Date of Award

1-1-2013

Document Type

Open Access Dissertation

Department

Biomedical Engineering

First Advisor

Jay D Potts

Abstract

Corneal transplantation and related surgical procedures are areas of tissue replacement which have seen promising advances. Currently, cornea transplants are one of the most common surgical procedures, with approximately 40,000 occurring each year in the United States. Still, problems exist with a 20% rejection rate, post surgical infections, and the need for a constant supply of donors. The ability to safely and quickly heal and regenerate corneal epithelia fills an area of advancement in regenerative medicine, with implications reaching beyond the scope of vision therapies toward healing of a wide range of tissues and wounds. In vivo corneal wounds were created in rats through a surgical procedure chemically loosening and mechanically removing the epithelium from the corneal surface. Our study applies a synthetically developed peptide capable of promotion of wound healing and epithelial regeneration. The alpha-carboxy terminus 1 (aCT1) peptide is a 25 amino acid peptide from the C-terminus of connexin 43, modified to promote cellular uptake. Previous studies applying aCT1 to excisional wounds in rats produced resulting tissue having an overall reduced level of scar tissue and decreased healing time, compared to controls. Upon entering the wound site, the peptide acts as a competitive inhibitor to the ZO-1-Cx43 interaction. In doing so, the formation of gap junctions during healing is altered. Here we first characterized the development and synthesis of alginate based microcapsules, designed to deliver aCT1 in a controlled and sustained release. We hypothesized sustained release of aCT1 would enhance the peptides innate wound healing ability beyond previous results. An alginate-poly-l-ornithine pH 4.3 microcapsule capable of delivery of 150µM of peptide over 48hrs was ultimately produced. Both directly applied aCT1 in a pluronic gel system and microencapsulated aCT1 were tested in two studies of in vivo corneal wound injuries using normal and STZ type I diabetic Sprague Dawley rats. Data derived from RT-PCR, confocal immunohistochemistry, inflammatory H&E scoring, western blotting, Elisa protein analysis, and fluorescent wound measurements indicated a significant increase in wound healing speed when aCT1 was applied at a 150µM concentration. The application of 150µM aCT1 alginate-poly-l-ornithine pH 4.3 microcapsules further increased corneal wound healing rate beyond those seen when using aCT1 alone.

Rights

© 2013, Keith Brian Moore

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