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Proteochondroitin sulfate synthesized in cartilages induced in vivo and in vitro by bone matrix gelatin
402
Biochimica et Biophysica Acta, 586 (1979) 4 0 2 - - 4 1 7 © E l s e v i e r / N o r t h - H o l l a n d Biomedical Press
BBA 2 8 9 7 8
P R O T E O C H O N D R O I T I N S U L F A T E SYNTHESIZED IN CARTILAGES INDUCED IN VIVO AND IN V I T R O BY BONE MATRIX GELATIN
A T S U H I K O O O H I R A a, H I R O S H I N O G A M I a, Y O S H I N O R I K U B O K I b a n d SATOSHI SASAKI b
a Department of Embryology, Institute for Developmental Research, Kasugai, Aichi 480-03 and b Department of Biochemistry, School of Dentistry, Tokyo Medical and Dental University, Tokyo 113 (Japan) ( R e c e i v e d J a n u a r y 9th, 1979)
Key words: Proteochondroitin sulfate; Bone matrix gelatin; Proteoglycan; Collagen synthesis; Chondrogenesis; Development; (Cartilage)
Summary Implanted "allogeneic demineralized bone matrix gelatin induced sequential development of cartilage and bone in the recipient rat muscle tissue. Proteoglycans of the implants labeled in vivo with [3SS]sulfate at different stages of development were analyzed by sucrose density gradient centrifugation. The major proteoglycan synthesized in day-5 implant, just prior to onset of chondrogenesis, was a dermatan sulfate-containing proteoglycan with relatively slow sedimentation rate. Additionally, a small amount of a faster sedimenting component could be detected. The faster sedimenting proteoglycan, in which chondroitin 4-sulfate accounted for 85% of total radioactivity, became predominant in day-10 sample when cartilage formation was maximal. By day 30, when cartilage had been replaced by newly formed bone, the synthesis of this faster sedimenting c o m p o n e n t had ceased. A similar, if not identical, proteoglycan was found to be a major one synthesized by the in vitro-induced cartilage. This proteoglycan was smaller in overall size and shorter in length of its chondroitin sulfate chains than a major proteoglycan c o m p o n e n t obtained from neonatal rat epiphyseal cartilage. Concurrent with these changes in proteoglycan type, there appeared to be a change in collagen type, since type II collagen, in addition to type I collagen, was synthesized in day-10 implant. These results indicate that the proteoglycan can be used as a molecular marker for chondrogenesis by bone matrix gelatin.
Abbreviations: PCS-BMG, the p r o t e o c h o n d r o t i n sulfate obtained in fraction X of the sucrose density gradient; PCS-H, hyaline cartilage-unique proteochondroitin sulfate; PCS, proteochondroitin sulfate.
403 Introduction Proteoglycans and collagens are two major constituents of extracellular matrices in skeletal tissues. Recently their role as molecular markers of cell differentiation has emerged more readily, in addition to a role in the morphogenesis of skeletal tissues as mediators of tissue interaction and cell migration. For example, temporal and spatial transitions in collagen types have been observed with development of an embryonic chick limb [1--3]. Changes in proteoglycan types have also been observed during development of limb bud chondrocytes in vivo and in vitro [4--6]. Implanted demineralized long bone has been shown by several investigators to induce in vivo an ectopic sequential formation of cartilage and bone, similar to that occurring in embryos. For example, Urist et al. [7,8] implanted demineralized bone matrix gelatin, which is prepared by sequential treatments of adult rat long bones with various extractant solutions into the abdominal musculature of adult, male Sprague-Dawley rats, and Reddi and Huggins [9] implanted bone matrix powders demineralized with 0.5 M HC1 subcutaneously in the thoracic region of young rats. Furthermore, Nogami and Urist [10] have demonsrated that bone matrix gelatin is able to induce the formation of hyaline cartilage when cultured in vitro with freshly excised skeletal muscle tissue of near-term fetal Sprague-Dawley rats. In view of these morphological observations, it is of interest to elucidate whether or not the bone matrixinduced cartilage and bone contain the same types of matrix macromolecule as those in other cartilages and bones, respectively. More recently, Reddi et al. [11,12] using the bone matrix powder system have revealed that transitions in types of matrix macromolecules occur during bone matrix-induced cartilage and bone formation. In the present paper, we will report the results of isolation and partial characterization of a proteochondroitin sulfate from the cartilage induced in vivo and in vitro by bone matrix gelatin. The study demonstrates that the molecule differs in physical and chemical characteristics from a major proteochondroitin sulfate component of neonatal rat epiphyseal cartilage, suggesting that the bone matrix gelatininduced cartilage has a molecular architecture in extracellular space somewhat different from that of other hyaline cartilages in rats. Materials and Methods Materials. The following commercial materials were used: carrier-free H23sSO4, from Japan Radioisotope Association, Tokyo; L-[U-~4C]proline, 205 Ci/mol, from New England Nuclear, Boston; chondroitinase-ABC and chondroitinase-AC from Seikagaku Kogyo Co., Tokyo; Pronase-P from Kaken Kagaku Co., Tokyo; Sepharose CL-2B and Sepharose CL-6B from Pharmacia, Uppsala; CM-cellulose (Whatman CM-32) from H. Reeve Angel and Co., London; Diaflo PM-10 membrane from Amicon Corp., Lexington; Falcon organ culture dish and grid from Falcon Plastics, Oxnard; chicken plasma from Difco Laboratories, Detroit; tissue culture medium 199, Eagle's minimum essential medium and newborn calf serum from Nakarai Chemicals, Ltd., Kyoto. Neutral salt-soluble collagen was prepared from rat skin by the method of
404 Piez et al. [13]. Insoluble cartilage was prepared from bovine mandibular cartilage by th.e m e t h o d of Miller [14]. Preparations of implants and explants. Bone matrix gelatin was prepared from diaphyses of long bones of adult Sprague-Dawley rats (4- to 6-monthold) by sequential treatments of 0.6 M HC1, 8 M LiC1 and at H20 55°C as described by Urist et al. [8]. Six pieces o f bone matrix gelatin {about 150 mg dry weight), which had been cut at a b o u t 1-mm intervals to form notches [10], were implanted into the abdominal wall muscle of each male SpragueDawley rat (3.5-month-old, weighing 395 ± 3 8 . 6 g ) . On designated days, Na23SSO4 (1 Ci/1 in 0.9% NaC1) was injected intraperitoneally at a dose of 2 pCi/g b o d y weight. After 3 h, the implants were dissected out and used for extraction of labeled proteoglycans. Methods for in vitro induction of cartilage by bone matrix gelatin described by Nogami and Urist [10] were used with a slight modification as follows. All the operations were performed under sterile conditions. Bone matrix gelatin was washed in 199 medium containing 15% n e w b o w n calf serum to reconstitute the matrix and split into hemicylinders with a No. 11 surgical blade. The hemicylinders were wetted with chicken plasma, and then cut halfway through at a b o u t 1-mm intervals transversely to form notches. Muscle from the triceps humerus of 2 0 ~ a y - o l d Sprague-Dawley rat fetus was minced in a drop of the medium, and the minced muscle was placed on the hemicylindrical segment of bone matrix gelatin. Two of these preparations were placed on a reversed grid in a Falcon organ culture dish containing 2 ml of the medium. A b o u t 75% of the medium was changed every 48 h. After 21 days of culture in a CO2 incubator (WJ-3C, Hirasawa Works, Tokyo), 10 explants were placed in 3 ml o f the fresh medium containing 60 pCi of [3SS]sulfate. After 3 h labeling, the explants were washed in ice-cold medium and used for extraction of labeled proteoglycans. Biochemical analysis. For the measurements of DNA content and hexuronate content in implants, the implants excised were placed in 10 vols. of 0.05 M Tris-HC1, pH 8.0, containing 0.1 M EDTA, 3% ethanol and Pronase-P (1 mg/ ml). The suspension was incubated at 50°C overnight. Under these conditions, all the samples at different stages could be digested. D N A and hexuronatecontaining materials in the digest were precipitated with 3 vols. of 95% ethanol/1.3% potassium acetate, and measured by the method of Burton [15] and by the m e t h o d of Bitter and Muir [16], respectively. Preparation ofproteoglycan samples. The labeled implants were immediately placed in 5 vols. of ice-cold, 4 M guanidine HC1/0.05 M Tris-HC1, pH 7.5, containing 0.1 M 6-aminohexanoic acid, 0.005 M benzamidine HC1 and 0.01 M EDTA in order to prevent proteolysis during extraction procedure [17]. The suspension was homogenized at 0°C in a loose fitting, Potter glass homogenizer. The homogenate was stirred at 4°C overnight, and the residue was removed by centrifugation at 20 000 × g for 30 min. The supernatant was used as a starting material for further analysis of proteoglycans. For separation of proteoglycans with sucrose density gradient centrifugation [ 18], each extraction was diluted with an appropriate volume of the guanidine HC1 solution to give the same concentration of carbazole-reacting hexuronate (0.5 mmol/1). This is necessary since the apparent sedimentation velocity
405 of a proteoglycan sample tends to decrease, as the concentration of the proteoglycan increases. A 1 ml portion of the solution was layered on a 14.5 ml linear sucrose gradient (5--20% in the guanidine HC1 solution) in a cellular nitrate tube, at the b o t t o m of which 0.5 ml of 40% sucrose in the guanidine HC1 solution had been placed. The t u b e was centrifuged in a RPS-27 rotor in a Hitachi 65 P ultracentrifuge at 25 000 rev./min for 30 h at 4°C. The t u b e was then punctured at the b o t t o m , and 1 ml fractions were collected. Each fraction was diluted with 2 ml of cold water, and proteoglycan was precipitated with 6 ml of 95% ethanol/1.3% potassium acetate. The ethanol precipitation from water was repeated at least four times, and the final precipitate was used for the analysis of the polysaccharide moiety and for the measurement of radioactivity. Analysis of polysaccharide moiety. Proteoglycan precipitated with ethanol was subjected to the sequential treatments of 0.3 M NaOH/5% trichloroacetic acid, and precipitation with ethanol in order to obtain polysaccharide moiety of the proteoglycan as described previously [19]. An aliquot (0.3 pmol of carbazole-reacting hexuronate) was digested either with chondroitinase-ABC or with chondroitinase-AC to determine the relative amounts of isomeric chondroitin sulfates by the m e t h o d of Saito et al. [20]. Another aliquot (about 2 pmol of hexuronate or more than 50 000 d p m of 3sS) was dissolved in 1 ml of 2 M guanidine HC1/0.02 M Tris-HC1, pH 7.5, and subjected to gel filtration on a Sepharose 6B column (1.5 × 50 cm) in the buffer at room temperature. A b o u t 2-ml fractions were collected, and analyzed for hexuronate and for radioactivity. Characterization of collagen. Day-10 implants (500 mg wet weight) were excised and sliced in 5 ml of Eagle's Minimal Essential Medium containing 500 ~g of ~-aminopropionitril fumarate, 250 pg of ascorbic acid and 15% newborn calf serum. The suspension was preincubated at 37°C with gentle shaking. After 20-min preincubation, the tissue was transferred to 5 ml of the fresh medium supplemented with 10 pCi of [14C]proline, incubated for 2 h at 37°C, and then replaced in 10 ml of the fresh medium containing 4 mM unlabeled proline. The incubation with unlabeled proline was carried o u t for another 1 h at 37°C for complete conversion of labeled procollagen to labeled collagen [ 21]. Under these conditions the incorporation of label was linear with time up to an 8 h exposure of label. The labeled tissue was homogenized in a Potter-Elvehjem glass homogenizer in 10 ml of 1 M NaC1/0.05 M Tris-HC1, pH 7.5, at 4°C. The homogenate was stirred overnight. After centrifugation at 20 000 × g for 30 min, the residue was re-extracted in 5 ml of the buffer. Collagen was precipitated from the combined supernatant fluid b y increasing the NaC1 concentration to 4.3 M. The precipitate was collected by centrifugation, washed t w o additional times with 4.3 M NaC1/0.05 M Tris-HC1, pH 7.5, and finally dissolved in 20 ml of 1 M NaC1/0.05 M Tris-HC1, pH 7.5, together with 10 mg of purified rat skin soluble collagen. This solution comprised a b o u t 70% of total [14C]hydroxyproline, which was determined b y the m e t h o d of Juva and Prockop [22]. The NaC1 concentration of this solution was increased to 2.4 M by adding a cold solution of 4.3 M NaC1/0.05 M Tris-HC1, pH 7.5. The precipitate thus formed was isolated by centrifugation. Additional fraction was obtained by raising the salt
406 concentration to 4.3 M by adding solid NaC1. These isolated collagens were chromatographed separately on a CM-cellulose column (1.6 X 3 cm) in urea/acetate buffer as described previously [23]. Labeled and carrier collagen a-chains isolated after CM-cellulose chromatography were mixed with an additional 10 mg of ~1(I) chain, and cleavage at methionyl residues was achieved in 70% formic acid containing 100 mg cyanogen bromide according to the m e t h o d of Kuboki and Mechanic [24]. The peptides produced by digestion with CNBr were chromatographed at 40°C on a CM~ellulose column (0.9 × 8 cm) in 0.02 M sodium citrate buffer, pH 3.6, with a linear gradient from 0.01 to 0.16 M NaC1 over a total volume of 200 ml at a flow rate of 20 ml/h. Fractions o f a b o u t 2 ml were collected and their absorbance at 230 nm and radioactivity were measured. Results
Light microscopy As the sequential cellular transitions have been described in detail previously [25], brief recapitulation will be described here to lead a better understanding of the following biochemical findings. On the fifth day after implantation of bone matrix gelatin, a large number of mesenchymal cells proliferating between muscle and implanted bone matrix gelatin were observed. Light microscopic examination could not reveal the existence of cartilaginous tissue at this stage of the development. By day 10, abundant hyaline cartilage had developed on the surface of bone matrix gelatin and inside of notches cut in bone matrix gelatin. F e w osseous areas were observed morphologically. By day 20, most of mesenchymal cell envelope had been transformed into fibrous tissue, and resorption of the cartilage and replacement with new bone had begun. By day 30, cartilaginous area had been replaced completely by osseous area with newly formed bone marrow.
Biochemical analysis of implants The amounts o f DNA and hexuronate in preimplanted bone matrix gelatin and implants excised at various stages were determined (Table I). The wet
TABLE I CONTENTS OF DNA AND HEXURONATE IN PREIMPLANTED BONE MATRIX GELATIN, ABDOMINAL WALL MUSCLE, AND IMPLANTS AT VARIOUS STAGES OF DEVELOPMENT Sample
Preimplanted bone matrix
gelatin Dry weight of bone matrix gelatin implanted (g) Wet w e i g h t o f t i s s u e e x c i s e d (g) DNA (pmol) * H e x u r o n a t e (/~mol) ** * E x p r e s s e d as d A M P [ 1 5 ] . * * Ex p r e s s e d as g l u c u r o n a t e [ 16 ].
Abdominal wall muscle
0.150 -0.540 0.908
1.083 0.948 0.699
Implant Day 5
D a y 10
D a y 20
D a y 30
0.1 50
0.1 50
0.1 50
0.1 50
1.083 0.993 1.286
0.798 1.121 2.226
0.554 1.121 1.920
0.434 1.041 1.271
407
T A B L E II 3$S-RADIOACTIV1TY INCORPORATED INTO ABDOMINAL WALL MUSCLE AND IMPLANTS AT V A R I O U S S T A G E S O F D E V E L O P M E N T , A N D E X T R A C T I O N OF T H E L A B E L L E D M A T E R I A L S WITH THE 4 M GUANIDINE-HC1 SOLUTION Sample
Total radioactivity ( c p m / p m o l DNA) * Extract Residue
Implant
Abdominal wall muscle
Day 5
D a y 10
D a y 20
Day 30
8891 (1.0) 7815 1076
77 1 8 6 (8.7) 70 6 2 5 6 561
1 0 6 351 (12.0) 99 013 7 338
56 2 1 5 (6.3) 51 9 4 3 4 272
34 983 (3.9) 31 4 5 0 3 533
* V a l u e s in parentheses indicate ratios o f r a d i o a c t i v i t y in v a r i o u s i m p l a n t s t o that in a b d o m i n a l wall muscle.
weight of the implants decreased with the developmental stages, while the D N A content was nearly constant during the period examined. Hexuronate content increased rapidly between day 5 and day 10, and then decreased gradually. It is n o t e w o r t h y that the preimplanted bone matrix gelatin used contained both DNA and hexuronate. There is a summarising table of the constituents of the ox bone matrix which may be similar to those of the rat bone matrix [26]. The main constituent of the bone matrix is collagen (about 90% of dry weight), and both proteoglycans and glycoproteins also exist in it (a few percent each). It is n o t obvious n o w that what c o m p o n e n t in bone matrix gelatin plays an important role in the induction of cartilage. Urist and Iwata [ 27], however, consider that proteoglycan in bone matrix gelatin does not have inductive activity because bone matrix gelatin treated either with chondrotinase-ABC or with hyaluronidase still maintain its inductive property. Recently, from their ultrastructural studies, Anderson and Griner [28] called the attention to the possibility that residual cellular material could play a role in the induction. Our finding that bone matrix gelatin contains a significant a m o u n t of DNA may also indicate this possibility. 3SS-radioactivity incorporated into the implants was measured (Table II). Incorporating activity of [aSS]sulfate in day 5 implant was approx. 9 times as much as that in muscle on the basis of the a m o u n t of DNA. The activity increased further with the developmental stages and reached a maximum on day 10; thereafter it declined to lower levels. These results are consistent with the morphological observation, indicating that the cartilage formation is maximal on day 10.
Proteoglycans in implants From each implant, a b o u t 90% of 3SS-labeled proteoglycans was extracted with 4 M guanidine HC1 under the conditions used (Table II). The yield of hexuronate was also approx. 90% in each case. The proteoglycans thus obtained were separated by ultracentrifugation in a sucrose density gradient (Fig. 1). Most of the labeled materials from muscle were recovered as a broad peak at the top of the gradient (designated as 'Y'). On day 5, just before the earliest morphological indication of chrondrogenesis,
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Fig. 1. S u c r o s e d e n s i t y g r a d i e n t c e n t r i f u g a t i o n o f t h e p r o t e o g l y c a n s isolated f r o m a b d o m i n a l wall m u s c l e (a), d a y - 5 i m p l a n t (b), d a y - 1 0 i m p l a n t (c), d a y - 2 0 i m p l a n t (d), a n d d a y - 3 0 i m p l a n t (e). Abscissa, t u b e n u m b e r ; o r d i n a t e , a r b i t r a r y u n i t o f b o t h r a d i o a c t i v i t y a n d h e x u r o n a t e . Solid bars a b o v e c u r v e s i n d i c a t e f r a c t i o n s o f c o m p o n e n t X a n d c o m p o n e n t Y w h i c h w e r e p o o l e d s e p a r a t e l y f o r f u r t h e r analyses. T h e d o t t e d line i n d i c a t e s t u b e 8.
in addition to fraction Y a small amount o f labeled material appeared in the fraction with high sedimentation velosity (designated as 'X'). The proportion of component X in the proteoglycan preparation was maximal on day 10. By day 30, the synthesis of component X had ceased. T A B L E III C H O N D R O I T I N A S E D I G E S T I O N P R O D U C T S OF 3 5 S - L A B E L E D P O L Y S A C C H A R I D E MOIETIES OF PROTEOGLYCAN FRACTIONS ISOLATED FROM ABDOMINAL WALL MUSCLE AND IMPLANTS AT V A R I O U S S T A G E S OF D E V E L O P M E N T T h e v a l u e s are e x p r e s s e d as t h e p e r c e n t a g e o f t o t a l 3 5 S - r a d i o a c t i v i t y . T h e n o m e n c l a t u r e used are: Resist a n t , r e s i s t a n t p a r t against t h e d i g e s t i o n w i t h c h o n d r o i t i n a s e - A B C ; A - u n i t , ADi-4S p r o d u c e d b y t h e digest i o n w i t h o n l y c h o n d r o i t i n a s e - A C , B-unit, A D i - 4 S w h i c h c a n n o t b e p r o d u c e d b y t h e d i g e s t i o n w i t h c h o n d r o i t i n a s e - A C b u t c a n b e p r o d u c e d w i t h c h o n d r o i t i n a s e - A B C ; C-unit, ADi-6S p r o d u c e d b y t h e d i g e s t i o n w i t h c h o n d r o i t i n a s e - A B C . O t h e r s ar~ m a i n l y c o m p o s e d o f o v e r s u l f a t e d - u n s a t u r a t e d d i s a c c h a r i d e s and Na c e t y l g a l a c t o s a m i n e - 4 - s u l f a t e ( f o r t h e s t r u c t u r e a n d a b b r e v i a t i o n o f t h e d i s a c e h a r i d e s , see Ref. 19). Fraction *
Product Resistant A-unit B-unit C-unit Others * See Fig. 1.
Muscle Y
22.9 12.3 52.5 3.5 8.8
Day 5
D a y 10
D a y 20
D a y 30 Y
X
Y
X
Y
X
Y
8.1 84.2 <~1.0 3.5 4.2
12.9 38.5 44.2 1.3 3.1
2.8 86.7 <1.0 5.5 5.0
10.5 57.8 28.3 1.1 2.3
3.3 87.2 <1.0 4.0 5.5
17.5 60.1 15.7 3.0 3.7
24.8 56.5 13.2 1.6 3.9
409 The relative amounts of isomeric chondroitin sulfates and chondroitinaseABC resistant part in the 3SS-labeled polysaccharide moiety from these fractions were shown in Table III. Of the products of chondroitinase
Proteoglycans in explants Bone matrix gelatin is able to induce the formation of hyaline cartilage in vitro [10]. If the cartilage formed in vitro by bone matrix gelatin has the same characteristics as the in vivo-induced cartilage, PCS-BMG must also be synthesized in the in vitro-induced cartilage. To examine this possibility, minced triceps muscle of near-term fetal rats was cultured on bone matrix gelatin in 199 medium containing newborn calf serum for 21 days. It has been shown by light microscopic examination that cartilage has been formed within the period. On the 21st day, [3SS]sulfate was added to the medium. After 3 h, the labeled proteoglycans were solubilized from the tissue with the guanidine HCl-protease inhibitor solution as described above. Most of the radioactivity incorporated (approx. 90%) was extracted under the conditions used. Fig. 3 shows the sedimentation profile of the labeled proteoglycans in the sucrose density gradient. It is obvious that the profile coincides with that of the sample from day-10
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Fig. 2. Gel c h r o m a t o g r a p h y o n S e p h a r o s e CL-6B o f t h e 35 S-labeled a n d u n l a b e l e d p o l y s a c c h a r i d e c h a i n s o f f r a c t i o n X i s o l a t e d f r o m d a y - 1 0 i m p l a n t . Abscissa, t u b e n u m b e r ; o r d i n a t e , a r b i t r a r y u n i t o f b o t h radioa c t i v i t y a n d h e x u r o n a t e . V0, void v o l u m e . Vt, t o t a l v o l u m e . Fig. 3. S u c r o s e d e n s i t y g r a d i e n t c e n t r i f u g a t i o n o f t h e 35 S-labeled p r o t e o g l y c a n s i s o l a t e d f r o m t h e e x p l a n t w h i c h o r g a n - c u l t u r e d in 1 9 9 m e d i u m f o r 21 d a y s . Abscissa, t u b e n u m b e r ; o r d i n a t e , a r b i t r a r y u n i t o f r a d i o a c t i v i t y . T h e d o t t e d line i n d i c a t e s t u b e 8.
implant (cf. Fig. lc). The composition o f isomeric chondroitin sulfates in the labeled polysaccharide moiety of the c o m p o n e n t with high sedimentation velocity was closely similar to that of PCS-BMG (Table IV). The length o f its polysaccharide chain (Kay = 0.54 on a Sepharose 6B column) was also equal to that of PCS-BMG. The polysaccharides from the fraction with lower sedimentation velocity were composed of chondroitin 4-sulfate (about 50% of total radioactivity), dermatan sulfate (about 25%) and chondroitinase resistant part (about 20%). From these results one can conclude that PCS-BMG is synthesized in b o t h cartilages induced in vivo and in vitro by bone matrix gelatin. Proteoglycans from other sources in rats
Many investigators have reported the occurrence of hyaline cartilage-unique
TABLE IV S U M M A R I S I N G T A B L E W H I C H C O M P A R E S SOME P R O P E R T I E S O F T H E 3 S S ° L A B E L E D P R O T E O CHONDROITIN SULFATES ISOLATED FROM VARIOUS SOURCES Proteochondroitin sulfate
PCS-BMG f r o m Day-10 implant PCS-BMG f r o m t h e explant R a t PCS-H
Overall size * ( K a y o n 2B)
" Length of polysac° e h a r i d e ** (Kay o n 6B)
C h o n d r o i t i n a s e d i g e s t i o n p r o d u c t *** (% o f t o t a l r a d i o a c t i v i t y ) Resistant
A-unit
B-unit
C-unit
Others
0.29
0.54
2.8
86.7
5.5
5.0
0.29 0.12
0.54 0.46
2.9 2.2
89.1 89.3
3.5 4.8
4.5 3.7
* See Fig. 5. ** See Fig. 2 a n d Fig. 6. *** F o r t h e a b b r e v i a t i o n o f t h e p r o d u c t s , see T a b l e I I I .
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Fig. 4. Sucrose density gradient centrifugation of the 3S S-labeled and unlabeled prot e ogl yc a ns isolated from epiphyseal cartilages (a) and diaphyseal bones (b). Abscissa, tube n u m b e r ; ordinate, arbitrary unit of b o t h radioactivity and h e x u r o n a t e . The d o t t e d line indicates tube 8 corresponding to the pos i t i on o f PCS-BMG. Fig. 5. Gel c h r o m a t o g r a p h y on Sepharose CL-2B of the major prot e ogl yc a ns isolated f~om the n e o n a t a l rat epiphyses (e e) and from day-10 i m p l a n t (o o). An a l i quot (approx. 50 000 d p m ) of each p r o t e o g l y c a n separated by sucrose density gradient was precipitated w i t h e t ha nol as described in the t e x t and dissolved in 1 ml of 2 M guanidine HCI/0.02 M Tris*HCl, pH 7.5. The solution was fractionated in a Sephazose CL-2B c o l u m n (1.5 X 50 cm), equilibrated w i t h the guanidine HC1 buffer at room temperature. Fractions of 2 ml were collected and assayed for radioactivity. Vo, void volume. Vt, t o t a l volume.
proteochondroitin sulfate (PCS-H) in embryonic chicks, based on the sucrose gradient separation [4,5,18]. To examine whether PCS-H exists in neonatal rat epiphyseal cartilages and, if so, whether the PCS-H can be distinguished from PCS-BMG, 3SS-labeled proteoglycans from neonatal rat epiphyseal cartilages were prepared by the essentially same m e t h o d as described above. The yields of labeled materials and hexuronate b y the extraction were more than 80%. Fig. 4a illustrates the sedimentation profile of the proteoglycans in the sucrose density gradient. There were t w o distinct components as it was the case of embryonic chick epiphyseal cartilages [18]; a heavier c o m p o n e n t and a lighter one. The heavier c o m p o n e n t comprised a b o u t 95% of total radioactivity and total hexuronate. When this major c o m p o n e n t was chromatographed on a Sepharose 2B column under dissociative condition, it was eluted as a somewhat broad peak (Kay = 0.12) near the void volume (Fig. 5). The polysaccharide obtained by alkaline ~-elimination of this c o m p o n e n t was chromatographed on a Sepharose 6B column (Fig. 6). Kay values of the labeled and unlabeled polysaccharide chains were both 0.46 +_0.02, similar to that (0.44) of the polysaccharide chain of PCS-H isolated from embryonic chick cartilage [5]. Digestion of the labeled polysaccharide with chondroitinases revealed that chondroitin 4-sulfate was the major (about 90% of total radioactivity) and that der-
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matan sulfate did not occur in the preparation (Table IV). These data indicate that the heavier c o m p o n e n t isolated from neonatal rat epiphysis has similar characteristics to chick PCS-H-except for the composition of isomeric chondroitin sulfates in the polysaccharide moiety. This proteochondroitin sulfate will be referred to as rat PCS-H hereinafter. Fig. l c and Fig. 4a indicate that PCS-BMG sedimented more slowly than rat PCS-H, suggesting that the overall size of PCS-BMG is smaller than that of rat PCS-H. Indeed, the peak for PCS-BMG isolated from day-10 implant on the Sepharose 2B column was more included (Fig. 5). PCS-BMG from the explant was eluted in the same position as that from the implant (Kay = 0.29). Some properties of these proteochondroitin sulfates were summarized in Table IV. The table shows that PCS-BMG is smaller in polysaccharide chain size as well as in overall size than rat PCS-H, while relative amounts of isomeric chondroitin sulfates in the polysaccharide moieties were similar. The present study provided an o p p o r t u n i t y to compare the proteoglycans synthesized by diaphyseal bones with the proteoglycans synthesized by epiphyseal cartilage of neonatal rats. 3SS-radioactivity incorporated in the bone was about one thirtieth of that in the cartilage, based on the tissue wet weight. The yields of the labeled materials and hexuronate were more than 75% under the conditions used. Most of the labeled materials were recovered as a somewhat broad peak at the top of the sucrose gradient (Fig. 4b). Little radioactivity was observed in the position corresponding to rat PCS-H. Of the chondroitinasedigestion products of the labeled polysaccharides of the materials, the A unit was the major (approx. 60% of total radioactivity). Less than 30% was proved to be resistant to the lyase reaction. These results show that the characteristics of the materials are similar to those of the proteoglycans from day-30 implant, in which the newly formed bone was predominant and hematopoietic bone marrow differentiation was evident.
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Fig. 7. Gel c h r o m a t o g r a p h y o n Bio-Gel A - 1 5 m o f t h e e x t r a c t f r o m d a y - 1 0 i m p l a n t i n c u b a t e d w i t h [ 14 C]p r o l i n e . A b o u t 2 m l of t h e g u a n i d i n e HC1 e x t r a c t w a s f r a c t i o n a t e d in a Bio-Gel A - 1 5 m c o l u m n (1.5 × 5 0 c m ) , e q u i l i b r a t e d w i t h 2 M g u a n i d i n e H C I / O . 0 2 M Tris-HCl, p H 7.5, at r o o m t e m p e r a t u r e . F r a c t i o n s o f 2 m l w e r e c o l l e c t e d a n d a s s a y e d f o r b o t h t o t a l r a d i o a c t i v i t y (e e) and [ 1 4 C ] h y d r o x y p r o l i n e (o-o). Solid bax above curve indicates the fractions corresponding to s-chains, which were subsequently pooled and subjected to chromatography on CM-cellulose (see Fig. 8a). V O, void volume. Vt, total volume. Fig. 8. CM-cellulose e l u t i o n p a t t e r n s of t h e l a b e l e d s - c h a i n s o b t a i n e d f r o m t h e g u a n i d i n e HC1 e x t r a c t b y gel c h r o m a t o g r a p h y (a), [ 1 4 C ] c o l l a g e n o u s c o m p o n e n t p r e c i p i t a t e d f r o m t h e NaC1 e x t r a c t w i t h 2.4 M NaC1 (b), a n d [ 1 4 C ] c o l l a g e n o u s c o m p o n e n t s u b s e q u e n t l y p r e c i p i t a t e d w i t h 4.3 M NaCI (c). E a c h s a m p l e was dissolved in 5 m l o f 4 M u r e a / O . 0 4 M s o d i u m a c e t a t e , p H 4.8, t o g e t h e r w i t h a b o u t 10 m g o f r a t skin soluble t y p e I collagen. T h e m i x t u r e w a s h e a t e d to 4 0 ° C for 1 0 rain a n d c h r o m a t o g z a p h e d in a CMcellulose c o l u m n (1.6 × 3 c m ) as d e s c r i b e d p r e v i o u s l y [ 2 3 ] . F r a c t i o n s o f 2 m l w e r e c o l l e c t e d a n d a s s a y e d for b o t h r a d i o a c t i v i t y a n d a b s o r b a n c e at 2 3 0 n m . Solid b a r s a b o v e c u r v e s i n d i c a t e t h e f r a c t i o n s c o r r e s p o n d i n g t o col-chains, w h i c h w e r e s u b s e q u e n t l y p o o l e d a n d s u b j e c t e d t o d i g e s t i o n w i t h CNBr.
Collagen synthesized in day-l O implant The major proteochondroitin sulfate (PCS-BMG) synthesized in bone matrix gelatin-induced hyaline cartilages is different in physical and chemical properties from rat PCS-H, namely the hyaline cartilage-unique proteochondroitin sulfate (see above). Then, do the cells in bone matrix gelatin-induced cartilages synthesize t y p e II collagen, or hyaline cartilage-unique collagen? To answer this question, the implants excised on day 10 was labeled with [14C]proline as described under Methods. 14C-labeled tissues were treated with the guanidine HCl-protease inhibitor solution by the same method used for proteoglycan extraction. Most of labeled materials (approx. 85% of total radioactivity) were solubilized by this method. When the extract was chromatographed on a Bio-Gel A-15m column as described previously [29], a large peak containing hydroxy[t4C]proline was eluted in the area expected for a-chains (Fig. 7). Additionally, there was a small, hydroxyproline-containing peak in the area expecting for triple-stranded collagens, which may be largely composed of t y p e III collagen [11]. The large peak fraction was chromatographed on a CM-cellulose column {Fig. 8a). Two
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Fig. 9. CM-ceHulose e l u t i o n p a t t e r n s o f t h e l a b e l e d CNBr p e p t i d e s d e r i v e d f r o m t h e c ~ l - c h a i n i s o l a t e d a f t e r p r e c i p i t a t i o n o f t h e c o l l a g e n at 2.4 M NaC1 (a) a n d f r o m t h e ~ l - c h a l n isolated a f t e r s u b s e q u e n t precipit a t i o n a t 4.3 M NaCI (b), a n d t h e CNBr p e p t i d e s d e r i v e d f r o m insoluble t y p e II c o l l a g e n p r e p a r e d f r o m b o v i n e m a n d i b u l a r cartilage (c). T h e C N B r p e p t i d e s w e r e d i s s o l v e d in 3 m l o f 0 . 0 2 M s o d i u m c i t r a t e b u f fer, p H 3.6, a t 4 0 ° C a n d c h r o m a t o g r a p h e d in a CM-cellulose c o l u m n (0.9 X 8 c m ) b y use o f a l i n e a r salt g r a d i e n t o f NaCI f r o m 0.01 to 0 . 1 6 M in t h e b u f f e r . T h e c a r r i e r w a s t h e C N B r p e p t i d e d e r i v e d f r o m ~ l ( I ) - c h a i n p r e p a r e d f r o m r a t skin. F r a c t i o n s o f 2 m l w e r e c o l l e c t e d a n d a s s a y e d f o r b o t h r a d i o a c t i v i t y a n d a b s o r b a n c e at 2 3 0 n m .
peaks were obtained; one is coeluted with rat skin a l chain and another with 2 chain. The'ratio of a 1 to a2 was 3.7:1, which is significantly higher than the 1 :a 2 ratio found in t y p e I collagen (2:1). This suggests that a mixture of type I and type II collagens is synthesized in day-10 implant. The corresponding ratios of the samples from day-5 implant and day-10 implant were 2.1:1 and 2.4:1, respectively. To separate the presumed collagen species by differential salt precipitation from neutral-salt solutions, the tissue labeled with ['4C]proline was treated with 1 M NaC1/0.05 M Tris-HC1, pH 7.5. The collagenous components were extracted and partially purified by the differential salt precipitation method. The CM-cellulose elution pattern of the collagen precipitated at 2.4 M NaC1 is presented in Fig. 8b, and a chromatogram illustrating the elution pattern of the collagen precipitated at 4.3 M NaC1 is shown in Fig. 8c. These chromatograms clearly indicate that two different collagens were separated. The ratios of a l to a2 for the 2.4 M precipitate was found to be 2.3:1, and that for the 4.3 M precipitate was 6.2:1. For further characterization of these °l-chains, each labeled chain was cleaved with CNBr in the presence of additional carrier a l ( I ) chain, and the resulting peptides were chromatographed on CM-cellulose. The [~4C]peptide profile of the 2.4 M NaCl-precipitated °l-chain was identical to a l ( I ) carrier collagen (Fig. 9a). On the other hand, the °l-chain that precipitated at 4.3 M NaC1 was identical to a l ( I I ) collagen from bovine mandibular cartilage (Fig. 9b and Fig. 9c). These results indicate that cells in day-10 implant synthesize both type I and t y p e II collagens. One may consider that the type II collagen may originate from the cartilaginous areas and the t y p e I collagen from the fibrous areas.
415 Discussion
The paper demonstrates the changes in proteoglycan types during the sequential cartilage and bone formation by bone matrix gelatin, based on sucrose density gradient separation. The synthetic activity for sulfated proteoglycans increased more than 8 times during first 5 days after implantation of bone matrix gelatin (Table II). The major proteoglycan synthesized in day-5 implant, when most cells were mesenchymal cells proliferating actively in contact with bone matrix gelatin, was a dermatan sulfate-rich, slowly sedimenting proteoglycan (Fig. 1 and Table III). The exact source of the proteoglycan is not clear, but some parts may originate from adhering soft tissues of the implants, such as muscle and fibrous tissue. Alternatively, one could consider that the mesenchymal cells which start to synthesize predominantly the dermatan sulfate-rich proteoglycan may serve as progenitor cells which eventually differentiate to chondroblasts. Although the idea has not been proposed to date, this may be supported by the reported occurrence of two different shifts in collagen types during the chondrogenesis; from type I to type II collagen synthesis during the chick limb chondrogenesis [1--3] and from type III to type II collagen synthesis during the bone matrix powder-induced chondrogenesis [ 11 ]. Synthesis of a new proteochondroitin sulfate, designated as PCS-BMG, starts about day 5 just prior to onset of the bone matrix gelatin-induced chondrogenesis. This molecule could be easily purified by the sucrose density gradient separation (Fig. 1 and Fig. 5). For separation of proteoglycans, CsC1 density gradient centrifugation is generally used [6,12,17,30,31]. This method can give satisfactory separation of proteins as a gel from some proteoglycans and good concentration of the proteoglycans to the bottom fraction of a centrifuge tube. It is apparent, however, that the bottom fraction contains many kinds of proteoglycan in some cases [12], judging from the subsequent gel chromatographic analysis of proteoglycans in the fraction. The sucrose density gradient method has an advantage in that a proteoglycan sample can be separated into at least four distinct fractions with one step procedure [4]. When the extract with guanidine HC1 solution contains enough proteoglycan for further analysis, the separation or purification procedure of the proteoglycans sometimes can be simplified as described in the present paper. Moreover, it should be stressed that some peculiar proteoglycans synthesized in embryonic chick epiphyseal cartilages can not be recovered in the bottom fraction of CsC1 density gradient [32]. The process of appearance and disappearance of PCS-BMG in implants bears a close resemblance to the morphological process that the hyaline cartilage is produced in muscle by implantation of bone matrix gelatin and then replaced with newly formed bone. A proteoglycan which could not be distinguished from PCS-BMG was shown to be a major component in the hyaline cartilage produced in near-term fetal rat triceps muscle cultured upon bone matrix gelatin. Nevertheless, it is remarkable that PCS-BMG has characteristics differing not only from those of muscle and bone proteoglycans but also from those of hyaline cartilage-unique proteoglycan, rat PCS-H, synthesized in neonatal rat epiphyseal cartilage. As PCS-BMG was proved to be smaller in the overall size as
416
well as in its polysaccharide chain size (Table IV), one may consider that rat PCS-H, synthesized in bone matrix gelatin-induced cartilage, has been converted into the form o f PCS-BMG by artificial enzymatic degradation during extraction procedure. To examine this possibility, we used a proteoglycanextracting solution of 4 M guanidine HC1/0.05 M Tris-HCl, pH 7.5, containing other kinds of enzyme-inhibitors such as 10 mM EDTA, 10 mM N-ethylmaleimide, 1 mM phenylmethanesulfonyl fluoride, and 0.36 mM pepstatin, which had been successfully used for procollagen extraction from embryonic chick epiphyseal cartilages [29]. However, the characteristics of the proteoglycan thus extracted were essentially the same as those of PCS-BMG (data not shown). These findings are consistent with the idea that there must be some functional differences for various hyaline cartilages in a single species because there is a heterogeneity with respect to molecular architecture of matrix macromolecules [12]. Although it is not clear in what ways PCS-BMG differs from rat PCS-H, at least two different possibilities can be considered: first, PCSBMG has the different protein core as well as the different polysaccharides from those of rat PCS-H, and second, PCS-BMG and rat PCS-H have the same protein core but the different polysaccharide chains. It is reasonable to regard that the protein core of PCS-BMG is the same as that of the PCS-H, namely the cartilage type o f proteoglycan, because bone matrix gelatin-induced cartilages have been proved to synthesize t y p e II collagen, namely the cartilage t y p e of collagen (Fig. 9). The difference in overall size between PCS-BMG and rat PCSH may largely be due to the difference in the length of their polysaccharide chains. Bone matrix gelatin-induced endochondral bone formation has been considered to be a useful experimental model for bone formation in fracture healing as well as in embryonic development. In fact, the sequential cellular transitions are similar to those observed in fracture healing [33]. Recently we isolated a proteochondroitin sulfate, which could not be distinguished from PCSBMG, from rat fibula-fracture callusat the stage when hyaline cartilage was predominant [34]. These findings, combined with the results shown in the present paper, suggest that bone matrix gelatin-induced bone formation is a useful experimental model for biochemical analysis of sequential process of fracture healing rather than of normal development of embryonic skeletal tissues.
Acknowledgements We thank Professor S. Suzuki, Nagoya university, for helPful suggestions during the work. We extend our appreciation to Miss K. Ozeki for excellent technical assistance.
References 1 2 3 4 5 6
L i n s e n m e y e r , T . F . , T o o l e , B.P. and Trelstad, R . L . ( 1 9 7 3 ) Dev. Biol. 3 5 , 2 3 2 - - 2 3 9 V o n d e r M a r k , H., y o n der Mark, K. and G a y , S. ( 1 9 7 6 ) Dev. Biol. 4 8 , 2 3 7 - - 2 4 9 V o n d e r M a r k , K., v o n d e r M a r k , H. a n d G a y , S. ( 1 9 7 6 ) Dev. Biol. 53, 1 5 3 - - 1 7 0 O k a y a m a , M.~ P a c i f i c i , M. a n d H o l t z e r , H. ( 1 9 7 6 ) P r o c . N a t l . Acad. Sci. U.S. 7 3 , 3 2 2 4 - - 3 2 2 8 K i t a m t t r a , K. a n d Y a m a g a t a , T. ( 1 9 7 6 ) F E B S L e t t . 7 1 , 3 3 7 - - 3 4 0 De L u c a , S., Heineg~.rd, D., H a s c a l l , V.C., K i m u r a , J . H . a n d C a p l a n , A . L ( 1 9 7 7 ) J. Biol. C h e m . 2 5 2 , 6600--6608
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"7 Urist, M.R. (1965) Science 150, 893--899 8 Urist, M.R., lwata, H., Ceccotti, P.L., Dorfman, R.L., Boyd, S.D., MeDowell, R.M. and Chien, C. (1973) Proc. Natl. Acad. Sei. U.S. 70, 3 5 1 1 - - 3 5 1 5 9 Reddi, A.H. and Huggins, C. (1972) Proc. Natl. Acad. Sci. U.S. 69, 1 6 0 1 - - 1 6 0 5 10 Nogami, H. and Urist, M.R. (1974) J. Cell Biol. 62, 510--519 11 Reddi, A.H., Gay, R., Gay, S. and Miller, E.J. (1977) Proc. Natl• Acad. Sci. U•S. 74, 5 5 8 9 - - 5 5 9 2 12 Reddi, A.H., Hascall, V.C. and Hascall, G.K• (1978) J. Biol• Chem. 253, 2429--2436 13 Piez, K.A., Eigner, E.A. and Lewis, M.S. (1963) Biochemistry 2, 58--66 1 4 Miller, E.J• (1972) Biochemistry 11, 4 9 0 3 - - 4 9 0 9 15 Burton, K. (1956) Bioehem. J. 62, 315---323 16 Bitter, T. and Muir, H.M. (1962) Anal. Biochem. 4 , 3 3 0 - - 3 3 4 17 Oegema, T.R., Jr•, Hascall, V•C. and D z i e w a l t k o w s k y , D.D. (1975) J. Biol• Chem. 250, 6 1 5 1 - - 6 1 5 9 18 Kimata, K., Okayama, M., Oohira, A. and Suzuki, S. (1974) J. Biol. Chem. 249, 1 6 4 6 - - 1 6 5 3 19 Oohira, A., Tamaki~ K., Terashima, Y., Chiba, A. and Nogami, H. (1977) Calcif. Tissue Res. 2 3 , 2 7 1 - 275 20 Saito, H., Yamagata, T. and Suzuki, S• (1968) J. Biol. Chem. 243, 1 5 3 6 - - 1 5 4 2 21 Oohira, A., Kimata, K., Suzuki, S., Takata, K•, Suzuki, L and Hoshino, M. (1974) J. Biol• Chem. 249, 1637--1645 22 Juva, K• and Prockop, D.J. (1966) Anal• Biochem. 15, 77--83 23 Oohira, A., Kusakabe, A• and Suzuki, S. (1975) Biochem. Biophys. Res. C ommun. 67, 1086--1092 24 Kuboki, Y. and Mechanic, G.L. (1974) Connect. Tissue Res. 2 , 2 2 3 - - 2 3 0 25 Nogami, H. and Urist, M.R. (1974) Clin. Orthop. 103, 235--251 26 lwata, H. and Urist, M.R• (1972) Clin. Orthop. 87, 257--274 27 Urist, M.R. and Iwata, H. (1973) J. TheoL Biol. 3 8 , 1 5 5 - - 1 6 7 28 Anderson, H.C. and Griner, S.A. (1977) Develop. Biol. 60, 351--358 29 Oohira, A., Nogami, H., Kusakabe, A., Kimata, K. and Suzuki, S. (1979) J. Biol. Chem., in the press 30 Hascall, V.C. and Sajdera, S.W. (1969) J• Biol. Chem. 244, 2384--2396 31 Hemegard, D. (1972) Biochim. Biophys. Acta 2 8 5 , 1 8 1 - - 1 9 2 32 Kimata, K., Oike, Y., Ito, K., Karasawa, K. and Suzuki, S. (1978) Biochem. Biophys. Res. Commtm. 85, 1 4 3 1 - - 1 4 3 9 33 Ham, A.W. and Harris, W.R. (1971) in The Biochemistry and Physiology of Bone (Bourne, G.H., ed.), Vol. 3, pp, 337--399, Academic Press, New York 34 Yo kobori, T., Oohira, A.~ Ozeki, K. and Nogami, H. (1978) Conn. Tissue (Tokyo) 10, Suppl., 28 (abstr.) •
o
Biochimica et Biophysica Acta, 586 (1979) 4 0 2 - - 4 1 7 © E l s e v i e r / N o r t h - H o l l a n d Biomedical Press
BBA 2 8 9 7 8
P R O T E O C H O N D R O I T I N S U L F A T E SYNTHESIZED IN CARTILAGES INDUCED IN VIVO AND IN V I T R O BY BONE MATRIX GELATIN
A T S U H I K O O O H I R A a, H I R O S H I N O G A M I a, Y O S H I N O R I K U B O K I b a n d SATOSHI SASAKI b
a Department of Embryology, Institute for Developmental Research, Kasugai, Aichi 480-03 and b Department of Biochemistry, School of Dentistry, Tokyo Medical and Dental University, Tokyo 113 (Japan) ( R e c e i v e d J a n u a r y 9th, 1979)
Key words: Proteochondroitin sulfate; Bone matrix gelatin; Proteoglycan; Collagen synthesis; Chondrogenesis; Development; (Cartilage)
Summary Implanted "allogeneic demineralized bone matrix gelatin induced sequential development of cartilage and bone in the recipient rat muscle tissue. Proteoglycans of the implants labeled in vivo with [3SS]sulfate at different stages of development were analyzed by sucrose density gradient centrifugation. The major proteoglycan synthesized in day-5 implant, just prior to onset of chondrogenesis, was a dermatan sulfate-containing proteoglycan with relatively slow sedimentation rate. Additionally, a small amount of a faster sedimenting component could be detected. The faster sedimenting proteoglycan, in which chondroitin 4-sulfate accounted for 85% of total radioactivity, became predominant in day-10 sample when cartilage formation was maximal. By day 30, when cartilage had been replaced by newly formed bone, the synthesis of this faster sedimenting c o m p o n e n t had ceased. A similar, if not identical, proteoglycan was found to be a major one synthesized by the in vitro-induced cartilage. This proteoglycan was smaller in overall size and shorter in length of its chondroitin sulfate chains than a major proteoglycan c o m p o n e n t obtained from neonatal rat epiphyseal cartilage. Concurrent with these changes in proteoglycan type, there appeared to be a change in collagen type, since type II collagen, in addition to type I collagen, was synthesized in day-10 implant. These results indicate that the proteoglycan can be used as a molecular marker for chondrogenesis by bone matrix gelatin.
Abbreviations: PCS-BMG, the p r o t e o c h o n d r o t i n sulfate obtained in fraction X of the sucrose density gradient; PCS-H, hyaline cartilage-unique proteochondroitin sulfate; PCS, proteochondroitin sulfate.
403 Introduction Proteoglycans and collagens are two major constituents of extracellular matrices in skeletal tissues. Recently their role as molecular markers of cell differentiation has emerged more readily, in addition to a role in the morphogenesis of skeletal tissues as mediators of tissue interaction and cell migration. For example, temporal and spatial transitions in collagen types have been observed with development of an embryonic chick limb [1--3]. Changes in proteoglycan types have also been observed during development of limb bud chondrocytes in vivo and in vitro [4--6]. Implanted demineralized long bone has been shown by several investigators to induce in vivo an ectopic sequential formation of cartilage and bone, similar to that occurring in embryos. For example, Urist et al. [7,8] implanted demineralized bone matrix gelatin, which is prepared by sequential treatments of adult rat long bones with various extractant solutions into the abdominal musculature of adult, male Sprague-Dawley rats, and Reddi and Huggins [9] implanted bone matrix powders demineralized with 0.5 M HC1 subcutaneously in the thoracic region of young rats. Furthermore, Nogami and Urist [10] have demonsrated that bone matrix gelatin is able to induce the formation of hyaline cartilage when cultured in vitro with freshly excised skeletal muscle tissue of near-term fetal Sprague-Dawley rats. In view of these morphological observations, it is of interest to elucidate whether or not the bone matrixinduced cartilage and bone contain the same types of matrix macromolecule as those in other cartilages and bones, respectively. More recently, Reddi et al. [11,12] using the bone matrix powder system have revealed that transitions in types of matrix macromolecules occur during bone matrix-induced cartilage and bone formation. In the present paper, we will report the results of isolation and partial characterization of a proteochondroitin sulfate from the cartilage induced in vivo and in vitro by bone matrix gelatin. The study demonstrates that the molecule differs in physical and chemical characteristics from a major proteochondroitin sulfate component of neonatal rat epiphyseal cartilage, suggesting that the bone matrix gelatininduced cartilage has a molecular architecture in extracellular space somewhat different from that of other hyaline cartilages in rats. Materials and Methods Materials. The following commercial materials were used: carrier-free H23sSO4, from Japan Radioisotope Association, Tokyo; L-[U-~4C]proline, 205 Ci/mol, from New England Nuclear, Boston; chondroitinase-ABC and chondroitinase-AC from Seikagaku Kogyo Co., Tokyo; Pronase-P from Kaken Kagaku Co., Tokyo; Sepharose CL-2B and Sepharose CL-6B from Pharmacia, Uppsala; CM-cellulose (Whatman CM-32) from H. Reeve Angel and Co., London; Diaflo PM-10 membrane from Amicon Corp., Lexington; Falcon organ culture dish and grid from Falcon Plastics, Oxnard; chicken plasma from Difco Laboratories, Detroit; tissue culture medium 199, Eagle's minimum essential medium and newborn calf serum from Nakarai Chemicals, Ltd., Kyoto. Neutral salt-soluble collagen was prepared from rat skin by the method of
404 Piez et al. [13]. Insoluble cartilage was prepared from bovine mandibular cartilage by th.e m e t h o d of Miller [14]. Preparations of implants and explants. Bone matrix gelatin was prepared from diaphyses of long bones of adult Sprague-Dawley rats (4- to 6-monthold) by sequential treatments of 0.6 M HC1, 8 M LiC1 and at H20 55°C as described by Urist et al. [8]. Six pieces o f bone matrix gelatin {about 150 mg dry weight), which had been cut at a b o u t 1-mm intervals to form notches [10], were implanted into the abdominal wall muscle of each male SpragueDawley rat (3.5-month-old, weighing 395 ± 3 8 . 6 g ) . On designated days, Na23SSO4 (1 Ci/1 in 0.9% NaC1) was injected intraperitoneally at a dose of 2 pCi/g b o d y weight. After 3 h, the implants were dissected out and used for extraction of labeled proteoglycans. Methods for in vitro induction of cartilage by bone matrix gelatin described by Nogami and Urist [10] were used with a slight modification as follows. All the operations were performed under sterile conditions. Bone matrix gelatin was washed in 199 medium containing 15% n e w b o w n calf serum to reconstitute the matrix and split into hemicylinders with a No. 11 surgical blade. The hemicylinders were wetted with chicken plasma, and then cut halfway through at a b o u t 1-mm intervals transversely to form notches. Muscle from the triceps humerus of 2 0 ~ a y - o l d Sprague-Dawley rat fetus was minced in a drop of the medium, and the minced muscle was placed on the hemicylindrical segment of bone matrix gelatin. Two of these preparations were placed on a reversed grid in a Falcon organ culture dish containing 2 ml of the medium. A b o u t 75% of the medium was changed every 48 h. After 21 days of culture in a CO2 incubator (WJ-3C, Hirasawa Works, Tokyo), 10 explants were placed in 3 ml o f the fresh medium containing 60 pCi of [3SS]sulfate. After 3 h labeling, the explants were washed in ice-cold medium and used for extraction of labeled proteoglycans. Biochemical analysis. For the measurements of DNA content and hexuronate content in implants, the implants excised were placed in 10 vols. of 0.05 M Tris-HC1, pH 8.0, containing 0.1 M EDTA, 3% ethanol and Pronase-P (1 mg/ ml). The suspension was incubated at 50°C overnight. Under these conditions, all the samples at different stages could be digested. D N A and hexuronatecontaining materials in the digest were precipitated with 3 vols. of 95% ethanol/1.3% potassium acetate, and measured by the method of Burton [15] and by the m e t h o d of Bitter and Muir [16], respectively. Preparation ofproteoglycan samples. The labeled implants were immediately placed in 5 vols. of ice-cold, 4 M guanidine HC1/0.05 M Tris-HC1, pH 7.5, containing 0.1 M 6-aminohexanoic acid, 0.005 M benzamidine HC1 and 0.01 M EDTA in order to prevent proteolysis during extraction procedure [17]. The suspension was homogenized at 0°C in a loose fitting, Potter glass homogenizer. The homogenate was stirred at 4°C overnight, and the residue was removed by centrifugation at 20 000 × g for 30 min. The supernatant was used as a starting material for further analysis of proteoglycans. For separation of proteoglycans with sucrose density gradient centrifugation [ 18], each extraction was diluted with an appropriate volume of the guanidine HC1 solution to give the same concentration of carbazole-reacting hexuronate (0.5 mmol/1). This is necessary since the apparent sedimentation velocity
405 of a proteoglycan sample tends to decrease, as the concentration of the proteoglycan increases. A 1 ml portion of the solution was layered on a 14.5 ml linear sucrose gradient (5--20% in the guanidine HC1 solution) in a cellular nitrate tube, at the b o t t o m of which 0.5 ml of 40% sucrose in the guanidine HC1 solution had been placed. The t u b e was centrifuged in a RPS-27 rotor in a Hitachi 65 P ultracentrifuge at 25 000 rev./min for 30 h at 4°C. The t u b e was then punctured at the b o t t o m , and 1 ml fractions were collected. Each fraction was diluted with 2 ml of cold water, and proteoglycan was precipitated with 6 ml of 95% ethanol/1.3% potassium acetate. The ethanol precipitation from water was repeated at least four times, and the final precipitate was used for the analysis of the polysaccharide moiety and for the measurement of radioactivity. Analysis of polysaccharide moiety. Proteoglycan precipitated with ethanol was subjected to the sequential treatments of 0.3 M NaOH/5% trichloroacetic acid, and precipitation with ethanol in order to obtain polysaccharide moiety of the proteoglycan as described previously [19]. An aliquot (0.3 pmol of carbazole-reacting hexuronate) was digested either with chondroitinase-ABC or with chondroitinase-AC to determine the relative amounts of isomeric chondroitin sulfates by the m e t h o d of Saito et al. [20]. Another aliquot (about 2 pmol of hexuronate or more than 50 000 d p m of 3sS) was dissolved in 1 ml of 2 M guanidine HC1/0.02 M Tris-HC1, pH 7.5, and subjected to gel filtration on a Sepharose 6B column (1.5 × 50 cm) in the buffer at room temperature. A b o u t 2-ml fractions were collected, and analyzed for hexuronate and for radioactivity. Characterization of collagen. Day-10 implants (500 mg wet weight) were excised and sliced in 5 ml of Eagle's Minimal Essential Medium containing 500 ~g of ~-aminopropionitril fumarate, 250 pg of ascorbic acid and 15% newborn calf serum. The suspension was preincubated at 37°C with gentle shaking. After 20-min preincubation, the tissue was transferred to 5 ml of the fresh medium supplemented with 10 pCi of [14C]proline, incubated for 2 h at 37°C, and then replaced in 10 ml of the fresh medium containing 4 mM unlabeled proline. The incubation with unlabeled proline was carried o u t for another 1 h at 37°C for complete conversion of labeled procollagen to labeled collagen [ 21]. Under these conditions the incorporation of label was linear with time up to an 8 h exposure of label. The labeled tissue was homogenized in a Potter-Elvehjem glass homogenizer in 10 ml of 1 M NaC1/0.05 M Tris-HC1, pH 7.5, at 4°C. The homogenate was stirred overnight. After centrifugation at 20 000 × g for 30 min, the residue was re-extracted in 5 ml of the buffer. Collagen was precipitated from the combined supernatant fluid b y increasing the NaC1 concentration to 4.3 M. The precipitate was collected by centrifugation, washed t w o additional times with 4.3 M NaC1/0.05 M Tris-HC1, pH 7.5, and finally dissolved in 20 ml of 1 M NaC1/0.05 M Tris-HC1, pH 7.5, together with 10 mg of purified rat skin soluble collagen. This solution comprised a b o u t 70% of total [14C]hydroxyproline, which was determined b y the m e t h o d of Juva and Prockop [22]. The NaC1 concentration of this solution was increased to 2.4 M by adding a cold solution of 4.3 M NaC1/0.05 M Tris-HC1, pH 7.5. The precipitate thus formed was isolated by centrifugation. Additional fraction was obtained by raising the salt
406 concentration to 4.3 M by adding solid NaC1. These isolated collagens were chromatographed separately on a CM-cellulose column (1.6 X 3 cm) in urea/acetate buffer as described previously [23]. Labeled and carrier collagen a-chains isolated after CM-cellulose chromatography were mixed with an additional 10 mg of ~1(I) chain, and cleavage at methionyl residues was achieved in 70% formic acid containing 100 mg cyanogen bromide according to the m e t h o d of Kuboki and Mechanic [24]. The peptides produced by digestion with CNBr were chromatographed at 40°C on a CM~ellulose column (0.9 × 8 cm) in 0.02 M sodium citrate buffer, pH 3.6, with a linear gradient from 0.01 to 0.16 M NaC1 over a total volume of 200 ml at a flow rate of 20 ml/h. Fractions o f a b o u t 2 ml were collected and their absorbance at 230 nm and radioactivity were measured. Results
Light microscopy As the sequential cellular transitions have been described in detail previously [25], brief recapitulation will be described here to lead a better understanding of the following biochemical findings. On the fifth day after implantation of bone matrix gelatin, a large number of mesenchymal cells proliferating between muscle and implanted bone matrix gelatin were observed. Light microscopic examination could not reveal the existence of cartilaginous tissue at this stage of the development. By day 10, abundant hyaline cartilage had developed on the surface of bone matrix gelatin and inside of notches cut in bone matrix gelatin. F e w osseous areas were observed morphologically. By day 20, most of mesenchymal cell envelope had been transformed into fibrous tissue, and resorption of the cartilage and replacement with new bone had begun. By day 30, cartilaginous area had been replaced completely by osseous area with newly formed bone marrow.
Biochemical analysis of implants The amounts o f DNA and hexuronate in preimplanted bone matrix gelatin and implants excised at various stages were determined (Table I). The wet
TABLE I CONTENTS OF DNA AND HEXURONATE IN PREIMPLANTED BONE MATRIX GELATIN, ABDOMINAL WALL MUSCLE, AND IMPLANTS AT VARIOUS STAGES OF DEVELOPMENT Sample
Preimplanted bone matrix
gelatin Dry weight of bone matrix gelatin implanted (g) Wet w e i g h t o f t i s s u e e x c i s e d (g) DNA (pmol) * H e x u r o n a t e (/~mol) ** * E x p r e s s e d as d A M P [ 1 5 ] . * * Ex p r e s s e d as g l u c u r o n a t e [ 16 ].
Abdominal wall muscle
0.150 -0.540 0.908
1.083 0.948 0.699
Implant Day 5
D a y 10
D a y 20
D a y 30
0.1 50
0.1 50
0.1 50
0.1 50
1.083 0.993 1.286
0.798 1.121 2.226
0.554 1.121 1.920
0.434 1.041 1.271
407
T A B L E II 3$S-RADIOACTIV1TY INCORPORATED INTO ABDOMINAL WALL MUSCLE AND IMPLANTS AT V A R I O U S S T A G E S O F D E V E L O P M E N T , A N D E X T R A C T I O N OF T H E L A B E L L E D M A T E R I A L S WITH THE 4 M GUANIDINE-HC1 SOLUTION Sample
Total radioactivity ( c p m / p m o l DNA) * Extract Residue
Implant
Abdominal wall muscle
Day 5
D a y 10
D a y 20
Day 30
8891 (1.0) 7815 1076
77 1 8 6 (8.7) 70 6 2 5 6 561
1 0 6 351 (12.0) 99 013 7 338
56 2 1 5 (6.3) 51 9 4 3 4 272
34 983 (3.9) 31 4 5 0 3 533
* V a l u e s in parentheses indicate ratios o f r a d i o a c t i v i t y in v a r i o u s i m p l a n t s t o that in a b d o m i n a l wall muscle.
weight of the implants decreased with the developmental stages, while the D N A content was nearly constant during the period examined. Hexuronate content increased rapidly between day 5 and day 10, and then decreased gradually. It is n o t e w o r t h y that the preimplanted bone matrix gelatin used contained both DNA and hexuronate. There is a summarising table of the constituents of the ox bone matrix which may be similar to those of the rat bone matrix [26]. The main constituent of the bone matrix is collagen (about 90% of dry weight), and both proteoglycans and glycoproteins also exist in it (a few percent each). It is n o t obvious n o w that what c o m p o n e n t in bone matrix gelatin plays an important role in the induction of cartilage. Urist and Iwata [ 27], however, consider that proteoglycan in bone matrix gelatin does not have inductive activity because bone matrix gelatin treated either with chondrotinase-ABC or with hyaluronidase still maintain its inductive property. Recently, from their ultrastructural studies, Anderson and Griner [28] called the attention to the possibility that residual cellular material could play a role in the induction. Our finding that bone matrix gelatin contains a significant a m o u n t of DNA may also indicate this possibility. 3SS-radioactivity incorporated into the implants was measured (Table II). Incorporating activity of [aSS]sulfate in day 5 implant was approx. 9 times as much as that in muscle on the basis of the a m o u n t of DNA. The activity increased further with the developmental stages and reached a maximum on day 10; thereafter it declined to lower levels. These results are consistent with the morphological observation, indicating that the cartilage formation is maximal on day 10.
Proteoglycans in implants From each implant, a b o u t 90% of 3SS-labeled proteoglycans was extracted with 4 M guanidine HC1 under the conditions used (Table II). The yield of hexuronate was also approx. 90% in each case. The proteoglycans thus obtained were separated by ultracentrifugation in a sucrose density gradient (Fig. 1). Most of the labeled materials from muscle were recovered as a broad peak at the top of the gradient (designated as 'Y'). On day 5, just before the earliest morphological indication of chrondrogenesis,
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Fig. 1. S u c r o s e d e n s i t y g r a d i e n t c e n t r i f u g a t i o n o f t h e p r o t e o g l y c a n s isolated f r o m a b d o m i n a l wall m u s c l e (a), d a y - 5 i m p l a n t (b), d a y - 1 0 i m p l a n t (c), d a y - 2 0 i m p l a n t (d), a n d d a y - 3 0 i m p l a n t (e). Abscissa, t u b e n u m b e r ; o r d i n a t e , a r b i t r a r y u n i t o f b o t h r a d i o a c t i v i t y a n d h e x u r o n a t e . Solid bars a b o v e c u r v e s i n d i c a t e f r a c t i o n s o f c o m p o n e n t X a n d c o m p o n e n t Y w h i c h w e r e p o o l e d s e p a r a t e l y f o r f u r t h e r analyses. T h e d o t t e d line i n d i c a t e s t u b e 8.
in addition to fraction Y a small amount o f labeled material appeared in the fraction with high sedimentation velosity (designated as 'X'). The proportion of component X in the proteoglycan preparation was maximal on day 10. By day 30, the synthesis of component X had ceased. T A B L E III C H O N D R O I T I N A S E D I G E S T I O N P R O D U C T S OF 3 5 S - L A B E L E D P O L Y S A C C H A R I D E MOIETIES OF PROTEOGLYCAN FRACTIONS ISOLATED FROM ABDOMINAL WALL MUSCLE AND IMPLANTS AT V A R I O U S S T A G E S OF D E V E L O P M E N T T h e v a l u e s are e x p r e s s e d as t h e p e r c e n t a g e o f t o t a l 3 5 S - r a d i o a c t i v i t y . T h e n o m e n c l a t u r e used are: Resist a n t , r e s i s t a n t p a r t against t h e d i g e s t i o n w i t h c h o n d r o i t i n a s e - A B C ; A - u n i t , ADi-4S p r o d u c e d b y t h e digest i o n w i t h o n l y c h o n d r o i t i n a s e - A C , B-unit, A D i - 4 S w h i c h c a n n o t b e p r o d u c e d b y t h e d i g e s t i o n w i t h c h o n d r o i t i n a s e - A C b u t c a n b e p r o d u c e d w i t h c h o n d r o i t i n a s e - A B C ; C-unit, ADi-6S p r o d u c e d b y t h e d i g e s t i o n w i t h c h o n d r o i t i n a s e - A B C . O t h e r s ar~ m a i n l y c o m p o s e d o f o v e r s u l f a t e d - u n s a t u r a t e d d i s a c c h a r i d e s and Na c e t y l g a l a c t o s a m i n e - 4 - s u l f a t e ( f o r t h e s t r u c t u r e a n d a b b r e v i a t i o n o f t h e d i s a c e h a r i d e s , see Ref. 19). Fraction *
Product Resistant A-unit B-unit C-unit Others * See Fig. 1.
Muscle Y
22.9 12.3 52.5 3.5 8.8
Day 5
D a y 10
D a y 20
D a y 30 Y
X
Y
X
Y
X
Y
8.1 84.2 <~1.0 3.5 4.2
12.9 38.5 44.2 1.3 3.1
2.8 86.7 <1.0 5.5 5.0
10.5 57.8 28.3 1.1 2.3
3.3 87.2 <1.0 4.0 5.5
17.5 60.1 15.7 3.0 3.7
24.8 56.5 13.2 1.6 3.9
409 The relative amounts of isomeric chondroitin sulfates and chondroitinaseABC resistant part in the 3SS-labeled polysaccharide moiety from these fractions were shown in Table III. Of the products of chondroitinase
Proteoglycans in explants Bone matrix gelatin is able to induce the formation of hyaline cartilage in vitro [10]. If the cartilage formed in vitro by bone matrix gelatin has the same characteristics as the in vivo-induced cartilage, PCS-BMG must also be synthesized in the in vitro-induced cartilage. To examine this possibility, minced triceps muscle of near-term fetal rats was cultured on bone matrix gelatin in 199 medium containing newborn calf serum for 21 days. It has been shown by light microscopic examination that cartilage has been formed within the period. On the 21st day, [3SS]sulfate was added to the medium. After 3 h, the labeled proteoglycans were solubilized from the tissue with the guanidine HCl-protease inhibitor solution as described above. Most of the radioactivity incorporated (approx. 90%) was extracted under the conditions used. Fig. 3 shows the sedimentation profile of the labeled proteoglycans in the sucrose density gradient. It is obvious that the profile coincides with that of the sample from day-10
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implant (cf. Fig. lc). The composition o f isomeric chondroitin sulfates in the labeled polysaccharide moiety of the c o m p o n e n t with high sedimentation velocity was closely similar to that of PCS-BMG (Table IV). The length o f its polysaccharide chain (Kay = 0.54 on a Sepharose 6B column) was also equal to that of PCS-BMG. The polysaccharides from the fraction with lower sedimentation velocity were composed of chondroitin 4-sulfate (about 50% of total radioactivity), dermatan sulfate (about 25%) and chondroitinase resistant part (about 20%). From these results one can conclude that PCS-BMG is synthesized in b o t h cartilages induced in vivo and in vitro by bone matrix gelatin. Proteoglycans from other sources in rats
Many investigators have reported the occurrence of hyaline cartilage-unique
TABLE IV S U M M A R I S I N G T A B L E W H I C H C O M P A R E S SOME P R O P E R T I E S O F T H E 3 S S ° L A B E L E D P R O T E O CHONDROITIN SULFATES ISOLATED FROM VARIOUS SOURCES Proteochondroitin sulfate
PCS-BMG f r o m Day-10 implant PCS-BMG f r o m t h e explant R a t PCS-H
Overall size * ( K a y o n 2B)
" Length of polysac° e h a r i d e ** (Kay o n 6B)
C h o n d r o i t i n a s e d i g e s t i o n p r o d u c t *** (% o f t o t a l r a d i o a c t i v i t y ) Resistant
A-unit
B-unit
C-unit
Others
0.29
0.54
2.8
86.7
5.5
5.0
0.29 0.12
0.54 0.46
2.9 2.2
89.1 89.3
3.5 4.8
4.5 3.7
* See Fig. 5. ** See Fig. 2 a n d Fig. 6. *** F o r t h e a b b r e v i a t i o n o f t h e p r o d u c t s , see T a b l e I I I .
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Fig. 4. Sucrose density gradient centrifugation of the 3S S-labeled and unlabeled prot e ogl yc a ns isolated from epiphyseal cartilages (a) and diaphyseal bones (b). Abscissa, tube n u m b e r ; ordinate, arbitrary unit of b o t h radioactivity and h e x u r o n a t e . The d o t t e d line indicates tube 8 corresponding to the pos i t i on o f PCS-BMG. Fig. 5. Gel c h r o m a t o g r a p h y on Sepharose CL-2B of the major prot e ogl yc a ns isolated f~om the n e o n a t a l rat epiphyses (e e) and from day-10 i m p l a n t (o o). An a l i quot (approx. 50 000 d p m ) of each p r o t e o g l y c a n separated by sucrose density gradient was precipitated w i t h e t ha nol as described in the t e x t and dissolved in 1 ml of 2 M guanidine HCI/0.02 M Tris*HCl, pH 7.5. The solution was fractionated in a Sephazose CL-2B c o l u m n (1.5 X 50 cm), equilibrated w i t h the guanidine HC1 buffer at room temperature. Fractions of 2 ml were collected and assayed for radioactivity. Vo, void volume. Vt, t o t a l volume.
proteochondroitin sulfate (PCS-H) in embryonic chicks, based on the sucrose gradient separation [4,5,18]. To examine whether PCS-H exists in neonatal rat epiphyseal cartilages and, if so, whether the PCS-H can be distinguished from PCS-BMG, 3SS-labeled proteoglycans from neonatal rat epiphyseal cartilages were prepared by the essentially same m e t h o d as described above. The yields of labeled materials and hexuronate b y the extraction were more than 80%. Fig. 4a illustrates the sedimentation profile of the proteoglycans in the sucrose density gradient. There were t w o distinct components as it was the case of embryonic chick epiphyseal cartilages [18]; a heavier c o m p o n e n t and a lighter one. The heavier c o m p o n e n t comprised a b o u t 95% of total radioactivity and total hexuronate. When this major c o m p o n e n t was chromatographed on a Sepharose 2B column under dissociative condition, it was eluted as a somewhat broad peak (Kay = 0.12) near the void volume (Fig. 5). The polysaccharide obtained by alkaline ~-elimination of this c o m p o n e n t was chromatographed on a Sepharose 6B column (Fig. 6). Kay values of the labeled and unlabeled polysaccharide chains were both 0.46 +_0.02, similar to that (0.44) of the polysaccharide chain of PCS-H isolated from embryonic chick cartilage [5]. Digestion of the labeled polysaccharide with chondroitinases revealed that chondroitin 4-sulfate was the major (about 90% of total radioactivity) and that der-
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Fig. 6. Gel c h r o m a t o g r a p h y o n S e p h a r o s e C L - 6 B o f t h e 3 S S - l a b e l e d a n d u n l a b e l e d p o l y s a c c h a r i d e chains of the major proteoglycan, rat PCS-H, from neonatal rat epiphyses. Abscissa, tube number;'ordinate, arbit r a r y u n i t o f b o t h r a d i o a c t i v i t y a n d h e x u r o n a t e . V0, void v o l u m e ; Vt, t o t a l v o l u m e . T h e d o t t e d line i n d i v a t e s t u b e 3 7 c o r r e s p o n d i n g t o t h e e l u t i o n site o f t h e p o l y s a c c h a r i d e c h a i n o f P C S - B M G .
matan sulfate did not occur in the preparation (Table IV). These data indicate that the heavier c o m p o n e n t isolated from neonatal rat epiphysis has similar characteristics to chick PCS-H-except for the composition of isomeric chondroitin sulfates in the polysaccharide moiety. This proteochondroitin sulfate will be referred to as rat PCS-H hereinafter. Fig. l c and Fig. 4a indicate that PCS-BMG sedimented more slowly than rat PCS-H, suggesting that the overall size of PCS-BMG is smaller than that of rat PCS-H. Indeed, the peak for PCS-BMG isolated from day-10 implant on the Sepharose 2B column was more included (Fig. 5). PCS-BMG from the explant was eluted in the same position as that from the implant (Kay = 0.29). Some properties of these proteochondroitin sulfates were summarized in Table IV. The table shows that PCS-BMG is smaller in polysaccharide chain size as well as in overall size than rat PCS-H, while relative amounts of isomeric chondroitin sulfates in the polysaccharide moieties were similar. The present study provided an o p p o r t u n i t y to compare the proteoglycans synthesized by diaphyseal bones with the proteoglycans synthesized by epiphyseal cartilage of neonatal rats. 3SS-radioactivity incorporated in the bone was about one thirtieth of that in the cartilage, based on the tissue wet weight. The yields of the labeled materials and hexuronate were more than 75% under the conditions used. Most of the labeled materials were recovered as a somewhat broad peak at the top of the sucrose gradient (Fig. 4b). Little radioactivity was observed in the position corresponding to rat PCS-H. Of the chondroitinasedigestion products of the labeled polysaccharides of the materials, the A unit was the major (approx. 60% of total radioactivity). Less than 30% was proved to be resistant to the lyase reaction. These results show that the characteristics of the materials are similar to those of the proteoglycans from day-30 implant, in which the newly formed bone was predominant and hematopoietic bone marrow differentiation was evident.
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Fig. 7. Gel c h r o m a t o g r a p h y o n Bio-Gel A - 1 5 m o f t h e e x t r a c t f r o m d a y - 1 0 i m p l a n t i n c u b a t e d w i t h [ 14 C]p r o l i n e . A b o u t 2 m l of t h e g u a n i d i n e HC1 e x t r a c t w a s f r a c t i o n a t e d in a Bio-Gel A - 1 5 m c o l u m n (1.5 × 5 0 c m ) , e q u i l i b r a t e d w i t h 2 M g u a n i d i n e H C I / O . 0 2 M Tris-HCl, p H 7.5, at r o o m t e m p e r a t u r e . F r a c t i o n s o f 2 m l w e r e c o l l e c t e d a n d a s s a y e d f o r b o t h t o t a l r a d i o a c t i v i t y (e e) and [ 1 4 C ] h y d r o x y p r o l i n e (o-o). Solid bax above curve indicates the fractions corresponding to s-chains, which were subsequently pooled and subjected to chromatography on CM-cellulose (see Fig. 8a). V O, void volume. Vt, total volume. Fig. 8. CM-cellulose e l u t i o n p a t t e r n s of t h e l a b e l e d s - c h a i n s o b t a i n e d f r o m t h e g u a n i d i n e HC1 e x t r a c t b y gel c h r o m a t o g r a p h y (a), [ 1 4 C ] c o l l a g e n o u s c o m p o n e n t p r e c i p i t a t e d f r o m t h e NaC1 e x t r a c t w i t h 2.4 M NaC1 (b), a n d [ 1 4 C ] c o l l a g e n o u s c o m p o n e n t s u b s e q u e n t l y p r e c i p i t a t e d w i t h 4.3 M NaCI (c). E a c h s a m p l e was dissolved in 5 m l o f 4 M u r e a / O . 0 4 M s o d i u m a c e t a t e , p H 4.8, t o g e t h e r w i t h a b o u t 10 m g o f r a t skin soluble t y p e I collagen. T h e m i x t u r e w a s h e a t e d to 4 0 ° C for 1 0 rain a n d c h r o m a t o g z a p h e d in a CMcellulose c o l u m n (1.6 × 3 c m ) as d e s c r i b e d p r e v i o u s l y [ 2 3 ] . F r a c t i o n s o f 2 m l w e r e c o l l e c t e d a n d a s s a y e d for b o t h r a d i o a c t i v i t y a n d a b s o r b a n c e at 2 3 0 n m . Solid b a r s a b o v e c u r v e s i n d i c a t e t h e f r a c t i o n s c o r r e s p o n d i n g t o col-chains, w h i c h w e r e s u b s e q u e n t l y p o o l e d a n d s u b j e c t e d t o d i g e s t i o n w i t h CNBr.
Collagen synthesized in day-l O implant The major proteochondroitin sulfate (PCS-BMG) synthesized in bone matrix gelatin-induced hyaline cartilages is different in physical and chemical properties from rat PCS-H, namely the hyaline cartilage-unique proteochondroitin sulfate (see above). Then, do the cells in bone matrix gelatin-induced cartilages synthesize t y p e II collagen, or hyaline cartilage-unique collagen? To answer this question, the implants excised on day 10 was labeled with [14C]proline as described under Methods. 14C-labeled tissues were treated with the guanidine HCl-protease inhibitor solution by the same method used for proteoglycan extraction. Most of labeled materials (approx. 85% of total radioactivity) were solubilized by this method. When the extract was chromatographed on a Bio-Gel A-15m column as described previously [29], a large peak containing hydroxy[t4C]proline was eluted in the area expected for a-chains (Fig. 7). Additionally, there was a small, hydroxyproline-containing peak in the area expecting for triple-stranded collagens, which may be largely composed of t y p e III collagen [11]. The large peak fraction was chromatographed on a CM-cellulose column {Fig. 8a). Two
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Fig. 9. CM-ceHulose e l u t i o n p a t t e r n s o f t h e l a b e l e d CNBr p e p t i d e s d e r i v e d f r o m t h e c ~ l - c h a i n i s o l a t e d a f t e r p r e c i p i t a t i o n o f t h e c o l l a g e n at 2.4 M NaC1 (a) a n d f r o m t h e ~ l - c h a l n isolated a f t e r s u b s e q u e n t precipit a t i o n a t 4.3 M NaCI (b), a n d t h e CNBr p e p t i d e s d e r i v e d f r o m insoluble t y p e II c o l l a g e n p r e p a r e d f r o m b o v i n e m a n d i b u l a r cartilage (c). T h e C N B r p e p t i d e s w e r e d i s s o l v e d in 3 m l o f 0 . 0 2 M s o d i u m c i t r a t e b u f fer, p H 3.6, a t 4 0 ° C a n d c h r o m a t o g r a p h e d in a CM-cellulose c o l u m n (0.9 X 8 c m ) b y use o f a l i n e a r salt g r a d i e n t o f NaCI f r o m 0.01 to 0 . 1 6 M in t h e b u f f e r . T h e c a r r i e r w a s t h e C N B r p e p t i d e d e r i v e d f r o m ~ l ( I ) - c h a i n p r e p a r e d f r o m r a t skin. F r a c t i o n s o f 2 m l w e r e c o l l e c t e d a n d a s s a y e d f o r b o t h r a d i o a c t i v i t y a n d a b s o r b a n c e at 2 3 0 n m .
peaks were obtained; one is coeluted with rat skin a l chain and another with 2 chain. The'ratio of a 1 to a2 was 3.7:1, which is significantly higher than the 1 :a 2 ratio found in t y p e I collagen (2:1). This suggests that a mixture of type I and type II collagens is synthesized in day-10 implant. The corresponding ratios of the samples from day-5 implant and day-10 implant were 2.1:1 and 2.4:1, respectively. To separate the presumed collagen species by differential salt precipitation from neutral-salt solutions, the tissue labeled with ['4C]proline was treated with 1 M NaC1/0.05 M Tris-HC1, pH 7.5. The collagenous components were extracted and partially purified by the differential salt precipitation method. The CM-cellulose elution pattern of the collagen precipitated at 2.4 M NaC1 is presented in Fig. 8b, and a chromatogram illustrating the elution pattern of the collagen precipitated at 4.3 M NaC1 is shown in Fig. 8c. These chromatograms clearly indicate that two different collagens were separated. The ratios of a l to a2 for the 2.4 M precipitate was found to be 2.3:1, and that for the 4.3 M precipitate was 6.2:1. For further characterization of these °l-chains, each labeled chain was cleaved with CNBr in the presence of additional carrier a l ( I ) chain, and the resulting peptides were chromatographed on CM-cellulose. The [~4C]peptide profile of the 2.4 M NaCl-precipitated °l-chain was identical to a l ( I ) carrier collagen (Fig. 9a). On the other hand, the °l-chain that precipitated at 4.3 M NaC1 was identical to a l ( I I ) collagen from bovine mandibular cartilage (Fig. 9b and Fig. 9c). These results indicate that cells in day-10 implant synthesize both type I and t y p e II collagens. One may consider that the type II collagen may originate from the cartilaginous areas and the t y p e I collagen from the fibrous areas.
415 Discussion
The paper demonstrates the changes in proteoglycan types during the sequential cartilage and bone formation by bone matrix gelatin, based on sucrose density gradient separation. The synthetic activity for sulfated proteoglycans increased more than 8 times during first 5 days after implantation of bone matrix gelatin (Table II). The major proteoglycan synthesized in day-5 implant, when most cells were mesenchymal cells proliferating actively in contact with bone matrix gelatin, was a dermatan sulfate-rich, slowly sedimenting proteoglycan (Fig. 1 and Table III). The exact source of the proteoglycan is not clear, but some parts may originate from adhering soft tissues of the implants, such as muscle and fibrous tissue. Alternatively, one could consider that the mesenchymal cells which start to synthesize predominantly the dermatan sulfate-rich proteoglycan may serve as progenitor cells which eventually differentiate to chondroblasts. Although the idea has not been proposed to date, this may be supported by the reported occurrence of two different shifts in collagen types during the chondrogenesis; from type I to type II collagen synthesis during the chick limb chondrogenesis [1--3] and from type III to type II collagen synthesis during the bone matrix powder-induced chondrogenesis [ 11 ]. Synthesis of a new proteochondroitin sulfate, designated as PCS-BMG, starts about day 5 just prior to onset of the bone matrix gelatin-induced chondrogenesis. This molecule could be easily purified by the sucrose density gradient separation (Fig. 1 and Fig. 5). For separation of proteoglycans, CsC1 density gradient centrifugation is generally used [6,12,17,30,31]. This method can give satisfactory separation of proteins as a gel from some proteoglycans and good concentration of the proteoglycans to the bottom fraction of a centrifuge tube. It is apparent, however, that the bottom fraction contains many kinds of proteoglycan in some cases [12], judging from the subsequent gel chromatographic analysis of proteoglycans in the fraction. The sucrose density gradient method has an advantage in that a proteoglycan sample can be separated into at least four distinct fractions with one step procedure [4]. When the extract with guanidine HC1 solution contains enough proteoglycan for further analysis, the separation or purification procedure of the proteoglycans sometimes can be simplified as described in the present paper. Moreover, it should be stressed that some peculiar proteoglycans synthesized in embryonic chick epiphyseal cartilages can not be recovered in the bottom fraction of CsC1 density gradient [32]. The process of appearance and disappearance of PCS-BMG in implants bears a close resemblance to the morphological process that the hyaline cartilage is produced in muscle by implantation of bone matrix gelatin and then replaced with newly formed bone. A proteoglycan which could not be distinguished from PCS-BMG was shown to be a major component in the hyaline cartilage produced in near-term fetal rat triceps muscle cultured upon bone matrix gelatin. Nevertheless, it is remarkable that PCS-BMG has characteristics differing not only from those of muscle and bone proteoglycans but also from those of hyaline cartilage-unique proteoglycan, rat PCS-H, synthesized in neonatal rat epiphyseal cartilage. As PCS-BMG was proved to be smaller in the overall size as
416
well as in its polysaccharide chain size (Table IV), one may consider that rat PCS-H, synthesized in bone matrix gelatin-induced cartilage, has been converted into the form o f PCS-BMG by artificial enzymatic degradation during extraction procedure. To examine this possibility, we used a proteoglycanextracting solution of 4 M guanidine HC1/0.05 M Tris-HCl, pH 7.5, containing other kinds of enzyme-inhibitors such as 10 mM EDTA, 10 mM N-ethylmaleimide, 1 mM phenylmethanesulfonyl fluoride, and 0.36 mM pepstatin, which had been successfully used for procollagen extraction from embryonic chick epiphyseal cartilages [29]. However, the characteristics of the proteoglycan thus extracted were essentially the same as those of PCS-BMG (data not shown). These findings are consistent with the idea that there must be some functional differences for various hyaline cartilages in a single species because there is a heterogeneity with respect to molecular architecture of matrix macromolecules [12]. Although it is not clear in what ways PCS-BMG differs from rat PCS-H, at least two different possibilities can be considered: first, PCSBMG has the different protein core as well as the different polysaccharides from those of rat PCS-H, and second, PCS-BMG and rat PCS-H have the same protein core but the different polysaccharide chains. It is reasonable to regard that the protein core of PCS-BMG is the same as that of the PCS-H, namely the cartilage type o f proteoglycan, because bone matrix gelatin-induced cartilages have been proved to synthesize t y p e II collagen, namely the cartilage t y p e of collagen (Fig. 9). The difference in overall size between PCS-BMG and rat PCSH may largely be due to the difference in the length of their polysaccharide chains. Bone matrix gelatin-induced endochondral bone formation has been considered to be a useful experimental model for bone formation in fracture healing as well as in embryonic development. In fact, the sequential cellular transitions are similar to those observed in fracture healing [33]. Recently we isolated a proteochondroitin sulfate, which could not be distinguished from PCSBMG, from rat fibula-fracture callusat the stage when hyaline cartilage was predominant [34]. These findings, combined with the results shown in the present paper, suggest that bone matrix gelatin-induced bone formation is a useful experimental model for biochemical analysis of sequential process of fracture healing rather than of normal development of embryonic skeletal tissues.
Acknowledgements We thank Professor S. Suzuki, Nagoya university, for helPful suggestions during the work. We extend our appreciation to Miss K. Ozeki for excellent technical assistance.
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