Multifunctional biomimetic materials for corneal regeneration

The cornea is the outermost layer of the eye, which is responsible for transmitting 95% of the incident light to the retina for vision and provides 70% of the focusing power of the eye. Corneal disease is a primary cause of blindness worldwide. Replacing the pathologic cornea with a donor cornea is the most accepted treatment, but there is a severe shortage of donor tissue, resulting in an extensive waiting list for transplantation of over 10 million people. In this thesis, we worked on the development of artificial corneas to solve the donor shortage issue. Although an artificial cornea made from carbodiimide crosslinked recombinant human collagen developed within our lab was successfully transplanted into 10 patients in a clinical trial, this material was not tough enough to withstand severe disease conditions where inflammation is present, and where enzymes secreted can cause premature implant degradation. To improve mechanical strength and material stability, a secondary network of 2-methacryloyloxyethyl phosphorylcholine (MPC) biopolymer was incorporated within the collagen hydrogel, forming an interpenetrating network (IPN). High resolution transmission electron microscopy showed that the implants comprised loosely bundled collagen filaments. X-ray scattering further revealed that the collagen fibrils within the implants were uniaxially oriented, whereas a biaxial alignment is present within the human cornea. This fibril arrangement resulted in highly transparent implants that transmitted virtually all incoming light of visible spectra together with a large proportion of UV light. This study is critical in a sense that it strongly suggests that all patients transplanted with this artificial cornea should take the precaution to use UV protection prior to re-growth of the epithelium, which is known to absorb harmful UV rays. To determine the utility of the implants for clinical use, we showed that they could be cut with a femtosecond laser. Laser excision of diseased patient tissue avoids damage to the surrounding healthy tissue, thereby circumventing excessive, undesirable inflammatory responses associated with the manual surgical technique while the cutting of a matched implant allows for precise host-graft apposition and seamless regeneration. We also showed that the surface of the implants could be modified to enhance rapid and stable epithelial growth. We demonstrated that we could pattern the implants surfaces using microcontact printing with fibronectin as “ink”. The dimensions of the patterned stripes were important in controlling corneal epithelial cell behavior including proliferation. This is important to ensure rapid wound healing and hence, an overall superior clinical outcome. In all of the above materials, the collagen was crosslinked with N-(3-dimethylaminopropyl)N'-ethylcarbodiimide (EDC)/N-hydroxysuccinimide (NHS). EDC is a zero-length crosslinker and while it produces a sufficiently robust hydrogel for clinical implantation, suturability was still an issue. To enhance suturability, we evaluated the effects of an epoxy-based crosslinker, 1,4-Butanediol diglycidyl ether (BDDGE), which has been shown to result in collagen hydrogels with enhanced elasticity. As neuronal ingrowth into the hydrogels and epithelial cell coverage are important considerations in achieving regeneration, we examined the effects of incorporation of short cell adhesive laminin peptides within the BDDGE-crosslinked hydrogels. We showed that incorporation of YIGSR and IKVAV peptides enhanced the proliferation of corneal epithelial cells and neuronal progenitor cells, respectively. Although artificial corneas made from collagen have been successfully tested in the clinic, animal-derived collagens, in general, come from very heterogeneous sources and carry a risk of pathogen transmission. Use of recombinant human collagens mitigates those issues but just like native collagens; they are large macromolecules, relatively inert and therefore difficult to chemically alter to design in new functionalities. They are difficult and hence expensive to produce. Collagen-like peptides (CLP), also known as collagen mimetic peptides, are relatively short sequences that have been designed to replicate and reproduce the function of full-length collagen. We examined the safety and efficacy of one such CLP that we had conjugated to polyethylene glycol-maleimide (PEG) as implants for promoting corneal regeneration in mini-pig models. This CLP-PEG implants promoted the regeneration of corneal epithelial and stromal cells from endogenous progenitors, as well as cornea nerves to form a stable neo-cornea. The use of fully synthetic materials that can be produced under a tightly controlled environment such as CLP-PEG mitigates safety issues associated with native collagen from animal or human sources, as well as makes production sufficiently costeffective to allow for future scale-up. LIST OF PUBLICATIONS This thesis is based on the following papers, which, will be referred to by their Roman numerals in the main text. I. The structural and optical properties of type III human collagen biosynthetic corneal substitutes. Sally Hayes, Phillip Lewis, M. Mirazul Islam, James Doutch, Thomas Sorensen, Tomas White, May Griffith, and Keith M. Meek. Acta Biomater. 2015 Oct 1; 25: 121–130. II. Functional fabrication of recombinant human collagen-phosphorylcholine hydrogels for regenerative medicine applications. M. Mirazul Islam*, Vytautas Cėpla*, Chaoliang He*, Joel Edin, Tomas Rakickas, Karin Kobuch, Živilė Ruželė, W. Bruce Jackson, Mehrdad Rafat, Chris P. Lohmann, Ramūnas Valiokas*, May Griffith*. Acta Biomater. 2015 Jan;12:70-80. *equal contribution. III. Epoxy cross-linked collagen and collagen-laminin Peptide hydrogels as corneal substitutes. Li Buay Koh*, Mohammad Mirazul Islam*, Debbie Mitra*, Christopher W. Noel*, Kimberley Merrett, Silvia Odorcic, Per Fagerholm, William. Bruce Jackson, Bo Liedberg, Jaywant Phopase*, and May Griffith*. J Funct Biomater. 2013 Aug 28;4(3):162-77. *equal contribution. IV. Cathelicidin LL-37 and HSV-1 Corneal Infection: Peptide Versus Gene Therapy. Chyan-Jang Lee, Oleksiy Buznyk,* Lucia Kuffova,* Vijayalakshmi Rajendran, John V. Forrester, Jaywant Phopase, Mohammad M. Islam, Mårten Skog, Jenny Ahlqvist, and May Griffith Transl Vis Sci Technol. 2014 May; 3(3): 4. *equal contribution. V. Self-assembled collagen-like-peptide implants as alternatives to human donor corneal transplantation. M. Mirazul Islam,* R. Ravichandran,* D. Olsen, M. K. Ljunggren, Per Fagerholm, C. J. Lee, M. Griffith* and J. Phopase. * Manuscript. *equal contribution. TABLE OF CONTENTS