Improve peripheral nerve regeneration through electric field stimulation of the substrate

Hieu NguyenNovember 29, 2011

PI: Christine Schmidt

Preliminary proposal

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Motivation

Motivation

• 11,000 Americans are affected by paralysis each year costing $7 billion

• An excess of 50,000 peripheral nerve repair procedures are performed annually

American Paralysis Association, 1997National Center for Health Statistics, Classification of Diseases, 9th Rev, 1995The Boston Globe. Afghanistan, September 2011. http://www.boston.com/bigpicture/2011/09/afghanistan_september_2011.htmlThe Animal Pet Doctor. http://animalpetdoctor.homestead.com/surg2.jpgDiabetic Foot Ulcer. Health.com 2009 http://www.health.com/health/static/hw/media/medical/hw/h9991432_001.jpg

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Current repair methods for large nerve defects

Motivation

Nerve graft

-Autologous (gold standard)

Biro , Ciuce et al. The repair of a 10 mm defect in the sciatic nerve with collagen tube. Timisoara Medical Journal 2004.

Autologous graft. Arrows show sutures at 1 cm apart.

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Motivation

Avance® Nerve Graft. AxoGen® http://www.axogeninc.com/ . Published internet 2011.

Nerve graft

-Autologous (gold standard)-Acellular

Current repair methods for large nerve defects

Axogen’s acellular nerve graft.

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Motivation

Biro , Ciuce et al. The repair of a 10 mm defect in the sciatic nerve with collagen tube. Timisoara Medical Journal 2004.

Nerve graft

-Autologous (gold standard)-Acellular-ECM/polymer-based

Current repair methods for large nerve defects

Collagen graft - arrows show sutures at 1 cm apart.

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Motivation

Schlosshauer, et al. Synthetic nerve guide implants in humans: a comprehensive survey. Neurosurgery 2006.

Nerve graft

-Autologous (gold standard)-Acellular-ECM/biological -based-Synthetic

Current repair methods for large nerve defects

PLA-PCL

PGA4mm

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Motivation

Implementing growth cues

-Physical cues

Lee JY, Schmidt CE, et al. Polypyrrole-coated electrospun PLGA nanofibers for neural tissue applications. Biomaterials 2009.

Technologies to improve nerve repair

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Motivation

Li, Hoffman-Kim, et al. Multi-molecular gradients of permissive and inhibitory cues direct neurite outgrowth. Ann Biomed Eng 2008.

Implementing growth cues

-Physical cues-Chemical cues

Technologies to improve nerve repair

LN

CSPG

40µm

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Motivation

Rajnicek, McCaig et al., Temporally and spatially… J. Cell Science 2006.

Implementing growth cues

-Physical cues-Chemical cues-Electrical cues

Technologies to improve nerve repair

Electric fields (EF) are endogenous

EF ranging 18 - 1600 mV/mm depending upon amphibian species and location on the neural tube.

McCaig CD, Rajnicek AM, Song B and Zhao M. Has electrical growth cone guidance found its potential? Trends Neurosci 2002.

Corneal epithelium layer and ionic flow created during tissue damage.

McCaig CD, Rajnicek AM, Song B and Zhao M. Controlling cell behavior electrically: current views and future potential. Physiol Rev 2005.

Background

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11Zhao, McCaig et al. Orientation and directed migration of cultured corneal epithelial cells… J. Cell Science 1996.

Background

EF through media controls cell migration

Cathodal steering of corneal epithelial cellsa) Before EF exposureb) 1 hr EF with cathode on leftc) 6 hr EF cathode on rightd) 8 hr EF on left

12Durgam, Schmidt, et al. Novel degradable co-polymers of polypyrrole support cell proliferation… J Biomat Sci 2009.

Background

EF through substrate increases neurite density

Neural-like PC12 cells grown on polypyrrole (PPy). Arrows point to neurites.

PPy stimulated 100 µA for 2 hrs. PC12 cells exhibit longer neurites and greater density.

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Improve nerve regeneration through EF stimulation of the substrate

Proposal

Reasons for substrate stimulation:

1. EF control of cell behavior through substrate is novel (PPy)

2. Provide scaffolding (glia and axons)

3. Local control of EF

4. Prolonged effects of EF

Aim 1 – Control cell growth by electrically stimulating a conductive substrate

Aims

Proposal

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Aim 2 – Model and explore environmental changes surrounding cells within EFs

Aim 3 - Optimize an electrically conductive polymer to supply an EF across a nerve injury for commercial use

1) Use one cell type to observe general reaction to changes in current, voltage, duration, and AC/DC EF through the media and through the substrate

2) Observe morphology of multiple cell types using the optimal conditions found above

3) Measure protein content to compare endogenous EF to substrate EF

Aim 1

Aim 1

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Aim 1 – Control cell growth by electrically stimulating a conductive substrate

16Substrate EF

Media EFMatrigel™ Hydrogel

Cell

(TC plate, PPy)

Aim 1

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Aim 1

EF through media

EF parameters

- Distance between electrodes: 10 cm- Voltage: 1 V/cm - Resistivity: 20 kΩ·cm- Current: 0.5 – 15 µA- Duration: 2 - 24 hrs

Schwann cells without EF.

Aim 1

EF through media aligns glial cells

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90°

0°

EF 1 V/cm

no EF EF stim0

10

20

30

40

50

60

Average cell alignment

Cell

angl

e (d

egre

es)

EF

Schwann cells exposed to EF align perpendicularly.

100µm

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EF Stimulation for whole DRG

- Dimensions of PPy: 0.5 x 2.0 cm2

- Voltage: 100 mV/cm DC or AC 60Hz peak-to-peak- Resistivity PPy: 5 kΩ·cm- Current: 0.5 – 15 µA- Duration: 2 hours

Aim 1

EF through substrate

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Aim 1

EF through substrate increases axon elongation

DRGs exposed to EF exhibit longer axons and growth parallel to EF (b III tubulin).

1mm

No EF

(axon length in µm) Control (n=506)

DC(n=358)

AC (n=281)

Average 820 927 (+13%) 996 (+21%)

Standard Deviation 302 278 343

Student’s t-test vs Control p<0.01 p<0.01

Axon lengths 3 days after 2 hr stimulation

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Aim 1

Protein analysis indicates changes in cell behavior

ELISA results http://www.biotech-weblog.com/50226711/enzymelinked_immunosorbent_assay_elisa_35_years_after.phpELISA Steps http://www.epitomics.com

ELISA assay for NGF, BDNF

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Aim 1

Aim 1 – Future work

1) Determine whether I or V is changing cell behavior. Continue to observe cell changes in AC/DC EFs

2) Use optimized conditions above to observe changes in DRGs and astrocytes (possible bridge for CNS)

3) Supernatant is being collected for protein analysis of cells stimulated through the media vs substrate

Aim 1 – Control cell growth by electrically stimulating a conductive substrate

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1) Model Debye length, induced current, and EF around a cell using Virtual Cell and COMSOL Multiphysics®

2) Can we create an electrical gradient inside a hydrogel

3) Examine cell behavior when placed on a stimulated hydrogel to see if there are any prolonged effects of EF to the environment

Aim 2

Aim 2

Aim 2 – Model and explore environmental changes surrounding cells within EFs

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Aim 2

Virtual Cell software can model ion movement and channel properties from empirical data

VCell Software 4.8. Center for Cell Analysis & Modeling (CCAM). University of Connecticut Health Center. http://www.nrcam.uchc.edu/index.html?current=one.

Model Ca+ distribution during depolarization.

Input stacked confocal image to create accurate 3D models.

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Aim 2

COMSOL Multiphysics® uses FEA to measure electromagnetics and physics

Appali R, et al. 3D-simulation of action potential propogation in a squid giant axon. Excerpt from COMSOL Conference 2009.

Tubular axon model. Axon model with drawn boundaries.

Cross section of EF surrounding axon during excitation (at various time points).

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Aim 2

DC EF changes Ca+ concentration within a gel

Gel

Stimulate gel for 20 hrs Section gel into 5 parts

Add Calcium green-1

Calcium Green -1 http://www.umsl.edu/~tsytsarev/tsytsarev_files/Lecture10.htm

1 2

3 4

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Aim 2

Control EF Stim0

10

20

30

40

50

60

Average cell alignment

Cell

angl

e (d

egre

es)90°

0°

EF 100 mV/cm

Schwann cells increase alignment on stimulated gels

Schwann cells were seeded after Matrigel was stimulated for 20 hrs.

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1) Model Debye length, induced current, and EF arround a cell using Virtual Cell and COMSOL Multiphysics®

2) We have created and measured ionic changes in gels electrically stimulated through the substrate

3) Prolonged affects of stimulation on gel will need to be analyzed using microscopy/spectroscopy

Aim 2

Aim 2 – Future work

Aim 2 – Model and explore environmental changes surrounding cells within EFs

1) Test stability and biocompatibility of TDA’s polymer substrate

2) Examine cell behavior on 2D substrate exposed to EF (film)

3) Examine cell behavior in 3D structure exposed to EF (conduit)

Aim 3

Aim 3

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Aim 3 – Optimize an electrically conductive polymer to supply an EF across a nerve injury for commercial use

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Nerve

Aim 3

Glass slide

PPy-PCL

1.0 x 1.5 cm2 Polycarbonate wells

100 mV/cm

PC12 cells (20,000 cells)

Seed PC12

Pre-stim

Stim Image

2Timeline 2

1st day 2nd day 3rd day

Aim 3

Experimental setup for PPy stability and biocompatibility

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NPS Control 1 2 PS Control 3 40

100

200

300

400

500

600

700

800

900

Total # of Live Cells on one PPy strip

Condition# Li

ve C

ells

24h

rs a

fter

stim

(w

/ st

.dev

.)

NPS = not pre-stimulated control (no stim after)1,2 = not pre-stimulated, stimulated

PS = pre-stimulated control (no stim after)3,4 = pre-stimulated, stimulated

Aim 3

PPy stimulation does not affect biocompatibility but does change cell adhesivity

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Aim 3

EF stimulation of DRGs on a 2D substrate

5-10 longest axons from 6 DRGs are stacked

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Aim 3

EF stimulation of DRGs on a 2D substrate enhances axon growth toward electrodes

35PPy conduit

6 uL gel 8 uL gel 6 uL gel

DRGlevel of media w/ NGF

200 mV (= 100mV/cm)

Aim 3

EF stimulation of DRGs in 3D

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Aim 3

EF stimulation of DRGs in 3D reduces axon density but may increase axon length

DRG in conduit with no EF DRG in conduit with EF 100 mV/cm for 2 hrs

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1) TDA’s polymer is stable and biocompatible

2) DRGs stimulated with EF on 2D films display longer axons and directed growth towards electrodes

3) Need to repeat experiment to determine if EF stimulation in 3D conduits enhances nerve growth

Aim 3

Aim 3 – Future work

Aim 3 – Optimize an electrically conductive polymer to supply an EF across a nerve injury for commercial use

Aim 1 – Control cell growth by electrically stimulating a conductive substrate.

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Aim 2 – Model and explore environmental changes surrounding cells within EFs

Aim 3 - Optimize an electrically conductive polymer to supply an EF across a nerve injury for commercial use

Summary

Summary of Aims

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Aug

Sept

Oct

Nov

Dec

Jan

Feb

Mar

April

May

June

July

Aug

Sept

Oct

Nov

Dec

2011 2012

Aim 1.1 – change resistance for ΔV or ΔI, for media and substrate

Aim 1.3 – collect protein samples

Aim 2.1 – model EF surrounding cell

Aim 2.3 – place cells on stimulated gel

Aim 3.3 – Continue 3D EF stim of conduit

Hieu’s timeline (Sept 2011)Summary

Aim 1.2 – observe optimal EF on DRG and astrocyte

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End

Acknowledgements

Schmidt groupChristine SchmidtJae Young LeeJon NickelsLeo ForcinitiZin KhaingJohn HardyCraig Milroy…and all other lab members!

My committeeRichard AldrichAaron BakerHenry RylanderLaura Suggs

Undergraduate AssistantsSung Ji AhnAlvin NguyenThomas MathewsDan WalkerJan NguyenJeff CoursenClaudia WeiJacque Chow

CollaborationsSilvia Luebben, TDAShawn Sapp, TDARobert Ross, VTI

FundingNDSEG FellowshipUndergraduate Research FellowshipIE Internship

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42Schmidt, Langer,et al. Stimulation of neurite outgrowth using an electrically conducting polymer. Proc Natl Acad Sci 1997.

Background

EF through substrate increases neurite density

Neural-like PC12 cells increase neurite formationa) PC12 cells grown on polypyrrole (PPy) before stimulationb) PPy stimulated at 100 mV for 2 hrs, image taken 24 hrs after

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Things to note

1. EF = electric field2. Current = movement of charged species3. Substrate = Polypyrrole (PPy)4. Cells used:

1. DRG = dorsal root ganglia2. Schwann cells3. Astrocytes

Proposal

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Schwann cells (top) and astrocytes (bottom) align perpendicularly to the EF.

EF

Aim 1

EF through media aligns glial cells

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Schwann cell alignment

no EF (48°)

EF stim(27°)

90°

0°

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Aim 1

AC and DC EF influences axon length

(axon length in µm) Control (n=506)

DC(n=358)

AC (n=281)

Average 820 927 996

Standard Deviation 302 278 343

Length inc vs Control 13% 21%

Student’s t-test vs Control p<0.01 p<0.01

Student’s t-test DC vs AC p<0.05

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Summary

Summary of Aims

Aim 1 - Determine how electrically stimulating a conductive substrate can control cell growth.(Characterization)

Aim 2 - Determine environmental changes around cells within EFs(Modeling and Exploratory)

Aim 3 - Optimize an electrically conductive polymer to supply an EF across a nerve injury(Translational)