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.

Corneal nerve structure and function as markers of diabetic neuropathy

Diabetic neuropathy is a significant clinical problem that currently has no effective therapy, and in advanced cases, leads to foot ulceration and lower limb amputation. The accurate detection, characterisation and quantification of this condition are important in order to define at-risk patients, anticipate deterioration, monitor progression and assess new therapies. This thesis evaluates novel corneal methods of assessing diabetic neuropathy. Over the past several years two new non-invasive corneal markers have emerged, and in cross-sectional studies have demonstrated their ability to stratify the severity of this disease. Corneal confocal microscopy (CCM) allows quantification of corneal nerve parameters and non-contact corneal aesthesiometry (NCCA), the presumed functional correlate of corneal structure, assesses the sensitivity of the cornea. Both these techniques are quick to perform, produce little or no discomfort for the patient, and with automatic analysis paradigms developed, are suitable for clinical settings. Each has advantages and disadvantages over established techniques for assessing diabetic neuropathy. New information is presented regarding measurement bias of CCM images, and a unique sampling paradigm and associated accuracy determination method of combinations is described. A novel high-speed corneal nerve mapping procedure has been developed and application of this procedure in individuals with neuropathy has revealed regions of sub-basal nerve plexus that dictate further evaluation, as they appear to show earlier signs of damage than the central region of the cornea that has to date been examined. The discriminative capacity of corneal sensitivity measured by NCCA is revealed to have reasonable potential as a marker of diabetic neuropathy. Application of these new corneal markers for longitudinal evaluation of diabetic neuropathy has the potential to reduce dependence on more invasive, costly, and time-consuming assessments, such as skin biopsy.

Current status and future prospects for cultured limbal tissue transplants in Australia and New Zealand

Cultured limbal tissue transplants have become widely used over the last decade as a treatment for limbal stem cell deficiency (LSCD). While the number of patients afflicted with LSCD in Australia and New Zealand is considered to be relatively low, the impact of this disease on quality of life is so severe that the potential efficacy of cultured transplants has necessitated investigation. We presently review the basic biology and experimental strategies associated with the use of cultured limbal tissue transplants in Australia and New Zealand. In doing so, we aim to encourage informed discussion on the issues required to advance the use of cultured limbal transplants in Australia and New Zealand. Moreover, we propose that a collaborative network could be established to maintain access to the technology in conjunction with a number of other existing and emerging treatments for eye diseases.

Silk fibroin in ocular surface reconstruction : what is its potential as a biomaterial in ophthalmics?

Hardly a month goes by within the scientific literature without some new material “X” being reported as a suitable material on which to grow cell type “Y”, for the potential purpose of treating disease “Z”. Thus when fibroin, a protein found in silk, was first proposed as a biomaterial for cell growth [1] it joined a long list of other materials of both natural as well as synthetic origin. Nevertheless, in the second decade of the Asian Century it is perhaps befitting that a material of so much importance to the continent’s cultural and economic history, should become the focus of cutting-edge biomedical research. Sentiments aside, however, silk fibroin possesses quite a unique combination of properties which make it a promising candidate for repairing the eye and especially for treating damage to the cornea, the transparent window at the front of the eye.

Evaluation of methods for cultivating limbal mesenchymal stromal cells

Background: Mesenchymal stromal cells (MSC) with similar properties to bone marrow derived mesenchymal stromal cells (BM-MSC) have recently been grown from the limbus of the human cornea. We presently contribute to this novel area of research by evaluating methods for culturing human limbal MSC (L-MSC). Methods: Four basic strategies are compared: serum-supplemented medium (10% foetal bovine serum; FBS), standard serum-free medium supplemented with B-27, epidermal growth factor, and fibroblast growth factor 2, or one of two commercial serum-free media including Defined Keratinocyte Serum Free Medium (Invitrogen), and MesenCult-XF (Stem Cell Technologies). The phenotype of resulting cultures was examined using photography, flow cytometry (for CD34, CD45, CD73, CD90, CD105, CD141, CD271), immunocytochemistry (α-sma), differentiation assays (osteogenesis, adipogenesis, chrondrogenesis), and co-culture experiments with human limbal epithelial (HLE) cells. Results: While all techniques supported to varying degrees establishment of cultures, sustained growth and serial propagation was only achieved in 10% FBS medium or MesenCult-XF medium. Cultures established in 10% FBS medium were 70-80% CD34-/CD45-/CD90+/CD73+/CD105+, approximately 25% α-sma+, and displayed multi-potency. Cultures established in MesenCult-XF were >95% CD34-/CD45-/CD90+/CD73+/CD105+, 40% CD141+, rarely expressed α-sma, and displayed multi-potency. L-MSC supported growth of HLE cells, with the largest epithelial islands being observed in the presence of MesenCult-XF-grown L-MSC. All HLE cultures supported by L-MSC widely expressed the progenitor cell marker ∆Np63, along with the corneal differentiation marker cytokeratin 3. Conclusions: We conclude that MesenCult-XF® is a superior culture system for L-MSC, but further studies are required to explore the significance of CD141 expression in these cells.

Wide-field assessment of the human corneal subbasal nerve plexus in diabetic neuropathy using a novel mapping technique

To develop a rapid optimized technique of wide-field imaging of the human corneal subbasal nerve plexus. A dynamic fixation target was developed and, coupled with semiautomated tiling software, a rapid method of capturing and montaging multiple corneal confocal microscopy images was created. To illustrate the utility of this technique, wide-field maps of the subbasal nerve plexus were produced in 2 participants with diabetes, 1 with and 1 without neuropathy. The technique produced montages of the central 3 mm of the subbasal corneal nerve plexus. The maps seem to show a general reduction in the number of nerve fibers and branches in the diabetic participant with neuropathy compared with the individual without neuropathy. This novel technique will allow more routine and widespread use of subbasal nerve plexus mapping in clinical and research situations. The significant reduction in the time to image the corneal subbasal nerve plexus should expedite studies of larger groups of diabetic patients and those with other conditions affecting nerve fibers. The inferior whorl and the surrounding areas may show the greatest loss of nerve fibers in individuals with diabetic neuropathy, but this should be further investigated in a larger cohort.

Utility of corneal confocal microscopy for assessing mild diabetic neuropathy: baseline findings of the LANDMark study

Background:  For those in the field of managing diabetic complications, the accurate diagnosis and monitoring of diabetic peripheral neuropathy (DPN) continues to be a challenge. Assessment of sub-basal corneal nerve morphology has recently shown promise as a novel ophthalmic marker for the detection of DPN. Methods:  Two hundred and thirty-one individuals with diabetes with predominantly mild or no neuropathy and 61 controls underwent evaluation of diabetic neuropathy symptom score, neuropathy disability score, testing with 10 g monofilament, quantitative sensory testing (warm, cold, vibration detection) and nerve conduction studies. Corneal nerve fibre length, branch density and tortuosity were measured using corneal confocal microscopy. Differences in corneal nerve morphology between individuals with and without DPN and controls were investigated using analysis of variance and correlations were determined between corneal morphology and established tests of, and risk factors for, DPN. Results:  Corneal nerve fibre length was significantly reduced in diabetic individuals with mild DPN compared with both controls (p < 0.001) and diabetic individuals without DPN (p = 0.012). Corneal nerve branch density was significantly reduced in individuals with mild DPN compared with controls (p = 0.032). Corneal nerve fibre tortuosity did not show significant differences. Corneal nerve fibre length and corneal nerve branch density showed modest correlations to most measures of neuropathy, with the strongest correlations to nerve conduction study parameters (r = 0.15 to 0.25). Corneal nerve fibre tortuosity showed only a weak correlation to the vibration detection threshold. Corneal nerve fibre length was inversely correlated to glycated haemoglobin (r = -0.24) and duration of diabetes (r = -0.20). Conclusion:  Assessment of corneal nerve morphology is a non-invasive, rapid test capable of showing differences between individuals with and without DPN. Corneal nerve fibre length shows the strongest associations with other diagnostic tests of neuropathy and with established risk factors for neuropathy.

Corneal sensitivity is related to established measures of diabetic peripheral neuropathy

Purpose:  The objective was to investigate the association between corneal sensitivity and established measures of diabetic peripheral neuropathy (DPN). Methods:  Corneal sensitivity was measured in 93 individuals with diabetes, 146 diabetic individuals without neuropathy and 61 control individuals without diabetes or neuropathy using a non-contact corneal aesthesiometer at the baseline visit of a five-year longitudinal natural history study of DPN. The correlation between corneal sensitivity and established measures of neuropathy was estimated and multi-dimensional scaling was used to represent similarities and dissimilarities between variables. Results:  The corneal sensitivity threshold was significantly correlated with a majority of established measures of DPN. Correlation coefficients ranged from -0.32 to 0.26. Using multi-dimensional scaling, non-contact corneal aesthesiometry was closer to the neuropathy disability score, diabetic neuropathy symptom score and Neuropad and most dissimilar to electrophysiological parameters and quantitative sensory testing. Conclusion:  Corneal sensitivity, although not strongly related, is associated with other functional measures of DPN and might provide a useful adjunct in identifying functional loss of small nerve fibre integrity.

Corneal changes following near work in myopic anisometropia

PURPOSE: To examine the symmetry of corneal changes following near work in the fellow eyes of non-amblyopic myopic anisometropes. METHODS: Thirty-four non-amblyopic myopic anisometropes (minimum 1 D spherical equivalent anisometropia) were recruited. Corneal topography was measured with the Medmont E300 Videokeratoscope before and after a controlled near task. Subjects were positioned to minimise head movements and read continuous text on a computer monitor for 10 minutes at an angle of 25 degrees downward gaze and an accommodation demand of 2.5 D. Measures of palpebral aperture morphology during primary and downward gaze were also obtained using digital photography and analysed with customised software. RESULTS: Significant changes in corneal topography were observed after ten minutes of reading. Localised superior regions of corneal topographical change (a hyperopic shift in corneal power) were typically exhibited in both eyes following the near task. The mean change in the corneal sphero-cylinder was +0.02/-0.11 x 113 and +0.02/-0.06 x 68 for the more and less myopic eyes respectively for a 6 mm corneal diameter. A significantly greater change in corneal astigmatism power vector J0 (a larger increase in against the rule astigmatism) was observed in the more myopic eyes (p < 0.01 for a 6 mm diameter). The more and less myopic eyes exhibited a high degree of interocular symmetry for measures of palpebral aperture morphology during both primary and downward gaze. Changes in corneal power vectors following reading were associated with eyelid position during downward gaze. CONCLUSIONS: Changes in corneal topography observed following a controlled reading task were highly symmetrical between the fellow eyes of myopic anisometropes due to the interocular symmetry of the palpebral aperture. However, the more myopic eye did exhibit a small but significantly greater magnitude of change in corneal astigmatism compared to the less myopic eye following reading. These findings may have implications for understanding the mechanism of development of non-amblyopic myopic anisometropia.

Genetic analysis of maize streak virus isolates from Uganda reveals widespread distribution of a recombinant variant

Maize streak virus (MSV) contributes significantly to the problem of extremely low African maize yields. Whilst a diverse range of MSV and MSV-like viruses are endemic in sub-Saharan Africa and neighbouring islands, only a single group of maize-adapted variants - MSV subtypes A1 -A6 - causes severe enough disease in maize to influence yields substantially. In order to assist in designing effective strategies to control MSV in maize, a large survey covering 155 locations was conducted to assess the diversity, distribution and genetic characteristics of the Ugandan MSV-A population. PCR-restriction fragment-length polymorphism analyses of 391 virus isolates identified 49 genetic variants. Sixty-two full-genome sequences were determined, 52 of which were detectably recombinant. All but two recombinants contained predominantly MSV-A1-like sequences. Of the ten distinct recombination events observed, seven involved inter-MSV-A subtype recombination and three involved intra-MSV-A1 recombination. One of the intra-MSV-A1 recombinants, designated MSV-A1 UgIII, accounted for >60% of all MSV infections sampled throughout Uganda. Although recombination may be an important factor in the emergence of novel geminivirus variants, it is demonstrated that its characteristics in MSV are quite different from those observed in related African cassava-infecting geminivirus species. © 2007 SGM.

Using optical coherence tomography to assess corneoscleral morphology after soft contact lens wear

Purpose. To evaluate the use of optical coherence tomography (OCT) to assess the effect of different soft contact lenses on corneoscleral morphology. Methods. Ten subjects had anterior segment OCT B-scans taken in the morning and again after six hours of soft contact lens wear. For each subject, three different contact lenses were used in the right eye on non-consecutive days, including a hydrogel sphere, a silicone hydrogel sphere and a silicone hydrogel toric. After image registration and layer segmentation, analyses were performed of the first hyper-reflective layer (HRL), the epithelial basement membrane (EBL) and the epithelial thickness (HRL to EBL). A root mean square difference (RMSD) of the layer profiles and the thickness change between the morning and afternoon measurements, was used to assess the effect of the contact lens on the corneoscleral morphology. Results. The soft contact lenses had a statistically significant effect on the morphology of the anterior segment layers (p <0.001). The average amounts of change for the three lenses (average RMSD values) for the corneal region were lower (3.93±1.95 µm for the HRL and 4.02±2.14 µm for the EBL) than those measured in the limbal/scleral region (11.24±6.21 µm for the HRL and 12.61±6.42 µm for the EBL). Similarly, averaged across the three lenses, the RMSD in epithelial thickness was lower in the cornea (2.84±0.84 µm) than the limbal/scleral (5.47±1.71 µm) region. Post-hoc analysis showed that ocular surface changes were significantly smaller with the silicone hydrogel sphere lens than both the silicone hydrogel toric (p<0.005) and hydrogel sphere (p<0.02) for the combined HRL and EBL data. Conclusions. In this preliminary study, we have shown that soft contact lenses can produce small but significant changes in the morphology of the limbal/scleral region and that OCT technology is useful in assessing these changes. The clinical significance of these changes is yet to be determined.

Corneal changes following short-term rigid contact lens wear

Purpose: The aim of this cross-over study was to investigate the changes in corneal thickness, anterior and posterior corneal topography, corneal refractive power and ocular wavefront aberrations, following the short term use of rigid contact lenses. Method: Fourteen participants wore 4 different types of contact lenses (RGP lenses of 9.5 mm and 10.5 mm diameter, and for comparison a PMMA lens of 9.5 mm diameter and a soft silicone hydrogel lens) on 4 different days for a period of 8 h on each day. Measures were collected before and after contact lens wear and additionally on a baseline day. Results: Anterior corneal curvature generally showed a flattening with both of the RGP lenses and a steepening with the PMMA lens. A significant negative correlation was found between the change in corneal swelling and central and peripheral posterior corneal curvature (all p ≤ 0.001). RGP contact lenses caused a significant decrease in corneal refractive power (hyperopic shift) of approximately 0.5 D. The PMMA contact lenses caused the greatest corneal swelling in both the central (27.92 ± 15.49 μm, p < 0.001) and peripheral (17.78 ± 12.11 μm, p = 0.001) corneal regions, a significant flattening of the posterior cornea and an increase in ocular aberrations (all p ≤ 0.05). Conclusion: The corneal swelling associated with RGP lenses was relatively minor, but there was slight central corneal flattening and a clinically significant hyperopic change in corneal refractive power after the first day of lens wear. The PMMA contact lenses resulted in significant corneal swelling and reduced optical performance of the cornea.

Effect of age on components of peripheral ocular aberrations

Purpose: To investigate the effect of age on the contributions of the anterior cornea and internal components to ocular aberrations in the peripheral visual field. Methods: Ocular aberrations were measured in 10 young emmetropes and 7 older emmetropes using a modified commercial Hartmann-Shack aberrometer across 42° x 32° of central visual field. Anterior corneal aberrations were estimated from anterior corneal topography using theoretical ray-tracing. Internal aberrations were calculated by subtracting anterior corneal aberrations from ocular aberrations. Results: Anterior corneal aberrations of young subjects were reasonably compensated by the internal aberrations, except for astigmatism for which the internal contribution was small out to the 21° field limit. The internal coma and spherical aberration of the older subjects were considerably smaller in magnitude than those of the young subjects such that the compensation for anterior corneal aberrations was poorer. This can be explained by age-related changes in the lens shape and refractive index distribution. Conclusion: oss of balance between anterior cornea and internal components of higher order aberrations with increasing age, found previously for on-axis vision, applies also to the peripheral visual field.