Engineering Multi-Functional Three-Dimensional Nervous Tissue Constructs Engineering Multi-Functional Three-Dimensional Nervous Tissue Constructs

Advisor: Dr. Bryan J. Pfister

Presenter: Mevan Siriwardane, C2PRISM Research Fellow

Advisor: Dr. Bryan J. Pfister

Presenter: Mevan Siriwardane, C2PRISM Research Fellow

Development and GrowthDevelopment and Growth

Axon Stretch GrowthMechanical tension in nerves is natural

during development in most species

Axon Stretch GrowthMechanical tension in nerves is natural

during development in most species

BackgroundBackground

Nerve Guidance Conduits for Treating Nerve Injury Hollow tubes used in support of nerve regeneration

Nerve Guidance Conduits for Treating Nerve Injury Hollow tubes used in support of nerve regeneration

Questions:

What is tissue engineering?

2D Axon Stretch-Growth2D Axon Stretch-Growth

Building a Nerve Tissue Transplant: “Axon Stretch-Growth” Building a Nerve Tissue Transplant: “Axon Stretch-Growth”

2-D Stretch-Growth

2D Axon Stretch-Growth2D Axon Stretch-Growth

2D Axon Stretch-Growth2D Axon Stretch-Growth

3D Axon Stretch-Growth3D Axon Stretch-Growth Building a Nerve Tissue Transplant: “Axon Stretch-Growth” Building a Nerve Tissue Transplant: “Axon Stretch-Growth”

3-D Stretch-Growth

3D Axon Stretch-Growth3D Axon Stretch-GrowthDesign Considerations

Sustaining stretch-grown axons in a gel

Design ConsiderationsSustaining stretch-grown axons in a gel

3D Axon Stretch-Growth3D Axon Stretch-GrowthAxons crossing through mesh!Axons crossing through mesh!

Axons also grow along the fibers of the nylon mesh!!!

Axons also grow along the fibers of the nylon mesh!!!

3D Axon Stretch-Growth3D Axon Stretch-Growth

Axon Outgrowth on 2D and 3D Collagen SubstratesAxon Outgrowth on 2D and 3D Collagen Substrates

Evaluation of Candidate Materials for Nerve Tissue Construct Rat Tail Collagen Type I Bovine Tendon Collagen Type I Study Axon Growth Behavior

2D coating on dishes (Collagen) 3D Hydrogels (Collagen)

Evaluation of Candidate Materials for Nerve Tissue Construct Rat Tail Collagen Type I Bovine Tendon Collagen Type I Study Axon Growth Behavior

2D coating on dishes (Collagen) 3D Hydrogels (Collagen)

Axon Outgrowth on 2D Rat Tail and Bovine Tendon Collagen Axon Outgrowth on 2D Rat Tail and Bovine Tendon Collagen

Neurite Outgrowth on 2D Rat Tail and Bovine Tendon Type I Collagen

0

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0 2 4 6 8 10

Days

Ou

tgro

wth

Dis

tan

ce (

m)

2D Rat Tail Collagen

2D Bovine TendonCollagen

307.37m/da y

228.03m/da y

Axon Outgrowth on 2D Rat Tail and Bovine Tendon CollagenAxon Outgrowth on 2D Rat Tail and Bovine Tendon Collagen Morphology Differences Morphology Differences

b) DRG on Rat Tail Collagen Type I

a) DRG on Bovine Tendon Collagen Type I

2D vs. 3D Axonal Outgrowth2D vs. 3D Axonal Outgrowth

DRG Neurite Outgrowth on Collagen Substrates

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0 2 4 6 8 10

Days

Ou

tgro

wth

Dis

tan

ce (

m)

Neurite Outgrowth in 2D246.32um/dayNeurite Outgrowth in 3D153.63um/day

HypothesisHypothesis

The incorporation of uni-axial fibers to soft 3D collagen gels will provided a rigid framework for guiding directional outgrowth for regenerating axons

The incorporation of uni-axial fibers to soft 3D collagen gels will provided a rigid framework for guiding directional outgrowth for regenerating axons

Axon Growth on FibersAxon Growth on Fibers

a) DRG on Collagen Fibers b) DRG on PLLA Fibers

Axon Growth on FibersAxon Growth on Fibers

PLLA Fiber

Collagen Fiber

Axon Outgrowth Rate on Collagen FibersAxon Outgrowth Rate on Collagen Fibers

DRG Neurite Outgrowth on Collagen Substrates

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7000

0 2 4 6 8 10

Day

Ou

tgro

wth

Dis

tan

ce

(m

)

Neurite Outgrowth in 2D246.32um/day

Neurite Outgrowth in 3D153.63um/day

Neurite Outgrowth onCollagen Fibers794.24um/day

Collagen Bulk Fibers vs. Collagen-Coated PLLA FibersCollagen Bulk Fibers vs. Collagen-Coated PLLA Fibers

Neurite Outgrowth on Collagen and Collagen-coated PLLA Fibers

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7000

0 2 4 6 8

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Ou

tgro

wth

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Collagen Fibers

Collagen-Coated PLLAFibers

Future DirectionsFuture Directions

a) DRG on ThickPLLA Fiber

b) DRG on ThickCollagen Fiber

c) DRG on ThinPLLA Fiber

d) DRG on ThinCollagen Fiber

Fiber Diameter and Molecular Orientation

Future DirectionsFuture Directions

a) DRG on ThickPLLA vs. Collagen Fiber

b) DRG on ThinPLLA vs. Collagen Fiber

Preferential Growth

Future DirectionsFuture Directions

a) DRG on Multi-filamentThin PLLA Fibers

b) DRG on Multi-filamentThin Collagen Fibers

c) DRG on Multi-filamentThin PLLA Fibers (Spaced)

d) DRG on Multi-filamentThin Collagen Fibers (Spaced)

Multi-filament and Spacing

Future DirectionsFuture Directions Protein coatings on collagen fibers Comparison of rat tail and bovine tendon

collagen hydrogels on 3D axon outgrowth Integration of living axon stretch-grown cultures

into hydrogel matrix with fiber-reinforced composites

Clinical evaluation of construct using in vivo rat models

Protein coatings on collagen fibers Comparison of rat tail and bovine tendon

collagen hydrogels on 3D axon outgrowth Integration of living axon stretch-grown cultures

into hydrogel matrix with fiber-reinforced composites

Clinical evaluation of construct using in vivo rat models

Questions?Questions?

3D Nervous Tissue Construct

Fibers Fibers