Matrix vesicles in embryonic chick bone: Considerations of their identification, number, distribution, and possible effects on calcification of extracellular matrices

Matrix Vesicles in Embryonic Chick Bone: Considerations of Their Identification, Number, Distribution, and Possible Effects on Calcification of Extracellular Matrices J. LANDIS,

WILLIAM

AND

MARY

C. PAINE,?

J.

MELVIN

KAREN

J. HODGENS,

GLIMCHER

Laboratory for the Study of Skeletal Disorders and Rehabilitation, Department of Orthopedic Surgery, Harvard Medical School, and The Children’s Hospital, Boston, Massachusetts 02115 Received December 19, 1986 Bone tissue from normal 5- to 2 1 -day-old embryonic chicks has been examined by transmission electron microscopy to identify extracellular matrix vesicles, their number, and distribution at beginning stages of tissue mineralization during early osteogenesis. Principal tissue treatment was fixation in glutaraldehyde and osmium, followed by staining with uranyl and lead salts. Some specimens were decalcified with EDTA and stained. In embryonic bone tissue prepared by these methods, it was rather difficult to identify structural organelles conclusively, but along the proximal and distal diaphyses of tibiae and femurs from chicks 5-l 2 days old, matrix vesicles were occasionally observed in the extracellular tissue spaces. Some vesicles appeared to contain or to be associated with mineral particles. Very few vesicles were found in these same regions in older chicks or along the periosteal surfaces of the long bones in chicks of any age examined. The vast majority of the extracellular mineral particles was associated with collagen fibrils in all chick bone tissues at all ages. The presence of only small numbers of vesicles, restricted to specific regions and ages of bone tissue undergoing active mineralization, argues against an obligatory requirement for matrix vesicles in the calcification of extracellular bone matrices. 0 1986 Academic PEE., II-C

Matrix vesicles have been suggested by a number of investigators as serving a critical role in the calcification of the extracellular matrices of bone, cartilage, and other mineralized tissues (Ali, 1976; Ali et al., 1970, 1977b; Anderson, 1969, 1976, 1978; Bernard, 1972; Bonucci, 1967, 1970, 1971, 1981; Eisenmann and Glick, 1972; Katchburian, 1973; Wuthier, 198 1). The evidence for such a role is supported in part by electron microscopic observations which indicate that matrix vesicles are located in tissue regions undergoing mineralization and may occasionally contain solid-phase calcium phosphate (Ca-P) particles in such forms as needles, rods, or platelets (Anderson, 1969, 1976, 1978; Anderson and Reynolds, 1973; Bernard, 1972; Bonucci, 1967, 1970, 1971; Eisenmann and Glick, 1972; Katchburian, 1973). Structurally, vesicles have been det Mary manuscript

Collyer Paine died is dedicated to her.

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scribed as generally circular in cross-sectional profile with a variable diameter, having moderate electron density and bound by a trilaminar membrane (Anderson, 1969, 1976; Anderson and Reynolds, 1973; Bonucci, 1967, 1970, 1971; Eisenmann and Glick, 1972; Katchburian, 1973; Thyberg, 1974). In epiphyseal cartilage, where they have been studied most, matrix vesicles are commonly observed with a distribution reported in early work as limited principally to proliferating and hypertrophic zones in which calcification occurs actively (Anderson, 1969; Anderson and Reynolds, 1973; Bonucci, 1967, 1970). More recent stereological studies have indicated a different vesicle distribution, however, unequal throughout the growth plate with the greatest number of vesicles to be found in the zone of resting cells (Reinholt et al., 1982). Vesicles have also been demonstrated during dentinogenesis and embryonic osteogenesis although only at earliest develop-

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mental stages and at discrete and isolated locations within the respective extracellular tissue spaces (Bernard, 1972; Bernard and Pease, 1969; Eisenmann and Glick, 1972; Glick, 1980; Glick and Edie, 1975). The central questions of whether matrix vesicles participate in a direct fashion in calcification and are obligatory to the process remain unresolved because of important physical-chemical, biological, and technical considerations. It is not clear, for instance, how the relatively few matrix vesicles seen by electron microscopy can affect the temporally and spatially discontinuous events of calcification which occur throughout the large volume of the extracellular regions of bone, cartilage, or other mineralizing tissues. Moreover, in bone and dentin, it is uncertain how the putative events of matrix vesicle calcification can influence these tissues at times other than early osteogenesis and dentinogenesis after which the number of vesicles is greatly reduced and in some cases absent. Finally, most electron microscopic studies of vesicles have relied on preparative methods which fail to avoid artifacts potentially altering the location, nature, and even formation and dissolution of Ca-P particles; any such changes which may result make it impossible to determine precisely whether the extracellular vesicles in fact contain mineral in viva Under these circumstances, a reexamination of embryonic chick bone was undertaken to identify matrix vesicles and determine their number and distribution in early osteogenesis. Implications of the results as they are related to the role of matrix vesicles in calcification of this as well as other vertebrate calcifying tissues are discussed. MATERIALS

AND METHODS

Methodology carefully followed that of Anderson and Reynolds (1973) who were among the first investigators to describe matrix vesicles in osteogenic tissue. The bone tissues observed in this study were the femurs and tibiae obtained from normal 5- to 2 lday-old embryonic chicks (Spafas, Inc., Norwich, CT), staged according to Hamburger and Hamilton (195 l), fixed at 4°C in cacodylate-buffered glutaraldehyde con-

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FIG. 1. Light micrograph of a stage 29 (Hamburger and Hamilton, 195 1) normal embryonic chick femur sectioned longitudinally to illustrate the regions of bone tissue (enclosed by boxes) examined by electron microscopy. The periosteal surfaces (PS) along the proximal (P), distal (D), and central (C) zones of the diaphysis (primary center of ossification) are those areas actively mineralizing as the tissue grows progressively in length and thickness. The density within the periosteal ring of bone is attributable to mineral deposition already occurring at this developmental stage. Cartilage is noted by CART. x 40. taining 3 mMCaClz, posttixed in Zetterquist’s buffered osmium tetroxide, dehydrated in graded ethanols, infiltrated in propylene oxide, and embedded in Epon. Sections (-80 nm) of chick femurs and tibiae were stained with uranyl acetate and lead citrate and examined in a JEOL 1OOC electron microscope, operated at 60 kV with a cold stage to limit temperature-induced artifacts and contamination of specimens. Longitudinal sections of bone tissue were examined from distal or proximal ends of the primary center of ossification toward the midshaft. In this manner, it was possible to follow temporal and spatial changes occurring in the nature of the relatively homogeneous population of newly deposited mineral phase particles and in their relationship to cellular and extracellular tissue components (Glimcher, 1976; Roufosse et al., 1979). Because the presence of solid-phase mineral might obscure underlying structures and thereby interfere with proper identification of extracellular tissue components such as matrix vesicles, some specimens were thoroughly decalcified with 10% EDTA in 2.51 glutaraldehyde for 7-14 days prior to fixation. Additionally, alternate serial sections were decalcified by floatinggrids on EDTA for 48 hr. Original photomicrographs were taken at magnificationsof x 5000-20 000. Matrix vesicles were identified by their size, shape, moderate electron density, and presence of an investing membrane. RESULTS

Figure 1 illustrates the regions studied in longitudinal thin sections of embryonic

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chick bone. The areas enclosed by boxes delineate the proximal and distal zones and the periosteal surface of the primary center of ossification in the chick femur and tibia. These anatomical portions of the bone tissue are known to undergo active mineralization and contain the earliest deposits of an extracellular solid phase of calcium phosphate (Glimcher and Krane, 1968; Landis et al., 1977b). The montage prints shown in Figs. 2-31 are enlargements of the representative ultrastructure from the proximal end (Figs. 2-23) and the central periosteum (Figs. 24-31) from tibiae of 12day-old embryonic chicks. There are few structural differences which have been observed in these same regions at other stages of embryonic chick bone development (521 days). Of special note in Figs. 2-3 1 are the following: (1) There are only a very few matrix vesicles recognizable on the basis of their size and other structural features including

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the presence of intact investing membranes. Matrix vesicles which are readily identifiable appear in the cartilaginous portions of the same specimen (Fig. 32). (2) Of the occasional matrix vesicles which can be identified definitively in proximal and distal portions of the tibia, it is not at all clear whether they are structural components within cartilage or bone at this relatively early developmental stage; such uncertainty is the result of the fact that the mineralization front which subsequently partitions, and hence distinguishes, cartilaginous tissue from bone tissue is in only the formative stages in these regions of the chick long bones. (3) Along the periosteal bone surface and where the mineralization front is clearly present in the tissue, there are a scant few vesicles apparent in either the osteoid (between the mineralization front and the adjacent palisade of osteoblasts) or the collagenous extracellular matrix between osteoblasts and their precursor cells. (4) There

FIGS. 2-3 1. Montage prints of electron micrographs from I2-day-old normal embryonic chick tibiae, fixed in glutaraldehyde and osmium tetroxide according to Anderson and Reynolds (1973) and stained with uranyl acetate and lead citrate. In these figures, the prominent ultrastructural features are designated as follows: osteoblast, OB; osteoblast process, OP, mitochondrion, M; ribosomes, R; endoplasmic reticulum, ER, filaments, F; collagen, C; putative matrix vesicles, MV, mineral-phase deposits, MIN. FIGS. 2-12. Osteoid from the periosteal surfaces along the proximal end of a tibia is composed principally of collagen with which the majority of the mineral phase is associated. Enlargements of selected features are outlined in boxes. There are few definitive extracellular matrix vesicles although some structures may be found with apparent investing membranes and resembling such vesicles (enlarged in Figs. 8, 9, and 11). Other ultrastructural components associated with putative mineral deposits are more likely collagen fibrils sectioned transversely (enlarged in Figs. 11 and 12). Globular densities (arrows in Figs. 7 and 8) of an unknown nature but somewhat similar in appearance to those reported by Doherty (1983) are present near osteoblast cell membranes; such globular densities are observed within relatively electron-dense structures of apparent cellular origin. Figs. 2-6, x 30 000; Figs. 7-12, x 80 000. FIGS. 13-23. Osteoid from the periosteal surfaces along the proximal end of a tibia from a 12-day-old normal embryonic chick different from that examined and shown in Figs. 2-12. Ultrastructural features are as described in Figs. 2-12. Certain components contain dense material and are either matrix vesicles or similar to matrix vesicles (Fig. 23). Mineral is again associated principally with collagen fibrils in these regions of the tissue (Figs. 18-22). Note that in numerous profiles, collagen fibrils sectioned transversely resemble matrix vesicles in size and shape and the differentiation between these two structures conceivably may be difficult. Figs. 13-17, x 30 000; Figs. 18-23, x 80 000. FIGS. 24-3 1. Osteoid from the periosteal surfaces along the central diaphysis of a tibia from a 12-day-old normal chick contains deposits of putative mineral associated principally with collagen fib&. Few definitive matrix vesicles are apparent even within enlargements of selected structures (Figs. 29-3 1). Such enlargements indicate more convincingly an association between mineral and collagen. Figs. 24-28, x 30 000; Figs. 29-3 1, x 80000.

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are a great many needle- and plate-like particles, the putative solid mineral phase deposits in the tissue, which are not found associated with any structures having a trilaminar membrane. (5) Even in early and initial calcification, the major proportion of the mineral phase deposits is located within, over, or on collagen fibrils. (6) Some mineral particles may be seen in proximity to structures which appear to be nearly completely disrupted with only suggestions of a mem-

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brane. (7) The regions of interest contain numbers of structures which are difficult, if not impossible, to identify definitively. (8) While longitudinal profiles of collagen and various profiles of the cell processes can be recognized most easily, it is conceivable that certain aspects of these structures-cross sections of collagen fibrils, for examplecould be mistaken for matrix vesicles. Figures 33-38 present the typical ultrastructure from a decalcified section of a

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FIG. 32. The cartilaginous portion of the tissue adjacent to the mineralized periosteal region of the tibia shown in Figs. 24-31. There are many clearly identifiable membrane-invested vesicles (MV) in the cartilage regions while similar vesicles are observed only infrequently in the adjacent osteoid. Cartilage collagen, C,,; small condensations of proteoglycans, PG, chondrocyte, CH. x 24 000.

proximal tibia of a 20-day-old embryonic chick. Similar to the observations in undecalcified tissue, well-defined matrix vesicles are not apparent in this region of bone although, again, there are numerous structures which resemble vesicles in size and shape, and other components are present with partially intact membranes. Indeed, following the decalcification of the tissue, this particular portion of developing tibiae or femurs, as well as distal and periosteal regions, now appears replete with an array of various small and large structures which are on the whole especially difficult to identify. DISCUSSION

IdentiJication of extracellular matrix vesicles in embryonic chick bone. On the basis

of considerable electron microscopy, the presence of extracellular matrix vesicles in epiphyseal growth plate cartilage from a variety of vertebrate tissues, including specimens prepared by both conventional aqueous (Ali et al., 1977a; Anderson, 1969, 1976, 1978; Anderson and Reynolds, 1973; Bonucci, 1967, 1970, 1971; Thyberg, 1974; Thyberg and Friberg, 1970, 1972) and anhydrous means (Landis and Glimcher, 1982; Morris et al., 1984), has been well documented. The microscopic identification of these same vesicular structures has also been reported in bone tissue, but the evidence establishing their presence unequivocally is seriously limited by a number of considerations. First, unlike studies of cartilage, only a relatively few investigations have examined the’ultrastructure of bone tissue with

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FIGS. 33-38. Portion of the proximal tibia from a 20day-old normal embryonic chick. The tissue was decalcified with 10% EDTA in 2.5% glutaraldehyde for 2 weeks, the solution being changed each 2-3 days and then stained with uranyl acetate and lead citrate. Decalcification does not reveal an increased number of clearly defined extracellular matrix vesicles in osteoid of periosteal regions of the tibia, but numerous other structures are apparent (enlargements, Figs. 36-38), resembling most closely the collagen fibrils horn bone (C). Cartilage, CART: cartilage collagen, C,,; osteoblast, OB, decalcified osteoid region, OS; grid bar supporting the section, GB. Figs. 33-35, x 11 500; Figs. 36-38, x 29 000.

respect to demonstrating matrix vesicles and, second, of those investigations, findings do not entirely agree. The present report attempts to elaborate and clarify the earlier work.

Among previous studies of bone tissues, there are those, on the one hand, in which extracellular matrix vesicles have ostensibly been identified. These include the work by cryomicrotomy with normal mineralizing

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avian bone (Gay, 1977; Gay and Schraer, 1975; Gay et al., 1978; Schraer and Gay, 1977) results with intramembranous bone tissue from fetal mice prepared by inert dehydration (Bernard and Pease, 1969), and examinations employing conventional aqueous procedures applied to bone from developing fetal and newborn rats and mice, embryonic chicks, and other species (Anderson, 1976; Bab et al., 1979; Bonucci, 1971; Muhlrad et al., 1981; Omoy et al., 1980, 1981; Scherft and Groot, 198 1; Sela and Bab, 1979). Other results utilizing conventional sample preparation have apparently shown matrix vesicles in bone from rachitic rats (Anderson et al., 1981) and in human osteomalacic bone (Anderson et al., 1980; Dopping-Hepenstal et al., 198 1). On the other hand, extracellular matrix vesicles have been noted only infrequently, if at all, in studies of bone from embryonic chicks and young mice, prepared for microscopy by cryomicrotomy (Landis et al., 1977a), anhydrous treatment with organic solvents (Landis et al., 1977b, 1980), or conventional techniques (Landis et al., 1977b). The discrepancies as to the presence and frequency of occurrence of matrix vesicles in bone determined in some of the previous work may be partially explained by variations in specimen treatment. With regard to certain aspects of cryomicrotomy, this demanding technique best maintains the biological and physical-chemical nature of both the organic and the inorganic phases of a given tissue during its microscopic preparation (Landis, 1979; Landis and Glimcher, 1978). The method, however, yields frozen thin tissue sections possessing very little inherent contrast in components other than those intrinsically electron dense themselves, such as the mineral-phase particles (Ali and Wisby, 1975; Boothroyd, 1975; Gay, 1977; Gay and Schraer, 1975; Landis and Glimcher, 1978, 1982; Landis et al., 1977a). The organic tissue architecture as a whole, however, and individual organic cellular and extracellular organelles in particular (especially those with investing

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cytomembranes) are not at all readily visualized following this preparative regime, a problem which has been discussed elsewhere with respect to bone (Ali and Wisby, 1975; Gay and Schraer, 1975; Landis et al., 1977a) and is no less obstructive in cartilage studies (Landis and Glimcher, 1982). The reliable identity, then, of matrix vesicles in frozen, unfixed, and unstained tissue sections cannot be made because of this reservation. Indeed, even if staining were used to enhance frozen section contrast, including that of cytomembranes, another difficulty likely to be encountered and which would also preclude positive identification is that individual vesicles would undoubtedly be practically indistinguishable from other extracellular membranous components, such as small cell processes sectioned transversely, or from collagen fibrils observed in cross-sectioned profiles. From certain of the present results, this can be no less a problem in stained sections of tissues treated with conventional aqueous means to provide an optimal ultrastructural appearance. Thus, even though the inorganic mineral-phase particles and organic tissue constituents may be retained with cryomicrotomy, the organic constituents are neither easily nor confidently recognizable. Hence, previous results from cryomicrotomy (Gay et al., 1978; Schraer and Gay, 1977) which reported that certain structures represented matrix vesicles in bone tissue, may not be conclusive evidence. With regard to the microscopic appearance of bone tissue treated by means other than cryomicrotomy, anhydrous organic solvents (Bernard and Pease, 1969; Landis et al., 1977b, 1980) and the more conventional approaches with aqueous fixatives (Anderson, 1969; Anderson and Reynolds, 1973; Bonucci, 1970) are far more advantageous in demonstrating matrix vesicles. It should be noted, however, that, while they are helpful in specific instances, these techniques, like cryomicrotomy, will also compromise attempts to make clear interpretations of certain ultrastructural features in

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the calcified tissues of interest. In comparison to cryomicrotomy, organic solvents maintain comparable integrity of mineralphase deposits and a better ultrastructural appearance of the organic components. Cytomembranes, nevertheless, are visualized in negative contrast (Landis et al., 1977b, 1980). Aqueous methodology provides the organic components with an appearance superior to that obtained with organic solvents, especially since the organic constituents can be stained by a variety of methods and cytomembranes appear positively contrasted. However, the use of water introduces the inorganic constituents of the tissue to a number of critical artifacts in addition to dissolution (Boothroyd, 1964; Glimcher and Krane, 1968; Landis, 1979, 1980, 1981; Landis et al., 1977b; Termine, 1972; Thorogood and Craig Gray, 1975). The relative merits of these procedures applied to calcified tissues, as well as detailed discussion of respective results, have been reported earlier (Boothroyd, 1975; Landis, 1979, 1980, 1981; Landis and Glimcher, 1978, 1982; Landis et al., 1977a,b; Thorogood and Craig Gray, 1975). Thus, the use of organic solvents or, better still, aqueous solvents in the fixation and preparation of bone tissue is advantageous for maintaining the ultrastructural appearance and ease of observing organic tissue components such as matrix vesicles. If one is not interested in the question of whether matrix vesicles contain mineral particles but rather the presence, frequency of occurrence, and distribution of vesicles, then decalcified stained sections are most suitable.

Even under optimal conditions with these methods, however, the identification of extracellular matrix vesicles may yet be di#icult (see below). This statement is to be emphasized as a principal conclusion of the present results, and its implications must be carefully understood in the context of matrix vesicle studies. Microscopically, matrix vesicles may be identified definitively by the histochemical demonstration of specific enzymes and by

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the presence of investing membranes (Thyberg, 1974; Thyberg and Friberg, 1970, 1972). As this and earlier papers (Bonucci, 1969, 1970, 1971; Landis et al., 1977b) illustrate, the latter may well be distinguished, but there is significant uncertainty in many instances. Among a number of factors which may preclude a clear identification of matrix vesicles are oblique sectioning and inadequate staining so that only a small portion of the vesicle membrane can be observed. Equally important is the fact that, in regions of bone tissue undergoing active mineralization, and especially at proximal and distal portions and along the periosteal surfaces of embryonic chick long bones, the elaboration of the extracellular osteoid matrices results in the synthesis and secretion of a host of organic constituents. These processes are then followed rapidly by the deposition of a mineral phase. The summary of events, viewed microscopically, is a complex milieu in which many extracellular components are effectively indistinguishable from each other. Indeed, some cannot be visualized except by specialized cytochemical methods (Poole et al., 1983, 1984). Even with membranes sharply delineated in a bone tissue section, it can still remain a problem, as mentioned previously, to differentiate between matrix vesicles and transverse profiles of small bone cell processes and those of cross sections of collagen fib&s which, although not membrane bound, have a similar general size, circular shape, and electron density. The identification of extracellular constituents in osteoid regions can be made even more complicated by the presence of highly electron-dense mineral-phase particles and/ or “crystal ghosts,” the stained putative organic counterparts of the mineral (Bonucci, 1967,1969, 1970, 1971,1981; Bonucci and Rem-ink, 1978). Their slightest effect will be to obscure the profiles and appearance of nearby organic structures and their most serious effect will be to blanket the extracellular spaces completely in heavy electron density. Indeed, it has been suggested that

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with respect to matrix vesicles, the mineral phase also disrupts and destroys their investing membranes, thereby obliterating their presence altogether (Bonucci, 198 1). Whether or not there is such membrane destruction, decalcification of whole embryonic chick bones or their thin tissue sections does not provide any clearer distinction between possible individual matrix vesicles or other extracellular osteoid components as the present results attest.

Number and distribution of extracellular matrix vesicles in embryonic chick bone. Although the problems which have been considered in the foregoing discussion are serious relative to vesicle identification, there are demonstrable organelles in certain portions of embryonic chick long bones which satisfy the physical criteria for matrix vesicles (appropriate size, shape, electron density, investing membranes). These vesicles appear by microscopy most commonly during the earliest stages (6-10 days) of osteogenesis and are very infrequent at later times (1 l-2 1 days). Such observations are in good agreement with previous studies in which matrix vesicles were reported during mantle dentinogenesis of the rat incisor (Bernard, 1972; Eisenmann and Glick, 1972; Glick, 1980; Glick and Edie, 1975) and the initial stages of intramembranous osteogenesis in the mouse calvarium (Bernard and Pease, 1969) but not at times thereafter. Of those matrix vesicles which can be identified in this study, there are only a few in total number found within the extracellular tissue spaces of the developing tibia or femur. This number is far less than that represented in typical electron photomicrographs of calcified cartilage (Anderson, 1969; Bonucci, 1967, 1969; Thyberg, 1974; Thyberg and Friberg, 1970, 1972) dentin (Bernard, 1972; Eisenmann and Glick, 1972; Katchburian, 1973) or intramembranous bone (Bernard and Pease, 1969), and it would not seem to be indicative of the relatively large numbers of matrix vesicles measured directly by stereology throughout the growth plate cartilage of the rat (Rein-

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holt et al., 1982). The matrix vesicles seen here occur characteristically as individual organelles, quite distant and isolated from one another in the extracellular space, similar to their distribution in intramembranous bone (Bernard and Pease, 1969) but unlike the usual appearance of vesicles in clusters or groups in the longitudinal septa of calcified cartilage (Anderson, 1969, 1976b; Bonucci, 1967, 1969, 1970, 1971) or in close proximity in mantle dentin (Bernard, 1972; Eisenmann and Glick, 1972; Katchburian, 1973). In addition, those vesicles presently identified are found almost exclusively in the proximal and distal regions of the long bones with only a few vesicles along the periosteal surfaces of the tissues. The reason is not known for this apparent difference in the number and distribution of matrix vesicles over the entirety of bone regions undergoing active mineralization.

Possible eflects of matrix vesicles on the calcification of the extracellular bone matrix. The present observations that matrix vesicles in bone are restricted to certain regions of the tissue and appear only during specific initial intervals of development may help explain the failure to observe vesicles in the chick tibia or femur examined previously (Landis et al., 1977b, 1980). On the other hand, these results raise a repeated and perplexing question (Glimcher, 1976, 198 1; Landis and Glimcher, 1982; Landis et al., 1977b) related to the suggested role of matrix vesicles in vertebrate tissue calcification: If matrix vesicles are associated with initial and subsequent deposition of a mineral phase, how is it possible for such vesicl+mineral associations to influence the calcification of extracellular spaces, first, in regions spatially distinct from those in which the few vesicles are observed and, second, at developmental times well beyond those at which vesicles occur? With respect to the basic premise of this important question-that matrix vesicles are associated with a mineral phase-the earlier remarks of this discussion must be under-

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scored, namely, that the identification of matrix vesicles in bone is not straightforward. Furthermore, from the photomicrographs in this study and under the circumstances discussed above and elsewhere (Glimcher, 1976, 198 1) relative to the appearance of extracellular mineral deposition and/or “crystal ghosts,” it is not clear that an association in fact exists between vesicles and the particles of a mineral phase in bone tissue. Indeed, recent ultrastructural studies made by cryomicrotomy (Landis and Glimcher, 1982), electron spectroscopic imaging (Arsenault and Ottensmeyer, 1983, 1984) and immunoelectron microscopy (Choi et al., 1983; Poole et al., 1983, 1984, 1986) of rat or bovine growth plate cartilage, the tissue in which matrix vesicles were first reported (Anderson, 1969; Bonucci, 1967, 1969) indicate that these vesicles are not the sites at which incipient extracellular mineralization occurs. On the other hand, there is now new evidence of vesicle-mineral interaction in certain vertebrate tissues, the tendons from some avian species (Landis, 1985, 1986). While the question of a specific relationship between vesicles and calcification has been addressed at length in other reports (Glimcher, 1976, 1981; Landis and Glimcher, 1982; Landis et al., 1977b), it should again be emphasized that the calcification of bone, cartilage, dentin, or tendon as a tissue is a spatially discontinuous process, wherein the mineralization of each of its components-collagen, matrix vesicles, or other constituents-occurs as an independent physical chemical event (Glimcher, 1976, 198 1; Landis and Glimcher, 1982; Landis et al., 1977b). In this context, the principal influences of matrix vesicle-mineral associations on extracellular mineral deposition in bone, or in any other calcified vertebrate tissue, are indirect, either facultative or regulatory (Glimcher, 198 1; Landis and Glimcher, 1982). It is conceivable, as one possibility, that the mineral particles within or associated with the vesicles dissolve and serve as an ion source to raise the

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metastability of the extracellular fluid until heterogeneous nucleation of a solid phase of calcium phosphate occurs in the extracellular tissue spaces. However, because of the relatively few vesicles in the tissues, as shown here and in other studies (Landis, 1985; Landis et al., 1977b, 1980), the small mass of mineral potentially available from the vesicles, and the low concentrations of the ions ultimately contributed by such mass, especially relative to the concentrations of calcium and phosphorus ions within the large fluid volume of the extracellular milieu, it is most likely that mineral dissolution from matrix vesicles would affect only the heterogeneous nucleation of calcium phosphate in local regions nearby or adjacent to the vesicles and would not significantly influence the massive and rapid calcification of extracellular matrices as a whole (J. Christoffersen and W. J. Landis, manuscript in preparation). In a somewhat similar regulatory role, but one which would require neither the presence of a solid mineral phase within vesicles nor high concentrations of calcium and phosphorus ions in solution, the matrix vesicles would act as ion pumps, again increasing the metastability of the extracellular fluids and facilitating the principal event of tissue calcification, the heterogeneous nucleation of calcium phosphate by collagen fibrils. This possible function for matrix vesicles is more attractive than the first described above in the sense that a continuously operating pump would provide a relatively constant ion source for the extracellular tissue spaces, rather than one critically limited by the mass or concentration of an initial calcium phosphate solid or solution phase associated with the vesicle. Other alternative, but still indirect, roles for matrix vesicles in vertebrate calcification include the suggestion that they would enzymatically degrade potential inhibitors of calcium phosphate nucleation and growth (Thyberg, 1974; Thyberg and Friberg, 1970, 1972; Thyberg et al., 1975) or they would offer a phospholipid surface stereochemi-

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tally compatible with interactions between vesicle phosphate groups, solution calcium ions, and other molecular species present in extracellular fluids (Landis and Glimcher, 1982). Regardless of any actual or ascribed involvement of matrix vesicles with bone calcification, the time span during which vesicles appear is very short in the developmental period of the chick tibia or femur. The present results, indicating that matrix vesicles occur only in the earliest stages of chick long bone formation, are similar in that regard to those describing mantle dentinogenesis (Eisenmann and Glick, 1972; Glick, 1980; Click and Edie, 197 5) and intramembranous osteogenesis (Bernard and Pease, 1969). These data, together with the apparent near absence of vesicles along the periosteal surfaces of bone from embryonic chicks of any age, imply that neither matrix vesicles nor a putative vesicle-mineral association are obligatory for subsequent extracellular bone matrix calcification. This conclusion is in agreement with that based on bone turnover studies showing biological evidence for the complete renewal in 48 to 72 hr of the tibia1 diaphysis of young embryonic chicks (Tanzer and Hunt, 1964). Under these circumstances, both the organic and inorganic tissue matrices, including the matrix vesicles, collagen fib&s, mineral, and other cellular and extracellular tissue components, are removed and replaced in their entirety. Thus, any influence that vesicles may have exerted on tissue calcification at earlier periods will have disappeared a short time later as a consequence of remodeling, a result again implying that matrix vesicles and vesicle-mineral interaction are unnecessary for calcification to begin and proceed during the growth and development of embryonic chick bone. The principal results of this investigation argue against a direct role for matrix vesicles in mineralization of embryonic chick bone. As such, they are consistent with data obtained in this and other laboratories in pre-

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