Precisely Assembled Nanofiber Arrays as a Platform to Engineer Aligned Cell Sheets for Biofabrication

A hybrid cell sheet engineering approach was developed using ultra-thin nanofiber arrays to host the formation of composite nanofiber/cell sheets. It was found that confluent aligned cell sheets could grow on uniaxially-aligned and crisscrossed nanofiber arrays with extremely low fiber densities. The porosity of the nanofiber sheets was sufficient to allow aligned linear myotube formation from differentiated myoblasts on both sides of the nanofiber sheets, in spite of single-side cell seeding. The nanofiber content of the composite cell sheets is minimized to reduce the hindrance to cell migration, cell-cell contacts, mass transport, as well as the foreign body response or inflammatory response associated with the biomaterial. Even at extremely low densities, the nanofiber component significantly enhanced the stability and mechanical properties of the composite cell sheets. In addition, the aligned nanofiber arrays imparted excellent handling properties to the composite cell sheets, which allowed easy processing into more complex, thick 3D structures of higher hierarchy. Aligned nanofiber array-based composite cell sheet engineering combines several advantages of material-free cell sheet engineering and polymer scaffold-based cell sheet engineering; and it represents a new direction in aligned cell sheet engineering for a multitude of tissue engineering applications

Biocompatible/ Bioresorbable Polymer Based Silver Nanaomaterial Coatng for Chronic Indwelling Medical Devices and Bioscaffolds for Tissue Regrowth

The objective of this study is to synthesize and characterize antimicrobial, bio-polymer based silver nanomaterials composite coatings, for use in chronic indwelling medical devices, and bioscaffolds. The coatings and bioscaffolds are comprised of novel biomass mediated silver nano particles (SNP) that are biocompatible, highly concentrated, highly pure, cost-effective, polydispersed and compatible with a range of polymer systems applicable for use with existing chronic indwelling medical devices. This thesis is divided into three main chapters. In Chapter 1, detailed review on the need for antimicrobial nanocomposite coatings for chronic indwelling medical devices along with different SNP synthesis and characterization methods is provided. In Chapter 2 a comprehensive description of biocompatible/bioresorbable poly (L-lactide) (PLLA) based thin film coatings comprised of novel 25-75 nm silver nano particles SNP is provided. The particle and film morphology is characterized using Transmission Electron Microscopy (TEM), Atomic Force Microscopy (AFM) and UV-Visible Spectroscopy. The release rate of SNP is profiled by Thermogravimetric Analysis (TGA) and Inductively Coupled Plasma Optical Emission Spectrometer (ICP-OES). These coatings, suitable for chronic indwelling devices, drastically reduce the microbial biofilm formed by Staphylococcus aureus and Escherichia coli by 3-5 log reduction. This chapter details the synthesis of PLLA cast-coatings and the procedure to embed SNP, an antimicrobial agent, at a range of concentrations to identify an optimal SNP concentration of 700-800 ppm that efficacious and non-cytotoxic to human epithelial carcinoma cells (HeLa). Chapter 3 explains the procedure of making biocompatible/bioresorbable PLLA-PEG co-polymer block bioscaffolds designed to degrade and resorb at a controlled rate while providing a suitable substrate for tissue regrowth. The antimicrobial properties of these porous bioscaffolds are tested across varying concentrations of biomass mediated SNP, to determine an efficacious antimicrobial concentration. The bioscaffolds are efficacious as it reduces the Staphylococcus aureus and Escherichia coli biofilm by 92.5- 99.9%, respectively, at an antimicrobial SNP concentration of 800ppm

Nanofibrous Surface Modification of Ultra-Thin PCL Membrane for Cardiovascular Tissue Engineering Application

Master'sMASTER OF SCIENC

Assessment of the biocompatibility, stability, and suitability of novel thermoresponsive films for the rapid generation of cellular constructs

Stimuli responsive polymers (SRP) are of great interest in the bioengineering community due to their use in applications such as drug delivery and tissue engineering. One example of an SRP is poly(N-isopropyl acrylamide) or pNIPAM. This SRP has the capability of changing its conformation with a slight temperature change: adherent mammalian cells spontaneously release as a confluent cell sheet, which can be harvested for cell sheet engineering purposes. Since its initial use in 1968, many researchers have used pNIPAM to obtain a cell sheet composed of their cell type of interest. The differing protocols used for these diverse cell types, such as the conditions used for cell detachment, and the varying methods used for derivatizing substrates with pNIPAM have all led to conflicting reports on the utility of pNIPAM for cell sheet engineering purposes, as well as the relative cytotoxicity of the polymer. In this work, some of the key inconsistencies in the literature and previously unaddressed challenges when utilizing pNIPAM films are overcome for the purpose of rapid generation of cellular constructs, specifically spheroids. Pertinent characteristics of low temperature detachment are investigated for their effect on the kinetics of cell detachment. In addition, a novel, inexpensive method for obtaining pNIPAM films for mammalian cell detachment, combining pNIPAM with a sol-gel, was optimized and compared to plasma polymerization deposition. Furthermore, proper storage conditions (e.g. temperature and relative humidity) for these films were investigated to increase stability of the films for using tissue culture conditions. To increase the speed of generation of cell sheets, electrospun mats and hydrogels with a high surface area-to-volume ratio were developed. The result is a platform appropriate for the rapid formation of cellular constructs, such as engineered tissues and spheroids for cancer cell research

POLY(N-ISOPROPYL ACRYLAMIDE)-COATED SURFACES: INVESTIGATION OF CYTOTOXICITY WITH MAMMALIAN CELLS AND THE MECHANISM OF CELL DETACHMENT

With the increase in life expectancy and with growing numbers of an aging population, there is a rising need worldwide for replacement tissues and organs. One way to address this growing need is through engineered tissues, such as those generated from stimulus-responsive polymers. Stimulus-responsive polymers undergo a physical or chemical change when a stimulus is applied. One such material is poly(N-isopropyl acrylamide), (pNIPAM), which undergoes a conformation change in a physiologically relevant temperature range to release intact mammalian cell monolayers capable of being used to engineer tissues. Two factors currently limit the use of cell sheets for this purpose: 1) although the NIPAM monomer is toxic, it is unclear (and highly contested) whether its polymerized form is toxic as well; 2) there is little understanding of the mechanism of how cells detach from pNIPAM, and whether the (possibly) cytotoxic polymer would be transferred to implanted engineered tissues. In this work, we present an investigation of the cytotoxicity of pNIPAM-grafted surfaces, as well as an investigation of the mechanism of cell detachment from pNIPAM. The cytotoxicity of substrates prepared using several polymerization and deposition techniques are evaluated using appropriate cytotoxicity tests (MTS, Live/Dead, plating efficiency). Endothelial, epithelial, smooth muscle, and fibroblast cells were used for the cytotoxicity testing. The mechanism of cell detachment from pNIPAM was investigated using endothelial cells and surfaces synthesized via surface-initiated atom transfer radical polymerization. The detachment experiments were performed at various temperatures with and without an ATP inhibitor. In addition, fluorescent pNIPAM surfaces were generated to determine if any pNIPAM is removed with the detached cells. We find that cell sheets obtained by detachment from pNIPAM films will be suitable for use in engineered tissues, provided that the pNIPAM films that the cells were obtained from are themselves robust (i.e., grafted, covalently linked, or similar). We also find that the cell detachment from pNIPAM is mostly a passive process, and that no pNIPAM is removed from the surfaces during the detachment. Our results therefore provide an important step to clearing the hurdles presently obstructing the generation of engineering tissues from pNIPAM films

FIBRONECTIN CONJUGATION ONTO THREE-DIMENSIONAL POROUS POLYURETHANE SCAFFOLDS FOR VASCULAR TISSUE ENGINEERING

In tissue engineering, scaffolds serve as the three-dimensional (3D) structural framework controlling cell behavior and ultimately the performance of the final construct. Cell interactions with synthetic scaffolds can be improved by attaching biomolecules such as proteins or peptides. Fibronectin (FN) is a protein that contains several domains including the cell adhesion tri-peptide, Arginine-Glycine-Aspartic Acid, allowing it to mediate cell attachment and proliferation on various substrates. In this work, FN was conjugated on 3D highly porous poly(carbonate) urethane (PCU) scaffolds through grafted poly(acrylic) acid (AA) spacers. Scaffolds were fabricated using a solvent casting-particulate leaching method. AA was grafted on the 3D scaffolds using a ceric ion initiator, and FN was conjugated using an N-hydroxysuccinimide intermediate. Scaffold pore structures were visualized using scanning electron microscopy and Fourier transform infrared spectroscopy was used to monitor reaction progress. A toluidine blue assay was used to quantify grafted AA groups. Survey and high-resolution X-ray photoelectron spectroscopy scans of scaffolds provided changes in atomic composition and chemical groups, respectively. Immunofluorescence studies showed FN to be evenly distributed over the scaffold surfac