Growth cartilage calcification and formation of bone trabeculae are late and dissociated events in the endochondral ossification of Rana catesbeiana

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1 Cell Tissue Res (2001) 306: DOI /s REGULAR ARTICLE Sérgio Luis Felisbino Hernandes F. Carvalho Growth cartilage calcification and formation of bone trabeculae are late and dissociated events in the endochondral ossification of Rana catesbeiana Received: 15 November 2000 / Accepted: 9 July 2001 / Published online: 13 September 2001 Springer-Verlag 2001 Abstract Endochondral ossification in the growth cartilage of long bones from the bullfrog Rana catesbeiana was examined. In stage-46 tadpoles and 1-year-old animals, the hypertrophic cartilage had a smooth contact with the bone marrow and the matrix showed no calcification or endochondral bone formation. In spite of showing no aspects of calcification, the chondrocytes exhibited alkaline phosphatase activity and some of them died by apoptosis. However, matrix calcification and endochondral ossification were observed in 2-year-old bullfrogs. Calcium deposits appeared as isolated or coalesced spherical structures in the extracellular matrix of hypertrophic cartilage. Bone trabeculae were restricted to the central area at the sites where the hypertrophic cartilage surface was exposed to the bone marrow. Cartilage matrix calcification and the formation of bone trabeculae were not dependent on each other. Osteoclasts were involved in calcified matrix resorption. These results demonstrate that the calcification of hypertrophic cartilage and the deposition of bone trabeculae are late events in R. catesbeiana and do not contribute to the development and growth of long bones in adults. These processes may play a role in reinforcing bony structures as the bullfrog gains weight in adulthood. In addition, the deposition of bone trabeculae is not dependent on cartilage matrix calcification. Keywords Hypertrophic chondrocyte Apoptosis Alkaline phosphatase Periosteal ossification Bone growth Osteoclast Bullfrog Rana catesbeiana (Anura) This work was supported by FAPESP S.L. Felisbino H.F. Carvalho ( ) Department of Cell Biology, Institute of Biology, State University of Campinas (UNICAMP), PO Box 6109, Campinas, SP, Brazil hern@unicamp.br Tel.: , Fax: Introduction The development and growth of bones via a cartilage model involves perichondral (or periosteal) and endochondral ossification. Periosteal ossification occurs outside and around cartilage and leads to the formation of the bone cortex. This process continues at the leading edge of the growing bone cortex, located at the Ranvier groove, and corresponds internally to the upper zone of hypertrophic cartilage (Gardner and Gray 1970; Shapiro et al. 1977; Osdoby and Caplan 1981). Endochondral ossification is a complex process by which the growth cartilage within the periosteal bone cortex is progressively replaced by bony tissue. Cellular events, such as cell proliferation and hypertrophy and matrix calcification, are coordinated events contributing to longitudinal expansion. Calcification is crucial for the recruitment of the osteoprogenitior cells stimulated by the invading blood vessels. Osteoblasts subsequently replace the calcified cartilage matrix with bone (Hunziker 1994; Cancedda et al. 1995; Erlenbacher et al. 1995; Bianco et al. 1998). In previous studies, we have described the structure of the epiphyseal cartilage of Rana catesbeiana (Felisbino and Carvalho 1999, 2000). This cartilage is plugged into the end of a tubular bone shaft and is divided into articular, lateral articular and growth cartilages. The growth cartilage is located within the bone tube. In the proliferative zone, there is a perpendicular separation of daughter cells and no columnar arrangement of the chondrocytes is observed. Neither hypertrophic matrix calcification nor endochondral ossification is seen in 1-year-old animals, an observation that suggests that long bone extension depends mostly on periosteal ossification. Similar findings have been reported for other anuran species (Dikson 1982; Dell Orbo et al. 1992). Based on these observations, we have investigated whether endochondral ossification and other events related to this process occur in bullfrog cartilage and have compared the results with current models for avian and mammalian growth plates. Our findings suggest that car-

2 320 tilage calcification and the formation of bone trabeculae in endochondral ossification are not essential for long bone development and growth in this anuran. Rather, these phenomena are late events that are probably involved in reinforcing the bone ends as the animals gain weight in adulthood. Materials and methods Animals Specimens of R. catesbeiana were obtained from a commercial farm in Atibaia (São Paulo State, Brazil) where they had been fed an artificial diet ad libitum. The bullfrogs were grouped into three age categories. The first group consisted of tadpoles at stage 46 of larval development (transformation completed) and were about 6 months old (Gosner 1960). The second group contained young 1-year-old animals (about 6 months post-transformation) and the third group consisted of 2-year-old animals (18 months post-transformation). The frogs were killed by decapitation after cold immobilization and the distal femoral (DF) epiphyseal cartilages were dissected out and immediately fixed by immersion as described below. Histochemistry Cartilage fragments were fixed in 4% formaldehyde in phosphate buffer, ph 7.4, containing 0.85% NaCl (PBS) for 24 h. Some samples were decalcified in 4% formaldehyde, 10% acetic acid and 0.85% NaCl solution for 15 days. The material was then dehydrated in a graded ethanol series, clarified in Cedar wood oil and embedded in Paraplast Plus embedding medium. Some fragments were embedded in JB-4 resin (Polysciences). Serial sagittal Paraplast (6 µm) or resin (2 µm) sections were stained with toluidine blue (TB) or picrosirius-hematoxylin (PSH; Junqueira et al. 1979) or were subjected to von Kossa s reaction for calcified matrix. Enzyme histochemistry Some tissue fragments were fixed in 4% formaldehyde in 0.1 M cacodylate buffer, ph 7.2, for 4 h, washed with the same buffer, treated with 70% ethanol and embedded in JB-4 resin. Alkaline phosphatase (AlkPase) activity was detected by using 0.1% α-naphthyl phosphate, 0.1% Fast red and 50 mm MgCl 2 in 0.1 M TRIS-malate buffer, ph 10. For the identification of osteoclasts, the same compounds were dissolved in 0.1 M acetate buffer, ph 5.0, plus 50 mm sodium tartrate for tartrate-resistant acid phosphatase (TRAP) activity (Minkin 1982; Cole and Wezeman 1987). Controls were prepared by using the same solutions without α-naphthyl phosphate. Sections were counter-stained with methyl green. DNA fragmentation DNA fragmentation was detected by labelling in situ with the terminal deoxynucleotidyl transferase (TUNEL) method (TdT-FragEl; Amershan Pharmacia Biotech). Paraplast sections were treated with proteinase K and 3'-end-labelled with fluorescein-conjugated dutp. An incubation step with a peroxidase-conjugated rabbit anti-fluorescein antibody was followed by the diamino benzidine reaction. A positive control was produced by DNase I digestion. Sections were counter-stained with methyl green. Fig. 1 Schematic drawing of the distal femoral (DF) epiphyseal cartilage of adult R. catesbeiana. The cartilage is inserted at the end of a tubular bone structure. The epiphyseal cartilage can be divided into: articular (1), lateral articular (2) and growth (3) cartilages (short arrows periosteal bone, bm bone marrow). Reprinted from Felisbino and Carvalho 1999, by permission of Churchill Livingstone Results The epiphyseal cartilage can be divided into articular, lateral articular and growth cartilages. The growth cartilage is inserted into the end of a tubular bone structure (Fig. 1). The hypertrophic zone of growth cartilage in stage-46 tadpoles and in 1-year-old bullfrogs is shown in Figs. 2 and 3, respectively. This cartilage showed no response to von Kossa s reaction for calcified matrix. The uncalcified matrix of hypertrophic cartilage was in direct contact with the bone marrow and showed a regular surface lined by mononucleated cells. No endochondral bone was observed in any of the animals of these two groups. Endochondral bone and matrix calcification were seen in the DF growth cartilage of 2-year-old frogs (Fig. 4). In these animals, the hypertrophic cartilage showed an irregular contact with the bone marrow, in contrast to the younger frogs. At the margins of the cartilage in contact with the bone marrow, bone trabeculae were identified by intense staining with PSH and the characteristic birefringence in polarizing microscopy (Fig. 4) and by the positive response to von Kossa s reaction. Von Kossa s reaction revealed bone trabeculae formation associated with uncalcified cartilage matrix in 2-year-old frogs (Fig. 5) and calcified hypertrophic cartilage not associated with the deposition of bone trabeculae (Fig. 6). These variable aspects were confirmed by inspection of serial sections of the full thickness of the cartilage and were unrelated to sex, size or any anatomical variation.

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4 322 The resorption of uncalcified cartilage matrix released calcified structures into the bone marrow where they were surrounded by multinucleated cells (Fig. 7). These cells were positive for TRAP activity and were associated with both bone trabeculae and calcified cartilage in 2-year-old animals (Fig. 8). Fig. 2 Stage-46 tadpole. PSH-stained section of decalcified DF growth cartilage. The hypertrophic cartilage (HC) shows a smooth contact with the bone marrow (M) and there is no endochondral ossification or matrix calcification. Note the absence of a columnar arrangement of the chondrocytes and the abundant extracellular matrix between these cells. 90 Fig. 3 One-year-old animal. PSH-stained section of decalcified DF growth cartilage. The hypertrophic cartilage (HC) shows a smooth contact with the bone marrow (M) and there is no endochondral ossification or matrix calcification. 90 Fig. 4 Two-year-old animal. PSH-stained section of decalcified DF growth cartilage. The hypertrophic cartilage (HC) shows an irregular border with the bone marrow (M). Strongly stained bone trabeculae (arrows) are seen associated with calcified (cc) and uncalcified (uc) cartilage. Calcified cartilage occurs in direct contact with bone marrow and not associated with bone trabeculae deposition (arrowheads). 45 Fig. 5 Two-year-old animal. Resin section of non-decalcified DF growth cartilage subjected to von Kossa s reaction. A positive von Kossa s reaction in the bone trabecula (arrow) along the border of hypertrophic cartilage (HC) with bone marrow. There is no calcification in the extracellular matrix of the hypertrophic cartilage. Methyl green counter-staining. 90 Fig. 6 Two-year-old animal. Resin section of non-decalcified DF growth cartilage subjected to von Kossa s reaction. This reaction reveals the presence of calcium deposits (black staining) in the extracellular matrix of hypertrophic cartilage (HC). Note that calcifed matrix (arrowheads) is released into bone marrow (M) but there are no associated bone trabeculae. Methyl green counterstaining. 90 Fig. 7 Two-year-old animal. TB-stained resin section of nondecalcified DF growth cartilage. Calcified cartilage matrix (arrow) released into the bone marrow is surrounded by multinucleated cells (arrowheads) with evident nucleoli. 360 Fig. 8 Two-year-old animal. Resin section of non-decalcified DF growth cartilage. Tartrate-resistant acid phosphatase activity was found in the cytoplasm (red staining) of a polynucleated osteoclast (oc) close to a bone trabecula (bt). Methyl green counter-staining. 900 Fig. 9 Stage-46 tadpole. Resin section of non-decalcified DF growth cartilage. Alkaline phosphatase activity is found in the plasma membrane (arrows) of chondrocytes from the lower proliferative zone (lpz). upz Upper proliferative zone. Methyl green counter-staining. 180 Fig. 10 Stage-46 tadpole. Resin section of non-decalcified DF growth cartilage. Alkaline phosphatase activity can be detected in the plasma membrane of hypertrophic chondrocytes (arrows). Methyl green counter-staining. 360 Fig. 11 Two-year-old animal. DNA fragmentation as revealed by TUNEL labelling. Positive reaction (arrow) is observed in chondrocytes to within three cell layers of the contact between the lower hypertrophic zone with the marrow. Methyl green counter-staining. 900 Fig. 12 Two-year-old animal. DNA fragmentation as revealed by TUNEL labelling. A DNase-digested section showing a positive reaction (arrows) in all chondrocyte nuclei. Methyl green counterstaining. 360 Table 1 Events associated with growth cartilage in bullfrogs of various ages (+ presence, absence) Event AlkPase activity was detected on the surface of chondrocytes from the lower proliferative zone (Fig. 9) to the lower hypertrophic zone (Fig. 10) in stage-46 tadpoles and in 1-year-old and 2-year-old animals (not shown). DNA fragmentation was observed in cells of the lower hypertrophic cartilage in animals from the various age groups (Fig. 11). These apoptotic chondrocytes occurred only a short distance (about three cell layers) from the transition to the marrow. All the cell nuclei in DNasedigested sections gave a positive reaction (Fig. 12) The proximal femoral and tibio-fibula growth cartilages were also examined and the results were similar to those described above for the distal femoral growth cartilage, except that bone trabeculae were rare in the proximal tibio-fibula. The results are summarized in Table 1. Discussion Age Stage-46 One year Two years tadpole old old Cell death AlkPase activity Matrix calcification + Endochondral ossification a + a Observed as the deposition of bone trabeculae on the surface of calcified or uncalcified cartilage Although the role of growth cartilage and endochondral ossification in long bone growth is well defined for avians and mammals (Hunziker and Schenk 1989; Breur et al. 1991, 1992; Barreto et al. 1993; Farnum and Wilsman 1993; Wilsman et al. 1996), the function of anuran growth cartilage is poorly understood, mainly because of its distinctive structural aspects compared with that of mammals and birds (Dikson 1982; Dell Orbo et al. 1992; Felisbino and Carvalho 1999). Endochondral ossification in R. catesbeiana was found to be a delayed process. Bone trabeculae originating from endochondral ossification were not observed until the animals were 2 years old. Thus, periosteal ossification in frogs up to 2 years of age was not followed by cartilage calcification and bone trabeculae deposition. Uncalcified cartilage was continuously resorbed and contributed to the formation of the marrow cavity. This finding is consistent with the idea that long bone development and growth up to adulthood depend mostly on periosteal ossification (Dikson 1982; Dell Orbo et al. 1992; Felisbino and Carvalho 1999). The formation of bone trabeculae is not dependent on cartilage calcification, since osteoblasts can deposit bone on the surface of uncalcified cartilage. Calcified cartilage is either resorbed by osteoclasts or used as a scaffold for the deposition of bone trabeculae. This control

5 of endochondral ossification in bullfrogs may represent an economic adaptation for bone growth. The resorption of calcified cartilage and bone by osteoclasts and the resorption of uncalcified cartilage by mononucleated cells are similar to those in birds and mammals (Schenk et al. 1967; Cole and Wezeman 1987). AlkPase activity is apparently constitutive in differentiating chondrocytes in bullfrog growth cartilage, with the reaction being detected from the proliferating zone to the hypertrophic zone. There is no spatial or temporal association with calcium deposition. Together with the observation that apoptotic chondrocytes are present in animals of the three studied age groups, this finding indicates that many events occurring during endochondral ossification are not necessarily related to each other. This conclusion agrees with an earlier proposition that the events occurring in growth cartilage (i.e. cell proliferation and hypertrophy) are not directly related to matrix calcification or to the formation of bone trabeculae but are certainly important for radial expansion as bones grow by periosteal ossification (Felisbino and Carvalho 1999). In conclusion, calcification of hypertrophic cartilage and the deposition of bone trabeculae are late events and do not play an essential role in the development and growth of long bones in R. catesbeiana. However, as the animals grow older and gain weight, reinforcement of the bone ends is necessary and requires the above events. This observation is consistent with the hypothesis that mechanical loading constitutes an important factor during skeletal morphogenesis (Carter and Wong 1988; van t Veen et al. 1995). 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