Type III collagen in normal human articular cartilage

Summary Type III collagen in normal human articular cartilage has been detected biochemically and its location in a diffuse area around the chondrocytes demonstrated by immunofluorescence. It can be found pericellularly throughout the depth of the cartilage and is evident in specimens ranging in age from 17 to 81 years.     

Postnatal development of collagen structure in ovine articular cartilage

Background  

Articular cartilage (AC) is the layer of tissue that covers the articulating ends of the bones in diarthrodial joints. Across species, adult AC shows an arcade-like structure with collagen predominantly perpendicular to the subchondral bone near the bone, and collagen predominantly parallel to the articular surface near the articular surface. Recent studies into collagen fibre orientation in stillborn and juvenile animals showed that this structure is absent at birth. Since the collagen structure is an important factor for AC mechanics, the absence of the adult Benninghoff structure has implications for perinatal AC mechanobiology. The current objective is to quantify the dynamics of collagen network development in a model animal from birth to maturity. We further aim to show the presence or absence of zonal differentiation at birth, and to assess differences in collagen network development between different anatomical sites of a single joint surface. We use quantitative polarised light microscopy to investigate properties of the collagen network and we use the sheep (Ovis aries) as our model animal.     

Biosynthesis of collagen crosslinks in rabbit articular cartilage in vivo

The biosynthesis in vivo of the two reducible aldimine crosslinks of immature rabbit articular collagen, hydroxylysinohydroxynorleucine and hydroxylysinonorleucine, is demonstrated. The peak amount of crosslink was detected 1–2 weeks following labeling of the cartilage with [¹⁴C]lysine. The subsequent diminution which occurred was due primarily to a decrease in the amount of hydroxylysinohydroxynorleucine. Natural reduction of the aldimine crosslinks in vivo did not occur. Glucosylgalactosyl hydroxylysine and galactosylhydroxylysine, in a $\text{1.45}\text{1.00}$ ratio, were synthesized. Seventy-three percent of the hydroxylysine residues were glycosylated. [³H]NaBH₄ reduction of non-¹⁴C-labeled cartilage showed diminished amounts of reducible crosslink with time and the presence of hexosyl lysines and hexosyl hydroxylysines in mature articular cartilage.     

Stresses in the local collagen network of articular cartilage: a poroviscoelastic fibril-reinforced finite element study

Osteoarthritis (OA) is a multifactorial disease, resulting in diarthrodial joint wear and eventually destruction. Swelling of cartilage, which is proportional to the amount of collagen damage, is an initial event of cartilage degeneration, so damage to the collagen fibril network is likely to be one of the earliest signs of OA cartilage degeneration. We propose that the local stresses and strains in the collagen fibrils, which cause the damage, cannot be determined dependably without taking the local arcade-like collagen-fibril structure into account. We investigate this using a poroviscoelastic fibril-reinforced FEA model. The constitutive fibril properties were determined by fitting numerical data to experimental results of unconfined compression and indentation tests on samples of bovine patellar articular cartilage. It was demonstrated that with this model the stresses and strains in the collagen fibrils can be calculated. It was also exhibited that fibrils with different orientations at the same location can be loaded differently, depending on the local architecture of the collagen network. To the best of our knowledge, the present model is the first that can account for these features. We conclude that the local stresses and strains in the articular cartilage are highly influenced by the local morphology of the collagen-fibril network.     

Postnatal development of depth-dependent collagen density in ovine articular cartilage

Background  

Articular cartilage (AC) is the layer of tissue that covers the articulating ends of the bones in diarthrodial joints. Adult AC is characterised by a depth-dependent composition and structure of the extracellular matrix that results in depth-dependent mechanical properties, important for the functions of adult AC. Collagen is the most abundant solid component and it affects the mechanical behaviour of AC. The current objective is to quantify the postnatal development of depth-dependent collagen density in sheep (Ovis aries) AC between birth and maturity. We use Fourier transform infra-red micro-spectroscopy to investigate collagen density in 48 sheep divided over ten sample points between birth (stillborn) and maturity (72 weeks). In each animal, we investigate six anatomical sites (caudal, distal and rostral locations at the medial and lateral side of the joint) in the distal metacarpus of a fore leg and a hind leg.     

Contribution of postnatal collagen reorientation to depth-dependent mechanical properties of articular cartilage

The collagen fibril network is an important factor for the depth-dependent mechanical behaviour of adult articular cartilage (AC). Recent studies show that collagen orientation is parallel to the articular surface throughout the tissue depth in perinatal animals, and that the collagen orientations transform to a depth-dependent arcade-like structure in adult animals. Current understanding on the mechanobiology of postnatal AC development is incomplete. In the current paper, we investigate the contribution of collagen fibril orientation changes to the depth-dependent mechanical properties of AC. We use a composition-based finite element model to simulate in a 1-D confined compression geometry the effects of ten different collagen orientation patterns that were measured in developing sheep. In initial postnatal life, AC is mostly subject to growth and we observe only small changes in depth-dependent mechanical behaviour. Functional adaptation of depth-dependent mechanical behaviour of AC takes place in the second half of life before puberty. Changes in fibril orientation alone increase cartilage stiffness during development through the modulation of swelling strains and osmotic pressures. Changes in stiffness are most pronounced for small stresses and for cartilage adjacent to the bone. We hypothesize that postnatal changes in collagen fibril orientation induce mechanical effects that in turn promote these changes. We further hypothesize that a part of the depth-dependent postnatal increase in collagen content in literature is initiated by the depth-dependent postnatal increase in fibril strain due to collagen fibril reorientation.     

A hyperelastic biphasic fibre-reinforced model of articular cartilage considering distributed collagen fibre orientations: continuum basis,computational aspects and applications

Cartilage is a multi-phase material composed of fluid and electrolytes (68–85% by wet weight), proteoglycans (5–10% by wet weight), chondrocytes, collagen fibres and other glycoproteins. The solid phase constitutes an isotropic proteoglycan gel and a fibre network of predominantly type II collagen, which provides tensile strength and mechanical stiffness. The same two components control diffusion of the fluid phase, e.g. as visualised by diffusion tensor MRI: (i) the proteoglycan gel (giving a baseline isotropic diffusivity) and (ii) the highly anisotropic collagenous fibre network. We propose a new constitutive model and finite element implementation that focus on the essential load-bearing morphology: an incompressible, poroelastic solid matrix reinforced by an inhomogeneous, dispersed fibre fabric, which is saturated with an incompressible fluid residing in strain-dependent pores of the collagen–proteoglycan solid matrix. The inhomogeneous, dispersed fibre fabric of the solid further influences the fluid permeability, as well as an intrafibrillar portion that cannot be ‘squeezed out’ from the tissue. Using representative numerical examples on the mechanical response of cartilage, we reproduce several features that have been demonstrated experimentally in the cartilage mechanics literature.     

Type IX collagen: a possible function in articular cartilage

The effect of type IX on in vitro fibrillogenesis of type II collagen indicated that, while not preventing fibrillogenesis, the presence of type IX collagen reduced the size of the type II fibre aggregates. This observation is consistent with the in vivo localisation studies of type IX collagen. Using the immunogold labelling technique, type IX collagen was shown to be located evenly on small fibrils which occur at higher concentration closer to the cell. Therefore type IX collagen may function as a regulator of fibre diameter in articular cartilage.     

Type X collagen expression in osteoarthritic and rheumatoid articular cartilage

Type X collagen is a short chain, non-fibrilforming collagen synthesized primarily by hypertrophic chondrocytes in the growth plate of fetal cartilage. Previously, we have also identified type X collagen in the extracellular matrix of fibrillated, osteoarthritic but not in normal articular cartilage using biochemical and immunohistochemical techniques (von der Mark et al. 1992 a). Here we compare the expression of type X with types I and II collagen in normal and degenerate human articular cartilage by in situ hybridization. Signals for cytoplasmic α1(X) collagen mRNA were not detectable in sections of healthy adult articular cartilage, but few specimens of osteoarthritic articular cartilage showed moderate expression of type X collagen in deep zones, but not in the upper fibrillated zone where type X collagen was detected by immunofluorescence. This apparent discrepancy may be explained by the relatively short phases of type X collagen gene activity in osteoarthritis and the short mRNA half-life compared with the longer half-life of the type X collagen protein. At sites of newly formed osteophytic and repair cartilage, α1(X) mRNA was strongly expressed in hypertrophic cells, marking the areas of endochondral bone formation. As in hypertrophic chondrocytes in the proliferative zone of fetal cartilage, type X collagen expression was also associated with strong type II collagen expression.     

Texture analysis of the 3D collagen network and automatic classification of the physiology of articular cartilage

A close relationship has been found between the 3D collagen structure and physiological condition of articular cartilage (AC). Studying the 3D collagen network in AC offers a way to determine the condition of the cartilage. However, traditional qualitative studies are time consuming and subjective. This study aims to develop a computer vision-based classifier to automatically determine the condition of AC tissue based on the structural characteristics of the collagen network. Texture analysis was applied to quantitatively characterise the 3D collagen structure in normal (International Cartilage Repair Society, ICRS, grade 0), aged (ICRS grade 1) and osteoarthritic cartilages (ICRS grade 2). Principle component techniques and linear discriminant analysis were then used to classify the microstructural characteristics of the 3D collagen meshwork and the condition of the AC. The 3D collagen meshwork in the three physiological condition groups displayed distinctive characteristics. Texture analysis indicated a significant difference in the mean texture parameters of the 3D collagen network between groups. The principle component and linear discriminant analysis of the texture data allowed for the development of a classifier for identifying the physiological status of the AC with an expected prediction error of 4.23%. An automatic image analysis classifier has been developed to predict the physiological condition of AC (from ICRS grade 0 to 2) based on texture data from the 3D collagen network in the tissue.     

Caprine articular,meniscus and intervertebral disc cartilage: An integral analysis of collagen network and chondrocytes

Cartilage is a tissue with only limited reparative capacities. A small part of its volume is composed of cells, the remaining part being the hydrated extracellular matrix (ECM) with collagens and proteoglycans as its main constituents. The functioning of cartilage depends heavily on its ECM. Although it is known that the various (fibro)cartilaginous tissues (articular cartilage, annulus fibrosus, nucleus pulposus, and meniscus) differ from one each other with respect to their molecular make-up, remarkable little quantitative information is available with respect to its biochemical constituents, such as collagen content, or the various posttranslational modifications of collagen. Furthermore, we have noticed that tissue-engineering strategies to replace cartilaginous tissues pay in general little attention to the biochemical differences of the tissues or the phenotypical differences of the (fibro)chondrocytes under consideration. The goal of this paper is therefore to provide quantitative biochemical data from these tissues as a reference for further studies. We have chosen the goat as the source of these tissues, as this animal is widely accepted as an animal model in orthopaedic studies, e.g. in the field of cartilage degeneration and tissue engineering. Furthermore, we provide data on mRNA levels (from genes encoding proteins/enzymes involved in the synthesis and degradation of the ECM) from (fibro)chondrocytes that are freshly isolated from these tissues and from the same (fibro)chondrocytes that are cultured for 18 days in alginate beads. Expression levels of genes involved in the cross-linking of collagen were different between cells isolated from various cartilaginous tissues. This opens the possibility to include more markers than the commonly used chondrogenic markers type II collagen and aggrecan for cartilage tissue-engineering applications.     

Experimental osteoarthritic articular cartilage is enriched in guanidine soluble type VI collagen

Experimental osteoarthritis was surgically induced in the right knee joint of dogs; the left knee served as a control. Articular cartilage was extracted with 4 M guanidinium chloride, 0.05 M sodium acetate, pH 6.0, containing proteinase inhibitors and the proteins purified by associative CsCl density gradient centrifugation. Equal quantities of protein were electrophoresed in agarose-acrylamide gradient gels and the high molecular weight type VI collagen bands detected in immunoblots with a polyclonal antiserum. Type VI collagen bands between 185 and 220 kDa were evident in the pathological specimens of dogs sacrificed 3, 5, and 7 months after surgery and were either absent or only very weakly visible in the controls. These results demonstrate that experimental osteoarthritic cartilage is enriched in 4 M guanidine-soluble type VI collagen.     

Localization of collagen type VI in articular cartilage of young and adult mice

Collagen type VI was demonstrated immunomorphologically in articular cartilage (distal femur) of young (2–8 weeks) and adult mice by fluorescence and electron microscopy (gold-labelled second antibody—sandwich method) using pre-and post-embedding techniques. This collagen type was mainly seen in the vicinity of chondrocytes, and in larger amounts in adult cartilage. Electron-microscopic inspection (pre-embedding technique) revealed labelling above plaques that were 40–160 nm in size, and from which up to 7 fine filaments ( 10 nm) per unit sectional plane radiated. Using the post-embedding technique, only labelled plaques could be demonstrated; fine filaments were not perceptible. This was partly a result of the low contrast. It is assumed that the globular ends of up to 20 of the fine type VI filaments are anchored in one plaque and that the antibodies bind to the non-collagenous globular domains. Filaments radiated from the plaques and formed a threedimensional network that stabilized the structures of the cartilaginous matrix. Antibodies against fibronectin also labelled similar plaques. The ends of the type VI filaments are possibly linked into the plaques by fibronectin.     

Association of the aggrecan keratan sulfate-rich region with collagen in bovine articular cartilage

Aggrecan, the predominant large proteoglycan of cartilage, is a multidomain macromolecule with each domain contributing specific functional properties. One of the domains contains the majority of the keratan sulfate (KS) chain substituents and a protein segment with a proline-rich hexapeptide repeat sequence. The function of this domain is unknown but the primary structure suggests a potential for binding to collagen fibrils. We have examined binding of aggrecan fragments encompassing the KS-rich region in a solid-phase assay. A moderate affinity (apparent Kd = 1.1 microM) for isolated collagen II, as well as collagen I, was demonstrated. Enzymatic digestion of the KS chains did not alter the capacity of the peptide to bind to collagen, whereas cleavage of the protein core abolished the interaction. The distribution of the aggrecan KS-rich region in bovine tarsometatarsal joint cartilage was investigated using immunoelectron microscopy. Immunoreactivity was relatively low in the superficial zone and higher in the intermediate and deep zones of the uncalcified cartilage. Within the pericellular and territorial matrix compartments the epitopes representing the aggrecan KS-rich region were detected preferentially near or at collagen fibrils. Along the fibrils, epitope reactivity was non-randomly distributed, showing preference for the gap region within the D-period. Our data suggest that collagen fibrils interact with the KS-rich regions of several aggrecan monomers aligned within a proteoglycan aggregate. The fibril could therefore serve as a backbone in at least some of the aggrecan complexes.     

Localization of collagen X in human fetal and juvenile articular cartilage and bone

The tissue localization was analysed of collagen X during human fetal and juvenile articular cartilagebone metamorphosis. This unique collagen type was found in the hypertrophic cartilage zone peri- and extracellularly and in cartilage residues within bone trabeculae. In addition, occasionally a slight intracellular staining reaction was found in prehypertrophic proliferating chondrocytes and in chondrocytes surrounding vascular channels. A slight staining was also seen in the zone of periosteal ossification and occasionally at the transition zone of the perichondrium to resting cartilage. Our data provide evidence that the appearance of collagen X is mainly associated with cartilage hypertrophy, analogous to the reported tissue distribution of this collagen type in animals. In addition, we observed an increased and often spotty distribution of collagen X with increasing cartilage degeneration associated with the closure of the growth plate. In basal hypertrophic cartilage areas, a co-distribution of collagens II and X was found with very little and spotty collagen III. In juvenile cartilage areas around single hypertrophic chondrocytes, co-localization of collagens X and I was also detected.     

The importance of superficial collagen fibrils for the function of articular cartilage

It has been proposed that the superficial tangential zone (STZ) of articular cartilage is essential to the tissue’s load-distributing function. However, the exact mechanism by which the STZ fulfills this function has not yet been revealed. Using a channel-indentation experiment, it was recently shown that compared to intact tissue, cartilage without STZ behaves slightly stiffer and deforms significantly different in regions adjacent to mechanically compressed areas (Bevill et al. in Osteoarthr Cartil 18:1310–1318, 2010). We aim to further explore the role of STZ in the load-transfer mechanism of AC by thorough biomechanical analysis of these experiments. Using our previously validated fibril-reinforced swelling model of articular cartilage, which accounts for the depth-dependent collagen structure and biochemical composition of articular cartilage, we simulated the above-mentioned channel-indenter compression experiments for both intact and STZ-removed cartilage. First, we show that the composition of the deep zone in cartilage is most effective in carrying cartilage compression, which explains the apparent tissue stiffening after STZ removal. Second, we show that tangential fibrils in the STZ are responsible for transferring compressive loads from directly loaded regions to adjacent tissue. Cartilage with an intact STZ has superior load-bearing properties compared to cartilage in which the STZ is compromised, because the STZ is able to recruit a larger area of deep zone cartilage to carry compressive loads.     

A computational remodeling approach to predict the physiological architecture of the collagen fibril network in corneo-scleral shells

Organized collagen fibrils form complex networks that introduce strong anisotropic and highly nonlinear attributes into the constitutive response of human eye tissues. Physiological adaptation of the collagen network and the mechanical condition within biological tissues are complex and mutually dependent phenomena. In this contribution, a computational model is presented to investigate the interaction between the collagen fibril architecture and mechanical loading conditions in the corneo-scleral shell. The biomechanical properties of eye tissues are derived from the single crimped fibril at the micro-scale via the collagen network of distributed fibrils at the meso-scale to the incompressible and anisotropic soft tissue at the macro-scale. Biomechanically induced remodeling of the collagen network is captured on the meso-scale by allowing for a continuous re-orientation of preferred fibril orientations and a continuous adaptation of the fibril dispersion. The presented approach is applied to a numerical human eye model considering the cornea and sclera. The predicted fibril morphology correlates well with experimental observations from X-ray scattering data.