Page 51 - ASME_NEMB_2016_Program

TRACK 4 TRACK 4 Technical Program

Small-conductance calcium-activated potassium (SK) channels are a family Technical Presentation. NEMB2016-5977
of potassium channels that are voltage independent and are activated solely
by intracellular calcium (Ca2+) through constitutively bound calmodulin. SK Se Jun Lee, George Washington University, Washington, DC, United
channels exist within microdomains of Ca2+ sources and mediate afterhy- States, Lijie Zhang, The George Washington University, Washing-
perpolarization and regulate neuronal excitability. The location and density ton, DC, United States
of ion channels in neurons are important determinants of their functional and
physiological impact. Although the three subtypes of SK channels, SK1, SK2
and SK3, have partially overlapping yet distinct distributions in the brain, SK2 Neural defect resulted from various traumas and diseases, represents a
subtype is the most abundant. SK2 channels reside primarily in dendrites critical clinical problem all over the world. Currently fully neural functional
with lower expression in the soma. However, it remains unclear if SK chan- recovery is very difficult to achieve. Traditional surgical procedures such as
nerve tissue graft may encourage and guide axonal regeneration between
nels are present on neuronal axonal membranes.
the ends of the severed axons and are effective for small nerve bundles
with short gap distance. However, the repair of more complex defects with
In this study, we employed single molecule atomic force microscopy (AFM)
combined with a natural toxin, to test for the presence of SK channels in larger nerve gap remains problematic. In order to overcome these limita-
axons. We exploited the selective block of SK channels by bee venom tions, synthetic nerve scaffolds are being developed to mimic natural neural
extracellular matrix that would encourage neuronal differentiation, growth
toxin, apamin, to detect SK channels on the axonal membrane. Single mol-
ecule AFM records force-distance curves of an approach/retract cycle of and axon elongation across the gap. Nerve scaffolds can be fabricated by
the apamin-functionalized AFM cantilever to/from the axonal surface. The various methods. Amongst them, 3D printing techniques have drawn great
unbinding forces obtained from the force-distance curves correspond to interest because they can prepare scaffolds with highly controlled spatial
the presence of a SK channel. When 1µm2 scan areas along the axon was architecture and complexity to meet the customized requirements. The main
objective of this study is to create an innovative nerve scaffold with a biomi-
probed using an apamin-functionalized AFM cantilever, we observed mean
unbinding forces of 20±8.0pN in about 5.6% of the sampled sites (n=6). In metic nano to micro architecture by integrating advanced 3D printing tech-
order to test whether the unbinding forces observed were indeed between nique and conductive multi walled carbon nanotubes (MWCNTs). MWCNTs
apamin and SK channel unbinding, we repeated the experiment on cells are widely investigated in neural interfacing applications due to its unique
pretreated with apamin. This would cause SK channels to be occupied by physical, chemical and electrical properties. Unlike other conductive poly-
mer materials, their electrical conductivity remains high over a long period of
apamin, thereby becoming unavailable for detection by the AFM probe and
presumably leading to lower frequency of unbinding forces. Indeed, we time under harsh condition. It is expected that MWCNT scaffolds may allow
observed a significant decrease in the frequency in the unbinding forces the electrical stimulation and promote excitability of neurons. In addition,
(0.58%, n=3) when the cells were pretreated with apamin. Additionally, we amine-functionalized MWCNTs will be used as the key component of our 3D
transfected HEK cells with SK2-S (short SK2 channel splice isoform) chan- printing inks and will add further controlled chemical signaling cues and me-
chanical strength to promote directed neural cell growth and differentiation
nels and found mean binding forces of 28±5pN in 4.5% of the sampled sites.
However, when the HEK cells were pretreated with apamin, the frequency of for our printed constructs.
unbinding forces decreased to 0.3 %. These experiments demonstrate the
specificity of AFM to detect SK channels. A series of mixture solutions of 0.01%, 0.025%, and 0.05% amine-functional-
ized MWCNTs and biocompatible poly (ethylene glycol) diacrylate (PEG-DA)
hydrogel were successfully printed by our custom made stereolithograpy
Next, we tested the effect of cyclic adenosine monophosphate (cAMP) ac-
tivated protein kinase A (PKA) on axonal SK channel surface expression as (SL) 3D bioprinter. SL printed scaffolds were designed as square pattern with
PKA controls SK channel expression in other neuronal compartments (soma small, medium, and large pores geometry (corresponding to 31%, 52%, 66%
and dendrites). When cells were pretreated with Rp-CAMPS (100µM for 30 porosity) using computer aided design software. Our results shows that the
min), a cAMP analog that prevents the activation of PKA by cAMP, we found MWCNTs were homogenously distributed inside the 3D printed scaffolds.
Neural stem cells (NSCs, ATCC) were further seeded onto prewetted scaf-
a higher mean frequency of unbinding events (µ=20±9pN, surface density =
10.1%, n= 5). Similar increase in surface density was obtained when the cells fold and evaluated for adhesion and proliferation study. 4 hour cell adhesion
were pretreated with PKA inhibitor KT5720 (1µM for 30 min). This indicates study showed the scaffolds with 52% porosity can significantly improve cell
that cAMP-PKA regulates the expression of axonal SK channels. attachment compared to scaffolds with smaller pores. Then three more
Therefore, we show that apamin-sensitive SK channels reside on the axon groups of scaffolds (52% porosity) with different concentration of MWCNTs
were evaluated for proliferation study. Compared to any other groups, NSCs
and are under the control of cAMP-PKA.
proliferate significantly on scaffolds with 0.01% MWCNTs after 5 day of cul-
ture. Through this study, amine-functionalized MWCNTs were effectively 3D
10:20am Movement of a voltage sensor within a bilayer bioprinted into a novel neural scaffold and greatly improved neural stem cell
adhesion and proliferation, thus promising for future neural regeneration
Technical Presentation. NEMB2016-6078 applications.

Mehdi Torbati, Vikash Chaurasia, University of Houston, Houston,
TX, United States, Kranthi Mandadapu, University of California, 4-4
Berkeley, Berkeley, CA, United States, Ashutosh Agrawal, Universi- SELF-ASSEMBLY
ty of Houston, Houston, TX, United States

Neurons communicate via a traveling wave of electrical excitation that prop- Harris 4:00pm - 5:30pm
agates down an axon at speeds in the range of 10-100 m/s. This electrical
wave is triggered and regulated by opening and closing of voltage-gated Session Organizer: Sinan Keten, Northwestern University, Evan-
ion channels. These channels have been traditionally believed to be sensi- ston, IL, United States
tive to the local electrochemical environment. A growing number of exper-
iments now conclusively show that lipid-associated mechanical stimuli can
control the channel response. We develop a quantitative model to elucidate 4:00pm Understanding and Manipulating Nano-scale Self-as-
the interaction of a voltage sensor domain with it’s neighboring lipids. We sembling in Living Systems for Regenerative Medicine
predict the impact of key lipid/protein properties on the transmembrane
movement of the sensor. Keynote. NEMB2016-5940
Mingjun Zhang, Ohio State University, Columbus, OH, United
10:40am 3D Bioprinting Conductive Nano Scaffold with Multi- States 51
walled Carbon Nanotube for Improved Nerve Regeneration