Diameter distributions of collagenous tissues in relation to sex. A quantitative ultrastructural study

A number of factors have been implicated in the regulation of collagen fibril diameter. Electron microscopy analysis was used to investigate the role of sex on fibril diameter. Female mouse skin collagen fibrils' mean diameter values were significantly smaller than those from the male, independent of age. In addition, the female rabbit collagen fibrils had a marked decrease in the mean diameter compared to male, in all the tissues examined (skin, liver, and bone). These data suggest that the collagen fibril diameter is related to sex.     

The role of collagen in bone structure: an image processing approach

Bone collagen structure in normal and pathological tissues is illustrated using techniques of thin section transmission electron microscopy and computer-assisted analysis. The normal bone collagen types, fibril architecture and diameter are described. In pathological tissue, deviations from normal fine structure are reflected in abnormal arrangements of collagen fibrils and abnormalities in fibril diameter. Computer analyses of normal fibril positive staining patterns are presented in order to provide a basis for comparing such patterns with pathological ones.     

Computer-assisted quantification of the multi-scale structure of films made of nanofibrillated cellulose

Films made of nanofibrillated cellulose (NFC) are most interesting for use in packaging applications. However, in order to understand the film-forming capabilities of NFC and their properties, new advanced methods for characterizing the different scales of the structures are necessary. In this study, we perform a comprehensive characterisation of NFC-films, based on desktop scanner analysis, scanning electron microscopy in backscatter electron imaging mode (SEM-BEI), laser profilometry (LP) and field-emission scanning electron microscopy in secondary electron imaging mode (FE-SEM-SEI). Objective quantification is performed for assessing the (i) film thicknesses, (ii) fibril diameters and (iii) fibril orientations, based on computer-assisted electron microscopy. The most frequent fibril diameter is 20–30 nm in diameter. A method for acquiring FE-SEM images of NFC surfaces without a conductive metallic layer is introduced. Having appropriate characterisation tools, the structural and mechanical properties of the films upon moisture were quantified.     

Collagen fibril formation in poly(vinyl alcohol) and poly(vinyl pyrrolidone) films

The formation of collagen fibrils in poly(vinyl alcohol) (PVA) and in poly(vinyl pyrrolidone) (PVP) was investigated using Atomic Force Microscopy (AFM). The water solutions of PVA and PVP containing 1%, 3% and 5% of collagen were cast onto glass plate. After slow solvent evaporation thin polymeric films were obtained. AFM images showed the fibril formation in both, PVA and PVP films containing collagen. The amount of collagen in PVA and PVP matrix has an important effect on the structure and size of collagen fibril formed. The diameter of collagen fibrils in PVA films is bigger than the diameter of collagen fibrils formed in PVP films.     

Collagen structure and functional implications

The bio-mechanical requirements to which the connective tissue is subjected suggest that a causal correlation exist between the substructure and the collagen fibril function. We discuss the relationship between the inner structure of collagen fibrils, their diameter, their spatial layout and the functional requirements they have to withstand, and suggest that collagen fibrils may belong to two different forms indicated as "T-type" and "C-type". The first class, consisting of large, heterogeneous fibrils, parallely tightly packed, subjected to tensile stress along their axis is found in highly tensile structures such as tendons, ligaments and bone. The other class, consisting of small, homogeneous fibrils, helically arranged, resisting multidirectional stresses, is mostly present within highly compliant tissues such as blood vessel walls, skin and nerve sheaths. What causes these architectures to appear is discussed in detail in this review.     

Atomic Force Microscopy of bulk tendon samples: affect of location and fixation on tissue ultrastructure

Atomic Force Microscopy (AFM) is a surface characterisation technique which analyses topology. To date, AFM studies of tissue ultrastructure have focussed on single collagen fibrils extracted from different tissues prior to analysis. Using sample preparation techniques used in electron microscopy studies, this work uses AFM to analyse the collagen ultrastructure of bulk samples from bovine deep digital flexor tendons (DDFTs). DDFT ultrastructure in regions of the tendon which experience different loading conditions are compared. Samples are analysed post-freezing and post-aldehyde fixation with either 10% formalin or 4% glutaraldehyde in order to investigate the affect of tissue preservation on tissue ultrastructure. The results demonstrate that both fibril diameter and repeat unit of the tendon vary between different regions in the dorsoventral plane, with regions subjected to both tensile and compressive forces exhibiting smaller fibril diameter and repeat unit compared to regions subjected to tensile forces alone. These differences are detectable regardless of the tissue preservation technique used. However these measured differences do vary with preservation techniques with aldehyde-fixed samples exhibiting smaller fibril diameters and larger repeat units compared to frozen samples. These results demonstrate that AFM is a highly suitable technique for the characterisation of different ultrastructures in bulk samples but that it is important to be consistent in the choice of preservation technique.     

Structural variations of collagen in normal and pathological tissues: role of electron microscopy

The spectrum of ultrastructural appearances assumed by collagen in normal and pathological tissues is illustrated using techniques of thin section transmission electron microscopy and computer-assisted analysis. The normal fibrillar collagen types are described in order to provide a basis for comparing other normal and abnormal forms. In normal tissues, the anchoring fibril and basal lamina (basement membrane) represent tissue structures largely containing collagen but differing significantly in organisation from normal types I to III fibrillar collagen. In pathological tissue, deviations from normal fine structure are reflected in abnormal aggregates of collagen fibrils (amianthoid and skeinoid fibres) and abnormalities in fibril diameter and cross-sectional profile. Fibrous and segment long-spacing collagen represent two further organisational variants of collagen, the former found widely in pathological tissues, the latter very rarely. Much remains to be discovered about these abnormal collagen variants-their mode of formation, the cells that produce them, and their roles. They also present a challenge for the collagen biologist formulating hypotheses of collagen fibril assembly and molecular organisation.     

Preferential Alignment of Hydroxyapatite Crystallites in Nanocomposites with Chemically Disintegrated Silk Fibroin

Hydroxyapatite (HAp) nanocrystals were prepared at room temperature by a coprecipitation method from Ca(OH)₂ and H₃PO₄, in the presence of chemically disintegrated silk fibroin (SF). Adsorbed amounts of cations on SF and crystallinity of HAp in the composite were increased by the chemical disintegration of SF higher order structure. Preferential alignment of c-axis of HAp crystallites along the longitudinal direction of ca. 150nm SF fibril was observed. These changes due to disintegration of SF were discussed in terms of the chemical interaction between HAp and SF. The resulted composite with preferential alignment of HAp nanocrystals is a good candidate as a starting material for bone substitutes.     

Resolution and reproducibility of measures of the diameter of small collagen fibrils by transmission electron microscopy--application to the rabbit corneal stroma

AIM: To assess the impact of measurements of different numbers of small collagen fibrils at different final magnification values on the resultant mean values for fibril diameter (FD). METHODS: Very high magnification (33,000x) electron micrographs were taken of the posterior-central zone of the corneal stroma from young adult rabbits (2 kg), printed at 46,000 or 50,000x, scanned at 400 d.p.i. and 35 mm slides prepared. These were projected to give final magnifications between 150,000x and 450,000x. An overlay of fibril outlines was prepared from the projected images and the fibril diameters (FD's) measured to within 0.5 mm. RESULTS: On the overlays, at different projection magnifications, the average FD's ranged from 4 to 13.5 mm to allow measures to be made at a real resolution of between 3.5 and 1 nm. Using a fixed sized region of interest (ROI) on the overlays, the average values of FD's ranged between 24.9 and 31.7 nm, and could vary (for any particular micrograph) by up to 3.6 nm according to the number of FD's measured (n=34-384/ROI). Using a fixed number of FD measures (n=100) at different projection magnifications yielded average FD values from different corneas of between 25.1 and 35.2 nm, which could vary by up to 4.3 nm depending on the magnification used. CONCLUSIONS: The results indicate that different average values for measures of fibril diameter of small collagen fibrils can easily be obtained according to the final magnification used and the number of fibrils measured, and that the overall averages can depend on whether the data sets are averaged or pooled. These aspects of the morphometry of small collagen fibrils therefore need to be carefully specified.     

显微红外光谱法研究豚鼠膝关节软骨下骨的增龄性改变

应用显微FTIR技术原位分析雌性Hartley豚鼠随月龄增加胫骨关节软骨下骨的化学变化。红外光谱测定三种月龄(1月、2月和3月)豚鼠软骨下3个不同吸收颜色的骨小梁区(a,b,c)和中央骨髓区(d)。结果显示：(1)随月龄增加,骨小梁总面积增加,与正常骨小梁谱图相似的a区减少,d区波形有逐渐向a区发展的趋势。(2)2月龄和3月龄的b和c区AmideⅢ红移以c区红移显著并且变成肩峰,代表核酸和多糖的吸收峰峰强是a区的7倍。(3)三种月龄的c区均出现了β型糖苷键吸收峰。(4)I_AmideⅠ/I_AmideⅡ在2月b区最大,3月各区最小,I_AmideⅢ/I_AmideⅡ 在 2月、3月由a到c依次降低,I_νsPO-₂/I_AmideⅡ在2月、3月的b和c区较相应a区高7倍以上。结果符合骨关节炎不同阶段软骨下骨的组织结构及化学组成的变化规律。初步研究表明,显微光谱成像技术结合FTIR可原位分析不同区域的软骨下骨小梁和骨髓的分子组成,为骨关节炎软骨下骨组织病理学的分子水平研究提供了可靠信息。     

Ultrasonic propagation properties of articular cartilage at 100 MHz

A pilot study on articular cartilage assessed the contributions of individual matrix components to ultrasound propagation. The influence of collagen fibril orientation and collagen cross linking was also assessed. Sections of adult bovine articular cartilage cut both parallel and perpendicular to the articular surface were examined using the scanning laser acoustic microscope (SLAM) operating at an ultrasonic frequency of 100 MHz. A set of samples was evaluated that had been sequentially treated by enzymes to (1) remove 85% of the chondroitin sulfate; (2) remove remaining glycosaminoglycans, glycoproteins, and other noncollagen proteins, leaving only the collagen fibril network; and (3) disrupt the collagen intermolecular cross links. Two striking observations were made: a profound effect of the "preferred" collagen fibril orientation on ultrasonic speed and a marked increase in attenuation coefficient when intermolecular cross links were broken in the collagen.     

Raman spectroscopy reveals differences in collagen secondary structure which relate to the levels of mineralisation in bones that have evolved for different functions

Bone is a composite material comprising a collagen fibril scaffold surrounded by crystals of carbonated‐hydroxyapatite mineral. It is well established that the relative proportions of mineral and collagen in mature bone are not definite and are adapted in order to ‘tune’ its mechanical properties. It is not known, however, how the mineral to collagen ratio is controlled. This paper uses Raman spectroscopy (which permits the probing of both the mineral and the collagen phases of bone) to explore the hypothesis that the control mechanism is related to the nature of the collagen and that bones with different levels of mineralisation have qualitatively different collagen. Raman spectra of functionally adapted bones with varying levels of mineralisation are presented and features that indicate the differences in the collagen's secondary structure (amide I band profiles) and post‐translational modification (hydroxyproline/proline ratios) are highlighted. The study demonstrates that Raman spectroscopy can provide a means to investigate the mechanisms that control the mineral to collagen ratio of bone. Understanding these mechanisms could pave the way towards the therapeutic alteration of the mineral to collagen ratio and, thus, the control of the mechanical properties of bone. Copyright © 2012 John Wiley & Sons, Ltd.     

Cholesterol binding to amyloid-beta fibrils: a TEM study

There is increasing interest in the role of brain cholesterol in Alzheimer's disease and the contribution of cholesterol to the formation of amyloid plaques. This paper presents a TEM study showing the binding of soluble 10 nm diameter cholesterol-PEG 600 micelles to amyloid-β_1-42 (Aβ_1-42) fibrils formed either in the presence of this cholesterol derivative or to preformed fibrils generated under four different fibrillogenesis conditions. Specimens negatively stained with uranyl acetate revealed that during 24 h fibrillogenesis at 37 °C the cholesterol-PEG micelles bound periodically to Aβ_1-42 protofibrils and apparently also formed a thin smooth unbroken coating on mature double helical fibrils. Preformed protofibrils, generated in water alone or in the presence of 0.1 mM cupric sulphate, also exhibited periodic binding of cholesterol-PEG micelles, indicating the inherently helical nature of the protofibril. Double helical mature Aβ_1-42 fibrils, generated in the presence of cholesterol microcrystals or hydrogen peroxide (1 mM), bound cholesterol-PEG micelles with no immediately apparent regularity and without creating a smooth coating. The differing capacities of the Aβ_1-42 protofibrils and mature double helical fibrils to bind cholesterol-PEG 600 may indicate differences in the accessibility of the micellar cholesterol to the purported Aβ_17-21 hydrophobic cholesterol-binding motif on the fibril surfaces.     

Biaxial Orientation Mechanism of Drawn Natural Rubber. II. Quantitative Analysis of the Biaxial Orientation

Natural rubber film was prepared from triple centrifuged latex. The film was uniaxially drawn at room temperature and the induced crystalline orientation was quantitatively studied by wide angle X‐ray diffraction. The intensity distribution of 200 and 120 reflections confirmed that the induced crystals have a biaxial orientation with the c‐axis parallel to the draw direction. The orientation of the a‐axis was evaluated by using an orientation function of the (200) plane. The function (F ₂₀₀) indicated that the crystalline a‐axis is parallel to the film surface depending on the draw ratio and on positions in the film. The experimental results are possible to explain by shish‐kebab‐like crystallization developed from a highly oriented fibril. The secondary crystallization grows perpendicularly to the draw direction along the a‐axis. The population of the secondary crystals is controlled by an ellipsoidal free space. The shape of the ellipsoid is changed by the fibril distribution depending on width and thickness of the sample film. In this study, the quantitative biaxial orientation is consistently explained by the shish‐kebab‐like crystallization and the ellipsoidal free space.     

Ultrastructural properties of the Theromyzon (Annelida: Hirudinae) cocoon membrane

The cocoon of the leech Theromyzon trizonare consists of fibrils packed into an arrangement that produces both C- and S-like patterns of bow-shaped lines in sections oblique through the membrane. Sections normal to the cocoon membrane show layers containing cross-sections of fibrils (approximately 16 nm dia.) that are separated by a center-to-center distance of approximately 23 nm. In cross-section, each fibril presents a central hole approximately 5 nm in diameter. A structureless layer covers most of the exterior surface of the cocoon membrane, and short protuberances are apparent in some zones.     

Magnetic properties of maghemite nanoparticle systems: surface anisotropy and interparticle interaction effects

The static and dynamic magnetic properties of powders of maghemite nanoparticles with average diameter D=2.7, 4.6 and 8.7 nm have been investigated by magnetisation, AC susceptibility at variable frequency (5<ν<10⁴ Hz) and Mössbauer spectroscopy measurements. The results provide an insight into the correlation between intra-particle and inter-particle effects.     

Assessment of bone structure and acoustic impedance in C3H and BL6 mice using high resolution scanning acoustic microscopy

Two hundred-MHz time-resolved scanning acoustic microscopy was applied for the investigation of acoustic and structural bone properties of mice from two inbred strains. Transverse sections of femur taken from 5 C57BL/6J@Ico and 5 C3H/HeJ@Ico mice were explored. Both strains had the same bone diameter, but the C3H/HeJ@Ico mice had greater cortical thickness, smaller cancellous diameter, and greater acoustic impedance values than C57BL/6J@Ico mice. The strong differences in the measured acoustic impedances among the two inbred strains indicate that the impedance is a good parameter to detect genetic variations of the skeletal phenotype in small animal models.     

Vertebrae length and ultra-structure measurements of collagen fibrils and mineral content in the vertebrae of lordotic gilthead seabreams (Sparus aurata)

Skeletal deformities of gilthead seabream (Sparus aurata) are a major factor affecting the production cost, the external morphology and survival and growth of the fish. Adult individuals of S. aurata were collected from a commercial fish farm in Greece and were divided into two groups: one with the presence of lordosis, a skeletal deformity, and one without any skeletal deformity. Fishes were X-rayed, and cervical, abdominal and caudal vertebrae lengths were measured. Vertebrae were taken from the site of the vertebral column where lordosis occurred. One part was decalcified and prepared for collagen examination with transmission electron microscopy, and the rest were incinerated, and the Ca and P contents were measured. The stoichiometries of the samples were obtained by EDS (Energy Dispersive Spectroscopy). The same procedure was followed for fish without skeletal deformities (vertebrae were taken from the middle region of the vertebral column). The decalcified vertebrae parts were examined with TEM, collagen micrographs were taken and the fibrils’ periods and diameters were measured. There were no significant differences for both Ca and P or the collagen fibrils’ periods between the two fish groups. The mean lengths of the cervical, abdominal and caudal vertebrae where lordosis occurred were similar to the lengths of the respective regions of the individuals without the skeletal deformity. The TEM examination showed a significantly smaller mean vertebrae collagen fibril diameter from the fishes with lordosis compared with those from the controls, revealing the significance of collagen to bone structure.     

低盐和高盐环境下α-Synuclein构象的~(19)F NMR研究

天然无结构的突触核蛋白α-synuclein(AS)与帕金森病密切相关.最近研究发现低盐与高盐环境下AS纤维化的速率不同,所形成的纤维结构,细胞毒性与传染性也不一样,但盐效应对AS聚集及纤维结构影响的具体分子机制仍不清楚.该文通过生物标记方法在AS的酪氨酸芳香环上引入19F标记的探针,利用19F核磁共振(NMR)方法研究了低盐与高盐环境下AS的构象差异,发现19F NMR对天然无结构蛋白构象变化非常灵敏,AS在低盐中的构象比较紧密,而在高盐下比较松散,这种在溶液中起始的构象差异可能是导致最终AS纤维结构与生物效应不同的原因.