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Regional distribution of water and glycosaminoglycan in immature articular cartilage
173
Biochimica et Biophysica Acta, 500 (1977) 1 7 3 - - 1 8 6 © E l s e v i e r / N o r t h - H o l l a n d Biomedical Press
BBA 28338
R E G I O N A L D I S T R I B U T I O N OF WATER AND GLYCOSAMINOGLYCAN IN IMMATURE A R T I C U L A R C A R T I L A G E
M.B.E. SWEET *, E.J.-M.A. T H O N A R and A.R. I M M E L M A N
Department of Orthopaedic Surgery, University of the Witwatersrand, Johannesburg (Republic of Sou th Africa) (Received March 14th, 1977)
Summary The distribution of water and glycosaminoglycan in different functional regions of bovine immature articular cartilage were studied. There was always more water in each articulating than in the corresponding growing zone, but there was less water in bot h zones in the areas of m a x i m u m contact. There was more h y alu r o n ate and much more keratan sulphate in the articulating areas of m a x imu m c o n t a c t than in the minimum c o n t a c t areas. In the growing zone the distribution of these two glycosaminoglycans did not vary as significantly but there was slightly more keratan sulphate in the area of m axi m um contact. Regional variations in chondroitin sulphate c o n t e n t were also present although n o t as striking as those of keratan sulphate. The results suggest that some keratan sulphate may be synthesized as a reaction to load.
Introduction The c h o n d r o c y t e s of articular cartilage lie in an abundant extracellular matrix co mp o s ed of collagen fibres and an a m o r p h o u s gel. The gel contains polyanionic substances of large molecular weight know n as proteoglycans; these substances are responsible for m a ny of the physical properties of cartilage. Proteoglycans consist of a central protein core to which side chains of glycosaminoglycans (chondroitin sulphate and keratan sulphate) are covalently attached [1]. One end of the protein core has no attached c a r b o h y d r a t e [2] but is capable o f aggregating with h y a l u r o n a t e know n to be present in cartilage [31. The glycosaminoglycan c o n t e n t of cartilage varies not only with dept h [4,5] but also in topographically different areas t hat are essential or non-essential for articulation [6]. This paper describes the topographical distribution of glycos* To whom correspondence should be addressed.
174 aminoglycans of both the articulating (superficial) and growing (deep) layers (zones) of immature bovine articular cartilage. Materials and Methods Tissue. The knee joints of four calves, 3 months of age, were obtained from the abattoir immediately after slaughter. After wiping clean the articular surfaces, full-thickness blocks of cartilage, 3 mm 2, were removed from the ridge forming the lateral border of the patellar surface (regions 1--3) and from the lateral condyle (regions 4--6) of the distal end of the femur as illustrated in Fig. 1. From each block one slice (1 mm thickness) was removed parallel to the free surface from both the articulating zone (including the free surface) and the growing zone 1 mm above the calcified layer. Care was taken to exclude all mineralized tissue. Tissue from each region and zone was pooled. Chemicals. Apart from the following, all chemicals used were analytical grade: galactosamine hydrochloride, glucosamine hydrochloride, glucuronolactone, carbazole and acetyl acetone. The last named was redistilled. A suspension of twice crystallized papain was purchased from British Drug Houses, Poole, England, as was testicular hyaluronidase. Chondroitinase AC from A r t h r o b a c t e r aurescens and hyaluronidase from S. h y a h i r o l i t i c u s were purchased
'~t
I
ANT,
,I LAT
MED i I
POST. F i g . 1. C a l f f e m u r . A b o v e : a r e a s 1 - - 6 a r e i n d i c a t e d i n l a t e r a l p r o j e c t i o n . B e l o w : t h e a r t i c u l a r s u r f a c e o f t h e d i s t a l f e m o r a l e p i p h y s i s i n d i a g r a m m a t i c f o r m w i t h i n d i c a t i o n o f t h e r e g i o n s s t u d i e d . A r e a s 1, 2 a n d 3 are situated on the ridge lateral to the patellar groove, while areas 4, 5 and 6 arc situated on the caudal summit. Areas of maximum contact are shaded.
175
from Miles Laboratories, Elkhardt, Ind., U.S.A. and from Sigma Chemicals Co., St. Louis, Mo., U.S.A. respectively. ECTEOLA-cellulose (capacity 0.31 mequiv./g, lot 92C-2170) was supplied by Sigma Chemicals. International Reference Standards of glycosaminoglycans were a gift of Dr. Martin B. Mathews, University of Chicago, Ill., U.S.A. Determination of water content. Slices of cartilage were weighed immediately following excision and again after dehydration in successive changes of absolute ethanol, ethanol/ether (1 : 1, v/v) and ether, and dessication in vacuo at 60°C. A possible source of error, removal of some of the lipid by this procedure, was ignored as lipid accounts for only about 1% of the wet weight of cartilage [7]. Analytical methods. Uronic acid was determined by an automated modification (Sweet, M.B.E. and Immelman, A.R., unpublished) of the carbazole method [8] using glucuronolactone as a standard. Galactose was determined enzymatically using galactose dehydrogenase according to the manufacturer's instructions (Boehringer, Mannheim). Hexosamine was determined by the method of Cessi and Piliego [9], using galactosamine hydrochloride and glucosamine hydrochloride as standards. Samples were hydrolysed in 4 m HC1 for 4 h at 100°C in sealed tubes under nitrogen. Galactosamine and glucosamine were determined on a Beckman Automatic Amino Acid Analyzer (15 cm column of Beckman M 81 resin). Samples were hydrolysed under similar conditions as above and corrected for losses occurring during hydrolysis. Excess acid was removed by lyophilisation. Hydroxyproline was measured by the m e t h o d of Woessner [10] after hydrolysis of tissue in 6 M HC1 at 100°C for 24 h. Excess acid was removed by evaporation over NaOH pellets in vacuo and the dry residue was dissolved in a known volume of water before analysis. Preparation o f glycosaminoglycans. Tissue was dehydrated as above, weighed and digested by crystalline papain (0.1 mg/100 mg dry tissue) in 0.1 M sodium acetate (pH 5.5) containing 5 mM L-cysteine and 0.05 M EDTA (disodium salt) for 4 h at 60°C [11]. Glycosaminoglycans were precipitated from the digest by the addition of three volumes of 0.5 M potassium acetate in ethanol following removal of the papain by precipitation with trichloroacetic acid. Chromatography on ECTEOLA-cellulose. Aliquots of the glycosaminoglycans were transferred quantitatively to columns (4 × 60 mm) of ECTEOLAcellulose for ion-exchange chromatography as described by Antonopoulos et al. [12]. The columns were eluted with 1 ml of water, 0.02 M HC1, 0.3 M NaC1, 2.5 M sodium formate, water and 6 M HCI. With the exception of the 6 M HC1 fraction which was dried in vacuo over NaOH, each fraction was precipitated by the addition of three volumes of 0.5 M potassium acetate in ethanol. All samples were analysed for glucosamine and galactosamine. For larger scale preparations ECTEOLA-cellulose columns of 1 × 16 cm were used and eluted by 30 ml of each solution listed above. Fractions were concentrated to 3 ml by filtration dialysis using an Amicon Diaflo UM 2 membrane (Amicon Corporation, Lexington, Mass., U.S.A.) before precipitating the glycosaminoglycans as before. Further fractionation of the glycosaminoglycans eluted by 2.5 M sodium formate and water and by 6 M HC1 was attempted by determining the proportion of galactosamine- and glucosamine-containing substances precipitable and non-precipitable by the addition of 10% cetylpyridinium chloride. The potassium salts of the glycosaminoglycans of the cetylpyridinium
176 chloride p r e c i p i t a t e and the s u p e r n a t a n t were r e c o v e r e d and t r e a t e d with 0.5 M K O H at 4°C u n d e r nitrogen for 24 h b e f o r e f u r t h e r p r e c i p i t a t i o n with cetylp y r i d i n i u m chloride or e t h a n o l . Electrophoresis. Samples o f f r a c t i o n a t e d g l y c o s a m i n o g l y c a n s were subjected to e l e c t r o p h o r e s i s on cellulose a c e t a t e strips b y a m o d i f i c a t i o n [13] o f the m e t h o d o f Seno et al. [ 1 4 ] . Enzymatic digestion. T h e i d e n t i t y o f the f r a c t i o n a t e d g l y c o s a m i n o g l y c a n s was c o n f i r m e d b y e l e c t r o p h o r e s i s b e f o r e and a f t e r specific e n z y m e digestion. T h e e n z y m e s testicular h y a l u r o n i d a s e [ 1 5 ] , h y a l u r o n i d a s e f r o m S. hyaliticus [16] and c h o n d r o i t i n a s e AC [17] were used according to published m e t h o d s . Results
Whole tissue analysis As described elsewhere [ 1 8 , 1 9 ] the articular and growing zones o f i m m a t u r e articular cartilage are m o r p h o l o g i c a l l y and b i o c h e m i c a l l y distinct. The results of the p r e s e n t investigation c o n f i r m and e x t e n d these findings. Thus the water c o n t e n t was c o n s i s t e n t l y higher in each area o f the articulating z o n e t h a n in the c o r r e s p o n d i n g areas o f the growing zone. However, the w a t e r c o n t e n t was n o t c o n s t a n t t h r o u g h o u t each zone; it was lowest in areas 5 and 6, the region o f m a x i m u m c o n t a c t (Table I). The results in Table I indicate t h a t the uronic acid and h e x o s a m i n e c o n t e n t o f t h e growing z o n e was higher t h a n t h a t of the articulating z o n e w h e n wet tissue was e x a m i n e d (Figs. 2a and 2b). The differences were r e d u c e d w h e n c a r b o h y d r a t e c o n t e n t was expressed as a f u n c t i o n o f d r y tissue (Table I and Figs. 2c and 2d). This was m o s t m a r k e d in the areas o f m a x i m u m c o n t a c t (4, 5 and 6). I n d e e d in area 5 t h e r e was m o r e h e x o s a m i n e in the d r y articulating z o n e t h a n in the growing zone. Thus the variable w a t e r c o n t e n t had an i m p o r t a n t bearing on the relative c o n c e n t r a t i o n s o f uronic acid and h e x o s a m i n e in the t w o zones. T h e m o l a r ratios o f h e x o s a m i n e to uronic acid were similar in all regions o f the growing z o n e and areas 1--3 o f the articulating zone, b u t s h o w e d an increase in the articulating areas 4, 5 and 6 (Fig. 2f). Similarly, little variation in the galactosamine to glucosamine m o l a r ratios was a p p a r e n t in the growing z o n e , b u t the ratio fell f r o m 13.95 in area 1 to 3.91 in area 5 o f the articulating z o n e (Fig. 2e). T h e results o f these relatively simple e x p e r i m e n t s indicate a fair degree o f tissue h e t e r o g e n e i t y over the joint.
Fractionation of glycosaminoglycans T h e g l y c o s a m i n o g l y c a n s were r e c o v e r e d f r o m the papain digests and fract i o n a t e d b y i o n - e x c h a n g e c h r o m a t o g r a p h y on ECTEOLA-cellulose. The i d e n t i t y o f the f r a c t i o n a t e d substances was established b y the analysis o f each f r a c t i o n for galactosamine and glucosamine and b y e l e c t r o p h o r e s i s o f individual fractions on cellulose acetate b e f o r e and a f t e r specific e n z y m a t i c digestion. The results o f these e x p e r i m e n t s are given in Table II per d r y weight as follows: F r a c t i o n s I and 2 ( H 2 0 and 0.02 M HC1) c o n t a i n g l y c o p r o t e i n s and traces o f g l y c o s a m i n o g l y c a n oligosaccharides [20] n e i t h e r o f which were d e t e c t a b l e by electrophoresis. T h e p r o p o r t i o n o f h e x o s a m i n e p r e s e n t was generally l o w e r in the growing zone. T h e r e were no significant differences b e t w e e n m i n i m u m and m a x i m u m c o n t a c t areas.
OF CARTILAGE
8.72 1.26
Hexosamine rag/100 mg dry weight rag/100 mg wet weight
6 . 7 1 _+ 0 . 2 3
6.97 0.91
6.94 0.91
86.9
6.80 0.79
6.60 0.77
88.4
* R e s u l t s o f i n d i v i d u a l a n a l y s e s o n f o u r c a l f legs.
Hydroxyproline * rag/100 mg dry weight
8.84 1.28
85.5
3
6.76 0.84
6.06 0.75
87.6
4
6 . 8 2 _+ 0 . 1 3
9.42 1.71
8.26 1.50
81.9
5
7.81 1.40
7.16 1.28
82.1
6
10.22 2.38
10.19 2.37
76.7
7 . 5 3 +_ 0 . 3 1
8.98 1.84
8.86 1.82
79.5
2
1
2
1
8.60 2.08
8.25 2.00
6.96 1.70
6.72 1.64
75.6
4
ARTICULAR
75.8
3
ZONES OF IMMATURE Growing zone areas
AND GROWING
Articulating zone areas
SAMPLES FROM THE ARTICULATING
Uronic acid rag/100 mg dry weight rag/100 mg wet weight
Water content percent of wet weight
ANALYSIS
TABLE I
7.66 + 0.18
8.10 2.46
7.80 2.36
69.7
5
CARTILAGE
7.80 2.28
7.60 2.22
70.8
6
b-* --.1
178
b
m
+; 12
k
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Fig. 2. Analysis content
per
of articulating
wet
weight
(d);
whole
digests.
weight
molar E7. . . . . .
and
(a) and
ratios
of
per
.
zones
weight
galaetosamine
i~, a x t i e u l a t i n g
zone;
.
1
growing dry
.
to •
.
2
3
of six areas
(e); hexosamine glucosamine •, growing
4
,
,
5
6
of immature content
(e) and
articular per
hexosamine
wet
cartilage: weight
to uronic
(b)
Uronic and
acid
per
acid dry
(f) of the
zone.
F r a c t i o n 3 (0.3 M NaC1). T h e g l u c o s a m i n e - c o n t a i n i n g g l y c o s a m i n o g l y c a n was identified as h y a l u r o n a t e w h i c h a c c o u n t e d for less than 3% o f total h e x o s a m i n e in all s a m p l e s and s h o w e d little variation b e t w e e n z o n e s or areas. The g a l a c t o s a m i n e - c o n t a i n i n g material was identified as c h o n d r o i t i n sulphate likely to be o f s h o r t - c h a i n or u n d e r - s u l p h a t e d f o r m . A s t u d y o f [3SS]sulphate-labelled
Galactosamine Glucosamine Molar ratio
Galactosamine Glucosamine Molar ratio
Galactosamine Glucosamine Molar ratio
Galactosamine Glucosamine Molar ratio
H20 and 0 . 0 2 M HCI
0.3 M NaCl
2.5 M s o d i u m formate and H2O
6 . 0 M HC1
* See t h e t e x t f o r d e t a i l s .
Hexosamine
Fraction
t o all e n z y m e s .
0.49 1.18 0.42
5 0.81 2.56 0.32
6
0.98 0.29 3.42
43.07 0.40 107.90
0.56 0.54 1.05
0.81 2.03 0.40
1.08 0.45 2.41
33.00 0.33 100.95
0.57 0.76 0.75
1.02 1.70 0.60
1.14 0.38 3.01
31.46 2.23 14.13
0.40 0.94 0.42
0.48 0.91 0.53
0.42 1.15 0.37
1.09 0.34 3.22
28.77 4.62 6.23
0.59 0.65 0.92
0.94 0.34 2.80
39.43 6.85 5.76
0.69 0.99 0.69
0.54 0.14 3.79
33.85 5.18 6.53
1.06 1.22 0.86
0.84 0.22 3.89
49.78 2.95 16.88
0.50 1.21 0.42
0.26 1.29 0.20 0.50 0.87 0.57
0.24 0.57 0.41
0.77 0.32 2.43
43.49 3.37 12.91
2
1
3
1
2
Growing zone areas
Articulating zone areas 4
0.50 0.88 0.57
0.55 1.46 0.38
1.44 0.63 2.29
39.56 3.00 13.18
3
0.83 0.92 0.90
0.32 0.65 0.49
1.98 0.55 3.61
31.08 2.51 12.37
4
0.76 0.95 0.80
0.38 0.86 0.45
0.77 0.31 2.50
37.59 3.61 10.43
5
0 . 3 5 C S 1'3 0.99 HA 2 0.35
0.14 0.66 0.21
1.06 4,* 0.41 4,* 2.56
3 6 . 8 0 C S 1'3 3.14 KS 4 11.72
6
F r a c t i o n a t i o n o n E C T E O L A - c e l l u l o s e o f g l y c o s a m i n o g l y c a n s p r e p a r e d f r o m p a p a i n d i g e s t s o f c a r t i l a g e s a m p l e s . T h e r e s u l t s are e x p r e s s e d as p m o l h e x o s a m i n e p e r 100 m g dry w e i g h t . The i d e n t i t y of each g l y c o s a m i n o g l y c a n was e s t a b l i s h e d by d e t e r m i n i n g the s e n s i t i v i t y to specific e n z y m a t i c d e g r a d a t i o n b e f o r e and a f t e r elect r o p h o r e s i s o n c e l l u l o s e a c e t a t e o r r e f r a c t i o n a t i o n o n E C T E O L A - c e l l u l o s e . I d e n t i t i e s arc i n d i c a t e d : H A , h y ' , d u r o n a t e ; C S , c h o n d r o i t i n s u l p h a t e ; K S , k e r a t a n sulp h a t e . S e n s i t i v i t i e s are i n d i c a t e d : l s e n s i t i v e t o t e s t i c u l a r h y a l u r o n i d a s e ; 2 s e n s i t i v e t o S t r e p t o m y c e s h y a l u r o n i d a s e ; 3 s e n s i t i v e t o c h o n d r o i t i n a s e A C ; 4 i n s e n s i t i v e
T A B L E II
¢D
180 glycosaminoglycans from similar cartilage has indicated an unsuspected degree of electrophoretic heterogeneity of the chondroitin sulphate present in this fraction (Lyons, G., Sweet, M.B.E. and T h o n a r E.J-M.A., unpublished work). As with hyaluronate, the concent r a t i on of this glycosaminoglycan did not vary significantly from sample to sample. Fractions 4 and 5 (2.5 M sodium formate and H20). Most of the chondroitin sulphate was recovered from these fractions. With the exception of area 5, there was always more chondr oi t i n sulphate in the respective growing zone, particularly in areas 1, 2 and 3. F u r t h e r m o r e , the chondroitin sulphate c o n t e n t varied from area to area in both zones. A variable a m o u n t of glucosaminecontaining material was recovered from each fraction; it was soluble in cetylpyridinium chloride (Table III) and insensitive to testicular hyaluronidase. F u r t h e r m o r e equimolar amounts of galactose were recovered with the glucosamine. Although the electrophoretic mobility of the material was similar to that o f chondroitin 6-sulphate, it may be identified as keratan sulphate. It is i m p o r t a n t to note that the c onc e nt r at i on of this " p o o l " of keratan sulphate varied. It was virtually absent from areas 1 and 2 of the articulating zone, but present in substantial amounts in the cerresponding m axi m um cont act areas, particularly area 5. On the ot her hand similar amounts were recovered from all regions o f the growth zone with the exception of area 5 where there was more. The ex tr emely small quantities of glucosamine recovered from the 2.5 M sodium f o r mate and H20 fraction of areas 1 and 2 (articulating zone) has led to the apparent paradox of a molar ratio of galactosamine : glucosamine > I in the c e t y lp y r id in iu m chloride supernatant. However, the small quantities involved make interpretation difficult. The galactosamine (11% of that eluted in this fraction) that remained in solution following the addition of cetylpyridinium chloride to the fraction of articulating area 2 (Table III) should lead to caution in attempting to calculate [21] from the molar ratios of galactosamine to glucosamine, the chain length of keratan sulphate present in the ot her samples. As the ratios stand, values of 2--3 disaccharide-repeating periods, low by any standards, would be given. F u r t h e r investigation of this material is required. Fraction 6 (6.0 M HC1). Keratan sulphate is eluted by 6.0 M HC1 from ECTEOLA-cellulose [20]. Small, relatively constant amounts of glucosamine were present in each sample (Table II) apart from in the growing zones of areas 3 and 4. Ab o u t three times as much galactosamine was eluted simultaneously in each case. A t t e m p t s to characterise the parent molecule of the galactosamine (part of which presumably formed the keratan sulphate linkage region) were hampered by the paucity of material. Almost all the glueosamine and galactosamine were soluble in cetylpyridinium chloride both before and after t r e a t m e n t with 0.5 M KOH. In addition prior digestion with testicular hyaluronidase did n o t affect the elution of either hexosamine from ECTEOLA-cellulose (Table IV). Table II shows that the concent r a t i on of galactosamine bore a quantitative relationship to the glucosamine concentration in this fraction, but not to the c o n t e n t of chondroitin sulphate or keratan sulphate in fractions 4 and 5. The variations in the glycosaminoglycan c o n t e n t of the different samples became more significant when the results were expressed on the basis of wet weight. This point is supported by the data presented in Fig. 3 which shows that the growing zone had a higher c o n t e n t of short-chain a n d / o r under-
ELUTED
FROM
ECTEOLA-CELLULOSE
(2.5
M SODIUM
FORMATE
AND
H20)
BY
33.00 (4.32)
43.49 (8.91)
39.43 (7.14)
37.59 (11.39)
G r o w i n g z o n e , area 2
A r t i c u l a t i n g z o n e , area 5
G r o w i n g z o n e , area 5
3.61 (1.09)
6.85 (1.24
3.37 (0.69)
0.33 (0.04)
10.4
5.8
12.9
100.9
36.63 (11.10)
36.89 (6.68)
42.47 (8.70)
29.34 (3.84)
GaINH 2
Molar ratio
GaINH 2
GIuNH 2
Precipitate
Untreated
A r t i c u l a t i n g z o n e , area 2
Cartilage
0.73 (0.22)
1.60 (0.29)
0.93 (0.19)
0.08 (0.01)
GIuNH 2
50.2
23.1
45.7
366.8
Molar • ratio
0.96 (0.29)
2.54 (0.46)
1.02 (0.21)
3.66 (0.48)
GalNH 2
2.88 (0.87)
5.25 (0.95)
2.44 (0.50)
0.25 (0.03)
GluNH 2
0.33
0.48
0.42
14.6
Molar ratio
Cetylpyridinium chloride supernatant
C h o n d r o i t i n a s e AC d e g r a d a t i o n c o n f i r m e d t h e p r e s e n c e o f c h o n d r o i t i n s u l p h a t e in t h e c e t y l p y r i d i n i u m c h l o r i d e p r e c i p i t a t e s . T h e m a t e r i a l in t h e s u p e r n a t a n t w a s r e s i s t a n t to t e s t i c u l a r h y a l u r o n i d a s e a p a r t f r o m t h a t in a r e a 2. T h e r e s u l t s arc e x p r e s s e d as p m o l / 1 0 0 m g d r y w e i g h t a n d (in p a r e n t h e s e s ) p m o l / 1 0 0 m g w e t w e i g h t .
FURTHER FRACTIONATION OF GLYCOSAMINOGLYCANS PRECIPITATION WITH CETYLPYRIDINIUM CHLORIDE
T A B L E III
FRACTIONATION
OF
GLYCOSAMINOGLYCANS
ELUTED
FROM
1.084 (0.142)
0.771 (0.158)
0.945 (0.171)
0.766 (0.232)
G r o w i n g zone, area 2
A r t i c u l a t i n g zone, area 5
G r o w i n g zone, a r e a 5
GaINH 2
Untreated
A r t i c u l a t i n g zone, area 2
Cartilage
0.307 (0.093)
0.337 (0.061)
0.317 (0.065)
0.450 (0.059)
GIuNH 2
GalNH 2
0.084 (0.011) 0.068 (0.014) 0.072 (0.013) 0.076 (0.023)
Molar ratio 2.41
2.43
2.80
2.50
Precipitate
0.007 (0.002)
0.006 (0.001)
0.020 (0.004)
0.015 (0.002)
GluNH 2
(6.0M
10.86
12.00
3.40
5.50
Molar ratio
ECTEOLA-CELLULOSE
T h e r e s u l t s are e x p r e s s e d a s p m o l / 1 0 0 m g d r y w e i g h t a n d (in p a r e n t h e s e s ) p m o l / 1 0 0 m g w e t w e i g h t .
PYRIDINIUM CHLORIDE (SEE TEXT)
FURTHER
T A B L E IV ADDITION
OF
CETYL-
0.690 (0.209)
0.873 (0.158)
0.702 (0.144)
1.000 (0.131)
GalNH 2
0.300 (0.091)
0.331 (0.060)
0.298 (0.061)
0.435 (0.057)
GluNH2
2.30
2.64
2.36
2.30
Molar ratio
Cetylpyridinium chloride supernatant
HC1) BY T H E
b~
183 ARTICULATING
ZONE
GROWING
ZONE
lOARTICULATIKG
ZONE
GROWING
ZONE
I
~
I
~
H20 +
~
~o-
i
~ - -
-- ~
----
---
=
--
_-- ~ -
~
I-
M
HC[
H2 O+
0.02 M Her
=
m
0.02
~
=
--
~
--
~
=
~
~
~
~
0.3M
NaCI
.~,-
~
215M
2.5 M H C O O N a
HCOONa
+ H20
÷ H20 20-
i-
10-
6.0M
HCI
~
-
-
-
-
l
i
-
-
I
6.0M
HCt
1o-
i-
1 2 3 4
56
1 2 3 4
AREAS
S
6
1 2 3 4
S
6
12
34
56
AREAS
Fig. 3. F r a c t i o n a t i o n o f g l y c o s a m i n o g l y c a n s o f e a c h s a m p l e on E C T E O L A - c e l l u l o s e . T h e r e s u l t s are r e p r e s e n t e d as p m o l h e x o s a m i n e p e r 1 0 0 m g w e t c a r t i l a g e . G a l a c t o s a m i n e (m) a b o v e t h e line; g l u c o s a m i n e (~) b e l o w t h e line. Fig. 4. F r a c t i o n a t i o n o f g l y c o s a m i n o g l y c a n s o f e a c h s a m p l e o n E C T E O L A - c e l l u l o s e . T h e r e s u l t s are r e p r e s e n t e d as p e r c e n t a g e s of t h e t o t a l h e x o s a m i n e in e a c h s a m p l e . G a l a c t o s a m i n e (m) a b o v e t h e line; g l u c o s a m i n e (~) b e l o w t h e line.
s u l p h a t e d c h o n d r o i t i n sulphate, h y a l u r o n a t e and c h o n d r o i t i n sulphate t h a n the articulating zone. F u r t h e r m o r e , a t r e n d n o t obvious in the d r y tissue, was the increased h y a l u r o n a t e c o n t e n t o f areas 5 and 6 o f the articulating zone. In contrast t o the results f o r the d r y tissue, t h e c o n t e n t o f k e r a t a n sulphate (eluted b y 2.5 M s o d i u m f o r m a t e and H~O) o f the t w o zones o f the m a x i m u m c o n t a c t areas (areas 4, 5 and 6) were virtually identical (Fig. 3).
Relative proportion of galactosamine and glucosamine in each glycosaminoglycan pool In c o m p a r i n g t h e p r o p o r t i o n s o f h e x o s a m i n e p r e s e n t in each fraction, certain t r e n d s n o t n o t i c e a b l e in t e r m s o f a b s o l u t e c o n c e n t r a t i o n s , b e c a m e clear (Fig. 4). T h e r e was very little d i f f e r e n c e b e t w e e n t h e t w o zones o f area 3. T h e p r o p o r t i o n o f c h o n d r o i t i n sulphate p r e s e n t was similar in the t w o zones o f the m i n i m u m c o n t a c t area, b u t greater in the growing z o n e o f t h e m a x i m u m c o n t a c t areas. T h e p r o p o r t i o n o f k e r a t a n sulphate present in all areas o f the growing z o n e s h o w e d little variation; in areas 1 and 2 o f the articulating z o n e t h e r e was p r o p o r t i o n a t e l y less, b u t in areas 4, 5 and 6, p r o p o r t i o n a t e l y m o r e .
184 Discussion The water and glycosaminoglycan c o n t e n t in the different regions was generally similar to that r e por t ed by o t h e r workers [4,5,22], but some obvious and o th er mo r e subtle differences between the individual samples studied were noted. The results show that variations in glycosaminoglycan and water c o n t e n t of the immature joint occur both with distance from the articular surface and from region to region. The degree of h y d r a t i o n of cartilage is the result of two opposing forces: water is attracted by the high osmotic pressure of the proteoglycans and their surrounding c o u n t e r ions, while the inflow of water is limited by the collagen f r a m ewo r k [23,24] and the degree of cross-linking therein. Variations in the water c o n t e n t may t her e f or e be due to variations in the glycosaminoglycan c o n t e n t , the degree of proteoglycan aggregation [25] or the elastic restraint of collagen. This last factor seems to be the most likely cause of the lower water c o n t e n t of the growing zone as it had a higher c o n t e n t of collagen. Furthermore the growing zone contained more chondroitin sulphate, particularly in the area of min im um contact, and there was no correlation between regional variations in water c o n t e n t and chondroitin sulphate c o n t e n t , although the c o n t e n t of these two substances did vary in parallel in the two zones. Considerable local variations in chondroitin sulphate c o n t e n t have been re p o r ted in cartilage of adult hum a n femoral condyle [6] but not in femoral head [24] or patella [26]. The regional variations report ed here may therefore be present at all ages in certain forms of cartilage, but not in others. The growing zone of articular cartilage has been regarded as analogous to epiphyseal cartilage [5], a view for which there is histological justification. Larsson et al. [13] r e por t ed the hyal ur ona te c o n t e n t of epiphyseal cartilage varied from 13% of the total hexosamine in the h y p e r t r o p h i c zone to 5% in the resting zone, the latter value being more than twice that of any of the results given here. This suggests a possible difference in the role of hyal uronat e in the two tissues and that the growing zone of articular cartilate is distinct from epiphyseal cartilage. This distinction is further emphasized by the presence of keratan sulphate in the growing zone and its virtual absence from epiphyseal cartilage [13,27--30]. These factors lead us to suggest that the dual function of the growing zone may result in a difference in gene expression. Indeed it has been said that mechanical factors influence the regional c o n t e n t of glycosaminoglycan [31]. The suggestion that the increase in keratan sulphate c o n t e n t o f the deepest layer of adult articular cartilate is the result of anoxia [32] is unlikely to apply to immature articular cartilage as the growing zone is nourished by vessels arising from the subchondral bone [33]. It is generally felt that the appearance and increasing c o n t e n t of skeletal keratan sulphate in cartilage is representative of maturation and ageing [34]. The p r o n o u n c e d regional variations in keratan sulphate concent rat i on between area 5 and areas 1 and 2 of the articulating zone may therefore indicate an earlier matu r atio n of the c h o n d r o c y t e s of area 5, possibly as a response to load. On the o t h e r hand any argument relating keratan sulphate c o n t e n t to load does n o t seem tenable in respect of the growing zone, particularly as this glycosaminoglycan has been d e m o n s t r a t e d in the corresponding zone of articular
185 cartilage of bovine foetuses from the second and third trimester of pregnancy (Thonar, E.J.M.A. and Sweet, M.B.E., unpublished). Recently, Hascall et al. [35] have reported keratan sulphate constituted about 7% of monomeric proteoglycan of chick limb buds from stage 23--24 embryos. Whether its presence in proteoglyean is indicative of a stage of maturity of chondrocytes or whether it has some other biological significance, is not clear at present. In the model proteoglycan proposed by Hascall and Heinegard [2] and by Rosenberg et al. [36], a proportion of the keratan sulphate is attached between clusters of chondroitin sulphate to the general polysaccharide-attachment region. Papain digestion of these substances yields a heterogeneous mixture of peptidoglycans which may contain chondroitin sulphate and keratan sulphate attached to a c o m m o n peptide apart from chondroitin sulphate-peptide and keratan sulphate-peptide [37]. This is the likely explanation for the presence of some of the glucosamine in the formate fractions and may be indicative of structural variations within the proteoglycans from different regions of the cartilage, particularly articulating area 2. In addition to the regional and zonal differences described, the results presented in this paper emphasise the importance of discrete sampling of tissue. This applied particularly to the articulating zone where the marked regional variations could be nullified by pooling or averaging the tissue or the results. The disadvantages of working with small quantities of material are obvious and often cannot be overcome by pooling tissue from different individuals. However, the development of micro- and ultramicromethods for proteoglycan isolation and characterization will hopefully lead to greater insight into the problems of local maturation or reaction to physiological load. Acknowledgements We acknowledge with thanks the financial assistance of the Faculty of Medicine Research Fund and of De Beers Industrial Diamonds Ltd. and the gift of International Reference Standards of glycosaminoglycans by Dr. Martin B. Mathews, Department of Pediatrics, University of Chicago, Ill. 60637, U.S.A. References 1 M a t h e w s , M.B. a n d L o z a i t y t e , I. ( 1 9 5 8 ) A r c h . B i o c h e m . Biophys. 74, 1 5 8 - - 1 7 4 2 Hascall, V.C. a n d H e i n e g a r d , D. ( 1 9 7 5 ) i n E x t r a c e l l u l a r Matrix I n f l u e n c e s on G c n e E x p r e s s i o n (Slavkin, H.C. a n d G r e u l i c h , R.C., eds.), pp. 4 2 3 - - 4 3 3 , A c a d e m i c Press, N e w Y o r k 3 H e i n e g a r d , D. a n d Hascall, V.C. ( 1 9 7 4 ) J. Biol. C h e m . 249, 4 2 5 0 - - 4 2 5 6 4 L e m p e r g , R . K . , L a r s s o n , S.-E. a n d H j e r t q u i s t , S.-O. ( 1 9 7 1 ) B i o c h e m . Biophys. A s p e c t s 7, 4 1 9 - - 4 2 0 5 L e m p e r g , R. a n d L a r s s o n , S.-E. ( 1 9 7 4 ) Calcif. Tissue Res. 15, 2 3 7 - - 2 5 1 6 Bjelle, A.O. ( 1 9 7 4 ) Scand. J. R h e u m a t o l . 3, 8 1 - - 8 8 7 M e a c h i m , G. a n d S t o c k w e l l , R . A . ( 1 9 7 3 ) in A d u l t A r t i c u l a r Cartilage ( F r e e m a n , M . A . R . , ed.), pp. 1-50, P i t m a n Medical, L o n d o n 8 Bitter, T. a n d Muir, H. ( 1 9 6 2 ) Anal. B i o c h e m . 4, 3 3 0 - - 3 3 4 9 Cessi, C. a n d Piliego, F. ( 1 9 6 0 ) B i o c h e m . J. 77, 5 0 8 - - 5 1 0 10 Woessner, J . F . ( 1 9 6 1 ) A r c h . B i o c h e m . 93, 4 4 0 - 4 4 7 11 S c o t t , J.E. ( 1 9 6 0 ) M e t h o d s B i o c h e m . A n a l . 8, 1 4 5 - - 1 9 7 12 A n t o n o p o u l o s , C.A., H e i n e g a r d , D. a n d Gardell, S. ( 1 9 6 7 ) B i o c h i m . Biophys. A c t a 148, 1 5 8 - - 1 6 3 13 L a r s s o n , S.-E., R a y , R.D. a n d K u e t t n e r , K . E . ( 1 9 7 3 ) Calcif. Tissue Res. 13, 2 7 1 - - 2 8 5 14 S e n o , N., A n n o , K., K o n d o , K., Nagase, S. a n d Saito, S. ( 1 9 7 0 ) Anal. B i o c h e m . 37, 1 9 7 - - 2 0 1 15 S w e e t , M.B.E., T h o n a r , E.J-M.A. a n d I m m e l m a n , A . R . ( 1 9 7 6 ) B i o c h i m . B i o p h y s . A c t a 4 3 7 , 7 1 - - 8 6
186 16 Sakaki, T., H o n d a , T., W a k u m o t o , N., Fugita, A., O k a d a , H., Ishizaki, Y., Onishi, Y., C h i k a m o r i , N. a n d H a m a g u c h i , R. ( 1 9 7 5 ) J. O s a k a D e n t . Univ. 9, 3 1 - - 4 6 17 Saito, H., Y a m a g a t a , T. a n d S u z u k i , S. ( 1 9 6 8 ) J. Biol. C h e m . 243, 1 5 3 6 - - 1 5 4 2 18 T h o n a r , E.J-M.A., S w e e t , M.B.E. a n d I m m e l m a n , A.R. ( 1 9 7 5 ) S. Afr. J. Sci. 71, 3 4 7 - - 3 4 8 19 S w e e t , M.B.E., T h o n a r , E.J-M.A. a n d I m m e l m a n , A . R . ( 1 9 7 6 ) S. Aft. J. Sci. 72, 1 6 - - 1 9 20 Bjelle, A . O . , A n t o n o p o u l o s , C.A., E n g f e l d t , B. a n d H j e r t q u i s t , S.-O. ( 1 9 7 2 ) Calcif. Tissue Res. 8, 237--246 21 L o h m a n d e r , S. ( 1 9 7 6 ) Thesis, K a r o l i n s k a I n s t i t u t e t , S t o c k h o l m 22 S i m u n e k , Z. a n d Muir, H. ( 1 9 7 2 ) B i o c h e m . J. 126, 5 1 5 - - 5 2 3 23 M a r o u d a s , A. ( 1 9 7 6 ) N a t u r e 260, 8 0 8 - - 8 0 9 24 M a r o u d a s , A., Evans, H. a n d A l m e i d a , L. ( 1 9 7 3 ) A n n . R h e u m . Dis. 32, 1--9 25 M c D e v i t t , C.A. a n d Muir, H. ( 1 9 7 6 ) J. Bone Jt. Surg. 58-B, 9 4 - - 1 0 1 26 Ficat, C. a n d M a r o u d a s , A. ( 1 9 7 5 ) A n n . R h e u m . Dis. 34, 5 1 5 - - 5 1 9 27 H j e r t q u i s t , S.-O. ( 1 9 6 4 ) A c t a Soc. Med. Upps. 69, 2 3 - - 4 0 28 D o r f m a n , A. ( 1 9 6 2 ) Fed. Proc. Fed. A m . Soc. Exp. Biol. 21, 1 0 7 0 - - 1 0 7 4 29 R o k o s o v a - C m u c h a l o v a , B. a n d B e n t l e y , J.P. ( 1 9 6 8 ) B i o c h e m . P h a r m a c o l . Suppl., pp. 3 1 5 - - 3 2 8 3 0 H a n d l e y , C.J. a n d Phelps, C.F. ( 1 9 7 2 ) B i o c h e m . J. 126, 4 1 7 - - 4 3 2 31 M a t h e w s , M.B. ( 1 9 7 5 ) C o n n e c t i v e Tissue: M a c r o m o l e c u l a r S t r u c t u r e a n d E v o l u t i o n , p. 1 5 7 , SpringerVerlag, Berlin 32 S t o c k w e l l , R.A. a n d S c o t t , J.E. ( 1 9 6 5 ) A n n . R h e u m . Dis. 24, 3 4 1 - - 3 5 0 33 M c K i b b i n , B. ( 1 9 7 3 ) in A d u l t A r t i c u l a r Cartilage ( F r e e m a n , M.A.R., ed.), pp. 2 7 7 - - 2 8 6 , P i t m a n Medical, L o n d o n 34 M a t h e w s , M.B. ( 1 9 7 3 ) in C o n n e c t i v e Tissue a n d Ageing (Vogel, H . G . , ed.), pp. 1 3 8 - - 1 3 9 35 Hascai1, V.C., O e g e m a , T . R . , B r o w n , M. a n d Caplan, A.I. ( 1 9 7 6 ) J. Biol. C h e m . 251, 3 5 1 1 - - 3 5 1 9 36 R o s e n b e r g , L., Margolis, R., W o l f e n s t e i n - T o d e l , C., Pal, S. a n d Strider, W. ( 1 9 7 5 ) in E x t r a c e l l u l a r Matrix I n f l u e n c e s o n G e n e E x p r e s s i o n (Slavkin, H.C. a n d G r e u l i c h , R.C., eds.), pp. 4 1 5 - - 4 2 1 , Acad e m i c Press, N e w Y o r k 37 S t u h l s a t z , H.W. a n d Greiling, H. ( 1 9 7 6 ) in T h e M e t h o d o l o g y of C o n n e c t i v e Tissue R e s e a r c h (Hall, D.A., ed.), pp. 1 2 9 - - 1 3 6 , J o y n s o n - B r u v v e r s , O x f o r d
Biochimica et Biophysica Acta, 500 (1977) 1 7 3 - - 1 8 6 © E l s e v i e r / N o r t h - H o l l a n d Biomedical Press
BBA 28338
R E G I O N A L D I S T R I B U T I O N OF WATER AND GLYCOSAMINOGLYCAN IN IMMATURE A R T I C U L A R C A R T I L A G E
M.B.E. SWEET *, E.J.-M.A. T H O N A R and A.R. I M M E L M A N
Department of Orthopaedic Surgery, University of the Witwatersrand, Johannesburg (Republic of Sou th Africa) (Received March 14th, 1977)
Summary The distribution of water and glycosaminoglycan in different functional regions of bovine immature articular cartilage were studied. There was always more water in each articulating than in the corresponding growing zone, but there was less water in bot h zones in the areas of m a x i m u m contact. There was more h y alu r o n ate and much more keratan sulphate in the articulating areas of m a x imu m c o n t a c t than in the minimum c o n t a c t areas. In the growing zone the distribution of these two glycosaminoglycans did not vary as significantly but there was slightly more keratan sulphate in the area of m axi m um contact. Regional variations in chondroitin sulphate c o n t e n t were also present although n o t as striking as those of keratan sulphate. The results suggest that some keratan sulphate may be synthesized as a reaction to load.
Introduction The c h o n d r o c y t e s of articular cartilage lie in an abundant extracellular matrix co mp o s ed of collagen fibres and an a m o r p h o u s gel. The gel contains polyanionic substances of large molecular weight know n as proteoglycans; these substances are responsible for m a ny of the physical properties of cartilage. Proteoglycans consist of a central protein core to which side chains of glycosaminoglycans (chondroitin sulphate and keratan sulphate) are covalently attached [1]. One end of the protein core has no attached c a r b o h y d r a t e [2] but is capable o f aggregating with h y a l u r o n a t e know n to be present in cartilage [31. The glycosaminoglycan c o n t e n t of cartilage varies not only with dept h [4,5] but also in topographically different areas t hat are essential or non-essential for articulation [6]. This paper describes the topographical distribution of glycos* To whom correspondence should be addressed.
174 aminoglycans of both the articulating (superficial) and growing (deep) layers (zones) of immature bovine articular cartilage. Materials and Methods Tissue. The knee joints of four calves, 3 months of age, were obtained from the abattoir immediately after slaughter. After wiping clean the articular surfaces, full-thickness blocks of cartilage, 3 mm 2, were removed from the ridge forming the lateral border of the patellar surface (regions 1--3) and from the lateral condyle (regions 4--6) of the distal end of the femur as illustrated in Fig. 1. From each block one slice (1 mm thickness) was removed parallel to the free surface from both the articulating zone (including the free surface) and the growing zone 1 mm above the calcified layer. Care was taken to exclude all mineralized tissue. Tissue from each region and zone was pooled. Chemicals. Apart from the following, all chemicals used were analytical grade: galactosamine hydrochloride, glucosamine hydrochloride, glucuronolactone, carbazole and acetyl acetone. The last named was redistilled. A suspension of twice crystallized papain was purchased from British Drug Houses, Poole, England, as was testicular hyaluronidase. Chondroitinase AC from A r t h r o b a c t e r aurescens and hyaluronidase from S. h y a h i r o l i t i c u s were purchased
'~t
I
ANT,
,I LAT
MED i I
POST. F i g . 1. C a l f f e m u r . A b o v e : a r e a s 1 - - 6 a r e i n d i c a t e d i n l a t e r a l p r o j e c t i o n . B e l o w : t h e a r t i c u l a r s u r f a c e o f t h e d i s t a l f e m o r a l e p i p h y s i s i n d i a g r a m m a t i c f o r m w i t h i n d i c a t i o n o f t h e r e g i o n s s t u d i e d . A r e a s 1, 2 a n d 3 are situated on the ridge lateral to the patellar groove, while areas 4, 5 and 6 arc situated on the caudal summit. Areas of maximum contact are shaded.
175
from Miles Laboratories, Elkhardt, Ind., U.S.A. and from Sigma Chemicals Co., St. Louis, Mo., U.S.A. respectively. ECTEOLA-cellulose (capacity 0.31 mequiv./g, lot 92C-2170) was supplied by Sigma Chemicals. International Reference Standards of glycosaminoglycans were a gift of Dr. Martin B. Mathews, University of Chicago, Ill., U.S.A. Determination of water content. Slices of cartilage were weighed immediately following excision and again after dehydration in successive changes of absolute ethanol, ethanol/ether (1 : 1, v/v) and ether, and dessication in vacuo at 60°C. A possible source of error, removal of some of the lipid by this procedure, was ignored as lipid accounts for only about 1% of the wet weight of cartilage [7]. Analytical methods. Uronic acid was determined by an automated modification (Sweet, M.B.E. and Immelman, A.R., unpublished) of the carbazole method [8] using glucuronolactone as a standard. Galactose was determined enzymatically using galactose dehydrogenase according to the manufacturer's instructions (Boehringer, Mannheim). Hexosamine was determined by the method of Cessi and Piliego [9], using galactosamine hydrochloride and glucosamine hydrochloride as standards. Samples were hydrolysed in 4 m HC1 for 4 h at 100°C in sealed tubes under nitrogen. Galactosamine and glucosamine were determined on a Beckman Automatic Amino Acid Analyzer (15 cm column of Beckman M 81 resin). Samples were hydrolysed under similar conditions as above and corrected for losses occurring during hydrolysis. Excess acid was removed by lyophilisation. Hydroxyproline was measured by the m e t h o d of Woessner [10] after hydrolysis of tissue in 6 M HC1 at 100°C for 24 h. Excess acid was removed by evaporation over NaOH pellets in vacuo and the dry residue was dissolved in a known volume of water before analysis. Preparation o f glycosaminoglycans. Tissue was dehydrated as above, weighed and digested by crystalline papain (0.1 mg/100 mg dry tissue) in 0.1 M sodium acetate (pH 5.5) containing 5 mM L-cysteine and 0.05 M EDTA (disodium salt) for 4 h at 60°C [11]. Glycosaminoglycans were precipitated from the digest by the addition of three volumes of 0.5 M potassium acetate in ethanol following removal of the papain by precipitation with trichloroacetic acid. Chromatography on ECTEOLA-cellulose. Aliquots of the glycosaminoglycans were transferred quantitatively to columns (4 × 60 mm) of ECTEOLAcellulose for ion-exchange chromatography as described by Antonopoulos et al. [12]. The columns were eluted with 1 ml of water, 0.02 M HC1, 0.3 M NaC1, 2.5 M sodium formate, water and 6 M HCI. With the exception of the 6 M HC1 fraction which was dried in vacuo over NaOH, each fraction was precipitated by the addition of three volumes of 0.5 M potassium acetate in ethanol. All samples were analysed for glucosamine and galactosamine. For larger scale preparations ECTEOLA-cellulose columns of 1 × 16 cm were used and eluted by 30 ml of each solution listed above. Fractions were concentrated to 3 ml by filtration dialysis using an Amicon Diaflo UM 2 membrane (Amicon Corporation, Lexington, Mass., U.S.A.) before precipitating the glycosaminoglycans as before. Further fractionation of the glycosaminoglycans eluted by 2.5 M sodium formate and water and by 6 M HC1 was attempted by determining the proportion of galactosamine- and glucosamine-containing substances precipitable and non-precipitable by the addition of 10% cetylpyridinium chloride. The potassium salts of the glycosaminoglycans of the cetylpyridinium
176 chloride p r e c i p i t a t e and the s u p e r n a t a n t were r e c o v e r e d and t r e a t e d with 0.5 M K O H at 4°C u n d e r nitrogen for 24 h b e f o r e f u r t h e r p r e c i p i t a t i o n with cetylp y r i d i n i u m chloride or e t h a n o l . Electrophoresis. Samples o f f r a c t i o n a t e d g l y c o s a m i n o g l y c a n s were subjected to e l e c t r o p h o r e s i s on cellulose a c e t a t e strips b y a m o d i f i c a t i o n [13] o f the m e t h o d o f Seno et al. [ 1 4 ] . Enzymatic digestion. T h e i d e n t i t y o f the f r a c t i o n a t e d g l y c o s a m i n o g l y c a n s was c o n f i r m e d b y e l e c t r o p h o r e s i s b e f o r e and a f t e r specific e n z y m e digestion. T h e e n z y m e s testicular h y a l u r o n i d a s e [ 1 5 ] , h y a l u r o n i d a s e f r o m S. hyaliticus [16] and c h o n d r o i t i n a s e AC [17] were used according to published m e t h o d s . Results
Whole tissue analysis As described elsewhere [ 1 8 , 1 9 ] the articular and growing zones o f i m m a t u r e articular cartilage are m o r p h o l o g i c a l l y and b i o c h e m i c a l l y distinct. The results of the p r e s e n t investigation c o n f i r m and e x t e n d these findings. Thus the water c o n t e n t was c o n s i s t e n t l y higher in each area o f the articulating z o n e t h a n in the c o r r e s p o n d i n g areas o f the growing zone. However, the w a t e r c o n t e n t was n o t c o n s t a n t t h r o u g h o u t each zone; it was lowest in areas 5 and 6, the region o f m a x i m u m c o n t a c t (Table I). The results in Table I indicate t h a t the uronic acid and h e x o s a m i n e c o n t e n t o f t h e growing z o n e was higher t h a n t h a t of the articulating z o n e w h e n wet tissue was e x a m i n e d (Figs. 2a and 2b). The differences were r e d u c e d w h e n c a r b o h y d r a t e c o n t e n t was expressed as a f u n c t i o n o f d r y tissue (Table I and Figs. 2c and 2d). This was m o s t m a r k e d in the areas o f m a x i m u m c o n t a c t (4, 5 and 6). I n d e e d in area 5 t h e r e was m o r e h e x o s a m i n e in the d r y articulating z o n e t h a n in the growing zone. Thus the variable w a t e r c o n t e n t had an i m p o r t a n t bearing on the relative c o n c e n t r a t i o n s o f uronic acid and h e x o s a m i n e in the t w o zones. T h e m o l a r ratios o f h e x o s a m i n e to uronic acid were similar in all regions o f the growing z o n e and areas 1--3 o f the articulating zone, b u t s h o w e d an increase in the articulating areas 4, 5 and 6 (Fig. 2f). Similarly, little variation in the galactosamine to glucosamine m o l a r ratios was a p p a r e n t in the growing z o n e , b u t the ratio fell f r o m 13.95 in area 1 to 3.91 in area 5 o f the articulating z o n e (Fig. 2e). T h e results o f these relatively simple e x p e r i m e n t s indicate a fair degree o f tissue h e t e r o g e n e i t y over the joint.
Fractionation of glycosaminoglycans T h e g l y c o s a m i n o g l y c a n s were r e c o v e r e d f r o m the papain digests and fract i o n a t e d b y i o n - e x c h a n g e c h r o m a t o g r a p h y on ECTEOLA-cellulose. The i d e n t i t y o f the f r a c t i o n a t e d substances was established b y the analysis o f each f r a c t i o n for galactosamine and glucosamine and b y e l e c t r o p h o r e s i s o f individual fractions on cellulose acetate b e f o r e and a f t e r specific e n z y m a t i c digestion. The results o f these e x p e r i m e n t s are given in Table II per d r y weight as follows: F r a c t i o n s I and 2 ( H 2 0 and 0.02 M HC1) c o n t a i n g l y c o p r o t e i n s and traces o f g l y c o s a m i n o g l y c a n oligosaccharides [20] n e i t h e r o f which were d e t e c t a b l e by electrophoresis. T h e p r o p o r t i o n o f h e x o s a m i n e p r e s e n t was generally l o w e r in the growing zone. T h e r e were no significant differences b e t w e e n m i n i m u m and m a x i m u m c o n t a c t areas.
OF CARTILAGE
8.72 1.26
Hexosamine rag/100 mg dry weight rag/100 mg wet weight
6 . 7 1 _+ 0 . 2 3
6.97 0.91
6.94 0.91
86.9
6.80 0.79
6.60 0.77
88.4
* R e s u l t s o f i n d i v i d u a l a n a l y s e s o n f o u r c a l f legs.
Hydroxyproline * rag/100 mg dry weight
8.84 1.28
85.5
3
6.76 0.84
6.06 0.75
87.6
4
6 . 8 2 _+ 0 . 1 3
9.42 1.71
8.26 1.50
81.9
5
7.81 1.40
7.16 1.28
82.1
6
10.22 2.38
10.19 2.37
76.7
7 . 5 3 +_ 0 . 3 1
8.98 1.84
8.86 1.82
79.5
2
1
2
1
8.60 2.08
8.25 2.00
6.96 1.70
6.72 1.64
75.6
4
ARTICULAR
75.8
3
ZONES OF IMMATURE Growing zone areas
AND GROWING
Articulating zone areas
SAMPLES FROM THE ARTICULATING
Uronic acid rag/100 mg dry weight rag/100 mg wet weight
Water content percent of wet weight
ANALYSIS
TABLE I
7.66 + 0.18
8.10 2.46
7.80 2.36
69.7
5
CARTILAGE
7.80 2.28
7.60 2.22
70.8
6
b-* --.1
178
b
m
+; 12
k
\'4
O O 29
O O i l
O
I
i
0 x
I
5L
/
J
L 4
:X.
d
c 55
~3
(2~ 5O © ©
,
~5
\
O O
\ C
t9
E
aO
o
i
35
I It
~ 0
O x
I[
30
f
e
I 3 . ~-
.o
O k)
00
u
x (9
d: ,
,
,
,
,
.
1
2
~
4
5
O
Fig. 2. Analysis content
per
of articulating
wet
weight
(d);
whole
digests.
weight
molar E7. . . . . .
and
(a) and
ratios
of
per
.
zones
weight
galaetosamine
i~, a x t i e u l a t i n g
zone;
.
1
growing dry
.
to •
.
2
3
of six areas
(e); hexosamine glucosamine •, growing
4
,
,
5
6
of immature content
(e) and
articular per
hexosamine
wet
cartilage: weight
to uronic
(b)
Uronic and
acid
per
acid dry
(f) of the
zone.
F r a c t i o n 3 (0.3 M NaC1). T h e g l u c o s a m i n e - c o n t a i n i n g g l y c o s a m i n o g l y c a n was identified as h y a l u r o n a t e w h i c h a c c o u n t e d for less than 3% o f total h e x o s a m i n e in all s a m p l e s and s h o w e d little variation b e t w e e n z o n e s or areas. The g a l a c t o s a m i n e - c o n t a i n i n g material was identified as c h o n d r o i t i n sulphate likely to be o f s h o r t - c h a i n or u n d e r - s u l p h a t e d f o r m . A s t u d y o f [3SS]sulphate-labelled
Galactosamine Glucosamine Molar ratio
Galactosamine Glucosamine Molar ratio
Galactosamine Glucosamine Molar ratio
Galactosamine Glucosamine Molar ratio
H20 and 0 . 0 2 M HCI
0.3 M NaCl
2.5 M s o d i u m formate and H2O
6 . 0 M HC1
* See t h e t e x t f o r d e t a i l s .
Hexosamine
Fraction
t o all e n z y m e s .
0.49 1.18 0.42
5 0.81 2.56 0.32
6
0.98 0.29 3.42
43.07 0.40 107.90
0.56 0.54 1.05
0.81 2.03 0.40
1.08 0.45 2.41
33.00 0.33 100.95
0.57 0.76 0.75
1.02 1.70 0.60
1.14 0.38 3.01
31.46 2.23 14.13
0.40 0.94 0.42
0.48 0.91 0.53
0.42 1.15 0.37
1.09 0.34 3.22
28.77 4.62 6.23
0.59 0.65 0.92
0.94 0.34 2.80
39.43 6.85 5.76
0.69 0.99 0.69
0.54 0.14 3.79
33.85 5.18 6.53
1.06 1.22 0.86
0.84 0.22 3.89
49.78 2.95 16.88
0.50 1.21 0.42
0.26 1.29 0.20 0.50 0.87 0.57
0.24 0.57 0.41
0.77 0.32 2.43
43.49 3.37 12.91
2
1
3
1
2
Growing zone areas
Articulating zone areas 4
0.50 0.88 0.57
0.55 1.46 0.38
1.44 0.63 2.29
39.56 3.00 13.18
3
0.83 0.92 0.90
0.32 0.65 0.49
1.98 0.55 3.61
31.08 2.51 12.37
4
0.76 0.95 0.80
0.38 0.86 0.45
0.77 0.31 2.50
37.59 3.61 10.43
5
0 . 3 5 C S 1'3 0.99 HA 2 0.35
0.14 0.66 0.21
1.06 4,* 0.41 4,* 2.56
3 6 . 8 0 C S 1'3 3.14 KS 4 11.72
6
F r a c t i o n a t i o n o n E C T E O L A - c e l l u l o s e o f g l y c o s a m i n o g l y c a n s p r e p a r e d f r o m p a p a i n d i g e s t s o f c a r t i l a g e s a m p l e s . T h e r e s u l t s are e x p r e s s e d as p m o l h e x o s a m i n e p e r 100 m g dry w e i g h t . The i d e n t i t y of each g l y c o s a m i n o g l y c a n was e s t a b l i s h e d by d e t e r m i n i n g the s e n s i t i v i t y to specific e n z y m a t i c d e g r a d a t i o n b e f o r e and a f t e r elect r o p h o r e s i s o n c e l l u l o s e a c e t a t e o r r e f r a c t i o n a t i o n o n E C T E O L A - c e l l u l o s e . I d e n t i t i e s arc i n d i c a t e d : H A , h y ' , d u r o n a t e ; C S , c h o n d r o i t i n s u l p h a t e ; K S , k e r a t a n sulp h a t e . S e n s i t i v i t i e s are i n d i c a t e d : l s e n s i t i v e t o t e s t i c u l a r h y a l u r o n i d a s e ; 2 s e n s i t i v e t o S t r e p t o m y c e s h y a l u r o n i d a s e ; 3 s e n s i t i v e t o c h o n d r o i t i n a s e A C ; 4 i n s e n s i t i v e
T A B L E II
¢D
180 glycosaminoglycans from similar cartilage has indicated an unsuspected degree of electrophoretic heterogeneity of the chondroitin sulphate present in this fraction (Lyons, G., Sweet, M.B.E. and T h o n a r E.J-M.A., unpublished work). As with hyaluronate, the concent r a t i on of this glycosaminoglycan did not vary significantly from sample to sample. Fractions 4 and 5 (2.5 M sodium formate and H20). Most of the chondroitin sulphate was recovered from these fractions. With the exception of area 5, there was always more chondr oi t i n sulphate in the respective growing zone, particularly in areas 1, 2 and 3. F u r t h e r m o r e , the chondroitin sulphate c o n t e n t varied from area to area in both zones. A variable a m o u n t of glucosaminecontaining material was recovered from each fraction; it was soluble in cetylpyridinium chloride (Table III) and insensitive to testicular hyaluronidase. F u r t h e r m o r e equimolar amounts of galactose were recovered with the glucosamine. Although the electrophoretic mobility of the material was similar to that o f chondroitin 6-sulphate, it may be identified as keratan sulphate. It is i m p o r t a n t to note that the c onc e nt r at i on of this " p o o l " of keratan sulphate varied. It was virtually absent from areas 1 and 2 of the articulating zone, but present in substantial amounts in the cerresponding m axi m um cont act areas, particularly area 5. On the ot her hand similar amounts were recovered from all regions o f the growth zone with the exception of area 5 where there was more. The ex tr emely small quantities of glucosamine recovered from the 2.5 M sodium f o r mate and H20 fraction of areas 1 and 2 (articulating zone) has led to the apparent paradox of a molar ratio of galactosamine : glucosamine > I in the c e t y lp y r id in iu m chloride supernatant. However, the small quantities involved make interpretation difficult. The galactosamine (11% of that eluted in this fraction) that remained in solution following the addition of cetylpyridinium chloride to the fraction of articulating area 2 (Table III) should lead to caution in attempting to calculate [21] from the molar ratios of galactosamine to glucosamine, the chain length of keratan sulphate present in the ot her samples. As the ratios stand, values of 2--3 disaccharide-repeating periods, low by any standards, would be given. F u r t h e r investigation of this material is required. Fraction 6 (6.0 M HC1). Keratan sulphate is eluted by 6.0 M HC1 from ECTEOLA-cellulose [20]. Small, relatively constant amounts of glucosamine were present in each sample (Table II) apart from in the growing zones of areas 3 and 4. Ab o u t three times as much galactosamine was eluted simultaneously in each case. A t t e m p t s to characterise the parent molecule of the galactosamine (part of which presumably formed the keratan sulphate linkage region) were hampered by the paucity of material. Almost all the glueosamine and galactosamine were soluble in cetylpyridinium chloride both before and after t r e a t m e n t with 0.5 M KOH. In addition prior digestion with testicular hyaluronidase did n o t affect the elution of either hexosamine from ECTEOLA-cellulose (Table IV). Table II shows that the concent r a t i on of galactosamine bore a quantitative relationship to the glucosamine concentration in this fraction, but not to the c o n t e n t of chondroitin sulphate or keratan sulphate in fractions 4 and 5. The variations in the glycosaminoglycan c o n t e n t of the different samples became more significant when the results were expressed on the basis of wet weight. This point is supported by the data presented in Fig. 3 which shows that the growing zone had a higher c o n t e n t of short-chain a n d / o r under-
ELUTED
FROM
ECTEOLA-CELLULOSE
(2.5
M SODIUM
FORMATE
AND
H20)
BY
33.00 (4.32)
43.49 (8.91)
39.43 (7.14)
37.59 (11.39)
G r o w i n g z o n e , area 2
A r t i c u l a t i n g z o n e , area 5
G r o w i n g z o n e , area 5
3.61 (1.09)
6.85 (1.24
3.37 (0.69)
0.33 (0.04)
10.4
5.8
12.9
100.9
36.63 (11.10)
36.89 (6.68)
42.47 (8.70)
29.34 (3.84)
GaINH 2
Molar ratio
GaINH 2
GIuNH 2
Precipitate
Untreated
A r t i c u l a t i n g z o n e , area 2
Cartilage
0.73 (0.22)
1.60 (0.29)
0.93 (0.19)
0.08 (0.01)
GIuNH 2
50.2
23.1
45.7
366.8
Molar • ratio
0.96 (0.29)
2.54 (0.46)
1.02 (0.21)
3.66 (0.48)
GalNH 2
2.88 (0.87)
5.25 (0.95)
2.44 (0.50)
0.25 (0.03)
GluNH 2
0.33
0.48
0.42
14.6
Molar ratio
Cetylpyridinium chloride supernatant
C h o n d r o i t i n a s e AC d e g r a d a t i o n c o n f i r m e d t h e p r e s e n c e o f c h o n d r o i t i n s u l p h a t e in t h e c e t y l p y r i d i n i u m c h l o r i d e p r e c i p i t a t e s . T h e m a t e r i a l in t h e s u p e r n a t a n t w a s r e s i s t a n t to t e s t i c u l a r h y a l u r o n i d a s e a p a r t f r o m t h a t in a r e a 2. T h e r e s u l t s arc e x p r e s s e d as p m o l / 1 0 0 m g d r y w e i g h t a n d (in p a r e n t h e s e s ) p m o l / 1 0 0 m g w e t w e i g h t .
FURTHER FRACTIONATION OF GLYCOSAMINOGLYCANS PRECIPITATION WITH CETYLPYRIDINIUM CHLORIDE
T A B L E III
FRACTIONATION
OF
GLYCOSAMINOGLYCANS
ELUTED
FROM
1.084 (0.142)
0.771 (0.158)
0.945 (0.171)
0.766 (0.232)
G r o w i n g zone, area 2
A r t i c u l a t i n g zone, area 5
G r o w i n g zone, a r e a 5
GaINH 2
Untreated
A r t i c u l a t i n g zone, area 2
Cartilage
0.307 (0.093)
0.337 (0.061)
0.317 (0.065)
0.450 (0.059)
GIuNH 2
GalNH 2
0.084 (0.011) 0.068 (0.014) 0.072 (0.013) 0.076 (0.023)
Molar ratio 2.41
2.43
2.80
2.50
Precipitate
0.007 (0.002)
0.006 (0.001)
0.020 (0.004)
0.015 (0.002)
GluNH 2
(6.0M
10.86
12.00
3.40
5.50
Molar ratio
ECTEOLA-CELLULOSE
T h e r e s u l t s are e x p r e s s e d a s p m o l / 1 0 0 m g d r y w e i g h t a n d (in p a r e n t h e s e s ) p m o l / 1 0 0 m g w e t w e i g h t .
PYRIDINIUM CHLORIDE (SEE TEXT)
FURTHER
T A B L E IV ADDITION
OF
CETYL-
0.690 (0.209)
0.873 (0.158)
0.702 (0.144)
1.000 (0.131)
GalNH 2
0.300 (0.091)
0.331 (0.060)
0.298 (0.061)
0.435 (0.057)
GluNH2
2.30
2.64
2.36
2.30
Molar ratio
Cetylpyridinium chloride supernatant
HC1) BY T H E
b~
183 ARTICULATING
ZONE
GROWING
ZONE
lOARTICULATIKG
ZONE
GROWING
ZONE
I
~
I
~
H20 +
~
~o-
i
~ - -
-- ~
----
---
=
--
_-- ~ -
~
I-
M
HC[
H2 O+
0.02 M Her
=
m
0.02
~
=
--
~
--
~
=
~
~
~
~
0.3M
NaCI
.~,-
~
215M
2.5 M H C O O N a
HCOONa
+ H20
÷ H20 20-
i-
10-
6.0M
HCI
~
-
-
-
-
l
i
-
-
I
6.0M
HCt
1o-
i-
1 2 3 4
56
1 2 3 4
AREAS
S
6
1 2 3 4
S
6
12
34
56
AREAS
Fig. 3. F r a c t i o n a t i o n o f g l y c o s a m i n o g l y c a n s o f e a c h s a m p l e on E C T E O L A - c e l l u l o s e . T h e r e s u l t s are r e p r e s e n t e d as p m o l h e x o s a m i n e p e r 1 0 0 m g w e t c a r t i l a g e . G a l a c t o s a m i n e (m) a b o v e t h e line; g l u c o s a m i n e (~) b e l o w t h e line. Fig. 4. F r a c t i o n a t i o n o f g l y c o s a m i n o g l y c a n s o f e a c h s a m p l e o n E C T E O L A - c e l l u l o s e . T h e r e s u l t s are r e p r e s e n t e d as p e r c e n t a g e s of t h e t o t a l h e x o s a m i n e in e a c h s a m p l e . G a l a c t o s a m i n e (m) a b o v e t h e line; g l u c o s a m i n e (~) b e l o w t h e line.
s u l p h a t e d c h o n d r o i t i n sulphate, h y a l u r o n a t e and c h o n d r o i t i n sulphate t h a n the articulating zone. F u r t h e r m o r e , a t r e n d n o t obvious in the d r y tissue, was the increased h y a l u r o n a t e c o n t e n t o f areas 5 and 6 o f the articulating zone. In contrast t o the results f o r the d r y tissue, t h e c o n t e n t o f k e r a t a n sulphate (eluted b y 2.5 M s o d i u m f o r m a t e and H~O) o f the t w o zones o f the m a x i m u m c o n t a c t areas (areas 4, 5 and 6) were virtually identical (Fig. 3).
Relative proportion of galactosamine and glucosamine in each glycosaminoglycan pool In c o m p a r i n g t h e p r o p o r t i o n s o f h e x o s a m i n e p r e s e n t in each fraction, certain t r e n d s n o t n o t i c e a b l e in t e r m s o f a b s o l u t e c o n c e n t r a t i o n s , b e c a m e clear (Fig. 4). T h e r e was very little d i f f e r e n c e b e t w e e n t h e t w o zones o f area 3. T h e p r o p o r t i o n o f c h o n d r o i t i n sulphate p r e s e n t was similar in the t w o zones o f the m i n i m u m c o n t a c t area, b u t greater in the growing z o n e o f t h e m a x i m u m c o n t a c t areas. T h e p r o p o r t i o n o f k e r a t a n sulphate present in all areas o f the growing z o n e s h o w e d little variation; in areas 1 and 2 o f the articulating z o n e t h e r e was p r o p o r t i o n a t e l y less, b u t in areas 4, 5 and 6, p r o p o r t i o n a t e l y m o r e .
184 Discussion The water and glycosaminoglycan c o n t e n t in the different regions was generally similar to that r e por t ed by o t h e r workers [4,5,22], but some obvious and o th er mo r e subtle differences between the individual samples studied were noted. The results show that variations in glycosaminoglycan and water c o n t e n t of the immature joint occur both with distance from the articular surface and from region to region. The degree of h y d r a t i o n of cartilage is the result of two opposing forces: water is attracted by the high osmotic pressure of the proteoglycans and their surrounding c o u n t e r ions, while the inflow of water is limited by the collagen f r a m ewo r k [23,24] and the degree of cross-linking therein. Variations in the water c o n t e n t may t her e f or e be due to variations in the glycosaminoglycan c o n t e n t , the degree of proteoglycan aggregation [25] or the elastic restraint of collagen. This last factor seems to be the most likely cause of the lower water c o n t e n t of the growing zone as it had a higher c o n t e n t of collagen. Furthermore the growing zone contained more chondroitin sulphate, particularly in the area of min im um contact, and there was no correlation between regional variations in water c o n t e n t and chondroitin sulphate c o n t e n t , although the c o n t e n t of these two substances did vary in parallel in the two zones. Considerable local variations in chondroitin sulphate c o n t e n t have been re p o r ted in cartilage of adult hum a n femoral condyle [6] but not in femoral head [24] or patella [26]. The regional variations report ed here may therefore be present at all ages in certain forms of cartilage, but not in others. The growing zone of articular cartilage has been regarded as analogous to epiphyseal cartilage [5], a view for which there is histological justification. Larsson et al. [13] r e por t ed the hyal ur ona te c o n t e n t of epiphyseal cartilage varied from 13% of the total hexosamine in the h y p e r t r o p h i c zone to 5% in the resting zone, the latter value being more than twice that of any of the results given here. This suggests a possible difference in the role of hyal uronat e in the two tissues and that the growing zone of articular cartilate is distinct from epiphyseal cartilage. This distinction is further emphasized by the presence of keratan sulphate in the growing zone and its virtual absence from epiphyseal cartilage [13,27--30]. These factors lead us to suggest that the dual function of the growing zone may result in a difference in gene expression. Indeed it has been said that mechanical factors influence the regional c o n t e n t of glycosaminoglycan [31]. The suggestion that the increase in keratan sulphate c o n t e n t o f the deepest layer of adult articular cartilate is the result of anoxia [32] is unlikely to apply to immature articular cartilage as the growing zone is nourished by vessels arising from the subchondral bone [33]. It is generally felt that the appearance and increasing c o n t e n t of skeletal keratan sulphate in cartilage is representative of maturation and ageing [34]. The p r o n o u n c e d regional variations in keratan sulphate concent rat i on between area 5 and areas 1 and 2 of the articulating zone may therefore indicate an earlier matu r atio n of the c h o n d r o c y t e s of area 5, possibly as a response to load. On the o t h e r hand any argument relating keratan sulphate c o n t e n t to load does n o t seem tenable in respect of the growing zone, particularly as this glycosaminoglycan has been d e m o n s t r a t e d in the corresponding zone of articular
185 cartilage of bovine foetuses from the second and third trimester of pregnancy (Thonar, E.J.M.A. and Sweet, M.B.E., unpublished). Recently, Hascall et al. [35] have reported keratan sulphate constituted about 7% of monomeric proteoglycan of chick limb buds from stage 23--24 embryos. Whether its presence in proteoglyean is indicative of a stage of maturity of chondrocytes or whether it has some other biological significance, is not clear at present. In the model proteoglycan proposed by Hascall and Heinegard [2] and by Rosenberg et al. [36], a proportion of the keratan sulphate is attached between clusters of chondroitin sulphate to the general polysaccharide-attachment region. Papain digestion of these substances yields a heterogeneous mixture of peptidoglycans which may contain chondroitin sulphate and keratan sulphate attached to a c o m m o n peptide apart from chondroitin sulphate-peptide and keratan sulphate-peptide [37]. This is the likely explanation for the presence of some of the glucosamine in the formate fractions and may be indicative of structural variations within the proteoglycans from different regions of the cartilage, particularly articulating area 2. In addition to the regional and zonal differences described, the results presented in this paper emphasise the importance of discrete sampling of tissue. This applied particularly to the articulating zone where the marked regional variations could be nullified by pooling or averaging the tissue or the results. The disadvantages of working with small quantities of material are obvious and often cannot be overcome by pooling tissue from different individuals. However, the development of micro- and ultramicromethods for proteoglycan isolation and characterization will hopefully lead to greater insight into the problems of local maturation or reaction to physiological load. Acknowledgements We acknowledge with thanks the financial assistance of the Faculty of Medicine Research Fund and of De Beers Industrial Diamonds Ltd. and the gift of International Reference Standards of glycosaminoglycans by Dr. Martin B. Mathews, Department of Pediatrics, University of Chicago, Ill. 60637, U.S.A. References 1 M a t h e w s , M.B. a n d L o z a i t y t e , I. ( 1 9 5 8 ) A r c h . B i o c h e m . Biophys. 74, 1 5 8 - - 1 7 4 2 Hascall, V.C. a n d H e i n e g a r d , D. ( 1 9 7 5 ) i n E x t r a c e l l u l a r Matrix I n f l u e n c e s on G c n e E x p r e s s i o n (Slavkin, H.C. a n d G r e u l i c h , R.C., eds.), pp. 4 2 3 - - 4 3 3 , A c a d e m i c Press, N e w Y o r k 3 H e i n e g a r d , D. a n d Hascall, V.C. ( 1 9 7 4 ) J. Biol. C h e m . 249, 4 2 5 0 - - 4 2 5 6 4 L e m p e r g , R . K . , L a r s s o n , S.-E. a n d H j e r t q u i s t , S.-O. ( 1 9 7 1 ) B i o c h e m . Biophys. A s p e c t s 7, 4 1 9 - - 4 2 0 5 L e m p e r g , R. a n d L a r s s o n , S.-E. ( 1 9 7 4 ) Calcif. Tissue Res. 15, 2 3 7 - - 2 5 1 6 Bjelle, A.O. ( 1 9 7 4 ) Scand. J. R h e u m a t o l . 3, 8 1 - - 8 8 7 M e a c h i m , G. a n d S t o c k w e l l , R . A . ( 1 9 7 3 ) in A d u l t A r t i c u l a r Cartilage ( F r e e m a n , M . A . R . , ed.), pp. 1-50, P i t m a n Medical, L o n d o n 8 Bitter, T. a n d Muir, H. ( 1 9 6 2 ) Anal. B i o c h e m . 4, 3 3 0 - - 3 3 4 9 Cessi, C. a n d Piliego, F. ( 1 9 6 0 ) B i o c h e m . J. 77, 5 0 8 - - 5 1 0 10 Woessner, J . F . ( 1 9 6 1 ) A r c h . B i o c h e m . 93, 4 4 0 - 4 4 7 11 S c o t t , J.E. ( 1 9 6 0 ) M e t h o d s B i o c h e m . A n a l . 8, 1 4 5 - - 1 9 7 12 A n t o n o p o u l o s , C.A., H e i n e g a r d , D. a n d Gardell, S. ( 1 9 6 7 ) B i o c h i m . Biophys. A c t a 148, 1 5 8 - - 1 6 3 13 L a r s s o n , S.-E., R a y , R.D. a n d K u e t t n e r , K . E . ( 1 9 7 3 ) Calcif. Tissue Res. 13, 2 7 1 - - 2 8 5 14 S e n o , N., A n n o , K., K o n d o , K., Nagase, S. a n d Saito, S. ( 1 9 7 0 ) Anal. B i o c h e m . 37, 1 9 7 - - 2 0 1 15 S w e e t , M.B.E., T h o n a r , E.J-M.A. a n d I m m e l m a n , A . R . ( 1 9 7 6 ) B i o c h i m . B i o p h y s . A c t a 4 3 7 , 7 1 - - 8 6
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