Improve peripheral nerve regeneration through electric field stimulation of the substrate Hieu Nguyen November 29, 2011 PI: Christine Schmidt Preliminary proposal

Motivation 2 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, 1997 National Center for Health Statistics, Classification of Diseases, 9th Rev, 1995 The Boston Globe. Afghanistan, September The Animal Pet Doctor. Diabetic Foot Ulcer. Health.com

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 Autologous graft. Arrows show sutures at 1 cm apart.

Motivation Avance® Nerve Graft. AxoGen® Published internet Nerve graft -Autologous (gold standard) -Acellular Current repair methods for large nerve defects 4 Axogen’s acellular nerve graft.

Motivation Biro, Ciuce et al. The repair of a 10 mm defect in the sciatic nerve with collagen tube. Timisoara Medical Journal Nerve graft -Autologous (gold standard) -Acellular -ECM/polymer-based Current repair methods for large nerve defects 5 Collagen graft - arrows show sutures at 1 cm apart.

Motivation Schlosshauer, et al. Synthetic nerve guide implants in humans: a comprehensive survey. Neurosurgery Nerve graft -Autologous (gold standard) -Acellular -ECM/biological -based -Synthetic Current repair methods for large nerve defects 6 PLA-PCL PGA 4mm

Motivation Implementing growth cues -Physical cues Lee JY, Schmidt CE, et al. Polypyrrole-coated electrospun PLGA nanofibers for neural tissue applications. Biomaterials Technologies to improve nerve repair 7

Motivation Li, Hoffman-Kim, et al. Multi-molecular gradients of permissive and inhibitory cues direct neurite outgrowth. Ann Biomed Eng Implementing growth cues -Physical cues -Chemical cues Technologies to improve nerve repair 8 LN CSPG 40µm

Motivation Rajnicek, McCaig et al., Temporally and spatially… J. Cell Science Implementing growth cues -Physical cues -Chemical cues -Electrical cues Technologies to improve nerve repair 9

Electric fields (EF) are endogenous EF ranging 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 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 Background 10

Zhao, McCaig et al. Orientation and directed migration of cultured corneal epithelial cells… J. Cell Science Background EF through media controls cell migration Cathodal steering of corneal epithelial cells a)Before EF exposure b)1 hr EF with cathode on left c)6 hr EF cathode on right d)8 hr EF on left 11

Durgam, Schmidt, et al. Novel degradable co-polymers of polypyrrole support cell proliferation… J Biomat Sci Background EF through substrate increases neurite density Neural-like PC12 cells grown on polypyrrole (PPy). Arrows point to neurites. 12 PPy stimulated 100 µA for 2 hrs. PC12 cells exhibit longer neurites and greater density.

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 13

Aim 1 – Control cell growth by electrically stimulating a conductive substrate Aims Proposal 14 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 15 Aim 1 – Control cell growth by electrically stimulating a conductive substrate

Substrate EF Media EF Matrigel™ Hydrogel Cell (TC plate, PPy) Aim 1 16

Aim 1 EF through media 17 EF parameters - Distance between electrodes: 10 cm - Voltage: 1 V/cm - Resistivity: 20 kΩ·cm - Current: 0.5 – 15 µA - Duration: hrs

Schwann cells without EF. Aim 1 EF through media aligns glial cells 18 90° 0° EF 1 V/cm EF Schwann cells exposed to EF align perpendicularly. 100µm

EF Stimulation for whole DRG - Dimensions of PPy: 0.5 x 2.0 cm 2 - 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 19

Aim 1 EF through substrate increases axon elongation 20 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 (+13%)996 (+21%) Standard Deviation Student’s t-test vs Control p<0.01 Axon lengths 3 days after 2 hr stimulation

Aim 1 Protein analysis indicates changes in cell behavior 21 ELISA results ELISA Steps ELISA assay for NGF, BDNF

Aim 1 Aim 1 – Future work 22 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

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

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 Model Ca+ distribution during depolarization. Input stacked confocal image to create accurate 3D models.

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 Tubular axon model.Axon model with drawn boundaries. Cross section of EF surrounding axon during excitation (at various time points).

Aim 2 DC EF changes Ca+ concentration within a gel Gel 26 Stimulate gel for 20 hrsSection gel into 5 parts Add Calcium green-1 Calcium Green

Aim ° 0° EF 100 mV/cm Schwann cells increase alignment on stimulated gels Schwann cells were seeded after Matrigel was stimulated for 20 hrs.

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 28 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 29 Aim 3 – Optimize an electrically conductive polymer to supply an EF across a nerve injury for commercial use

Nerve Aim 3 30

Glass slide PPy-PCL 1.0 x 1.5 cm 2 Polycarbonate wells 100 mV/cm PC12 cells (20,000 cells) Seed PC12 Pre-stim Stim Image 2 Timeline 2 1st day2nd day 3rd day Aim 3 Experimental setup for PPy stability and biocompatibility 31

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 32

Aim 3 EF stimulation of DRGs on a 2D substrate 5-10 longest axons from 6 DRGs are stacked 33

Aim 3 34 EF stimulation of DRGs on a 2D substrate enhances axon growth toward electrodes

PPy conduit 6 uL gel8 uL gel6 uL gel DRG level of media w/ NGF 200 mV (= 100mV/cm) Aim 3 EF stimulation of DRGs in 3D 35

Aim 3 EF stimulation of DRGs in 3D reduces axon density but may increase axon length 36 DRG in conduit with no EF DRG in conduit with EF 100 mV/cm for 2 hrs

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 37 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. 38 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

Aug Sept Oct Nov Dec Jan Feb Mar April May June July Aug Sept Oct Nov Dec Aim 1.1 – change resistance for ΔV or ΔI, for media and substrate Aim 1.3 – collect protein samplesAim 2.1 – model EF surrounding cellAim 2.3 – place cells on stimulated gelAim 3.3 – Continue 3D EF stim of conduit Hieu’s timeline (Sept 2011) 39 Summary Aim 1.2 – observe optimal EF on DRG and astrocyte

End Acknowledgements 40 Schmidt group Christine Schmidt Jae Young Lee Jon Nickels Leo Forciniti Zin Khaing John Hardy Craig Milroy …and all other lab members! My committee Richard Aldrich Aaron Baker Henry Rylander Laura Suggs Undergraduate Assistants Sung Ji Ahn Alvin Nguyen Thomas Mathews Dan Walker Jan Nguyen Jeff Coursen Claudia Wei Jacque Chow Collaborations Silvia Luebben, TDA Shawn Sapp, TDA Robert Ross, VTI Funding NDSEG Fellowship Undergraduate Research Fellowship IE Internship

41

Schmidt, Langer,et al. Stimulation of neurite outgrowth using an electrically conducting polymer. Proc Natl Acad Sci Background EF through substrate increases neurite density Neural-like PC12 cells increase neurite formation a)PC12 cells grown on polypyrrole (PPy) before stimulation b)PPy stimulated at 100 mV for 2 hrs, image taken 24 hrs after 42

Things to note 1.EF = electric field 2.Current = movement of charged species 3.Substrate = Polypyrrole (PPy) 4.Cells used: 1.DRG = dorsal root ganglia 2.Schwann cells 3.Astrocytes Proposal 43

44

Schwann cells (top) and astrocytes (bottom) align perpendicularly to the EF. EF Aim 1 EF through media aligns glial cells 45 no EF (48°) EF stim(27°) 90° 0°

Aim 1 AC and DC EF influences axon length (axon length in µm) Control (n=506) DC (n=358) AC (n=281) Average Standard Deviation Length inc vs Control13%21% Student’s t-test vs Controlp<0.01 Student’s t-test DC vs ACp<

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) 47