Matrix Vol. 12/1992, pp. 362 - 368 © 1992 by Gustav Fischer Verlag, Stuttgart
Effects of Proteoglycan on Hydroxyapatite Formation Under Non-Steady-State and Pseudo-Steady-State Conditions GRAEME K. HUNTER 1 and SUSAN K. SZIGETY Department of Oral Biology, Faculty of Dentistry, University of Alberta, Edmonton, Alberta, T6G 2N8, Canada.
Abstract Addition of chondroitin sulfate (CS) or cartilage proteoglycan to metastable calcium phosphate solutions inhibits the formation of hydroxyapatite (HA). However, pre-equilibration of CS or proteoglycan with calcium prior to the addition of phosphate results in higher levels of HA precipitation compared to control solutions of identical calcium and phosphate activity. These findings indicate that the inhibition of HA formation by proteoglycans and CS is largely due to calcium binding. Further, its ability to bind calcium ions reversibly suggests that proteoglycan may act as a promoter, not an inhibitor, of calcification in cartilage. Key words: calcification, cartilage, hydroxyapatite, proteoglycan, steady-state.
Introduction The calcification of cartilage that occurs at the epiphyseal growth plate of long bones involves the deposition of crystals of hydroxyapatite (HA) in an extracellular matrix consisting principally of Type II collagen and proteoglycan (Brighton, 1984). In cartilage, unlike bone, collagen is not thought to playa specific role in the calcification process. Proteoglycan, however, has been implicated in the calcification of cartilage both as a promoter and an inhibitor. The view of proteoglycans as inhibitors of calcification is based largely on studies of the effects of proteoglycan on HA formation and growth in metastable calcium phosphate solutions (Cuervo et aI., 1973; Blumenthal et aI., 1979; Chen et aI., 1984; Chen and Boskey, 1985; Chen and Boskey, 1986). As recently pointed out, however, most such studies have been performed under non-physiological conditions of limited calcium availability (Hunter, 1991). This is an important qualification, because the chondroitin sulfate (CS) chains of proteoglycans can bind I calcium ions 1 Present address: Division of Oral Biology, Faculty of Dentistry, University of Western Ontario, London, Ontario, N6A SCI, Canada.
(Hunter, 1987 a), and this binding has been correlated with the ability of CS to inhibit HA formation (Hunter et aI., 1985). In vivo, however, binding of calcium to proteoglycan should not reduce the free calcium concentration, as cartilage is in equilibrium with an essentially infinite reservoir of calcium in the rest of the body. In other words, physiological conditions are steady-state, whereas in vitro conditions are non-steady-state. The view that proteoglycans act as promoters of calcification originated from early studies in which cartilage preparations immersed in physiological salt solutions were shown to take up calcium ions (Pfaundler, 1904; Freudenberg and Gyorgy, 1923). The term Kalksalzfanger (lime salt catcher) was coined for the calcium-concentrating agent, now known to be proteoglycan. A means by which a Kalksalz{dnger could promote calcification was suggested by studies showing that immersion of cartilage preparations sequentially in calcium- and then phosphate-containing solutions (but not vice versa) resulted in the formation of mineral deposits in the tissue (Freudenberg and Gyorgy, 1923; Boyd and Neuman, 1951; Sobel and Burger, 1954). A mechanism of cartilage calcification consistent with these observations has recently been proposed (Hunter, 1987b). Calcium is sequestered into non-calcified cartilage
Proteoglycans and Cartilage Calcification by binding to the fixed negative charges of proteoglycans. At sites of calcification, a local increase in extracellular phosphate concentration causes the displacement of some of the bound calcium ions by an ion-exchange effect. This raises the Ca x P0 4 product above the precipitation threshold for HA. The ion-exchange mechanism of cartilage calcification predicts that proteoglycan-calcium complexes exposed to an increased phosphate concentration will act as promoters of calcification. In the present study, this prediction has been tested by quantifying formation of HA in the presence and absence of proteoglycan and CS pre-equilibrated with calcium. Under these pseudo-steady-state conditions, proteoglycan and CS increase the rate and amount of HA precipitation. Methods Purification ofproteoglycan from porcine nasal septum cartilage
Pig heads were obtained fresh from a local abbatoir, and the nasal septum cartilage removed. Proteoglycan aggregate and monomer preparations were prepared by a modification of the method of Hascall and Kimura (1982). Cartilage was cleaned, sliced finely and lyophilized. Freeze-dried tissue was ground in a Thomas-Wiley freezer mill at liquid nitrogen temperature, and then added at 30 mglml to a solution of 4 M guanidine-HCl/50 mM Tris-HCl, pH 7.4 containing proteinase inhibitors (5 mM N-ethyl maleimide, 10 mM ethylene diamine tetra-acetic acid, 0.1 M 6aminohexanoic acid, 1 mM phenyl methyl sulfonyl fluoride). After stirring at 4°C for 24 h, the extract was centrifuged at 28,000 x g for 40 min. The supernatant was retained, and the tissue residue re-extracted for a further 24 h prior to recentrifugation. The supernatants were pooled, and then concentrated approximately 4-fold by ultrafiltration using a Millipore "Minitan" concentrator with a 10,000 MW cut-off membrane. The extract was then dialyzed versus 7 volumes of distilled water (dH 2 0) plus inhibitors to reduce the guanidine-HCl concentration to 0.5 M. CsCI was added at 1.1 gig to give a density of 1.62 gI ml, and samples centrifuged at 149,000 x g for 48 - 72 h at 10 °C using a Beckman Ti 70 rotor. Gradients were harvested by needle puncture and the lower one-quarter dialyzed against dH 2 0 and lyophilized (A1). Half the sample was redissolved in 4 M guanidine-HCl containing 0.55 gig CsC! (dissociative conditions), the remainder redissolved in 0.5 M guanidine-HCl containing 1.1 gig CsC! (associative conditions), and both ultracentrifuged as described above. Fractions corresponding to the lower one-quarter of both associative (A1A1) and dissociative (A1D1) gradients were pooled, dialyzed against 1 M NaC! to convert proteoglycans to the sodium salt, then dialyzed against dH 2 0 and lyophilized.
363
Pseudo-steady-state system
Chondroitin 4-sulfate (CS) from bovine trachea was obtained from the Sigma Chemical Company. This was dissolved at 50 mglml in 100 ml of 2.2 mM CaCh/150 mM NaC!, sterilized by passage through a 0.451lm membrane filter, and dialyzed versus 3.91 of the same CaC! 2 /NaCI solution at 37°C for 16 h. Following dialysis, the volume of the CS solution was measured (some increase in volume typically occurred, due to osmotic effects). To determine the amount of calcium bound to CS, the calcium concentrations in the CS solution ("dialyzate") and the CaCh/NaCI solution against which the CS had been dialyzed ("diffusate") were measured by inductively-coupled plasma emission spectroscopy. 50-ml aliquots of dialyzate and diffusate were lyophilized, then redissolved in 25 ml of distilled water (dH 2 0). CS, at the same concentration as in the dialyzate, was added to one aliquot of diffusate prior to addition of dH 2 0. Redissolved dialyzate, diffusate and diffusate plus CS were added to equal volumes of 2.64 mM sodium phosphate, pH 7.4. Triplicate 15-ml aliquots of these final solutions were then added to thermostatable beakers (Mettler) fitted with custom-made perspex inserts and incubated for 1000 min at 37°C on Mettler DL-21 autotitrators operating in the pH stat mode, using 50 mM NaOH as titrant. The mineral formed was shown to be HA by powder X-ray diffraction (Department of Geology, University of Alberta). Experiments involving proteoglycan were performed using a modification of the method used for CS. The proteoglycan concentrations used were 10 and 25 mg/m!. At these concentrations, no changes in volume occurred during dialysis. Aliquots of 5 or 6 ml were run on autotitrators. CS used for comparison in proteoglycan experiments was extracted from porcine nasal septum cartilage by alkaline hydrolysis and purified by ion-exchange chromatography on Dowex 1-X2 (Mathews, 1976).
Results A pseudo-steady-state system for determining the effect ofproteoglycan and chondroitin sulfate on hydroxyapatite formation
The method used to compare the effects of proteoglycan and CS on HA formation under non-steady-state and pseudo-steady-state conditions is shown in Fig.1. To achieve pseudo-steady-state conditions, HA formation was initiated by addition of phosphate to a solution of proteoglycan or CS pre-equilibrated with calcium ("dialyzate"), and compared to HA formed by addition of phosphate to an aliquot of the solution against which the proteoglycan or CS had been dialyzed ("diffusate"). Dialyzate and diffusate solutions should therefore contain the same free calcium concentration, but dialyzate will also contain calcium
364
G. K. Hunter and S. K. Szigety
bound to proteoglycan or CS. To compare results from this pseudo-steady-state system with those previously obtained under non-steady-state conditions, proteoglycan and CS (Na salts) were added directly to additional aliquots of diffusate solution prior to addition of phosphate. Titration data were quantitated in terms of three parameters: the maximal rate of addition of NaOH (A max ), the time at which half the final volume of NaOH was added (to.s), and the total volume of NaOH added at 1000 min (V 1000)' Amax , to.s and V1000 are therefore measures of the maximal rate of HA formation, the precipitation lag time, and the total amount of HA formed, respectively. Effect ofchondroitin sulfate on hydroxyapatite formation under non-steady-state and pseudo-steadystate conditions
In initial studies, the effects on HA formation of CS were determined under non-steady-state and pseudo-steadystate conditions. The initial CS concentration used was 50 mg/ml, but this decreased to 38 mg/ml during dialysis. Equilibration of CS with CaCh/NaCI resulted in a amount of calcium binding equivalent to approximately twice the free calcium concentration (Table I). When added to phosEffect of Proteoglycan on Hydroxyapatite Formation Under Non-Steady-State and Pseudo-Steady-State Conditions Semi-Permeable Membrane
i/
CaCI 2 /NaCI : I I
---PG
I
I I I I
CaF ~CaF"'Ca-PG
!
/\
- Lyophyllze sample - redissolve in H2O • add P0 4 • measure HA formed
~
Control
!
"dialysate"
"diffusate"
- Lyophyllze sample - redissolve In H2O
~
- Lyophyllze sample - redissolve In H2O
- add P04 • add PG • measure HA formed
- add P04 • measure HA formed
Non Steady-State
Pseudo Steady-State
!
A
0.500
00400 0.300 0.200 0.100
E
0.000 0.600
"0
0.500
"0 "0
00400
I
0.300
CIl
0.200
Q)
« 0
Z
"0
>
B
0.100 0.000 0.600 0.500
00400 0.300 0.200 0.100 0.000 0
100
200
300
400 500
600 700
800
900 1000
Time (min.) Fig. 2. Effect of chondroitin sulfate on hydroxyapatite formation under pseudo-steady-state and non-steady-state conditions. (a). Diffusate (control). (b). Diffusate plus 38 mg/ml CS (non-steady-state). (c). Dialyzate (pseudo-steady-state).
phate, the CS dialyzate resulted in a rate and amount of HA precipitation significantly greater, and a lag time slightly shorter, than the corresponding diffusate (Figure 2 a and 2c, Table I). Addition of diffusate plus non-pre-equilibrated CS to phosphate, in contrast, resulted in no formation of HA (Figure 2 b).
[37"C' 16" I
0.600
!
Fig. 1. Method used to study effects of proteoglycan on hydroxyapatite formation under non-steady-state and pseudo-steady-state conditions. PG - proteoglycan; CaF - free calcium; Ca-PG - calcium bound to proteoglycan.
Effect ofcartilage proteoglycan on hydroxyapatite formation under non-steady-state and pseudo-steadystate conditions
Proteoglycan monomers (AlDl) and aggregates (AlAI) were prepared from porcine nasal septum cartilage. The AlAI preparation used was shown to contain approximately 70% aggregate by chromatography on Sepharose CL-2B under non-dissociative conditions (not shown). The effects of proteoglycan monomers and aggregates on HA formation at concentrations of 10 mg/ml were determined using the pseudo-steady-state system described above. As shown in Table II, pre-equilibration of 10 mg/ml proteoglycan with CaCl2 /NaCI resulted in binding of calcium, although less so than 38 mg/ml CS. When added to
Proteoglycans and Cartilage Calcification
365
Table 1. Titration parameters for the effect of chondroitin sulfate on hydroxypatite formation under pseudo-steady-state and nonsteady-state conditions.
diffusate: diffusate + CS: dialyzate:
Ca(mM)
Amax (mlJh)
VIQOO (ml)
to.s (min)
2.28 2.28 6.20
0.0047 ± 0.001 0.002 ± 0 0.D17 ± 0.001 (P
E
0.250
"0
0200
"0 "0
0.150
Q)
i i
0.050 .,'
-
B
0.0000
3
.....j /
/ :
."'-
100
200 300 400
500 600 700 800 900 1000
Time (min) Fig. 4. Effect of proteoglycan monomer and chondroitin sulfate on hydroxyapatite formation under pseudo-steady-state conditions. Line 1: CS diffusate (2.34 mM Cal. Line 2: AlDl diffusate (2.38 mM Cal. Line 3: A1Dl (25 mg/ml) dialyzate (5.22 mM Cal. Line 4: CS (25 mg/ml) dialyzate (5.22 mM Cal. The Ca values given in parenthesis are the total Ca concentrations measured in diffusates and dialyzates by inductively coupled plasma emission spectroscopy.
0.100 0.050 0.000
c
0.250 0.200 0.150 0.100 0.050 0.000
----------------------------
o
100
200 300
400
500 600
700
800 900 1000
Time (min) Fig.3. Effect of cartilage proteoglycan monomer and aggregate preparations on hydroxyapatite formation under pseudo-steadystate and non-steady-state conditions. (a). Diffusate. (b). A1Dl (10 mg/ml) dialyzate. (c). AlAI (10 mg/ml) dialyzate. Dashed lines in (b) and (c) represent diffusate solutions to which were added 10 mg/ml AID1 and AlAI, respectively, prior to the addition of phosphate.
Maroudas et al. (1988). These findings indicate that substantial amounts of calcium binding to proteoglycan occur under physiological conditions. Addition of phosphate to control solutions containing non-pre-equilibrated CS at 38 mg/ml and proteoglycan at 10 mg/ml (non-steady-state condition) resulted in complete inhibition of HA formation. Taken together with the calcium analyses described above, this suggests strongly that the ability of proteoglycan and CS to inhibit HA formation and growth under non-steady-state conditions is largely due to calcium binding effects, despite what has been claimed by others (Cuervo et aI., 1983; Chen and Boskey,
1985; Blumenthal, 1981). In marked contrast, addition of phosphate to pre-equilibrated CS and proteoglycan solutions resulted in maximum rates and final amounts of HA precipitation that were significantly higher than those obtained with the corresponding diffusate control (solutions of identical calcium and phosphate activity). Based on these results, it seems reasonable to suggest that proteoglycan at physiological concentration (approximately 50 mg/ml) will act as a promoter of HA formation under pseudo-steady-state conditions. There are two possible mechanisms by which proteoglycan may promote the formation of HA under the conditions used in this study. First, phosphate may displace calcium bound to proteoglycan by competing with the proteoglycan anionic groups, thereby raising the (free) Ca X P0 4 product (Hunter, 1987b; Hunter and Bader, 1989). Second, precipitation of HA may decrease the free calcium concentration sufficiently that the calcium-proteoglycan equilibrium is affected, causing release of bound calcium (Hunter, 1991). The former effect should decrease the precipitation lag time, but only slightly increase the final amount of precipitation; the latter effect should not affect the lag time, but significantly increase the amount of precipitation. As pre-equilibrated proteoglycan and CS increase the amount of HA formation, it appears that the calcium reservoir effect occurs in this system. However, the precipitation lag time is sufficiently variable that it is not possible to determine whether the calcium displacement effect is also occurring. These findings suggest that proteoglycans may act as promoters, not inhibitors, of cartilage calcification. This conclusion assumes that the proteoglycans of pre-calcified cartilage are in equilibrium with the calcium of surrounding
Proteoglycans and Cartilage Calcification tissues and fluids. A large body of literature, discussed in Hunter and Bader (1989), indicates that the calcium content of cartilage is approximately 10-20 mmole/kg wet weight, 4 to 8-fold higher than that of serum. The detailed studies of Maroudas and co-workers have shown that the calcium content of cartilage correlates with the fixed negative charge density of proteoglycans, and that similar calcium-sequestering activites are observed with cartilage in vivo and in vitro, and with purified proteoglycans (Maroudas, 1980; Maroudas et aI., 1988). There seems little reason to doubt that cartilage matrix is in ionic equilibrium with the surrounding fluids, and therefore contains a large reservoir of proteoglycan-bound calcium. In order for proteoglycan to act as a promoter of calcification, it is also necessary to postulate an local elevation in extracellular phosphate concentration, presumably by release from chondrocytes. Such a phosphate-elevating mechanism could involve alkaline phosphatase, which is present on the plasma membrane of hypertrophic chondrocytes, or matrix vesicles, extracellular membrane-bounded bodies found in calcifying tissues. The presence of this mechanism might also explain why growth plate, but not other cartilages, undergoes calcification. Rather than acting as promoters of the primary calcification event, it is also possible that proteoglycans supply calcium ions for the growth of HA crystals nucleated by another mechanism. However, it should be noted that the studies reported above deal only with de novo crystal formation, and that the effect of pre-equilibrated proteoglycan on seeded growth of HA has not been determined. In conclusion, the findings reported above provide a molecular-level explanation for the observation that calcification occurs in cartilage preparations exposed sequentially to calcium- and phosphate-containing solutions (Freudenberg and Gyorgy, 1923; Boyd and Neuman, 1951; Sobel and Burger, 1954). Addition of cartilage to calcium solution causes binding of calcium ions to proteoglycan; subsequent addition of cartilage to phosphate solution causes displacement of calcium ions and formation of HA. That a similar sequence of events may occur in vivo is suggested by the observation that accumulation of calcium in the matrix of growth plate cartilage preceeds the accumulation of phosphate (Althoff et aI., 1982; Shapiro and Boyd, 1984). Therefore, proteoglycan may function as a cation-exchanging calcium reservoir in the calcification of cartilage. Footnote 1 The terms "bind" and "binding" are used here without distinction between atmospheric (territorial) binding and site (specific) binding (Manning, 1969).
Acknowledgements We thank Sophie Lehocky for expert technical assistance. This work was supported by the Medical Research Council of Canada.
367
References Althoff, J., Quint, P., Krefting, E.-R. and Hohling, H.J.: Morphological studies on the epiphyseal growth plate combined with biochemical and X-ray microprobe analyses. Histochemistry 74: 541-552, 1982. Blumenthal, N.C.: Mechanism of proteoglycan inhibition of hydroxyapatite formation. In: The Chemistry and Biology of Mineralized Connective Tissues, ed. by Veis, A., Elsevier, Holland, 1981, pp. 509-515. Blumenthal, N.C., Posner, A.S., Silverman, A.D. and Rosenberg, L. c.: Effect of proteoglycans on in vitro hydroxyapatite formation. Calcif. Tissue Int. 27: 75-82, 1979. Boyd, E. S. and Neuman, W. F.: The surface chemistry of bone. V. The ion-binding properties of cartilage. j. Bioi. Chem. 193: 243-251,1951. Brighton, C. T.: The growth plate. Orthopedic Clinics N. America 15: 571-595, 1984. Chen, c.-c. and Boskey, A.L.: Mechanisms of proteoglycan inhibition of hydroxyapatite growth. Calcif. Tissue Int. 37: 395-400,1985. Chen, c.-c. and Boskey, A. L.: The effects of proteoglycans from different cartilage types on in vitro hydroxyapatite proliferation. Calcif. Tissue Int. 39: 324-327, 1986. Chen, c.-c., Boskey, A. L. and Rosenberg, L. c.: The inhibitory effects of cartilage proteoglycans on hydroxyapatite growth. Calcif. Tissue Int. 36: 285 - 290, 1984. Cuervo, L. A., Pita, J. C. and Howell, D.S.: Inhibition of calcium phosphate mineral growth by proteoglycan aggregate fractions in a synthetic lymph. Calc. Tiss. Res. 13: 1-10,1973. Freudenberg, E. and Gyorgy, P.: Der Verkalkungsvorgang bei der Entwicklung des Knochens. Ergebnisse d. inn. Med. 24: 17- 28, 1923. Hascall, V. C. and Kimura, J. H.: Proteoglycans: isolation and characterization. Meth. Enzymol. 82: 769-800, 1982. Hunter, G. K.: Chondroitin sulfate-derivatized agarose beads: A new system for studying cation binding to glycosaminoglycans. Anal. Biochem. 165: 435-441, 1987a. Hunter, G. K.: An ion-exchange mechanism of cartilage calcification. Connect. Tissue Res. 16: 111-120, 1987b. Hunter, G. K.: Role of proteoglycan in the provisional calcification of cartilage. A review and reinterpretation. Clin. Orthop. Rei. Res. 262: 256-280, 1991. Hunter, G.K., Allen, B.L., Grynpas, M.D. and Cheng, P.-T.: Inhibition of hydroxyapatite formation in collagen gels by chondroitin sulfate. Biochem. J. 228: 463 -469, 1985. Hunter, G.K. and Bader, S.M.: A mathematical modelling study of epiphyseal cartilage calcification. j. Theor. BioI. 138: 195-211,1989. Manning, G.S.: Limiting laws and counterion condensation in polyelectrolyte solutions. I. Colligative properties. j. Chem. Phys. 51: 924-933, 1969. Maroudas, A.: Physical chemistry of articular cartilage and the intervertebral disc. In: The Joints and Synovial Fluid, Vol. 2, ed. by Sokoloff, L., Academic Press, New York, 1980, pp.239-291. Maroudas, A., Weinberg, P.D., Parker, K.H. and Winlove, c.P.: The distributions and diffusivities of small ions in chondroitin sulphate, hyaluronate and some proteoglycan solutions. Biophys. Chem. 32: 257-270, 1988. Mathews, M. B.: Purification of skeletal keratan sulfate. In: The Methodology of Connective Tissue Research, ed. by Hall, D. S., Joynson-Bruvvers, 1976,pp.147-151. pfaundler, M.: Dber die Elemente der Gewebsverkalkung und ihre
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Beziehung zur Rachitisfrage. Jahrb. Kinderheilk. 60: 123 -177, 1904. Read, R., Sutherland, J. and Gosh, P.: The matrix components of the epiphyseal growth plate and articular cartilages from dogs treated with ammonium tetrathiomolybdate, a copper antagonist. Aust. J. Exp. Bioi. Med. Sci. 64: 545 - 562, 1986. Shapiro, I. M. and Boyde, A.: Microdissection-elemental analysis of the mineralizing growth cartilage of the normal and rachitic chick. Metab. Bone Dis. Rei. Res. 5: 317-326, 1984. Sobel, A.E. and Burger, M.: Calcification. XIV. Investigation of
the role of chondroitin sulfate in the calcifying mechanism. Proc. Soc. Exp. Bioi. Med. 87: 7-13, 1954. Wuthier, R.E.: A zonal analysis of inorganic and organic constituents of the epiphysis during endochondral calcification. Calc. Tiss. Res. 4: 20-38, 1969. Dr. Graeme K. Hunter, Department of Oral Biology, Faculty of Dentistry, University of Alberta, Edmonton, Alberta, T6G 2N8, Canada.
Effects of Proteoglycan on Hydroxyapatite Formation Under Non-Steady-State and Pseudo-Steady-State Conditions GRAEME K. HUNTER 1 and SUSAN K. SZIGETY Department of Oral Biology, Faculty of Dentistry, University of Alberta, Edmonton, Alberta, T6G 2N8, Canada.
Abstract Addition of chondroitin sulfate (CS) or cartilage proteoglycan to metastable calcium phosphate solutions inhibits the formation of hydroxyapatite (HA). However, pre-equilibration of CS or proteoglycan with calcium prior to the addition of phosphate results in higher levels of HA precipitation compared to control solutions of identical calcium and phosphate activity. These findings indicate that the inhibition of HA formation by proteoglycans and CS is largely due to calcium binding. Further, its ability to bind calcium ions reversibly suggests that proteoglycan may act as a promoter, not an inhibitor, of calcification in cartilage. Key words: calcification, cartilage, hydroxyapatite, proteoglycan, steady-state.
Introduction The calcification of cartilage that occurs at the epiphyseal growth plate of long bones involves the deposition of crystals of hydroxyapatite (HA) in an extracellular matrix consisting principally of Type II collagen and proteoglycan (Brighton, 1984). In cartilage, unlike bone, collagen is not thought to playa specific role in the calcification process. Proteoglycan, however, has been implicated in the calcification of cartilage both as a promoter and an inhibitor. The view of proteoglycans as inhibitors of calcification is based largely on studies of the effects of proteoglycan on HA formation and growth in metastable calcium phosphate solutions (Cuervo et aI., 1973; Blumenthal et aI., 1979; Chen et aI., 1984; Chen and Boskey, 1985; Chen and Boskey, 1986). As recently pointed out, however, most such studies have been performed under non-physiological conditions of limited calcium availability (Hunter, 1991). This is an important qualification, because the chondroitin sulfate (CS) chains of proteoglycans can bind I calcium ions 1 Present address: Division of Oral Biology, Faculty of Dentistry, University of Western Ontario, London, Ontario, N6A SCI, Canada.
(Hunter, 1987 a), and this binding has been correlated with the ability of CS to inhibit HA formation (Hunter et aI., 1985). In vivo, however, binding of calcium to proteoglycan should not reduce the free calcium concentration, as cartilage is in equilibrium with an essentially infinite reservoir of calcium in the rest of the body. In other words, physiological conditions are steady-state, whereas in vitro conditions are non-steady-state. The view that proteoglycans act as promoters of calcification originated from early studies in which cartilage preparations immersed in physiological salt solutions were shown to take up calcium ions (Pfaundler, 1904; Freudenberg and Gyorgy, 1923). The term Kalksalzfanger (lime salt catcher) was coined for the calcium-concentrating agent, now known to be proteoglycan. A means by which a Kalksalz{dnger could promote calcification was suggested by studies showing that immersion of cartilage preparations sequentially in calcium- and then phosphate-containing solutions (but not vice versa) resulted in the formation of mineral deposits in the tissue (Freudenberg and Gyorgy, 1923; Boyd and Neuman, 1951; Sobel and Burger, 1954). A mechanism of cartilage calcification consistent with these observations has recently been proposed (Hunter, 1987b). Calcium is sequestered into non-calcified cartilage
Proteoglycans and Cartilage Calcification by binding to the fixed negative charges of proteoglycans. At sites of calcification, a local increase in extracellular phosphate concentration causes the displacement of some of the bound calcium ions by an ion-exchange effect. This raises the Ca x P0 4 product above the precipitation threshold for HA. The ion-exchange mechanism of cartilage calcification predicts that proteoglycan-calcium complexes exposed to an increased phosphate concentration will act as promoters of calcification. In the present study, this prediction has been tested by quantifying formation of HA in the presence and absence of proteoglycan and CS pre-equilibrated with calcium. Under these pseudo-steady-state conditions, proteoglycan and CS increase the rate and amount of HA precipitation. Methods Purification ofproteoglycan from porcine nasal septum cartilage
Pig heads were obtained fresh from a local abbatoir, and the nasal septum cartilage removed. Proteoglycan aggregate and monomer preparations were prepared by a modification of the method of Hascall and Kimura (1982). Cartilage was cleaned, sliced finely and lyophilized. Freeze-dried tissue was ground in a Thomas-Wiley freezer mill at liquid nitrogen temperature, and then added at 30 mglml to a solution of 4 M guanidine-HCl/50 mM Tris-HCl, pH 7.4 containing proteinase inhibitors (5 mM N-ethyl maleimide, 10 mM ethylene diamine tetra-acetic acid, 0.1 M 6aminohexanoic acid, 1 mM phenyl methyl sulfonyl fluoride). After stirring at 4°C for 24 h, the extract was centrifuged at 28,000 x g for 40 min. The supernatant was retained, and the tissue residue re-extracted for a further 24 h prior to recentrifugation. The supernatants were pooled, and then concentrated approximately 4-fold by ultrafiltration using a Millipore "Minitan" concentrator with a 10,000 MW cut-off membrane. The extract was then dialyzed versus 7 volumes of distilled water (dH 2 0) plus inhibitors to reduce the guanidine-HCl concentration to 0.5 M. CsCI was added at 1.1 gig to give a density of 1.62 gI ml, and samples centrifuged at 149,000 x g for 48 - 72 h at 10 °C using a Beckman Ti 70 rotor. Gradients were harvested by needle puncture and the lower one-quarter dialyzed against dH 2 0 and lyophilized (A1). Half the sample was redissolved in 4 M guanidine-HCl containing 0.55 gig CsC! (dissociative conditions), the remainder redissolved in 0.5 M guanidine-HCl containing 1.1 gig CsC! (associative conditions), and both ultracentrifuged as described above. Fractions corresponding to the lower one-quarter of both associative (A1A1) and dissociative (A1D1) gradients were pooled, dialyzed against 1 M NaC! to convert proteoglycans to the sodium salt, then dialyzed against dH 2 0 and lyophilized.
363
Pseudo-steady-state system
Chondroitin 4-sulfate (CS) from bovine trachea was obtained from the Sigma Chemical Company. This was dissolved at 50 mglml in 100 ml of 2.2 mM CaCh/150 mM NaC!, sterilized by passage through a 0.451lm membrane filter, and dialyzed versus 3.91 of the same CaC! 2 /NaCI solution at 37°C for 16 h. Following dialysis, the volume of the CS solution was measured (some increase in volume typically occurred, due to osmotic effects). To determine the amount of calcium bound to CS, the calcium concentrations in the CS solution ("dialyzate") and the CaCh/NaCI solution against which the CS had been dialyzed ("diffusate") were measured by inductively-coupled plasma emission spectroscopy. 50-ml aliquots of dialyzate and diffusate were lyophilized, then redissolved in 25 ml of distilled water (dH 2 0). CS, at the same concentration as in the dialyzate, was added to one aliquot of diffusate prior to addition of dH 2 0. Redissolved dialyzate, diffusate and diffusate plus CS were added to equal volumes of 2.64 mM sodium phosphate, pH 7.4. Triplicate 15-ml aliquots of these final solutions were then added to thermostatable beakers (Mettler) fitted with custom-made perspex inserts and incubated for 1000 min at 37°C on Mettler DL-21 autotitrators operating in the pH stat mode, using 50 mM NaOH as titrant. The mineral formed was shown to be HA by powder X-ray diffraction (Department of Geology, University of Alberta). Experiments involving proteoglycan were performed using a modification of the method used for CS. The proteoglycan concentrations used were 10 and 25 mg/m!. At these concentrations, no changes in volume occurred during dialysis. Aliquots of 5 or 6 ml were run on autotitrators. CS used for comparison in proteoglycan experiments was extracted from porcine nasal septum cartilage by alkaline hydrolysis and purified by ion-exchange chromatography on Dowex 1-X2 (Mathews, 1976).
Results A pseudo-steady-state system for determining the effect ofproteoglycan and chondroitin sulfate on hydroxyapatite formation
The method used to compare the effects of proteoglycan and CS on HA formation under non-steady-state and pseudo-steady-state conditions is shown in Fig.1. To achieve pseudo-steady-state conditions, HA formation was initiated by addition of phosphate to a solution of proteoglycan or CS pre-equilibrated with calcium ("dialyzate"), and compared to HA formed by addition of phosphate to an aliquot of the solution against which the proteoglycan or CS had been dialyzed ("diffusate"). Dialyzate and diffusate solutions should therefore contain the same free calcium concentration, but dialyzate will also contain calcium
364
G. K. Hunter and S. K. Szigety
bound to proteoglycan or CS. To compare results from this pseudo-steady-state system with those previously obtained under non-steady-state conditions, proteoglycan and CS (Na salts) were added directly to additional aliquots of diffusate solution prior to addition of phosphate. Titration data were quantitated in terms of three parameters: the maximal rate of addition of NaOH (A max ), the time at which half the final volume of NaOH was added (to.s), and the total volume of NaOH added at 1000 min (V 1000)' Amax , to.s and V1000 are therefore measures of the maximal rate of HA formation, the precipitation lag time, and the total amount of HA formed, respectively. Effect ofchondroitin sulfate on hydroxyapatite formation under non-steady-state and pseudo-steadystate conditions
In initial studies, the effects on HA formation of CS were determined under non-steady-state and pseudo-steadystate conditions. The initial CS concentration used was 50 mg/ml, but this decreased to 38 mg/ml during dialysis. Equilibration of CS with CaCh/NaCI resulted in a amount of calcium binding equivalent to approximately twice the free calcium concentration (Table I). When added to phosEffect of Proteoglycan on Hydroxyapatite Formation Under Non-Steady-State and Pseudo-Steady-State Conditions Semi-Permeable Membrane
i/
CaCI 2 /NaCI : I I
---PG
I
I I I I
CaF ~CaF"'Ca-PG
!
/\
- Lyophyllze sample - redissolve in H2O • add P0 4 • measure HA formed
~
Control
!
"dialysate"
"diffusate"
- Lyophyllze sample - redissolve In H2O
~
- Lyophyllze sample - redissolve In H2O
- add P04 • add PG • measure HA formed
- add P04 • measure HA formed
Non Steady-State
Pseudo Steady-State
!
A
0.500
00400 0.300 0.200 0.100
E
0.000 0.600
"0
0.500
"0 "0
00400
I
0.300
CIl
0.200
Q)
« 0
Z
"0
>
B
0.100 0.000 0.600 0.500
00400 0.300 0.200 0.100 0.000 0
100
200
300
400 500
600 700
800
900 1000
Time (min.) Fig. 2. Effect of chondroitin sulfate on hydroxyapatite formation under pseudo-steady-state and non-steady-state conditions. (a). Diffusate (control). (b). Diffusate plus 38 mg/ml CS (non-steady-state). (c). Dialyzate (pseudo-steady-state).
phate, the CS dialyzate resulted in a rate and amount of HA precipitation significantly greater, and a lag time slightly shorter, than the corresponding diffusate (Figure 2 a and 2c, Table I). Addition of diffusate plus non-pre-equilibrated CS to phosphate, in contrast, resulted in no formation of HA (Figure 2 b).
[37"C' 16" I
0.600
!
Fig. 1. Method used to study effects of proteoglycan on hydroxyapatite formation under non-steady-state and pseudo-steady-state conditions. PG - proteoglycan; CaF - free calcium; Ca-PG - calcium bound to proteoglycan.
Effect ofcartilage proteoglycan on hydroxyapatite formation under non-steady-state and pseudo-steadystate conditions
Proteoglycan monomers (AlDl) and aggregates (AlAI) were prepared from porcine nasal septum cartilage. The AlAI preparation used was shown to contain approximately 70% aggregate by chromatography on Sepharose CL-2B under non-dissociative conditions (not shown). The effects of proteoglycan monomers and aggregates on HA formation at concentrations of 10 mg/ml were determined using the pseudo-steady-state system described above. As shown in Table II, pre-equilibration of 10 mg/ml proteoglycan with CaCl2 /NaCI resulted in binding of calcium, although less so than 38 mg/ml CS. When added to
Proteoglycans and Cartilage Calcification
365
Table 1. Titration parameters for the effect of chondroitin sulfate on hydroxypatite formation under pseudo-steady-state and nonsteady-state conditions.
diffusate: diffusate + CS: dialyzate:
Ca(mM)
Amax (mlJh)
VIQOO (ml)
to.s (min)
2.28 2.28 6.20
0.0047 ± 0.001 0.002 ± 0 0.D17 ± 0.001 (P
E
0.250
"0
0200
"0 "0
0.150
Q)
i i
0.050 .,'
-
B
0.0000
3
.....j /
/ :
."'-
100
200 300 400
500 600 700 800 900 1000
Time (min) Fig. 4. Effect of proteoglycan monomer and chondroitin sulfate on hydroxyapatite formation under pseudo-steady-state conditions. Line 1: CS diffusate (2.34 mM Cal. Line 2: AlDl diffusate (2.38 mM Cal. Line 3: A1Dl (25 mg/ml) dialyzate (5.22 mM Cal. Line 4: CS (25 mg/ml) dialyzate (5.22 mM Cal. The Ca values given in parenthesis are the total Ca concentrations measured in diffusates and dialyzates by inductively coupled plasma emission spectroscopy.
0.100 0.050 0.000
c
0.250 0.200 0.150 0.100 0.050 0.000
----------------------------
o
100
200 300
400
500 600
700
800 900 1000
Time (min) Fig.3. Effect of cartilage proteoglycan monomer and aggregate preparations on hydroxyapatite formation under pseudo-steadystate and non-steady-state conditions. (a). Diffusate. (b). A1Dl (10 mg/ml) dialyzate. (c). AlAI (10 mg/ml) dialyzate. Dashed lines in (b) and (c) represent diffusate solutions to which were added 10 mg/ml AID1 and AlAI, respectively, prior to the addition of phosphate.
Maroudas et al. (1988). These findings indicate that substantial amounts of calcium binding to proteoglycan occur under physiological conditions. Addition of phosphate to control solutions containing non-pre-equilibrated CS at 38 mg/ml and proteoglycan at 10 mg/ml (non-steady-state condition) resulted in complete inhibition of HA formation. Taken together with the calcium analyses described above, this suggests strongly that the ability of proteoglycan and CS to inhibit HA formation and growth under non-steady-state conditions is largely due to calcium binding effects, despite what has been claimed by others (Cuervo et aI., 1983; Chen and Boskey,
1985; Blumenthal, 1981). In marked contrast, addition of phosphate to pre-equilibrated CS and proteoglycan solutions resulted in maximum rates and final amounts of HA precipitation that were significantly higher than those obtained with the corresponding diffusate control (solutions of identical calcium and phosphate activity). Based on these results, it seems reasonable to suggest that proteoglycan at physiological concentration (approximately 50 mg/ml) will act as a promoter of HA formation under pseudo-steady-state conditions. There are two possible mechanisms by which proteoglycan may promote the formation of HA under the conditions used in this study. First, phosphate may displace calcium bound to proteoglycan by competing with the proteoglycan anionic groups, thereby raising the (free) Ca X P0 4 product (Hunter, 1987b; Hunter and Bader, 1989). Second, precipitation of HA may decrease the free calcium concentration sufficiently that the calcium-proteoglycan equilibrium is affected, causing release of bound calcium (Hunter, 1991). The former effect should decrease the precipitation lag time, but only slightly increase the final amount of precipitation; the latter effect should not affect the lag time, but significantly increase the amount of precipitation. As pre-equilibrated proteoglycan and CS increase the amount of HA formation, it appears that the calcium reservoir effect occurs in this system. However, the precipitation lag time is sufficiently variable that it is not possible to determine whether the calcium displacement effect is also occurring. These findings suggest that proteoglycans may act as promoters, not inhibitors, of cartilage calcification. This conclusion assumes that the proteoglycans of pre-calcified cartilage are in equilibrium with the calcium of surrounding
Proteoglycans and Cartilage Calcification tissues and fluids. A large body of literature, discussed in Hunter and Bader (1989), indicates that the calcium content of cartilage is approximately 10-20 mmole/kg wet weight, 4 to 8-fold higher than that of serum. The detailed studies of Maroudas and co-workers have shown that the calcium content of cartilage correlates with the fixed negative charge density of proteoglycans, and that similar calcium-sequestering activites are observed with cartilage in vivo and in vitro, and with purified proteoglycans (Maroudas, 1980; Maroudas et aI., 1988). There seems little reason to doubt that cartilage matrix is in ionic equilibrium with the surrounding fluids, and therefore contains a large reservoir of proteoglycan-bound calcium. In order for proteoglycan to act as a promoter of calcification, it is also necessary to postulate an local elevation in extracellular phosphate concentration, presumably by release from chondrocytes. Such a phosphate-elevating mechanism could involve alkaline phosphatase, which is present on the plasma membrane of hypertrophic chondrocytes, or matrix vesicles, extracellular membrane-bounded bodies found in calcifying tissues. The presence of this mechanism might also explain why growth plate, but not other cartilages, undergoes calcification. Rather than acting as promoters of the primary calcification event, it is also possible that proteoglycans supply calcium ions for the growth of HA crystals nucleated by another mechanism. However, it should be noted that the studies reported above deal only with de novo crystal formation, and that the effect of pre-equilibrated proteoglycan on seeded growth of HA has not been determined. In conclusion, the findings reported above provide a molecular-level explanation for the observation that calcification occurs in cartilage preparations exposed sequentially to calcium- and phosphate-containing solutions (Freudenberg and Gyorgy, 1923; Boyd and Neuman, 1951; Sobel and Burger, 1954). Addition of cartilage to calcium solution causes binding of calcium ions to proteoglycan; subsequent addition of cartilage to phosphate solution causes displacement of calcium ions and formation of HA. That a similar sequence of events may occur in vivo is suggested by the observation that accumulation of calcium in the matrix of growth plate cartilage preceeds the accumulation of phosphate (Althoff et aI., 1982; Shapiro and Boyd, 1984). Therefore, proteoglycan may function as a cation-exchanging calcium reservoir in the calcification of cartilage. Footnote 1 The terms "bind" and "binding" are used here without distinction between atmospheric (territorial) binding and site (specific) binding (Manning, 1969).
Acknowledgements We thank Sophie Lehocky for expert technical assistance. This work was supported by the Medical Research Council of Canada.
367
References Althoff, J., Quint, P., Krefting, E.-R. and Hohling, H.J.: Morphological studies on the epiphyseal growth plate combined with biochemical and X-ray microprobe analyses. Histochemistry 74: 541-552, 1982. Blumenthal, N.C.: Mechanism of proteoglycan inhibition of hydroxyapatite formation. In: The Chemistry and Biology of Mineralized Connective Tissues, ed. by Veis, A., Elsevier, Holland, 1981, pp. 509-515. Blumenthal, N.C., Posner, A.S., Silverman, A.D. and Rosenberg, L. c.: Effect of proteoglycans on in vitro hydroxyapatite formation. Calcif. Tissue Int. 27: 75-82, 1979. Boyd, E. S. and Neuman, W. F.: The surface chemistry of bone. V. The ion-binding properties of cartilage. j. Bioi. Chem. 193: 243-251,1951. Brighton, C. T.: The growth plate. Orthopedic Clinics N. America 15: 571-595, 1984. Chen, c.-c. and Boskey, A.L.: Mechanisms of proteoglycan inhibition of hydroxyapatite growth. Calcif. Tissue Int. 37: 395-400,1985. Chen, c.-c. and Boskey, A. L.: The effects of proteoglycans from different cartilage types on in vitro hydroxyapatite proliferation. Calcif. Tissue Int. 39: 324-327, 1986. Chen, c.-c., Boskey, A. L. and Rosenberg, L. c.: The inhibitory effects of cartilage proteoglycans on hydroxyapatite growth. Calcif. Tissue Int. 36: 285 - 290, 1984. Cuervo, L. A., Pita, J. C. and Howell, D.S.: Inhibition of calcium phosphate mineral growth by proteoglycan aggregate fractions in a synthetic lymph. Calc. Tiss. Res. 13: 1-10,1973. Freudenberg, E. and Gyorgy, P.: Der Verkalkungsvorgang bei der Entwicklung des Knochens. Ergebnisse d. inn. Med. 24: 17- 28, 1923. Hascall, V. C. and Kimura, J. H.: Proteoglycans: isolation and characterization. Meth. Enzymol. 82: 769-800, 1982. Hunter, G. K.: Chondroitin sulfate-derivatized agarose beads: A new system for studying cation binding to glycosaminoglycans. Anal. Biochem. 165: 435-441, 1987a. Hunter, G. K.: An ion-exchange mechanism of cartilage calcification. Connect. Tissue Res. 16: 111-120, 1987b. Hunter, G. K.: Role of proteoglycan in the provisional calcification of cartilage. A review and reinterpretation. Clin. Orthop. Rei. Res. 262: 256-280, 1991. Hunter, G.K., Allen, B.L., Grynpas, M.D. and Cheng, P.-T.: Inhibition of hydroxyapatite formation in collagen gels by chondroitin sulfate. Biochem. J. 228: 463 -469, 1985. Hunter, G.K. and Bader, S.M.: A mathematical modelling study of epiphyseal cartilage calcification. j. Theor. BioI. 138: 195-211,1989. Manning, G.S.: Limiting laws and counterion condensation in polyelectrolyte solutions. I. Colligative properties. j. Chem. Phys. 51: 924-933, 1969. Maroudas, A.: Physical chemistry of articular cartilage and the intervertebral disc. In: The Joints and Synovial Fluid, Vol. 2, ed. by Sokoloff, L., Academic Press, New York, 1980, pp.239-291. Maroudas, A., Weinberg, P.D., Parker, K.H. and Winlove, c.P.: The distributions and diffusivities of small ions in chondroitin sulphate, hyaluronate and some proteoglycan solutions. Biophys. Chem. 32: 257-270, 1988. Mathews, M. B.: Purification of skeletal keratan sulfate. In: The Methodology of Connective Tissue Research, ed. by Hall, D. S., Joynson-Bruvvers, 1976,pp.147-151. pfaundler, M.: Dber die Elemente der Gewebsverkalkung und ihre
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Beziehung zur Rachitisfrage. Jahrb. Kinderheilk. 60: 123 -177, 1904. Read, R., Sutherland, J. and Gosh, P.: The matrix components of the epiphyseal growth plate and articular cartilages from dogs treated with ammonium tetrathiomolybdate, a copper antagonist. Aust. J. Exp. Bioi. Med. Sci. 64: 545 - 562, 1986. Shapiro, I. M. and Boyde, A.: Microdissection-elemental analysis of the mineralizing growth cartilage of the normal and rachitic chick. Metab. Bone Dis. Rei. Res. 5: 317-326, 1984. Sobel, A.E. and Burger, M.: Calcification. XIV. Investigation of
the role of chondroitin sulfate in the calcifying mechanism. Proc. Soc. Exp. Bioi. Med. 87: 7-13, 1954. Wuthier, R.E.: A zonal analysis of inorganic and organic constituents of the epiphysis during endochondral calcification. Calc. Tiss. Res. 4: 20-38, 1969. Dr. Graeme K. Hunter, Department of Oral Biology, Faculty of Dentistry, University of Alberta, Edmonton, Alberta, T6G 2N8, Canada.
