Arch. Histol. Cytol., Vol. 53, No. 2 (1990) p. 187-192

Endochondral Calcification by Hypertrophic Chondrocytes

in the Meckel's Cartilage Grafted into the Isogenic Mouse

Spleen

Kiyoto ISHIZEKI, Naoki FUJIWARA, Mitsutaka SUGAWARA and Tokio NAWA

Department of Oral Anatomy, Iwate Medical University School of Dentistry, Morioka, Japan

Received September 8, 1989

Summary. We found that when the midsection of Meckel's cartilage bars obtained from mice on the eighteenth day of gestation were grafted into isogenic mouse spleen, chondrocytes induced an endochondral calcification. Concurrent with the onset of calcification throughout Meckel's cartilage matrix, periodic banded thick collagen fibrils and matrix vesicles were observed around the chondrocytes. Although most of the chondrocytes prior to grafting were hypertrophic cells, they survived for seven days in the splenic tissue and had well-developed secretory organelles. The cells which were surrounded by calcified matrix were relatively small, spherical, and showed a morphology closely resembling that of osteocytes. These findings suggest that the life span of hypertro-

phic chondrocytes is influenced by the microenviron-ment of the spleen.

Meckel's cartilage, known as a fetal organ, is ini-tially formed as the main support of the mandible. It is divided into three distinct subdivisions by the regional transformation of the cartilage (BHASKER et al., 1953; FROMMER and MARGOLIES, 1971). Among these regions, the rostral process (distal portion) and auricular end (proximal portion) of the Meckel's cartilage have been thought to undergo endochondral-type ossification, but not the midsection (BHASKER et al., 1953; GORET-NICAISE and PILET, 1983; RICHMAN and DIEWRT, 1988). The hypertrophic chondrocytes in the midsection of the normal Meckel's cartilage degenerate and are replaced by invading cells accompanied by mem-brane bone formation around the cartilage. Recently, it has been proposed that some of the hypertrophic cells following morphological changes transform into osteogenic cells (HOLTROP, 1972; SHIMOMURA and

RAY, 1973; RICHMAN and DIEWERT, 1988; YOSHIOKA and YAGI, 1988). Thus, there is still some confusion regarding the ultimate fate of the terminal chon-drocytes. The present study examined morphologically the

fate of the hypertrophic chondrocytes in the middle area of Meckel's cartilage grafted into the spleen.

MATERIALS AND METHODS

Meckel's cartilage bars obtained from mouse (ddy strain) embryos on the eighteenth day of gestation

(day of vaginal plug =day 0) were used because carti-lage in this stage contains a large number of hyper-trophic chondrocytes in the absence of degenerated chondrocytes. The mandibular arches containing Meckel's cartilage were dissected aseptically and

placed in Hanks' balanced salt solution supplemented with Kanamycin (1 mg/ml). After they were immersed in 10 mM EDTA in magnesium-calcium free-phosphate buffer for 10 min to remove excessive cellular elements of the connec-tive tissue from Meckel's cartilage bars, the Meckel's cartilage was removed mechanically. These spicules were immediately transferred to fresh Hanks' solu-tion and one-third were cut away using scissors. The midsection was used for grafting because it was known that this region does not have endochondral calcification. These explants were grafted into the spleen of isogenic adult mice as previously reported (ISHIZEKI et al., 1987). Briefly, using a Venula-V-2 needle (Top Co. Ltd, Tokyo) the grafts were carefully transplanted to the exposed spleen of the recipients which were anes-thetized intraperitoneally with Nembutal (sodium

187

188 K. ISHIZEKI et al.:

pentobarbiturate, 0.6mg/10gm body weight). The woundsin the splenic capsule and the skin were closed with a drop of surgical Aron Alpha-A (Sankyo, Tokyo). The grafted explants were examined after 7 days. The animals were sacrified by a overdose of ether and immediately perfused with a modified Karmov-sky's fixative, which contained 2.0% paraformalde-hyde and 1.25% glutaraldehyde solution. Dissected Meckel's cartilage, together with splenic tissue, were further fixed in 2.5% glutaraldehyde with cacodylate buffer (pH 7.4) for 2-4h. The specimens were post-fixed in 1.0% 0504 for 1 h, dehydrated in a graded series of ethanol, and embedded in Epon 812. For electron microscopy, ultrathin sections were

cut in the absence of decalcification with a diamond knife on an LKB-Ultratome and stained with uranyl and lead citrate prior to observation with a JEM-100B electron microscope. For light microscopic observations, the semi-thin

sections (1 sum) were stained with toluidine blue (pH 3.0). The same specimens, after obtaining ultrathin sections, were decalcified en bloc with Plank-Rychlo solution (PLANK and RYCHLO,1952) for 2 h according to our previous report (ISHIZEKI et al., 1990) for electron microscopy.

RESULTS

Control explant

The Meckel's cartilage in the 18-day-old embryos was divided into three distinct layers. In the outer-most layer, a few fibroblastic perichondrial cells could be noted, whereas the innermost layer consist-ed of a maturating zone containing relatively large spindle-shaped cells. The central area of the Meckel's cartilage bar was a hypertrophic zone, occupied by cellular lacunae containing hypertrophic chondrocytes

(Fig. la, b). The hypertrophic chondrocytes were char-acterized by large spherical cells, cytoplasmic vacuoles of various sizes, and a light round nucleus. They were surrounded by a large amount of pericel-lular matrix which was stained metachromatically with toluidine blue. In the cartilage matrix, evidence of calcification was not observed (Fig, ib). Meckel's cartilage in this stage did not contain degenerative chondrocytes. Ultrastructurally, the hypertrophic chondrocytes

were characterized by a regular spherical nucleus containing a prominent nucleolus, well-developed secretory organelles, several cytoplasmic vacuoles, and many small mitochondria (Fig. 1c). The nuclear

chromatin was pale and euchromatic, and the nu-cleolus was in contact with the nuclear membrane. The cisternae of the rough endoplasmic reticulum were relatively short and were located on both sides of the cell body. The Golgi apparatus was divided into several groups; many vesicles containing floc-culent material were present near the stacks. Some of cytoplasmic vacuoles contained lysosome-like dense bodies. The outline of these cells was usually round or oval, and many thin cytoplasmic processes extended into the fine collagenous fibrils composing the pericellular matrix. These fibrillar matrices were deposited densely; collagen fibrils having clear peri-odic structures and matrix vesicles were not observ-ed in the Meckel's cartilage of this stage.

Grafted Meckel's cartilage

Endochondral calcification was observed throughout the Meckel's cartilage matrix 7 days after grafting

(Fig. 2a). Calcification of the cartilage matrix occur-red usually around the hypertrophic chondrocytes, but occasionally also in peripheral areas including maturating chondrocytes. Early calcification appear-ed on the collagenous matrix as spotted deposits. The calcifying area was stained deep blue with toluidine blue and could be easily distinguished from other uncalcified extracellular matrices. Most of the hyper-trophic chondrocytes were encircled by calcified lacunae and revealed a large, euchromatic nucleus and a pale cytoplasm with a deeply stained pericel-lular matrix. The chondrocytes containing larger vacuoles could also be seen intermingling among the hypertrophic cells, but few degenerated cells were recognizable. During the 7-day period, the invasion of capillaries or splenic cells from recipient tissues into Meckel's cartilage matrix was not observed. The ultrastructures clearly showed that the hyper-

trophic chondrocytes surrounded by calcified Meckel's cartilage still contained a large regular round nucleus with a prominent nucleolus, well-developed Golgi apparatus and rough endoplasmic reticulum, mito-chondria, several dense bodies, and occasionally, abundant free ribosome clusters (Fig. 2b). Such cells were relatively small and the nucleo-

cytoplasmic ratio decreased vs. hypertrophic chondro-cytes prior to graftings. The outline of the cells was smooth and usually possessed several elongated cyto-

plasmic processes penetrating into the calcified carti-lage matrix. Uncalcified perichondral areas surround-ing these cells were occupied by thick collagen fibrils with periodic structures, which could not be seen in the matrix of the intact Meckel's cartilages. Also,

Meckel's Cartilage Calcification 189

matrix vesicles were found in association with the first mineral deposits (Fig. 2c). The initial calcification of the cartilage matrix appeared as calcospherites which consisted of aggre-

gates of needle-like crystals associated with collagen fibrils (Fig. 2d). The cells surviving during the presentexperimental period retained secretory organelles of the Golgi-ER system and the cells which were sur-rounded completely by calcified matrix had a morpho-logy resembling that of osteocytes.

DISCUSSION

The present results indicated that the hypertrophicchondrocytes in the grafted Meckel's cartilage sur-vived for a long period and, furthermore, induced the formation of calcified cartilage matrix.

In normal Meckel's cartilage, endochondral ossifi-cation takes place in the auricular and rostral ends, but chondrocytes in the midsection degenerate and die in the absence of participation in the formation of mandibular bone (BHASKAR et al., 1953; FROMMER and MARGOLIES, 1971). Therefore, the middle area of normal Meckel's cartilage bar does not form calcified

Fig. 1. a. A survey of longitudinal sections through the midsection of Meckel's cartilage (MC) in an 18-day-old embryonic mouse. Toluidine blue staining. X95. b. Enlargement of the midsection which is composed of a few

perichondrial cells (FC) and maturating cells (MC), and many large round hypertrophic chondrocytes (HC) which are surrounded by a strong metachromatic cartilage matrix. Toluidine blue staining. X420. c. An electron micrograph of a typical chondrocyte. The cells have a large round nucleus, well-developed Golgi-ER system, small mitochondria (MT), and cytoplasmic vacuoles (V). Some of cytoplasmic vacuoles contain lysosomal dense bodies (arrowheads). GA Golgi apparatus, rER rough endoplasmic reticulum. X 7,000

a b c

190 K. ISHIZEKI et al.:

Fig. 2. Legend on the opposite page.

a b

c

d

Meckel's Cartilage Calcification 191

cartilage. Essentially, since endochondral ossification including mandibular condyle (SILBERMANN and FROMMER, 1972, 1974) or the growth plate (EGGLI et al.,1985; JEE,1988) usually occurs in the hypertrophic chondrocyte- associated areas of the chondro-osseous

junction, the calcification throughout hypertrophic zone or, occasionally, maturating zone cells-such as observed in the present study-was a unique pheno-menon. When Meckel's cartilage of the same stage was cultivated in vivo in the subcutaneous, muscle, or peritoneal cavity, hypertrophic chondrocytes de-

generated rapidly and no calcified cartilage matrix was formed (ISHIZEKI, unpublished data). In addition, RICHMAN and DIEWERT (1988) have reported that, on

grafting of Meckel's cartilage into the anterior eye chamber of the rat, endochondral ossification did not appear in the midsection. The experimental results demonstrated that for survival of chondrocytes, an excellent blood supply is necessary so that Meckel's cartilage calcification is not induced by degenerated cells. Furthermore, another reason why calcification does not occur in the permanent cartilage tissues may be the presence of glycosaminoglycans or proteo-

glycans within the cartilage matrix which inhibit crystal growth for calcification. SHIMOMURA et al.

(1975) and SUZUKI et al. (1981) reported that when some enzymes were released from recipient-derived blood cells, probably macrophages, such substances were removed from the matrix, and cartilage calcification was initiated. The present study present-ed the distinct possibility that the survival of the hypertrophic chondrocytes was prolonged by the abundant supply of nutrients and oxygen. This made

possible the cooperation between matrix vesicles (which were secreted by surviving chondrocytes, associated with extracellular matrix) and quite pos-sibly blood-derived factors, inducing calcification in the Meckel's cartilage.

It is unclear, as yet, why the chondrocytes fall into hypertrophy. Recent studies have demonstrated that the terminal hypertrophic chondrocytes are not degenerating or effete cells but viable as active cells

(FARNUM and WILSMAN, 1987). Furthermore, it has been suggested that some of these transform into bone cells (HOLTROP, 1972; SHIMOMURA and RAY, 1973; RICHMAN and DIEWERT, 1988). In the present study, in spite of the increase in number of hypertro-

phic cells during the 7 days after transplantation, few degenerated chondrocytes were seen. Moreover, it was confirmed at the electron microscopic level that the surviving chondrocytes retained the secretory organelles of the Golgi-ER system. The chondrocytes containing a large round heterochromatic nucleus completely surrounded by a calcified matrix were relatively small, and extended several elongated

processes into its matrix. These cells had an ultra-structural appearance closely resembling that of osteocytes as described by TEN CATS (1980), JEE

(1988), and MARKS and POPOFF (1988). COLE and COLE (1989) suggested the possibility that the perivascular cells in the epiphyseal cartilage transform into chon-drocytes, but in the present study, since capillary invasion failed to appear, it was clear that original chondrocytes transformed into osteocyte-like cells. Thus, our findings demonstrated that the chondro-cytes in the Meckel's cartilage can survive according to the surrounding microenvironment of the spleen, and they are both viable and metabolically active as described above. Further study is needed on the ultimate fate of

these hypertrophic cells. In should also be noted that the present experimental system consisting of pure chondrocyte population is useful for the biological study of endochondral calcification in the absence of osteogenic cells.

Fig. 2. a. Longitudinal section through Meckel's cartilage (MC) grafted for 7 days. In the lacunae, hypertrophic chondrocytes of various sizes are seen and several contain large cytoplasmic vacuoles. Note the extensive calcification throughout the extracellular matrix surrounding the cellular lacunae. Decalcified specimen. Toluidine blue staining. ST splenic tissue. x570. b. An electron micrograph of an osteocyte-like chondrocyte in Meckel's cartilage grafted for 7 days. The cell still retains a Golgi apparatus (GA) and rough endoplasmic reticulum (rER), and frequent lysosomal dense bodies (arrowheads) can also be seen throughout the cytoplasm. Note the cytoplasmic processes (arrows) penetrating into the calcified matrix. Undecalcified specimen. N nucleus, NO nucleolus, FR free ribosomes. x5,600. c. Higher magnification of area outlined in b. Arrowheads indicate matrix vesicles in association with initial mineral deposits and collagenous matrix. Note the thick collagen fibrils with regular periodicity intermingling in the pericellular matrix (arrows). x56,000. d. Enlarged electron micrograph of the lower portion in b. The calcified pericellular region is formed by the fusion of the calcospherites (CS) which consist of aggregates of needle-like crystals. Also, note the cluster of free ribosomes

(FR) which are distributed throughout the cytoplasm around the nucleus (N). x11,000

192 K. ISHIZEKI et al.

Ackowledgements. We are grateful to Dr. Tatsuo USHIKI, Department of Anatomy, Iwate Medical University School of Medicine, for his useful comments, and to Assistant Professor Paul LANGMAN, Department of Eng-lish, Iwate Medical University, for advice concerning English usage.

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Dr. Kiyoto ISHIZEKI Department of Oral Anatomy Iwate Medical University School of Dentistry 1-3-27 Chuou-dori Morioka, 020 Japan

石 関 清 人

020盛 岡市 中央 通1-3-27

岩手 医科大 学歯 学部

口腔解 剖学 第二 講座