Tissue & Cell, 2001 33 (4) 318±325 ß 2001 Harcourt Publishers Ltd DOI: 10.1054/tice.2001.0179, available online at http://www.idealibrary.com
Apoptotic bone cells may be engulfed by osteoclasts during alveolar bone resorption in young rats F. Boabaid,1 P. S. Cerri,2 E. Katchburian1 Abstract. The alveolar bone is a suitable in vivo physiological model for the study of apoptosis and interactions of bone cells because it undergoes continuous, rapid and intense resorption/remodelling, during a long period of time, to accommodate the growing tooth germs. The intensity of alveolar bone resorption greatly enhances the chances of observing images of the extremely rapid events of apoptosis of bone cells and also of images of interactions between osteoclasts and osteocytes/ osteoblasts/bone lining cells. To find such images, we have therefore examined the alveolar bone of young rats using light microscopy, the TUNEL method for apoptosis, and electron microscopy. Fragments of alveolar bone from young rats were fixed in Bouin and formaldehyde for morphology and for the TUNEL method. Glutaraldehyde±formaldehyde fixed specimens were processed for transmission electron microscopy. Results showed TUNEL positive round/ovoid structures on the bone surface and inside osteocytic lacunae. These structures ± also stained by hematoxylin ± were therefore interpreted, respectively, as osteoblasts/lining cells and osteocytes undergoing apoptosis. Osteoclasts also exhibited TUNEL positive apoptotic bodies inside large vacuoles; the nuclei of osteoclasts, however, were always TUNEL negative. Ultrathin sections revealed typical apoptotic images ± round/ ovoid bodies with dense crescent-like chromatin ± on the bone surface, corresponding therefore to apoptotic osteoblasts/lining cells. Osteocytes also showed images compatible with apoptosis. Large osteoclast vacuoles often contained fragmented cellular material. Our results provide further support for the idea that osteoclasts internalize dying bone cells; we were however, unable to find images of osteoclasts in apoptosis. ß 2001 Harcourt Publishers Ltd Keywords: alveolar bone, apoptosis, bone resorption, osteoclasts, bone remodelling
Introduction Bone is a living, continuously renewing tissue that depends upon a multitude of interacting systemic and local 1 Department of Morphology, Federal University of SaÄo Paulo (UNIFESP), S.P., Brasil 2 Department of Morphology, SaÄo Paulo State University (UNESP), S.P., Brasil
Received 5 October 2000 Accepted 23 January 2001 Correspondence to: Professor Eduardo Katchburian, Department of Morphology, School of Medicine, Federal University of SaÄo Paulo, Rua Botucatu, 740, 04023±900 SaÄo Paulo, S.P., Brasil. Tel: 55 11 5575±2881; Fax: 55 11 5576 4328; E-mail: [email protected]
factors for its homeostasis. Systemic factors that have a direct effect on bone include parathyroid hormone, calcitonin, 1, 25 ± dihydroxy vitamin D3 and other hormones such as estrogens. Local factors include molecules such as prostanoids, growth factors (BMPs, TGF-b, TNF-a) interleukins (IL-1, IL-6, IL-11) and other cytokines (Raisz & Rodan, 1998). The combined action of these factors, often simultaneous, regulate bone cell proliferation, differentiation, function and survival (Boyce et al., 1999). More recently, however, it has been shown that some of these factors are capable of switching on the chain of molecular events leading to apoptosis or programmed cell death of bone cells (Hill et al., 1997). Thus, accumulated
APOPTOSIS AND ALVEOLAR BONE RESORPTION
evidence over recent years points in the direction that apoptosis might in fact play a signi®cant role in the overall maintenance of bone mass and structure (Hughes & Boyce, 1997; Noble et al., 1997; Jilka et al., 1998; Boyce et al., 1999). However, the fate of apoptotic osteocytes and/ or osteoblasts during bone resorption remains poorly understood (Elmardi et al., 1990). It has been suggested that osteoclasts engulf and destroy dying bone cells (Taniwaki & Katchburian, 1998) and, more recently, it has been proposed that osteoclasts are in fact attracted to resorption sites by signals originating from dying (apoptotic) osteocytes or osteoblasts/bone lining cells (Bronckers et al., 1996; Tomkinson et al., 1997; Tomkinson et al., 1998). The alveolar bone is a suitable in vivo physiological model to study apoptosis and interactions of bone cells because it undergoes continuous, rapid and intense resorption/remodelling, during a long period of time, to accommodate the growing tooth germs (Marks & Schroeder, 1996; Ten Cate, 1998). The intensity of alveolar bone resorption greatly enhances the chances of observing images of the extremely rapid events of apoptosis of bone cells and also of images of interactions between osteoclasts and osteocytes/osteoblasts/bone lining cells (Taniwaki & Katchburian, 1998). In an attempt to ®nd such images, we have therefore examined the alveolar bone of young rats using light microscopy, the TUNEL method for apoptosis, and transmission electron microscopy.
Materials and Methods Animals Alveolar bone from the upper maxilla of 1-, 3-, 5-, 14- and 29 day-old Wistar rats from both sexes, obtained from the Federal University of SaÄo Paulo animal house, were used in the experiments. The animals were anaesthetized with ether, decapitated, and fragments of the alveolar bone overlaying the upper ®rst molars were removed with the help of a dissecting microscope and placed in the ®xative solution (see below). Principles of laboratory animal care (NIH publication 85±23 1985) and national laws on animal use were observed. Light microscopy The specimens were ®xed in Bouin at room temperature for 24 hours and dehydrated in graded ethanols, embedded in paraf®n and 5-mm sections were obtained. Sections (5-mm thick) were stained with hematoxylin and eosin (HE) and examined in a Olympus BX-50 light microscope. Tartrate resistant acid phosphatase (TRAP) For demonstration of TRAP activity, we used the azo dye method (Burstone, 1965). The specimens were ®xed in 4% formaldehyde (from paraformaldehyde) for 24 hours, at
room temperature, decalci®ed in 7% EDTA solution for 7 days, at 48C. After decalci®cation, the specimens were dehydrated in graded ethanols, embedded in paraf®n and 5-mm sections were obtained. The incubating solution was prepared by dissolving 70 mg of naphtol AS-BI (Sigma, St Louis, MO, USA) in 250-ml of N.N-dimethylformamide, followed by addition of 50 ml of 0.2 mM sodium acetate buffer (pH 5.2) and 70 mg of Fast Red Salt TR (Sigma, St Louis, MO, USA) as the coupling agent; 115 mg of sodium tartrate dehydrate was added to the solution. The sections were incubated at 378C for 2 hours, washed in distilled water and stained with hematoxylin. TUNEL method The Oncor Apoptosis Detection Kit peroxidase (Apoptag-Plus Oncor, Gaithersburg, MD, USA) was used (Gavrieli et al., 1992). The specimens were ®xed in 4% formaldehyde (from paraformaldehyde) for 24 hours, at room temperature, decalci®ed in 7% EDTA solution for 7 days, at 48C. After decalci®cation, the specimens were dehydrated in graded ethanols, embedded in paraf®n, and 5-mm sections were obtained and adhered to silanized slides. Some specimens were ®xed in Bouin's solution, embedded in paraf®n and 5-mm sections were obtained. Deparaf®nized sections were washed in PBS (50-mM sodium phosphate, pH 7.4, 200mM NaCl) for 5 minutes and pre-digested with proteinase K solution (20mg/ml in PBS) for 15 minutes at room temperature to expose the DNA strands. The sections were subsequently treated with 3% hydrogen peroxide for 15 minutes to block endogenous peroxidase and immersed in a equilibration buffer for 5 minutes. This was followed by incubation in a solution containing terminal deoxynucleotidyl transferase (TdT) at 378C for 1 hour in a humid chamber. The reaction was stopped by incubation in the stop/wash solution at 378C for 30 minutes. The sections were then incubated in the anti-digoxigenin±peroxidase solution in a humid chamber for 30 minutes. After washing in PBS, they were treated with 0.06% 3.3 diaminobenzidine (Sigma, St Louis, MO, USA) in PBS for 5 minutes at room temperature. The sections were counterstained with methyl green or Carazi's hematoxylin and dehydrated, rinsed in xylol and mounted in Entellan medium. The same procedure as above was used on sections of mammary gland (positive control) provided by the manufacturer of the kit. Negatives controls were prepared by replacing TdT enzyme by distilled water. Transmission electron microscopy The specimens were ®xed in a mixture of 4% glutaraldehyde and 4% formaldehyde (from paraformaldehyde) buffered at pH 7.4 (0.1M sodium cacodylate) (Katchburian & Holt, 1972) for 24 hours, at 48C. The specimens were decalci®ed in 7% EDTA buffered at pH 7.4 (0.1M sodium cacodylate) for 7 days, at 48C, washed in cacodylate
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M L S L
Fig. 1 (A±E) Light micrographs of portions of the alveolar bone stained by hematoxylin±eosin (HE), with the exception of 1B, which was stained by the
TRAP method. In A, a low magnification view shows two large osteoclasts (OC) apposed to the bone surface. Osteoblasts/lining cells (arrowheads) are often located between the osteoclasts and the matrix (M) surface. Some osteocytes (S) are also observed within lacunae of the matrix (M). X330. In B, Tartrateresistant acid phosphatase (TRAP) activity is observed in an osteoclast (OC) located in a superficial lacuna. M, matrix; S, osteocytes. X660. In C, two round/ ovoid structures (V1, V2), containing several small bodies (arrows) densely stained by hematoxylin appear to be inside vacuolar structures of osteoblasts/
APOPTOSIS AND ALVEOLAR BONE RESORPTION
lining cells. X1700. In D, a large osteocytic lacuna contains an irregularly shaped cell (S1) exhibiting several round dense bodies stained by hematoxylin (arrows); an oval-shaped cell (S2) with a condensed nucleus is also observed. M, matrix. X1700. In E, a multinucleated osteoclast (OC), located inside a deep excavation of the bone matrix (M) surface, exhibits a large vacuole (V) containing a dense round/ovoid body (arrow) strongly stained by hematoxylin. X1700. Fig. 2 (A±F) Light micrographs of portions of the alveolar bone stained by the TUNEL method and counterstained with methyl green (A, C, D, F) or hematoxylin (B, E). All TUNEL-positive structures exhibit varying degrees of yellow±brown colour. In 2A, a round/ovoid TUNEL-positive structure (arrow), apposed to the bone surface is in contact with a neighbouring osteoblast/lining cell (B). M, matrix; S, osteocytes. X1700. In B, a small flat and elongated TUNEL-positive structure (arrow) is located between an osteoclast (OC) and the matrix (M) surface. X1700. In C, two osteocytic lacunae (L) contain fragmented and irregularly distributed TUNEL-positive material that may appear condensed (arrows) in some regions. M, matrix. X1700. In D, another osteocytic lacuna (L) shows a TUNEL-positive round/ovoid body (arrow) and a somewhat diffuse staining at the periphery (arrowheads). M, matrix. X1700. In E, a round/ovoid TUNEL-positive body (arrow) is observed inside a large vacuole near the TUNEL-negative nuclei (N) of an osteoclast. Compare with Fig. 1E. M, matrix. X1700. In F, a large round/ovoid TUNEL-positive body (arrow) is almost entirely surrounded by a portion of the cytoplasm of an osteoclast (OC) at the bone surface. Another smaller round TUNEL-positive body (arrowhead) is observed in the region. M, matrix. X1700.
buffer and transferred to a solution of cacodylate buffered 1% osmium tetroxide for 1 hour at 48C. After dehydration in graded ethanols, the specimens were trated with propilene oxide prior to embedding in Araldite. Toluidine-stained semithin sections were examined with a light microscope for selection of regions to be trimmed. Ultrathin sections were collect onto grids and stained with uranyl acetate and lead citrate before examination in a ZEISS EM 900 electron microscope.
Results The alveolar bone revealed the typical appearance of bone undergoing rapid resorption with numerous osteoclasts of variable shapes apposed to the matrix surface, osteocytes within lacunae and osteoblasts and/or lining cells on the surface (Fig. 1A). Occasionally, some osteoblasts/lining cells were located between elongated osteoclasts and the matrix surface (Fig. 1A). The osteoclasts exhibited conspicuous TRAP activity (Fig. 1B). After extensive examination of the preparations, we detected the presence of small round/ovoid bodies, intensely stained by hematoxylin, on the bone surface (Fig. 1C). Similarly, dense round/ovoid bodies were detected within large osteocytic lacunae, apparently associated with irregularly shaped osteocytes. Sometimes, the same lacuna was shared by another osteocyte, often with a condensed nucleus (Fig. 1D). In regions of intense resorptive activity, we detected some osteoclasts, often located within a deep excavation, exhibiting a large vacuole containing a dense round/ovoid body strongly stained by hematoxylin (Fig. 1E). Examination of the TUNEL-stained sections showed that some structures within the matrix and on its surface were positive, exhibiting thus various degrees of yellow±brown colour. Round/ovoid TUNEL positive structures were detected on the bone surface, in close proximity to TUNEL negative osteoblasts/lining cells (Fig. 2A). Sometimes, an elongated TUNEL positive structure was found between an osteoclast (TUNEL negative) and the bone surface (Fig. 2B). Within osteocytic lacunae, small round/ovoid TUNEL positive bodies, densely stained, were observed in association with
fragmented, irregularly distributed and somewhat diffuse TUNEL-positive material (Figs 2C and D). All osteoclasts showed TUNEL-negative nuclei. Some osteoclasts, however, contained a round/ovoid densely stained TUNEL-positive body inside a large vacuole (Fig. 2E), similar to that observed Fig. 1E In other instances, we observed a large TUNEL-positive round/ovoid body almost entirely surrounded by a portion of an osteoclast (Fig. 2F). Controls for the TUNEL method using involuting mammary gland sections revealed positive structures. Sections incubated in medium lacking TdT enzyme were negative. Examination of ultrathin sections revealed that some regions of the matrix contained round/ovoid dense bodies on its surface instead of the typical osteoblasts/lining cells (Fig. 3). These bodies, located inside large vacuoles, exhibited crescent-like images of condensed peripheral chromatin. Furthermore, these bodies were frequently observed in close contact with the cytoplasm of a neighbouring osteoclast (Fig. 3) that sometimes emitted cytoplasmic projections that partially surrounded the dense body (Fig. 4). However, some vacuoles occasionally showed only membrane whorls (Fig. 3). In other regions, we observed shrunken cells, within opened up lacunae, containing irregularly shaped nuclei and long thin cytoplasmic projections (Fig. 5). In similar regions, however, we also observed cells with an apparently intact cytoplasm, but with a nucleus almost completely ®lled by condensed chromatin. These cells, presumably immature osteocytes or late osteoblasts, were partially surrounded by a portion of the cytoplasm of an osteoclast (Fig. 6). We also observed that some osteoclasts exhibited a large vacuole containing membranous and granular fragments of cytoplasm and an irregularly shaped nucleus with blocks of condensed peripheral chromatin (Fig. 7).
Discussion Our results con®rm and extend previous reports that showed apoptosis of osteocytes and osteoblasts and/or lining cells during alveolar bone resorption. Apoptosis in the alveolar bony crypt coexisted with numerous multishaped TRAP-positive osteoclasts ± a speci®c marker for
BOABAID ET AL.
V2 OC RER VA
N VA VE
Fig. 3 Electron micrograph of a portion of the alveolar bone surface. Three large vacuolar structures (V1, V2, V3) are present between the matrix (M)
surface and a portion of an osteoclast (OC). Vacuole V1 contains a large round/ovoid body which shows dense structures of varied sizes: two of them (arrows) exhibit crescent-like structures typical of condensed peripheral chromatin. Vacuole V2 shows a dense body (arrow) containing a crescent-like structure typical of condensed peripheral chromatin. Vacuole V3 shows whorls of membrane profiles. The osteoclast cytoplasm shows several mitochondria (MT), vacuoles (VA) and rough endoplasmic reticulum (RER). X11000. Fig. 4 Electron micrograph of a portion of the alveolar bone. A large round/ovoid body on the left (P), presumably a portion of a cell nucleus, exhibits a conspicuous mass of crescent-like condensed peripheral chromatin (arrow). A portion of a multinucleated osteoclast (OC), containing mitochondria (MT), vacuoles (VA) and vesicles (VE), shows cytoplasmic projections (arrowheads) that
APOPTOSIS AND ALVEOLAR BONE RESORPTION
partially surround the body (P). N, nuclei. X7800. Fig. 5 Electron micrograph of a portion of the alveolar bone. An osteocyte (S), within an opened up lacuna of the matrix (M), shows a highly convoluted nucleus (N) containing irregular masses of condensed peripheral chromatin (arrows). The shrunken cell cytoplasm, exhibiting apparently intact organelles, shows several irregular thin surface projections (arrowheads). X8400.
these cells (Hughes et al., 1995; Kameda et al., 1997). Preparations stained by HE revealed round/ovoid dense bodies ± strongly stained by hematoxylin ± on some regions of the matrix surface, inside osteocytic lacunae and also within osteoclast vacuoles. Likewise, TUNELpositive bodies were present on the matrix surface, inside osteocytic lacunae and in conspicuous osteoclast vacuoles, indicating therefore DNA fragmentation. The diffuse TUNEL staining observed in osteocytic lacunae was probably due to leakage of DNA fragments from osteocyte nuclei (Wheeldon et al., 1995; Rojo & Gonzalez, 1998). The above results may therefore be interpreted as indicating apoptosis of osteocytes and osteoblasts/lining cells and also that apoptotic cells or bodies may be internalized by osteoclasts. These results were supported by our ultrastructural observations that showed dense round/ovoid bodies containing crescent-like condensed peripheral chromatin on the matrix surface and often in close juxtaposition to osteoclasts. These ultrastructural images are characteristic of apoptotic bodies as described in other tissues (Kerr et al., 1972; Wyllie et al., 1980; Majno & Joris, 1995). We believe that our results have shown for the ®rst time, in vivo, typical ultrastructural images of apoptotic bodies on the bone surface. We also observed ultrastructural images of osteocytes or late osteoblasts in what appeared to be initial stages of apoptosis. Apoptotic cells/bodies, mostly observed in close apposition to osteoclasts, were often surrounded by osteoclast cytoplasm processes. Some osteoclasts exhibited large vacuoles containing fragmented membranous cell constituents and apoptotic nuclei. As expected, the images of apoptosis were not frequent and were therefore dif®cult to ®nd, particularly in electron micrographs. There was no observable pattern in the distribution of apoptotic images and they were not present in all sections examined. This is consistent with observations in other tissues where TUNEL-positive structures and images of apoptosis were not frequently observed (Gavrieli et al., 1992; Coles et al., 1993; Vaahtokari et al., 1996; Baratella et al., 1999; Cerri et al., 2000). In tissue culture (in vitro) experiments, however, images of apoptosis are abundant (Dempster et al., 1997; Kameda et al., 1995; Kameda et al., 1997; Noble et al., 1997); in tissue sections (in vivo) they are not readily observed because apoptotic bodies are quickly removed by neighbouring cells or macrophages (Majno & Joris, 1995; Raff, 1998). For the reasons aforementioned, we felt that quanti®cation was unlikely to produce meaningful results since the number of apoptotic bodies present in a given time is variable for different tissues and also depends upon the speed in which apoptotic bodies are
removed. In the thymus, for example, 97% of the developing thymocytes die but only 0, 2% appear TUNELpositive (Coles et al., 1993). For tissues such as bone and cartilage, where most cells are inside lacunae, apoptotic cells/bodies can only be phagocytosed when the lacunae open up at the surface. The fate of apoptotic cells enclosed inside lacunae located deep in the tissue is not understood (Gibson et al., 1995; Roach et al., 1995; Blanco et al., 1998; Roach et al., 2000). In addition to osteocyte apoptosis (Kaneko et al., 1997; Noble et al., 1997; Tomkinson et al., 1997; Tomkinson et al., 1998; Boyce et al., 1999), our results strongly support the view that osteoblasts/lining cells also undergo apoptosis during bone degradation (Hill et al., 1997; Kaneko et al., 1997; Landry et al., 1997; Jilka et al., 1998). Images of apoptosis were more frequent for osteocytes than osteoblasts/lining cells probably because osteocytes, as mentioned earlier, are enclosed inside lacunae and cannot be removed quickly (Bronckers et al., 1996; Tomkinson et al., 1997). Osteocytes and osteoblasts/lining cells communicate with each other via canaliculi and by means of well-developed and extensive gap junctions (Lanyon, 1993; Raisz & Rodan, 1998). It has been proposed by some authors that the initial signals for apoptosis are captured by osteocytes, thereafter spreading to osteoblasts and possibly to osteoclasts during bone remodelling (Bronckers et al., 1996; Noble et al., 1997; Boyce et al., 1999). However, it has also been suggested that osteoblasts possess receptors for apoptosis-inducing molecules or signals (Hill et al., 1997; Jilka et al., 1998). Our results provide further support for the idea that osteoclasts engulf and destroy apoptotic bone cells or bodies, i.e. osteocytes and possibly osteoblasts, as previously suggested (Bronckers et al., 1996; Noble et al., 1997; Tomkinson et al., 1998). We cannot exclude, however, the possibility that neighbouring osteoblasts/lining cells may also be able to engulf apoptotic bodies since in most other tissues apoptotic bodies are removed by the combined action of neighbouring same-type cells and macrophages (Majno & Joris, 1995; Raff, 1998). Our results do not support the strongly held view that osteoclasts undergo apoptosis during bone resorption, being therefore in agreement with the ®ndings of Jilka et al. (1998). We never observed apoptotic changes in osteoclast nuclei; apoptotic structures were always found inside large osteoclast vacuoles. However, images of apoptotic osteoclasts have been described, mainly in culture (Kameda et al., 1995), particularly after exposure to biphosphonates (Hughes et al., 1995), glucocorticoids (Dempster et al., 1997) and estrogens (Hughes et al., 1996; Kameda et al., 1997; Suda et al., 1997).
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OC VA N
M N MT
OC M VE
7 Fig. 6 Electron micrograph of a portion of the alveolar bone. A cell, presumably an immature osteocyte or late osteoblast (B), inside an opened up lacuna, exhibits a nucleus (N) which is almost entirely filled by condensed chromatin (arrows), and a cytoplasm with apparently intact organelles. A portion of an osteoclast (OC), containing mitochondria (MT), vacuoles (VA) and vesicles (VE), apposed to the bone surface, appears to make contact with this cell (B), partially surrounding it. M, matrix; CZ, clear zone. X9000. Fig. 7 Electron micrograph of a portion of the alveolar bone. A portion of the cytoplasm of an osteoclast (OC) shows a very large vacuole (V) containing membranous and granular fragments and an irregularly shaped cell nucleus (NU) with blocks of condensed peripheral chromatin (arrows). A osteoblast/lining cell (B) is present between the osteoclast and the bone matrix (M). N, nuclei; MT, mitochondria; VA, vacuoles; VE, vesicles. X8100.
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