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Fine structure of bone matrix calcification
Journal of Oral Biosciences 54 (2012) 19–24
Contents lists available at SciVerse ScienceDirect
Journal of Oral Biosciences journal homepage: www.elsevier.com/locate/job
Review
Fine structure of bone matrix calcification Kazuto Hoshi n Departments of Cartilage & Bone Regeneration (Fujisoft), Graduate School of Medicine, The University of Tokyo, Hongo 7-3-1, Bunkyo-ku, Tokyo 113-0033, Japan
a r t i c l e i n f o
abstract
Article history: Received 16 September 2011 Received in revised form 26 October 2011 Accepted 26 October 2011 Available online 24 February 2012
Various functions of bones are derived from the characteristics that they calcify. In bone calcification, hydroxyapatite is crystallized on the type I collagen-based organic matrices. The organic components of those bone matrices are commonly shared with other fibrous tissues. As extracellular fluid supersaturates hydroxyapatite, the reasons why bones are specifically calcified remains enigmatic. With the bone matrix calcification, the structural and the spatial changes of those organic substances seem to be closely associated, and participate in the complicated regulation of hydroxyapatite multiplication. The present paper overviews the morphological findings of bone calcification, and discusses the mineral and organic environments in bone matrix calcification. & 2012 Published by Elsevier B.V. on behalf of Japanese Association for Oral Biology.
Keywords: Bone matrix Calcification Collagen Calcified nodule Osteoblast
Contents 1. 2.
3.
Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 19 Contents . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 20 2.1. Formation of calcified nodules . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 20 2.2. Crystallization in the calcified nodules . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 23 2.3. Expansion of calcified nodules. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 23 Conclusion remarks . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 23 Conflict of interest . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 23 Acknowledgments . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 23 References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 23
1. Introduction Bone is a typical example of hard tissue. The bones work to shape, support, and protect body structures. They also aid body movement, house bone marrow, and store various minerals essential for the preservation of life. All these functions are derived from the characteristics that the bones calcify. The hardness of bones is derived from the specific composition. Compared with in soft tissues, large part of water is replaced by inorganic materials in bones. Calcium (Ca) and phosphorus (P) are major components of the inorganic compositions of human bones. The Ca/P ratio indicates high value of 2.33. It suggests that crystals in bones almost consist in hydroxyapatite (HA). Bones contain not only inorganic materials, but also organic substances, including collagen, proteoglycan, and other non-collagenous matrix proteins [1]. Those components are not unique for bones,
n
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but are commonly shared with other fibrous tissues including skin, tendon, or ligaments. It is well known that extracellular fluid supersaturates for forming HA [2]. As the entire body is filled with the fluid containing high concentration of calcium and phosphorus, it is always suffering from pathological calcification. However, fibrous tissues other than bones keep the uncalcified situation in fact. The reasons why only bones are calcified remains enigmatic. The previous findings from morphological view demonstrated that matrix vesicles start the primary calcification in bones [3]. Osteoblasts bud the 30- to 100-nm-wide matrix vesicles, which are surrounded by a lipid bilayer. Within the matrix vesicles, crystallized HA appears and grows, showing the primary calcification (Fig. 1, top). As those matrix vesicles were found, not only in the bones, but also in cartilage, dentin, or other hard tissues [4,5], the HA crystallization in the matrix vesicles is regarded as the pivotal event for biological calcification. Otherwise, HA crystals subsequently multiply, forming calcified nodules that expand, fuse, and then calcify expansive matrix
1349-0079/$ - see front matter & 2012 Published by Elsevier B.V. on behalf of Japanese Association for Oral Biology. doi:10.1016/j.job.2012.01.004
20
K. Hoshi / Journal of Oral Biosciences 54 (2012) 19–24
2. Contents 2.1. Formation of calcified nodules
Fig. 1. In the new bone, round osteoblasts and immature calcifying matrix are present. In immature bone matrix, calcification advanced as the distance from osteoblasts increased. Within membrane-bound matrix vesicles (MV), crystallization occurs (top, arrowheads). Subsequently, calcified nodules (CN) formed, followed by focal calcification along collagen fibrils (Co), which are referred to as collagen calcification (top, arrows). Finally, calcified matrix was expanded (bottom). The images were rearranged from Ref. [5].
(Fig. 1, bottom). The expansion of calcification in matrix is another clue to solve the molecular mechanisms of bone calcification. With this process, the structural and the spatial changes of various organic substances in bone matrix seem to be closely associated, and participate in the complicated regulation of HA multiplication. The present paper overviews the morphological findings of bone calcification, and especially focuses on the interaction between matrix molecules and bone minerals in the stage of expansive calcification.
The immature bone matrix, uncalcified osteoid, consists of many organic substances, including proteoglycans, non-collagenous matrix proteins, phospholipids, and alkaline phosphatase (ALP). Those substances can interact with calcium or phosphate, both of which are components of HA. However, those organics can be detected in both bone and soft tissue. Only the presence of those substances in bones cannot explain the mechanisms of bone matrix calcification. More important issue seems their structural and localizational alterations triggered by biological action of osteoblasts. Those organics interacting with minerals were reported to change their structures and localizations, during the transition from the uncalcified to the calcified phase, which affected the mineral microenvironment and initiate calcification [1]. Energyfiltering transmission electron microscopy (EFTEM) is useful to observe the morphological changes of both molecular structures and mineral localizations. Conventional transmission electron microscopy (TEM) enables us to visualize images through the contrast produced by elastic electron scattering. Electrons that pass through the elements include inelastically scattered types, which lose a measurable amount of energy, after their collision with inner-shell electrons, identified as being the principal causes of the aberration typically encountered in this method. EFTEM, on the other hand, can visualize the distribution of those inelastically scattered electrons that are marked by the loss of varying amounts of energy. By setting energy loss at 0 eV, most of the inelastically scattered electrons are eliminated, thus revealing images of a higher resolution than any obtainable via conventional TEM (Fig. 2, top left). In addition, certain elements are mapped at that particular level, when the electrons exhibiting energy loss typical of the elements are localized. Because the spatial resolution of the conventional electron probe X-ray microanalysis depends on the spot size of the electron beam, it usually is confined to a range between a few micrometers and some hundreds of nanometers. However, as resolution of EFTEM during elemental analysis corresponds to that of conventional TEM, mapping of Ca or P at the molecule level becomes possible. In order to analyze bone matrix calcification, alterations of the mineral microenvironment according to the ultrastructural and localizational changes in organic substances during bone calcification were visualized by the application of EFTEM. When observing the calcifying bone in the intramenbraneous ossification, Ca was localized in certain osteoid and throughout areas of calcification. Similarly, P was localized in some osteoid and all calcified areas. However, their localization patterns in the osteoid were not the same. Under higher magnification, a calcified nodule, collagen fibrils, or proteoglycan meshworks among them were clearly observed by EFTEM (Fig. 2, top), and Ca and P also colocalized in calcified nodules (Fig. 2, bottom). However, the Ca of osteoid tended to localize at proteoglycan meshwork among collagen fibrils (Fig. 2, bottom left), whereas P often maps to collagen fibril structures (Fig. 2, bottom right). Ca and P were preserved in different areas of uncalcified matrix, while both colocalize after the formation of calcified areas. In order to discuss the mineral localization in and around the calcified nodules, the organics interacting with minerals were localized at TEM level [1]. Progeoglycan, possessing high affinity with Ca, such as decorin and chondroitin sulfate, are immunohistochemically found along the osteoid at light microscopic level, while Cuprolinic blue stain can enhance proteoglycans in TEM images. CB-based TEM observation highlighted the fact that positively stained structures tended to be smaller the further they
K. Hoshi / Journal of Oral Biosciences 54 (2012) 19–24
21
0eV
Co
CN
Co Co
Fig. 2. Ca and P mapping in the area around a calcified nodule (bar 100 nm). The 0-eV loss (top, left), 250-eV loss (top, right), Ca localizations (bottom, left), and P localizations (bottom, right). Asterisks represent the same area of each image. Ca (blue) and P (green) also colocalized in a calcified nodule (CN). However, in uncalcified areas, Ca tends to localize at proteoglycan sites, while P often maps to collagen fibril (Co) structures. The images were rearranged from Ref. [1]. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)
undecalcified
UA/Pb staining
Fig. 3. (A) In undecalcified ultrathin sections (left), the crustal like structure are visible. Those crystals showed a hexagonal lattice under the high resolution observation (inset). In the staining with uranyl acetate and lead citrate (right), crystal sheath became visible (arrowheads). (B) In the calcified nodules, both BSP (10 nm) and osteopontin (20 nm) were densely localized (left). Under a higher magnification (right), both were arranged linearly (arrows, top) and located in the electron dense line (bottom). The images were rearranged from Ref. [7].
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K. Hoshi / Journal of Oral Biosciences 54 (2012) 19–24
were situated from osteoblasts. Large branching or fibrous CB-positive structures were seen in the osteoid, whereas smaller structures were concentrated in early stage-calcified nodules. When the calcified nodule expanded, the small structures were dispersed along the periphery of the calcified nodules [1].
Otherwise, the localizations of ALP that can boost phosphate anions were observed in osteoblasts and osteoid by both enzyme histochemistry and immunohistochemistry. Enzyme activity was detected by TEM on dorsal surfaces of osteoblasts and some areas of the matrix. Under higher magnification, enzyme activities were
Fig. 4. (A) Calcification progresses, when the distance from osteoblasts (OB) increases. (B) Under higher magnification, collagen fibrils were visible. The collagen fibrils at the diameter of about 50 nm (Co) possessed clear and round contour in cross-section (top). In the parallel with progression of calcification, some collagen fibrils showed the bifurcation, suggesting side-to-side fusion (middle). In the expansive calcified areas, the network of fine filamentous structure disappeared around collagen fibrils (bottom). MV, matrix vesicles; CN, calcified nodules; asterisks, proteoglycans. (C) Immunocytochemistry of decorin. Green areas indicate immunolocalization of decorin. (D) Gold particles indicated decorin localization. As calcification progresses, decorin surrounding collagen fibrils (Co) decreases. Top, osteoid containing matrix vesicles (MV); bottom, calcifying areas including calcified nodules (CN). The images were rearranged from Ref. [13]. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)
K. Hoshi / Journal of Oral Biosciences 54 (2012) 19–24
localized not only in matrix vesicles, but also in calcified nodules. ALP immunolocalizations also were observed on the surface of matrix vesicles, as well as in and around calcified nodules [1]. In vivo organic substances, including proteoglycans and collagen fibrils, formed complicated meshwork, in the osteoid. Because extracellular fluid is present in those narrow spaces, liberated ions may be influenced heavily by the inherent electrical charge of organic substances and limited in dynamic behavior. Taking those morphological findings into consideration, the difference in Ca and P localization patterns in the osteoid may limit the local [Ca2][PO4] product, leading to the general inhibition of hydroxyapatite crystallization. In contrast, colocalizations of Ca and P were observed in areas where calcified nodules were expanding. Proteoglycans were thought to play important roles in the accumulation of calcium to those areas undergoing calcification. Cytohistochemical localizations of ALP protein were found not only in matrix vesicles [6], but also in and around the calcified nodules [1]. Because enzyme cytochemical examination of calcified nodules revealed that an enzymatic reaction had occurred, ALP localized in the calcified nodules is thought to possess the activity to boost local concentrations of phosphate anions. Thus, both ALP and structurally altered proteoglycans were localized in the calcified nodules undergoing further calcification. These two substances may colocalize Ca and P to promote bone calcification, although elemental distribution patterns were not the same, in uncalcified areas. 2.2. Crystallization in the calcified nodules In the calcified nodules, the crystal possesses needle- and plate-like shapes and are approximately 80 nm long, 30 nm wide, and 8 nm thick (Fig. 3A). The constancy of the shapes and sizes of the crystal suggests cellular and organics-based control for crystal formation and growth. The consistent formation of crystals may facilitate biological regulation of calcification, because the crystals with the small and identical sizes have advantage for the expansive calcification during development or growth of body, and for the rapid resorption when calcium recruitment is needed for the homeostatic reasons. By the fine observation at the TEM level, organic crystal sheaths are observed in the vicinity of crystal [7] (Fig. 3A, right). Because these structures are closely associated with crystals, they are thought to participate in the crystal formation and growth. Bone sialoprotein (BSP) or osteopontin are regarded as the candidates consisting of the crystal sheath [7] (Fig. 3B). Those proteins are rich in acidic amino acids, such as glutamate or aspartate, and possess high affinity for calcium [8–10]. Those proteins could also interact with calcification crystals. In vitro, BSP promotes hydroxyapatite nucleation, although ostepoitin functions negatively. The latter also inhibits the crystal elongation. The co-operation of those two proteins, in spite of their conflicting nature, would effectively promote or retard crystallization, as well as regulate the growth speed and size of crystals. 2.3. Expansion of calcified nodules After calcified nodules form, they come into contact with adjacent collagen fibrils, and eventually calcify an expansive matrix of collagen. However, inhibitors of HA crystal formation and growth, such as pyrophosphate or proteoglycans, can be abundantly found around the collagen fibrils [11]. Collagen, by itself, is known to inhibit HA crystallization as well [12]. Therefore, as a whole, the environment surrounding collagen fibrils is not conducive to HA crystal formation and growth. In such a
23
situation, osteoblasts may remotely control the collagen calcification by secretion of various organic substances. Collagen fibrils are synthesized by the osteoblasts. Immediately after secretion, the diameter of collagen fibrils was demonstrated as a diameter of approximately 50 nm (Fig. 4A,B). However, once calcification begins, the collagen fibrils become fused, revealing complicated contours and widths greater than 400 nm (Fig. 4B). As fused collagen fibrils tended to gather in dense bundles, those took on a mosaic-like appearance, contrasted against non-collagenous area (Fig. 4B). Such collagen fibril fusion is speculated to promote rapid calcification, because partial localization of collagen fibrils provides vacant spaces where HA crystals growth physicochemically [13]. Decorin is regarded to be associated with the molecular mechanisms of this collagen fibril fusion. Decorin is the major proteoglycan in bones. It consists of a single GAG chain (chondroitin sulfate or dermatin sulfate) and about 40 kDa of core protein. Dense localization of decorin was noted around the thin collagen fibrils of uncalcified osteoid (Fig. 4C,D). However, when calcification progressed and collagen fibrils diameter increased, decorin was prominently decreased (Fig. 4D). As GAG chains possessing a negative charge inhibit calcification, the removal of decorin acts to promote calcification. Moreover, decorin also controls the formation and growth of type I collagen fibrils. Although amino propeptides of type I collagen, as well as other kinds of collagen, participate in fibrollogensis in the early stage of self-assembly, decorin is speculated to adjust the diameter of collagen fibrils by binding to d bands of mature fibrils [14,15]. Proteoglycan-degrading metalloproteinases secreted by osteoblasts may be involved in this process.
3. Conclusion remarks The structural and the localizational changes of various organic substances in bone matrix are deeply involved in the bone calcification. Those changes are probably regulated by osteoblasts. The remote controls of bone matrix organics by osteoblasts may be essential mechanisms in bone calcification. Further studies are needed to elucidate the remote factors from osteoblasts or the transcriptional regulation of those factors. Conflict of interest No potential conflicts of interest are disclosed.
Acknowledgments I would like to express my sincere thanks to emeritus Prof. Hidehiro Ozawa, Niigata University Graduate School of Medical and Dental Sciences, Prof. Sadakazu Ejiri, Asahi University, School of Dentistry, and Prof. Norio Amizuka, Graduate School of Dental Medicine, Hokkaido University, for kind instruction and invaluable advices. I also thank Mr. Tomoaki Sakamoto for his useful support. References [1] Hoshi K, Ejiri S, Ozawa H. Localizational alterations of calcium, phosphorus, and calcification-related organics such as proteoglycans and alkaline phosphatase during bone calcification. J Bone Miner Res 2001;16(2):289–98. [2] Neuman, FN, Neuman, NW: Mechanisms of calcification. In: The chemical dynamics of bone mineral. Chicago, IL, USA: The University of Chicago Press; 1958. p. 169–87. [3] Bernard GW, Pease DC. An electron microscopic study of initial intramembraneous osteogenesis. Am J Anat 1969;125:271–90. [4] Anderson HC. Molecular biology of matrix vesicles. Clin Orthop 1995;314: 266–80.
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[5] Hoshi K, Amizuka N, Kurokawa T, Ozawa H. Ultrastructure and immunolocalization of transforming growth factor-beta in chondrification of murine ligamentous fibroblasts and endochondral calcification induced by recombinant human bone morphogenetic protein-2. Acta Histochem Cytochem 1997;30:371–9. [6] Matsuzawa T, Anderson HC. Phosphatases of epiphyseal cartilage studied by electron microscopic cytochemical methods. J Histochem Cytochem 1971;19: 801–8. [7] Hoshi K, Ejiri S, Ozawa H. Organic components of crystal sheaths in bones. J Electron Microsc (Tokyo) 2001;50(1):33–40. [8] Oldberg A, Franzen A, Heinegard D. Cloning and sequence analysis of rat bone sialoprotein (osteopontin) cDNA reveals an Arg–Gly–Asp cell-binding sequence. Proc Natl Acad Sci USA 1986;83:8819–23. [9] Young MF, Kerr JM, Ibaraki K, Heegaard AM, Robey PG. Structure, expression, and regulation of the major noncollagenous matrix proteins of bone. Clin Orthop Rel Res 1992;281:275–94.
[10] Young MF, Ibaraki K, Kerr JM, Lyu MS, Kozak CA. Murine bone sialoprotein (BSP): cDNA cloning, mRNA expression, and genetic mapping. Mamm Genome 1994;5:108–11. [11] Chen CC, Boskey AL. Mechanisms of proteoglycan inhibition of hydroxyapatite growth. Calcif Tissue Inter 1985;37:395–400. [12] Katz EP. The kinetics of mineralization in vitro. I. The nucleation properties of 640-angstrom collagen at 25 degrees. Biochim Biophys Acta 1969;194: 121–9. [13] Hoshi K, Kemmotsu S, Takeuchi Y, Amizuka N, Ozawa H. The primary calcification in bones follows removal of decorin and fusion of collagen fibrils. J Bone Miner Res 1999;14:273–80. [14] Birk DE, Nurminskaya MV, Zycband EI. Collagen fibrillogenesis in situ: fibril segments undergo post-depositional modifications resulting in linear and lateral growth during matrix development. Dev Dyn 1995;202:229–43. [15] Scott JE, Orford CR. Dermatan sulphate-rich proteoglycan associates with rat tail-tendon collagen at the d band in the gap region. Biochem J 1981;197:213–6.
Contents lists available at SciVerse ScienceDirect
Journal of Oral Biosciences journal homepage: www.elsevier.com/locate/job
Review
Fine structure of bone matrix calcification Kazuto Hoshi n Departments of Cartilage & Bone Regeneration (Fujisoft), Graduate School of Medicine, The University of Tokyo, Hongo 7-3-1, Bunkyo-ku, Tokyo 113-0033, Japan
a r t i c l e i n f o
abstract
Article history: Received 16 September 2011 Received in revised form 26 October 2011 Accepted 26 October 2011 Available online 24 February 2012
Various functions of bones are derived from the characteristics that they calcify. In bone calcification, hydroxyapatite is crystallized on the type I collagen-based organic matrices. The organic components of those bone matrices are commonly shared with other fibrous tissues. As extracellular fluid supersaturates hydroxyapatite, the reasons why bones are specifically calcified remains enigmatic. With the bone matrix calcification, the structural and the spatial changes of those organic substances seem to be closely associated, and participate in the complicated regulation of hydroxyapatite multiplication. The present paper overviews the morphological findings of bone calcification, and discusses the mineral and organic environments in bone matrix calcification. & 2012 Published by Elsevier B.V. on behalf of Japanese Association for Oral Biology.
Keywords: Bone matrix Calcification Collagen Calcified nodule Osteoblast
Contents 1. 2.
3.
Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 19 Contents . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 20 2.1. Formation of calcified nodules . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 20 2.2. Crystallization in the calcified nodules . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 23 2.3. Expansion of calcified nodules. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 23 Conclusion remarks . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 23 Conflict of interest . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 23 Acknowledgments . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 23 References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 23
1. Introduction Bone is a typical example of hard tissue. The bones work to shape, support, and protect body structures. They also aid body movement, house bone marrow, and store various minerals essential for the preservation of life. All these functions are derived from the characteristics that the bones calcify. The hardness of bones is derived from the specific composition. Compared with in soft tissues, large part of water is replaced by inorganic materials in bones. Calcium (Ca) and phosphorus (P) are major components of the inorganic compositions of human bones. The Ca/P ratio indicates high value of 2.33. It suggests that crystals in bones almost consist in hydroxyapatite (HA). Bones contain not only inorganic materials, but also organic substances, including collagen, proteoglycan, and other non-collagenous matrix proteins [1]. Those components are not unique for bones,
n
Tel./fax: þ81 3 5800 9891. E-mail address: [email protected]
but are commonly shared with other fibrous tissues including skin, tendon, or ligaments. It is well known that extracellular fluid supersaturates for forming HA [2]. As the entire body is filled with the fluid containing high concentration of calcium and phosphorus, it is always suffering from pathological calcification. However, fibrous tissues other than bones keep the uncalcified situation in fact. The reasons why only bones are calcified remains enigmatic. The previous findings from morphological view demonstrated that matrix vesicles start the primary calcification in bones [3]. Osteoblasts bud the 30- to 100-nm-wide matrix vesicles, which are surrounded by a lipid bilayer. Within the matrix vesicles, crystallized HA appears and grows, showing the primary calcification (Fig. 1, top). As those matrix vesicles were found, not only in the bones, but also in cartilage, dentin, or other hard tissues [4,5], the HA crystallization in the matrix vesicles is regarded as the pivotal event for biological calcification. Otherwise, HA crystals subsequently multiply, forming calcified nodules that expand, fuse, and then calcify expansive matrix
1349-0079/$ - see front matter & 2012 Published by Elsevier B.V. on behalf of Japanese Association for Oral Biology. doi:10.1016/j.job.2012.01.004
20
K. Hoshi / Journal of Oral Biosciences 54 (2012) 19–24
2. Contents 2.1. Formation of calcified nodules
Fig. 1. In the new bone, round osteoblasts and immature calcifying matrix are present. In immature bone matrix, calcification advanced as the distance from osteoblasts increased. Within membrane-bound matrix vesicles (MV), crystallization occurs (top, arrowheads). Subsequently, calcified nodules (CN) formed, followed by focal calcification along collagen fibrils (Co), which are referred to as collagen calcification (top, arrows). Finally, calcified matrix was expanded (bottom). The images were rearranged from Ref. [5].
(Fig. 1, bottom). The expansion of calcification in matrix is another clue to solve the molecular mechanisms of bone calcification. With this process, the structural and the spatial changes of various organic substances in bone matrix seem to be closely associated, and participate in the complicated regulation of HA multiplication. The present paper overviews the morphological findings of bone calcification, and especially focuses on the interaction between matrix molecules and bone minerals in the stage of expansive calcification.
The immature bone matrix, uncalcified osteoid, consists of many organic substances, including proteoglycans, non-collagenous matrix proteins, phospholipids, and alkaline phosphatase (ALP). Those substances can interact with calcium or phosphate, both of which are components of HA. However, those organics can be detected in both bone and soft tissue. Only the presence of those substances in bones cannot explain the mechanisms of bone matrix calcification. More important issue seems their structural and localizational alterations triggered by biological action of osteoblasts. Those organics interacting with minerals were reported to change their structures and localizations, during the transition from the uncalcified to the calcified phase, which affected the mineral microenvironment and initiate calcification [1]. Energyfiltering transmission electron microscopy (EFTEM) is useful to observe the morphological changes of both molecular structures and mineral localizations. Conventional transmission electron microscopy (TEM) enables us to visualize images through the contrast produced by elastic electron scattering. Electrons that pass through the elements include inelastically scattered types, which lose a measurable amount of energy, after their collision with inner-shell electrons, identified as being the principal causes of the aberration typically encountered in this method. EFTEM, on the other hand, can visualize the distribution of those inelastically scattered electrons that are marked by the loss of varying amounts of energy. By setting energy loss at 0 eV, most of the inelastically scattered electrons are eliminated, thus revealing images of a higher resolution than any obtainable via conventional TEM (Fig. 2, top left). In addition, certain elements are mapped at that particular level, when the electrons exhibiting energy loss typical of the elements are localized. Because the spatial resolution of the conventional electron probe X-ray microanalysis depends on the spot size of the electron beam, it usually is confined to a range between a few micrometers and some hundreds of nanometers. However, as resolution of EFTEM during elemental analysis corresponds to that of conventional TEM, mapping of Ca or P at the molecule level becomes possible. In order to analyze bone matrix calcification, alterations of the mineral microenvironment according to the ultrastructural and localizational changes in organic substances during bone calcification were visualized by the application of EFTEM. When observing the calcifying bone in the intramenbraneous ossification, Ca was localized in certain osteoid and throughout areas of calcification. Similarly, P was localized in some osteoid and all calcified areas. However, their localization patterns in the osteoid were not the same. Under higher magnification, a calcified nodule, collagen fibrils, or proteoglycan meshworks among them were clearly observed by EFTEM (Fig. 2, top), and Ca and P also colocalized in calcified nodules (Fig. 2, bottom). However, the Ca of osteoid tended to localize at proteoglycan meshwork among collagen fibrils (Fig. 2, bottom left), whereas P often maps to collagen fibril structures (Fig. 2, bottom right). Ca and P were preserved in different areas of uncalcified matrix, while both colocalize after the formation of calcified areas. In order to discuss the mineral localization in and around the calcified nodules, the organics interacting with minerals were localized at TEM level [1]. Progeoglycan, possessing high affinity with Ca, such as decorin and chondroitin sulfate, are immunohistochemically found along the osteoid at light microscopic level, while Cuprolinic blue stain can enhance proteoglycans in TEM images. CB-based TEM observation highlighted the fact that positively stained structures tended to be smaller the further they
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0eV
Co
CN
Co Co
Fig. 2. Ca and P mapping in the area around a calcified nodule (bar 100 nm). The 0-eV loss (top, left), 250-eV loss (top, right), Ca localizations (bottom, left), and P localizations (bottom, right). Asterisks represent the same area of each image. Ca (blue) and P (green) also colocalized in a calcified nodule (CN). However, in uncalcified areas, Ca tends to localize at proteoglycan sites, while P often maps to collagen fibril (Co) structures. The images were rearranged from Ref. [1]. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)
undecalcified
UA/Pb staining
Fig. 3. (A) In undecalcified ultrathin sections (left), the crustal like structure are visible. Those crystals showed a hexagonal lattice under the high resolution observation (inset). In the staining with uranyl acetate and lead citrate (right), crystal sheath became visible (arrowheads). (B) In the calcified nodules, both BSP (10 nm) and osteopontin (20 nm) were densely localized (left). Under a higher magnification (right), both were arranged linearly (arrows, top) and located in the electron dense line (bottom). The images were rearranged from Ref. [7].
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were situated from osteoblasts. Large branching or fibrous CB-positive structures were seen in the osteoid, whereas smaller structures were concentrated in early stage-calcified nodules. When the calcified nodule expanded, the small structures were dispersed along the periphery of the calcified nodules [1].
Otherwise, the localizations of ALP that can boost phosphate anions were observed in osteoblasts and osteoid by both enzyme histochemistry and immunohistochemistry. Enzyme activity was detected by TEM on dorsal surfaces of osteoblasts and some areas of the matrix. Under higher magnification, enzyme activities were
Fig. 4. (A) Calcification progresses, when the distance from osteoblasts (OB) increases. (B) Under higher magnification, collagen fibrils were visible. The collagen fibrils at the diameter of about 50 nm (Co) possessed clear and round contour in cross-section (top). In the parallel with progression of calcification, some collagen fibrils showed the bifurcation, suggesting side-to-side fusion (middle). In the expansive calcified areas, the network of fine filamentous structure disappeared around collagen fibrils (bottom). MV, matrix vesicles; CN, calcified nodules; asterisks, proteoglycans. (C) Immunocytochemistry of decorin. Green areas indicate immunolocalization of decorin. (D) Gold particles indicated decorin localization. As calcification progresses, decorin surrounding collagen fibrils (Co) decreases. Top, osteoid containing matrix vesicles (MV); bottom, calcifying areas including calcified nodules (CN). The images were rearranged from Ref. [13]. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)
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localized not only in matrix vesicles, but also in calcified nodules. ALP immunolocalizations also were observed on the surface of matrix vesicles, as well as in and around calcified nodules [1]. In vivo organic substances, including proteoglycans and collagen fibrils, formed complicated meshwork, in the osteoid. Because extracellular fluid is present in those narrow spaces, liberated ions may be influenced heavily by the inherent electrical charge of organic substances and limited in dynamic behavior. Taking those morphological findings into consideration, the difference in Ca and P localization patterns in the osteoid may limit the local [Ca2][PO4] product, leading to the general inhibition of hydroxyapatite crystallization. In contrast, colocalizations of Ca and P were observed in areas where calcified nodules were expanding. Proteoglycans were thought to play important roles in the accumulation of calcium to those areas undergoing calcification. Cytohistochemical localizations of ALP protein were found not only in matrix vesicles [6], but also in and around the calcified nodules [1]. Because enzyme cytochemical examination of calcified nodules revealed that an enzymatic reaction had occurred, ALP localized in the calcified nodules is thought to possess the activity to boost local concentrations of phosphate anions. Thus, both ALP and structurally altered proteoglycans were localized in the calcified nodules undergoing further calcification. These two substances may colocalize Ca and P to promote bone calcification, although elemental distribution patterns were not the same, in uncalcified areas. 2.2. Crystallization in the calcified nodules In the calcified nodules, the crystal possesses needle- and plate-like shapes and are approximately 80 nm long, 30 nm wide, and 8 nm thick (Fig. 3A). The constancy of the shapes and sizes of the crystal suggests cellular and organics-based control for crystal formation and growth. The consistent formation of crystals may facilitate biological regulation of calcification, because the crystals with the small and identical sizes have advantage for the expansive calcification during development or growth of body, and for the rapid resorption when calcium recruitment is needed for the homeostatic reasons. By the fine observation at the TEM level, organic crystal sheaths are observed in the vicinity of crystal [7] (Fig. 3A, right). Because these structures are closely associated with crystals, they are thought to participate in the crystal formation and growth. Bone sialoprotein (BSP) or osteopontin are regarded as the candidates consisting of the crystal sheath [7] (Fig. 3B). Those proteins are rich in acidic amino acids, such as glutamate or aspartate, and possess high affinity for calcium [8–10]. Those proteins could also interact with calcification crystals. In vitro, BSP promotes hydroxyapatite nucleation, although ostepoitin functions negatively. The latter also inhibits the crystal elongation. The co-operation of those two proteins, in spite of their conflicting nature, would effectively promote or retard crystallization, as well as regulate the growth speed and size of crystals. 2.3. Expansion of calcified nodules After calcified nodules form, they come into contact with adjacent collagen fibrils, and eventually calcify an expansive matrix of collagen. However, inhibitors of HA crystal formation and growth, such as pyrophosphate or proteoglycans, can be abundantly found around the collagen fibrils [11]. Collagen, by itself, is known to inhibit HA crystallization as well [12]. Therefore, as a whole, the environment surrounding collagen fibrils is not conducive to HA crystal formation and growth. In such a
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situation, osteoblasts may remotely control the collagen calcification by secretion of various organic substances. Collagen fibrils are synthesized by the osteoblasts. Immediately after secretion, the diameter of collagen fibrils was demonstrated as a diameter of approximately 50 nm (Fig. 4A,B). However, once calcification begins, the collagen fibrils become fused, revealing complicated contours and widths greater than 400 nm (Fig. 4B). As fused collagen fibrils tended to gather in dense bundles, those took on a mosaic-like appearance, contrasted against non-collagenous area (Fig. 4B). Such collagen fibril fusion is speculated to promote rapid calcification, because partial localization of collagen fibrils provides vacant spaces where HA crystals growth physicochemically [13]. Decorin is regarded to be associated with the molecular mechanisms of this collagen fibril fusion. Decorin is the major proteoglycan in bones. It consists of a single GAG chain (chondroitin sulfate or dermatin sulfate) and about 40 kDa of core protein. Dense localization of decorin was noted around the thin collagen fibrils of uncalcified osteoid (Fig. 4C,D). However, when calcification progressed and collagen fibrils diameter increased, decorin was prominently decreased (Fig. 4D). As GAG chains possessing a negative charge inhibit calcification, the removal of decorin acts to promote calcification. Moreover, decorin also controls the formation and growth of type I collagen fibrils. Although amino propeptides of type I collagen, as well as other kinds of collagen, participate in fibrollogensis in the early stage of self-assembly, decorin is speculated to adjust the diameter of collagen fibrils by binding to d bands of mature fibrils [14,15]. Proteoglycan-degrading metalloproteinases secreted by osteoblasts may be involved in this process.
3. Conclusion remarks The structural and the localizational changes of various organic substances in bone matrix are deeply involved in the bone calcification. Those changes are probably regulated by osteoblasts. The remote controls of bone matrix organics by osteoblasts may be essential mechanisms in bone calcification. Further studies are needed to elucidate the remote factors from osteoblasts or the transcriptional regulation of those factors. Conflict of interest No potential conflicts of interest are disclosed.
Acknowledgments I would like to express my sincere thanks to emeritus Prof. Hidehiro Ozawa, Niigata University Graduate School of Medical and Dental Sciences, Prof. Sadakazu Ejiri, Asahi University, School of Dentistry, and Prof. Norio Amizuka, Graduate School of Dental Medicine, Hokkaido University, for kind instruction and invaluable advices. I also thank Mr. Tomoaki Sakamoto for his useful support. References [1] Hoshi K, Ejiri S, Ozawa H. Localizational alterations of calcium, phosphorus, and calcification-related organics such as proteoglycans and alkaline phosphatase during bone calcification. J Bone Miner Res 2001;16(2):289–98. [2] Neuman, FN, Neuman, NW: Mechanisms of calcification. In: The chemical dynamics of bone mineral. Chicago, IL, USA: The University of Chicago Press; 1958. p. 169–87. [3] Bernard GW, Pease DC. An electron microscopic study of initial intramembraneous osteogenesis. Am J Anat 1969;125:271–90. [4] Anderson HC. Molecular biology of matrix vesicles. Clin Orthop 1995;314: 266–80.
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