athy -- Calcification. Pyrophosphate arthropathy (also known as calcium pyrophosphate deposition disease or pseudogout) is. Send reprint requests to Graeme ...
Calcif Tissue Int (1987)41:164-170
Calcified Tissue International 9 1987 Springer-Verlag New York Inc.
Effect of Glycosaminoglycans on Calcium Pyrophosphate Crystal Formation in Collagen Gels Graeme K. Hunter, Marc D. Grynpas, Pei-Tak Cheng, and Kenneth P. H. Pritzker Department of Pathology, University of Toronto; and ConnectiveTissue Research Group, Mount Sinai Hospital, 600 University Avenue, Toronto, Ontario, M5G 1X5, Canada
Summary. Formation of calcium pyrophosphate dihydrate (CPPD) crystals in native collagen gels represent an in vitro model system for the study of pathological cartilage calcification. The conditions under which CPPD forms in collagen gels have been determined. At low Ca x pyrophosphate product, CPPD forms directly. At high Ca x pyrophosphate product, CPPD forms via the amorphous intermediate calcium magnesium pyrophosphate (CMPP). Chondroitin sulfate (CS) inhibits formation of CPPD by both pathways, but apparently by different mechanisms. Direct CPPD formation is inhibited with low potency by CS, apparently by binding of Ca 2+ ions. Indirect formation of CPPD is inhibited with high potency by CS, apparently by stabilization of the CMPP intermediate. Comparison of the inhibition of direct CPPD formation by the two glycosaminoglycan species occurring in cartilage proteoglycan showed that CS is a more potent inhibitor than keratan sulfate (KS), in agreement with the greater Ca2+-binding affinity of CS. The increase in KS/CS ratio which occurs in human hyaline cartilage with aging may facilitate deposition of CPPD crystals by decreasing the exclusion of pyrophosphate anions.
Key words: Calcium pyrophosphate - - Collagen gel - - Glycosaminoglycans - - Pyrophosphate arthropathy - - Calcification.
Pyrophosphate arthropathy (also known as calcium pyrophosphate deposition disease or pseudogout) is Send reprint requests to Graeme K. Hunter, Ph.D, Department
of Pathology, Mount Sinai Hospital, 600 UniversityAvenue, Toronto, M5G IX5, Canada.
a degenerative arthritis characterized by the deposition of calcium pyrophosphate dihydrate (CPPD) crystals in articular cartilage, meniscus, and intervertebral disc [1, 2]. Like osteoarthritis, pyrophosphate arthropathy exhibits a distinct age dependence, afflicting approximately 5% of individuals aged 60 years or greater, but the pathogenesis of this disease is poorly understood. The availability of p y r o p h o s p h a t e is probably rate limiting for CPPD formation, as the normal synovial fluid pyrophosphate concentration is only 1-2 p~M [3], but far higher levels occur in the synovial fluid of individuals suffering from pyrophosphate arthropathy [4]. Chondrocyte biosynthetic processes, particularly glycosyl transferase reactions involved in proteoglycan synthesis, may generate large amounts of pyrophosphate in cartilage. In agreement with this, alterations in pyrophosphate metabolism in the cartilage of pyrophosphate arthropathy patients have been demonstrated [5]. Cartilage proteoglycan may be involved in another way in the pathogenesis of pyrophosphate arthropathy. Human hyaline cartilage undergoes a series of changes with age characterized by increasing fibrillarity and decreased hydration [6]. In particular, proteoglycan from older individuals has a higher content of KS and lower content of CS [7]. As CS contains both sulfate and carboxylate groups, but KS only sulfates, the fixed negative charge density of cartilage decreases with age. The incidence of pyrophosphate arthropathy therefore appears to exhibit a reciprocal correlation with the fixed charge density of the affected tissues. In addition, cartilage from individuals afflicted with pyrophosphate arthropathy has been reported to contain less CS and more KS than cartilage from agematched controls [8]. B e c a u s e proteoglycans confer cartilage with its cation-binding and anionexcluding properties, it seems reasonable to specu-
G. K. Hunter et al.: Calcium Pyrophosphate Formation in Collagen Gels l a t e t h a t c h a n g e s in g l y c o s a m i n o g l y c a n ( G A G ) composition with aging may facilitate the pathological calcification of cartilage. I n t h e p r e s e n t s t u d y , t h e effects o f C S a n d K S o n CPPD crystal formation have been determined. P r e v i o u s s t u d i e s p e r f o r m e d in this l a b o r a t o r y a n d by Mandel and Mandel showed that CPPD and other (nonphysiological) calcium pyrophosphates c a n b e g r o w n in gels c o m p o s e d o f g e l a t i n o r s o d i u m m e t a s i l i c a t e [9, 10]. To s i m u l a t e m o r e c l o s e l y t h e extracellular matrix of cartilage, native collagen gels h a v e n o w b e e n u s e d to s u p p o r t t h e g r o w t h o f c a l c i u m p y r o p h o s p h a t e c r y s t a l s . U s i n g this s y s t e m , it h a s b e e n s h o w n t h a t C S is a m o r e p o t e n t i n h i b i t o r of CPPD formation than KS.
165
Analysis of Precipitates Formed in Collagen Gels Precipitates were harvested from gels after removal of the overlay solution and freezing at -20~ Tubes were cracked open and the crystal-bearing region of the gel was cut out. After thawing, expressed solution was removed and the precipitate was dried at 37~ For X-ray diffraction, precipitates were powdered and placed in glass capillary tubes. Samples were analyzed with a DebyeScherrer powder camera using Ni-filtered Cu-K~x radiation (0.15418 nm) at 40 kV and 25 mA with an exposure time of 2 hours. For scanning electron microscopy, precipitates were carbon coated under vacuum and examined with a Hitachi $520 scanning electron microscope. For elemental analysis, precipitates were ashed in hot nitric/ perchloric acid (4:1) and analyzed by inductively coupled plasma emission spectroscopy [13].
Materials and Methods Results Chondroitin 4-sulfate (whale cartilage) and keratan sulfate (bovine cornea) were obtained from Sigma Chemical Co. Antibiotic mixture (penicillin/streptomycin/amphotericin B) was obtained from Gibco. 45CAC12(CES-3, 10-40 mCi/mg) was obtained from Amersham International.
Growth of Calcium Pyrophosphate Crystals in Native Collagen Gels Direct formation of calcium pyrophosphate in collagen gels was achieved by modifying the method previously described for hydroxyapatite [11]. Collagen was dissolved at 5 mg/ml in 10 mM HC1, then added to appropriate volumes of 5 M NaC1, antibiotic solution, and 0.1 M sodium pyrophosphate, pH 7.4. The pH was adjusted to 7.4 with 0.1 M NaOH, and the solution then diluted and degassed. If required, GAGs were added to collagen solution as an aqueous concentrate immediately prior to dilution. Following degassing, 1.5 ml gels were poured in 12 x 75 mm polystyrene snap-cap tubes and allowed to set at 37~ overnight. Final concentrations were 2 mg/ml collagen, 0.15 M NaCI, 100 U/ml penicillin G, 100 p.g/ml streptomycin sulfate, and 0.25 ~g/ml amphotericin B. Pyrophosphate was used in the range 0.4-0.9 raM. Gels were overlayed with 1.5 ml of solution containing CaC12 in the range 0.8-1.8 raM, with or without MgC1z in the range 0.16-0.36 mM, NaC1 and antibiotics as above, and 45CAC12 in the range 1-2 ~Ci/ml, and incubated at 37~ The molar ratio of Ca to pyrophosphate was maintained at 2:1, as in CPPD. The Ca/Mg ratio was maintained at 5:1 to favor formation of CPPD rather than calcium pyrophosphate tetrahydrate [12]. To quantitate precipitation of calcium pyrophosphate, 10 ~xl aliquots of overlay solution were removed at intervals throughout the incubation period and 45Ca activity was determined by liquid scintillation counting. To distinguish between loss of 45Ca due to diffusion and precipitation, pyrophosphate-containing gels were compared with pyrophosphate-free gels (in groups of 3). Formation of CPPD at high Ca x pyrophosphate product was achieved as follows: 8 ml gels containing 25 mM pyrophosphate without NaC1 were overlayed with 4 ml of 40 mM CAC12/8 mM MgCI2 and incubated at 37~
Direct Formation of Calcium Pyrophosphate Crystals in Native Collagen Gels Calcium pyrophosphate crystals were grown by d i f f u s i o n o f C a 2+ i o n s i n t o t y p e I c o l l a g e n g e l s d o p e d w i t h s o d i u m p y r o p h o s p h a t e . To s t i m u l a t e p h y s i o l o g i c a l c o n d i t i o n s as f a r a s p o s s i b l e , gels c o n t a i n e d 0.15 M NaC1, w e r e b u f f e r e d at p H 7.4, a n d w e r e i n c u b a t e d at 37~ Calcium pyrophosphate precipitation was quantitated by uptake of 45Ca f r o m t h e o v e r l a y s o l u t i o n , u s i n g p y r o p h o s p h a t e - f r e e gels to d i s t i n g u i s h p r e c i p i t a t i o n f r o m diff u s i o n , as p r e v i o u s l y d e s c r i b e d [11]. F o l l o w i n g inc u b a t i o n , p r e c i p i t a t e s w e r e h a r v e s t e d f r o m gels a n d characterized by powder X-ray diffraction. As s h o w n in F i g u r e l a , in t h e a b s e n c e o f M g t h e p r o d u c t f o r m e d is c a l c i u m p y r o p h o s p h a t e t e t r a h y d r a t e ( o r t h o r h o m b i c ) [ C P P T ( O ) ] . A d d i t i o n o f M g at a C a / M g r a t i o o f 5:1 r e s u l t e d in t h e f o r m a t i o n o f C P P D (Fig. l b ) , as p r e v i o u s l y d e m o n s t r a t e d in sol u t i o n [12]. A t t h e s a m e C a • p y r o p h o s p h a t e product, higher amounts of preciPitation of C P P T ( O ) (in t h e a b s e n c e o f Mg) o c c u r r e d t h a n p r e c i p i t a t i o n o f C P P D (in t h e p r e s e n c e o f Mg) (Fig. 2). Under these conditions, formation of CPPT(O) commenced after 3 days and formation of CPPD a f t e r 4 d a y s ; in b o t h c a s e s , a p l a t e a u v a l u e w a s reached by 12-13 days.
Effect of Glycosaminoglycans on Direct Formation of CPPD in Collagen Gels To d e t e r m i n e w h e t h e r G A G s i n h i b i t f o r m a t i o n o f CPPD, as previously shown for hydroxyapatite [11], C S w a s i n c o r p o r a t e d i n t o c o l l a g e n gels at c o n -
166
G.K. Hunter et al.: Calcium PyrophosphateFormation in Collagen Gels
Fig. 1. PowderX-ray diffractionof calcium pyrophosphateprecipitates formed in collagengels. A. 0.8 mM pyrophosphate, 1.6 mM Ca; B. 0.8 mM pyrophosphate, 1.6 mM Ca, 0.32 mM Mg; C. 25 mM pyrophosphate, 40 mM Ca, 8 mM Mg; D. 25 mM pyrophosphate,40 mM Ca, 8 mM Mg, 1 mg/mlCS.
centrations of 2, 5, and 10 mg/ml. Calcium pyrophosphate precipitation was quantitated by incorporation of 45Ca, as described above. As shown in Figure 3, amount of CPPD formation decreased with increasing CS concentration. The amount of calcium binding to CS was determined from the 45Ca remaining in the overlay solution of control (pyrophosphate-free) gels following incubation. This apparent binding is unlikely to be due to Gibbs-Donnan effects since the ionic strength is much higher than the calcium concentration. A reciprocal relationship exists between amount of calcium binding and amount of CPPD formation, suggesting that CS may inhibit crystal formation by reducing the available calcium concentration. Direct formation of CPPD was quantitated in the presence of CS and KS at a concentration of 2.5 mg/ml. As shown in Figure 4, there is little inhibition of CPPD formation by KS at this concentration, although the difference between control and KS curves early in the incubation period suggests a delay effect. However, the same concentration of CS results in substantial inhibition of CPPD formation. The rate of 45Ca uptake into CPPD is approximately 40% lower in CS-containing gels than in KS-containing gels. From the pyrophosphate-free gels, it was calculated that there was approximately
twofold higher binding of Ca 2+ to CS than to KS (not shown). Therefore, CS is a more potent inhibitor of direct CPPD formation than KS.
Effect o f Chondroitin Sulfate on CPPD Formation at High Ca x Pyrophosphate P r o d u c t At high Ca x p y r o p h o s p h a t e product (40 mM CaCI 2, 8 mM MgCI2, 25 mM sodium pyrophosphate), formation of a flocculent precipitate occurs in the upper part of the gel over the first 24 hours of incubation. Over the succeeding days, aggregates of crystals surrounded by clear zones appear within the flocculent precipitate. By 3 - 4 weeks, the inital precipitate has completely transformed into crystal aggregates. The elemental compositions of the initial and final precipitates are shown in Table 1. The composition of the final precipitates is consistent with CPPD. This was confirmed by X-ray diffraction (Fig. lc). The initial precipitate, however, contains a higher amount of Mg, corresponding to the Ca/Mg ratio initially present in the overlay solution (Table 1). This material gave an amorphous X-ray diffraction pattern (not shown). By scanning electron microscopy, the initial phosphate exhibited no periodicity, but the final precipitate consisted of
G. K. Hunter et al.: Calcium PyrophosphateFormation in CollagenGels
8
A
6
4 6" .A," 2
E -
9A'~
i
E
.o- . . . . . . . .
"
0 i
i
i
i
i
i
i
i
1
i
i
t
B
8
-o
..." ...-"
. .......
-o
.(2,-------
i
INCUBATION TIME ( d )
Fig. 2. Uptake of 45Ca into calcium pyrophosphate in collagen gels. Precipitationof calcium pyrophosphatewas quantitated by uptake of 45Causing pyrophosphate-freegels as control(mean of 3 determinations). A. 9 ..... 9 0.4 mM pyrophosphate,0.8 mM Ca; ~ - - - A 0.5 mM pyrophosphate, 1.0 mM Ca; 9 9 0.6 mM pyrophosphate, 1.2 mM Ca. B. 9 ..... 9 0.6 mM pyrophosphate, 1.2 mM Ca, 0.24 mM Mg; A - - - A 0.8 mM pyrophosphate, 1.6 mM Ca, 0.32 mM Mg; 9 - 9 1.0 mM pyrophosphate, 2.0 mM Ca, 0.36 mM Mg. spherical clusters of needle-shaped crystals (Fig. 5). Therefore, the initial precipitate is probably an a m or p h o u s calcium magnesium p y r o p h o s p h a t e (CMPP). This phase is kinetically favored under these conditions, but on longer incubation, transforms by redissolution into the thermodynamically favored CPPD. CS was incorporated into high Ca x pyrophosphate gels at a concentration of 1 mg/ml. X-ray diffraction patterns of precipitates formed in gels with and without CS after 4 weeks of incubation are shown in Figure lc and d. CS has no effect on the initial precipitation of CMPR However, the transformation of CMPP into CPPD is inhibited by CS (Fig. ld). Therefore, low concentrations of CS stabilize the amorphous CMPP intermediate. Discussion
Conditions have been established which permit the growth of calcium pyrophosphate crystals in native type I collagen gels under approximately physiolog-
167
ical conditions. Type I collagen was used because of the relative ease of preparation of this collagen type, although CPPD crystals in vivo form in both type I and type II collagen-containing tissues [2]. As previously shown in solution 112], in the absence of Mg the crystal phase formed is CPPT(O), but at a Ca/Mg ratio of 5:1 the crystal phase formed is CPPD. The Ca/pyrophosphate ratio occurring in CPPD (2:1) was used in the present study. Under these conditions, CPPD is formed at calcium concentrations close to the serum level. However, the calcium concentration in hyaline cartilage is much higher, due to Gibbs-Donnan effects and binding of Ca 2+ ions to proteoglycan fixed negative charges [14-16]. The pyrophosphate concentrations used in the present study (0.4-0.9 mM) are far higher than the normal synovial fluid concentration ( I - 2 ~xM), but the pyrophosphate concentration in cartilage has never been measured accurately. At a calcium concentration of 1 mM, at least 100 txM pyrophosphate is necessary for CPPD formation in solution [171. At high Ca x pyrophosphate product (but the same Ca/Mg/pyrophosphate ratio), CPPD forms by a different mechanism. An amorphous precipitate of CMPP forms almost immediately, and this slowly transforms into CPPD by redissolution. CS inhibits formation of CPPD by both pathways, but apparently by different mechanisms. Direct formation of CPPD is inhibited by CS with low potency, and this inhibition is correlated with an apparent binding of Ca z+ ions to the GAG anionic groups. At high Ca x pyrophosphate product, the formation of amorphous CMPP is not affected by CS, but the transformation of CMPP into CPPD is inhibited with high potency. The amount of CS needed to completely inhibit formation of CPPD from CMPP is sufficient to bind only 5% of the total calcium, suggesting that CS inhibits this transformation by a different mechanism. Therefore, at low Ca • pyrophosphate product, CS inhibits CPPD formation by binding of calcium; at high Ca x pyrophosphate product, CS inhibits CPPD formation by stabilizing the amorphous intermediate CMPE Comparison of the inhibition of direct formation of CPPD by the two GAGs present in cartilage proteoglycan showed that CS is a more potent inhibitor than KS. As shown previously, CS binds calcium with higher affinity than KS and has approximately twice the number of calcium-binding sites (G. K. Hunter et al, unpublished). The biological implications of this latter observation are not clear. Since there is a decrease in CS (a more potent inhibitor of CPPD formation) and an increase in KS (a less potent inhibitor) in human hyaline cartilage with aging, it seems reasonable to
G . K . Hunter et al.: Calcium Pyrophosphate Formation in Collagen Gels
168
6-" 6 x
-~ g
5
E
o= 14
s 3
o_. lO 8
g
6
t
l
4
0
1
I
I
I
2
3
4
/
[Chondroitin
5 sulfate]
I
I
i
I
i
6
7
8
9
10
(mg/ml)
-~
Fig. 3. Effect of chondroitin sulfate concentration on direct formation of CPPD in collagen gels. 0.8 mM pyrophosphate, 1.6 mM Ca, 0.32 mM Mg. Precipitation was quantitated as described in the legend to Figure 2. Calcium binding was determined using control (pyrophosphate-free) gels. Incubation time was 14 days.
Table 1. Elemental compositions of precipitates formed in collagen gels 8 .o
7
s •
~.
P "~
6
,_E
~s
E E
j~
...~
4 1"
Incubation time (d)
Weight
Weight
Weight
% Ca
% Mg
% P
Ca/P molar ratio
1 28
17.8 25.1
1.59 0.44
16.4 19.4
0.839 1.00
Initial concentrations: 40 mM Ca, 8 mM Mg, 25 mM P (pyrophosphate) Values represent the mean of two experiments, each analyzed in
triplicate
... r ""
3
8 2
;
;
,'o
1'2
11
,'6
INCUBATION TIME (d)
Fig. 4. Effect of chondroitin sulfate and keratan sulfate on direct formation of CPPD in collagen gels. 0.8 mM pyrophosphate, 1.6 mM Ca, 0.32 mM Mg. GAG concentration used was 2.5 mg/ml. Precipitation was quantitated as described in the legend to Figure 2. 9 - 9 no GAG; /~ - - - A CS-containing gels; 9 ..... 9 KS-containing gels.
speculate that the age dependence of pyrophosphate arthropathy is due, at least in part, to these matrix changes. However, as recently suggested by us, inhibition of crystal formation by calcium binding in vitro may not correspond to inhibition in vivo [18]. That is, GAGs may inhibit CPPD formation under in vitro conditions of limited calcium availability, but cartilage in vivo is in equilibrium with an essentially infinite reservoir of calcium. Therefore, it was suggested that proteoglycan func-
tions as a source of calcium for epiphyseal cartilage calcification rather than as an inhibitor. The relevance of these changes in cartilage proteoglycan composition to pathological cartilage calcification may be in the availability of pyrophosphate. Anions are partially excluded from cartilage because of the proteoglycan fixed negative charges. As the KS/CS ratio in cartilage increases with age, the fixed charge density decreases, and pyrophosphate (presumably generated endogenously) will be excluded to a lesser extent. Conversely, calcium will be present at lower concentration in aged cartilage, but should still be at high enough concentration that the increased availability of pyrophosphate may suffice to trigger the formation of CPPD crystals, and hence the pathological calcification of cartilage in aged individuals.
Acknowledgments. The
authors wish to thank Maria Torontali, Julie Richards, and Marina Fuller for their skilled technical assistance. Electron microscopy was performed by Douglas P. Holmyard. This work was supported by the Medical Research Council of Canada and the Canadian Arthritis Society. G. K. Hunter was the recipient of a Research Fellowship from the Gerontology Research Council of Ontario.
G. K. Hunter et al.: Calcium Pyrophosphate Formation in Collagen Gels
169
Fig. 5. Scanning electron microscopy of precipitates formed in collagen gels. a. Incubation t i m e - - 1 day; b. incubation t i m e - - 4 weeks.
References 1. McCarty DJ, Kohn NN, Faires JS (1962) The significance of calcium phosphate crystals in the synovial fluid of arthritis patients: the "pseudogout syndrome." Ann Intern Med 56:711-737 2. Resnik CS, Resnick D (1983) Crystal deposition disease. Semin Arthritis Rheum 12:390-403 3. Altman RD, Muniz O, Pita JC (1973) Microanalysis of inorganic pyrophosphate in synovial fluid and plasma. Arthritis Rheum 16:171-178 4. Silcox DC, McCarty DJ (1974) Elevated inorganic pyr ophosphate concentrations in synovial fluids in osteoarthritis and pseudogout. J Lab Clin Med 83:518-531
5. Tenenbaum J, Muniz O, Schumacher HR, Good AE, Howell DS (1981) Comparison of phosphohydrolase activities from articular cartilage in calcium pyrophosphate deposition disease and primary osteoarthritis. Arthritis Rheum 24:492500 6. Roughley PJ, White RJ (1980) Age-related changes in the structure of proteoglycan subunits from human articular cartilage. J Biol Chem 255:217-224 7. Mathews MB, Glagov S (1966) Acid mucopolysaccharide patterns in aging human cartilage. J Clin Invest 45:11031111 8. Bjelle AO (1973) The glycosaminoglycans of articular cartilage in calcium pyrophosphate dihydrate (CPPD) crystal deposition disease. Calcif Tissue Res 12:37-46
170
G . K . Hunter et al.: Calcium Pyrophosphate Formation in Collagen Gels
9. Pritzker KPH, Cheng P-T, Adams ME, Nyburg SC (1978) Calcium pyrophosphate dihydrate crystal formation in model hydrogels. J Rheumatol 5:469-473 10. Mandel NS, Mandel GS (1984) Calcium pyrophosphate crystallization kinetics in a gelatin matrix: a model for pyrophosphate arthropathy. Arthritis Rheum 27:789-798 11. Hunter GK, Allen BL, Grynpas MD, Cheng P-T (1985) Inhibition of hydroxyapatite formation in collagen gels by chondroitin sulfate. Biochem J 228:463-469 12. Cheng P-T, Pritzker KPH (1981) The effect of calcium and magnesium ions on calcium pyrophosphate crystal formation in aqueous solutions. J Rheumatol 8:772-781 13. Jones JW, Capan SG, O'Haver TC (1982) Critical evaluation of a multi-element scheme using plasma emission anhydride evolution atomic absorption spectroscopy for the analysis of plant and animal tissues. The Analyst 107:353-377 14. Maroudas A (1980) Physical chemistry of articular cartilage
15.
16.
17.
18.
and the intervertebral disc. In: Sokoloff L (ed), The joints and synovial fluid, Vol II. Academic Press, pp 239-291 Wuthier RE (1969) A zonal analysis of inorganic and organic constituents of the epiphysis during endochondral calcification. Calcif Tissue Res 4:20-38 Althoff J, Quint J, Krefting E-R, Hohling HJ (1982) Morphological studies on the epiphyseal growth plate combined with biochemical and X-ray microprobe analysis. Histochemistry 74:541-552 Cheng P-T, Pritzker KPH, Adams ME, Nyburg SC, Omar SA (1980) Calcium pyrophosphate crystal formation in aqueous solutions. J Rheumatol 7:609-616 Hunter GK (1987) An ion-exchange mechanism of cartilage calcification. Connect Tissue Res 16:111-120
Received October 3, 1986, and in revised form December 18, 1986.
