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DIVISION AND DEATH OF CELLS IN DEVELOPING SYNOVIAL JOINTS AND LONG BONES
Cell Biology International 2002, Vol. 26, No. 8, 679–688 doi:10.1006/cbir.2002.0918, available online at http://www.idealibrary.com on
DIVISION AND DEATH OF CELLS IN DEVELOPING SYNOVIAL JOINTS AND LONG BONES EMMA KAVANAGH†, MERSEDEH ABIRI, YVETTE S. BLAND and DOREEN E. ASHHURST* Department of Anatomy, St George’s Hospital Medical School, Tooting, London SW17 0RE, U.K. Received 26 March 2001; revised 4 April 2002; accepted 22 April 2002 Articular chondrocytes are a unique set of cells from the time the cellular condensations that become the anlagen of the long bones develop in the embryo. In the presumptive joint the cells of the opposing bones are packed very closely together, but at cavitation, the central, flattened cells move apart to form the articular surfaces. As the articular cartilage develops the cells are pushed further apart by the cartilaginous matrix. To determine the contributions of cell proliferation and death to cavitation and the subsequent development and growth of articular cartilage, direct observations were made to identify mitotic cells and those with apoptotic bodies in haematoxylin-stained sections of developing joints, and growing and ageing articular cartilage of the rabbit knee. These observations were extended using antibodies to the proliferating cell nuclear antigen (PCNA) and TdT-mediated dUTP nick end labelling (TUNEL) on corresponding sections. Low levels of cell division do occur in the articular cartilage up to 6 weeks postnatally, but matrix formation makes the major contribution to the increase in size of the cartilage. Cell death is not observed during cavitation, nor during the development of the articular cartilage proper. Apoptosis is essential, however, for the removal of the epiphyseal cartilage during ossification of the epiphyses and in the growth plate. 2002 Elsevier Science Ltd. All rights reserved.
K: rabbit knee joint; cell division; cell death/apoptosis; cavitation; articular cartilage; development. A: BSA, bovine serum albumin; PBS, phosphate buffered saline; PCD, programmed cell death; PCNA, proliferating cell nuclear antigen; TdT, terminal deoxytransferase; TUNEL, TdT-mediated dUPT nick end labelling.
INTRODUCTION The major event in the formation of a synovial joint is cavitation. The cartilaginous anlagen of the bones are separated by the interzone which has three layers; these are the chondrogenous layers and the intermediate layer of spindle-shaped cells. During cavitation the cells of the intermediate layer separate to form the surface of the articular cartilage; no loss of cells by apoptosis was seen (see for example, Andersen, 1961; Bland and Ashhurst, 1996; Ito and Kida, 2000). At the time of cavitation the chondrogenous layers are composed of closely packed cells with little matrix. None of the cells in To whom correspondence should be addresed: Tel.: 020 8725 5224; Fax: 020 8725 3326; E-mail [email protected] †Present address: Department of Cell Biology, Institute of Ophthalmology, 11–24, Bath Street, London EC1V 9EL, U.K. 1065–6995/02/$-see front matter
the interzone has the characteristics of chondrocytes. They synthesize type V collagen, but neither type II collagen, nor matrilin-1, both of which are products of the underlying epiphyseal cartilage at this time (Bland and Ashhurst, 1996; Kavanagh and Ashhurst, 1999). After cavitation, there is rapid growth and matrix formation as the articular cartilage develops from the chondrogenous layers and the epiphyses enlarge. The cells become widely separated as matrix formation proceeds. Cell division and death have been implicated in cavitation, subsequent joint formation and in the growth and development of articular cartilage. In 8-week human foetal joints, antibodies to PCNA reveal that mitotic cells are confined to the perichondrium and the proximal and distal chondrogenous layers of the interzone; very few are present in the intermediate layer (Edwards et al., 1994). 2002 Elsevier Science Ltd. All rights reserved.
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During the early development of the marsupial Monodelphis knee joint, PCNA positive cells are distributed throughout the cartilaginous anlagen, but later become restricted to the articular surface where they persist into the adult (Archer et al., 1994). Mankin (1962, 1963) using 3H-thymidine incorporation described two bands of proliferating cells in the articular cartilage of neonatal rabbit femora, one above the subchondral plate and the other under the articular surface. The upper band only was lost before the epiphyseal growth plates closed and no proliferating cells were present in the cartilage of skeletally mature rabbits. PCD occurs in many sites during embryonic development. It occurs in the interdigital regions to separate the digits (Zakeri et al., 1994; Mori et al., 1995; Gan˜an et al., 1996). Both hammer toe and fused toe mutants in man result from disruption of interdigital PCD (Zakeri et al., 1994; van der Hoeven, 1994). Cell necrosis and apoptosis were observed in the very early stages of joint formation in the chicken and rat, but not during cavitation (Mitrovic, 1977, 1978; Mori et al., 1995; Nalin et al., 1995; Kimura and Shiota, 1996). In contrast, Abu-Hijleh et al. (1997) claim that cell death is important in cavitation in the chick. PCD occurs in the hypertrophic chondrocytes in growth plates and secondary ossification centres of the epiphyses (Farnum and Wilsman, 1989). Two questions arise. First, does cell proliferation play a large part in the growth of articular cartilage, or is growth brought about primarily by matrix accumulation? Second, does cell death occur during cavitation? To answer these questions morphological observations of mitotic and apoptotic cells were combined with PCNA localization of dividing cells, and TUNEL localization of apoptotic cells. The observed distributions of dividing and dying cells in the developing and ageing rabbit knee joint enabled the contributions of cell proliferation (versus matrix formation) to the growth of the articular cartilage before and after cavitation, and of PCD during cavitation and the subsequent development and growth of the epiphyses to be assessed.
The tibial plateau and underlying bone were taken from 3-, 6- and 12- to 14-week, 8-month and 2-year-old rabbits. At least two rabbits were used at each stage. The tissues were fixed in 4% paraformaldehyde in 0.05 M Tris-HCl buffer, pH 7.3, for 18 h before being washed extensively in buffer. With the exception of 17-day joints, the tissue was decalcified in 14.3% EDTA, pH 7.0, until radiographically free from calcium. After washing, the tissue was dehydrated in graded ethanols, cleared in methyl salicylate and embedded in paraffin wax. Sagittal serial longitudinal sections were cut at 7 m. Groups of sections at different levels in the tissue were subjected to the tests or stained. The number of groups varied between 3 and 15 depending on age and size. For histological observations sections were stained with either haematoxylin and eosin, or Alcian Blue (pH 2.5) followed by haematoxylin and eosin.
MATERIALS AND METHODS
Detection of apoptotic cells
Preparation of tissue for microscopy Hindlimbs distal to the mid-femoral region were removed from New Zealand White rabbit foetuses aged 17, 20 and 25 days. Knee joints were dissected from newborn and 1-week-old neonatal rabbits.
Detection of dividing cells 1. Morphological detection. Sections were stained with iron haematoxylin and dividing cells were identified by the presence of mitotic figures. 2. PCNA localization. The sections were dewaxed and rehydrated through graded ethanols and then rinsed in PBS. The sections were then incubated in mouse monoclonal PC10 antibody (Dako, U.K.) at a dilution of 1:50 in 1% BSA in PBS for 1 h at room temperature. After rinsing in PBS the sections were incubated in goat anti-mouse IgG conjugated to alkaline phosphatase (Sigma, Poole, U.K.) at 1:50 dilution in 1% BSA in PBS for 1 h at room temperature. The bound antibodies were detected with the following substrate; 5 mg naphthol AS-BI phosphate was dissolved in one drop dimethyl formamide and added to 5 mg Fast Red TR in 10 ml veronal acetate buffer, pH 9.2, with levamisol added at 1 mg/ml to inhibit endogenous alkaline phosphatase activity. Incubation was for 20 to 30 min at room temperature. The sections were then washed and mounted in glycerine jelly. For controls the primary antibody was omitted; there was no staining of control sections.
1. Morphological detection. Sections were stained with iron haematoxylin and cells with apoptotic bodies were located. 2. TUNEL. The TUNEL procedure was carried out using a kit according to the manufacturer’s
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instructions (Roche, Lewes, U.K.), with some modifications. The sections were dewaxed and rehydrated through graded ethanols. The sections were permeablized with proteinase K (20 mg/ml) for 10 min at 37C. After washing in PBS the TUNEL reaction mixture with TdT was added to the slides and incubated at 37C for 1 h. They were washed in PBS and blocked in Roche blocking solution for 30 min. Converter-AP was added to the slides and incubated at 37C for 30 min, before detection with the substrate for 3 min in the dark. After washing in PBS, the sections were mounted in glycerin jelly. For control sections TdT was omitted from the TUNEL reaction mixture. At no time was there any staining in control sections. Presentation of the results The number of PCNA-positive cells per section of the chondrogenous layers or articular cartilage was very low, that is, only 1 or 2 cells per section in the foetus and scattered cells in the neonate. Thus, a high magnification photomicrograph shows only 1 or 2 positive cells. The results are, therefore, presented in diagrams which show the distribution of dividing cells compiled from sections taken throughout the joint. The distribution of TUNELpositive cells is shown in the same way. The variation in the numbers of cells indicated in the neonatal tissues suggests the greater activity and growth at the early postnatal stages. RESULTS The results of the localization of PCNA- and TUNEL-positive cells are summarized in Figures 1 to 7. The line drawings show the distributions of dividing and apoptotic cells. The distributions in each section are combined from sections through the joint. Individual sections rarely have more than five PCNA-positive cells in the chondrogenous layers or articular cartilage. Cell division At 17 days, the developing foetal femur and tibia are separated at the joint by the three layers of the interzone, the two chondrogenous layers and the intermediate layer (Fig. 1a). The very few PCNA-positive cells (estimate not more than 1 per 1000) are located in the chondrogenous layers (Fig. 1b). At 20 days the distribution is similar (Fig. 2a and b). A similar number of cells with
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mitotic spindles are seen (Fig. 10). At 25 days, cavitation in the knee joint is nearing completion and the chondrogenous layers are developing into the articular cartilage (Fig. 3a). Some PCNA positive cells are present in the developing articular cartilage (Figs 3b, 8 and 9), but none are present in the foetal epiphyseal cartilage. Between birth and about 6 weeks postnatal the secondary ossification centre is developing in the epiphyses and ossification continues until about 6 weeks (Figs 4a and 5a). From birth to 6 weeks, the very few PCNA-positive or mitotic cells are in the superficial and middle zones of the articular cartilage particularly along the sides (Figs 4b and 5b). From 12 weeks onwards, no PCNA-positive or mitotic cells are found in the articular cartilage (Figs 6a, b, 7a and b). The growth plate is fully developed on its diaphyseal side from birth. Only isolated, very weakly PCNA-positive cells are seen in the proliferative zone. There are many PCNA-positive or mitotic osteoblasts on the surface of the bony trabeculae in the secondary ossification centre in the epiphysis (Figs 11, 12 and 14) and in the diaphyseal bone (Figs 4b, 5b and 6b). Their numbers are much reduced in the epiphyseal bone at 12 weeks. Many PCNA-positive cells are found in the cleft of Ranvier (Figs 4b, 5b, 16 and 17). PCNA-positive cells are also found in the periosteum, growing ligaments and their insertions, menisci and tendons throughout the foetal and neonatal period (not shown). Cell death Neither TUNEL-positive cells, nor cells with apoptotic bodies, are seen in any region of the interzone before or during cavitation (Figs 3b and 19). None can be seen in the developing or mature articular cartilage (Figs 1 to 7). In the 17- and 20-day foetuses, hypertrophic chondrocytes in the centre of the diaphysis where ossification is starting are TUNEL-positive (not shown) and some have apoptotic bodies. At 25 days, some of the cells of the epiphyseal cartilage are hypertrophic and TUNEL-positive (Fig. 3b). TUNEL-positive hypertrophic chondrocytes are present until ossification of the epiphyseal cartilage is complete at around 6 weeks (Figs 3b, 4b, 5b, 13 and 15). TUNEL-positive cells are also located in the hypertrophic region of the growth plate (Figs 4b, 5b and 6b). Many TUNEL-positive and apoptotic osteoblasts are found in the newly formed bone in the diaphysis and epiphysis (Figs 4b, 5b, 6b and 13).
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Figs. 1–3 show foetal knee joints. (a) is a section showing the femur (F) and tibia (T). In Figures 1 and 2 the interzone (IZ) consists of the chondrogenous layers and intermediate layer. The joint in Figure 3 has cavitated and the articular cartilage (AC) is developing. Menisci (M) and patellar tendon (PT) are also visible. The small arrowheads on Figure 3a indicate the position at which the width of the tibial plateau was measured. (b) is a diagram of the joint in which the chondrogenous layers or putative articular cartilage are shaded. The distributions of dividing cells (o) and apoptotic cells (x) are shown. Figure 1—17-day foetal joint (H&E). (Magnification 98.) Figure 2—20-day foetal joint (H&E). (Magnification 98.) Figure 3—25-day foetal joint (H&E). (Magnification 38.)
No TUNEL-positive osteoblasts are seen at 8 months and 2 years (Fig. 7b). Development and growth of the articular cartilage The width of the central region of the tibial epiphysis at different ages was measured across its widest point (see Figs 3a and 4a) on sections such as those in Figures 3 to 7. The widths of the tibial epiphyses are as follows—25-day foetus 1.5 mm, newborn 3.25 mm, 1-week neonate 4.25 mm, 3-week 9 mm, 6-week 10 mm, 12-week 11 mm, 2-year 10 mm. These measurements demonstrate that the greatest growth of the tibial epiphysis occurs before 3 weeks postnatal, during which time
the epiphysis is extensively remodelled to achieve the adult shape. The growth plates close at about 6 months which suggests that longitudinal growth continues after the epiphyses have reached their full size. Before cavitation the chondrogenous layers contain closely packed cells with little matrix between them (Fig. 18). Between cavitation at around 25 days and 6 weeks postnatal the number of cells per unit area in the articular cartilage decreases while the amount of matrix increases (Figs 19 to 22). Between 3 and 6 weeks the cells in the middle region of the cartilage form columns. Concomitant with these changes, the epiphyseal cartilage is removed; ossification of the epiphysis is completed
Figs. 4–7 show the epiphysis of the tibia. (a) is a section showing the articular cartilage (AC) which is a distinct layer. The epiphyseal bone is separated from the diaphyseal bone by the growth plate (GP). (b) is a diagram of the tibia; the articular cartilage, plus any remaining epiphyseal cartilage, is shaded. The distributions of dividing cells (o) and apoptotic cells (x) are shown. Figure 4—3-week neonatal tibia (AB/H&E). Ossification is still progressing along the boundary of the articular cartilage and epiphyseal bone (arrows). The cleft of Ranvier is present (large arrowheads). The small arrowheads indicate the position at which the width of the tibial plateau was measured. (Magnification 7.) Figure 5—6-week neonatal tibia (AB/H&E). The cartilage is now thinner and ossification is almost complete at the boundary of the articular cartilage and epiphyseal bone (arrows). (Magnification 6.5.) Figure 6—12-week tibia (AB/H&E). A plate of bone is present under the articular cartilage (arrows). The growth plate is still open. (Magnification 5.5.) Figure 7—2-year adult tibia (AB/H&E). The articular cartilage lies on a dense plate of bone (arrows). (Magnification 6.)
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between 6 and 12 weeks postnatal (Figs 5, 6, 22 and 23). Apart from the development of the tidemark, there are no obvious changes, such as in the number of chondrocytes, in the articular cartilage between 12 weeks and 2 years (Figs 23 and 24). DISCUSSION
Figs. 8–9 The recently cavitated joint of a 25-day foetal knee joint to show dividing cells in the chondrogenous layer of the femur. The only PCNA-positive cells are indicated by arrows. None is present in the underlying epiphyseal cartilage (EC). A few PCNA-positive cells (arrows) occur in the meniscus (M). (Magnification Fig. 8 103, Fig. 9 260.)
In the present study, the sites at which cells divide and those at which cells undergo PCD during joint formation and the subsequent growth of the articular cartilage were determined. The great variability in the results reported using the PCNA and TUNEL techniques suggests that caution must be exercised in their interpretation. This point has been made by histopathologists using these methods for clinical diagnosis (McCormick and Hall, 1992; Yu and Filipe, 1993). In this study observations were made on histological preparations to verify that dividing and apoptotic cells are present in the regions of positive results. Cell proliferation PCNA is an acidic nuclear protein that is present primarily during the S-phase of the cell cycle. It has
Figs. 10–13 Figure 10. The interzone of a 20-day foetal knee joint. The intermediate layer of flattened cells is arrowed. Two mitotic figures (arrowheads) are seen in the chondrogenous layer of the femur; these are the only mitotic figures present in the chondrogenous layers in this section. (Magnification 460.) Figures 11, 12 and 13. The junction between the cartilage (C) and bone (B) in a 3-week-old neonate. Figure 11—the articular cartilage is lying on the remnants of the epiphyseal cartilage containing hypertrophic chondrocytes. The bony trabeculae are lined with osteoblasts (arrows). AB/H&E. Figure 12—the only PCNA-positive cells (arrows) are osteoblasts on the bony trabeculae. Figure 13—TUNEL-positive hypertrophic chondrocytes (arrows) are present in the remaining epiphyseal cartilage and TUNEL-positive osteoblasts (arrows) in the bone. (Magnification Figs 11, 12 and 13 90.)
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Figs. 14–17 Figure 14. Developing bone in the secondary ossification centre in the epiphysis of a 3-week neonatal tibia. PCNA-positive osteoblasts (arrows) are associated with the cartilaginous trabeculae under the epiphyseal cartilage (EC). (Magnification 400.) Figure 15.The epiphyseal cartilage in a 3-week neonatal tibia; the ossification centre (O) is at the bottom of the figure. TUNEL-positive, hypertrophic chondrocytes are present in all the lacunae. (Magnification 400.) Figures 16 and 17.The cleft of Ranvier in a 6-week-old neonate. Figure 16—the cleft (arrowed) and its relation to the growth plate (GP) and periosteum (P). AB/H&E. Figure 17—PCNA-positive cells in the cleft (arrowed). (Magnification Figs 16 and 17 90.)
a half-life of 20 h and may still be present and capable of being immunostained after the cell has left the cycle. A form of PCNA may be found in non-dividing cells that remain capable of division (Yu et al., 1992; Yu and Filipe, 1993). The main application of methods for the detection of PCNA is to assess proliferation in neoplastic tissues, but they have also been used in normal tissues. The results are affected by fixation and other preparative factors. These must be taken into account in the interpretation of the results. Mitotic figures were observed in histological preparations of the regions in which PCNA-positive cells were found (see Bland and Ashhurst, 1996).
Programmed cell death PCD, or apoptosis, were identified here using the TUNEL procedure that recognizes DNA fragmentation. This is the end-point of a sequence that lasts 12 to 24 h. The method is prone to producing false positive and negative results and the results depend on factors such as fixation time, the efficiency of the enzymatic pretreatments, etc. (Huppertz et al., 1999; Saraste, 1999). For this reason, sections with haematoxylin-stained nuclei serial to those used for the TUNEL procedure were used as controls to identify apoptotic cells morphologically. In instances in which the morphological distribution of apoptotic cells did not coincide with the TUNEL
positive cells, the morphological distribution was regarded as the more reliable. Cavitation In the foetal rabbit knee interzone, the few dividing cells are located in the highly cellular chondrogenous layers. These cells are primarily the result of division at the time of initial condensation formation at around 15 days; that stage was not examined. This localization is similar to those found in the human and Monodelphis precavitational joints (Archer et al., 1994; Edwards et al., 1994). During cavitation of the rabbit knee joint the flattened cells of the intermediate layer of the interzone split apart without any cell death or necrosis (see Fig. 19) (Bland and Ashhurst, 1996). That apoptosis is not involved is confirmed by the absence of TUNEL-positive cells in any part of the interzone. The intermediate layer cells form the superficial layer of the articular cartilage (Bland and Ashhurst, 1996). This is supported by the observations of human, rat and chicken joints (Mitrovic, 1977, 1978; Craig et al., 1987; Edwards et al., 1994; Ito and Kida, 2000). Further development and growth of the long bones After cavitation at 25 days there is rapid 3-dimensional growth of the femur and tibia
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Figs. 18–20 Figure 18. The interzone (IZ) between the femur (F) and tibia (T) of a 20-day foetus. The chondrogenous layers with closely packed cells are separated by the intermediate layer. H&E (Magnification 225.) Figure 19. The site of cavitation in a 25-day foetal knee joint. The spindle-shaped cells (arrows) of the intermediate layer through which cavitation will continue are clearly seen. No apoptotic cells are present. (H&E) (Magnification 400.) Figure 20. The articular cartilage (AC) and epiphyseal cartilage (EC) of a 25-day foetus in which cavitation is complete (AB/H&E). (Magnification 225.)
especially between the 25-day foetus and 3-week neonate. Matrix formation makes a major contribution to the increasing size of the articular cartilage and leaves the cells widely separated. As few scattered PCNA-positive cells are found in the superficial and middle regions of the articular cartilage between the 25-day foetus and 6-week neonate, it is suggested that cell proliferation contributes little to the growth of articular cartilage during ossification of the epiphysis. The localization of the dividing cells in the rabbit during the period of growth agrees in general with that observed in the rabbit by Mankin (1962, 1963) and Monodelphis by Archer et al. (1994), but disagrees
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with Archer et al. in that no mitotic cells were observed after the cessation of growth. Apoptotic body-containing and TUNELpositive hypertrophic chondrocytes are seen under articular cartilage in the remnants of the epiphyseal cartilage as it is removed; none is seen after ossification is complete. All the epiphyseal chondrocytes with their matrix are removed. The chondrocytes become hypertrophic before death, as do those of the growth plate. Whether all the cells become apoptotic or die by other mechanisms (see Roach and Clark, 2000) was not investigated. Apoptotic cells were found in both calcified and uncalcified regions of adult mouse articular cartilage, but only in calcified regions of adult rat cartilage (Adams and Horton, 1998). Using flow cytometry on isolated adult human articular chondrocytes, Hashimoto et al. (1998) estimated that 4.8% of chondrocytes in normal cartilage, but 22.3% in osteoarthritic cartilage, were apoptotic, whereas Blanco et al. (1998) found that 11% of normal, but 51% of osteoarthritic chondrocytes, were apoptotic. No evidence was seen for a ‘growth-plate-like’ region at the junction of the articular and epiphyseal cartilages that would add to the external dimensions of the articular cartilage and hence of the epiphysis. The epiphyseal cartilage is distinct from the articular cartilage in that the matrix contains matrilin-1 (Kavanagh and Ashhurst, 1999). All the hypertrophic chondrocytes are surrounded by matrilin-1 containing matrix and only this matrix is removed (Kavanagh and Ashhurst, 1999). The mitotic cells observed are above this region. Many PCNA-positive and TUNEL-positive osteoblasts are seen in the developing and remodelling bone of both the diaphyses and epiphyses of the rabbit tibia during the neonatal period. The number of osteoblasts on bone surfaces is far in excess of the number of osteocytes that are found in the bone matrix. A low rate of apoptosis has been calculated to be sufficient to account for the loss of 50 to 70% of the osteoblasts when the lining cells and osteocytes are counted at the end of remodelling in the mouse (Jilka et al., 1998). No quantification of TUNEL-positive cells was attempted, but their prevalence in the rabbit appears far greater than the 0.6% found by Jilka et al. (1998), who used 12-week-old mouse femora, i.e. bone from almost skeletally mature animals, and assumed an osteoblast life-span of about 12 days, or 300 h. In contrast, Owen (1963) estimated that differentiating osteoblasts in growing femora of 2-week-old Dutch rabbits remain on the
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Figs. 21–24 The postnatal development of the articular cartilage (AC) and its relationship to the epiphyseal cartilage (EC) and bone (B). Figure 21—3-week neonate (AB/H&E), the dashed line indicates the approximate boundary between the articular and epiphyseal cartilage. Figure 22—6-week neonate (AB/H&E). Figure 23—12-week juvenile (AB/H&E). Figure 24—2-year adult; the tidemark (TM) is visible (AB/H&E). (Magnification Figs 21 to 24 120.)
bone surface for only 2 to 3 days before they move into the matrix. Between 3 and 18 days postnatal the cross-sectional area of the tibial diaphysis of a NZW rabbit increases 6 to 7 times (Critchlow et al., 1994). None of the bone present at 3 days remains at 18 days and it was estimated that osteoblasts spend only about 36 h on the bone surfaces. Thus, both cell division and cell death must occur at a much higher rate in neonatal rabbits than that suggested by Jilka et al. (1998) in 12-week mouse femora and would account for the large numbers of PCNA-positive and TUNEL-positive cells seen. Concluding remarks The low frequency of cell division in the developing joint and articular cartilage suggests that a large proportion of the cells in the mature articular cartilage are already present in the interzone before cavitation. Thus, matrix formation, which pushes the cells apart, is probably the major contributor to growth. The contribution of PCD, or apoptosis, appears from the present results to be confined to the death of hypertrophic chondrocytes in the epiphyseal cartilage and growth plate. No evidence was seen for PCD at the joint line around the time of cavitation. It is indeed difficult to postulate how unwanted, dying or dead cells and other debris would be eliminated from the developing joint cavity before the formation of the synovium.
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DIVISION AND DEATH OF CELLS IN DEVELOPING SYNOVIAL JOINTS AND LONG BONES EMMA KAVANAGH†, MERSEDEH ABIRI, YVETTE S. BLAND and DOREEN E. ASHHURST* Department of Anatomy, St George’s Hospital Medical School, Tooting, London SW17 0RE, U.K. Received 26 March 2001; revised 4 April 2002; accepted 22 April 2002 Articular chondrocytes are a unique set of cells from the time the cellular condensations that become the anlagen of the long bones develop in the embryo. In the presumptive joint the cells of the opposing bones are packed very closely together, but at cavitation, the central, flattened cells move apart to form the articular surfaces. As the articular cartilage develops the cells are pushed further apart by the cartilaginous matrix. To determine the contributions of cell proliferation and death to cavitation and the subsequent development and growth of articular cartilage, direct observations were made to identify mitotic cells and those with apoptotic bodies in haematoxylin-stained sections of developing joints, and growing and ageing articular cartilage of the rabbit knee. These observations were extended using antibodies to the proliferating cell nuclear antigen (PCNA) and TdT-mediated dUTP nick end labelling (TUNEL) on corresponding sections. Low levels of cell division do occur in the articular cartilage up to 6 weeks postnatally, but matrix formation makes the major contribution to the increase in size of the cartilage. Cell death is not observed during cavitation, nor during the development of the articular cartilage proper. Apoptosis is essential, however, for the removal of the epiphyseal cartilage during ossification of the epiphyses and in the growth plate. 2002 Elsevier Science Ltd. All rights reserved.
K: rabbit knee joint; cell division; cell death/apoptosis; cavitation; articular cartilage; development. A: BSA, bovine serum albumin; PBS, phosphate buffered saline; PCD, programmed cell death; PCNA, proliferating cell nuclear antigen; TdT, terminal deoxytransferase; TUNEL, TdT-mediated dUPT nick end labelling.
INTRODUCTION The major event in the formation of a synovial joint is cavitation. The cartilaginous anlagen of the bones are separated by the interzone which has three layers; these are the chondrogenous layers and the intermediate layer of spindle-shaped cells. During cavitation the cells of the intermediate layer separate to form the surface of the articular cartilage; no loss of cells by apoptosis was seen (see for example, Andersen, 1961; Bland and Ashhurst, 1996; Ito and Kida, 2000). At the time of cavitation the chondrogenous layers are composed of closely packed cells with little matrix. None of the cells in To whom correspondence should be addresed: Tel.: 020 8725 5224; Fax: 020 8725 3326; E-mail [email protected] †Present address: Department of Cell Biology, Institute of Ophthalmology, 11–24, Bath Street, London EC1V 9EL, U.K. 1065–6995/02/$-see front matter
the interzone has the characteristics of chondrocytes. They synthesize type V collagen, but neither type II collagen, nor matrilin-1, both of which are products of the underlying epiphyseal cartilage at this time (Bland and Ashhurst, 1996; Kavanagh and Ashhurst, 1999). After cavitation, there is rapid growth and matrix formation as the articular cartilage develops from the chondrogenous layers and the epiphyses enlarge. The cells become widely separated as matrix formation proceeds. Cell division and death have been implicated in cavitation, subsequent joint formation and in the growth and development of articular cartilage. In 8-week human foetal joints, antibodies to PCNA reveal that mitotic cells are confined to the perichondrium and the proximal and distal chondrogenous layers of the interzone; very few are present in the intermediate layer (Edwards et al., 1994). 2002 Elsevier Science Ltd. All rights reserved.
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During the early development of the marsupial Monodelphis knee joint, PCNA positive cells are distributed throughout the cartilaginous anlagen, but later become restricted to the articular surface where they persist into the adult (Archer et al., 1994). Mankin (1962, 1963) using 3H-thymidine incorporation described two bands of proliferating cells in the articular cartilage of neonatal rabbit femora, one above the subchondral plate and the other under the articular surface. The upper band only was lost before the epiphyseal growth plates closed and no proliferating cells were present in the cartilage of skeletally mature rabbits. PCD occurs in many sites during embryonic development. It occurs in the interdigital regions to separate the digits (Zakeri et al., 1994; Mori et al., 1995; Gan˜an et al., 1996). Both hammer toe and fused toe mutants in man result from disruption of interdigital PCD (Zakeri et al., 1994; van der Hoeven, 1994). Cell necrosis and apoptosis were observed in the very early stages of joint formation in the chicken and rat, but not during cavitation (Mitrovic, 1977, 1978; Mori et al., 1995; Nalin et al., 1995; Kimura and Shiota, 1996). In contrast, Abu-Hijleh et al. (1997) claim that cell death is important in cavitation in the chick. PCD occurs in the hypertrophic chondrocytes in growth plates and secondary ossification centres of the epiphyses (Farnum and Wilsman, 1989). Two questions arise. First, does cell proliferation play a large part in the growth of articular cartilage, or is growth brought about primarily by matrix accumulation? Second, does cell death occur during cavitation? To answer these questions morphological observations of mitotic and apoptotic cells were combined with PCNA localization of dividing cells, and TUNEL localization of apoptotic cells. The observed distributions of dividing and dying cells in the developing and ageing rabbit knee joint enabled the contributions of cell proliferation (versus matrix formation) to the growth of the articular cartilage before and after cavitation, and of PCD during cavitation and the subsequent development and growth of the epiphyses to be assessed.
The tibial plateau and underlying bone were taken from 3-, 6- and 12- to 14-week, 8-month and 2-year-old rabbits. At least two rabbits were used at each stage. The tissues were fixed in 4% paraformaldehyde in 0.05 M Tris-HCl buffer, pH 7.3, for 18 h before being washed extensively in buffer. With the exception of 17-day joints, the tissue was decalcified in 14.3% EDTA, pH 7.0, until radiographically free from calcium. After washing, the tissue was dehydrated in graded ethanols, cleared in methyl salicylate and embedded in paraffin wax. Sagittal serial longitudinal sections were cut at 7 m. Groups of sections at different levels in the tissue were subjected to the tests or stained. The number of groups varied between 3 and 15 depending on age and size. For histological observations sections were stained with either haematoxylin and eosin, or Alcian Blue (pH 2.5) followed by haematoxylin and eosin.
MATERIALS AND METHODS
Detection of apoptotic cells
Preparation of tissue for microscopy Hindlimbs distal to the mid-femoral region were removed from New Zealand White rabbit foetuses aged 17, 20 and 25 days. Knee joints were dissected from newborn and 1-week-old neonatal rabbits.
Detection of dividing cells 1. Morphological detection. Sections were stained with iron haematoxylin and dividing cells were identified by the presence of mitotic figures. 2. PCNA localization. The sections were dewaxed and rehydrated through graded ethanols and then rinsed in PBS. The sections were then incubated in mouse monoclonal PC10 antibody (Dako, U.K.) at a dilution of 1:50 in 1% BSA in PBS for 1 h at room temperature. After rinsing in PBS the sections were incubated in goat anti-mouse IgG conjugated to alkaline phosphatase (Sigma, Poole, U.K.) at 1:50 dilution in 1% BSA in PBS for 1 h at room temperature. The bound antibodies were detected with the following substrate; 5 mg naphthol AS-BI phosphate was dissolved in one drop dimethyl formamide and added to 5 mg Fast Red TR in 10 ml veronal acetate buffer, pH 9.2, with levamisol added at 1 mg/ml to inhibit endogenous alkaline phosphatase activity. Incubation was for 20 to 30 min at room temperature. The sections were then washed and mounted in glycerine jelly. For controls the primary antibody was omitted; there was no staining of control sections.
1. Morphological detection. Sections were stained with iron haematoxylin and cells with apoptotic bodies were located. 2. TUNEL. The TUNEL procedure was carried out using a kit according to the manufacturer’s
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instructions (Roche, Lewes, U.K.), with some modifications. The sections were dewaxed and rehydrated through graded ethanols. The sections were permeablized with proteinase K (20 mg/ml) for 10 min at 37C. After washing in PBS the TUNEL reaction mixture with TdT was added to the slides and incubated at 37C for 1 h. They were washed in PBS and blocked in Roche blocking solution for 30 min. Converter-AP was added to the slides and incubated at 37C for 30 min, before detection with the substrate for 3 min in the dark. After washing in PBS, the sections were mounted in glycerin jelly. For control sections TdT was omitted from the TUNEL reaction mixture. At no time was there any staining in control sections. Presentation of the results The number of PCNA-positive cells per section of the chondrogenous layers or articular cartilage was very low, that is, only 1 or 2 cells per section in the foetus and scattered cells in the neonate. Thus, a high magnification photomicrograph shows only 1 or 2 positive cells. The results are, therefore, presented in diagrams which show the distribution of dividing cells compiled from sections taken throughout the joint. The distribution of TUNELpositive cells is shown in the same way. The variation in the numbers of cells indicated in the neonatal tissues suggests the greater activity and growth at the early postnatal stages. RESULTS The results of the localization of PCNA- and TUNEL-positive cells are summarized in Figures 1 to 7. The line drawings show the distributions of dividing and apoptotic cells. The distributions in each section are combined from sections through the joint. Individual sections rarely have more than five PCNA-positive cells in the chondrogenous layers or articular cartilage. Cell division At 17 days, the developing foetal femur and tibia are separated at the joint by the three layers of the interzone, the two chondrogenous layers and the intermediate layer (Fig. 1a). The very few PCNA-positive cells (estimate not more than 1 per 1000) are located in the chondrogenous layers (Fig. 1b). At 20 days the distribution is similar (Fig. 2a and b). A similar number of cells with
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mitotic spindles are seen (Fig. 10). At 25 days, cavitation in the knee joint is nearing completion and the chondrogenous layers are developing into the articular cartilage (Fig. 3a). Some PCNA positive cells are present in the developing articular cartilage (Figs 3b, 8 and 9), but none are present in the foetal epiphyseal cartilage. Between birth and about 6 weeks postnatal the secondary ossification centre is developing in the epiphyses and ossification continues until about 6 weeks (Figs 4a and 5a). From birth to 6 weeks, the very few PCNA-positive or mitotic cells are in the superficial and middle zones of the articular cartilage particularly along the sides (Figs 4b and 5b). From 12 weeks onwards, no PCNA-positive or mitotic cells are found in the articular cartilage (Figs 6a, b, 7a and b). The growth plate is fully developed on its diaphyseal side from birth. Only isolated, very weakly PCNA-positive cells are seen in the proliferative zone. There are many PCNA-positive or mitotic osteoblasts on the surface of the bony trabeculae in the secondary ossification centre in the epiphysis (Figs 11, 12 and 14) and in the diaphyseal bone (Figs 4b, 5b and 6b). Their numbers are much reduced in the epiphyseal bone at 12 weeks. Many PCNA-positive cells are found in the cleft of Ranvier (Figs 4b, 5b, 16 and 17). PCNA-positive cells are also found in the periosteum, growing ligaments and their insertions, menisci and tendons throughout the foetal and neonatal period (not shown). Cell death Neither TUNEL-positive cells, nor cells with apoptotic bodies, are seen in any region of the interzone before or during cavitation (Figs 3b and 19). None can be seen in the developing or mature articular cartilage (Figs 1 to 7). In the 17- and 20-day foetuses, hypertrophic chondrocytes in the centre of the diaphysis where ossification is starting are TUNEL-positive (not shown) and some have apoptotic bodies. At 25 days, some of the cells of the epiphyseal cartilage are hypertrophic and TUNEL-positive (Fig. 3b). TUNEL-positive hypertrophic chondrocytes are present until ossification of the epiphyseal cartilage is complete at around 6 weeks (Figs 3b, 4b, 5b, 13 and 15). TUNEL-positive cells are also located in the hypertrophic region of the growth plate (Figs 4b, 5b and 6b). Many TUNEL-positive and apoptotic osteoblasts are found in the newly formed bone in the diaphysis and epiphysis (Figs 4b, 5b, 6b and 13).
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Figs. 1–3 show foetal knee joints. (a) is a section showing the femur (F) and tibia (T). In Figures 1 and 2 the interzone (IZ) consists of the chondrogenous layers and intermediate layer. The joint in Figure 3 has cavitated and the articular cartilage (AC) is developing. Menisci (M) and patellar tendon (PT) are also visible. The small arrowheads on Figure 3a indicate the position at which the width of the tibial plateau was measured. (b) is a diagram of the joint in which the chondrogenous layers or putative articular cartilage are shaded. The distributions of dividing cells (o) and apoptotic cells (x) are shown. Figure 1—17-day foetal joint (H&E). (Magnification 98.) Figure 2—20-day foetal joint (H&E). (Magnification 98.) Figure 3—25-day foetal joint (H&E). (Magnification 38.)
No TUNEL-positive osteoblasts are seen at 8 months and 2 years (Fig. 7b). Development and growth of the articular cartilage The width of the central region of the tibial epiphysis at different ages was measured across its widest point (see Figs 3a and 4a) on sections such as those in Figures 3 to 7. The widths of the tibial epiphyses are as follows—25-day foetus 1.5 mm, newborn 3.25 mm, 1-week neonate 4.25 mm, 3-week 9 mm, 6-week 10 mm, 12-week 11 mm, 2-year 10 mm. These measurements demonstrate that the greatest growth of the tibial epiphysis occurs before 3 weeks postnatal, during which time
the epiphysis is extensively remodelled to achieve the adult shape. The growth plates close at about 6 months which suggests that longitudinal growth continues after the epiphyses have reached their full size. Before cavitation the chondrogenous layers contain closely packed cells with little matrix between them (Fig. 18). Between cavitation at around 25 days and 6 weeks postnatal the number of cells per unit area in the articular cartilage decreases while the amount of matrix increases (Figs 19 to 22). Between 3 and 6 weeks the cells in the middle region of the cartilage form columns. Concomitant with these changes, the epiphyseal cartilage is removed; ossification of the epiphysis is completed
Figs. 4–7 show the epiphysis of the tibia. (a) is a section showing the articular cartilage (AC) which is a distinct layer. The epiphyseal bone is separated from the diaphyseal bone by the growth plate (GP). (b) is a diagram of the tibia; the articular cartilage, plus any remaining epiphyseal cartilage, is shaded. The distributions of dividing cells (o) and apoptotic cells (x) are shown. Figure 4—3-week neonatal tibia (AB/H&E). Ossification is still progressing along the boundary of the articular cartilage and epiphyseal bone (arrows). The cleft of Ranvier is present (large arrowheads). The small arrowheads indicate the position at which the width of the tibial plateau was measured. (Magnification 7.) Figure 5—6-week neonatal tibia (AB/H&E). The cartilage is now thinner and ossification is almost complete at the boundary of the articular cartilage and epiphyseal bone (arrows). (Magnification 6.5.) Figure 6—12-week tibia (AB/H&E). A plate of bone is present under the articular cartilage (arrows). The growth plate is still open. (Magnification 5.5.) Figure 7—2-year adult tibia (AB/H&E). The articular cartilage lies on a dense plate of bone (arrows). (Magnification 6.)
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between 6 and 12 weeks postnatal (Figs 5, 6, 22 and 23). Apart from the development of the tidemark, there are no obvious changes, such as in the number of chondrocytes, in the articular cartilage between 12 weeks and 2 years (Figs 23 and 24). DISCUSSION
Figs. 8–9 The recently cavitated joint of a 25-day foetal knee joint to show dividing cells in the chondrogenous layer of the femur. The only PCNA-positive cells are indicated by arrows. None is present in the underlying epiphyseal cartilage (EC). A few PCNA-positive cells (arrows) occur in the meniscus (M). (Magnification Fig. 8 103, Fig. 9 260.)
In the present study, the sites at which cells divide and those at which cells undergo PCD during joint formation and the subsequent growth of the articular cartilage were determined. The great variability in the results reported using the PCNA and TUNEL techniques suggests that caution must be exercised in their interpretation. This point has been made by histopathologists using these methods for clinical diagnosis (McCormick and Hall, 1992; Yu and Filipe, 1993). In this study observations were made on histological preparations to verify that dividing and apoptotic cells are present in the regions of positive results. Cell proliferation PCNA is an acidic nuclear protein that is present primarily during the S-phase of the cell cycle. It has
Figs. 10–13 Figure 10. The interzone of a 20-day foetal knee joint. The intermediate layer of flattened cells is arrowed. Two mitotic figures (arrowheads) are seen in the chondrogenous layer of the femur; these are the only mitotic figures present in the chondrogenous layers in this section. (Magnification 460.) Figures 11, 12 and 13. The junction between the cartilage (C) and bone (B) in a 3-week-old neonate. Figure 11—the articular cartilage is lying on the remnants of the epiphyseal cartilage containing hypertrophic chondrocytes. The bony trabeculae are lined with osteoblasts (arrows). AB/H&E. Figure 12—the only PCNA-positive cells (arrows) are osteoblasts on the bony trabeculae. Figure 13—TUNEL-positive hypertrophic chondrocytes (arrows) are present in the remaining epiphyseal cartilage and TUNEL-positive osteoblasts (arrows) in the bone. (Magnification Figs 11, 12 and 13 90.)
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Figs. 14–17 Figure 14. Developing bone in the secondary ossification centre in the epiphysis of a 3-week neonatal tibia. PCNA-positive osteoblasts (arrows) are associated with the cartilaginous trabeculae under the epiphyseal cartilage (EC). (Magnification 400.) Figure 15.The epiphyseal cartilage in a 3-week neonatal tibia; the ossification centre (O) is at the bottom of the figure. TUNEL-positive, hypertrophic chondrocytes are present in all the lacunae. (Magnification 400.) Figures 16 and 17.The cleft of Ranvier in a 6-week-old neonate. Figure 16—the cleft (arrowed) and its relation to the growth plate (GP) and periosteum (P). AB/H&E. Figure 17—PCNA-positive cells in the cleft (arrowed). (Magnification Figs 16 and 17 90.)
a half-life of 20 h and may still be present and capable of being immunostained after the cell has left the cycle. A form of PCNA may be found in non-dividing cells that remain capable of division (Yu et al., 1992; Yu and Filipe, 1993). The main application of methods for the detection of PCNA is to assess proliferation in neoplastic tissues, but they have also been used in normal tissues. The results are affected by fixation and other preparative factors. These must be taken into account in the interpretation of the results. Mitotic figures were observed in histological preparations of the regions in which PCNA-positive cells were found (see Bland and Ashhurst, 1996).
Programmed cell death PCD, or apoptosis, were identified here using the TUNEL procedure that recognizes DNA fragmentation. This is the end-point of a sequence that lasts 12 to 24 h. The method is prone to producing false positive and negative results and the results depend on factors such as fixation time, the efficiency of the enzymatic pretreatments, etc. (Huppertz et al., 1999; Saraste, 1999). For this reason, sections with haematoxylin-stained nuclei serial to those used for the TUNEL procedure were used as controls to identify apoptotic cells morphologically. In instances in which the morphological distribution of apoptotic cells did not coincide with the TUNEL
positive cells, the morphological distribution was regarded as the more reliable. Cavitation In the foetal rabbit knee interzone, the few dividing cells are located in the highly cellular chondrogenous layers. These cells are primarily the result of division at the time of initial condensation formation at around 15 days; that stage was not examined. This localization is similar to those found in the human and Monodelphis precavitational joints (Archer et al., 1994; Edwards et al., 1994). During cavitation of the rabbit knee joint the flattened cells of the intermediate layer of the interzone split apart without any cell death or necrosis (see Fig. 19) (Bland and Ashhurst, 1996). That apoptosis is not involved is confirmed by the absence of TUNEL-positive cells in any part of the interzone. The intermediate layer cells form the superficial layer of the articular cartilage (Bland and Ashhurst, 1996). This is supported by the observations of human, rat and chicken joints (Mitrovic, 1977, 1978; Craig et al., 1987; Edwards et al., 1994; Ito and Kida, 2000). Further development and growth of the long bones After cavitation at 25 days there is rapid 3-dimensional growth of the femur and tibia
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Figs. 18–20 Figure 18. The interzone (IZ) between the femur (F) and tibia (T) of a 20-day foetus. The chondrogenous layers with closely packed cells are separated by the intermediate layer. H&E (Magnification 225.) Figure 19. The site of cavitation in a 25-day foetal knee joint. The spindle-shaped cells (arrows) of the intermediate layer through which cavitation will continue are clearly seen. No apoptotic cells are present. (H&E) (Magnification 400.) Figure 20. The articular cartilage (AC) and epiphyseal cartilage (EC) of a 25-day foetus in which cavitation is complete (AB/H&E). (Magnification 225.)
especially between the 25-day foetus and 3-week neonate. Matrix formation makes a major contribution to the increasing size of the articular cartilage and leaves the cells widely separated. As few scattered PCNA-positive cells are found in the superficial and middle regions of the articular cartilage between the 25-day foetus and 6-week neonate, it is suggested that cell proliferation contributes little to the growth of articular cartilage during ossification of the epiphysis. The localization of the dividing cells in the rabbit during the period of growth agrees in general with that observed in the rabbit by Mankin (1962, 1963) and Monodelphis by Archer et al. (1994), but disagrees
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with Archer et al. in that no mitotic cells were observed after the cessation of growth. Apoptotic body-containing and TUNELpositive hypertrophic chondrocytes are seen under articular cartilage in the remnants of the epiphyseal cartilage as it is removed; none is seen after ossification is complete. All the epiphyseal chondrocytes with their matrix are removed. The chondrocytes become hypertrophic before death, as do those of the growth plate. Whether all the cells become apoptotic or die by other mechanisms (see Roach and Clark, 2000) was not investigated. Apoptotic cells were found in both calcified and uncalcified regions of adult mouse articular cartilage, but only in calcified regions of adult rat cartilage (Adams and Horton, 1998). Using flow cytometry on isolated adult human articular chondrocytes, Hashimoto et al. (1998) estimated that 4.8% of chondrocytes in normal cartilage, but 22.3% in osteoarthritic cartilage, were apoptotic, whereas Blanco et al. (1998) found that 11% of normal, but 51% of osteoarthritic chondrocytes, were apoptotic. No evidence was seen for a ‘growth-plate-like’ region at the junction of the articular and epiphyseal cartilages that would add to the external dimensions of the articular cartilage and hence of the epiphysis. The epiphyseal cartilage is distinct from the articular cartilage in that the matrix contains matrilin-1 (Kavanagh and Ashhurst, 1999). All the hypertrophic chondrocytes are surrounded by matrilin-1 containing matrix and only this matrix is removed (Kavanagh and Ashhurst, 1999). The mitotic cells observed are above this region. Many PCNA-positive and TUNEL-positive osteoblasts are seen in the developing and remodelling bone of both the diaphyses and epiphyses of the rabbit tibia during the neonatal period. The number of osteoblasts on bone surfaces is far in excess of the number of osteocytes that are found in the bone matrix. A low rate of apoptosis has been calculated to be sufficient to account for the loss of 50 to 70% of the osteoblasts when the lining cells and osteocytes are counted at the end of remodelling in the mouse (Jilka et al., 1998). No quantification of TUNEL-positive cells was attempted, but their prevalence in the rabbit appears far greater than the 0.6% found by Jilka et al. (1998), who used 12-week-old mouse femora, i.e. bone from almost skeletally mature animals, and assumed an osteoblast life-span of about 12 days, or 300 h. In contrast, Owen (1963) estimated that differentiating osteoblasts in growing femora of 2-week-old Dutch rabbits remain on the
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Figs. 21–24 The postnatal development of the articular cartilage (AC) and its relationship to the epiphyseal cartilage (EC) and bone (B). Figure 21—3-week neonate (AB/H&E), the dashed line indicates the approximate boundary between the articular and epiphyseal cartilage. Figure 22—6-week neonate (AB/H&E). Figure 23—12-week juvenile (AB/H&E). Figure 24—2-year adult; the tidemark (TM) is visible (AB/H&E). (Magnification Figs 21 to 24 120.)
bone surface for only 2 to 3 days before they move into the matrix. Between 3 and 18 days postnatal the cross-sectional area of the tibial diaphysis of a NZW rabbit increases 6 to 7 times (Critchlow et al., 1994). None of the bone present at 3 days remains at 18 days and it was estimated that osteoblasts spend only about 36 h on the bone surfaces. Thus, both cell division and cell death must occur at a much higher rate in neonatal rabbits than that suggested by Jilka et al. (1998) in 12-week mouse femora and would account for the large numbers of PCNA-positive and TUNEL-positive cells seen. Concluding remarks The low frequency of cell division in the developing joint and articular cartilage suggests that a large proportion of the cells in the mature articular cartilage are already present in the interzone before cavitation. Thus, matrix formation, which pushes the cells apart, is probably the major contributor to growth. The contribution of PCD, or apoptosis, appears from the present results to be confined to the death of hypertrophic chondrocytes in the epiphyseal cartilage and growth plate. No evidence was seen for PCD at the joint line around the time of cavitation. It is indeed difficult to postulate how unwanted, dying or dead cells and other debris would be eliminated from the developing joint cavity before the formation of the synovium.
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