Stanford Cornea Project

Stanford Cornea Project

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Laura Hartman, Dale Waters, Rachel Parke-Houben,

Curtis W. Frank

Stayce Beck, Luo Luo Zheng, Yuhua Hu

Jennifer Cochran

Resmi Charalel, Phil Huie, Vijay VanchinathanRoopa 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

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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, 2005. 123(10): p. 1319-29.

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

hydrogelStroma

hydrogelStroma

The Stanford Approach

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high diffusion + stable, optically clear hydrogel “invisible” material

1.protein modification(Collagen and EGF) Epithelium grows back

2.

Material

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Single Network Double Network

Single Network Double Network

1st network swollen in monomeric building blocks of

2nd network

polymerization of 2nd network

• no chemical linkage• two interpenetrating networks (IPNs)• highly improved mechanical properties

J.P. Gong, et al., Advanced Materials 2003

Mechanical Stability

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1PEG 3.4K PAA PEG 8.0K PEG 14K PAADN (PEG 3.4K-PAA)

DN (PEG 8.0K-PAA)

PAA DN (PEG 14K-PAA)

1st network: Poly(ethylene glycol) (PEG)

2nd network: Poly(acrylic acid) (PAA)IP

N

HO

OH

OHO

0

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3

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Max

imum

Ten

sile

Str

ess

[MPa

]

PEG

(4.6

kDa)

PEG

(8kD

a)

PEG

(14k

Da)

PAA

PAA

PAA

IPN

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

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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

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O OH O OH O O O OH

O O OHNH

R

hydrogel hydrogel

hydrogel

+NHS/EDC

phosphate buffer

NO O

R NH2+

R = protein, polypeptide, growth factor, amino acid, carbohydrate, phosphate-containing moiety, hormone, neurotransmitter or nucleic acid

• no de-swelling of the gel• washing in buffer possible• no denaturation of proteins

Protein Tethering: Qualitative

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• 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

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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

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rabbit # 2 - post-op 50 days

Future Work

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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 EY016987– NIH Grant 5T90 DK070103-03.

• Singapore Eye Research Institute (SERI)• BioX• Stanford Office of Technology Licensing• Stanford MedScholar Program• Fight for Sight• Visx

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