Biomedical Engineering Reference
In-Depth Information
between PO and MWCNT, these substrates highly likely also differed in their surface
topology. In contrast, in the Hu et al. study (Hu et al.
2004
), the same MWCNT topol-
ogy was used to display different charges. Thus, these data may point to the possibility
that, besides electrostatic charge, other physical qualities of the substrate could affect
neuronal growth, a topic that we further discuss below.
As alluded to above, substrate qualities play a role in the process of growth cone
motility and neurite branching (Lustgarten et al.
1991
; Mattson et al.
2000
) . First,
we discuss “dilution” of electrostatic charge and then the effects of roughness/topol-
ogy and conductivity on neuronal growth. Since the “fl avor” (negative, zwitterionic/
neutral, and positive) of electrostatic charge of substrate matters, it is possible that
reducing the charge of the substrate could be used to further modulate neuronal
growth and neurite outgrowth. One approach is to reduce PEI cationic charge with
bare CNTs that are neutral. Hu et al. (Hu et al.
2005
) did just that by synthesizing a
polymer, where branched PEI was grafted onto SWCNTs. In this study, commer-
cially available SWCNT-COOH material was treated with oxalyl chloride to form
the intermediate product SWCNT-COCl. In turn, branched PEI was reacted with
SWCNT-COCl to generate the graft copolymer SWCNT-PEI. Quantitative assess-
ment of the SWCNT percent weight in the graft copolymer using thermogravimet-
ric analysis and near-IR spectroscopy revealed that SWCNTs contribute 18-19% of
SWCNT-PEI weight. When deposited onto glass coverslips and visualized under
bright-fi eld microscopy, as expected, this material showed intermediate transpar-
ency to that of PEI and AP-MWCNT substrates (Fig.
2a-c
). Comparison of mor-
phological characteristics of calcein-loaded hippocampal neurons in cultures
(Fig.
2d-f
), prepared from 0- to 2-day-old rats, revealed that neurons grown on
SWCNT-PEI display intermediate growth and neurite outgrowth characteristics to
those of neurons grown on AP-MWCNT- or PEI-coated coverslips (Fig.
2g
). Thus,
it appears that the intermediate growth of neurons on SWCNT-PEI can be attributed
to the reduced positive charge of the PEI as it is copolymerized with SWCNTs.
Whether graded dilution of charge would yield graded neuronal growth and neurite
outgrowth awaits further experimentation.
Roughness of the substrate can affect neuronal growth and interactions between
neurons and the substrate. For instance, in a non-CNT system with SiO
2
as the sub-
strate, it was demonstrated that roughness of the material dictated growth of sub-
stantia nigra neurons cultured after isolation from prenatal Wistar rats (Fan et al.
2002
). It was reported that after 5 days in culture about as twice as many neurons
per area adhered to and grew on a SiO
2
surface having an average roughness (Ra) of
20-50 nm, when compared to SiO
2
surfaces with their Ra less than 10 nm or greater
than 70 nm. A later study employing CNTs as scaffolds for neural cells had made
similar observations that topology/roughness of the substrate matter. Hence, Sorkin
et al. (
2009
) cultured dissociated neurons that originated from embryonic rat corti-
ces or from locust frontal ganglion on quartz micropatterned with AP-CNT (most
likely MWCNTs) islands made by CVD. Neurons grown on patterned substrates
were visualized after fi xation using SEM. While neurons from either species were
unable to adhere to SiO
2
, they grew on CNT islands. Although not systematically
studied, this effect was attributed to surface roughness of CNT islands. Rat neurons,
