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Mitogenic effects of hydroxyapatite and calcium pyrophosphate dihydrate crystals on cultured mammalian cells.

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668
MITOGENIC EFFECTS OF HYDROXYAPATITE AND
CALCIUM PYROPHOSPHATE DIHYDRATE CRYSTALS
ON CULTURED MAMMALIAN CELLS
HERMAN S. CHEUNG. MICHAEL T. STORY, and DANIEL J. McCARTY
Synthetic hydroxyapatite (HA) crystals in 1 %
serum stimulated 'H thymidine uptake into quiescent
canine synovial fibroblasts and human foreskin fibroblast cultures, as did 10% serum. The onset of stimulation and peak uptake of thymidine after crystal addition
were delayed by 2-3 hours as compared with the effects
produced by 10% serum. Stimulation of 'H thymidine
uptake was proportional to the serum concentration
used. HA crystals (50 pglml) stimulated nuclide uptake
at each serum concentration used. 3H thymidine uptake
was also proportional to the dose of HA or calcium
pyrophosphate dihydrate crystals, although larger doses
of the latter crystal were required to produce equivalent
effects. Not all particulates were effective mitogenic
agents. Latex beads and diamond crystals had no effect.
Monosodium urate crystals modestly stimulated and
calcium urate crystals markedly stimulated nuclide uptake. The more complex crystals found in a naturally
occurring condition (calcinosis) were as mitogenic as the
pure synthetic HA. The synovial cell hyperplasia sometimes associated with crystals might be explained in part
by their mitogenic activity.
-
From the Division of Rheumatology, Department of Medicine and the Department of Surgery, Medical College of Wisconsin,
Milwaukee.
Supported by USPHS grants AM26062 and AM18074,
RCDA award AM1065, and a grant from the Kroc Foundation.
Herman S. Cheung, PhD: Associate Professor of Medicine,
Biochemistry and Orthopedic Surgery, and recipient of a Research
Career Development Award; Michael T. Story, PhD: Assistant
Professor of Surgery and Biochemistry; Daniel J. McCarty, MD:
Professor and Chairman, Department of Medicine.
Address reprint requests to Herman S. Cheung, PhD,
Division of Kheumatology, Department of Medicine, Medical College of Wisconsin, 8700 West Wisconsin Avenue, Milwaukee, WI
53226.
Submitted for publication April 21, 1983; accepted in revised form January 26, 1984.
Arthritis and Rheumatism, Vol. 27, No. 6 (June 1984)
Synovial thickening is a common clinical finding in the affected joints of patients with calcium
pyrophosphate dihydrate (CPPD) crystal deposition
(1). Such thickening can resemble that seen in rheumatoid arthritis. Histologically, synovial cell proliferation
and infiltration by mononuclear inflammatory cells are
seen (1,2). CPPD crystals are phagocytosed by synovia1 lining cells (2). Clearance of radiolabeled synthetic
CPPD from rabbit joints appears to occur entirely by
this route, i.e., all crystal dissolution occurs within
synovial cells (3,4).
We have recently described a condition characterized by microspheroidal clumps of apatite crystals,
activated collagenase, and neutral protease activities
in nearly acellular fluids from patients with shoulder
joint degeneration (5,6). Clumps of crystals were also
found in synovial lining cells in a hyperplastic synovium (7). Addition of either synthetic CPPD or hydroxyapatite crystals to canine and human synovial
cells in tissue culture induced the release of collagenase and neutral protease (8), as had been shown
previously. by Werb and Reynolds using latex beads
and other particulates (9). During these experiments, it
was noted that cell cultures exposed to apatite crystals
consistently contained more cells than did control
cultures, suggesting that these particles were mitogenic .
Data are presented here in support of a mitogenic effect of hydroxyapatite, CPPD and, to a lesser
extent, urate crystals.
MATERIALS AND METHODS
All reagents were prepared in doubly deionized,
distilled, charcoal-filtered water.
MITOGENIC EFFECTS OF HA AND CPPD
Cell cultures. Synovial cells were obtained from stifle joints of normal, anesthetized mongrel dogs by the
method of Clarris et a1 (10). Briefly, the joints were aspirated
and rinsed with physiological saline, and fibrin and free cells
removed with a syringe. Ten ml of saline containing 20 mg
trypsin (Worthington Biochemicals, Freehold, NJ) was introduced into the joints and left in situ for 20 minutes. The
cell suspension was withdrawn and collected by centrifugation. Isolated cells included varying populations of macrophage-like and fibroblast-like cells.
About los cells were inoculated into 2 100-mm plastic
petri dishes (Scientific Products, McGaw Park, IL) and
grown to confluence in Dulbecco's modified Eagle's medium
(DMEM) supplemented with 10% horse serum and containing 1% penicillin, streptomycin, and amphotericin B (all
from Gibco, Grand Island, NY). Cells were then released
with trypsin and transferred to culture dishes containing 24
wells, each 15 mm in diameter and containing 1 ml medium
(Multiwell, Gibco). Secondary cultures which consisted entirely of fibroblast-like cells were fed once as above, reaching approximately 80% confluence in 4 days. Cells were then
rendered quiescent by removing the medium, washing once
with 1 ml of DMEM containing 1% horse serum, and
subsequent incubation in this medium. After 24 hours, the
various particulates were added. Unless otherwise specified.
each experiment was run in quadruplicate.
Human foreskin fibroblast cultures were established
from explants according to a previously described procedure
(11). They were grown and maintained in DMEM supplemented with 10% fetal calf serum containing 1% penicillin,
streptomycin, and amphotericin B. All cultures used were
the fifth passage cells. They were transferred to Multiwell
culture dishes and rendered quiescent in a procedure identical to that used with the dog synovial fibroblasts.
669
Particulates. Synthetic h ydroxyapatite (HA) crystals
were obtained as a gift from Dr. M. D. Francis of the Proctor
and Gamble Co., Cincinnati, OH. These had a molar C d P
ratio of 1.67. X-ray powder diffraction was kindly performed
by Dr. Neil S. Mandel of the Department of Medicine,
Medical College of Wisconsin, and showed interplanar spacings of hydroxyapatite (1 2).
High-resolution infrared spectrophotometry (kindly
performed by J. R. Lehr of the National Fertilizer Center,
Muscle Shoals, AL) showed absorption bands characteristic
of hydroxyapatite with about 1% carbonate substitution (12).
Triclinic calcium pyrophosphate dihydrate was synthesized
and characterized as previously described (3,4). Monosodium urate monohydrate (MSU) was prepared from twicerecrystallized uric acid (Sigma Co., St. Louis, MO) and
sodium hydroxide as previously described (13). X-ray diffraction showed only the interplanar spacings of MSU. Two
preparations were used. Preparation A consisted of MSU
crystals stored in suspension since 1964. These were 1-5 pm
in length. Preparation B consisted of a fresh preparation of
MSU crystals sonicated (Bransonic 200, Scientific Products)
for 2-5 minutes to produce crystals 5-30 pm long.
Calcium urate (CaU) was synthesized from recrystallized uric acid and calcium hydroxide. The molar uric acid/
calcium ratio of the product was 4/3 as calculated from
spectrophotometric measurements of uric acid by a uricase
method (14) and of calcium by atomic adsorption spectrophotometry. The same molar ratio and identical x-ray
diffraction powder diagrams were obtained in crystals prepared under neutral or alkaline conditions and despite variation of the starting ratio from 2/1 to 1 4 . These crystals could
not be distinguished from MSU by compensated polarized
light microscopy (15). They were strongly negatively birefringent and showed axial extinction. They were 3.3 times
I
B
35i
301
25-
0
x
E, 200
Q)
3
E
2
1510-
I
c)
5-
6
Ib
T
1;
20
TIME (Hours)
A
I
1-
i5
30
35
I
5
I
10
15
20 25
30
TIME (Hours)
35
40
45
B
Figure 1. A, Uptake of 'H thymidine by canine synovial fibroblasts as a function of time after addition of 10% horse serum (A-A)
or 100 pg
hydroxyapatite crystals suspended in I% horse serum (x-x).
Control is 1% horse serum (C-0). B, Uptake of 'H thymidine by human
or 100 pg hydroxyapatite suspended in 1% fetal calf seforeskin fibroblasts as a function of time after addition of 10% fetal calf serum (A-A)
rum (x-x).
Control is 1% fetal calf serum (0-0). All values mean t SEM; n - 4.
CHEUNG ET AL
670
more soluble than MSU in DMEM after equilibration for 24
hours at 37°C with excess crystals (28.5 mg/dl versus 8.75
mg/dl). These were sonicated to 5-30 pm as described
above.
Diamond crystals (1-5 pm long) were obtained from
A. Landau Co., Philadelphia, PA. Latex beads, 0.5 pm
in diameter, were obtained from Dow Chemical Co., Indianapolis, IN as a suspension. One ml contained 140 mg of beads.
A milky suspension of crystals obtained from subcutaneous
tissues of a girl with dermatomyositis/calcinosis was concentrated by centrifugation at 1,OOOg for 30 minutes, washed
twice with distilled water, once with 95% alcohol and once
with acetone, and air-dried. Fourier Transform Infrared
Analysis (12) showed this material to be a mixture of
partially carbonate-substituted hydroxyapatite and octacalcium phosphate.
Treatment of particulates. The clumps of synthetic
HA and single CPPD crystals were sieved to desired sizes
after gentle crushing in an agate mortar (Endecott test
sieves, London, England). HA crystal aggregates, 10-20
pm, were used throughout. Aliquants of HA, CPPD, diamond, MSU, and CaU were heated to 180°C for 2 hours to
destroy possible pyrogenic contamination. Mandel has demonstrated that a phase change, presumably water loss,
occurs when urate crystals are heated, but only at 187°C or
hotter (16). Five mg of the calcific material from the patient
was added to 1 ml DMEM containing 2 mg trypsin (Worthington Biochemicals) and incubated for 1 hour at 37°C.
After centrifugation and washing with DMEM, 2.5 mg of
calcified material was heated 3 s described above.
16h
40h
64h
112h
?
9x
T’
E
0
u
.r
t
5
I
0
100
4
TC
Figure 3. The duration of mitogenic stimulation of hydroxyapatite
crystals (100 d w e l l ) in I% serum was compared with serum alone
by determination of ’H thymidine uptake into washed cells at 16.40,
64, and 112 hours. Cell protein per well was determined at the same
time. All values mean 2 SEM. $ = P < 0.001 versus control; n
values are denoted in histogram.
HS(*/o)
HAorg/ml)
I
1 2 2
0 50 0
1 0 1 0
50
0 50
Figure 2. Uptake of ’H thymidine into washed cells 16 hours after
addition of serum with or without hydroxyapatite (HA) crystals. All
values mean ? SEM; n = 4. P < 0.05 versus control horse serum
(HS)at I%, 2%, and 10%. Control uptake = 3.2 2 0.1 x lo-’ cpm/
well.
Determination of time course and level of uptake. 3H
thymidine (5 pCi, specific activity 30 Ci/mmole; New England Nuclear, Boston, MA) was added to wells at various
intervals after addition of 100 pg HA crystals in 1% serum or
after addition of 10% serum, to determine the time of peak
uptake into cellular DNA. After pulse labeling for 60 minutes, the cells were washed 3 times with Hanks’ balanced
salt solution (HBSS), released with 2 ml of 20 mM EDTA,
and precipitated with an equal volume of 20% trichloroacetic
acid solution. The precipitate was collected by centrifugation and dissolved in 2 ml of 0. IN NaOH. Cell-associated ’H
content was determined in duplicate in a liquid scintillation
counter (Packard Instruments, Downer’s Grove, IL). Protein content was determined using the method of Lowry et a1
(17). Both canine synovial fibroblasts and human foreskin
fibroblasts were tested in subsequent experiments; only data
from canine synovial fibroblasts are reported here.
The effect of 3H thymidine uptake with increasing
concentrations of horse serum (1, 2 , and 10%) with and
MITOGENIC EFFECTS OF HA AND CPPD
5001
.+-
67 1
addition, 6 wells of each set were harvested and cells were
released with 20 mM EDTA. Cell counts were done with a
hemocytometer and cell protein was determined by Lowry
assay (17).
Statistical analysis. Significance was calculated using
Student’s I-test (18).
0
$ 300
RESULTS
Y
0)
C
.-
E 200
)r
5
2 loo
n
Y
Figure 4. Effect of varying doses of hydroxyapatite (HA) crystals
on uptake of ’H thymidine into washed cells at 16 hours. All values
mean 2 SEM. $ = P < 0.001 versus control horse serum (HS).
Control uptake = 3.9 t 0.2 x lo-.’ cpm/well; n values are denoted in
histogram.
without 50 pg/ml HA crystals was determined. Synovial
fibroblasts were pulsed for 1 hour with 3H thymidine 16
hours after addition of serum or serum plus crystals in this
and all subsequent experiments.
Series of wells were run in parallel after addition of
50 pg/ml HA crystals in DMEM with I% horse serum. Cells
were pulsed with 3H thymidine for 1 hour after 16, 40, 64,
and 112 hours. Uptake of label and cell protein per well was
determined as before.
Dose-response measurements. The effects of increasing doses of HA crystals from 10-100 pdml and of CPPD
crystals (10-20 pm) from 10-400 pg/ml were examined. The
effects of larger CPPD crystals (>50 p n ) in doses of 100-400
&ml were also studied. MSU crystals (preparation A) were
used in doses of 10-820 pg/ml; diamond crystals were added
in doses ranging from 100-400 Fg/ml; latex beads were used
in amounts rafiging from 29-466 pg/ml.
Determination of effects of calcific material from a
patient with calcinosis. The effect on 3H thymidine uptake of
adding the hydroxyapatite-octacalcium phosphate containing material (either heated, unheated, or trypsin-treated) to
quiescent synovial cell cultures was examined.
To determine the effect on cell number and protein,
50,000 synovial fibroblasts were seeded per well of 2
Multiwell growth dishes in the presence of 10% serum. After
24 hours incubation, the wells were drained and rinsed twice
with DMEM to remove unattached cells and residual serum.
They were then divided into 4 sets of 12 wells.
Of the 2 control sets (sets 1 and 2). 1 received DMEM
supplemented with 1% serum and the other received the
same media with 10% serum. DMEM with 1% serum containing 100 pg/ml HA and identical media but with 100 pg/ml
patient’s calcified material were added to wells of sets 3 and
4, respectively. On the third and fifth days after crystal
Time course and serum enhancement of 3H thymidine uptake by quiescent cell cultures. The addition
of 100 pg/ml HA crystals in 1% serum DMEM stimulated 3H thymidine incorporation as much as did 10%
serum (Figures 1A and B). The onset of this effect and
time of peak thymidine uptake were delayed by approximately 2 hours for canine cells and 3 hours for
human foreskin fibroblasts. All subsequent pulse labeling was done 16 hours after addition of particulates
or serum to canine synovial fibroblasts. Uptake was
proportional to the serum concentration and was enhanced at each concentration by the presence of 50 pg/
ml HA crystals (Figure 2). The effect of 50 pg/ml HA
crystals persisted for at least 112 hours (Figure 3). Cell
protein per well also increased more than threefold as
compared with control (1% serum) by 112 hours.
Dose-response relationships. The effect of 10
pg/ml HA crystals was approximately twice that of
control (Figure 4). A further increase in ’H thymidine
uptake occurred as the dose of crystals was increased
to 100 pg/ml. The square of the lowest dose (10 pg/ml)
produced approximately a twofold increase in incorporation. The CPPD crystals (10-20 pm) also showed a
dose-response relationship, although no effect was
found at the lowest concentration used (Figure 5).
Larger CPPD crystals (>50 pm) failed to stimulate 3H
thymidine incorporation.
e
-5 300
200
.%
-z loo
f
I
HS(%)
CPPO(jlq/ill)
I
0
I
loo*
I
I
I
200* 400* 10
I*
50
I
*
loo
I*
200
Figure 5. Effect of varying doses of calcium pyrophosphate dihydrate (CPPD) crystals of different sizes on uptake of ’H thymidine
into washed cells at 16 hours. All values mean 2 SEM. * = CPPD >
50 p n ; $ = P < 0.001 versus control horse serum (HS). Control
uptake = 3.5 5 0.3 x lo-’ cpm/well; n values are denoted in
histogram.
CHEUNG ET AL
Table 1. Uptake of 'H thymidine by synovial cells exposed to
various concentrations of hydroxyapatite (HA) crystals,
monosodium urate monohydrate (MSU) crystals (5-10 pm),
diamond crystals (1-5 pm), or latex beads (0.5pm)*
Additiofi
None (control)
HA
MSC
Diamond
Latex bead
Concentration
(d m l )
50
10
20
40
80
215
410
820
100
200
400
29
58
116
233
466
cpm (74
control)
100 2
328 2
120 c
140 2
120 2
145 2
n
30
7t
20
I5
20
20
16
4
4
4
4
8
4
4
4
4
4
4
4
4
4
4
10
190 ? 23$
155 2 20
110 12
120 2 5
125 ? 15
130 5 5
90 2 20
100 2 2
120 2
110 c 5
105 2 10
*
*
* Values expressed as % of control (1% serum) uptake
Control uptake 3.7 2 0.8 x lo-' cpdwell.
+ P < 0.001 versus control.
$ P < 0.05 versus control.
2
SEM.
Likewise, no stimulation by either diamond
crystals or latex beads was found (Table I). MSU
crystals (preparation A) modestly stimulated 3H thymidine uptake at doses ranging from 80-410 pg/ml, but
this effect was not seen at the lowest and highest
concentrations used (10 and 820 pg/ml).
Effect of calcific material from a patient with
calcinosis. The naturally occurring crystals stimulated
'H thymidine uptake nearly as much as did thc synthetic HA crystals (Table 2). The effects of heating the
crystals and of trypsin treatment were negligible. Cells
incubated with HA, the calcified material, or 10%
serum showed highly significant increases in both cell
number and cell protein on days 3 and 5 , compared
with cells incubated with 1% serum only (Table 3).
DISCUSSION
Synovial hyperplasia often occurs in joints containing calcium pyrophosphate dihydrate ( I ,2) or hydroxyapatite crystal deposits (7). Data shown here
indicate that these crystals act as mitogens when
added to cells in monolayer culture.
Synthetic HA in 1% serum stimulated 3H thymidine uptake into quiescent cell cultures to about the
same degree as did 10% serum. The 2-hour and 3-hour
delays in the onset and peak time of uptake in experiments using canine synovial fibroblasts and human
foreskin fibroblasts, respectively, may be related to a
requirement for internalization. Endocytosis of both
HA and CPPD crystals certainly occurs (8).
HA crystals, 50 pg/ml, stimulated nuclide uptake at each serum concentration used. The mitogenic
effect of HA crystals persisted for at least 5 days,
during which time cell protein per well increased
approximately threefold. The reason(s) for such persistence are unclear.
There is no difference between heat-inactivated
(56°C for 45 minutes) serum and non-heat-inactivated
serum in our cell proliferation assay (data not shown).
Thus, the mitogenic effect of the crystals does not
involve any interaction between the crystals and heatlabile serum components.
'H thymidine uptake was approximately proportional to the dose of HA or CPPD (Figures 4 and 3,
although larger doses of the latter crystal were required. Increased nuclide uptake was not found with
10 pg/ml CPPD, nor when very large (>50 pm) CPPD
crystals were used. Whether this is due to the larger
surface area per mass of the smaller crystals or to
decreased internalization of the larger crystals is presently unclear.
Not all particulates were effective mitogenic
agents. Latex beads and diamond crystals had no
effect, despite extensive internalization demonstrated
by transmission electron microscopy (data not
shown). Monosodium urate crystals modestly stimulated 'H thymidine uptake. However, the reasons for
the lack of stimulation at both the lowest and highest
doses used are unclear. The more complex crystals
Table 2. Effect of calcific material from patient with calcinosis on
'H thymidine uptake*
Addition
Concentration
(~drnl)
+
100 2 9
None
HA
50
HA
C
C
C
C
100
* Control uptake
cpm (% control
SEM)
50
LOO
100 (T + H)
100 (TI
345 2
390 1260 2
340 2
310 5
315 2
18t
20t
17t
15;
217
24t
= 3.8 ? 0.4 x lo-' cpmiwell; n -- 4. HA =
hydroxyapatite; C = calcific material; T = trypsin treated; H =
heated.
t P < 0.001 versus control.
MITOGENIC EFFECTS OF HA AND CPPD
673
Table 3. Effect of hydroxyapatite (HA) crystals and calcific material from a patient with calcinosis
on cell number and cell protein
Day 5
Day 3
Addition*
1% HS
10% HS
H A (100 pg)
c (100 pg)
Cell no.
x lo-5
0.44 2
0.99 2
0.82 2
0.77 2
0.1 I
0.16t
0.13t
0.17t
* HS = control horse serum; C
t P < 0.001 versus 1%. HS.
=
Protein
Cell no.
(UP)
x 10-5
23.6
48.3
42.1
38.8
2
2
2
-t
4.0
3.2t
8.9t
3.St
0.51 2
1.50 2
1.24 2
1.15 ?
0.09
0.18t
0.20t
0.16t
Protein
(!a)
27.4 i 4.4
74.9 2 6.17
64.2 2 5.8t
56.6 2 6.2t
calcific material.
found in a naturally occurring condition (calcinosis)
stimulated 3H thymidine uptake nearly as well as did
the pure synthetic hydroxyapatite, with nuclide uptake
unchanged by treatment of the calcinosis crystals with
heat or trypsin. The range of concentrations of synthetic and calcified crystals used in these experiments
was similar to the concentrations measured by direct
chemical analysis of the washed joint fluid pellets of
patients with the Milwaukee shoulder syndrome (1245 pghl joint fluid), in previous studies (6).
Three and 5 days after crystal addition, cell
number and protein were significantly increased in
cells incubated with HA, calcinosis material, or 10%
serum, versus those incubated with 1% serum only.
An earlier observation demonstrated that canine synovial fibroblasts incubated with HA or CPPD crystals
released similar amounts of lactic dehydrogenase as
control cells incubated without crystal (8). These data
suggest that calcium complexes stimulate growth,
rather than stimulating death of a small percentage of
cells with subsequent replicatiOn and replacement by
others.
Rubin and Sanui described stimulation of ’H
thymidine uptake into mouse 3T3 cells in culture by
precipitates of calcium and inorganic pyrophosphate
(PPi) (19). Orthophosphate in higher concentrations
could substitute for PPi. This effect was partially
blocked by increasing magnesium concentrations and
appeared to be somewhat specific, since no such effect
on chicken embryo fibroblasts was found. Strontium
could be substituted for calcium with similar but less
pronounced effects. Barnes and Colowick also showed
increased 3H thymidine uptake by 3T3 cells exposed to
precipitates of calcium phosphate (20). Precipitates of
barium sulfate showed similar but less marked stimulation. Both investigators suggested that divalent cations
were probably the critical factor.
Scher et al (21) have classified mitogens into
two groups: (a) factors which do not stimulate DNA
synthesis per se, but render cells in Go or G I phase
“competent” to do so; and (b) factors which stimulate
DNA synthesis in competent cells. Platelet-derived
growth factor, fibroblast growth factor, and calcium
phosphate precipitates fall into the first group (21,22),
while somatomedin C is an example of an agent in the
second group. It seems likely that the mitogenic effects
of the calcium-containing crystals on cultured synovial
cells described here are due to their ability to confer
“competence.” Experiments to further delineate the
mechanism of this phenomenon are in progress. We
speculate that calcium is released intracellularly by
digestion of crystals after endocytosis.
All in vivo dissolution of synthetic triclinic
CPPD crystals appears to take place within cells (3,4).
Addition of 45calcium-labeled HA crystals to cultured
synovial cells is associated with (a) uptake by the cells
and (b) release of nuclide into tissue culture medium
(23). The important role of ambient calcium concentration in promotion of growth of cultured fibroblasts was
described by Balk in 1971, in the same paper that first
described the growth-promoting effects of serum versus plasma (24). Under certain conditions, calcium
ionophore A23187 promotes mitosis (25). Evidence
cited above suggests that other divalent cations, such
as strontium or barium, may be able to substitute for
calcium.
If HA or CPPD crystals are indeed “competence”-promoting factors, they mpy be responsible for
synovial cell proliferation in the crystal deposition
diseases, since the factors in plasma needed for “progression” (such as somatomedin) are present in synovial fluid (26,27), which is largely a dialysis of plasma
(28).
ACKNOWLEDGMENTS
The authors wish to thank Ms Janine Struve and Mr.
Thomas Nichols for their excellent technical assistance and
Dr. Lawrence Ryan for helpful discussion.
CHEUNG ET AL
674
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DJ McCarty. Philadelphia, Lea & Febiger, 1979, pp
1276-1 299
2. Schumacher HR Jr: The synovitis of pseudogout: electron microscopic observations. Arthritis Rheum 1 1:426435, 1968
3. McCarty DJ, Palmer DW, Halverson PB: Clearance of
calcium pyrophosphate dihydrate crystals in vivo. I.
Studies using I6%b labeled triclinic crystals. Arthritis
Rheum 22:718-727, 1979
4. McCarty DJ, Palmer DW, James C: Clearance of calcium pyrophosphate dihydrate crystals in vivo. 11. Studies using triclinic crystals doubly labeled with 45Ca and
”Sr. Arthritis Rheum 22: 1122-1 131, 1979
5. McCarty DJ, Halverson PB, Carrera GF, Brewer BJ,
Kozin F: “Milwaukee shoulder”-association of microspheroids containing hydroxyapatite crystals, active collagenase, and neutral protease with rotator cuff defects.
I. Clinical aspects. Arthritis Rheum 24:464-473, 1981
6. Halverson PB, Cheung HS, McCarty DJ, Garancis J ,
Mandel N: “Milwaukee shoulder”-association of microspheroids containing hydroxyapatite crystals, active
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7. Garancis JC, Cheung HS, Halverson PB, McCarty DJ:
“Milwaukee shoulder”-association of microspheroids
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and neutral protease with rotator cuff defects. 111. Morphologic and biochemical studies of an excised synovium showing chondromatosis. Arthritis Rheum 24:484491, 1981
8. Cheung HS, Halverson PB, McCarty DJ: Release of
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9. Werb Z, Reynolds JJ: Stimulation of endocytosis of the
secretion of collagenase and neutral protease from rabbit
synovial fibroblasts. J Exp Med 140:1482-1497, 1974
10. Clarris BT, Fraser JR, Moran CJ, Muirden KD: Rheumatoid synovial cells from intact joints: morphology,
growth and polykaryocytosis. Ann Rheum Dis 36:293301, 1977
11. Cheung HS: An improved method of establishing human
fibroblast cultures from explants. J Tissue Culture Meth
6:39-40, 1980
12. McCarty DJ, Lehr JR, Halverson PB: Crystal populations in human synovial fluid: identification of apatite,
octacalcium phosphate, and tricalcium phosphate. Arthritis Rheum 26:1220-1224, 1983
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5:284-290, 1963
14. Praetorius E: An enzymatic method for the determination of uric acid by ultraviolet spectrophotometry.
Scand J Clin Lab Invest 1:222-230, 1949
15. McCarty DJ, Hollander JL: Identification of sodium
urate crystals in gouty synovial fluid. Ann Intern Med
54:452-460, 1962
16. Mandel NS: Structural changes in sodium urate crystals
on heating. Arthritis Rheum 23:772-776, 1980
17. Lowry OH, Rosebrough NJ, Fan AL, Randall RJ:
Protein measurement with the folin phenol reagent. J
Biol Chem 193265-275, 1951
18. Mendenhall W: Introduction to Probability and Statistics. Third edition. Belmont, California, Duxbury Press,
1971, pp 230-235
19. Rubin H, Sanui H: Complexes of inorganic pyrophosphate orthophosphate and calcium as stimulants of 3T3
cell multiplication. Proc Natl Acad Sci USA 74:50265030, 1977
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crystals, effect, dihydrate, mammalia, pyrophosphate, hydroxyapatite, culture, calcium, mitogenic, cells
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