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Ultrastructural cytochemistry of proteoglycans associated with calcification of shark cartilage.

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THE ANATOMICAL RECORD 208:149-158 (1984)
Ultrastructural Cytochernistry of Proteoglycans Associated
With Calcification of Shark Cartilage
MINORU TAKAGI, RICHARD T. PARMLEY, FRANCIS R. DENYS,
HIROSHI YAGASAKI, AND YOSHIHISA TODA
Department of Anatomy, Nihon University School of Dentistry, Tokyo 101,
Japan (M. T , H. Y,Y.T), Institute of Dental Research, University of
Alabama in Birmingham, Birmingham, A L 35294 fM. T,R. TP.,R R.D.),
and Departments of Pediatrics and Pathology, University of Texas Health
Science Center at S a n Antonio, T X 78284 (R.T P )
ABSTRACT
Proteoglycans (PGs) as well as sulfated glycosaminoglycans
(GAGS) are closely associated with cartilage calcification. An inner zone of
endoskeletal tesserae of sharks is composed of a unique calcified hyaline
cartilage. Initial calcification can be seen in the cartilage close to the inner
zone.We have ultrastructurally examined shark, Triakis scyllia, noncalcifying,
calcifying, and calcified cartilage using the tannic acid-ferric chloride (TA-Fe),
the high iron diamine (HID), and the HID-thiocarbohydrazide-silver proteinate (HID-TCH-SP)methods for localization of sulfated complex carbohydrates.
In noncalcifying cartilage, TA-Fe and HID strongly stained matrix granules
which were round, ovoid, elongated, or irregularly shaped and presumably
represented PG monomers. The size and staining intensity of the reactive
matrix granules progressively decreased in calcifying cartilage toward the
calcification front of the calcified cartilage. Similarly, a progressive decrease
in the size of the HID-TCH-SP stain deposits in the matrix granules was
observed in the calcifying cartilage close to the calcification front and was
interpreted a s a decrease in length of sulfate containing GAG chains. In the
calcified cartilage, the highly calcified areas were often localized in the calcification front and contained few or no small KID-TCH-SPstain deposits, whereas
the weakly calcified regions contained more stain deposits. These results indicate that partial and complete degradation of sulfated GAGs and/or PGs may
be a requisite for calcification of shark cartilage.
Proteoglycans (PGs) as well as glycosaminoglycans (GAGS) are thought to influence
the calcification of cartilage, bone, and dentin. PGs, whose sulfated GAG molecules possess a strong bond formation with calcium,
maintain calcium phosphate in a stable colloidal state and inhibit biological calcification (Di Salvo and Schubert, 1967). The
removal of these PGs or their partial degradation by lysosomal enzymes would then result in calcification (Campo, 1970; Baylink et
al., 1972; Glimcher, 1976). Alternatively,
some degraded PGs may constitute the nucleating site for calcification (Campo, 1970;
Baylink et al., 1972). Previous biochemical
(Lohmander and Hjerpe, 19751, histochemical (Van den Hooff, 1964; Quintarelli and
0 1984 ALAN R. LISS, INC.
Dellovo, 1965; Silbermann and Frommer,
1974), immunohistochemical (Poole et al.,
1982), ultrastructural-cytochemical (Matukas and Krikos, 1968; Bonucci, 1969; Smith,
1970; Thyberg et al., 1973; Silbermann and
Frommer, 1974; Davis et al., 1982; Shepard
and Mitchell, 1982; Takagi et al., 1983b1, and
high-resolution electron spectroscopic imaging (Arsenault and Ottensmeyer, 1983) studies of the changes of PGs in cartilage calcification have been conducted, primarily with
mammalian and avian connective tissue
sources.
Received June 20, 1983; accepted September 29, 1983.
Address reprint requests to Minoru Takagi, D.D.S., PhD.,
Department of Anatomy, Nikon University School of Dentistry,
1-8-13,Kanda-Surugadai,Chiyoda-Ku, Tokyo, Japan.
150
M. TAKAGI ET AL.
routinely dehydrated, and embedded in Spurn
(1969) low-viscosity resin.
Sharks possess a n internal skeleton of cartilage which contains sulfated GAGS including chondroitin sulfate A, C, D, and variable
proportions of keratan sulfates (Mathews,
1966,1975). The oversulfated nature of chondroitin sulfates in the shark cartilage presumably prevents its calcification. However,
this cartilage frequently contains variable
amounts of calcium salt deposits which are
called endoskeletal tesserae (Kemp et al.,
1975; Kemp and Westrin, 1979). Ultrastructural examination of PGs in shark cartilage
may well enlighten our understanding of the
role of PGs in calcification of cartilage in
several species. However, only a few ultrastructural studies of shark endoskeletal tesserae have been reported (Kemp et al., 1975;
Kemp and Westrin, 1979). To our knowledge,
no one has demonstrated the changes in the
localization of GAG containing PGs in shark
cartilage calcification. The present study of
shark jaw cartilage was undertaken to identify the ultrastructural localization of PGs in
calcification, using the tannic acid-uranyl
acetate (TA-UA)method (Sannes et al., 1978;
Takagi et al., 1983a) for anionic complex
carbohydrates, the TA-ferric chloride (TAFe) method (Sannes et al., 1978; Takagi et
al., 1983a1, the high iron diamine (HID)method (Spicer, 1965; Spicer et al., 19671, and
the HID-thiocarbohydrazide-silver proteinate (HID-TCH-SP) method (Sannes et al.,
1979)for sulfated complex carbohydrates.
TA-UA Method for Anionic Complex
Carbohydrates
The aldehyde-fixed, rinsed specimens with
and without TA were routinely dehydrated
in graded alcohols and propylene oxide and
embedded in Spurr (1969)low-viscosityresin.
Unosmicated thin sections mounted on stainless steel grids were treated for 10 min with
a filtered fresh TA solution, pH 2.6-2.8, containing 5 g tannic acid (J.T. Baker Chemical
Co., Phillipsburg, NJ) in 95 ml of distilled
water, rinsed three times in distilled water,
and treated for 5 min with a filtered fresh
UA solution (pH 4.1-4.3) containing 1g uranyl acetate in 99 ml of distilled water (Sannes
et al., 1978; Takagi et al., 1983a). Subsequently, stained sections were rinsed six
times in distilled water. Control specimens
were also examined without the TA-UA
treatment.
TA-Fe Method for Sulfated Complex
Carbohydrates
The aldehyde-fixed, rinsed specimens with
and without TA were routinely dehydrated
in graded alcohols and propylene oxide and
embedded in Spurr (1969)low-viscosityresin.
Unosmicated thin sections mounted on stainless steel grids were treated for 10 min with
a filtered fresh TA solution as described
above, rinsed three times in distilled water,
and treated for 1min with a filtered fresh 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., 1983a). Subsequently, stained sections
were rinsed six times in distilled water and
examined. Control specimens were also examined without the TA-Fe treatment.
MATERIALS AND METHODS
Tissue Preparation
Endoskeletal tissues from leopard sharks,
each approximately 50 cm in length, were
used in this study. Cartilage specimens including endoskeletal tesserae were taken
from the shark, Triakis scyllia, jaws. Some
specimens were fixed in 2.7% glutaraldehyde
in 0.1 M cacodylate buffer (pH 7.35) for 2 h
at 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)
for 2 h at 22°C (Takagi et al., 1983a). Subsequently the specimens were rinsed several
times in 0.1 M cacodylate buffer (pH 7.35)
containing 7% sucrose and then stained or
postfixed as outlined below. Thin sections
were examined without counterstains with a
Philips 300 or Hitachi H-500 electron microscope.
Routine Morphology
The aldehyde-fixed, rinsed specimens without TA were postfixed for 1 h in 1%0 ~ 0 4
,
HID and HID-TCH-SP Methods for Sulfated
Complex Carbohydrates
The aldehyde-fixed, rinsed specimens without TA were stained for 18 h a t 22°C in a n
HID solution (Spicer, 1965; Spicer et al.,
1967), which was prepared by adding 1.4 ml
of 40% FeCl3 (Fisher Scientific Co., Fair
Lawn, NJ) to a fresh diamine solution containing 120 mg of N,N-dimethyl-m-phenylenediamine (HCI)B (Eastman Kodak Co.,
Rochester, NY) and 20 mg of N,N-dimethylp-phenylenediamine (HC1) (Fisher Scientific)
in 50 ml of HzO. Control specimens (for evaluation of intrinsic density) were incubated
SHARK CARTILAGE PROTEOGLYCANS
for 18 h a t 22°C in a MgC12 solution, pH 1.4,
prepared by adding 1.4 ml of 40% MgClz to
50 ml of H2O and adjusting the pH with HC1.
Some specimens were then postfixed in 1%
Os04, routinely dehydrated, and embedded
inspurr (1969) low-viscosity resin. The postosmication step was omitted for other
specimens.
To enhance HID staining, some thin sections were stained with a thiocarbohydrazide
(Eastman Kodak)-silver proteinate (strong
silver protein, Roboz Surgical Instrument
Co., Washington, D.C.) sequence (TCH-SP)as
described previously (Thiery, 1967; Sannes et
al., 1979).The TCH-diamine andor iron complex presumably reduce SP to form electrondense stain deposits. SP background staining
was eliminated by filtering (Whatman filter
No. 2) the SP solution twice before use. Acid
MgClz controls were similarly processed.
RESULTS
Morphological Observations
Endoskeletal tesserae, localized in the peripheral part of shark jaw cartilage, were
calcified tissues which contained a n outer
zone presumed to be bone tissue and a n inner
zone composed of calcified cartilage (Kemp et
al., 1975; Kemp and Westrin, 1979).Contour
lines, called Liesegang rings, ran in the matrix of the inner zone or the calcified cartilage matrix (Kemp et al., 1975; Kemp and
Westrin, 1979) and were often concentric
around chondrocytes which appeared to be
trapped by calcification of cartilage. Chondrocytes, which were spherical or ovoid, were
observed in the calcified and noncalcified matrix (Fig. 1). Most of these chondrocytes appeared to be engaged in the calcification of
the extracellular matrix around them and
did not appear degenerated. Rare degenerative chondrocytes were observed. Calcifying
globular bodies or clusters of needle-like
crystals were observed in the vicinity of a
junction between calcifying and calcified cartilage (Fig. 1).
TA-UA Staining for Anionic Complex
Carbohydrates
TA-UA stained matrix granules and collagen fibrils in cartilage matrix as well as intracellular glycogen (Fig. 2). The calcified
cartilage included highly or weakly calcified
matrix (Fig. 2), identified by contour lines
which were presumably formed by a difference in mineralization degree of the calcified
matrix. The weakly calcified matrix demonstrated the presence of the sparse organic
151
material after weak demineralization with
TA-UA treatment at acidic pH (Fig. 2).
TA-Fe Staining for Sulfated Complex
Carbohydrates
TA-Fe diffusely stained matrix granules in
the noncalcifying extracellular matrix which
was distantly located from the calcification
front. Staining was not observed on plasmalemma, glycogen, lysosomes, ribosomes, mitrochondria, nucleus, and collagen fibrils. TAFe-positive matrix granules were round,
ovoid, elongated, or irregularly shaped and
varied in size, having a diameter of 20-50
nm. The size and staining intensity of TA-Fepositive matrix granules were similar in the
noncalcifying matrix distant from or near to
the calcification front (Fig. 31, but progressively decreased in calcifying cartilage close
to the calcification front (Fig. 4). Often, calcified areas did not contain TA-Fe-reactive
matrix granules.
HID and HID-TCH-SP Staining for Sulfated
Complex Carbohydrates
The acidic nature of the HID solution resulted in decalcification and staining of the
specimens. HID strongly stained matrix
granules which varied considerably in their
size and shape. The size of HID-reactive matrix granules progressively decreased from
calcifying cartilage to the calcification front
(Fig. 5). The osmiophilic appearance of the
calcification front prevented evaluation of
HID-positive material in this area. Consequently HID-TCH-SP staining was examined
in unosmicated specimens as described below. HID-TCH-SP produced stain deposits of
several sizes (6-20 nm in diameter) in matrix
granules (Figs. 6-9). HID-TCH-SP staining
was observed neither in membrane limited
vesicles presumed to be matrix vesicles nor
in demineralized globular bodies (Figs. 6, 7).
However, HID-TCH-SP-stained material was
adherent to the outer surfaces of the limiting
membrane of the vesicles. In addition, a progressive decrease in the size of HID-TCH-SP
stain deposits in the matrix granules was
observed in the calcifying cartilage matrix
close to the calcification front, which frequently lacked stain deposits (Figs. 6, 8). In
the calcified cartilage, highly calcified areas
contained few or no small HID-TCH-SP stain
deposits (6-8 nm in diameter), whereas more
abundant HID-TCH-SP-positivematerial was
localized in the weakly calcified areas (Fig.
9). HID-TCH-SP-reactive matrix granules in
the weakly calcified areas were distributed
152
M. TAKAGI ET AL
Fig. 1. Calcifying globular bodies (arrows) in the shark
jaw cartilage can be seen in the vicinity of a junction
between calcifying and calcified matrix. Chondrocytes,
which are localized in these sites, appear intact. Contour
lines (arrowhead) are frequently observed in calcified
cartilage matrix. Osmicated specimen; not counterstained. x 1,800;bar = 5 pm.
Fig. 2. TA-UA stains the extracellular matrix gran-
ules (arrowheads, enlarged in inset), collagen fibrils (Co,
and elnarged in inset), and intracellular glycogen (gly).
TA-UA treatment at acidic pH partly demineralizes calcified matrix and renders contour lines more obvious
when compared to those without TA-UA. Nucleus (N).
Calcifying globular bodies (arrows). Unosmicated specimen; not counterstained. ~ 9 , 2 0 0 bar
;
= 1 pm. Inset
~28,000;bar = 0.5 km.
SHARK CARTILAGE PROTEOGLYCANS
Fig. 3. In noncalcifying cartilage, strong TA-Fe staining of the extracellular matrix is localized in matrix
granules (arrowheads). The size and staining intensity
of TA-Fe reactive matrix granules were similarly observed in the cartilage matrix. Chondrocyte (C). Thin
section from TA-glutaraldehyde-fixedspecimen without
; = 1fim.
postosmication; not counterstained. ~ 2 0 , 2 5 0bar
Fig. 4. In the calcifying cartilage, the size and staining intensity of TA-Fe-positive matrix granules (arrowheads) progressively decreased toward the calcification
front of the calcified cartilage matrix (CM). TA-Fe treat-
153
ment at acidic pH results in demineralization of calcified
matrix, which often lacks staining. Thin section from
TA-glutaraldehyde-fixed specimen without postosmication; not counterstained. Chondrocyte (C); Nucleus (N).
~20,250;bar = 1I m .
Fig. 5. The size and staining intensity of HID-reactive
matrix granules (arrowheads) progressively decreased
from the calcifying cartilage to the osmiophilic calcified
matrix (CM) in this osmicated specimen. HID treatment
at acidic pH resulted in demineralization and staining
of specimens. Chondrocyte (C); Nucleus (N). Not
counterstained. ~20,250;bar = 1pm.
more sparsely than those in the noncalcify- ground staining was not present in unosmicated specimens.
ing and calcifying cartilage (Fig. 9).
The aldehyde-fixed specimens without TAUA or TA-Fe treatment lacked staining. In
Cytochemical Controls
addition, the TA-glutaraldehyde-fixed speciHID control specimens comparably incu- mens without TA-UA or TA-Fe treatment
bated in a n acid MgC12 solution lacked the demonstrated a minimal increase in matrix
stain density observed in HID specimens. granule density (Fig. 111, which was attribAcid MgClz controls of unosmicated speci- uted to better preservation of the complex
mens exposed to TCH-SP lacked staining carbohydrates during fixation rather than
(Fig. 101, except for some fine TCH-SP stain true TA staining (Takagi et al., 1983a). The
deposits (< 6 nm in diameter) which were addition of cationic reagents to fixatives has
observed on a few membrane structures of previously been shown to improve cartilage
osmicated control specimens. This back- preservation (Hunziker et al., 1982).
Fig. 6. HID-TCH-SP stain deposits are observed in matrix granules (arrowheads, and enlarged in lower inmatrix granules but are not discernible in membrane- set) can be seen in calcifying cartilage matrix close t o
limited vesicles (thick arrows, and enlarged in upper the calcification front of the calcified matrix (CM). 0 s insets) and globular bodies (thin arrow). A progressive micated specimen; not counterstained. X17,ZOO; bar = 1
decrease in the size of HID-TCH-SP stain deposits in pm. Insets X40,OOO; bar = 0.5 pm.
Fig. 7. The size of HID-TCH-SP stain deposits also
decreases close to the globular body (arrow) which lacks
staining. Osmicated specimen; not counterstained.
~40,000;
bar = 0.5pm.
Fig. 8. The size of HID-TCH-SP stain deposits in the
matrix granules (arrowheads, and enlarged in inset) decreases close to the calcification front of the calcified
cartilage matrix (CM) which lacks staining in this unosmicated specimen. Nucleus (N). Not counterstained.
x 17,200;bar = 1pm. Inset ~40,000;
bar = 0.2 pm.
Fig. 9. Calcified cartilage contains variable amounts
of HID-TCH-SP staining which was graded from no
staining to moderate staining. The variety of the staining possibly results from differences in the degree of
mineralization in the calcified matrix. Areas presumed
to be highly calcified matrix (thin arrows) include or
lack fine HID-TCH-SPstain deposits whereas those areas
presumed to be weakly calcified matrix (thick arrows)
contain more HID-TCH-SP stain deposits. Intense staining can be seen in noncalcified matrix (EM) in the vicinity of the calcification front. Osmicated specimen; not
counterstained. x 17,200;bar = 1 pm.
156
M. TAKAGI ET AL.
The present studies have demonstrated the
changes in the matrix granule morphology
and sulfated GAG content associated with
calcification of shark jaw cartilage, utilizing
ultrastructural cytochemical methods specific for sulfated complex carbohydrates. The
decrease in the size of TA-Fe- and HIDstained matrix granules and the size of HIDTCH-SP stain deposits in calcifying cartilage
and the complete loss of both stained materials in calcified cartilage indicate that PGs
and sulfated GAGS are partially or completely removed during calcification. The decrease in the size of matrix granules (Matukas and Krikos, 1968; Thyberg et al., 1973;
Takagi et al., 1983b) and HID-TCH-SPstain
deposits (Takagi et al., 1983b) in calcifying
cartilage is similar to that observed in mammalian cartilage. The complete loss of staining in calcified shark cartilage differs from
rat epiphyseal cartilage, where complete or
overall loss of HID-TCH-SPstaining does not
occur during calcification (Takagi et al.,
1983b). Thus in this respect shark cartilage
appears distinct, and data obtained from this
system are a requisite for our understanding
of the role of PGs in calcification of cartilage
in several species.
The morphology, size, and staining properties (i.e., TA-Fe, HID, and HID-TCH-SP
staining) of matrix granules in noncalcifying
shark cartilage are very similar to those observed in the proliferative and upper hypertrophic zone of rat epiphyseal cartilage
(Takagi et al., 1982b).This suggests that the
matrix granule may represent the PG monomer(s) observed in previous ultrastructural
studies of mammalian cartilage (Hascall,
1980;Poole et al., 1982;Takagi et al., 1982b).
Previous studies (Takagi et al., 1982a,b)
have correlated the variation in size of the
HID-TCH-SPstain deposits in a given specimen with the length or degree of sulfation of
GAG chains. In the present study we demonstrate a decrease in the size of HID-TCH-SP
stain deposits in calcifying cartilage and the
calcification front. This HID-TCH-SP result
suggests that, concomitant with the decrease
Fig. 10. No TCH-SP stain deposits are present in this
unosmicated control specimen treated with acid MgClz
rather than HID. Nucleus (N); calcified cartilage matrix
(CM). Thin section from glutaraldehyde-fixed specimen;
not counterstained. x 17,000;bar = 1pm.
Fig. 11. This TA-glutaraldehyde-fixedspecimen lacks
the density observed in TA-UA- and TA-Fe-stained thin
sections (cf. Figs. 2, 3). Nucleus (N); calcified cartilage
(CM). Unosmicated specimen without counterstains.
~17,000;
bar = 1 pm.
DISCUSSION
SHARK CARTILAGE PROTEOGLYCANS
in matrix granule size demonstrated with
TA-Fe staining, the GAGs in calcifying areas
are decreased in size or degree of sulfation.
This observation is consistent with previous
ultrastructural studies which demonstrate a n
accumulation of small Ruthenium Red-positive material (Silbermann and Frommer,
1974) and small HID-TCH-SP stain deposits
(Takagi et al., 1983b) in calcification of mammalian cartilage. Thus in both mammalian
and shark cartilage degradation of PGs andl
or sulfated GAGs appears necessary for cartilage calcification.
Matrix vesicles which might be initial calcification sites have been identified in
previous ultrastructural studies of shark cartilage (Kemp and Westrin, 1979).The present
studies identified membrane-limited bodies
presumed to be matrix vesicles a t the calcification front. These vesicles and globular bodies lacked HID-TCH-SP staining so that we
could not clarify whether the vesicles were
related to removal of PGs and sulfated GAGs.
In contrast, small HID-TCH-SP stain deposits accumulated in disrupted matrix vesicles
and globular bodies in calcification of rat epiphyseal cartilage, indicating a role for matrix vesicles in sulfated GAG degradation
(Takagi et al., 198313).
The fact that in some areas partial and in
other areas complete loss of PGs or sulfated
GAGs could be observed in shark cartilage
calcification indicates the presence of different types of calcified cartilage. This interpretation is consistent with previous studies
describing the presence of three principal
kinds of calcified cartilage in elasmobranchs
(Qrvig, 1967). To our knowledge, complete
removal of PGs and/or sulfated GAGs has not
been biochemically or ultrastructurally demonstrated in calcification of mammalian cartilage, bone, and dentin. The loss of all
stainable sulfated GAGs clearly distinguished
cartilage calcification in sharks from that
previously described in mammals, and suggests unique mechanisms of calcification in
this system.
ACKNOWLEDGMENTS
The authors thank Ms. Barbara A. Woolley
and Ms. Shoko Ogura for their secretarial
assistance. This work was supported in part
by National Institutes of Health Grant DE02670.
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ultrastructure, proteoglycans, associates, calcification, cytochemistry, cartilage, sharp
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