Quantitative determination of the mineral distribution in different collagen zones of calcifying tendon using high voltage electron microscopic tomography

High voltage electron microscopic tomography was used to make the first quantitative determination of the distribution of mineral between different regions of collagen fibrils undergoing early calcification in normal leg tendons of the domestic turkey, Meleagris gallopavo. The tomographic 3-D reconstruction was computed from a tilt series of 61 different views spanning an angular range of +/- 60 degrees in 2 degrees intervals. Successive applications of an interactive computer operation were used to mask the collagen banding pattern of either hole or overlap zones into separate versions of the reconstruction. In such 3-D volumes, regions specified by the mask retained their original image density while the remaining volume was set to background levels. This approach was also applied to the mineral crystals present in the same volumes to yield versions of the 3-D reconstructions that were masked for both the crystal mass and the respective collagen zones. Density profiles from these volumes contained a distinct peak corresponding only to the crystal mass. A comparison of the integrated density of this peak from each profile established that 64% of the crystals observed were located in the collagen hole zones and 36% were found in the overlap zones. If no changes in crystal stability occur once crystals are formed, this result suggests the possibilities that nucleation of mineral is preferentially and initially associated with the hole zones, nucleation occurs more frequently in the hole zones, the rate of crystal growth is more rapid in the hole zones, or a combination of these alternatives. All lead to the conclusion that the overall accumulation of mineral mass is predominant in the collagen hole zones compared to overlap zones during early collagen fibril calcification.     

An X-ray diffraction analysis of rat tail tendons treated with Cupromeronic Blue

Cupromeronic Blue was used to stain selectively the proteoglycans in rat tail tendons under ‘critical electrolyte’ conditions. Earlier electron microscopical observations indicated that at least one type of proteoglycan filament is associated with tendon collagen fibrils at the positive staining band ‘d’. To ensure that this was not an artefact caused by specimen preparation or the subsequent positive staining of the collagen fibrils, we have analysed low angle meridional diffraction patterns from stained but not dehydrated, embedded or counterstained tissues. Axial electron density profiles of Cupromeronic Blue-stained compared with unstained rat tail tendons revealed the axial locations and relative amounts of dye in both mature and young wet specimens. In mature tendons, the difference electron density profile contained a broad peak centred near residue 180 along the 234-residue D-period. This corresponds to the electron-optical staining band ‘d’. In young tendons a similar distribution of stain was observed although in this case there was evidence of a doublet of peaks, one centred near residue 182 (band ‘d’) and the other near residue 165 (midway between bands d and e₁). The wet proteoglycan-Cupromeronic Blue complexes distribute over about 30 nm along the collagen fibril axis. Comparison with the images of filaments seen in the electron microscope suggests that the dye complexes collapse significantly on dehydration and embedding.     

Irregular Shaped,Assumably Semi‐Crystalline Calciumphosphate Platelet Deposition at the Mineralization Front of Rabbit Femur Osteotomy: A HR‐TEM Study

Although bone minerals have been widely studied by various techniques in previous studies, crystal structures, morphology of bone minerals and its building pathway remained still controversy. In this work, the ultrastructure of the mineralization front of rabbit femur has been studied by conventional and high‐resolution (HR) transmission electron microscopy (TEM). In order to induce a healing and demineralization process the animals were subjected to a standardized osteotomy stabilized with titan screws and sonic pins. After 84 days follow‐up time the newly build bone was investigated. The mineralization front of rabbit femur osteotomy contains partly mineralized collagen fibrils with a pronounced striped pattern together with a large number of agglomerated apatite platelets. The striation is caused by mineralization in the hole zones of the collagen fibrils, corresponding to the early stage of mineralization. In the TEM micrographs, the mineralization zone appears denser and compact when compared with fully mineralized bone, although most of the collagen fibrils are completely mineralized in the latter (higher concentration of interfibrillar apatite platelets within the mineralization zone). In bone some partly mineralized collagen fibrils are also observed, revealing the same arrangement, regular shape, and size of apatite platelets as collagen fibrils in the mineralization zone. Apatite platelets with irregular shapes are observed at the vortex‐shaped outer boundary of the mineralization zone, i.e. at the interfaces with nonmineralized collagen or osteoblasts. HR TEM micrographs reveal that the platelets are assumably semicrystalline and that within the platelet nanocrystalline domains of apatite are embedded in an amorphous calciumphosphate matrix. SCANNING 35: 169‐182, 2013. © 2012 Wiley Periodicals, Inc.     

AFM and STM study of beta-amyloid aggregation on graphite

Atomic force microscopy (AFM) and scanning tunneling microscopy (STM) have been employed in situ and ex situ to directly study the aggregation of beta-amyloid(1-42) (Abeta42) peptide on hydrophobic graphite.From in situ AFM images, Abeta42 peptides were seen to aggregate into the sheets that preferred to three orientations with characteristic 3-fold symmetry (Proc. Natl. Acad. Sci. USA 96 (1999) 3688). The sheets were formed by parallel narrow lines with a height of 0.8-1.0nm and a width of 12-14nm. The narrow lines looked like beaded chains and have a right-handed axial periodicity.The high-resolution ex situ AFM and STM images showed that some fibrils of beta-amyloid had a characteristic domain texture, indicating they were formed through the association of protofibrils and monomers. The fibril containing lateral associated filaments that exhibited right-handed twist was clearly observed in the STM image.These results provide important clues to study the detailed structure of beta-amyloid aggregates and the mechanism of the Abeta fibrils formation on hydrophobic surface.     

Papain‐gel degrades intact nonmineralized type I collagen fibrils

Papain‐gel has been utilized as a chemo‐mechanical material for caries removal due to its ability to preserve underlying sound dentin. However, little is known about the effect of the papain enzyme on intact type I collagen fibrils that compose the dentin matrix. Here we sought to define structural changes that occur in intact type I collagen fibrils after an enzymatic treatment with a papain‐gel. Intact and nonmineralized type I collagen fibrils from rat tail were obtained and treated with a papain‐gel (Papacarie) for 30 s, rinsed with water and imaged using an atomic force microscope (AFM). Additionally, polished healthy dentin specimens were also treated using the same protocol described above and had their elastic modulus (E) and hardness (H) measured by means of AFM‐based nanoindentation. AFM images showed that the papain‐gel induced partial degradation of the fibrils surface, yet no rupture of fibrils was noticed. The distinction between gap and overlap zones of fibrils vanished in most regions after treatment, and overlap zones appeared to be generally more affected. Mechanical data suggested a gradual decrease in E and H after treatments. A significant two‐fold drop from the values of normal dentin (E=20±1.9, H=0.8±0.08 GPa) was found after four applications (E=9.7±3.2, H=0.24±0.1 GPa) (P<0.001), which may be attributed to the degradation of proteoglycans of the matrix. In summary, this study provided novel evidence that intact nonmineralized type I collagen fibrils are partially degraded by a papain‐gel. SCANNING 31: 253–258, 2009. © 2010 Wiley Periodicals, Inc.     

Assessment of collagen fibril spacing in relation to selected region of interest (ROI) on electron micrographs--application to the mammalian corneal stroma

AIMS: To evaluate measurements of collagen fibril spacing using different shaped regions of interest (ROI) on transmission electron micrograph (TEM) images of rabbit corneal stroma. METHODS: Following glutaraldehyde fixation and phosphotungstic acid staining, TEM images of collagen fibrils in cross section were projected at a final magnification close to 250,000 × to obtain overlays. Interfibril distances (IFDs; center‐to‐center spacing) were measured within different ROIs of the same nominal area (0.25 μm²) but different shape (with the length to width, L:W, ratio from 1:1 to 6:1). The IFD distribution was analyzed, and the 2D organization assessed using a radial distribution analysis. RESULTS: The fibrils had an average diameter of 35.3 ± 3.8 (SD) nm, packing density of 393 ± 4 fibrils / μm² and a fibril volume fraction of 0.39 ± 0.02. IFDs ranged from 29 to 1400 nm depending on the shape of the ROI, with average values ranging from 263 to 443 nm. By artificially selecting IFD data only to a radial distance of 250 nm, the average IFDs were just 145–157 nm. The radial distributions, to 250 nm, all showed a nearest neighbors first peak which shifted slightly from predominantly at 45–54 nm with more rectangular ROIs. The radial distribution profiles could be shown to be statistically different if the ROI L:W ratio was 2:1 or greater. CONCLUSION: Selection of an ROI for assessment of packing density and interfibril distances should be standardized for comparative assessments of TEMs of collagen fibrils. Microsc. Res. Tech., 2011. © 2011 Wiley‐Liss, Inc.     

Imaging and 3D morphological analysis of collagen fibrils

The recent booming of multiphoton imaging of collagen fibrils by means of second harmonic generation microscopy generates the need for the development and automation of quantitative methods for image analysis. Standard approaches sequentially analyse two-dimensional (2D) slices to gain knowledge on the spatial arrangement and dimension of the fibrils, whereas the reconstructed three-dimensional (3D) image yields better information about these characteristics. In this work, a 3D analysis method is proposed for second harmonic generation images of collagen fibrils, based on a recently developed 3D fibre quantification method. This analysis uses operators from mathematical morphology. The fibril structure is scanned with a directional distance transform. Inertia moments of the directional distances yield the main fibre orientation, corresponding to the main inertia axis. The collaboration of directional distances and fibre orientation delivers a geometrical estimate of the fibre radius. The results include local maps as well as global distribution of orientation and radius of the fibrils over the 3D image. They also bring a segmentation of the image into foreground and background, as well as a classification of the foreground pixels into the preferred orientations. This accurate determination of the spatial arrangement of the fibrils within a 3D data set will be most relevant in biomedical applications. It brings the possibility to monitor remodelling of collagen tissues upon a variety of injuries and to guide tissues engineering because biomimetic 3D organizations and density are requested for better integration of implants.     

A nanostructured look of collagen apatite porosity into human mineralized collagen fibril

Bone tissue is a hierarchical material characterized at nanoscale by the mineralized collagen fibril, a recurring structure mainly composed of apatite minerals, collagen and water. Bone nanostructure has a fundamental role in determining the mechanical behavior of the tissue and its mass transport properties. Diffusion phenomenon allows to maintain an adequate supply of metabolites in the mechanisms of bone remodeling, adaptation and repair. Several analytical and computational models have been developed to analyze and predict bone tissue behavior. However, the fine replication of the natural tissue still represents a challenge. Insights on the structural organization at nanoscale and on the influence of apatite mineral crystals on the diffusion coefficient lead to outline the functional conditions for the development of biomimetic strategies for bone tissue engineering. Thorough understanding of bone nanostructure is essential to improve longevity of bioscaffolds and to decrease the risk of failure by controlling their mechanical and biological performance.     

Nanostructural investigation of frontalis sling biomaterial surfaces

This study examined the nanostructural surface of three frontalis sling biomaterials: autogenous fascia lata, preserved fascia lata and silicone rod. The morphological characteristics of the sling biomaterial surfaces were examined qualitatively and quantitatively by scanning electron microscopy and atomic force microscopy, respectively. The autogenous fascia lata showed well‐arranged nanostructures of parallel fascia collagen fibrils with clear 67 nm axial periodicity, whereas the preserved fascia lata showed tangled nanostructures of damaged collagen fibril bundles. The silicone rod showed a substantial amount of debris with some scratches and the smoothest roughness compared with the other sling biomaterials, followed by preserved fascia lata. Autogenous fascia lata showed the highest surface roughness. The association between the roughness and cell adhesion suggests that the nanostructure of autogenous fascia lata biomaterials is the best for frontalis sling and that of the silicone rod biomaterials is the worst. SCANNING 33:419–425, 2011. © 2011 Wiley Periodicals, Inc.     

Orientation fields of nonlinear biological fibrils by second harmonic generation microscopy

The orientation of fibrils within biological tissues is of primary importance. In this study, we propose a simple method based on second harmonic generation (SHG) microscopy to map, pixel by pixel, the orientation of the symmetry axis of the second‐order nonlinear susceptibility tensor of fibrils that produce SHG. The method uses only four images acquired at specific polarizations of the input laser beam, and can be easily and cheaply implemented on a confocal microscope. In addition to orientation informations, the method also provides polarization independent images and estimations of the ratio of the nonlinear susceptibility components. We demonstrate the relevance of our concept by studying the orientation fields of the collagen meshwork in a healthy rat liver that provides well separated fibrils. By correlating the mean orientation of the nonlinear susceptibility to the fibril orientation itself for many fibril segments, and using circular statistics, it is shown that both orientations are truly parallel at the fibril scale. Our polarimetric method allows to map fibril orientation fields, independently of individual fibril contrast in the SHG image.     

Observation of the ultrastructure of anterior cruciate ligament graft by atomic force microscopy

This study presented the fibril ultrastructure of retrieved grafts from the reconstruction of anterior cruciate ligament (ACL) using atomic force microscopy (AFM). The tapping mode images of the AFM were taken from different areas of the longitudinally cut grafts. Regular arrangement of collagen fibrils was found in certain areas of the graft. In many areas, however, the fibrils were not well arranged in a single direction, with some smaller fibrils oriented vertically to larger parallel fibrils. The crossing and tangling of fibrils in ACL grafts was well represented in the three‐dimensional AFM image. This abnormality of graft ultrastructure might indicate the possible alteration of the mechanical environment after ACL reconstruction. This study demonstrated the suitability and importance of ultrastructure observation of ACL grafts by AFM. SCANNING 31: 19–23, 2009. © 2009 Wiley Periodicals, Inc.     

Mechanical and micromorphological evaluation of chlorhexidine-mediated dentin remineralization

Chlorhexidine (CHX) has been reported to reduce self-degradation of collagen fibrils by inhibiting host-derived protease activity in demineralized dentin. Theoretically, if the collagen fibril scaffold of demineralized dentin maintains its original crosslinkage pattern on treatment with CHX and appropriate supplementation with necessary mineral sources, dentin remineralization may occur in demineralized lesions. In this study, we provide direct mechanical and micromorphological evidence for the ability of CHX to promote remineralization of demineralized dentin. Specifically, with respect to demineralized dentin blocks treated with different concentrations of CHX (0.02-2%) and stored in simulated body fluid, we have observed a significant increase in the elastic modulus of dentin treated with relatively high concentrations of CHX (0.2 and 2%) as storage time increased, whereas the elastic modulus of the non-CHX treated control group decreased. We have also observed a dense mineral deposition along collagen fibrils in the dentin group treated with 0.2 and 2% CHX via field emission scanning electron microscopy.     

Imaging of collagen type III in fluid by atomic force microscopy

Type III collagen is a component of the basement membrane of endothelial cells, and may play a role in the interaction between hemostatic system proteins and the basement membrane of blood vessels. To begin to investigate these structural interactions, we have imaged type III collagen in solution by atomic force microscopy. A 20 microg/ml solution of type III collagen in bicarbonate buffer (pH 9.5) from calf skin was deposited onto a freshly cleaved mica substrate. Atomic force microscopy images were acquired using a fluid cell and tapping mode with oxide-sharpened silicon nitride probes 2, 3, and 4 hours after deposition of the collagen onto the mica. Two-hour preparations displayed fibrillar networks with well-defined sites of nucleation and lateral growth. At 3 and 4 hour polymerizations, more mature fibrils of increasing lengths, diameters, and complexity were observed. Fibrils appeared to be aligning and twisting (helical formation) to form a mature fibril with a higher mass per unit area. Interestingly, the mature fibrils appeared larger centrally with tapered ends displaying declining slopes. These observations compare favorably with those previously published on collagen type I assembly [Gale et al. (1995) Biophys. J. 68:2124-2128]. High resolution atomic force microscopy images of type III collagen in solution should provide a template for observation of the interactions between basement membrane components and hemostatic system proteins present in cardiovascular disease.     

Collagen types I, III, and V constitute the thick collagen fibrils of the mouse decidua

A mammal's endometrium is deeply remodeled while receiving and implanting an embryo. In addition to cell proliferation and growth, endometrial remodeling also comprises synthesis and degradation of several molecular components of the extracellular matrix. All of these events are orchestrated by a precise sequence of ovarian hormones and influenced by several types of cytokines. As we have previously reported, an intriguing and rapid increase in collagen fibril diameter occurs in the decidualized areas of the endometrium, surrounding the implantation crypt, whereas collagen fibrils situated far from the embryo remain unchanged. Collagen fibrilogenesis is a complex molecular process coordinated by a number of factors, such as the types and amounts of glycosaminoglycans and proteoglycans associated with collagen molecules. Collagen genetic type, mechanical stress, aging, and other factors not yet identified also contribute to this development. A recent study suggests that thick fibrils from mouse decidua are formed, at least in part, by aggregation of thin fibrils existing in the stroma before the onset of decidualization. In the present ultrastructural study using single and double immunogold localization, we showed that both thin and thick collagen fibrils present in the mouse pregnant endometrium endometrium are heterotypic structures formed at least by type I, type III, and type V collagens. However, type V collagen predominates in the thick collagen fibrils, whereas it is almost absent of the thin collagen fibrils. The putative role of type V homotrimer in the rapid increase of the diameter of collagen fibrils of the mouse decidua is discussed.     

The effect varied scanning electron microscopy desiccation techniques has on demineralized dentin

The study objective was to assess (a) the effect of a rubbing‐application of ethylenediaminetetraacetic acid (EDTA) or citric acid (CA) has on the ultrastructure of surface dentin and (b) the effect of two scanning electron microscopy (SEM) desiccation preparation techniques have on the collagen surface produced. Treatment regions on proximal root surfaces of extracted human teeth were root planned to expose dentin. Cotton pellets soaked in either 30% CA or 24% EDTA solution were rubbed on the treatment region then processed for SEM using one of two desiccation techniques, that is, (a) critically point dried from liquid CO₂ (control) or (b) air‐dried from tetramethylsilane (experimental). Specimens were coated with gold/palladium and viewed/photographed with an SEM. Specimens of the control groups displayed tufted fibrils (CA > EDTA) with many dentin tubules being partially obscured by overhanging fibrils. Air‐dried specimens of both treatment groups displayed a flat intact monolayer devoid of a matted meshwork of fibrous collagen. Discrete fibril “sprigs,” emanating from the surface monolayer, were characteristic of the EDTA group only. The rubbing‐application of EDTA on dentin produces a tufted fibril surface somewhat similar to that produced by CA. Air‐drying desiccation of both resulted in marked distortion with fibril collapse/coalescence of the tufted collagen matrix.     

Arrangement and fine structure of collagen fibrils in the decidualized mouse endometrium

The adaptations of the mouse uterus to pregnancy include extensive modifications of the cells and extracellular matrix of the endometrial connective tissue that surround the embryos. Around each implanted embryo this tissue redifferentiates into a transient structure called decidua, which is formed by polygonal cells joined by intercellular junctions. In the mouse, thick collagen fibrils with irregular profile appear in decidualized areas of the endometrium but not in the nondecidualized stroma and interimplantation sites. The fine organization of these thick fibrils has not yet been established. This work was addressed to understand the arrangement and fine structure of collagen fibrils of the decidua of pregnant mice during the periimplantation stage. Major modifications occurred in collagen fibrils that surrounded decidual cells: (1) the fibrils, which were arranged in parallel bundles in nonpregnant animals, became organized as baskets around decidual cells; (2) very thick collagen fibrils with very irregular profiles appeared around decidual cells. Analysis of replicas and serial sections suggests that the thick collagen fibrils form by the lateral aggregation of thinner fibrils to a central fibril resulting in very irregular profile observed in cross sections of thick fibrils. The sum of modifications of the collagen fibrils seem to represent an adaptation of the endometrium to better support the decidual cells while they hold the embryos during the beginning of their development. The deposition of thick collagen fibrils in the decidua may contribute to form a barrier that impedes leukocyte migration within the decidua, preventing immunological rejection of genetically dissimilar embryonic tissues.     

Postoperative effect of radiofrequency treatments on the rabbit dermal collagen fibrillary matrix

This study quantitatively examined the short and mid‐long term effects of radiofrequency (RF) treatment on the normal dermal collagen fibrils of live rabbits. Effects were evaluated by histology and scanning probe microscopy analysis of dermal tissues treated using three RF energy levels (10, 20, and 30 W) and either a single‐ or multiple‐pass procedure. Progressive changes in the morphology of rabbit dermal collagen fibrils were investigated over a 30‐day post‐treatment period. All RF‐treated groups, except for the low‐energy group (10 W), displayed more prominent inflammatory responses compared to the control. This inflammatory response was more prominent a day after treatment. Dermal tissues 30‐days after RF treatment exhibited prominent myofibroblast activity associated with collagen contractile activity during wound healing in addition to chronic inflammation. A decrease in the morphology of dermal collagen fibrils after RF treatment continued until seven days postoperatively. The collagen fibril diameter increased to near baseline at 30 days postoperatively. Low‐energy and multi‐pass treatments resulted in greater collagen fibril contraction and recovery at the nanostructural level at 30 days postoperatively than did a single high‐energy treatment. Microsc. Res. Tech. 76:219–224, 2013. © 2012 Wiley Periodicals, Inc.     

Age dependence of collagen fibril and subfibril diameters revealed by transverse freeze-fracture and -etching technique

Collagenous tissues taken from tail tendons and cornea of freshly killed rats and rabbits, respectively, were fractured transversely and longitudinally at 80 K without any chemical pretreatment. The tissues were subsequently etched at 195 K for 30 min under a vacuum in the 130 μPa range. Carbon-platinum replicas of the tissues were examined by transmission electron microscopy. It is demonstrated that the collagen fibrils are very closely packed and are composed of subfibrils whose size may vary with each tissue. Fibrils and subfibrils grow in diameter during growth and maturation but their size remains unchanged during the ageing process. The subfibril is suggested to be the growing unit in the collagen fibril based on the observation that the ratio of the diameter of fibril to subfibril does not change through growth, maturation and ageing. The ratio is approximately 10 for rat tail tendon and 5 for rabbit cornea and ear cartilage, suggesting the tissues from the two animals contain on the order of 100 and 25 subfibrils per fibril, respectively. It is proposed that the usual chemical fixation and dehydration processes used in conventional electron microscopy cause significant shrinkage in the diameters of subfibrils and fibrils.     

Atomic force microscopy of collagen structure in bone and dentine revealed by osteoclastic resorption

Mineralised tissues such as bone consist of two material phases: collagen protein fibrils, secreted by osteoblasts, form model structures for subsequent deposition of mineral, calcium hydroxyapatite. Collagen and mineral are removed in a three-dimensional manner by osteoclasts during bone turnover in skeletal growth or repair. Bone active drugs have recently been developed for skeletal diseases, and there is revived interest in changes in the structure of mineralised tissues seen in disease and upon treatment. The resolution of atomic force microscopy and use of unmodified samples has enabled us to image bone and dentine collagen exposed by the natural process of cellular dissolution of mineralised matrix. The morphology of bone and dentine has been analysed when fully mineralised and after osteoclast-mediated bone resorption, and compared with results from other microscopy techniques. Banded type I collagen, with 66.5+/-1.4 nm axial D-periodicity and 62.2+/-7.0 nm diameter, has been identified within resorption lacunae in bone and 69.4+/-4.3 nm axial D-periodicity and 140.6+/-12.4 nm diameter in dentine substrates formed by human and rabbit osteoclasts, respectively. This observation suggests a route by which the material and morphological properties of bone collagen can be analysed in situ, compared with collagen from non-skeletal sites, and contrasted in diseases of medical importance, such as osteoporosis, where skeletal tissue is mechanically weakened.