The role of mechanical and chemical cues on corneal cells function

Abstract

This thesis describes the role of mechanical and chemical cues to preserve corneal cells function. Specifically, the use of decellularised cornea scaffolds is explored to look at the effects of mechanical stiffness in determining the fate of corneal limbal stem cells when they repopulate the acellular scaffolds. In addition, retinoic acid is introduced into the serum-free culture medium either in 2D or 3D environment to evaluate its potential in modulating the phenotype of both normal and diseased human corneal keratocytes. The greatest challenge in developing a bio-engineered cornea has always been to recapitulate the corneal stromal biology within the artificial scaffold. The idea to use decellurised corneas is brought forward as it holds both the structure and composition of native cornea, which is what the bio-engineered cornea is lacking. Here, different methods of decellularisation are used to identify the best method which maintains the structural integrity of scaffolds as well as allowing recellularisation of limbal stem cells to model corneal epithelial growth. Further manipulation to the substrate stiffness via collagenase treatment is adapted to explore how stiffness affects limbal stem cell behaviour. The quest to establish and promote the use of serum-free culture medium for corneal cell culture work is continued in this thesis. It is hypothesized that retinoic acid supplementation in culture medium is able to modulate specific chemical cues especially the matrix metalloproteases (MMPs) to retain quiescent properties of corneal keratocytes previously exposed to serum in vitro. Similar effect is also postulated onto fibroblast taken from keratoconic corneas, in which modulation of the MMPs enables the keratoconic fibroblast to de-differentiate into a more normal, quiescent keratocyte. This work therefore demonstrates the influence of mechanical and chemical conditionings on corneal cells function and may provide a greater understanding in the study of corneal biology and tissue engineering application.