Stanford Cornea Project 1

2 Laura Hartman, Dale Waters, Rachel Parke- Houben, Curtis W. Frank Stayce Beck, Luo Luo Zheng, Yuhua Hu Jennifer Cochran Resmi Charalel, Phil Huie, Vijay Vanchinathan Roopa Dalal, Michael Carrasco, Jaan Noolandi Christopher N. Ta

Who needs an artificial cornea? In the United States, over 33,000 corneal transplants are performed each year Worldwide, 10 million people are blind due to corneal disease. Most of these people do not have access to corneal transplants and remain blind due to a lack of donor tissue supply and distribution. trachoma corneal ulcer trachoma corneal ulcer

Current available keratoprosthesis 4 Boston keratoprosthesis (PMMA) AlphaCor device (PHEMA) ≈1,200 devices implanted to date (still requires donor corneas) ≈ 300 devices implanted to date; limited use Osteo-odonto keratoprosthesis 224 devices implanted with surprisingly high success rates. requires complex surgery and is only performed by a select few surgeons throughout the world Falcinelli, G., et al. Arch Ophthalmol, (10): p

Properties of an Artificial Cornea Biocompatible Optically clear centrally Nutrient permeable Mechanically strong Surface epithelialization Peripheral tissue integration CAD model acknowledgement: L. Kourtis, Stanford Dept. of Mechanical Engineering

Epithelium hydrogel Stroma hydrogel Stroma The Stanford Approach 6 high diffusion + stable, optically clear hydrogel  “invisible” material 1. protein modification (Collagen and EGF)  Epithelium grows back 2.

Material 7 Single NetworkDouble Network Single NetworkDouble Network 1 st network swollen in monomeric building blocks of 2 nd network polymerization of 2 nd network no chemical linkage two interpenetrating networks (IPNs) highly improved mechanical properties J.P. Gong, et al., Advanced Materials 2003

Mechanical Stability 8 1 st network: Poly(ethylene glycol) (PEG) ‏ 2 nd network: Poly(acrylic acid) (PAA)‏ IPN Maximum Tensile Stress [MPa] PEG (4.6kDa)‏PEG (8kDa)‏PEG (14kDa)‏ PAA IPN water content: ~90% tunable material mechanical stability (contact lens vs. inlay) pore size: diffusion (nutrient vs. drug delivery) longterm stability (implant vs. tissue scaffold)

Diffusion 9 glucose ascorbic acid sodium bicarbonate lactic acid urea amino acids A high rate of small molecule diffusion through the hydrogel is required to maintain a healthy epithelium Hydrogel (~100 μm) Flap edge Epithelium Stroma

Protein Tethering: Cell Re-Growth 10 no de-swelling of the gel washing in buffer possible no denaturation of proteins

Protein Tethering: Qualitative 11 Using fluorescently-labeled collagen, we have shown that this tethering method supports a stable binding of ECM protein. PEG Diacrylamide Hydrogel Control (adsorption) BINDING OF COLLAGEN NHS/EDC (covalent linkage) PEG Diacrylamide Hydrogel

12 Phalloidin (red) and Nuclear (DAPI(blue)) staining of primary rabbit corneal fibroblast cells grown on PEGacrylate/PAA Hydrogel tethered with A)Control, B) Collagen, C)Fibronectin, D)Laminin and E) 1:1 mixture of collagen and Fibronectin. D E Protein Tethering: Cell Re-Growth Corneal Fibroblast Cells Attach to ECM-Tethered Hydrogels A) control DC B

In vivo experiments 13 rabbit # 2 - post-op 50 days

Future Work 14 MATERIAL Determine diffusion coefficients for other proteins through human cornea Apply principles to development of artificial cornea Modify refractive index for inlay application (presbyopia) DEVICE Protein tethering Optimize the ECM content tethered to the hydrogel Use time-lapse microscopy to study cell migration on the hydrogel Addition of enhanced growth factor (EGF) to the protein layer Tissue Integration Fine-tuning is still needed to reduce the pore diameter to 50 – 100 μm Confocal fluorescence microscopy will be used to demonstrate that the channels are interconnected Tether proteins to the channel walls and test for fibroblast growth IN VIVO EXPERIMENTS Implant hydrogel-onlays/inlays Implant artificial cornea

Funding National Institutes of Health / National Eye Institute – R01 EY – NIH Grant 5T90 DK Singapore Eye Research Institute (SERI) BioX Stanford Office of Technology Licensing Stanford MedScholar Program Fight for Sight Visx 15

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