Document Type

Thesis - Open Access

Award Date

2019

Degree Name

Doctor of Philosophy (PhD)

Department / School

Pharmaceutical Sciences

First Advisor

Gudiseva Chandrasekher

Keywords

Biomaterial, Construct, Cornea, Epithelium/Stroma, Extracellular matrix, Regeneration

Abstract

Transparent cornea, at the front of the eye, mediates vision by refracting incoming light. Due to its anatomical position, cornea is susceptible to a variety of physical and chemical insults. Severe damage caused to cornea due to trauma or disease conditions can impair vision. While the cornea’s inherent repair mechanism restores partial vision loss, transplantation is the only viable treatment option if its transparency is completely lost. A global shortage of transplantable donor tissues necessitates the need for the development of functional corneal prosthetics. As cornea mostly is made up of extracellular matrix (ECM), several studies have explored the utility of ECM components such as collagens and proteoglycans to fabricate corneal replacements. However, replaceable devices made of these materials so far did not produce desirable outcomes in patients due to a variety of reasons. Employment of a biomaterial that closely mimics the native corneal composition is very critical for the development of corneal replacements. An ideal corneal substitute should also be able to harbor the epithelium which is critical to maintaining tissue function. Therefore, we have considered it worthwhile to utilize the ECM of cornea for preparing a therapeutic platform that can be useful not only for producing corneal replacements but also for treating multiple vision impairing corneal disorders. The work presented in this thesis shows that corneal ECM components can promote epithelial cell regeneration. Further, a biomaterial is prepared from the cornea tissue itself, referred to as cornea ECMIX, and it is employed to design a construct whose characteristics and functionality is evaluated. The content included in Chapter 1 (Introduction) describes the relevant literature related to research presented in this thesis. The topics covered include a description of ECM components and how ECM components may vary between tissue to tissue. This chapter also discusses the corneal ECM microenvironment, and its importance for maintaining tissue health. List of various materials, chemicals and descriptions of general methods employed to perform the experiments are presented in Chapter 2. The results presented in Chapter 3 shows the secretion kinetics of three corneal ECM components (Collagen I, Collagen IV and Fibronectin) in response to corneal epithelial injury. The medium containing these components also promoted epithelial cell growth. The observations from this chapter signify the importance of cornea-derived ECM components on epithelial cell regeneration. In order to utilize intrinsic corneal ECM for therapeutic applications, we have described a method to liquefy cornea in its entirety (ECMIX), that is described in Chapter 4. To the best of our knowledge, currently there is no other method available to completely solubilize the cornea in this manner. The process of this method is proprietary, and a U.S. patent has been filed (October 2017-Tissue-Derived Biomaterial composition and methods for Ocular and other Therapeutic Applications - US Patent Pending). ECMIX stimulated growth promoting intracellular signaling mechanisms and upregulated cell cycle protein expression in corneal epithelium. Further, we demonstrated the ability of ECMIX to regenerate epithelium on human corneas. ECMIX growth promoting potential can be attributed to the collective influence of multiple native corneal ECM components. Optical (transmittance and refractive index), rheological (viscosity and viscoelasticity) properties of ECMIX and its employment for the fabrication of a corneal replacement are described in Chapter 5. The fibrillar collagen arrangement in ECMIX construct and human corneas was comparable as evidenced by scanning electron microscope analysis. Further, the prepared constructs were evaluated for temperature stability, susceptibility to proteolytic degradation, swelling capacity, and glucose permeability. Constructs supported the growth of corneal cells (epithelium and fibroblasts). In conclusion, our studies highlight that engineering of ECM-based therapeutic devices for regeneration of damaged ocular surface is worthy of consideration. Corneal ECMIX and ECMIX-construct that are developed in this research work are beneficial platforms to treat recurrent corneal epithelial erosions and other corneal dystrophies where the presence of a healthy ECM is very critical. ECMIX-based therapeutic devices could regenerate epithelial cells on ocular surfaces and replace damaged human corneas. In the era of 3D printing of organs, ECMIX, whose composition approximates that of the native corneal tissue could serve as bio-ink to print corneas for transplantation purposes.

Library of Congress Subject Headings

Cornea -- Diseases -- Treatment.
Extracellular matrix.
Epithelial cells.
Regeneration (Biology)
Tissue engineering.

Format

application/pdf

Number of Pages

174

Publisher

South Dakota State University

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Rights Statement

In Copyright