RESEARCH ARTICLES

14. A. Dai, T. M. L. Wigley, B. A. Boville, J. T. Kiehl, L. E.

Buja, J. Clim. 14, 485 (2001).
15. D. V. Hoyt, K. H. Schatten, J. Geophys. Res. 98, 18895
(1993).
16. G. A. Meehl, W. M. Washington, T. M. L. Wigley, J. M.
Arblaster, A. Dai, J. Clim. 16, 426 (2003).
17. C. Ammann, G. A. Meehl, W. M. Washington, C. S.
Zender, Geophys. Res. Lett. 30, 1657 (2003) (DOI
10.1029/2003GL016875).
18. The G, A, and O forcings in the PCM experiments are
identical to those used in integrations performed
with the NCAR Climate System Model (13, 14). Total
solar irradiance changes were prescribed according to
Hoyt and Schatten (15) and updated as in (16), with
no wavelength dependence of the forcing. Volcanic
forcing was based on estimates of total sulfate loading and a simplied model of aerosol distribution and
decay (17).
19. E. Kalnay et al., Bull. Am. Meteorol. Soc. 77, 437
(1996).
20. J. K. Gibson et al., ECMWF Re-Analysis Project Reports
Series 1 (European Centre for Medium-Range Weather Forecasts, Reading, UK, 1997), 66 pp.
21. S. Pawson, M. Fiorino, Clim. Dyn. 15, 241 (1999).
22. B. D. Santer et al., J. Geophys. Res. 104, 6305 (1999).
23. World Meteorological Organization ( WMO), MeteorologyA Three-Dimensional Science: Second Session of the Commission for Aerology [WMO Bulletin
IV(4), WMO, Geneva, 1957], pp. 134 138.
24. T. Reichler, M. Dameris, R. Sausen, D. Nodorp, A
Global Climatology of the Tropopause Height Based
on ECMWF Analyses (Institut fur Physik der Atmosphare, Report 57, Deutsche Forschungsanstalt fur
Luft und Raumfahrt, Wessling, Germany, 1996).
25. We estimate pLRT from reanalysis and model data by
interpolation of the lapse rate in a p coordinate system, where p denotes pressure, R/cp, and R and cp
are the gas constant for dry air and the specic heat
capacity of dry air at constant pressure (24). The algorithm identies the threshold model level at which the
lapse rate falls below 2C/km and then remains less
than this critical value for a specied vertical distance
(23). The exact pressure at which the lapse rate attains
the critical value is determined by linear interpolation of
lapse rates in the layers immediately above and below
the threshold level. This denition of tropopause height
is robust under most conditions. Exceptions include
situations where the atmosphere is relatively isothermal or where multiple stable layers are present (24). To
avoid unrealistically high or low pLRT values, search
limits are restricted to pressure levels between roughly
550 and 75 hPa. Decadal-scale trends in pLRT are relatively insensitive to the temporal resolution of the input
temperature data (3, 6).
26. The NCEP pLRT calculation performed here used
monthly mean temperature data at 17 model pressure levels. A similar calculation used the 6-hourly
temperature data available at 28 model sigma levels,
with higher resolution in the vicinity of the tropical
tropopause (6).
27. C. A. Mears, M. C. Schabel, F. W. Wentz, J. Clim., in
press.
28. J. R. Christy, R. W. Spencer, W. D. Braswell, J. Atmos.
Ocean. Tech. 17, 1153 (2000).
29. The radiative emissions contributing to the T4 and T2
measurements peak at roughly 74 and 595 hPa,
respectively. In the tropics, emissions from the troposphere make a substantial contribution to estimated T4 temperatures.
30. B. D. Santer et al., Science 300, 1280 (2003).
31. The total linear change in T4 in NCEP is 1.65C over
1979 1999 (32). This is considerably larger than the
simulated T4 change of 0.73C in ALL or the estimated observed MSU T4 changes of 0.93C (27) and
1.07C (28). NCEPs excessive stratospheric cooling is
due to biases in both the satellite-derived temperature
retrievals and the assimilated radiosonde data (6, 21).
Because T2 receives a small (roughly 10% globally)
contribution from the stratosphere, any error in NCEPs
T4 trends will propagate into T2. This partly explains
why NCEPs troposphere cools markedly over 1979
1999, with a total linear change in T2 of 0.22C.
32. The total linear change is dened as b n, where b is
the slope parameter of the linear trend (in hPa per

33.

34.
35.

36.
37.

38.

month or C per month) tted by the standard leastsquares method over a specied period of n months.
For forcing component x, this is calculated as (x/y)
100, where x is the total linear change in pLRT over a
stipulated time interval (due to x) and y is the linear
change in pLRT (over the same interval) due to ALL.
This denition is not applicable if the linear change in
ALL is close to zero, a situation that never arises here.
V. Ramaswamy, M. D. Schwarzkopf, W. Randel, Nature 382, 616 (1996).
Because the O experiment does not separate tropospheric and stratospheric ozone changes, we cannot
quantify their individual contributions to overall
changes in pLRT. Historical increases in tropospheric
ozone are expected to warm T2, thereby increasing
tropopause height. Such warming is not evident in
the O integration (Fig. 2C) because of the cooling
effect of stratospheric ozone depletion on the upper
troposphere. Figure 2 indicates that in PCM, ozoneinduced increases in tropopause height arise primarily through stratospheric ozone depletion and the
resultant cooling of T4, not through tropospheric
ozone changes.
P. A. Stott et al., Science 290, 2133 (2000).
Linear changes in tropopause height over 1979 1999
are the sole exception: SUM results are 19% smaller
than in ALL. This difference is largely attributable to
noise differences. SUM is a linear combination of ve
climate responses plus ve different (ensemble-averaged) noise estimates, and must therefore be noisier
than ALL. ALL versus SUM noise differences are likely to
have the greatest impact on trends calculated over
relatively short periods.
K. Hasselmann, in Meteorology of Tropical Oceans,
D. B. Shaw, Ed. (Royal Meteorological Society of
London, London, UK, 1979), pp. 251259.

39. B. D. Santer et al., J. Geophys. Res. 100, 10693

(1995).
40. E. Roeckner, L. Bengtsson, J. Feichter, J. Lelieveld, H.
Rodhe, J. Clim. 12, 3004 (1999).
41. Two reanalyses (NCEP and ERA) combined with two
noise data sets for calculation of natural variability
statistics (PCM and ECHAM) yields four nonoptimized detection time estimates.
42. W. J. Randel, F. Wu, W. R. Rios, J. Geophys. Res. 108,
4024 (2003) (DOI 10.1029/2002JD002595).
43. Work at Lawrence Livermore National Laboratory
was performed under the auspices of the U.S. Department of Energy (DOE), Environmental Sciences Division, contract W-7405-ENG-48. T.M.L.W. was supported by the National Oceanic and Atmospheric
Administration Ofce of Global Programs (Climate
Change Data and Detection) grant no. NA87GP0105
and by DOE grant no. DE-FG02-98ER62601. A portion of this study was supported by the DOE Ofce of
Biological and Environmental Research, as part of its
Climate Change Prediction Program. The MSU
weighting functions were provided by J. Christy
(Univ. of Alabama in Huntsville). E. Roeckner (MaxPlanck Institut fur Meteorologie, Hamburg, Germany)
supplied ECHAM control run data. Review comments
from B. Hoskins and S. Pawson substantially improved the manuscript. We also thank R. Anthes, W.
Randel, and J. Bates for bringing GPS-based monitoring of tropopause height to our attention.
Supporting Online Material
www.sciencemag.org/cgi/content/full/301/5632/479/
DC1
SOM Text
Fig. S1
4 March 2003; accepted 30 May 2003

Dense Packing and Symmetry in

Small Clusters of Microspheres
Vinothan N. Manoharan,1 Mark T. Elsesser,1 David J. Pine1,2*
When small numbers of colloidal microspheres are attached to the surfaces of liquid
emulsion droplets, removing uid from the droplets leads to packings of spheres
that minimize the second moment of the mass distribution. The structures of the
packings range from sphere doublets, triangles, and tetrahedra to exotic polyhedra
not found in innite lattice packings, molecules, or minimumpotential energy
clusters. The emulsion system presents a route to produce new colloidal structures
and a means to study how different physical constraints affect symmetry in small
parcels of matter.
What defines an optimal packing of a set of n
identical spheres? Although centuries old,
this question remains both pertinent and pervasive in mathematics and science (1). For
packings of an infinite number of spheres, the
obvious measure of optimality is the bulk
density. As Kepler conjectured and Hales
proved (2, 3), the optimal infinite packing is
the face-centered cubic (fcc) arrangement,
which maximizes the density or, equivalently, minimizes the volume per sphere. But for
a finite group of spheres there is no compelling definition of density, and optimality in
1
Department of Chemical Engineering, 2Department
of Materials, University of California, Santa Barbara,
CA 93106, USA.

*To whom correspondence should be addressed. Email: pine@mrl.ucsb.edu

finite sphere packings can be defined by the

minimization of any physically reasonable
variable, such as potential energy or surface
area, for example. Different minimization criteria can lead to dramatically different sequences of packings (4), with symmetries
that are rarely consistent with that of an
infinite (bulk) packing.
In nature, differences in symmetry between finite and bulk packings manifest
themselves in a variety of phenomena. The
interactions between atoms or particles in
many systems, including metals, noble gases,
and colloids, are nearly spherically isotropic,
and the equilibrium structures at high density
are fcc, a consequence of Keplers conjecture. But finite packings of such particles, as
found in isolated clusters, complexes, or local
arrangements of bulk phases, frequently

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RESEARCH ARTICLES
adopt point symmetries that are inconsistent
with fcc order. For example, local polytetrahedral packings with fivefold symmetry are
energetically favored in undercooled metallic
liquids (57). The incompatibility of these
packings with long-range order inhibits crystal nucleation and induces the formation of
metastable phases (7). Similar arguments involving conflicting optimal packing criteria
at the local and bulk scales are central to
theories of glass formation (8) and jamming
(9, 10). A better understanding of how finite
groups of spheres organize themselves may
therefore help us decipher and control the
structure of matter at many different length
scales. To this end, a primary scientific challenge is to determine the minimization principle that governs the shapes of finite packings for a given set of physical constraints.
We show that under a compressive force,
small numbers (n 2 to 15) of hard spheres
pack into distinct and identical polyhedra for
each value of n. These configurations (clusters)
are related to a fundamental minimization principle that was only recently discovered within
the mathematics literature (11). The clusters are
formed in a three-phase colloidal system consisting of evaporating oil droplets suspended in
water, with n micrometer-sized polymer spheres
(microspheres) attached to the droplet surfaces.
In this system, capillary forces provide a compressive force that is spherically symmetric until
packing constraints break the symmetry. A key
feature of our approach is that for a given value
of n, the packing process is reproducible and can
be examined dynamically. In addition, we are
able to isolate clusters of a given n in macroscopic quantities, thus providing a new class of
nonspherical colloidal particles with which dif-

ferent packing and crystallization motifs can

be studied.
Packing process. The principal components of our packings are equal-sized, crosslinked polystyrene microspheres, 844 nm in
diameter [2.5% polydispersity (12)], with sulfate groups covalently bonded to the surface
(13). In pure water, the sulfate groups dissociate, charging the surfaces of the spheres and
preventing van der Waals attractions from
bringing them together. In an organic solvent
such as toluene, which is a good solvent for
polystyrene, the dissociation of sulfate
groups is limited, but the van der Waals
forces between the particles are much smaller
than in water (14). The particles swell with
the solvent and interact only through a shortranged steric (entropic) repulsion. To a good
approximation, they act as hard spheres (15).
To pack these spheres together, we use a
system that contains both liquids (13). We disperse the spheres in toluene (Fig. 1A), add water, and mix to create an oil-in-water emulsion
consisting of small droplets of toluene ranging
from 1 to 10 m in diameter (Fig. 1B). The
particles are strongly bound to the droplet interfaces by surface tension (16). We preferentially
evaporate the toluene from the system, forcing
the hard-spherelike particles in each droplet to
pack together (Fig. 1C). As Fig. 1D shows, the
critical feature of the evaporation process is a
mechanically stable intermediate stage called a
spherical packing, formed when the particles
touch one another on the surface of the droplet.
Removing more oil at this stage causes the
droplet to deform, generating capillary forces
that ultimately lead to a rapid (33 ms) rearrangement of the particles. When the last of the
toluene evaporates, the particles deswell, at

Fig. 1. Optical micrographs and diagrams (insets) of the packing process.

(A) Polymer particles swollen and dispersed in toluene (shown in blue).
(B) Emulsion of toluene droplets with particles, still swollen, bound to the
interfaces. (C) Clusters after toluene evaporation. These are stabilized
against further aggregation by the dissociation of charged groups on the
surfaces of the particles. (D) A time series of micrographs taken during

484

which point the interparticle van der Waals attractions increase and the particles stick to one
another, forming a small colloidal aggregate.
Capillary forces thus pack the particles into their
final configuration while they act as hard
spheres, and van der Waals forces subsequently
cement the spheres together. A side effect of this
packing method is that the surfaces of the particles on the outside of the cluster are exposed to
water at an earlier stage of evaporation. The
dissociation of surface charges on these outer
particles prevents the cluster as a whole from
aggregating with other clusters.
Geometry of microsphere packings. After all the toluene has evaporated, we are left
with a suspension of clusters of different sizes.
Each cluster comes from a single droplet containing n spheres, but n varies from cluster to
cluster because the initial droplets are not uniform in size. Centrifugation in a density gradient (13) allows us to separate the clusters on the
basis of differences in sedimentation velocity,
or average hydrodynamic radius (17). Although
all of our microspheres have the same mass, the
effective surface area and the sedimentation
velocity of a cluster of these particles vary
greatly with their configuration. The separation
yields a set of sharp, well-separated bands (Fig.
2), showing that only specific configurations
emerge from our packing process.
In fact, the separation reveals a remarkable result: Clusters of a given n are all
identical, as determined by extracting the
bands from the density gradient (insets, Fig.
2) and examining the clusters with the optical
microscope. The structures of the clusters are
shown in Fig. 3. If we treat the center of each
sphere as a vertex of a polyhedron, the first
few observed configurations are the line seg-

evaporation of the toluene, showing the evolution of the system between (B) and (C). Particles freely diffuse about the surface of the droplet
until, as more toluene evaporates, they touch one another, forming a
spherical packing (blue arrowhead). Deformation of the interface then
leads to a rapid rearrangement (orange arrowhead) to a cluster. The nal
conguration of this seven-sphere cluster is also shown in Fig. 3.

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RESEARCH ARTICLES
ment (n 2), triangle (n 3), tetrahedron
(n 4), triangular dipyramid (n 5), and
octahedron (n 6). These familiar structures
occur in many common molecules or coordination complexes. For example, these are the
packings of fluorine atoms in diatomic fluorine, boron trifluoride, carbon tetrafluoride,
phosphorus pentafluoride, and sulfur hexafluoride, respectively. The polyhedra are also
subelements (local packings) of the fcc lattice
or its stacking variants.
The larger clusters are an unusual and
perhaps unfamiliar set of packings. None of
these can be decomposed into combinations
of regular tetrahedra and octahedra, and
thus none is found in close-packed lattices.
Clusters containing 7 to 10 particles are
members of a set of highly symmetric polyhedra known as convex deltahedra (18),
some of which belong to noncrystallo-

Fig. 2. Photograph of a test tube containing a

cluster suspension separated by centrifugation
in a density gradient (left). Fourteen distinct
bands, each representing a region of high concentration of clusters, are visible in the tube,
showing that the cluster formation process
leads to specic, well-dened congurations of
particles. The number and sharpness of the
bands starkly contrast with those of fractally
aggregated clusters (30), where increasing
congurational variations prevent banding
for n 3. Each band shown contains clusters
of the same n, with n 1 at the top (see
electron micrographs at right). Each of the
upper bands contains between 108 and 1010
identical colloidally stable clusters, making
the separation process a useful method for
preparing macroscopic suspensions of particles with various shapes.

graphic point groups (see classifications in

Fig. 3) such as D5h (fivefold symmetry, n
7) and D4d (n 10). Bernal (19) observed
that these polyhedra cannot fill space, and
proposed that they might represent local
packings of atoms in liquid phases. Deltahedral geometry also occurs in certain boron hydride molecules (20), but this is a
result of the trivalent bonding of boron
rather than of a sphere-packing process.
Compared to the deltahedral packings, the
11-sphere cluster is particularly striking, as
it is the lowest-n cluster that is not convex.
It belongs to the low-symmetry point group Cs.
Atoms interacting through a spherically
symmetric Lennard-Jones potential form
clusters similar to ours up to n 7 (21), but
for n 7 Lennard-Jones clusters minimize
energy by maximizing the number of contacts
between particles, which leads to polytetrahedral packings. Although our seven-sphere
cluster can be viewed as an aggregate of five
distorted tetrahedra (22), the other clusters
are not polytetrahedral, and they generally
have higher symmetry than the LennardJones packings. Overall, the sequence of
sphere packings we obtain is distinct from
others observed in matter.
The unique structures of the packings for
each n suggest that they are optimal arrangements in the context of some minimization
principle. The simplest candidate for minimization is the potential energy. In our system,
though, the only attractive interactions are the
van der Waals forces that bind the particles
together. These short-ranged interactions are
insignificant compared to the thermal energy
when the distance between the sphere surfaces is greater than about 50 nm, given a Hamaker constant of 1020 J for the polystyrenewater-polystyrene system (23). Thus, if van
der Waals forces were responsible for the
clustering, we would expect the clusters to
adopt the structures of Lennard-Jones clusters. They do not, implying that our packings
are not a result of attractive forces.
The clusters do not minimize their volume
either, at least not for any simple definition of
volume. Minimizing the volume of the convex hull of a set of spheres leads to a sequence of clusters known as sausage packings, or linear arrangements of spheres for
n 56 (24, 25). Minimizing the volume of
the smallest spherical hull that encapsulates
the particles leads to a different set of packings as well (26).
Because attractions do not cause the clustering and because density cannot be precisely defined for our packings, we examine the
next simplest quantity to minimize: the second moment of the mass distribution
M 2 ni 1 ri r02, where ri is the center
coordinate of the ith sphere and r0 is the
center of mass of the cluster. Minimizing the
second moment is perhaps the most conve-

nient way to define density without resorting

to artificial definitions of cluster volume. Using global optimization techniques, Sloane et
al. (11) have calculated the packings that
minimize M2 (minimal-moment packings),
obtaining a sequence of clusters with unique
configurations for each n. These theoretical
configurations exactly correspond to those of
our packings (Fig. 3A). Although the minimization principle is quite fundamental, we
know of no observations of this type of packing in nature.
Relation between cluster packings and
spherical packings. In analyzing the mechanism by which the minimal-moment clusters
form, we find that the essential element is the
spherical packing: the shell of particles on the
surface of the droplet that causes the interface
to deform (stage three in Fig. 1D). After the
spherical packing forms, Brownian motion
no longer plays a role in the development of
the cluster, because capillary and contact
forces now move the particles. The final cluster configurations are thus determined solely
by the positions of particles in the spherical
packing. At this stage, there is a well-defined
density: the number of particles per unit of
surface area. We believe each n-sphere cluster is identical because each n-particle droplet
system must proceed through a spherical
packing that maximizes this density. Our
clusters may therefore represent collapsed
states of optimal spherical packings. In this
scenario, the second moment of the mass
distribution is a reasonable quantity to minimize, because the capillary forces acting on
the particles in the spherical packing are directed toward the center of the droplet.
The configurations of clusters with n 11
support the notion of clusters as collapsed
optimal spherical packings. The higher-order
clusters are not minimal-moment clusters
(Fig. 3B), although for n 12 and n 13
they differ only in the positions of one or two
spheres. Our 12-sphere cluster is a pure icosahedron, a configuration that is known to
have optimal spherical packing (27). Symmetry appears to prevent rearrangement in this
case, because all sphere positions are indistinguishable, so there is no weak spot to
initiate the collapse. However, when we dry
suspensions of icosahedra completely, we do
observe minimal-moment structures, likely
because the higher surface tension of airwater interfaces, compared to that of toluenewater interfaces, leads to much greater capillary forces.
The spherical packings for 13 spheres and
more do rearrange after oil evaporation, but
they do not minimize the second moment.
Compared to the lower-order clusters, a critical difference is that the minimal-moment
structures contain an internal sphere, or a
sphere coordinated fully by 12 others. In our
system, the particles in the spherical packing

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RESEARCH ARTICLES
Fig. 3. Cluster congurations at
each n, as determined by optical
and electron microscopy. Some
shrinkage and deformation of the
spheres occur during drying and exposure to the electron beam, but
the congurations of particles remain the same as those observed in
situ optically. (A) Left columns
show electron micrographs of the
clusters. All clusters of a given n
have identical structures. Right columns illustrate the polyhedra
formed by drawing lines from the
center of each particle to its neighbors. Below are the names of the
polyhedra [from (18)] and Schonies point groups. Middle columns
show computer renderings of
sphere congurations that minimize the second moment of the
mass distribution (11), which
match our packings. Not shown are
the line segment (n 2, Dh) and
the triangle (n 3, D3h). (B) Clusters with n 11 differ increasingly from minimal second moment
structures. We observe some small
variations in the packing from cluster to cluster for n 12.

are bound to a continuous and smooth surface, the droplet interface, and there does not
appear to be a route to an internal sphere
upon collapse. The second moment does,
however, decrease in the rearrangement, and
the configurations we observe are consistent
at each n, albeit with many more small variations in the packing than at lower n. Minimization of higher-order moments, defined
by M k ni 1 ri r0k, where k is any integer greater than 2, does not account for the
n 12 configurations. For these cases, Mk (at
least up to k 7) is smaller for the minimal
second moment packings than for the packings we observe. For n 12, minimization of
higher moments appears to favor structures
that are more isotropic, so that a spherical
packing of eight spheres has a lower M3 than
our eight-sphere cluster, and a spherical packing of 11 spheres has a lower M4 than our
11-sphere cluster. The second moment thus
remains the most appropriate measure of density. Minimization of the second moment
subject to a constraint (for example, that no
internal spheres may exist) might account for

486

all the cluster configurations we observe. In

any case, the configurations for n 11 are an
even more unusual set of packings, with a
notable lack of symmetry for the 13- and
14-sphere clusters, both of which are chiral,
and high symmetry for the 12-sphere (Ih point
group) and 15-sphere (D3h) clusters.
Colloidal dispersions of clusters. From
a technological perspective, the salient feature of our process is that it consistently
yields a specific set of structures with unique
and interesting symmetries. We can exploit
this feature using the separation procedure we
describe above. In the density gradient tube
shown in Fig. 2, there are billions (108 to
1010, or 0.1 to 10 mg) of each type of cluster.
Thus, by separating the bands, we can produce entire colloidal dispersions of sphere
doublets, tetrahedra, or any of the larger,
more exotic polyhedra in macroscopic quantities. These dispersions remain stable indefinitely, with no cluster breakup or intercluster
aggregation. Static light-scattering measurements on dilute cluster suspensions show excellent agreement with rigorous calculations

of scattering from idealized sphere packings

of the same symmetries (Fig. 4), indicating
that each cluster fraction is stable and well
separated. There are other methods for making colloidal clusters (28, 29), but because
these rely on adsorption onto patterned substrates, they have intrinsically small yields (105
clusters) and require that the packings be constructed in a layer-by-layer fashion, which cannot reproduce all the structures shown here.
Concluding remarks. Our observations
support the idea of the second moment of the
mass distribution as a simple, useful definition of optimal packing in certain colloidal
systems. This minimization principle may
provide some insights into the local processes
underlying the densification of bulk powder
slurries or wet granular materials. Beyond
these applications, though, lies a new role for
colloidal microspheres: as model systems for
studying packing itself. Optimal finite sphere
packings are still poorly understood mathematically and scientifically, and even our relatively simple system produces a sequence of
packings predicted only within the past dec-

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RESEARCH ARTICLES
18.
19.
20.
21.
22.
23.
24.
25.
26.
27.
28.

Fig. 4. Intensity of scattered light (488 nm) as

a function of scattering angle for dispersions of
the rst four clusters. Solid lines are calculations of the theoretical scattering from randomly oriented idealized packings of 844-nm
spheres (31). The data and curves are offset for
clarity. Curve 1, single spheres; 2, sphere doublets; 3, triangles; 4, tetrahedra. The agreement
between theory and experiment shows that the
clusters are well separated and stable.

ade (11). The varied and unusual polyhedra

of this sequence illustrate how certain symmetries, including fivefold rotational symmetry, can arise solely from compression and
packing constraints and need not require interparticle attractions. It may well be possible
to define other types of constraints in microsphere systems and in so doing to discover
new sphere-packing motifs.
References and Notes

1. J. H. Conway, N. J. A. Sloane, Sphere Packings, Lattices, and Groups (Springer, New York, ed. 3, 1999).
2. T. C. Hales, Discrete Comput. Geom. 17, 1 (1997).
3. T. C. Hales, Discrete Comput. Geom. 18, 135 (1997).
4. L. Fejes-To
th, Regular Figures (Macmillan, New York,
1964).
5. H. Reichert et al., Nature 408, 839 (2000).
6. T. Schenk, D. Holland-Moritz, V. Simonet, R. Bellissent, D. M. Herlach, Phys. Rev. Lett. 89, 075507
(2002).
7. K. F. Kelton et al., Phys. Rev. Lett. 90, 195504 (2003).
8. D. R. Nelson, F. Spaepen, Solid State Phys. 42, 1
(1989).
9. A. J. Liu, S. R. Nagel, Nature 396, 21 (1998).
10. C. S. OHern, S. A. Langer, A. J. Liu, S. R. Nagel, Phys.
Rev. Lett. 86, 111 (2001).
11. N. J. A. Sloane, R. H. Hardin, T. D. S. Duff, J. H.
Conway, Discrete Comput. Geom. 14, 237 (1995).
12. Polydispersity is dened as the relative standard deviation in the particle size distribution.
13. Materials and methods are available as supporting
material on Science Online.
14. B. R. Saunders, B. Vincent, Adv. Colloid Interface Sci.
80, 1 (1999).
15. M. S. Wolfe, C. Scopazzi, J. Colloid Interface Sci. 133,
265 (1989).
16. S. Levine, B. D. Bowen, S. J. Partridge, Colloids Surf.
38, 325 (1989).
17. R. Hinton, M. Dobrota, Density Gradient Centrifugation, vol. 6, part 1, of Laboratory Techniques in Bio-

chemistry and Molecular Biology (Elsevier/NorthHolland, New York, 1976).

N. W. Johnson, Can. J. Math. 18, 169 (1966).
J. D. Bernal, Nature 185, 68 (1960).
R. E. Williams, Inorg. Chem. 10, 210 (1971).
M. R. Hoare, P. Pal, Adv. Phys. 20, 161 (1971).
F. Spaepen, Nature 408, 781 (2000).
lvarez et al., Adv. Colloid Interface Sci.
R. Hidalgo-A
67, 1 (1996).
U. Betke, M. Henk, J. M. Wills, Discrete Comput.
Geom. 13, 297 (1995).
J. M. Wills, Math. Intell. 20, 16 (1998).
A. Boerdijk, Philips Res. Rep. 7, 303 (1952).
B. W. Clare, D. L. Kepert, Proc. R. Soc. London Ser. A
A405, 329 (1986).
J. Aizenberg, P. V. Braun, P. Wiltzius, Phys. Rev. Lett.
84, 2997 (2000).

29. Y. D. Yin, Y. N. Xia, Adv. Mater. 13, 267 (2001).

30. M. M. Takayasu, F. Galembeck, J. Colloid Interface Sci.
202, 84 (1998).
31. D. W. Mackowski, M. I. Mishchenko, J. Opt. Soc. Am.
A 13, 2266 (1996).
32. We thank V. Breedveld and D. Roux for helpful discussions. Supported by NSF under grant numbers
CTS-0221809 and DGE-9987618 and by Unilever
Corporation.
Supporting Online Material
www.sciencemag.org/cgi/content/full/301/5632/483/
DC1
Materials and Methods
References
28 April 2003; accepted 18 June 2003

Cell Therapy of -Sarcoglycan Null

Dystrophic Mice Through IntraArterial Delivery of Mesoangioblasts
Maurilio Sampaolesi,1 Yvan Torrente,2 Anna Innocenzi,1
Rossana Tonlorenzi,1 Giuseppe DAntona,3
M. Antonietta Pellegrino,3 Rita Barresi,4 Nereo Bresolin,2,5
M. Gabriella Cusella De Angelis,1,3 Kevin P. Campbell,4
Roberto Bottinelli,3 Giulio Cossu1,6,7*
Preclinical or clinical trials for muscular dystrophies have met with modest
success, mainly because of inefcient delivery of viral vectors or donor cells
to dystrophic muscles. We report here that intra-arterial delivery of wildtype mesoangioblasts, a class of vessel-associated stem cells, corrects morphologically and functionally the dystrophic phenotype of virtually all
downstream muscles in adult immunocompetent -sarcoglycan (-SG) null
mice, a model organism for limb-girdle muscular dystrophy. When mesoangioblasts isolated from juvenile dystrophic mice and transduced with a
lentiviral vector expressing -SG were injected into the femoral artery of
dystrophic mice, they reconstituted skeletal muscle in a manner similar to
that seen in wild-type cells. The success of this protocol was mainly due to
widespread distribution of donor stem cells through the capillary network,
a distinct advantage of this strategy over previous approaches.
There are currently three main experimental
approaches to therapy of muscular dystrophies (1). Gene therapy focuses on the development of new vectors capable of delivering
1
Stem Cell Research Institute, H. S. Raffaele, Via
Olgettina 58, 20132 Milan, Italy. 2Department of
Neurological Science, Istituto di Ricovero e Cura a
Carattere Scientico (IRCCS), Centro Dino Ferrari,
Ospedale Maggiore Policlinico, Via F. Sforza 35,
20122 Milan, Italy. 3Department of Experimental
Medicine, University of Pavia, Via Forlanini 6,
27100 Pavia, Italy. 4Howard Hughes Medical Institute, Department of Physiology, Biophysics and
Neurology, University of Iowa College of Medicine,
Iowa City, IA 52242, USA. 5IRCCS E. Medea, Bosisio
Parini, 23842 Lecco, Italy. 6Department of Histology and Medical Embryology, University of Rome La
Sapienza, Via A. Scarpa 14, 00161 Rome, Italy.
7
Institute of Cell Biology and Tissue Engineering,
San Raffaele Biomedical Science Park of Rome, Via
Castel Romano 100, 00128 Rome, Italy.

*To whom correspondence should be addressed. Email: cossu.giulio@hsr.it

efficiently the missing gene to the postmitotic

nuclei of the muscle fibers in vivo. The pharmacological approach aims to restore the protein complex that is altered in many forms of
muscular dystrophy through different strategies ranging from skipping mutated exons to
increasing the synthesis of cognate proteins
such as utrophin. The cell therapy approach
aims to functionally rescue the tissue by delivery of cells (2); these may be satellite cells
(which regenerate new fibers after damage to
the muscle) or pluripotent stem cells (which
have been shown to differentiate into skeletal muscle both in vitro and in vivo).
However, the limited self-renewal and migratory capacity of dystrophic satellite cells
and our modest knowledge of stem cell
biology have so far hampered the success
of the cell therapy approach.
We recently identified a class of vesselassociated fetal stem cells that can differ-

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