Hieu Nguyen November 20, 2012 PI: Christine Schmidt

1. Hieu NguyenNovember 20, 2012PI: Christine Schmidt Controlling neural cell behavior with electric field stimulation across a conductive substrate

2. Motivation Motivation • 11,000 Americans are affected by paralysis from spinal damage 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 2011. http://www.boston.com/bigpicture/2011/09/afghanistan_september_2011.html The Animal Pet Doctor. http://animalpetdoctor.homestead.com/surg2.jpg Diabetic Foot Ulcer. Health.com 2009 http://www.health.com/health/static/hw/media/medical/hw/h9991432_001.jpg

3. Background Current repair methods for large nerve defects Nerve graft • Autologous (gold standard) Autologous graft. Arrows show sutures at 1 cm apart. Biro , Ciuce et al. The repair of a 10 mm defect in the sciatic nerve with collagen tube. Timisoara Medical Journal 2004.

4. Background Current repair methods for large nerve defects Nerve graft • Autologous (gold standard) • Acellular Axogen’sacellular nerve graft. Avance® Nerve Graft.AxoGen® http://www.axogeninc.com/ . Published internet 2011.

5. Background Current repair methods for large nerve defects Nerve graft • Autologous (gold standard) • Acellular • Biological/synthetic Collagen graft - arrows show sutures at 1 cm apart. 4mm PGA Biro , Ciuce et al. The repair of a 10 mm defect in the sciatic nerve with collagen tube. Timisoara Medical Journal 2004. Schlosshauer, et al. Synthetic nerve guide implants in humans: a comprehensive survey. Neurosurgery 2006.

6. Background Technologies to improve nerve repair Implementing growth cues • Physical cues Lee JY, Schmidt CE, et al. Polypyrrole-coated electrospun PLGA nanofibers for neural tissue applications. Biomaterials 2009.

7. Background Technologies to improve nerve repair Implementing growth cues • Physical cues • Chemical cues LN CSPG 40µm Li, Hoffman-Kim, et al. Multi-molecular gradients of permissive and inhibitory cues direct neurite outgrowth. Ann Biomed Eng 2008.

8. Background Technologies to improve nerve repair Implementing growth cues • Physical cues • Chemical cues • Electrical cues Rajnicek, McCaig et al., Temporally and spatially… J. Cell Science 2006.

9. Background Electric fields (EF) are endogenous EF ranging 18 - 1600 mV/mm depending upon amphibian species and location on the neural tube. Corneal epithelium layer and ionic flow created during tissue damage. McCaig CD, Rajnicek AM, Song B and Zhao M. Has electrical growth cone guidance found its potential? Trends Neurosci 2002. McCaig CD, Rajnicek AM, Song B and Zhao M. Controlling cell behavior electrically: current views and future potential. Physiol Rev 2005.

10. Background EF through media EF through substrate = Electric Field (EF) = Ionic current (I)

11. Background EF through media controls cell migration EF through substrate increases neurite density Control Stimulated 100 uA 50 µm PPy stimulated 100 µA for 2 hrs. PC12 cells exhibit longer neurites and greater density. Cathodal steering of corneal epithelial cells Zhao, McCaiget al. Orientation and directed migration of cultured corneal epithelial cells… J. Cell Science 1996. Durgam, Schmidt, et al. Novel degradable co-polymers of polypyrrole support cell proliferation… J BiomatSci 2009.

12. Method Improve nerve regeneration through EF stimulation of the substrate • Reasons for substrate stimulation: • 1. EF control of cell behavior through substrate is novel • 2. Local control of EF • 3. Provide scaffolding (glia and axons)

13. Method Cell model of the peripheral nervous system • Schwann cells • Precede axon growth • Support axon viability • Dorsal root ganglia • Sensory neuron • Project axons to body • Source: • SC: rat sciatic nerve • DRG: isolation from p2 SD rats Navarro et al. Neural plasticity after peripheral nerve injury and regeneration. ProgNeurobiol 2007.

14. Aims • Aim 3 • Develop an electrically conductive nerve conduit to improve nerve regeneration • Aim 2 • Examine changes in extracellular environment when exposed to substrate EF Aim 1 Observe Schwann cell morphology when exposed to an EF through media and substrate • vs

15. Aim 1 Aim 1– Observe Schwann cell morphology when exposed to an EF through media and substrate Al-Majed, Gordon et al. Brief electrical stimulation promotes the speed and accuracy… J Cell Neurosci 2000. Durgam, Schmidt, et al. Novel degradable co-polymers of polypyrrole support cell proliferation… J BiomatSci 2009. Erickson and Nuccitelli. Embryonic fibroblast motility and orientation… J Cell Biol 1984. Zhao, McCaig et al. Orientation and directed migration of cultured corneal epithelial cells… J Cell Science 1996

16. Aim 1 EF through media EF through substrate 1. 1. 2. 2. 3. • Cell culture: • - Schwann cells P6-12 • - DMEM Hi Glu, 10% FBS Song, Zhao et al. Application of direct current electric fields to cells and tissues... Nature Protocol 2007.

17. Aim 1 Measuring Schwann cell orientation EF 90° 90 300 Cell length (um) 0° 45 200 100µm 100 EF Schwann cells without EF. Schwann cells exposed to EF align perpendicularly. 0

18. Aim 1 Schwann cell orientation Values describe cell angle orientation (in degrees).

19. Aim 1 Observations of EF through substrate EF High EF - 110 mV/mm Control AC EF EF AC EF 60 Hz EF 24 hours 100 µm

20. Aim 1 Aim 1– Observe Schwann cell morphology when exposed to an EF through media and substrate Summary: • EF through media • Increasing EF increases cell alignment • Maximum alignment is obtained within 6 – 8 hours • AC signals cause cells to spread radially • EF through substrate • AC signals cause cells to spread radially • EF cause ECM to change

21. Aim 2 • Aim 2 – Examine changes in extracellular environment when exposed to substrate EF ECM stimulation ECM gel: Matrigel EF: 10 mV/mm Current: 3 mA Duration: 20 hrs Cells: Seed SC after stimulation

22. Aim 2 Schwann cells align on EF stimulated gels 90° 0° EF 10 mV/mm Schwann cells were seeded after Matrigel was stimulated for 20 hrs. EF stimulated gels induce Schwann cell alignment

23. Aim 2 DC EF changes Ca+ distribution within a gel Gel sections 1 2 3 4 5 1 2 Stimulate gel for 2 hrs Freeze and section gel into 5 parts - + 10 mV/mm - + 4 3 Add Calcium green-1 Calcium Green -1 http://www.umsl.edu/~tsytsarev/tsytsarev_files/Lecture10.htm

24. Aim 2 EF have long term effects on ECM gels • EF parameters • EF: 10 mV/mm • Current: 3 mA • Duration: 20 hrs Control EF EF 10 mV/mm ECM environment retains memory of EF effects 100 µm

25. Aim 2 Collagen I alignment on EF stimulated ITO EF 50 µm 0.2 mg/ml Collagen I fibril aligns perpendicular to substrate EF

26. Aim 2 EF increases collagen fibril bundle formation EF 5 µm Control 24 hr, EF10 mV/mm Fibril bundle: >5 fibrils densely packed >10 µm EF stimulation increases prevalence of collagen I fibril bundles

27. Aim 2 EF increases collagen fibril bundle formation EF 5 µm Control 24 hr, EF10 mV/mm Fibril bundle: >5 fibrils densely packed >10 µm EF stimulation increases prevalence of collagen I fibril bundles

28. Aim 2 • Aim 2 – Examine changes in extracellular environment when exposed to substrate EF Summary: • ECM gel exposed to substrate EF • EF changes charge distribution in ECM • EF has long term effects on ECM • EF aligns dilute collagen I fibrils • EF increases collagen fibril bundle formation

29. Aim 3 • Aim 3 – Develop an electrically conductive nerve conduit to improve nerve regeneration Substrate stimulation ECM gel: Matrigel EF: 10 mV/mm (DC and AC) Current: 15 µA Duration: 2 hrs Cells: DRG

30. Aim 3 Experimental setup for PPy stability and biocompatibility DRG PPy-PCL 10 mV/mm Glass slide 1.0 x 1.5 cm2 Polycarbonate wells • Cell culture: • - DRGs from P2 SD rats • - 60 ul of Matrigel • - RPMI, 10% FBS, 1% PSA, 50 ng/ml NGF

31. Aim 3 EF through PPy increases axon elongation through gel No EF Axon lengths 3 days after 2 hr EF 10 mV/mm 1mm DC and AC EF stimulation through PPy increases axon length 200 µm DRGs exposed to EF extend longer axons (stained with β-III-tubulin).

32. Aim 3 EF stimulation of DRGs on PPy enhances axon growth toward electrodes

33. Aim 3 EF stimulation of DRGs in 3D 140 mV (= 10mV/mm) level of media w/ NGF PPy conduit DRG 4 mm 6 mm 4 mm

34. Aim 3 EF stimulation of DRGs in 3D may reduce axon density but increase axon length 100 µm DRG in conduit with no EF DRG in conduit with DC 10 mV/mm for 2 hrs Need to develop another model to find conclusive observations

35. Aim 3 • Aim 3 – Develop an electrically conductive nerve conduit to improve nerve regeneration Summary: • DRG stimulation on PPy • DC / AC stimulation increases DRG axon length 13 – 21% • DRG axons extend towards nearest electrode • DRGs may extend longer axons in EF stimulated PPy conduits (further investigation is needed)

36. Summary Summary of Aims • Aim 3 • Aim 2 Aim 1

37. End Acknowledgements Schmidt group Christine Schmidt Jae Young Lee Jon Nickels Leo Forciniti John Fonner John Hardy Craig Milroy ZinKhaing Scott Zawko Derek Hernandez Sydney Geissler Ryan Nagao Sarah Mayes Chase Cornelison Richelle Thomas HymaDurgam Diana Gutierrez Collaborations and Assistance Silvia Luebben (TDA) Shawn Sapp (TDA) Robert Ross (VTI) John Pearce (ECE Dept.) Jason Cook (Emelianov) Alex Hannah (Emelianov) Mary Nguyen (Suggs) Julie Rytlewski (Suggs) Tushar Sharma (Zhang) My committee Richard Aldrich Aaron Baker H. Grady Rylander Laura Suggs Undergraduate Assistants Claudia Wei Jacque Chow Lindsey Nguy Hieu K. Nguyen Jeff Coursen Alvin Nguyen Jan Nguyen Dan Walker Thomas Mathews Sung JiAhn Imaging Shouliang Zhang Julie Hayes Dwight Romanovicz Funding NDSEG Fellowship NIH SBIR (TDA) Undergraduate Research Fellowship IE Internship

38. Note • Add stastics explanation, hyp, std dv • Remake data with 0-180 • Explain how EF is not uniform across cell surface • Explain EF around electrodes better • Quantitative how to find which cells to measure/or not measure…. L:w ratios • Statistician to find mix model test, non parametric tests • 31 slide, stats • Dissert pg 53. change rho/ r = a/l

39. Aim 1 EF through substrate penetrates into media

40. Aim 3 PPy stimulation does not affect biocompatibility but does change cell adhesivity