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substrates for neuronal growth. Fixed neurons were investigated using SEM while
live cells loaded with calcein were subjected to fl uorescence microscopy. The mor-
phological characteristics of neurons grown on zwitterionic MWCNT-PABS were
similar to those of neurons grown on AP-MWCNT. Neurite outgrowth was depen-
dent on the surface charge carried by functionalized MWCNTs. Whereas there was
no signifi cant difference in the number of neurites between neurons grown on the
various MWCNTs, neurons grown on the positively charged MWCNT-EN and the
zwitterionic MWCNT-PABS had signifi cantly greater number of growth cones
than the neurons grown on the negatively charged MWCNT-COOH. In addition,
neurons grown on positively charged MWCNT-EN produced signifi cantly longer
neurites and greater neurite branching than neurons grown on zwitterionic or nega-
tively charged MWCNTs. Neurite branching was graded, since neurons grown on
zwitterionic MWCNT-PABS showed less branches than neurons grown on
MWCNT-EN, but more than neurons grown on negatively charged MWCNT-
COOH (Fig. 1e ). Taken together, the results of this study suggest that MWCNT
modifi cations provide a means to modulate the growth of neurons based on elec-
trostatic charge that can be introduced to MWCNTs by the attachment of various
chemical compounds.
Several additional research groups made use of MWCNTs as a substrate/scaffold
for growth on neurons in primary culture. In a technical note, Xie et al. ( 2006 )
implicated that functionalized MWCNTs supported greater neurite growth than
nonfunctionalized MWCNTs. In this study, MWCNTs were obtained by microwave
CVD (Varadan and Xie 2002 ) . After refl uxing MWCNTs in the mixture of sulfuric
and nitric acids, an oxidation method that introduces carboxyl groups mainly at the
end of MWCNTs (Liu et al. 1998 ), the resulting MWCNT-COOH material was
dispersed in water and then vacuum fi ltered to generate MWCNT mats on top of a
track-etched polycarbonate membrane. These mats were seeded with dissociated
dorsal root ganglion (DRG) neurons from newborn rats. Although the pH of the
standard culture medium used (DMEM) was not explicitly stated, it should be ~7.4,
which implicates that MWCNT-COOH mats display negative surface charge as
opposed to neutrally charged unmodifi ed/bare MWCNT mats. Using SEM, Xie
et al. qualitatively assessed neuronal growth on bare and functionalized MWCNTs.
They observed growth cone formation and neurites, which branched and some also
bundled, extending from DRG neurons grown on MWCNT-COOH mats. DRG neu-
rons cultured on bare mats showed similar morphological characteristics, but shorter
neurites were observed. Furthermore, neurites readily intertwined with functional-
ized, but not with bare MWCNTs, implicating that there might be preferential/
stronger neurite interactions with functionalized rather than with bare MWCNTs.
These qualitative observations in respect to neurite outgrowth using DRG neurons
are in disagreement with the quantitative analysis done on live hippocampal neu-
rons as described by Hu et al. (Hu et al. 2004 ). Such seemingly disparate fi ndings
could be a result of: (1) differences in the experimental approach: qualitative SEM
on fi xed (Xie et al. 2006 ), as opposed to quantitative fl uorescence microscopy of
live neurons (Hu et al. 2004 ); (2) reporting on variability in growth by neurons iso-
lated from different regions: peripheral (DRG) vs. central (hippocampus) nervous
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