ARTICLE IN PRESS

Biomaterials 28 (2007) 344353

www.elsevier.com/locate/biomaterials

Review

Carbon nanotube applications for tissue engineering

Benjamin S. Harrison, Anthony Atala
Wake Forest Institute for Regenerative Medicine, Wake Forest University, Medical Center Boulevard, Winston-Salem, NC 27157, USA
Received 19 April 2006; accepted 18 July 2006
Available online 28 August 2006

Abstract

As the eld of tissue engineering advances, new tools for better monitoring and evaluating of engineered tissues along with new
biomaterials to direct tissue growth are needed. Carbon nanotubes may be an important tissue engineering material for improved
tracking of cells, sensing of microenvironments, delivering of transfection agents, and scaffolding for incorporating with the hosts body.
Using carbon nanotubes for optical, magnetic resonance and radiotracer contrast agents would provide better means of evaluating tissue
formation. In addition, monitoring and altering intra and intercellular processes would be useful for design of better engineered tissues.
Carbon nanotubes can also be incorporated into scaffolds providing structural reinforcement as well as imparting novel properties such
as electrical conductivity into the scaffolds may aid in directing cell growth. Potential cytotoxic effects associated with carbon nanotubes
may be mitigated by chemically functionalizing the surface. Overall, carbon nanotubes may play an integral role as unique biomaterial
for creating and monitoring engineered tissue.
r 2006 Elsevier Ltd. All rights reserved.

Keywords: Carbon nanotubes; Nanocomposite; Biosensor; Gene transfer; Scaffold

Contents

1. Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 344
2. Cell tracking and labeling. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 346
2.1. Optical labeling. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 346
2.2. MRI contrast agents . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 346
2.3. Radiotracers . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 347
3. Sensing cellular behavior . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 347
4. Augmenting cellular behavior . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 348
5. Matrix enhancement . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 349
6. Cytotoxicity . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 350
7. Conclusions and future outlook . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 351
References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 351

1. Introduction of cell and organ transplantation, as well as advances in

The goal of tissue engineering is to replace diseased or
damaged tissue with biologic substitutes that can restore
and maintain normal function. Major advances in the areas
Corresponding author. Tel.: +1 336 713 7292; fax: +1 336 713 7290.
E-mail address: bharriso@wfubmc.edu (B.S. Harrison).
materials science and engineering, have aided in the
continuing development of tissue engineering and regen-
erative medicine. As the eld of tissue engineering
advances, there is a growing need for better monitoring
and evaluating tools of engineered tissues along with new
biomaterials to facilitate tissue growth. Like tissue en-
gineering, which involves directing the growth of cells to

0142-9612/$ - see front matter r 2006 Elsevier Ltd. All rights reserved.
doi:10.1016/j.biomaterials.2006.07.044
 ARTICLE IN PRESS
B.S. Harrison, A. Atala / Biomaterials 28 (2007) 344353
form higher ordered structures, nanotechnology is also a
bottom-up approach that focuses on assembling simple
elements to form complex structures. Nanotechnology
involves utilizing materials which possess at least one
physical dimension between 1 and 100 nm to construct
structures, devices, and systems that have novel properties.
In fact, many biological components, such as DNA,
involve some aspect of nano-dimensionality.
The nano-dimensionality of nature has logically given
rise to the interest in using nanomaterials for tissue
engineering. These materials have the potential to have a
signicant impact on tissue engineering. Already, iron
oxide superparamagnetic nanoparticles and quantum dots
have been used to track the biodistribution of cells [1,2].
Interestingly, nanomaterials can also be multifunctional
capable of both targeting and imaging [3]. One nanoma-
terial that has the potential for multiple uses in tissue
engineering is the carbon nanotube.
Following the discovery of carbon nanotubes by lijima
[4], carbon-based nanotechnology has been rapidly devel-
oping as a platform technology for a variety of uses
including biomedical applications. Carbon nanotubes are
cylindrical carbon tubes possessing nanometer diameters
with much longer lengths (4100 nm) resulting in very large
aspect ratios (Fig. 1). They possess a very broad range of
electronic, thermal, and structural properties dened by
diameter, length, and chirality or twist. Carbon nanotubes
can be composed of a single tubecommonly called a
single-walled carbon nanotube (SWNT)or concentric
cylinders of carboncommonly referred to as multi-walled
carbon nanotubes (MWNTs).
Carbon nanotubes are generally prepared via three
methods: arc-discharge [5], laser ablation [6], and chemical
vapor deposition (CVD) [7]. CVD is the most widely used
commercial method of producing carbon nanotubes. This
process typically involves reacting a metal catalyst with a
hydrocarbon feedstock at high temperatures (4700 1C) to
Fig. 1. Scanning electron micrographs of multi-walled carbon nanotubes
show the tubular structure is nanometer in diameter and microns in length
(courtesy of NanoTechLabs Inc.).
345
produce carbon nanotubes which depending on the
reaction conditions can create a wide variety of lengths
(nanometers to millimeters) and widths (1100 nm). Nano-
tubes produced using this method commonly have metal
catalysts or carboneous deposits on the outside of the
nanotube. Since metal catalysts such as nickel can be used
for growing carbon nanotubes, there is concern about
carbon nanotubes being cytotoxic. Thus a purication step
is usually required before carbon nanotubes can be used for
biomedical applications.
There are several approaches to purifying carbon
nanotubes [811]. Reuxing carbon nanotubes in an
oxidizing acid such as nitric acid is one of the most
popular methods of purication. This process oxidizes and
removes the metal catalysts and carboneous deposits from
the inside and outside of the tube. In addition, acids can
attack the more reactive ends of the carbon nanotubes
opening the tube ends and creating carboxylic acid groups.
In addition, any defects in the tube may also be oxidized
creating additional carboxylic acids groups along the
length of the tube. These carboxylic acid groups can be
further functionalized allowing tuning of the surface
chemistry of the nanotube.
With its carbon composition, high aspect ratio, electrical
and physical properties, there has been growing interest
in using carbon nanotubes for biomedical applications.
Since the year 2000, there has been an approximate
doubling each year of articles related to carbon nanotubes
for use in biomedical applications with 2004 marking the
beginning of applying carbon nanotubes to tissue engineer-
ing (Fig. 2). This review focuses on research involving
carbon nanotubes which are relevant to the tissue
engineering eld. There are four areas that carbon
nanotubes can be used in which are relevant for tissue
engineeringcell tracking and labeling, sensing cellular
behavior, augmenting cellular behavior and enhancing
tissue matrices.
Fig. 2. Number of articles per year published regarding carbon nanotubes
for biomedical applications.
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346 B.S. Harrison, A. Atala / Biomaterials 28 (2007) 344353
2. Cell tracking and labeling
The ability to track implanted cells, and to monitor the
progress of tissue formation in vivo and non-invasively is
important especially in tissue-engineered constructs of
clinically relevant sizes. Labeling implanted cells would
not only help in evaluating the viability of the engineered
tissue but would also help in understanding of the
biodistribution and migration pathways of transplanted
cells. Non-invasive methods are particularly attractive
because traditional methods such as intravital microscopy
or ow cytometry are extremely time consuming and
challenging. However, contrasting agents in vivo need to
have good biocompatibility, high contrasting ability, and
stability. Examination of the literature thus far shows
carbon nanotubes are feasible as imaging contrast
agents for optical, magnetic resonance, and radiotracer
modalities.
2.1. Optical labeling
Optical imaging in vivo is attractive because optical
detection can be a simple and direct method for diagnostics
and biomedical imaging. However, there are many
problems with optical methods such as tissue absorbing
or scattering light, autouorescence, and photobleaching.
In many cases the optical absorption bands are narrow and
the resulting emissions are broad. This can limit the
simultaneous detection of multiple uorophores. Carbon
nanotubes possess many properties desirable for optical
detection. They possess optical transitions in the near-
infrared (NIR) that minimizes these interferences. The
infrared spectrum between 900 and 1300 nm is an
important optical window for biomedical applications
because of the lower optical absorption (greater penetra-
tion depth of light) and small auto-uorescent background.
In addition, carbon nanotubes display good photostability.
Raman scattering and uorescence spectroscopy may be
promising methods for tracking carbon nanotubes in cells
over long durations of time. For instance, SWNTs
Fig. 3. Murine myoblast stem cells incubated with DNA-encapsulated nanotubes: (a) area map of uorescence intensity overlaid onto optical micrograph
of incubated cells; (b) selected spectra from highlighted locations in area map showing Raman scattering (R) and uorescence (F) bands (courtesy of D.
Heller and M. StranoUniversity of Illinois at Urbana-Champaign).
dispersed in a Pluorinc surfactant incubated with mouse
peritoneal macrophage-like cells and can be imaged using
uorescence techniques. It was estimated that 70,000
nanotubes were ingested per cell at the rate of one
nanotube per second [12]. Excitation at 660 nm resulted
in a structured uorescence spectrum characteristic of
SWNT. Nucleic-acid encapsulated SWNTs have also been
shown to be stable for weeks when ingested by 3T3
broblasts and murine myoblast stems cells and be
monitored using Raman spectroscopy [13]. The nanotubes
remained in the cells during repeated cells divisions
suggesting that such probes could be used for studying
cell proliferation and stem-cell differentiation. The reason
they remain so well in the cell may be due to their
hydrophobic nature. Fig. 3 clearly illustrates that carbon
nanotubes are internalized by a myoblast stem cell and
concentration within the cell can be determined using the
signal intensity. Since Raman spectroscopy is very sensitive
to functional groups in a molecule, this technique may
provide useful information about the microenvironment of
the cell. This is one of the most promising methods for
using carbon nanotubes as optical biosensors in vivo and
can serve as a platform technique for more complicated
sensors. This added sophistication can be introduced by
modifying the nanotubes with additional probes [14] or
targeting agents.
2.2. MRI contrast agents
To date, several types of nanomaterials have shown
signicant impact as clinical MRI contrast agents and the
next generation contrast agents. These include iron-oxide
superparamagnetic nanoparticles, gadolinium-based nano-
particles, and quantum dots [1517]. While fullerenes such
as buckminsterfullerene and carbon nanotubes are com-
posed entirely of carbon, and thus provide poor contrast in
MRI, they can be functionalized to make them more
readily detected. Typically, this is done by attaching a
heavy atom such as gadolinium to the surface of the
fullerene or caging the heavy atom [18,19]. While many
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B.S. Harrison, A. Atala / Biomaterials 28 (2007) 344353
examples of C60 derivatives have been successfully imaged
using MRI, there are relatively few examples for tubular
fullerenes. One notable report demonstrated that gadoli-
nium could be loaded into ultra short (20100 nm long)
nanotubes through side-wall defects or end-of-tube open-
ings [20]. Such gadonanotubes displayed proton relaxivities
40 times greater than Gd[(DTPA)(H2O)]
2a standard
clinical contrast agent. The large proton relaxivities
exhibited by the Gd3+@US-tubes are due to water
molecules have easy access to the high concentration of
coordinated paramagnetic ions. With such a large con-
trasting ability such materials could be dispersed through-
out an engineered tissue allowing monitoring the process of
the engineered tissue.
2.3. Radiotracers
Carbon nanotubes can also be modied with radio-
tracers for gamma scintigraphy. In one case, 111In was
covalently bound to SWNTs and administered to BALB/c
mice to measure the biodistribution of nanotubes [21].
Functionalized SWNTs (f-SWNT) were shown not to
accumulate in any specic organ and were rapidly cleared
from the blood system through the renal excretion route
without any toxic side effects or mortality and could be
visually observed in the urine. Therefore, heavy elements
functionalized to nanotubes could serve as X-ray contrast
agents.
Carbon nanotube-based materials can provide image
enhancement for a variety of modalities and may be used
as NIR uorescent labels, MRI contrast agents, and
radiolabels. The above-mentioned work also suggests that
nanotubes have good biocompatibility and can be functio-
nalized to serve as a vehicle for carrying imaging agents.
Such nanomaterials could be useful for visualization of
dynamic structural and functional properties of engineered
tissues. With continued effort in this area, the full potential
of carbon nanotubes for imaging will be realized.
3. Sensing cellular behavior
While tracking cells aids in understanding cell migration,
monitoring the cellular microenvironment would provide
even more knowledge needed for successfully engineering
tissue. The ability to monitor cellular physiology such as
ion transport, enzyme/cofactor interactions, protein and
metabolite secretion and cellular behavior such as matrix
adhesion could help design better engineered tissues.
Nanosensors could be used to provide continuous mon-
itoring of the performance of the engineered tissues.
Already, tissue and cellular responses have been observed
using other nanoparticle contrast agents such as inamma-
tion of pancreatic islets [22], apoptosis [2325], and
angiogenesis [26,27]. The ability to monitor apoptosis
and angiogenesis with relatively high spatial resolution
would be advantageous not only to tissue engineering but
also disease progress and therapy response.
347
One method of monitoring engineered tissues would be
to use implantable sensors capable of relaying information
extracorporeally. Such a sensor would provide real time
data related to the physiological relevant parameters such
as pH, pO2, and glucose levels. There are several
advantages to using nanosensors for evaluating engineered
tissues in vivo. First, because the sensing element is
nanosized, miniaturization of the probe and implanting
throughout an engineered tissue would not adversely
perturb the system. Second, due to their enormous surface
area, the active area is large for immobilizing numerous
biological and chemical compounds including DNA [28]
and proteins [29] for improved sensitivity. Carbon nano-
tubes provide several attractive features that make them
ideal components for nanosensors including their electrical
properties, large surface area, and the capacity to
immobilize DNA or other proteins.
Because of their unique electronic structures, carbon
nanotube electrochemical sensors can potentially simplify
the analysis of redox-active proteins and amino acids
allowing cell monitoring in engineered tissues. By assem-
bling carbon nanotubes between two electrodes, a eld
effect transistor (FET) forms with the nanotubes serving as
the gate (Fig. 4). It has been shown that by applying a bias
across a nanotube, the electrical properties of carbon
nanotubes become exceedingly sensitive to the proximity of
electrical charges to the nanotubes [30]. By attaching a
sensing element to the nanotube surface, perturbations in
the electrical current passing through the nanotubes occur
upon binding which can be detected.
While relaying sensed information extracorporeally from
nanosensors is still in its infancy, carbon nanotube-based
sensors have already been demonstrated to measure many
biological relevant factors. For example, the electrooxida-
tion process of insulin and amperometric determination of
Fig. 4. Scanning electron microscopy (SEM) micrograph of electrode-
posited SWNT ropes between gold electrodes. These ropes can be
selectively burned out leaving only semiconducting tubes that then exhibit
FET transistor characteristics (courtesy of R. CzerwNanoTechLabs
Inc.).
 ARTICLE IN PRESS
348 B.S. Harrison, A. Atala / Biomaterials 28 (2007) 344353
insulin can be monitored using MWNT electrodes; this
suggesting a possible method to evaluate the quality of
pancreatic islets prior to their transplantation. Inorganic
particles can also be deposited onto the surface of carbon
nanotubes. Platinum microparticles have been deposited
on MWNTs and were capable of sensing thiols containing
amino acids such as L-cysteine and glutathione in rat
striatal microdialysates [31]. MWNTs have also been
modied with putrescine oxidase for detection of putres-
cine [32]. The possibility of placing the sensor onto a
exible biocompatible substrate is also appealing for in vivo
sensing. For instance, free cholesterol in blood can be
measured using a multiwalled carbon nanotube electrode
placed on a biocompatible substrate [33]. Polyaniline and
nanotube composite sensors have been shown to form
exible pH sensors [34].
One challenge facing any implantable sensor is the
formation of biolms caused by the non-specic binding of
serum proteins resulting in attenuate sensor response.
Several strategies have been developed to resist protein
adsorption, including coating the surface with highly
hydrophilic, neutrally charged and sterically hindered
polymers such as polyethylene glycol [35]. Tween 20 for
example has been coated on top of the nanotube surface
prevents proteins from adhering to the surface however the
interface between the nanotube and metal contact may be
more problematic [29]. While coating the polymer may
prevent non-specic binding to the nanotube, another
possible approach involves cycling the potential across the
surface to minimize biolm formation. For example, by
biasing the nanotube sensor to negative potentials, inter-
ferences from endogenous ascorbic and uric acids can be
avoided [32].
An alternative approach to electrochemical sensing is
through the use of optical methods. Taking advantage of
the optical window between 900 and 1300 nm, modied
SWNT have been used to measure b-D-glucose concentra-
tions [36]. This method relied on monitoring the NIR
uorescence from the SWNT that changed upon binding of
glucose. The optical window between 900 and 1300 nm is
important for biomedical applications because of the lower
optical absorption, hence greater penetration depths of
light and small auto-uorescent background. This ap-
proach in conjunction with others may provide novel
sensing strategies for tissue engineering.
Monitoring the cellular microenvironment using nano-
sensors will provide benecial information for designing
and evaluating engineered tissues. Carbon nanotubes may
prove to be important components for such nano-
sensors taking advantage of the electrical properties
possessed by this carbon-based material. Eventual ad-
vances in nanotube sensors will produce arrays of
nanotubes with several thousand sensing elements capable
of simultaneous monitoring of an engineered tissue. Such
data could provide a highly detailed map of cellular or
tissue behavior which would be invaluable for the
construction of tissue.
4. Augmenting cellular behavior
A third area where carbon nanotubes can make an
impact in tissue engineering is controlling the production of
or delivery of tissue-inducing substances such as growth
factors. Carbon nanotubes have already been used for a
number of cell altering applications including localized
drug delivery [37] and transfection [28]. They have also
been proposed as ion channel blockers [38]. Many of these
methods take advantage of the large aspect ratio and ease
of functionalization of carbon nanotubes. Thus, carbon
nanotubes could be components of drug delivery systems.
A signicant advantage of carbon nanotubes over
spherical nanoparticles is that they can be heterogeneously
functionalized. The end and sidewalls of carbon nanotubes
possess different chemical reactivities that can facilitate a
dual functionalized drug delivery platform (Fig. 5). For
instance, amine containing targeting agents such as
antibodies could be attached to the ends of the nanotube.
Drugs can then be linked with biodegradable linkers to the
sidewall of the carbon nanotube and a targeting or imaging
moiety can be attached to the ends.
Carbon nanotubes are not limited as a vehicle for
pharmaceutical delivery as they have been demonstrated
for delivery of genetic material. There are many ap-
proaches to delivering genetic materials to cells including
the use of viral (retroviral, lentiviral, or adenoviral)
vectors, and non-viral methods such as cationic organic
molecules and polymers. While viral methods have high
transfection efciencies, they have inherent risk factors
such as immunogenicity and pathogenicity [39]. Cationic
polymers such as polyethylenimine (PEI) also have high
efciency but are cytotoxic [40]. Carbon nanotubes are
promising materials because their synthetic origin mitigates
the risks associated with viral vectors, and they can easily
be tuned to reduce any cytotoxic effects.
Carbon nanotubes can be used to facilitate delivery of
DNA or any bioactive agent to cells. While they can be
functionalized to attach either electrostatically or cova-
lently to DNA and RNA, the remaining unfunctionalized
and hydrophobic portions of the nanotubes can be
attracted to the hydrophobic regions of the cells. This
hydrophobicity explains non-specic binding between
nanotubes and proteins [41]. In addition, they can be
functionalized with bioactive proteins to help cross the
cellular membrane. For example, biotin functionalized
carbon nanotubes were bound to uorescent dyes were
capable of intercellular transport of uorescent streptavi-
din [42]. Nanotubes can also be functionalized with
cationic components capable of penetrating human and
murine cell types. Such nanotubes can be complexed with
plasmid DNA to facilitate transfection of cells [28].
In addition, DNA or siRNA can be bound to nanotubes
with cleavable disulde bonds and delivered to mammalian
cells [43].
Besides heterogeneous functionalization, carbon nano-
tubes could provide localized delivery of therapeutic agents
 ARTICLE IN PRESS
B.S. Harrison, A. Atala / Biomaterials 28 (2007) 344353
Fig. 5. Chemical functionalization of carbon nanotubes showing both tip and side wall functionalization.
triggered by external sources. Previously, it was shown that
carbon nanotubes absorb NIR light at wavelengths that
are optically transparent to native tissue. For example,
irradiation with a 880 nm laser pulses can induce local
heating of SWNTs in vitro thereby releasing its molecular
cargo without harming cells or can be internalized within a
cancer cell and with sufcient heating kill the cell [44]. This
could allow selective delivery of drugs to certain cell types,
helping to control the distribution of such cells throughout
the engineered tissue.
5. Matrix enhancement
A fourth area that carbon nanotubes will impact tissue
engineering is for structural support. The matrix plays a
critical role in tissue engineering. It is responsible for
dening the space the engineered tissue occupies and aiding
the process of tissue development. While popular synthetic
polymers such as PLGA and PLA have been used for tissue
engineering, they lack the necessary mechanical strength.
In addition, such polymers cannot easily be functionalized
in contrast to carbon nanotubes which can be readily
functionalized. These advantages however have to be
weighed against the drawback that carbon nanotubes are
not biodegradable.
Carbon nanotubes have the potential for providing the
needed structural reinforcement for tissue scaffolding. By
dispersing a small fraction of carbon nanotubes into a
polymer, signicant improvements in the mechanical
strength of the composite have been observed. Carbon
nanotubes have been put into a host of synthetic polymers
and more recently, in biopolymers. For example, MWNTs
blended with chitosan showed signicant improvement in
mechanical properties compared with those of chitosan
[45]. Nanocomposites composed of 2% MWNT more than
doubled the Youngs modulus and tensile strength
349
compared to neat chitosan. Tuning of the mechanical
properties of the polymer can be adjusted depending on
nanotube loading and with the need of very small amounts
may counterbalance their non-degradable nature.
In vitro work has shown that several different cells types
have been successfully grown on carbon nanotubes or
nanocomposites. For example, blends of SWNT with
collagen support smooth muscle cell growth [46]. L929
mouse broblasts have been successfully grown on carbon
nanotube scaffolds [47]. Because MWNTs can have
diameters approximately 100 nm, they can possibly be
used to mimic neural bers for neuronal growth. It has
been shown that hippocampal neurons from 0- to 2-day-
old Sprague-Dawley rats were able to grow on carbon
nanotubes coated with 4-hydroxynonenal [48].
If the nanotubes are placed in an array, then they can be
used to form neural networks. For example, cortical tissue
from 1-day-old Charles River rats were cultured on a
nanopatterned substrate containing carbon nanotubes [49].
Arrangements of dissociated neuronal cells on patterned
surfaces consisting of CNT islands will self-organize into
ordered, compactly wired networks. Scanning electron
microscopy of neuronal networks on CNT templates
clearly reveal preferential adhesion of neurons and glia
cells to the CNT-coated regions and form ordered net-
works with geometries determined by the arrangement of
the CNT islands (Fig. 6A). Glia-like cells appear to form
an intermediate support layer on which many of the
neurons adhere. Direct attachment of neural cells on CNT
surface is also apparent (Fig. 6B). The exact effect of
carbon nanotubes on neurons is still unclear. Neurons
deposited on CNTs showed a signicant increase in the
frequency of spontaneous postsynaptic currents (771 Hz
for CNT vs. 1.170.2 Hz for control n 15) [50]. However,
no other general electrophysiological characteristics were
observed compared to neurons grown on glass. This
 ARTICLE IN PRESS
350 B.S. Harrison, A. Atala / Biomaterials 28 (2007) 344353
Fig. 6. (A) Microscopic images of neuron bridging an array of carbon
nanotubes thereby creating neural networks. The nanotube coating
consists of a pristine carbon nanotube lm, which is grown using a
chemical vapor deposition process. Dissociated cortical cells from 1-day-
old Charles River rats were plated for several days onto patterned quartz
or oxidized silicon substrates at a density of 1  103 cells/mm2. The bar
represents 100 mm. For scanning electron microscopy observation, samples
were xated, dried and, thereafter, gold coated. (B) Increased magnica-
tion of one nanotube pad shows neurons are generally conned to the
nanotubes surface except where neurons extend to adjacent nanotube
pads. Bar represents 10 mm (courtesy of Y. HaneinTel Aviv University,
Israel).
difference between the two groups may be due to the
electrical conductivity of the substrate. Thus carbon
nanotubes can be used as structural supports for en-
gineered tissue. In addition to tissue engineering, net-
worked patterns of neurons can be used to build advanced
neuro-chips for bio-sensing applications (e.g. drug and
toxin detection) [51].
Utilizing the electrical conductivity of carbon nanotubes
may prove to be a useful tool for directed cell growth. For
example when an alternating current is applied to the
substrate, nanocomposites of polylactic acid and MWNTs
have been shown to increase osteoblast proliferation by
46% and a greater than 300% increase in calcium
production [52]. Also, an upregulation of collagen I (a
major component in organic bone formation), osteonectin
and osteocalcin was observed. This suggests that nano-
composites may used to stimulate bone formation.
In addition to structural reinforcement, carbon nano-
tubes can be functionalized to provide additional capabil-
ities for tissue engineering. During the early stages of bone
formation, collagen serves as a nucleation site for the
deposition of hydroxyapatite, the principle component of
bone. Phosphate-substituted carbon nanotubes can sub-
stitute for collagen to direct the crystallization of hydro-
xyapatite reaching a thickness of 3 mm after 14 days of
mineralization [53]. The high aspect ratio of carbon
nanotubes should allow the scaffold to be aligned to more
closely mimic bone in vivo.
Carbon nanotubes can also be functionalized to release
bioactive factors. It has been shown that such factors, for
example glucose oxidase, can be attached to nanotubes and
still retain enzymatic activity [54]. This may allow a
mixture of different functionalized carbon nanotubes to
be added to a scaffold to create increasingly sophisticated
structures. Potentially, enzymatic or protein functionaliza-
tion, coupled with the electrochemical properties men-
tioned previously, may allow for matrices to provide not
only the mechanical integrity for cell growth but also to
monitor simultaneously the growth progress.
Beyond random orientation blends of nanotubes and
polymers, carbon nanotubes can be arranged into ordered
biomaterials which can provide mechanical support against
in vivo forces, such that the predened 3D structure is
maintained during tissue development. To create 3D
arrays, carbon nanotubes can be grown on a patterned
bed of catalysts. This permits control of the size, spacing,
and periodicity of the nanotubes to produce sophisticated
architectures. For example, L929 mouse broblasts have
been grown on such scaffolds [47]. Neurons can be grown
on patterned arrays of carbon nanotubes creating neural
networks [49]. In addition, carbon nanotubes can be
electrospun with biopolymers to form aligned matrices [55].
Carbon nanotubes have the ability to serve as a
multifunctional structural materials. Carbon nanotubes
may be able to provide the initial structural reinforcement
needed for newly created tissue scaffolds. Though not
biodegradable, carbon nanotubes can be rapidly removed
from the body [21]. This means that as engineered scaffolds
degrade, they can be readily cleared. While, mechanical
reinforcement was the initial motivation to using carbon
nanotubes, there is evidence that carbon nanotubes can
impart, accelerate and direct growth of cells. It is
particularly interesting that carbon nanotubes appear to
be good substrates from neuron growth.
6. Cytotoxicity
However promising a new technology or material might
be for biomedical applications, it must be safe. Any time
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B.S. Harrison, A. Atala / Biomaterials 28 (2007) 344353
a foreign substance is introduced into the body, under-
standing the organisms response to it is crucial. There is
debate in the literature regarding the cytotoxicity of
fullerene nanomaterials such as buckyballs and carbon
nanotubes. Thus a review would not be complete without
reviewing the cytotoxicity of carbon nanotubes. Some
studies have indicated that carbon nanotubes are cytotoxic
while others have shown nanotubes to be excellent
substrates for cellular growth.
Several in vitro studies report that carbon nanotubes are
cytotoxic. For example, when SWNTs and MWNTs were
incubated with alveolar macrophages (AM), signicant
(35% increase) in cytotoxicity was observed after 6 h of
exposure [56]. SWNTs appear to signicantly impair
phagocytosis of AM at low doses (0.38 mg/cm2) in vitro.
Increasing the dosage to 3 mg/cm2 for MWNT resulted in
characteristic features of necrosis and degeneration. It is
worth noting that the nanotubes used were only of 90%
purity; the remaining balance being amorphous carbon and
residual catalyst. Surprisingly, C60 was also tested and
showed relatively little toxicity. Molecular characterization
of the cytotoxic mechanism of MWNTs and nano-onions
(MWNO) on human broblasts shows that there is a dose-
dependent effect of gene expression [57]. At concentrations
of 0.6 mg/ml for nanotubes and 6 mg/ml for nano-onions,
serious impact on the cellular functions in maintenance,
growth and differentiation occurred. MWNT appear to
cause more stress than MWNO. Interestingly, the observed
effects on the expression of p38/ERK and EGFR from
broblasts in contact with these fullerenes suggest that they
may be useful nanomedicine platform for cancer therapy,
especially epithelially derived cancers. Others have re-
ported on the toxicity of human keratinocytes by SWNTs
[58,59]. The reason for the observed cytotoxicity is possibly
oxidative stress and activation of nuclear transcription
factor-kB in human keratinocytes [60]. However, carbon
nanotubes have also been reported to improve neural
signal transfer and support dendrite elongation and cell
adhesion in vitro [50] as well as support other cell types
such as smooth muscle [46], broblasts [47], osteoblast [52].
This suggests that a combination of several factors are
responsible for the observed effects [61].
More experimental data is beginning to be produced
regarding the in vivo effects of carbon nanotubes. For
example, introducing 5 mg/kg SWNT into the intratracheal
regions of rats produced mortality of 15% within 24 h
compared to control rats with carbonyl iron or graphite
particles (310 mm). It was concluded that the mortality
was due to the impact of agglomerating the major airways
in the rat and not due to inherent toxicity of SWNT [62].
The pulmonary toxicity of nanotubes in mice 7 and 90 days
after intratracheal instillation has been investigated. 0.1 mg
of material showed no overt clinical signs though over 50%
of the mice died when exposed to 0.5 mg of material.
However, over time the mice with intratracheal instillation
of carbon nanotubes showed grandulomas along with
other lung lesions. It was shown that metal catalysts were
351
not the cause of the grandulomas nor were graphite
impurities remaining on the nanotubes. Such data is
contrasted by reports that carbon nanobers implanted
into the subcutaneous tissue of rats showed no severe
inammatory response [63]. And in vivo studies have
shown that single walled carbon nanotubes containing
soot did not induce measurable skin irritation or pulmon-
ary toxicity [64].
While concerns that carbon nanotubes may be cytotoxic
dampens the enthusiasm of this material for biomedical
applications, new approaches are being developed to
mitigate their toxicity. Culturing cells with carbon nano-
tubes functionalized with glycopolymers, for example,
were indistinguishable from cells grown in the absence of
carbon nanotubes [65]. Such strategies may prove to make
carbon nanotubes safer and also more useful for tissue
engineering.
7. Conclusions and future outlook
Carbon nanotubes appear well suited as a biomaterial
and may become a useful tool for tissue engineering.
Carbon nanotubes have the capacity to be used in cellular
imaging, chemical and biological sensing, bioactive agent
delivery, and matrix engineering. While new uses of carbon
nanotubes for biomedical applications are being developed
concerns about cytotoxicity may be mitigated by chemical
fuctionalization. However, there will be some limitations to
this nanomaterial since it is not biodegradable. Yet, it has
been shown to be excreted in vivo and so could be cleared
from the body once it is no longer needed. A promising
new approach to non-invasive imaging by exploiting the
NIR optical properties of carbon nanotubes may create
signicant advances in in vivo monitoring of engineered
tissues. In addition, the capability of creating nanosized
sensors may provide key information regarding tissue
microenvironments thus yielding new insight into the
cellmatrix interface. Demonstrations of drug and DNA
intercellular delivery using carbon nanotubes show promise
for carbon nanotubes to be used as alternatives to viruses
for transfection. Finally, carbon nanotubes may serve as an
important component to imparting novel properties to the
biomatrix for directing cell growth. Ultimately, the use of
carbon nanotubes in combination with other biomaterials
may be necessary to achieve the goals of tissue engineering.
In conclusion, the use of carbon nanotubes for tissue
engineering represent a challenging but potentially reward-
ing opportunity to develop the next generation of
engineered biomaterials.
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