Cartilage calcification in the human thoracic column

A histological and microradiological study of the cartilage calcification processes in the human thoracic column of an old man has been performed. Two different types of cartilage mineralization have been identified. The first corresponds to a calcification of the hyaline cartilage ground substance where chondrocytes are apparently intact. The second is a real mineralization of the chondrocyte lacunae in an uncalcified matrix, which we have called cartilaginous necrosis.     

In vitro formation of mineralized cartilagenous tissue by articular chondrocytes

Summary Study of the deep articular cartilage and adjacent calcified cartilage has been limited by the lack of an in vitro culture system which mimics this region of the cartilage. In this paper we describe a method to generate mineralized cartilagenous tissue in culture using chondrocytes obtained from the deep zone of bovine articular cartilage. The cells were plated on Millipore CM^R filters. The chondrocytes in culture accumulated extracellular matrix and formed cartilagenous tissue which calcified when β-glycerophosphate was added to the culture medium. The cartilagenous tissue generated in vitro contains both type II and type X collagens, large sulfated proteoglycans, and alkaline phosphatase activity. Ultrastructurally, matrix vesicles were seen in the extracellular matrix. Selected area electron diffraction confirmed that the calcification was composed of hydroxyapatite crystals. The chondrocytes, as characterized thus far, appear to maintain their phenotype under these culture conditions which suggests that these cultures could be used as a model to examine the metabolism of cells from the deep zone of cartilage and mineralization of cartilagenous tissue in culture.     

The biomechanics of hyaline cartilage under distension stress]

The mechanical properties of costal cartilage specimens from cattle were tested under periodical compressive loads. The specimens were put between the compression plates of an electronic materials testing machine and were exposed to defined deformations and deformation rates. On the basis of these induced periodical deformation-time curves as input functions we obtained force-time curves as output functions. Results: (1) The cyclic force-time curves generated by deformation-time input functions with constant amplitudes initially showed a general decrease of force peaks until a force-time curve with constant amplitudes gradually arose. (2) If periodically loaded after a constant deformation had taken place the force peaks increased, at first pronouncedly but later diminishing. We named this the 'peak increase phenomenon'. (3) The mechanorheological curves of hyaline cartilage can be explained approximately by the viscoelastic behaviour of microfibrils in a parallel arrangement with the elastoviscous properties of cartilaginous ground substance.     

Histologic studies of cartilaginous structures in the anterior region of the head of the sperm whale Physeter macrocephalus

Recently discovered cartilaginous structures in the forehead of the sperm whale (Behrmann and Klima 1985) were investigated histologically. The largest and most important of these structures is the nasal roof cartilage which can be derived from the tectum nasi, a part of the embryonic nasal capsule (Klima et al. 1986). In the investigated sperm whale fetuses, this structure consists of embryonic hyaline cartilage which is well suited for morphogenetic processes and fast growth. In the investigated adult sperm whale, the originally hyaline cartilage has been transformed into a special kind of elastic cartilage. The arrangement of cells, territories, and extracellular substance resembles hyaline cartilage. This component represents an adaptation to pressure load. The appearance and arrangement of elastic fibres resembles elastic cartilage. This component is an adaptation to distortion forces. Obviously, pressure and distortion are the strongest mechanical strains that the nasal roof cartilage is exposed. We see the function of this cartilage structure therein that, being a pressure-elastic skeletal support and following the left nasal meatus along its whole extension through the massive and soft forehead, it secures the only direct respiratory passage. Additionally, fibre bundles of transversely striated muscles are anchored in the perichondrium of the nasal roof cartilage. The function of this delicately interwoven muscle system is seen by us in the fine tuning of contraction and dilatation of the respiratory passage. Moreover, a possible function as a sound conducting cartilaginous structure serving the echolocation system is considered (c.f. Pilleri et al. 1983).     

Chondrocytes inhibit endothelial sprout formation in vitro: evidence for involvement of a transforming growth factor-beta.

Using a quantitative in vitro model of spontaneous endothelial sprout formation, we have attempted to define physiological inhibitors of angiogenesis from hyaline cartilage, a tissue whose antiangiogenic properties have been well described. The model consists of embedding bovine microvascular endothelial cell aggregates into fibrin or collagen gels, which results in the formation of radially growing sprouts. When chondrocytes derived from the permanent cartilagenous region of the chick embryo sternum are cocultured with the endothelial cell aggregates, sprout formation is markedly inhibited. Addition of anti-TGF-beta antibodies to the cocultures significantly reduced the inhibitory effect of chondrocytes on sprout formation. Chondrocyte-conditioned medium or exogenously added TGF-beta 1 have a similar albeit transient inhibitory effect. Depletion of TGF-beta from chondrocyte conditioned medium with anti-TGF-beta antibodies and solid-phase protein-A significantly decreases the inhibition of sprout formation. These results demonstrate that a chondrocyte-derived TGF-beta-like molecule inhibits capillary sprout formation in vitro and suggest that the antiangiogenic properties of cartilage may at least in part, be mediated by TGF-beta.     

A system of interlacunar network and thick fibrils in human hyaline cartilage

Summary A system consisting of an interlacunar network and thick fibrils was demonstrated in the matrix of human fetal and neonatal hyaline cartilage, using an osmium-ferrocyanide mixture as a second fixative. The network appeared as irregular strands consisting of hyaluronidase-sensitive, amorphous and fine fibrillar material. The thick fibrils measured 75–125 μm in diameter, each appearing to consist of several collagen fibrils twisted into a cable and cemented by dense amorphous material. Strands of the network were seen to cross and focally distort the thick fibrils, suggesting that the strands exert some tensile forces on the thick fibrils. During the first year of life the network rapidly became undemonstrable, but the thick fibrils persisted into adulthood. This system of interlacunar network and thick fibrils appears to form an integral functional unit which may play an organizational tole in the formation of cartilagenous matrix during development. Furthermore, it may contribute to the mechanical strength of the coflagen framework in hyaline cartilage.     

The aging alterations of the arytenoid cartilage

The aging processes of the arytaenoid cartilage were described. We inspected 58 arytaenoid cartilage at the age of 0 to 91 years. The arytaenoid cartilage consists mainly of hyaline cartilage, whereas the apex, colliculus and the vocal processus consist of elastic cartilage. The cartilage of larynx exhibits changes concerning the cells and the intercellular substance. Chondrocytes show fatty degeneration, the cell density decreased. During the age intercellular substance shows the following changes: albuminoid degeneration, partly loss of intercellular substance with exhibition of well visible collagen fibers, calcification and ossification. Intercellular substance shows basophilic reaction up to the 4th decennium, later the reaction becomes more and more eosinophilic.     

Effect of articular cartilage resection on the growth and formation of the femoral and acetabular epiphyses]

Different parts of the articular cartilage were resected in 46 rabbits at the age of 2.5 months. The resected narrow stripe of the articular cartilage completely restored by the 60--90th day and the growth of the condyles was not disturbed. Resection of considerable areas of the articular cartilage on the condyles and on the femoral head was accompanied by a certain disturbance of the osseous tissue growth in these areas with resulted impression of the condyles, deformation of the head and further formation of coxa vara. The removal of 1/3 of the articular cartilage of the cotyloid cavity resulted in a certain increase of its diamter, uneven development at the site of resection; the femoral head of this joint increased, its spherical shape was altered. The restored cartilage did not restore its original structure characteristic for a growing bone. The newly formed articular cartilage lost its ability to participate in endochondral bone formation during the growth of the animal.     

Toward regeneration of articular cartilage

Articular cartilage is classified as permanent hyaline cartilage and has significant differences in structure, extracelluar matrix components, gene expression profile, and mechanical property from transient hyaline cartilage found in the epiphyseal growth plate. In the process of synovial joint development, articular cartilage originates from the interzone, developing at the edge of the cartilaginous anlagen, and establishes zonal structure over time and supports smooth movement of the synovial joint through life. The cascade actions of key regulators, such as Wnts, GDF5, Erg, and PTHLH, coordinate sequential steps of articular cartilage formation. Articular chondrocytes are restrictedly controlled not to differentiate into a hypertrophic stage by autocrine and paracrine factors and extracellular matrix microenvironment, but retain potential to undergo hypertrophy. The basal calcified zone of articular cartilage is connected with subchondral bone, but not invaded by blood vessels nor replaced by bone, which is highly contrasted with the growth plate. Articular cartilage has limited regenerative capacity, but likely possesses and potentially uses intrinsic stem cell source in the superficial layer, Ranvier's groove, the intra‐articular tissues such as synovium and fat pad, and marrow below the subchondral bone. Considering the biological views on articular cartilage, several important points are raised for regeneration of articular cartilage. We should evaluate the nature of regenerated cartilage as permanent hyaline cartilage and not just hyaline cartilage. We should study how a hypertrophic phenotype of transplanted cells can be lastingly suppressed in regenerating tissue. Furthermore, we should develop the methods and reagents to activate recruitment of intrinsic stem/progenitor cells into the damaged site. Birth Defects Research (Part C) 99:192–202, 2013 . © 2013 Wiley Periodicals, Inc .     

Requirement of fibroblast growth factor signaling for regeneration of epiphyseal morphology in rabbit full-thickness defects of articular cartilage

The involvement of fibroblast growth factor-2 (FGF-2) during the repair process in rabbit full-thickness defects of articular cartilage was studied. Fibroblast growth factor-2 (50 pg/h) was administered for 2 weeks in a 5mm defect of articular cartilage, which is large enough not to repair spontaneously. The administration of FGF-2 resulted in the regeneration of the articular cartilage and the subchondral bone within 8 weeks. In these defects, undifferentiated mesenchymal cells initiated chondrogenic differentiation coupled with replacement by subchondral bone, resulting in the resurfacing of the defects with hyaline cartilage and the recovery of subchondral bone up to the original bone–articular cartilage junction. In rabbits, full-thickness defects are capable of regenerating articular cartilage as long as the defect size is limited to ≤3 mm in diameter. In the defects, strong immunoreactivity for FGF-2 was observed in the granulation tissue filling the defects in the early stage of repair, in association with the expression of FGF-2 mRNA shown by in situ hybridization. Once the undifferentiated mesenchymal cells had differentiated into chondrocytes, both the immunoreactivity and the in situ hybridization signal declined significantly. Upon the local administration of a monoclonal antibody against FGF-2 (bFM-1, 50ng/h), the defects were filled with fibrous tissue and no resurfacing hyaline cartilage was formed. Compared to the non-treated defects, there were marked increases in FGF-2 immunoreactivity and the overexpression of FGF-2 mRNA in the reparative tissue in the bFM-1 -treated defects. This rebound phenomenon indicates that the autocrine FGF-2 signaling is critically important for the regeneration of articular cartilage.     

Localization of collagens and alkaline phosphatase activity during mineralization and ossification of human first rib cartilage

The localization of type X collagen and alkaline phosphatase activity was examined in order to gain a better understanding of tissue remodelling during development of human first rib cartilage. First rib cartilages from children and adolescents showed no staining for type X collagen and alkaline phosphatase activity. After onset of mineralization in the late second decade, a peripheral ossification process preceded by mineralized fibrocartilage could be distinguished from a more central one preceded by mineralized hyaline cartilage. No immunostaining for type X collagen was found in either type of cartilage. However, strong staining for alkaline phosphatase activity was detected around chondrocyte-like cells within fibrocartilage adjacent to the peripheral mineralization front, while a weaker staining pattern was observed around chondrocytes of hyaline cartilage near the central mineralization front. In addition, the territorial matrix of some chondrocytes within the hyaline cartilage revealed staining for type I collagen, suggesting that these cells undergo a dedifferentiation process, which leads to a switch from type II to type I collagen synthesis. The study provides evidence that mineralization of the hyaline cartilage areas in human first rib cartilage occurs in the absence of type X collagen synthesis but in the presence of alkaline phosphatase. Thus, mineralization of first rib cartilage seems to follow a different pattern from endochondral ossification in epiphyseal discs.     

Untersuchungen Zur Eosinophilie Hyalinen Knorpels

Zusammenfassung Während der chemischen Fixierung von Geweben und der nachfolgenden Behandlung in Alkoholreihen geht Hämoglobin in Lösung und diffundiert in das Gewebe. Die Diffusion des Hämoglobins ist besonders gut in der Grundsubstanz hyaliner Knorpel zu verfolgen. Das eingedrungene Hämoglobin wurde mit der Ultramikrospektrographie nachgewiesen. Die Hämoglobindiffusion läßt sich durch die Kryostatschnittechnik und durch eine Fixierung in 4%igem Formalin mit Zusatz von 2,5% Ferricyankalium verhindern bzw. mit Zusatz von Natriumchlorid oder Phosphat zu Formalin einschränken. Besonders säurehaltige Fixierungslösungen trennen die Häm- von der Globinkomponente. Der Globinanteil verbleibt in der Grundsubstanz des Knorpels und kann das Vorliegen eines ortsständigen Eiweiß-körpers vortäuschen. Die Einlagerung des vollständigen Hämoglobins oder des Globins allein bedingen die diffuse Eosinophilie der Grundsubstanz im fixierten Knorpel und blockiert die Darstellung der sauren Mucopolysaccharide (Alcianblaufärbung) sowie die metachromatische Reaktion der Grundsubstanz.

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Summary In the course of chemical fixation of tissues and the subsequent treatment with alcohol hemoglobin dissolves and diffuses into the tissue. The diffusion of hemoglobin can be observed particularly well in the ground substance of hyaline cartilage. There the hemoglobin is demonstrable by means of ultramicrospectrography. The diffusion of the hemoglobin can be prevented through the use of the cryostat technique and fixation in 4% formalin containing 2.5% ferricyankalium, NaCl or phosphate. Acid containing fixation media separate the hem from the globin component. The globin remains in the ground substance of the cartilage and may be mistaken for a sessil protein. The storage of either the complete hemoglobin molecule or the globin fraction causes the diffuse eosinophily in the ground substance of fixed cartilage which also blocks the determination of acid mucopolysaccharides (alcian blue staining method) as well as the metachromatic reaction of the ground substance.

     

Electron microscopy of human articular cartilage]

Scanning and transmission microscopy of the articular cartilage was performed in femoral condyles of persons at the age of 30-50 years. It was demonstrated that hyaline cartilage is covered with a protective fibrillar layer consisting of tightly pressed collagenous fibrillae with an underlying layer of fibroblastic cells. In the intracellular substance of the hyaline cartilage fibrillar structures form a complex reticular web with vertical arrangement of the main collagenous fasiculi. In the superficial layer of the hyaline cartilage the collagenous fibrillae and their fasciculi form arcade-like structures. Lacunar chondrocytes have a rough villose surface, cellular secrete is discharged as round granules through cytoplasmic membrane. Ultrastructural changes in chondrocytes are observed simultaneously with their degenerative-dystrophic changes.     

Study on the Microstructure of Human Articular Cartilage/Bone Interface

For improving the theory of gradient microstructure of cartilage/bone interface, human distal femurs were studied. Scanning Electron Microscope (SEM), histological sections and MicroCT were used to observe, measure and model the microstructure of cartilage/bone interface. The results showed that the cartilage/bone interface is in a hierarchical structure which is composed of four different tissue layers. The interlocking of hyaline cartilage and calcified cartilage and that of calcified cartilage and subchondral bone are in the manner of “protrusion-pore” with average diameter of 17.0 μm and 34.1 μm respectively. In addition, the cancellous bone under the cartilage is also formed by four layer hierarchical structure, and the adjacent layers are connected by bone trabecula in the shape of H, I and Y, forming a complex interwoven network structure. Finally, the simplified structure model of the cartilage/bone interface was proposed according to the natural articular cartilage/bone interface. The simplified model is a 4-layer gradient biomimetic structure, which corresponds to four different tissues of natural cartilage/bone interface. The results of this work would be beneficial to the design of bionic scaffold for the tissue engineering of articular cartilage/bone.     

Analysis of the permeation of cryoprotectants in cartilage

Some tissues, such as cartilage and cornea, carry an internal fixed negative charge, leading to a swelling pressure that is balanced by tensile stress in the tissue matrix. During the addition and removal of cryoprotectants the changes in osmotic pressure will cause the tissue to deform. Because of the fixed charge and osmotic deformation, the permeation process in such tissues differs from ordinary diffusion processes. In this paper a biomechanical multi-solute theory is introduced to describe this process in cartilage tissue. Typical values for the physiological and biomechanical properties are used in the simulation. Several parameters - the aggregate modulus, the fixed charge density and the frictional parameter - are analyzed to show their impact on the process. It is shown that friction between water and cryoprotectant has the greatest influence but the fixed charge density is also important. The aggregate modulus and the frictional parameter between the cryoprotectant and the solid matrix have the least influence. Both the new biomechanical model and the conventional diffusion model were fitted to published experimental data concerning the time course of mean tissue cryoprotectant concentration when cartilage is immersed in solutions of dimethyl sulphoxide or propylene glycol: in all cases and with both models a good fit was obtained only when a substantial amount of non-solvent water was assumed.     

CHONDROGENESIS, STUDIED WITH THE ELECTRON MICROSCOPE

The role of the cells in the fabrication of a connective tissue matrix, and the structural modifications which accompany cytodifferentiation have been investigated in developing epiphyseal cartilage of fetal rat by means of electron microscopy. Differentiation of the prechondral mesenchymal cells to chondroblasts is marked by the acquisition of an extensive endoplasmic reticulum, enlargement and concentration of the Golgi apparatus, the appearance of membrane-bounded cytoplasmic inclusions, and the formation of specialized foci of increased density in the cell cortex. These modifications are related to the secretion of the cartilage matrix. The matrix of young hyaline cartilage consists of groups of relatively short, straight, banded collagen fibrils of 10 to 20 mµ and a dense granular component embedded in an amorphous ground substance of moderate electron density. It is postulated that the first phase of fibrillogenesis takes place at the cell cortex in dense bands or striae within the ectoplasm subjacent to the cell membrane. These can be resolved into sheaves of "primary" fibrils of about 7 to 10 mµ. They are supposedly shed (by excortication) into the matrix space between the separating chondroblasts, where they may serve as "cores" of the definitive matrix fibrils. The diameter of the fibrils may subsequently increase up to threefold, presumably by incorporation of "soluble" or tropocollagen units from the ground substance. The chondroblast also discharges into the matrix the electrondense amorphous or granular contents of vesicles derived from the Golgi apparatus, and the mixed contents of large vacuoles or blebs bounded by distinctive double membranes. Small vesicles with amorphous homogeneous contents of moderate density are expelled in toto from the chondroblasts. In their subsequent evolution to chondrocytes, both nucleus and cytoplasm of the chondroblasts undergo striking condensation. Those moving toward the osteogenic plate accumulate increasingly large stores of glycogen. In the chondrocyte, the enlarged fused Golgi vesicles with dense contents, massed in the juxtanuclear zone, are the most prominent feature of the cytoplasm. Many of these make their way to the surface to discharge their contents. The hypertrophied chondrocytes of the epiphyseal plate ultimately yield up their entire contents to the matrix.     

Structuro-functional organization of the fibrillar component and ground substance of human rib cartilage

A complex morphological investigation (histology, histochemistry, scanning and transmissive electron microscopy, electron histochemistry) has been performed to study the intercellular substance of the costal hyalinous cartilage. It has been demonstrated that the fibrillar framework of the costal cartilage consists of branching collagenous fibrillae, chaotically scattering. The fibrillae are surrounded with the ground substance; one of its components is the reticular ruthenium-positive structure.     

Immunoelectron microscopic analysis of chondroitin sulfates during calcification in the rat growth plate cartilage

The proximal growth plate cartilage of rat tibia was fixed in the presence of ruthenium hexamine trichloride (RHT) in order to preserve proteoglycans in the tissue. Quantitative changes of chondroitin sulfates during endochondral calcification were investigated by immunoelectron microscopy using mouse monoclonal antibodies 1-B-5, 2-B-6, and 3-B-3, which recognize unsulfated, 4-sulfated, and 6-sulfated chondroitin sulfates, respectively. The content of chondroitin-4-sulfate in the cartilage matrix increased from the proliferative zone to the calcifying zone, while that of unsulfated chondroitin sulfate decreased. Chondroitin-6-sulfate remained constant from the proliferative zone to the upper hypertrophic zone, then decreased in the calcifying zone. The immunoreaction to each antibody increased conspicuously in the cartilagenous core of metaphysial bone trabeculae. The changes of sulfation in chondroitin sulfate chains of proteoglycans may play an important role in inducing and/or promoting calcification in growth plate cartilage.     

Real-time observation of fluid flows in tissue during stress relaxation using Raman spectroscopy

This paper outlines a technique to measure fluid levels in articular cartilage tissue during an unconfined stress relaxation test. A time series of Raman spectrum were recorded during relaxation and the changes in the specific Raman spectral bands assigned to water and protein were monitored to determine the fluid content of the tissue. After 1000 s unconfined compression the fluid content of the tissue is reduced by an average of 3.9% ± 1.7%. The reduction in fluid content during compression varies between samples but does not significantly increase with increasing strain. Further development of this technique will allow mapping of fluid distribution and flows during dynamic testing making it a powerful tool to understand the role of interstitial fluid in the functional performance of cartilage.