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Immunoelectron microscopy of type X collagen: Supramolecular forms within embryonic chick cartilage
I)E~EI,OI’ME:NTAL
BIOLOGY
138,
lmmunoelectron
53-62 (1990)
Microscopy of Type X Collagen: Supramolecular within Embryonic Chick Cartilage
Forms
THOMAS M. SCHMID* AND THOMAS F. LINSENMAYERt
Accepted October 2.2, 19X.9 To determine the supramolecular forms in which avian type X collagen molecules assemble within the matrix of hypertrophic cartilage, we performed immunoelectron microscopy with colloidal gold-labeled monoclonal antibodies. In addition double-labeled analyses were performed for the molecule and type II collagen, employing two monoclonal antibodies attached to different size gold particles. Both in situ limb cartilages and the extracellular matrix of chondrocyte cultures were examined. We observed in both systems that type X collagen is present in two forms. One is as fine filaments (~5 nm in diameter) within mats which are found predominantly in the pericellular matrix of the hypertrophic chondrocytes. The second form is in association with the fibrils 110-20 nm in diameter) which also react with the antibody for type II collagen. It seems that the Iilamentous mats represent a form in which the type X collagen is initially secreted from the cell. The type X associated with the striated fibrils most likely represents a secondary association of the molecule with preexisting type II/IX/XI fibrils. The data are consistent with our previously proposed hypothesis that type X collagen is involved in, and perhaps even “targets,” certain matrix components for degradation and removal. I( 1990 Academic Press, In, INTRODIJCTION
of type X in the development of endochondral bones must depend on the type(s) of supramolecular aggregate(s) in which it assembles or with which it becomes associated. In the present investigation we have performed an immunoelectron microscopic investigation of type X collagen in situ in embryonic chick limb cartilage and the extracellular matrix of chondrocyte cultures. We have observed that type X is present in two different supramolecular forms: one is in amorphous mats, and the other is associated with type II collagen fibrils.
During endochondral bone formation, individual chondrocytes undergo a progression from young cells undergoing rapid division to mature cells having the greatest capacity for synthesizing matrix components to hypertrophic cells which become progressively enlarged and eventually are removed. In young, prehypertrophic cartilage, the chondrocytes synthesize a mixture of collagen types II, IX, and XI (Burgeson and Hollister, 1979; Eyre et al., 198713; Mayne, 1989) which coassemble into heterotypic fibrils (Eyre et al., 1987a; van der Rest and Mayne, 1988; Vaughan et al., 1988; Mendler et al., 1989). Once the cells have initiated hypertrophy, they add type X to their biosynthetic repertoire (Schmid and Conrad, 1982a,b; Schmid and Linsenmayer, 1985; Kielty et al, 1985). As the hypertrophic program progresses, the synthesis of this collagen increases, with a concomitant decrease in synthesis of the other types (Gibson and Flint, 1985). Hypertrophy and subsequent changes in the cartilage matrix include (a) enlargement of lacunae to accommodate the great increase in volume of the individual chondrocytes (Hunziker et al., 1987), (b) calcification of the cartilage matrix, providing a substratum for bone deposition, and (c) removal of calcified and uncalcified cartilage matrix resulting in formation of a marrow cavity. It is during these processes when deposition of type X collagen occurs within the matrix. Like other collagen molecules, the structural function
METHODS
Labeled Antibody
Prepamfion
and Testing
Monoclonal antibodies against type X collagen (X-AC9) (Schmid and Linsenmayer, 1985) and type II collagen (II,B,) (Linsenmayer and Hendrix, 1980) were obtained from ascites fluid of hybridoma-containing nude mice. They were purified by ammonium sulfate precipitation (50% saturation) followed by chromatography on Protein A-Sepharose (loading buffer-O.1 M sodium phosphate buffer, pH 8.5; elution buffer-50 mM sodium phosphate, 50 mM citrate, pH 5.0). As a control antibody we employed a myeloma IgGl protein (MOPC 21) obtained from Sigma. Fifteen-nanometer colloidal gold particles (G15) were prepared by reducing chloroauric acid with citrate (Frens, 1973). Five-nanometer gold particles (G5) were purchased from Janssen (Ted Pella, Inc., Tustin, CA). 53
0012-1606/90 $3.00 (‘opyriyht All rights
!C 1990 by Academic Press, Inc. of reproduction in any form reserved.
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The anti-collagen antibodies and the control IgG Tissue sections or cultured cells were treated with 5 were directly adsorbed to colloidal gold. Purified anti- mg/ml hyaluronidase for 30 min in 0.1 M cacodylate bodies were added to 20 ml of colloidal gold in 2 m&I buffer, pH 7.2 (tissue sections), or in DME (cells). Covborate buffer, pH 9.0 (final antibody concentration, 50 erslips were reacted overnight with colloidal gold preppg/ml). After 1 hr, BSA was added to a concentration of arations (Ass0 of 0.5-0.6) and the washed 4X with 1% 1%. The preparations were washed three times with BSA in 0.1 M cacodylate buffer (pH 7.2) for 30 min. The BSA-Tris saline buffer (1% BSA, 20 mM Tris, 0.13 M coverslips were fixed in 2.5% glutaraldehyde in cacodylNaCl, pH 8.2) and collected by centrifugation at 50,OOOg ate buffer for 30 min and were washed and treated with (G5) or 14,000g(G15) for 45 min at 4°C. The pellets were 1% 0~0~ in cacodylate buffer. After being washed with suspended in BSA-Tris saline buffer and centrifuged water, the coverslips were stained in block with 1% through a stepwise glycerol gradient consisting of 10, uranyl acetate for an hour, dehydrated through a 15, 20, 25, and 30% glycerol in BSA-Tris saline buffer. graded series of ethanols, and embedded in Epon-AralThe desired antibody-gold complexes were recovered dite in inverted Beem capsules. The plastic blocks were from the middle of the gradient (Slot and Geuze, 1981). detached from the coverslips after freezing in liquid The specificities of the antibody-gold probes were nitrogen (Larramendi, 1988). Thin sections were cut, tested by ELISA as described for the monoclonal anti- placed onto uncoated copper grids, and stained with bodies (Schmid and Linsenmayer, 1985). Plates coated 2.5% uranyl acetate in 50% ethanol and lead citrate. with either type X or type II collagen were incubated They were examined and photographed on a JEOL 100 with serial dilutions of the gold probes for 2 hr and CX electron microscope. washed. Before dilution of the antibody-gold preparaRESULTS tions had an AssOof 0.5-0.6. A goat anti-mouse IgG peroxidase conjugate (Hyclone Laboratories, Logan UT) Controls for Specificity of Antibody-Gold Complexes was used as the second antibody and incubated for 1 hr. The peroxidase substrate containing 200 yl 0.01% oThe identification of type X collagen and its relationphenylenediamine and 0.1% Hz02 in water was incu- ship to fibrils containing type II collagen in embryonic bated for 30 min. The reaction was stopped by the addi- chick cartilage were examined using a monoclonal antition of 50 ~1 of 50% HaSO, and the absorbance was body specific for each collagen type adsorbed directly to determined at 492 nm on a Dynatech ELISA plate different size gold particles. This approach has been reader. used successfully for the detection of type I and type V collagen within the same fibril in the chicken cornea Tissue Preparation (Birk et ab, 1988). The procedure maximizes resolution Tibiotarsi were dissected from 14-day chick embryos by creating the smallest possible distance between the and the sheaths of periosteal bones were removed. The antigen and the marker gold particle and affords the cartilage pieces were treated by one of the following capability of performing simultaneous double-labeling methods. (a) The tissue was not fixed before frozen sec- for two different collagen types. tions were cut. (b) The tissue was fixed in 1% paraforThe specificity of the preparations of antibody-gold maldehyde in 0.1 M cacodylate buffer, pH 7.2, for 5 min was tested (a) by ELISA on plates coated with purified at 4°C. (c) The tissue was fixed in 4% paraformaldecollagen preparations and (b) by immunohistochemical hyde in cacodylate buffer for 30 min at 4°C. Formalde- reactivity at the light microscopic level. In ELISA the hyde-fixed tissues were incubated for another 30 min in anti-type X collagen-gold conjugate and the anti-type 0.1 M Tris, 50 mM glycine buffer, pH 7.5, to quench free II collagen conjugate displayed the same reactivity aldehydes. Fixed or unfixed tissues were embedded in previously reported for the anti-type X (Schmid and OCT compound and 12-pm frozen sections were cut and Linsenmayer, 1985) and anti-type II (Linsenmayer and dried onto albuminized Thermanox coverslips (ICN, Hendrix, 1980) monoclonal antibodies. The control Lisle, IL). IgG-gold bound to neither collagen-coated plate (data Chondrocytes were isolated by enzymatic digestion of not shown). 12-day chick embryonic tibiotarsi as previously deImmunohistochemical reactivity of the anti-collascribed (Schmid and Linsenmayer, 1983) and plated gen-gold probes was tested on cryostat sections of 14onto Thermanox coverslips. The medium consisted of day tibiotarsi. The distributions of the anti-collagenDulbecco’s Modified Eagle’s medium (DME) containing gold complexes were the same as those reported by indi10% calf serum (Hyclone, defined-supplemented), 50 rect immunofluorescent methods. The anti-type X-gold pg/ml ascorbic acid, and 100 pg/ml fl-aminopropionicomplex stained the hypertrophic cartilage near the trile fumarate. Chondrocyte cultures were not fixed be- marrow cavity, the anti-type II-gold complex reacted fore incubation with antibody-gold probes. with the entire cartilage rudiment, and the control
SCHMII) AND LINSENMAYER
MOPC21 gold complex showed no detectable (data not shown). Single-Label
Immunoelectron
Type X Colkzgeri Vltrastructmz1
reactivity
Microscopy
At the electron microscopic level, within the hypertrophic cartilage matrix the anti-type X collagen-gold reacted with two distinct structures (Fig. 1). The most intense reactivity (>35 particles/pm’) was found over mats of fine filamentous material (Fig. 1A and marked with asterisk in Fig. 1B). The fine filaments appeared to have diameters less than 5 nm. Gold particles were also found associated with fibrils lo-20 nm in diameter, the major fibrillar type in cartilage, but these were less intensely labeled. As can be seen in Fig. lC, the filamentous mats are most evident in the matrix closest to the cell surface. In this figure the mats abut a gap (g) between the cell surface and matrix. Such gaps between the chondrocyte surface and the adjacent matrix were typically observed, and probably represent a shrinkage artifact created during processing of the tissue with conventional fixatives (Hunziker et ah, 1984; Eggli et ah, 1985). Also seen in Fig. lC, the fine filamentous mats decrease in number, or at least are less evident, as the distance from the cell surface increases. In regions of the matrix lacking the fine filamentous material, the type X collagen label is usually associated with the loto ‘LO-nm-diameter fibrils. The distribution and density of fibril-associated, anti-type X collagen label is illustrated in the low power micrograph shown in Fig. 2A. Although the density of gold particles observed in such sections was not high (approximately I2 particles/pm’), it was much greater than in identically treated sections of tarsus (CO.29 particles/pm” shown in Fig. 2B), a nonhypertrophic cartilage containing no type X collagen. Typical photographic fields of the latter were almost devoid of gold particles. Also, in nonhypertrophic cartilage, there was an absence of the type X collagen-reactive filamentous mats, which were so prominent in the hypertrophic zone. This can be appreciated in Fig. 2B, which shows a portion of a chondrocyte (c) with no adjacent filamentous mats. Double-Label
Immu,noelectron
Microscopy
Since the anti-type X-gold particles were associated with lo- to 20-nm-diameter fibrils, we performed double-label analyses for collagen types X and II to determine possible associations between them. In these experiments hypertrophic cartilage was labeled with anti-type X collagen antibody complexed to 15-nm gold (X-G15) and anti-type II collagen antibody complexed to 5-nm gold (II-G5).
Foms
55
The salient features observed in this study can be seen in Fig. 3. In pericellular regions (Fig. 3A), the filamentous material (asterisk) shows strong reactivity for type X collagen, but for type II the reactivity is slight or nonexistent. However, examination of many lo- to 20-nm-diameter fibrils shows some reactivity for both collagen types. Some fibrils which are decorated with both anti-type X collagen-gold particles and anti-type II collagen-gold particles are marked with arrowheads. Although the great majority of fibrils fall within the lo-20 nm size range, sometimes fibrils with a larger diameter and more distinct banding pattern were observed. An example of such a fibril with a diameter of about 40 pm is shown in Fig. 3B. Such fibrils show strong reactivity for type II collagen which is labeled at periodic intervals of 60-70 nm. The anti-type II reactivity is greater for the large diameter fibrils than for the smaller lo- to 20-nm-diameter fibrils. The anti-type Xgold particles also show some reactivity with the large diameter fibrils. A third component of potential interest to the present investigation was vesicles. These were found both near the lacunar wall (Fig. 3A) and among the fibrils of the interterritorial matrix (not shown). The majority of the vesicles were not labeled with either anti-collagen-gold probes; however, as shown in Fig. 3A, some gold particles can be detected adjacent to the vesicles. Chondrocyte Cultures Chondrocytes were maintained in monolayer culture for 8 weeks and SDS-PAGE analysis showed that they synthesized a high proportion of their collagen as type X. The cultures were examined by immunoelectron microscopy using the X-G15 gold to compare the distribution of type X in cultures to that found in cartilage tissue. One advantage to the analysis of the cell cultures is that the cells are labeled with the antibody-gold particles in cell culture medium. This should avoid potential artifacts caused during the drying of cryostat tissue sections onto coverslips before antibody-gold staining. The distribution of type X collagen was quite similar to that observed for the matrix of the hypertrophic cartilage tissue in situ. As shown in Fig. 4, the gold complexes were found associated with both filamentous material (Fig. 4A) and fibrils (Figs. 4A and 4B). Again, the mats were more evident near the cell-matrix interface, but the gradient of reactivity from this interface into the deeper matrix was not as obvious as in the hypertrophic cartilage tissue. We also noted that the cultures contained several different types and sizes of membrane-bound vesicles and multivesicular bodies. Some of these are probably artifacts of preparation, since they contain myelin fig-
FIG. 1. Immunogold staining of 14-day embryonic chick hypertrophic cartilage with an anti-type X collagen antibody adsorbed to 15-nm gold particles (X-G15). The tissue was fixed in 4% paraformaldehyde for 30 min at 4°C. (A) High magnification micrograph of a fine filamentous mat. Note the high density of anti-type X-gold label associated with these mats. Bar, 0.2 Wm. (B) Filamentous mat (asterisk) heavily labeled with the anti-type X-gold. Adjacent to the mat are to lo- to 20-nm-diameter fibrils which show little gold label. This partitioning of gold label in the filamentous pericellular mats was typical for tissue fixed in 4% paraformaldehyde. Bar, 0.2 pm. (C) Low magnification micrograph showing the anti-type X gold label associated with fine filamentous material on the wall of the lacunae. A gap (g) separates the chondrocyte (c) from the adjacent matrix. Bar, 0.25 pm. 56
SCHMII) AND LINSENMAYER
Type X Collagen
Ultrastructural
Forms
FIG. 2. Low magnification micrographs of 14-day embryonic chick cartilage derived from the hypertrophic cavity (A) and from the tarsus (B), a region of nonhypertrophic cartilage. Both are stained with the anti-type X cartilage tissue was fixed in 1% paraformaldehyde for 5 min. (A) Distribution of type X-gold label in the matrix shows that the same anti-type X collagen/l&nm gold probe essentially does not label cartilage in the tarsus. adjacent to the chondrocyte lacks the fine filamentous mats seen in hypertrophic cartilage. Bar, 0.25 Gm.
57
region adjacent to the marrow collagen/Snm gold probe. The between the cells. Bar, 1 pm. B Note also in B that the matrix
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DEVELOPMENTAL BIOLOGY
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FIG. 3. Double labeling of hypertrophic cartilage with an anti-type X collagen/l5-nm gold and an anti-type II collagen/5-nm gold. The tissue is unfixed. (A) Distribution of the two labels near the boundary of the lacunae. Filamentous material in upper left is marked with an asterisk and some fibrils containing both types X and II are marked with arrowheads. Bar, 0.2 pm. (B) Distribution of the two antibody-gold probes deeper in the matrix. Ten- to twenty nanometer fibrils predominate in this region. Also seen is a 40-nm-diameter collagen fibril with well-defined periodicity. Bar, 0.1 pm.
SCHMI~ ANI) LINSENMAYER
Type X Collugen
UltrustrwYural
Fwms
FIG. 4. Chondrocyte cell cultures labeled with the anti-type X collagen 15nm gold particles. (A) Decoration of the gold particles on the fine hlamentous material near the lacunar wall. (B) Anti-type X-gold label is also associated with the lO- to 20-nm fibrils in the cell cultures. (C) Anti-tvptx X collagen-gold probe decorating filamentous material associated with a ruptured vesicle. Magnification is the same for the three micrographs. Bar, 0.1 em.
ures (not shown). Others, however, contain material morphologically similar to that found in the filamentous mats and thus may represent transport/secretory vesicles of type X collagen. Occasionally one of these is found in a ruptured state (Fig. 4C), in which case the filamentous contents react with the anti-type X collagen antibody. DISCIJSSION
In the present investigation, we observed by singleand double-label immunoelectron microscopy that type
X collagen occurs in two forms, a filamentous one found predominantly in mats located in the matrix immediately adjacent to the cell surface, and a fibrillar one in which the molecule is associated with fibrils of type II collagen. Both forms were found within the matrix of hypertrophic cartilage tissue and of chondrocytes in culture. The greatest density of anti-type X collagen-gold complexes was over the mats of filamentous material. The small diameter of the filaments in the mats suggests an arrangement of individual molecules or, at the
60
DEVEL~PMENTALBIOLOGY
most, a lateral association of only a few molecules. It is possible that the filamentous mats may represent a recently secreted form of type X collagen. Molecules may dissociate from the aggregates and subsequently diffuse deeper into the matrix to become associated with the lo- to 20-nm-diameter fibrils. The small size of the type X molecule should facilitate its diffusion. The preferential location of the mats in the pericellular region and their absence from matrix farther away from the cell is consistent with this, as is the observation that sometimes similar, if not identical, material can be detected near ruptured vesicles. In light microscopic studies with immunoperoxidase staining Gibson et al. (1986) have described a pericellular distribution of type X collagen in regions of hypertrophic cartilage where type X synthesis had just been initiated. In ultrastructural studies on the proximal growth plate of avian tibiotarsus, Howlett (1979) reported the presence of “an extremely sparse and finely delicate network of microfibrils” which radiated across the chondrocytic lacunae from the cellular membrane to the septal wall. This delicate network was only apparent in the zones of hypertrophy and degenerating hypertrophic cartilage. The mats were best preserved in preparations which had been fixed, but they also could be detected in unfixed tissues and cultures. Thus, they are not an artifact of fixation. We cannot be completely certain, however, that the large differential in type X collagen reactivity noted between the filamentous mats and the fibril-associated material is a true representation of the quantitative distribution of the molecule. The filamentous mats, while strongly reactive for type X collagen, usually showed very little staining for type II collagen. If the mats represent secretory packages of matrix material which has recently been released from the cell, it suggests that such cells are synthesizing a high percentage of their collagen as type X or that types II and X may be synthesized and/or secreted by separate routes. Both possibilities may occur. We know from double-label immunofluorescence analyses of permeabilized chondrocytes in vitro that individual cells can contain both types of collagens (Solursh et al., 1986) and sometimes intracellular compartments seem to contain one of these collagen types but little if any of the other (unpublished observations). Besides differences in the packaging or secretion of type X and type II collagens, their mRNA levels are differentially affected by incubation of the cells with dimethyl sulfoxide (Manduca et al., 1988). The other major location for type X collagen was in association with lo- to 20-nm-diameter fibrils, a result also obtained by A. R. Poole (personal communication). We speculate that these represent a secondary associa-
VOLUME138,199O
tion of newly secreted type X collagen molecules or filaments with preexisting type II-type IX-type XI collagen fibrils (Vaughan et al., 1988; Mendler et al., 1989). From double-labeling we know the fibrils have epitopes for both anti-type II collagen and anti-type X collagen antibodies. Also, when one compares the sizes and periodicities of these to the collagen fibrils in a nonhypertrophic cartilage (such as the tarsus), there are no detectable differences in diameters and banding pattern. The only difference is that in the hypertrophic matrix they have type X collagen associated with them. The association of type X collagen with lo- to 20-nmdiameter fibrils occurs largely, if not entirely, on the fibril surface. In certain favorable cases the antibodygold complexes appear to decorate fine filamentous material associated with the fibrils. In itself, the accessibility of the anti-type X-gold complexes to their epitopes suggests a surface association. Epitopes on collagens buried within fibrils are antigenically “masked” (Linsenmayer et ah, 1983; Fitch et al., 1984; Birk et al, 1988; Mendler et al, 1989). Consistent with the diffusion of type X into the preexisting matrix, studies on the synthesis of type X and II collagens by chondrocytes in monolayer (Schmid and Conrad, 1982b) or in collagen gels (Gibson et al., 1986) showed that the majority of the type X is secreted into the medium, while a high proportion (>50%) of the type II remained in the cell layer. Only after a lag period did the type X eventually become deposited in the matrix surrounding the cells (Gibson et ah, 1986). One working hypothesis we have raised previously is that type X might facilitate degradation and removal of the hypertrophic matrix. In solution studies, type X collagen was degraded more rapidly than type II collagen by human skin collagenase (Schmid et ab, 1986) and elastase cleaves type X at sites near the collagenase cleavage sites (Gadher et al., 1988, 1989). Our data suggest that type X collagen may be secreted separately from type II and possible in its complete absence. As it diffuses deeper into the matrix, it becomes associated with preexisting type II collagen fibrils. The coating of type X collagen, in addition to itself being readily degradable, may sequester elevated concentrations of collagenase or elastase in the vicinity of the fibrils, thus facilitating their removal. The fate of at least some of the type X collagen within the filamentous mats may also be degradation. We have already presented arguments that the type X collagen within the filamentous mats near the cell surface represents newly secreted material which may be transported further into the matrix. And, while this is likely, it is our impression that the rates of synthesis and/or diffusion of the molecule must be such that a considerable amount of filamentous type X also accumulates at
SCHMIU AND LINSENMAYER
Type X Collu,yen Ultrustructurul
this site. At this time the cells are undergoing rapid hypertrophy (Hunziker et al., 1987), requiring an equally rapid increase in volume of the investing lacuriae-a process undoubtedly requiring matrix degradation. Dean et al. (1985) have reported that a fourfold increase in collagenase activity accompanied a sixfold increase in hypertrophic cell volume in rachitic rat growth plates. The loose pericellular meshwork of filamentous type X collagen may be readily degraded, thus facilitating expansion of the lacunae and allowing cell enlargement. The correlative evidence supporting a role for type X in the calcification of cartilage is strong, yet a direct role for type X in the process remains to be established. The molecule is synthesized by chondrocytes before calcification is initiated (Schmid and Linsenmayer, 1985) and it persists in regions of calcified cartilage (Grant et ab, 1985; Kwan et ab, 1986; Gibson et al, 1986; Love11 and Eyre, 1988). Habuchi et al. (1985) found a coordinate regulation between collagen and alkaline phosphatase syntheses in chondrocyte cultures. The addition of organic phosphates to chondrocyte cultures stimulates the synthesis of type X collagen and mineralization of the cultures (Morris and Balian, 1985; Gerstenfeld and Lian, 1988). The evidence presented here that type X collagen modifies the surface of cartilage collagen fibrils may be important for the subsequent calcification of the tissue. We thank Paloma Larramendi and Frances Roche for their excellent technical assistance and John Fitch for critical reading of the manuscript. Supported by an Arthritis Investigator Award from the National Arthritis Foundation, NIH Grants HD20715 and AR39239 (T.M.S.). and NIH Grant HD23681 (T.F.L.). REFERENCES BIRI<, D. E., FITCH, J. M., BA~IAKZ, J. P., and LINSENMAYER, T. F. (1988). Collagen type I and type V are present in the same fibril in the avian cornea1 stroma. J. Cell Bid. 106, 999-1008. BIIR(:ESON, R. E., and HOI,I,ISTF.R, D. W. (1979). Collagen heterogeneity in human cartilage: identification of several new chains. Biro cl/c,nl. Rioph ys. Res. Con, I,Lu?~.87, 1124-1131. DEAN, D. D., MIINIZ, 0. E., BERMAN, I., PITA, J. C., CARRENO, M. R., WOESSN~::K,J. F. W., JR., and HOWELL, D. S. (1985). Localization of collagenasc in the growth plate of rachitic rats. J. Clin. Invest. 76, 716-722. W., HUNZIKER, E. B., and SCHENK, R. K. EGGLI, P. S., HERRMANN, (1985). Matrix compartments in the growth plate of the proximal tibia of rats. An&. Rec. 211, 246-257. EYRE, D. R., APON, S., W~J, J.-J., ERICSSON, L. H., and WALSH, K. A. (1987a). Collagen type IX: Evidence for covalent cross-linkages to type II collagen in cartilage. FEBS Lett. 220, 337-341. EYRE, D. R., W~J, J.-J., and APON, S. (1987b). A growing family of collagens in articular cartilage: Identification of 5 genetically distinct types. J. Rhe?~mutol. 14, 25-27. FELL, H. (1925). The histogenesis of cartilage and bone in the long bones of embryonic fowl. J. Mwphol. Physiol. 40, 417-459. FITCH, J. M., Guess, J., MAYNE, R., JOHNSON-WINT, B., and LINSEN-
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MAYER, T. F. (1984). Organization of collagen types I and V in the embryonic chicken cornea: Monoclonal antibody studies. Proc. Nut1 Acad. Sci. USA 81,2791-2’795. FRENS, G. (1973). Controlled nucleation for the regulation of the particle size in monodisperse gold suspension. Nufure (London) Phys. Sci. 241 20-22. GADHER, ‘S. J., EYRE, D. R., DUANCE, V. C., WOTTON, S. F., HECK, L. W., SCHMID, T. M., and WOOLLEY, D. L. (1988). Susceptibility of cartilage collagens type II, IX, X, and XI to human synovial collagenase and neutrophil elastase. Eur. J. Biochem. 175,1-i’. GADHE:R, S. J., SCHMID, T. M., HECK, L. W., and WOOLLEY, D. L. (1989). Cleavage of collagen type X by human synovial collagenase and neutrophil elastase. M&rix 9,109-115. GERSTENFELD, L. C., and LIAN, L. B. (1988). In vitro calcification of chondrocyte cultures: Induction of alkaline phosphatase enzyme activity and the expression of type X collagen. Trans. Orthop. RPS. S/JC 13, 290. GIHSON, G. J., BEARMAN, C. H., and FLINT, M. H. (1986). The immunoperoxidase localization of type X collagen in chick cartilage and lung. Collrcyrr~ R&f. RPS. 6, 163-l 84. GIBSON, G. J., and FLINT, M. H. (1985). Type X collagen synthesis by chick sternal cartilage and its relationship to endochondral development. J. Cdl Bid. 101, 2’77-284. GRANT, W. T., SIJSSMAN, M. D., and BAI~IAN, G. (1985). A disulfidebonded short chain collagen synthesized by degenerative and calcifying zones of bovine growth plate cartilage. J. BioL Chem. 260, 3798-3803. HAUIJCIII, H., CONRAD,
H. E., and GLASEK, J. H. (1985). Coordinate regulation of collagen and alkaline phosphatase levels in chick embryo chondrocytes. J. Bid. Chrm. 260, 13,029-13,034. HOWLETT, C. R. (1979). The fine structure of the proximal growth plate of the avian tibia. J rlrccrt. 128, 377-399. HIINZIKE:K, E. B., HERRMANN, W., S(:HENK, R. K., MUELLER, M., and MOOR, H. (1984). Cartilage ultrastructure after high pressure freezing, freeze substitution, and low temperature embedding. I. Chondrocyte ultrastructure~Implications for the theories of mineralization and vascular invasion. J. Cell Bio/. 98, 267-276. HUN~IKEK, E. B., SCHENK, R. K., and CRUZ-ORIVE, L-M. (1987). Quantitation of chondrocyte performance in growth-plate cartilage during longitudinal bone growth. J. Rune J. Sure. 69A, 162-173. KIELTY, C. M., KWAN, A. P. L., HOLMES, D. F., SCHOR, S. L., and GRAN’I’, M. E. (1985). Type X collagen, a product of hypertrophic chondrocytes. Biochcm. .I 27, 545-554. KWAN, A. P. L., FREEMONT, A. J., and GRANT, M. E. (1986). Immunoperoxidase localization of type X collagen in chick tibiae. Biosci. Rep. 6,155-162. LARRAMEN~I, P. C. H. (1988). A good support for the thick frozen sections for immunolabeling when post embedding in plastic for EM is required. J. Elwtrox Microsc. Tech:ch. 10, 123-124. LINSENMAYER, T. F., FITCH, J. M., SCIIMII), T. M., ZAK, N. B., GIBNEY, E., SANI~SKSON, R. D., and MAYN~:, R. (1983). Monoclonal antibodies against chicken type V collagen: Production, specificity, and use for immunocytochemical localization in embryonic cornea and other organs. J. Q>l/ Biol. 96, 124-132. LINSENMAYER, T. F., and HENIYJRIX, M. J. C. (1980). Monoclonal antibodies to connective tissue macromolecules: Type II collagen. Biothem. Biophys. Res. Commun. 92, 440-446. LOVELL, T. P., and EYRE, D. R. (1988). Unique biochemical characteristics of the calcified zone of articular cartilage. Trans. Orthop Res. .soc. 13, 511. MANI~JCA, P., CASTAC,NOI,A, P., and CANCEUI~A, R. (1988). Dimethyi sulfoxide interferes with ia vitro differentiation of chick embryo endochondral chondrocytes. I)ea Bid. 125, 234-236. MAYNE, R. (1989). Cartilage collagcns. Arthritis Rh,eum. 32, 241-246. MGNI~LER, M., EICII-BENDER, S. G., VAUGHAN, I,., WINTERHALTER,
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K. H., and BRUCKNER, P. (1989). Cartilage contains mixed fibrils of collagen types II, IX, and XI. J. Cell Biol. 108, 191-197. MORRIS, d. B., and BALIAN, G. (1985). Treatment of chondrocytes with fl-glgcerophosphate increases synthesis of short chain (type X) collagen. Ann. N. Y Acad. Sci. 460,475-47’7. SCHMID, T. M., and CONRAD, H. E. (1982a). A unique low molecular weight collagen secreted by cultured chick embryo chondrocytes. J. Biol. Chem. 257, 12,444-12,450. SCHMID, T. M., and CONRAD, H. E. (1982b). Metabolism of low molecular weight collagen by chondrocytes obtained from histologically distinct zones of the chick embryo tibiotarsus. J. Bid. Ch,em. 257, 12,451-12,457. SCHMID, T. M., and LINSENMAYER, T. F. (1983). A short chain (pro)collagen from aged endochondral chondrocytes. J. Biol. Chem. 258,9504-9509. SCHMID, T. M., and LINSENMAYER, T. F. (1985). Immunochemical localization of short chain cartilage collagen (type X) in avian tissues. J. Cell Bid. 100, 598-605. SCHMID, T. M., MAYNE, R., JEFFREY, J. J., and LINSENMAYER, T. F.
VOLUME 138, 1990
(1986). Type X collagen contains two cleavage sites for a vertebrate collagenase. J. Rio/. Chem. 261,4184-4189. SLOT, J. W., and GETJZE,H. J. (1981). Sizing of protein A-colloidal gold probes for immunoelectron microscopy. J. Cell Biol. 90,533-536. SOLURSII, M., JE:NSF:N, K. L., REITER, R. S., SCHMID, T. M., and LINSENMAYER, T. F. (1986). Environmental regulation of type X collagen production by cultures of limb mesenchyme, mesectoderm, and sternal chondrocytes. Del). Biol. 117, 80-101. STOCUM, D. L., DAVIS, R. M., LEC;ER, M., and CONRAD, H. E. (1979) Development of the tibiotarsus in the chick embryo: Biochemical activity of histologically distinct zones. J. Emlnyol. Eql. Mwrphol. 54,l.s170. VAN DER REST, M., and MAYNE, R. (1988). Type IX collagen-proteoglycan from cartilage is covalently crosslinked to type II collagen. J. Biol. CIIPW. 263, 1615-1618. VAUGHAN, L., MENDLER, M., HIJBER, S., BRTJCKNER, P., WINTERHALTER, K., IRWIN, M., and MAYNE, R. (1988). D-Periodic distribution of collagen type IX along cartilage fibrils. J. Cell Biol. 106, 991L997.
BIOLOGY
138,
lmmunoelectron
53-62 (1990)
Microscopy of Type X Collagen: Supramolecular within Embryonic Chick Cartilage
Forms
THOMAS M. SCHMID* AND THOMAS F. LINSENMAYERt
Accepted October 2.2, 19X.9 To determine the supramolecular forms in which avian type X collagen molecules assemble within the matrix of hypertrophic cartilage, we performed immunoelectron microscopy with colloidal gold-labeled monoclonal antibodies. In addition double-labeled analyses were performed for the molecule and type II collagen, employing two monoclonal antibodies attached to different size gold particles. Both in situ limb cartilages and the extracellular matrix of chondrocyte cultures were examined. We observed in both systems that type X collagen is present in two forms. One is as fine filaments (~5 nm in diameter) within mats which are found predominantly in the pericellular matrix of the hypertrophic chondrocytes. The second form is in association with the fibrils 110-20 nm in diameter) which also react with the antibody for type II collagen. It seems that the Iilamentous mats represent a form in which the type X collagen is initially secreted from the cell. The type X associated with the striated fibrils most likely represents a secondary association of the molecule with preexisting type II/IX/XI fibrils. The data are consistent with our previously proposed hypothesis that type X collagen is involved in, and perhaps even “targets,” certain matrix components for degradation and removal. I( 1990 Academic Press, In, INTRODIJCTION
of type X in the development of endochondral bones must depend on the type(s) of supramolecular aggregate(s) in which it assembles or with which it becomes associated. In the present investigation we have performed an immunoelectron microscopic investigation of type X collagen in situ in embryonic chick limb cartilage and the extracellular matrix of chondrocyte cultures. We have observed that type X is present in two different supramolecular forms: one is in amorphous mats, and the other is associated with type II collagen fibrils.
During endochondral bone formation, individual chondrocytes undergo a progression from young cells undergoing rapid division to mature cells having the greatest capacity for synthesizing matrix components to hypertrophic cells which become progressively enlarged and eventually are removed. In young, prehypertrophic cartilage, the chondrocytes synthesize a mixture of collagen types II, IX, and XI (Burgeson and Hollister, 1979; Eyre et al., 198713; Mayne, 1989) which coassemble into heterotypic fibrils (Eyre et al., 1987a; van der Rest and Mayne, 1988; Vaughan et al., 1988; Mendler et al., 1989). Once the cells have initiated hypertrophy, they add type X to their biosynthetic repertoire (Schmid and Conrad, 1982a,b; Schmid and Linsenmayer, 1985; Kielty et al, 1985). As the hypertrophic program progresses, the synthesis of this collagen increases, with a concomitant decrease in synthesis of the other types (Gibson and Flint, 1985). Hypertrophy and subsequent changes in the cartilage matrix include (a) enlargement of lacunae to accommodate the great increase in volume of the individual chondrocytes (Hunziker et al., 1987), (b) calcification of the cartilage matrix, providing a substratum for bone deposition, and (c) removal of calcified and uncalcified cartilage matrix resulting in formation of a marrow cavity. It is during these processes when deposition of type X collagen occurs within the matrix. Like other collagen molecules, the structural function
METHODS
Labeled Antibody
Prepamfion
and Testing
Monoclonal antibodies against type X collagen (X-AC9) (Schmid and Linsenmayer, 1985) and type II collagen (II,B,) (Linsenmayer and Hendrix, 1980) were obtained from ascites fluid of hybridoma-containing nude mice. They were purified by ammonium sulfate precipitation (50% saturation) followed by chromatography on Protein A-Sepharose (loading buffer-O.1 M sodium phosphate buffer, pH 8.5; elution buffer-50 mM sodium phosphate, 50 mM citrate, pH 5.0). As a control antibody we employed a myeloma IgGl protein (MOPC 21) obtained from Sigma. Fifteen-nanometer colloidal gold particles (G15) were prepared by reducing chloroauric acid with citrate (Frens, 1973). Five-nanometer gold particles (G5) were purchased from Janssen (Ted Pella, Inc., Tustin, CA). 53
0012-1606/90 $3.00 (‘opyriyht All rights
!C 1990 by Academic Press, Inc. of reproduction in any form reserved.
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DEVELOPMENTALBIOLOGY
VOLUME138.1990
The anti-collagen antibodies and the control IgG Tissue sections or cultured cells were treated with 5 were directly adsorbed to colloidal gold. Purified anti- mg/ml hyaluronidase for 30 min in 0.1 M cacodylate bodies were added to 20 ml of colloidal gold in 2 m&I buffer, pH 7.2 (tissue sections), or in DME (cells). Covborate buffer, pH 9.0 (final antibody concentration, 50 erslips were reacted overnight with colloidal gold preppg/ml). After 1 hr, BSA was added to a concentration of arations (Ass0 of 0.5-0.6) and the washed 4X with 1% 1%. The preparations were washed three times with BSA in 0.1 M cacodylate buffer (pH 7.2) for 30 min. The BSA-Tris saline buffer (1% BSA, 20 mM Tris, 0.13 M coverslips were fixed in 2.5% glutaraldehyde in cacodylNaCl, pH 8.2) and collected by centrifugation at 50,OOOg ate buffer for 30 min and were washed and treated with (G5) or 14,000g(G15) for 45 min at 4°C. The pellets were 1% 0~0~ in cacodylate buffer. After being washed with suspended in BSA-Tris saline buffer and centrifuged water, the coverslips were stained in block with 1% through a stepwise glycerol gradient consisting of 10, uranyl acetate for an hour, dehydrated through a 15, 20, 25, and 30% glycerol in BSA-Tris saline buffer. graded series of ethanols, and embedded in Epon-AralThe desired antibody-gold complexes were recovered dite in inverted Beem capsules. The plastic blocks were from the middle of the gradient (Slot and Geuze, 1981). detached from the coverslips after freezing in liquid The specificities of the antibody-gold probes were nitrogen (Larramendi, 1988). Thin sections were cut, tested by ELISA as described for the monoclonal anti- placed onto uncoated copper grids, and stained with bodies (Schmid and Linsenmayer, 1985). Plates coated 2.5% uranyl acetate in 50% ethanol and lead citrate. with either type X or type II collagen were incubated They were examined and photographed on a JEOL 100 with serial dilutions of the gold probes for 2 hr and CX electron microscope. washed. Before dilution of the antibody-gold preparaRESULTS tions had an AssOof 0.5-0.6. A goat anti-mouse IgG peroxidase conjugate (Hyclone Laboratories, Logan UT) Controls for Specificity of Antibody-Gold Complexes was used as the second antibody and incubated for 1 hr. The peroxidase substrate containing 200 yl 0.01% oThe identification of type X collagen and its relationphenylenediamine and 0.1% Hz02 in water was incu- ship to fibrils containing type II collagen in embryonic bated for 30 min. The reaction was stopped by the addi- chick cartilage were examined using a monoclonal antition of 50 ~1 of 50% HaSO, and the absorbance was body specific for each collagen type adsorbed directly to determined at 492 nm on a Dynatech ELISA plate different size gold particles. This approach has been reader. used successfully for the detection of type I and type V collagen within the same fibril in the chicken cornea Tissue Preparation (Birk et ab, 1988). The procedure maximizes resolution Tibiotarsi were dissected from 14-day chick embryos by creating the smallest possible distance between the and the sheaths of periosteal bones were removed. The antigen and the marker gold particle and affords the cartilage pieces were treated by one of the following capability of performing simultaneous double-labeling methods. (a) The tissue was not fixed before frozen sec- for two different collagen types. tions were cut. (b) The tissue was fixed in 1% paraforThe specificity of the preparations of antibody-gold maldehyde in 0.1 M cacodylate buffer, pH 7.2, for 5 min was tested (a) by ELISA on plates coated with purified at 4°C. (c) The tissue was fixed in 4% paraformaldecollagen preparations and (b) by immunohistochemical hyde in cacodylate buffer for 30 min at 4°C. Formalde- reactivity at the light microscopic level. In ELISA the hyde-fixed tissues were incubated for another 30 min in anti-type X collagen-gold conjugate and the anti-type 0.1 M Tris, 50 mM glycine buffer, pH 7.5, to quench free II collagen conjugate displayed the same reactivity aldehydes. Fixed or unfixed tissues were embedded in previously reported for the anti-type X (Schmid and OCT compound and 12-pm frozen sections were cut and Linsenmayer, 1985) and anti-type II (Linsenmayer and dried onto albuminized Thermanox coverslips (ICN, Hendrix, 1980) monoclonal antibodies. The control Lisle, IL). IgG-gold bound to neither collagen-coated plate (data Chondrocytes were isolated by enzymatic digestion of not shown). 12-day chick embryonic tibiotarsi as previously deImmunohistochemical reactivity of the anti-collascribed (Schmid and Linsenmayer, 1983) and plated gen-gold probes was tested on cryostat sections of 14onto Thermanox coverslips. The medium consisted of day tibiotarsi. The distributions of the anti-collagenDulbecco’s Modified Eagle’s medium (DME) containing gold complexes were the same as those reported by indi10% calf serum (Hyclone, defined-supplemented), 50 rect immunofluorescent methods. The anti-type X-gold pg/ml ascorbic acid, and 100 pg/ml fl-aminopropionicomplex stained the hypertrophic cartilage near the trile fumarate. Chondrocyte cultures were not fixed be- marrow cavity, the anti-type II-gold complex reacted fore incubation with antibody-gold probes. with the entire cartilage rudiment, and the control
SCHMII) AND LINSENMAYER
MOPC21 gold complex showed no detectable (data not shown). Single-Label
Immunoelectron
Type X Colkzgeri Vltrastructmz1
reactivity
Microscopy
At the electron microscopic level, within the hypertrophic cartilage matrix the anti-type X collagen-gold reacted with two distinct structures (Fig. 1). The most intense reactivity (>35 particles/pm’) was found over mats of fine filamentous material (Fig. 1A and marked with asterisk in Fig. 1B). The fine filaments appeared to have diameters less than 5 nm. Gold particles were also found associated with fibrils lo-20 nm in diameter, the major fibrillar type in cartilage, but these were less intensely labeled. As can be seen in Fig. lC, the filamentous mats are most evident in the matrix closest to the cell surface. In this figure the mats abut a gap (g) between the cell surface and matrix. Such gaps between the chondrocyte surface and the adjacent matrix were typically observed, and probably represent a shrinkage artifact created during processing of the tissue with conventional fixatives (Hunziker et ah, 1984; Eggli et ah, 1985). Also seen in Fig. lC, the fine filamentous mats decrease in number, or at least are less evident, as the distance from the cell surface increases. In regions of the matrix lacking the fine filamentous material, the type X collagen label is usually associated with the loto ‘LO-nm-diameter fibrils. The distribution and density of fibril-associated, anti-type X collagen label is illustrated in the low power micrograph shown in Fig. 2A. Although the density of gold particles observed in such sections was not high (approximately I2 particles/pm’), it was much greater than in identically treated sections of tarsus (CO.29 particles/pm” shown in Fig. 2B), a nonhypertrophic cartilage containing no type X collagen. Typical photographic fields of the latter were almost devoid of gold particles. Also, in nonhypertrophic cartilage, there was an absence of the type X collagen-reactive filamentous mats, which were so prominent in the hypertrophic zone. This can be appreciated in Fig. 2B, which shows a portion of a chondrocyte (c) with no adjacent filamentous mats. Double-Label
Immu,noelectron
Microscopy
Since the anti-type X-gold particles were associated with lo- to 20-nm-diameter fibrils, we performed double-label analyses for collagen types X and II to determine possible associations between them. In these experiments hypertrophic cartilage was labeled with anti-type X collagen antibody complexed to 15-nm gold (X-G15) and anti-type II collagen antibody complexed to 5-nm gold (II-G5).
Foms
55
The salient features observed in this study can be seen in Fig. 3. In pericellular regions (Fig. 3A), the filamentous material (asterisk) shows strong reactivity for type X collagen, but for type II the reactivity is slight or nonexistent. However, examination of many lo- to 20-nm-diameter fibrils shows some reactivity for both collagen types. Some fibrils which are decorated with both anti-type X collagen-gold particles and anti-type II collagen-gold particles are marked with arrowheads. Although the great majority of fibrils fall within the lo-20 nm size range, sometimes fibrils with a larger diameter and more distinct banding pattern were observed. An example of such a fibril with a diameter of about 40 pm is shown in Fig. 3B. Such fibrils show strong reactivity for type II collagen which is labeled at periodic intervals of 60-70 nm. The anti-type II reactivity is greater for the large diameter fibrils than for the smaller lo- to 20-nm-diameter fibrils. The anti-type Xgold particles also show some reactivity with the large diameter fibrils. A third component of potential interest to the present investigation was vesicles. These were found both near the lacunar wall (Fig. 3A) and among the fibrils of the interterritorial matrix (not shown). The majority of the vesicles were not labeled with either anti-collagen-gold probes; however, as shown in Fig. 3A, some gold particles can be detected adjacent to the vesicles. Chondrocyte Cultures Chondrocytes were maintained in monolayer culture for 8 weeks and SDS-PAGE analysis showed that they synthesized a high proportion of their collagen as type X. The cultures were examined by immunoelectron microscopy using the X-G15 gold to compare the distribution of type X in cultures to that found in cartilage tissue. One advantage to the analysis of the cell cultures is that the cells are labeled with the antibody-gold particles in cell culture medium. This should avoid potential artifacts caused during the drying of cryostat tissue sections onto coverslips before antibody-gold staining. The distribution of type X collagen was quite similar to that observed for the matrix of the hypertrophic cartilage tissue in situ. As shown in Fig. 4, the gold complexes were found associated with both filamentous material (Fig. 4A) and fibrils (Figs. 4A and 4B). Again, the mats were more evident near the cell-matrix interface, but the gradient of reactivity from this interface into the deeper matrix was not as obvious as in the hypertrophic cartilage tissue. We also noted that the cultures contained several different types and sizes of membrane-bound vesicles and multivesicular bodies. Some of these are probably artifacts of preparation, since they contain myelin fig-
FIG. 1. Immunogold staining of 14-day embryonic chick hypertrophic cartilage with an anti-type X collagen antibody adsorbed to 15-nm gold particles (X-G15). The tissue was fixed in 4% paraformaldehyde for 30 min at 4°C. (A) High magnification micrograph of a fine filamentous mat. Note the high density of anti-type X-gold label associated with these mats. Bar, 0.2 Wm. (B) Filamentous mat (asterisk) heavily labeled with the anti-type X-gold. Adjacent to the mat are to lo- to 20-nm-diameter fibrils which show little gold label. This partitioning of gold label in the filamentous pericellular mats was typical for tissue fixed in 4% paraformaldehyde. Bar, 0.2 pm. (C) Low magnification micrograph showing the anti-type X gold label associated with fine filamentous material on the wall of the lacunae. A gap (g) separates the chondrocyte (c) from the adjacent matrix. Bar, 0.25 pm. 56
SCHMII) AND LINSENMAYER
Type X Collagen
Ultrastructural
Forms
FIG. 2. Low magnification micrographs of 14-day embryonic chick cartilage derived from the hypertrophic cavity (A) and from the tarsus (B), a region of nonhypertrophic cartilage. Both are stained with the anti-type X cartilage tissue was fixed in 1% paraformaldehyde for 5 min. (A) Distribution of type X-gold label in the matrix shows that the same anti-type X collagen/l&nm gold probe essentially does not label cartilage in the tarsus. adjacent to the chondrocyte lacks the fine filamentous mats seen in hypertrophic cartilage. Bar, 0.25 Gm.
57
region adjacent to the marrow collagen/Snm gold probe. The between the cells. Bar, 1 pm. B Note also in B that the matrix
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DEVELOPMENTAL BIOLOGY
VOLUME
138,199O
FIG. 3. Double labeling of hypertrophic cartilage with an anti-type X collagen/l5-nm gold and an anti-type II collagen/5-nm gold. The tissue is unfixed. (A) Distribution of the two labels near the boundary of the lacunae. Filamentous material in upper left is marked with an asterisk and some fibrils containing both types X and II are marked with arrowheads. Bar, 0.2 pm. (B) Distribution of the two antibody-gold probes deeper in the matrix. Ten- to twenty nanometer fibrils predominate in this region. Also seen is a 40-nm-diameter collagen fibril with well-defined periodicity. Bar, 0.1 pm.
SCHMI~ ANI) LINSENMAYER
Type X Collugen
UltrustrwYural
Fwms
FIG. 4. Chondrocyte cell cultures labeled with the anti-type X collagen 15nm gold particles. (A) Decoration of the gold particles on the fine hlamentous material near the lacunar wall. (B) Anti-type X-gold label is also associated with the lO- to 20-nm fibrils in the cell cultures. (C) Anti-tvptx X collagen-gold probe decorating filamentous material associated with a ruptured vesicle. Magnification is the same for the three micrographs. Bar, 0.1 em.
ures (not shown). Others, however, contain material morphologically similar to that found in the filamentous mats and thus may represent transport/secretory vesicles of type X collagen. Occasionally one of these is found in a ruptured state (Fig. 4C), in which case the filamentous contents react with the anti-type X collagen antibody. DISCIJSSION
In the present investigation, we observed by singleand double-label immunoelectron microscopy that type
X collagen occurs in two forms, a filamentous one found predominantly in mats located in the matrix immediately adjacent to the cell surface, and a fibrillar one in which the molecule is associated with fibrils of type II collagen. Both forms were found within the matrix of hypertrophic cartilage tissue and of chondrocytes in culture. The greatest density of anti-type X collagen-gold complexes was over the mats of filamentous material. The small diameter of the filaments in the mats suggests an arrangement of individual molecules or, at the
60
DEVEL~PMENTALBIOLOGY
most, a lateral association of only a few molecules. It is possible that the filamentous mats may represent a recently secreted form of type X collagen. Molecules may dissociate from the aggregates and subsequently diffuse deeper into the matrix to become associated with the lo- to 20-nm-diameter fibrils. The small size of the type X molecule should facilitate its diffusion. The preferential location of the mats in the pericellular region and their absence from matrix farther away from the cell is consistent with this, as is the observation that sometimes similar, if not identical, material can be detected near ruptured vesicles. In light microscopic studies with immunoperoxidase staining Gibson et al. (1986) have described a pericellular distribution of type X collagen in regions of hypertrophic cartilage where type X synthesis had just been initiated. In ultrastructural studies on the proximal growth plate of avian tibiotarsus, Howlett (1979) reported the presence of “an extremely sparse and finely delicate network of microfibrils” which radiated across the chondrocytic lacunae from the cellular membrane to the septal wall. This delicate network was only apparent in the zones of hypertrophy and degenerating hypertrophic cartilage. The mats were best preserved in preparations which had been fixed, but they also could be detected in unfixed tissues and cultures. Thus, they are not an artifact of fixation. We cannot be completely certain, however, that the large differential in type X collagen reactivity noted between the filamentous mats and the fibril-associated material is a true representation of the quantitative distribution of the molecule. The filamentous mats, while strongly reactive for type X collagen, usually showed very little staining for type II collagen. If the mats represent secretory packages of matrix material which has recently been released from the cell, it suggests that such cells are synthesizing a high percentage of their collagen as type X or that types II and X may be synthesized and/or secreted by separate routes. Both possibilities may occur. We know from double-label immunofluorescence analyses of permeabilized chondrocytes in vitro that individual cells can contain both types of collagens (Solursh et al., 1986) and sometimes intracellular compartments seem to contain one of these collagen types but little if any of the other (unpublished observations). Besides differences in the packaging or secretion of type X and type II collagens, their mRNA levels are differentially affected by incubation of the cells with dimethyl sulfoxide (Manduca et al., 1988). The other major location for type X collagen was in association with lo- to 20-nm-diameter fibrils, a result also obtained by A. R. Poole (personal communication). We speculate that these represent a secondary associa-
VOLUME138,199O
tion of newly secreted type X collagen molecules or filaments with preexisting type II-type IX-type XI collagen fibrils (Vaughan et al., 1988; Mendler et al., 1989). From double-labeling we know the fibrils have epitopes for both anti-type II collagen and anti-type X collagen antibodies. Also, when one compares the sizes and periodicities of these to the collagen fibrils in a nonhypertrophic cartilage (such as the tarsus), there are no detectable differences in diameters and banding pattern. The only difference is that in the hypertrophic matrix they have type X collagen associated with them. The association of type X collagen with lo- to 20-nmdiameter fibrils occurs largely, if not entirely, on the fibril surface. In certain favorable cases the antibodygold complexes appear to decorate fine filamentous material associated with the fibrils. In itself, the accessibility of the anti-type X-gold complexes to their epitopes suggests a surface association. Epitopes on collagens buried within fibrils are antigenically “masked” (Linsenmayer et ah, 1983; Fitch et al., 1984; Birk et al, 1988; Mendler et al, 1989). Consistent with the diffusion of type X into the preexisting matrix, studies on the synthesis of type X and II collagens by chondrocytes in monolayer (Schmid and Conrad, 1982b) or in collagen gels (Gibson et al., 1986) showed that the majority of the type X is secreted into the medium, while a high proportion (>50%) of the type II remained in the cell layer. Only after a lag period did the type X eventually become deposited in the matrix surrounding the cells (Gibson et ah, 1986). One working hypothesis we have raised previously is that type X might facilitate degradation and removal of the hypertrophic matrix. In solution studies, type X collagen was degraded more rapidly than type II collagen by human skin collagenase (Schmid et ab, 1986) and elastase cleaves type X at sites near the collagenase cleavage sites (Gadher et al., 1988, 1989). Our data suggest that type X collagen may be secreted separately from type II and possible in its complete absence. As it diffuses deeper into the matrix, it becomes associated with preexisting type II collagen fibrils. The coating of type X collagen, in addition to itself being readily degradable, may sequester elevated concentrations of collagenase or elastase in the vicinity of the fibrils, thus facilitating their removal. The fate of at least some of the type X collagen within the filamentous mats may also be degradation. We have already presented arguments that the type X collagen within the filamentous mats near the cell surface represents newly secreted material which may be transported further into the matrix. And, while this is likely, it is our impression that the rates of synthesis and/or diffusion of the molecule must be such that a considerable amount of filamentous type X also accumulates at
SCHMIU AND LINSENMAYER
Type X Collu,yen Ultrustructurul
this site. At this time the cells are undergoing rapid hypertrophy (Hunziker et al., 1987), requiring an equally rapid increase in volume of the investing lacuriae-a process undoubtedly requiring matrix degradation. Dean et al. (1985) have reported that a fourfold increase in collagenase activity accompanied a sixfold increase in hypertrophic cell volume in rachitic rat growth plates. The loose pericellular meshwork of filamentous type X collagen may be readily degraded, thus facilitating expansion of the lacunae and allowing cell enlargement. The correlative evidence supporting a role for type X in the calcification of cartilage is strong, yet a direct role for type X in the process remains to be established. The molecule is synthesized by chondrocytes before calcification is initiated (Schmid and Linsenmayer, 1985) and it persists in regions of calcified cartilage (Grant et ab, 1985; Kwan et ab, 1986; Gibson et al, 1986; Love11 and Eyre, 1988). Habuchi et al. (1985) found a coordinate regulation between collagen and alkaline phosphatase syntheses in chondrocyte cultures. The addition of organic phosphates to chondrocyte cultures stimulates the synthesis of type X collagen and mineralization of the cultures (Morris and Balian, 1985; Gerstenfeld and Lian, 1988). The evidence presented here that type X collagen modifies the surface of cartilage collagen fibrils may be important for the subsequent calcification of the tissue. We thank Paloma Larramendi and Frances Roche for their excellent technical assistance and John Fitch for critical reading of the manuscript. Supported by an Arthritis Investigator Award from the National Arthritis Foundation, NIH Grants HD20715 and AR39239 (T.M.S.). and NIH Grant HD23681 (T.F.L.). REFERENCES BIRI<, D. E., FITCH, J. M., BA~IAKZ, J. P., and LINSENMAYER, T. F. (1988). Collagen type I and type V are present in the same fibril in the avian cornea1 stroma. J. Cell Bid. 106, 999-1008. BIIR(:ESON, R. E., and HOI,I,ISTF.R, D. W. (1979). Collagen heterogeneity in human cartilage: identification of several new chains. Biro cl/c,nl. Rioph ys. Res. Con, I,Lu?~.87, 1124-1131. DEAN, D. D., MIINIZ, 0. E., BERMAN, I., PITA, J. C., CARRENO, M. R., WOESSN~::K,J. F. W., JR., and HOWELL, D. S. (1985). Localization of collagenasc in the growth plate of rachitic rats. J. Clin. Invest. 76, 716-722. W., HUNZIKER, E. B., and SCHENK, R. K. EGGLI, P. S., HERRMANN, (1985). Matrix compartments in the growth plate of the proximal tibia of rats. An&. Rec. 211, 246-257. EYRE, D. R., APON, S., W~J, J.-J., ERICSSON, L. H., and WALSH, K. A. (1987a). Collagen type IX: Evidence for covalent cross-linkages to type II collagen in cartilage. FEBS Lett. 220, 337-341. EYRE, D. R., W~J, J.-J., and APON, S. (1987b). A growing family of collagens in articular cartilage: Identification of 5 genetically distinct types. J. Rhe?~mutol. 14, 25-27. FELL, H. (1925). The histogenesis of cartilage and bone in the long bones of embryonic fowl. J. Mwphol. Physiol. 40, 417-459. FITCH, J. M., Guess, J., MAYNE, R., JOHNSON-WINT, B., and LINSEN-
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