Disaggregation of bone into crystals

Summary The sizes, shapes, and organizational states of the crystals in bone are studied by systematic disaggregation of the mineral phase. This is achieved by oxidizing the organic phase with sodium hypochlorite, dispersing the resultant particles by sonication, and separating the crystal aggregates from the crystal monomers by gravity setting in ethanol. Six different bones are compared. Bones in which crystals are intimately associated with the collagen fibrils mostly disaggregate into crystal monomers. In dense bones, where the crystals are mostly located between fibrils, they tend to persist as “fused” aggregates. All the crystals are tabular or plate-shaped. In bones in which the majority of crystals are associated with the collagen fibrils, just less than 90% of the crystals are shorter than about 450 ? in length. Their widths are on the average about 250 ?, almost an order of magnitude larger than the diameters of individual gap regions within the collagen fibril. The notion that one crystal is located in one gap region is therefore untenable and a reevaluation of the relations between collagen and mineral in bone is necessary.     

X-Ray Diffraction on Cyclically Loaded Osteons

The results of a study on the fine structural distortion due to the two previously observed types of degradation in cyclically loaded single osteons (i.e., stiffness degradation and pinching effect) are presented. Fully calcified longitudinal and alternate osteons were isolated from 350-μm-thick longitudinal sections of human femoral cortical bone. The samples were prepared from 500-μm-long central cylindrical portions of an osteon, whose two ends were penetrating into rectangular lugs for fixation to an electromechanical device that cyclically loaded the samples. This device was connected to a microwave micrometer and a recorder. The structural distortions induced by cyclic loading were investigated by high- and low-angle X-ray diffraction on conventional and synchrotron radiation sources. Cyclic loading results in a reduction in the degree of orientation of apatite crystallites, especially in longitudinal osteons, in which the most abundant longitudinal lamellae are not protected against buckling by transverse lamellae as they are in alternate osteons. In contrast, the degree of orientation of collagen fibrils does not seem to be affected by cycling loading in the two osteon types, possibly because the disorientation of collagen fibrils is, within limits, a reversible process. Finally, the contrast between the disorientation of inorganic crystallites and the apparently unaltered distribution of collagen fibrils suggests that the degradation of cyclically loaded osteons may be due to a separation of the crystallites from the fibrils. Received: 6 June 1996 / Accepted: 7 August 1997     

Collagen production of osteoblasts revealed by ultra-high voltage electron microscopy

In the bone, collagen fibrils form a lamellar structure called the “twisted plywood-like model.” Because of this unique structure, bone can withstand various mechanical stresses. However, the formation of this structure has not been elucidated because of the difficulty of observing the collagen fibril production of the osteoblasts via currently available methods. This is because the formation occurs in the very limited space between the osteoblast layer and bone matrix. In this study, we used ultra-high-voltage electron microscopy (UHVEM) to observe collagen fibril production three-dimensionally. UHVEM has 3-MV acceleration voltage and enables us to use thicker sections. We observed collagen fibrils that were beneath the cell membrane of osteoblasts elongated to the outside of the cell. We also observed that osteoblasts produced collagen fibrils with polarity. By using AVIZO software, we observed collagen fibrils produced by osteoblasts along the contour of the osteoblasts toward the bone matrix area. Immediately after being released from the cell, the fibrils run randomly and sparsely. But as they recede from the osteoblast, the fibrils began to run parallel to the definite direction and became thick, and we observed a periodical stripe at that area. Furthermore, we also observed membrane structures wrapped around filamentous structures inside the osteoblasts. The filamentous structures had densities similar to the collagen fibrils and a columnar form and diameter. Our results suggested that collagen fibrils run parallel and thickly, which may be related to the lateral movement of the osteoblasts. UHVEM is a powerful tool for observing collagen fibril production.     

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.     

A low-angle X-ray diffraction analysis of osteonic inorganic phase using synchrotron radiation

Summary Using synchrotron radiation the lowangle X-ray diffraction method has been applied to single osteon samples to yield new data on the texture of the inorganic bone fraction. Two sample types—cylindrically shaped osteonic samples and osteonic radial hemisections—were prepared from longitudinal and alternate osteons at both the initial and final stages of calcification. The results indicate that the diffraction pattern is due to the inorganic phase, which reveals the same axial periodicity as native collagen fibrils and fits into the main band. No change is appreciable as osteons pass from the initial to the final stage of calcification. This means that when crystallites covering much more than a collagen axial period are observed under the electron microscope, they do not appreciably affect the calcified banding of collagen fibrils. The osteonic axis corresponds to the main direction of collagen orientation both in longitudinal and alternate osteons. The degree of orientation, however, is lower in alternate osteons than in longitudinal ones, where only few thin, incomplete transversal lamellae are found.     

X-ray diffraction and electron microscope study of osteons during calcification

Summary To obtain information on the changes in the inorganic bone fraction during calcification, low- and wide-angle X-ray diffraction techniques and electron microscopy have been applied to single osteon samples. The samples were cylindrically shaped and their axes corresponded to the axes of the Haversian canals. The selection was made according to the degree of calcification and the orientation of collagen bundles and inorganic particles. Osteons at both the initial and final stages of calcification were chosen. Arrangements of fiber bundles and inorganic particles in successive lamellae characteristic of three types of osteon were selected, that is, longitudinally structured osteons, transversely structured osteons, and alternately structured osteons. The results indicate that in osteonic lamellar bone there are two types of inorganic particles: (1) granules arranged in linear or needle-shaped entities with maximum width 40–45 Å, which are regularly distributed at the level of the main band of the collagen fibrils where their maximum length reaches the length of the main band itself; that is, about 400 Å; and (2) very long crystallites, with a diameter of 40–45 Å, which grow with their crystallographicc-axis parallel to the collagen fibrils and cover much more than a major collagen period.     

SEM and TEM study of the hierarchical structure of C57BL/6J and C3H/HeJ mice trabecular bone

Transmission electron microscopy (TEM) and scanning electron microscopy (SEM) were used to study the hierarchical structure of trabecular bone from C57BL/6J (low bone mass) and C3H/HeJ mice (high bone mass). Bone was harvested from two different anatomical locations: femoral metaphysis and L5 vertebra. This investigation focused on three structural scales: the mesostructural (porous network of trabecular struts), the microstructural (collagen fibril arrangements in trabecular packets), and the nanostructural (collagen fibril and apatite crystals) levels. At the mesostructural level, no distinct differences were found in the trabecular structure of femoral metaphysis but thinner trabecular struts were observed in L5 vertebra for C57BL/6J mice strain. At the microstructural level, the collagen fibrils forming the rotated, twisted, and orthogonal plywood arrangements were distinguished as well as atypical arrangements. At the nanostructural level, the shape and size of apatite crystals, and their arrangement with respect to collagen fibrils were studied. In spite of very different bone mass densities, both mice strains had similar structures at the nanostructural and microstructural levels.     

Matrix vesicles and focal proteoglycan aggregates are the nucleation sites revealed by the lanthanum incubation method: A correlated study on the hypertrophic zone of the rat epiphyseal cartilage

Correlated studies were performed with light and electron microscopy, and backscattered electron image in conjunction with X-ray microanalysis, of lanthanum-incubated epiphyseal cartilage of the young rat. The hallmark of this procedure is the appearance of LaP electrondense deposits (not present in control sections) in precise sites of the hypertrophic zone. The ultrastructural study revealed a dual nature of these sites: “dense matrix vesicles” and “focal filament aggregates.” The dense matrix vesicles are a specific type of matrix vesicle with the intrinsic capacity of precipitating LaP mineral, as soon as they originate from the hypertrophic chondrocytes. Furthermore, the matrix vesicles were found to be heterogeneous because lanthanum-devoid, “light matrix vesicles” were also present. The focal filament aggregates, which were not recognized in unstained sections and in controls, are apparently focal concentrations of proteoglycans with high lanthanum binding capacity, although the presence in them of other components (e.g., type X collagen, C-propeptide of type II collagen) cannot be excluded. They were in close connection with the light matrix vesicles in the upper hypertrophic zone, and were loaded with a variable quantity of LaP irregular electron-dense deposits in the lower hypertrophic zone. These irregular deposits are similar to, but distinct from, calcification nodules. The lanthanum incubation method indirectly detects the matrix Ca-binding components (which bind La ions), and the calcification initiation sites (which precipitate a LaP-mineral phase). A sequence is proposed of successive steps of LaP nucleation within the focal filament aggregates, which possibly mimics calcium phosphate deposition. Such a sequence seems to require the participation not only of dense matrix vesicles, but also of the filamentous components of the focal aggregates, possibly together with the activity of alkaline phosphatase.     

Tensile properties of antler bone

Summary The tensile deformation characteristics of compact bone from deer antler were measured in both the “dry” and “wet” states and compared with published values for bovine compact bone. The tensile strength in the wet state (108±5.1 MN/m²) was comparable to the value for bovine compact bone tested at the same strain rate. The modulus value was very low: 7.5±0.9 GN/m². The work to fracture was comparatively high, about 3 times that for bovine compact bone. Fractographic examination revealed fibrillar and osteonal shear for samples fractured in the dry state. In the samples tested in the wet state, some regions exhibited pull-out of lamellar segments from within a Haversian system. The results are explained in terms of the higher collagen content and lesser degree of mineralization in the antler.     

Fine powdering exposes the mineral-protected collagen of bone to protease digestion

Summary The method of heat-denaturation and trypsin digestion was used to dissect bone biochemically into mineral-protected and mineral-unprotected pools of collagenous matrix. It was found that varying the particle size of the bone powder had a profound effect on the results. Using mature bovine cortical bone, the observed pool of “unmineralized” (mineral-unprotected) collagen could be varied from 2% to more than 60% of the total bone collagen simply by decreasing the particle size of the bone sample from greater than 1 mm to less than 38 μm. No major differences were seen in the contents of hydroxypyridinium cross-links between the collagens of the trypsin-soluble and trypsin-insoluble pools from the fine powders, contrary to earlier reports. A trend to a higher content of these cross-links was evident, however, in the very small collagen pool extracted from the coarsest bone particles. Similar extraction differences were noted using bacterial collagenase to probe for mineral-protected vs. mineral-unprotected domains of bone collagen. In summary, the biochemical dissection results appear largely to be an artifact of the powdering technique, the shear energies of which presumably destroy the intimate physical relationship between the mineral crystallites and the collagen fibrils at the fractured surfaces of the bone particles. As the fractured surface area increases with decreasing particle size so the fraction of protease degradable collagen increases. Since powdering is routinely adopted for many structural studies on both the mineral and organic phases of bone, the findings on finely powdered bone should be interpreted cautiously.     

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.     

Load-induced proteoglycan orientation in bone tissuein vivo andin vitro

Summary Previous studies of Alcian blue-induced birefringence in adult avian cortical bone showed that a short period of intermittent loading rapidly produces an increased level of orientation of proteoglycans within the bone tissue. In the absence of further loading, this persists for over 24 hours. We have proposed that this phenomenon could provide a means for “capturing” the effects of transient strains, and so provide a persistent, constantly updated strain-related influence on osteocyte populations related to the bones' averaged recent strain history, in effect, a “strain memory” in bone tissue. In our present study, we use the Alcian blue-induced birefringence technique to demonstrate that proteoglycan orientation also occurs after intermittent loading of both cortical and cancellous mammalian bonein vivo andin vitro. We also show that the change in birefringence is proportional to the magnitude of the applied strain, and that the reorientation occurs rapidly, reaching a maximal value after only 50 loading cycles. Examination of electron micrographs of bone tissue after staining with cupromeronic blue allows direct visualization and quantification of the change in proteoglycan orientation produced by loading. This shows that intermittent loading is associated with a realignment of the proteoglycan protein cores, bringing them some 5 degrees closer to the direction of collagen fibrils in the bone matrix.     

Spectroscopic markers of bone quality in alendronate-treated postmenopausal women

Summary  Comparison of infrared spectroscopic images of sections from biopsies of placebo-treated post-menopausal women and women treated for 3 years with 10 mg/day alendronate demonstrated significant increases in cortical bone mineral content, no alterations in other spectroscopic markers of “bone quality,” but a decrease in tissue heterogeneity. Methods  The material properties of thick sections from iliac crest biopsies of seven alendronate-treated women were compared to those from ten comparably aged post-menopausal women without bone disease, using infrared spectroscopic imaging at ∼7 μm spatial resolution. Parameters evaluated were mineral/matrix ratio, crystallinity, carbonate/amide I ratio, and collagen maturity. The line widths at half maximum of the pixel histograms for each parameter were used as measures of heterogeneity. Results  The mineral content (mineral/matrix ratio) in the cortical bone of the treated women’s biopsies was higher than that in the untreated control women. Crystallinity, carbonate/protein, and collagen maturity indices were not significantly altered; however, the pixel distribution was significantly narrowed for all cortical and trabecular parameters with the exception of collagen maturity in the alendronate treatment group. Conclusions  The increases in mineral density and decreased fracture risk associated with bisphosphonate treatment may be counterbalanced by a decrease in tissue heterogeneity, which could impair tissue mechanical properties. These consistent data suggest that alendronate treatment, while increasing the bone mass, decreases the tissue heterogeneity.     

Analysis of hydroxyproline by high performance liquid chromatography and its application to collagen turnover studies in bone cultures

Summary We describe a high performance liquid chromatography (HPLC) technique for separating and quantitating hydroxyproline in calvarial cultures. Using a reverse-phase Nova-Pak C₁₈ column and a 140 mM sodium acetate, 0.05% triethylamine (TEA), 6% acetonitrile solvent system, we obtained a complete separation of hydroxyproline. Recovery of added standards ranged from 89 to 103% and intraassay variability was <8%. [³H]hydroxyproline measurements were used to examine changes in collagen turnover in rat calvariae labeled with [³H]proline and “chased” in the presence of 10 mM unlabeled proline. The addition of parathyroid hormone (PTH) during a 24–48 hour “chase” period increased the release of acid-soluble [³H]hydroxyproline into the culture medium, indicating an increase of fully degraded collagen. This method offers a sensitive and reproducible technique for monitoring changes in bone matrix degradation and in studying agents that modify this process.     

A mixed packing model for bone collagen

Summary A wide variety of physical properties, including sonic velocity, dimensional changes between wet and dried stages, anisotropy of the tissue properties, density, X-ray diffraction, differential microcalorimetry, dielectric constant, and composition (water, mineral, organic content) for the mineralized and demineralized tissue was used to develop a model for the superlattice structure of bone collagen. A mixed model is suggested where the collagen molecules are in register as in SLS type of aggregation within the microfibril, and the microfibrils are staggered in D unit steps according to the Hodge-Petruska scheme. A square packing model with 4 or more molecules per microfibril best fits the HP scheme with the effective molecular diameter of the wet collagen molecule, and allows for the regular array of axial gap filling microcrystallites of 5 nm or larger diameter. It is concluded that: 1. Macroscopic dimensional changes of adult bovine bone matrix closely match molecular dimensional changes of collagen superlattice. 2. Effective molecular diameter of dry collagen is 1.09 nm and that of wet bone collagen is 1.42 – 1.45 nm. 3. Water layer of the wet bone collagen molecule is 0.16 nm thick. 4. Water in the bone collagen molecule is distributed in 5 regimes much like in the tendon collagen molecule. 5. “Hidden” water, 0.10 g water per dry collagen of regimes I and II, is within the triple helix. 6. “External” water incorporated in the collagen molecule provides transition between the highly structured collagen molecule and the intermolecular medium. 7. Water incorporated in the mineralized bone collagen molecule is less than in demineralized bone matrix. 8. For adult bovine cortical bone, 25% by volume is water, 32% dry organic, 43% mineral; 28% by volume of the mineral is axial gap filling, 58% radial intrafibrillar, and 14% radial extrafibrillar.     

Assessment of bone mineral and matrix using backscatter electron imaging and FTIR imaging

The resistance of bone to fracture is determined by its geometric and material properties. The geometry and density can be determined by radiographic methods, but material properties such as collagen structure, mineral composition, and crystal structure currently require analysis by invasive techniques. Backscatter electron imaging provides quantitative information on the distribution of the mineral within tissue sections, and infrared and other vibrational spectroscopic methods can supplement these data, providing site-specific information on mineral content as well as information on collagen maturity and distributions of crystal size and composition. This information contributes to the knowledge of “bone quality.”     

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.     

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.     

Size and Shape of Mineralites in Young Bovine Bone Measured by Atomic Force Microscopy

Atomic force microscopy (AFM) was used to obtain three-dimensional images of isolated mineralites extracted from young postnatal bovine bone. The mean mineralite size is 9 nm × 6 nm × 2.0 nm, significantly shorter and thicker than the mineralites of mature bovine bone measured by the same technique. Mineralites of the young postnatal bone can be accommodated within the hole zone regions of a quasi-hexagonally packed collagen fibril in the fashion described by Hodge [9] in which laterally adjacent hole zone regions form continuous channels across the diameter of a fibril for a distance of at least 10 nm. Deposition of mineralites of the size noted above in this void volume of the fibrils would result in little or no distortion of the collagen molecules or supramolecular structure of the collagen fibril. The new AFM data supporting this claim is consistent with findings obtained by electron microscopy and low-angle x-ray and neutron diffraction that mineralites formed within collagen fibrils during initial stages of calcification occur within the hole zone region. However, the deposition of additional mineralites in the intermolecular spaces between collagen molecules in the overlap region of the fibrils would significantly distort the fibrils since the space available between adjacent molecules is considerably less than even the smallest dimension of the mineralites.     

Mechanism by which MLO-A5 Late Osteoblasts/Early Osteocytes Mineralize in Culture: Similarities with Mineralization of Lamellar Bone

The mechanisms whereby bone mineralizes are unclear. To study this process, we used a cell line, MLO-A5, which has highly elevated expression of markers of the late osteoblast such as alkaline phosphatase, bone sialoprotein, parathyroid hormone type 1 receptor, and osteocalcin and will mineralize in sheets, not nodules. In culture, markers of osteocytes and dendricity increase with time, features of differentiation from a late osteoblast to an early osteocyte. Mineral formation was examined using transmission electron microscopy, scanning electron microscopy with energy-dispersive X-ray analysis, and atomic force microscopy. At 3–4 days of culture, spheres of approximately 20–50 nm containing calcium and phosphorus were observed budding from and associated with developing cellular projections. By 5–6 days, these calcified spheres were associated with collagen fibrils, where over time they continued to enlarge and to engulf the collagen network. Coalescence of these mineralized spheres and collagen-mediated mineralization were responsible for the mineralization of the matrix. Similar calcified spheres were observed in cultured fetal rat calvarial cells and in murine lamellar bone. We propose that osteoid-osteocytes generate spherical structures that calcify during the budding process and are fully mineralized on their developing cellular processes. As the cellular process narrows in diameter, these mineralized structures become associated with and initiate collagen-mediated mineralization.