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LETTERS
nature materials | VOL 3 | APRIL 2004 |
www.nature.com/naturematerials 249
T issue engineering aims to replace, repair or
regeneratetissue/organ function, by delivering signalling
molecules andcells on a three-dimensional (3D) biomaterials
scaffold that
supports cell infiltration and tissue organization1,2. To control
cell
behaviour and ultimately induce structural and functional tissue
formation on surfaces, planar substrates have been patterned
with
adhesion signals that mimic the spatial cues to guide cell
attachment
and function3–5. The objective of this study is to create
biochemical
channels in 3D hydrogel matrices for guided axonal growth. An
agarose
hydrogel modified with a cysteine compound containing a
sulphydryl
protecting group provides a photolabile substrate that can be
patterned with biochemical cues. In this transparent hydrogel
we
immobilized the adhesive fibronectin peptide fragment,
glycine–
arginine–glycine–aspartic acid–serine (GRGDS),in selected
volumes of
the matrix using a focused laser.We verified in vitro the
guidance effects
of GRGDS oligopeptide-modified channels on the 3D cell

migration
and neurite outgrowth. This method for immobilizing
biomolecules in
3D matrices can generally be applied to any optically clear
hydrogel,
offering a solution to construct scaffolds with programmed
spatial
features for tissue engineering applications.
Hydrogels have been widely studied as tissue scaffolds because
they
are biocompatible and non-adhesive to cells, allowing cell
adhesion
to be programmed in6–8. Current microfabrication methods for
3D hydrogel matrices with controlled intrinsic structure mainly
include photolithographic patterning9–11, microfluidic
patterning12,
electrochemical deposition13 and 3D printing14. Notably,
although these
layering techniques can conveniently shape the hydrogel on X–
Y planes,
they have limited control over both the coherence of the layers
along the
z direction and the local chemistry. Combining photolabile
hydrogel
matrices with focused light provides the possibility of
eliminating the
layering process and directly modifying the local physical or
chemical
properties in 3D. This results in a promising (and perhaps
facile) way to
fabricate novel tissue constructs15,16, as is described herein to
control cell
behaviour by controlling the local chemical properties of gels.
Reconstituting adhesive biomolecules into biomaterials is of

great
importance to understanding cell–substrate interactions that can
be
translated to tissue-regeneration designs. Using 2D lithographic
techniques, adhesive biomolecules can be localized in arbitrary
shapes
and sizes17,18. For example, patterning narrow strips of the
extracellular
matrix (ECM) adhesion protein, laminin, on non-cell-adhesive
2D
substrates elicited oriented neurite growth through integrin-
receptor
mediated processes19,20. Similar results were obtained using
oligopeptides derived from laminin–integrin active sites, such
as
YIGSR, IKVAV and RGD (the letters are the standard amino
acid
symbols), among others4,21. The fidelity of neurons to the
peptide
regions was attributed to both cell-adhesive and non-adhesive
regions
that mimic the in vivo attractive and repulsive haptotactic (that
is,
contact-mediated) cues, respectively, present during
development22.
This inspired us to investigate ways to create isolated
biomolecular
channels in cell invasive, yet non-adhesive, matrices, thereby
extending 2D to 3D patterning, and better approximating
functional
ECM analogues.
In this study, we used a soft thermosensitive agarose hydrogel
as a
model material because it is optically transparent, facilitating

bulk
modification using light. Dilute low-gelling-temperature
agarose
hydrogel is permissive to 3D neurite outgrowth and has been
used as
spinal cord surrogates23,24.A general scheme for producing
oligopeptide
channels in the agarose gel matrices is illustrated in Fig. 1.
We hypothesized that the spatial resolution between adhesive
and
non-adhesive volumes in the agarose matrix would promote
guidance
within the peptide-modified channels.
To prepare a photolabile matrix for 3D photo-fabrication, the
photosensitive S-2-nitrobenzyl-cysteine (S-NBC), which can be
efficiently cleaved in aqueous environments to release free
nucleophile on irradiation by ultraviolet (UV) light25,26, was
first
bound to the dissolved agarose polymer through a 1,1′-
carbonyldiimidazole (CDI) activation process. There was a
direct
correlation between the amount of S-NBC-modified agarose and
the
amount of CDI activation. Moderately modified agarose (that is,
up
to 5 wt%) remained thermally reversible, easily dissolving at
high
temperatures, and forming optically transparent hydrogels on
cooling. S-NBC-modified agarose had a UV absorption peak at
266 nm, which was attributed to the 2-nitrobenzyl groups in the
gel
(Supplementary Information, S1). Photo-irradiating S-NBC-
modified agarose hydrogel matrices (using a UV lamp) resulted
in
free sulphydryl groups, which were then available to react with

sulphydryl-reactive biomolecules. The S-NBC-modified
hydrogel
matrices were proven to be safe cell substrates, showing no
toxicity or
A photolabile hydrogel for guided three-
dimensional cell growth and migration
YING LUO1,2 AND MOLLY S. SHOICHET1,2,3
1Department of Chemical Engineering and Applied Chemistry,
University of Toronto, 200 College Street, Toronto, Ontario
M5S 3E5, Canada
2Institute of Biomaterials and Biomedical Engineering,
University of Toronto, 4 Taddle Creek Road, Toronto, Ontario
M5S 3G9, Canada
3Department of Chemistry, University of Toronto, 80 St.
George Street, Toronto, Ontario M5S 3H6, Canada
*e-mail: [email protected]
Published online: 21 March 2004; doi:10.1038/nmat1092
nmat1092PRINT 3/11/04 11:26 AM Page 249
© 2004 Nature Publishing Group
LETTERS
250 nature materials | VOL 3 | APRIL 2004 |
www.nature.com/naturematerials
inhibitory effects on cell adhesion or neurite growth
(Supplementary
Information, S2).
To fabricate the free sulphydryl channels in the gel, the S-NBC-
modified agarose gel was irradiated with a He–Cd 325 nm laser

(4.0 mW).
A 0.3-mm-diameter laser beam, focused by a convex lens with a
focal
length of 10 cm,was used to irradiate a 1.5-mm-thick,0.5 wt%
agarose gel
sample for 1 s. Free sulphydryl functional channels were
created on
irradiation, providing the template for biomolecule
immobilization.
To demonstrate the universality of this technique, we coupled
agarose with the GRGDS sequence, an oligopeptide capable of
promoting integrin-mediated cell adhesion27,28. A maleimide-
terminated GRGDS, (N-α-(3-maleimidopropionyl)-N-ε-
fluorescein) lysine-GRGDS, was prepared by solid-state peptide
synthesis, with the fluorescein added simply to facilitate the
visualization of the oligopeptide. The distribution of the peptide
in the
gel after laser fabrication was analysed by confocal microscopy
and the
peptide channels were observed penetrating the 1.5-mm-thick
sample.
Figure 2a shows a typical XY cross-section micrograph of the
oligopeptide distribution at a given depth within the agarose
matrix:
GRGDS peptides were conjugated to the gel in isolated circular
channels defined by the shape of the laser beam. These circular
peptide
domains were observed continuously at all depths in the gel.
The diameter of the channels was between 150 and 170 µm over
a 1 mm
depth, corresponding to the focal spot size of the focused laser
beam of
160 µm. The XY cross-section micrographs at all levels were
reconstructed by confocal microscopy to provide the 3D stereo
channel

image (Fig. 2b).
The fluorescence intensity profile of the cross-sections of two
oligopeptide channels in the bulk hydrogel was analysed (Fig.
2c).
The intensity increased sharply at the irradiated regions,
demonstrating
that homogenous agarose hydrogels cause minimal lateral
scattering
during irradiation and such gel matrices are suitable for 3D
photo-fabrication.
The transverse diameter of the biochemical channels is
determined
by the focal spot size of the focused laser, but their longitudinal
dimensions are affected by two factors: (i) the depth of focus of
the
focused beam (usually defined as the distance over which the
focal
spot size changes ±5%)29; and (ii) the transmittance of light
through
the hydrogel material, which depends on the concentration of
2-nitrobenzyl photolabile moieties and not on agarose, which
absorbs
only 1.2% of the incident 325-nm light across a 1.5-mm gel.
The theoretical depth of focus using the convex lens with a
focal
length of 10 cm is 7 cm (ref. 29; see Supplementary
Information, S3).
Thus the longitudinal dimensions of our channels are mainly
limited by
the concentration of the photolabile moiety, S-NBC. The
variation in
longitudinal fluorescent intensity in the channel is shown in
Fig. 2d,

where the relative fluorescence intensity decreases with depth
of
fluorescein-labelled GRGDS as a result of the S-NBC
absorption of light
travelling through the 3D hydrogel matrix. The transmittance of
light through S-NBC-modified agarose hydrogels decreases with
gel
thickness and S-NBC concentration (Fig. 3). The light intensity
at
different depths can be calculated using the Beer–Lambert law
with the
corresponding extinction coefficient for the 2-nitrobenzyl
moiety
of 1,409 L mol–1 cm–1. Generally, the lower the concentration
of
biomolecular ligands (or photolabile moiety) required, the
thicker the
photofabricated gel.
We found that irradiating agarose hydrogel matrices with the
laser
beam for more than 1 s did not further increase the
immobilization
of peptides at the gel surface region, which received the highest
dose of
energy longitudinally30.We approximated the GRGDS
concentration at
Figure 1 The general strategy used to create adhesive
biochemical channels in agarose hydrogel matrices relies on
modifying agarose with photolabile groups, focused
laser light sources and biomolecule coupling. Specifically, a,
bulk agarose is first activated with CDI followed by reaction
with 2-nitrobenzyl-protected cysteine. On exposure to UV
light, the 2-nitrobnezyl group is cleaved leaving free sulphydryl
groups that react with maleimido-terminated biomolecules.

b,The strategy to create biomolecular channels in agarose
hydrogel matrices uses the chemical modification strategy in (a)
and a focused laser, resulting in alternating volumes of cell-
adhesive (peptide) channels separated by non-adhesive
(agarose) volumes.
OH
OH
OH
OH
OH
OH
OH
OH
OH
NN
N NO
O
O
O
O

O
O
O
O
C C
C
C
Agarose
νh
NH CHC2 SH
COOH
NH2CHCH2S CH2
COOH NHCHCH2S CH2
COOH
S-NBC-agarose
NH CHCH2S
COOH
N-biomolecule
NO2
O

O
N- biomolecule
NO2
-SH
-SH
-SH
Focused laser
O
O
N- biomolecule
Laser penetrates hydrogel matrix,
producing free sulphhydryl groups
2-nitrobenzyl-protected region
Biomolecule-modified region
a
b

LETTERS
the top surface of the channel at 41.5 ± 2.2 µM (0.5 wt% gel,
0.4 mM
S-NBC) (Supplementary Information, S4). By comparing the
local
irradiation intensity on the surface to that within the channel
(Fig. 3)
and assuming a linear relationship between the irradiation
intensity and
photochemical yield31, we estimated GRGDS concentration to
vary
from 41.5 ± 2.2 µM at the gel surface to 33.0 ± 2.0 µM at 1.5
mm depth.
This chemical gradient may be useful for guided regeneration3;
alternatively, it could be minimized by irradiating samples from
both
top and bottom.
To determine how neural cells responded to the GRGDS
adhesion
domains in the hydrogel, we used agarose matrices (0.5 wt%,
0.4 mM
S-NBC), which have been shown to permit neurite growth21.
Dissociated cells from explants of E9 chick dorsal root ganglia
were
plated on top (that is, the same side that had been irradiated) of
the
hydrogel matrices containing biochemical channels.Owing to
generally
poor adhesion properties of the agarose surfaces, cells formed
clusters on top of the oligopeptide channels. Intriguingly (from

day
three onwards after cell plating), cell clusters that aggregated
on
oligopeptide channels were observed to extend thick processes
into the
biochemical channels; the migration of cells and elongation of
cell
processes were often found to turn at the edge of the adhesive
biochemical domain, indicating the guidance effect of the
channel
chemistry. Representative images of cell behaviour inside the
hydrogel
matrices show that the GRGDS channel promoted neurite
extension
and cell migration (Fig. 4): Fig. 4a (optical image) and 4b
(fluorescent
image of green fluorescein-tagged peptide channel and red F-
actin
rhodamine–phalloidin cytoskeletal staining) show a single
process
emerging from a cell cluster. In Fig. 4c, the blue DAPI nuclear
staining
shows that multiple cells migrated into the GRGDS channel. At
day six
of cell culture, a majority of the channels (76 ± 16%, mean ±
standard
deviation, n = 50 channels) were observed to contain neurites
and cellular assemblies, having an average migration distance of
633 ± 181 µm (mean ± standard deviation, n = 50 channels).
Cell invasion into non-degradable physically crosslinked
matrices
are probably affected by both the mechanical and biochemical
properties of the hydrogel material. (Supplementary
Information, S5).
By dynamic rheological analysis, we found that there was a

small
difference in the local mechanical properties of modified
(storage
modulus, G′ = 17.0 ± 0.7 Pa, n = 4) and un-modified (G′ = 14.8
± 0.6 Pa)
agarose due to laser-activated chemistry. Despite a slightly
higher G′ for
the modified agarose, this difference did not affect cell
penetration,
confirming that the chemical differences between the channels
and the
surrounding volumes are responsible for the observed cellular
response.
To understand further the interaction between the GRGDS
oligopeptide channels and cells, neural cells were cultured on
matrices
modified with scrambled oligopeptide channels containing the
inactive sequence GRDGS. Neither neurite outgrowth nor cell
migration was observed inside the scrambled oligopeptide
channels.
Therefore, the guided axonal growth and cell migration
observed in
a b
c
0 200 400 600 800 1,000
Position (µm)
R
el
at
iv

e
flu
or
es
ce
nc
e
in
te
ns
ity
R
el
at
iv
e
flu
or
es
ce
nc
e
in
te

ns
ity
0 500 1,000 1,500
Depth ( µm)
d
0 1 2 3 4 5
90
80
70
50
40
30
20
10
60
Gel thickness (mm)
Tr
as
m
itt

an
ce
(%
)
= 0.4 mM
= 0.8 mM
Figure 2 Biochemical channels synthesized in agarose hydrogels
were
characterized with a fluorescein-tagged GRGDS peptide. a,A
representative XY cross-
section image of green fluorescently labelled oligopeptide
channels (scale bar: 200 µm);
b,A representative image of the green fluorescently labelled
oligopeptide channels
constructed from a series of XY cross-sectional micrographs
over a 0.5 mm depth (scale
bar: 200 µm); c,The relative fluorescence intensity profile of a
line pass through the centre
of the cross-sections of two oligopeptide channels shows
intensity contrast between the
peptide modified and non-modified regions; d,The longitudinal
fluorescence intensity
profile along the central axis of the channel shows a decrease in
fluorescent intensity with
depth, indicating a concentration gradient of oligopeptide.
Figure 3 The transmittance of laser light is affected by both the
thicknesses of the
S-NBC-modified agarose hydrogel matrices and the
concentration of S-NBC of
0.4 mM and 0.8 mM (mean ± standard deviation, n = 5). The

data indicate that the
concentration of the S-NBC in the matrix affects the depth (or
length) of modified channels.
In those channels used for guided cell growth (0.4 mM S-NBC,
1.5 mm), the concentration
of peptide on the bottom surface may decrease to 80% of what it
was on the top (laser
exposed) surface.
LETTERS
252 nature materials | VOL 3 | APRIL 2004 |
GRGDS channels probably resulted from the activation of
integrin
receptors in the cell membrane and not from local mechanical
property
variations or non-specific depositions of proteins in the
oligopeptide-
modified regions. The unreacted S-NBC-modified agarose
(found
between the biochemical channels) is non-adhesive and non-
cytotoxic
(Supplementary Information, S2).
Neurons are able to respond to a wide range of adhesion ligand
concentrations on substrates by regulating the expression of
integrin
receptors32.We observed a similar cellular response from DRG

cells,with
respect to the neurite number and length, when the GRGDS
concentration was varied from 13 µM to 70 µM on agarose
surfaces30.
This suggests that the ligand concentration variation in
chemical
channels (of 41.5 µM to 33 µM) observed within the 1.5-mm-
thick
channels probably had a minimal (if any) effect on cell
behaviour.
The axonal guidance that we observed in our GRGDS channels
may
result from either direct interaction between neurons and
GRGDS, or
the integrated crosstalk of multiple cell types found in the DRG
explants. Additional experiments are necessary to identify the
cellular
components inside the GRGDS channels to reveal the
mechanisms
leading to the oriented cellular assemblies.
In summary, we demonstrate a paradigm to immobilize
biomolecules in selected volumes in a 3D hydrogel matrix using
laser
fabrication techniques and photochemistry. The cellular
guidance
observed is achieved by chemical (and not physical) channels.
Provided that the hydrogel materials are cell invasive, matrices
with
heterogeneous biochemical domains are novel platforms to
facilitate
the elucidation of fundamental cell–substrate interactions.
Adhesion channels elicited oriented axonal growth in hydrogels,
suggesting that the incorporation of the biochemical channels
into
biodegradable hydrogel matrices may find clinical applications

for
guided nerve regeneration or be extended to other tissues.
METHODS
All chemicals were purchased from Sigma and used as received
unless otherwise specified.
MODIFICATION OF ULTRA-LOW-GELLING-
TEMPERATURE (ULGT) AGAROSE WITH S-NBC
S-NBC was synthesized by reacting L-cysteine and 2-
nitrobenzyl bromide (1:1 molar ratio) in NaOH
solution at room temperature for 2 h. The pure product was
obtained by recrystallization in water.
To conjugate S-NBC to the agarose polymer, the ULGT agarose
polymer was first dissolved in dimethyl
sulphoxide (DMSO). Under an inert nitrogen environment, CDI
was added to partially activate the
hydroxyl groups in agarose polymer. After 1 h activation, a
solution of S-NBC in DMSO was added.
The mixture was kept at room temperature overnight before it
was extensively dialysed against water to
remove the unreacted S-NBC. The substitution level of S-NBC
in the resulting agarose was determined by
measuring the absorbance at 266 nm using an ultraviolet
spectrometer (Ultraspec 4000, Biopharmacia).
CREATING PHYSIOCHEMICAL CHANNELS IN ULGT
AGAROSE HYDROGEL MATRIX
(N-α-(3-maleimidopropionyl)-N-ε-fluorescein)-lysine-GRGDS

was prepared by solid-state peptide
synthesis based on Fmoc chemistry. N-ε-fluorescein-lysine-
GRGDS was first synthesised without
cleaving the side-chain protecting groups on a peptide
synthesizer (Pioneer, BioApplied Systems).
3-Maleimidopropionic acid was activated using dicyclohexyl
carbodiimide in dichloromethane and
reacted with the amine terminal of the peptide on the resin. The
maleimide-activated peptide was de-
protected and cleaved from the resin using 95% aqueous
trifluoroacetic acid and lyophilised. Scrambled
maleimide-activated oligopeptide, (N-α-(3-
maleimidopropionyl)-N-ε-fluorescein)-lysine-GRDGS, was
synthesised using the same method described to synthesize
activated GRGDS.
Irradiation of the hydrogel was performed using a 325 nm He-
Cd laser (Omnichrome 3074R-S-
A03, Melles Griot). The laser was focused by a convex fused-
silica lens. 0.5 wt% S-NBC agarose solution
prepared in phosphate buffer (pH 7.4) was refrigerated at 4 °C
for 3 h to form a hydrogel sample of
~1–5 mm thick. The gel was placed at the focal spot of the
focused laser for irradiation and moved using
an XY stage. To obtain the transmittance of light through the
hydrogel matrices, the laser-beam energy
was measured before and after it penetrated through the sample

volume. The irradiated sample was
immersed and shaken in phosphate buffer containing maleimide-
activated GRGDS oligopeptide for 8 h.
Unreacted GRGDS peptide was removed by washing the gel for
2 days. Peptide distribution in the
hydrogel matrix was imaged using a laser scanning confocal
microscope (FV300, Olympus).
RHEOLOGICAL CHARACTERIZATION
To investigate how the photo-modification affected the
mechanical stiffness of the hydrogel, dynamic
rheological tests were performed to measure the shear modulus
of the modified and unmodified agarose
hydrogels. S-NBC agarose solutions (0.5 wt%), irradiated (1
min, 0.4 W, X-cite, EFOS) or non-irradiated,
were mixed with maleimide-GRGDS at 1 mg per ml for 1 h at
room temperature. To measure the complex
shear moduli of the modified and un-modified agarose hydrogel,
0.5 ml of solution was loaded between
the cone and plate geometry on a dynamic rheometer (AR 2000,
TA Instruments) and cooled down at
a b c
Figure 4 Primary rat dorsal root ganglia cells were plated on 3D
patterned GRGDS oligopeptide-modified, 0.5 wt% agarose gels.
Three days after plating, DRG cells grew within
GRGDS-oligopeptide-modified agarose channels only, and not

in surrounding volumes.A cell cluster on top of a GRGDS
channel shows cell migration into the channel and extension of
a
process into the oligopeptide-modified channel as viewed by: a,
optical microscopy; b, confocal fluorescent microscopy, where
the channel is green (due to a fluorescein-labelled
oligopeptide) and the cells are red (due to the cytoskeletal F-
actin rhodamine–phalloidin stain); and c, fluorescent
microscopy, where a nuclear DAPI stain (blue) confirms cell
migration into
the oligopeptide-modified channel. (Scale bars: 100 µm)
LETTERS
4 °C for 90 min to solidify. The temperature was raised to 37 °C
(to simulate the cell-culture temperature)
and equilibrated for 5 min. The frequency sweep was performed
from 0.1 to 100 rad s–1 with a 1% shear
strain applied to each sample.
NEURAL CELL RESPONSE TO BIOCHEMICAL CHANNELS
OF GRGDS
To study the guidance effect of GRGDS oligopeptide channels
on axonal growth, the cells dissociated
from the dorsal root ganglia explanted from E9 chicks, were

plated on top of the hydrogel matrices
containing either active GRGDS or scrambled GRDGS
oligopeptide channels. After culturing the cells in
the media containing 10% horse serum and 50 ng per ml nerve
growth factor for 6 days (37 °C, 5% CO2),
the samples were imaged on their side by phase
contrast/fluorescence microscopy (Zeiss LM410) and
confocal microscopy (FV300, Olympus) so as to observe the
cell behaviour inside the hydrogel matrices.
Rhodamine–phalloidin (red) and DAPI (blue) fluorescent
markers (Molecular Probes) were used to
identify cellular skeletons and nuclei, respectively, in the gel
using standard methods.
Received 2 July 2003; accepted 3 February 2004; published 21
March 2004.
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26. McCray, J. A. & Trentham, D. R. Properties and uses of
photoreactive caged compounds. Annu. Rev.
Biophys. Biophys. Chem. 18, 239–270 (1989).
27. Yamada, Y. & Kleinman, H. K. Functional domains of cell
adhesion molecules. Curr. Opin. Cell Biol. 4,
819–823 (1992).
28. Ruoslahti, E. RGD and other recognition sequences for
integrins. Annu. Rev. Cell. Dev. Biol. 12,

697–715 (1996).
29. Siegman, A. E. Lasers (University Science Books, Mill
Valley, California, 1986).
30. Luo, Y. in Department of Chemical Engineering and
Applied Chemistry 95–96, 129–132 (University of
Toronto, Toronto, Ontario, Canada, 2003).
31. Silbey, R. J. & Alberty, R. A. Physical chemistry (Wiley,
New York, 2001).
32. Condic, M. L. & Letourneau, P. C. Ligand-induced changes
in integrin expression regulate neuronal
adhesion and neurite outgrowth. Nature 389, 852–856 (1997).
Acknowledgements
We are grateful to the Natural Sciences and Engineering
Research Council of Canada, Ontario Graduate
Scholarship and Connaught for funding and thank Ying-Fang
Chen, Patricia Musoke-Zawedde and
David Martens for their assistance.
Correspondence and requests for materials should be addressed
to M.S.
Supplementary Information accompanies the paper on
Competing financial interests
The authors declare that they have no competing financial …
Axonal chemotaxis is believed to be important in wiring up the

developing and regenerating nervous system, but little is known
about how axons actually respond to molecular gradients. We
report a new quantitative assay that allows the long-term
response of axons to gradients of known and controllable shape
to be examined in a three-dimensional gel. Using this assay, we
show that axons may be nature’s most-sensitive gradient
detectors, but this sensitivity exists only within a narrow range
of ligand concentrations. This assay should also be applicable
to other biological processes that are controlled by molecular
gradients, such as cell migration and morphogenesis.
A key hypothesis in developmental neuroscience is that axons
are often
guided to their targets by sensing molecular gradients1–4.
Precise
measurements of axonal sensitivity to gradients in
physiologically rel-
evant environments are essential for understanding how
gradients
direct axons along particular trajectories in vivo, and for
designing
effective therapeutic interventions using guidance factors.
Several
quantitative gradient assays have greatly aided the study of
chemotaxis
in fibroblasts, leukocytes and neutrophils5–8. These assays,
however,
are generally unsuitable for studying axonal guidance, and
previous
approaches to quantifying axonal sensitivity to gradients have
so far
provided only limited information9–14. Here we present a new
technol-
ogy that is compatible with the demands of long-term neuronal
cell
culturing and that allows for the efficient generation of precise,

repro-
ducible and arbitrarily shaped gradients of diffusible molecules.
Using
this technology, we show that growth cones, the motile sensing
struc-
tures at the tips of developing axons, are capable of detecting a
concen-
tration difference as small as about one molecule across their
spatial
extent. Furthermore, we show that this sensitivity exists across
only a
relatively small range of ligand concentrations, indicating that
adapta-
tion in these growth cones is limited.
RESULTS
Gradient generation
We established gradients by ‘printing’ drops of solution in a a
series
of ten lines 1 mm apart with increasing amounts of chemotropic
molecules onto the surface of a thin collagen gel (Fig. 1a). After
a
relatively short time, molecules diffuse to fill in the gaps
between the
printed lines and to create a profile that is independent of the
depth.
The resulting smooth gradient can be quite stable, as in general
the
time τ required for significant diffusion over a distance L scales
as
the square of distance, τ ≈ L2/D, where D is the diffusion
coeffi-
cient15. For nerve growth factor (NGF), the guidance factor
used in
these experiments, we have measured D = 8 × 10–7cm2/s in

collagen
(see Methods), which gives τ ≈ 52 min for L = 0.5 mm, the
distance
over which molecules must diffuse to fill in the space in
between the
lines or to reach the midplane of the gel (where the axons are
grow-
ing), but τ ≈ 3.6 d for L = 5 mm, the horizontal distance from
the
center to the end of the printed pattern. The time-dependent
con-
centration profile can be calculated using finite element
modeling of
the diffusion equation. Figure 1b,c shows the result of a two-
dimen-
sional simulation of an exponential gradient applied to a gel,
appro-
priate to the situation depicted in Figure 1a, assuming no
significant
variation in the direction parallel to the printed lines. The
printed
molecules diffused quickly into the thin gel, and the
concentration
rapidly became independent of depth (Fig. 1b). The initial
oscilla-
tions along the length of the block quickly died away, followed
by a
long period of a relatively stable gradient, particularly at the
low-
concentration end (Fig. 1c).
The actual concentration gradients produced by this method can
be measured with quantitative fluorescence imaging.
Concentration
profiles of fluorescently labeled casein for exponential and
linear

patterns are shown (Fig. 2a,b; casein is of similar molecular
weight to
NGF but is relatively inexpensive and easy to label). The actual
time-
dependent concentration profiles of casein that were extracted
from
the fluorescence imaging show a good match with the results of
the
finite element modeling using an independently determined
value of
D = 6 × 10–7cm2/s (Fig. 2c). This gradient generation method
has
several important advantages over previous approaches: large
num-
bers of identical gradients can be generated quickly, the often
pre-
cious chemotropic molecules are required in only very limited
quantities, the gradients are established in a short time but can
remain stable for a day or more, nonlinear gradient shapes can
be
used and gradients of multiple factors with different shapes and
arbi-
trary spatial relationships can be generated. This technique
could
also be used to provide a source of factor while axons are
growing, as
no direct contact is made with the gel.
1Department of Neuroscience, Georgetown University Medical
Center, Washington, DC, 20007, USA. 2Department of Physics,
Georgetown University, Washington, DC
20057, USA. 3Department of Anatomy and Neurobiology, and
The Program in Neuroscience, University of Maryland School
of Medicine, Baltimore, Maryland 21201,
USA. 4These authors contributed equally to this work.
Correspondence should be addressed to G.J.G.

([email protected]).
Published online: 25 May 2004; doi:10.1038/nn1259
A new chemotaxis assay shows the extreme sensitivity
of axons to molecular gradients
William J Rosoff1,4, Jeffrey S Urbach2,4, Mark A Esrick2,
Ryan G McAllister2, Linda J Richards3 & Geoffrey J Goodhill1
T E C H N I C A L R E P O R T
6 7 8 VOLUME 7 | NUMBER 6 | JUNE 2004 NATURE
NEUROSCIENCE
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Application to axon guidance
We have applied this technology to a well-characterized model
system,
the response of the rat dorsal root ganglion (DRG) to NGF9,16–
18. The
sensitivity of DRG axons was measured by culturing explants in
a
three-dimensional collagen gel in the presence of an exponential
NGF
concentration gradient. This gradient shape produced a
percentage
change in concentration across the growth cone that was
independent
of its position in the gradient. Several explants were placed in a
row
that was parallel to the printed lines, between the third and
fourth line
of the pattern. This region of the gradient (between 1.2 and 1.3
cm) is
expected to be the most stable (Fig. 1c). The overall NGF
concentra-
tion was adjusted to be 1 nM at the location of the explants,

independ-
ent of the steepness of the gradient (see Methods). As a simple
way of
quantifying the guidance response, we defined the guidance
ratio as
the number of bright pixels representing axons on the high side
of the
gradient minus the number on the low side, normalized by the
total
number of bright pixels representing axons (ref. 19; see
Methods). If all
neurites emerged from the explant on the high-concentration
side, the
guidance ratio would be 1.
We have measured the width of rat DRG growth cones in three-
dimensional collagen to be no larger than 10 µm including
filopodia
(data not shown; see also ref. 20). We therefore defined the
gradient
steepness s as the percentage change in concentration across 10
µm.
The value of the guidance ratio after 36–40 h in culture for s =
0, 0.1,
0.2 and 0.4% is shown (Fig. 3a). There was an increase in the
guidance
ratio as the steepness of the gradient increased, and significant
guid-
ance occurred even for s = 0.1%. This was despite the fact that
DRG
explants are comprised of a heterogeneous population of
neurons, not
all of which are responsive to NGF21. Representative explants
with
guidance ratios close to the mean for each steepness are shown
(Figs. 3d–f). As the gradient steepness increased, the asymmetry

increased markedly.
These explants did not show obvious turning of neurites up the
gra-
dient (Figs. 3d–f), and a statistical analysis of turning for the
data set
reported in Figure 3a found no significant neurite turning
overall. To
determine whether the asymmetry in outgrowth as measured by
the
guidance ratio was simply the result of a trophic effect, whereby
the
higher concentration of NGF on the up-gradient side of the
explant
caused more undirected outgrowth than on the down-gradient
side, we
measured total neurite outgrowth as a function of absolute
concentra-
tion of NGF (Fig. 3b). The curve is bell shaped with a peak at
about
1 nM, consistent with earlier studies22. There was little change
in out-
growth between 0.3 nM and 1 nM, but there was a significant
decrease
in outgrowth between 1 nM and 3 nM (P < 10–5). In our
gradient
experiments, the difference in concentration between the two
sides of
each explant varied between about 10% (s = 0.1%) and 50% (s =
0.4%),
which is small compared with that required for significant
variations in
outgrowth. In any case, the decrease in outgrowth between 1 nM
and
3 nM indicates that the trophic effect of NGF by itself would
actually

produce a negative guidance ratio for concentrations around 1
nM.
To determine the range of concentrations for which growth
cones are
sensitive to shallow gradients, we established exponential NGF
gradi-
ents of steepness s = 0.2% but with NGF concentrations at the
explants
in the range of 0.0001–100 nM (Fig. 3c). The guidance ratio
curve is
bell shaped with a peak at 1–10 nM, where the guidance is
highly signif-
icant (P < 10–7). The guidance ratio is more weakly significant
for
0.1 nM (P < 0.005) and is not significant for 100 nM and for
0.01 nM
and below. Unlike the 1 nM case described above, in the 10 nM
case we
saw clear neurite turning (turning strength = 0.023; P < 0.0001;
Fig. 3g). Weaker turning was also observed in the 100 nM
condition
(turning strength = 0.015; P < 0.02) and the 0.1 nM condition
(turning
NATURE NEUROSCIENCE VOLUME 7 | NUMBER 6 | JUNE
2004 6 7 9
Figure 1 Gradient generation technique. (a) Lines of
chemotropic factor
were printed onto the surface of a gel using a computer-
controlled pump.
By increasing the density of droplets from one line to the next
and allowing

the factor to diffuse into the gel, a smooth gradient was created.
(b,c) Concentration profile at various times in a block of gel 3.5
cm long by
1 mm deep generated by finite element modeling. The initial
pattern
consisted of ten equally spaced lines 1 mm apart. The quantity
of factor in
each subsequent line was increased by a constant factor of 1.22
in this
case, producing an exponential concentration gradient with a
percentage
change of 0.2% every 10 µm. (b) Profile through the vertical
thickness of
the collagen. (c) Profile along the length of the collagen (least-
concentrated
line at 1.5 cm). After the initial transients have died away, a
smooth profile
remains. Although the concentration at the high end decays, at
the low end
both the concentration and gradient steepness remain relatively
stable. The
time for stabilization and eventual decay of the gradient is
inversely
proportional to the diffusion coefficient. For gradients steeper
than 0.2%,
the concentration at the low end of the gradient increases slowly
over time
as a result of diffusion from the region of higher concentration,
and the
gradient flattens somewhat.
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strength = 0.010; P < 0.09, not significant). No significant
turning was
seen in other conditions. We conclude that these growth cones
are
unable to adapt their sensitivity to shallow gradients over a

concentra-
tion range of more than about two to three orders of magnitude.
DISCUSSION
The minimum concentration difference across a motile cell that
is
required for directional sensing is typically quoted as 1–2%
(refs.
23,24), although statistically significant responses for values as
low as
0.5% have been found for neutrophils6,7. Our results show that
axons
can be guided by gradients that are substantially shallower than
this.
This sensitivity is particularly remarkable considering the noisy
envi-
ronment in which the growth cone must operate. Statistical
fluctua-
tions in receptor binding limit the accuracy of any instantaneous
measurement of concentration25,26. For a growth cone with a
diame-
ter of 10 µm in a 0.1% gradient at 1 nM, the number of
molecules in
the vicinity of half of the growth cone is on the order of 1,000,
so the
average difference in the number of molecules between the
high- and
low-concentration sides of the growth cone is about one
molecule.
We also found a guidance ratio significantly different from zero
for
s = 0.2% at 0.1 nM, an average difference of about one-fifth of
a mol-
ecule across the growth cone. By contrast, the effects of
Brownian
motion will produce instantaneous fluctuations in the number of

molecules of approximately the square root of 1,000 or about 30
at
1 nM. Overcoming this noise requires averaging over many
independ-
ent measurements. A simple calculation indicates that growth
cones
would need to average concentration measurements for at least
sev-
eral minutes to achieve the sensitivity we have measured25,26,
but spe-
cific mechanisms by which this might be accomplished are
unknown.
Turning of neurites in the gradient at a concentration of 1 nM
was
not seen. We believe that we have, however, observed a
guidance effect
in this case because the degree of asymmetry in outgrowth from
the
explants is much larger than one would expect given the small
differ-
ence in NGF concentration between the two sides of the explant.
One
possibility is that turning predominately occurs before the
neurites
leave the explant. Neurite turning outside the explant was,
however,
strongly significant at 10 nM and more weakly significant at
100 nM.
Thus, surprisingly, measureable turning and biased outgrowth
are not
always correlated and seem to be related in a nontrivial way.
Dissociated neurons can also be used in our assay, which will
allow
the dependence of individual axon trajectories on gradient
steepness

to be probed more directly.
A recent study examining adaptation of gradient sensitivity of
Xenopus laevis spinal axons to external ligand concentrations27
meas-
ured the response of axons growing on a two-dimensional
substrate
to very steep gradients (5–10% over 10 µm) over a relatively
short
period of time (1–2 h). Adaptation was probed by introducing
rela-
tively small step changes in ligand concentration in the fluid
above the
growth cone. In contrast, we have examined mammalian axons
guided by very shallow gradients for long periods of time in a
three-
dimensional assay, more akin to the in vivo situation, and have
varied
ligand concentration at the growth cone over seven orders of
magni-
tude. We find a similar bell-shaped dependence on
concentration to
that previously measured for leukocytes5, which can be
explained the-
oretically by the effect of stochastic concentration
fluctuations28. As
the peak is expected to be roughly centered at the dissociation
con-
stant, Kd, this provides indirect evidence that the effective Kd
mediat-
ing guidance responses for the binding of NGF to its receptors
trkA
and p75 is about 1 nM (refs. 29,30). The relatively narrow
window of
concentration for effective guidance of axons indicates that

there
must be tight in vivo regulation of the absolute concentration of
guid-
ance factors present. Based on the less-quantitative
experimental
measurements of gradient sensitivity and the absolute
concentration
range that was previously available, we estimated the maximum
dis-
tance an axon could be guided by a single gradient of
exponential
shape to be about 1 cm (ref. 31). Results from our more
quantitative
assay now extend this estimate to about 2 cm.
Our results show the power of the technology we have
developed to
quickly and reliably produce molecular gradients that can
robustly
guide axons. The exquisite sensitivity of axonal gradient
detection
6 8 0 VOLUME 7 | NUMBER 6 | JUNE 2004 NATURE
NEUROSCIENCE
Figure 2 Fluorescence imaging of casein concentration
gradients.
(a) Concentration profile determined from fluorescence
intensity 1 h after
printing a pattern of lines of exponentially increasing strength
(s = 0.4%;
ten lines 2 cm wide and 1 mm apart). (b) Concentration profile
for a pattern
of lines of linearly increasing strength 1 h after printing (ten

lines 2 cm
wide and 1 mm apart). (c) Comparison of the gradient shown in
b with a
numerical simulation. Points, measured concentration along the
center of
the linear gradients; lines, simulated values; 3 h, red; 24 h,
blue.
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demonstrates that axons have developed a perhaps uniquely
effective
mechanism for integrating guidance cues. This sensitivity, and
its
variation with absolute concentration, provide important
constraints
for the in vitro control of axonal trajectories and therapies for
axonal
regeneration after injury, and for hypotheses about how ligand
gradi-
ents are transduced into signals for directed motion in growth
cones.
METHODS
Tissue preparation. DRGs were removed from the lumbar region
of P2 rat
pups, trimmed and washed in DMEM and enzymatically
digested for 12 min
in 0.25% trypsin/10 µg/ml DNAse1/Ca2+- and Mg2+-free Hanks
Balanced
Salt
Solution

to loosen the capsule. The reaction was stopped by addition of
FBS, and the explants were centrifuged and resuspended in
DMEM three
times. Explants were then equilibrated in 0.2% liquid collagen
at 4 °C before
being plated.
Dry collagen gels. A 0.2% collagen gel solution was prepared
under sterile
conditions by mixing on ice type I rat tail collagen stock
(Collaborative
Biomedical Products) diluted with water to contain 0.2 mg/ml
collagen, 27 µl
of a 7.5% sodium bicarbonate solution per ml of original
collagen stock, 10×
Optimem (Invitrogen) to a final concentration of 1×, 50 U
penicillin,
50 µg/ml streptomycin. We uniformly spread 750 µl of the
solution over the
bottom of a 35-mm dish and allowed it to set, forming the
bottom layer of
collagen (∼ 0.5 mm thick). We added a fresh layer of 750 µl of
collagen and
then arranged six explants in a line in the dish before the

collagen set
(Fig. 3g). The final collagen culture was approximately 1 mm
thick. The
dishes were returned to the incubator for 15 min for the top
layer of collagen
to set and were then ready for molecular printing. No fluid
culture medium
was added on top of the collagen, as this would provide a short
circuit for the
gradient; we therefore refer to these as dry gels. No serum was
added to the
collagen. Dishes were returned for 36–40 h to a 37 °C incubator
with 5% CO2.
We chose this time interval because it is typical of more
standard collagen gel
coculture experiments; it is unlikely that axons are exposed to a
single gradi-
ent for this length of time in vivo.
Fixation and immunohistochemistry. Explants were fixed by
covering the
collagen with 1.5 ml of 10% formaldehyde and 0.1% Triton X-
100 (J.T.
Baker) in PBS for several hours. Explants were washed five
times with PBS

for 1 h each and then incubated overnight in an antibody
directed against
neuronal β-tubulin (TUJ1; Babco; 1:1,000), followed by a
further five washes
in PBS for 1 h each. The explants were then incubated overnight
in the sec-
ondary antibody Alexafluor 488–conjugated goat anti-mouse
IgG
(Molecular Probes; 1:1,000), washed five times in PBS for 1 h
each and pho-
tographed with a CCD (charge-coupled device) camera on a
Nikon TE300
inverted fluorescence microscope.
NATURE NEUROSCIENCE VOLUME 7 | NUMBER 6 | JUNE
2004 6 8 1
Figure 3 Guidance of DRG axons by NGF gradients. (a)
Guidance ratio of DRG explants as a function of gradient
steepness. Error bars are s.e.m. For
s = 0.1%, the guidance ratios were significantly different from
zero (P < 10–8), and 80% of explants (43 of 54) had positive
guidance ratios. Responses for

s = 0.2% and 0.4% were also significantly different (P < 0.005).
For s = 0.4%, 53 of 54 explants had positive guidance ratios (54
explants per condition
pooled over three separate experiments; similar results were
seen in each experiment). (b) Outgrowth in response to NGF
after 36–40 h in culture, as
measured by the number of bright pixels representing neurites,
divided by the area of the explant (36 explants per condition
pooled over three separate
experiments). (c) Guidance as a function of absolute NGF
concentration at the explants for s = 0.2%. The probability that
the mean guidance ratio is zero
in each case is given above each data point (54–108 explants
per condition pooled over six separate experiments). Although
the 1 nM condition represents
equivalent conditions to the 0.2% condition in a, the guidance
ratios in these two cases are not exactly the same as they are
derived from a different series
of experiments. (d–f) Typical explants from experiment in a for
s = 0.1%, 0.2% and 0.4%, respectively (guidance ratios within
0.01, that is 5–10%, of the
mean). The NGF gradient points upwards. (g) Complete row of
explants for one dish from experiment in c, s = 0.2%, 10 nM.
Clear turning of the neurites is
apparent. Scale bars, 500 µm.©

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Molecular printing. For printing, we used a commercially
available pump
(Gesim) that can deliver precisely repeatable nanoliter droplets

at rates of up to
1,000 drops/s. A dish containing the gel was mounted on a
computer-con-
trolled commercially available high-precision x–y translation
stage. The spac-
ing between drops can be controlled by varying the rate of
droplet ejection and
the rate of translation of the x–y stage. Gradients were
established within the
middle 10 mm × 20 mm of a 35-mm culture dish in the manner
suggested in
Figure 1a. The gradient was positioned so that the line of
explants was between
the third and fourth line of the gradient. To produce an
exponential gradient
with steepness s per 10 µm, the number of drops in each
successive line
increased by a factor of e100s. This factor is 1, 1.11, 1.22 and
1.49 for steepnesses
s = 0, 0.1, 0.2 and 0.4%, respectively. The droplet volume was
measured to be 1.4
nl. For Figure 3a, the solution in the pump was 117 nM NGF
(Roche
Diagnostics) in PBS. The number of drops in the first line of the
gradient was

adjusted so that the concentration at the explant position was 1
nM after the
initial concentration variations smoothed out. The total volume
of fluid
deposited on each line for every condition was equalized by the
application of
the appropriate amount of the vehicle solution (PBS) after the
deposition of the
gradient of NGF solution. In addition, a 5-mm plateau of the
vehicle solution
was added adjacent to the low-concentration side of the
gradient. For Figure 3c,
procedures were the same, except that the concentration of NGF
in the pump
was varied to achieve the desired concentration at the explant
position.
Statistical analysis. Fluorescent images of TUJ1-stained
neurites were
processed using Scion Image (Scion Corporation) to outline the
explant. The
image containing neurites outside the explant was then
thresholded to give a
binary image. The total number of nonzero pixels, divided by
the number of

pixels contained in the explant, was used to calculate outgrowth
(Fig. 3b). The
guidance ratio R (Fig. 3a,c) was determined from the number of
nonzero pix-
els on the high-concentration side of the explant, H, and the
number on the
low-concentration side, L, according to R = (H – L)/(H + L).
Because this is
normalized by total neurite outgrowth, it is expected to be
relatively insensitive
to total outgrowth. We quantified turning by detecting neurite
segments in the
explant images using a ridge-tracking algorithm that is similar
to one pre-
sented previously32, although we developed ours independently.
The turning
vector associated with each neurite segment was calculated as
the difference
vector between a unit vector that is parallel to the segment and
the radial unit
vector measured from the explant center. The deflection of the
neurite up the
gradient was measured as the component of the turning vector
along the gra-
dient, and the average of these components across all explants

was defined as
the turning strength for that condition. Significance values
quoted for the
guidance ratio and turning strength give the probability that the
mean of the
distribution is zero, as determined by a t-test.
Measurement of diffusion coefficients. Using the micropump, a
single line of
the diffusant was delivered onto a 1-mm layer of collagen in a
35-mm petri
dish. The concentration profile along the length of the gel
perpendicular to the
line was measured by fluorescence imaging of either directly
labeled protein
(casein, Sigma; labeled with Oregon Green488 FluoReporter kit,
Molecular
Probes) or immunofluorescence labeling of fixed protein (NGF).
The narrow
line spreads out into a Gaussian concentration profile and the
width of the
Gaussian evolves linearly in time, with a slope determined by
D. The profile
measured at each time was fit to a Gaussian curve, and D was
determined from

a linear fit to the width versus time.
Finite element modeling. The simulations shown in Figures 1b,c
and 2c were
produced by two-dimensional finite element modeling
(PDEase2D, Macsyma
Inc.) of the diffusion equation in a domain of 3.5 cm × 0.1 cm,
with no flux
boundary conditions. The printing of the lines was modeled by a
constant flux
for a brief period (10 s) in narrow regions (0.01 cm) along one
of the long
edges of the domain.
ACKNOWLEDGMENTS
We thank J. Savich, R. Arevalo, S. Mittar, C. Fleury and J.
Torri for their
assistance with technology development. Supported by the
National Institutes
of Health, the National Science Foundation, the Department of
Defense and
the Whitaker Foundation.
COMPETING INTERESTS STATEMENT
The authors declare that they have no competing financial

interests.
Received 13 January; accepted 23 April 2004
Published online at http://www.nature.com/natureneuroscience/
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