Histochemistry (1992) 98:275-281
Histochemistry © Springer-Verlag 1992
Localization of collagen X in human fetal and juvenile articular cartilage and bone A.G. Nerlich 1, T. Kirsch 2, I. Wiest a, p. Betz 3, K. von der Mark 2
t Pathologisches Institut der UniversitS.t Mfinchen, Thalkirchnerstrasse 36, W-8000 M/inchen 2, Federal Republic of Germany 2 Max-Planck Arbeitsgruppe ffir klinische Rheumatologie an der Universitfit Erlangen-Nfirnberg, Schwabachanlage 10, W-8520 Erlangen, Federal Republic of Germany 3 Institut ftir Rechtsmedizin der Universit/it Miinchen, Frauenlobstrasse 6, W-8000 Mfinchen 2, Federal Republic of Germany Accepted: 2 September 1992 Abstract. The tissue localization was analysed of collagen X during human fetal and juvenile articular cartilagebone metamorphosis. This unique collagen type was found in the hypertrophic cartilage zone peri- and extracellularly and in cartilage residues within bone trabeculae. In addition, occasionally a slight intracellular staining reaction was found in prehypertrophic proliferating chondrocytes and in chondrocytes surrounding vascular channels. A slight staining was also seen in the zone of periosteal ossification and occasionally at the transition zone of the perichondrium to resting cartilage. Our data provide evidence that the appearance of collagen X is mainly associated with cartilage hypertrophy, analogous to the reported tissue distribution of this collagen type in animals. In addition, we observed an increased and often "spotty" distribution of collagen X with increasing cartilage "degeneration" associated with the closure of the growth plate. In basal hypertrophic cartilage areas, a co-distribution of collagens II and X was found with very little and "spotty" collagen III. In juvenile cartilage areas around single hypertrophic chondrocytes, co-localization of collagens X and I was also detected.
Introduction
During the development of long bones, chondrocytes differentiate along a series of morphologically different stages, which show a unique pattern of synthesized collagen types (for review see von der Mark 1986). While the undifferentiated precursor cells of limb cartilage produce collagen I, the pattern of synthesis is changing during chondrogenesis to collagen II synthesis by differentiated chondrocytes (vonder Mark et al. 1976), and further to collagen X synthesis (Schmid and Linsenmayer 1987). The discovery of collagen X synthesis by hypertrophic chondrocytes in vitro (Gibson et al. 1982; Capasso et al. 1982) and in the zone of hypertrophic cardCorrespondence to." A. Nerlich
lage in the growth plate in vivo (Schmid and Conrad 1982) provided a specific experimental tool for unequivocal identification of hypertrophic chondrocytes in situ and in vitro. Using monoclonal antibodies (Schmid and Linsenmayer 1985 a, b; Summers et al. 1988) and cDNA probes (Ninomiya et al. 1986) chick collagen X was specifically located in hypertrophic cartilage of growth plates, in the calcifying cartilage of sternal and rib cartilage, and in the developing notochord (Linsenmayer et al. 1986; Iyama et al. 1991). In addition, residues of collagen X were found within cartilage inclusions of bone trabeculae (Schmid and Linsenmayer 1985a, b). Rather extensive studies have been performed using animal models, in particular in developing chicken cartilage. Far less information is available on the distribution of collagen X in mammalian and in particular in human cartilaginous tissues. A comparison of the known data from the experimental systems with the human zone of cartilage-bone transition is furthermore hampered by the different anatomical structures of endochondral ossification. Recently Kirsch and vonder Mark (1990, 1991 a) developed an antibody directed against bovine and human collagen X and demonstrated the specificity of this antibody by various techniques, including immunohistochemistry. In these studies, collagen X was localized in the lower hypertrophic zone of chondrocytes of the fetal epiphyseal growth plate and in the sternum (Kitsch and yon der Mark 1991a). In our present report we extend these studies and investigate the localization of collagen X in the developing human growth plate in relation to collagens I, II and III to obtain further insight into the various stages of chondrocyte differentiation along long bone development.
Materials and methods Tissue specimens
Tissue specimens were obtained at autopsy from i5 fetuses (12-40 weeks of gestation) and 8 children (12 days-16 years of age). These
276 results were compared to the findings in 3 young, previously healthy adults (18-21 years). Autopsy had been performed 6-12 h after death. In all cases the femoral head was removed, and sagitally divided into parallel sections providing axial orientation of cartilage and bone. The specimens were fixed in buffered formaldehyde (4-8%) for 24 h and then decalcified in 0.1 M EDTA, pH 7.2. Following embedding into paraffin tissue sections of 2 Ixm in thickness were prepared. In 4 cases unfixed tissue sections were decalcified as indicated above (0.1 M EDTA, pH 7.2, 4° C) for the preparation of frozen sections.
Antibodies Rabbit antibodies specific for human collagen X were prepared as previously described (Kirsch and von der Mark 1991a). Briefly, purified collagen X coupled to haemocyanin was subcutaneously injected into rabbits, and the resulting antiserum was purified by cross-absorption with human collagen I, II, VI and XI, bovine collagen IX and human fibronectin. The specificity of the antiserum was tested by enzyme-linked immunosorbant assay (ELISA) and using Western blot technique. Furthermore, the addition of the purified antigen (collagen X) to the antibody incubation was shown specifically to abolish the immunoreaction (Kirsch and von der Mark 1991a). Antibodies against the interstitial collagens I, II, III and V had been prepared as described (Timpl et al. 1977).
Immunohistochemistry Appropriate tissue sections were deparaffinized and subsequently enzymatically pretreated to enhance immunoreactivity. For this purpose, a mixture of 0.2% trypsin and 0.1% testicular hyaluronidase (both from Sigma Chemical Co., Deisenhofen, FRG) was applied for 30 rain. The unfixed tissue sections were pretreated only with hyaluronidase as described above. Following several washing steps (phosphate buffered saline) the sections were incubated with the primary antibody. The antigen-antibody complex was then visualized using secondary anti-rabbit antibodies coupled with avidin-biotin complexes (ABC; Hsu et al. 1981) or the alkaline phosphatase-antialkaline phosphatase complex (APAAP; Cordell et al. 1984) using commercial kits (ABC: Vector, Burlingame, Calif., USA; APAAP: Dako, Hamburg, FRG). As chromogen we used either diaminobenzidine (Dako, Hamburg, FRG) or fast red (Sigma). Endogenous alkaline phosphatase was inactivated with levamisole. The staining procedure for collagens I, II, III and V was performed as described recently (Nerlich et al. 1995). For double-labelling experiments we combined the ABC and the APAAP methods as reported (Nerlich and Schleicher 1991).
Results
A comparison of the results obtained for unfixed and fixed tissue samples revealed the very same staining pattern, although the staining intensity was less homogeneous in the unfixed samples. Control experiments using pre-immune I g G yielded negative results.
Fetal ossification zone The immunohistochemical analysis of fetal cartilage and bone tissue revealed in all cases a distinct localization of collagen X in the extracellular matrix of hypertrophic cartilage with an accentuated pericellular and a somewhat reduced and spotty distribution in the interterritor-
ial matrix (Figs. 1 D and 2). In addition, residual cartilage islands within bone trabeculae contained collagen X. The bone matrix remained unstained. A small rim of collagen X, however, was also noted at the periosteal ossification zone in close association with osteoblastic cells (not shown). In the normal resting cartilage no collagen X deposition was found. In cartilage of some, but not all, cases of mid-fetal stages (18th-28th week) a faint, but distinct, positive staining for collagen X was noted in a small zone adjacent to the fibrous perichondrium, resembling the " g e r m i n a l z o n e " of cartilage (Fig. 3 C). The cartilage cells here were, however, negatively stained. The cells of the cartilage proliferation zone showed a positive intracellular staining to varying degrees (Figs. 1 D and 2C, D). In addition, a few chondrocytes surrounding vascular canals of the resting cartilage showed positive intracellular staining for collagen X (Fig. 3A). These observations were similar in all specimens analysed between the 12th week of gestation and the 40th week. The staining intensity, however, seemed to increase with advancing gestational age. Staining of the fetal ossification zone with antibodies against the interstitial collagens I, II, III and V revealed a specific distribution of these collagen types. Collagen I was restricted to the bone matrix and the periosteum and perichondrium. Collagen II was exclusively found in the cartilage of all zones. Collagen I I I was present in the connective tissue of the vascular channels in the resting cartilage, in the primary osteoid and some lacunae of basal hypertrophic chondrocytes, and in the endosteum of bone trabeculae (Fig. 1 A C ) .
Juvenile cartilage The immunohistochemical finding in the juvenile ossification zone was similar to that of fetal stages. Accordingly, collagen X was mainly found in the zone of cartilage hypertrophy of the epiphyseal cartilage. With advancing age, however, the epiphysis contained increasing collagen X deposits in an irregular, often spotty pattern (Fig. 2E) deposited in the interterritorial matrix. This finding paralleled t h e light microscopic appearance of " d e g e n e r a t i o n " of the epiphysis. In early juvenile cartilage of the femoral head (5 months of age) within the resting cartilage and apparently in the surroundings of a larger vascular channel, we noted an extensive extracel-
Fig. 1 A-D. Immunohistochemical localization of various intersti-
tial collagens in the fetal osteo-chondral junction zone (36 weeks), A Collagen I is exclusively found in the primary spongy bone, but not in the cartilage region. B Collagen II is present in residual, proliferating and hypertrophic cartilage, but not in the osteoid matrix. Note the collagen II-positive remnants of cartilage within bone trabeculae. C Collagen III occurs as a thin endosteal layer at the surface of bone trabeculae, as well as around basal hypertrophic ehondrocytes. D Collagen X is found around hypertrophic chondrocytes and in the adjacent matrix. Note the slightly positive staining for this collagen type in some proliferating chondrocytes intracellularly and in cartilage residues within the bone. A-D x 400
277
278
279 lular deposition of collagen X. This area is presumably a centre of secondary ossification (Fig. 3 B). At that stage, the adjacent cartilage contained, as expected, no extracellular collagen X deposition (not shown). Between the age of a few months and the closure of the epiphyseal growth plate at about 18 years, focal and extensive extracellular collagen X deposition is found in the epiphyseal growth plate (Figs. 2E and 3D, E), while the respective articular cartilage zone contains no significant collagen X deposition. This observation coincided with the light microscopic appearance of strongly reduced chondrocyte proliferation and hypertrophy in the articular cartilage in comparison to the proliferating and hypertrophic growth plate. Adult articular cartilage (18 years of age and above) contained no intra- or extracellular collagen X staining. The interstitial collagens I, II, III and V showed a tissue distribution similar to that found in the fetal tissue. In addition, with advancing degeneration of the epiphyseal cartilage, spotty deposits of collagens I, III and V were noted within the cartilage matrix. Using doublelabelling procedures we furthermore observed the simultaneous deposition of collagens I and X around some of the irregularly hypertrophic chondrocytes in older epiphyseal cartilages (Fig. 3 D and F).
Discussion In our present report we describe the tissue distribution in situ of collagen X during human fetal and juvenile endochrondral ossification of the femoral head. Our results provide evidence that this collagen type is mainly found in the hypertrophic cartilage zone. These findings are in agreement with previous observations made by Schmid and Linsenmayer (1985b) and Gibson etal. (1986) in the avian system, although the morphology of avian and human endochondral ossification zone is somewhat different. In our report we also found a slight intracellular collagen X-positive reaction in some prehypertrophic proliferating chondrocytes. A similar phenomenon has already been described by Gibson et al. (1986) and Schmid and Linsenmayer (1987) in the animal system. In addition we found a slight, but distinctive, positive staining for collagen X at the periosteal ossification layer
Fig. 2A-D. Immunolocalizationof collagenX in fetal and juvenile cartilage of various ages. A Early fetal cartilage (13 weeks) shows strongly positive reaction in the hypertrophic cartilage zone. B At the same age, hypertrophic chondrocytes are present at the transition to the perichondrium.Note the diffuselypositive staining for collagen X in the cartilage matrix. C Osteo-chondraljunction at the fetal age of 38 weeks with typical collagen X localization in hypertrophiccartilage. Note the positivityof some, but not all, prehypertrophic chondrocytes. D Typical collagen X localization in a chondro-osseousjunction at the age of 7 months. Note the staining of the cartilage core in the bone trabeculae. E Growth plate from a 3-year-oldinfant showingthe focal, intensivecollagen X stainingin the "degeneratively"transformedcartilage. A x 250, B x400, C-E x 100
(Schmid and Linsenmayer 1985, 1987). This finding indicates that collagen X may also be synthesized by certain mesenchymal cells at their transition to osteoblasts. Furthermore, a similar slight staining reaction for collagen X was observed at the border of the perichondrium and resting cartilage. Since the appropriate controls were negative, we feel that an unspecific staining pattern is excluded. Therefore, it is conceivable that collagen X may also temporarily be expressed at the early transition of mesenchymal stem cells differentiating to resting hyaline chondrocytes. Since we found such a transitional staining for collagen X only in those cases between the 18th and the 28th week of gestation, this observation needs to be substantiated by further analysis. The previously established distribution pattern of collagen X attributed this collagen type exclusively to the hypertrophic cartilage. From the present studies in proliferating chondrocytes the intracellular staining may represent a "precursor" stage before this collagen type is secreted and deposited in the matrix. In the light of our findings in the developing human articular system, one may speculate that this collagen type is not only associated with chondrocyte proliferation and hypertrophy, but also with the initial proliferation and differentiation of mesenchymal stem cells to resting chondrocytes. Recent in-situ hybridization experiments using a human DNA fragment encoding most of the collagen X C-terminal globular domain (vonder Mark et al. 1992), however, demonstrated no significant positive signal in the fetal zone of proliferating and resting cartilage; nevertheless a positive reaction was shown for a collagen II-specific RNA probe. These conflicting data, which argue against collagen X production in proliferating chondrocytes might be due to a lower detection level of the in-situ hybridization technique used. In our study we found an increase in the staining intensity from early fetal to postnatal hypertrophic cartilage. This apparent increase in collagen X deposition may be related to the morphologically evident increase and "maturation" of ossification. Recently, it has been proposed that collagen X acts as a calcium-binding protein (Kitsch and yon der Mark 1991b), thus facilitating calcification of the hypertrophic cartilage matrix (Schmid et al. 1990). In the light of our present findings it is thus conceivable that the increasing collagen X deposition with ongoing gestational age may be a prerequisite for proper bone formation in human long bones. This assumption, however, needs further confirmation. Thus far there exists no biochemical quantitative estimation of the amount of collagen X in the human fetal ossification zone due to the low amounts present. An additional finding in this study was the observation that extensive collagen X deposits occurred in areas of presumably secondary ossification, thus confirming the assumption that collagen X deposition precedes ossification. In juvenile epiphyseal cartilage we observed a similar spotty deposition of collagen X within the matrix, which even increased with ongoing morphological degeneration of the epiphyseal cartilage. In addition, we demonstrate that this degeneration (which finally leads to the bony closure of the epiphyseal growth plate) is
280
281
Fig. 3. A Intracellular positive staining for collagen X in chondrocytes surrounding a vascular channel. The matrix itself is devoid of collagen X (38 weeks). B Intensive matrix staining around an obliquely sectioned vascular channel for collagen X at the age of 5 months, in a zone of presumable formation of a secondary ossification centre. C Perichondrial staining for collagen X (blue) at the chondro-fibrous transition zone of a fetus aged 18 weeks, demonstrating intense collagen X staining. D, E Transverse section through a juvenile growth plate (6 years) for the localization of both collagen I (brown) and collagen X (blue) in double-labelling experiments. Collagen I is mostly restricted to the osseous matrix and also occurs around isolated hypertrophic chondrocytes (E), which are pericellularly positive for collagen X. A, B x 250, C ×600, D x60, E x600
also p a r a l l e l e d b y a n i n c r e a s i n g m a t r i x d e p o s i t i o n o f collagens I a n d III, which u s u a l l y are n o t f o u n d w i t h i n the c a r t i l a g i n o u s m a t r i x . D o u b l e - l a b e l l i n g studies indicate c o - l o c a l i z a t i o n o f collagens I a n d X, thus i n d i c a t i n g the p o s s i b i l i t y t h a t a t r a n s i t i o n o f " h y p e r t r o p h i c " c h o n d r o c y t e s to " d e d i f f e r e n t i a t e d " c h o n d r o c y t e s m a y o c c u r in vivo u n d e r c e r t a i n c i r c u m s t a n c e s . T h i s o b s e r v a t i o n m a y p l a y s o m e crucial role in the u n d e r s t a n d i n g o f p a t h o logical processes o f c a r t i l a g e d e g e n e r a t i o n , especially in osteoarthrosis.
Acknowledgement. The present study was supported by a grant from the Bundesministerium fiir Forschung und Technologie (VM 8619/2)
References Capasso O, Gionti E, Pontarelli G, Ambesi-Impiobato FS, Tajana G, Cancedda R (1982) The culture of chick embryo chondrocytes and the control of their differentiated functions in vitro. Exp Cell Res 142:197-206 Cordell JL, Falini B, Erber WN, Ghosh AK, Abdulaziz Z, MacDonald S, Pulford AF, Stein H, Mason DY (1984) Immunoenzymatic labeling of monoclonal antibodies using immune complexes of alkaline phosphatase and monoclonal anti-alkaline phosphatase. J Histochem Cytochem 32:219-225 Gibson GJ, Schor SL, Grant ME (1982) The identification of a new cartilage collagen synthesized by chondrocytes cultured within collagen gels. Connect Tissue Res 9:207-208 Gibson G J, Bearman CH, Flint MH (l 986) The immunoperoxidase localization of type X collagen in chick cartilage and lung. Coll Relat Res 6:163-184 Hsu SM, Raine L, Fanger H (1981) A comparative study of the peroxidase-antiperoxidase method and an avidin-biotin complex method for studying polypeptide hormones with radio immunoassay antibodies. Am J Clin Pathol 75 : J 34-139 Iyama KI, Ninomiya Y, Olsen BR, Linsenmayer TF, Trelstad RL,
Hayashi M (1991) Spatiotemporal pattern of type X collagen gene expression and collagen deposition in embryonic chick vertebrae undergoing endochondral ossification. Anat Rec 229: 462-472 Kirsch T, Mark K yon der (1990) Isolation of bovine type X collagen and immunolocalization in growth-plate cartilage. Biochem J 265:453-459 Kirsch T, Mark K v o n d e r (1991 a) Isolation of human type X collagen and immunolocalization in fetal human cartilage. Eur J Biochem 196: 575-580 Kirsch T, Mark K yon der (1991 b) Ca-binding properties of type X collagen. FEBS Lett 294:149-152 Linsenmayer TF, Gibney E, Schmid TM (1986) Segmental appearance of type X collagen in the developing avian notochord. Dev Biol 113:467-473 Mark K yon der (I 986) Differentiation, modulation and dedifferentiation of chondrocytes. In : Kiihn K, Krieg T (eds) Connective tissue: biological and clinical aspects. Karger, Basel, pp 272 315 Mark K vonder, Mark H yon der, Gay S (1976) Study of differential collagen synthesis during development of the chick embryo by immunofluorescence. Dev Biol 48:237 249 Mark K yon der, Kirsch T, Aigner T, Reichenberger E, Nerlich A, Weseloh G, St613 H (1992) The fate of chondrocytes in osteoarthritic cartilage: regeneration, dedifferentiation or hypertrophy? In: Kuettner KE, Schleyerbach R, Peyron JG, Hascall VC (eds) Articular cartilage and osteoarthritis. Raven Press, New York, pp 221-234 Nerlich A, Schleicher E (1991) Immunohistochemical localization of extracellular matrix components in human diabetic glomerular Iesions. Am J Pathol 139:889 899 Nerlich A, Wiest I, Kantimm S, Brenner R, Mark K vonder (1991) Die immunhistochemische Analyse der normalen und pathologischen Knochen- und Knorpelmatrix als Methode in der Osteologie. In: Werner E, Matthias HH (eds) Osteologie - interdisziplin/ir. Springer, Heidelberg Berlin New York, pp 41-45 Ninomiya Y, Gordon M, Rest M van der, Schmid TM, Linsenmayer TF, Olsen BR (1986) The developmentally regulated type X colIagen gene contains a long open reading frame without introns. J Biol Chem 261:5041-5050 Schmid TM, Conrad HE (1982) A unique low molecular weight collagen secreted by cultured chick embryo chondrocytes. J Biol Chem 257:12444-12450 Schmid TM, Linsenmayer TF (1985 a) Immunohistochemical localization of short chain cartilage collagen (type X) in avian tissues. J CelI Biol 100:598-605 Schmid TM, Linsenmayer TF (1985b) Developmental acquisition of type X collagen in the embryonic chick tibiotarsus. Dev Biol 107: 373-38l Schmid TM, Linsenmayer TF (1987) Type X collagen. In: Mayne R, Burgeson RE (eds) Structure and function of collagen types. Academic Press, New York London, pp 223-259 Schmid TM, Popp RG, Linsenmayer TF (1990) Hypertrophic cartilage matrix. Ann New York Acad Sci 580 : 64-73 Summers TA, Irwin MH, Mayne R, Balian G (1988) Monoclonal antibodies to type X collagen. J Biol Chem 263:581-587 Timpl R, Wick G, Gay S (1977) Antibodies to distinct types of collagens and procollagens and their application in immunohistology. J Immunol Methods 18:165-175
Histochemistry © Springer-Verlag 1992
Localization of collagen X in human fetal and juvenile articular cartilage and bone A.G. Nerlich 1, T. Kirsch 2, I. Wiest a, p. Betz 3, K. von der Mark 2
t Pathologisches Institut der UniversitS.t Mfinchen, Thalkirchnerstrasse 36, W-8000 M/inchen 2, Federal Republic of Germany 2 Max-Planck Arbeitsgruppe ffir klinische Rheumatologie an der Universitfit Erlangen-Nfirnberg, Schwabachanlage 10, W-8520 Erlangen, Federal Republic of Germany 3 Institut ftir Rechtsmedizin der Universit/it Miinchen, Frauenlobstrasse 6, W-8000 Mfinchen 2, Federal Republic of Germany Accepted: 2 September 1992 Abstract. The tissue localization was analysed of collagen X during human fetal and juvenile articular cartilagebone metamorphosis. This unique collagen type was found in the hypertrophic cartilage zone peri- and extracellularly and in cartilage residues within bone trabeculae. In addition, occasionally a slight intracellular staining reaction was found in prehypertrophic proliferating chondrocytes and in chondrocytes surrounding vascular channels. A slight staining was also seen in the zone of periosteal ossification and occasionally at the transition zone of the perichondrium to resting cartilage. Our data provide evidence that the appearance of collagen X is mainly associated with cartilage hypertrophy, analogous to the reported tissue distribution of this collagen type in animals. In addition, we observed an increased and often "spotty" distribution of collagen X with increasing cartilage "degeneration" associated with the closure of the growth plate. In basal hypertrophic cartilage areas, a co-distribution of collagens II and X was found with very little and "spotty" collagen III. In juvenile cartilage areas around single hypertrophic chondrocytes, co-localization of collagens X and I was also detected.
Introduction
During the development of long bones, chondrocytes differentiate along a series of morphologically different stages, which show a unique pattern of synthesized collagen types (for review see von der Mark 1986). While the undifferentiated precursor cells of limb cartilage produce collagen I, the pattern of synthesis is changing during chondrogenesis to collagen II synthesis by differentiated chondrocytes (vonder Mark et al. 1976), and further to collagen X synthesis (Schmid and Linsenmayer 1987). The discovery of collagen X synthesis by hypertrophic chondrocytes in vitro (Gibson et al. 1982; Capasso et al. 1982) and in the zone of hypertrophic cardCorrespondence to." A. Nerlich
lage in the growth plate in vivo (Schmid and Conrad 1982) provided a specific experimental tool for unequivocal identification of hypertrophic chondrocytes in situ and in vitro. Using monoclonal antibodies (Schmid and Linsenmayer 1985 a, b; Summers et al. 1988) and cDNA probes (Ninomiya et al. 1986) chick collagen X was specifically located in hypertrophic cartilage of growth plates, in the calcifying cartilage of sternal and rib cartilage, and in the developing notochord (Linsenmayer et al. 1986; Iyama et al. 1991). In addition, residues of collagen X were found within cartilage inclusions of bone trabeculae (Schmid and Linsenmayer 1985a, b). Rather extensive studies have been performed using animal models, in particular in developing chicken cartilage. Far less information is available on the distribution of collagen X in mammalian and in particular in human cartilaginous tissues. A comparison of the known data from the experimental systems with the human zone of cartilage-bone transition is furthermore hampered by the different anatomical structures of endochondral ossification. Recently Kirsch and vonder Mark (1990, 1991 a) developed an antibody directed against bovine and human collagen X and demonstrated the specificity of this antibody by various techniques, including immunohistochemistry. In these studies, collagen X was localized in the lower hypertrophic zone of chondrocytes of the fetal epiphyseal growth plate and in the sternum (Kitsch and yon der Mark 1991a). In our present report we extend these studies and investigate the localization of collagen X in the developing human growth plate in relation to collagens I, II and III to obtain further insight into the various stages of chondrocyte differentiation along long bone development.
Materials and methods Tissue specimens
Tissue specimens were obtained at autopsy from i5 fetuses (12-40 weeks of gestation) and 8 children (12 days-16 years of age). These
276 results were compared to the findings in 3 young, previously healthy adults (18-21 years). Autopsy had been performed 6-12 h after death. In all cases the femoral head was removed, and sagitally divided into parallel sections providing axial orientation of cartilage and bone. The specimens were fixed in buffered formaldehyde (4-8%) for 24 h and then decalcified in 0.1 M EDTA, pH 7.2. Following embedding into paraffin tissue sections of 2 Ixm in thickness were prepared. In 4 cases unfixed tissue sections were decalcified as indicated above (0.1 M EDTA, pH 7.2, 4° C) for the preparation of frozen sections.
Antibodies Rabbit antibodies specific for human collagen X were prepared as previously described (Kirsch and von der Mark 1991a). Briefly, purified collagen X coupled to haemocyanin was subcutaneously injected into rabbits, and the resulting antiserum was purified by cross-absorption with human collagen I, II, VI and XI, bovine collagen IX and human fibronectin. The specificity of the antiserum was tested by enzyme-linked immunosorbant assay (ELISA) and using Western blot technique. Furthermore, the addition of the purified antigen (collagen X) to the antibody incubation was shown specifically to abolish the immunoreaction (Kirsch and von der Mark 1991a). Antibodies against the interstitial collagens I, II, III and V had been prepared as described (Timpl et al. 1977).
Immunohistochemistry Appropriate tissue sections were deparaffinized and subsequently enzymatically pretreated to enhance immunoreactivity. For this purpose, a mixture of 0.2% trypsin and 0.1% testicular hyaluronidase (both from Sigma Chemical Co., Deisenhofen, FRG) was applied for 30 rain. The unfixed tissue sections were pretreated only with hyaluronidase as described above. Following several washing steps (phosphate buffered saline) the sections were incubated with the primary antibody. The antigen-antibody complex was then visualized using secondary anti-rabbit antibodies coupled with avidin-biotin complexes (ABC; Hsu et al. 1981) or the alkaline phosphatase-antialkaline phosphatase complex (APAAP; Cordell et al. 1984) using commercial kits (ABC: Vector, Burlingame, Calif., USA; APAAP: Dako, Hamburg, FRG). As chromogen we used either diaminobenzidine (Dako, Hamburg, FRG) or fast red (Sigma). Endogenous alkaline phosphatase was inactivated with levamisole. The staining procedure for collagens I, II, III and V was performed as described recently (Nerlich et al. 1995). For double-labelling experiments we combined the ABC and the APAAP methods as reported (Nerlich and Schleicher 1991).
Results
A comparison of the results obtained for unfixed and fixed tissue samples revealed the very same staining pattern, although the staining intensity was less homogeneous in the unfixed samples. Control experiments using pre-immune I g G yielded negative results.
Fetal ossification zone The immunohistochemical analysis of fetal cartilage and bone tissue revealed in all cases a distinct localization of collagen X in the extracellular matrix of hypertrophic cartilage with an accentuated pericellular and a somewhat reduced and spotty distribution in the interterritor-
ial matrix (Figs. 1 D and 2). In addition, residual cartilage islands within bone trabeculae contained collagen X. The bone matrix remained unstained. A small rim of collagen X, however, was also noted at the periosteal ossification zone in close association with osteoblastic cells (not shown). In the normal resting cartilage no collagen X deposition was found. In cartilage of some, but not all, cases of mid-fetal stages (18th-28th week) a faint, but distinct, positive staining for collagen X was noted in a small zone adjacent to the fibrous perichondrium, resembling the " g e r m i n a l z o n e " of cartilage (Fig. 3 C). The cartilage cells here were, however, negatively stained. The cells of the cartilage proliferation zone showed a positive intracellular staining to varying degrees (Figs. 1 D and 2C, D). In addition, a few chondrocytes surrounding vascular canals of the resting cartilage showed positive intracellular staining for collagen X (Fig. 3A). These observations were similar in all specimens analysed between the 12th week of gestation and the 40th week. The staining intensity, however, seemed to increase with advancing gestational age. Staining of the fetal ossification zone with antibodies against the interstitial collagens I, II, III and V revealed a specific distribution of these collagen types. Collagen I was restricted to the bone matrix and the periosteum and perichondrium. Collagen II was exclusively found in the cartilage of all zones. Collagen I I I was present in the connective tissue of the vascular channels in the resting cartilage, in the primary osteoid and some lacunae of basal hypertrophic chondrocytes, and in the endosteum of bone trabeculae (Fig. 1 A C ) .
Juvenile cartilage The immunohistochemical finding in the juvenile ossification zone was similar to that of fetal stages. Accordingly, collagen X was mainly found in the zone of cartilage hypertrophy of the epiphyseal cartilage. With advancing age, however, the epiphysis contained increasing collagen X deposits in an irregular, often spotty pattern (Fig. 2E) deposited in the interterritorial matrix. This finding paralleled t h e light microscopic appearance of " d e g e n e r a t i o n " of the epiphysis. In early juvenile cartilage of the femoral head (5 months of age) within the resting cartilage and apparently in the surroundings of a larger vascular channel, we noted an extensive extracel-
Fig. 1 A-D. Immunohistochemical localization of various intersti-
tial collagens in the fetal osteo-chondral junction zone (36 weeks), A Collagen I is exclusively found in the primary spongy bone, but not in the cartilage region. B Collagen II is present in residual, proliferating and hypertrophic cartilage, but not in the osteoid matrix. Note the collagen II-positive remnants of cartilage within bone trabeculae. C Collagen III occurs as a thin endosteal layer at the surface of bone trabeculae, as well as around basal hypertrophic ehondrocytes. D Collagen X is found around hypertrophic chondrocytes and in the adjacent matrix. Note the slightly positive staining for this collagen type in some proliferating chondrocytes intracellularly and in cartilage residues within the bone. A-D x 400
277
278
279 lular deposition of collagen X. This area is presumably a centre of secondary ossification (Fig. 3 B). At that stage, the adjacent cartilage contained, as expected, no extracellular collagen X deposition (not shown). Between the age of a few months and the closure of the epiphyseal growth plate at about 18 years, focal and extensive extracellular collagen X deposition is found in the epiphyseal growth plate (Figs. 2E and 3D, E), while the respective articular cartilage zone contains no significant collagen X deposition. This observation coincided with the light microscopic appearance of strongly reduced chondrocyte proliferation and hypertrophy in the articular cartilage in comparison to the proliferating and hypertrophic growth plate. Adult articular cartilage (18 years of age and above) contained no intra- or extracellular collagen X staining. The interstitial collagens I, II, III and V showed a tissue distribution similar to that found in the fetal tissue. In addition, with advancing degeneration of the epiphyseal cartilage, spotty deposits of collagens I, III and V were noted within the cartilage matrix. Using doublelabelling procedures we furthermore observed the simultaneous deposition of collagens I and X around some of the irregularly hypertrophic chondrocytes in older epiphyseal cartilages (Fig. 3 D and F).
Discussion In our present report we describe the tissue distribution in situ of collagen X during human fetal and juvenile endochrondral ossification of the femoral head. Our results provide evidence that this collagen type is mainly found in the hypertrophic cartilage zone. These findings are in agreement with previous observations made by Schmid and Linsenmayer (1985b) and Gibson etal. (1986) in the avian system, although the morphology of avian and human endochondral ossification zone is somewhat different. In our report we also found a slight intracellular collagen X-positive reaction in some prehypertrophic proliferating chondrocytes. A similar phenomenon has already been described by Gibson et al. (1986) and Schmid and Linsenmayer (1987) in the animal system. In addition we found a slight, but distinctive, positive staining for collagen X at the periosteal ossification layer
Fig. 2A-D. Immunolocalizationof collagenX in fetal and juvenile cartilage of various ages. A Early fetal cartilage (13 weeks) shows strongly positive reaction in the hypertrophic cartilage zone. B At the same age, hypertrophic chondrocytes are present at the transition to the perichondrium.Note the diffuselypositive staining for collagen X in the cartilage matrix. C Osteo-chondraljunction at the fetal age of 38 weeks with typical collagen X localization in hypertrophiccartilage. Note the positivityof some, but not all, prehypertrophic chondrocytes. D Typical collagen X localization in a chondro-osseousjunction at the age of 7 months. Note the staining of the cartilage core in the bone trabeculae. E Growth plate from a 3-year-oldinfant showingthe focal, intensivecollagen X stainingin the "degeneratively"transformedcartilage. A x 250, B x400, C-E x 100
(Schmid and Linsenmayer 1985, 1987). This finding indicates that collagen X may also be synthesized by certain mesenchymal cells at their transition to osteoblasts. Furthermore, a similar slight staining reaction for collagen X was observed at the border of the perichondrium and resting cartilage. Since the appropriate controls were negative, we feel that an unspecific staining pattern is excluded. Therefore, it is conceivable that collagen X may also temporarily be expressed at the early transition of mesenchymal stem cells differentiating to resting hyaline chondrocytes. Since we found such a transitional staining for collagen X only in those cases between the 18th and the 28th week of gestation, this observation needs to be substantiated by further analysis. The previously established distribution pattern of collagen X attributed this collagen type exclusively to the hypertrophic cartilage. From the present studies in proliferating chondrocytes the intracellular staining may represent a "precursor" stage before this collagen type is secreted and deposited in the matrix. In the light of our findings in the developing human articular system, one may speculate that this collagen type is not only associated with chondrocyte proliferation and hypertrophy, but also with the initial proliferation and differentiation of mesenchymal stem cells to resting chondrocytes. Recent in-situ hybridization experiments using a human DNA fragment encoding most of the collagen X C-terminal globular domain (vonder Mark et al. 1992), however, demonstrated no significant positive signal in the fetal zone of proliferating and resting cartilage; nevertheless a positive reaction was shown for a collagen II-specific RNA probe. These conflicting data, which argue against collagen X production in proliferating chondrocytes might be due to a lower detection level of the in-situ hybridization technique used. In our study we found an increase in the staining intensity from early fetal to postnatal hypertrophic cartilage. This apparent increase in collagen X deposition may be related to the morphologically evident increase and "maturation" of ossification. Recently, it has been proposed that collagen X acts as a calcium-binding protein (Kitsch and yon der Mark 1991b), thus facilitating calcification of the hypertrophic cartilage matrix (Schmid et al. 1990). In the light of our present findings it is thus conceivable that the increasing collagen X deposition with ongoing gestational age may be a prerequisite for proper bone formation in human long bones. This assumption, however, needs further confirmation. Thus far there exists no biochemical quantitative estimation of the amount of collagen X in the human fetal ossification zone due to the low amounts present. An additional finding in this study was the observation that extensive collagen X deposits occurred in areas of presumably secondary ossification, thus confirming the assumption that collagen X deposition precedes ossification. In juvenile epiphyseal cartilage we observed a similar spotty deposition of collagen X within the matrix, which even increased with ongoing morphological degeneration of the epiphyseal cartilage. In addition, we demonstrate that this degeneration (which finally leads to the bony closure of the epiphyseal growth plate) is
280
281
Fig. 3. A Intracellular positive staining for collagen X in chondrocytes surrounding a vascular channel. The matrix itself is devoid of collagen X (38 weeks). B Intensive matrix staining around an obliquely sectioned vascular channel for collagen X at the age of 5 months, in a zone of presumable formation of a secondary ossification centre. C Perichondrial staining for collagen X (blue) at the chondro-fibrous transition zone of a fetus aged 18 weeks, demonstrating intense collagen X staining. D, E Transverse section through a juvenile growth plate (6 years) for the localization of both collagen I (brown) and collagen X (blue) in double-labelling experiments. Collagen I is mostly restricted to the osseous matrix and also occurs around isolated hypertrophic chondrocytes (E), which are pericellularly positive for collagen X. A, B x 250, C ×600, D x60, E x600
also p a r a l l e l e d b y a n i n c r e a s i n g m a t r i x d e p o s i t i o n o f collagens I a n d III, which u s u a l l y are n o t f o u n d w i t h i n the c a r t i l a g i n o u s m a t r i x . D o u b l e - l a b e l l i n g studies indicate c o - l o c a l i z a t i o n o f collagens I a n d X, thus i n d i c a t i n g the p o s s i b i l i t y t h a t a t r a n s i t i o n o f " h y p e r t r o p h i c " c h o n d r o c y t e s to " d e d i f f e r e n t i a t e d " c h o n d r o c y t e s m a y o c c u r in vivo u n d e r c e r t a i n c i r c u m s t a n c e s . T h i s o b s e r v a t i o n m a y p l a y s o m e crucial role in the u n d e r s t a n d i n g o f p a t h o logical processes o f c a r t i l a g e d e g e n e r a t i o n , especially in osteoarthrosis.
Acknowledgement. The present study was supported by a grant from the Bundesministerium fiir Forschung und Technologie (VM 8619/2)
References Capasso O, Gionti E, Pontarelli G, Ambesi-Impiobato FS, Tajana G, Cancedda R (1982) The culture of chick embryo chondrocytes and the control of their differentiated functions in vitro. Exp Cell Res 142:197-206 Cordell JL, Falini B, Erber WN, Ghosh AK, Abdulaziz Z, MacDonald S, Pulford AF, Stein H, Mason DY (1984) Immunoenzymatic labeling of monoclonal antibodies using immune complexes of alkaline phosphatase and monoclonal anti-alkaline phosphatase. J Histochem Cytochem 32:219-225 Gibson GJ, Schor SL, Grant ME (1982) The identification of a new cartilage collagen synthesized by chondrocytes cultured within collagen gels. Connect Tissue Res 9:207-208 Gibson G J, Bearman CH, Flint MH (l 986) The immunoperoxidase localization of type X collagen in chick cartilage and lung. Coll Relat Res 6:163-184 Hsu SM, Raine L, Fanger H (1981) A comparative study of the peroxidase-antiperoxidase method and an avidin-biotin complex method for studying polypeptide hormones with radio immunoassay antibodies. Am J Clin Pathol 75 : J 34-139 Iyama KI, Ninomiya Y, Olsen BR, Linsenmayer TF, Trelstad RL,
Hayashi M (1991) Spatiotemporal pattern of type X collagen gene expression and collagen deposition in embryonic chick vertebrae undergoing endochondral ossification. Anat Rec 229: 462-472 Kirsch T, Mark K yon der (1990) Isolation of bovine type X collagen and immunolocalization in growth-plate cartilage. Biochem J 265:453-459 Kirsch T, Mark K v o n d e r (1991 a) Isolation of human type X collagen and immunolocalization in fetal human cartilage. Eur J Biochem 196: 575-580 Kirsch T, Mark K yon der (1991 b) Ca-binding properties of type X collagen. FEBS Lett 294:149-152 Linsenmayer TF, Gibney E, Schmid TM (1986) Segmental appearance of type X collagen in the developing avian notochord. Dev Biol 113:467-473 Mark K yon der (I 986) Differentiation, modulation and dedifferentiation of chondrocytes. In : Kiihn K, Krieg T (eds) Connective tissue: biological and clinical aspects. Karger, Basel, pp 272 315 Mark K vonder, Mark H yon der, Gay S (1976) Study of differential collagen synthesis during development of the chick embryo by immunofluorescence. Dev Biol 48:237 249 Mark K yon der, Kirsch T, Aigner T, Reichenberger E, Nerlich A, Weseloh G, St613 H (1992) The fate of chondrocytes in osteoarthritic cartilage: regeneration, dedifferentiation or hypertrophy? In: Kuettner KE, Schleyerbach R, Peyron JG, Hascall VC (eds) Articular cartilage and osteoarthritis. Raven Press, New York, pp 221-234 Nerlich A, Schleicher E (1991) Immunohistochemical localization of extracellular matrix components in human diabetic glomerular Iesions. Am J Pathol 139:889 899 Nerlich A, Wiest I, Kantimm S, Brenner R, Mark K vonder (1991) Die immunhistochemische Analyse der normalen und pathologischen Knochen- und Knorpelmatrix als Methode in der Osteologie. In: Werner E, Matthias HH (eds) Osteologie - interdisziplin/ir. Springer, Heidelberg Berlin New York, pp 41-45 Ninomiya Y, Gordon M, Rest M van der, Schmid TM, Linsenmayer TF, Olsen BR (1986) The developmentally regulated type X colIagen gene contains a long open reading frame without introns. J Biol Chem 261:5041-5050 Schmid TM, Conrad HE (1982) A unique low molecular weight collagen secreted by cultured chick embryo chondrocytes. J Biol Chem 257:12444-12450 Schmid TM, Linsenmayer TF (1985 a) Immunohistochemical localization of short chain cartilage collagen (type X) in avian tissues. J CelI Biol 100:598-605 Schmid TM, Linsenmayer TF (1985b) Developmental acquisition of type X collagen in the embryonic chick tibiotarsus. Dev Biol 107: 373-38l Schmid TM, Linsenmayer TF (1987) Type X collagen. In: Mayne R, Burgeson RE (eds) Structure and function of collagen types. Academic Press, New York London, pp 223-259 Schmid TM, Popp RG, Linsenmayer TF (1990) Hypertrophic cartilage matrix. Ann New York Acad Sci 580 : 64-73 Summers TA, Irwin MH, Mayne R, Balian G (1988) Monoclonal antibodies to type X collagen. J Biol Chem 263:581-587 Timpl R, Wick G, Gay S (1977) Antibodies to distinct types of collagens and procollagens and their application in immunohistology. J Immunol Methods 18:165-175
