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?