Biomedical Engineering Reference
In-Depth Information
which have thinner processes than locust neurons, showed extensive curling and
intertwining of neurites around CNTs. A similar phenotype was seen with thin, but
not thick, neurites of locust neurons; thick neurites show entanglement between
themselves rather than with CNTs. It appears that CNTs with diameters relatively
similar to that of thin neurites allowed entanglement, which could represent an
anchoring mechanism for neurite attachment to rough surfaces. These results sug-
gest that surface topology of CNT scaffolds should be considered when designing
scaffolds for neuronal growth.
Conductivity is another important CNT attribute which can modulate neuronal
growth and neurite outgrowth in cell culture. Malarkey et al. (Malarkey et al.
2009
)
seeded hippocampal neurons, prepared from 0- to 2-day-old rats, onto retainable
CNT fi lms deposited on glass coverslips. Such fi lms were made by spraying water-
soluble SWCNTs on heated glass coverslips. Water-soluble SWCNTs were obtained
by functionalization of SWCNTs with polyethylene glycol (PEG). Here, the
SWCNT-COCl intermediate was reacted with PEG to generate SWCNT-PEG.
Malarkey et al. sprayed different amounts of the SWCNT-PEG aqueous solution to
generate retainable uncharged substrates/fi lms with the thickness of 10, 30, or 60 nm
(Fig.
3a
), which corresponded to the increasing fi lm conductivity of 0.3 S/cm, 28 S/
cm, and 42 S/cm, respectively. While the conductivities increased with increasing
thickness, the surface roughness of these fi lms was not signifi cantly different from
one another as characterized by atomic force microscopy. However, their surface
was signifi cantly rougher that that of the standard, positively charged, and noncon-
ductive substrate PEI. Thus, effects on neuronal growth achieved on the various
SWCNT-PEG fi lms result from their differences in conductivity, not surface rough-
ness or charge. It should be noted that when the neuronal growth on various SWCNT
fi lms is compared to that of neurons grown on PEI, any difference observed could
be the outcome of roughness, charge, and/or conductivity. To assess possible effects
on cellular morphology, hippocampal neurons were cultured on PEI and SWCNT-
PEG fi lms for 3 days. At that time, live neurons were loaded with calcein and visu-
alized using fl uorescence microscopy (Fig.
3b-e
). The total number of neurites
decorating individual neurons remained the same regardless of the conductivity of
the fi lm. However, the total length of all processes and their branches, as well as the
mean neurite length, was signifi cantly greater in neurons grown on the 10-nm thick
SWCNT-PEG fi lms than in neurons grown on coverslips coated with the PEI or
with the thicker fi lms (30 and 60 nm) with higher conductivity, but comparable
roughness to the 10-nm SWCNT-PEG fi lm (Fig.
3f
). These fi ndings indicate that
certain SWCNT-PEG conductivity (0.3 S/cm) could promote neurite outgrowth as
compared to other SWCNT fi lms. Additionally, there was a signifi cant increase in
the average area of the neuronal cell body at 28 S/cm when compared to standard
PEI and to 42 S/cm substrate, but not when compared to 0.3 S/cm substrate, on
which neurons showed only a trend of body enlargement (Fig.
3f
). There was a
signifi cantly higher number of growth cones on neurons grown on 60-nm SWCNT
fi lms when compared to measurements on 30-nm SWCNT fi lms. Since the rough-
ness of these two conductive fi lms is similar, it appears that the higher conductivity
caused an increase in the number of growth cones. Neurons grown on the smoother
