Audra Shadforth

Treatment of Silk Fibroin with Poly(ethylene glycol) for the Enhancement of Corneal Epithelial Cell Growth

A silk protein, fibroin, was isolated from the cocoons of the domesticated silkworm (Bombyx mori) and cast into membranes to serve as freestanding templates for tissue-engineered corneal cell constructs to be used in ocular surface reconstruction. In this study, we sought to enhance the attachment and proliferation of corneal epithelial cells by increasing the permeability of the fibroin membranes and the topographic roughness of their surface. By mixing the fibroin solution with poly(ethylene glycol) (PEG) of molecular weight 300 Da, membranes were produced with increased permeability and with topographic patterns generated on their surface. In order to enhance their mechanical stability, some PEG-treated membranes were also crosslinked with genipin. The resulting membranes were thoroughly characterized and compared to the non-treated membranes. The PEG-treated membranes were similar in tensile strength to the non-treated ones, but their elastic modulus was higher and elongation lower, indicating enhanced rigidity. The crosslinking with genipin did not induce a significant improvement in mechanical properties. In cultures of a human-derived corneal epithelial cell line (HCE-T), the PEG treatment of the substratum did not improve the attachment of cells and it enhanced only slightly the cell proliferation in the longer term. Likewise, primary cultures of human limbal epithelial cells grew equally well on both non-treated and PEG-treated membranes, and the stratification of cultures was consistently improved in the presence of an underlying culture of irradiated 3T3 feeder cells, irrespectively of PEG-treatment. Nevertheless, the cultures grown on the PEG-treated membranes in the presence of feeder cells did display a higher nuclear-to-cytoplasmic ratio suggesting a more proliferative phenotype. We concluded that while the treatment with PEG had a significant effect on some structural properties of the B. mori silk fibroin (BMSF) membranes, there were minimal gains in the performance of these materials as a substratum for corneal epithelial cell growth. The reduced mechanical stability of freestanding PEG-treated membranes makes them a less viable choice than the non-treated membranes.

A Bruch's membrane substitute fabricated from silk fibroin supports the function of retinal pigment epithelial cells in vitro.

Silk fibroin provides a promising biomaterial for ocular tissue reconstruction, including the damaged outer blood–retinal barrier of patients afflicted with age‐related macular degeneration (AMD). The aim of the present study was to evaluate the function of retinal pigment epithelial (RPE) cells in vitro, when grown on fibroin membranes manufactured to a thickness similar to that of Bruchs membrane (3 µm). Confluent cultures of RPE cells (ARPE‐19) were established on fibroin membranes and maintained under conditions designed to promote maturation over 4 months. Control cultures were grown on polyester cell culture well inserts (Transwell®). Cultures established on either material developed a cobblestone morphology, with partial pigmentation, within 12 weeks. Immunocytochemistry at 16 weeks revealed a similar distribution pattern between cultures for F‐actin, ZO‐1, ezrin, cytokeratin pair 8/18, RPE‐65 and Na+/K+‐ATPase. Electron microscopy revealed that cultures grown on fibroin displayed a rounder apical surface with a more dense distribution of microvilli. Both cultures avidly ingested fluorescent microspheres coated with vitronectin and bovine serum albumin (BSA), but not controls coated with BSA alone. VEGF and PEDF were detected in the conditioned media collected from above and below the two membrane types. Levels of PEDF were significantly higher than for VEGF on both membranes and a trend was observed towards larger amounts of PEDF in apical compartments. These findings demonstrated that RPE cell functions on fibroin membranes are equivalent to those observed for standard test materials (polyester membranes). As such, these studies support advancement to studies of RPE cell implantation on fibroin membranes in a preclinical model. Copyright

Improving the cellular invasion into PHEMA sponges by incorporation of the RGD peptide ligand: The use of copolymerization as a means to functionalize PHEMA sponges

A monomer that contained the RGD ligand motif was synthesized and copolymerized with 2-hydroxyethyl methacrylate using polymerization-induced phase separation methods to form poly(2-hydroxyethyl methacrylate)-based hydrogel sponges. The sponges had morphologies of aggregated polymer droplets and interconnected pores, the pores having dimensions in the order of 10 μm typical of PHEMA sponges. RGD-containing moieties appeared to be evenly distributed through the polymer droplets. Compared to PHEMA sponges that were not functionalized with RGD, the new sponges containing RGD allowed greater invasion by human corneal epithelial cells, by advancing the attachment of cells to the surface of the polymer droplets.

Incorporation of Human Recombinant Tropoelastin into Silk Fibroin Membranes with the View to Repairing Bruch’s Membrane

Bombyx mori silk fibroin membranes provide a potential delivery vehicle for both cells and extracellular matrix (ECM) components into diseased or injured tissues. We have previously demonstrated the feasibility of growing retinal pigment epithelial cells (RPE) on fibroin membranes with the view to repairing the retina of patients afflicted with age-related macular degeneration (AMD). The goal of the present study was to investigate the feasibility of incorporating the ECM component elastin, in the form of human recombinant tropoelastin, into these same membranes. Two basic strategies were explored: (1) membranes prepared from blended solutions of fibroin and tropoelastin; and (2) layered constructs prepared from sequentially cast solutions of fibroin, tropoelastin, and fibroin. Optimal conditions for RPE attachment were achieved using a tropoelastin-fibroin blend ratio of 10 to 90 parts by weight. Retention of tropoelastin within the blend and layered constructs was confirmed by immunolabelling and Fourier-transform infrared spectroscopy (FTIR). In the layered constructs, the bulk of tropoelastin was apparently absorbed into the initially cast fibroin layer. Blend membranes displayed higher elastic modulus, percentage elongation, and tensile strength (p < 0.01) when compared to the layered constructs. RPE cell response to fibroin membranes was not affected by the presence of tropoelastin. These findings support the potential use of fibroin membranes for the co-delivery of RPE cells and tropoelastin.

Hydrogels with lotus leaf topography: Investigating surface properties and cell adhesion

The interactions of cells with the surface of materials is known to be influenced by a range of factors that include chemistry and roughness; however, it is often difficult to probe these factors individually without also changing the others. Here we investigate the role of roughness on cell adhesion while maintaining the same underlying chemistry. This was achieved by using a polymerization in mold technique to prepare poly(hydroxymethyl methacrylate) hydrogels with either a flat topography or a topography that replicated the microscale features of lotus leaves. These materials were then assessed for cell adhesion, and atomic force microscopy and contact angle analysis were then used to probe the physical reasons for the differing behavior in relation to cell adhesion.

Biomaterial templates for the culture and transplantation of retinal pigment epithelial cells: A critical review

The idea of retinal cell transplantation as a potential treatment for age-related retinal degeneration, a leading cause of blindness in the Western world, has been around for a number of decades. To date, however, it has not been entirely successful; one of the main reasons for this is the lack of an ideal substratum for the retinal cells, specifically for the growth of retinal pigment epithelial cells prior to transplantation. This chapter reviews the reasoning behind this potential treatment, the development of animal transplantation models for human trials, the prerequisites of an ideal substratum, the past and current research on substratum materials, and the potential for future developments in this area.

Effect of changes in the surface chemistry and topography of poly(2-hydroxyethyl methacrylate) on the in vitro attachment of human corneal epithelial cells

The effects on cell adhesion induced by changes in the topography and chemistry of poly(2-hydroxyethyl methacrylate) hydrogel surfaces were investigated in vitro using the human corneal epithelial cell line, HCE-T. Poly(2-hydroxyethyl methacrylate) surfaces with a lotus-leaf-like topography and poly(2-hydroxyethyl methacrylate) surfaces with a flat topography, but functionalized with the cell-adhesive peptide sequence Arg–Gly–Asp, both enhanced attachment of HCE-T cells as compared to flat, non-functionalized poly(2-hydroxyethyl methacrylate) surfaces. However, the simultaneous existence on the same poly(2-hydroxyethyl methacrylate) surface of Arg–Gly–Asp motifs and of lotus-leaf-like topographical patterns led to an apparently antagonistic effect reflected in reduced cell attachment. The study provided additional evidence of the complexity of the cell–biomaterial interactions.

Characterization of Human iPSC-RPE on a Prosthetic Bruch's Membrane Manufactured From Silk Fibroin

Purpose RPE cell transplantation as a potential treatment for AMD has been extensively investigated; however, in AMD, ultrastructural damage affects both the RPE and its underlying matrix support, the Bruchs membrane (BrM). An RPE monolayer supported by a surrogate scaffold could thus provide a more effective approach to cell-based therapy for AMD. Toward this goal, we aimed to establish a functional human induced pluripotent stem cell–derived (hiPSC)-RPE monolayer on a Bombyx mori silk fibroin (BMSF) scaffold. Methods RPE differentiated from five distinct hiPSC lines were cultured on BMSF membrane coated with extracellular matrix (ECM, COL1), and either regular tissue culture plastic or Transwell coated with ECM (LAM-TCP). Morphologic, gene and protein expression, and functional characteristics of the hiPSC-RPE cultured on different membranes were compared in longitudinal experiments spanning 1 day to ≥3 months. Results The hiPSC-RPE monolayers on ECM-coated BMSF and TCP could be maintained in culture for ≥3 months and displayed RPE-characteristic morphology, pigmentation, polarity, and expression of RPE signature genes and proteins. Furthermore, hiPSC-RPE on both ECM-coated BMSF and TCP displayed robust expression and secretion of several basement membrane proteins. Importantly, hiPSC-RPE cells on COL1-BMSF and LAM-TCP showed similar efficacy in the phagocytosis and degradation of photoreceptor outer segments. Conclusions A biomaterial scaffold manufactured from silk fibroin supports the maturation and long-term survival of a functional hiPSC-RPE monolayer. This has significant implications for both in vitro disease modeling and in vivo cell replacement therapy.

The current state of stem cell therapy for ocular disease

Herein, we review the safety, efficacy, regulatory standards and ethical implications of the use of stem cells in ocular disease. A literature review was conducted, registered clinical trials reviewed, and expert opinions sought. Guidelines and codes of conduct from international societies and professional bodies were also reviewed. Collated data is presented on current progress in the field of ocular regenerative medicine, future challenges, the clinical trial process and ethical considerations in stem cell therapy. A greater understanding of the function and location of ocular stem cells has led to rapid advances in possible therapeutic applications. However, in the context of significant technical challenges and potential long-term complications, it is imperative that stem cell practices operate within formal clinical trial frameworks. While there remains broad scope for innovation, ongoing evidence-based review of potential interventions and the development of standardized protocols are necessary to ensure patient safety and best practice in ophthalmic care.

Mounting of Biomaterials for Use in Ophthalmic Cell Therapies

When used as scaffolds for cell therapies, biomaterials often present basic handling and logistical problems for scientists and surgeons alike. The quest for an appropriate mounting device for biomaterials is therefore a significant and common problem. In this review, we provide a detailed overview of the factors to consider when choosing an appropriate mounting device including those experienced during cell culture, quality assurance, and surgery. By way of example, we draw upon our combined experience in developing epithelial cell therapies for the treatment of eye diseases. We discuss commercially available options for achieving required goals and provide a detailed analysis of 4 experimental designs developed within our respective laboratories in Australia, the United Kingdom, and Belgium.