amyloid fibrils is an important first step toward understanding the molecular basis of neurodegenerative diseases. For β-amyloid (Aβ) fibrils, conventional solid-state NMR structure determination using uniform labeling is limited by extensive peak overlap. We describe the characterization of a distinct structural polymorph of Aβ using solid-state NMR, transmission electron microscopy (TEM), and Rosetta model building. First, the overall fibril arrangement is established using mass-per-length measurements from TEM. Then, the fibril backbone arrangement, stacking registry, and “steric zipper” core interactions are determined using a number of solid-state NMR techniques on sparsely 13C-labeled samples. Finally, we perform Rosetta structure calculations with an explicitly symmetric representation of the system. We demonstrate the power of the hybrid Rosetta/NMR approach by modeling the in-register, parallel “Iowa” mutant (D23N) at high resolution (1.2Å backbone rmsd). The final models are validated using an independent set of NMR experiments that confirm key features

The JOREK non-linear extended MHD code and applications to large-scale instabilities and their control in magnetically confined fusion plasmas

JOREK is a massively parallel fully implicit non-linear extended magneto-hydrodynamic (MHD) code for realistic tokamak X-point plasmas. It has become a widely used versatile simulation code for studying large-scale plasma instabilities and their control and is continuously developed in an international community with strong involvements in the European fusion research programme and ITER organization. This article gives a comprehensive overview of the physics models implemented, numerical methods applied for solving the equations and physics studies performed with the code. A dedicated section highlights some of the verification work done for the code. A hierarchy of different physics models is available including a free boundary and resistive wall extension and hybrid kinetic-fluid models. The code allows for flux-surface aligned iso-parametric finite element grids in single and double X-point plasmas which can be extended to the true physical walls and uses a robust fully implicit time stepping. Particular focus is laid on plasma edge and scrape-off layer (SOL) physics as well as disruption related phenomena. Among the key results obtained with JOREK regarding plasma edge and SOL, are deep insights into the dynamics of edge localized modes (ELMs), ELM cycles, and ELM control by resonant magnetic perturbations, pellet injection, as well as by vertical magnetic kicks. Also ELM free regimes, detachment physics, the generation and transport of impurities during an ELM, and electrostatic turbulence in the pedestal region are investigated. Regarding disruptions, the focus is on the dynamics of the thermal quench (TQ) and current quench triggered by massive gas injection and shattered pellet injection, runaway electron (RE) dynamics as well as the RE interaction with MHD modes, and vertical displacement events. Also the seeding and suppression of tearing modes (TMs), the dynamics of naturally occurring TQs triggered by locked modes, and radiative collapses are being studied.Peer ReviewedPostprint (published version

The 3D Bioprinted Scaffolds for Wound Healing

Skin tissue engineering and regeneration aim at repairing defective skin injuries and progress in wound healing. Until now, even though several developments are made in this field, it is still challenging to face the complexity of the tissue with current methods of fabrication. In this review, short, state-of-the-art on developments made in skin tissue engineering using 3D bioprinting as a new tool are described. The current bioprinting methods and a summary of bioink formulations, parameters, and properties are discussed. Finally, a representative number of examples and advances made in the field together with limitations and future needs are provided

Methods for protein complex prediction and their contributions towards understanding the organization, function and dynamics of complexes

Complexes of physically interacting proteins constitute fundamental functional units responsible for driving biological processes within cells. A faithful reconstruction of the entire set of complexes is therefore essential to understand the functional organization of cells. In this review, we discuss the key contributions of computational methods developed till date (approximately between 2003 and 2015) for identifying complexes from the network of interacting proteins (PPI network). We evaluate in depth the performance of these methods on PPI datasets from yeast, and highlight challenges faced by these methods, in particular detection of sparse and small or sub- complexes and discerning of overlapping complexes. We describe methods for integrating diverse information including expression profiles and 3D structures of proteins with PPI networks to understand the dynamics of complex formation, for instance, of time-based assembly of complex subunits and formation of fuzzy complexes from intrinsically disordered proteins. Finally, we discuss methods for identifying dysfunctional complexes in human diseases, an application that is proving invaluable to understand disease mechanisms and to discover novel therapeutic targets. We hope this review aptly commemorates a decade of research on computational prediction of complexes and constitutes a valuable reference for further advancements in this exciting area.Comment: 1 Tabl

Radially Aligned, Electrospun Nanofibers as Dural Substitutes for Wound Closure and Tissue Regeneration Applications

This paper reports the fabrication of scaffolds consisting of radially aligned poly(ε-caprolactone) nanofibers by utilizing a collector composed of a central point electrode and a peripheral ring electrode. This novel class of scaffolds was able to present nanoscale topographic cues to cultured cells, directing and enhancing their migration from the periphery to the center. We also established that such scaffolds could induce faster cellular migration and population than nonwoven mats consisting of random nanofibers. Dural fibroblast cells cultured on these two types of scaffolds were found to express type I collagen, the main extracellular matrix component in dural mater. The type I collagen exhibited a high degree of organization on the scaffolds of radially aligned fibers and a haphazard distribution on the scaffolds of random fibers. Taken together, the scaffolds based on radially aligned, electrospun nanofibers show great potential as artificial dural substitutes and may be particularly useful as biomedical patches or grafts to induce wound closure and/or tissue regeneration

Controlling Ionic Transport in RRAM for Memory and Neuromorphic Computing Applications

Resistive random-access memory, based on a simple two-terminal device structure, has attracted tremendous interest recently for applications ranging from non-volatile data storage to neuromorphic computing. Resistive switching (RS) effects in RRAM devices originate from internal, microscopic ionic migration and the associated electrochemical processes which modify the materials’ chemical composition and subsequently their electrical and other physical properties. Therefore, controlling the internal ionic transport and redox reaction processes, ideally at the atomic scale, is necessary to optimize the device performance for practical applications with large-size arrays. In this thesis we present our efforts in understanding and controlling the ionic processes in RRAM devices. This thesis presents a comprehensive study on the fundamental understanding on physical mechanism of the ionic processes and the optimization of materials and device structures to achieve desirable device performance based on theoretical calculations and experimental engineering. First, I investigate the electronic structure of Ta2O5 polymorphs, a resistive switching material, and the formation and interaction of oxygen vacancies in amorphous Ta2O5, an important mobile defect responsible for the resistive switching process, using first-principles calculations. Based on the understanding of the fundamental properties of the switching material and the defect, we perform detailed theoretical and experimental analyses that reveal the dynamic vacancy charge transition processes, further helping the design and optimization of the oxide-based RRAM devices. Next, we develop a novel structure including engineered nanoporous graphene to control the internal ionic transport and redox reaction processes at the atomic level, leading to improved device performance. We demonstrate that the RS characteristics can be systematically tuned by inserting a graphene layer with engineered nanopores at a vacancy-exchange interface. The amount of vacancies injected in the switching layer and the size of the conducting filaments can be effectively controlled by the graphene layer working as an atomically-thin ion-blocking material in which ionic transports/reactions are allowed only through the engineered nanosized openings. Lastly, better incremental switching characteristics with improved linearity are obtained through optimization of the switching material density. These improvements allow us to build RRAM crossbar networks for data clustering analysis through unsupervised, online learning in both neuromorphic applications and arithmetic applications in which accurate vector-matrix multiplications are required. We expect the optimization approaches and the optimized devices can be used in other machine learning and arithmetic computing systems, and broaden the range of problems RRAM based network can solve.PHDMaterials Science and EngineeringUniversity of Michigan, Horace H. Rackham School of Graduate Studieshttps://deepblue.lib.umich.edu/bitstream/2027.42/146119/1/jihang_1.pd

Engineering of Fibrous Scaffolds for use in Regenerative Medicine

Tissue engineering with fibrous scaffolds is emerging as a major research area in the field of regenerative medicine. The major themes pursued in this thesis are the study of the cellular response to nanofibrous constructs, the role of nanofibres in the engineering of synthetic scaffolds, and the development of technology to facilitate the fabrication of nanofibrous scaffolds with controlled architectures. Cells cultured on multi‐walled carbon nanotubes displayed reduced proliferation and altered cytoskeletal development, thought to be due to the undermining of the maturation of focal adhesions. Development of an electrospinning chamber enabled the creation of poly(methyl methacrylate), poly(lactic acid) and poly(caprolactone) fibres for the study of cellular response to nano‐ and macro‐fibrous scaffolds. Cell attachment and organisation on the electrospun fibres was visualised using scanning electron microscopy, oblique microscopy and live cell microscopy. It was found that the incorporation of nanofibres into scaffolds restricts the maturation of focal adhesions which modulates cytoskeletal formation. This can be used to restrict the migration and the proliferation of attachment dependant cells such as osteoblasts or maintain the differentiation of cells such as chodrocytes. To scale up electrospun fibre production, use of rotating collectors, multi‐jet spinning and secondary electrodes to focus the spinning were investigated. Further to this, development of an array of focusing electrodes to control, stabilise and deflect the jet was also investigated towards the creation of a rapid‐prototype electrospinning system. The secondary electrode array was found to reduce the spreading of the jet to a spot diameter of 10mm and charged deflection plates successfully redirecting the position of the jet as it arrived at the collector

Signal and power integrity co-simulation using the multi-layer finite difference method

Mixed signal system-on-package (SoP) technology is a key enabler for increasing functional integration, especially in mobile and wireless systems. Due to the presence of multiple dissimilar modules, each having unique power supply requirements, the design of the power distribution network (PDN) becomes critical. Typically, this PDN is designed as alternating layers of power and ground planes with signal interconnects routed in between or on top of the planes. The goal for the simulation of multi-layer power/ground planes, is the following: Given a stack-up and other geometrical information, it is required to find the network parameters (S/Y/Z) between port locations. Commercial packages have extremely complicated stack-ups, and the trend to increasing integration at the package level only points to increasing complexity. It is computationally intractable to solve these problems using these existing methods. The approach proposed in this thesis for obtaining the response of the PDN is the multi-layer finite difference method (M-FDM). A surface mesh / finite difference based approach is developed, which leads to a system matrix that is sparse and banded, and can be solved efficiently. The contributions of this research are the following: 1. The development of a PDN modeler for multi-layer packages and boards called the the multi-layer finite difference method. 2. The enhancement of M-FDM using multi-port connection networks to include the effect of fringe fields and gap coupling. 3. An adaptive triangular mesh based scheme called the multi-layer finite element method (MFEM) to address the limitations of M-FDM 4. The use of modal decomposition for the co-simulation of signal nets with the PDN. 5. The use of a robust GA-based optimizer for the selection and placement of decoupling capacitors in multi-layer geometries. 6. Implementation of these methods in a tool called MSDT 1.Ph.D.Committee Chair: Madhavan Swaminathan; Committee Member: Andrew F. Peterson; Committee Member: David C. Keezer; Committee Member: Saibal Mukhopadyay; Committee Member: Suresh Sitarama