Ashleigh Cooper

Chitosan-based nanofibrous membranes for antibacterial filter applications

Nanofibrous membranes have drawn considerable interest for filtration applications due to their ability to withstand high fluid flux while removing micro- and nano-sized particulates from solution. The desire to introduce an antibacterial function into water filter applications presents a challenge to widespread application of fibrous membranes because the addition of chemicals or biocides may produce harmful byproducts downstream. Here, we report the development of chitosan-polycaprolactone (PCL) nanofibrous membranes to utilize the natural antibacterial property of chitosan for antibacterial water filtration. Chitosan-PCL fibers with diameters of 200-400 nm and chitosan contents of 25, 50 and 75 wt% were prepared by electrospinning. In a series of bacterial challenge tests, chitosan-PCL fibrous membranes significantly reduced Staphylococcus aureus adhesion compared to PCL fibrous membranes. In water permeability and particulate size removal tests, fibrous membranes with 25% chitosan supported the greatest water flux (∼7000 L/h/m(2)) with 100% removal of 300-nm particulates, while maintaining the membrane integrity. This study demonstrates the potential of chitosan-PCL nanofibrous membranes as pre-filters for water filtration systems that demonstrate combinatorial filtration and intrinsic antibacterial advantages.

Aligned chitosan-based nanofibers for enhanced myogenesis

Tissue-engineered nanofibrous matrices can potentially serve as an implantable scaffold for the reconstruction of damaged or lost tissue by regulating cell proliferation, organization, and function. In this study, we developed a polyblend chitosan-polycaprolactone (PCL) nanofibrous scaffold with unidirectional fiber orientation by electrospinning for skeletal muscle tissue reconstruction and investigated the effect of the fiber alignment on cell organization and differentiation in comparison with randomly oriented nanofibers and 2D films of the same material. The chitosan-PCL material was shown to support skeletal muscle cell attachment and proliferation, and the fiber alignment promoted skeletal muscle cell morphogenesis and aligned myotube formation in the nanofiber orientation. Reverse-transcription PCR analyses revealed an up-regulation of differentiation-specific genes, troponin T and myosin heavy chain, in muscle cells on the aligned nanofiber scaffolds, confirming the ability of aligned chitosan-PCL nanofibers to enhance muscle cell differentiation. These results suggest that chitosan-PCL nanofibrous scaffolds with unidirectional fiber orientation can significantly enhanced muscle cell development making it a potential scaffold for enhanced skeletal myogenesis.

A facile bottom-up route to self-assembled biogenic chitin nanofibers

A facile bottom-up strategy affords cytocompatible self-assembled biogenic chitin nanofibers with diameter control. Ultrafine (3 nm) nanofibers are easily obtained from drying a chitin/hexafluoro 2-propanol solution, and larger (10 nm) nanofibers are precipitated from LiCl/N,N-dimethylacetamide upon addition of water.

Centrifugal electrospinning of highly aligned polymer nanofibers over a large area

Well-ordered one-dimensional nanostructures are enabling important new applications in textiles, energy, environment and bioengineering owing to their unique and anisotropic properties. However, the production of highly aligned nanofibers in a large area remains a significant challenge. Here we report a powerful, yet economical approach that integrates the concepts of the parallel-electrode electrospinning with centrifugal dispersion to produce nanofibers with a high degree of alignment and uniformity at a large scale. We first demonstrated this approach with polyvinylidene fluoride to show how experimental parameters regulate fiber properties, and then with chitosan, a natural polymer, and polyethylene oxide, a synthetic polymer, to illustrate the versatility of the system. As a model application, we then demonstrated the significance of fiber alignment in improving the piezoelectric effect for voltage generation. The technique presented here may be used for mass production of aligned nanofibers of various polymers for a myriad of applications.

Self-assembled chitin nanofiber templates for artificial neural networks

Self-assembled chitin nanofibers were applied as a biomimetic extracellular matrix for the attachment of primary neuronsin vitro. Chitin nanofiber surfaces were deacetylated to form 4 nm and 12 nm diameter chitosan nanofibers that were coupled with poly-D-lysine (PDL) to examine combinatory effects and structurally analyzed by atomic force microscopy. The chitosan substrates were then employed for mouse cortical neuron cultures to examine their capabilities to support cell attachment, neurite coverage and survival. The 4 nm chitosan nanofibers improved single cortical neuron attachment compared to the 12 nm chitosan fibers and bare glass substrates, illustrating the improved adhesive properties of the surface. Importantly, the 4 nm chitosan nanofibers with PDL supported 37.9% neuron viability compared to only 13.5% on traditional PDL surfaces after a 7-day culture period, illustrating significantly improved long-term cell viability. The nanofibrillar chitosan surface could provide an alternative substrate for in vitroprimary neuron cultures to serve as artificial neural networks for diagnostics and therapeutics.

On-site alginate gelation for enhanced cell proliferation and uniform distribution in porous scaffolds.

High cell density and uniformity in a tissue-engineered construct is essential to expedite the formation of a uniform extracellular matrix. In this study, we demonstrated an on-site gelation approach to increase cellular population and uniformity through porous scaffolds using alginate as gelling material. The on-site gelation was triggered during cell seeding and was shown to effectively restrain the cells in the porous scaffold during subsequent cell cultivation. The initial demonstration of the effectiveness of this system was made with chondrocyte cells, targeted at functional restoration of damaged or dysfunctional cartilage. By limiting cellular mobility, cell population increased by 89% after 7 days of cell culture in scaffolds encapsulating alginate gel as opposed to a 36% increase in scaffolds without gel. The cell distribution throughout the gelled scaffold was found to be more uniform than in the nongelled scaffold. SEM analysis revealed that the cells exhibited typical chondrocytic morphology. Improved cellular functionality was verified by low levels of collagen type I gene expression and steady gene activity levels of collagen type II over 3 weeks of cell cultivation. Alternatively, cells seeded in scaffolds with the conventional cell-seeding method demonstrated increased levels of collagen type I gene expression, indicating the possibility of cell dedifferentiation over long-term cell culture. Success with the chitosan-alginate scaffold model suggested that this flexible on-site gelation method could be potentially applied to other cell and tissue types for enhanced tissue engineering development.

Nanofiber-based in vitro system for high myogenic differentiation of human embryonic stem cells.

Myogenic progenitor cells derived from human embryonic stem cells (hESCs) can provide unlimited sources of cells in muscle regeneration but their clinical uses are largely hindered by the lack of efficient methods to induce differentiation of stem cells into myogenic cells. We present a novel approach to effectively enhance myogenic differentiation of human embryonic stem cells using aligned chitosan-polycaprolactone (C-PCL) nanofibers constructed to resemble the microenvironment of the native muscle extracellular matrix (ECM) in concert with Wnt3a protein. The myogenic differentiation was assessed by cell morphology, gene activities, and protein expression. hESCs grown on C-PCL uniaxially aligned nanofibers in media containing Wnt3a displayed an elongated morphology uniformly aligned in the direction of fiber orientation, with increased expressions of marker genes and proteins associated with myogenic differentiation as compared to control substrates. The combination of Wnt3a signaling and aligned C-PCL nanofibers resulted in high percentages of myogenic-protein expressing cells over total treated hESCs (83% My5, 91% Myf6, 83% myogenin, and 63% MHC) after 2 days of cell culture. Significantly, this unprecedented high-level and fast myogenic differentiation of hESC was demonstrated in a culture medium containing no feeder cells. This study suggests that chitosan-based aligned nanofibers combined with Wnt3a can potentially act as a model system for embryonic myogenesis and muscle regeneration.

Electrospinning of chitosan derivative nanofibers with structural stability in an aqueous environment

We report a simple method to produce stable chitosan derivative nanofibers via electrospinning. A chitosan solution with lactate salt was electrospun to produce nanofibers, followed by thermal treatment to enhance fiber stability. Chemical and morphological analyses demonstrated that the resulting nanofibers were crosslinked via amidation between chitosan and lactate salt. These fibers exhibited sustained morphological and structural stabilities to serve as a scaffold for biomedical applications.

Aligned Chitosan‐Polycaprolactone Polyblend Nanofibers Promote the Migration of Glioblastoma Cells

In vitro models that accurately mimic the microenvironment of invading glioblastoma multiform (GBM) cells will provide a high-throughput system for testing potential anti-invasion therapies. Here, the ability of chitosan-polycaprolactone polyblend nanofibers to promote a migratory phenotype in human GBM cells by altering the nanotopography of the nanofiber membranes is investigated. Fibers are prepared with diameters of 200 nm, 400 nm, and 1.1 μm, and are either randomly oriented or aligned to produce six distinct nanotopographies. Human U-87 MG GBM cells, a model cell line commonly used for invasion assays, are cultured on the various nanofibrous substrates. Cells show elongation and alignment along the orientation of aligned fibers as early as 24 h and up to 120 h of culture. After 24 h of culture, human GBM cells cultured on aligned 200 nm and 400 nm fibers show marked upregulation of invasion-related genes including β-catenin, Snail, STAT3, TGF-β, and Twist, suggesting a mesenchymal change in these migrating cells. Additionally, cells cultured on 400 nm aligned fibers show similar migration profiles as those reported in vivo, and thus these nanofibers should provide a unique high-throughput in vitro culture substrate for developing anti-migration therapies for the treatment of GBM.

Polymeric fibrous matrices for substrate-mediated human embryonic stem cell lineage differentiation.

Chitosan-based fibrous matrices are prepared to mimic the ECM architecture and elucidate substrate-mediated hESC differentiation due to topographical scale and anisotropy without exogenic morphogens. Fibrous matrices support fewer pluripotent hESCs than films but enable topography-mediated hESC differentiation. Matrices composed of 400 nm and 1.1 µm diameter fibers support increased expression of neural markers indicative of ectodermal commitment while matrices of 200 nm diameter fibers increase expression of osteogenic and hepatic markers indicative of endodermal and mesodermal commitment. The fibrous-mediated hESC differentiation highlights the significant implication of tailored ECM-like substrates for hESC-based therapies.