ARTICLE |
Correspondence to: Helmtrud I. Roach, University Orthopaedics, CF86, MP 817, Southampton General Hospital, Southampton SO16 6YD, UK. E-mail: hr@soton.ac.uk
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Summary |
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Despite the continued presence of growth plates in aged rats, longitudinal growth no longer occurs. The aims of this study were to understand the reasons for the cessation of growth. We studied the growth plates of femurs and tibiae in Wistar rats aged 6280 weeks and compared these with the corresponding growth plates from rats aged 216 weeks. During skeletal growth, the heights of the plates, especially that of the hypertrophic zone, reflected the rate of bone growth. During the period of decelerating growth, it was the loss of large hydrated chondrocytes that contributed most to the overall decrease in the heights of the growth plates. In the old rats we identified four categories of growth plate morphology that were not present in the growth plates of younger rats: (a) formation of a bone band parallel to the metaphyseal edge of the growth plate, which effectively sealed that edge; (b) extensive areas of acellularity, which were resistant to resorption and/or remodeling; (c) extensive remodeling and bone formation within cellular regions of the growth plate; and (d) direct bone formation by former growth plate chondrocytes. These processes, together with a loss of synchrony across the plate, would prevent further longitudinal expansion of the growth plate despite continued sporadic proliferation of chondrocytes. (J Histochem Cytochem 51:373383, 2003)
Key Words: bone growth, age, growth plate, histology, acellularity, remodeling, transdifferentiation, rat
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Introduction |
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Longitudinal growth of long bones depends on a functional growth plate with precise coordination, both temporally and spatially, of a multitude of processes. Overall growth is regulated systemically by growth hormone, with important contributions from glucocorticoids and thyroid hormone (
Despite all the advances made in recent years, understanding longitudinal bone growth is still a challenge to the cell biologist because it is difficult, in the growth plate of rapidly growing animals, to separate the multitudinous events temporally and spatially. In larger mammals (rabbit upwards), the growth plates close at skeletal maturity and longitudinal growth ceases. Smaller rodents (rats, mice), however, maintain a growth plate into old age (26 weeks of age, after which growth virtually ceases in rats (
We studied the age-related changes in growth plate morphology during the period of 216 weeks, which included periods of accelerating growth, skeletal maturity and decelerating growth. In the growing animals, we could relate the height and morphology of the growth plates to the rate of longitudinal growth, in agreement with previous studies. In the aged rats, we identified four categories of age-related changes in the growth plate, each of which altered growth plate function and could potentially explain and contribute to the observed cessation of growth.
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Materials and Methods |
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Animals
All animal experimentation was performed under license from the Home Office in accordance with the . Wistar rats, bred in the University of Southampton animal facility, were housed in plastic boxes in rooms maintained at 22C with a 12hr light cycle. All animals had free access to water and food. For studies of the growth plates of younger rats, rats were culled at 2, 4, 8, 12, or 16 weeks (two to four rats in each group). In addition, 10 rats were allowed to live without intervention until they died from natural causes or were culled if there was evidence of pain or distress. The time of death (or culling) ranged from 62 to 87 weeks.
Tissue Processing and Histology
Tibiae and femurs were removed and fixed in 4% paraformaldehyde for 48 hr and decalcified in 5% EDTA in 0.1 M Tris at 4C, which was replaced on a weekly basis until decalcification was complete. The proximal and distal parts of the bones were cut longitudinally. The proximal part of the femur was cut through the femoral head, whereas the distal part of the femur and the proximal portion of the tibia were cut coronally through the midline such that two symmetrical anterior and posterior portions remained. These bone samples were dehydrated in graded ethanols, cleared in chloroform, and embedded in paraffin. Longitudinal sections (6 µm) were obtained from the proximal and distal parts. These were mounted on poly-L-lysine-coated slides and stained by one of the following methods. Alcian blue and Sirius red were used (
Immunocytochemistry was carried out for proliferating cell antigen (PCNA), a marker of cell proliferation, the S-100 protein, a marker of cells of the chondrocytic lineage, and type I collagen, the typical bone-type collagen. The anti-PCNA (monoclonal anti-rat, 1:100 dilution) was obtained from DAKO (Glostrup, Denmark); the anti-S-100 antiserum (rabbit anti-bovine, 1:200 dilution) was obtained from Sigma (Poole, Dorset, UK), and the anti-type I collagen (LF-67, rabbit anti-human, crossreacts with rat, 1:300 dilution) was a gift from Dr. Larry Fisher (NIH, Bethesda, MD). For PCNA immunocytochemistry, sections were covered with the primary antibody overnight at 4C, followed by visualization with the avidinbiotin method with peroxidase and 3-amino-9-ethylcarbazole (AEC), yielding a brown reaction product. These sections were counterstained with 0.2% light green and 1% Alcian blue. Control sections were incubated with mouse serum (negative control; Sigma), and then treated as above. No staining was found in control sections. For double immmunocytochemistry of type I collagen and the S100 protein, sections were incubated with the anti-type I antibody for 2 hr at room temperature, followed by visualization with avidinbiotinperoxidase and AEC as above. The sections were then incubated with the anti-S100 antibody overnight and visualized using the alkaline phosphatase anti-alkaline phosphatase method (APAAP) with Fast blue, yielding a blue reaction product (
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Results |
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Changes in the Growth Plates Up to and Beyond Skeletal Maturity (216 Weeks)
Using the Alcian blueSirius red stain, the growth plate cartilage could be clearly distinguished from bone and appeared as a blue band between the secondary ossification center of the epiphysis and the metaphyseal spongiosa (Fig 1a1d). At 2 weeks, approximately half of the cartilaginous epiphysis (chondroepiphysis) had been replaced by the secondary ossification center (Fig 1a). At this age, the height of the growth plate could not be taken as the distance between the primary spongiosa and the secondary ossification center (bony epiphysis), because approximately one third of that distance still represented the epiphyseal cartilage of the chondroepiphysis rather than growth plate cartilage. The latter could be discerned from the morphology of the chondrocytes and is marked by the bar in Fig 1e. At 4 weeks, the secondary ossification center almost, but not quite, filled the chondroepiphysis. The height of the growth plate was now equal to the distance between the secondary center of ossification and the metaphysis, at least in the central part of the plate. By 8 weeks (not shown) and 12 weeks (Fig 1c), the height of the growth plate had started to decrease, with a further slight decrease evident at 16 weeks (not shown). In old rats, only a very thin band of growth plate cartilage remained (arrows in Fig 1d).
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To identify ongoing resorption, sections were stained for TRAP activity, which identifies resorbing cells, such as osteoclasts (
A more detailed examination of the growth plate structure at the various ages demonstrated that the heights of the metaphyseal growth plates changed considerably with age, as illustrated in Fig 2. Maximal growth plate height was achieved at 2 and 4 weeks (Fig 2a and Fig 2b) and at this stage the various zones of the growth plate, i.e., the reserve zone (RZ), proliferative zone (PZ), upper hypertrophic zone (UHZ), and lower hypertrophic zone (LHZ), were clearly distinguished. The lower hypertrophic zone was wide and included chondrocytes with cell heights up to 40 µm. The decrease in the heights of the growth plates was mostly due to loss of the lower hypertrophic zone (12 weeks; Fig 2c) and in old rats all cells were of similar size, which corresponded to the size of the proliferative cells of the younger growth plates (Fig 2d).
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To determine the incidence and location of cell proliferation, sections were stained for PCNA, a marker for proliferating cells (
Growth Plate Morphology of Old Rats
Although the heights of the growth plates changed with age until at least 16 weeks, the height within a given growth plate was fairly uniform, as illustrated by the 8-week-old tibial growth plate shown in Fig 3a, where the blue-staining band of growth plate cartilage separated the bony epiphysis from the endochondral bone of the spongiosa. In contrast, the growth plates of old rats were irregular and the spongiosa consisted of a few fairly thick spicules. In addition, several features were found that were not present in younger animals. These were (a) a thin band of bone matrix apposed horizontally to the growth plate cartilage (1 in Fig 3c, also seen as a red-staining band in Fig 3d), (b) large regions of cartilage that did not contain cells (2 in Fig 3c, better illustrated in Fig 4a4c) alternating with regions of high cellularity, (c) partial resorption of the core growth plate cartilage and replacement with bone (3 in Fig 3c, better seen in Fig 4g4i), and (d) apparent bone formation within the lacunae of growth plate chondrocytes (4 in Fig 3d, better seen in Fig 5). These features were found in all growth plates of aged rats, although the extent of each varied among animals.
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Horizontal Bone Band. During endochondral ossification in young growth plates, bone matrix is deposited onto struts of non-resorbed calcified cartilage that project into the marrow space at a right angle to the growth plate disc (Fig 1a1c and Fig 3a). In the aged rats, spongiosa was absent in some regions, presumably as a consequence of resorption (Fig 3c). In such areas, bone matrix was directly apposed to the cartilage, parallel to the growth plate (Fig 3d), effectively sealing the growth plate with bone at the metaphyseal border.
Acellularity and High Cellularity. In the growth plates of young rats, the cartilage core, which forms the center of the spicules of primary and secondary spongiosa, is usually thin and is only detectable only at higher magnification. By contrast, some spicules of aged rats contained a wide core of cartilage that was evident even at low magnifications and often persisted well below the average height of the growth plate (* in Fig 4a, Fig 4d4f, and Fig 4i). Closer examination of these cartilage cores revealed that no cells were present (Fig 4b and Fig 4c). Such acellular regions could extend from the former reserve zone to the vascular front and beyond into the spongiosa (Fig 4a), suggesting that the cartilage matrix was more resistant to resorption than adjacent cellular matrix. Acellular areas were also identified in the growth plates of 12- and 16-week-old animals, although less frequently (not shown). Adjacent to acellular regions, regions of high cellularity were frequently present (Fig 4d and Fig 4e). They were characterized morphologically by the deeper Alcian blue staining, which is indicative of recently synthesized proteoglycans (Fig 4e and Fig 4f), and by the fact that cells were present in clusters. The close proximity of the cells within a cluster (Fig 4f) suggested that the increased cellularity had resulted from cell division. However, PCNA was not detectable by immunocytochemistry (not shown) within these clusters, indicating that the cells had left the cell cycle, i.e., that the cell division had happened some time ago. Often the cellular regions appeared confined by adjacent acellular regions, suggesting that the expansion of the chondrocytes had been limited by external constraints.
Remodeling of Core Growth Plate Cartilage. In the growth plates of young rats, resorption of cartilage takes place at the vascular front in a coordinated and uniform manner, so that the overall height of the growth plate (the core cartilage) remains constant within an individual growth plate. Although the growth plates of old rats were irregular, it was still possible to approximate an average height of core cartilage by viewing the whole growth plate at low magnification, (see Fig 3c, shown at higher magnification in Fig 4h and Fig 4i). Acellular regions, as mentioned above, clearly extended below this average core into the spongiosa. In addition, vascular invasion from the marrow space had taken place into this core of growth plate cartilage in many samples from the aged rats (Fig 4g4i) and bone matrix had been deposited on the edges of the resorbed cartilage cavities (Fig 4h and Fig 4i). In location, this was different from the normal process of endochondral ossification, in which bone is deposited onto struts of cartilage that project below the height of the growth plate. We shall refer to this process as remodeling of core cartilage. In general, this remodeling was confined to the lower half of the growth plate, but could reach towards the upper third of the growth plate (Fig 4h and Fig 4i).
Intralacunar Bone Formation by Former Chondrocytes? A rarer feature than the previous three was the finding that a Sirius red-positive matrix surrounded some individual chondrocytes within the growth plate cartilage (Fig 5a5c). In the examples illustrated, the cells were found at some distance from vascular channels. In some lacunae, the matrix stained both red (typical for bone) and blue (typical for cartilage, arrows in Fig 5b). Occasionally cells were surrounded by a very thin line of red-staining matrix only (arrowheads in Fig 5b), which probably indicated a very early stage. More frequently, the Sirius red-positive matrix filled the lacuna or extended over several lacunae (* in Fig 5b).
The Sirius red-positive intralacunar matrix displayed the typical birefringence of bone matrix (Fig 5d) and was immunopositive for type I collagen (arrows in Fig 5e and Fig 5f), suggesting that bone matrix had been formed by former chondrocytes. In some cases (not shown), vascular invasion was closely associated with this intralacunar bone formation. Hence, the possibility that a lacuna that appeared intact in one section was connected to a vascular channel in the third dimension could not be excluded, and it was therefore possible that in these instances vascular-derived osteoblasts had deposited the bone matrix. However, in many cases (illustrated by Fig 5) bone matrix-containing lacunae were at a considerable distance from the vascular spaces, suggesting that the lacuna had remained intact and that the bone matrix had been synthesized by former chondrocytes.
To determine whether the type I collagen matrix co-localized with the chondrocyte-specific marker S100 (
Extensive intralacunar bone, such as that illustrated in Fig 5, was observed only in 2/10 animals, which raised the question of what was special about these two animals. Although we could not provide a definitive answer, we noted more evidence of anabolic bone formation within the medullary space in these animals compared with the rest, suggesting that these two rats had received some unknown anabolic stimulus.
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Discussion |
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This study first described the changes in rat growth plates that relate to the overall rate of longitudinal growth up to skeletal maturity, then identified those changes that occurred within the growth plates after growth had ceased. The overall height of the growth cartilage was an indication of the rate of longitudinal bone growth, in agreement with previous studies (
After 2830 weeks, growth virtually ceases in rats (
The presence of large acellular regions suggested that the chondrocytes, which had formed this cartilage, had undergone programmed cell death and that the cell remnants had been eliminated in situ. In apoptosis, the major type of programmed cell death (type I cell death), the end stage is the formation of apoptotic bodies, which are removed by heterophagocytosis (reviewed by
Acellular regions were observed to alternate with regions of high cellularity. Because ongoing proliferation could not be demonstrated in the old rats, the high cellularity suggested that cell proliferation had occurred at some stage after growth had ceased but that insufficient matrix synthesis had failed to separate the daughter cells. Morphologically, such regions resembled the proliferative zone of younger growth plates in some places, but the crucial difference was the lack of synchrony across the plate. Linear growth would be possible only if proliferation of one column of chondrocytes occurred in synchrony with its lateral neighbors across the entire plate. Because this was not the case in the aged rat growth plates, expansion, if it occurred at all, was localized and was also constricted by adjacent acellular regions as well as by bone formation at the metaphyseal edge.
The second feature found only in the growth plates of aged rats was remodeling within the core of growth plate cartilage, where the core is defined as that cartilage that was present in the growth plate when longitudinal growth ceased. The process was similar to endochondral ossification in that resorption of cartilage matrix was followed by bone deposition onto the walls of the resorbed cavities. The crucial difference was the location of this ossification, in that it was found within the core of growth plate cartilage, not within the vascular/marrow space as would be the case in younger rats. The degree of within-GP remodeling was related to the degree of overall bone remodeling (unpublished observations), suggesting that the growth plate was subject to the same factors that stimulated bone remodeling.
In addition to remodeling of core cartilage, a bone-like matrix was also observed within chondrocytic lacunae, suggesting that some chondrocytes in aged rat growth plates had become bone-forming cells. There has been considerable debate over the past 40 years about whether chondrocytes can become bone-forming cells under certain circumstances (
In summary, the present studies provide new insights into the mechanisms of restricting longitudinal growth in old rats despite the presence of a growth plate. The overall lack of synchrony of cellular events, the increase in the acellular areas combined with remodeling of the core cartilage, and sealing of the growth plate at the metaphyseal border resulted in a growth plate that was no longer functional so that longitudinal growth was restricted or impossible.
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Acknowledgments |
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We are grateful to Dr S. LangleyEvans and Dr A. AhieSayer (MRC Environmental Epidemiology Unit, University of Southampton) for providing the rat bones. We also wish to acknowledge the superb technical expertise of Ms Stefanie Inglis.
Received for publication March 21, 2002; accepted October 16, 2002.
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