The LysosomalSystem in Endochondral Growth

The Lysosomal System in Endochondral Growth

J. THYBERG

.

U. FRIBERG

With 35 Figures

GUSTAV FISCHER VERLAG STUTTGART . NEW YORK . 1978

Ph. D. M. D., Ph. D.

JOHAN THYBERG,

ULF FRIBERG,

Department of Histology, Karolinska Institutet S-104 01 Stockholm (Sweden)

Acknowledgements Research grants were obtained from the Swedish Medical Research Council (proj. no. 12X-03355), the Swedish Cancer Society (proj. no. 100-K71-05XK), the King Gustaf V 80th Birthday Fund, the Harald and Greta Jeansson Foundations, the C. B. Nathhorst Foundation, the A. O. Sward Foundation, and from the funds of Karolinska Institutet. The skilful technical assistance of Ms. Eva Lundberg and Ms. Karin Blomgren and the secretarial assistance of Ms. Ingrid Waalma are gratefully acknowledged.

CIP-Kuntitelaufnahme dec Deutschen Bibliothek Thyberg, Johann The lysosomal system in endochondral growth / J. Thyberg ; U. Friberg. - 1. Auf!. - Stuttgart, New York: Fischer, 1978. (Progress in histochemistry and cytochemistry ; Vol. 10, No.4) ISBN 3-437-10524-8 NE: Friberg, Ulf: ISBN 089574-002-8 (NY) Progress in Histochemistry and Cytochemistry ISSN 0079-6336 © Gustav Fischer Verlag' Stuttgart· New York 1978 AIle Rechte vorbehalten Satz: Bauer & B6keler, Denkendorf Druck und Einband: H. Laupp jun., Tiibingen Printed in Germany

Contents Introduction

2 2.1 2.2 2.3 2.4 2.5 2.6 3 3.1

............

Lysosomes in epiphyseal cartilage. Biochemical properties . . Cytochemical properties . Ultrastructural properties Role in heterophagy . . . Role in autophagy and cell degeneration ........ Extracellular release of lysosomal enzymes and matrix mineralization

3 3 4 7 16

Lysosomes in metaphyseal bone. Biochemical properties . . . . . .

24 24

3.2 3.3 3.4 3.4.1 3.4.2 3.5 3.6

35

4

Summary

37

5

References

39

6

Subject index

16 17

24 26 30 30 33 34

Cytochemical properties . Ultrastructural properties Role in heterophagy ... Resorption of epiphyseal cartilage . Bone resorption. . . . . . . . . . Role in autophagy ........ Extracellular release of lysosomal enzymes and bone mineralization

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Lysosomes and Endochondral Growth

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1 Introduction The longitudinal growth of long bones is effected by a complex mechanism in which calcified epiphyseal cartilage is replaced by metaphyseal bone (BLOOM and FAWCETf 1975). This process involves multiplication of chondrocytes in the growth plate followed by cellular maturation and hypertrophy with subsequent calcification of the intercellular matrix or, more precisely, of the longitudinal bars separating adjacent cell columns (Fig. 1). It is widely recognized that the calcification of the cartilaginous matrix is preceded by, or connected with, a chemical modification and partial removal of its constituent proteoglycans and it has been suggested that this change is a prerequisite for mineralization (CAMPO 1970; KOBAYASHI 1971). In association with the calcification of the matrix the chondrocytes disintegrate and, at the metaphyseal end of the epiphyseal plate, the lacunae are broken up and invaded by capillary sprouts accompanied by a proliferating mesenchyme. After removal of the degenerated chondrocytes and the surrounding unmineralized matrix, osteoblasts appear at the mineralized longitudinal septa and lay down an osteoid matrix which thereafter calcifies. The primary bone trabeculae formed in this way later undergo an extensive reorganization as the longitudinal growth of the bone continues, necessitating means for resorption of the bony tissue. Timelapse studies on tissue cultures of bone (GAILLARD 1959; GOLDHABER 1960) have shown that osteoclasts are actively involved in this process. Since the number of primary bone trabeculae is only about half the number of longitudinal septa in the epiphyseal plate, there must also be a mechanism for resorption of calcified cartilage. It was proposed by SCHENK et al. (1967) that this process is mediated by chondroclasts, the fine structure of which was described as identical with that of osteoclasts. Most mammalian cells have in their cytoplasm a unique class of organelles-lysosomes-contain-

ing a collection of acid hydrolases capable of digesting almost all known biological macromolecules. The lysosomes constitute part of a complex, dynamic membranous system which participates in the intracellular digestion of exogenous material ingested by endocytosis, as well as of endogenous cell components segregated by autophagy (DE DUVE and W ATfIAUX 1966; DINGLE and FELL 1969; HERS and VAN HOOF 1973). Furthermore, there is abundant evidence that lysosomal enzymes have an important function in the extracellular digestion of proteoglycans and other macromolecules in the intercellular substance of cartilage and other connective tissues. A substantial contribution to this field of lysosome research has been the establishment of the decisive role of acid proteases in the retinol-induced breakdown of the matrix of chick limb-bone rudiments grown in vitro (DINGLE 1969; MORRISON 1970). It has generally been assumed that reverse endocytosis, i. e. exocytosis, plays a key role in the cellular secretion of lysosomal enzymes (DINGLE 1969). However, as further discussed below, the presence of matrix vesicles of lysosomal nature in calcifying cartilage indicates that other possibilities may also exist. It is evident that the longitudinal growth of long bones included several steps where lysosomes and lysosomal enzymes may be involved. Among these the three most important are: (i) the removal of proteoglycans from the cartilaginous matrix in connection with the start of calcification; (ii) the resorption of degenerated chondrocytes and unmineralized matrix at the erosion line; and (iii) the resorption of mineralized cartilage and bone matrix. The object of this report is to review current information regarding the presence of lysosomes and lysosomal enzymes in the cells ofthe endochondral growth apparatus and to discuss the evidence which suggests that the lysosomal system participates in the abovementioned processes under physio-

J. THYBERG . U. FRIBERG

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Figure ( Part of two chondrocyte columns in thtepiphyseal plate. Ne';'ly ivided cells are lying in groups of two within the same lacuna. Otherwise, the individual cells in the columns are separated by transversal septa (ts) and the columns by longitudinal septa (Is). The cells have large nuclei with prominent nucleoli. Even at this low magnification numerous cisterriae of gtanular'endoplasmic reticulum can be discerned in the cytoplasm. Some cytoplasmic dense bodies (arrows) are also seen. Afew similar bodies are present in the longitudinal septa (arrowheads). - Magnification X 1,800.

Lysosomes and Endochondral Growth logical conditions. Special emphasis is given to work done in our laboratory during the latest years. Lysosomal functions or alterations which are mainly of pathophysiological significance will not be considered in any detail.

2 Lysosomes in epiphyseal cartilage 2.1 Biochemical properties The cells in cartilage and bone are surrounded by large amounts of intercellular substance containing a dense network of collagen fibrils, an amorphous ground substance and a mineral phase. The physical properties of this matrix lead to difficulties in preparing homogenates for fractionation and isolation of subcellular components. Hence, few studies of lysosomes isolated from cartilage and bone have been presented in the literature. The aforementioned problems may, however, be at least partly overcome by using tissue from young animals, which is softer and more easily homogenized, or cells separated from the extracellular matrix by enzymatic digestion. A lysosome-enriched fraction was isolated from homogenates of embryonic chick epiphyseal cartilage by AMADO et al. (1974) and WASTESON et al. (1975) and shown to contain acid hydrolases which degraded chondroitin sulf. ate. The degradation seemed to be initiated by an endopolysaccharidase cleaving hexosaminidic

". The figures show upper tibial epiphyseal cartilage or metaphyseal bone from young guinea pigs, chondrocyte cultures or transplants of chondrocytes isolated from fetal guinea pig epiphyses. Unless otherwise indicated the specimens were fixed in buffered glutaraldehyde followed by osmium tetroxide, dehydrated in ethanol and embedded in epoxy resin. The thin sections were stained with uranyl acetate followed by lead citrate.

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bonds, followed by the concerted action of a ~­ glucuronidase, a chondrosulfatase and a ~­ N -acetylgalactosaminidase. ARSENIS et al. (1971) isolated chondrocytes from embryonic chick cartilage by trypsin-collagenase digestion, then sonicated and fractionated by differential centrifugation according to DE DUVE et al. (1955). The distribution of various enzyme activities among the fractions resembled that found in rat liver (DE DUVE et al. 1955). All acid hydrolases showed similar distribution patterns with the highest specific activity in the , i. e. lysosomal, fraction. Furthermore, structure-linked latency was demonstrated for acid phosphatase and cathepsin. Taken together the data indicate that cartilage contains considerable amounts of lysosomal acid hydrolase activity capable of degrading both cell constituents and extracellular matrix components. Biochemical methods have also been used to demonstrate and estimate enzyme activities in slices, homogenates and extracts of growth cartilage. KUHLMAN and McNAMEE (1971) and GRANDA and POSNER (1971) studied material collected from the different zones of epiphyseal plates of rabbits and calves, respectively. Although the results differed somewhat between the two studies and with respect to different enzymes, acid hydrolase activity, presumably of lysosomal origin (BARRETT 1972), tended to increase from the zone of resting toward the zone of hypertrophic cartilage. Similar observations have been reported by JIBRIL (1967) and HAVIVI and TAL (1973). These results suggest that acid hydrolases may be important in the processes preceding the mineralization of the cartilage. Among the lysosomal enzymes particular interest has been paid to the cathepsins and their role in the degradation of the cartilage matrix. FELL and DINGLE (1963) found that embryonic chick cartilaginous limb-bone rudiments grown in the presence of vitamin A released an acid protease into the culture medium. A similar protease was present in a particulate

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fraction of the rudiments. It was subsequently demonstrated that a specific antiserum to liver cathepsin D inhibited both the autolytic and vitamin A-induced breakdown of the cartilage matrix in organ culture (MORRISON 1970; DINGLE 1971). Likewise, pepstatin, a potent inhibitor of cathepsin D (BARRETI and DINGLE 1972), was found to prevent the autolytic degradation of proteoglycans in cartilage (DINGLE et al. 1972). Human cathepsins B1 and D have also been shown to degrade isolated proteoglycans, giving endproducts similar to those produced by the action of papain and trypsin, respectively (MORRISON et al. 1973). Cathepsin B 1 has further been found to be capable of attacking native collagen (BURLEIGH et al. 1974). Whereas the above studies have established the presence and function of cathepsin D in cartilage indirectly by using antisera against the liver enzyme, WOESSNER (1973) purified and characterized cathepsin D from cartilage. This isolated enzyme digested proteoglycans with an optimum at about pH 4.0 and showed no activity at pH 7.2. The presence of four different cathepsins in rabbit ear cartilage has been demonstrated by ALI et al. (1967, 1969) using specific synthetic substrates.

2.2 Cytochemical properties The cytochemical localization of lysosomal enzyme activities at the electron microscopic level has been of great importance in providing a link between biochemistry and cell morphology. However, good cytochemical methods are available only for a few lysosomal enzyme activities, mainly utilizing a Gomori-type of medium (GOMORI 1952). Moreover, a number of methodological problems are connected with this type of work. Thus, the enzyme may be partially inactivated by either the primary fixative, usually glutaraldehyde and/or formaldehyde (MILLER and PALADE 1964; ]ANIGAN 1965; Hopsu-HAVU et al. 1967; FAHIMI and DROCHMANS 1968; ARBORGH et al. 1971 ;

BRUNK and ERICSSON 1972), or the capturing reagent of the incubation medium (HopsUHAVU et al. 1967). The substrate and/or the capturing reagent may not penetrate freely and uniformly into the tissue and formed precipitate may be lost during postfixation in osmium tetroxide (REALE and LUCIANO 1964) as well as during staining of thin sections with heavy metal salt solutions (KALIMO et al. 1968). Hence, negative results must be considered with great care. Similarly, proper controls must be performed to exclude false positive precipitations formed, for example, by non-enzymatic hydrolysis of the substrate or unspecific binding of the capturing reagent to tissue components (for further refs. see BECK et al. 1972; DAEMS et al. 1972). Cytoplasmic dense bodies. The fine structural location of acid phosphatase and aryl sulfatase activity in the guinea pig epiphyseal plate has been determined in our laboratory (THYBERG and FRIBERG 1970, 1972; THYBERG 1972). These studies helped to establish the lysosomal nature of membrane-bound dense bodies in the cytoplasm of the chondrocytes (Figs. 2 and 3). In favorable sections, all the dense bodies revealed a positive staining. Reaction product was occasionally present also in the Golgi complex. This finding conforms to the belief that this organelle, or perhaps rather the associated GERL system, is responsible for the formation of lysosomes (for reviews see DE DUVE and WATIIAUX 1966; COHN and FEDORKO 1969; NOVIKOFF 1976). Matrix vesicles. Acid phosphatase and aryl sulfatase activity was further demonstrated extracellularly in lysosome-like vesicles (Figs. 4 and 5; see further section 2.3). Many of these matrix vesicles were, nevertheless, unreactive. The reason for this may be multifold. For example, the enzyme activity in particular vesicles may be low or nonexistent due to an inherent heterogeneity among the vesicles or release of enzyme through a defective limiting membrane (d. Fig. 10). The possibility of enzyme inactivation or loss of reaction product during the

Lysosomes and Endochondral Growth

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Figures 2 and 3. Parts of two chondrocytes from the proliferative zone after incubation of glutaraldehyde-fixed tissue for demonstration of acid phosphatase and aryl sulfatase activity, respectively. In Fig. 2 the internal structure of the lysosomes is obscured by the precipitate. In Fig. 3 the precipitate is mainly confined to the periphery of the lysosome and the characteristic dense, homogeneous matrix of the organelle is evident (d. Figs. 6 and 7). The
bodies but were unable to demonstrate reaction product for this enzyme in the matrix vesicles. Considering the abovementioned methodological problems and the fact that many vesicles had a morphology resembling that of lysosomes (ANDERSON 1969), it may be difficult to know whether this was due to absence of enzyme activity in the vesicles or shortcomings of the cytochemical technique employed. The studies of ALI et al. (1970) and ALI (1976) have been taken as evidence against the existence of lysosomal matrix vesicles in growth cartilage. In these studies were isolated from fetal bovine or rabbit epiphyseal cartilage by digestion with collagenase and subsequent differential centrifugation of the digest. The fractions so obtained were studied ultrastructurally and assayed for various enzyme activities. By far the greater part (amounting up to 85 %)

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5 Figures 4 and 5. Type I matrix vesicles in longitudinal septa from the hypertrophying (Fig. 4) and proliferating (Fig. 5) zones of epiphyseal cartilage incubated for demonstration of acid phosphatase and aryl sulfatase activity, respectively. Varying amounts of lead precipitate are present in the vesicles (d. Figs. 2 and 3). In Fig. 4 some precipitate also appears to be present immediately outside a few vesicles (arrows; d. Fig. 10). Thin collagen fibrils otherwise constitute the main structural component of the extracellular matrix (cross-sectioned in Fig. 5). Magnifications X 33,000 (Fig. 4), X 22,000 (inset Fig. 4), X 114,000 (Fig. 5).

of the aCtiVIties of alkaline phosphatase, inorganic pyrophosphatase, ATPase and 5'-nucleotidase were reported to occur in extracellular, vesicle-containing fractions. These results could

hardly reflect the in vivo conditions and contrast with enzyme cytochemical observations in other epiphyseal cartilages, which show prominent amounts of reaction product for alkaline phos-

Lysosomes and Endochondral Growth phatase and ATPase on the surface of the chondrocytes (MATSUZAWA and ANDERSON 1971 ; THYBERG and FRIBERG 1972). Since at least three of the enzymes mentioned above are believed to be located on the outside of the plasma membrane (TRAMS and LAUTER 1974), it is possible that they were disengaged from the cells during the digestion of the cartilage (d. DE DUVE 1971) and subsequently adsorbed unspecifically to cell membrane fragments in the digest. Thus, a crude collagenase preparation, containing peptidase and trypsinlike activities, was used; it has been shown that commercially available collagenases, even after extensive purification, are able to digest molecules other than collagen (PETERKOFSKY and DIEGELMANN 1971; MIYOSHI and ROSENBLOOM 1974). Moreover, the incubation conditions were such that damage and breakdown of many chondrocytes could be expected. Finally, although in lesser quantities than alkaline phosphatase and related enzymes, significant amounts of lysosomal hydrolases were present in the vesicle-containing fractions. PERESS et al. (1974) and WUTHIER (1975, 1976) studied the lipid content of
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whether this staining pattern is due to a possible association of the enzymes with matrix vesicles. Using various specific antisera, this technique (WESTON and POOLE 1973), together with the new methodology for tissue proteinases developed by SMITH and VAN FRANK (1975), will be helpful in studying the cellular and extracellular locations of lysosomal enzymes other than those for which precipitation reactions according to GOMORI (1952) are available. BONUCCI (1967, 1970) reported matrix vesicles of guinea pig and rat epiphyseal cartilage to be PAS-positive, a staining quality also characterizing lysosomes (BECK et al. 1972).

2.3 Ultrastructural properties Cytoplasmic dense bodies. In addition to the most prominent cytoplasmic organelles, i. e. mitochondria, endoplasmic reticulum and the Golgi complex, all of which have been described in considerable detail already in the fine structural study on developing epiphyseal cartilage of GODMAN and PORTER (1960), chondrocytes contain a characteristic but quantitatively less significant group of structures designated because of the high electron density of their matrix (PALFREY and DAVIES 1966; THYBERG et al. 1970, 1971, 1973a, 1975a; BRIGHTON et al. 1973; MEIKLE 1975). In the cells of guinea pig epiphyseal cartilage (THYBERG and FRIBERG 1970) the cytoplasmic dense bodies were rounded or slightly irregular in shape and generally had a diameter of 0.2 0.4 f.tm (total range about 0.05 - 1 f.tm). They were surrounded by a triple-layered membrane with a total thickness of about 9 nm. The plasma membrane and the membrane of the Golgi vacuoles were of similar dimensions, whereas the nuclear, endoplasmic reticulum and mitochondrial membranes were distinctly thinner (6 - 7 nm). This is in accordance with observations in other tissues (e. g. YAMAMOTO 1963) and indicates that the dense bodies constitute part of

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the exoplasmic space of the chondrocytes (DE

DUVE 1969). Excepting the frequent occurrence of a thin electron-lucent rim just beneath the limiting membrane (d. DAEMS et al. 1972; MEIKLE 1975), an osmiophilic, homogeneous matrix, showing a fine granularity at high magnification, completely filled the cytoplasmic dense bodies (Figs. 6 and 7). Small membranous fragments or other inclusions of varying appearance were sometimes found within this matrix (see further sect-

ions 2.4 and 2.5). Nevertheless, in individual chondrocytes as well as throughout the different zones of the epiphyseal plate, the dense bodies as a whole constituted a structurally uniform group of structures. In the resting and proliferative zones up to about 3 - 5 dense bodies were seen per cell and section. However, as in the other zones, a considerablenumber of cell sections showed no dense bodies at all. The hypertrophying chondrocytes were, on the average, most numerous in dense

Figures 6 and 7. Portions of two chondrocytes from the hypertrophying zone demonstrate the fine structure of the cytoplasmic dense bodies. These are limited by a triple-layered membrane of similar dimensions to the plasma membrane (pm) and filled with a uniformly dense matrix. The inner dense layer of the membrane is difficult to see clearly because of its close apposition to the matrix. - Magnifications X 63,000 and X 77,000.

Lysosomes and Endochondral Growth

bodies, and as many as 15 - 20 such structures per cell and section were occasionally observed. In the calcifying zone, where the chondrocytes regularly displayed signs of degeneration, the number of dense bodies per cell was lower. In the morphometric study of BRIGHTON et al. (1973), the content of lysosome-like dense bodies was similarly found to reach a maximum in the zone of hypertrophic cells. Likewise, condylar cartilage showed the largest number of dense hodies in the hypertrophic chondrocytes (MEIKLE 1975). The cytochemical demonstration of acid phosphatase (THYBERG and FRIBERG 1970, 1972; MATSUZAWA and ANDERSSON 1971; MEIKLE 1975) and aryl sulfatase (THYBERG 1972; MEIKLE 1975) activity within the cytoplasmic dense bodies (see further section 2.2) provide strong evidence for their identification as lysosomes (BARRETT 1972; BECK et al. 1972; DAEMS et al. 1972). This conclusion is in full conformity with the morphological characteristics of the bodies (DAEMS et al. 1972). Moreover, there seems to be good agreement between the morphological distribution of cytoplasmic dense bodies, as described above, and the levels

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of acid hydrolase activities in different zones of epiphyseal and other calcifying cartilages (JIBRIL 1967; KUHLMAN and McNAMEE 1970; GRANDA and POSNER 1971; HAVIVI and TAL 1973 - see further section 2.1). Matrix vesicles. Considerable interest has lately been focused on the existence of a heterogeneous population of vesicular structures in the intercellular substance of calcifying cartilage, and their possible involvement in the mineralization process. Attention was drawn to these structures simultaneously by ANDERSON (1967) in a study of induced cartilage development and by BONUCCI (1967) in a study of guinea pig and rat epiphyseal cartilage. BONUCCI (1967, 1970) described the vesicles as membrane-bound, rounded having an amorphous, osmiophilic and PAS-reactive content. They were concentrated in the longitudinal bars separating adjacent cell columns, being most numerous close to the calcification front and believed to be formed either from fragments of cytoplasm of degenerating chondrocytes or from fragments of cell processes. In an investigation on chick embryonic cartilage MATUKAS and KRIKOS (1968) observed membrane-bound

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dense bodies in condensed areas of matrix undergoing mineralization, and raised the possibility that they represented lysosomes. ANDERSON (1969), in studying mouse epiphyseal cartilage, introduced the term matrix vesicles for membrane-bound structures occurring within the longitudinal septa with the highest concentration in the hypertonic zone. It was suggested that some or all of the vesicles represented lysosomes since they resembled lysosomes. This view was later abandoned because of the inability to demonstrate acid phosphatase activity in the vesicles (MATSUZAWA and ANDERSON 1971). However, as outlined in the pre-

ceding section, negatIve enzyme cytochemical results are generally inconclusive (d. DAEMS et al. 1972). Neither can the results of ALI et al. (1970) and ALI (1976) be taken as good evidence against the existence of lysosomal matrix vesicles, as also discussed in the preceding section. Two distinct types of vesicular structures, differing both in fine structure of their contents and in enzyme cytochemical properties, were regularly observed in the intercellular substance of guinea pig epiphyseal cartilage by THYBERG and FRIBERG (1970, 1972) and THYBERG (1972). They were both limited by a triple-layered membrane with a total thickness

Figures 8 - 10. Matrix vesicles in longitudinal septa of growth plate cartilage. All vesicles are limited by a triplelayered membrane. Those of type I (mv I) are rounded in shape and contain a dense matrix which sometimes appears to be in the process of leaking into the extracellular space proper (arrow Fig. 10). They are thus morphologically indistinguishable from the lysosomes in the chondrocytes (d. Figs. 1, 6 and 7). Most of the type II vesicles (mv II) are irregularly shaped. Their matrix is similar in appearance to the ground cytoplasm of the chondrocytes (d. Fig. 6). The extracellular matrix consists of thin collagen fibrils with attached rounded or polygonal granules. - Magnifications X 32,000 (Fig. 8), X 108,000(inset Fig. 8), x 75,000 (Fig. 9), x 65,000 (Fig. 10).

Lysosomes and Endochondral Growth

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of about 9 nm. A further similarity between the two types of vesicles was that they occurred at all levels of the cartilage and were most numerous in the hypertrophying and calcifying zones. Matrix vesicles type I were morphologically indistinguishable from the cytoplasmic dense bodies. Thus, these structures were of similar shape, ranged within the same size and had a content of identical appearance (Figs. 8 - 10; d. Figs. 6 and 7). The membranous coat of the vesicles was frequently defective, however, and leakage of their contents into the intercellular substance proper was sometimes evident (Fig. 10). Type I vesicles were almost exclusively found in the longitudinal septa, particularly in their central parts, but occasionally occurred also in the transversal septa and in the pericellular zones. Acid phosphatase and aryl sulfatase activity was demonstrated cytochemically within these vesicles (see section 2.2). Matrix vesicles type II had an irregular form and maximal dimensions of about 0.05 - 0.5 ftm. Their contents were less electron-dense than that of vesicles type I and similar in appearance to the ground cytoplasm of the chondrocytes, including ribosome-like structures (Figs. 8 and 10). The type II vesicles were most numerous in the pericellular zones but were also found in the longitudinal septa, where they often occurred in close relationship to vesicles of type I (Figs. 8 and 10). After incubation for demonstration of alkaline phosphatase and ATPase activity, reaction product appeared on the surface of the

type II vesicles as well as on the surface of the chondrocytes (see section 2.2). From the morphological and enzyme cytochemical observations dealt with above it seems resonable to conclude that matrix vesicles type II represent fragments either of budding cytoplasmic processes or of degenerating cells, and that their limiting membrane is equivalent to the plasma membrane. This is in accordance with the concentration of the vesicles in the hypertrophying and calcifying zones, where the chondrocytes ultimately disintegrate (SCOTI and PEASE 1956; GOODMAN and PORTER 1960; TAKUMA 1960; ENGFELDT 1969; THYBERG and FRIBERG 1971; BRIGHTON et al. 1973). Similar conclusions regarding the cellular origin of matrix vesicles have been reached by other investigators (ANDERSON 1969; Au et al. 1970; BONUCCI 1970, 1971; MATSUZAWA and ANDERSON 1971; RABINOVITCH and ANDERSON 1976). Concerning the nature of matrix vesicles type I, the results described above indicate that these vesicles represent cytoplasmic dense bodies, i. e. lysosomes, released from the chondrocytes. This interpretation is supported by observations of several other studies (MATUKAS and KRIKOS 1968; DEARDEN 1974; MEIKLE 1975). MEIKLE (1975) suggested that lysosomes are released from the chondrocytes at an early stage in their maturation, well before they begin to degenerate. It is then difficult, however, to explain the high frequency of lysosome-like matrix vesicles in the zones of hypertrophy and early calcificat-

Figure 11. Chondrocyte from a 20 hr monolayer culture of cells isolated from fetal epiphyseal cartilage by collagenase digestion. The isolated cells were first allowed to adhere to the plastic dish for 4 hr in normal culture medium and then transferred to fresh medium containing colloidal thorium dioxide particles. After exposure to these particles for 16 hr the cells were harvested by trypsinization and prepared for electron microscopy. The cell on the micrograph contains several lysosomes heavily loaded with marker particles. No unlabeled lysosomes are seen. The inset shows part of a chondrocyte exposed to thorium dioxide for 16 hr but at a concentration 10 times lower than that for the cell in the main figure. The number of marker particles in the lysosomes are now more moderate and their characteristic dense matrix is clearly seen. - Magnifications X 13,000 and X 27,000 (inset). Figure 12. Photomicrograph of a transplant removed 3 weeks after intramuscular injection of isolated chondrocytes. The tissue formed by the injected cells looks like normal cartilage with cells located in lacunae and surrounded by a basophilic matrix. In the periphery the cells are smaller and more flattened and the matrix more weakly stained than in the central parts. Hematoxylin and eosin. - Magnification X 150. Figures 13 and 14. Cartilage formed by thorium dl
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ion as well as the finding that the vesicles were often grouped together in large clusters. In our opinion, and as further discussed below, it seems more probablechatmatrixvesicles type I originate from lysosomes in disintegrating chondrocytes (THYBERG and FRIBERG 1970, 1972). The exact way in which these vesicles reach their location in the central parts of the longitudinal septa is still unclear. One possibility is that the lysosomes, after being released from a chondrocyte, follow the flow of matrix macromolecules secreted from the chondrocyte or its neighboring cells. This flow must be directed mainly toward the longitudinal septa, where the largest amounts of organized matrix occur. An uncertain point in this connection is whether and how the lysosomes are capable of finding a way into the preexisting intercellular substance, and if this is at all necessary for their location to the center of the septa. To answer these questions detailed knowledge of the pattern of organization and turnover of the extracellular matrix in the epiphyseal plate would be required. Another possibility is that matrix vesicles type I emanate from chondrocytes located more or less midway between two cell columns; such cells were often found' both in the proliferative and hypertrophying zones. If such a cell underwent extensive disintegration, it seems likely that the former lacuna would then be filled up by matrix secreted from the adjacent chondrocytes. Lysosomes and cytoplasmic fragments remaining could in this way become matrix vesicles of type I and II respectively. Moreover, these vesicles could, at least initially, be expected to be

grouped together in the longitudinal septum (see Figs. 8 and 10). A corroboration and extension of the ideas discussed above was obtained by THYBERG et al. (1975 a) after labeling of the lysosomes in the chondrocytes. Pilot experiments showed that the electron-dense markers ferritin and colloidal thorium dioxide did not penetrate to any significant extent, either in vivo or in vitro, into intact epiphyseal plates, or thin slices thereof. The following experimental design was therefore adopted. Briefly, chondrocytes from cartilaginous epiphyses of 40 - 50-day-old guinea pig fetuses were dissociated from the surrounding matrix by collagenase digestion. The isolated cells were then cultured in monolayers in the presence of colloidal thorium dioxide, fixed after varying intervals, and studied ultrastructurally. It was thereby demonstrated that the exogenous marker particles, after initial uptake by endocytosis, accumulated in the lysosomes (Fig. 11; see also section 2.4). In the continued experimentation, chondrocytes labeled with thorium dioxide particles were injected intramuscularly into young guinea pigs. Here the transplanted cells reconstituted a typical hyaline cartilage (Figs. 12 - 14) within which cellular hypertrophy and degeneration as well as matrix calcificatibn occurred after 2 - 3 weeks. In connection with the degeneration of the chondrocytes, the lysosomes remained structurally intact and retained the incorporated marker (Fig. 15). Moreover, thorium dioxidelabeled lysosomes were found not only in the lacunae of more or less completely fragmented

somes are seen. m mitochondria, er granular endoplasmic reticulum, mvb multivesicular body. Fig. 13 uranyl acetate staining, Fig. 14 unstained. - Magnifications X 18,000 and X 50,000. Figure 15. Portion of a degenerating chondrocyte in a 2.5-week-old transplant formed by thorium dioxidelabeled cells. Vesiculation and disintegration of the cytoplasm is evident and with the exception of an apparently intact lysosome filled with marker particles no normal organelles are seen. The inset demonstrates a thorium dioxide-containing matrix vesicle of type I in the extracellular matrix of the same type of transplant. Only uranyl acetate staining. - Magnifications X 36,000 and X 59,000 (inset). Figure 16. Part of a chondrocyte from a monolayer culture exposed to Dextran T150 (weight average molecular weight = 150,000). Visualization of glycogen (arrows) and ingested Dextran was obtained by a one-step fixationblock staining of the cells with a mixture containing formaldehyde, glutaraldehyde, osmium tetroxide and lead citrate in phosphate buffer, pH 7.4 (SIMIONESCU et al. 1972). G Golgi complex, llysosomes with densely stained Dextran. Section staining with lead citrate. - Magnifications X 22,000 and X 41,000 (inset).

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cells, but also in the intercellular substance proper, i. e. midway between neighboring chondrocytes. Altogether the observations suggested that these latter bodies largely represented remnants of disintegrated cells, the lacunae of which had been replaced by matrix produced by adjacent chondrocytes. Summing up, the observations reported by THYBERG et al. (1975 a) give firm support to the concept that, in guinea pig epiphyseal cartilage, matrix vesicles type I represent lysosomes arising from disintegrating chondrocytes. Moreover, together with the results given by THYBERG and FRIBERG (1970,1972) and THYBERG (1972), they point to the possibility that the lysosomes remain intact during the process of cellular disintegration, and r~lease their contents only after having settled in their position in the intercellular substance. This interpretation is in accordance with the findings of HAWKINS et al. (1972). In an experimental study on cultured Chang liver cells, these authors concluded that the lysosomes appeared to be stable organelles, bursting first when the cells are already irreversibly dead.

2.4 Role in heterophagy The participation of the lysosomal system in ingestion and intracellular digestion of exogenous material has been well established in a large number of cell types (for reviews see DE DUVE and WATTIAUX 1966; DINGLE and FELL 1969; HERS and VAN HOOF 1973). However, excepting macrophages, few studies of chondrocytes and other connective tissue cells have been made. The endocytotic ability of cartilage cells was first inferred in the study of GLAUERT et al. (1969) in which exposure of cultured chick limbbone rudiments to sucrose was shown to cause cytoplasmic vacuolation. More direct evidence was subsequently provided by THYBERG et al. (1975 a), who studied the uptake and intracellular accumulation of colloidal thorium dioxide particles in cultured chondrocytes. The marker

particles were initially found in invaginations of the plasma membrane, then in endocytic vesicles, and finally accumulated in cytoplasmic dense bodies, i. e. lysosomes (Fig. 11). All dense bodies were generally labeled. This finding, together with observations made after transplantation of the cells, indicated that a continuous exchange of material occurred within the lysosomal system of the cells (d. BRUNK 1973). Cultured chondrocytes have also been shown to endocytize dextrans of varying molecular weights (Fig. 16; THYBERG and MOSKALEWSKI, unpublished observations). It was suggested by DINGLE (1969) and DINGLE et al. (1969) that lysosomes are involved in the catabolism of the intercellular substance of cartilage and other connective tissues by a two-stage process including partial extracellular breakdown of the matrix by enzymes secreted from the cells, followed by endocytosis of the partially degraded material, the digestion of which is completed within the lysosomal system. It seems improbable, however, that such a mechanism is of any substantial importance in the epiphyseal growth plate in view of the fast turnover of this tissue (KEMBER 1960) and the relative scarcity of lysosomes in the chondrocytes. Moreover, while a distinct decrease in the concentration of proteoglycans occurs in the zone of early calcification (see further section 2.6), the cells are degenerated or start to degenerate here (SCOTT and PEASE 1956; GODMAN and PORTER 1960; TAKUMA 1960; ENGFELDT 1969; THYBERG and FRIBERG 1971; BRIGHTON et al. 1973) and can therefore not be very active metabolically. The exact physiological role of heterophagy, if any, in epiphyseal cartilage therefore remains to be elucidated.

2.5 Role in autophagy and cell degeneration Autophagy constitutes a mechanism by which cells are able to segregate and degrade portions of their own cytoplasm in organelles called

Lysosomes and Endochondral Growth autophagosomes and autolysosomes or, to use a more general term, secondary lysosomes (DE DUVE and WATIIAUX 1966; ERICSSON 1969). In normal cells this may represent a means of disposing of worn-out organelles. At the same time valuable chemical constituents (amino acids, etc.) are formed which may be reutilized in the cell metabolism. As discussed by ERICSSON (1969), increased autophagy is noted in cells deprived of essential nutrients and as a response to various pathological influences. Signs of autophagy are seldom seen in the normal growth plate cartilage. Considering the fast turnover of this tissue (KEMBER 1960) and the belief that all chondrocytes are irreversibly dead when reached by the erosion line (SCOTI and PEASE 1956; GODMAN and PORTER 1960; TAKUMA 1960; ENGFELDT 1969; THYBERG and FRIBERG 1971; BRIGHTON et al. 1973), it also seems unlikely that there is any significant need for autophagy here. Nevertheless, like most other cells, chondrocytes have the ability to carry out this process. MOSKALEWSKI et al. (1975), for example, have shown that exposure of cultured chondrocytes to antimicrotubular agents results in augmented autophagy. Similar observations have been hade on other cell types (HIRSIMAKI et al. 1975, 1976; KOVACS et al. 1975). Of more interest is what role lysosomes play in cell degeneration and autolysis in epiphyseal cartilage. As pointed out by DE DUVE (1969), rupture of lysosomes and subsequent release of acid hydrolases into the ground cytoplasm could be a significant factor in causing cell death. It may be difficult, however, to decide which of the two, lysosome rupture or cell death, precedes the other. In the study of HAWKINS et al. (1972) the time of bursting of lysosomal membranes relative to other changes of cell injury and necrosis in cultured Chang liver cells was estimated. Before injury, induced by inhibition of energy metabolism or immunologic damage to the plasma membrane, the cells were allowed to form many lysosomes containing both enzymes and exogenous markers which could be observed morphologically. The results indicated that

17

lysosomes are stable organelles which, following injury of the types studied, burst first in the postmortem phase of cellular necrosis, acting then as scavengers which help to clear the cellular debris. Support for this view was obtained by THYBERG et al. (1975 a) in studying cartilage formed by thorium dioxide-labeled chondrocytes. Thus, in connection with the degeneration of the chondrocytes, occurring after 2 - 3 weeks, the lysosomes remained structurally intact and did not release the incorporated marker (for further details see the description given in section 2.3). Applying these observations to the epiphyseal plate, it is suggested that lysosomes do not cause cell death but may be involved in digesting the disintegrated cells, a process enabling the growth of metaphyseal capillary sprouts into the former chondrocyte columns (d. MEIKLE 1975; see also section 3.4.1).

2.6 Extracellular release of lysosomal enzymes and matrix Q1ineralization Early fine structural studies on epiphyseal plate cartilage revealed that the calcification of the intercellular substance is restricted to the longitudinal bars separating adjacent cell columns (ROBINSON and CAMERON 1956; SCOTI and PEASE 1956). It was later shown that the deposition of mineral begins in the central parts of these bars (ENGFELDT 1969). Thus, the location of initial mineralization appears to coincide with the location of the largest number of matrix vesicles (Figs. 17 and 18). In guinea pig epiphyseal cartilage (THYBERG 1974), the first mineral deposits were found in the central parts of the longitudinal septa, and appeared in the form of single, needle-like crystals, or small clusters thereof. This crystalline material was usually located in the close vicinity of matrix vesicles type I, but not within them (Figs. 19 and 20). Growth of crystals into the vesicles seemed to be a secondary phenomenon, taking place during later stages of mineralization. When mineral crystals were seen apparently inside

J. THYBERG . U. FRIBERG

18

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Figures 17 and 18. Longitudinal septa from the calcifying zone of the epiphyseal growth plate cartilage showing moderate and heavy mineralization, respectively. The mineral deposits are located in the central parts of the septa where numerous matrix vesicles type I (mv I) also are present. - Magnifications X 32,000 and x 37,000.

Lysosomes and Endochondral Growth matrix vesicles, these were mostly of type II. Such vesicles were, however, only rarely found in the areas of initial calcification. No direct relationship between the early mineral crystals and the organic, macromolecular components of the intercellular substance, i. e. collagen fibrils and proteoglycan granules, was noted. In cartilage formed by isolated chondrocytes injected into skeletal muscular tissue of young guinea pigs (THYBERG et al. 1975 a), the pattern of initial calcification was analogous to that described above. Thus, the mineralization began in midposition between neighboring cells and the early crystal deposits often lay in close proximity to matrix vesicles of type 1. A similar spatial relationship between extracellular vesicles and initial calcification has been demonstrated in other cartilages (MATUKAS and KRIKOS 1968; DEARDEN 1974; DEARDEN and ESPINOSA 1974; MEIKLE 1975;- see also ANDERSON 1969; ANDERSON et al. 1975). It has also been claimed, however, that the very first crystallites are laid down inside the vesicles (ALI et al. 1970; ANDERSON et al. 1970; BoNUCCI 1970, 1971; ANDERSON 1973; SIMON et al. 1973; HOHLING et al. 1976). Although this is not the proper place for an extended discussion of this point, we would like to raise a few questions regarding this latter view. First, when mineral crystals are seen both inside and outside matrix vesicles it is difficult, if not impossible, to judge which of them were laid down first. Moreover, no electron diffraction data have been presented which show that hydroxyapatite, the predominating crystalline mineral of cartilage and bone (POSNER 1969; TERMINE 1972), is present within matrix vesicles. Hence, it is not possible to exclude for certain that precipitation of mineral ions inside the vesicles can have taken place during the preparation of the tissue. Finally, the early calcification of cartilage usually occurs as multiple, scattered foci of crystal clusters, many of which obviously must lack direct contact with matrix vesicles (Fig. 21). It is then difficult to imagine that crystals initially deposited within

a

.

19

matrix vesicles should provide, in principle, the nuclei from which continued crystal growth occurs (d. ANDERSON 1973). The abovementioned close morphological connection between matrix vesicles type I and early mineral deposits suggests a functional role of these vesicles in the processes which make the cartilaginous matrix calcifiable. Since type I vesicles were found at all levels of the epiphyseal plate, the mere presence of such vesicles is not a sufficient requirement for initiation of the mineralization. The occurrence of the largest numbers of type I vesicles at about that level in the cartilage where the calcification starts indicates that a quantitative requirement may also be involved. Furthermore, the finding that the first crystals were located around and not within the vesicles suggests that the latter structures are implicated in the mineralization process by releasing agents which assist in rendering the intercellular substance calcifiable (d. MEIKLE 1975). In view of the lysosomal nature of matrix vesicles type I (see sections 2.2 and 2.3), these agents probably correspond to one or more hydrolytic enzymes. One problem in this context is that lysosomal enzymes have acid pH optima, and if they were supposed to digest any extracellular material, would probably require a pH lower than 7. In this context it is interesting, however, to note the findings of AVILA and CONVIT (1976). They isolated lysosomes from leucocytes and measured activities of a large number of acid hydrolases in the presence of sulfated glycosaminoglycans, e. g. chondroitin sulfate, the predominating glycosaminoglycan in hyaline cartilage proteoglycans. In general, the glycosaminoglycans induced a strong pHdependent inhibition of the lysosomal enzymes at pH values lower than 5.0, with full activity at higher pH values. In an extracellular fluid phase, aspirated by micropuncture technique from the hypertrophying zone of rat epiphyseal cartilage, the pH was reported to be about 7.6 (HOWELL et al. 1968). The lysosomal concept, as advanced by DE DUVE (1963), postulates the existence of an

20

.

]. THYBERG . U. FRIBERG

Figures 19 'and 20. Details from longitudinal septa in early stages of mineralization demonstrating the relationship between mineral crystals and type I matrix vesicles (mv I). c collagen fibrils, mg matrix granules (proteoglycans).Magnifications X 87,000 (Fig. 19), X 82,000 (Fig. 20), X 188,000 (inset Fig. 20).

Lysosomes and Endochondral Growth

acid milieu within the Iysosomes. Accordingly, the pH of the interior of Iysosomes isolated from rat liver has been found to be up to 1.5 pH units lower than in the surrounding medium (REIjNGaUD and TAGER 1973; HENNING 1975). It has been suggested that the presence of nondiffusible anions inside the lysosomes produces a Donnan equilibrium, which could serve as an energy-independent means to maintain an acid intralysosomal pH (COFFEY and DE DUVE 1968; HENNING et al. 1973; HENNING 1975). Moreover, an energy-dependent proton pump may exist in the lysosome membrane (MEGa et al. 1972; MEGa 1975). After disintegration of a chondrocyte in the epiphyseal plate, and release of lysosomes into the extracellular space, an acid pH could conceivably be preserved within the freed Iysosomes (matrix vesicles type I), as long as their limiting membrane remains intact, by way of the first or perhaps of both the mechanisms mentioned above. In connection with disruption of the vesicles and leakage of their content into the surrounding intercellular substance proper, a more acid milieu could then, at least for a short time interval, be established locally in the matrix. This in turn might allow the lysosomal hydrolases to carry out their catalytic activity. In view of the results of AVILA and CONVIT (1976) this might even occur if the pH is in the neutral regIOn. As mentioned in the introduction, the calcification of cartilage is preceded by, or connected with, a chemical alteration and partial loss of proteoglycans. For example, HIRSCHMAN and DZIEWIATKOWSKI (1966), in an immunohistochemical study on rat epiphyseal cartilage, found a markedly diminished staining, with fluorescein-labeled antibodies, of proteoglycans in the zone of provisional calcification, compared with the proliferative and the upper hypertrophying zone. Similarly, a clear reduction in number and/or size of the proteoglycan granules in areas of incipient mineralization has been observed ultrastructurally (MATUKAS and KRIKO'i 1968; SMITH 1970; THYBERG et al.

.

21

1973 b). Using a dissection procedure which permitted zonal analysis of the epiphyseal plate of fetal calf bone, WUTHIER (1969) found the proteoglycan content measured as hexosamine and expressed as percentage of organic matrix to be at a maximum in the proliferating cartilage and then to be approximately halved from the hypertrophic to the calcified cartilage. PITA et al. (1970) reported that a proteoglycan fraction, thought to represent proteoglycan aggregates, was almost completely absent from whole calcified cartilage, as well as from extracellular fluid aspirated from calcified cartilage in vivo; in vitro this fraction functioned as an inhibitor of mineral growth (see also HOWELL and PITA 1976). These results have been corroborated and extended by LOHMANDER and HjERPE(1975), who studied the proteoglycans in young guinea pig rib and Beagle puppy epiphyseal cartilage. By centrifuging finely ground material in acetonebromoform density gradients three fractions were produced, representing non-, low and highly mineralized tissue. Chemical analyses of the different fractions indicated that, in both cartilages, about half of the proteoglycan content was lost before or during the early phases of calcification. Concomitantly, the proportion of proteoglycan aggregates decreased. In this context it is interesting to note that proteoglycan aggregates, but not monomers, have been demonstrated to inhibit the growth of seeding minerals in a synthetic lymph (CUERVO et al. 1973). The degradation of proteoglycans at the calcification front of epiphyseal and other mineralizing cartilages is most probably enzymatic, and should, considering the degenerate state of the chondrocytes in these areas, predominantly occur extracellularly. Although studies on cartilage are few, a number of enzymes which could be involved in the above-mentioned degradative process have in many other tissues been shown to be lysosomal. Among these enzymes are hyaluronidase and cathepsin D (BARRETT 1972). Thus, hyaluronidase digests hyaluronic acid, which in hyaline cartilage constitutes the back-

22

J. THYBERG . U. FRIBERG

Lysosomes and Endochondral Growth bone of the proteoglycan aggregates (HARDINGHAM and MUIR 1972; HASCALL and HEINEGARD 1974a, b). Together with other lysosomal glycosidases, hyaluronidase is also able to attack the chondroitin sulfate side chains of the proteoglycan monomers (BARRETT 1972; W ASTESON et al. 1975). It has been demonstrated that cathepsin D is present in cartilage and is capable of splitting the protein core of the proteoglycan monomers, yielding endproducts similar to those resulting from the action of trypsin (MORRISON et al. 1973; WOESSNER 1973). In summary, the limitation of this process to a narrow zone of the cartilage could be explained by the involvement of lysosomal enzymes, released from matrix vesicles type I, in the degradation of proteoglycans in the epiphyseal plate. Since various mechanisms by which a loss or chemical alteration of proteoglycans could promote calcification have been indicated (for a review see CAMPO 1970), this may account for the appearance of the first mineral deposits in close proximity to type I vesicles. Nevertheless, there are also other mechanisms by which enzymes liberated from the latter structures could influence the formation and further growth of crystal seeds. For example, such enzymes may be engaged in the conversion of inorganic pyrophosphate to orthophosphate. In vitro, inorganic pyrophosphate inhibits the transformation of amorphous calcium phosphate into crystalline apatite (RUSSELL and FLEISCH 1970) and, in vivo, there is in the epiphyseal plate a dramatic decrease in the ratio of inorganic pyrophosphate to inorganic phosphate during early phases of calcification (WUTHIER 1972). Acid pyrophosphatase activity has been demonstrated to be present in lysosomes (BARRETT 1972). Moreover, in calf scapula cartilage the pyrophosphat-

23

ase activity was found to have a pH-optimum around pH 4.4, and to increase several times in the calcifying zone (JIBRIL 1967). It was recently suggested that lysozyme is involved in deaggregation of proteoglycans in the epiphyseal plate, and in that way plays an important role in the initiation and regulation of the calcification process (KUETTNER et al. 1974). However, the widespread occurrence of lysozyme in epiphyseal cartilage (GUENTHER et al. 1974) would in that case imply that it is normally inactive, and is in some way activated prior to the start of mineralization. It may be hypothesized that lysosomal enzymes are implicated in such an activation. Mechanisms by which extracellular vesicles, equivalent to our type II vesicles, could be implicated in the mineralization process have been presented by ANDERSON (1973). These theories are not incompatible per se with the ideas discussed above, but no direct spatial relationship between matrix vesicles type II and the initial laying down of mineral was observed by THYBERG (1974) and THYBERG et al. (1975a). Since there is a clear structural heterogeneity among the matrix vesicles, it seems probable that there could also be a functional heterogeneity. Admittedly, the above discussion is partly speculative. It is hoped, nevertheless, that it will provide a framework for future studies on the presence and functions of lysosomal vesicles in the intercellular substance of epiphyseal and other calcifying cartilages.

Figure 21. Initial calcification in a longitudinal septum showing separate clusters of mineral crystals. c collagen fibrils, mg matrix granules (proteoglycans) stained with ruthenium red. The inset shows the very first signs of mineralization in a 2.5-week-old transplant formed by isolated chondrocytes injected intramuscularly (only uranyl acetate staining). - Magnifications X 79,000 and X 17,000 (inset). Figures 22 and 23. Osteoblasts in metaphyseal bone after incubation of glutaraldehyde-fixed tissue for demonstration of acid phosphatase and aryl sulfatase activity, respectively. Excluding small amounts of precipitate in the Golgi complex (G in Fig. 22), reaction product is exclusively found in Iysosomes. Omission of the postosmication

24

J. THYBERG

.

U. FRIBERG

3. Lysosomes in metaphyseal bone 3.1 Biochemical properties As far as we know, no biochemical investigations of lysosomes isolated from bone formed by endochondral ossification are present in the literature. Nevertheless, the studies of VAES and collaborators have clearly demonstrated the existence of lysosomes in bone cells (for a review see VAES 1969). VAES and JACQUES (1965 a) and LiEBERHERR et al. (1973) assayed and partly characterized a large number of acid hydrolases in homogenates of infant rat calvaria. By differential centrifugation they further demonstrated that these enzymes belonged to cytoplasmic particles showing the main characteristics of lysosomes, i. e. specific sedimentability and structure-linked latency (VAES and JACQUES 1965b; VAES 1965, 1967; VREVEN et al. 1973). This contrasted to the occurrence of cytochrome oxidase In the mitochondrial fraction and of the various alkaline phosphatases tested in the microsomal fraction.

3.2 Cytochemical properties Acid phosphatase activity has been demonstrated in lysosomal bodies in rat metaphyseal bone (DoTY et al. 1968; LUCHT 1971; DoTY and SCHOFIELD 1972, 1976; SCHOFIELD et al. 1974) and in fracture callus (GOTHLIN and ERICSSON 1971, 1973a; GOTHLIN et al. 1973). LUCHT (1971) and DoTY and SCHOFIELD (1972) further reported acid phosphatase activity to be present extracellularly in the ruffled

border region of the osteoclasts. On the other hand, GOTHLIN and ERICSSON (1971) found reaction product over tubular elements, vesicles and vacuoles located subjacent to the ruffled border, but not extracellularly. The fine structural location of acid phosphatase and aryl sulfatase activity in guinea pig metaphyseal bone was determined by THYBERG et al. (1975b). To avoid the risk of achieving false positive precipitations due to the high mineral and the high free phosphate content in bone (ASHTON et al. 1973) the incubations were made on demineralized tissue. Reaction product for the above-mentioned lysosomal enzymes was present in cytoplasmic dense bodies in all cells types, i. e. endothelial cells, different types of perivascular cells, osteoblasts and chondroclasts/osteoclasts (Figs. 22 and 23). Reaction product was occasionally found in the cisternae and small vesicles of the Golgi complex and, in the chondroclasts/osteoclasts, also in cytoplasmic vacuoles. No significant amounts of precipitate were, however, observed in the channels between the cytoplasmic processes of the ruffled border. DoTY and SCHOFIELD (1972, 1976), using various substrates, located several other acid hydrolase activities in rat metaphyseal bone. However, excepting aryl sulfatase and inorganic trimetaphosphatase activity, a non-specific acid phosphatase was suggested to be responsible for the reactions. Staining was further found in lysosomes after incubation at neutral pH for ATPase, phosphoamidase and p-nitrophenyl phosphatase. The two latter enzyme activities could not be differentiated from each other and it was proposed that the lysosomal staining for ATPase could be due to phosphoamidase (d. BARRETT 1972). A magnesium independent

account for the
Lysosomes and Endochondral Growth

25

26

.

J. THYBERG . U. FRIBERG

Figure 25. Detail from the cytoplasm of an osteoblast demonstrating the fine structure of a lysosome. It is limited by a triple-layered membrane beneath which a thin electron-lucent rim is present. Otherwise the lysosome is filled with a dense matrix. er granular endoplasmic reticulum, r free polyribosomes. - Magnification X 83,000. neutral ATPase has also been found in the lysosomes of the cells in fracture callus (GOTHLIN and ERICSSON 1973 b).

3.3 Ultrastructural properties The fine structure of different cell types in the metaphysis and at other sites of bone formation has been described by a large number of authors (for a recent review see CAMERON 1972). The proliferation and specialization of cells in the fetal rat metaphysis was studied by SCOTT (1967 a) using electron microscopic autoradiography with tritiated thymidine. Two cell types could be distinguished among the labeled osteogenic cells, a spindle-shaped type (A), and a rounded type (B). The type A cells had characteristics generally associated with matrix formation and were regarded as pre-osteoblasts. The type B cells were reported to resemble de-

veloping neutrophilic granulocytes and were tentatively interpreted as representing preosteoclasts. This concept of two cell lines has been corroborated by ultrastructural observations on the young guinea pig metaphysis (THYBERG et al. 1975 b) and on callus formation in the adult rat (GOTHLIN 1973; GOTHLIN and ERICSSON 1973c). Thus, in our material (THYBERG et al. 1975 b) two well-defined cell populations could be distinguished similar to those described by SCOTT (1967 a). The perivascular cells type A were evidently closely related to the osteoblasts. Admittedly, the former cells varied structurally within wide limits. Some of them were immature with a large nucleus and only small amounts of cytoplasm with scant organelles. However, at the other end of the line the type A cells were indistinguishable from the osteoblasts, the latter then defined by their association with a collagenous matrix. Hence, it seems well founded to

Lysosomes and Endochondral Growth consider the perivascular cells type A as precursors of the osteoblasts. Perivascular cells type A and osteoblasts were distinguished by a well developed granular endoplasmic reticulum and Golgi complex (Fig. 24), i. e. organelle systems involved in synthetic and secretory functions (PALADE 1975). Among the other organelles a few cytoplasmic dense bodies were found. They were surrounded by a 9 nm thick, triple-layered membrane, beneath which a thin electron-lucent rim frequently occurred (Fig. 25; d. DAEMS et al. 1972). A dense homogeneous matrix, sometimes mixed with vesicular structures and/or other inclusions, filled the bodies. Together with the positive staining for acid phosphatase and aryl sulfatase (see section 3.2) and the ability to accumulate exogenous marker particles (see section 3.4) these morphological characteristics identify the dense bodies as lysosomes (BARRETI 1972;

27

BECKetal.1972;DAEMSetal.1972). In contrast to the findings of SCOTI (1967 a), perivascular cells type B were macrophage-like rather than granulocyte-like (THYBERG et al. 1975 b). Because of their resemblance to the chondroclasts and osteoclasts it seems reasonable to assume that they are pre-osteoclasts. It has been suggested that osteoclasts may arise by coalescence of monocytes from the blood (VAN FURTH et al. 1972). Experimental evidence supporting this hypothesis has been presented by GOTHUN and ERICSSON (1973d) in a report on callus formation in parabiotic rats. On the basis of labeling experiments with tritiated thymidine and thorium dioxide particles they concluded that at least a proportion of the osteoclasts were formed from blood-born precursors. Similar conclusions have come from studies of mammalian osteopetrosis (MARKS and WALKER 1976). In the metaphysis of

Figure 26. Juxtanuclear area of an osteoclast showing stacks of Golgi cisternae (G) surrounded by numerous vacuoles (v) and lysosomes (I). The latter have a dense homogeneous content and just beneath the limiting membrane a characteristic electron-lucent rim is present. er granular endoplasmic reticulum, m mitochondria, n nucleus, r free polyribosomes. - Magnification X 28,000.

28

J. THYBERG . U. FRIBERG

growing animals it seems equally probable, however, that type B cells and thus osteoclasts develop from the undifferentiated mesenchyme accompanying the invading capillaries. Further studies will be required to elucidate the particulars of the development of multinucleated
the advancing capillary sprouts. They contained numerous membrane-bound cytoplasmic dense bodies showing lysosomal enzyme activity (see section 3.2), a prominent Golgi complex and large numbers of mitochondria and free polyribosomes. As compared to type A cells and osteoblasts, the granular endoplasmic reticulum was poorly developed (Figs. 27 and 28). The lysosomes were of highly variable size and inclusions of different types were frequently seen within them (see further section 3.4). ANDERSON and PARKER (1966) and SCHENK

Figure 27. Part of a type B perivascular cell closely attached to a cartilaginous septum (cs). An intravenous injection of colloidal thorium dioxide particles was given one day before killing the animal. Varying amounts of ingested marker particles are found in the numerous Iysosomes (I). No unlabeled lysosomes are seen. G Golgi complex, m mitochondria, n nucleus, r free polyribosomes. Tissue decalcified with EDTA after the glutaraldehyde fixation. - Magnification X 19,000. Figure 28. Perivascular cell of type B (B) located just outside the wall of an invading capillary sprout in the metaphysis. An intravenous injection of colloidal thorium dioxide was given two days before killing the animal. The type B cell contains several Iysosomes with ingested marker particles. In two of the Iysosomes (I) other inclusions are also evident. A perivascular cell type A, cl capillary lumen, ec endothelial cell, n nucleus. - Magnification X 16,000. Figures 29 - 31. Osteoclasts in the metaphysis of a young guinea pig which received an intravenous injection of colloidal thorium dioxide two days before being killed. Figs. 29 and 30 show in low magnification, the ruffled borders (rb) of two cells which surround the blind ends of two cartilaginous septa (cs) in the process of resorption.

Lysosomes and Endochondral Growth

29

30

J. THYBERG . U. FRIBERG

et al. (1967, 1968) likewise noted the presence of lysosome-like bodies in perivascular cells in rat metaphyseal bone. Chondroclasts and osteoclasts were identical in structure and will from now on be referred to collectively as osteoclasts. These cells were large, multinucleated and distinctly polarized. On one side they showed a ruffled border region opposed to the surface of calcified cartilage and/or bone matrix in the process of being resorbed (Figs. 29 and 30). This ruffled border, described in detail already by SCOTT and PEASE (1956), consisted of numerous thin cytoplasmic projections, delineating narrow channels or saccules penetrating towards the interior of the cell. On both sides of the ruffled border region the cell surface lay in direct contact with the matrix, sealing off and perhaps allowing the establishment of a special microenvironment in the former, resorptive region (d. VAES 1969). In this transitional zone the peripheral cytoplasm contained large numbers of microfilaments but essentially no other organelles (Figs. 29 and 30). Below the ruffled border the cytoplasm of the osteoclasts showed many vacuoles of largely varying size and, somewhat deeper into the cell, numerous cytoplasmic dense bodies. The latter were more uniform in size than the vacuoles and had morphological characteristics of lysosomes with an electron-lucent rim just inside the limiting membrane and an otherwise dense, homogeneous content, generally without identifiable inclusions (Fig. 26). As discussed above (section 3.2), the dense bodies also displayed lysosomal enzyme activity. In addition to the location beneath the ruffled border, cytoplasmic dense bodies were numerous in more central parts of the osteoclasts, i. e. in the area around the nuclei (Figs. 26, 29 - 31). Here they often showed a close relationship to the dictyosomes of the Golgi complex (Fig. 26). Detailed descriptions of cytoplasmic vacuoles and bodies in rat metaphyseal bone have been given by SCOTT (1967b) and LUCHT (1972 a). Our observations in the guinea pig (THYBERG et al. 1975 b), referred to above, are in general agreement with those of these authors.

Narrow cisternae of granular endoplasmic reticulum were in the osteoclasts concentrated to that side of the nuclei situated opposite to the ruffled border region. Mitochondria were numerous in the spaces between the cisternae of the reticulum as well as in the central perinuclear regions of the cells.

3.4 Role in heterophagy The ability of osteogenic cells to ingest and accumulate material within lysosomes has been demonstrated using exogenous markers of different types (GOTHLIN and ERICSSON 1972, 1973c; LUCHT 1972b; THYBERG 1975). In the following sections these and other observations will be discussed in relation to the resorptive processes occurring in connection with endochondral bone formation.

3.4.1 Resorption of epiphyseal cartilage After intravenous injection of colloidal thorium dioxide to young guinea pigs marker particles were present in the lysosomes of all cell types in the metaphysis (THYBERG 1975). The most active uptake was observed in perivascular type B cells and osteoclasts whereas perivascular type A cells and osteoblasts ingested more moderate amounts of the tracer (Figs. 27 - 32, 34, 35). Similar findings have been made by GOTHLIN and ERICSSON (1973c) in studies on the uptake of colloidal thorium dioxide in different cell types of rat fracture callus. In addition to the marker particles, the phagolysosomes of perivascular type B cells contained other inclusions, including recognizable fragments of cells and, more occasionally, cartilage matrix (Figs. 27 and 28). Together with the frequent location close to the erosion line, these findings strongly suggest that, in the endochondral growth apparatus, perivascular type B cells are of fundamental importance for the resorption of degenerated chondrocytes and

Lysosomes and Endochondral Growth

31

In Fig. 30 a thin layer of bone matrix (bm) has been deposited on the cartilage surface and is obviously being removed together with the cartilage. Below the ruffled borders the osteoclasts demonstrate numerous vacuoles (v) of varying size. The cells further contain large numbers of lysosomes with ingested thorium dioxide particles (I).

32

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.

U. FRIBERG

.

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•

er granular endoplasmic reticulum,m mitochondria, tz transitional zone of peripheral cytoplasm closely adjoining the cartilaginous septa just above the . Tissue decalcified with EDTA after glutaraldehyde fixation. - Magnifications X 5,000, X 7,000 and X 16,000. surrounding unmineralized matrix, and that their lysosomal system plays a major role in this context. The cellular uptake and intracellular digestion of cell remnants and extracellular matrix are probably preceded by a partial extracellular digestion mediated by lysosomal or other lytic enzymes released from disintegrating chondrocytes, perivascular type B cells and possibly also endothelial cells of the capillary sprouts growing into the former chondrocyte lacunae. Concerning perivascular type B cells it is interesting to note in this context that in macrophages and polymor-phonucIear leucocytes phagocytosis has been found to be associated with selective release of lysosomal hydrolases, probably by the phenomenon of (WEISMANN et al. 1971). Since the advancing capillary sprouts were generally found to come into direct contact with the unmineralized transversal septa (d. ANDERSON and PARKER 1966; SCHENK et al. 1967,

1968) it seems probable that the capillaries, by longitudinal growth, are capable of penetrating into the last encapsulated chondrocyte in each cell column. As observed by SCHENK et al. (1968) the vessels expand within a recently opened lacuna, approaching the transversal septum delimiting the next one. The remnants of the disintegrated chondrocyte are then most likely displaced behind the tip of the capillary, reaching the perivascular type B cells (d. section 3.3), by which they may then be phagocytized. The model for resorption of unmineralized components of epiphyseal cartilage presented above constitutes an extension of ideas discussed previously (BROOKES and LANDON 1964; ANDERSON and PARKER 1966; SCHENK et al. 1967, 1968). Similarly, studies on fracture callus have indicated that macrophage-like cells are important elements during the remodeling of bone tissue (GOTHLIN 1973; GOTHLIN and ERICSSON 1973c).

Lysosomes and Endochondral Growth

33

Figure 32. Part of an osteocyte in metaphyseal bone of a young guinea pig which received an intravenous mjection of colloidal thorium dioxide two days before being killed. I lysosomes with ingested marker particles, a autophagosome/autolysosome, bm bone matrix. The inset demonstrates an autophagosome/autolysosome (a) containing fragments of granular endoplasmic reticulum. I Iysosomes with a few thorium dioxide particles. Tissue decalcified with EDTA after glutaraldehyde fixation. - Magnifications X 25,000 and X 29,000 (inset).

3.4.2 Bone resorption By way of the resorptive process just discussed, the calcified longitudinal septa, which are apparently resistant to the process, are freed from other cartilage components and may subsequently serve as cores on whose surface osteoblasts appear and deposit bone matrix which later calcifies. The number of primary bone trabeculae formed in this way is, however, only about half the number of longitudinal septa in the epiphyseal plate. Moreover, as the longitudinal growth of the bone continues, the bone trabeculae go through an extensive reorganization. Consequently, there must exist means for the removal of calcified cartilage and bone matrix. It is well known today that osteoclasts are responsible for bone resorption (for reviews see HANCOX 1972; CAMERON 1972) and it has

been indicated by SCHENK et al. (1967) that resorption of calcified cartilage is mediated by chondroclasts, the fine structure of which is identical to that of osteoclasts. Although there are striking differences in composition of the organic matrices of cartilage and bone, there is probably no reason to consider these cells as separate entities. Thus, as shown in Fig. 30, one and the same cell is apparently able to remove cartilage and bone matrix simultaneously. Extensive biochemical studies of the involvement of lysosomes and lysosomal enzymes in the process of bone resorption have been performed by VAES (1965 b, 1968, 1969) using tissue cultures of calvarial bone stimulated with parathyroid hormone. By integrating the results of these studies with histochemical and ultrastructural data, a working hypothesis of osteoclastic bone resorption was put forward (VAES

34

J. THYBERG . U. FRIBERG

1969). According to this scheme lysosomal hydrolases are secreted in bulk by exocytosis in the ruffled border region of the osteoclast and, extracellularly, possibly together with a nonlysosomal collagenase (VAES 1969, 1972 a, b), bring about a partial digestion of the organic matrix. The progression of this process is secured by enchanced lysosomal enzyme synthesis in the cells. Simultaneous release of acid in the resorption zone, made possible mainly through a stimulation of aerobic glycolysis, was proposed to account for the solubilization of the mineral. This lays the organic matrix open to attack by enzymes and at the same time helps to create a proper environment for the action of the acid hydrolases. It was further suggested that matrix fragments produced by the extracellular resorption finally are taken up in the osteoclasts by endocytosis, to be further digested in the lysosomal system of the cells. The above model has been confirmed and extended by electron microscopic studies. Thus, LUCHT (1971) and DolY and SCHOFIELD (1972) noted reaction product for acid phosphatase extracellularly in the ruffled border region of the osteoclasts. Moreover, using electron microscopic autoradiography, LUCHT and NORGAARD (1976) showed that proteins, suggested largely to represent lysosomal enzymes, are secreted in this region of the cells. The endocytotic ability of osteoclasts has been demonstrated after intravenous injections of the markers peroxidase (LUCHT 1972b) and colloidal thorium dioxide (GOTHLIN and ERICSSON 1972, 1973c; THYBERG 1975). The most extensive uptake was found to occur in the ruffled border region and the ingested material accumulated in lysosomes located beneath this region as well as perinuclearly, close to the dictyosomes of the Golgi complex (Figs. 29 - 31). The lysosomes otherwise contained no or only few morphologically identifiable inclusions. Hence, it seems possible that the extracellular digestion of the matrix results in a finely dissociated material, which after uptake into the cells is difficult to recognize in the electron microscope. Another possibility

is that part of this material is not ingested and further digested by the osteoclasts but removed in other ways, for example by diffusion away from the resorption zone and uptake into neighboring cells or into the blood. Further support for the model of osteoclastic bone resorption discussed above has been obtained by studying the effects of parathyroid hormone and calcitonin on osteoclasts both in vivo and in vitro. Thus, parathyroid hormone, a hormone inducing bone resorption and causing a rise in plasma calcium (for a review see COPP 1970), has been found to increase the number of osteoclasts (TATEVOSSIAN 1973), to increase both the frequency of occurrence and development of the ruffled border (HOLTROP et aI1974), and to stimulate the lysosomal system of the 'cells (LUCHT and MAUNSBACH 1973; d. VAES 1969). On the other hand, the hypocalcemic hormone calcitonin (HIRSCH and MUNSON 1969; Copp 1970) has been found to cause a decrease in size of the osteoclasts (KALLIO et al. 1972; LUCHT 1973), disappearance of rypical ruffled borders (KALLIO et al. 1972; LUCHT 1973; HOLTROP et al. 1974), decreased endo- and exocytotic activity (VAES 1972c; LUCHT 1973), and signs of increased autophagy (LUCHT 1973). It thus seems probable that the hormonal control of bone resorption includes a regulation of the activities of the lysosomal system in the osteoclast.

3.5 Role in autophagy As mentioned in the previous section, LUCHT (1973) noted increased numbers of autophagic vacuoles in osteoclasts in rats treated with calcitonin, Autolysosomes containing acid phosphatase activity and partly digested organelles were also observed. This raises the possibiliry that the decreased size of the osteoclasts seen after treatment with calcitonin both in vitro (KALLIO et al. 1972) and in vivo (LUCHT 1973) at least partly may be due to a stimulation of the autophagic actions of the lysosomal system in these cells.

Lysosomes and Endochondral Growth

In studies of the guinea pig metaphysis we have frequently noted the presence of autophagosomes in newly formed osteocytes (Fig. 32). Conceivably, this could represent a means for the conversion of osteoblasts to osteocytes with a concomitant smaller size and lower content of cytoplasmic organelles.

3.6 Extracellular release of lysosomal enzymes and bone mineralization The mineralization of bone differs in several ways from that of cartilage. For example, in contrast to the epiphyseal chondrocytes, the osteoblasts do not normally disintegrate in connection with the calcification of the matrix but transform into and persist as osteocytes. This may explain why no matrix vesicles with morphological and/or cytochemical properties of lysosomes occur in the osteoid matrix (THYBERG et al. 1975b; d. section 2.2). On the other hand, vesicular structures believed to represent cross-sections and/or fragments of cytoplasmic processes are present in the bone matrix. Like the plasma membrane of the osteoblasts, the limiting membrane of these vesicles displays alkaline phosphatase activity. In studies on embryonic chick bone ANDERSSON and REYNOLDS (1973) suggested that vesicles of this type concentrate calcium and phosphate ions by an enzymatic process and thus initiate crystal formation. However, when investigating early stages of mineralization in embryonic mouse and guinea pig bone, we found that the initial mineral deposits were generally laid down outside the (Fig. 33; THYBERG, unpublished observations). As in cartilage (d. section 2.6) the early calcification of bone occurs as multiple, scattered foci of crystal clusters, not all of which could be in direct contact with (d. DECKER 1966; BERNARD and PEASE 1969; ANDERSSON and REYNOLDS 1973). These observations do not conform to the idea that mineral crystals are first deposited inside matrix vesicles and that further mineral-

.

35

ization takes place by epitactic crystal growth from these nuclei (ANDERSON 1973; ANDERSON and REYNOLDS 1973). Moreover, it is wellknown that numerous cytoplasmic processes extend from the surface of the osteocytes, running through fine tubular passages (canaliculi) in the calcified bone matrix (BLOOM and FAWCETI 1975). Since the vesicles found in osteoid matrix are believed to be formed by budding from the osteoblast processes (ANDERSON and REYNOLDS 1973; THYBERG et al. 1975 b), in order for the former to playa major role in bone mineralization, there must exist definite functional differences between them and the processes from which they arise. Such differences are not easily conceived and remain to be clearly demonstrated. As in the case of cartilage (d. section 2.6), the mineralization of bone is preceded by modification of its organic matrix. PUGLIARELLO et al. (1970), analyzing osteoid and osteons with different degrees of calcification, found that more than 80 % of the non-collagenous nitrogen disappeared during calcification. Moreover, the proteoglycan content, measured as hexosamines, decreased by about 30 % at the onset of calcification. Collagen, on the contrary, remained practically constant in all samples. Recently ENGFELDT and HJERPE (1976) used ultracentrifugation in acetone-bromoform density gradients to separate powdered human bone into three fractions having different degrees of mineralization. Analyses of the proteoglycans in the different fractions supported and extended the observations of PUGLIARELLO et al. (1970). The content of proteoglycans decreased when the degree of mineralization increased and the glycosaminoglycans (chondroitin sulfate) shifted toward lower average molecular weight. Evidence of loss of proteoglycans at sites where bone mineralization is initiated 'Was also obtained by BAYLINK et al. (1972) in a histochemical study. It seems likely that the mechanism for the loss of proteoglycans at the mineralizing front in bone is enzymatic. In view of their wide speci-

J. THYBERG . U. FRIBERG

36

!

•

I ~.

'>Iv

\

Lysosomes and Endochondral Growth

ficities, lysosomal hydrolases could well be involved in this digestive activity (d. section 2.6). However, no matrix vesicles of lysosomal character are present in the bone matrix. This would then require that enzymes be secreted from the lysosomes in the osteoblasts by exocytosis. No definitive evidence thereof exists but we have repeatedly noted that lysosomes are located very close to that surface of the osteoblasts facing the bone matrix (Figs. 34 and 35).

37

4 Summary In the epiphyseal growth plate the chondrocytes have been found to contain a structurally rather homogeneous group of membrane-bound cytoplasmic dense bodies, identified as lysosomes by the demonstration of acid phosphatase and aryl sulfatase activity within their matrix. The largest number of dense bodies occurs in the

Figure 33. Early calcification in fetal guinea pig bone. Mineral crystals (mc) are clustered in many small separate foci. The organic matrix is built up of a network of collagen fibrils (c) seen both in cross- and longitudinal section. Numerous matrix vesicles (mv) and/or osteoblast processes are also evident. No mineral deposits can be detected inside these structures. 0 peripheral part of an osteoblast. - Magnification X 25,000. Figures 34 and 35. Peripheral portion of os teo blasts and adjoining bone matrix in metaphyseal bone of young guniea pigs given an intravenous injection of colloidal thorium dioxide one or two days before being killed. Lysosomes, identified by the presence of ingested marker particles, are located immediately adjacent to the plasma membrane. - Magnifications X 23,000 (Fig. 34), X 41,000 (inset Fig. 34), X 32,000 (Fig. 35), X 23,000 (inset Fig. 35).

38

.

J. THYBERG . U. FRIBERG

cells of the hypertrophying zone and, likewise, marized above, this indicates that type I vesicles lysosomal enzyme activities have been found to may be implicated in the processes which make increase toward the calcification front. the cartilaginous matrix calcifiable. In view of Lysosomal structures are also present extrathe lysosomal nature of these vesicles their effect cellularly in epiphyseal cartilage. Thus, two is most probably mediated by hydrolytic enzydistinct types of membrane-bound matrix mes released into the intercellular substance vesicles have been distinguished in our labores released into the intercellular substance atory. Matrix vesicles of type I were morphoproper. Such enzymes could, for example, partilogically indistinguishable from cytoplasmic cipate in the digestion and removal of extradense bodies and like them showed positive cellular proteoglycans which have been demonreactions for lysosomal enzyme activities. They strated to be associated with the start of were generally located in the central parts of calcification. Ultrastructurally, these changes the longitudinal bars separating adjacent cell were visualized as a drop in the number and size columns and were most numerous in the hyperof ruthenium red-stained granules from the trophying and calcifying zones. These findings middle of the hypertrophying zone to the upper parts of the calcifying zone. suggest that type I vesicles represent lysosomes released from the chondrocytes. Similarly, Two major types of osteogenic cells appear observations have been made which indicate the around the capillary sprouts which invade the persistence of lysosomes and their liberation metaphyseal aspect of the epiphyseal growth from other cell components in connection with plate cartilage. Type A perivascular cells mordegeneration and disintegration of the chondrophologically resemble the osteoblasts and are cytes. Further support of this interpretation was thus regarded as representing an early stage in obtained in experimental studies dealing with their specialization. The endoplasmic reticulum the distribution of labeled lysosomes in hyaline and the Golgi complex are dominant organelles cartilage formed by transplanted chondrocytes in these cells whereas lysosomes usually are few previously exposed to exogenous marker partiin number. On the other hand type B perivascular cells contain large numbers of phagocles in monolayer culture. Matrix vesicles of type II were most numerous somes and phagolysosomes, the latter showing in the pericellular zones but were also found in positive reactions for acid phosphatase and the longitudinal septa. Their contents were aryl sulfatase activity, and have been shown to similar in appearance to the ground cytoplasm actively ingest exogenous marker particles. of the chondrocytes, sometimes including ribo- Type B cells are closely related to the osteoclasts some-like structures. After incubation for demon- ,and have been suggested as precursors of these stration of alkaline phosphatase and ATPase multinucleated cells. Moreover, they may play activity, reaction product appeared on the sur- a major role in the elimination of cell remnants face of the type II vesicles and on the plasma and unmineralized matrix as the metaphyseal membrane of the chondrocytes. It therefore capillaries grow into the former chondrocyte seems probable that matrix vesicles of type II lacunae. The calcified longitudinal septa, on the represent fragments either of budding cytoplas- other hand, are apparently resistant to the remic processes or of disintegrating cells. sorptive action of the type B perivascular cells. In the calcifying zone of epiphyseal growth Removal of such septa as well as mineralized cartilage the initial deposits of inorganic crystals bone matrix is accomplished by osteoclasts. have been found to occur in the central parts of With a specialized surface region, the ruffled the longitudinal septa, in our studies in close border, these cells surround the blind lower proximity to but not usually within matrix ends of some longitudinal septa. Underneath vesicles of type I. Together with the data sum- the ruffled border, the cytoplasm contains

Lysosomes and Endochondral Growth numerous vacuoles and lysosomes. Activities of acid phosphatase and aryl sulfatase have been demonstrated within these lysosomes, which also accumulate exogenous marker particles. Conceivably, the osteoclasts secrete acid and hydrolytic enzymes in the ruffled border region, the

extracellular action

of which

leads

to

demineralization and partial digestion of the organic matrix. The resulting residues may then be taken up by endocytosis for further degradation within the lysosomal system.

5 References ALI, S. Y.: Analysis of matrix vesicles and their role in the calcification of epihpyseal cartilage. - Fed. Proc. 35,135-142 (1976). ALI, S. Y., SAJDERA, S. W., ANDERSON, H. C: Isolation and characterization of calcifying matrix vesicles from epiphyseal cartilage. - Proc. nat. Acad. Sci. (Wash.) 67, 1513-1520 (1970). AMADO, R., INGMAR, B., LINDAHL, D., WASTESON, A.: Depolymerisation and desulphation of chondroitin sulphate by enzymes from embryonic chick cartilage. - Febs. Lett. 39, 49-52 (1974). ANDERSON, C E., PARKER, J.: Invasion and resorption in enchondral ossification: an electron microscopic study. - J. Bone Joint Surg. 48A, 899-914 (1966). ANDERSON, H. C: Electron microscopic studies of induced cartilage development and calcification. - J. Cell Bio!. 35, 81-101 (1967). -: Vesicles associated with calcification in the matrix of epiphyseal cartilage. - J. Cell Bio!. 41, 59-72 (1969). -: Calcium-accumulating vesicles in the intercellular matrix of bone. - In: Hard Tissue Growth, Repair and Remineralization. Ciba Found. Symp. 11, pp. 213-246. - Associated Scientific Pub!., Amsterdam 1973. ANDERSON, H. C, CECIL, R., SAJDERA, S. W.: Calcification of rachitic rat cartilage in vitro by extracellular matrix vesicles. - Amer. J. Path. 79, 237-254 (1975). ANDERSON, H. C, MATSUZAWA, T., SAJDERA, S. W., ALI, S. Y.: Membranous particles in calcifying cartilage matrix. - Trans. N.Y. Acad. Sci. 32, 619-630 (1970). ANDERSON, H. C, REYNOLDS, J. J.: Pyrophosphate stimulation of calcium uptake into cultured em-

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bryonic bones. Fine structure of matrix vesicles and their role in calcification. - Develop. Bio!. 34, 211227 (1973). ARBORGH, B., ERICSSON,J. L. E., HELMINEN, H.: Inhibition of renal acid phosphatase and aryl sulfatase activity by glutaraldehyde fixation. - J. Histochem. Cytochem. 19,449-451 (1971). ARSENIS, C, EISENSTEIN, R., SOBLE, L. W., KUETTNER, K. E.: Enzyme activities in chick embryonic cartilage: their subcellular distribution in isolated chondrocytes. - J. Cell Bio!. 49, 459-467 (1971). ASHTON, B., HOHLING, H. J., NICHOLSON, W. A. R., ZESSACK, D., KRIZ, W., BOYDE, A.: Quantitative analysis of Ca, P and S in mineralizing and nonmineralizing tissues. - Naturwissenschaften 60, 392-393 (1973). AVILA, J. L., CONVIT, J: Physicochemical characteristics of the glycosaminoglycan-lysosomal enzyme interaction in vitro: a model of control of leucocytic lysosomal activity. - Biochem. J. 160, 129-136 (1976). BARRETT,A.J.: Lysosomal enzymes.-In: DINGLE,J.T. (Ed.), Lysosomes, a Laboratory Handbook, pp. 46-135. - North-Holland Pub!., AmsterdamLondon 1972. BARRETT, A. J., DINGLE, J. T.: The inhibitKln of tissue acid proteinases by pepstatin. - Biochem. J. 127, 439-441 (1972). BAYLINK, D., WERGEDAL, J., THOMPSON, E.: Loss of proteinpolysaccharides at sites where bone mineralization is initiated. - J. Histochem. Cytochem. 20, 279- 292 (1972). BECK, F., LLOYD, J. B., SQUIER, C A.: Histochemistry. In: DINGLE, J. T. (Ed.), Lysosomes, a Laboratory Handbook, pp. 200-239. - North-Holland Pub!., Amsterdam- London 1972. BERNARD,G.W., PEASE, D.C.: An electron microscopic study of initial intramembranous osteogenesis. Amer. J. Anat. 125,271-290 (1969). BLOOM, W., FAWCETT, D. W.: A textbook of histology. - W. B. Saunders, Philadelphia 1975. BONUCCI, E.: Fine structure of early cartilage calcification. - J. Dltrastruct. Res. 20,33- 50 (1967). -: Fine structure and histochemistry of «calcifying globules" in epiphyseal cartilage. - Z. Zellforsch. 103,192-217 (1970). -: The locus of initial calcification in cartilage and bone. - Clin. Orthop. re!. Res. 78, 108-139 (1971). BRIGHTON, CT., SUGIOKA, Y., HUNT, R. M.: Cytoplasmic structures of epiphyseal plate chondrocytes. - J. Bone Joint Surg. 55 A, 771-784 (1973). BROOKES, M., LANDON, D. N.: The juxta-epiphysial vessels in the long bones of foetal rats. - J. Bone Joint Surg. 46B, 336- 345 (1964). BRUNK, D.: Distribution and shifts of ingested marker particles in residual bodies and other lysosomes.

40

.

J. THYBERG

. U. FRIBERG

Studies on in vitro cultivated human glia cells in phase II and III.- Exp. Cel!. Res. 79, 15-27 (1973). BRUNK, U. T., ERICSSON, J. L. E.: The demonstration of acid phosphatase in in vitro cultured tissue cells. Studies on the significance of fixation, tonicity and permeability. - Histochem. J. 4, 349-363 (1972). BURLEIGH, M. c., BARRETI, A. J., LAZARUS, G. S.: Cathepsin Bl: a lysosomal enzyme that degrades native collagen. - Biochem. J. 137, 387-398 (1974). CAMERON, D. A.: The ultrastructure of bone. - In: BOURNE, G. H. (Ed.), The Biochemistry and Physiology of Bone, vo!. 1, pp. 191-236. - Academic Press, New York 1972. CAMPO, R. D.: Protein-polysaccharides of cartilage and bone in health and disease. - Clin. Orthop. re!. Res. 68, 182-209 (1970). COFFEY, J. W., DE DUVE, c.: Digestive activity of lysosomes. The digestion of proteins by extracts of rat liver lysosomes. - J. bio!' Chern. 243, 32553263 (1968). COHN, Z. A., FEDORKO, M. E.: The formation and fate of lysosomes. - In: DINGLE, J. T. and FELL, H. B. (Eds.), Lysosomes in Biology and Pathology, vol. 1, pp. 43-63. - North-Holland Pub!., AmsterdamLondon 1969. Copp, D. H.: Endocrine regulation of calcium metabolism. - Ann. Rev. Physio!. 32, 61-86 (1970). CUERVO, L. A., PITA, J. c., HOWELL, D. S.: Inhibition of calcium phosphate mineral growth by proteoglycan aggregate fractions in a synthetic lymph. - Calcified Tiss. Tes. 13, 1-10 (1973). DAEMS, W. TH., WISSE, E., BREDEROO, P.: Electron microscopy of the vacuolar apparatus. - In: DINGLE, J. T. (Ed.), Lysosomes, a Laboratory Handbook, pp. 150-199. - North-Holland Pub!., AmsterdamLondon 1972. DEARDEN, L. c.: Enhanced mineralization of the tibial epiphyseal plate in the rat following propylthiouracil treatment: a histochemical, light, and electron microscopic study. - Anat. Rec. 178, 671-689 (1974). DEARDEN, L. c., ESPINOSA, T.: Comparison of mineralization of the tibial epiphyseal plate in immature rats following treatment with cortisone, propylthiouracil or after fasting. - Calcified Tiss. Res. 15, 93-110 (1974). DECKER, J. D.: An electron microscopic investigation of osteogenesis in the embryonic chick. - Amer. J. Anat. 118,591-614 (1966). DE DUVE, c.: The lysosome concept. - In: DE REUCK, A. V. S. and CAMERON, M. P. (Eds.), Ciba Foundation Symposium Lysosomes, pp. 1-35. - J. & A. Churchill Ltd., London 1963. -: The lysosome in retrospect. - In: DINGLE, J. T. and FELL, H. B. (Eds.), Lysosomes in Biology and

Pathology, vo!. 1, pp. 3-40. - North-Holland Pub!., Amsterdam-London 1969. -: Tissue fractionation: past and present. - J. Cell Bioo. 50, 20 D - 55 D (1971). DE DUVE,C., PRESSMAN,B.C., GIANETIO,R., WATDE DUVE, c., PRESSMAN, B. c., GIANETIO, R., WATTIAUX, R., ApPELMANS, F.: Tissue fractionation studies. 6. Intracellular distribution patterns of enzymes in rat-liver tissue. - Biochem. J. 60,604-617 (1955). DE DUVE, c., WATIIAUX,R.: Functions of lysosomes.Ann. Rev. Physio!' 28,435-492 (1966). DINGLE, J. T.: The extracellular secretion of lysosomal enzymes. - In: DINGLE, J. T. and FELL, H. B. (Eds.), Lysosomes in Biology and Pathology, vo!. 2, pp. 421-436. - North-Holland Pub!., AmsterdamLondon 1969. -: The immunoinhibition of cathepsin D-mediated cartilage degradation. - In: BARRETI, A. J. and DINGLE, J. T. (Eds.), Tissue Proteinases, pp. 313326. - North-Holland Pub!., Amsterdam-London 1971. DINGLE, J. T., BARRETI, A. J., POOLE, A. R., STOVIN, P.: Inhibition by pepstatin of human cartilage degradation. - Biochem. J. 127,443-444 (1972). DINGLE, J. T., FELL, H. B.: Lysosomes in Biology and Pathology, Vols. 1-2. - North-Holland Pub!., Amsterdam-London 1969. DINGLE, J. T., FELL, H. B., GLAUERT, A. M.: Endocytosis of sugars in embryonic skeletal tissues in organ culture. IV. Lysosomal and other biochemical effects: general discussion. - J. Cell Sci. 4, 139-154 (1969). DoTY, S. B., SCHOFIELD, B. H.: Electron microscopic localization of hydrolytic enzymes in osteoclasts. Histochem. J. 4, 245-258 (1972). -: Enzyme histochemistry of bone and cartilage cells. - Progr. Histochem. Cytochem. 811, 1-38 (1976). DoTY, S. B., SCHOFIELD, B. H., ROBINSON, R. A.: The electron microscopic identification of acid phosphatase and adenosinetriphosphatase in bone cells following parathyroid extract or thyrocalcitonin administration. - Excerpta Medica, Amsterdam 14, 169181 (1968). ENGFELDT, B.: Studies on the epiphysial growth zone. Electronmicroscopic studies on the normal epiphysial growth zone. - Acta path. microbio!. scand. 75, 201-219 (1969). ENGFELDT, B., HJERPE, A.: Glycosaminoglycans and proteoglycans of human bone tissue at different stages of mineralization. - Acta path. microbio!. scand. Sect. A 84, 95-106 (1976). ERICSSON, J. L. E.: Mechanism of cellular autophagy.In: DINGLE, J. T. and FELL, H. B. (Eds.), Lysosomes in Biology and Pathology, vo!. 2, pp. 345-394. North-Holland Pub!., Amsterdam-London 1969.

Lysosomes and Endochondral Growth FAHIMI, H. D., DROCHMANS, P.: Purification of glutaraldehyde, its significance for preservation of acid phosphatase activity. - J. Histochem. Cytochem. 16,199-204 (1968). FELL, H. B., DINGLE, J. T.: Studies on the mode of action of excess of vitamin A. 6. Lysosomal protease and the degradation of cartilage matrix. - Biochern. J. 87,403-408 (1963). GAILLARD, P. J.: Parathyroid gland and bone in vitro.Develop. BioI. 1, 152-181 (1959). GLAUERT, A. M., FELL, H. B., DINGLE, J. T.: Endocytosis of su gars in embryonic skeletal tissue in organ culture. II. Effect of sucrose on cellular fine structure. - J. Cell. Sci. 4, 105-131 (1969). GODMA , G. c., PORTER, K. R.: Chondrogenesis, studied with the electron microscope. - J. biophys. biochem. Cytol. 8, 719-760 (1960). GOLDHABER, P.: Behavior of bone in tissue culture. In: SOGNNAES, R. F. (Ed.), Calcification in Biological Systems, pp. 349-372. - Amer. Ass. Adv. Sci. (Wash.) 1960. GOMORI, G.: Microscopic histochemistry. Principles and practice. - The University of Chicago Press, Chicago 1952. GOTHLlN, G.: Electron microscopic observations on fracture repair in the rat. - Acta pathol. micro bioI. scand. Sect. A 81, 507-522 (1973). GOTHLlN, G. ARBORGH, B., ERICSSON, J. L. E., HELMINEN, H.: Histochemical and biochemical studies on the localization and activity of lysosomal enzymes in fracture callus. - Histochemie 35, 97110 (1973). GOTHLl , G.,ERICSSON,J. L. E.: Fine structurallocalization of acid phosphomonoesterase in the brush border region of osteoclasts. - Histochemie 28, 337-344 (1971). -: Observations on the mode of uptake of thorium dioxide particles by osteoclasts in fracture callus. Calcified Tiss. Res. 10,216-222 (1972). -: Fine structural localization of acid phosphomonesterase in the osteoblasts and osteocytes of fracture callus. - Histochemic 35, 81-91 (1973 a). -: Studies on the ultrastructural localization of adenosine triphosphatase activity in fracture callus. - Histochemie 35,111-126 (1973 b). -: Electron microscopic studies on the uptake and storage of thorium dioxide molecules in different cell types of fracture callus. - Acta pathol. microbioI. scand. Sect. A 81, 523-542 (1973c). -: On the histogenesis of the cells in fracture callus: electron microscopic autoradiographic observations in parabiotic rats and studies on labeled monocytes. - Virchows Arch. Abt. B Zellpath. 12, 318-329 (1973 d). GRANDA, J. L., POSNER, A. S.: Distribution of four

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hydrolases in the epiphyseal plate. - Clin. Orthop. reI. Res. 74,269-272 (1971). GUENTHER, H. L., SORGENTE, N., GUENTHER, H. E., ElSE STEIN, R., KUEITNER, K. E.: Lysozyme in preosseous cartilage. VI. Purification, characterization and localization of mammalian cartilage lysozyme. - Biochim. biophys. Acta (Arnst.) 372, 321334 (1974). HA cox, N. M.: The osteoclast. - In: BOURNE, G. H. (ed.), The Biochemistry and Physiology of Bone, vol. 1, pp. 45- 67. - Academic Press, New York 1972. HARDINGHAM, T. E., MUIR, H.: The specific interaction of hyaluronic acid with cartilage proteoglycans. - Biochim. biophys. Acta 279, 401-405 (1972). HASCALL, V. c., HEINEGARD, D.: Aggregation of cartilage proteoglycans. I The role of hyaluronic acid. J. bioI. Chern. 249,4232-4241 (1974a). -: Aggregation of cartilage proteoglycans. Oligosaccharide competitors of the proteoglycan-hyaluronic acid interaction. - ]. bioI. Chern. 249, 42424249 (1974 b). HAVIVI, E., TAL, R.: Phosphatase activity in ephiphyseal cartilage of the chicken (Gallus gallus). Compo Biochem. Physiol. 46B, 709-716 (1973). HAWKINS, H. K., ERICSSON, J. L. E., BIBERFELD, P., TRUMP, B. F.: Lysosome and phagosome stability in lethal cell injury. - Amer. J. Path. 68, 255-288 (1972). HENNING, R.: pH gradient across the lysosomal membrane generated by selective cation permeability and Donnan equilibrium. - Biochim. biophys. Acta (Arnst.) 401, 307-316 (1975). HENNING, R., PLATTNER, H., STOFFEL, W.: Nature and localization of acidic groups on lysosomal membranes. - Biochim. biophys. Acta (Arnst.) 330, 6175 (1973). HERS, H. G., VAN HOOF, F.: Lysosomes and Storage Diseases. - Academic Press, New York 1973. HIRSCH, P. F., MUNSON, P. L.: Thyrocalcitonin. Physiol. Rev. 49,548-622 (1969). HIRSCHMAN, A., DZIEWIATKOWSKI, D. D.: Proteinpolysaccharide loss during endochondral ossification: immunochemical evidence. - Science 154, 393-395 (1966). HIRSIMAKI, Y., ARSTILA, A. u., TRUMP, B. F.: Autophagocytosis: in vitro induction by microtubule poisons. - Exp. Cell Res. 92, 11-14 (1975). HIRSIMAKI, P., TRUMP, B. F., ARSTILA, A. U.: Studies on vinblastine-induced autophagocytosis in the mouse liver. I. The relation of lysosomal changes to general injurious effects. - Virchows Arch. B. Cell Path. 22,89-109 (1976). HOHLlNG, H. J., STEFFENS, H., STAMM, G., MAYS, U.: Transmission microscopy of freeze dried, unstained

42

]. THYBERG . U. FRIBERG

epiphyseal cartilage of the guinea pig. - Cell Tiss. alkaline phosphatases, inorganic pyrophosphatases Res. 167,243-263 (1976). and phosphoprotein phosphatase of bone. I. ChaHOLTROP, M. E., RAISZ, L. G., SIMMONS, H. A.: The racterization and assay. - Biochim. biophys. Acta effects of parathyroid hormone, colchicine, and (Arnst.) 293,160-169 (1973). calcitonin on the ultrastructure and the activity of LOHMANDER, S., HJERPE, A.: Proteoglycans of minerosteoclasts in organ culture. - ]. Cell BioI. 60, 346alizing rib and epiphyseal cartilage. - Biochim. 355 (1974). biophys. Acta (Amst.)- 404, 93-109 (1975). LUCHT, U.: Acid phosphatase of osteoclasts demonHopsu-HAvu, V. K., ARSTILA, A. U., HELMINEN, H. J., strated by electron microscopic histochemistry. KALIMO, H. 0.: Improvement in the method for the Histochemie 28,103-117 (1971). electron microscopic localization of arylsulphatase acitivity.- Histochemie 8, 54-64 (1967). -: Cytoplasmic vacuoles and bodies of the osteoclast. HOWELL, D. S., PITA, ]. c.: Calcification of growth An electron microscopic study. - Z. Zellforsch. 135,229-244 (1972 a). plate cartilage with special reference to studies on micropuncture fluids. - Clin. Orthop. reI. Res. 118, -: Absorption of peroxidase by osteoclasts as studied by electron microscope histochemistry. - Histo208-229 (1976). chemie 29, 274-286 (1972 b). HOWELL, D. S., PITA,]. c., MARQUES, J. F., MADRUGA, J. E.: Partition of calcium, phosphate and protein -: Effects of calcitonin on osteoclasts in vivo: an in the fluid phase aspirated at calcifying sites in ultrastructural and histochemical study. - Z. Zellforsch.145, 75-87 (1973). epiphyseal cartilage. - J. clin. Invest. 47, 1121LUCHT, U., MAUNSBACH, A.B.: Effects of parathyroid 1132 (1968). JANIGAN, D. T.: The effects of aldehyde fixation on hormone on osteoclasts in vivo: an ultrastructural acid phosphatase activity in tissue blocks. - ]. and histochemical study. - Z. Zellforsch. 141, 529Histochem. Cytochem. 13,476-483 (1965). 544 (1973). JIBRIL, A. 0.: Phosphates and phosphatases in preosLUCHT, U., NIl>RGAARD,]. 0.: Export of protein from seous cartilage. - Biochim. biophys. Acta (Arnst.) the osteoclast as studied by electron microscopic 141,605-613 (1967). autoradiography. - Cell Tiss. Res. 168, 89-99 KALIMO, H. 0., HELMINEN, H. J., ARSTILA, A. U., (1976). Hopsu-HAVU, V. K.: The loss of enzyme reaction MARKS, S. c., Jr., WALKER, D. G.: Mammalian osteoproducts from ultrathin sections during the staining , petrosis - a model for studying cellular and humoral for electron microscopy. - Histochemie 14, 123factors in bone resorption. - In: BOURNE, G. H. 130 (1968). (Ed.), The Biochemistry and Physiology of Bone, KALLIO, D. M., GARANT, P. R., MINKIN, c.: Ultravol. IV, pp. 227-301. - Academic Press, New York structural effects of calcitonin on osteoclasts in 1976. tissue culture. - ]. Ultrastruct. Res. 39, 205-216 MATSUZAWA, T., ANDERSON, H. c.: Phosphatases of (1972). epiphyseal cartilage studied by electron microscopic KEMBER, N. F.: Cell division in endochondral ossificcytochemical methods. - J. Histochem. Cytochem. ation: a study of cell proliferation in rat bones by 19,801-808 (1971). the method of tritiated thymidine autoradiography. MATUKAS, V.J., KRIKOS, G.A.: Evidence for changes in J. Bone Joint Surg. 42B, 824-839 (1960). protein polysaccharide associated with the onset of KOBAYASHI, S.: Acid mucopolysaccharides in calcified calcification in cartilage. - J. Cell BioI. 39, 43-48 tissues. - Int. Rev. Cytol. 30, 257-371 (1971). (1968). KOVACS, J., REZ, G., KISS, A.: Vinblastine-induced MEGO, J. L.: Further evidence for a proton pump in autophagocytosis and its prevention by cyclomouse kidney phagolysosomes: effect of nigericin heximide and emetine in mouse pancreatic acinar and 2,4-dinitrophenol on the stimulation of intracells in vivo. - Cytobiologie 11, 309- 313 (1975). lysosomal proteolysis by ATP. - Biochem. biophys. KUETTNER, K. E., SORGENTE, N., CROXEN, R. L., Res. Commun. 67,571-575 (1975). HOWELL, D. S., PITA, J. c.: Lysozyme in preosseous cartilage. VII. Evidence for physiological role of MEGO, J. L., FARB, R. M., BARNES, ].: An adenosine triphosphate-dependent stabilization of proteolytic lyzozyme in normal endochondral calcification. activity in heterolysosomes. Evidence for a proton Biochim. biophys. Acta (Arnst.) 372, 335-344 pump. - Biochem. ]. 128,763-769 (1972). (1974). KUHLMAN, R.E., McNAMEE, E.].: The biochemical MEIKLE, M. c.: The distribution and function of lysosomes in condylar cartilage. - J. Anat. (Lond.) importance of the hypertrophic cartilage cell area to 119,85-96 (1975). enchondral bone formation. - J. Bone Joint Surg. 52A, 1025-1032 (1970). MILLER, F., PALADE, G. E.: Lytic activities in renal LIEBERHERR, M., VREVEN, J., VAES, G.: The acid and protein absorption droplets. An electron micro-

Lysosomes and Endochondral Growth scopical cytochemical study. - J. Cell BioI. 23, 519-552 (1964). MIYOSHI, M., ROSENBLOOM, ].: General proteolytic activity of highly purified preparations of clostridial collagenase. - Connect. Tiss. Res. 2,77- 84 (1974). MORRISON, R. I. G.: The breakdown of proteoglycans by lysosomal enzymes and its specific inhibition by an antiserum to cathepsin D. - In: BALAZS, E. A. (Ed.), Chemistry and Molecular Biology of the Intercellular Substance. Structural Organization and Function of the Matrix, vol. 3, pp. 1683-1706. Academic Press, London- New York 1970. MORRISON, R. I. G., BARRETT, A. J., DINGLE, J. T., PRIOR, D.: Cathepsins B1 and D action on human cartilage proteoglycans. - Biochim. biophys. Acta (Arnst.) 302, 411-419 (1973). MOSKALEWSKI, S., THYBERG, J., LOHMANDER, S., fRIBERG, U.: Influence of colchicine and vinblastine on the Golgi complex and matrix deposition in chondrocyte aggregates: an ultrastructural study. - Exp. Cell Res. 95,440-454 (1975). NOVIKOFF, A. B.: The endoplasmic reticulum: a cytochemist's view (a review). - Proc. nat. Acad. Sci. (Wash.) 73, 2781-2787 (1976). PALADE, G.: Intracellular aspects of the process of protein synthesis. - Science 189, 347-358 (1975). PALFREY, A. ]., DAVIES, D. V.: The fine structure of chondrocytes. - J. Anat. (Lond.) 100, 213-226 (1966). PERESS, N. S., ANDERSON, H. c., SAJDERA, S. W.: The lipids of matrix vesicles from bovine fetal epiphyseal cartilage. - Calcified Tiss. Res. 14, 275-281 (1974). PETERKOFSKY, B., DIEGELMANN, R.: Use of a mixture of proteinase-free collagenases for the specific assay of radioactive collagen in the presence of other proteins. - Biochemistry 10, 988- 994 (1971). PITA, J. c., CUERVO, L. A., MADRUGA, J. E., MULLER, F. J., HOWELL, D. S.: Evidence for a role of proteinpolysaccharides in regulation of mineral phase separation in calcifying cartilage. - J. din. Invest. 49,2188-2197 (1970). POOLE, A. R., HEMBRY, R. M., DINGLE, J. T.: Cathepsin D in cartilage: the immunohistochemical demonstration of extracellular enzyme in normal and pathological conditions. - J. Cell Sci. 14, 139-161 (1974). POSNER, A. S.: Crystal chemistry of bone mineral. Physiol. Rev. 49, 760-792 (1969). PUGLIARELLO, M. c., VITTUR, F., DE BERNARD, B., BONUCCI, E., ASCENZI, A.: Chemical modifications in osteones during calcification. - Calcified Tiss. Res. 5, 108-114 (1970). RABINOVITCH, A. L., ANDERSON, H. c.: Biogenesis of matrix vesicles in cartilage growth plates. - Fed. Proc. 35,112-116 (1976).

43

REALE, E., LUCIANO, L.: A possible source of errors in electron-histochemistry. - J. Histochem. Cytochem. 12,713-715 (1964). REIJNGOUD, D. J., TAGER, J. M.: Measurement of intralysosomal pH. - Biochim. biophys. Acta (Arnst.) 297, 174-178 (1973). ROBINSON, R.A., CAMERON, D.A.: Electron microscopy of cartilage and bone matrix at the distal epiphyseal line of the femur in the newborn infant. - J. biophys. biochem. Cytol2, 253-263 (1956). RUSSELL, R. G. G., FLEISCH, H.: Inorganic pyrophosphate and pyrophosphatases in calcification and calcium homeostasis. - Clin. Orthop. reI. Res. 69, 101-117 (1970). SCHENK, R. K., SPIRO, D., WIENER, J.: Cartilage resorption in the tibial epiphyseal plate of growing rats. J. Cell BioI. 34, 275-291 (1967). SCHENK, R. K., WIENER, J., SPIRO, D.: Fine structural aspects of vascular invasion of the tibial epiphyseal plate of growing rats. - Acta anat. (Basel) 69, 1-17 (1968). SCHOFIELD, B. H., LEVIN, L. S., DOTY, S. B.: Ultrastructure and lysosomal histochemistry of ia rat osteoclasts. - Calcified Tiss. Res. 14, 153-160 (1974). SCOTT, B. L.: Thymidine- 3 H electron microscope radioautography of osteogenic cells in the fetal rat. - J. Cell Biol.35, 115-126 (1967 a). -: The occurrence of specific cytoplasmic granules in the osteoclast. - J. Ultrastruct. Res. 19,417-431 (1967b). SCOTT, B. L., PEASE, D. c.: Electron microscopy of the epiphyseal apparatus. - Anat. Rec. 126, 465495 (1956). SIMIONESCU, N., SIMIONESCU, M., PALADE, G. E.: Permeability of intestinal capillaries. Pathway followed by dextrans and glycogens. - J. Cell BioI. 53, 365-392 (1972). SIMON, D. R., BERMAN, 1., HOWELL, D. S.: Relationship of extracellular matrix vesicles to calcification in normal and healing rachitic epiphyseal cartilage. Anat. Rec. 176,167-179 (1973). SMITH, J. W.: The disposition of proteinpolysaccharide in the epiphysial plate cartilage of the young rabbit. - J. Cell Sci. 6, 843-864 (1970). SMITH, R. E., VAN FRANK, R. M.: The use of amino acid derivatives of 4-methoxy-~-naphthylaminefor the assay and subcellular localization of tissue proteinases. - In: DINGLE, J. T. and DEAN, R. T. (Eds.), Lysosomes in Biology and Pathology, vol. 4, pp. 193-249. - North-Holland Publ., AmsterdamLondon 1975. TAKUMA, S.: Electron microscopy of the developing cartilagenous epiphysis. - Arch. oral BioI. 2, 111119 (1960). TATEVOSSIAN, A.: Effect of parathyroid extract on

44

J. THYBERG . U. FRIBERG

blood calcium and osteoclast count in mIce. Calcified Tiss. Res. 11, 251- 257 (1973). TERMINE, J. D.: Mineral chemistry and skeletal biology. - Clin. Orthop. reI. Res. 85, 207-241 (1972). THINES-SEMPOUX, D.: A comparison between. the lysosomal and the plasma membrane. In: DINGLE, J. T. (Ed.), Lysosomes in Biology and Pathology, vol. 3, pp. 278-299. - North-Holland Publ., Amsterdam- London 1973. THYBERG, J.: Ultrastrucmrallocalization of aryl sulfatase activity in the epiphyseal plate. - J. Ultrastruct. Res. 38, 332- 342 (1972). -: Electron microscopic smdies on the initial phases of calcification in guinea pig epiphyseal cartilage. J. Ultrastruct. Res. 46,206-218 (1974). -: Electron microscopic smdies on the uptake of exogenous marker particles by different cell types in the guinea pig metaphysis. - Cell Tiss. Res. 156, 301-315 (1975). THYBERG, J., FRIBERG, U.: Ultrastrucmre and acid phosphatase activity of matrix vesicles and cytoplasmic dense bodies in the epiphyseal plate. - J. Ultrastruct. Res. 33, 554- 573 (1970). -: Ultrastructure of the epiphyseal plate of the normal guinea pig. - Z. Zellforsch. 122, 254- 272 (1971). -: Electron microscopic enzyme histochemical studies on the cellular genesis of matrix vesicles in the epiphyseal plate. - J. Ultrastruct. Res. 41, 43-59 (1972). THYBERG, J., LOHMANDER, 5., FRIBERG, U.: Electron microscopic demonstration of proteoglycans in guinea pig epiphyseal cartilage. - J. Ultrastruct. Res. 45,407-427 (1973b). THYBERG, J., MOSKALEWSKI, 5., FRIBERG, U.: Electron microscopic studies on the uptake of colloidal thorium dioxide particles by isolated fetal guinea pig chondrocytes and the distribution of labeled Iysosomes in cartilage formed by transplanted chondrocytes. - Cell Tiss. Res. 156, 317-344 (1975 a). THYBERG, J., NILSSON,S., FRIBERG, U.: Electron microscopic studies on -guinea pig rib cartilage. Structural heterogeneity and effects of extraction with guanidine-HCI. - Z. Zellforsch. 146, 83-102 (1973 a). -: Electron nUcroscopic and enzyme cytochemical smdies on the guinea pig metaphysis with special reference to the lysosomal system of different cell types. - Cell Tiss. Res. 156,273-299 (1975 b). TRAMS, E. G., LAUTER, C. J.: On the sidedness of plasma membrane enzymes. - Biochim. biophys. Acta (Amst.)345, 180-197(1974). VAES, G.: Studies on bone enzymes. The activation and release of latent acid hydrolases and catalase in

bone-tissue homogenates. - Biochem J. 97, 393402 (1965 a). -: Excretion of acid and of lysosomal hydrolytic enzymes during bone resorption induced in tissue culture by parathyroid extract. - Exp. Cell Res. 39,470-474 (1965 b). ~: Hyaluronidase activity in lysosomes of bone tissue. - Biochem. J.I03, 802-804 (1967). -: On the mechanisms of bone resorption. The action of parathyroid hormone on the excretion and synthesis of lysosomal enzymes and on the extracellular release of acid by bone cells. - J. Cell BioI. 39, 676- 697 (1968). -: Lysosomes and the cellular physiology of bone resorption. - In: DINGLE, J. T. and FELL, H. B. (Eds.), Lysosomes in Biology and Pathology, vol. 1, pp. 217-253. - North-Holland Publ., AmsterdamLondon 1969. -: The release of collagenase as an inactive proenzyme by bone explants in culture. - Biochem. J. 126, 275- 289 (1972 a). -: Multiple steps in the activation of the inactive precursor of bone collagenase by trypsin. - Febs. Lett. 28, 198- 200 (1972 b). -: Inhibitory actions of calcitonin on resorbing bone explants in culture and on their release of lysosomal hydrolases.- J. dent. Res. 51,362-366 (1972c). VAES, G., JACQUES, P.: Studies on bone enzymes: distribution of acid hydrolases, alkaline phenylphosphatase, cytochrome oxidase and catalase in subcellular fraction of bone tissue homogenates. Biochem. J. 97, 389-392 (1965 a). -: Studies on enzymes: the assay of acid hydrolases and other enzymes in bone tissue. - Biochem. J. 97,380-388 (1965 b). VAN FURTH, R., COHN, Z. A., HIRSCH, J. G., HUMPHREY, J. H., SPECTOR, W. G., LANGEVOORT, H. L.: The mononuclear phagocyte system: a new classification of macrophages, monocytes, and their precursor cells. - Bull. WHO 46, 845- 852 (1972). VREVEN, ]., LIEBERHERR, M., VAES, G.: The acid and alkaline phosphatases, inorganic pyrophosphatases and phosphoprotein phosphatase of bone. II. Distribution in subcellular fractions of bone tissue homogenates and structure-linked latency. - Biochim. biophys. Acta (Amst.) 293, 170-177 (1973). WASTESON, A., AMADO, R., INGMAR, B., HELDIN, c.H.: Degradation of chondroitin sulphate by lysosomal enzymes from embryonic chick cartilage. In: PEETERS, H. (Ed.), Protides of the Biological Fluids. - Proceedings of the XXllnd colloquium, pp. 431-435. - Pergamon Press, Oxford 1975. WEISSMANN, G., DUKoR, P., ZURIER, B.: Effect of cyclic AMP on release of lysosomal enzymes from phagocytes. - Nature New BioI. 231, 131-135 (1971).

Lysosomes and Endochondral Growth WESTON, P. D., POOLE, A. R.: Antibodies to enzymes and their uses with specific reference to cathepsin D and other lysosomal enzymes. - In: DINGLE, J. T. (ed.), Lysosomes in Biology and Pathology, vol. 3, pp. 425-464. - North-Holland Publ., Amsterdam 1973. WOESSNER, J. F.: Purification of cathepsin D from cartilage and uterus and its action on the proteinpolysaccharide complex of cartilage. - J. bioI. Chern. 248, 1634-1642 (1973). WUTHIER, R. E.: A zonal analysis of inorganic and organic constituents of the epiphysis during endochondral calcification. - Calcified Tiss. Res.4, 20-38 (1969).

45

-: Relationship between pyrophosphate, amorphous calcium phosphate and other factors in the sequence of calcification in vivo. - Calcified Tiss. Res. 10, 198206 (1972). -: Lipid composition of isolated epiphyseal cartilage cells, membranes and matrix vesicles. - Biochim. biophys. Acta (Arnst.) 409,128-143 (1975). -: Lipids of matrix vesicles. - Fed. Proc. 35, 117121 (1976). YAMAMOTO, T.: On the thickness of the unit membrane. - J. Cell BioI. 17,413-421 (1963).