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Schwann Cell Response on Polypyrrole Substrates Upon Electrical Stimulation
Leandro Forciniti, Jose Ybarra III, John Maldonado, Muhammad H. Zaman, Christine E. Schmidt
Chemical Engineering Doctoral Candidate, University of Texas, Austin
Causes of peripheral neuropathy (PN)
DiseaseInjury
Injury
PN due to injury a growing concern
Easy to injure Trauma
Incident rate 2.4 % of the U.S. population
Expected to grow to 8.3% of the population due to an aging1
War 35% troops
extremities wounded in combat2
1 Gale Encyclopedia of Neurological Disorders 2Journal of Craniofacial Surgery. 21(4):998-1001, July 2010.
Peripheral nerve anatomy and types of injuries
Fourth Degree
Fifth Degree
Injuries Requiring Treatment
Peripheral NerveAxon
Endoneurium where Schwann cells are
Perineurium
Epineurium
Third Degree
Second Degree
First Degree
Injuries That Autologously Recover
Three treatment options for peripheral neuropathy (PN) due to injury
IntraluminalChannels
Oriented NerveSubstratum
ElectricalActivity
Biochemical Signals
Nerve Nerve Conduit
Incorporation ofSupport Cells
Biodegradability/Porosity
IntraluminalChannels
Oriented NerveSubstratum
ElectricalActivity
Biochemical Signals
Nerve Nerve Conduit
Incorporation ofSupport Cells
Biodegradability/Porosity
IntraluminalChannels
Oriented NerveSubstratum
ElectricalActivity
Biochemical Signals
Nerve Nerve Conduit
Incorporation ofSupport Cells
Biodegradability/Porosity
IntraluminalChannels
Oriented NerveSubstratum
ElectricalActivity
Biochemical Signals
Nerve Nerve Conduit
Incorporation ofSupport Cells
Biodegradability/Porosity
IntraluminalChannels
Oriented NerveSubstratum
ElectricalActivity
Biochemical Signals
Nerve Nerve Conduit
Incorporation ofSupport Cells
Biodegradability/Porosity
IntraluminalChannels
Oriented NerveSubstratum
ElectricalActivity
Biochemical Signals
Nerve Nerve Conduit
Incorporation ofSupport Cells
Biodegradability/Porosity
Biomaterials for PN requires proper integration of stimuli
Chemical Contact Electrical
Lee et al. (2002) Gomez et al. (2006) Huang et al. (2008)
For more information on stimuli integration see Forciniti et al. ABME. (2008).
Polypyrrole (PPy) contains all three types of stimuli
Electrical Stimulation (Estim) of PC-121
Nerve Growth Factor (NGF)-Stim
of PC-122
Controllable Topology3
1 Schmidt, C.E. et al. PNAS. 1997; 2 Lee, J.Y. et al. J.R. Soc. Interface 2009; 3Ateh, D.D. et al. J.R. Soc. Interface. 2006.
-
+-
+
Methods of synthesizing PPy
Chemical Synthesis Electrochemical Synthesis
Platinum Gauze
SlideReferenceElectrode
Pyrrole + Dopant
Bi-potentiostat
Current integrator
Multimeter
Platinum Gauze
SlideReferenceElectrode
Pyrrole + Dopant
Bi-potentiostat
Current integrator
Multimeter
HN -e
HN
NH
HN *
*
X-
X- = Cl -, pTS, etc SO3-H3C
para-toluene sulfate (pTS)
Polypyrrole a tunable material
Forciniti et al. Biomedical Materials. 2008.
PPy material properties affect cell viability
Forciniti et al. Biomedical Materials. (2008).
Schwann cell (SC) behavior on PPy:PSS upon electrical stimulation
Copper tape
Petri Dish
Film
Fibronectin (FN), Laminin (LN) or Nerve Growth Factor (NGF)
PDMS Glue
Petri Dish
Cell Culture Media
Film
10,000 SC
10 X Magnification
Phase Contrast
10 min/frame
From Counter and Reference Electrode socket (cathode)
Cell Culture Media
Film
10,000 SC
10 X Magnificatio
n Phase
Contrast10
min/frame
e-
From Working electrode socket
(anode)
Electrical currents pass through both the PPy:PSS organic polymer and the
underlying ITO slide
• 0.1 V + 10% FBS
•Electric Field seen in media ~ 80 mV @ 2 mm distance from surface
Indium Tin Oxide (ITO) PPy:PSS on ITO
300-600 Å
1,090 Å
Current profiles for electrical stimulation of Schwann cells
Laminin (LN) adsorbed to surface
PPy:PSS No protein PPy:PSS 100 ug/mL LN PPy:PSS 100 ug/mL LN + Estim
Time lapse movies allow us to monitor SC behavior in the presence of e-fields
PPy:PSS + 100 ng/mL NGF (18 hours)
PPy:PSS + 100 ng/mL NGF + Estim
(18 hours)
W.E.
Electrical stimulation orients SC but does not affect migration speed
a b
cc
ab
c
d
Number of * denotes statistically similarity (p<0.05)
a aa a
b bb
b
Electrical stimulation orients SC
NGF
FN
LN
Anode
Cathode
Top
Bottom
Center
NGF secretion up regulated by protein adsorption and changes in surface
topology upon e-stim
a
b b
cc
a
NGF secretion up regulated by protein adsorption and changes in surface topology
upon e-stim
a,ba a
c
d
b
NGF secretion up regulated by protein adsorption and changes in surface topology
upon e-stim
a
b
ab
cb
E-stim increases surface nanoscale surface roughness in DPBS
PPy:PSS Pre-stimulation PPy:PSS Post-stimulation
Conclusions
Electrical stimulation affects only the directionality of the migration rather than cell migration speed.
Adsorption levels of stimulatory proteins affect cell migration speed.
Adsorption level and changes in surface topology may affect secretion levels of NGF by Schwann cells.
Future Directions
We would like to determine ion flux passing by the cell upon electrical stimulation. We propose to monitor the flux of a charged dye upon electrical stimulation.
We would like to determine the effect increase protein adsorption upon electrical stimulation has on surface topology. We propose to get AFM roughness data.
We would like to determine whether electrical stimulation is reversible. We propose to monitor cell migration upon reversible electrical gradients.
Acknowledgements
Dr. Christine E. Schmidt Dr. Muhammad H. Zaman Dr. Schmidt’s Lab Dr. Zaman’s Lab Zin Khaing Jae Y. Lee Funding Resources:
▪ UT Graduate Continuing Fellowship
▪ NIH R21
Questions?