use of electrical stimulation to improve nerve regeneration jan nguyen, claudia wei, jeff coursen...
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Use of Electrical Stimulation to Improve Nerve
Regeneration
Jan Nguyen, Claudia Wei, Jeff CoursenHieu Nguyen Group
April 2010
Hieu Data Blitz (04.29.2010)
Overview:
1.PPy conduit from TDA2.Fill with hydrogel3.Embed DRGs4.EF stimulation
• Why hydrogels? – Structural rigidity provides contact guidance for cell
adhesion & growth• Prevents conduit from collapsing
• Applications: o Allows DRGs to adhere to surface & not float in mediao Test how effective hydrogels are since goal is to have in body
• Provides 3D scaffoldo Representation of body o Allows for testing multiple layers of cells or organso Medium that serves as drug delivery device
PureCollagen
Matrigel Collagen gel with HEPES
Collagen gel with 10x DMEM
Collagen / HA hydrogel
[1] [2] [3] [4] [5]
• 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
• 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
• 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
• 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.htmlhttp://images.google.com/imgres?imgurl=http://www.abcam.com/ps/CMS/Images/immuno_staining2.jpg&imgrefurl=http://www.abcam.com/index.htmlhttp://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
• Biomedical Applicationso Biosensor o Drug delivery device
• Useso Antistatic Coatingo Protective coating for photoelectrochemical cellso Battery
• Polypyrrole is a electrically conducting polymer
“Geetha, S. et al. "Biosensing and Drug Delivery by Polypyrrole." Analytica Chimica Acta (2006): 119-25. Web.
PPy was chosen for this study because it is1.Electrically conductive2.Easy to synthesize3.Flexible4.Shown to be biocompatible with mammalian cells5.Shown to promote neurite growth
Drawbacks1.NOT biodegradable2.Fragile and brittle
Huang, Yi-Cheng, and Yi-You Huang. "Biomaterials and Strategies for Nerve Regeneration." Artificial Organs 30.7 (2006): 514-22.
•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
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)
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.
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
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.
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.)
into Hydrogel onto PPy surface
no neurites
Collagen control II IIIII
Collagen stim
Matrigel control I IIIIIII
Matrigel stim II III IIIII
Tally for neurite extension
Columns = where the neurite extended toRows = type of hydrogel, and EF stimulation of PPy or no stimulation (control)
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
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
AcknowledgementsWe 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