JOURNAL OF PATHOLOGY, VOL. 180: 430433 (1996)

MORPHOLOGICAL AND BIOCHEMICAL EVIDENCE FOR APOPTOSIS IN THE TERMINAL HYPERTROPHIC

CHONDROCYTES OF THE GROWTH PLATE

MICHIHISA ZENMYO, SETSURO KOMIYA, RIKIMARU KAWABATA*, YASUYUKI S A S A G U R I ~ , AKIO INOUE AND MINORU MORIMATSU*

Departments of Orthopaedic Surgery and *Pathology, Kurume University School of Medicine, Kururne 830, Japan; ?Department of Pathology, University of Occupational and Environmental Health, School of Medicine, Yahatu Nishi-ku, Kitakyushu 807, Japan

SUMMARY

The purpose of this study was to investigate the mechanism of cell death in chondrocytes of the growth plate. In the degenerative chondrocyte zone of the growth plate, apoptotic chondrocytes were defeated by the in situ nick end labelling method, by DNA analysis in agarose gel, and by electron microscopy. The results of the in situ nick end labelling method and the occurrence of a ladder pattern of DNA in agarose gel analysis indicated the activation of endogenous endonucleases, resulting in DNA fragmentation. Electron micrographs showed the early morphological changes associated with apoptosis. This report presents both morphological and biochemical evidence for apoptosis in the terminal hypertrophic chondrocytes of the growth plate. These data suggest that apoptosis of degenerative chondrocytes may play an important role in the control of normal and pathological endochondral ossification.

KEY WORDS-apOptOSiS; chondrocytes; growth plate

INTRODUCTION

Chondrocytes of the growth plate cartilage, through the process of endochondral ossification, are responsible for controlling both the rate and the extent of long bone growth during postnatal development.' The functional unit within the growth plate consists of a column of chondrocytes that pass through a sequence of phases including proliferation, hypertrophy, degeneration, and death. The ultimate fate of the terminal hypertrophic chondrocytes (degenerative chondrocytes) is an import- ant problem, because they reside at the critical interface between cartilage and bone at the last transverse septum.

Although some previous investigators have reported that the terminal hypertrophic chondrocytes die at the last transverse septum,l there is no report that refers to the mechanism of death of these cells. Mechanisms of cell death in cartilage development are essentially uncharacterized. Farnum et al. showed ultrastructural evidence of cellular condensation in the terminal hyper- trophic chondrocytes, which they considered to be a form of programmed cell death, or apoptosis,s but they did not present direct evidence as to the mechanism of chondrocyte death.

In this study, we have demonstrated clearly for the first time that the terminal hypertrophic chondrocytes can undergo apoptosis, characterized by both ultrastruc- tural changes and DNA fragmentation. The mechanism by which the terminal chondrocytes ultimately die is central to our understanding of the conversion of calci- fied cartilage to bone in the final step of endochondral ossification.

Addressee for correspondence: M. Zenmyo, MD, Department of Orthopaedic Surgery, Kurume University School of Medicine, Kurume 830. Japan.

CCC 0022-341 7/96/120430-04 <c) 1996 by John Wiley & Sons, Ltd.

MATERIALS AND METHODS

In situ nick end labelling method for apoptosis Specimens were obtained from 3-day-old pigs. They

were killed with chloroform and the growth plates and associated regions of the proximal tibia were quickly exposed. The tissue was fixed in 4 per cent neutral buffered formaldehyde for 18 h and embedded in paraf- fin. Several 5 p m serial sections were obtained from the paraffin block. Before staining, the sections were routinely deparaffinized through xylene and graded alcohol.

Apoptosis (DNA fragmentation) was detected by the modified terminal deoxynucleotidyl transferase (TdT)- mediated deoxyuridine triphosphate (dUTP)-biotin nick end labelling (TUN EL) method, according to Gavrieli et ~ 1 . ~ In brief, after proteinase K digestion (20pglml) (Boehringer Mannheim GmbH, Mannheim, Germany) and removal of endogenous peroxidase with 2 per cent H202, the sections were incubated at 37°C for 1 h in a solution containing TdT and digoxigenin-labelled dUTP and deoxyadenosine triphosphate (dATP). The sections were then treated with the peroxidase-labelled anti- digoxigenin antibody solution for 30 min. The reaction products were developed with 3,3'-diaminobenzidine tetrahydochloride and counterstained with methyl green. As a negative control, phosphate-buffered saline (PBS) was substituted for TdT containing digoxigenin- labelled dUTP and dATP, which resulted in no staining. All the reagents were purchased froin Oncor Ltd. (Gaithersburg, MD, U.S.A.).

Electron microscopy

The tissue was minced into small pieces, fixed for 5 h at room temperature in 2.5 per cent glutaraldehyde in

Received 15 November 1995 Accepted 7 June 1996

APOPTOSIS IN GROWTH PLATE CHONDROCYTES 43 1

Fig. 1-Identification of apoptotic cells in the growth plate by the in situ nick end labelling method. (A) Negative control. (B) Apoptotic chondrocytes are detected at the degenerative zone of the growth plate

0.1 mol/l cacodylate buffer, post-fixed in 1 per cent osmium tetroxide, dehydrated in graded ethanol, and embedded in Epon 8 12. One-micrometre sections were stained with toluidine blue and thin sections of selected areas were stained with uranyl acetate-lead citrate and examined under a JEM-2000 electron microscope (JEOL Ltd., Tokyo, Japan).

Analysis of DNA in agarose gel For the preparation of DNA for agarose electro-

phoresis, the growth plate of the proximal tibia and articular cartilage was dissected out under aseptic con- ditions. The tissue was immediately minced, placed in a baked glass homogenizer, and homogenized with 10 ml of digestion buffer [lo0 mM NaC1, 1 0 m ~ Tris-HC1 (pH 8.0), 25 mM EDTA (pH 8.0), 0.5 per cent SDS, 0.1 mg/ml proteinase K] per g tissue, until thoroughly dispersed. The sample was incubated for 12-18 h at 50"C, extracted with an equal volume of phenol/ chloroforndisoamyl alcohol (2524: l), and centrifuged at 1700 g for 10 min. The aqueous phase was transferred to a fresh tube and incubated with 50-100 pg/ml RNase A at 37°C for 1 h. The sample was re-extracted with phenol/chloroform (1: 1). DNA was precipitated over- night with 2.5 volumes of 100 per cent ethanol and 0.02 volumes of 5 M NaCl at - 20°C. DNA pellets were recovered by centrifugation (13 000 g for 10 min), washed with 70 per cent ethanol, air-dried, and resus- pended in 1 ml of TE buffer. A lop1 sample was supplemented with 2pl of sample buffer (0.25 per cent bromophenol blue, 30 per cent glyceric acid) and

electrophoretically separated on a 2 per cent agarose gel containing lpg/ml ethidium bromide for 1 h at 50V. Pictures were taken by UV transillumination.

RESULTS In situ nick end labelling method for apoptosis

The growth plates consisted of layers of resting, proliferating, hypertrophic, and degenerating chondro- cytes and calcified septum. Apoptosis was identified as nuclear staining by the modified TUNEL method. Apoptotic cells were detected most strongly in the last transverse septum of the growth plate. Degenerative chondrocytes were also stained. No apoptotic cells were found in the proliferative or hypertrophic chondrocyte zone (Fig. 1) or in articular cartilage (Fig. 2).

Electron microscopy The main ultrastructural criteria used for the

definition of apoptotic cells were margination and con- densation of chromatin, enlargement of endoplasmic reticulum cisternae, and convolution of the cell surface. In chondrocytes of the degenerative chondrocyte zone, early morphological changes associated with apoptosis were observed, such as nuclear shrinkage accompanied by margination and condensation of the chromatin (Fig. 3).

Analysis of DNA in agarose gel DNA analysis of chondrocytes in the growth plate

showed the ladder pattern due to internucleosomal

432 M. ZENMYO ET AL.

Fig. 2-Apoptotic cells are not detected by the in situ nick end labelling method in articular cartilage

cleavage, characteristic of apoptosis. A banding pattern with peaks around 340 and 170 bp could be distin- guished. On the contrary, DNA extracted from the articular cartilage showed a smear-like pattern and did not demonstrate the ladder pattern (Fig. 4).

DISCUSSION

‘Apoptotic’ cell death is now widely accepted as an active process, resulting from a decision of the cell based on information from its own environment, internal metabolism, developmental history, and Apoptosis has several distinctive feature^,^.^ differing from necrosis in morphology, in the process of cell death, and in other respects. It is thought to involve the expression of an endogenous endonuclease which frag- ments the DNA into oligonucleosomal fragments of

150-200 bp. This process is the earliest and most characteristic biochemical event common to all forms of apoptosis. Typical morphological changes are observed during this process, including condensation and fragmentation of the nucleus and modification of the cytoplasmic organelle^.^

The growth plate is conveniently divided into the reserve zone of resting cells, the proliferative zone, the hypertrophic zone of mature cells, and the degenerative zone, which coincides with the calcified septum region. 1 3 , 1 4 In the lower hypertrophic zone, the longitu- dinal septa display crystals of apatite that subsequently enlarge and coalesce to calcify the whole longitudinal septum in the zone of provisional calcification. Between the calcified septa, the hypertrophic chondrocytes swell further, appear to degenerate and die, and are invaded by capillaries approaching from the diaphyseal side. 15- I 7

Little is known about the mechanism of cellular death of the terminal hypertrophic chondrocytes in the growth plate. In spite of difficulty in the detection of apoptotic cells at the electron microscopic level,l8 Farnum et al. have shown cellular condensation of the terminal hyper- trophic chondrocytes, which they suggested might rep- resent a form of programmed cell death, or apopto~is .~ We also observed some apoptic cells with early morpho- logical changes, such as nuclear shrinkage accompanied by margination and condensation of the chromatin, but our present study also showed biochemical evidence for apoptosis in the terminal hypertrophic chondrocytes. The results of the iui situ nick end labelling method and the occurrence of a ladder pattern of DNA in agarose gel analysis indicate the activation of endogenous endonuclease, resulting in DNA fragmentation.

Cell death is an essential event in the development, growth, and tissue homeostasis of multicellular

Fig. 3-Electron microscopy of chondrocytes of the growth plate. (A, B) In chondrocytes undergoing apoptosis, the early stages of chromatin condensation are visible and the nuclear membrane is ill-defined. Scale bar= 1 pm

APOPTOSIS IN GROWTH PLATE CHONDROCYTES 43 3

Fig. &DNA fragmentation in chondrocytes of the growth plate. Lane 1 is a digest of (0x174 DNA with Hae 111; the sizes of relevant marker bands are indicated in base pairs. Lane 2: DNA extracted from the growth plates; the ladder pattern is seen. Lane 3: DNA extracted from the articular cartilage; the ladder pattern is not seen

organisms.'9-21 In the growth plate, the rate at which the cells proliferate, hypertrophy, degenerate, and die deter- mines the growth rate or elongation rate of the bone involved.' The mechanism of cell death in the terminal hypertrophic chondrocytes is therefore important to our understanding of the biology of the growth plate. Some previous papers have shown that apoptotic chondro- cytes are seen in embryonic bone generation (or endo- chondral o s ~ i f i c a t i o n ) . ~ ~ . ~ ~ Our present study suggests that cell death, particularly apoptosis, may play a key role in the control of normal and pathological

endochondral ossification, not only in bone generation but also in long bone development. Although this study presents both morphological and biochemical evidence for apoptosis in the terminal hypertrophic chondrocytes, the stimulus that leads to apoptosis during endo- choiidral ossification remains u n k n o ~ n . ~ ~ - ~ ~ Additional studies are necessary to resolve this problem.

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