ARTICLE |
Correspondence to: H. Clarke Anderson, Dept. of Pathology and Laboratory Medicine, U. of Kansas Medical Center, Kansas City, KS 06160. E-mail: handerso@kumc.edu
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Summary |
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We assessed the distribution and relative staining intensity of bone morphogenetic protein (BMP)-17 by immunohistochemistry in tibial growth plates, epiphyses, metaphyses, and articular cartilage in one 21-week and one 22-week human fetus and in five 10-week-old SpragueDawley rats. In the rats, articular cartilage was also examined. BMP proteins were mostly cytoplasmic, with negligible matrix staining. Highest BMP levels were seen in (a) hypertrophic and calcifying zone chondrocytes of growth plate (BMP-17), (b) osteoblasts and/or osteoprogenitor fibroblasts and vascular cells of the metaphyseal cortex and medulla (BMP-16), (c) osteoclasts of the metaphysis and epiphysis (BMP-1,-4,-5, and -6), and (d) mid to deep zone articular chondrocytes of weanling rats (BMP-17). BMP staining in osteoclasts, an unexpected finding, was consistently strong with BMP-4, -5, and -6 but was variable and dependent on osteoclast location with BMP-2,-3, and -7. BMP-17 were moderately to intensely stained in vascular canals of human fetal epiphyseal cartilage by endothelial cells and pericytes. BMP-1,-3,-5,-6, and -7 were localized in hypertrophic chondrocytes adjacent to cartilage canals. We conclude that BMP expression is associated with maturing chondrocytes of growth plate and articular cartilage, and may play a role in chondrocyte differentiation and/or apoptosis. BMP appears to be expressed by osteoclasts and might be involved in the intercellular "cross-talk" between osteoclasts and neighboring osteoprogenitor cells at sites of bone remodeling. (J Histochem Cytochem 48:14931502, 2000)
Key Words: apoptosis, articular cartilage, bone, bone morphogenetic protein, calcification, cartilage, endochondral ossification, growth plate, osteoblast, osteoclast
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Introduction |
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CHONDROCYTES of the active growth plate exist in an ordered temporal and spatial array. Most cell division occurs in the proliferative zone of the growth plate, which contains mother chondrocytes with daughter cells stacked in columns beneath them. By coordinated cell birth in the proliferative zone and cell death in the lower hypertrophic cell zone, a constant distance is maintained between proliferating cells and the underlying bony metaphysis (
Chondrocyte maturation and vesiculation are likely to be controlled by external molecular cues as well as by timed expression of internal genes. Expression by growth plate chondrocytes of parathyroid hormone-related peptide (PTHrp) (
In recent years a growing list of BMPs has been isolated, cloned, and sequenced. The osteoinductive BMPs, especially BMP-27, which were shown to induce ectopic bone formation after subcutaneous implantation in rats, are all a part of the TGF-ß superfamily of proteins (
An accumulating body of evidence indicates that BMPs not only function to promote osseous differentiation but also are involved in triggering apoptosis. In addition to the evidence linking BMP-7 and the BMP-IB receptor to apoptosis of growth plate chondrocytes (
Because of the apparent role of BMPs in promoting both differentiation and apoptosis of chondrocytes, it is not surprising that the first reports of BMP localization in growth plate have indicated greatest expression of BMP-17 and BMP receptors in hypertrophic cells where both processes occur (
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Materials and Methods |
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Tissue Preparation
Upper tibial growth plates with attached metaphyseal and epiphyseal bone and articular cartilage were obtained from five weanling SpragueDawley rats, sliced once longitudinally, and fixed for 2448 hr in 4% paraformaldehyde. The same region of upper tibia from two 21-week gestation human fetuses with normal limb development was removed and fixed in 4% paraformaldehyde. In both human cases, death occurred spontaneously in utero at approximately 12 hr before fixation. After fixation, the tissues were decalcified in 10% EDTA in distilled water for approximately 1 week at room temperature with daily agitation. The decalcified samples were dehydrated in changes of ethanol at increasing concentration, embedded in low melting temperature paraffin, and sectioned at 5-µm thickness.
Immunoperoxidase Staining
Embedding medium was removed from the sections by exposure to xylene, followed by incubation in ethanol solutions (100% ethanol followed by 95%) to begin rehydration. Deparaffinized, rehydrated sections were incubated in 3% Triton X-100 in PBS to permeabilize and were blocked with DAKO (Carpinteria, CA) blocking solution before incubation overnight in primary antibody solution. Primary antibodies were diluted in PBS as follows: anti-BMP-1, 1:2000; anti-BMP-2, -3, -5, -6, and -7, 1:100; anti-BMP-4, 1:5. After incubation in primary antibody, sections were briefly incubated in the appropriately diluted secondary antibody (biotinylated anti-rabbit, anti-mouse, or anti-goat immunoglobulins), followed by incubation in streptavidinperoxidase and buffered substrate solution (pH 7.5) containing hydrogen peroxide and 3,3-diaminobenzidine chromogen solution. (All of the above reagents were purchased from DAKO). The sections were counterstained in 0.5% Mayers' hematoxylin (DAKO) or, in some instances, in 5% toluidine blue in 100% ethanol.
Primary Antibodies
Anti-BMP-1/tolloid rabbit antibody was a polyclonal raised against recombinant human BMP and crossreacts with BMP-1 and tolloid but not with other BMPs or TGF-ß (kindly provided by Dr. Paul Reynolds, University of Rochester). Rabbit anti-BMP-2 and -3 were polyclonal antibodies raised against recombinant human BMP-2 (serum ID W12) and -3 (serum ID W22) (kindly provided by Dr. I.K. Moutsatsos, Genetics Institute, Inc.). Anti-BMP-2 is specific for BMP-2 and anti-BMP-3 crossreacts with BMP-2 and -5. Anti-BMP-4 was a monoclonal raised against recombinant human BMP-4 and is monospecific for BMP-4 (kindly provided by Dr. K. Masuhara, Osaka University). Rabbit anti-BMP-6 polyclonal antibodies, raised against the precursor and mature forms of recombinant human BMP-6, are specific for these forms of BMP-6 and do not react with other BMPs (kindly provided by Dr. S. Gitelman, University of California, San Francisco). Anti-BMP-5 and -7 were goat polyclonal antibodies obtained from Santa Cruz Biotechnology (Santa Cruz, CA). Both anti-BMP-5 and -7 were raised against C-terminal peptides of the mature regions of BMP-5 and -7 and do not crossreact with other BMPs.
Human and Animal Subjects
Human embryonic tissues were obtained at autopsy under authorization by the University of Kansas Medical Center Human Subjects Review Board. Rat tissues were obtained after humane sacrifice using procedures authorized by the University of Kansas Medical Center Institutional Animal Care and Use Committee, ACUP No. 86-02-03-01.
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Results |
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Growth Plate
There was a similar relative distribution of BMP-17 in rat and human growth plates (Table 1 and Table 2). BMP staining was highest in the cytoplasm of maturing and late hypertrophic chondrocytes, with perhaps a slight predominance during maturation and early hypertrophy (Fig 1 and Fig 2). Staining of all BMPs was light and variable in reserve and proliferative zone chondrocytes. The BMPs were not detected to any significant degree in cartilage or bone matrix.
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Metaphysis
Osteoblasts showed moderate to intense staining for all the BMPs (Table 1 and Table 2). However, with some BMPs (especially BMP-2, -3, -5, -6, and -7), endosteal osteoblast staining varied from intense to undetectable depending on their location (Fig 3). Osteoblasts of the metaphyseal cortex were more uniformly positive. With BMP-2, -3, -5, -6, and -7 in rat and BMP-3 and 7 in human, marrow stromal cells and/or vascular cells adjacent to endosteal osteoblasts were often positively stained (Fig 3B).
Osteoclasts
These resorptive cells were identified by their multinuclearity and their location close to trabecular and subperiosteal bone surfaces of cortex, metaphysis, and epiphyseal secondary ossification centers. BMP-17 were stained in the cytoplasm of rat and human osteoclasts (Fig 4). Staining for BMP-1, -4, -5, and -6 was moderate to intense in virtually all osteoclasts (Table 1 and Table 2; Fig 4). Immunostaining for BMP-2 and 3 was more variable, with some negatively-reacting osteoclasts. BMP-7 staining was minimal in rat osteoclasts (Table 1).
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Articular Cartilage and Vascular Canals
In weanling rat, BMP-17 immunostaining was most intense in the cytoplasm of mid-zonal (maturing) and mid- to deep-zonal (hypertrophic) chondrocytes of articular cartilage (Table 3; Fig 5A). BMP-1 staining was less intense and more variable in surface articular chondrocytes (Table 3).
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In human fetal epiphyses, the secondary ossification centers had not yet formed and vascular canals penetrated these regions, which were composed primarily of relatively undifferentiated cartilage. Chondrocytes located 510 cell layers deep to the cartilage canals were larger in diameter, suggesting the onset of hypertrophy, and they stained moderately to intensely for BMP-17 (Table 3; Fig 5B). An unexpected finding was the presence of significant cytoplasmic immunostaining for BMP-17 in endothelial cells and/or pericytes of the vascular canals (Fig 5B).
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Discussion |
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This study has confirmed earlier reports of localization of BMP-1, -4, -6, and -7 in maturing hypertrophic and apoptotic chondrocytes of growth plate (
Much experimental evidence already reported indicates a role for BMPs in the promotion of skeletal cell differentiation. BMP-2, -4, -6, and -7 can induce heterotopic cartilage and bone formation when implanted subcutaneously into rats and mice (
In growth plate, however, chondrocyte differentiation and maturation overlap and blend into the subsequent phase of programmed cell death, and both phases are associated with high intracellular levels of BMPs. Given the continuum between differentiation and programmed cell death in growth plate chondrocytes, and the overlap of high BMP expression levels during both phases, it may prove difficult to define and experimentally separate the differentiation-promoting from the cell death-promoting activities of BMPs in these cells. Certainly, there is evidence for a role of BMPs in promoting apoptosis during skeletal development (
To our knowledge, this study presents the first demonstration of BMPs in chondrocytes associated with epiphyseal cartilage vascular canals. Moderate to intense staining of BMP-17 was seen in perivascular hypertrophic chondrocytes and of BMP-17 in endothelium and pericytes of cartilage vessels (Table 3; Fig 5). Cartilage canals are believed to participate in the development of secondary ossification centers of the epiphysis. However, the interaction between these penetrations and the coincident hypertrophy of chondrocytes and the mineralization of juxtaposed cartilage matrix is poorly understood. As recently demonstrated by
It is not widely recognized that osteoclasts (as well as osteoblasts) contain significant amounts of BMPs. In this study we have demonstrated staining of BMP-17 by osteoclasts in human fetal and weanling rat metaphysis, a site at which active osteoclast-mediated bone remodeling occurs. Our finding of BMPs in osteoclasts could possibly be due to an artifact related to immunostaining procedures and does not prove that the presence of BMP proteins in osteoclasts is necessarily due to local BMP expression. It could, for example, be due to phagocytosis of extracellular BMPs by osteoclasts. Nevertheless, this supports our preliminary reports of BMP-1, -4, and -6 in growth plate osteoclasts (
It also has been suggested that BMPs, deposited in bone matrix by osteoblasts, might be released for paracrine osteogenic activity from osteoclastic resorption sites of bone remodeling (
Received for publication January 5, 2000; accepted May 16, 2000.
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