Date of Award

12-8-2024

Document Type

Thesis

School

School of Arts, Sciences, Humanities & Education

Programme

Ph.D.-Doctoral of Philosophy

First Advisor

Dr.Krishnakumar Subramanian

Second Advisor

Dr.K.Umamaheswari

Keywords

Stem Cells, Tissue Engineering, CORNEA, SCAFFOLDS, Epithelial Mesenchymal Transition

Abstract

A clear healthy cornea is essential for visual acuity. As a self-renewing and regenerating tissue, the corneal epithelium has a stem cell pool, the limbus that acts as its proliferative reservoir. When limbal epithelial stem cells are destroyed or become dysfunctional, a pathological state known as limbal stem cell deficiency (LSCD) manifests. Damaged corneas have been successfully reconstructed by transplanting the cultured limbal epithelial stem cells. Limbal stem cell transplantation (LSCT) is one of the applicable and most challenging procedures in clinical ophthalmology for the reconstruction of the stratified ocular surface epithelium in patients with LSCD. Given the above challenge, the present thesis aims to report a few approaches to the LSCT procedure.

Auto and allograft limbal transplantations are effective in restoring the corneal epithelium, inhibiting inflammation and neovascularization. The combination of limbal and denuded Human Amniotic Membrane (dHAM) transplantation has been shown to improve the surgical outcome in patients with total LSCD. However, a few problems in HAM remained unresolved, such as the sterile storage for longer periods, the thinness of the membrane that affects the suture strength, wrinkling while transplanting, and early degradation of the membrane. The HAM is a collagenous sheet and requires cross-linkers to increase its strength. This study reports on a novel and simple base chemical – aluminium sulphate (Al2So4) as a cross-linking agent. The Al2So4 cross-linked HAM was compared for the physical, chemical, mechanical, and biocompatible properties with dHAM. A better result of the properties in comparison to the conventional scaffold was obtained and with the approval of institutional ethics the cross-linked HAM is used currently for corneal reconstruction.

The ideal properties of the scaffold that can be used for limbal stem cell transplantation should be optically clear, low cost of production, durable, suturable, provide structural integrity to the ocular surface, gas permeable, allow the diffusion of glucose, and proteins, and ions, have antimicrobial properties, biocompatible and biomimetic scaffold. As an alternative is required in place of dHAM, a novel method of isolation and preparation of a collagen based scaffold from fish scales (Fish Scale Collagen - FSC), a renewable biowaste material was reported in this study. The physico-chemical, mechanical, and culture characteristics of FSC were compared with dHAM. The results suggested optically clear with sufficient mechanical properties, biocompatible, and marker gene expression for the growth characterization of limbal epithelial cells. Thus, the use of this biopolymeric, biocompatible collagen based material from a novel biosource for the growth of corneal epithelial cells offers several advantages in terms of eliminating the risk of disease transmission, reducing the inconsistency in tissue composition associated with biological substrates, being able to be custom fabricated to suit specific requirements, and possibly providing a readily available alternative tissue material for clinical use.

A limbal stem cell transplantation clinical trial was performed on a 29-year-old male patient with chemical injury by ex vivo explant cultured corneal epithelial cells on HAM. The graft was rejected after a few days of post-transplantation. The pathological sections of the pannus showed the presence of dense vascular channels with an impression of vascularisation and hemorrhage. Stromal fibroblasts with chronic inflammatory cells and lymphocytes were seen. We strongly believe that the epithelial-mesenchymal transition (EMT) could have happened due to the explant culture method of cultivating the corneal epithelial cells. Thus, we propose a culture method by using collagenase enzyme on the limbal tissue collected from the donor cadaver eyeball and isolating the pigmented limbal epithelial cells.

A comparative study between the conventional explant culture method and collagenase based culture method was conducted. This method has significant growth kinetics, colony forming efficiency, and proliferation capacity. The EMT upregulating genes were found to be downfolded in this novel culture method indicating the absence of EMT. Putative stem cell markers were maintained throughout the culture for a longer period in comparison to the explants culture method. Our technique achieved the isolation of purified corneal epithelial progenitor cells without contamination by fibroblasts, which often interfere with the primary culture of cells isolated from tissues. Thus, this alternative novel method of isolating limbal epithelial cells would avoid corneal graft rejection that happens due to EMT in the LSCT procedure.

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