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Ultrastructural distribution of sulfated complex carbohydrates in elastic cartilage of the young rabbit.

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THE ANATOMICAL RECORD 207547-556 (1983)
Ultrastructural Distribution of Sulfated Complex
Carbohydrates in Elastic Cartilage of the
Young Rabbit
Department ofdnatomy, Nihon University School ofDentistry, Tokyo,
Japan (M.T, M.K., H.Y), and the Institute ofDental Research (M.
R.TP., ER.D.) and the Departments of Pediatrics and Pathology (R.T P ) ,
University ofAlabama in Birmingham, Birmingham, AL 35294
Sulfated glycosaminoglycans are a n integral component of
elastic cartilage. We have investigated the ultrastructural distribution of sulfated complex carbohydrates (CC) in the mature cartilage and the perichondrium of young rabbit auricles using the high iron diamine-thiocarbohydrazidesilver proteinate (HID-TCH-SP) and the tannic acid-ferric chloride (TA-Fe)
methods. In the mature cartilage, HID-TCH-SP stained intracellular Golgi
saccules of the mature face, secretory granules, and the extracellular matrix
granules, but staining was not discernible in collagen fibrils and osmiophilic
elastic fibers consisting of only amorphous elastin. The HID and TA-Fe staining were similarly observed in matrix granules, whereas the elastic fibers and
collagen fibrils lacked the staining. The pericellular matrix granules had a
diameter of 34 5 nm (mean SD; n = 30). Thiery’s periodate-TCH-SP (PATCH-SP) method stained vicinal glycol-containing CC in collagen fibrils but
failed to stain matrix granules and elastic fibers. In the perichondrium, HIDTCH-SP staining of the organelles was less intense in the flattened chondrocytes when compared with those in large mature chondrocytes. The extracellular HID and HID-TCH-SP staining were observed in the matrix granules.
The diameter of pericellular matrix granules (19 4 nm, mean k SD; n = 30)
was significantly smaller when compared to those in the mature cartilage (P
< 0.001). The HID-TCH-SP staining was closely associated with collagen
fibrils. However, the staining was not seen in collagen fibrils and osmiophilic
elastic fibers consisting of elastin and microfibrils. The PA-TCH-SP method
stained collagen fibrils and microfibrils but did not stain the amorphous
elastin. Thus these studies demonstrate that sulfated CC are packaged in
chondrocyte secretory granules and are released into the extracellular matrix
to form matrix granules, but are not incorporated into collagen fibrils and
elastic fibers.
Elastic cartilage contains glycosaminoglycans (GAGs), collagen fibrils, and elastic fibers (Serafini-Fracassini and Smith, 1974).
The content of elastic fibers makes this cartilage tissue unique in its physiochemical
Biochemical studies (Wusteman and Gillard, 1977) have indicated that the GAGs of
ear elastic cartilage are composed of chondroitin sulfate and hyaluronic acid, but the definitive location of each type of GAG is not
known. Recent light microscopic histochemical studies (Yamada et al., 1982) have demonstrated the presence of keratanase
cc) 1983 ALAN
(Pseudomonas sp.1-digestible keratan sulfate
in ear elastic cartilage, in addition to chondroitinase ABC- and AC-digestible chondroitin sulfate. Several ultrastructural cytochemical methods have been utilized to localize GAGs in elastic cartilage and have included ruthenium red (Myers, 1976; Nielsen,
19761, colloidal thorium (Yamada and HoshReceived J u n e 2, 1983; accepted July 27, 1983
Address reprint requests to Dr. Minoru Takagi, Department of
Anatomy, Nihon University School of Dentistry, 1-8-13,KandaSurugadai, Chiyoda-ku, Tokyo, Japan.
ino, 1973),and bismuth nitrate (Serafini-Fracassini and Smith, 1974). However, more
specific stains for sulfated GAGS have not
been utilized. The present study of ear elastic
cartilage was undertaken utilizing various
specific stains for complex carbohydrates to
determine the subcellular route of the sulfated complex carbohydrate synthesis and secretion, and its subsequent distribution in
the extracellular space, especially in relationship to collagen fibrils and elastic fibers.
Tissue Preparation
Tissue from the external ear of 1.5-kg rabbits was used in this study. Specimens were
taken from the tip of the ear under anesthesia. Some specimens were fixed in 2.7%
glutaraldehyde in 0.1 M cacodylate buffer
(pH 7.35) for 2 hours a t 4°C or 22"C, whereas
others were fixed in 2.7% glutaraldehyde in
0.1 M cacodylate buffer (pH 6.8) containing
2% tannic acid (TA) (Futaesaku et al., 1972)
for 2 hours a t 22°C. Subsequently, the specimens were rinsed several times in 0.1 M
cacodylate buffer (pH 7.35) containing 7% sucrose and then stained a s outlined below.
described previously (Thiery, 1967; Sannes et
al., 1979).The TCH-diamine and/or iron complex presumably reduces SP to form a n electron-dense stain deposit. SP background
staining was eliminated by filtering (Whatman filter #2) the SP solution twice before
use. Acid MgC12 controls were similarly
TA-Fe method
The aldehyde-TA-fixed, rinsed specimens
were routinely dehydrated in graded alcohols
and propylene oxide and embedded in Spurr
low-viscosity resin. Thin sections mounted on
stainless steel grids were treated for 10 minutes with a filtered fresh TA solution, pH
2.6-2.8, containing 5 gm tannic acid (J.T.
Baker Chemical Co., Phillipsburg, NJ) in 95
ml of distilled water, rinsed three times in
distilled water, and treated for 1minute with
a filtered fresh ferric chloride (Fe) solution
(pH 1.4-1.6) which was prepared by adding 5
ml of 40% ferric chloride (Fisher Scientific
Co., Fair Lawn, NJ) to 95 ml of distilled water
(Sannes et al., 1978; Takagi et al., 1983).Subsequently, stained sections were rinsed six
times in distilled water and examined. Control specimens were also examined without
the TA-Fe treatment or after exposing only
to the TA or the Fe solution.
Sulfated Complex Carbohydrate Staining
HID-TCH-SP method
Vicinal Glycol-Containing Complex
The aldehyde-fixed, rinsed specimens withCarbohydrate Staining
out TA were stained for 18 hours a t 22°C in
a high iron diamine (HID) solution (Spicer, PA-TCH-SP method
1965; Spicer et al., 19671, which was prepared
The aldehyde-fixed, rinsed specimens withby adding 1.4 ml of 40% FeC13 (Fisher Scien- out TA were routinely dehydrated and
tific Co., Fair Lawn, NJ) to a fresh diamine
solution containing 120 mg of N,N-dimethylm-phenylenediamine (HC1)z (Eastman Kodak Co., Rochester, NY) and 20 mg of N,NFig. 1. In mature auricle cartilage high iron diaminedimethyl-p-phenylenediamine(HCl) (Fisher thiocarbohydrazide-silverproteinate (HID-TCH-SPjstain
Scientific Co., Fair Lawn, NJ) in 50 ml of deposits are visible in chondrocyte Golgi expanded sacH20. Control specimens (for evaluation of cules (ES), condensing vacuoles (CV), intermediate vacand mature secretory granules (SG) but are
uoles (IW,
intrinsic density) were incubated for 18 hours not
present in mitochondria (Mt), rough endoplasmic
at 22°C in a MgCl2 solution, pH 1.4, pre- reticulum (ER), flattened Golgi saccules (S), distended
pared by adding 1.4 ml of 40% MgC12 to 50 portions (DS), coated vesicles (arrows), and the nucleus
ml of HzO and adjusting the pH with HC1. (N). Unosmicated specimen, not counterstained. X 31,250.
Some specimens were then postfixed in 1%
Fig. 2. HID staining of osmicated specimens can be
Os04, buffered with 0.1 M cacodylate, rouin elastic cartilage matrix granules (arrows)around
tinely dehydrated, and embedded in Spurr seen
a mature chondrocyte (C). HID-stained matrix granules
(1969) low-viscosity resin. The post-osmica- attach to the osmiophilic elastic fiber (El. Not counterstained. ~31,250.
tion step was omitted for other specimens.
To enhance HID staining, some thin secFig. 3. HID-TCH-SP stain deposits are observed in
tions were stained (by immersion) with a
thiocarbohydrazide (Eastman Kodak Co., elastic cartilage matrix granules (arrows) in this unosmicated specimen. Elastic fibers (El lack staining; howRochester, NY)-silver proteinate (strong sil- ever,
the stain deposits (arrowheads) can be occasionally
ver protein, Roboz Surgical Instrument Co., found within the interstices of the elastic fibers. Not
Inc., Washington, DC) sequence (TCH-SP) as counterstained. x 31,250.
embedded in Spurr low-viscosity resin. Cytochemical localization of periodate (PA)-reactive complex carbohydrates was accomplished by staining thin sections of the unosmicated specimens according to the PA-TCHSP method of Thiery (1967). Sections were
mounted on stainless steel grids, oxidized in
1%periodic acid (G. Frederick Smith Chemical Co., Columbus, OH) for 45 minutes, rinsed
five times for 10 minutes each in distilled
water, and treated for 40 minutes with 2%
thiocarbohydrazide in 20% acetic acid. After
rinsing in 10% acetic acid and four times in
distilled water, they were exposed for 30 minutes to 1%silver proteinate in the dark and
rinsed six times in distilled water. As a control, periodic acid oxidation was omitted from
the sequence.
Ultrastructural details of ear elastic cartilage have been described previously (Sheldon
and Robinson, 1958; Anderson, 1964; Serafini-Fracassini and Smith, 1974; Cox and Peacock, 1977). In this study we have mainly
described ultrastructural cytochemical results which were obtained from the mature
cartilage and the perichondrium.
The Mature Cartilage
Mature chondrocytes were round, ovoid
shaped, and contained a prominent Golgi apparatus consisting of several stacks of flattened saccules (Fig. 1).Distended portions of
the first saccules of the stack were seen and
lacked HID-TCH-SP staining. Toward the
maturing face, a few saccules appeared more
expanded than the others. At close proximity
to those saccules, condensing and intermediate vacuoles, which were derived from the
Golgi apparatus, could be observed and demonstrated minimal or moderate HID-TCH-SP
staining. Mature secretory granules demonstrated intense HID-TCH-SP staining. Mitochondria, rough endoplasmic reticulum, and
the nucleus lacked HID-TCH-SP staining
(Fig. 1).
In the extracellular matrix, matrix granules believed to represent proteoglycan monomer(s) (Hascall, 1980; Poole et al., 1982;
Takagi et al., 1982) demonstrated intense
HID staining. These granules were round,
ovoid, elongated, or irregularly shaped and
often adhered to collagen fibrils and elastic
fibers consisting of only amorphous elastin
without microfibrils (Figs. 2 , 7). The HIDreactive matrix granules, which were local-
ized in the pericellular matrix, appeared to
represent newly synthesized and secreted
proteoglycans. These pericellular matrix
granules had a diameter of 34 5 nm (mean
+ SD; n = 30; Fig. 2). HID-TCH-SP stain
deposits were observed in matrix granules
but were not seen in collagen fibrils and elastic fibers. However, HID-TCH-SP-positive
sulfated material, which was entrapped in
elastic fibers, was found within the interstices of the elastic fibers (Figs. 3, 4). HIDTCH-SP staining was similar in unosmicated
and osmicated specimens; however, elastic
fibers demonstrated intense osmiophilia a s
previously reported by Thyberg et al. (1979).
Acid MgC12 controls of unosmicated specimens exposed to TCH-SP lacked staining,
whereas some fine TCH-SP stain deposits
( < 6 nm in diameter) were observed on a few
membrane structures of osmicated control
specimens. Elastic fibers demonstrated intense osmiophilia in the osmicated control
specimens (Fig. 5).
TA-Fe-stained matrix granules but did not
stain collagen fibrils and elastic fibers (Fig.
6). Unosmicated control specimens without
TA-Fe treatment lacked similar density.
PA-TCH-SP staining was observed in collagen fibrils whereas matrix granules and
elastic fibers lacked staining. Microfibrils
were not associated with the amorphous component, elastin (Fig. 7). Control specimens
Fig. 4. HID-TCH-SP stain deposits can be seen in
elastic cartilage matrix granules (arrows) in this osmicated specimen whereas osmiophilic elastic fibers (El
lack staining. The stain deposits (arrowhead) are occasionally seen within the interstices of the elastic fibers.
Not counterstained. x 31,250.
Fig. 5. In osmicated MgCla control specimens treated
with the TCH-SP sequence, matrix granules, collagen
fibrils, and elastic fibers in the extracellular cartilage
matrix lack stain deposits. However, elastic fibers (El
demonstrate intense osmiophilia. Not counterstained.
Fig. 6. Tannic acid-ferric chloride (TA-Fe) staining of
the elastic cartilage matrix in this unosmicated specimen is localized in matrix granules (arrows) which are
round, ovoid, elongated, or irregularly shaped, whereas
elastic fibers lack staining. Not counterstained. x 31,250.
Fig. 7. Periodate (PA)-TCH-SPstains collagen fibrils
(Co) in the mature cartilage matrix whereas matrix
granules and elastic fibers (E) lack staining. PA-TCHSP-positive microfihrils are not associated with the elastin (cf. Fig. 12). Unosmicated specimen, not counterstained. ~31,250.
without PA lacked TCH-SP staining of the
extracellular cartilage matrix.
The Perichondrium
Immature chondrocytes, which were localized in the perichondrium, had a flattened
shape and contained a few HID-TCH-SPreactive secretory granules (Figs. 8,9).
In the extracellular cartilage matrix, HID
and HID-TCH-SP methods stained the matrix granules which were localized between
and on the collagen fibrils. The diameter of
HID-positive matrix granules (19 k 4 nm,
mean SD; n = 30),which were localized in
the pericellular matrix, was significantly
smaller when compared to those in the mature cartilage (P < 0.001). Most collagen fibrils with a diameter of about 50-100 nm
were more densely packed when compared to
those in the mature cartilage (Figs. 9-11).
HID and HID-TCH-SP staining were observed in the longitudinal sections of collagen fibrils, whereas transverse sections of
these fibrils always lacked staining (Figs. 10,
11).Elastic fibers in this region mainly consisted of microfibrils and also possibly amorphous elastin, and demonstrated intense
osmiophilia but lacked HID-TCH-SP staining (Figs. 8,9).
Acid MgC12 control specimens of unosmicated specimens exposed to TCH-SP lacked
staining, whereas some fine TCH-SP stain
deposits were seen on a few membrane structures of osmicated specimens in which elastic
fibers demonstrated intense osmiophilia.
PA-TCH-SP moderately to intensely
stained microfibrils with a diameter of about
10-15 nm and collagen fibrils, whereas the
central amorphous component, elastin, and
matrix granules lacked staining (Fig. 12).
Control specimens without PA lacked TCHSP staining in these sites.
The present study has utilized ultrastructural cytochemical methods to localize sulfated and vicinal glycol-containing complex
carbohydrates in elastic cartilage of young
rabbit auricles. The more intense intracellular HID-TCH-SP staining observed in mature cartilage compared to the perichondrium
indicates a gradual increase in synthesis and
secretion of sulfated complex carbohydrates
during chondrocyte differentiation from the
perichondrium to the mature cartilage. The
staining of intracellular sulfated glycoconju-
gates in Golgi saccules and secretory granules of mature chondrocytes is similar to the
staining of sulfated material in these sites in
the hypertrophic chondrocytes from rat epiphyseal cartilage (Takagi et al., 1981).Extracellular HID and HID-TCH-SP staining in
the matrix granule is in agreement with previous ultrastructural cytochemical and immunocytochemical studies (Takagi et al.,
1982) that have demonstrated chondroitin
sulfate and keratan sulfate in this site in rat
epiphyseal cartilage, indicating that the matrix granule may represent proteoglycan
monomer(s). Similarly, previous studies suggest that the matrix granule is formed by the
collapsed proteoglycan(s) after tissue fixation, staining, and dehydration (Hascall,
1980; Poole et al., 1982).The lack of PA-TCHSP staining of vicinal glycols in matrix granules suggests that the sulfated material represents a glycosaminoglycan rather than a
glycoprotein, consistent with biochemical
isolation of chondroitin sulfate from this tissue (Wusteman and Gillard, 1977).
The larger size of HID- and HID-TCH-SPpositive matrix granules in the mature cartilage compared with those in the perichondrium suggests that chondrocytes may
secrete two different (i.e., small and large)
proteoglycan monomers at both sites or that
each matrix granule increases its content of
sulfate and proteoglycan monomers during
cartilage differentiation. The possibility of
two different proteoglycan monomers is supported by previous biochemical studies
(Franien et al., 1981)demonstrating that the
superficial and the middle zone do not contain the larger proteoglycan monomers found
Fig. 8. HID-TCH-SP staining in this osmicated specimen can be seen in the presumed secretory granule (SG,
and enlarged in inset) of a flattened chondrocyte in the
perichondrium. The extracellular staining is localized in
the matrix granules but is not discernible in the collagen
fibrils (Co) and the elastic fibers (El. Not counterstained.
~ 9 , 9 0 0Inset,
Fig. 9. In the perichondrium HID-TCH-SP stain deposits can be seen in the presumed secretory granule
(SG) in an immature chondrocyte and the extracellular
matrix granules (arrows). Elastic fibers (E, and enlarged
in inset) mainly consisting of microfibrils and also possibly amorphous elastin lack staining but demonstrate
the intense osmiophilia. Osmicated specimen, not
counterstained. ~31,250.Inset, ~64,000.
in the deeper zone of articular cartilage.
These results indicate that smaller proteoglycans probably contain less chondroitin
sulfate (Franien et al., 1981). On the other
hand, previous ultrastructural cytochemical
studies indicate that each matrix granule
represents one or several proteoglycan monomers (Hascall, 19801, and conceivably
smaller matrix granules, such as those in the
perichondrium, could contain fewer monomers than the larger matrix granules in mature cartilage.
The lack of HID and HID-TCH-SP staining
in elastic fibers, including amorphous elastin
and/or microfibrils, is in contrast to previous
light microscopic studies (Spicer, 1965; Gad
and Sylven, 1969) demonstrating HID reactivity of elastic fibers. Our findings are in
agreement with biochemical studies of elastic fibers indicating that elastin and microfibrils do not contain sulfated material (Ross,
1973; Serafini-Fracassini and Smith, 1974).
However, HID-positive material was closely
associated with elastic fibers and collagen
fibrils in the present ultrastructural studies.
This observation suggests that distinct resolution of elastic fibers, collagen fibrils, and
sulfated material may not be possible at the
light microscopic level.
Anionic material, which is clearly localized
outside collagen fibrils, has been ultrastructurally demonstrated around these fibrils in
ear elastic cartilage (Myers, 1976) and other
sites (Ruggeri et al., 1975; Shepard and
Mitchell, 1977; Behnke and Zelander, 1970;
Takusagawa et al., 1982) using cationic reagents. Immunocytochemical studies (Poole
et al., 1982) of articular cartilage have localized particulate reaction products for proteoglycan monomers in close association with
collagen fibrils. In contrast, recent ultrastructural cytochemical studies (Butler and
Heap, 1982)have localized Alcian blue staining inside the collagen fibrils. The HID and
HID-TCH-SP methods are more specific for
sulfated complex carbohydrates and clearly
provide adequate stain penetration in
densely packed collagen fibrils. Although
HID-TCH-SP stain deposits were observed in
the longitudinal sections of collagen fibrils,
their transverse sections always lacked the
HID-TCH-SP staining. This observation suggests that sulfated complex carbohydrates
are very closely associated with collagen fibrils; however, they are localized outside collagen fibrils. This interpretation is consistent
with previous biochemical results (see review
by Lindahl and Hook, 1978) utilizing a variety of techniques indicating that all glycosaminoglycans except hyaluronic acid, which
lacks sulfate groups, and keratan sulfate,
bind to collagen by electrostatic interaction
at physiological pH and ionic strength.
The PA-TCH-SP staining of microfibrils
and collagen fibrils may represent localization of vicinal glycol-containing glycoproteins, which have been biochemically
identified in microfibrils (see review by Ross,
1973; Sear et al., 1978) as well as in collagen
fibrils (Nimni, 1974).The PA-TCH-SPmethod
was useful in distinguishing the positive microfibrils from the unreactive amorphous
elastin in the present study. Microfibrils were
predominantly localized in the perichondrium, whereas the amorphous elastin without microfibrils was present in the mature
cartilage. This observation indicates that the
appearance of the microfibrils precedes the
appearance of the amorphous elastin during
the cartilage maturation or differentiation
from the perichondrium to the mature cartilage. This interpretation is consistent with
the previous light and electron microscopic
studies demonstrating the presence of abundant preelastic fibers or microfibrils with
small foci of amorphous elastin in the perichondrium (Bradamante et al., 1975) and
elastin without preelastic fibers in the ma-
Fig. 10. In the perichondrium, HID staining of osmicated specimens is present in matrix granules (arrows)
that are closely associated with collagen fibrils. However, HID-reactive matrix granules are not visible in
transverse sections of collagen fibrils. The diameter of
these granules is significantly decreased when compared
to those in the mature cartilage (cf. Fig. 2). The banding
of collagen fibrils is evident. Not counterstained.
Fig. 11. HID-TCH-SP stain deposits are observed in
the longitudinal sections of collagen fibrils (enlarged in
right upper inset) in the perichondrium, but are not
always seen in transverse sections of these fibrils (left
lower inset). Osmicated specimen, not counterstained. x
31,260. Insets, ~62,500.
Fig. 12. PA-TCH-SP staining is localized in collagen
fibrils (Co) and microfibrils (Mf, and enlarged in inset),
whereas amorphous elastin (arrowheads) and matrix
granules lack staining. Unosmicated specimen, not
counterstained. ~ 3 1 , 2 5 0Inset,
ture cartilage (Serafini-Fracassini and Smith,
The authors thank Ms. Barbara A. Woolley
€or her secretarial assistance. This work was
supported in part by National Institutes of
Health grant No. DE-02670.
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