The Essential Role of Fetuin in the Serum-Induced Calcification of Collagen

The mineral in bone is located primarily within the collagen fibril, and during mineralization the fibril is formed first and then water within the fibril is replaced with mineral. Our goal is to understand the mechanism of fibril mineralization, and as a first step we recently determined the size exclusion characteristics of the fibril. This study indicates that apatite crystals up to 12 unit cells in size can access the water within the fibril while molecules larger than a 40-kDa protein are excluded. We proposed a novel mechanism for fibril mineralization based on these observations, one that relies exclusively on agents excluded from the fibril. One agent generates crystals outside the fibril, some of which diffuse into the fibril and grow, and the other selectively inhibits crystal growth outside of the fibril. We have tested this mechanism by examining the impact of removing the major serum inhibitor of apatite growth, fetuin, on the serum-induced calcification of collagen. The results of this test show that fetuin determines the location of serum-driven mineralization: in fetuin’s presence, mineral forms only within collagen fibrils; in fetuin’s absence, mineral forms only in solution outside the fibrils. The X-ray diffraction spectrum of serum-induced mineral is comparable to the spectrum of bone crystals. These observations show that serum calcification activity consists of an as yet unidentified agent that generates crystal nuclei, some of which diffuse into the fibril, and fetuin, which favors fibril mineralization by selectively inhibiting the growth of crystals outside the fibril.     

Organization of apatite crystals in human woven bone

The organization of collagen fibrils differs in woven bone and lamellar bone, and it reflects certain aspects of the nature of the mineral crystals associated with them. In order to investigate the morphology and distribution of apatite crystals in woven bone, mineralized collagen fibrils and isolated crystals from the mid-diaphyses of human fetal femurs were observed with scanning and transmission electron microscopy and high-resolution electron microscopy. A number of features of woven bone were observed for the first time by these means. Similar to mature crystals from lamellar bone, the apatite crystals in woven bone are also platelet-shaped. However, most likely because of a high rate of old bone resorption and new bone formation in woven material, the average crystal dimensions are considerably smaller than those of mature crystals in lamellar bone. Apatite crystals were noted on the surface of collagen fibrils in woven bone. In densely packed woven bone, the periodicity of mineral deposited on individual fibrils is in registration over many fibrils. In addition to their association with collagen surfaces, crystals also appear distributed in both extrafibrillar and intrafibrillar collagen regions. In both cases, the minerals are crystalline and defect-free. These characteristics provide insight into the spatial and temporal relation between collagen and mineral that is the basis for the structure and organization of the mineral comprising human woven bone.     

The organic-inorganic relationships in bone matrix undergoing osteoclastic resorption

The organic-inorganic relationships in bone matrix undergoing osteoclastic resorption have been studied in rat tibial diaphyses using electron microscope techniques in an attempt to identify the steps of the resorption process. Results suggest that bone resorption occurs in two phases: the first, an extracellular phase, leads to bone matrix fragmentation and partial dissolution, and the second, an intracellular phase, to complete digestion of the breakdown products of the bone matrix. The first component of the bone matrix to be attacked by the osteoclast is the ground substance. This induces the release of the crystals lying between, and on, the collagen fibrils; any crystals lying within fibrils are released later, when the fibrils break up. As this stage proceeds, the collagen fibrils retain their normal intrinsic texture, but gradually loose their lateral aggregation, appearing as individual fibrils (some of them uncovered by crystals), mixed with fragments of fibrils and many free crystals. The loosened but otherwise structurally normal collagen fibrils, and their fragments, are strongly argyrophilic. Complete dissolution of the disaggregated fibrils occurs outside the cell, both in the resorption zone and in the initial portion of the channels of the ruffled border. The free crystals present in the resorption zone and those phagocytosed in cytoplasmic vacuoles are organic-inorganic structures, whose organic component (the crystal ghost) is, at least in part, of proteoglycan nature. Dissolution of inorganic material occurs within the cytoplasmic vacuoles of the osteoclasts. Results are viewed in relation to the process of bone resorption and, as far as crystal ghosts are concerned, to that of bone calcification. A tentative summary of the various steps involved in the mechanism of bone resorption is given.     

Crystal-collagen relationships in calcified turkey leg tendons visualized by selected-area dark field electron microscopy

Summary The distribution and orientation of biological apatite crystals in calcified turkey leg tendons were studied by selected-area dark field electron microscopy. This imaging technique enables the direct visualization of apatite and the specific determination of the crystallographic axes (a, b-axes or c-axis) within calcified collagen fibrils. This study shows that at early stages of mineralization, rod-shaped apatite crystals (5–20 nm in length) were localized and dispersed within gap zones bordering both the collagen molecule C- and N-terminal regions. At later stages of mineral deposition the crystals were more extensive, occupying greater areas of the gap zone and, in addition, apatite crystals were found to occur in the overlap zones. The orientation of apatite crystals was observed to be an alternating and interlocking distribution of a, b-axes and c-axis along the axial period of collagen fibrils. This distribution is interpreted as representing a continuous rotation of apatite axial orientation along the collagen period.     

Crystals in calcified epiphyseal cartilage and cortical bone of the rat

Summary The size of hydroxyapatite (HAP) crystals in calcified cartilage and cortical bone of the rat has been studied and compared with that of synthetic poorly crystalline hydroxyapatite (PCHA). Crystal size was determined by X-ray diffraction and selected-area dark field imaging, and their elemental compositions were determined by emission spectroscopy. The crystal size of cartilage, bone, and PCHA were found to be between 120 and 170 ? in length by 50 ? in width as determined by both X-ray diffraction and dark field imaging. Cartilage had a lower ash weight than bone but both have the same Ca/P ratio of 1.6. These findings, though in agreement with the X-ray diffraction literature, differ from observations made by conventional bright field electron microscopy. We conclude that mineral sites in cartilage and bone, which exist as platelike structures, are in fact aggregates of small 120–170×50 ? HAP crystals. The consequences of these findings are discussed in view of crystal relationships with collagen and other macromolecules.     

The dentino-enamel junction: A structural and microanalytical study of early mineralization

Summary The spatial localization of enamel and dentin apatite crystals of the rat tooth has been studied by electron microscopic methods—bright field, selected-area dark field, and electron spectroscopic imaging. The sequential events of dentin calcification followed by the formation and growth of enamel crystals were determined and compared to previous studies. In dentin, initial sites of mineral deposition occur in areas subjacent to the dentino-enamel junction (DEJ). The subsequent expansion of these deposits progresses towards the DEJ to the terminal ends of dentin collagen fibrils. Concomitantly, an electron-dense enamel matrix is released by ameloblasts; with the presence of this matrix, the growth of enamel crystals occurs from the underlying calcified dentin. Enamel crystal growth continues to within close proximity of the plasma membrane of ameloblasts. A close spatial relationship between enamel and the crystals of calcified dentin collagen fibrils was observed by selected-area dark field imaging. Such areas of crystal intimacy show a co-localization of calcium and phosphorus extending from calcified collagen fibrils to enamel sheaths which encase enamel crystals. A working model of the spatial relationship between crystals of dentin and enamel is presented and discussed in light of mechanisms by which calcified dentin may promote the formation of enamel crystals.     

Twisted plywood architecture of collagen fibrils in human compact bone osteons

Summary Ultrathin sections of decalcified human compact bone, observed by transmission electron microscopy, reveal that collagen fibrils can be distributed in the form of a superimposed series of nested arcs. This characteristic pattern has never been interpreted in previous works on compact bone structure. We demonstrate, by goniometric observations at the ultrastructural level, that such series of nested arcs are a consequence of the “twisted plywood” architecture of collagen fibrils in the compact bone matrix. In the same specimens, an “orthogonal plywood” disposition of collagen fibrils is also observed; a transition exists between these two types of orders. We show that the “twisted plywood structure” accounts well for certain optical properties of osteons, observed in polarizing microscopy, described as “intermediate osteons.” The particular geometry of collagen fibrils, leading to nested arcs in oblique sections, is analogous to the distribution of molecules in certain liquid crystals (called cholesteric liquid crystals). The principle of a liquid crystalline self-assembly of the collagen matrix in bone is therefore discussed.     

The role of magnesium on the structure of biological apatites

Summary X-ray diffraction, infrared absorption spectroscopy, and chemical investigation have been carried out on deproteinated samples of turkey leg tendon at different degrees of calcification. The inorganic phase consists of poorly crystalline B carbonated apatite. On increasing calcification, the apatite crystal size, as well as its thermal stability, increase while the relative magnesium content is reduced. On the other hand, synchrotron X-ray diffraction data clearly indicate that apatite lattice parameters do not change as the crystals get larger. At the last stage of calcification the crystal size, chemical composition, and thermal conversion of the apatite crystallites approximate those of bone samples, which have been examined for comparison. The results provide a quantitative relationship between relative magnesium content and extent of apatite conversion into B-tricalcium phosphate by heat treatment. Furthermore, they suggest that the smaller crystallites laid down inside the gap region of the collagen fibrils are richer in magnesium than the longer ones that fill the space between collagen fibrils.     

Age- and genotype-dependence of bone material properties in the osteogenesis imperfecta murine model (oim).

Cortical mineralization of long bones was studied in collagen alpha2(I)-deficient mice (oim) used as a model for human osteogenesis imperfecta. Aspects of the age development of the mice were characterized by combining nanometer- to micrometer-scale structural analysis with microhardness measurements. Bone structure was determined from homozygous (oim/oim) and heterozygous (oim/+) mice and their normal (+/+) littermates as a function of animal age by small-angle X-ray scattering (SAXS) and quantitative backscattered electron imaging (qBEI) measurements. SAXS studies found anomalies in the size and arrangement of bone mineral crystals in both homozygous and heterozygous mice aged 1-14 months. Generally, the crystals were smaller in thickness and less well aligned in these mice compared with control animals. An increase in the mean crystal thickness of the bone was found within all three genotypes up to an age of 3 months. Vicker's hardness measurements were significantly enhanced for oim bone (homozygotes and heterozygotes) compared with controls. The microhardness values were correlated directly with increased mineral content of homozygous and heterozygous compared with control bone, as determined by qBEI analysis. There was also a significant increase of mineral content with age. Two possibilities for collagen-mineral association are discussed for explaining the increased hardness and mineral content of oim/oim bone, together with its decreased toughness and thinner mineral crystals. As a consequence of the present measurements, one model for oim bone could incorporate small and densely packed mineral crystals. A second model for possible collagen-mineral association in oim material would consist of two families of mineral crystals, one being smaller and the other being much larger than the crystals found in normal mouse long bones.     

Characterization of bone mineral crystals in horse radius by small-angle X-ray scattering

The size and the orientation of the bone salt (mineral) crystals in the cranial and caudal zones in the transverse midshaft section of the equine radius were investigated by small-angle X-ray scattering (SAXS). The results are interpreted as indicating that the crystals had an elongated shape with an average thickness of T=3.79±0.20 nm in the cranial region. Their orientation was predominantly in the longitudinal direction of the bone. There was no preferential orientation within the transverse plane. The distribution of tilt angles with respect to the longitudinal direction was determined directly from the SAXS data: the average angle was about 30° for the cranial region and 45° for the caudal region. Assuming that the needle-like crystals are parallel with the collagen fibrils, the angular distribution of the crystals is in good agreement with previous measurements of collagen orientation using circularly polarized light microscopy.     

Microfocus Small Angle X-ray Scattering Reveals Structural Features in Archaeological Bone Samples; Detection of Changes in Bone Mineral Habit and Size

Microfocus X-ray scattering provides a powerful nondestructive technique capable of providing important information about the size, habit, and arrangement of mineral crystals in bone. The technique is capable of probing textural differences in a sample at a micron scale resolution. The study presented here involved the analysis of a number of archaeological bones by microfocus X-ray scattering at the ESRF Grenoble in order to determine local changes in mineral durability. The results showed that regions of bone with a modified microscopic morphology contained a greater dispersion of crystal shape when compared with more intact regions and control contemporary bone samples, but the crystal thickness values showed similar consistency. We speculate that the persistence of collagen in the archaeological bone may allow diagenetic remodeling of bone in terms of crystallite shape but defines the size of remodelled crystallites. The ability to detect such local changes in texture has wide potential for determining crystal characteristics in healthy and diseased bone samples.     

The role of collagen in bone strength

Bone is a complex tissue of which the principal function is to resist mechanical forces and fractures. Bone strength depends not only on the quantity of bone tissue but also on the quality, which is characterized by the geometry and the shape of bones, the microarchitecture of the trabecular bones, the turnover, the mineral, and the collagen. Different determinants of bone quality are interrelated, especially the mineral and collagen, and analysis of their specific roles in bone strength is difficult. This review describes the interactions of type I collagen with the mineral and the contribution of the orientations of the collagen fibers when the bone is submitted to mechanical forces. Different processes of maturation of collagen occur in bone, which can result either from enzymatic or nonenzymatic processes. The enzymatic process involves activation of lysyl oxidase, which leads to the formation of immature and mature crosslinks that stabilize the collagen fibrils. Two type of nonenzymatic process are described in type I collagen: the formation of advanced glycation end products due to the accumulation of reducible sugars in bone tissue, and the process of racemization and isomerization in the telopeptide of the collagen. These modifications of collagen are age-related and may impair the mechanical properties of bone. To illustrate the role of the crosslinking process of collagen in bone strength, clinical disorders associated with bone collagen abnormalities and bone fragility, such as osteogenesis imperfecta and osteoporosis, are described.     

Do more highly organized collagen fibrils increase bone mechanical strength in loss of mineral density after one-year running training?

The aim of this study was to evaluate the effects of long-term running training on the structural properties of bone. Ten beagle dogs ran according to a strenuous progressive program (up to 40 km/day) for 1 year. At the end of the training program, there was a significant reduction in bone mineral density (up to 9.7%) in the vertebrae of the runner dogs as compared with 10 sedentary control dogs. Polarized light microscopy of the vertebral trabecular bone, however, displayed proportionally higher retardation values of the collagen network of the runner dogs than of the sedentary dogs, suggesting a reorganization in a more parallel manner in the collagen fibrils. The concentration and cross-linking of collagen in the bones remained similar in both groups. No differences were observed in the force to failure of bones of the two groups nor in the histomorphometric analysis of the bones. We suggest that the collagen network in the bones accounted for the maintenance of the strength properties in the bones of the runner dogs despite the loss of mineral density.     

Calcificationin vitro of demineralized bone matrix

Collagen was prepared from compact sheep bone by decalcification with EDTA and from rat tail tendons by acetic acid extraction and reconstitution with NaCl. The deposition of apatite in sheep bone collagen in a metastable calcification solution was studied chemically and by electron microscopy. The bone collagen was shown to be a good nucleation catalyst for mineral deposition, while rat tail collagen was a poor catalyst. Mineral deposition in bone collagen occured in two separate kinetic phases, a rapid phase of nucleation and crystal growth, giving rise to small calcified islands, and a second slow phase, ascribed to growth in regions not involving the catalytic sites. This second phase of mineral deposition is considered to be the result of impaired ion diffusion through the closely-aligned collagen fibrils, thus leaving large areas of the collagen free of mineral even though the buffer remains highly supersaturated. Electron micrographs suggested that the catalytic sites might be in some relationship to the 640 Å periodicity of collagen, but a role for non-collagenous material bound to the collagen has not been excluded.The poor catalytic activity of reconstituted collagen was not due to the presence of loosely-bound inhibitors, although inhibitors could be strongly bound to this type of collagen and be absent from bone collagen. The differences in catalytic activity may have a bearing on physiological calcification. A more general hypothesis for nucleation of a mineral phase in biological systems is required.This work was supported in part by the European Atomic Energy Community (EURA-TOM), Brussels, Belgium.     

A correlation between the distribution of biological apatite and amino acid sequence of type I collagen

Summary We have determined the localization of apatite within type I collagen fibrils of calcifying turkey leg tendons by both bright field and selectedarea dark field (SADF) electron microscopy and have compared this to computer-modeled, chick type I collagen amino acid sequence data. Apatite crystals occur in both the gap and overlap zones at early stages of mineralization in an asymmetric pattern that corresponds to the polarity, N-to C-orientation, of the collagen molecule. Based on comparisons with computer-generated models of known amino acid sequence of collagen, it was determined for early stages of mineral deposition that apatite is restricted by areas of high hydrophobicity. The gap zone is less hydrophobic than the overlap zone on average but each of these zones had areas of high hydrophobicity that correlated with sites of low localization of mineral. Possible interactions between hydrophobic regions and the process of mineral deposition are discussed.     

Relationships between bone protein and mineral in developing porcine long bone and calvaria

Several proteins in the bone matrix have been implicated in the regulation of mineral crystal formation and growth. To investigate the relationships between these proteins and the mineral phase at various stages of mineral maturation, fetal porcine calvariae and long bones were fragmented and the particles (20 microm) separated by density gradient sedimentation into fractions of increasing density (1.8 to >2.2 g/cm3). Samples from each fraction were analyzed by X-ray diffraction to obtain the average crystal size/strain and chemical composition. Other samples were sequentially extracted, first with 4.0 mol/L guanidium hydrochloride (GuHCl) (G1), then with 0.5 mol/L ethylene-diamine tetraacetic acid (EDTA) (E), and again with 4.0 mol/L Gu-HCI (G2), for analysis of proteins in different tissue compartments. Based on the mineral density distribution and crystal size, fetal porcine bone protein content was determined for tissue residue and each extract and the protein composition analyzed by sodium dodecyl-polyacrylamide gel electrophoresis (SDS-PAGE). Although the insoluble organic matrix decreased with mineral density the collagen and protein content remained fairly constant, representing approximately 10% of the tissue weight, except in the highest density fraction. Whereas the total extractable protein, representing predominantly noncollagenous proteins, did not show density-related differences, differences were observed for individual proteins on SDS-PAGE. Consistent with their presence in osteoid, the content of bone sialoprotein (BSP), tyrosine-rich acidic matrix protein (TRAMP), and a series of small proteins with cell attachment properties in the G1 extract decreased with mineral density, whereas TRAMP and BSP were increased in G2 extracts. Mineral-associated proteins, including alpha2HS-glycoprotein, BSP, osteopontin (OPN), and osteocalcin, increased with mineral density, whereas secreted protein acidic and rich in cysteine (SPARC)/osteonectin, and some minor proteins, appeared to decrease. Differences of individual proteins within and between the calvarial and long bones could be related to the role of these proteins in the formation and maturation of hydroxyapatite crystals. Collectively, these studies demonstrate mineral density-associated changes in protein composition that reflect a rapid maturation of mineral crystals in embryonic porcine bones.     

Ultra-structural defects cause low bone matrix stiffness despite high mineralization in osteogenesis imperfecta mice

Bone is a complex material with a hierarchical multi-scale organization from the molecule to the organ scale. The genetic bone disease, osteogenesis imperfecta, is primarily caused by mutations in the collagen type I genes, resulting in bone fragility. Because the basis of the disease is molecular with ramifications at the whole bone level, it provides a platform for investigating the relationship between structure, composition, and mechanics throughout the hierarchy. Prior studies have individually shown that OI leads to: 1. increased bone mineralization, 2. decreased elastic modulus, and 3. smaller apatite crystal size. However, these have not been studied together and the mechanism for how mineral structure influences tissue mechanics has not been identified. This lack of understanding inhibits the development of more accurate models and therapies. To address this research gap, we used a mouse model of the disease (oim) to measure these outcomes together in order to propose an underlying mechanism for the changes in properties. Our main finding was that despite increased mineralization, oim bones have lower stiffness that may result from the poorly organized mineral matrix with significantly smaller, highly packed and disoriented apatite crystals. Using a composite framework, we interpret the lower oim bone matrix elasticity observed as the result of a change in the aspect ratio of apatite crystals and a disruption of the crystal connectivity.     

Vectorial sequence of mineralization in the turkey leg tendon determined by electron microscopic imaging

Summary Turkey leg tendons were used as a model tissue to study the spatial and temporal relationships of mineral deposition between matrix vesicles and collagen fibrils by various electron microscopic techniques—bright field, selected-area dark field (SADF), and electron spectroscopic imaging (ESI). These latter imaging techniques enabled the direct localization and spatial distributions of both apatite crystals and atomic elements (Ca, P) within matrix vesicles and collagen. In longitudinal planes of section, a consistent vectorial gradient of mineralization was observed which started with the first localization of apatite mineral in matrix vesicles; with further development, the mineral spread from the vesicle to the extravesicular interstices and then into the adjacent collagen fibrils. Once intrafibrillar, the mineral was observed to advance both laterally and axially. The association of vesicle/collagen mineral was examined by ESI analysis of Ca and P elemental maps and appeared as a continuum between the vesicles and the adjacent collagen fibrils. Similarly, an intimate spatial relationship was observed between the mineral of vesicles and collagen in transversely cut sections of tendon. The sequential development of this mineralized matrix is discussed in light of matrix vesicle/collagen interactions.     

Nucleation and growth of mineral crystals in bone studied by small-angle X-ray scattering

Summary The mechanism of calcification in bone and related tissues is a matter of current interest. The mean size and the arrangement of the mineral crystals are important parameters difficult to obtain by electron microscopy. Furthermore, most studies have been carried out on poorly calcified model systems or chemically treated samples. In the work presented here, native bone was studied as a function of age by a quantitative small-angle X-ray scattering method (SAXS). Bone samples (calvariae and ulnae) from rats and mice were investigated. Measurements were performed on native bone immediately after dissection for samples up to 1 mm thick. The size, shape, and predominant orientation of the mineral crystals in bone were obtained for embryonal, young, and adult animals. The results indicate that the mineral nucleates as thin layers of calcium phosphate within the hole zone of the collagen fibrils. The mineral nuclei subsequently grow in thickness to about 3 nm, which corresponds to maximum space available in these holes.     

TEM analysis of the nanostructure of normal and osteoporotic human trabecular bone

Transmission electron microscopy (TEM) was used to investigate the crystal-collagen interactions in normal and osteoporotic human trabecular bone at the nanostructural level. More specifically, two-dimensional TEM observations were used to infer the three-dimensional information on the shape, the size, the orientation, and the alignment of apatite crystals in collagen fibrils in normal and osteoporotic bone. We found that crystals were of platelet shape with irregular edges and that there was no substantial difference in crystal length or crystal thickness between normal and osteoporotic trabecular bone. The crystal arrangement in cross-sectioned fibrils did not neatly conform to the parallel arrangement of crystals seen in longitudinally-sectioned fibrils. Instead, the crystal arrangement in both normal and osteoporotic trabecular bone took on more of a random, undulated arrangement, with certain localized areas demonstrating circular oriented patterns. The TEM imaging was done using bright fields only. Thus, the results presented are within the limitations of this approach.