Structure and function of bone collagen fibrils

The intermolecular volume of fully hydrated collagen fibrils from a number of mineralized and non-mineralized tissues of adult rats has been determined both by an exclusion technique and by a method which involves the monitoring of specific X-ray diffraction parameters. The intermolecular volume of either bone or dentinal fibrils is approximately twice that of either tail or achilles tendon, and the most frequent intermolecular distance in bone or dentine fibrils is approximately 3 Å larger than of the tendons.A number of fibrillar structures are most compatible with the intermolecular volume of rat tail tendon. These include hexagonal molecular packing and orthogonal arrays of microfibrils comprising seven parallel molecular strands. The intermolecular volume of bone or dentinal collagen fibrils, on the other hand, appears to arise from structures having a disordered or pseudo-hexagonal molecular packing, in which the most frequent intermolecular distance is about 19 Å.The space associated with collagen fibrils in adult bone is such that 70 to 80% of the mineral is located within the intermolecular space of the fibrils—approximately equal amounts of mineral being in spaces having lateral dimensions of 25 to 75 Å and 6 to 12 Å, respectively. Particles located in the latter kind of intermolecular space probably constitute, to a large extent, the non-crystalline mineral phase of adult bone.The stereo-chemical constraints on the transport of mineral ions into and within collagen fibrils of bone and tendon support the postulate that bone collagen is an in vivo catalyst for mineral deposition and further suggests that its catalytic activity may be partially regulated through its molecular packing.     

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.     

Neutron diffraction studies of collagen in fully mineralized bone

Neutron diffraction measurements have been made of the equatorial and meridional spacings of collagen in fully mineralized mature bovine bone and demineralized bone collagen, in both wet and dry conditions. The collagen equatorial spacing in wet mineralized bovine bone is 1.24 nm, substantially lower than the 1.53 nm value observed in wet demineralized bovine bone collagen. Corresponding spacings for dry bone and demineralized bone collagen are 1.16 nm and 1.12 nm, respectively. The collagen meridional long spacing in mineralized bovine bone is 63.6 nm wet and 63.4 nm dry. These data indicate that collagen in fully mineralized bovine bone is considerably more closely packed than had been assumed previously, with a packing density similar to that of the relatively crystalline collagens such as wet rat tail tendon. The data also suggest that less space is available for mineral within the collagen fibrils in bovine bone than had previously been assumed, and that the major portion of the mineral in this bone must be located outside the fibrils.     

Hierarchical assembly and the onset of banding in fibrous long spacing collagen revealed by atomic force microscopy.

The mechanism of formation of fibrillar collagen with a banding periodicity much greater than the 67 nm of native collagen, i.e. the so-called fibrous long spacing (FLS) collagen, has been speculated upon, but has not been previously studied experimentally from a detailed structural perspective. In vitro, such fibrils, with banding periodicity of approximately 270 nm, may be produced by dialysis of an acidic solution of type I collagen and alpha(1)-acid glycoprotein against deionized water. FLS collagen assembly was investigated by visualization of assembly intermediates that were formed during the course of dialysis using atomic force microscopy. Below pH 4, thin, curly nonbanded fibrils were formed. When the dialysis solution reached approximately pH 4, thin, filamentous structures that showed protrusions spaced at approximately 270 nm were seen. As the pH increased, these protofibrils appeared to associate loosely into larger fibrils with clear approximately 270 nm banding which increased in diameter and compactness, such that by approximately pH 4.6, mature FLS collagen fibrils begin to be observed with increasing frequency. These results suggest that there are aspects of a stepwise process in the formation of FLS collagen, and that the banding pattern arises quite early and very specifically in this process. It is proposed that typical 4D-period staggered microfibril subunits assemble laterally with minimal stagger between adjacent fibrils. alpha(1)-Acid glycoprotein presumably promotes this otherwise abnormal lateral assembly over native-type self-assembly. Cocoon-like fibrils, which are hundreds of nanometers in diameter and 10-20 microm in length, were found to coexist with mature FLS fibrils.     

Observing growth steps of collagen self-assembly by time-lapse high-resolution atomic force microscopy

Insights into molecular mechanisms of collagen assembly are important for understanding countless biological processes and at the same time a prerequisite for many biotechnological and medical applications. In this work, the self-assembly of collagen type I molecules into fibrils could be directly observed using time-lapse atomic force microscopy (AFM). The smallest isolated fibrillar structures initiating fibril growth showed a thickness of approximately 1.5 nm corresponding to that of a single collagen molecule. Fibrils assembled in vitro established an axial D-periodicity of approximately 67 nm such as typically observed for in vivo assembled collagen fibrils from tendon. At given collagen concentrations of the buffer solution the fibrils showed constant lateral and longitudinal growth rates. Single fibrils continuously grew and fused with each other until the supporting surface was completely covered by a nanoscopically well-defined collagen matrix. Their thickness of approximately 3 nm suggests that the fibrils were build from laterally assembled collagen microfibrils. Laterally the fibrils grew in steps of approximately 4 nm, indicating microfibril formation and incorporation. Thus, we suggest collagen fibrils assembling in a two-step process. In a first step, collagen molecules assemble with each other. In the second step, these molecules then rearrange into microfibrils which form the building blocks of collagen fibrils. High-resolution AFM topographs revealed substructural details of the D-band architecture of the fibrils forming the collagen matrix. These substructures correlated well with those revealed from positively stained collagen fibers imaged by transmission electron microscopy.     

Early mineral deposition in calcifying tendon characterized by high voltage electron microscopy and three-dimensional graphic imaging.

Extracellular matrix organization and the spatial relationship between collagen fibrils, vesicular structures, and the first deposits of mineral in the calcifying leg tendon from the domestic turkey, Meleagris gallopavo, have been investigated by high voltage electron microscopy and three-dimensional computer graphic imaging of serial thick tissue sections. The work demonstrates that the tendon extracellular matrix is a complex assembly of somewhat flexible, highly aligned collagen fibrils with different diameters and occasionally opposite directionality. Smaller collagen fibrils appear to branch from larger fibrils or to aggregate to form those of greater size. While the matrices are dominated by fibrils, space exists between adjacent packed fibrils. The three-dimensional perspective indicates that approximately 60% of the total tendon volume is extrafibrillar over the regions examined. The first observable mineral in this tissue is extrafibrillar and appears to derive from vesicles. This view of three-dimensional matrix-mineral spatial relations supports earlier two-dimensional results that mineral is initially associated with membrane-invested vesicles and is deposited between collagen fibrils, but it is distinct in showing the mineral at different depths in the matrix rather than at a single depth as deduced from two-dimensional conventional electron microscopy. These results are important in the onset and development of tendon calcification in that they suggest, first, that collagen fibrils appear to be aligned three-dimensionally such that their hole zones are in contiguous arrangement. This situation may create channels or grooves within the collagen volume to accommodate extensive mineral deposition in association with the fibrils. Second, the results indicate that there are widely dispersed sites of vesicle-mediated mineralization in the tendon matrix, that the bulk of mineralization in this tissue is collagen-mediated, and that, while vesicles may possibly exert some local influence temporally on mineralization of neighboring collagen, vesicle- and collagen-mediated mineralization arise at spatially and structurally distinct sites by independent nucleation phenomena. Such concepts are fundamental in considerations of possible mechanisms of mineralization of tendon and potentially of other normally calcifying vertebrate tissues in general.     

Three-dimensional spatial relationship between the collagen fibrils and the inorganic calcium phosphate crystals of pickerel (Americanus americanus) and herring (Clupea harengus) bone

High-voltage (1.0 MV) electron microscopy and stereomicroscopy, electron probe microanalysis, electron diffraction and three-dimensional computer reconstruction, have been used to examine the spatial relationship between the inorganic crystals of calcium phosphate and the collagen fibrils of pickerel and herring bone. High-voltage stereo electron-micrographs were obtained of cross-sections of the cylinder-shaped intramuscular bones in uncalcified regions, in regions where only one or only several crystals had been deposited in some of the fibrils, and in successive sections containing progressively more mineral crystals until the stage of full mineralization was reached. High-resolution electron probe microanalysis confirmed that the electron-dense particles contained calcium and phosphorus. In the earliest stages of mineralization and progressing throughout the mineralization process, the crystals are located only within the collagen fibrils; crystals are not observed free in the extracellular spaces between collagen fibrils. The progressive increase in the mass of mineral deposited in the bone tissue with time occurs, essentially, completely within the collagen fibrils including the stage of full mineralization. At this stage, cross-sectional profiles of collagen fibrils are completely obliterated by mineral. A small number of crystals that are located on or close to the surface of the fibrils appear to extend a very short distance into the spaces between the fibrils. These ultrastructural observations of the very onset of calcification in which nucleation of the calcium phosphate crystals is clearly shown to begin within specific volumes of collagen fibrils, and of the subsequent temporal and spatial sequences of this phenomenon, which shows that calcification continues wholly within the collagen fibrils until maximum calcification is achieved, add important information on the basic physical chemical mechanism of the calcification and the structural elements that are involved. The spatial and temporal independence of the sites where mineralization is initiated establishes that such ultrastructural locations within individual collagen fibrils represent independent, physical chemical nucleation loci. The findings are totally inconsistent with the proposal that crystals must first be deposited in matrix vesicles, or other components such as mitochondria, and subsequently released and propagated in the interfibrillar space, until they eventually reach and impregnate the hole zone regions of the collagen fibrils. Three-dimensional computer reconstruction of serial transverse and longitudinal sections demonstrates periodic swellings along the collagen fibrils, corresponding to the hole zone region of their axial period as mineralization proceeds.(ABSTRACT TRUNCATED AT 400 WORDS)     

A comparative study of clinically well-characterized human atherosclerotic plaques with histological, chemical, and ultrastructural methods

Atherosclerotic plaques (six cases) with well-documented clinical history were analysed using histology, scanning electron microscopy (SEM), transmission electron microscopy (TEM), energy-dispersive X-ray (EDX) spectroscopy, infrared spectroscopy (IR), thermogravimetry (TG), and high-resolution synchrotron X-ray diffraction. All samples contained about 60-70 wt% biological carbonated apatite (in dry state) in a nanocrystalline form with particle sizes of about 20 nm. Structurally, there are strong similarities to bone mineral. Ultrastructural investigations documented typical calcospherites, mineralisation processes starting at collagen fibrils and ring-shaped crystalline mineralised structures. There were no significant ultrastructural or chemical differences between the calcifications of individual patients.     

Aspects of Mineral Structure in Normally Calcifying Avian Tendon

Structural characteristics of normally calcifying leg tendons of the domestic turkey Meleagris gallopavo have been observed for the first time by tapping mode atomic force microscopy (TMAFM), and phase as well as corresponding topographic images were acquired to gain insight into the features of mineralizing collagen fibrils and fibers. Analysis of different regions of the tendon has yielded new information concerning the structural interrelationships in vivo between collagen fibrils and fibers and mineral crystals appearing in the form of plates and plate aggregates. TMAFM images show numerous mineralized collagen structures exhibiting characteristic periodicity (54-70 nm), organized with their respective long axes parallel to each other. In some instances, mineral plates (30-40 nm thick) are found interspersed between and in intimate contact with the mineralized collagen. The edges of such plates lie parallel to the neighboring collagen. Many of these plates appear to be aligned to form larger aggregates (475-600 nm long x 75-90 nm thick) that also retain collagen periodicity along their exposed edges. Intrinsic structural properties of the mineralizing avian tendon have not previously been described on the scale reported in this study. These data provide the first visual evidence supporting the concept that larger plates form from parallel association of smaller ones, and the data fill a gap in knowledge between macromolecular- and anatomic-scale studies of the mineralization of avian tendon and connective tissues in general. The observed organization of mineralized collagen, plates, and plate aggregates maintaining a consistently parallel nature demonstrates the means by which increasing structural complexity may be achieved in a calcified tissue over greater levels of hierarchical order.     

Amelogenin-Collagen Interactions Regulate Calcium Phosphate Mineralization in Vitro

Collagen and amelogenin are two major extracellular organic matrix proteins of dentin and enamel, the mineralized tissues comprising a tooth crown. They both are present at the dentin-enamel boundary (DEB), a remarkably robust interface holding dentin and enamel together. It is believed that interactions of dentin and enamel protein assemblies regulate growth and structural organization of mineral crystals at the DEB, leading to a continuum at the molecular level between dentin and enamel organic and mineral phases. To gain insight into the mechanisms of the DEB formation and structural basis of its mechanical resiliency we have studied the interactions between collagen fibrils, amelogenin assemblies, and forming mineral in vitro, using electron microscopy. Our data indicate that collagen fibrils guide assembly of amelogenin into elongated chain or filament-like structures oriented along the long axes of the fibrils. We also show that the interactions between collagen fibrils and amelogenin-calcium phosphate mineral complexes lead to oriented deposition of elongated amorphous mineral particles along the fibril axes, triggering mineralization of the bulk of collagen fibril. The resulting structure was similar to the mineralized collagen fibrils found at the DEB, with arrays of smaller well organized crystals inside the collagen fibrils and bundles of larger crystals on the outside of the fibrils. These data suggest that interactions between collagen and amelogenin might play an important role in the formation of the DEB providing structural continuity between dentin and enamel.     

An axial periodic fibrillar arrangement of antigenic determinants for fibronectin and procollagen on ascorbate treated human fibroblasts

Fibronectin and collagens are major constituents of the cell matrix of fibroblasts. Fibronectin is a 220,000 dalton glycoprotein that mediates a variety of adhesive functions of cells examined in vitro. Fibronectin is secreted in a soluble form and interacts with collagen to form extracellular filaments. Fibronectin and procollage type I were localized using the peroxidase anti-peroxidase method. Under standard culture conditions, fibronectin and procollagen were localized to non-periodic 10 nm extracellular fibrils, the cell membrane and plasma membrane vesicles. Ascorbate treatment of cells leads to a new larger fibril with a diameter of approximately 40 nm. Antibodies to fibronectin and procollagen I react to these native collagen fibrils with an axial periodicity of approximately 70 nm. Fibronectin is clearly associated with native collagen fibrils produced by ascorbate treated cells and there is an asymetric distribution or segregation of fibronectin on these collagen fibrils with a 70 nm axial repeat.     

In situ atomic force microscopy of partially demineralized human dentin collagen fibrils

Dentin collagen fibrils were studied in situ by atomic force microscopy (AFM). New data on size distribution and the axial repeat distance of hydrated and dehydrated collagen type I fibrils are presented. Polished dentin disks from third molars were partially demineralized with citric acid, leaving proteins and the collagen matrix. At this stage collagen fibrils were not resolved by AFM, but after exposure to NaOCl(aq) for 100-240 s, and presumably due to the removal of noncollagenous proteins, individual collagen fibrils and the fibril network of dentin connected to the mineralized substrate were revealed. High-aspect-ratio silicon tips in tapping mode were used to image the soft fibril network. Hydrated fibrils showed three distinct groups of diameters: 100, 91, and 83 nm and a narrow distribution of the axial repeat distance at 67 nm. Dehydration resulted in a broad distribution of the fibril diameters between 75 and 105 nm and a division of the axial repeat distance into three groups at 67, 62, and 57 nm. Subfibrillar features (4 nm) were observed on hydrated and dehydrated fibrils. The gap depth between the thick and thin repeating segments of the fibrils varied from 3 to 7 nm. Phase mode revealed mineral particles on the transition from the gap to the overlap zone of the fibrils. This method appears to be a powerful tool for the analysis of fibrillar collagen structures in calcified tissues and may aid in understanding the differences in collagen affected by chemical treatments or by diseases.     

Assembly of collagen into microribbons: effects of pH and electrolytes

Collagen represents the major structural protein of the extracellular matrix. Elucidating the mechanism of its assembly is important for understanding many cell biological and medical processes as well as for tissue engineering and biotechnological approaches. In this work, conditions for the self-assembly of collagen type I molecules on a supporting surface were characterized. By applying hydrodynamic flow, collagen assembled into ultrathin ( approximately 3 nm) highly anisotropic ribbon-like structures coating the entire support. We call these novel collagen structures microribbons. High-resolution atomic force microscopy topographs show that subunits of these microribbons are built by fibrillar structures. The smallest units of these fibrillar structures have cross-sections of approximately 3 x 5nm, consistent with current models of collagen microfibril formation. By varying the pH and electrolyte of the buffer solution during the self-assembly process, the microfibril density and contacts formed within this network could be controlled. Under certain electrolyte compositions the microribbons and microfibers display the characteristic D-periodicity of approximately 65 nm observed for much thicker collagen fibrils. In addition to providing insight into the mechanism of collagen assembly, the ultraflat collagen matrices may also offer novel ways to bio-functionalize surfaces.     

Are mineralized tissues open crystal foams reinforced by crosslinked collagen? Some energy arguments

Which of the elementary components (hydroxyapatite (HA) crystals, collagen, non-collagenous organic matter, water) do significantly contribute to the ultrastructural elastic stiffness magnitude and anisotropy of mineralized tissues; and how, i.e. through which shapes and assemblages (which micromechanical morphology)? We suggest answers to these questions by analyzing stiffness-volume fraction relationships of wet and dry tissue specimens in the framework of strain energy considerations. Radial stiffness values of both isotropic and anisotropic tissues are found to depend linearly to quadratically on only the mineral volume fraction. This suggests the isotropic contribution of HA to the ultrastructural stiffness. An energy-based analysis of the difference between the axial and radial stiffness values of anisotropic, collagen-rich tissues allows us to assess the collagen elasticity contribution, which is found to depend linearly on the extra-collagenous mineral concentration. These results suggest that collagen and hydroxyapatite are the elementary components governing the ultrastructural elastic stiffness magnitude and anisotropy of bone and mineralized tendons. The elastic stiffness of water and non-collagenous organic matter does not play a significant role. As for the morphological issue, we suggest that mineralized tissues are isotropic open crystal foams; and that these foams are reinforced unidirectionally by collagen molecules which are mechanically activated through tight links between these molecules and HA-crystals. The HA crystals are mechanically activated through stretching and bending in long bone tissues, they are predominantly stretched in mineralized tendons, and bent in hyperpycnotic tissues.     

Layered water in crystal interfaces as source for bone viscoelasticity: arguments from a multiscale approach

Extracellular bone material can be characterised as a nanocomposite where, in a liquid environment, nanometre-sized hydroxyapatite crystals precipitate within as well as between long fibre-like collagen fibrils (with diameters in the 100 nm range), as evidenced from neutron diffraction and transmission electron microscopy. Accordingly, these crystals are referred to as ‘interfibrillar mineral’ and ‘extrafibrillar mineral’, respectively. From a topological viewpoint, it is probable that the mineralisations start on the surfaces of the collagen fibrils (‘mineral-encrusted fibrils’), from where the crystals grow both into the fibril and into the extrafibrillar space. Since the mineral concentration depends on the pore spaces within the fibrils and between the fibrils (there is more space between them), the majority of the crystals (but clearly not all of them) typically lie in the extrafibrillar space. There, larger crystal agglomerations or clusters, spanning tens to hundreds of nanometers, develop in the course of mineralisation, and the micromechanics community has identified the pivotal role, which this extrafibrillar mineral plays for tissue elasticity. In such extrafibrillar crystal agglomerates, single crystals are stuck together, their surfaces being covered with very thin water layers. Recently, the latter have caught our interest regarding strength properties (Fritsch et al. 2009 J Theor Biol. 260(2): 230–252) – we have identified these water layers as weak interfaces in the extrafibrillar mineral of bone. Rate-independent gliding effects of crystals along the aforementioned interfaces, once an elastic threshold is surpassed, can be related to overall elastoplastic material behaviour of the hierarchical material ‘bone’. Extending this idea, the present paper is devoted to viscous gliding along these interfaces, expressing itself, at the macroscale, in the well-known experimentally evidenced phenomenon of bone viscoelasticity. In this context, a multiscale homogenisation scheme is extended to viscoelasticity, mineral-cluster-specific creep parameters are identified from three-point bending tests on hydrated bone samples, and the model is validated by statistically and physically independent experiments on partially dried samples. We expect this model to be relevant when it comes to prediction of time-dependent phenomena, e.g. in the context of bone remodelling.     

Elastic deformation of mineralized collagen fibrils: an equivalent inclusion based composite model

Mineralized collagen fibrils are the basic building blocks of bone tissue at the supramolecular level. Several disease states, manipulation of the expression of specific proteins involved in biomineralization, and treatment with different agents alter the extent of mineralization as well as the morphology of mineral crystals which in turn affect the mechanical function of bone tissue. An experimental assessment of mineralized fibers' mechanical properties is challenged by their small size, leaving analytical and computational models as a viable alternative for investigation of the fibril-level mechanical properties. In the current study the variation of the elastic stiffness tensor of mineralized collagen fibrils with changing mineral volume fraction and mineral aspect ratios was predicted via a micromechanical model. The partitioning of applied stresses between mineral and collagen phases is also predicted for normal and shear loading of fibrils. Model predictions resulted in transversely isotropic collagen fibrils in which the modulus along the longer axis of the fibril was the greatest. All the elastic moduli increased with increasing mineral volume fraction whereas Poisson's ratios decreased with the exception of v12 (=v21). The partitioning of applied stresses were such that the stresses acting on mineral crystals were about 1.5, 15, and 3 times greater than collagen stresses when fibrils were loaded transversely, longitudinally, and in shear, respectively. In the overall the predictions were such that: (a) greatest modulus along longer axis; (b) the greatest mineral/collagen stress ratio along the longer axis of collagen fibers (i.e., greatest relief of stresses acting on collagen); and (c) minimal lateral contraction when fibers are loaded along the longer axis. Overall, the pattern of mineralization as put forth in this model predicts a superior mechanical function along the longer axis of collagen fibers, the direction which is more likely to experience greater stresses.     

Stress-strain experiments on individual collagen fibrils

Collagen, a molecule consisting of three braided protein helices, is the primary building block of many biological tissues including bone, tendon, cartilage, and skin. Staggered arrays of collagen molecules form fibrils, which arrange into higher-ordered structures such as fibers and fascicles. Because collagen plays a crucial role in determining the mechanical properties of these tissues, significant theoretical research is directed toward developing models of the stiffness, strength, and toughness of collagen molecules and fibrils. Experimental data to guide the development of these models, however, are sparse and limited to small strain response. Using a microelectromechanical systems platform to test partially hydrated collagen fibrils under uniaxial tension, we obtained quantitative, reproducible mechanical measurements of the stress-strain curve of type I collagen fibrils, with diameters ranging from 150-470 nm. The fibrils showed a small strain (epsilon < 0.09) modulus of 0.86 +/- 0.45 GPa. Fibrils tested to strains as high as 100% demonstrated strain softening (sigma(yield) = 0.22 +/- 0.14 GPa; epsilon(yield) = 0.21 +/- 0.13) and strain hardening, time-dependent recoverable residual strain, dehydration-induced embrittlement, and susceptibility to cyclic fatigue. The results suggest that the stress-strain behavior of collagen fibrils is dictated by global characteristic dimensions as well as internal structure.     

Post-yield nanomechanics of human cortical bone in compression using synchrotron X-ray scattering techniques

The ultrastructural response to applied loads governs the post-yield deformation and failure behavior of bone, and is correlated with bone fragility fractures. Combining a novel progressive loading protocol and synchrotron X-ray scattering techniques, this study investigated the correlation of the local deformation (i.e., internal strains of the mineral and collagen phases) with the bulk mechanical behavior of bone. The results indicated that the internal strains of the longitudinally oriented collagen fibrils and mineral crystals increased almost linearly with respect to the macroscopic strain prior to yielding, but markedly decreased first and then gradually leveled off after yielding. Similar changes were also observed in the applied stress before and after yielding of bone. However, the collagen to mineral strain ratio remained nearly constant throughout the loading process. In addition, the internal strains of longitudinal mineral and collagen phases did not exhibit a linear relationship with either the modulus loss or the plastic deformation of bulk bone tissue. Finally, the time-dependent response of local deformation in the mineral phase was observed after yielding. Based on the results, we speculate that the mineral crystals and collagen fibrils aligned with the loading axis only partially explain the post-yield deformation, suggesting that shear deformation involving obliquely oriented crystals and fibrils (off axis) is dominant mechanism of yielding for human cortical bone in compression.     

Immunocytochemical localization of fibronectin in human fibroblast cultures using a cell surface replica technique

The pericellular fibronectin-containing matrices of human foreskin fibroblasts cultured in ascorbate-supplemented medium were examined using surface replicas. An extensive filamentous network is present over and between adjacent cells, with a considerable amount at points of cell-to-cell contact. Indirect immunocytochemical localization of the distribution of fibronectin and procollagen type III within the matrix was done using the peroxidase-antiperoxidase (PAP) sandwich technique. The PAP molecule with the surrounding diaminobenzidine reaction product appears as a globular particle of approximately 39 nm in surface replicas. The apparent size of the marker was larger (60-80 nm) when bound to pericellular fibronectin, due presumably to the binding of more than one PAP complex to each fibronectin molecule. The immunocytochemical data suggest that fibronectin is a component of most, if not all, matrix fibrils. Some of the smallest filaments of the matrix (5-10 nm) exhibit a periodic, beaded appearance, with a repeat distance of approximately 70-100 nm. After either anti-fibronectin or anti-procollagen type III labeling, the filaments were decorated at regular 70-100 nm intervals with the globular marker. We suggest that the periodicity may be due to fibronectin molecules bound to collagen microfibrils at regular intervals. Our results demonstrate the usefulness of combined surface replica and immunocytochemical techniques for analysis of matrix components of cultured cells.     

Atomic force microscopy and FT-IR spectroscopy investigations of human heart valves

Human aortic, mitral, tricuspid and pulmonary heart valves were investigated by the contact mode atomic force microscopy (AFM) in air, and using FT-IR spectroscopy in the frequency range 950-4000 cm(-1). Heart valves were collected post mortem from 65-78 years old patients who died from non-cardiac diseases. All of the examined valves showed considerable heterogeneity in the surface topography of collagen fibrils as well as in their organization on the tissue surface. The AFM images revealed areas with significantly different spatial organization of the collagen fibril bundles. We observed zones with multidirectional, stacked collagen fibrils as well as areas of thin fibrils packed regularly, densely and "in phase". The majority of the collagen fibrils reproduced the typical transverse D-banding pattern, with the band interval varying in rather wide range of 70-90 nm. Using AFM imaging, objects that correspond to some pathological states of heart valves at their early stages, i.e. some forms of mineral deposits, were observed. The FT-IR spectra allowed us to recognize main components, i.e. collagen and elastin, in di.erent layers (ventricularis, fibrosa) of the valve leaflets as well as they gave also support for the presence of mineral deposits on the valve surface. The presented results showed, that the AFM imaging and FT-IR spectroscopy can be applied as a complementary methods for structural characterization of heart valves at the molecular and supramolecular levels.