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.     

Haemopoietic long-term bone marrow cultures from adult mice show osteogenic capacity in vitro on 3–dimensional collagen sponges

Abstract. Adult murine bone marrow cells, cultured under conditions for long-term haemopoietic marrow cultures, produce bone matrix proteins and mineralized tissue in vitro , but only after the adherent stromal cells were loaded on a 3-dimensional collagen sponge. Provided more than 8 × 10⁶ cells are loaded, mineralization as measured by ⁸⁵Sr uptake from the culture medium, occurred in this 3-dimensional configuration (3-D) within 6 days. In contrast if undisrupted marrow fragments (containing more than 10⁷ cells) are placed directly on a collagen sponge, then it requires more than 10 days before significant mineralization can similarly be detected. The 2-dimensional (2-D) long-term marrow culture system allows prior expansion of the stromal cells and some differentiation in an osteogenic direction within the adherent stromal layer. This is suggested by the presence of type I collagen and alkaline phos-phatase positive cells. However, synthesis of osteonectin and a bone specific protein, osteocalcin, as well as calcification are only observed in 3-D cultures. Electron microscopy demonstrated hydroxyapatite mineral on collagen fibres, osteoblast-like cells, fibroblasts, cells which accumulated lipids, and macrophages which were retained on the collagen matrices. Irradiation of confluent long-term bone marrow cultures, prior to their loading on the collagen sponge showed that haemopoietic stem cells are not necessary for the mineralization.     

Demonstration of the capacity of nacre to induce bone formation by human osteoblasts maintained in vitro.

Nacre implanted in vivo in bone is osteogenic suggesting that it may possess factor(s) which stimulate bone formation. The present study was undertaken to test the hypothesis that nacre can induce mineralization by human osteoblasts in vitro. Nacre chips were placed on a layer of first passage human osteoblasts. None of the chemical inducers generally required to obtain bone formation in vitro was added to the cultures. Osteoblasts proliferated and were clearly attracted by nacre chips to which they attached. Induction of mineralization appeared preferentially in bundles of osteoblasts surrounding the nacre chips. Three-dimensional nodules were formed by a dense osteoid matrix with cuboidal osteoblasts at the periphery and osteocytic-like cells in the center. These nodules contained foci with features of mineralized structures and bone-like structures, both radiodense to X-ray. Active osteoblasts (e.m.) with abundant rough endoplasmic reticulum, extrusion of collagen fibrils and budding of vesicles were observed. Matrix vesicles induced mineral deposition. Extracellular collagen fibrils appeared cross-banded and electrodense indicating mineralization. These results demonstrate that a complete sequence of bone formation is reproduced when human osteoblasts are cultured in the presence of nacre. This model provides a new approach to study the steps of osteoblastic differentiation and the mechanisms of induction of mineralization.     

Development and morphofunctional characterization of the osteoblastic phenotype in cell culture in vitro

The objective of this research was to study osteogenic properties of cultured rabbit bone marrow stromal cells, newborn rat cranium bone cells and rat osteocarcoma ROS 17-2/8 cells. For this purpose cytochemical reaction for alkaline phosphatase was performed by the Lowry method, mineral deposition was assessed by staining of the cultures after von Kossa. Cranium bone cells were shown to synthesize alkaline phosphatase (34 +/- 7 nmol/min/10(6) cells), the matrix mineralization being found. Bone marrow stromal cells displayed a lower activity alkaline phosphatase level than did cranium bone cells (4 +/- 0.6 nmol/min/10(6) cells). However, cell cultivation in the presence of dexamethasone in the medium (10(-8) M) induced a higher activity of alkaline phosphatase (9 +/- 1 nmol/min/10(6) cells), mineralization of the extracellular matrix being the case. The highest level of alkaline phosphatase activity was found for ROS 17-2/8 cells (60 +/- 12 nmol/min/10(6) cells) but no matrix mineralization was determined. According to these data, matrix calcification and formation of bone-like nodules are the most important properties of osteoblastic differentiation in vitro.     

Structural characterization of the mandibular condyle in human fetuses: light and electron microscopy studies.

Mandibular condyles from 18- to 20-week-old human fetuses were examined in the light and electron microscope with particular attention to intratissue organization and extracellular matrix. In the human fetus the condyle has been divided into five layers: (1) the most superficial, articular layer, (2) chondroprogenitor cell layer, (3) condroblast cell layer, (4) nonmineralized hypertrophic cell layer, and (5) mineralized hypertrophic cell layer. The articular layer is rich in collagen fibers (mostly of the type I collagen), but the cells seldom divide. By contrast, in the chondroprogenitor cell layer and upper part of the chondroblastic cell layer mitosis gives rise to new cells. The matrix in the latter layer is composed of thick banded 'lucent' fibrils in a loose feltwork of granules representing cartilage proteoglycans. The daughter cells in the progenitor cell layer undergo differentiation which is apparently completed along the lower border of the mineralized hypertrophic cell layer--the ossification front. The matrix in the hypertrophic cell layer reveals distinct matrix vesicles that undergo mineralization and subsequently coalesce to form larger sheets of mineralized extracellular matrix. Mineralized cartilage serves as a backbone for new bone formation as marrow-derived osteoblasts and osteoclasts attach to remnants of mineralized cartilage, which enables the turning on of the remodeling cycles involved in new bone formation. It can be concluded that the process of endochondral ossification as has been reported in lower animals is recapitulated in the human fetus, thus the dynamics associated with condylar morphogenesis is maintained through phylogeny.     

Localization of calcium and phosphorus in early predentin-matrix components by electron spectroscopic imaging (ESI)-analysis in rat molars

Summary The subcellular distribution of the inorganic elements calcium (Ca) and phosphorus (P) was studied in the first-formed dentin matrix during initial mineralization in neonatal rat molars. This most peripheral matrix region is comprised of a proteoglycan-rich ground substance, interwoven by a collagenous network, matrix vesicles, aperiodic fibrils derived from the dental basal lamina, and apical odontoblastic cell processes. All matrix components may possibly serve as templets for mineral deposition during initial calcification of first-formed mantle dentin and predentin. By means of the very sensitive ESI-analysis we studied the subcellular localization of Ca and P and their possible association with distinct organic extracellular matrix components and odontoblasts. Ca-signals were found in the ground substance, at striated collagen fibrils and plasma membranes of odontoblasts in the cuspal early matrix region, but occurred only sparsely in the ground substance of the more distal matrix region where odontoblast processes attach to aperiodic fibrils of the dental basal lamina. Ca was generally absent in matrix vesicles. In contrast, P-signals were found in matrix vesicles, at aperiodic fibrils and at the plasma membranes of odontoblasts. Ca and P co-localized at striated collagen fibrils (type I or II). These results suggest that striated collagen fibrils might serve as primary deposition sites for calcium phosphate during early biological calcification of organic extracellular macromolecules.     

MV-mimicking micelles loaded with PEG-serine-ACP nanoparticles to achieve biomimetic intra/extra fibrillar mineralization of collagen in vitro

Since their discovery, matrix vesicles (MVs) containing minerals have received considerable attention for their role in the mineralization of bone, dentin and calcified cartilage. Additionally, MVs' association with collagen fibrils, which serve as the scaffold for calcification in the organic matrix, has been repeatedly highlighted. The primary purpose of the present study was to establish a MVs–mimicking model (PEG-S-ACP/micelle) in vitro for studying the exact mechanism of MVs-mediated extra/intra fibrillar mineralization of collagen in vivo. In this study, high-concentration serine was used to stabilize the amorphous calcium phosphate (S-ACP), which was subsequently mixed with polyethylene glycol (PEG) to form PEG-S-ACP nanoparticles. The nanoparticles were loaded in the polysorbate 80 micelle through a micelle self-assembly process in an aqueous environment. This MVs–mimicking model is referred to as the PEG-S-ACP/micelle model. By adjusting the pH and surface tension of the PEG-S-ACP/micelle, two forms of minerals (crystalline mineral nodules and ACP nanoparticles) were released to achieve the extrafibrillar and intrafibrillar mineralization, respectively. This in vitro mineralization process reproduced the mineral nodules mediating in vivo extrafibrillar mineralization and provided key insights into a possible mechanism of biomineralization by which in vivo intrafibrillar mineralization could be induced by ACP nanoparticles released from MVs. Also, the PEG-S-ACP/micelle model provides a promising methodology to prepare mineralized collagen scaffolds for repairing bone defects in bone tissue engineering.     

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.     

Immunoelectron microscopic studies of glycosaminoglycans in the metaphyseal bone trabeculae of growing rats

Summary The types and distribution of glycosaminoglycans (GAGs) were studied immunocytochemically in osteoid, mineralized bone matrix, and cartilage matrix of growing rat metaphyseal bone after aldehyde fixation and EDTA demineralization, using four monoclonal antibodies (mAbs 1-B-5, 2-B-6, 3-B-3 and 5-D-4). These mAbs specifically recognize epitopes in non-sulphated chondroitin (C0-S); chondroitin 4-sulphate (C4-S) and dermatan sulphate (DS); chondroitin 6-sulphate (C6-S) and C0-S; and keratan sulphate (KS) respectively. In osteoid, all mAbs except 1-B-5 weakly stained matrix material on and between collagen fibrils, and moderately stained organic material corresponding to bone nodules, which are known sites of mineralization. However, the staining of osteoid abruptly decreased at the mineralization front; weak staining was confined mostly to the organic material of bone nodules in mineralized bone matrix, with very weak or no staining of the rest of the bone matrix. This staining progressively decreased toward the mineralized cartilage matrix and became negative. The mineralized cartilage matrix and lamina limitans reacted strongly with all mAbs except 5-D-4. These results indicate that osteoid contains sulphated proteoglycans containing C4-S and/or DS, C6-S and KS, and subsequent bone matrix mineralization appears to require accumulation of these macromolecules within bone nodules and eventual loss of these substances for complete mineralization, whereas proteoglycans containing C0-S, C4-S and/or DS, and C6-S, still exist in mineralized cartilage matrix and lamina limitants.     

Osteoblast-related gene expression of bone marrow cells during the osteoblastic differentiation induced by type I collagen

Bone marrow contains multipotent cells that differentiate into fibroblasts, adipocytes, and osteoblasts. Recently we found that type I collagen matrix induced the osteoblastic differentiation of bone marrow cells. Three weeks after cells were cultured with type I collagen, they formed mineralized tissues. In this study, we investigated the expression of osteoblast-related genes (alkaline phosphatase, osteocalcin, bone sialoprotein, osteopontin, and cbfa-1) during the osteoblastic differentiation. The expression of alkaline phosphatase and osteopontin genes increased time-dependently during the osteoblastic differentiation. Osteocalcin and bone sialoprotein genes were expressed in cells that formed mineralized tissues, and both were expressed only after cells reached the mineralized tissue-formation stage. On the other hand, the cbfa-1 gene was expressed from the early differentiation stage. The Asp-Gly-Glu-Ala (DGEA) amino acid domain of type I collagen interacts with the alpha2beta1 integrin receptor on the cell membrane and mediates extracellular signals into cells. When the collagen-integrin interaction was interrupted by the addition of DGEA peptide to the culture, the expression of osteoblastic phenotypes of bone marrow cells was inhibited. These findings imply that the collagen-alpha2beta1 integrin interaction is an important signal for the osteoblastic differentiation of bone marrow cells.     

Ultrastructural aspects after cryoultramicrotomy of bone tissue and sutural cartilage in neonatal mice calvaria

Using transmission electron microscopy after cryoultramicrotomy, mineralized as well as unmineralized bone tissues and sutural cartilage were observed in neonatal mice calvaria. A good definition of osteoblasts (nucleus, membranes, organelles) and extracellular constituents (collagen fibrils, matrix vesicles, mineral substance) was obtained. The sutural zone was composed of an unmineralized cartilaginous tissue with more or less hypertrophic cells surrounded by a finely fibrillar matrix.     

Three-dimensional spatial relationship between the collagen fibrils and the inorganic calcium phosphate crystals of pickerel (Americanus americanus) and herring (Clupea harengus) bone

High-voltage (1.0 MV) electron microscopy and stereomicroscopy, electron probe microanalysis, electron diffraction and three-dimensional computer reconstruction, have been used to examine the spatial relationship between the inorganic crystals of calcium phosphate and the collagen fibrils of pickerel and herring bone. High-voltage stereo electron-micrographs were obtained of cross-sections of the cylinder-shaped intramuscular bones in uncalcified regions, in regions where only one or only several crystals had been deposited in some of the fibrils, and in successive sections containing progressively more mineral crystals until the stage of full mineralization was reached. High-resolution electron probe microanalysis confirmed that the electron-dense particles contained calcium and phosphorus. In the earliest stages of mineralization and progressing throughout the mineralization process, the crystals are located only within the collagen fibrils; crystals are not observed free in the extracellular spaces between collagen fibrils. The progressive increase in the mass of mineral deposited in the bone tissue with time occurs, essentially, completely within the collagen fibrils including the stage of full mineralization. At this stage, cross-sectional profiles of collagen fibrils are completely obliterated by mineral. A small number of crystals that are located on or close to the surface of the fibrils appear to extend a very short distance into the spaces between the fibrils. These ultrastructural observations of the very onset of calcification in which nucleation of the calcium phosphate crystals is clearly shown to begin within specific volumes of collagen fibrils, and of the subsequent temporal and spatial sequences of this phenomenon, which shows that calcification continues wholly within the collagen fibrils until maximum calcification is achieved, add important information on the basic physical chemical mechanism of the calcification and the structural elements that are involved. The spatial and temporal independence of the sites where mineralization is initiated establishes that such ultrastructural locations within individual collagen fibrils represent independent, physical chemical nucleation loci. The findings are totally inconsistent with the proposal that crystals must first be deposited in matrix vesicles, or other components such as mitochondria, and subsequently released and propagated in the interfibrillar space, until they eventually reach and impregnate the hole zone regions of the collagen fibrils. Three-dimensional computer reconstruction of serial transverse and longitudinal sections demonstrates periodic swellings along the collagen fibrils, corresponding to the hole zone region of their axial period as mineralization proceeds.(ABSTRACT TRUNCATED AT 400 WORDS)     

Constitutive expression of thrombospondin 1 in MC3T3-E1 osteoblastic cells inhibits mineralization

Thrombospondin 1 (TSP1) is a multifunctional extracellular glycoprotein present mainly in the fetal and adult skeleton. Although an inhibitory effect of TSP1 against pathological mineralization in cultured vascular pericytes has been shown, its involvement in physiological mineralization by osteoblasts is still unknown. To determine the role of TSP1 in biomineralization, mouse osteoblastic MC3T3-E1 cells were cultured in the presence of antisense phosphorothioate oligodeoxynucleotides complementary to the TSP1 sequence. The 18- and 24-mer antisense oligonucleotides caused concentration-dependent increases in the number of mineralized nodules, acid-soluble calcium deposition in the cell/matrix layer, and alkaline phosphatase activity within 9 days, without affecting cell proliferation. The corresponding sense or scrambled oligonucleotides did not affect these parameters. In the antisense oligonucleotide-treated MC3T3-E1 cells, thickened extracellular matrix, well-developed cell processes, increased intracellular organelles, and collagen fibril bundles were observed. On the other hand, the addition of TSP1 to the culture decreased the production of a mineralized matrix by MC3T3-E1 cells. Furthermore, MC3T3-E1 clones overexpressing mouse TSP1 were established and assayed for TSP1 protein and their capacity to mineralize. TSP1 dose-dependently inhibited mineralization by these cells both in vitro and in vivo. These results indicate that TSP1 functions as an inhibitory regulator of bone mineralization and matrix production by osteoblasts to sustain bone homeostasis.     

Type I collagen-induced osteoblastic differentiation of bone-marrow cells mediated by collagen-alpha2beta1 integrin interaction

Bone marrow cells are multipotent cells. When bone marrow cells were cultured with type I collagen matrix gels, they showed high alkaline phosphatase activity, collagen synthesis, and formed mineralized tissues. Furthermore, cells expressed osteocalcin and bone sialoprotein genes, which are osteoblast-specific genes. These findings indicate that type I collagen matrix gels induce osteoblastic differentiation of bone marrow cells. Type I collagen interacts with the alpha 2 beta 1 integrin receptor on the cell membrane and mediates extracellular signals into cells. DGEA peptide is a cell-binding domain of type I collagen molecule. When collagen-integrin interaction was interrupted by the addition of Asp-Gly-Glu-Ala (DGEA) peptide to the culture, the expression of osteoblastic phenotypes of bone marrow cells was inhibited. Furthermore, anti-alpha 2 integrin antibody, which interacts with alpha subunit of integrin and blocks the binding of integrin with collagen, suppressed the expression of osteoblastic phenotypes. These findings imply that collagen-alpha 2 beta 1 integrin interaction is an important signal for the osteoblastic differentiation of bone marrow cells.     

Vitamin K2, a gamma-carboxylating factor of gla-proteins, normalizes the bone crystal nucleation impaired by Mg-insufficiency

It has been reported that the Mg-insufficient bone is fragile upon mechanical loading, despite its high bone mineral density, while vitamin K2 (MK-4: menatetrenone) improved the mechanical strength of Mg-insufficient bone. Therefore, we aimed to elucidate the ultrastructural properties of bone in rats with dietary Mg insufficiency with and without MK-4 supplementation. Morphological examinations including histochemistry, transmission electron microscopy, electron probe microanalysis (EPMA) and X-ray diffraction were conducted on the femora and tibiae of 4-week-old Wistar male rats fed with 1) a normal diet (control group, 0.09% Mg), 2) a Mg-insufficient diet (low Mg group, 0.006% Mg), or 3) a Mg-insufficient diet supplemented with MK-4 (MK-4 group, 0.006% Mg, 0.03% MK-4). MK-4 appeared to inhibit the osteoclastic bone resorption that is stimulated by Mg insufficiency. EPMA analysis, however, revealed an increased concentration of Ca paralleling Mg reduction in the low Mg group. Assessment by X-ray diffraction revealed an abundance of a particular synthetic form of hydroxyapatite in the low Mg group, while control bones featured a variety of mineralized crystals. In addition, Mg-deficient bones featured larger mineral crystals, i.e., crystal overgrowth. This crystalline aberration in Mg-insufficient bones induced collagen fibrils to mineralize easily, even in the absence of mineralized nodules, which therefore led to an early collapse of the fibrils. MK-4 prevented premature collagen mineralization by normalizing the association of collagen fibrils with mineralized nodules. Thus, MK-4 appears to rescue the impaired collagen mineralization caused by Mg insufficiency by promoting a re-association of the process of collagen mineralization with mineralized nodules.     

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.     

Osteoblasts isolated from mouse calvaria initiate matrix mineralization in culture

A method is presented for isolating osteoblasts from newborn mouse calvaria without the use of digestive enzymes. The procedure is based on the ability of osteoblasts to migrate from bone onto small glass fragments (Jones, S.J., and A. Boyde, 1977, Cell Tissue Res., 184:179- 193). The isolated cells were cultured for up to 14 d in Dulbecco's modified Eagle's medium supplemented with 10% fetal calf serum and 50 micrograms/ml of ascorbic acid. 7-d cultures were incubated for 24 h with [3H]proline. High levels of collagen synthesis relative to total protein were found, as measured by collagenase digestion of medium and cell layer proteins. Analysis of pepsin-digested proteins from the same cultures by SDS PAGE showed that type I collagen was predominantly produced with small amounts of type III and V (alpha 1 chains) collagens. Osteoblasts grown in the presence of beta-glycerophosphate were able to initiate mineral deposition in culture. Electron microscopic analysis of the cultures revealed the presence of needle- shaped apatite-like crystals associated with collagen fibrils and vesicles in the extracellular space. Mouse skin fibroblasts cultured under identical conditions failed to initiate mineralization. Electron histochemical studies revealed the presence of alkaline phosphatase activity, associated with osteoblast membranes, matrix vesicles and on or near collagen fibrils. Thus these isolated osteoblasts retained in culture their unique property of initiating mineralization and therefore represent a model of value for studying the mineralization process in vitro.     

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.     

Ultrastructural study of the long-term development of two experimental cutaneous calcinoses (topical calciphylaxis and topical calcergy) in the rat

Summary Skin calcification induced by topical calciphylaxis was provoked by a subcutaneous injection of iron chloride in rats previously sensitized by dihydrotachysterol. A cutaneous topical calcergy was induced by an injection of potassium permanganate. An electron-microscopical study of the long-term evolution of both these models of calcification was made. After the initial stages, mineralization of the connective tissue continued by a secondary nucleation process without matrix vesicles. The mineral composed of needle-like structures, apatite in nature, was mainly deposited between and around collagen fibrils, and showed various arrangements in calcified plaques. Intrafibrillar calcification was rarely observed and appeared only in the later stages. The extension of calcified deposits then stopped. Finally, there was a fragmentation of the mineralized area which was progressively surrounded by uncalcified collagen fibrils. A demineralization process, caused by cells such as macrophages and multinucleated giant cells, rather than a resorption of the calcified deposits, was noted. It is important to emphasize that, in both models of ectopic calcification, an evolution toward ectopic ossification was never observed, which is perhaps due to the absence of extensive resorption mechanisms.     

无巨核细胞条件下促血小板生成素对骨髓基质细胞纤维形成的影响

目的：观察无巨核细胞存在的条件下促血小板生成素能否刺激骨髓基质细胞纤维形成。方法：用改良Dexter培养法进行体外不同浓度促血小板生成素（TPO）作用下的基质细胞培养,在培养过程中检测基质细胞相对增殖指数,纤维连接蛋白、层粘素和Ⅳ型胶原的表达,以及Ⅲ型前胶原蛋白的合成。结果：TPO可刺激基质细胞增殖,相对增殖指数随TPO浓度增加而增强,但不随作用时间延长而增强;纤维连接素、层粘素和Ⅳ型胶原在对照组与实验组均有阳性表达,但实验组强于对照组,但阳性强度不随培养时间的延长而增强;标记的Ⅲ型前胶原蛋白平均荧光强度实验组高于对照组,差异明显,但这种作用的强弱与TPO浓度相关性不强。结论：无巨核细胞存在的条件下,TPO可直接刺激骨髓基质细胞产生细胞外基质和胶原,促进其纤维形成。