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Comments on the ultrastructural morphology of the calcification process: an attempt to reconcile matrix vesicles, collagen fibrils, and crystal ghosts
Bone and Mineral,
17 (1992) 219-222
219
Elsevier This paper was presented at the Fifth International Conference on Cell-Mediated Calcification and MatrixVesicles, held Novembe: 16-20.1991, Hilton Head, South Carolina.
onucci Depnrtmenl
ofHuman
Riopathology,
Se&on of Pathological Anatomy,
Universiry La Sapienza,
Rotne, Italy
The ultrastructural morphology of cartilage, bone, and other calcifying matrices shows that during the early stage of the calcification process the inorganic substance is in relationship with two principal structures: matrix vesicles (MVs) and collagen fibrils (CFs). The available data suggest that both these structures might have a primary role in the process. There are at present no doubts that MVs are the sites of initial calcification in epiphyseal cartilage, primary bone, mantle dentin, and other calcifying matrices [1,2]. They have the biochemical machinery, including alkaline phosphatase, which leads to deposition of hydroxyapatite crystals in their matrix and on the inner side of their membrane [3), and the earliest aggregates of crystals are formed within them. Numerous investigations in the last twenty years have shown the constant presence of MVs in the areas of epiphyseal cartilage which successively will calcify. They are present also in bone; however, in this tissue their numbers are variable and areas of early calcification can be found at the same time in connection with The suggestion that CFs can induce calcification was advanced since the first electron microscope studies on developing bone [4], chiefly because it was soon evident that in this tissue the mineral substance is in close relationship with the collagen periodic banding. Chiefly on the basis of this observation, it was suggested that early crystals are formed in the hole zones of the fibrils by a process of heterogeneous nucleation [4,5]. However, electron microscopy shows that two mineral phases can be found in bone and other calcifying tissues [6]: an ‘amorphous’ phase (AP) which probably consists of extremely small crystals and is in relationshi the collagen periodic banding, and needle-, filament-, or plate-like crystals which do not show this relationship and are more or less parallel to CFs. A number of ultrastructural and biophysical studies show that [6]: both AP and CRs correspond to poorly crystalline hydroxyapatite; AP is present in matrices with compact collagen texture (compact bone, calcified tendons), whereas CRs are abundant in
220 matrices with loose collagen texture (embryonic bone, calcified cartilage); AP forms electron-dense bands perpendicular to the axis of CFs, whereas CRS are predominantly parallel to CFs. These findings suggest that AP is contained within the holes of the collagen fibrils, whereas CRs are contained in the interfibrillary space. This is in agreement with the observation that [6,7]: first, because of its small dimensions, AP can be easily accommodated into the intermolecular spaces of the fibrils without disrupting their organization, whereas, second, this cannot be true for CRs which are thicker and longer than intermolecular spaces, even if the supposed presence of intrafibrillar pores and grooves [8,9] is accepted. On the other hand, the extrafibrillar location of CRs is confirmed by electron microscopy. This shows that numbers of CRs are proportional to the amount of interfibrillary substance, and that in areas of initial calcification CRs often surround uncalcified collagen fibrils, can be contained in MVs, and form the interfibrillary calcification nodules [6]. The presence of AP within CFs, and of CRs between CFs and in MVs, showv not only that the early stages of the calcification process occur in relationship with different structures in different tissues, but also that the different organization of the matrix can lead to development of different mineral phases. Thus, it is important to establish if each of the calcifying structures has its own calcification mechanism, that is, if there are as many mechanisms of calcification as calcifying matrices, or if there are factors which are common to all of them, that is, if the calcification process always occurs according to the same basic mechanism. Several ultrastructural findings suggest that a common calcification factor might be represented by the so-called ‘crystal ghosts’ [7]. Crystal ghasts (CGs) are organic structures which can be recognized under the electron microscope in sections treated by the Post-Embedding Decalcification and Staining method (BEDS) [lo]. Structures similar to CGs can be shown in undecalcified sections treated with chromium sulfate [ 111,and in sections from specimens decalcified before embedding in which anionic molecules are stabilized by addition of cationic dyes to the fixatives [12]. CGs have the same shape and location as, and appear to be components of, untreated CRs [6]. This suggests that they are intimately bound to the mineral substance, thus appearing the morphological equivalent of the biochemically-detected ‘crystal-bound’ proteins because these, like CGs, are linked to the mineral substance, and the link is of such a degree that they become soluble only after decalcification. CGs are characterized histochemically by the presence of acidic, probably sulphate groups [13,14]. However, their composition, which might be different in different calcifying matrices, is not known. Proteoglycans, glycoproteins and proteolipids, and other calcium-binding molecules, seem the most frequent components. Nothing is known with certainty about the origin of CGs. Considering not only the findings obtained from cartilage and bone, but also those drawn from studies of other biological systems, such for instance molluscan shells, two hypotheses can be suggested: CGs might originate during development of CRs by adsorption of specific proteins on crystal surface, a process which could be followed either by inhibition of further crystal growth [15], or by further crystal nucleation and protein incorporation into growing CRs [ 161; or, they could be preformed structures which are present in the calcifying tissues as normal constituents
of their matrix and are ‘unmasked’ and ma e reactive with mineral ions just before calcification [6,7]. Whatever their precise origin and composition might be, the constant presence of CGs in areas of early calcification cannot be fortuitous. Tentatively, they might be considered as organic structures of different shape and composition which, because of their Ca-binding properties, are able to initiate and/or regulate cal assembling calcium and phosphate ions in an apatite-like configuration organic-inorganic, crystal-like structures could be formed, the shape of which is filament-like (as that of CRs) when the shape of CGs is filamentous, or can assume other shape according to that of other types of CGs (as could happen, for instance, in the hole zones of the CFs). Several molecules with potential properties of CGs can be found in calcifying structures: phospholipids and proteolipids, which are in membrane and matrix of Vs; phosphoproteins, which are comlponents Fs and are probably cantain in their hole zones; proteoglycans, which are found in interfibrillary spaces of cartilage and other calcifying tissues. The alkaline pro phosphatase itself has characteristics of 66s because it h calcium-binding rated and inactivat ty, is constantly present in calcifying areas, and is incor calcified matrix [ 171. It is possible to speculate that calcification is induced by immobilization (in CFs, MVs or other structures) of organic molecules (morphologically identifiable as CGs) having the common property of binding calcium ions, and that this pro rather than the molecular organization of CGs, might represent the common factor responsible for calcification of different tissues. It might explain why early deposition of mineral substance with different appearance can occur, even at the same time, in structures having very different organization, and might reconcile the role that Cl3 have in calcification with that proper of MVs.
References 1 2 3 4 5
6 7 8
AndersonHC. Matrix vesicles in cartilage and bone. In: Bourne GH, ed. The biochemistry and physiology of bone, v. IV,2nd ed. New York, San Francisco,London 1976;135-157. Bonucci E. Matrixvesicles: their role in calcification. In: Linde A, ed. Dentin and dentinogenesis, v. 1. Boca Raton, CRC Press 1984;136-154. Wuthier RE. A review of the primarymechanismof endochondral calcificationwith special emphasis on the role of cells, mitochondriaand matrixvesicles. Clin Orthop 1982;169:219-242. GlimcherMJ. Molecular biology of mineralizedtissues with particularreferenceto bone. Rev Modern Phys 1959;31:359-393. GlimcherMJ. Composition, structureand organization of bone and other mineralizedtissues and the mechanismof calcification. In: Greep RO, Astwood EB, eds. Handbook of physiology 7: Endocrinology, v’.VII. Washington, Am Physiol Sot 1976;25-116. Bonucci E. The structural basis of calcification. In: Ruggeri A, Motta PM, eds. Ultrastructureof connective tissue matrix, Boston, M Nijhoff Pub1 1984;165-191. Bonucci E. Is there a calcification factor common to all calcifying matrices7 Scann Microsc 1987;1:1089-1102. Traub W, Arad T, Weiner S. Three-dimensional ordered distribution of crystals in turkey tendon collagen fibers. Proc Natl Acad Sci USA 1989;86:9822-9826.
222 9 Katz EP, Li S-T. The intermolecular space of reconstituted collagen fibrils. J Molec Biol 1973;73: 351-369. 10 Bonucci E, Reurink J. The fine structure of decalcified cartilage and bone: a comparison between decalcification procedures performed before and after embedding. C&if Tiss Res 1978;25:17911 Appleton J. Ultrastructural observations on the inorganic-organic relationships in early cartilage calcification. Calcif Tiss Res 1971;7:307-317. 12 Shepard N, Mitchell N. Acridine orange stabilitiation of glycosaminoglycans in beginning endochondral ossification, A comparative light and electron microscoptc study. Histochem 1981;70:lO7- 114. 13 Bonucci E, Silvestrini G, Di Grezia R. The ultrastructure of the organic phase associated with the inorganic substance in calcified tissues. Clin Grthop 1988;233:243-261. 14 Bonucci , Silvestrini G, Di Grezia R. Histochemical properties of the ‘crystal ghosts’ of calcifying epiphyseal cartilage. Connect Tiss Res 1989;22:43-SO. 15 Wheeler AP, Rusenko KW, Sikes CS. Qrganic matrix from carbonate biomineral as a regulator of mineralixatioa, In: Sikes CS, Wheeler AP, eds. Alabama, Univers of South Alabama Pub1 Serv, 1988;9. 16 Addadi L, Berman A, Moradian Gldak J, Weiner S. Structural and stereochemicalrelations between acidicmacromoleculesof organicmatricesand crystals.Connect Tiss Res 1989;21:127-135.
17 De Bernard B, Bianco P, BonucciE, Costantini M, Lunazzi GC, Martinuzzi P, Modricky 6, More L, Panfili E, Pollesello P, Stagni N, Vittur F. Biochemical and immuno-histochemicalevidence that in
cartilage an alkaline phosphatase is a Ca*+-bindingglycoprotein. J Cell Biol 1986;103:1615-1623.
17 (1992) 219-222
219
Elsevier This paper was presented at the Fifth International Conference on Cell-Mediated Calcification and MatrixVesicles, held Novembe: 16-20.1991, Hilton Head, South Carolina.
onucci Depnrtmenl
ofHuman
Riopathology,
Se&on of Pathological Anatomy,
Universiry La Sapienza,
Rotne, Italy
The ultrastructural morphology of cartilage, bone, and other calcifying matrices shows that during the early stage of the calcification process the inorganic substance is in relationship with two principal structures: matrix vesicles (MVs) and collagen fibrils (CFs). The available data suggest that both these structures might have a primary role in the process. There are at present no doubts that MVs are the sites of initial calcification in epiphyseal cartilage, primary bone, mantle dentin, and other calcifying matrices [1,2]. They have the biochemical machinery, including alkaline phosphatase, which leads to deposition of hydroxyapatite crystals in their matrix and on the inner side of their membrane [3), and the earliest aggregates of crystals are formed within them. Numerous investigations in the last twenty years have shown the constant presence of MVs in the areas of epiphyseal cartilage which successively will calcify. They are present also in bone; however, in this tissue their numbers are variable and areas of early calcification can be found at the same time in connection with The suggestion that CFs can induce calcification was advanced since the first electron microscope studies on developing bone [4], chiefly because it was soon evident that in this tissue the mineral substance is in close relationship with the collagen periodic banding. Chiefly on the basis of this observation, it was suggested that early crystals are formed in the hole zones of the fibrils by a process of heterogeneous nucleation [4,5]. However, electron microscopy shows that two mineral phases can be found in bone and other calcifying tissues [6]: an ‘amorphous’ phase (AP) which probably consists of extremely small crystals and is in relationshi the collagen periodic banding, and needle-, filament-, or plate-like crystals which do not show this relationship and are more or less parallel to CFs. A number of ultrastructural and biophysical studies show that [6]: both AP and CRs correspond to poorly crystalline hydroxyapatite; AP is present in matrices with compact collagen texture (compact bone, calcified tendons), whereas CRs are abundant in
220 matrices with loose collagen texture (embryonic bone, calcified cartilage); AP forms electron-dense bands perpendicular to the axis of CFs, whereas CRS are predominantly parallel to CFs. These findings suggest that AP is contained within the holes of the collagen fibrils, whereas CRs are contained in the interfibrillary space. This is in agreement with the observation that [6,7]: first, because of its small dimensions, AP can be easily accommodated into the intermolecular spaces of the fibrils without disrupting their organization, whereas, second, this cannot be true for CRs which are thicker and longer than intermolecular spaces, even if the supposed presence of intrafibrillar pores and grooves [8,9] is accepted. On the other hand, the extrafibrillar location of CRs is confirmed by electron microscopy. This shows that numbers of CRs are proportional to the amount of interfibrillary substance, and that in areas of initial calcification CRs often surround uncalcified collagen fibrils, can be contained in MVs, and form the interfibrillary calcification nodules [6]. The presence of AP within CFs, and of CRs between CFs and in MVs, showv not only that the early stages of the calcification process occur in relationship with different structures in different tissues, but also that the different organization of the matrix can lead to development of different mineral phases. Thus, it is important to establish if each of the calcifying structures has its own calcification mechanism, that is, if there are as many mechanisms of calcification as calcifying matrices, or if there are factors which are common to all of them, that is, if the calcification process always occurs according to the same basic mechanism. Several ultrastructural findings suggest that a common calcification factor might be represented by the so-called ‘crystal ghosts’ [7]. Crystal ghasts (CGs) are organic structures which can be recognized under the electron microscope in sections treated by the Post-Embedding Decalcification and Staining method (BEDS) [lo]. Structures similar to CGs can be shown in undecalcified sections treated with chromium sulfate [ 111,and in sections from specimens decalcified before embedding in which anionic molecules are stabilized by addition of cationic dyes to the fixatives [12]. CGs have the same shape and location as, and appear to be components of, untreated CRs [6]. This suggests that they are intimately bound to the mineral substance, thus appearing the morphological equivalent of the biochemically-detected ‘crystal-bound’ proteins because these, like CGs, are linked to the mineral substance, and the link is of such a degree that they become soluble only after decalcification. CGs are characterized histochemically by the presence of acidic, probably sulphate groups [13,14]. However, their composition, which might be different in different calcifying matrices, is not known. Proteoglycans, glycoproteins and proteolipids, and other calcium-binding molecules, seem the most frequent components. Nothing is known with certainty about the origin of CGs. Considering not only the findings obtained from cartilage and bone, but also those drawn from studies of other biological systems, such for instance molluscan shells, two hypotheses can be suggested: CGs might originate during development of CRs by adsorption of specific proteins on crystal surface, a process which could be followed either by inhibition of further crystal growth [15], or by further crystal nucleation and protein incorporation into growing CRs [ 161; or, they could be preformed structures which are present in the calcifying tissues as normal constituents
of their matrix and are ‘unmasked’ and ma e reactive with mineral ions just before calcification [6,7]. Whatever their precise origin and composition might be, the constant presence of CGs in areas of early calcification cannot be fortuitous. Tentatively, they might be considered as organic structures of different shape and composition which, because of their Ca-binding properties, are able to initiate and/or regulate cal assembling calcium and phosphate ions in an apatite-like configuration organic-inorganic, crystal-like structures could be formed, the shape of which is filament-like (as that of CRs) when the shape of CGs is filamentous, or can assume other shape according to that of other types of CGs (as could happen, for instance, in the hole zones of the CFs). Several molecules with potential properties of CGs can be found in calcifying structures: phospholipids and proteolipids, which are in membrane and matrix of Vs; phosphoproteins, which are comlponents Fs and are probably cantain in their hole zones; proteoglycans, which are found in interfibrillary spaces of cartilage and other calcifying tissues. The alkaline pro phosphatase itself has characteristics of 66s because it h calcium-binding rated and inactivat ty, is constantly present in calcifying areas, and is incor calcified matrix [ 171. It is possible to speculate that calcification is induced by immobilization (in CFs, MVs or other structures) of organic molecules (morphologically identifiable as CGs) having the common property of binding calcium ions, and that this pro rather than the molecular organization of CGs, might represent the common factor responsible for calcification of different tissues. It might explain why early deposition of mineral substance with different appearance can occur, even at the same time, in structures having very different organization, and might reconcile the role that Cl3 have in calcification with that proper of MVs.
References 1 2 3 4 5
6 7 8
AndersonHC. Matrix vesicles in cartilage and bone. In: Bourne GH, ed. The biochemistry and physiology of bone, v. IV,2nd ed. New York, San Francisco,London 1976;135-157. Bonucci E. Matrixvesicles: their role in calcification. In: Linde A, ed. Dentin and dentinogenesis, v. 1. Boca Raton, CRC Press 1984;136-154. Wuthier RE. A review of the primarymechanismof endochondral calcificationwith special emphasis on the role of cells, mitochondriaand matrixvesicles. Clin Orthop 1982;169:219-242. GlimcherMJ. Molecular biology of mineralizedtissues with particularreferenceto bone. Rev Modern Phys 1959;31:359-393. GlimcherMJ. Composition, structureand organization of bone and other mineralizedtissues and the mechanismof calcification. In: Greep RO, Astwood EB, eds. Handbook of physiology 7: Endocrinology, v’.VII. Washington, Am Physiol Sot 1976;25-116. Bonucci E. The structural basis of calcification. In: Ruggeri A, Motta PM, eds. Ultrastructureof connective tissue matrix, Boston, M Nijhoff Pub1 1984;165-191. Bonucci E. Is there a calcification factor common to all calcifying matrices7 Scann Microsc 1987;1:1089-1102. Traub W, Arad T, Weiner S. Three-dimensional ordered distribution of crystals in turkey tendon collagen fibers. Proc Natl Acad Sci USA 1989;86:9822-9826.
222 9 Katz EP, Li S-T. The intermolecular space of reconstituted collagen fibrils. J Molec Biol 1973;73: 351-369. 10 Bonucci E, Reurink J. The fine structure of decalcified cartilage and bone: a comparison between decalcification procedures performed before and after embedding. C&if Tiss Res 1978;25:17911 Appleton J. Ultrastructural observations on the inorganic-organic relationships in early cartilage calcification. Calcif Tiss Res 1971;7:307-317. 12 Shepard N, Mitchell N. Acridine orange stabilitiation of glycosaminoglycans in beginning endochondral ossification, A comparative light and electron microscoptc study. Histochem 1981;70:lO7- 114. 13 Bonucci E, Silvestrini G, Di Grezia R. The ultrastructure of the organic phase associated with the inorganic substance in calcified tissues. Clin Grthop 1988;233:243-261. 14 Bonucci , Silvestrini G, Di Grezia R. Histochemical properties of the ‘crystal ghosts’ of calcifying epiphyseal cartilage. Connect Tiss Res 1989;22:43-SO. 15 Wheeler AP, Rusenko KW, Sikes CS. Qrganic matrix from carbonate biomineral as a regulator of mineralixatioa, In: Sikes CS, Wheeler AP, eds. Alabama, Univers of South Alabama Pub1 Serv, 1988;9. 16 Addadi L, Berman A, Moradian Gldak J, Weiner S. Structural and stereochemicalrelations between acidicmacromoleculesof organicmatricesand crystals.Connect Tiss Res 1989;21:127-135.
17 De Bernard B, Bianco P, BonucciE, Costantini M, Lunazzi GC, Martinuzzi P, Modricky 6, More L, Panfili E, Pollesello P, Stagni N, Vittur F. Biochemical and immuno-histochemicalevidence that in
cartilage an alkaline phosphatase is a Ca*+-bindingglycoprotein. J Cell Biol 1986;103:1615-1623.