- Email: [email protected]
Ultrastructural Assessment of Mineral Crystallization and Collagen Mineralization in Bone
J. Oral Biosci. 52 （2）：94−99, 2010
REVIEW（For the Bright Future of Calcified Tissue Research）
Ultrastructural Assessment of Mineral Crystallization and Collagen Mineralization in Bone Minqi Li§, Tomoka Hasegawa, Hideo Masuki, Zhusheng Liu, Ying Guo, Reiko Suzuki, Tsuneyuki Yamamoto, Paulo HL Freitas and Norio Amizuka Department of Developmental Biology of Hard Tissue, Graduate School of Dental Medicine, Hokkaido University Kita−13, Nishi−7, Kita−ku, Sapporo, Japan 〔Received on February 1, 2010, Accepted on April 8, 2010〕 Key words：bone mineralization／matrix vesicle／collagen microfibrils／magnesium／mineralized nodule Abstract：Mineralization of bone matrix starts with the formation of matrix vesicles secreted by osteoblasts, which subsequently grow into mineralized nodules. These nodules are globular structures formed by mineral crystals assembled in a radial fashion, and appear to be the final inducers of collagen mineralization. Despite its apparent simplicity, the course of bone mineralization is tightly controlled by many factors in the microenvironment. In this review, we will present our recent experiments on the general processes of bone mineralization. First, we will discuss the ultrastructural alteration of mineral crystals in the context of magnesium（Mg）insufficiency, which causes an elevation of calcium（Ca）concentrations in bone and of the pure form of hydroxyapatite. Such elevated Ca concentrations also induced premature collagen mineralization without the mediation of mineralized nodules. This could mean that the availability of elements, such as Mg, in the bone microenvironment is important for the in vivo generation of the pure form of hydroxyapatite. Next, we will examine collagen mineralization in the context of ascorbic acid insufficiency. Ascorbic acid is essential for collagen formation and, when it is not available, the typical, stout collagen microfibrils with striations cannot be identified. In their place, filamentous fibrils of collagen without striations, i. e., immature collagen microfibrils, are observed. In the osteoid, however, many mineralized nodules feature some sort of“mineral extensions”towards the immature microfibrils. It seems that collagen microfibrils, even if immature, serve as a scaffold for the mineralization of collagen fibrils. These findings reaffirm that a sophisticated set of regulatory steps is necessary for proper mineralization in bone.
Introduction Bone mineralization is believed to start with the formation of matrix vesicles, small extracellular vesicles secreted by osteoblasts into osteoid. Matrix vesicles are surrounded by plasma membranes of approximately 40―200 nm in diameter1,2）, and provide a con§
Corresponding author E−mail：[email protected]
ductive microenvironment for the nucleation and subsequent growth of calcium phosphates. Hydroxyapatite crystals subsequently expand out of the matrix vesicles in order to form mineralized nodules, globular structures formed by ribbon−like （needle−like） mineral crystals assembled in a radial fashion. Even though the plasma membrane of the matrix vesicles is torn during the process of crystal growth, mineralized nodules might not lose all membrane−associated enzymes, such as alkaline phosphatase（ALP）3）, membrane Ca2＋ transport ATPase4）and others. Therefore,
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calcium and phosphate ions might accumulate onto the mineralized nodules and foment their growth. This growth of mineralized nodules does not seem to be merely a function of the chemical accumulation of calcium and phosphate ions, and might also be regulated by other organic components. The organic sheath surrounding hydroxyapatite crystals from mineralized nodules is called a“crystal ghost”5）, which contains many organic substances that may be involved in controlling bone mineralization6,7）. Hunter reported that non−collagenous proteins, such as osteocalcin and osteopontin, inhibit the nucleation and growth of mineral crystals, respectively8）. Although calcium and phosphate ions are the main constituents of hydroxyapatite, other ions, such as magnesium, sodium, fluorine and chlorine, are also present in the bone matrix and may help to regulate crystal size and growth. Mineralization of the collagen fibrils, which conprise more than 90％ organic materials in bone matrix, is an important step in bone matrix mineralization. Collagen microfibrils observable under transmission electron microscopy （TEM） are conformed as a superhelix, also referred to as tropocollagen, the smallest unit of a collagen microfibril. Tropocollagen fibrils parallel each other, forming characteristic striations within certain intervals. Hodge has demonstrated the presence of a ‘hole zone’ between neighboring tropocollagens, and suggested that collagen mineralization would start in this zone9）. It is generally believed that small proteoglycans, such as decorin and biglycan, are present in the hole zone, acting on the inhibition of mineral nucleation until collagen microfibrils are ready to undergo mineral；however, how and when proteoglycans in ization10,11） hole zones are eliminated in the course of collagen mineralization is still an ongoing discussion. In this review, we will present our recent experiments on bone mineralization, namely, 1）ultrastructural alteration of mineral crystals in the context of magnesium insufficiency, and 2）collagen mineralization in the context of ascorbic acid insufficiency.
Ultrastructural Alteration of Mineral Crystals in the Context of Magnesium Insufficiency Mineral crystals in bone consist mainly of calcium phosphates, such as hydroxyapatite, which often show substitution of the calcium（Ca）ion by magnesium ion （Mg2＋）, sodium ion（Na−）and strontium ion（Sr2＋）, among other cations. Kobayashi and his group have found that Mg−insufficient rats formed bones that, despite high bone mineral density（BMD）values, are fragile under mechanical loading （2004）12）. This means that Mg−insufficient bones have lower bone strength, implying lower bone quality. Bone quality is presently a focal term related to osteoporosis prevention and treatment, and bone regeneration. The concept established at the NIH consensus meeting suggests that bone strength depends on many factors, including mineralization, architecture, bone turnover, and the properties of organic proteins in bone13）. In order to verify the function of Mg for mineral crystallization in bone, we employed 4−week−old Wistar male rats fed with Mg−insufficient diet（low Mg group：0.006％ Mg, 0.5％ Ca, 0.66％ Pi）relative to the normal diet（control group：0.09％ Mg, 0.5％ Ca, 0.66％ Pi） for 4 weeks14）. Ultrastructural examinations by TEM, electron probe microanalysis（EPMA）, and X−ray diffraction were conducted on the femora and tibiae of the rats. When we observed mineralized bone covered by mature osteoblasts under TEM, mineral crystals in Mg−insufficient bones seemed larger than those found in controls. EPMA area analysis demonstrated that Mg concentration was markedly decreased in the metaphyses and diaphyses of the low Mg group, while the Ca concentration was dynamically elevated. Phosphorus concentration did not change between groups. Employing X−ray diffraction analysis, we verified that peak patterns from control samples were different from the synthetic（pure form）hydroxyapatite, indicating the variety of mineral crystals in in vivo bone. Low Mg samples, on the other hand, revealed peak patterns resembling those of synthetic hydroxyapatite. These findings indicate that Mg insufficiency appears to elevate Ca concentrations, which results in an increased prevalence of the
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Fig. 1 Scheme of abnormal mineralization in the microenvironment of low magnesium Surrounding the hydroxyapatite molecule there is a hydrogen layer that maintains a state of dynamic equilibrium among the many mineral elements present in the environment. The low magnesium（Mg）diet reduces the absolute number of Mg cations in the hydrogen layer, which results in higher calcium（Ca）concentration, according to Aoba et al23）. Such replacement of Mg by Ca increases the proportion of the non−biological pure form of hydroxyapatite and of larger mineral crystals. Furthermore, collagen microfibrils undergo direct mineralization as Mg concentration decreases.
pure form of hydroxyapatite crystals. In the osteoid of the controls, mineralized nodules provoke appositional mineralization of the surrounding collagen microfibrils. Alternatively, the collagen microfibrils displayed direct mineralization despite the absence of mineralized nodules in Mg−insufficient bones. This abnormal mineralization led to an early collapse of the microfibrils, suggesting that Mg insufficiency increases the content of pure hydroxyapatite and causes the premature collagen mineralization that is responsible for the weaker mechanical properties 1）. of Mg−insufficient bones（Fig. Previous works have demonstrated that Mg is a disruptor of crystal growth15―17）, which seems consistent with our findings of larger mineral crystals in the low Mg group. Mg has been also reported to act as an inhibitor of apatite nucleation15―17）, and we showed that Ca concentration was elevated in the low Mg group. It seems reasonable that, without the antago-
nistic presence of Mg ions, the increased Ca content promotes binding with the abundantly pre−existing phosphates in bone and triggers a faster process of collagen mineralization that is not mediated by mineralized nodules. Based on our findings, we postulate that marginal elements, such as Mg, play an important role in the control and refinement of mineral crystallization in bone. Although more research is needed to completely explain why bones that undergo mineralization mediated by mineralized nodules have better mechanical properties than those that mineralize differently, a general scheme can be proposed. Mg insufficiency seems to increase the content of the pure form of hydroxyapatite, and may also promote crystal growth. Such larger crystals would then induce faster, disorganized mineralization of immature collagen fibrils, which are more susceptible to mechanical failure. These precocious and faulty processes seem to be
97
Fig. 2 Assumed designs of bone mineralization mediating matrix vesicle mineralization and collagen mineralization Bone mineralization begins with the secretion of matrix vesicles by osteoblasts. The matrix vesicle provides a suitable microenvironment for the nucleation and growth of calcium phosphates, which will lead to the formation of mineralized nodules. Marginal elements, such as Mg, appear to be involved in generating in vivo forms of hydroxyapatite in bone by regulating the structure of mineral crystals. Although non−collagenous proteins may regulate the growth of mineral crystals in the mineralized nodules, these are usually found adjacent to collagen microfibrils. Collagen fibrils appear to be mineralized by the assumed mechanism of the hole zone theory or another process through which immature collagen microfibrils serve as a scaffold for mineralization.
responsible for the weaker mechanical properties verified in Mg−insufficient bones. Collagen Mineralization in Ascorbic Acid−insufficient Rats Spatial contact between mineralized nodules and collagen microfibrils might be the very event that initiates collagen mineralization. Interestingly, the area of collagen microfibrils associated with mineralized nodules sometimes swells and is devoid of the characteristic striations. As Ozawa and colleagues suggested, this swelling may implicate focal degradation or digestion of collagen microfibrils and surrounding organic materials, which is mediated by biological
events, i. e., mineralized nodule−mediated mineraliza18） tion . According to the“hole zone theory”, the hole zone between neighboring tropocollagens is the locus of crystal mineralization. In a physiological state, however, typeⅠ collagen microfibrils are exposed to calcium／phosphate supersaturated tissue fluid even in non−mineralizing tissues, such as tendons and ligaments. The explanation for this flaw in the theory is that small proteoglycans occupy the‘hole zone’and inhibit the nucleation of minerals until the collagen is ready to undergo mineralization10）. This hypothesis was strengthened by Fisher et al., who demonstrated that small proteoglycans, including decorin and biglycan, could attach to collagen fibrils11）. During collagen synthesis, ascorbic acid is necessary for the genera-
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M. Li, et al：Ultrastructural Assessment on Bone Mineralization
tion of hydroxyproline and hydroxylysine19―21）. Disruption of hydroxylysine and hydroxyproline may disturb the formation of striated collagen microfibrils. In order to verify these assumptions, we examined 13− week−old ODS／ShiJcl−od／od male rats, which are unable to synthesize ascorbic acid, and were given either an ascorbic acid−insufficient diet（ascorbic acid− insufficient group：0.3 mg／kg）or a normal diet（control group：200 mg／kg） for 4 weeks22）. Ultrastructural and histochemical examinations were conducted on the femora and tibiae of these rats. In the ascorbic acid−insufficient group, collagen microfibrils were fine filamentous structures without the typical striations, similar to immature collagen microfibrils. The control group displayed stout collagen microfibrils with the typical striations. Matrix vesicles and mineral nodules were present in the osteoid of both groups, which indicates that ascorbic acid insufficiency does not affect the formation of either matrix vesicles or mineralized nodules. At higher magnification, needle−like fine mineral crystals extended from mineralized nodules onto the filamentous, striation−free fibrils of collagen. Based on these findings, we postulate that striation−free fibrils might serve as a scaffold for mineralization（Fig. 2）. It is still premature to draw any definite conclusions, however, and defining the precise mechanism through which collagen mineralizes will require a substantial amount of additional work. Concluding Remarks Generally speaking, bone mineralization starts with the osteoblastic release of matrix vesicles, which provide a suitable microenvironment for the nucleation and subsequent growth of calcium phosphates that will lead to the formation of mineralized nodules. Marginal elements, such as Mg, appear to be involved in generating in vivo forms of hydroxyapatite in bone by regulating the structure of mineral crystals. In the absence of Mg, there were elevations of Ca concentration, of the synthetic form of hydroxyapatite, and of premature collagen mineralization, accompanied by early fibril collapse. In the context of ascorbic acid insufficiency, immature filamentous fibrils of collagen underwent mineralization mediated by mineralized
nodules, strongly suggesting their role as a scaffold for mineralization.
References 1）Anderson, H. C.：Electron microscopic studies of induced cartilage development and calcification. J. Cell Biol. 35：81―101, 1967. 2）Bonucci, E.：Fine structure of early cartilage calcification. J. Ultrastruct. Res. 20：33―50, 1967. 3）Oda, K., Amaya, Y., Fukushi−Irie, M., Kinameri, Y., Ohsuye, K., Kubota, I., Fujimura, S. and Kobayashi, J.：A general method for rapid purification of soluble versions of glycosylphosphatidylinositol−anchored proteins expressed in insect cells：An application for human tissue nonspecific alkaline phosphatase. J. Biochem. 126：694―699, 1999. 4）Nakano, Y., Beertsen, W., van den Bos, T., Kawamoto, T., Oda, K. and Takano, Y.：Site−specific localization of two distinct phosphatases along the osteoblast plasma membrane：tissue non−specific alkaline phosphatase and plasma membrane calcium ATPase. Bone 35：1077―1085, 2004. 5）Bianco, P., Hayashi, Y., Silvestrini, G. Termine, J. D. and Bonucci, E.：Osteonectin and Gla−protein in calf bone：ultrastructural immunohistochemical localization using the Protein A−gold method. Calcif. Tissue Int. 37：684―686, 1985. 6）Mark, M. P., Butler, W. T., Prince, C. W., Finkelman, R. D. and Ruch, J. V.：Development expression of 4− kDa bone phosphoprotein（osteopontin）and bone gamma−carboxyglutamic acid （Gla）−containing protein （osteocalcin）in calcifying tissues of rat. Differentiation 37：123―136, 1988. 7）Romberg, R. W., Werness, P. G., Riggs, B. L. and Mann, K. G.：Inhibition of hydroxyapatite crystal growth by bone−specific and other calcium−binding proteins. Biochemistry 25：1176―1180, 1986. 8）Hunter, G. K., Huaschka, P. V., Poole, A. R., Rosenberg, L. C. and Goldberg, H. A.：Nucleation and inhibition of hydroxyapatite formation by mineralized tissue proteins. Biochem. J. 317：59―64, 1996. 9）Hodge, A. J.：Molecular models illustrating the possible distributions of ‘hole’ in simple systematically staggered arrays of typeⅠ collagen molecules in native−type fibrils. Connect. Tissue Res. 21：137― 147, 1989. 10）Glimcher, M. J. and Krane, S. M.：The organization and structure of bone, and the mechanism of
99 calcification. In：A treatise on collagen, biology of collagen, ⅡB, pp.68―251, Academic press, New York, 1976. 11）Fisher, L. W., Whitson, S. W., Avioli, L. V. and Termine, J. D.：Matrix sialoprotein of developing bone. J. Biol. Chem. 258：12723―12727, 1983. 12）Kobayashi, M., Hara, K. and Akiyama, Y.：Effects of Vitamin K2（menatetrenone）and alendronate on bone mineral density and bone strength in rats fed a low− magnesium diet. Bone 35：1136―1143, 2004. 13）NIH Consensus Development Panel on Osteoporosis Prevention, Diagnosis and Therapy：Osteoporosis prevention, diagnosis, and therapy. JAMA 285：785― 795, 2001. 14）Amizuka, N., Li, M., Kobayashi, M., Hara, K., Akahane, S., Takeuchi, K., Freitas, P. H., Ozawa, H., Maeda, T. and Akiyama, Y.：Vitamin K2, a gamma−carboxylating factor of gla−proteins, normalizes the bone crystal nucleation impaired by Mg−insufficiency. Histol. Histopathol. 23：1353―1366, 2008. 15）Blumenthal, N. C., Betts, F. and Posner, A. S.：Stabilization of amorphous calcium phosphate by Mg and ATP. Calcif. Tissue Res. 23：245―250, 1977. 16）Bigi, A., Foresti, E., Gregorini, R., Ripamonti, A., Roveri, N. and Shah, J. S.：The role of magnesium
on the structure of biological apatites. Calcif. Tissue Int. 50：439―444, 1992. 17）Sojka, J. E. and Weaver, C. M.：Magnesium supplementation and osteoporosis. Nutr. Rev. 53：71―74, 1995. 18）Ozawa, H., Hoshi, K. and Amizuka, N.：Current concepts of bone biomineralization. J. Oral Biosci. 50：1 ―14, 2008. 19）Chatterjee, I. B.：Ascorbic acid metabolism. World Rev. Nutr. Diet. 30：69―87, 1978. 20）Wallerstein, R. O. and Wallerstein, R. O. Jr：Scurvy. Semin. Hematol. 13：211―218, 1976. 21）Cardinale, G. J. and Udenfriend, S.：Prolyl hydroxylase. Adv. Enzymol. Relat. Areas Mol. Biol. 41： 245―300, 1974. 22）Li, M., Hara, K., Akiyama, Y. and Amizuka, N.：Histochemical examinations of bone morphological abnormity in ODS／ShiJcl−od／od rats lacked synthesis ability of ascorbic acid. Acta Anat. Nippon. 83：160, 2008. 23）Aoba, T., Moreno, E. C. and Shimoda, S.：Competitive adsorption of magnesium and calcium ions onto synthetic and biological apatites. Calcif. Tissue Int. 51：143―150, 1992.
REVIEW（For the Bright Future of Calcified Tissue Research）
Ultrastructural Assessment of Mineral Crystallization and Collagen Mineralization in Bone Minqi Li§, Tomoka Hasegawa, Hideo Masuki, Zhusheng Liu, Ying Guo, Reiko Suzuki, Tsuneyuki Yamamoto, Paulo HL Freitas and Norio Amizuka Department of Developmental Biology of Hard Tissue, Graduate School of Dental Medicine, Hokkaido University Kita−13, Nishi−7, Kita−ku, Sapporo, Japan 〔Received on February 1, 2010, Accepted on April 8, 2010〕 Key words：bone mineralization／matrix vesicle／collagen microfibrils／magnesium／mineralized nodule Abstract：Mineralization of bone matrix starts with the formation of matrix vesicles secreted by osteoblasts, which subsequently grow into mineralized nodules. These nodules are globular structures formed by mineral crystals assembled in a radial fashion, and appear to be the final inducers of collagen mineralization. Despite its apparent simplicity, the course of bone mineralization is tightly controlled by many factors in the microenvironment. In this review, we will present our recent experiments on the general processes of bone mineralization. First, we will discuss the ultrastructural alteration of mineral crystals in the context of magnesium（Mg）insufficiency, which causes an elevation of calcium（Ca）concentrations in bone and of the pure form of hydroxyapatite. Such elevated Ca concentrations also induced premature collagen mineralization without the mediation of mineralized nodules. This could mean that the availability of elements, such as Mg, in the bone microenvironment is important for the in vivo generation of the pure form of hydroxyapatite. Next, we will examine collagen mineralization in the context of ascorbic acid insufficiency. Ascorbic acid is essential for collagen formation and, when it is not available, the typical, stout collagen microfibrils with striations cannot be identified. In their place, filamentous fibrils of collagen without striations, i. e., immature collagen microfibrils, are observed. In the osteoid, however, many mineralized nodules feature some sort of“mineral extensions”towards the immature microfibrils. It seems that collagen microfibrils, even if immature, serve as a scaffold for the mineralization of collagen fibrils. These findings reaffirm that a sophisticated set of regulatory steps is necessary for proper mineralization in bone.
Introduction Bone mineralization is believed to start with the formation of matrix vesicles, small extracellular vesicles secreted by osteoblasts into osteoid. Matrix vesicles are surrounded by plasma membranes of approximately 40―200 nm in diameter1,2）, and provide a con§
Corresponding author E−mail：[email protected]
ductive microenvironment for the nucleation and subsequent growth of calcium phosphates. Hydroxyapatite crystals subsequently expand out of the matrix vesicles in order to form mineralized nodules, globular structures formed by ribbon−like （needle−like） mineral crystals assembled in a radial fashion. Even though the plasma membrane of the matrix vesicles is torn during the process of crystal growth, mineralized nodules might not lose all membrane−associated enzymes, such as alkaline phosphatase（ALP）3）, membrane Ca2＋ transport ATPase4）and others. Therefore,
95
calcium and phosphate ions might accumulate onto the mineralized nodules and foment their growth. This growth of mineralized nodules does not seem to be merely a function of the chemical accumulation of calcium and phosphate ions, and might also be regulated by other organic components. The organic sheath surrounding hydroxyapatite crystals from mineralized nodules is called a“crystal ghost”5）, which contains many organic substances that may be involved in controlling bone mineralization6,7）. Hunter reported that non−collagenous proteins, such as osteocalcin and osteopontin, inhibit the nucleation and growth of mineral crystals, respectively8）. Although calcium and phosphate ions are the main constituents of hydroxyapatite, other ions, such as magnesium, sodium, fluorine and chlorine, are also present in the bone matrix and may help to regulate crystal size and growth. Mineralization of the collagen fibrils, which conprise more than 90％ organic materials in bone matrix, is an important step in bone matrix mineralization. Collagen microfibrils observable under transmission electron microscopy （TEM） are conformed as a superhelix, also referred to as tropocollagen, the smallest unit of a collagen microfibril. Tropocollagen fibrils parallel each other, forming characteristic striations within certain intervals. Hodge has demonstrated the presence of a ‘hole zone’ between neighboring tropocollagens, and suggested that collagen mineralization would start in this zone9）. It is generally believed that small proteoglycans, such as decorin and biglycan, are present in the hole zone, acting on the inhibition of mineral nucleation until collagen microfibrils are ready to undergo mineral；however, how and when proteoglycans in ization10,11） hole zones are eliminated in the course of collagen mineralization is still an ongoing discussion. In this review, we will present our recent experiments on bone mineralization, namely, 1）ultrastructural alteration of mineral crystals in the context of magnesium insufficiency, and 2）collagen mineralization in the context of ascorbic acid insufficiency.
Ultrastructural Alteration of Mineral Crystals in the Context of Magnesium Insufficiency Mineral crystals in bone consist mainly of calcium phosphates, such as hydroxyapatite, which often show substitution of the calcium（Ca）ion by magnesium ion （Mg2＋）, sodium ion（Na−）and strontium ion（Sr2＋）, among other cations. Kobayashi and his group have found that Mg−insufficient rats formed bones that, despite high bone mineral density（BMD）values, are fragile under mechanical loading （2004）12）. This means that Mg−insufficient bones have lower bone strength, implying lower bone quality. Bone quality is presently a focal term related to osteoporosis prevention and treatment, and bone regeneration. The concept established at the NIH consensus meeting suggests that bone strength depends on many factors, including mineralization, architecture, bone turnover, and the properties of organic proteins in bone13）. In order to verify the function of Mg for mineral crystallization in bone, we employed 4−week−old Wistar male rats fed with Mg−insufficient diet（low Mg group：0.006％ Mg, 0.5％ Ca, 0.66％ Pi）relative to the normal diet（control group：0.09％ Mg, 0.5％ Ca, 0.66％ Pi） for 4 weeks14）. Ultrastructural examinations by TEM, electron probe microanalysis（EPMA）, and X−ray diffraction were conducted on the femora and tibiae of the rats. When we observed mineralized bone covered by mature osteoblasts under TEM, mineral crystals in Mg−insufficient bones seemed larger than those found in controls. EPMA area analysis demonstrated that Mg concentration was markedly decreased in the metaphyses and diaphyses of the low Mg group, while the Ca concentration was dynamically elevated. Phosphorus concentration did not change between groups. Employing X−ray diffraction analysis, we verified that peak patterns from control samples were different from the synthetic（pure form）hydroxyapatite, indicating the variety of mineral crystals in in vivo bone. Low Mg samples, on the other hand, revealed peak patterns resembling those of synthetic hydroxyapatite. These findings indicate that Mg insufficiency appears to elevate Ca concentrations, which results in an increased prevalence of the
96
M. Li, et al：Ultrastructural Assessment on Bone Mineralization
Fig. 1 Scheme of abnormal mineralization in the microenvironment of low magnesium Surrounding the hydroxyapatite molecule there is a hydrogen layer that maintains a state of dynamic equilibrium among the many mineral elements present in the environment. The low magnesium（Mg）diet reduces the absolute number of Mg cations in the hydrogen layer, which results in higher calcium（Ca）concentration, according to Aoba et al23）. Such replacement of Mg by Ca increases the proportion of the non−biological pure form of hydroxyapatite and of larger mineral crystals. Furthermore, collagen microfibrils undergo direct mineralization as Mg concentration decreases.
pure form of hydroxyapatite crystals. In the osteoid of the controls, mineralized nodules provoke appositional mineralization of the surrounding collagen microfibrils. Alternatively, the collagen microfibrils displayed direct mineralization despite the absence of mineralized nodules in Mg−insufficient bones. This abnormal mineralization led to an early collapse of the microfibrils, suggesting that Mg insufficiency increases the content of pure hydroxyapatite and causes the premature collagen mineralization that is responsible for the weaker mechanical properties 1）. of Mg−insufficient bones（Fig. Previous works have demonstrated that Mg is a disruptor of crystal growth15―17）, which seems consistent with our findings of larger mineral crystals in the low Mg group. Mg has been also reported to act as an inhibitor of apatite nucleation15―17）, and we showed that Ca concentration was elevated in the low Mg group. It seems reasonable that, without the antago-
nistic presence of Mg ions, the increased Ca content promotes binding with the abundantly pre−existing phosphates in bone and triggers a faster process of collagen mineralization that is not mediated by mineralized nodules. Based on our findings, we postulate that marginal elements, such as Mg, play an important role in the control and refinement of mineral crystallization in bone. Although more research is needed to completely explain why bones that undergo mineralization mediated by mineralized nodules have better mechanical properties than those that mineralize differently, a general scheme can be proposed. Mg insufficiency seems to increase the content of the pure form of hydroxyapatite, and may also promote crystal growth. Such larger crystals would then induce faster, disorganized mineralization of immature collagen fibrils, which are more susceptible to mechanical failure. These precocious and faulty processes seem to be
97
Fig. 2 Assumed designs of bone mineralization mediating matrix vesicle mineralization and collagen mineralization Bone mineralization begins with the secretion of matrix vesicles by osteoblasts. The matrix vesicle provides a suitable microenvironment for the nucleation and growth of calcium phosphates, which will lead to the formation of mineralized nodules. Marginal elements, such as Mg, appear to be involved in generating in vivo forms of hydroxyapatite in bone by regulating the structure of mineral crystals. Although non−collagenous proteins may regulate the growth of mineral crystals in the mineralized nodules, these are usually found adjacent to collagen microfibrils. Collagen fibrils appear to be mineralized by the assumed mechanism of the hole zone theory or another process through which immature collagen microfibrils serve as a scaffold for mineralization.
responsible for the weaker mechanical properties verified in Mg−insufficient bones. Collagen Mineralization in Ascorbic Acid−insufficient Rats Spatial contact between mineralized nodules and collagen microfibrils might be the very event that initiates collagen mineralization. Interestingly, the area of collagen microfibrils associated with mineralized nodules sometimes swells and is devoid of the characteristic striations. As Ozawa and colleagues suggested, this swelling may implicate focal degradation or digestion of collagen microfibrils and surrounding organic materials, which is mediated by biological
events, i. e., mineralized nodule−mediated mineraliza18） tion . According to the“hole zone theory”, the hole zone between neighboring tropocollagens is the locus of crystal mineralization. In a physiological state, however, typeⅠ collagen microfibrils are exposed to calcium／phosphate supersaturated tissue fluid even in non−mineralizing tissues, such as tendons and ligaments. The explanation for this flaw in the theory is that small proteoglycans occupy the‘hole zone’and inhibit the nucleation of minerals until the collagen is ready to undergo mineralization10）. This hypothesis was strengthened by Fisher et al., who demonstrated that small proteoglycans, including decorin and biglycan, could attach to collagen fibrils11）. During collagen synthesis, ascorbic acid is necessary for the genera-
98
M. Li, et al：Ultrastructural Assessment on Bone Mineralization
tion of hydroxyproline and hydroxylysine19―21）. Disruption of hydroxylysine and hydroxyproline may disturb the formation of striated collagen microfibrils. In order to verify these assumptions, we examined 13− week−old ODS／ShiJcl−od／od male rats, which are unable to synthesize ascorbic acid, and were given either an ascorbic acid−insufficient diet（ascorbic acid− insufficient group：0.3 mg／kg）or a normal diet（control group：200 mg／kg） for 4 weeks22）. Ultrastructural and histochemical examinations were conducted on the femora and tibiae of these rats. In the ascorbic acid−insufficient group, collagen microfibrils were fine filamentous structures without the typical striations, similar to immature collagen microfibrils. The control group displayed stout collagen microfibrils with the typical striations. Matrix vesicles and mineral nodules were present in the osteoid of both groups, which indicates that ascorbic acid insufficiency does not affect the formation of either matrix vesicles or mineralized nodules. At higher magnification, needle−like fine mineral crystals extended from mineralized nodules onto the filamentous, striation−free fibrils of collagen. Based on these findings, we postulate that striation−free fibrils might serve as a scaffold for mineralization（Fig. 2）. It is still premature to draw any definite conclusions, however, and defining the precise mechanism through which collagen mineralizes will require a substantial amount of additional work. Concluding Remarks Generally speaking, bone mineralization starts with the osteoblastic release of matrix vesicles, which provide a suitable microenvironment for the nucleation and subsequent growth of calcium phosphates that will lead to the formation of mineralized nodules. Marginal elements, such as Mg, appear to be involved in generating in vivo forms of hydroxyapatite in bone by regulating the structure of mineral crystals. In the absence of Mg, there were elevations of Ca concentration, of the synthetic form of hydroxyapatite, and of premature collagen mineralization, accompanied by early fibril collapse. In the context of ascorbic acid insufficiency, immature filamentous fibrils of collagen underwent mineralization mediated by mineralized
nodules, strongly suggesting their role as a scaffold for mineralization.
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