Electron microscopical microprobe analysis of freeze dried and unstained mineralized epiphyseal cartilage

Summary Electron micrographs have been taken of unfixed, freeze dried, unstained epiphyseal cartilage. In the mineralized long septa round to elliptic clusters (up to 0.6 m in diameter) consisting mainly of dots and needles could be observed. The clusters were surrounded by microareas with a low contrast consisting mainly of ribbon plate-like crystallites. With the aid of scanning mode (STEM) of a transmission electron microscope, equipped with a Si(Li)detector system, both regions were analyzed for calcium and phosphorus by electronprobe X-ray microanalysis. In ten series of 106 measurements in each region, it could be determined by registration of the CaK and PKX-ray counts that the mineral content in the clusters was in the range of 30–100 % higher than in the light regions. The question of the sequence of the epiphyseal plate mineralization is discussed and whether the dense clusters represent the mineralized matrix vesicles.The authors thank the Deutsche Forschungsgemeinschaft for financial supportDedicated to Professor Dr. G. Pfefferkorn on the occasion of his 65th birthday     

Analysis of the crystal arrangement in collagen fibrils of mineralizing turkey tibia tendon

Summary Mineralized pieces of tendons from the tibio-tarsus of turkeys were (i) shock-frozen, freeze-dried, embedded and cut without staining, or (ii) fixed, embedded and stained after sectioning. Micrographs were taken with an electron microscope on longitudinally cut sections. The center-to-center distances of neighboring apatitic needles within collagen fibrils were measured. For shock-frozen and freeze-dried specimens, the average of these distances is 4.7 nm and the most frequent value 4.2 nm; for fixed and stained specimens, 3.8 nm and 3.6 nm, respectively. Laser diffraction of the electron micrographs showed a dumbbell-like intensity pattern (two diffuse maxima of intensity on the equator, one on each side of the central spot), giving an average distance of about 6 nm. This value represents the upper range of the direct measurements. The measurements demonstrate that the arrangement of the collagen microfibrils is mainly preserved during mineralization. However, using laser diffraction, distances of 9–11 nm were also observed. Such large distances can also be demonstrated by X-ray diffraction on collagen fibrils stained under special conditions. This may indicate that special conditions of apatitic mineralization or staining may alter the arrangement of the microfibrils.The authors thank the Deutsche Forschungsgemeinschaft for financial support     

Elektronenmikroskopie und Laserbeugungs-Untersuchungen zur Charakterisierung der organischen Matrix im Speichelstein und Hartgewebe

Zusammenfassung Elektronenmikroskopische Ultradünnschnittbilder vom Speichelstein zeigten — wie solche vom Dentin, Knochen usw. — nadelbzw. kettenförmige Ca-Phosphatbildungen mit globulären Keimen. Wir bestimmten in der Grundsubstanz a) die Länge der Nadeln als Werte für die Länge der kettenartigen Matrix, b) die Abstände zwischen den punktförmigen Keimen innerhalb der Nadeln als Wiedergabe der Abstände zwischen den aktiven Baugruppeneinheiten, die auf der Matrix die Mineralkeimbildung einleiten, und c) die Seitenabstände zwischen dicht zusammenliegenden, parallelen Ketten als Wiedergabe der Seitenabstände zwischen den Hauptsträngen der Matrix (s. Abb. 2). Die Bestimmungen zu b) und c) wurden sowohl über morphologische Vermessungen wie Laserbeugung an den elektronenmikroskopischen Platten durchgeführt. Die unter a) bezeichneten langen Nadeln (damit die Kettenlänge der Matrixleitstruktur) erreichten Werte über 1000 Å. Die unter b) bezeichneten morphologischen Vermessungen zwischen den Keimen ergaben Werte zwischen 40 und 85 Å und die unter c) bezeichneten Seitenabstände Werte zwischen 37 und 75 Å; die Laserbeugung führte zu Werten zwischen 37 und 54 Å, die also bevorzugt im unteren Bereich der morphologischen Vermessungen zu b) und c) lagen. Anhand dieser Meßdaten wurden erste Überlegungen darüber angestellt, ob die Leitstruktur in der organischen Matrix die Eiweiß- oder Polysaccharidkette im Proteinpolysaccharid bzw. Glykoprotein ist. Entsprechende Untersuchungen wurden auch innerhalb der Bakterien und der Bakterienmembran durchgeführt.

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Electron microscopic and laser diffraction measurement and study of the organic matrix of salivary concretions and hard body tissues

Summary Electron micrographs of ultrathin sections of salivary concretions showed needle-like Ca-phosphate formations with globular nuclei. Similar formations were observed in dentine, bone, and other hard tissues. We determined in the ground substance (a) the lengths of the needles as values for the length of the chain-like matrix backbone, (b) the distances between the dotlike nuclei within the needles as distances between the active sites of the chain-like matrix which induce the phosphate nucleation, and (c) the side distances between the close-packed, parallel needles as values for the distances between the parallel matrix backbones. The investigation of (b) and (c) was carried out by morphological measurement as well as by laser diffraction method on electron micrographs. The long needles of (a) (the length of the matrix backbone) reached values of more than 1,000 Å. Measurement of the distances between the small globular nuclei within the needles (b) gave values of 40 to 85 Å, and of those on the side distances of 37–75 Å. The values for (b) and (c) derived by laser diffraction appeared in the range of 37–54 Å, which was the lower range of the morphological values of (b) and (c). The results were analysed to discuss whether the main chain of the matrix is the proteinor the polysaccharide chain in the protein-polysaccharide or the glycoprotein. Corresponding measurements were carried out in areas within the bacteria and in the bacterial membrane.

Herrn Prof. Dr. L. Reimer und Herrn cand. phys. H. G. Heine, Physikalisches Institut der Universität Münster, danken wir für die Durchführung der Laserbeugungs-Untersuchungen und für wertvolle Diskussionen, Fräulein Gisela Rehsöft für sorgfältige Assistenz. Mit Unterstützung durch die Deutsche Forschungsgemeinschaft.     

Decorin modulates matrix mineralization in vitro

Decorin (DCN), a member of small leucine-rich proteoglycans, is known to modulate collagen fibrillogenesis. In order to investigate the potential roles of DCN in collagen matrix mineralization, several stable osteoblastic cell clones expressing higher (sense-DCN, S-DCN) and lower (antisense-DCN, As-DCN) levels of DCN were generated and the mineralized nodules formed by these clones were characterized. In comparison with control cells, the onset of mineralization by S-DCN clones was significantly delayed; whereas it was markedly accelerated and the number of mineralized nodules was significantly increased in As-DCN clones. The timing of mineralization was inversely correlated with the level of DCN synthesis. In these clones, the patterns of cell proliferation and differentiation appeared unaffected. These results suggest that DCN may act as an inhibitor of collagen matrix mineralization, thus modulating the timing of matrix mineralization.     

Ultrastructure of mineralized nodules formed in rat bone marrow stromal cell culture in vitro.

Rat bone marrow stromal cells were cultured in vitro. At days 14-15 of culture, dense clusters of polygonal cells were formed, and they mineralized 2-3 days later. The cells resembling osteoblasts or young osteocytes were histologically observed to be embedded in mineralized or unmineralized extracellular matrices of the nodules. Next, these mineralized nodules were electron-microscopically examined. The osteoblastic cells associated with the nodules had a well-developed rough endoplasmic reticulum, an evident Golgi apparatus and some mitochondria as their intracellular organellae. Some lysosomes and microfilaments were also visible in the cytoplasms. Moreover, some cells protruded cell processes toward the neighboring cells through the extracellular matrix. The extracellular matrix consisted of numerous collagen fibrils which were striated with 60-70 nm axial periodicity and which was similar to bone tissue collagen. A large number of matrix vesicles were scattered among the collagen fibrils in the unmineralized area of the nodules. In contrast, in the mineralized area, numerous matrix vesicles at different stages of maturation and many calcified spherules were observed. That is the mineralization in this culture system was considered to be initiated in association with the matrix vesicles and to progress along the collagen fibrils. From these findings, it was confirmed by the present study that the mineralized nodules formed in this bone marrow stromal cell culture were ultrastructurally similar to bone and that the mineralization also proceeded by going through the normal calcification process. This culture system is considered to be available to study osteogenic differentiation and calcification mechanisms.     

Electron microscopic microprobe analysis of mineralized collagen fibrils and extracollagenous regions in Turkey leg tendon

Summary Bundles of tibia tendon from 19 week-old turkeys were deep frozen, freeze dried and embedded in styrol methacrylate or Epon. In the distal mineralized region, bundles of unmineralized collagen fibrils as well as mineralized regions consisting of round microcompartments with low contrast surrounded by a mineral sheath with high contrast were found. The inner regions with low contrast corresponded to the mineralized collagen fibrils, while the contrast-rich peripheral zones corresponded to the mineralized collagen-free ground substance. Using electron microscopic microprobe analysis, it was shown that the peripheral mineralized region, consisting mainly of closely packed needles, often contained 100% more mineral substance than the central, mineralized collagen zone, which consisted mainly of plate-like crystallites. Possible reasons for this difference in mineral content are discussed on the molecular level.The authors would like to express their gratitude to the Deutsche Forschungsgemeinschaft for financial support and to Fräulein Christine Dörnen for valuable technical assistance     

Autophagy in osteoblasts is involved in mineralization and bone homeostasis

Bone remodeling is a tightly controlled mechanism in which osteoblasts (OB), the cells responsible for bone formation, osteoclasts (OC), the cells specialized for bone resorption, and osteocytes, the multifunctional mechanosensing cells embedded in the bone matrix, are the main actors. Increased oxidative stress in OB, the cells producing and mineralizing bone matrix, has been associated with osteoporosis development but the role of autophagy in OB has not yet been addressed. This is the goal of the present study. We first show that the autophagic process is induced in OB during mineralization. Then, using knockdown of autophagy-essential genes and OB-specific autophagy-deficient mice, we demonstrate that autophagy deficiency reduces mineralization capacity. Moreover, our data suggest that autophagic vacuoles could be used as vehicles in OB to secrete apatite crystals. In addition, autophagy-deficient OB exhibit increased oxidative stress and secretion of the receptor activator of NFKB1 (TNFSF11/RANKL), favoring generation of OC, the cells specialized in bone resorption. In vivo, we observed a 50% reduction in trabecular bone mass in OB-specific autophagy-deficient mice. Taken together, our results show for the first time that autophagy in OB is involved both in the mineralization process and in bone homeostasis. These findings are of importance for mineralized tissues which extend from corals to vertebrates and uncover new therapeutic targets for calcified tissue-related metabolic pathologies.     

Autophagy in osteoblasts is involved in mineralization and bone homeostasis

Bone remodeling is a tightly controlled mechanism in which osteoblasts (OB), the cells responsible for bone formation, osteoclasts (OC), the cells specialized for bone resorption, and osteocytes, the multifunctional mechanosensing cells embedded in the bone matrix, are the main actors. Increased oxidative stress in OB, the cells producing and mineralizing bone matrix, has been associated with osteoporosis development but the role of autophagy in OB has not yet been addressed. This is the goal of the present study. We first show that the autophagic process is induced in OB during mineralization. Then, using knockdown of autophagy-essential genes and OB-specific autophagy-deficient mice, we demonstrate that autophagy deficiency reduces mineralization capacity. Moreover, our data suggest that autophagic vacuoles could be used as vehicles in OB to secrete apatite crystals. In addition, autophagy-deficient OB exhibit increased oxidative stress and secretion of the receptor activator of NFKB1 (TNFSF11/RANKL), favoring generation of OC, the cells specialized in bone resorption. In vivo, we observed a 50% reduction in trabecular bone mass in OB-specific autophagy-deficient mice. Taken together, our results show for the first time that autophagy in OB is involved both in the mineralization process and in bone homeostasis. These findings are of importance for mineralized tissues which extend from corals to vertebrates and uncover new therapeutic targets for calcified tissue-related metabolic pathologies.     

Dentin matrix protein 1 immobilized on type I collagen fibrils facilitates apatite deposition in vitro

During bone and dentin mineralization, the crystal nucleation and growth processes are considered to be matrix regulated. Osteoblasts and odontoblasts synthesize a polymeric collagenous matrix, which forms a template for apatite initiation and elongation. Coordinated and controlled reaction between type I collagen and bone/dentin-specific noncollagenous proteins are necessary for well defined biogenic crystal formation. However, the process by which collagen surfaces become mineralized is not understood. Dentin matrix protein 1 (DMP1) is an acidic noncollagenous protein expressed during the initial stages of mineralized matrix formation in bone and dentin. Here we show that DMP1 bound specifically to type I collagen, with the binding region located at the N-telopeptide region of type I collagen. Peptide mapping identified two acidic clusters in DMP1 responsible for interacting with type I collagen. The collagen binding property of these domains was further confirmed by site-directed mutagenesis. Transmission electron microscopy analyses have localized DMP1 in the gap region of the collagen fibrils. Fibrillogenesis assays further demonstrated that DMP1 accelerated the assembly of the collagen fibrils in vitro and also increased the diameter of the reconstituted collagen fibrils. In vitro mineralization studies in the presence of calcium and phosphate ions demonstrated apatite deposition only at the collagen-bound DMP1 sites. Thus specific binding of DMP1 and possibly other noncollagenous proteins on the collagen fibril might be a key step in collagen matrix organization and mineralization.     

Matrix mineralization as a trigger for osteocyte maturation.

The morphology of the osteocyte changes during the cell's lifetime. Shortly after becoming buried in the matrix, an osteocyte is plump with a rich rough endoplasmic reticulum and a well-developed Golgi complex. This "immature" osteocyte reduces its number of organelles to become a "mature" osteocyte when it comes to reside deeper in the bone matrix. We hypothesized that mineralization of the surrounding matrix is the trigger for osteocyte maturation. To verify this, we prevented mineralization of newly formed matrix by administration of 1-hydroxyethylidene-1,1-bisphosphonate (HEBP) and then examined the morphological changes in the osteocytes in rats. In the HEBP group, matrix mineralization was disturbed, but matrix formation was not affected. The osteocytes found in the unmineralized matrix were immature. Mature osteocytes were seen in the corresponding mineralized matrix in the control group. The immature osteocytes in the unmineralized matrix failed to show immunoreactivity with anti-sclerostin antibody, whereas mature osteocytes in the mineralized matrix showed immunoreactivity in both control and HEBP groups. These findings suggest that mineralization of the matrix surrounding the osteocyte is the trigger for cytodifferentiation from a plump immature form to a mature osteocyte. The osteocyte appears to start secreting sclerostin only after it matures in the mineralized bone matrix.     

In vitro differentiation and mineralization of cartilaginous nodules from enzymatically released rat nasal cartilage cells

Nasal cartilage cells from 21-day-old rat fetuses were cultured at high density in the presence of ascorbic acid and β-glycerophosphate over a 12-day period. Immediately after plating, the cells exhibited a fibroblastic morphology, lost their chondrocyte phenotype and expressed type I collagen. On day 3, clusters of enlarged polygonal cells were found. These cell clusters synthetised type II collagen and formed an alcian-blue-positive matrix. The following days, a progressive increase in the number of cells positive for type 11 collagen was noted and, on day 8, typical cartilaginous nodules were formed. These nodules increased in size and number, spreading outward, laying down a dense matrix which mineralized. Light and electron microscopy observations of cross-sections of nodules confirmed the cartilaginous nature of this tissue formed in vitro with typical chondrocytes embedded in a hyaline matrix. Furthermore, at the electron microscopic level, matrix vesicles were seen in extracellular matrix associated with the initiation of mineralization. Typical rod-like crystals were present in the intercellular spaces along the collagen fibers. These results indicated that in a specific environment, dedifferentiated chondrocytes were able to redifferentiate and to form nodular structures with morphological ultrastructure of calcified cartilage observed in vivo.     

Long-chain polyphosphate in osteoblast matrix vesicles: Enrichment and inhibition of mineralization

Background

Inorganic polyphosphate (polyP) is a fundamental and ubiquitous molecule in prokaryotes and eukaryotes. PolyP has been found in mammalian tissues with particularly high levels of long-chain polyP in bone and cartilage where critical questions remain as to its localization and function. Here, we investigated polyP presence and function in osteoblast-like SaOS-2 cells and cell-derived matrix vesicles (MVs), the initial sites of bone mineral formation.

In situ Raman spectroscopic studies of the teeth of the chiton Acanthopleura hirtosa

 In situ Raman spectroscopy, in combination with energy dispersive spectroscopy, has been used for the first time to determine the identities and locations, at the micron level, of mineral phases present in single chiton teeth that have been extensively mineralized. At the later stages of development the major lateral teeth of the chiton Acanthopleura hirtosa show characteristic spectroscopic evidence for the presence of lepidocrocite (γ-FeOOH), magnetite (Fe₃O₄), and an apatitic calcium phosphate. Goethite (α-FeOOH) and ferrihydrite (5 Fe₂O₃·9 H₂O), which have been detected previously in teeth at the early stages of mineralization, were not detected in this mature tooth. The spatial distribution of these phases was determined, providing evidence for the presence of a discrete layer of lepidocrocite between the magnetite and apatite regions, illustrating the complexity of the biomineralization process. The technique of laser Raman microscopy is shown to be ideal for the examination of small biomineralized structures in situ, such as chiton teeth. Received: 6 July 1998 / Accepted: 19 August 1998     

Quantitative electron microscopic investigations of mineral nucleation in collagen

Summary It has been previously shown that the distances between the nuclei within the collagen bundles of mineralizing tissues were in good agreement with the repeat distances of the cross-banding pattern of collagen, which supports the assumption that the distances between the mineral deposits reflect to a good approximation the distances between nucleation centres on the collagen macromolecule. However, the lateral separation of the nuclei were significantly higher than the distances between close-packed triple helices.Recently a new model of collagen aggregation has been proposed in which the smallest morphological units are subfibrils (Ø approx. 39 Å) packed in tetragonal array. This led us to measure once again the lateral separation between a) close-packed calcium phosphate needles lying in bundles at (1) the mineralizing front of mantle dentine and (2) at the mineralizing front of rat tail bone, and b) between the uranyl-lead nuclei produced in the staining of rat tail tendon.The mean lateral distances separating these nuclei fell within the range of 39–47 Å, which is a little higher than the distances of 39 Å which separate the microholes between the subfibrils in the tetragonal packing model, which are regarded as the likely sites of nucleation. If, however, it is assumed that the forces generated during mineralization can cause the collagen fibres to swell, then the lateral separation of the nuclei and the distances between the microholes would correspond very closely.We thank the Deutsche Forschungsgemeinschaft for financial support. We thank Prof. Dr. K. Heckmann and Dr. U. Mays, Dept. of Zoology, Münster, for allowing us to use their Siemens-Elmiskop 101 sponsored by Stiftung Volkswagenwerk, and Frau Dr. Weichan, Applikationslabor Siemens, Berlin, for performing the tilting experiments at their Siemens-Elmiskop 102. We thank Fräulein Ute Sporman for valuable technical help.     

Ultrastructural observations on chondroid bone in the teleost fish Hemichromis bimaculatus

This paper presents transmission electron microscopical observations on the chondroid bone (CB) supporting the neurocraniad articulation facet of the upper pharyngeal jaws of juvenile specimens of Hemichromis bimaculatus (an acellular-boned teleost fish). Chondroid bone, a skeletal tissue morphologically intermediate between cartilage and bone, is composed of a dense mineralized collagenous matrix, resembling that of woven-fibred bone, and large chondrocyte-like cells. The latter vary considerably in their morphological features (functional cells, cells containing a large vacuole and degenerating cells). The CB is mineralized except for its upper layer. Mineralization is initiated in matrix vesicles. Clusters of apatite crystals coalesce at the mineralization front. Distally, the tissue grows by incorporation of cells which exhibit the features of osteoblasts, and which derive from less differentiated fibroblast-like cells located in the outermost layer of the tissue. Proximally, the CB is subjected to erosion by multinucleated clastic cells. The deposition of bone against the wall of lacunae which have been opened by clastic resorption may suggest a possible active involvement of the CB cells. Further studies should point out whether this bone substantially contributes to the acellular dermal dentigerous bone located below.     

Ultrastructural studies of initial stages of mineralization of long bones and vertebrae in human fetuses

We studied 27 embryos of 5-12 weeks gestational age where pregnancy was interrupted due to paramedical reasons, in order to find the developmental stages at which matrix vesicles appear in cartilage, and whether they are involved in the mineralization process. Specimens of long bones, lumbar and thoracic vertebral column were prepared for light, transmission and scanning electron microscopic studies. In the cartilaginous models of long bones, matrix vesicles were found amongst maturing and hypertrophic chondrocytes already by the 6th week after fertilization. By that stage, bone rudiments consisted of only cartilage that was not yet mineralized. In the vertebral column matrix, vesicles were found in the vertebral bodies amongst maturing and hypertrophic chondrocytes at the beginning of the 8th week. At that stage, although hypertrophy of chondrocytes was observed, mineralization was still absent. No matrix vesicles were found in the perichondrium, investing mesenchyme and intervertebral discs. Mineralization of cartilage in long bone rudiments started in the form of hydroxyapatite crystals within or around the matrix vesicles at 7 weeks of age and in the vertebral column at 11 weeks. As mineralization progressed, more hydroxyapatite crystals were observed around the matrix vesicles, forming typical calcospherites . Mineralization then progressed in the form described in other animals.     

Degeneration of osteoblasts involved in intramembranous ossification of fetal rat calvaria

Summary Ossification of calvariae from day-21 rat fetuses was reinvestigated by electron microscopy using different fixation techniques (glutaraldehyde/OsO₄, tannic acid, ruthenium red, K-pyroantimonate). An osteoid layer with scattered mineral deposits was found at the mineralization front. Directly beyond this layer, a sheet of one to two layers of necrotic and degenerating osteoblasts was present. Above this sheet, normal and healthy cells were seen, formed by six to eight layers of flattened cells, embedded in a collagenous matrix. The osteoblasts on the less mineralizing opposite side of the calcified cavariae and the osteocytes embedded in the calcified calvariae appeared healthy. Closer inspection of the necrotic zone revealed apatite crystal in vesicles which most probably originated from mitochondria of the degenerated cells. Large K-pyroantimonate deposits were found throughout the osteoid and the necrotic zone, whereas only small granules were scattered in the cytoplasm and at the plasma membrane of the healthy cells directly adjacent to the necrotic zone. A concept of intramembranous mineralization is outlined, according to which osteoblasts store enormous amounts of calcium, which are liberated by physiological cell death in the vicinity of the mineralizing front.     

In vitro differentiation and mineralization of human dental pulp cells induced by dentin extract

Summary In this study, the progenitor cells isolated from the human dental pulp were used to study the effects of ethylenediaminetetraacetic acid-soluble dentin extract (DE) on their differentiation and mineralization to better understand tissue injury and repair in the tooth. Mineralization of the matrix was increasingly evident at 14, 21, and 28 d after treatment with a mineralization supplement (MS) (ascorbic acid [AA], β-glycerophosphate [β-GP]) and MS+DE. Real-time polymerase chain reaction results showed type I collagen upregulation after the addition of MS+DE at 7 d. Alkaline phosphatase was downregulated after the mineralization became obvious at 14 d. Bone sialoprotein was shown to be upregulated in the mineralized cell groups at all time points and dentin sialophosphoprotein after 7 d. Core binding factor a 1 was upregulated by the treatment of MS and DE at 7, 14, and 21 d. These results indicated that the MS of AA, β-GP, and DE synergistically induced cell differentiation of pulp progenitor cells into odontoblast-like cells and induced in vitro mineralization.     

Regulated Production of Mineralization-competent Matrix Vesicles in Hypertrophic Chondrocytes

Matrix vesicles have a critical role in the initiation of mineral deposition in skeletal tissues, but the ways in which they exert this key function remain poorly understood. This issue is made even more intriguing by the fact that matrix vesicles are also present in nonmineralizing tissues. Thus, we tested the novel hypothesis that matrix vesicles produced and released by mineralizing cells are structurally and functionally different from those released by nonmineralizing cells. To test this hypothesis, we made use of cultures of chick embryonic hypertrophic chondrocytes in which mineralization was triggered by treatment with vitamin C and phosphate. Ultrastructural analysis revealed that both control nonmineralizing and vitamin C/phosphatetreated mineralizing chondrocytes produced and released matrix vesicles that exhibited similar round shape, smooth contour, and average size. However, unlike control vesicles, those produced by mineralizing chondrocytes had very strong alkaline phosphatase activity and contained annexin V, a membrane-associated protein known to mediate Ca²⁺ influx into matrix vesicles. Strikingly, these vesicles also formed numerous apatite-like crystals upon incubation with synthetic cartilage lymph, while control vesicles failed to do so. Northern blot and immunohistochemical analyses showed that the production and release of annexin V-rich matrix vesicles by mineralizing chondrocytes were accompanied by a marked increase in annexin V expression and, interestingly, were followed by increased expression of type I collagen. Studies on embryonic cartilages demonstrated a similar sequence of phenotypic changes during the mineralization process in vivo. Thus, chondrocytes located in the hypertrophic zone of chick embryo tibial growth plate were characterized by strong annexin V expression, and those located at the chondro–osseous mineralizing border exhibited expression of both annexin V and type I collagen. These findings reveal that hypertrophic chondrocytes can qualitatively modulate their production of matrix vesicles and only when induced to initiate mineralization, will release mineralization-competent matrix vesicles rich in annexin V and alkaline phosphatase. The occurrence of type I collagen in concert with cartilage matrix calcification suggests that the protein may facilitate crystal growth after rupture of the matrix vesicle membrane; it may also offer a smooth transition from mineralized type II/type X collagen-rich cartilage matrix to type I collagen-rich bone matrix.     

Patterns of mineralization in vitro

Summary Various patterns of mineralization are found in the organism during fetal and postnatal development. Different findings and theories have been published in the literature with regard to the mechanisms of mineralization, many of which are controversely discussed. In the present study the different patterns of mineralization observed in the organoid culture system of fetal rat calvarial cells were investigated by electron microscopy. In organoid culture, calvarial cells grow and differentiate at high density, and deposition of osteoid and mineralization of the matrix occur to a very high extent. Different types of mineralization could be observed more or less simultaneously. It was found that hydroxyapatite crystals were formed at collagen fibrils as well as in the interfibrillar space. Mineralization was frequently seen in necrotic cells and cellular remnants as well as in extra-and intracellular vesicles. Addition of bone or dentin matrices or the artificial hydroxyapatite Interpore 200 to the cells caused an increased mineralization in the vicinity and on the surface of the matrices with and without participation of collagen. On previously formed mineralized nodules, an apposition of mineralizing material appeared due to matrix secretion by osteoblasts. It is concluded that initiation of mineralization occurs-at least in vitro-at every nucleation point under appropriate conditions. These mineralization foci enlarge by further apposition as well as by cellular secretion of a mineralizing matrix. Furthermore, cell necroses may liberate mineralizable vesicles. All these patterns of mineralization are the result of different activities of one cell type.