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ORIGINAL PAPER
Morphology of crystals in calcium oxalate monohydrate
kidney stones
S. Sandersius Æ P. Rez
Received: 6 December 2006 / Accepted: 6 September 2007 / Published online: 26 September 2007
Springer-Verlag 2007
Abstract Both scanning electron microscopy and atomic
force microscopy (AFM) have shown that calcium oxalate
monohydrate kidney stones are made up from arrange-
ments of sub micron crystals. The purpose of this
investigation was to determine the morphology of these
crystals which was obscured by the presence of organic
matrix in our earlier study. Sections of stones were treated
to remove the protein component of the matrix and then
imaged using AFM. Images obtained after proteolysis
show that the crystals are in the form of plates stacked on
(100) surfaces. These results were confirmed by scanning
electron microscopy observations from selected regions of
calcium oxalate kidney stone surfaces. The observed
crystal sizes are consistent with both the known matrix
mass fraction and crystallite growth in the passage through
the collecting duct.
Keywords Calcium oxalate Crystal morphology
Scanning electron microscopy Atomic force microscopy
Introduction
Kidney stone disease has become relatively common in
developed countries with an incidence of about 12% [1].
Most stones have calcium oxalate as their main component,
usually in its monohydrate form [2]. Calcium oxalate
dihydrate (COD), mineral name weddellite, can also be
found in mixed stones. It is easily recognizable as dis-
tinctive bipyramids corresponding to the (101) facets of the
tetragonal structure, space group I 4/m. The COD bipyra-
mids are usually found on the outside of a core of calcium
oxalate monohydrate (COM) [3]. Calcium oxalate mono-
hydrate, sometimes known by its mineral name whewellite,
has a monoclinic cell described by space group P2
1
/c.
Crystal structures have been given by both Tazzoli and
Domeneghetti [4] and Deganello [5] who used different
conventions to describe the unit cell, the (100) plane of
Tazzoli and Domeneghetti being the ð10
1Þ plane of De-
ganello. Tazzoli and Domeneghetti [4] also distinguish
between a high temperature and a low temperature form of
whewellite where the unit cell is doubled along [010] and
there are slight differences in rotations of the oxalate
group. When crystallized from solution COM crystals grow
in a plate-like morphology whose faces are (100) in Tazzoli
and Domeneghetti [4] notation, whose convention we fol-
low. The sides are bounded by (010) and (021) and (121)
facets, shown schematically in Fig. 1.
Pure COM stones usually are spherulitic in nature,
sometimes with multiple spherules joined together. Occa-
sionally a small dimple is seen at the point of attachment to
the renal papilla. Optical microscopy of stone sections
reveals concentric rings and radial striations reminiscent of
growth rings of a tree [6]. Sometimes dumbbell arrange-
ments are also observed. Kidney stones are composites
made up from the calcium oxalate mineral phase and an
organic matrix phase. At some level the mineral phase
must be organized as single crystals bounded by recog-
nizable facets that are usually low index crystallographic
planes under normal growth conditions. A characteristic of
single crystals is that they give spot diffraction patterns and
there is continuity of lattice planes from one side to the
other. This does not exclude inclusions, point defects, line
defects such as dislocations or planar defects such as
stacking faults. The single crystals can clump together in a
S. Sandersius P. Rez (&)
Department of Physics, Arizona State University,
Tempe, AZ 85287-1504, USA
e-mail: peter.rez@asu.edu
123
Urol Res (2007) 35:287–293
DOI 10.1007/s00240-007-0115-3
seemingly random arrangement, possibly with organic
matrix between them, to form a polycrystalline aggregate.
The size of these crystals [we sometimes refer to sub micron
crystals whose presence is only detectable by scanning
electron microscope (SEM), atomic force microscopy
(AFM) or high angle resolution synchrotron X-ray diffrac-
tion (XRD) as crystallites] is significant for stone formation
as it sets limits on where they could form, given a knowl-
edge of crystal growth rates and urinary transit times
through various parts of the nephron. High-resolution
scanning electron microscopy and AFM showed that the
fundamental single crystal sizes were less than 1 lm and
they appeared to be stacked together as a compact aggregate
[7]. Similar results were also found in TEM of demineral-
ized stones induced in rats [8] and SEM studies of
demineralized human stones [9]. However, the expected
crystal facets for single crystals, as seen in crystals grown
from solution, were not observed.
We hypothesized that remnants of organic matrix pre-
vented the imaging of the true single crystal morphology.
The matrix accounts for about 2–3% of the weight of the
stone [10, 11] and is reported to be about 70% protein, 20%
lipid and 10% other organic macromolecules [12]. How-
ever, later work suggested that lipid might account for a
higher proportion of matrix in calcium oxalate stones [13].
If the stone consists of single crystals of COM surrounded
by matrix macromolecules, then removing a major com-
ponent of the matrix by proteolysis should help reveal the
true morphology of the single crystals making up the
mineral phase. The aim of this investigation is to identify
single crystal mineral components and relate them to pos-
sible stone growth mechanisms.
Materials and methods
Seven COM stones no larger than 2 mm were selected
based on XRD analysis. A small part of the stone was
scraped away and crushed for powder diffractometry in a
Rigaku D/MAX-IIB diffractometer. Stones that showed
only COM peaks (a conservative estimate would put the
presence of other constituents at less than 5%) were
selected for further analysis. The stones were mounted in
capsules in epoxy and polished using a succession of
grinding powders to ensure a flat surface, following the
procedure outlined in Shaapur et al. [14]. We need to polish
the stones so that they are smooth enough for AFM
examination. It is certainly a concern that the procedure
alters the morphology of large single crystals. For this
reason we have also examined exterior surfaces and, more
recently, surfaces after fracture under liquid N
2
by SEM.
The fact that the general morphologies are the same gives
us confidence that our procedure does not bring about gross
morphological changes.
The proteolysis was performed following the procedure
of Ryall et al. [15] described in their study of intracrys-
talline protein content of COM and COD crystals. A
12.5 mM Tris solution was prepared and HCl added to
make the pH 6.0, typical of urine. Five units of Cathepsin
D (Sigma) were added to 500 ll of distilled water. This
was divided into five aliquots of 100 ll. To achieve a final
dilution of 1 U per 200 ll, 100 ll of Tris buffer was added
to each of these aliquots. The stones were immersed in the
buffer and incubated for 3 days at 37C. They were then
removed from the solution, mounted on stubs and exam-
ined in a Digital Instruments Nanoscope II AFM. A 50·
optical microscope was used to search for suitable flat
regions and initial survey scans were taken as 256 · 256
pixel images of regions between 50 and 100 lm across.
Higher resolution images were taken with 512 · 512 pixels
from regions approximately 1 · 1 lm.
Two other COM stones whose identification was
checked by XRD were mounted in the as-received state on
aluminum stubs and the surfaces were examined using
3.0 kV beam in a Hitachi S 550 field emission SEM.
Previous investigations [7] have shown that there is mini-
mal charging of calcium oxalate at this accelerating voltage
since the incident beam current is balanced by the com-
bination of backscattered and secondary electron current.
There was therefore no need to deposit a conductive
coating on the specimens. Angles were measured from both
AFM and SEM images using the ImageJ image analysis
program [16].
Fig. 1 Calcium oxalate morphology showing prominent facets
indexed according to Tazzoli and Domeneghetti [4]
288 Urol Res (2007) 35:287–293
123
Results
Figure 2 is a typical AFM image from a stone section
surface. It shows particles ranging in size from 50 to
160 nm. After proteolysis the crystallite shapes became
more visible and appeared to have a plate-like morphology
as seen in Fig. 3. In these images it is still not possible to
distinguish individual facets since the crystals are oriented
almost perpendicular to the surface. In Fig. 4 there appears
to be a stacked arrangement of flat crystals. The boundaries
of the facets are not very distinct though those marked as A
and B were measured as 103 and 106, respectively, close
to the angle between (021) and ð0
21Þ faces, while C and D
were measured as 125 and 121, respectively, approxi-
mately the same as the 121 angle between (010) and ð12
1Þ
planes. SEM images (Fig. 5) of exposed surfaces show
rough plate-like morphology, with serrated edges very
similar to those observed by Ryall et al. [17] after prote-
olysis of COM crystals grown in urine. In Fig. 6a, which is
also an SEM image of a surface region, the crystals again
appear to be stacked. The obtuse angle between the edges
of the plates is approximately 120. To measure the dihe-
dral angles between planes more precisely the specimen
was tilted as shown in Fig. 6b. The measurements are
summarized in Table 1 and the mean is 119 ±2. This is
close to 121, the angle between (010) and ð12
1Þ type
planes, though it is not that different from 116, the angle
between ð12
1Þ and ð1
2
1Þ planes. A similar micrograph has
been published by Holden [18] on the Herring web site.
Although the size of the plates varied between about 0.5
and 1.5 lm for different specimens, the stacking was a
common feature of all observations.
Low resolution SEM and AFM images, given in Fig. 7
,
show that crystals are only stacked in the same orientation
over relatively small regions, about 5 lm across. Small
differences in crystal size or shape allow for changes in the
relative orientation of arrays of plates. Any XRD experi-
ment would therefore sample many random orientations
which is why COM stones give polycrystalline diffraction
patterns.
Fig. 2 AFM image of the surface of a COM stone section, bar is
1 lm
Fig. 3 AFM image of COM stone section surfaces after proteolysis,
bar is 1 lm
Fig. 4 AFM image showing COM crystal morphology in a stone
section after proteolysis, bar is 1 lm. Angles A is 103 and B is 106
close to the angle between (021) and ð0
21Þ faces, while C is 125 and
D is 121, approximately the same as the 121 between (010) and
ð12
1Þ planes. The angle measurement error is approximately 3
Urol Res (2007) 35:287–293 289
123
Discussion
Both the AFM images after proteolysis and the SEM
images of selected surface region suggest that COM stones
are formed from the aggregation of stacked plate-like sin-
gle crystals. If growth in the kidney is similar to crystal
growth from solution or nucleation of crystals on Langmuir
Blodgett films [19–21] the face on which stacking occurs is
(100), as this is the largest face and the general morphology
supports this conclusion. The crystals appear to be about
1 lm across, which is larger than the objects identified as
possible crystals in our earlier study [7]. It is possible,
especially for the surface observations, that there is a bias
towards larger sizes as these would be more prominent.
Alternatively the earlier observations could have mistak-
enly identified some of the serrations observed at the plate
edges (see Fig. 5) as separate crystals. The crystallite sizes
are the same as those derived from the peak widths in
synchrotron XRD studies of COM crystals grown in ul-
trafiltered urine and various solutions of aspartic and
glutamic acids and their dimers [22].
The stacking on (100) planes is consistent with the
observations of Sheng et al. [23] who investigated the
forces between various molecular groups and COM sur-
faces by linking molecules to AFM tips with the relevant
group exposed. They showed greater adhesion of carbox-
ylate and amidinium groups on COM (100) which would
imply that macromolecules have a positive role in
enhancing adhesion.
Fig. 5 SEM image showing plate-like crystals on COM stone
surface, bar is 0.5 lm
Fig. 6 a SEM image showing COM crystal morphology on a stone
surface, bar is 1.0 lm. Angles close to 120 are marked. b The same
region tilted for more accurate measurement of angles a, b, c, d, and e.
Values are given in Table 1. Bar is again 1.0 lm
Table 1 Angles marked in Fig. 6b
Angle (degrees)
a 116.8
b 121.2
c 121.7
d 117.8
e 119.2
Fig. 7 a AFM image from section after proteolysis, bar is 2 lm, and
b SEM image from surface, bar is 5 lm, showing stacking
290 Urol Res (2007) 35:287–293
123
The crystal size can be estimated for various cases
where a monolayer of an organic macromolecule surrounds
the crystal, as proposed by Leal and Finalyson [24]. For
simplicity consider a cuboid crystal, dimensions a, b and d,
where d is the shortest dimension. The volume of organic
macromolecule is
V
org
¼ 2 ab þad þ bdðÞl ð1Þ
where l is the monolayer thickness. The volume fraction
f
vol
of organic material is
f
vol
¼ 2
ab þ ad þ bdðÞl
abd
¼ 2
1
d
þ
1
b
þ
1
a
l ð2Þ
and the mass fraction f
m
is
f
m
¼ 2
1
d
þ
1
b
þ
1
a
l
q
prot
q
ox
ð3Þ
where q
prot
and q
ox
are the densities of protein (1.3 gm/cc)
and COM (2.2 gm/cc), respectively. The mass fraction can
be set equal to 0.03, the observed average macroscopic
weight fraction of matrix in COM stones. There are two
limiting cases, the first when the crystallite is almost cubic
and b = a = d. The mass fraction is, which is proportional
to the ratio of surface area to volume, is
f
m
¼
6l
d
q
prot
q
ox
ð4Þ
and the crystal size, d,is
d ¼
6l
0:03
1:3
2:2
ð5Þ
This is the size estimate given in the third column of
Table 2 and is shown schematically as Fig. 8a. It is
interesting to note that the expression for the mass fraction
would be the same if it were assumed that the crystallites
were spheres coated with one monolayer, since the ratio of
surface area to volume is identical. In this case the mass
fraction of organic matrix is
f
m
¼
4p
d
2
2
l
4p
3
d
2
3
q
prot
q
ox
¼
6l
d
q
prot
q
ox
ð6Þ
As it is likely that the interstices between the spheres will
be filled with organic material the mass fraction of organic
material in practice would be 0:363
q
prot
q
ox
for identical
close packed spheres, independent of the size of spheres
when their diameters are much larger than a monolayer.
The other limiting case, which is probably closer to the
observations, is shown in Fig. 8b and assumes that the
crystallites are thin plates such that a or b are much greater
than the thickness d. The organic matrix mass fraction
becomes
f
m
¼
2l
d
q
prot
q
ox
ð7Þ
However, this derivation assumes a stacking of one
monolayer associated with each side of the plate, so that
there are two macromolecule layers between each
crystallite. If there were only one macromolecule layer
between continuous plates, the mass fraction would be
f
m
¼
l
d
q
prot
q
ox
ð8Þ
which leads to a plate thickness d
d ¼
l
0:03
1:3
2:2
ð9Þ
If the plates were not continuous and there were regions of
organic matter between them, total area a, as shown
schematically in Fig. 8a then Eq. 8 would be modified as
f
m
¼
l
d
q
prot
q
ox
1 þ
a
A
ð10Þ
where A is the plate area. From micrographs such as Fig. 6
the area between plates is small, probably less than 10% of
the crystallite area, so f
m
is only marginally bigger than the
value given by Eq. 8. Furthermore Eq. 8 still defines the
limiting case.
Estimates for various macromolecules between the
plates in this case are given in the fourth column of
Table 2. The third and fourth column of Table 2 can be
viewed as size bounds for a crystallite in the limiting case
of the organic matrix being present as a single molecule
layer. The observations of plate thickness are between 0.2
and 0.5 lm (see Fig. 7) which implies that there is more
than one monolayer even for the large macromolecules.
A possible mechanism for stone formation would be
heterogeneous nucleation of the crystals in the nephron,
with subsequent aggregation/agglomeration on a favored
site such as the renal papilla. This is in some ways similar
to a model suggested by Vermeulen [25] who postulated
that growth occurs by agglomeration around a crystal
blocking a duct of Bellini on the renal papilla. The crystal
sizes would then also be consistent with what is known
about crystal growth rates. Kok and Khan [26] and Fin-
layson and Reid [27] give growth rates for COM as 1–
2 lm/min while Werness [28] gives a lower estimate of
0.14 lm/min. According to Kok and Khan [26] the transit
time through the nephron is 3–4 min and 30–45 s through
the collecting duct. If the higher growth rate is to be
believed, crystals nucleating throughout the nephron could
Urol Res (2007) 35:287–293 291
123
have sizes up to 3–8 lm, larger than what we have
observed. For the lower growth rate the size would be 0.4–
0.6 lm. It is entirely plausible that crystal nucleation only
takes place where calcium oxalate reaches its urinary super
saturation in the collection duct after water resorption. In
this case the higher growth rate would imply crystal sizes
approximately 0.5–1.5 lm, in agreement with our obser-
vations. The range of sizes from the lower growth rate in
the collecting duct would be 0.07–1 lm which is smaller
than what we observed. It is therefore plausible, if the
higher growth rate is correct, that calcium oxalate kidney
stones form by the disordered stacking of plate-like single
crystals of COM that nucleate and grow in the collecting
duct.
Acknowledgments We should like to acknowledge funding from
the College of Liberal Arts and Sciences, Arizona State University
and the use of facilities in the Center for Solid State Science and the
Center for Solid State Electronics Research. We would also like to
thank Magali Chauvet and Dr. Rosemary Ryall for making available
details of their proteolysis protocol. Helpful discussions with Dr.
J. Wesson are also acknowledged.
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Macromolecule
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Surrounded crystal size
(see Fig. 8a and text) (lm)
Slab thickness
(see Fig. 8b and text) (lm)
10 kDa protein 2.7 0.32 0.053
Phospholipid or 30 kDa protein 4 0.47 0.079
100 kDa protein 6 0.71 0.120
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