1. Use of Electrical Stimulation to Improve Nerve Regeneration Jan Nguyen, Claudia Wei, Jeff Coursen Hieu Nguyen Group April 2010

2. Hieu Data Blitz (04.29.2010) Overview: 1.PPy conduit from TDA 2.Fill with hydrogel 3.Embed DRGs 4.EF stimulation

3. Final semester project DRG body

4. Final semester project DRG body Hydrogel

5. Final semester project PPy conduit DRG body Hydrogel

6. Final semester project PPy conduit DRG body Hydrogel Electric field

7. Why hydrogels? – Structural rigidity provides contact guidance for cell adhesion & growth Prevents conduit from collapsing

8. Applications: o Allows DRGs to adhere to surface & not float in media o Test how effective hydrogels are since goal is to have in body Provides 3D scaffold o Representation of body o Allows for testing multiple layers of cells or organs o Medium that serves as drug delivery device

9. Pure Collagen MatrigelCollagen gel with HEPES Collagen gel with 10x DMEM Collagen / HA hydrogel [1][2][3][4][5]

10. PC12 cells in collagen hydrogel

11. 1. Curt’s hydrogel – Pros: optimized in Schmidt’s lab to have effect on neurite extension Can be put in final product  human body – Cons: our success rate with this hydrogel < matrigel Hydrogel’s current f(x) is to evaluate cell’s response to EF  type doesn’t matter 2. Matrigel – Pros: Know works in vitro b/c our success rate > other hydrogels Preparing matrigel is not necessary -Cons: -Can’t put into final product  human body

12. A. Silicone – Used as model with PC 12 cells B. PLGA from TDA – Used as model with PC 12 cells C. Ppy from TDA – Used in final project with explanted DRGs Hydrogel Silicone conduit PC12 cells with NGF

13. A. Cryostat Sectioning – i. fix – ii. freeze in Isopentane – iii. embed in OTC freezing medium – iv. section at -20C – v. sectioned at 40um and at 160um http://www.bio.miami.edu/~cmallery/255/255hist/mcb5.43.microtome.jpg http://www.chadie.de/cryotomee.jpg Cryotome sectioning for imaging cut thin slices to image cells inside opaque PPy

14. B. Immunostaining – i. fix with 4% Paraformaldehyde – ii. Wash with PBS – iii. permeabilize with 0.02% TritonX – iv. block with goat serum – v. stained with primary and secondary antibodies http://images.google.com/imgres?imgurl=http://www.abcam.com/ps/CMS/Images/immuno_staining2.jpg&imgrefurl=http://www.abcam.com/index.html ://www.abcam.com/index.html

15. C. Imaging – Under fluorescent lighting DRG in Ppy conduit

16. Biomedical Applications o Biosensor o Drug delivery device Uses o Antistatic Coating o Protective coating for photoelectrochemical cells o Battery Polypyrrole is a electrically conducting polymer “Geetha, S. et al. "Biosensing and Drug Delivery by Polypyrrole." Analytica Chimica Acta (2006): 119-25. Web.

17. PPy was chosen for this study because it is 1.Electrically conductive 2.Easy to synthesize 3.Flexible 4.Shown to be biocompatible with mammalian cells 5.Shown to promote neurite growth Drawbacks 1.NOT biodegradable 2.Fragile and brittle Huang, Yi-Cheng, and Yi-You Huang. "Biomaterials and Strategies for Nerve Regeneration." Artificial Organs 30.7 (2006): 514-22.

18. Synthesized on stainless steel Doped with PSS (oxidative doping) 0.7 mV at 40 min Detox methods: prestimulation vs soaking J.M. Sansiñena, V. Olazábal, T.F. Otero, C.N. Polo da Fonseca, M-A. De Paoli, Chem. Commun., (1997) 2217

19. Soak Soak for 1 day in DI water

20. Prestimulation 0.1 mV per cm (0.2 mV total) 2hours Prestimulation= best method of detoxification

22. Endogenous Electric Fields Transepithelial potential Break in epithelium allows low resistance pathway for ions Resulting Electric field: 100-1000 mV cm -1 (Nuccitelli, R. 2003. Endogenous Electric Fields in Embryos During Development, Regeneration, and Wound Healing. Radiation Protection Dosimetry Vol. 106, pp. 375-383)

23. Role of Endogenous Electric Fields Development Wound Healing Nerve regeneration Cell migration Mechanisms? Song, Bing et al. Nerve regeneration and wound healing are stimulated and directed by endogenous electric field in vivo. Journal of Cell Sciences 117 (2004):4681-4690.

24. Stimulation of PPy in vitro Electrodes: alligator clips or gold-plated mini-clips Stimulation: 200 mV for two hours PPy resistance: 10-40 kΩ Media (F12K) resistance: 1500 kΩ

25. Stimulation of Conduits in vivo Puck is surgically implanted near injury site Magnetic coil induces current in puck Electrodes sutured to conduit Confidential. Virginia Technologies Inc.

26. Using Magnetic Fields Directly Why? Less invasive Extended nerve growth found in direction of induced current (Macias Y. Melissa, et al. Directed and Enhanced Nuerite Growth with Pulsed Magnetic Field Stimulation. Bioelectromagnetics 21 (2000):272-286.)

27. Research Fall 2010 Research Fall 2010: Stimulate rat DRGs in PPy conduit with Hydrogel.

28. Final semester project DRG body Hydrogel PPy conduit Electric field

29. Experimental setup PPy conduit 6 uL gel8 uL gel6 uL gel DRG level of media 200 mV0 mV

30. DRG in Matrigel - stimulated DRG PPy conduit extended neurites onto conduit

31. DRG in Matrigel - control (not stimulated) DRG PPy conduit extended neurites into hydrogel

32. into Hydrogelonto PPy surfaceno neurites Collagen controlIIIIIII Collagen stim Matrigel controlIIIIIIII Matrigel stimIIIIIIIIII Tally for neurite extension Columns = where the neurite extended to Rows = type of hydrogel, and EF stimulation of PPy or no stimulation (control)

33. Quick summary Matrigel performed better in neurite promotion and structural support (compared to Collagen gel) Need more samples to draw conclusive data Do not use cryosectioning in order to preserve gel structure

34. 1. Song, Bing et al. Nerve regeneration and wound healing are stimulated and directed by endogenous electric field in vivo. Journal of Cell Sciences 117 (2004):4681-4690. 2.Hou, S. "The Repair of Brain Lesion by Implantation of Hyaluronic Acid Hydrogels Modified with Laminin." Journal of Neuroscience Methods 148 (2005): 60-70. Web. 3. Kotwal, A., and CE Schmidt. "Electrical stimulation alters protein adsorption and nerve cell interactions with electrically conducting biomaterials." Biomaterials 22 (2001): 1055-064. Print. 4. Macias Y. Melissa, et al. Directed and Enhanced Nuerite Growth with Pulsed Magnetic Field Stimulation. Bioelectromagnetics 21 (2000):272-286.) 5. Nuccitelli, R. Endogenous Electric Fields in Embryos During Development, Regeneration, and Wound Healing. Radiation Protection Dosimetry Vol. 106 (2003): 375-383. 6. Pearson, R. et al. "Spatial Confinement of Neurite Regrowth from Dorsal Root Ganglia within Nonporous Microconduits." Phillips, J. et al.. "A Self-Organizing Collagen Guidance Conduit." Tissue Engineering 11 (2005). Web. 7. Vernitskaya, T. "Polypyrrole: a Conducting Polymer; Its Synthesis, Properties and Applications." Russian Chemical Reviews 66.5 (1997): 443-57. Web. Literature Cited

35. Acknowledgements We would like to thank: Dr. Schmidt- for granting us the opportunity to participate in research Hyma- for informing us with proper lab safety techniques and procedures Zin, Jae, Leo, and other grad students- for their wealth of knowledge & advice Hieu- for taking us under your wings and providing not only bountiful knowledge, but guidance and support in our research endeavors