345
J. Anat (1992) 181, pp. 345-356, with 16 figures Printed in Great Britain
Lanthanum tracer and freeze-fracture studies suggest that compartmentalisation of early bone matrix may be related to initial mineralisation ANA MARIA V. SOARES', VICTOR E. ARANA-CHAVEZ', ANDREW R. REID2 AND EDUARDO KATCHBURIAN2'3 1 Department of Histology and Embryology, Institute of Biomedical Sciences, University of Sdo Paulo, Brazil, 2Department of Anatomy and Histology, The London Hospital Medical College, London, UK, and 3 Department of Histology and Electron Microscopy Centre, Escola Paulista de Medicina, Sao Paulo, Brazil
(Accepted 24 August 1992)
ABSTRACT
In adult bone the calcified matrix and enclosed osteocytes are separated from the extracellular space by a continuous layer of bone lining cells. It thus appears that bone matrix is compartmentalised and, as such, may constitute a 'milieu interieur' which is different from the general extracellular space. Since adult bone matrix is compartmentalised and matrix vesicles also form a microcompartment, it is conceivable that compartmentalisation, in early osteogenesis, may be a requirement for the initial events of the mineralisation process. We have therefore conducted an ultrastructural, tracer, and freeze-fracture study to determine the stage in which bone matrix becomes compartmentalised and also to find out whether there are tight junctions between osteoblasts. The results show that in early nonmineralised stages and in incipient mineralisation, lanthanum penetrates all intercellular spaces and the newly forming bone matrix which is rich in matrix vesicles and collagen. With the progression of mineralisation, when all matrix vesicles appear mineralised and calcification is 'spreading' to the surrounding matrix, lanthanum is restricted to intercellular spaces and conspicuous macular tight junctions are present between osteoblasts. We suggest that matrix vesicles act as microcompartments for calcification when the early bone matrix is in continuity with the surrounding extracellular space. In later stages, when lanthanum fails to penetrate the matrix, matrix vesicles may no longer be necessary because the bone matrix itself is compartmentalised, thus allowing for localised changes in composition that might favour mineral deposition. INTRODUCTION
In adult bone the mineralised matrix and enclosed osteocytes are separated from the general extracellular space by a continuous layer of bone lining cells (Jones et al. 1985). As early as 1956, Howard observed some differences in the ionic content between blood and bone fluid in patients with parathyroid diseases. Neuman & Neuman (1958) also reported an unbalanced state between the mineral phase of bone and the extracellular fluid (ECF) in healthy organisms. Finally, Neuman (1969) showed differences in ionic composition in the mineralised matrix of bone and the ECF, suggesting the existence of a 'bone membrane' which establishes the 'milieu interieur' of bone tissue by a compartmentalisation phenomenon.
In early developing bone, as well as other collagencontaining calcifying matrices, one of the most important events is the onset of the mineralisation process. Collagen and/or collagen-associated molecules were proposed as possible initiators of the process (Glimcher, 1959, 1976, 1989). However, the discovery of matrix vesicles as the initial sites of mineral nucleation in calcifying cartilage (Anderson, 1967; Bonucci, 1967; Ali et al. 1970), bone (Bernard & Pease, 1969) and dentine (Katchburian, 1973) introduced the concept that vesicles form an enclosed and protective microcompartment for nascent mineral formation (Anderson, 1989) in immature bone. Since adult bone matrix is compartmentalised and matrix vesicles also form a microcompartment, it is conceivable that compartmentalisation, in early osteo-
Correspondence to Dr E. Katchburian, Departamento de Histologia, Escola Paulista de Medicina, Rua Botucatu 740, 04023.062 Sao Paulo, S.P., Brazil.
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Figs 1-4. Light micrographs of developing calvaria from a rat embryo aged 17 days showing the sequence of development. Darkly stained osteoblasts (B) are present in all figures. In Figures 2-4, osteoblasts surround portions of early matrix (light grey). Fig. 1, early nonmineralised matrix; Fig. 2, incipient mineralising matrix; Figs 3 and 4, late mineralising matrix. Toluidine blue stained 1 gm resin sections. Bar, 10 gm.
Fig. 5. Electron micrograph of early nonmineralised matrix showing portions of adjacent early developing osteoblasts (B) and matrix (M). Electron opaque deposits of lanthanum are present in intercellular spaces (arrowheads) and in the early matrix (arrows). N, nucleus; C, collagen fibrils. Lead citrate staining. Bar, 1 gm.
genesis, may be a requirement for the initial events of the mineralisation process. We have therefore con-
ducted an ultrastructural, tracer, and freeze-fracture study to determine the stage in which bone matrix
347
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Figs 6-8. Early nonmineralised matrix. Fig. 6. Electron micrograph of early nonmineralised matrix in a more advanced stage of development than in Fig. 5. Numerous black dots of lanthanum are present between the early developing osteoblasts (arrowheads) and in the matrix (arrows). Matrix vesicles are not present but numerous osteoblast processes (P) are observed. B, osteoblasts; M, matrix; C, collagen fibrils. Lead citrate staining. Bar, 1 jim. Figs 7 and 8. Higher magnification views of early nonmineralised matrix in the same stage as that of Fig. 6. Electron opaque deposits of lanthanum (arrows) are mostly associated with collagen fibrils (C) which are observed in transverse (Fig. 7) and longitudinal (Fig. 8) sections. B, portions of osteoblasts. Lead citrate staining. Bar, 0.1 jIm.
348
Ana Maria V. Soares and others
Figs 9-11. Incipient mineralising matrix. Fig. 9. Electron micrograph showing incipient mineralising matrix. Numerous black dots of lanthanum are observed between osteoblasts (arrowheads) and also within the matrix (arrows), mostly in association with collagen fibrils (C) and osteoblast processes (P). Nonmineralised (V1) and mineralised matrix vesicles (V2) are present throughout the matrix. Some nonmineralised matrix vesicles (V1) exhibit lanthanum associated with their membrane. Lanthanum containing invaginations of the osteoblast surface observed on the right lower corner of the figure occasionally appear as though inside the cell (X). B, osteoblasts; M, matrix. Lead citrate staining. Bar, 0.5 ,um. Figs 10 and 11. Higher magnification views of Fig. 9 showing deposits of lanthanum (arrows) in association with a nonmineralised matrix vesicle (V") and an osteoblast process (P). The vesicle (V2) in Fig. 10 shows a disrupted membrane and crystal-like inclusions (S). In Fig. 11 a fully mineralised matrix vesicle (V2) is observed. Lead citrate staining. Bar, 0.1 pIm.
. . . . .*
Fig. 12. Electron micrograph showing incipient mineralising matrix in a more advanced stage of development than in Fig. 9. Deposits of lanthanum are clearly observed between 2 adjacent osteoblasts (arrowheads) but are rarely present in the matrix (arrows). V2, mineralised matrix vesicles; M, matrix; C, collagen fibrils; B, osteoblasts; P, osteoblast process. Lead citrate staining. Bar, 0.5 gm.
becomes compartmentalised and also to find out whether there are tight junctions between osteoblasts.
MATERIALS AND METHODS
We examined different stages of bone formation in calvaria of Wistar and Sprague-Dawley rat embryos aged between 17 and 19 d. For ultrathin sections, some specimens were fixed in 2.5 % glutaraldehyde containing lanthanum nitrate in 0.1 M sodium caco-
dylate buffer, pH 7.7 (lanthanum nitrate tracer kit, Emscope), for 4 h at room temperature. After washing for 10 min in 0.1 M sodium cacodylate buffer, they were postfixed in 1 % osmium tetroxide containing lanthanum nitrate in 0.1 M sodium cacodylate, pH 7.7, for 3 h. Other specimens were fixed in 2% glutaraldehyde-2.5 % formaldehyde (Sigma Chemical Co.) containing 1 % tannic acid (Baker Chemical Co.) in 0.1 M sodium cacodylate, pH 7.4, for 4 h at room temperature. After washing for 1 h in the same buffer, they were postfixed in buffered 1 % osmium tetroxide
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Figs 13-15. Incipient mineralising matrix. Fig. 13. Focal tight junction (arrow) between 2 osteoblast processes (P) before (a) and after (b) tilting the ultrathin section. In 13 a, the closely apposed plasma membranes appear as a 'fuzz' and are not therefore clearly discerned. In 13 b the section was tilted by approximately 150 and the unit membrane structure becomes clearly visible. A typical tight junction exhibits the outer leaflets of the adjoining membranes fused. Tannic acid-aldehyde fixation. Lead citrate/uranyl acetate staining. Bar, 0.05 gm. Fig. 14. Electron micrograph showing a focal tight junction (arrow) between 2 osteoblasts (B). Collagen fibrils (C) and a matrix vesicle (V) are present in the matrix (M). Bar, 0.5 gm. The inset shows a higher magnification view of the tight junction (arrow) which exhibits a similar appearance to that seen in Figure 13b. Bar, 0.05 gm. Tannic acid-aldehyde fixation. Lead citrate/uranyl acetate staining. Fig. 15. Freezefracture replica of developing bone in the same stage as in Figures 13 and 14 showing the P-fracture face of the plasma membrane of an osteoblast containing typical intramembranous particles. Rows of elongated (arrows) and globular (arrowheads) particles form images of tight junctions which run in different directions and merge with existing gap junctions (GJ). Bar, 0.1 tm.
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Fig. 16. Electron micrograph showing late mineralising matrix. Virtually all matrix vesicles (V2) are mineralised. Deposits of lanthanum (arrowheads), present between osteoblasts (B) do not reach the distal portion of the intercellular space and are absent from the matrix (M). C, collagen fibrils. Lead citrate staining. Bar, 1 gm.
for 2h. All specimens were dehydrated in graded concentrations of ethanol after which they were embedded in Premix (TAAB Laboratories) or Spurr (Electron Microscopic Sciences) resins. Toluidine blue stained thick sections were examined with a light microscope and carefully selected regions trimmed for ultrathin sectioning. Unstained and lead citrate or lead citrate/uranyl acetate stained ultrathin sections were collected onto copper grids. For freeze-fracture replication, specimens were fixed in 2% glutaraldehyde-2.5 % formaldehyde in 0.1 M sodium cacodylate, pH 7.4, for 4 h at room temperature. After washing for 30 min in the same buffer, they were treated overnight with cacodylatebuffered 30 % glycerol. Freeze-fracture replicas were
prepared using a Balzers BAF 301 unit, as previously described (Katchburian & Severs, 1982). Both the ultrathin sections and the freeze-fracture replicas were examined in Hitachi H300 or JEOL 100 CX II electron microscopes.
RESULTS
Early developing calvaria in stages prior to and at late stages of mineralisation show 3 regions which are disposed in sequence and represent different stages of development. From the periphery of the calvaria to the initial centre of ossification the following sequence was observed: early nonmineralised matrix (Fig. 1);
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Ana Maria V. Soares and others
Fig. 17. Electron micrograph showing late mineralising matrix in a more advanced stage of calcification than in Fig. 16. Similarly to Figure 16, deposits of lanthanum (arrowheads) present between osteoblasts (B) do not reach the distal portion of the intercellular space and are completely absent from the calcified matrix (M). Lead citrate staining. Bar, 0.5 gm.
incipient mineralising matrix (Fig. 2); late mineralising matrix (Figs 3, 4). Early nonmineralised matrix At this stage osteoblasts and/or preosteoblasts appear in close proximity, but no occluding junctions are observed in ultrathin sections or freeze-fracture replicas. Together with their processes, osteoblasts surround areas of collagenous early bone matrix (Fig. 5). Electron opaque deposits of lanthanum are readily seen in the newly forming bone matrix (Figs 5, 6). The deposits appear as electron opaque dots which are often associated with the irregularly arranged collagen fibrils of the matrix (Figs 5-8). Matrix vesicles are not present at this stage.
Incipient mineralising matrix With the progression of osteogenesis, matrix vesicles appear within the matrix. Deposits of lanthanum, as
in the previous stage, are present in the intercellular spaces between osteoblasts and within the matrix where they are observed in association with collagen fibrils and matrix vesicles. Some matrix vesicles show electron opaque deposits in their interior while others are devoid of them (Figs 9-1 1). These electron opaque deposits are interpreted, from previous literature, as mineral (Anderson, 1967; Bonucci, 1967). With further development, when most of the matrix vesicles appear calcified, lanthanum deposits are still present in the intercellular spaces between osteoblasts but only in certain areas of the matrix where a trickle of the tracer is observed (Fig. 12). Collagen fibrils are free of visible mineral deposits (Fig. 12). Osteoblasts are closer together and there are several regions of approximations between their adjoining plasma membranes. Most of these regions appear diffuse, often giving the impression of superimposed images (Fig. 13a). However, after tilting the ultrathin sections, some of these regions revealed typical images of focal tight junctions in which the outer leaflets of the
Compartmentalisation of early mineralising bone matrix
353
Fig. 18. Unstained electron micrograph of a lanthanum treated specimen also in the stage of late mineralising matrix. *Distribution of lanthanum (arrowheads) is similar to that observed in Figure 17. M, calcified matrix; B, osteoblasts. Unstained. Bar, 0.5 gm.
adjoining membranes appear fused (Fig. 13b). These junctions are present between osteoblast processes (Fig. 13 b) and between their cell bodies (Fig. 14) and are always present singly. Freeze-fracture replicas confirmed the presence of typical tight junction particles. These appear as rows of fused intramembranous particles (IMPs) which are often in continuity with small gap junctions (Fig. 15).
in the previous stage, but they continue to be of the focal type and may also appear between an osteoblast cell body and an osteoblast process (Fig. 19). Freezefracture replicas confirmed the presence of tight junction particles which appear to be more extensive, therefore occupying large areas of the plasma membranes of osteoblasts (Fig. 20). DISCUSSION
Late mineralising matrix
When all matrix vesicles exhibit mineral deposits, lanthanum is no longer observed within the matrix but is still present in the intercellular spaces between osteoblasts (Fig. 16). In a more advanced stage, when matrix vesicles are absent and mineral deposits are observed throughout the matrix, lanthanum remains excluded from the matrix but it is still present between osteoblasts, filling approximately the basal two thirds of the intercellular spaces (Figs 17, 18). Tight junctions between osteoblasts are more conspicuous than
Our experiments with lanthanum show that the tracer, when incorporated into the fixative, fully penetrates bone matrix in stages prior to and at the beginning of the mineralisation process. With the progression of mineralisation there is a gradual decline in tracer penetration which eventually fails to enter the bone matrix when all matrix vesicles are obliterated by mineral deposits. From this stage onwards, lanthanum is only observed filling approximately the basal two thirds of the intercellular spaces between osteoblasts. Thus the calcified matrix is completely free of tracer.
354
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Figs 19 and 20. Late mineralising matrix. Fig. 19. Electron micrograph showing a focal tight junction (arrow) between an osteoblast (B) and a small osteoblast process (P). At this stage, electron opaque deposits of mineral (*) are present in the matrix (M) which also contains collagen fibrils (C). Bar, 0.5 gtm. The inset shows a higher magnification view of the tight junction (arrow). Bar, 0.05 gm. R, portions of osteoblast processes. Tannic acid-aldehyde fixation. Lead citrate/uranyl acetate staining. Fig. 20. Freeze-fracture replica of developing bone in the same stage as in Figure 19 showing the P fracture face of the plasma membrane of an osteoblast exhibiting rows of tight junction particles (arrows), most of which are elongated. Bar, 0.1 Igm.
The present results also show that osteoblasts of early developing bone possess regions of membrane approximations that appear to form tight junctions. Such
regions were observed as fusion of the outer leaflets of the plasma membranes when sections were tilted in the electron microscope. Freeze-fracture replicas have
Compartmentalisation of early mineralising bone matrix confirmed the existence of single and branched rows of IMPs which often appear to be fused and are thus interpreted as tight junction particles. These rows of particles, however, did not form belt-like structures around the osteoblasts and were often in continuity with gap junctions. Such structures are interpreted as focal or macular tight junctions which have also been described in other tissues. Although the role of macular tight junctions is not understood, it has been shown that they prevent, at least partially, the passage of tracers between cells (Smith & Raviola, 1983; Smith et al. 1985). The fact that in the stage of early developing matrix (nonmineralised) the existing intercellular spaces between osteoblasts allow free access of lanthanum to the forming matrix shows that early bone matrix is not compartmentalised, i.e. it is not isolated from the surrounding extracellular space. It is therefore likely that constituents of the ECF have access to the early bone matrix and vice versa. The same situation occurs in the stage of incipient mineralisation. The matrix, at this stage, contains matrix vesicles which gradually increase in number and become calcified but no discernible signs of mineralisation are observed in relation to collagen; mineral deposits are exclusively associated with matrix vesicles. It is therefore possible that matrix vesicles act as microcompartments in which conditions for calcium phosphate precipitation may occur, perhaps by intervention of phosphatases (Ali, 1976; De Bernard et al. 1986) or other calciumbinding molecules present in the matrix vesicles (Bonucci, 1989). The fact that collagen remains unmineralised until virtually all matrix vesicles appear calcified is consistent with the idea that matrix vesicles may only be responsible for the mineralisation of the nonfibrillar phase of the bone matrix, in a manner similar to that suggested for dentine matrix (Katchburian, 1977). Also, it is claimed that it is not possible for hydroxyapatite crystals to grow out of matrix vesicles and as such promote calcification of collagen at great distances (Christoffersen et al. 1978; Christoffersen & Landis, 1991). Our results show that when virtually all matrix vesicles appear calcified, osteoblasts possess conspicuous areas of focal tight junctions and lanthanum no longer passes through their intercellular spaces, thus failing to reach the matrix which becomes compartmentalised. It is only when all matrix vesicles are calcified and bone matrix compartmentalised that mineral deposits begin to spread throughout the matrix. The existence of macular tight junctions, in addition to contributing to the closing up of the matrix, may be related to late polarisation events occurring in the osteoblasts. In
355
epithelial cells, tight junction formation is associated with stimulation of exocytosis, formation of an almost instantaneously overt apical membrane domain, and redistribution of internal organelles (Gumbiner, 1990). Polarisation of osteoblasts, together with compartmentalisation of the matrix, may at these later stages allow for compositional changes of the matrix, such as localised secretion of specific matrix molecule(s) (Turksen & Aubin, 1991) not found in noncalcifying collagen-containing matrices. These molecules may be phosphoproteins (Glimcher, 1989) or others (see Christoffersen & Landis, 1991, for review) which bind to collagen and as such provide for nucleation sites. From the foregoing considerations, it appears that matrix vesicles may be required only to initiate the mineralisation process when bone matrix is in some form of continuity with the general extracellular space. Once the matrix is compartmentalised, mineralisation proceeds without vesicles, probably in relation to collagen associated molecules, as proposed by Glimcher (1989). Finally, our results are also consistent with the concept of compartmentalisation of adult bone matrix proposed by Neuman (1969) who suggested the existence of a 'milieu interieur' in bone which is different from the general extracellular fluid. ACKNOWLEDGEMENTS
The authors thank Dr Antonio Sesso and Dr Edna Haapalainen for their advice and help. Technical assistance by Mr Gaspar F. de Lima and Mr Helio Correa is gratefully acknowledged. The work was supported by grants from CNPq and CAPES (Brazil). REFEREN CES
ALI SY (1976) Analysis of matrix-vesicles and their role in the calcification of epiphyseal cartilage. Federation Proceedings 35, 135-142. ALI SY, SAJDERA JW, ANDERSON HC (1970) Isolation and characterization of calcifying matrix-vesicles from epiphyseal cartilage. Proceedings of the National Academy of Sciences of the USA 67, 1513-1520. ANDERSON HC (1967) Electron microscopy studies of induced cartilage development and calcification. Journal of Cell Biology 35, 81-102. ANDERSON HC (1989) Biology of disease: mechanism of mineral formation in bone. Laboratory Investigation 60, 320-330. BERNARD GW, PEASE DC (1969) An electron microscopic study of initial intramembranous osteogenesis. American Journal of Anatomy 125, 271-290. BONUccI E (1967) Fine structure of early cartilage calcification. Journal of Ultrastructure Research 20, 33-50. BONUCCIlE (1989) Electron microscope studies of the early stage of the calcification process: role of matrix-vesicles. Progress in Clinical and Biological Research 295, 109-114.
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CHRISTOFFERSEN J, CHRISTOFFERSEN MR, KJAERGAARD N (1978) The kinetics of dissolution of calcium hydroxyapatite in water at constant pH. Journal of Crystal Growth 57, 21-26. CHRISTOFFERSEN J, LANDIS WJ (1991) A contribution with review to the description of mineralization of bone and other calcified tissues in vivo. Anatomical Record 230, 435-450. DE BERNARD B, BIANCO P, BONUccI E, CONSTANTINI M, LUNAZZI GC, MARTINUZZI P ET AL. (1986) Biochemical and immunohistochemical evidence that in cartilage an alkaline phosphatase is a Ca-binding glycoprotein. Journal of Cell Biology 103, 1615-1623. GLIMCHER MJ (1959) The molecular biology of mineralized tissues with particular reference to bone. Reviews of Modern Physics 31, 359-393. GLIMCHER MJ (1976) Composition, structure, and organization of bone and other mineralized tissues and the mechanisms of calcification. In Handbook of Physiology, section 7, vol. vii (ed. R. 0. Greep & E. B. Astwood), pp. 25-116. Washington: American Physiological Society. GLIMCHER MJ (1989) Mechanism of calcification: role of collagen fibrils and collagen-phosphoproteins complexes in vitro and in vivo. Anatomical Record 224, 139-153. GUMBINER B (1990) Generation and maintenance of epithelial cell polarity. Current Opinion in Cell Biology 2, 881-887. HOWARD JE (1956) Present knowledge of parathyroid function with
especial emphasis on its limitations. In Ciba Symposium on Bone Structure and Metabolism (ed. C. E. W. Wolstenholme & C. M. O'Connor), pp. 206-217. Boston: Little Brown. JONES SJ, BOYDE A, ALI NN, MACONNACHIE E (1985) A review of bone cell and substrate interaction. An illustration of the role of scanning electron microscopy. Scanning 7, 5-24. KATCHBURIAN E (1973) Membrane-bound bodies as initiators of mineralization of dentine. Journal of Anatomy 116, 285-302. KATCHBURIAN E (1977) Initiation of mineral deposition in dentine. Calcified Tissue Research Suppl. 22, 179-184. KATCHBURIAN E, SEVERS NJ (1982) Membranes of matrix-vesicles in early developing dentine. A freeze-fracture study. Cell Biology International Reports 6, 941-950. NEUMAN WF (1969) The 'milieu interieur' of bone: Claude Bernard revisited. Federation Proceedings 28, 1846-1850. NEUMAN WF, NEUMAN MW (1958) Chemical Dynamics of Bone Mineral. Chicago: University of Chicago Press. SMITH RL, RAVIOLA G (1983) The structural basis of the bloodaqueous barrier in the chicken eye. Investigative Ophthalmology and Visual Science 24, 326-338. SMITH RL, NISHIMURA Y, RAVIOLA G (1985) Interreceptor junction in the double cone of the chicken retina. Journal of Submicroscopic Cytology 17, 183-186. TURKSEN K, AUBIN JE (1991) Positive and negative immunoselection for enrichment of two classes of osteoprogenitor cells. Journal of Cell Biology 114, 373-384.
J. Anat (1992) 181, pp. 345-356, with 16 figures Printed in Great Britain
Lanthanum tracer and freeze-fracture studies suggest that compartmentalisation of early bone matrix may be related to initial mineralisation ANA MARIA V. SOARES', VICTOR E. ARANA-CHAVEZ', ANDREW R. REID2 AND EDUARDO KATCHBURIAN2'3 1 Department of Histology and Embryology, Institute of Biomedical Sciences, University of Sdo Paulo, Brazil, 2Department of Anatomy and Histology, The London Hospital Medical College, London, UK, and 3 Department of Histology and Electron Microscopy Centre, Escola Paulista de Medicina, Sao Paulo, Brazil
(Accepted 24 August 1992)
ABSTRACT
In adult bone the calcified matrix and enclosed osteocytes are separated from the extracellular space by a continuous layer of bone lining cells. It thus appears that bone matrix is compartmentalised and, as such, may constitute a 'milieu interieur' which is different from the general extracellular space. Since adult bone matrix is compartmentalised and matrix vesicles also form a microcompartment, it is conceivable that compartmentalisation, in early osteogenesis, may be a requirement for the initial events of the mineralisation process. We have therefore conducted an ultrastructural, tracer, and freeze-fracture study to determine the stage in which bone matrix becomes compartmentalised and also to find out whether there are tight junctions between osteoblasts. The results show that in early nonmineralised stages and in incipient mineralisation, lanthanum penetrates all intercellular spaces and the newly forming bone matrix which is rich in matrix vesicles and collagen. With the progression of mineralisation, when all matrix vesicles appear mineralised and calcification is 'spreading' to the surrounding matrix, lanthanum is restricted to intercellular spaces and conspicuous macular tight junctions are present between osteoblasts. We suggest that matrix vesicles act as microcompartments for calcification when the early bone matrix is in continuity with the surrounding extracellular space. In later stages, when lanthanum fails to penetrate the matrix, matrix vesicles may no longer be necessary because the bone matrix itself is compartmentalised, thus allowing for localised changes in composition that might favour mineral deposition. INTRODUCTION
In adult bone the mineralised matrix and enclosed osteocytes are separated from the general extracellular space by a continuous layer of bone lining cells (Jones et al. 1985). As early as 1956, Howard observed some differences in the ionic content between blood and bone fluid in patients with parathyroid diseases. Neuman & Neuman (1958) also reported an unbalanced state between the mineral phase of bone and the extracellular fluid (ECF) in healthy organisms. Finally, Neuman (1969) showed differences in ionic composition in the mineralised matrix of bone and the ECF, suggesting the existence of a 'bone membrane' which establishes the 'milieu interieur' of bone tissue by a compartmentalisation phenomenon.
In early developing bone, as well as other collagencontaining calcifying matrices, one of the most important events is the onset of the mineralisation process. Collagen and/or collagen-associated molecules were proposed as possible initiators of the process (Glimcher, 1959, 1976, 1989). However, the discovery of matrix vesicles as the initial sites of mineral nucleation in calcifying cartilage (Anderson, 1967; Bonucci, 1967; Ali et al. 1970), bone (Bernard & Pease, 1969) and dentine (Katchburian, 1973) introduced the concept that vesicles form an enclosed and protective microcompartment for nascent mineral formation (Anderson, 1989) in immature bone. Since adult bone matrix is compartmentalised and matrix vesicles also form a microcompartment, it is conceivable that compartmentalisation, in early osteo-
Correspondence to Dr E. Katchburian, Departamento de Histologia, Escola Paulista de Medicina, Rua Botucatu 740, 04023.062 Sao Paulo, S.P., Brazil.
346
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Figs 1-4. Light micrographs of developing calvaria from a rat embryo aged 17 days showing the sequence of development. Darkly stained osteoblasts (B) are present in all figures. In Figures 2-4, osteoblasts surround portions of early matrix (light grey). Fig. 1, early nonmineralised matrix; Fig. 2, incipient mineralising matrix; Figs 3 and 4, late mineralising matrix. Toluidine blue stained 1 gm resin sections. Bar, 10 gm.
Fig. 5. Electron micrograph of early nonmineralised matrix showing portions of adjacent early developing osteoblasts (B) and matrix (M). Electron opaque deposits of lanthanum are present in intercellular spaces (arrowheads) and in the early matrix (arrows). N, nucleus; C, collagen fibrils. Lead citrate staining. Bar, 1 gm.
genesis, may be a requirement for the initial events of the mineralisation process. We have therefore con-
ducted an ultrastructural, tracer, and freeze-fracture study to determine the stage in which bone matrix
347
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Figs 6-8. Early nonmineralised matrix. Fig. 6. Electron micrograph of early nonmineralised matrix in a more advanced stage of development than in Fig. 5. Numerous black dots of lanthanum are present between the early developing osteoblasts (arrowheads) and in the matrix (arrows). Matrix vesicles are not present but numerous osteoblast processes (P) are observed. B, osteoblasts; M, matrix; C, collagen fibrils. Lead citrate staining. Bar, 1 jim. Figs 7 and 8. Higher magnification views of early nonmineralised matrix in the same stage as that of Fig. 6. Electron opaque deposits of lanthanum (arrows) are mostly associated with collagen fibrils (C) which are observed in transverse (Fig. 7) and longitudinal (Fig. 8) sections. B, portions of osteoblasts. Lead citrate staining. Bar, 0.1 jIm.
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Ana Maria V. Soares and others
Figs 9-11. Incipient mineralising matrix. Fig. 9. Electron micrograph showing incipient mineralising matrix. Numerous black dots of lanthanum are observed between osteoblasts (arrowheads) and also within the matrix (arrows), mostly in association with collagen fibrils (C) and osteoblast processes (P). Nonmineralised (V1) and mineralised matrix vesicles (V2) are present throughout the matrix. Some nonmineralised matrix vesicles (V1) exhibit lanthanum associated with their membrane. Lanthanum containing invaginations of the osteoblast surface observed on the right lower corner of the figure occasionally appear as though inside the cell (X). B, osteoblasts; M, matrix. Lead citrate staining. Bar, 0.5 ,um. Figs 10 and 11. Higher magnification views of Fig. 9 showing deposits of lanthanum (arrows) in association with a nonmineralised matrix vesicle (V") and an osteoblast process (P). The vesicle (V2) in Fig. 10 shows a disrupted membrane and crystal-like inclusions (S). In Fig. 11 a fully mineralised matrix vesicle (V2) is observed. Lead citrate staining. Bar, 0.1 pIm.
. . . . .*
Fig. 12. Electron micrograph showing incipient mineralising matrix in a more advanced stage of development than in Fig. 9. Deposits of lanthanum are clearly observed between 2 adjacent osteoblasts (arrowheads) but are rarely present in the matrix (arrows). V2, mineralised matrix vesicles; M, matrix; C, collagen fibrils; B, osteoblasts; P, osteoblast process. Lead citrate staining. Bar, 0.5 gm.
becomes compartmentalised and also to find out whether there are tight junctions between osteoblasts.
MATERIALS AND METHODS
We examined different stages of bone formation in calvaria of Wistar and Sprague-Dawley rat embryos aged between 17 and 19 d. For ultrathin sections, some specimens were fixed in 2.5 % glutaraldehyde containing lanthanum nitrate in 0.1 M sodium caco-
dylate buffer, pH 7.7 (lanthanum nitrate tracer kit, Emscope), for 4 h at room temperature. After washing for 10 min in 0.1 M sodium cacodylate buffer, they were postfixed in 1 % osmium tetroxide containing lanthanum nitrate in 0.1 M sodium cacodylate, pH 7.7, for 3 h. Other specimens were fixed in 2% glutaraldehyde-2.5 % formaldehyde (Sigma Chemical Co.) containing 1 % tannic acid (Baker Chemical Co.) in 0.1 M sodium cacodylate, pH 7.4, for 4 h at room temperature. After washing for 1 h in the same buffer, they were postfixed in buffered 1 % osmium tetroxide
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Figs 13-15. Incipient mineralising matrix. Fig. 13. Focal tight junction (arrow) between 2 osteoblast processes (P) before (a) and after (b) tilting the ultrathin section. In 13 a, the closely apposed plasma membranes appear as a 'fuzz' and are not therefore clearly discerned. In 13 b the section was tilted by approximately 150 and the unit membrane structure becomes clearly visible. A typical tight junction exhibits the outer leaflets of the adjoining membranes fused. Tannic acid-aldehyde fixation. Lead citrate/uranyl acetate staining. Bar, 0.05 gm. Fig. 14. Electron micrograph showing a focal tight junction (arrow) between 2 osteoblasts (B). Collagen fibrils (C) and a matrix vesicle (V) are present in the matrix (M). Bar, 0.5 gm. The inset shows a higher magnification view of the tight junction (arrow) which exhibits a similar appearance to that seen in Figure 13b. Bar, 0.05 gm. Tannic acid-aldehyde fixation. Lead citrate/uranyl acetate staining. Fig. 15. Freezefracture replica of developing bone in the same stage as in Figures 13 and 14 showing the P-fracture face of the plasma membrane of an osteoblast containing typical intramembranous particles. Rows of elongated (arrows) and globular (arrowheads) particles form images of tight junctions which run in different directions and merge with existing gap junctions (GJ). Bar, 0.1 tm.
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Fig. 16. Electron micrograph showing late mineralising matrix. Virtually all matrix vesicles (V2) are mineralised. Deposits of lanthanum (arrowheads), present between osteoblasts (B) do not reach the distal portion of the intercellular space and are absent from the matrix (M). C, collagen fibrils. Lead citrate staining. Bar, 1 gm.
for 2h. All specimens were dehydrated in graded concentrations of ethanol after which they were embedded in Premix (TAAB Laboratories) or Spurr (Electron Microscopic Sciences) resins. Toluidine blue stained thick sections were examined with a light microscope and carefully selected regions trimmed for ultrathin sectioning. Unstained and lead citrate or lead citrate/uranyl acetate stained ultrathin sections were collected onto copper grids. For freeze-fracture replication, specimens were fixed in 2% glutaraldehyde-2.5 % formaldehyde in 0.1 M sodium cacodylate, pH 7.4, for 4 h at room temperature. After washing for 30 min in the same buffer, they were treated overnight with cacodylatebuffered 30 % glycerol. Freeze-fracture replicas were
prepared using a Balzers BAF 301 unit, as previously described (Katchburian & Severs, 1982). Both the ultrathin sections and the freeze-fracture replicas were examined in Hitachi H300 or JEOL 100 CX II electron microscopes.
RESULTS
Early developing calvaria in stages prior to and at late stages of mineralisation show 3 regions which are disposed in sequence and represent different stages of development. From the periphery of the calvaria to the initial centre of ossification the following sequence was observed: early nonmineralised matrix (Fig. 1);
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Fig. 17. Electron micrograph showing late mineralising matrix in a more advanced stage of calcification than in Fig. 16. Similarly to Figure 16, deposits of lanthanum (arrowheads) present between osteoblasts (B) do not reach the distal portion of the intercellular space and are completely absent from the calcified matrix (M). Lead citrate staining. Bar, 0.5 gm.
incipient mineralising matrix (Fig. 2); late mineralising matrix (Figs 3, 4). Early nonmineralised matrix At this stage osteoblasts and/or preosteoblasts appear in close proximity, but no occluding junctions are observed in ultrathin sections or freeze-fracture replicas. Together with their processes, osteoblasts surround areas of collagenous early bone matrix (Fig. 5). Electron opaque deposits of lanthanum are readily seen in the newly forming bone matrix (Figs 5, 6). The deposits appear as electron opaque dots which are often associated with the irregularly arranged collagen fibrils of the matrix (Figs 5-8). Matrix vesicles are not present at this stage.
Incipient mineralising matrix With the progression of osteogenesis, matrix vesicles appear within the matrix. Deposits of lanthanum, as
in the previous stage, are present in the intercellular spaces between osteoblasts and within the matrix where they are observed in association with collagen fibrils and matrix vesicles. Some matrix vesicles show electron opaque deposits in their interior while others are devoid of them (Figs 9-1 1). These electron opaque deposits are interpreted, from previous literature, as mineral (Anderson, 1967; Bonucci, 1967). With further development, when most of the matrix vesicles appear calcified, lanthanum deposits are still present in the intercellular spaces between osteoblasts but only in certain areas of the matrix where a trickle of the tracer is observed (Fig. 12). Collagen fibrils are free of visible mineral deposits (Fig. 12). Osteoblasts are closer together and there are several regions of approximations between their adjoining plasma membranes. Most of these regions appear diffuse, often giving the impression of superimposed images (Fig. 13a). However, after tilting the ultrathin sections, some of these regions revealed typical images of focal tight junctions in which the outer leaflets of the
Compartmentalisation of early mineralising bone matrix
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Fig. 18. Unstained electron micrograph of a lanthanum treated specimen also in the stage of late mineralising matrix. *Distribution of lanthanum (arrowheads) is similar to that observed in Figure 17. M, calcified matrix; B, osteoblasts. Unstained. Bar, 0.5 gm.
adjoining membranes appear fused (Fig. 13b). These junctions are present between osteoblast processes (Fig. 13 b) and between their cell bodies (Fig. 14) and are always present singly. Freeze-fracture replicas confirmed the presence of typical tight junction particles. These appear as rows of fused intramembranous particles (IMPs) which are often in continuity with small gap junctions (Fig. 15).
in the previous stage, but they continue to be of the focal type and may also appear between an osteoblast cell body and an osteoblast process (Fig. 19). Freezefracture replicas confirmed the presence of tight junction particles which appear to be more extensive, therefore occupying large areas of the plasma membranes of osteoblasts (Fig. 20). DISCUSSION
Late mineralising matrix
When all matrix vesicles exhibit mineral deposits, lanthanum is no longer observed within the matrix but is still present in the intercellular spaces between osteoblasts (Fig. 16). In a more advanced stage, when matrix vesicles are absent and mineral deposits are observed throughout the matrix, lanthanum remains excluded from the matrix but it is still present between osteoblasts, filling approximately the basal two thirds of the intercellular spaces (Figs 17, 18). Tight junctions between osteoblasts are more conspicuous than
Our experiments with lanthanum show that the tracer, when incorporated into the fixative, fully penetrates bone matrix in stages prior to and at the beginning of the mineralisation process. With the progression of mineralisation there is a gradual decline in tracer penetration which eventually fails to enter the bone matrix when all matrix vesicles are obliterated by mineral deposits. From this stage onwards, lanthanum is only observed filling approximately the basal two thirds of the intercellular spaces between osteoblasts. Thus the calcified matrix is completely free of tracer.
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Figs 19 and 20. Late mineralising matrix. Fig. 19. Electron micrograph showing a focal tight junction (arrow) between an osteoblast (B) and a small osteoblast process (P). At this stage, electron opaque deposits of mineral (*) are present in the matrix (M) which also contains collagen fibrils (C). Bar, 0.5 gtm. The inset shows a higher magnification view of the tight junction (arrow). Bar, 0.05 gm. R, portions of osteoblast processes. Tannic acid-aldehyde fixation. Lead citrate/uranyl acetate staining. Fig. 20. Freeze-fracture replica of developing bone in the same stage as in Figure 19 showing the P fracture face of the plasma membrane of an osteoblast exhibiting rows of tight junction particles (arrows), most of which are elongated. Bar, 0.1 Igm.
The present results also show that osteoblasts of early developing bone possess regions of membrane approximations that appear to form tight junctions. Such
regions were observed as fusion of the outer leaflets of the plasma membranes when sections were tilted in the electron microscope. Freeze-fracture replicas have
Compartmentalisation of early mineralising bone matrix confirmed the existence of single and branched rows of IMPs which often appear to be fused and are thus interpreted as tight junction particles. These rows of particles, however, did not form belt-like structures around the osteoblasts and were often in continuity with gap junctions. Such structures are interpreted as focal or macular tight junctions which have also been described in other tissues. Although the role of macular tight junctions is not understood, it has been shown that they prevent, at least partially, the passage of tracers between cells (Smith & Raviola, 1983; Smith et al. 1985). The fact that in the stage of early developing matrix (nonmineralised) the existing intercellular spaces between osteoblasts allow free access of lanthanum to the forming matrix shows that early bone matrix is not compartmentalised, i.e. it is not isolated from the surrounding extracellular space. It is therefore likely that constituents of the ECF have access to the early bone matrix and vice versa. The same situation occurs in the stage of incipient mineralisation. The matrix, at this stage, contains matrix vesicles which gradually increase in number and become calcified but no discernible signs of mineralisation are observed in relation to collagen; mineral deposits are exclusively associated with matrix vesicles. It is therefore possible that matrix vesicles act as microcompartments in which conditions for calcium phosphate precipitation may occur, perhaps by intervention of phosphatases (Ali, 1976; De Bernard et al. 1986) or other calciumbinding molecules present in the matrix vesicles (Bonucci, 1989). The fact that collagen remains unmineralised until virtually all matrix vesicles appear calcified is consistent with the idea that matrix vesicles may only be responsible for the mineralisation of the nonfibrillar phase of the bone matrix, in a manner similar to that suggested for dentine matrix (Katchburian, 1977). Also, it is claimed that it is not possible for hydroxyapatite crystals to grow out of matrix vesicles and as such promote calcification of collagen at great distances (Christoffersen et al. 1978; Christoffersen & Landis, 1991). Our results show that when virtually all matrix vesicles appear calcified, osteoblasts possess conspicuous areas of focal tight junctions and lanthanum no longer passes through their intercellular spaces, thus failing to reach the matrix which becomes compartmentalised. It is only when all matrix vesicles are calcified and bone matrix compartmentalised that mineral deposits begin to spread throughout the matrix. The existence of macular tight junctions, in addition to contributing to the closing up of the matrix, may be related to late polarisation events occurring in the osteoblasts. In
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epithelial cells, tight junction formation is associated with stimulation of exocytosis, formation of an almost instantaneously overt apical membrane domain, and redistribution of internal organelles (Gumbiner, 1990). Polarisation of osteoblasts, together with compartmentalisation of the matrix, may at these later stages allow for compositional changes of the matrix, such as localised secretion of specific matrix molecule(s) (Turksen & Aubin, 1991) not found in noncalcifying collagen-containing matrices. These molecules may be phosphoproteins (Glimcher, 1989) or others (see Christoffersen & Landis, 1991, for review) which bind to collagen and as such provide for nucleation sites. From the foregoing considerations, it appears that matrix vesicles may be required only to initiate the mineralisation process when bone matrix is in some form of continuity with the general extracellular space. Once the matrix is compartmentalised, mineralisation proceeds without vesicles, probably in relation to collagen associated molecules, as proposed by Glimcher (1989). Finally, our results are also consistent with the concept of compartmentalisation of adult bone matrix proposed by Neuman (1969) who suggested the existence of a 'milieu interieur' in bone which is different from the general extracellular fluid. ACKNOWLEDGEMENTS
The authors thank Dr Antonio Sesso and Dr Edna Haapalainen for their advice and help. Technical assistance by Mr Gaspar F. de Lima and Mr Helio Correa is gratefully acknowledged. The work was supported by grants from CNPq and CAPES (Brazil). REFEREN CES
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