Cell and Tissue Project Presentation

Engineering Stromal Tissue For Use In Clinical Cornea Transplantation

Kelsey Henderson, Quinn Otte, Matthew Lopez

“Where there is no vision, there is no hope.” –George Washington Carver

The EyeSensory organPart of the

nervous system

http://www.buzzle.com/articles/causes-of-absent-red-reflex-in-newborns.html

The CorneaFunctions: • Protection• Oxygen diffusion • Refraction of light

http://www.aafp.org/afp/2013/0115/p114.html

The StromaRelatively thick- 465μmKeratocyctes

Cornea Diseases/ProblemsFuch’s dystrophy: KeratoconisMacular corneal dystrophyCorneal ulcers Injury

http://www.uhbristol.nhs.uk/patients-and-visitors/your-hospitals/bristol-eye-hospital/bristol-laser-vision/what-is-keratoconus/

Current TreatmentDonor transplant: keratoplasty

Penetrating Endothelial Deep Anterior Lamellar

Problems:Lack of

donorsLimited

lifespan of tissue

Potential for disease transfer http://www.patienteducationcenter.org/articles/corneal-transplant/

Engineering Considerations

Biocompatibility

Degradation

Cell Sources

Material Choice

Optical Clarity

Oxygen Diffusion

Major ConsiderationsOxygen Diffusion

No vascular network Oxygen is supplied to cornea by air

Optical Clarity Parallel fibrils within lamella Orthogonally aligned lamellae Constant diameter of lamellae Water content of stroma

Degradation Material should degrade at the same rate that cells are

proliferating

Current synthetic optionsBiocompatibility

Clarity Degradation

Cell adhesionandproliferation

Oxygen diffusion

Mechanical strength

ACMP ✔ ✖ ✔ -- -- ✔PVA-AM ✔ ✔ -- -- -- ✖PVA-COL ✔ ✔ -- ✔ -- ✔CV ✔ ✔ ✔ ✔ -- ✔Silk Fibroin

✔ ✔ ✔ ✔ ✔ ✔

Our proposed optionHarvest DPCs from

patientSynthesize silk fibroin

sheetsCouple silk fibroin

with RGDhttp://www.prolifebiobank.com/dental_stem_cells.php

Short time water vapor annealing followed by enzymatic pretreatment with protease XIV

Our proposed optionDifferentiate DPCs then seed cells onto silk fibroinCulture silk sheets and layer to stromal thicknessReady for implantation by clinician

http://www.techautos.com/2012/10/02/researchers-announce-artificial-cornea-clinical-trials-under-way/

Testing our strategyIn vitro:

Biocompatibility testsPresence of corneal fibroblasts and scar tissueVascularization and haze

Differentiation testsGene and protein expression

Degradation tests

Testing our strategyIn vitro:

Cell compatibility/seeding testsProduction of ECM components such as types I, V,

and VI collagen, proteoglycans, keratocan, lumican and keratan sulfate

Live/dead assayClarity tests

Uniform size/spacing of lamellaeParallel orientation of fibrilsOrthogonal orientation of lamellae

Gas diffusion tests

Testing our strategyIn vivo:

BiocompatibilityECM productionDegradation

https://www.jax.org/jax-mice-and-services/find-and-order-jax-mice

ConclusionsThe need for cornea tissue is continually increasing

Current treatment involves autologous donor sourcesStromal layer is most difficult to engineerMajor considerations include oxygen perfusion,

optical clarity and degradationA silk biomaterial is proposed along with dental

pulp stem cells differentiated into keratocytesEventual goal is to create an engineered corneal

tissue that can be used in clinical applications

Questions?Thank you for your attention