Multimaterial tandem electrospinning for spatially modulated neural guidance

The goal of this work is the creation of an in vitro platform to investigate the combined effects of patterned topographical and bioactive cues towards achieving the spatially controlled growth of peripheral sensory neurons

Into the groove:instructive conductive silk films with topological guidance cues

Instructive biomaterials capable of controlling the behaviour of the cells are particularly interesting scaffolds for tissue engineering and regenerative medicine. Novel biomaterials are particularly important in societies with rapidly aging populations, where demand for organ/tissue donations is greater than their supply. Herein we describe the preparation of electrically conductive silk film-based nerve tissue scaffolds that are manufactured using all aqueous processing. Aqueous solutions of Bombyx mori silk were cast on flexible polydimethylsiloxane substrates with micrometer-scale grooves on their surfaces, allowed to dry, and annealed to impart β-sheets to the silk which assures that the materials are stable for further processing in water. The silk films were rendered conductive by generating an interpenetrating network of polypyrrole and polystyrenesulfonate in the silk matrix. Films were incubated in an aqueous solution of pyrrole (monomer), polystyrenesulfonate (dopant) and iron chloride (initiator), after which they were thoroughly washed to remove low molecular weight components (monomers, initiators, and oligomers) and dried, yielding conductive films with sheet resistances of 124 ± 23 kΩ square-1. The micrometer-scale grooves that are present on the surface of the films are analogous to the natural topography in the extracellular matrix of various tissues (bone, muscle, nerve, skin) to which cells respond. Dorsal Root Gangions (DRGs) adhere to the films and the grooves in the surface of the films instruct the aligned growth of processes extending from the DRGs. Such materials potentially enable the electrical stimulation of cells cultured on them, and future in vitro studies will focus on understanding the interplay between electrical and topographical cues on the behaviour of cells cultured on them

Nanotopography-Induced Structural Anisotropy and Sarcomere Development in Human Cardiomyocytes Derived from Induced Pluripotent Stem Cells

This document is the Accepted Manuscript version of a Published Work that appeared in final form in ACS Appl Mater Interfaces, copyright © American Chemical Society after peer review and technical editing by the publisher. To access the final edited and published work see http://doi.org/10.1021/acsami.5b11671.Understanding the phenotypic development of human induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs) is a prerequisite to advancing regenerative cardiac therapy, disease modeling, and drug screening applications. Lack of consistent hiPSC-CM in vitro data can be largely attributed to the inability of conventional culture methods to mimic the structural, biochemical, and mechanical aspects of the myocardial niche accurately. Here, we present a nanogrid culture array comprised of nanogrooved topographies, with groove widths ranging from 350 to 2000 nm, to study the effect of different nanoscale structures on the structural development of hiPSC-CMs in vitro. Nanotopographies were designed to have a biomimetic interface, based on observations of the oriented myocardial extracellular matrix (ECM) fibers found in vivo. Nanotopographic substrates were integrated with a self-assembling chimeric peptide containing the Arg-Gly-Asp (RGD) cell adhesion motif. Using this platform, cell adhesion to peptide-coated substrates was found to be comparable to that of conventional fibronectin-coated surfaces. Cardiomyocyte organization and structural development were found to be dependent on the nanotopographical feature size in a biphasic manner, with improved development achieved on grooves in the 700–1000 nm range. These findings highlight the capability of surface-functionalized, bioinspired substrates to influence cardiomyocyte development, and the capacity for such platforms to serve as a versatile assay for investigating the role of topographical guidance cues on cell behavior. Such substrates could potentially create more physiologically relevant in vitro cardiac tissues for future drug screening and disease modeling studies

FABRICATION OF GROOVED HOLLOW FIBER MEMBRANE FOR NERVE REGENERATION: PROCESS MODELING AND PERFORMANCE EVALUATION

Nerve injury is a general but intractable disease in traumatic injuries, leading to a significant reduction of functions in the nervous system. Extensive efforts are made on nerve injury rehabilitation. Since the appropriate connections between neurons and their targets are necessary, guiding axonal outgrowth is an essential step for neuron outgrowth in nervous system development, functioning, and regeneration. Besides the direct surgical nerve connection, an artificial means of guiding nerve regeneration called nerve conduits is widely applied in nerve injury rehabilitation. The main function of nerve conduits is to bridge the nerve gap, to help regenerating axons across damaged regions and guide them to appropriate targets. Recently, polymeric hollow fiber membranes (HFMs) have been studied as a potential nerve conduit for nerve regeneration and repair. In order to further improve the efficiency of HFMs, micropatterns such as aligned grooves are usually introduced on the inner surface of HFMs as an effective topographical guidance cue. The goal of this study is to fabricate HFMs with aligned grooves on the inner surface and understand their effect on nerve regeneration and repair. Consequently, there is a need, first, to carefully design the fabrication process of HFMs introducing aligned grooves on inner surface and understand the groove formation mechanism; second, to better understand the role of defined grooves on the inner surface of HFMs as topographical guidance cues promoting axonal outgrowth. The grooved HFMs were fabricated by means of a phase inversion-based spinning technique with a smooth and annular spinneret by carefully controlling the fabrication conditions. The effects of different operating conditions were experimentally studied, and the fabricated HFMs were also characterized. In order to explain the formation of grooves on the HFM inner surface, two different instability mechanisms were introduced: a hydrodynamic or Marangoni instability and an elastic or buckling instability. The results obtained between the experimental and the theoretical studies were compared in terms of the number of grooves under different operating conditions. Then, the fabricated HFMs with textured inner surface were used as nerve conduits. The effect of the geometry of the grooved inner surface on the axonal outgrowth was studied. A numerical model describing the motion and deformation of an axon moving on the grooved HMF inner surface was developed to study the effect of substrate geometry on axonal outgrowth. This work developed the first theoretical model for the groove formation mechanism during the HFM fabrication. In this model, the Marangoni instability was first used to investigate the onset of instability in the HFM fabrication, and the buckling of instability magnification was also studied. This work also presented the numerical simulation of axonal outgrowth on a three-dimensional substrate, where the influence of the substrate geometry was taken into account. The work covered by this thesis will help to fabricate nerve conduits for better nerve regeneration and repair

Neuronal cell growth on polymeric scaffolds studied by CARS microscopy

For studies of neuronal cell integration and neurite outgrowth in polymeric scaffold materials as a future alternative for the treatment of damages in the neuronal system, we have developed a protocol employing CARS microscopy for imaging of neuronal networks. The benefits of CARS microscopy come here to their best use; (i) the overall three-dimensional (3D) arrangement of multiple cells and their neurites can be visualized without the need for chemical preparations or physical sectioning, potentially affecting the architecture of the soft, fragile scaffolds and (ii) details on the interaction between single cells and scaffold fibrils can be investigated by close-up images at sub-micron resolution. The establishment of biologically more relevant 3D neuronal networks in a soft hydrogel composed of native Extra Cellular Matrix (ECM) components was compared with conventional two-dimensional networks grown on a stiff substrate. Images of cells in the hydrogel scaffold reveal significantly different networking characteristics compared to the 2D networks, raising the question whether the functionality of neurons grown as layers in conventional cultivation dishes represents that of neurons in the central and peripheral nervous systems

Simple and Novel Three Dimensional Neuronal Cell Culture Using a Micro Mesh Scaffold

Conventional method of cell culture studies has been performed on two-dimensional substrates. Recently, three-dimensional (3D) cell culture platforms have been a subject of interest as cells in 3D has significant differences in cell differentiation and behavior. Here we report a novel approach of 3D cell culture using a nylon micro mesh (NMM) as a cell culture scaffold. NMM is commonly used in cell culture laboratory, which eliminates the requirement of special technicality for biological laboratories. Furthermore, it is made of a micro-meter thick nylon fibers, which was adequate to engineer in cellular scales. We demonstrate the feasibility of the NMM as a 3D scaffold using E18 rat hippocampal neurons. NMM could be coated with cell adhesive coatings (polylysine or polyelectrolyte) and neurons showed good viability. Cells were also encapsulated in an agarose hydrogel and cultured in 3D using NMM. In addition, the 3D pattern of NMM could be used as a guidance cue for neurite outgrowth. The flexible and elastic properties of NMMs made it easier to handle the scaffold and also readily applicable for large-scale tissue engineering applications

Supracolloidal assemblies as sacrificial templates for porous silk-based biomaterials

Tissues in the body are hierarchically structured composite materials with tissue-specific properties. Urea self-assembles via hydrogen bonding interactions into crystalline supracolloidal assemblies that can be used to impart macroscopic pores to polymer-based tissue scaffolds. In this communication, we explain the solvent interactions governing the solubility of urea and thereby the scope of compatible polymers. We also highlight the role of solvent interactions on the morphology of the resulting supracolloidal crystals. We elucidate the role of polymer-urea interactions on the morphology of the pores in the resulting biomaterials. Finally, we demonstrate that it is possible to use our urea templating methodology to prepare Bombyx mori silk protein-based biomaterials with pores that human dermal fibroblasts respond to by aligning with the long axis of the pores. This methodology has potential for application in a variety of different tissue engineering niches in which cell alignment is observed, including skin, bone, muscle and nerve

Decreased astroglial cell adhesion and proliferation on zinc oxide nanoparticle polyurethane composites

Nanomaterials offer a number of properties that are of interest to the field of neural tissue engineering. Specifically, materials that exhibit nanoscale surface dimensions have been shown to promote neuron function while simultaneously minimizing the activity of cells such as astrocytes that inhibit central nervous system regeneration. Studies demonstrating enhanced neural tissue regeneration in electrical fields through the use of conductive materials have led to interest in piezoelectric materials (or those materials which generate a transient electrical potential when mechanically deformed) such as zinc oxide (ZnO). It has been speculated that ZnO nanoparticles possess increased piezoelectric properties over ZnO micron particles. Due to this promise in neural applications, the objective of the present in vitro study was, for the first time, to assess the activity of astroglial cells on ZnO nanoparticle polymer composites. ZnO nanoparticles embedded in polyurethane were analyzed via scanning electron microscopy to evaluate nanoscale surface features of the composites. The surface chemistry was characterized via X-ray photoelectron spectroscopy. Astroglial cell response was evaluated based on cell adhesion and proliferation. Astrocyte adhesion was significantly reduced on ZnO nanoparticle/polyurethane (PU) composites with a weight ratio of 50:50 (PU:ZnO) wt.%, 75:25 (PU:ZnO) wt.%, and 90:10 (PU:ZnO) wt.% in comparison to pure PU. The successful production of ZnO nanoparticle composite scaffolds suitable for decreasing astroglial cell density demonstrates their potential as a nerve guidance channel material with greater efficiency than what may be available today