A model for the ultrastructure of bone based on electron microscopy of ion-milled sections

The relationship between the mineral component of bone and associated collagen has been a matter of continued dispute. We use transmission electron microscopy (TEM) of cryogenically ion milled sections of fully-mineralized cortical bone to study the spatial and topological relationship between mineral and collagen. We observe that hydroxyapatite (HA) occurs largely as elongated plate-like structures which are external to and oriented parallel to the collagen fibrils. Dark field images suggest that the structures ("mineral structures") are polycrystalline. They are approximately 5 nm thick, 70 nm wide and several hundred nm long. Using energy-dispersive X-ray analysis we show that approximately 70% of the HA occurs as mineral structures external to the fibrils. The remainder is found constrained to the gap zones. Comparative studies of other species suggest that this structural motif is ubiquitous in all vertebrates.     

Transthyretin fibrillogenesis entails the assembly of monomers: a molecular model for in vitro assembled transthyretin amyloid-like fibrils

Extracellular accumulation of transthyretin (TTR) variants in the form of fibrillar amyloid deposits is the pathological hallmark of familial amyloidotic polyneuropathy (FAP). The TTR Leu55Pro variant occurs in the most aggressive forms of this disease. Inhibition of TTR wild-type (WT) and particularly TTR Leu55Pro fibril formation is of interest as a potential therapeutic strategy and requires a thorough understanding of the fibril assembly mechanism. To this end, we report on the in vitro assembly properties as observed by transmission electron microscopy (TEM), atomic force microscopy (AFM) and quantitative scanning transmission electron microscopy (STEM) for both TTR WT fibrils produced by acidification, and TTR Leu55Pro fibrils assembled at physiological pH. The morphological features and dimensions of TTR WT and TTR Leu55Pro fibrils were similar, with up to 300 nm long, 8 nm wide fibrils being the most prominent species in both cases. Other species were evident; 4-5 nm wide fibrils, 9-10 nm wide fibrils and oligomers of various sizes. STEM mass-per-length (MPL) measurements revealed discrete fibril types with masses of 9.5 and 14.0(+/-1.4) KDa/nm for TTR WT fibrils and 13.7, 18.5 and 23.2(+/-1.5) kDa/nm for TTR Leu55Pro fibrils. These MPL values are consistent with a model in which fibrillar TTR structures are composed of two, three, four or five elementary protofilaments, with each protofilament being a vertical stack of structurally modified TTR monomers assembled with the 2.9 nm axial monomer-monomer spacing indicated by X-ray fibre diffraction data. Ex vivo TTR amyloid fibrils were examined. From their morphological appearance compared to these, the in vitro assembled TTR WT and Leu55Pro fibrils examined may represent immature fibrillar species. The in vitro system operating at physiological pH for TTR Leu55Pro and the model presented for the molecular arrangement of TTR monomers within fibrils may, therefore, describe early fibril assembly events in vivo.     

Collagen II containing a Cys substitution for Arg-alpha1-519. Analysis by atomic force microscopy demonstrates that mutated monomers alter the topography of the surface of collagen II fibrils.

A recombinant human procollagen II was prepared that contained a substitution of Cys for Arg at alpha1-519 and that was found in five families with early onset generalized osteoarthritis with or without features of a mild chondrodysplasia. Previously, the presence of mutated monomers in mixtures with wildtype collagen II was shown to increase the lag period for fibril assembly. Also, the fibrils were more loosely packed and some thick fibrils lacked a D-periodic banding pattern. Here we re-examined the fibrils using a combination of transmission electron microscopy and atomic force microscopy. The presence of the mutated monomers increased the diameter of the thin filaments that were consistently formed in association with the thick fibrils of collagen II. In addition, the presence of the mutated monomers increased the depth of the gap regions in all fibrils with a distinct D-periodic banding pattern. The results, therefore, may indicate that the mutated monomers formed two or three additional outer layers of monomers in 0D-period staggers on the surface of the fibrils. Apparently, the mutated monomers were bound on the surface through intermolecular disulfide bonds.     

Two forms of apatite deposited during mineralization of the hen tendon.

Old hen tendon provides a model suitable for the study of calcification in an extracellular matrix. In the present study, we observed the mineralizing substances of hen tendon by scanning electron microscopy of plasma-osmium-coated specimens and by transmission electron microscopy of those processed by a plasma-polymerization film replica method. The mineralizing front area revealed a number of elliptical particles fused to each other and forming rod-like structures oriented parallel to collagen fibrils. The area of advanced mineralization possessed non-mineralizing cavities, in which tendon cells were likely to exist. At this site, we recognized a second form of mineral structure, one in which the crystals had a scale-like morphology and were deposited onto the major first-form mineral component. This crystal form was similar to hydroxyapatite synthesized under wet reaction conditions. These findings strongly suggest that the second form of mineral formed independent of collagen fibrils existed together with the predominant, collagen-dependent form of mineral. We speculate that cell membranes and an extremely slow mineralization process may contribute to the formation of this form of mineral during the mineralization process in the hen tendon.     

Microstructural Features of Non-Union of Human Humeral Shaft Fracture

Microstructures of non-unions of human humeral shaft fractures were investigated by using scanning electron microscopy, transmission electron microscopy, and X-ray microdiffraction. The non-union has a trabeculae structural framework similar to woven bone. Among the trabeculae are cavities that are subdivided into small chambers by thin plates of collagen fibrils. Some chambers are filled with variously shaped mineralized particles several micrometers in size. The collagen fibrils in both the trabeculae and the thin plates were only slightly mineralized by hydroxyapatite. Vesicles loaded with noncrystalline calcium phosphate (NCP) were observed in most mineralized particles, and brushite crystals with special morphology were seen to be embedded in some particles in irregular shapes. X-ray microdiffraction results indicated that the mineral phases in the non-unions were mainly NCP in addition to small amounts of hydroxyapatite and brushite. NCP deposition and insufficient mineralization of the collagen fibrils may be two important microstructural features of the non-unions of human humeral shaft fractures different from normally repaired bone callus.     

Polymer manipulation and nanofabrication in real time using transmission electron microscopy

Here we present time-resolved in situ transmission electron microscopy (TEM) observations and real-time manipulation of nematic ordered cellulose and ultradrawn polyethylene films. Drawn films of these two polymers exhibited a unique response to the low-dose electron beam. Electron beam damage was minimal based on retention of an organized electron diffraction pattern. Increased electron dosage appeared to melt the polymer with subsequent movement and attraction toward preferred electron concentrations within the beam. This discovery allowed the preferential, directed manipulation of polymer chain aggregates in two dimensions. These findings provide a basis for a new technique to manipulate and simultaneously observe dynamic assembly at the molecular level of structures using TEM.     

Amyloid fibril formation from crude protein mixtures

Amyloid fibrils have potential as bionanomaterials. A bottleneck in their commercial use is the cost of the highly purified protein typically needed as a starting material. Thus, an understanding of the role of heterogeneity in the mixtures from which amyloid fibrils are formed may inform production of these structures from readily available impure starting materials. Insulin, a very well understood amyloid-forming protein, was modified by various reagents to explore whether amyloid fibrils could still form from a heterogeneous mixture of insulin derivatives. Aggregates were characterized by thioflavin T fluorescence and transmission electron microscopy. Using acetylation, reduction carboxymethylation, reduction pyridylethylation, trypsin digestion and chymotrypsin digestion, it was shown that amyloid fibrils can form from heterogeneous mixtures of modified insulin. The modifications changed both the rate of reaction and the yield of the final product, but led to fibrillar structures, some with interesting morphologies. Well defined, long, unbranched fibrils were observed in the crude reduced carboxymethylated insulin mixture and the crude reduced pyridylethylated insulin revealed the formation of "wavy" fibrils, compared with the straighter native insulin amyloid fibrils. Although trypsin digestion inhibited fibrils formation, chymotrypsin digestion of insulin produced a mixture of long and short fibrils under the same conditions. We conclude that amyloid fibrils may be successfully formed from heterogeneous mixtures and, further, that chemical modification may provide a simple means of manipulating protein fibril assembly for use in bionanotechnological applications, enabling some design of overall morphology in the bottom-up assembly of higher order protein structures from amyloid fibrils.     

Electron diffraction studies of the calcareous skeletons of bryozoans

Electron microscopy and electron diffraction were used to investigate mineral crystallites dissociated from the skeletal walls of six species belonging to the Bryozoa, a phylum of predominantly marine colony-forming invertebrate animals. Four cheilostome bryozoans (Flustra foliacea, Membranipora membranacea, Thalamoporella novaehollandiae and Cellarinella foveolata) and two cyclostomes (Fasciculipora ramosa and Hornera robusta) were analysed. In each case, an attempt was made to relate the crystal morphology imaged in situ by scanning electron microscopy with the crystallographic orientation of isolated crystals determined by electron diffraction analysis in the transmission electron microscope. The results showed that the calcitic cheilostome and cyclostome skeletons consisted of closely packed arrays of plate-like Mg-containing calcite crystallites, and that the crystallographic a-axis was preferentially aligned perpendicular to the top and bottom surfaces of the flattened particles. The results suggest that calcite biomineralization occurs under similar crystallographic constraints in the five species studied even though the origins of cheilostomes and cyclostomes are separated by over 300 million years in the fossil record of the bryozoans. Similar studies for the aragonite crystallites in skeletons of M. membranacea indicated that the crystallographic b-axis was preferentially oriented perpendicular to the basal surfaces of irregular plate-like particles.     

A preliminary study of human pineal gland concretions: structural and chemical analysis

Acervuli and fragments of pineal gland obtained from 33 subjects of both sexes and age ranging from 1 to 87 years, (30 autopsy and 3 biopsy specimens) were analyzed by light microscopy, transmission and scanning electron microscopy, X-ray diffraction and X-ray energy dispersive microanalysis. It was found that primary mineralization occurs in an organic matrix formed by pinealocytes and that hydroxyapatite also takes place in mineral deposition. From our analysis, the formation of acervuli appears to be age and sex independent and can be possibly related to the secretory activity of the gland.     

Structure and composition of teleost scales from snakehead Channa argus (Cantor) (Perciformes: Channidae)

The structure of teleost scales from snakehead Channa argus was investigated using thermogravimetric analysis (TG), scanning electron microscopy (SEM), transmission electron microscopy (TEM), energy-dispersive analysis of X-rays (EDAX), Fourier transform infra-red spectroscopy (FTIR) and X-ray diffraction (XRD). Thermal treatment of fish scales indicates that the fibrillary plate is partially calcified. SEM shows two kinds of scale denticles, arranged along the circuli in the anterior field and the lateral fields, respectively. TEM indicates the stratum laxum with abundant fibrils, chromatophores and capillary blood vessels within the scale covering, and shows the fibrillary plate as an 'orthogonal plywood structure' of stratified lamellae, consisting of 80–100 nm diameter collagen fibres co-aligned in individual lamellae and alternated by c. 90° of the fibre alignment between adjacent lamellae. EDAX, FTIR and XRD show that the mineral phase of the scales is a carbonated hydroxyapatite with a Ca:P molar ratio of 1·85.     

Morphology-Dependent HIV-Enhancing Effect of Semen-Derived Enhancer of Viral Infection

PAP248–286 is a 39-residue fragment (residues 248 to 286) derived from protease cleavage of prostatic acidic phosphatase in semen. The amyloid fibrils formed in vitro by PAP248–286 can dramatically enhance human immunodeficiency virus (HIV) infection. To our knowledge, we present the first report that the HIV-enhancing potency of fibrils formed by PAP248–286 is morphology dependent. We identified pleomorphic fibrils by transmission electron microscopy in two buffer conditions. Our solid-state NMR data showed that these fibrils consist of molecules in distinct conformations. In agreement with NMR, fluorescence measurements confirmed that they are assembled along different pathways, with distinct molecular structures. Furthermore, our cell-based infectivity tests detected distinct HIV-enhancing potencies for fibrils in distinct morphologies. In addition, our transmission electron microscopy and NMR results showed that semen-derived enhancer of viral infection fibrils formed in sodium bicarbonate buffer remain stable over time, but semen-derived enhancer of viral infection fibrils formed in phosphate buffered saline keep evolving after the initial 7 days incubation period. Given time, most of the assemblies in phosphate buffered saline will turn into elongated thin fibrils. They have similar secondary structure but different packing than thin fibrils formed initially after 7 days incubation.     

Intrinsic linear heterogeneity of amyloid β protein fibrils revealed by higher resolution mass-per-length determinations

Amyloid β proteins spontaneously form fibrils in vitro that vary in their thermodynamic stability and in morphological characteristics such as length, width, shape, longitudinal twist, and the number of component filaments. It is vitally important to determine which variant best represents the type of fibril that accumulates in Alzheimer disease. In the present study, the nature of morphological variation was examined by dark-field and transmission electron microscopy in a preparation of seeded amyloid β protein fibrils that formed at relatively low protein concentrations and exhibited remarkably high thermodynamic stability. The number of filaments comprising these fibrils changed frequently from two to six along their length, and these changes only became apparent when mass-per-length (MPL) determinations are made with sufficient resolution. The MPL results could be reproduced by a simple stochastic model with a single adjustable parameter. The presence of more than two primary filaments could not be discerned by transmission electron microscopy, and they had no apparent relationship to the longitudinal twist of the fibrils. However, the pitch of the twist was strongly affected by the pH of the negative stain. We conclude that highly stable amyloid fibrils may form in which a surprising amount of intrinsic linear heterogeneity may be obscured by MPL measurements of insufficient resolution, and by the negative stains used for imaging fibrils by electron microscopy.     

Structure and texture of fibrous crystals formed by Alzheimer's abeta(11-25) peptide fragment

Amyloid fibril deposition is central to the pathology of Alzheimer's disease. X-ray diffraction from amyloid fibrils formed from full-length Abeta(1-40) and from a shorter fragment, Abeta(11-25), have revealed cross-beta diffraction fingerprints. Magnetic alignment of Abeta(11-25) amyloid fibrils gave a distinctive X-ray diffraction texture, allowing interpretation of the diffraction data and a model of the arrangement of the peptides within the amyloid fiber specimen to be constructed. An intriguing feature of the structure of fibrillar Abeta(11-25) is that the beta sheets, of width 5.2 nm, stack by slipping relative to each other by the length of two amino acid units (0.70 nm) to form beta ribbons 4.42 nm in thickness. Abeta(1-40) amyloid fibrils likely consist of once-folded hairpins, consistent with the size of the fibers obtained using electron microscopy and X-ray diffraction.     

Chitinous fibrils in the lorica of the flagellate chrysophyte Poteriochromonas stipitata (syn. Ochromonas malhamensis)

Ordered microfibrils are formed on the membrane of the cytoplasmic tail of the alga Poteriochromonas after attachment to a substrate. The ultrastructure of native and extracted stalk fibrils was studied with electron microscope methods. In addition, the structural polysaccharide was characterized by hydrolyses, separation of the monomers by thin- layer chromatography, gas-liquid chromatography and amino acid analysis, and by X-ray diffraction. The alkali-resistant fibrils yielded mostly glucosamine upon extensive hydrolysis, and showed X-ray diffraction patterns similar to those of fugal chitin. It is concluded that the resistant core of the fibrils is chitinous.     

The glucan–chitin complex in Saccharomyces cerevisiae. IV. The electron diffraction of crustacean and yeast cell wall chitin

Crustacean and yeast cell wall chitin were analyzed by means of transmission electron microscopy and selected-area diffraction. Single fibrils 8–25 nm wide have been observed in the micrographs of crustacean chitin. Analysis of a series of diffraction patterns obtained from thin crustacean chitin platelets yielded results which were in a better agreement with the theoretical structural model than those measured earlier. In this respect electron diffraction is shown to be superior to the more commonly used x-ray diffraction. Yeast cell wall chitin had a less perfect structure than the crustacean chitin. Single fibrils were not observed on the micrographs and electron diffraction patterns did not show any preferred fiber orientation. The evaluation of electron-diffraction patterns of both the primary septum and the adjacent circular zone of scar ring led to the conclusion that α-chitin is present in both these parts of the mother bud scar.     

Conformational Switching in PolyGln Amyloid Fibrils Resulting from a Single Amino Acid Insertion

The established correlation between neurodegenerative disorders and intracerebral deposition of polyglutamine aggregates motivates attempts to better understand their fibrillar structure. We designed polyglutamines with a few lysines inserted to overcome the hindrance of extreme insolubility and two D-lysines to limit the lengths of β-strands. One is 33 amino acids long (PolyQKd-33) and the other has one fewer glutamine (PolyQKd-32). Both form well-dispersed fibrils suitable for analysis by electron microscopy. Electron diffraction confirmed cross-β structures in both fibrils. Remarkably, the deletion of just one glutamine residue from the middle of the peptide leads to substantially different amyloid structures. PolyQKd-32 fibrils are consistently 10–20% wider than PolyQKd-33, as measured by negative staining, cryo-electron microscopy, and scanning transmission electron microscopy. Scanning transmission electron microscopy analysis revealed that the PolyQKd-32 fibrils have 50% higher mass-per-length than PolyQKd-33. This distinction can be explained by a superpleated β-structure model for PolyQKd-33 and a model with two β-solenoid protofibrils for PolyQKd-32. These data provide evidence for β-arch-containing structures in polyglutamine fibrils and open future possibilities for structure-based drug design.     

Ten to fourteen residue peptides of Alzheimer's disease protein are sufficient for amyloid fibril formation and its characteristic xray diffraction pattern

The molecular basis of fibril formation in Alzheimers disease was explored by electron micrographic and x-ray diffraction analysis of a series of synthetic peptides corresponding to portions of the amino acid sequence of beta protein and that of its putative precursor. A minimum 14 residue peptide was identified that formed typical amyloid fibrils under physiological conditions. Of these 14 residues, 10 were sufficient to give an identical 4.76 A and 10.6 A diffraction pattern as that recently described for isolated neurofibrillary tangles, amyloid plaque cores and leptomeningeal amyloid fibrils.     

Apolipoprotein C-II amyloid fibrils assemble via a reversible pathway that includes fibril breaking and rejoining

Alzheimer's and several other diseases are characterized by the misfolding and assembly of protein subunits into amyloid fibrils. Current models propose that amyloid fibril formation proceeds via the self-association of several monomers to form a nucleus, which then elongates by the addition of monomer to form mature fibrils. We have examined the concentration-dependent kinetics of apolipoprotein C-II amyloid fibril formation and correlated this with the final size distribution of the fibrils determined by sedimentation velocity experiments. In contrast to predictions of the nucleation-elongation model, the final size distribution of the fibrils was found to be relatively independent of the starting monomer concentration. To explain these results, we extended the nucleation-elongation model to include fibril breaking and rejoining as integral parts of the amyloid fibril assembly mechanism. The system was examined under conditions that affected the stability of the mature fibrils including the effect of dilution on the free pool of monomeric apolipoprotein C-II and the time-dependent recovery of fibril size following sonication. Antibody-labelling transmission electron microscopy studies provided direct evidence for spontaneous fibril breaking and rejoining. These studies establish the importance of breaking and rejoining in amyloid fibril formation and identify prospective new therapeutic targets in the assembly pathway.     

Growth of Collagen Fibril Seeds from Embryonic Tendon: Fractured Fibril Ends Nucleate New Tip Growth

Collagen fibrils are the principal tensile element of vertebrate tissues where they occur in the extracellular matrix as spatially organised arrays. A major challenge is to understand how the mechanisms of nucleation, growth and remodelling yield fibrils of tissue-specific diameter and length. Here we have developed a seeding system whereby collagen fibrils were isolated from avian embryonic tendon and added to purified collagen solution, in order to characterise fibril surface nucleation and growth mechanisms. Fragmentation of tendon in liquid nitrogen followed by Dounce homogenisation generated fibril length fragments. Most (> 94%) of the fractured ends of fibrils, which show an abrupt square profile, were found to act as nucleation sites for further growth by molecular accretion. The mechanism of this nucleation and growth process was investigated by transmission electron microscopy, atomic force microscopy and scanning transmission electron microscopy mass mapping. Typically, a single growth spur occurred on the N-terminal end of seed fibrils whilst twin spurs frequently formed on the C-terminal end before merging into a single tip projection. The surface nucleation and growth process generated a smoothly tapered tip that achieved maximum diameter when the axial extension reached ∼ 13 μm. Lateral growth also occurred along the entire length of all seed fibrils that contained tip projections. The data support a model of collagen fibril growth in which the broken ends of fibrils are nucleation sites for propagation in opposite axial directions. The observed fibril growth behaviour has direct relevance to tendon matrix remodelling and repair processes that might involve rupture of collagen fibrils.     

Microstructures of External Periosteal Callus of Repaired Femoral Fracture in Children

In order to understand further the mechanism of bone fracture repair, and thus to innovate better operative treatment for bone fracture and to design new implant materials for bone repair, microstructures of external periosteal callus (EPC) of repaired femoral fracture in both children and adults were investigated by using a scanning electron microscope, transmission electron microscopy, and an X-ray microdiffractometer. The repair time after the fractures in children and adults is on average 155 and 370 days, respectively. Collagen fibrils making up children's EPC (CEPC) are underdeveloped and insufficiently mineralized by hydroxyapatite (HA), while those from adults' EPC (AEPC) are similar to normal bone. A lot of particles loaded by brushite (DCPD) minerals were found among the collagen fibrils of CEPC. The main mineral phases in CEPC consist of DCPD and HA, while only HA exists in AEPC. Deposition of DCPD minerals could have compensated for the insufficient mineralization of the collagen fibrils of CEPC, thereby making fractured bone repair more rapidly in children than in adults.