Evaluation of fibroin-based scaffolds for ocular tissue reconstruction

The epithelium of the corneolimbus contains stem cells for regenerating the corneal epithelium. Diseases and injuries affecting the limbus can lead to a condition known as limbal stem cell deficiency (LSCD), which results in loss of the corneal epithelium, and subsequent chronic inflammation and scarring of the ocular surface. Advances in the treatment of LSCD have been achieved through use of cultured human limbal epithelial (HLE) grafts to restore epithelial stem cells of the ocular surface. These epithelial grafts are usually produced by the ex vivo expansion of HLE cells on human donor amniotic membrane (AM), but this is not without limitations. Although AM is the most widely accepted substratum for HLE transplantation, donor variation, risk of disease transfer, and rising costs have led to the search for alternative biomaterials to improve the surgical outcome of LSCD. Recent studies have demonstrated that Bombyx mori silk fibroin (hereafter referred to as fibroin) membranes support the growth of primary HLE cells, and thus this thesis aims to explore the possibility of using fibroin as a biomaterial for ocular surface reconstruction. Optimistically, the grafted sheets of cultured epithelium would provide a replenishing source of epithelial progenitor cells for maintaining the corneal epithelium, however, the HLE cells lose their progenitor cell characteristics once removed from their niche. More severe ocular surface injuries, which result in stromal scarring, damage the epithelial stem cell niche, which subsequently leads to poor corneal re-epithelialisation post-grafting. An ideal solution to repairing the corneal limbus would therefore be to grow and transplant HLE cells on a biomaterial that also provides a means for replacing underlying stromal cells required to better simulate the normal stem cell niche. The recent discovery of limbal mesenchymal stromal cells (L-MSC) provides a possibility for stromal repair and regeneration, and therefore, this thesis presents the use of fibroin as a possible biomaterial to support a three dimensional tissue engineered corneolimbus with both an HLE and underlying L-MSC layer. Investigation into optimal scaffold design is necessary, including adequate separation of epithelial and stromal layers, as well as direct cell-cell contact. Firstly, the attachment, morphology and phenotype of HLE cells grown on fibroin were directly compared to that observed on donor AM, the current clinical standard substrate for HLE transplantation. The production, transparency, and permeability of fibroin membranes were also evaluated in this part of the study. Results revealed that fibroin membranes could be routinely produced using a custom-made film casting table and were found to be transparent and permeable. Attachment of HLE cells to fibroin after 4 hours in serum-free medium was similar to that supported by tissue culture plastic but approximately 6-fold less than that observed on AM. While HLE cultured on AM displayed superior stratification, epithelia constructed from HLE on fibroin maintained evidence of corneal phenotype (cytokeratin pair 3/12 expression; CK3/12) and displayed a comparable number and distribution of ÄNp63+ progenitor cells to that seen in cultures grown on AM. These results confirm the suitability of membranes constructed from silk fibroin as a possible substrate for HLE cultivation. One of the most important aspects in corneolimbal tissue engineering is to consider the reconstruction of the limbal stem cell niche to help form the natural limbus in situ. MSC with similar properties to bone marrow derived-MSC (BM-MSC) have recently been grown from the limbus of the human cornea. This thesis evaluated methods for culturing L-MSC and limbal keratocytes using various serum-free media. The phenotype of resulting cultures was examined using photography, flow cytometry for CD34 (keratocyte marker), CD45 (bone marrow-derived cell marker), CD73, CD90, CD105 (collectively MSC markers), CD141 (epithelial/vascular endothelial marker), and CD271 (neuronal marker), immunocytochemistry (alpha-smooth muscle actin; á-sma), differentiation assays (osteogenesis, adipogenesis and chrondrogenesis), and co-culture experiments with HLE cells. While all techniques supported to varying degrees establishment of keratocyte and L-MSC cultures, sustained growth and serial propagation was only achieved in serum-supplemented medium or the MesenCult-XF„¥ culture system (Stem Cell Technologies). Cultures established in MesenCult-XF„¥ grew faster than those grown in serum-supplemented medium and retained a more optimal MSC phenotype. L-MSC cultivated in MesenCult-XFR were also positive for CD141, rarely expressed £\-sma, and displayed multi-potency. L-MSC supported growth of HLE cells, with the largest epithelial islands being observed in the presence of L-MSC established in MesenCult-XF„¥ medium. All HLE cultures supported by L-MSC widely expressed the progenitor cell marker £GNp63, along with the corneal differentiation marker CK3/12. Our findings conclude that MesenCult-XFR is a superior culture system for L-MSC, but further studies are required to explore the significance of CD141 expression in these cells. Following on from the findings of the previous two parts, silk fibroin was tested as a novel dual-layer construct containing both an epithelium and underlying stroma for corneolimbal reconstruction. In this section, the growth and phenotype of HLE cells on non-porous versus porous fibroin membranes was compared. Furthermore, the growth of L-MSC in either serum-supplemented medium or the MesenCult-XFR culture system within fibroin fibrous mats was investigated. Lastly, the co-culture of HLE and L-MSC in serum-supplemented medium on and within fibroin dual-layer constructs was also examined. HLE on porous membranes displayed a flattened and squamous monolayer; in contrast, HLE on non-porous fibroin appeared cuboidal and stratified closer in appearance to a normal corneal epithelium. Both constructs maintained CK3/12 expression and distribution of £GNp63+ progenitor cells. Dual-layer fibroin scaffolds consisting of HLE cells and L-MSC maintained a similar phenotype as on the single layers alone. Overall, the present study proposed to create a three dimensional limbal tissue substitute of HLE cells and L-MSC together, ultimately for safe and beneficial transplantation back into the human eye. The results show that HLE and L-MSC can be cultivated separately and together whilst maintaining a clinically feasible phenotype containing a majority of progenitor cells. In addition, L-MSC were able to be cultivated routinely in the MesenCult-XF® culture system while maintaining a high purity for the MSC characteristic phenotype. However, as a serum-free culture medium was not found to sustain growth of both HLE and L-MSC, the combination scaffold was created in serum-supplemented medium, indicating that further refinement of this cultured limbal scaffold is required. This thesis has also demonstrated a potential novel marker for L-MSC, and has generated knowledge which may impact on the understanding of stromal-epithelial interactions. These results support the feasibility of a dual-layer tissue engineered corneolimbus constructed from silk fibroin, and warrant further studies into the potential benefits it offers to corneolimbal tissue regeneration. Further refinement of this technology should explore the potential benefits of using epithelial-stromal co-cultures with MesenCult-XF® derived L-MSC. Subsequent investigations into the effects of long-term culture on the phenotype and behaviour of the cells in the dual-layer scaffolds are also required. While this project demonstrated the feasibility in vitro for the production of a dual-layer tissue engineered corneolimbus, further studies are required to test the efficacy of the limbal scaffold in vivo. Future in vivo studies are essential to fully understand the integration and degradation of silk fibroin biomaterials in the cornea over time. Subsequent experiments should also investigate the use of both AM and silk fibroin with epithelial and stromal cell co-cultures in an animal model of LSCD. The outcomes of this project have provided a foundation for research into corneolimbal reconstruction using biomaterials and offer a stepping stone for future studies into corneolimbal tissue engineering.

YBa2Cu3O7/NdGaO3/YBa 2Cu3O7 trilayers by modified off-axis sputtering

High-quality epitaxial YBa2Cu3O7-δ (YBCO) thin films were achieved by a modified off-axis sputtering technique with high deposition rates (3.3 nm/min). The film quality and the deposition rate depended crucially on the target-to-substrate separation. Epitaxial YBCO/NdGaO3(NGO)/YBCO trilayers were successfully grown onto SrTiO3, Y-ZrO2, and LaAlO3 substrates by dc and rf sputtering. The epitaxial relations were found to be [001] YBCO//[001]NGO, [100]YBCO, or [010] YBCO//[110]NGO and [001]YBCO//[110] NGO, [100]YBCO, or [010]YBCO//[001] NGO, where the latter orientation relationship was dominating. Subsequent top YBCO layers grew c axis oriented independently of the two epitaxial orientations of the NGO. The orientation relationships between YBCO and NGO were the same. Auger electron depth profiles and transmission electron microscopy indicated that the interdiffusion at the interface between the YBCO and NGO layers was not strong even at 740°C. The superconducting transition temperatures of the top and bottom YBCO layers were about the same as that of YBCO single layers, i.e., 87-90 K. Scanning electron microscopy of the surface morphologies of the YBCO and the NGO showed that a smaller substrate-target distance resulted in smoother films.

Single crystal Raman spectroscopy of brandholzite Mg[Sb(OH)6]2•6H2O and bottinoite Ni[Sb(OH)6]2•6H2O and the polycrystalline Raman spectrum of mopungite Na[Sb(OH)6]

The single crystal Raman spectra of minerals brandholzite and bottinoite, formula M[Sb(OH)6]2•6H2O, where M is Mg+2 and Ni+2 respectively, and the non-aligned Raman spectrum of mopungite, formula Na[Sb(OH)6], are presented for the first time. The mixed metal minerals comprise of alternating layers of [Sb(OH)6]-1 octahedra and mixed [M(H2O)6]+2 / [Sb(OH)6]-1 octahedra. Mopungite comprises hydrogen bonded layers of [Sb(OH)6]-1 octahedra linked within the layer by Na+ ions. The spectra of the three minerals were dominated by the Sb-O symmetric stretch of the [Sb(OH)6]-1 octahedron, which occurs at approximately 620 cm-1. The Raman spectrum of mopungite showed many similarities to spectra of the di-octahedral minerals informing the view that the Sb octahedra gave rise to most of the Raman bands observed, particularly below 1200 cm-1. Assignments have been proposed based on the spectral comparison between the minerals, prior literature and density field theory calculations of the vibrational spectra of the free [Sb(OH)6]-1 and [M(H2O)6]+2 octahedra by a model chemistry of B3LYP/6-31G(d) and lanl2dz for the Sb atom. The single crystal data spectra showed good mode separation, allowing the majority of the bands to be assigned a symmetry species of A or E.

Catalyst-free growth and optical properties of vertically aligned ZnO Nanorod Arrays on Si substrates covered with thin buffer layers

Vertically aligned ZnO nanorods have been grown on silicon substrates pre-coated with thin, less than 10 nm, textured ZnO seeding layers via a vapor-solid mechanism. The ZnO seeding layers, which were essential for vertical alignment of ZnO nanorods without using any metal catalyst, were prepared by decomposing zinc acetate. The structure and the luminescence properties of the ZnO nanorods synthesized onto ZnO seeding layers were investigated and their morphologies were compared with those of single-crystalline GaN substrates and silicon substrates covered with sputtered ZnO flms. Patterning of ZnO seed layers using photolithography allowed the fabrication of patterned ZnO-nanorod arrays.

Kinetics of low-pressure, low-temperature graphene growth : toward single-layer, single-crystalline structure

Graphene grown on metal catalysts with low carbon solubility is a highly competitive alternative to exfoliated and other forms of graphene, yet a single-layer, single-crystal structure remains a challenge because of the large number of randomly oriented nuclei that form grain boundaries when stitched together. A kinetic model of graphene nucleation and growth is developed to elucidate the effective controls of the graphene island density and surface coverage from the onset of nucleation to the full monolayer formation in low-pressure, low-temperature CVD. The model unprecedentedly involves the complete cycle of the elementary gas-phase and surface processes and shows a precise quantitative agreement with the recent low-energy electron diffraction measurements and also explains numerous parameter trends from a host of experimental reports. These agreements are demonstrated for a broad pressure range as well as different combinations of precursor gases and supporting catalysts. The critical role of hydrogen in controlling the graphene nucleation and monolayer formation is revealed and quantified. The model is generic and can be extended to even broader ranges of catalysts and precursor gases/pressures to enable the as yet elusive effective control of the crystalline structure and number of layers of graphene using the minimum amounts of matter and energy.

Thin single-walled carbon nanotubes with narrow chirality distribution : constructive interplay of plasma and Gibbs–Thomson effects

Multiscale, multiphase numerical modeling is used to explain the mechanisms of effective control of chirality distributions of single-walled carbon nanotubes in direct plasma growth and suggest effective approaches to further improvement. The model includes an unprecedented combination of the plasma sheath, ion/radical transport, species creation/loss, plasma–surface interaction, heat transfer, surface/bulk diffusion, graphene layer nucleation, and bending/lift-off modules. It is shown that the constructive interplay between the plasma and the Gibbs–Thomson effect can lead to the effective nucleation and lift-off of small graphene layers on small metal catalyst nanoparticles. As a result, much thinner nanotubes with narrower chirality distributions can nucleate at much lower process temperatures and pressures compared to thermal CVD. This approach is validated by a host of experimental results, substantially reduces the amounts of energy and atomic matter required for the nanotube growth, and can be extended to other nanoscale structures and materials systems, thereby nearing the ultimate goal of energy- and matter-efficient nanotechnology.

Plasma-assisted self-sharpening of platelet-structured single-crystalline carbon nanocones

A mechanism and model for the vertical growth of platelet-structured vertically aligned single-crystalline carbon nanostructures by the formation of graphene layers on a flat top surface are proposed and verified experimentally. It is demonstrated that plasma-related effects lead to self-sharpening of tapered nanocones to form needlelike nanostructures, in a good agreement with the predicted dependence of the radius of a nanocone's flat top on the incoming ion flux and surface temperature. The growth mechanism is relevant to a broad class of nanostructures including nanotips, nanoneedles, and nanowires and can be used to improve the predictability of nanofabrication processes. © 2007 American Institute of Physics.

Towards a single crystal Raman spectrum of kaolinite at 77 K

The Raman spectra at 77 K of the hydroxyl stretching of kaolinite were obtained along the three axes perpendicular to the crystal faces. Raman bands were observed at 3616, 3658 and 3677 cm−1 together with a distinct band observed at 3691 cm−1 and a broad profile between 3695 and 3715 cm−1. The band at 3616 cm−1 is assigned to the inner hydroxyl. The bands at 3658 and 3677 cm−1 are attributed to the out-of-phase vibrations of the inner surface hydroxyls. The Raman spectra of the in-phase vibrations of the inner-surface hydroxyl-stretching region are described in terms of transverse and longitudinal optic splitting. The band at 3691 cm−1 is assigned to the transverse optic and the broad profile to the longitudinal optic mode. This splitting remained even at liquid nitrogen temperature. The transverse optic vibration may be curve resolved into two or three bands, which are attributed to different types of hydroxyl groups in the kaolinite.