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Studies on glycosaminoglycans of regenerating rabbit ear cartilage
DEVELOPMENTAL
BIOLOGY
86, 198-205
(1981)
Studies
on Glycosaminoglycans of Regenerating Rabbit Ear Cartilage
KAREN
A.
HASTY,
GERALD
N. SMITH,
JR., AND ANDREW
H. KANG
Departments of Anatomy, Biochemistry and Medicine, University of Tennessee Center for the Health Sciences, and the Veterans Administration Medical Center, Memphis, Tennessee 38163 Received December 15, 1980; accepted in revised form March 26, 1981 Cartilage regeneration in the adult rabbit ear was examined with respect to glycosaminoglycan (GAG) synthesis at various stages of the regeneration process. Increased hyaluronic acid and chondroitin sulfate synthesis was first seen 31 days after wounding, when a metachromatic cartilage matrix could be distinguished from blastemal cells. Analysis of cartilage and the overlying skin separately showed that 90% of the labeled chondroitin sulfate was found in the cartilage being regenerated. DEAE-cellulose chromatography of GAG preparations from 35-day regenerating cartilages showed hyaluronic acid and chondroitin sulfate peaks eluting in the same position as those isolated from normal cartilages. The identity of the hyaluronic acid and chondroitin sulfate peaks was confirmed by their susceptibility to Streptomyces hyaluronidase and chondroitinase ABC, respectively. Although the degree of sulfation in normal and regenerated cartilages was similar, the ratio of chondroitin B-sulfate to chondroitin I-sulfate was increased in regenerated cartilages. GAG preparations from unlabeled cartilages were digested with chondroitinase ABC and the disaccharide digestive products were identified and quantitated. Normal cartilage had a ADi-GS/ADi-4S ratio of 0.27; the same ratio for the regenerated cartilage was 1.58.
INTRODUCTION
Rabbit ears are unique among mammalian tissues in their ability to regenerate cartilage as well as skin and other connective tissue elements. A full thickness hole punched through a rabbit ear re-epithelializes and a proliferating mass of cells accumulates subepidermally. This blastemal tissue, consisting of fibroblasts and undifferentiated mesenchymal cells, grows inward and fills the defect. Then an organized cartilage matrix forms within the ingrowth, contiguous with the old cartilage (Vorontsova and Liosner, 1960; Joseph and Dyson, 1966; Goss and Grimes, 1972). The morphologic differentiation of recognizable cartilage matrix from blastemal tissues during regeneration in the rabbit ear makes this a particularly valuable system for study of adult cartilage regeneration and repair. Extracellular matrix components, primarily proteoglycans, hyaluronic acid, and collagen, are responsible for many of the properties of cartilage. In normal cartilage, the collagen is type II collagen and the proteoglycan component is largely chondroitin sulfate covalently bound to a protein core. We have investigated the genetically distinct types of collagen in the rabbit ear regeneration system and confirmed that the intact cartilage contains type II collagen. The undifferentiated regeneration blastema synthesizes type I and the unususual collagen, [(~1(1)]~, the type I trimer, but not type II. As cartilage differentiation begins, type II is syn0012-1606/81/110198-08$02.00/O Copyright All rights
0 1981 by Academic Press, Inc. of reproduction in any form reserved.
198
thesized and deposited in the regenerate (Smith and Kang, 1981). In this report, GAG’ synthesis and accumulation is correlated with the morphological stages of rabbit ear regeneration. GAG from normal cartilage, the undifferentiated cells of the blastema, and the differentiating cartilage of the regenerate are compared. MATERIALS
AND
METHODS
Surgical Procedures
Male New Zealand rabbits (Charles River) weighing 2.5 kg were anesthetized with Rompun Xylazine at 5 mg/kg combined with Ketamine hydrochloride at 35 mg/kg. The ears, shaved and cleaned with 40% ethanol, were punched using an Osbourne saddle punch (0.7-cm diameter) sterilized with ethanol. One hole per ear was positioned in the proximal half of the ear, taking care to avoid the major blood vessels. Pressure was applied to prevent excessive bleeding and ears were allowed to heal without further treatment. At various periods after wounding, the outgrowth and surrounding tissues were harvested together using a l.Zcm-diameter punch. 1 Abbreviations. GAG, glycosaminoglycan; CTAB, cetyltrimethylammonium bromide; DEAE, diethylamino ethyl. ADi-4S, ADi-GS, and ADi-OS, the unsaturated disaccharides produced by digestion of chondroitin I-sulfate, chondroitin 6-sulfate, or chondroitin, respectively, with chondroitinase ABC.
HASTY,
SMITH,
AND KANG
In studies of the regenerated cartilage matrix, the new cartilage was dissected from the integument and normal cartilage.
199
Rabbit Ear Glycosaminoglycans
Tissues were fixed overnight in ethanolic Bouin’s fluid, dehydrated in alcohols, cleared in toluene, and embedded in paraffin. Six-micrometer sections were stained with toluidine blue.
250 pg each of hyaluronate and chondroitin sulfate were added as carrier, and 0.5 mg CTAB was added to precipitate undigested GAGS. Precipitates were collected by centrifugation and washed twice with 0.05% CTAB/ 0.05 M NaCl, solubilized in absolute methanol, and counted in Aquasol. A comparison of the precipitated radioactivity in the buffer control, and the various enzyme digests, therefore, identified the proportion of label incorporated into hyaluronic acid, chondroitin sulfate, or dermatan sulfate.
Glycosaminoglycan
DEAE
Histological
Preparation
Isolation
Harvested tissues were minced and incubated for 5 hr at 3’7°C in 2 ml of Dulbecco’s medium supplemented with 50 pg/ml ascorbic acid, 300 pg/ml of glutamine, and 50 &i/ml of [3H]acetate. Pieces were preincubated for 30 min in medium without the radioactive precursor. Incubations took place in a humidified atmosphere of 10% COJ90% air. After incubation, medium was removed and tissues were homogenized in 0.05 M Tris/ HCl buffer, pH 7.5, containing 14% ethanol. An aliquot was removed for DNA determination (Burton, 1956). Samples were boiled for 3 min to inactivate endogenous polysaccharidases, then Protease (Sigma, Type VI, 1 mg per sample), preincubated for 30 min, was added and the samples were incubated overnight at 50°C. The Protease was inactivated by boiling for 3 min and the samples were dialyzed against distilled water and centrifuged to remove insoluble materials. Supernatants were frozen for further GAG analysis. In some experiments, the regenerating tissues were dissected into a central core region containing the cartilage and an inner and outer integument region. These tissues were incubated with radioactive precursor and the GAG isolated as described above. Similarly, unlabeled GAG preparations were made from 35-day regenerated cartilages and from normal cartilages.
Enzyme
Susceptibility
Assay
Labeled GAGS were assayed by enzyme susceptibility according to the method of Toole (1976). Enzymes were tested against known standards for specificity. Aliquots (0.5 ml) of isolated GAGS in enriched Tris/HCl buffer, pH 7.2, were incubated at 37°C for 5 hr with buffer alone or with 50 ~1 of buffer containing Streptomyces hyaluronidase (100 TRU/ml, Calbiochem) which degrades hyaluronic acid; chondroitinase ABC (2 U/ml, Miles Laboratories) which degrades hyaluronic acid, chondroitin sulfate, and dermatan sulfate; or chondroitinase AC II (3.1 U/ml, Miles Laboratories) which degrades hyaluronic acid and chondroitin sulfate (Saito et al., 1968; Smith and Newsome, 1978). After incubation, the samples were boiled to inactivate the enzyme,
Chromatography
Isolated GAGS prepared as for enzyme assay were chromatographed at room temperature on a 1 X lo-cm column of DEAE-cellulose (Sigma) equilibrated with 0.05 M Tris/HCl buffer, pH 7.2 (Orkin et al., 1976). GAGS were eluted with a 250-ml linear NaCl gradient (O-l M). Fractions of 3 ml were collected and aliquots were either counted for radioactivity or assayed for uranic acid. Isolated peaks were pooled, dialyzed against distilled water, and assayed for enzyme susceptibility. Chondroitin sulfate peaks were digested with chondroitinase ABC or AC-II and the disaccharide digestive products were separated by thin-layer chromatography. The radioactive disaccharides were quantitated by scintillation counting. Unlabeled disaccharides were assayed by N-acetylhexosamine assay (Reissig et al. 1955).
Thin-Layer
Chromatography
Isolated GAG preparations from labeled regenerating tissues, dissected normal and regenerated cartilage, or peaks from DEAE-cellulose chromatography were digested with chondroitinase ABC for 5 hr at 37°C. The disaccharide digestion products, ADi-4S, ADi-GS, and ADi-OS were chromatographed on cellulose thin-layer plates (Merck). Samples were spotted and desalted overnight in a solvent of butanol:ethanol:distilled water (523216; v:v:v). Disaccharides were separated using a butanol:acetic acid:ammonia (3:2:1; v:v:v) solvent for 6 hr (Saito et al., 1968; Wasserman, 1977). Labeled GAGS were run in the presence of carrier disaccharides (2.5 hg each, Miles Laboratories). The disaccharides were located under ultraviolet light and cut out for liquid scintillation counting or N-acetylhexosamine assay.
Enzymatic
Determinations
of Unlabeled
GAGS
Aliquots of isolated GAG preparations from labeled normal and regenerating tissues, dissected normal and regenerating cartilages, or DEAE-cellulose peaks were assayed by the method of Reissig et al. (1955) after digestion with chondroitinase AC-II. The products of these enzymes are the unsaturated disaccharides, ADi-
200
DEVELOPMENTAL
BIOLOGY
6S, ADi-OS, and ADi-4s. The color yields from ADi-6S and ADi-OS are identical, but ADi-4S cannot be measured by this technique (Orkin and Toole, 1978). Treating ADi-4S with 0.05 units of chondro-&sulfatase (Miles Laboratories) allows this material to be assayed (Saito et al., 1968). Therefore, assays performed without the sulfatase measure the ADi-6S and ADi-OS, while inclusion of chondro-4-sulfatase in the assay allows all three disaccharides to be measured. In some experiments, disacchrides separated on thin-layer chromatography plates and located by uv absorption were scraped off the plate and assayed directly. The cellulose was removed by centrifugation before spectrophotometric measurements were made. Uranic
Acid Assay
Uranic acid was assayed by the method of Bitter and Muir (1962) as modified by Benya and Nimni (1978). RESULTS
Histological
Studies
Regeneration in the rabbit ear proceeds through several histologically distinct stages (Fig. 1). The initial phase of epithelialization and wound healing is characterized by the migration of the wound epithelium from the dorsal and ventral surfaces of the ear over the cut surface (Fig. la). The wound epithelia from both surfaces usually meet between Days 10 and 14. At this point, a proliferative phase begins with mitotic cells accumulating under the wound epithelium. This homogeneous blastemal tissue accumulates and fills a 0.7cm hole in about 3 weeks (Figs. lb, c). The following phase of differentiation is marked by the appearance of new metachromatic cartilage matrix adjacent to the old cartilage during the fourth week (Fig. Id). By 35 days, substantial new cartilage matrix is seen, but is more loosely organized than the normal cartilage. By 55 days, however, the regenerated cartilage matrix is more compact than the early cartilage matrix. This mature cartilage persists, as seen in g-month regenerate (Fig. le), and closely resembles normal cartilage (Fig. If). Time Course
of GAG Synthesis
Tissue explants from the various stages of regeneration showed an increase in incorporation of E3H]acetate into GAG with time after wounding (Fig. 2). Incorporation was increased in both hyaluronic acid and chondroitin sulfate. Cultures labeled with ?S04 showed a similar increase in chondroitin sulfate synthesis with time (data not shown). By correlating the histological stages of regeneration with the incorporation experi-
VOLUME
86, 1981
ments, it can be seen that the increased GAG accumulation occurs during the differentiative phase rather than the epithelialization and proliferation periods. The large deviations during the differentiative phase probably reflect the varying times of the onset of cartilage differentiation between animals. During this differentiative phase, it became possible to isolate the newly differentiating cartilage from the dorsal and ventral integument tissues. A 38-day regenerate was manually dissected into a core region, which was beginning to deposit cartilage matrix, and an integument region. Each tissue was then incubated with [3H]acetate and the radioactive GAG determined. The core region synthesizes 90% of the counts incorporated into chondroitin sulfate (Table 1). The integument, although comprising the larger percentage of the composite regenerate, synthesizes only 10% of the labeled chondroitin sulfate, but incorporates four times as many counts into hyaluronic acid as does the core region. DEAE
Chromatography
of Isolated
GAGS
Labeled GAGS from normal and 35-day regenerating tissues were applied to DEAE-cellulose columns and eluted with a NaCl gradient. The elution patterns of labeled hyaluronic acid and chondroitin sulfate from normal and 35-day regenerates are similar (Fig. 3). The hyaluronic acid peak was digested by Streptomyces hyaluronidase, an enzyme which degrades only hyaluronic acid, eliminating the possibility that significant quantities of chondroitin, which co-elutes with hyaluronic acid, are present. Similarly, the chondroitin sulfate peak was digested to the same extent with either chondroitinase AC II, which degrades chondroitin 4-sulfate and chondroitin 6-sulfate but not dermatan sulfate, or chondroitinase ABC, which degrades all three, showing the presence of only chondroitin sulfate. Since 90% of the labeled chondroitin sulfate in a 35-day regenerate is in the core region, the differentiating chondrocytes appear to be synthesizing largely chondroitin 4-sulfate and chondroitin 6-sulfate. Because net accumulation of extracellular matrix components is the result of ongoing synthesis and degradation, we confirmed and extended the short-term synthesis study by direct analysis of unlabeled GAG accumulated during the regeneration process. Although high levels of hyaluronate were predicted on the basis of the incorporation experiments, very little hyaluronate was deposited in the matrix (Fig. 4) relative to the chrondroitin sulfate present. This unlabeled chondroitin sulfate was considered to be from regenerated cartilage matrix adjacent normal cartilage. To analyze the regenerated cartilage matrix by itself, it was necessary
FIG. 1. Morphological studies on rabbit Cells may be seen accumulating between Homogeneous blastemal cells accumulate phase-Day 23 on. New cartilage matrix Normal cartilage.
ear regeneration. (a) Epithelialization phase-Days O-12. Epithelium migrates across the wound. the cut surfaces of the cartilage and the wound epithelium. (b) Proliferation phase-Days 13-22. and the hole fills centripetally. (c) Blastema cells near cut surface of cartilage. (d) Differentiation appears, contiguous with the old cartilage matrix. (e) 8 month. Mature, regenerated cartilage. (f)
to dissect the regenerated cartilage from the integument and the adjacent normal cartilage. When the total accumulated GAG of the regenerated dissected cartilage was analyzed directly on 35-day regenerates and compared with normal cartilages, no hyaluronic acid was detected within the matrix (Fig. 5). The chondroitin sulfate from the regenerated cartilage yielded a broader peak than that from the normal cartilages, indicating the presence of a slightly undersulfated component in a mixture that included large amounts of normally sulfated chondroitin sulfate. The total chondroitin sulfate peak was pooled for further analysis.
Position
and Degree of Sulfa&m
To identify the degree and position of sulfation of the chondroitin chain, GAG preparations and chondroitin sulfate peaks isolated on DEAE-cellulose columns from normal and 35-day regenerated cartilages were digested with chondroitinase ABC and the unsaturated disaccharide digestive products were quantitated by N-acetylhexosamine assay. There was no difference in the amount of disaccharide per DNA in cartilages from normal and 35-day regenerates (Table 2). However, the postion of sulfation was different. In normal cartilage,
202
DEVELOPMENTAL
BIOLOGY
VOLUME
86,198l
HA
600
A NORMAL
TISSUES
il
t
06
L
z 04 ‘p a 02 = 8 -l’
ZO,,
t
25
A
L
30
35
40
45
B 35 DAY REGENERATING TISSUES
t
06
% g s --I T I j/
c 04 FIG. 2. Time course of incorporation of [3H]acetate into hyaluronate (HA) and chondroitin sulfate (CS) by regenerating rabbit ear tissues. Increased synthesis of labeled glycosaminoglycans was seen in tissues where cartilage was differentiating, Days 31 and 37, as opposed to normal, blastemal, and completed regenerates. Each point represents the mean of triplicate samples from two to five rabbits. Bars show standard error of the mean. Days O-12, epithelialization phase. Days 13-22, proliferation phase. Days 23 on, differentiation phase.
the ratio of chondroitin g-sulfate + chondroitin O-sulfate to chondroitin 4-sulfate is 0.31 + 0.14. In contrast, this ratio in 35day regenerated cartilage is 2.63 f 0.4 (Table 2). This assay does not distinguish between chondroitin 6-sulfate and chondroitin O-sulfate disaccharides. To extend this observation, the chondroitinase digestion products were separated by thin-layer chromatography, located by their uv absorption, and assayed for N-acetylhexosamine. Normal cartilage shows the expected predominance of ADi-4S, whereas early regen-
02
’
25
30 35 FRACTION
40 45 NUMBER
50
FIG. 3. DEAE-cellulose chromatography of rH]acetate labeled glycosaminoglycans from (A) normal and (B) regenerating tissues of the adult rabbit ear. Both tissues synthesized hyaluronic acid (HA) and chondroitin sulfate (CS) during the labeling period. The CS from normal and regenerating tissues elute at the same ionic strength (as measured by conductivity of fractions).
erated cartilage is enriched with respect to ADI-6S (Table 3). In both types of matrix, ADi-OS is a minor component, varying from 9% in the normal to 16% in the regenerate. The primary difference, therefore, between GAGS synthesized by the early differentiating chondrocytes and those of normal cartilage is the relHA
[~HIACETATE
TABLE INCORPORATION
Total pg DNA/tissue Integument Cartilage
17.6 10.4
wm HA//e DNA 3791 1787
35 DAY REGENERATING
1 OF 38 DAY REGENERATE Percentage total (Integument and Cartilage) cpm CS/ pg DNA
HA
cs
384 5630
78 22
10 90
Note. [3H]Acetate incorporation by a 38-day regenerate. The differentiating tissues from a 38-day regenerate were removed and dissected into the core region, which was beginning to deposit cartilage matrix (cartilage) and the inner and outer integument regions (integument). Dissected tissues were labeled in vitro and proportions of labeled hyaluronic acid (HA) and labeled chondroitin sulfate (CS) were determined by susceptibility to Streptomyces hyaluronidase or ehondroitinase ABC.
t
IOOL
TISSLQ
c 5 2 0 g 2 - 2o 0T
j>-hf$ 20
25
30 35 FRACTION
40 45 NUMBER
50
55
FIG. 4. DEAE-cellulose chromatography of [aH]acetate labeled glycosaminoglycans from 35-day regenerating tissue, at a time when substantial differentiating cartilage matrix is seen. Although a high level of incorporation of radioactivity into hyaluronic acid (HA) is seen, a uranic acid assay of the same fractions show little unlabeled HA present.
HASTY,
A. NORMAL
SMITH,
AND KANG
cs
CARTILAGE
HA i CS
6. 35 DAY CARTILAGE
I
I
I
I
I
1
1
I
25
30
35
40
45
50
55
FRACTION
NUMBER
FIG. 5. DEAE-cellulose chromatography of unlabeled glycosaminoglycans of isolated cartilages from normal and regenerating rabbit ear. Fractions were assayed for uranic acid to locate GAG. The chondroitin sulfate (CS) elutes in the same position from both tissues, although the regenerating matrix CS peak is broad, indicating more heterogeneity. The elution position of standard hyaluronic acid (HA) is shown, but very little is seen in either sample.
atively high level of chondroitin 6-sulfate synthesized with respect to chondroitin 4-sulfate. To determine if increased chondroitin 6-sulfate accumulation was seen in undifferentiated blastemal tissues or appeared as the cartilage matrix differentiated, labeled GAG preparations were prepared from early blastemal tissues at Days 7,15, and 22, where the cartilage component is normal cartilage from the surrounding wound tissue, and later differentiating stages at Days 31, 35, and 37, composed of normal adjacent cartilage plus the regenerating cartilage. Each sample was digested with chondroitinase AC II before thin-layer chromatography with carrier disaccharides. Disaccarides were located by ultraviolet absorption and cut out for scintillation counting. The ratio of ADi-6S/ ADi-4S is low in the blastemal tissues, and only increases during cartilage matrix differentiation (Table 4). Thus, chondroitin sulfate populations rich in chondroitin 6-sulfate are associated with the production of early cartilage matrix and not with blastema formation. DISCUSSION
During regeneration in the rabbit major glycosaminoglycan deposited
ear system, the in the cartilage
Rabbit
Ear Glycosaminoglycans
203
matrix is chondroitin sulfate. Chondroitin sulfates from normal and regenerating cartilage show similar chromatographic behavior on DEAE-cellulose columns. This suggests little variation in the degree of sulfation, since, under identical conditions, undersulfated chondroitin sulfate from brachymorphic mice elutes from DEAE-cellulose columns at a lower ionic strength than chondroitin sulfate from normal mice (Orkin et al., 1976). We have confirmed, by analysis of disaccharides from exhaustive chondroitinase ABC digests, that the chondroitin sulfate of early regenerates contains only 16% chondroitin, as compared to 9% in the normal cartilage. Although the degree of sulfation does not change significantly during regeneration in the rabbit ear, the position of sulfation does change. During the times of epithelialization and cellular proliferation of the blastema, chondroitin 4-sulfate predominates as the matrix component. Regenerates with differentiating cartilage matrix have an increased chondroitin 6-sulfate to chondroitin 4-sulfate ratio. This suggests a change in the position of sulfation in the differentiating cartilage matrix, since cartilage, isolated from the integument, is responsible for 90% of the labeled chondroitin sulfate synthesized at this time. The shift in the sulfation position of chondroitin sulfate synthesized during in vitro incubation by the differentiating cartilage matrix as compared to normal cartilage, was confirmed directly on regenerated cartilages. Unlabeled differentiating cartilage showed a predominance of chondroitin 6-sulfate in the matrix. This chondroitin 6-sulfate rich matrix may be a transient initial state in cartilage differentiation since preliminary evidence indicates a return to a chondroitin 4-sulfate rich matrix in older regenerates. The chondroitin 6-sulfate enrichment in the cartilage matrix of differentiating cartilage may, therefore, reCHONDROITINASE
GAG
TABLE 2. ABC DIGESTION OF UNLABELED CARTILAGES NORMAL AND REGENERATED TISSUES Total disaccharides (ADi-6s + ADi-OS + ADi-IS) as pg N-acetylhexosamine/ /tic DNA
preparations
Normal Cartilages 35-Day regenerated cartilages (6)
(8)
ADi-6S
FROM
+ ADi-OS/ ADi-IS
2.52
f 0.80
0.31
f 0.14
2.58
IL 0.33
2.63
f 0.4
Note. Disaccharides were quantitated by N-acetylhexosamine assay in the absence (measures chondroitin 6-sulfate and chondroitin Osulfate) or the presence (also measures chondroitin 4-sulfate) of chondro-4-sulfatase. Number of observations are shown in parentheses. Data are expressed as the mean + the standard deviation.
204
DEVELOPMENTAL
BIOLOGY
VOLUME
TABLE CHONDROITINASE
Normal cartilage Regenerated cartilage Nofe.
Disaccharides
were
located
ABC
DIGESTION
86,
1981
3
PRODUCTS
ISOLATED
ON THIN-LAYER
Percentage ADi-6S
Percentage
ADi-4S
ADi-OS
19.6 51.3
71.5 32.5
9.0 16.3
by uv absorption
and assayed
PLATES
Percentage
ADi-GS/ADi-4S 0.27 1.58
for N-acetylhexosamine.
capitulate the ontogeny of fetal cartilage in the rabbit. Mathews (1967) reported high initial levels of chondroitin g-sulfate relative to chondroitin 4-sulfate in fetal rabbit cartilage. The chondroitin 4-sulfate increases with age and soon reaches a chondroitin 4-sulfate/chondroitin 6-sulfate ratio of 2/l. A similar developmental sequence is seen in embryonic chick cartilage (Robinson and Dorfman, 1969). In embryonic chick epiphyseal cartilage between 10 and 19 days, the synthesis of chondroitin and chondroitin 6-sulfate progressively decreases and the synthesis of chondroitin 4-sulfate concomitantly increases. These developmental relationships between the levels of chondroitin 4- and chondroitin g-sulfate, therefore, appear to be a factor in cartilage differentiation embryologically, as well as in cartilage regeneration in the adult rabbit. Another model of cartilage regeneration in the rabbit ear has been developed using transplants of perichondrium taken from the auricular cartilage. The perichondrial membrane responded by proliferating and synthesizing new cartilage matrix (Skoog et al., 1972; Wasteson and Ohlien, 197’7). The Sod-labeled GAGS from the new matrix were 95% susceptible to chondroitinase AC II, indicating the presence of only chondroitin sulfate. Similarly, our analysis of chondroitin sulfate peaks on DEAE-cellulose from unlabeled normal and regenerated cartilages shows identical degradation with chondroitinase ABC or chondroitinase AC II, thus eliminating the possibility of dermatan sulfate being present. When the isomers of chondroitin sulfate were examined in the cartilage regenerated from the perichondrium, the ratio of chondroitin 6-sulfate to chondroitin 4-sulfate was increased, but not to the extent seen in our system. The times of regeneration in the two systems are difficult to compare, but conceivably the perichondrial regenerate might have an even higher chondroitin 6-sulfate:chondroitin 4-sulfate ratio if examined earlier. The increased chondroitin 6sulfate may be characteristic of a proliferating chondrocyte just beginning to synthesize cartilage matrix. A model of cartilage matrix organization involving the association of proteoglycan subunits with hyaluranic acid has been proposed for hyaline and articular cartilages (Hardingham and Muir, 1972, 1974; Hascall
and Heinegard, 1974). The major proteoglycan of cartilage consists of a central protein core, with chondroitin sulfate and keratan sulfate side chains covalently bound along its length. A number of these proteoglycan molecules are then noncovalently linked to a common hyaluronic acid molecule, via a specialized linkage region of the protein core. In the elastic cartilage of the rabbit ear, a similar organization is probable. Proteoglycans produced by rabbit ear chondrocytes during short-term culture form a high proportion of aggregates with added hyaluronic acid. These proteoglycans contain chondroitin 4-sulfate and chondroitin 6-sulfate in a 2/l ratio, as well as a small amount of keratan sulfate (Madsen and Lohmander, 1979). Studies are underway in our laboratory to determine whether the chondroitin 6-sulfate rich proteoglycan of the newly regenerated cartilage matrix differs in physical properties from the chondroitin 4-sulfate rich proteoglycan of the mature cartilage. The second major GAG synthesized during regeneration in the rabbit ear system is hyaluronic acid. We had predicted high levels of hyaluronate synthesis during the outgrowth phase of regeneration, but a peak of hyaluronic acid synthesis was not seen until cartilage differentiation began. This is in contrast with the model of newt limb regeneration (Toole and Gross, 1971) where incorporation of [3H]acetate into hyaluronic acid peaked early in regeneration and declined with the onset of differentiation. In the newt, however, regenera-
CHONDROITINASE
TABLE 4 AC DIGESTION PRODUCTS
SUES, DAYS 7, 15, 22, BEFORE REGENERATING SEEN AND DIFFERENTIATING TISSUES, DAYS TENSIVE MATRIX IS FOUND
FROM
3H cpm
GAG
preparation
Blastemal tissues Differentiating tissues
BLASTEMAL
CARTILAGE MATRIX 31,35 AND 37, WHEN
TISIs Ex-
ADi-6S/
3H cpm ADi-IS 0.52 0.94
f 0.09 f 0.07
Note. The labeled disaccharides were isolated by thin-layer chromatography in the presence of carrier disaccharides, located by uv absorption and cut out for scintillation counting. Data are expressed as the mean + standard deviation.
HASTY,
SMITH,
AND KANG
tion is marked by extensive dedifferentiation of ,muscle and connective tissue during the first 10 days. Further hyaluronic acid synthesis was shown to be higher in the stump tissues where muscle and connective tissues were dedifferentiating, than in the outgrowth tissues, were proliferation predominates. In contrast, the rabbit ear has no muscle at the level where the punch is made and shows limited dedifferentiation of the original cartilage during blastemal formation. A regenerating tissue may have increased hyaluronic acid synthesis only where extensive dedifferentiation is in progress. Most of the hyaluronate synthesis in the rabbit ear occurs in the integument portions of the regenerate, but in short-term incubations in vitro, appreciable levels of hyaluronate synthesis were seen in regenerated cartilage samples. However, assay of unlabeled GAG from the new cartilage shows very little matrix accumulation of the hyaluronic acid. This may reflect high turnover of hyaluronic acid during the regeneration process. High levels of hyaluronidase activity are seen in the regenerating newt limb during cartilage differentiation (Toole and Gross, 1971; Smith et al., 1975). Similar correlations are seen in a variety of developmental situations (Toole, 1976). The negligible amounts of hyaluronate accumulated in the differentiating rabbit ear cartilage may reflect the balance between hyaluronic acid synthesis and degradation. Supported by New Faculty Research Grant HO0033 and funds from the Veteran’s Administration. We would like to thank Dr. Bryan P. Toole for valuable discussion. These results are included in the senior author’s Ph.D. dissertation (University of Tennessee Graduate School of Medical Sciences).
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205
Rabbit Ear Glycosaminoglycans hyaluronic
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with
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Acta 279, 401-405. HARDINGHAM, T. E., and MUIR, H. (1974). Hyaluronic acid in cartilage and proteoglycan aggregation. Biochem. J. 139, 565-581. HASCALL, V. C., and HEINEGARD, D. (1975). The structure of cartilage proteoglycans. In “Extracellular Matrix Influences on Gene Expression” (H. C. Slavkin and R. C. Greulich, eds.) pp. 423-433. Academic Press, New York. JOSEPH, J., and DYSON, M. (1966). Tissue replacement in the rabbit ear. Brit. J. Surg. 53, 372-380. MATHEWS, M. B. (1967). Macromolecular evolution of connective tissue. Biol. Rev. 42, 499-551. ORKIN, R. W., and TOOLE, B. P. (1978). Hyaluronidase activity and hyaluronate content of the developing chick embryo heart. Develop.
Biol. 66, 208-320. ORKIN, R. W., PRATT, chondroitin sulfate
R. M., and MARTIN, G. R., (1976). Undersulfated in the cartilage matrix of brachymorphic mice.
Develop. Biol. 50, 82-94. RESSIG, J. L., STROMINGER, J. L., and LELOIR, L. R. (1955). A modified calorimetric method for the estimation of N-acetylamino sugars. J. Biol. Chem. 217,959-966. ROBINSON, H. C., and DORFMAN, A. (1969). The sulfation of chondroitin sulfate in embryonic chick cartilage epiphysis. J. Biol. Chem. 244, 348-352. SAITO, H., YAMAGATA, T., and SUZUKI, S. (1968). Enzymatic methods for the determination of small quantities of isomeric chondroitin sulfates. J. Biol. Chem. 243, 1536-1542. SKOOG, T., OHLSEN, L., and SOHN, S. A. (1972). Perichondrial potential for cartilaginous regeneration. Stand. J. Plast. Reconstr. Surg. 6, 123-125. SMITH, G. N., JR., and KANG, A. H. (1981). Collagen synthesis by regenerating rabbit ear tissue. Develop. Biol., in press. SMITH, G. N., JR., and NEWSOME, D. A. (1975). of the glycosaminoglycans of the embryonic
The nature and origin chick vitreous body.
Develop. Biol. 62, 65-77. SMITH, G. N., JR., TOOLE, B. P., and GROSS, J. (1975). Hyaluronidase activity and glycosaminoglycan synthesis in the amputated newt limb. Comparison of denervated, nonregenerating limbs with regenerates. Develop. Biol. 43, 221-232. TOOLE, B. P. (1976). Morphogenetic role of glycosaminoglycans (acid mucopolysaccharides) in brain and other tissues. In “Neuronal Recognition” (S. H. Baronides, ed.), pp. 275-329. Plenum, New York. TOOLE, B. P., and GROSS, J. (1971). The extracellular matrix of the regenerating newt limb: Synthesis and removal of hyaluronate prior to differentiation. Develop. Biol. 25, 57-77. VORONTSOVA, M. A., and LIOSNER, L. D. (1960). “Asexual Propagation and Regeneration.” Pergamon, London. WASSERMAN, L., BER A., and ALLALOUF, D. (1977). Use of thin layer chromatography in the separation of disaccharides resulting from digestion of chondroitin sulphates with chondroitinases. J. Chromatogr. 136,342-347. WASTESON, A., and OHLSEN, L. (1977). Biosynthesis of chondroitin sulfate in cartilage regenerated from perichondrium. Stand. J. Plast. Reconstr. Surg. 11, 17-22.
BIOLOGY
86, 198-205
(1981)
Studies
on Glycosaminoglycans of Regenerating Rabbit Ear Cartilage
KAREN
A.
HASTY,
GERALD
N. SMITH,
JR., AND ANDREW
H. KANG
Departments of Anatomy, Biochemistry and Medicine, University of Tennessee Center for the Health Sciences, and the Veterans Administration Medical Center, Memphis, Tennessee 38163 Received December 15, 1980; accepted in revised form March 26, 1981 Cartilage regeneration in the adult rabbit ear was examined with respect to glycosaminoglycan (GAG) synthesis at various stages of the regeneration process. Increased hyaluronic acid and chondroitin sulfate synthesis was first seen 31 days after wounding, when a metachromatic cartilage matrix could be distinguished from blastemal cells. Analysis of cartilage and the overlying skin separately showed that 90% of the labeled chondroitin sulfate was found in the cartilage being regenerated. DEAE-cellulose chromatography of GAG preparations from 35-day regenerating cartilages showed hyaluronic acid and chondroitin sulfate peaks eluting in the same position as those isolated from normal cartilages. The identity of the hyaluronic acid and chondroitin sulfate peaks was confirmed by their susceptibility to Streptomyces hyaluronidase and chondroitinase ABC, respectively. Although the degree of sulfation in normal and regenerated cartilages was similar, the ratio of chondroitin B-sulfate to chondroitin I-sulfate was increased in regenerated cartilages. GAG preparations from unlabeled cartilages were digested with chondroitinase ABC and the disaccharide digestive products were identified and quantitated. Normal cartilage had a ADi-GS/ADi-4S ratio of 0.27; the same ratio for the regenerated cartilage was 1.58.
INTRODUCTION
Rabbit ears are unique among mammalian tissues in their ability to regenerate cartilage as well as skin and other connective tissue elements. A full thickness hole punched through a rabbit ear re-epithelializes and a proliferating mass of cells accumulates subepidermally. This blastemal tissue, consisting of fibroblasts and undifferentiated mesenchymal cells, grows inward and fills the defect. Then an organized cartilage matrix forms within the ingrowth, contiguous with the old cartilage (Vorontsova and Liosner, 1960; Joseph and Dyson, 1966; Goss and Grimes, 1972). The morphologic differentiation of recognizable cartilage matrix from blastemal tissues during regeneration in the rabbit ear makes this a particularly valuable system for study of adult cartilage regeneration and repair. Extracellular matrix components, primarily proteoglycans, hyaluronic acid, and collagen, are responsible for many of the properties of cartilage. In normal cartilage, the collagen is type II collagen and the proteoglycan component is largely chondroitin sulfate covalently bound to a protein core. We have investigated the genetically distinct types of collagen in the rabbit ear regeneration system and confirmed that the intact cartilage contains type II collagen. The undifferentiated regeneration blastema synthesizes type I and the unususual collagen, [(~1(1)]~, the type I trimer, but not type II. As cartilage differentiation begins, type II is syn0012-1606/81/110198-08$02.00/O Copyright All rights
0 1981 by Academic Press, Inc. of reproduction in any form reserved.
198
thesized and deposited in the regenerate (Smith and Kang, 1981). In this report, GAG’ synthesis and accumulation is correlated with the morphological stages of rabbit ear regeneration. GAG from normal cartilage, the undifferentiated cells of the blastema, and the differentiating cartilage of the regenerate are compared. MATERIALS
AND
METHODS
Surgical Procedures
Male New Zealand rabbits (Charles River) weighing 2.5 kg were anesthetized with Rompun Xylazine at 5 mg/kg combined with Ketamine hydrochloride at 35 mg/kg. The ears, shaved and cleaned with 40% ethanol, were punched using an Osbourne saddle punch (0.7-cm diameter) sterilized with ethanol. One hole per ear was positioned in the proximal half of the ear, taking care to avoid the major blood vessels. Pressure was applied to prevent excessive bleeding and ears were allowed to heal without further treatment. At various periods after wounding, the outgrowth and surrounding tissues were harvested together using a l.Zcm-diameter punch. 1 Abbreviations. GAG, glycosaminoglycan; CTAB, cetyltrimethylammonium bromide; DEAE, diethylamino ethyl. ADi-4S, ADi-GS, and ADi-OS, the unsaturated disaccharides produced by digestion of chondroitin I-sulfate, chondroitin 6-sulfate, or chondroitin, respectively, with chondroitinase ABC.
HASTY,
SMITH,
AND KANG
In studies of the regenerated cartilage matrix, the new cartilage was dissected from the integument and normal cartilage.
199
Rabbit Ear Glycosaminoglycans
Tissues were fixed overnight in ethanolic Bouin’s fluid, dehydrated in alcohols, cleared in toluene, and embedded in paraffin. Six-micrometer sections were stained with toluidine blue.
250 pg each of hyaluronate and chondroitin sulfate were added as carrier, and 0.5 mg CTAB was added to precipitate undigested GAGS. Precipitates were collected by centrifugation and washed twice with 0.05% CTAB/ 0.05 M NaCl, solubilized in absolute methanol, and counted in Aquasol. A comparison of the precipitated radioactivity in the buffer control, and the various enzyme digests, therefore, identified the proportion of label incorporated into hyaluronic acid, chondroitin sulfate, or dermatan sulfate.
Glycosaminoglycan
DEAE
Histological
Preparation
Isolation
Harvested tissues were minced and incubated for 5 hr at 3’7°C in 2 ml of Dulbecco’s medium supplemented with 50 pg/ml ascorbic acid, 300 pg/ml of glutamine, and 50 &i/ml of [3H]acetate. Pieces were preincubated for 30 min in medium without the radioactive precursor. Incubations took place in a humidified atmosphere of 10% COJ90% air. After incubation, medium was removed and tissues were homogenized in 0.05 M Tris/ HCl buffer, pH 7.5, containing 14% ethanol. An aliquot was removed for DNA determination (Burton, 1956). Samples were boiled for 3 min to inactivate endogenous polysaccharidases, then Protease (Sigma, Type VI, 1 mg per sample), preincubated for 30 min, was added and the samples were incubated overnight at 50°C. The Protease was inactivated by boiling for 3 min and the samples were dialyzed against distilled water and centrifuged to remove insoluble materials. Supernatants were frozen for further GAG analysis. In some experiments, the regenerating tissues were dissected into a central core region containing the cartilage and an inner and outer integument region. These tissues were incubated with radioactive precursor and the GAG isolated as described above. Similarly, unlabeled GAG preparations were made from 35-day regenerated cartilages and from normal cartilages.
Enzyme
Susceptibility
Assay
Labeled GAGS were assayed by enzyme susceptibility according to the method of Toole (1976). Enzymes were tested against known standards for specificity. Aliquots (0.5 ml) of isolated GAGS in enriched Tris/HCl buffer, pH 7.2, were incubated at 37°C for 5 hr with buffer alone or with 50 ~1 of buffer containing Streptomyces hyaluronidase (100 TRU/ml, Calbiochem) which degrades hyaluronic acid; chondroitinase ABC (2 U/ml, Miles Laboratories) which degrades hyaluronic acid, chondroitin sulfate, and dermatan sulfate; or chondroitinase AC II (3.1 U/ml, Miles Laboratories) which degrades hyaluronic acid and chondroitin sulfate (Saito et al., 1968; Smith and Newsome, 1978). After incubation, the samples were boiled to inactivate the enzyme,
Chromatography
Isolated GAGS prepared as for enzyme assay were chromatographed at room temperature on a 1 X lo-cm column of DEAE-cellulose (Sigma) equilibrated with 0.05 M Tris/HCl buffer, pH 7.2 (Orkin et al., 1976). GAGS were eluted with a 250-ml linear NaCl gradient (O-l M). Fractions of 3 ml were collected and aliquots were either counted for radioactivity or assayed for uranic acid. Isolated peaks were pooled, dialyzed against distilled water, and assayed for enzyme susceptibility. Chondroitin sulfate peaks were digested with chondroitinase ABC or AC-II and the disaccharide digestive products were separated by thin-layer chromatography. The radioactive disaccharides were quantitated by scintillation counting. Unlabeled disaccharides were assayed by N-acetylhexosamine assay (Reissig et al. 1955).
Thin-Layer
Chromatography
Isolated GAG preparations from labeled regenerating tissues, dissected normal and regenerated cartilage, or peaks from DEAE-cellulose chromatography were digested with chondroitinase ABC for 5 hr at 37°C. The disaccharide digestion products, ADi-4S, ADi-GS, and ADi-OS were chromatographed on cellulose thin-layer plates (Merck). Samples were spotted and desalted overnight in a solvent of butanol:ethanol:distilled water (523216; v:v:v). Disaccharides were separated using a butanol:acetic acid:ammonia (3:2:1; v:v:v) solvent for 6 hr (Saito et al., 1968; Wasserman, 1977). Labeled GAGS were run in the presence of carrier disaccharides (2.5 hg each, Miles Laboratories). The disaccharides were located under ultraviolet light and cut out for liquid scintillation counting or N-acetylhexosamine assay.
Enzymatic
Determinations
of Unlabeled
GAGS
Aliquots of isolated GAG preparations from labeled normal and regenerating tissues, dissected normal and regenerating cartilages, or DEAE-cellulose peaks were assayed by the method of Reissig et al. (1955) after digestion with chondroitinase AC-II. The products of these enzymes are the unsaturated disaccharides, ADi-
200
DEVELOPMENTAL
BIOLOGY
6S, ADi-OS, and ADi-4s. The color yields from ADi-6S and ADi-OS are identical, but ADi-4S cannot be measured by this technique (Orkin and Toole, 1978). Treating ADi-4S with 0.05 units of chondro-&sulfatase (Miles Laboratories) allows this material to be assayed (Saito et al., 1968). Therefore, assays performed without the sulfatase measure the ADi-6S and ADi-OS, while inclusion of chondro-4-sulfatase in the assay allows all three disaccharides to be measured. In some experiments, disacchrides separated on thin-layer chromatography plates and located by uv absorption were scraped off the plate and assayed directly. The cellulose was removed by centrifugation before spectrophotometric measurements were made. Uranic
Acid Assay
Uranic acid was assayed by the method of Bitter and Muir (1962) as modified by Benya and Nimni (1978). RESULTS
Histological
Studies
Regeneration in the rabbit ear proceeds through several histologically distinct stages (Fig. 1). The initial phase of epithelialization and wound healing is characterized by the migration of the wound epithelium from the dorsal and ventral surfaces of the ear over the cut surface (Fig. la). The wound epithelia from both surfaces usually meet between Days 10 and 14. At this point, a proliferative phase begins with mitotic cells accumulating under the wound epithelium. This homogeneous blastemal tissue accumulates and fills a 0.7cm hole in about 3 weeks (Figs. lb, c). The following phase of differentiation is marked by the appearance of new metachromatic cartilage matrix adjacent to the old cartilage during the fourth week (Fig. Id). By 35 days, substantial new cartilage matrix is seen, but is more loosely organized than the normal cartilage. By 55 days, however, the regenerated cartilage matrix is more compact than the early cartilage matrix. This mature cartilage persists, as seen in g-month regenerate (Fig. le), and closely resembles normal cartilage (Fig. If). Time Course
of GAG Synthesis
Tissue explants from the various stages of regeneration showed an increase in incorporation of E3H]acetate into GAG with time after wounding (Fig. 2). Incorporation was increased in both hyaluronic acid and chondroitin sulfate. Cultures labeled with ?S04 showed a similar increase in chondroitin sulfate synthesis with time (data not shown). By correlating the histological stages of regeneration with the incorporation experi-
VOLUME
86, 1981
ments, it can be seen that the increased GAG accumulation occurs during the differentiative phase rather than the epithelialization and proliferation periods. The large deviations during the differentiative phase probably reflect the varying times of the onset of cartilage differentiation between animals. During this differentiative phase, it became possible to isolate the newly differentiating cartilage from the dorsal and ventral integument tissues. A 38-day regenerate was manually dissected into a core region, which was beginning to deposit cartilage matrix, and an integument region. Each tissue was then incubated with [3H]acetate and the radioactive GAG determined. The core region synthesizes 90% of the counts incorporated into chondroitin sulfate (Table 1). The integument, although comprising the larger percentage of the composite regenerate, synthesizes only 10% of the labeled chondroitin sulfate, but incorporates four times as many counts into hyaluronic acid as does the core region. DEAE
Chromatography
of Isolated
GAGS
Labeled GAGS from normal and 35-day regenerating tissues were applied to DEAE-cellulose columns and eluted with a NaCl gradient. The elution patterns of labeled hyaluronic acid and chondroitin sulfate from normal and 35-day regenerates are similar (Fig. 3). The hyaluronic acid peak was digested by Streptomyces hyaluronidase, an enzyme which degrades only hyaluronic acid, eliminating the possibility that significant quantities of chondroitin, which co-elutes with hyaluronic acid, are present. Similarly, the chondroitin sulfate peak was digested to the same extent with either chondroitinase AC II, which degrades chondroitin 4-sulfate and chondroitin 6-sulfate but not dermatan sulfate, or chondroitinase ABC, which degrades all three, showing the presence of only chondroitin sulfate. Since 90% of the labeled chondroitin sulfate in a 35-day regenerate is in the core region, the differentiating chondrocytes appear to be synthesizing largely chondroitin 4-sulfate and chondroitin 6-sulfate. Because net accumulation of extracellular matrix components is the result of ongoing synthesis and degradation, we confirmed and extended the short-term synthesis study by direct analysis of unlabeled GAG accumulated during the regeneration process. Although high levels of hyaluronate were predicted on the basis of the incorporation experiments, very little hyaluronate was deposited in the matrix (Fig. 4) relative to the chrondroitin sulfate present. This unlabeled chondroitin sulfate was considered to be from regenerated cartilage matrix adjacent normal cartilage. To analyze the regenerated cartilage matrix by itself, it was necessary
FIG. 1. Morphological studies on rabbit Cells may be seen accumulating between Homogeneous blastemal cells accumulate phase-Day 23 on. New cartilage matrix Normal cartilage.
ear regeneration. (a) Epithelialization phase-Days O-12. Epithelium migrates across the wound. the cut surfaces of the cartilage and the wound epithelium. (b) Proliferation phase-Days 13-22. and the hole fills centripetally. (c) Blastema cells near cut surface of cartilage. (d) Differentiation appears, contiguous with the old cartilage matrix. (e) 8 month. Mature, regenerated cartilage. (f)
to dissect the regenerated cartilage from the integument and the adjacent normal cartilage. When the total accumulated GAG of the regenerated dissected cartilage was analyzed directly on 35-day regenerates and compared with normal cartilages, no hyaluronic acid was detected within the matrix (Fig. 5). The chondroitin sulfate from the regenerated cartilage yielded a broader peak than that from the normal cartilages, indicating the presence of a slightly undersulfated component in a mixture that included large amounts of normally sulfated chondroitin sulfate. The total chondroitin sulfate peak was pooled for further analysis.
Position
and Degree of Sulfa&m
To identify the degree and position of sulfation of the chondroitin chain, GAG preparations and chondroitin sulfate peaks isolated on DEAE-cellulose columns from normal and 35-day regenerated cartilages were digested with chondroitinase ABC and the unsaturated disaccharide digestive products were quantitated by N-acetylhexosamine assay. There was no difference in the amount of disaccharide per DNA in cartilages from normal and 35-day regenerates (Table 2). However, the postion of sulfation was different. In normal cartilage,
202
DEVELOPMENTAL
BIOLOGY
VOLUME
86,198l
HA
600
A NORMAL
TISSUES
il
t
06
L
z 04 ‘p a 02 = 8 -l’
ZO,,
t
25
A
L
30
35
40
45
B 35 DAY REGENERATING TISSUES
t
06
% g s --I T I j/
c 04 FIG. 2. Time course of incorporation of [3H]acetate into hyaluronate (HA) and chondroitin sulfate (CS) by regenerating rabbit ear tissues. Increased synthesis of labeled glycosaminoglycans was seen in tissues where cartilage was differentiating, Days 31 and 37, as opposed to normal, blastemal, and completed regenerates. Each point represents the mean of triplicate samples from two to five rabbits. Bars show standard error of the mean. Days O-12, epithelialization phase. Days 13-22, proliferation phase. Days 23 on, differentiation phase.
the ratio of chondroitin g-sulfate + chondroitin O-sulfate to chondroitin 4-sulfate is 0.31 + 0.14. In contrast, this ratio in 35day regenerated cartilage is 2.63 f 0.4 (Table 2). This assay does not distinguish between chondroitin 6-sulfate and chondroitin O-sulfate disaccharides. To extend this observation, the chondroitinase digestion products were separated by thin-layer chromatography, located by their uv absorption, and assayed for N-acetylhexosamine. Normal cartilage shows the expected predominance of ADi-4S, whereas early regen-
02
’
25
30 35 FRACTION
40 45 NUMBER
50
FIG. 3. DEAE-cellulose chromatography of rH]acetate labeled glycosaminoglycans from (A) normal and (B) regenerating tissues of the adult rabbit ear. Both tissues synthesized hyaluronic acid (HA) and chondroitin sulfate (CS) during the labeling period. The CS from normal and regenerating tissues elute at the same ionic strength (as measured by conductivity of fractions).
erated cartilage is enriched with respect to ADI-6S (Table 3). In both types of matrix, ADi-OS is a minor component, varying from 9% in the normal to 16% in the regenerate. The primary difference, therefore, between GAGS synthesized by the early differentiating chondrocytes and those of normal cartilage is the relHA
[~HIACETATE
TABLE INCORPORATION
Total pg DNA/tissue Integument Cartilage
17.6 10.4
wm HA//e DNA 3791 1787
35 DAY REGENERATING
1 OF 38 DAY REGENERATE Percentage total (Integument and Cartilage) cpm CS/ pg DNA
HA
cs
384 5630
78 22
10 90
Note. [3H]Acetate incorporation by a 38-day regenerate. The differentiating tissues from a 38-day regenerate were removed and dissected into the core region, which was beginning to deposit cartilage matrix (cartilage) and the inner and outer integument regions (integument). Dissected tissues were labeled in vitro and proportions of labeled hyaluronic acid (HA) and labeled chondroitin sulfate (CS) were determined by susceptibility to Streptomyces hyaluronidase or ehondroitinase ABC.
t
IOOL
TISSLQ
c 5 2 0 g 2 - 2o 0T
j>-hf$ 20
25
30 35 FRACTION
40 45 NUMBER
50
55
FIG. 4. DEAE-cellulose chromatography of [aH]acetate labeled glycosaminoglycans from 35-day regenerating tissue, at a time when substantial differentiating cartilage matrix is seen. Although a high level of incorporation of radioactivity into hyaluronic acid (HA) is seen, a uranic acid assay of the same fractions show little unlabeled HA present.
HASTY,
A. NORMAL
SMITH,
AND KANG
cs
CARTILAGE
HA i CS
6. 35 DAY CARTILAGE
I
I
I
I
I
1
1
I
25
30
35
40
45
50
55
FRACTION
NUMBER
FIG. 5. DEAE-cellulose chromatography of unlabeled glycosaminoglycans of isolated cartilages from normal and regenerating rabbit ear. Fractions were assayed for uranic acid to locate GAG. The chondroitin sulfate (CS) elutes in the same position from both tissues, although the regenerating matrix CS peak is broad, indicating more heterogeneity. The elution position of standard hyaluronic acid (HA) is shown, but very little is seen in either sample.
atively high level of chondroitin 6-sulfate synthesized with respect to chondroitin 4-sulfate. To determine if increased chondroitin 6-sulfate accumulation was seen in undifferentiated blastemal tissues or appeared as the cartilage matrix differentiated, labeled GAG preparations were prepared from early blastemal tissues at Days 7,15, and 22, where the cartilage component is normal cartilage from the surrounding wound tissue, and later differentiating stages at Days 31, 35, and 37, composed of normal adjacent cartilage plus the regenerating cartilage. Each sample was digested with chondroitinase AC II before thin-layer chromatography with carrier disaccharides. Disaccarides were located by ultraviolet absorption and cut out for scintillation counting. The ratio of ADi-6S/ ADi-4S is low in the blastemal tissues, and only increases during cartilage matrix differentiation (Table 4). Thus, chondroitin sulfate populations rich in chondroitin 6-sulfate are associated with the production of early cartilage matrix and not with blastema formation. DISCUSSION
During regeneration in the rabbit major glycosaminoglycan deposited
ear system, the in the cartilage
Rabbit
Ear Glycosaminoglycans
203
matrix is chondroitin sulfate. Chondroitin sulfates from normal and regenerating cartilage show similar chromatographic behavior on DEAE-cellulose columns. This suggests little variation in the degree of sulfation, since, under identical conditions, undersulfated chondroitin sulfate from brachymorphic mice elutes from DEAE-cellulose columns at a lower ionic strength than chondroitin sulfate from normal mice (Orkin et al., 1976). We have confirmed, by analysis of disaccharides from exhaustive chondroitinase ABC digests, that the chondroitin sulfate of early regenerates contains only 16% chondroitin, as compared to 9% in the normal cartilage. Although the degree of sulfation does not change significantly during regeneration in the rabbit ear, the position of sulfation does change. During the times of epithelialization and cellular proliferation of the blastema, chondroitin 4-sulfate predominates as the matrix component. Regenerates with differentiating cartilage matrix have an increased chondroitin 6-sulfate to chondroitin 4-sulfate ratio. This suggests a change in the position of sulfation in the differentiating cartilage matrix, since cartilage, isolated from the integument, is responsible for 90% of the labeled chondroitin sulfate synthesized at this time. The shift in the sulfation position of chondroitin sulfate synthesized during in vitro incubation by the differentiating cartilage matrix as compared to normal cartilage, was confirmed directly on regenerated cartilages. Unlabeled differentiating cartilage showed a predominance of chondroitin 6-sulfate in the matrix. This chondroitin 6-sulfate rich matrix may be a transient initial state in cartilage differentiation since preliminary evidence indicates a return to a chondroitin 4-sulfate rich matrix in older regenerates. The chondroitin 6-sulfate enrichment in the cartilage matrix of differentiating cartilage may, therefore, reCHONDROITINASE
GAG
TABLE 2. ABC DIGESTION OF UNLABELED CARTILAGES NORMAL AND REGENERATED TISSUES Total disaccharides (ADi-6s + ADi-OS + ADi-IS) as pg N-acetylhexosamine/ /tic DNA
preparations
Normal Cartilages 35-Day regenerated cartilages (6)
(8)
ADi-6S
FROM
+ ADi-OS/ ADi-IS
2.52
f 0.80
0.31
f 0.14
2.58
IL 0.33
2.63
f 0.4
Note. Disaccharides were quantitated by N-acetylhexosamine assay in the absence (measures chondroitin 6-sulfate and chondroitin Osulfate) or the presence (also measures chondroitin 4-sulfate) of chondro-4-sulfatase. Number of observations are shown in parentheses. Data are expressed as the mean + the standard deviation.
204
DEVELOPMENTAL
BIOLOGY
VOLUME
TABLE CHONDROITINASE
Normal cartilage Regenerated cartilage Nofe.
Disaccharides
were
located
ABC
DIGESTION
86,
1981
3
PRODUCTS
ISOLATED
ON THIN-LAYER
Percentage ADi-6S
Percentage
ADi-4S
ADi-OS
19.6 51.3
71.5 32.5
9.0 16.3
by uv absorption
and assayed
PLATES
Percentage
ADi-GS/ADi-4S 0.27 1.58
for N-acetylhexosamine.
capitulate the ontogeny of fetal cartilage in the rabbit. Mathews (1967) reported high initial levels of chondroitin g-sulfate relative to chondroitin 4-sulfate in fetal rabbit cartilage. The chondroitin 4-sulfate increases with age and soon reaches a chondroitin 4-sulfate/chondroitin 6-sulfate ratio of 2/l. A similar developmental sequence is seen in embryonic chick cartilage (Robinson and Dorfman, 1969). In embryonic chick epiphyseal cartilage between 10 and 19 days, the synthesis of chondroitin and chondroitin 6-sulfate progressively decreases and the synthesis of chondroitin 4-sulfate concomitantly increases. These developmental relationships between the levels of chondroitin 4- and chondroitin g-sulfate, therefore, appear to be a factor in cartilage differentiation embryologically, as well as in cartilage regeneration in the adult rabbit. Another model of cartilage regeneration in the rabbit ear has been developed using transplants of perichondrium taken from the auricular cartilage. The perichondrial membrane responded by proliferating and synthesizing new cartilage matrix (Skoog et al., 1972; Wasteson and Ohlien, 197’7). The Sod-labeled GAGS from the new matrix were 95% susceptible to chondroitinase AC II, indicating the presence of only chondroitin sulfate. Similarly, our analysis of chondroitin sulfate peaks on DEAE-cellulose from unlabeled normal and regenerated cartilages shows identical degradation with chondroitinase ABC or chondroitinase AC II, thus eliminating the possibility of dermatan sulfate being present. When the isomers of chondroitin sulfate were examined in the cartilage regenerated from the perichondrium, the ratio of chondroitin 6-sulfate to chondroitin 4-sulfate was increased, but not to the extent seen in our system. The times of regeneration in the two systems are difficult to compare, but conceivably the perichondrial regenerate might have an even higher chondroitin 6-sulfate:chondroitin 4-sulfate ratio if examined earlier. The increased chondroitin 6sulfate may be characteristic of a proliferating chondrocyte just beginning to synthesize cartilage matrix. A model of cartilage matrix organization involving the association of proteoglycan subunits with hyaluranic acid has been proposed for hyaline and articular cartilages (Hardingham and Muir, 1972, 1974; Hascall
and Heinegard, 1974). The major proteoglycan of cartilage consists of a central protein core, with chondroitin sulfate and keratan sulfate side chains covalently bound along its length. A number of these proteoglycan molecules are then noncovalently linked to a common hyaluronic acid molecule, via a specialized linkage region of the protein core. In the elastic cartilage of the rabbit ear, a similar organization is probable. Proteoglycans produced by rabbit ear chondrocytes during short-term culture form a high proportion of aggregates with added hyaluronic acid. These proteoglycans contain chondroitin 4-sulfate and chondroitin 6-sulfate in a 2/l ratio, as well as a small amount of keratan sulfate (Madsen and Lohmander, 1979). Studies are underway in our laboratory to determine whether the chondroitin 6-sulfate rich proteoglycan of the newly regenerated cartilage matrix differs in physical properties from the chondroitin 4-sulfate rich proteoglycan of the mature cartilage. The second major GAG synthesized during regeneration in the rabbit ear system is hyaluronic acid. We had predicted high levels of hyaluronate synthesis during the outgrowth phase of regeneration, but a peak of hyaluronic acid synthesis was not seen until cartilage differentiation began. This is in contrast with the model of newt limb regeneration (Toole and Gross, 1971) where incorporation of [3H]acetate into hyaluronic acid peaked early in regeneration and declined with the onset of differentiation. In the newt, however, regenera-
CHONDROITINASE
TABLE 4 AC DIGESTION PRODUCTS
SUES, DAYS 7, 15, 22, BEFORE REGENERATING SEEN AND DIFFERENTIATING TISSUES, DAYS TENSIVE MATRIX IS FOUND
FROM
3H cpm
GAG
preparation
Blastemal tissues Differentiating tissues
BLASTEMAL
CARTILAGE MATRIX 31,35 AND 37, WHEN
TISIs Ex-
ADi-6S/
3H cpm ADi-IS 0.52 0.94
f 0.09 f 0.07
Note. The labeled disaccharides were isolated by thin-layer chromatography in the presence of carrier disaccharides, located by uv absorption and cut out for scintillation counting. Data are expressed as the mean + standard deviation.
HASTY,
SMITH,
AND KANG
tion is marked by extensive dedifferentiation of ,muscle and connective tissue during the first 10 days. Further hyaluronic acid synthesis was shown to be higher in the stump tissues where muscle and connective tissues were dedifferentiating, than in the outgrowth tissues, were proliferation predominates. In contrast, the rabbit ear has no muscle at the level where the punch is made and shows limited dedifferentiation of the original cartilage during blastemal formation. A regenerating tissue may have increased hyaluronic acid synthesis only where extensive dedifferentiation is in progress. Most of the hyaluronate synthesis in the rabbit ear occurs in the integument portions of the regenerate, but in short-term incubations in vitro, appreciable levels of hyaluronate synthesis were seen in regenerated cartilage samples. However, assay of unlabeled GAG from the new cartilage shows very little matrix accumulation of the hyaluronic acid. This may reflect high turnover of hyaluronic acid during the regeneration process. High levels of hyaluronidase activity are seen in the regenerating newt limb during cartilage differentiation (Toole and Gross, 1971; Smith et al., 1975). Similar correlations are seen in a variety of developmental situations (Toole, 1976). The negligible amounts of hyaluronate accumulated in the differentiating rabbit ear cartilage may reflect the balance between hyaluronic acid synthesis and degradation. Supported by New Faculty Research Grant HO0033 and funds from the Veteran’s Administration. We would like to thank Dr. Bryan P. Toole for valuable discussion. These results are included in the senior author’s Ph.D. dissertation (University of Tennessee Graduate School of Medical Sciences).
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