Control of scar tissue formation in the cornea: strategies in clinical and corneal tissue engineering

Corneal structure is highly organized and unified in architecture with structural and functional integration which mediates transparency and vision. Disease and injury are the second most common cause of blindness affecting over 10 million people worldwide. Ninety percent of blindness is permanent due to scarring and vascularization. Scarring caused via fibrotic cellular responses, heals the tissue, but fails to restore transparency. Controlling keratocyte activation and differentiation are key for the inhibition and prevention of fibrosis. Ophthalmic surgery techniques are continually developing to preserve and restore vision but corneal regression and scarring are often detrimental side effects and long term continuous follow up studies are lacking or discouraging. Appropriate corneal models may lead to a reduced need for corneal transplantation as presently there are insufficient numbers or suitable tissue to meet demand. Synthetic optical materials are under development for keratoprothesis although clinical use is limited due to implantation complications and high rejection rates. Tissue engineered corneas offer an alternative which more closely mimic the morphological, physiological and biomechanical properties of native corneas. However, replication of the native collagen fiber organization and retaining the phenotype of stromal cells which prevent scar-like tissue formation remains a challenge. Careful manipulation of culture environments are under investigation to determine a suitable environment that simulates native ECM organization and stimulates keratocyte migration and generation

Facilitating the operational readiness of the NHS for the in-house manufacture and delivery of autologous cell therapy [210]

Facilitating the operational readiness of the NHS for the in-house manufacture and delivery of autologous cell therapy [210

Mechanical characterization of hydrogels and its implications for cellular activities

Mechanical characterization of hydrogels and its implications for cellular activitie

Isolation of mesenchymal stem cells from bone marrow aspirate

Isolation of mesenchymal stem cells from bone marrow aspirat

Chemical and topographical effects on cell differentiation and matrix elasticity in a corneal stromal layer model

Control and maintenance of the keratocyte phenotype is vital to developing in vitro tissue engineered strategies for corneal repair. In this study the influence of topographical and chemical cues on the mechanical, phenotypical and genotypical behaviour of adult human derived corneal stromal (AHDCS) cells in three dimensional (3D) multi-layered organised constructs is examined. Topographical cues are provided via multiple aligned electrospun nanofiber meshes, which are arranged orthogonally throughout the constructs and are capable of aligning individual cells and permitting cell migration between the layers. The influence of chemical cues is examined using different supplements in culture media. A non-destructive indentation technique and optical coherence tomography are used to determine the matrix elasiticity (elastic modulus) and dimensional changes, respectively. These measurements were indicative of changes in cell phenotype from contractile fibroblasts to quiescent keratocytes over the duration of the experiment and corroborated by qPCR. Constructs containing nanofibers have a higher initial modulus, reduced contraction and organised cell orientation compared to those without nanofibers. Cell-seeded constructs cultured in serum-containing media increased in modulus throughout the culture period and underwent significantly more contraction than constructs cultured in serum-free and insulin-containing media. This implies that the growth factors present in serum promote a fibroblast-like phenotype; qPCR data further validates these observations. These results indicate that the synergistic effect of nanofibers and serum-free media plus insulin supplementation provide the most suitable topographical and chemical environment for reverting corneal fibroblasts to a keratocyte phenotype in a 3D construct

Influence of cell number and collagen concentration on the mechanical behaviour of collagen hydrogel constructs [Abstracts]

Influence of cell number and collagen concentration on the mechanical behaviour of collagen hydrogel constructs [Abstracts

Non-destructive monitoring of the effect of conditions on corneal stromal cell differentiation in hydrogels

Non-destructive monitoring of the effect of conditions on corneal stromal cell differentiation in hydrogel

Controlling and online monitoring in a corneal stromal model [Abstract]

Controlling and online monitoring in a corneal stromal model [Abstract

Corneal stromal cell plasticity: in vitro regulation of cell phenotype through cell-cell interactions in a three-dimensional model

In vivo, epithelial cells are connected both anatomically and functionally with stromal keratocytes. Co-culturing aims at recapturing this cellular anatomy and functionality by bringing together two or more cell types within the same culture environment. Corneal stromal cells were activated to their injury phenotype (fibroblasts) and expanded before being encapsulated in type I collagen hydrogels constructs. Three different epithelial-stromal co-culture methods were then examined: epithelial explant; transwell; and the use of conditioned media. The aim was to determine whether the native, inactivated keratocyte cell phenotype could be restored in vitro. Media supplementation with transforming growth factor beta-1 (TGF-b1) was then used to determine whether the inactivated stromal cells retained their plasticity in vitro and could be re-activated to the fibroblast phenotype. Finally, media supplementation with wortmannin was used to inhibit epithelial–stromal cell interactions. Two different nondestructive techniques, spherical indentation and optical coherence tomography, were used to reveal how epithelial-stromal co-culturing with TGF-b1, and wortmannin media supplementation, respectively, affect stromal cell behavior and differentiation in terms of construct contraction and elastic modulus measurement. Cell viability, phenotype, morphology, and protein expression were investigated to corroborate our mechanical findings. It was shown that activated stromal cells could be inactivated to a keratocyte phenotype via co-culturing and that they retained their plasticity in vitro. Activated corneal stromal cells that were fibroblastic in phenotype were successfully reverted to a nonactivated keratocyte cell lineage in terms of behavior and biological properties; and then back again via TGF-b1 media supplementation. It was then revealed that epithelial–stromal interactions can be blocked via the use of wortmannin inhibition. A greater understanding of stromal–epithelial interactions and what mediates them offers great pharmacological potential in the regulation of corneal wound healing, with the potential to treat corneal diseases and injury by which such interactions are vital

Chemical and spatial influence on corneal stromal cell phenotype [Abstract]

Chemical and spatial influence on corneal stromal cell phenotype [Abstract