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.     

Doublecortin is expressed in articular chondrocytes

Articular cartilage and cartilage in the embryonic cartilaginous anlagen and growth plates are both hyaline cartilages. In this study, we found that doublecortin (DCX) was expressed in articular chondrocytes but not in chondrocytes from the cartilaginous anlagen or growth plates. DCX was expressed by the cells in the chondrogenous layers but not intermediate layer of joint interzone. Furthermore, the synovium and cruciate ligaments were DCX-negative. DCX-positive chondrocytes were very rare in tissue engineered cartilage derived from in vitro pellet culture of rat chondrosarcoma, ATDC5, and C3H10T1/2 cells. However, the new hyaline cartilage formed in rabbit knee defect contained mostly DCX-positive chondrocytes. Our results demonstrate that DCX can be used as a marker to distinguish articular chondrocytes from other chondrocytes and to evaluate the quality of tissue engineered or regenerated cartilage in terms of their "articular" or "non-articular" nature.     

Immunohistochemical distribution of connexin 43 in the cartilage of rats and mice

Using fluorescence immunohistochemistry, the distribution of connexin 43 was examined in hyaline cartilage and in the perichondrium of mouse and rat knee joints. In addition, rat chondrocytes were shown to be coupled in dye transfer studies with Lucifer Yellow. Connexin 43 was detected between chondrocytes in the outer layer of knee joint cartilage, between chondrocytes of the growth plate and between fibrocartilage-like cells at tendon and ligament insertions and in the tendons and ligaments proper. However, in the hyaline cartilage of the hind limbs of mature rats, the degree of connexin 43 immunoreactivity was diminished. These data suggest a possible involvement of connexins in cartilage development. © 1998 Chapman & Hall     

Structuro-functional organization of the fiber network in the human Achilles tendon

Owing to a complex morphological investigation of the human Achilles tendon, it was possible to distinguish four levels of the structural-functional organization of its fibrous elements and to reveal some regularities of their structure that recur at all the levels. Thus, collagenous molecules, microfibrillae, fibrillae and fibers have a wavy-spiral conformation. This spatial form is stabilized by a complex or a system of transversal connections corresponding to the given level of the organization. In order to maintain integrity (the structural-functional unity) of each level, certain substances of polysaccharide nature take part. Along the course of the long tendinous axis, a re-distribution (branching) of the fibrillar elements is observed at all the levels of the structural-functional organization.     

Collagen XXVII organises the pericellular matrix in the growth plate

In order to characterise the function of the novel fibrillar type XXVII collagen, a series of mice expressing mutant forms of the collagen were investigated. Mice harboring a glycine to cysteine substitution in the collagenous domain were phenotypically normal when heterozygote and displayed a mild disruption of growth plate architecture in the homozygous state. Mice expressing an 87 amino acid deletion in the collagenous domain of collagen XXVII were phenotypically normal as heterozygotes whereas homozygotes exhibited a severe chondrodysplasia and died perinatally from a lung defect. Animals expressing the 87 amino acid deletion targeted specifically to cartilage were viable but severely dwarfed. The pericellular matrix of proliferative chondrocytes was disrupted and the proliferative cells exhibited a decreased tendency to flatten and form vertical columns. Collagen XXVII plays an important structural role in the pericellular extracellular matrix of the growth plate and is required for the organisation of the proliferative zone.     

Mammalian chondrocytes expanded in the presence of fibroblast growth factor 2 maintain the ability to differentiate and regenerate three-dimensional cartilaginous tissue

The differentiated phenotype of chondrocytes from hyaline cartilage is gradually lost during expansion in monolayers. Chondrocytes can reexpress their differentiated phenotype by transfer into an environment that prevents cell flattening, but serially passaged cells never completely recover their chondrogenic potential. We report that chondrocytes expanded (up to 2000-fold) in the presence of fibroblast growth factor 2 (FGF-2) dedifferentiated, but fully maintained their potential for redifferentiation in response to environmental changes. After seeding onto three-dimensional polymer scaffolds, chondrocytes expanded in the presence of FGF-2 formed cartilaginous tissue that was histologically and biochemically comparable to that obtained using primary chondrocytes, in contrast to chondrocytes expanded to the same degree but in the absence of FGF-2. The presence of FGF-2 inhibited the formation of thick F-actin structures, which otherwise formed during monolayer expansion, were maintained during tissue cultivation, and were associated with reduced ability of chondrocytes to reexpress their differentiated phenotype. This study provides evidence that FGF-2 maintains the chondrogenic potential during chondrocyte expansion in monolayers, possibly due to changes in the architecture of F-actin elements and allows more efficient utilization of harvested tissue for cartilage tissue engineering.     

Can low concentrations of Papain help repair articular cartilage defects?

In the present study, we investigate the capability of low concentrations of Papain to stimulate cartilage mesenchymal cells proliferation and transformation to chondrocytes and evaluate the healing capability of partial thickness defects in medial condyle cartilage of 30 rabbits’ knee joints. Papain 0.1 mg/ml and Ringer saline l ml each were injected intra-articularly to rabbits of experimental and control groups (15 animals each). Healthy cartilage from lateral condyle and cartilage from medial condyle where the surgical defect was created were studied histologically and by TEM. The study revealed that 0.1 mg/ml Papain activates proliferation and spreading of mesenchymal stem cells to young forms of chondrocyte from perichondrium to the upper layers of healthy cartilage. In only 22.27% cases of the experimental group, surgical defects filled with cartilaginous tissue on the background of distinct destruction of collagenous matrix in the native cartilage. However, in 55.5% of the control group the defect was spontaneously healed by hyaline cartilaginous tissue completely or partially on the basis of slight destruction of collagenous matrix. The defect site was filled with activated chondrocyte-like cells from the subchondral plate (not perichondrium) in both groups, which acquired some cisterns of rough endoplasmic reticulum (RER) and produced matrix proteins. The results suggest that Papain did not ameliorate the recovery of cartilage defects acquired through surgically-induced injury of collagenous matrix in native cartilage. We observed that articular cartilage is the source of mesenchymal stem cells which have the ability to transform into young forms of chondrocytes. This transformation process depends on the level of destruction of native cartilage collagen matrix induced by the defect or by Papain.     

Chondrocyte differentiation and bone growth during the development of the cartilaginous skeleton of chickens]

Certain local alterations in functional and reproductive activity of chondrocytes were stated at the development of the cartilage skeleton. In epiphyses chondrocytes gradually pass into the phase of rest (G0) with subsequent multiplication during the process of skeletal development. In these structures biosynthesis of nonsulfated proteoglycans predominate, in time, while in other cartilage zones--that of sulfated ones. Proofs are furnished on gradual transition of epiphyseal chondrocytes into the state peculiar for cells of the proliferative zone accompanied by an intensified biosynthesis of sulfated proteoglycans and collagenous proteins. Owing to these peculiarities they can be compared with the cells of the reserve zone in the mammalian metaepiphyseal cartilage. It was stated that intensity of chondrogenesis and growth of bones are affected by several processes: intensity of chondrocyte multiplication, the rate of their repeated division in the proliferative zone, the velosity with which the cells transfer into the state of hypertrophy and the rate of the periostal bone formation at the border-line of metaphysis and diaphysis.     

Regulation of insulin secretion by energy metabolism in pancreatic B-cell mitochondria. Studies with a non-metabolizable leucine analogue.

This study compares the collagen types present in rabbit ear cartilage with those synthesized by dissociated chondrocytes in cell culture. The cartilage was first extracted with 4M-guanidinium chloride to remove proteoglycans. This step also extracted type I collagen. After pepsin solubilization of the residue, three additional, genetically distinct collagen types could be separated by fractional salt precipitation. On SDS (sodium dodecyl sulphate)/polyacrylamide-gel electrophoresis they were identified as type II collagen, (1 alpha, 2 alpha, 3 alpha) collagen and M-collagen fragments, a collagen pattern identical with that found in hyaline cartilage. Types I, II, (1 alpha, 2 alpha, 3 alpha) and M-collagen fragments represent 20, 75, 3.5, and 1% respectively of the total collagen. In frozen sections of ear cartilage, type II collagen was located by immunofluorescence staining in the extracellular matrix, whereas type I collagen was closely associated with the chondrocytes. Within 24h after release from elastic cartilage by enzymic digestion, auricular chondrocytes began to synthesize type III collagen, in addition to the above-mentioned collagens. This was shown after labelling of freshly dissociated chondrocytes with [3H]proline 1 day after plating, fractionation of the pepsin-treated collagens from medium and cell layer by NaCl precipitation, and analysis of the fractions by CM(carboxymethyl)-cellulose chromatography and SDS/polyacrylamide-gel electrophoresis. The 0.8 M-NaCl precipitate of cell-layer extracts consisted predominantly of type II collagen. The 0.8 M-NaCl precipitate obtained from the medium contained type I, II, and III collagen. In the supernatant of the 0.8 M-NaCl precipitation remained, both in the cell extract and medium, predominantly 1 alpha-, 2 alpha-, and 3 alpha-chains and M-collagen fragments. These results indicate that auricular chondrocytes are similar to chondrocytes from hyaline cartilage in that they produce, with the exception of type I collagen, the same collagen types in vivo, but change their cellular phenotype more rapidly after transfer to monolayer culture, as indicated by the prompt onset of type III collagen synthesis.     

INFLUENCE OF EXTERNAL POTASSIUM ON THE SYNTHESIS AND DEPOSITION OF MATRIX COMPONENTS BY CHONDROCYTES IN VITRO

The effect of a high external potassium concentration on the synthesis and deposition of matrix components by chondrocytes in cell culture was determined. There is a twofold increase in the amount of chondroitin 4- and 6-sulfate accumulated by chondrocytes grown in medium containing a high potassium concentration. There is also a comparable increase in the production of other sulfated glycosaminoglycans (GAG) including heparan sulfate and uncharacterized glycoprotein components. The twofold greater accumulation of GAG in the high potassium medium is primarily the result of a decrease in their rate of degradation. In spite of this increased accumulation of GAG, the cells in high potassium fail to elaborate appreciable quantities of visible matrix, although they do retain the typical chondrocytic polygonal morphology. Although most of the products are secreted into the culture medium in the high potassium environment, the cell layer retains the same amount of glycosaminoglycan as the control cultures. The inability of chondrocytes grown in high potassium to elaborate the typical hyaline cartilage matrix is not a consequence of an impairment in collagen synthesis, since there is no difference in the total amount of collagen synthesized by high potassium or control cultures. There is, however, a slight increase in the proportion of collagen that is secreted into the medium by chondrocytes in high potassium. Synthesis of the predominant cartilage matrix molecules is not sufficient in itself to ensure that these molecules will be assembled into a hyaline matrix.     

The head cartilage of cephalopods. I. Architecture and ultrastructure of the extracellular matrix

The morphology of head cartilage of the cephalopods Sepia officinalis and Octopus vulgaris has been studied on samples fixed and embedded for light- and electron microscopy and on fresh frozen sections viewed by polarizing microscopy. The organization of extracellular matrix (ECM) varies in different regions of the head cartilage. Superficial zones are made up of densely packed collagenous laminae parallel to the cartilage surface, while radially arranged laminae form a deeper zone where territorial and interterritorial areas are present. A compact arrangement of banded collagen fibrils (10-25 nm in diameter) forms the laminae of the superficial zones and of the interterritorial areas; a loose three-dimensional network of fibrils (10-20 nm) with many proteoglycan aggregates forms the territorial areas. A pericellular matrix surrounds the bodies of extremely branched territorial chondrocytes. Peculiar anchoring devices (ADs) are dispersed with variable orientation within the ECM. A perichondrium is present, but often connectival and muscular bundles are fused with the superficial layers of cartilage. Some vessels were also observed within the superficial inner zone and clusters of hemocyanin molecules were demonstrated both in the ECM and in some cells. The cephalopod head cartilage seems to share some morphological characteristics with both hyaline cartilage and bone tissue of vertebrates.     

Localization of type IX collagen in chondrons isolated from porcine articular cartilage and rat chondrosarcoma

Summary Chondrocytes, each with their pericellular matrix bounded by a fibrous capsule, can be extracted singly or in groups from both mature pig articular cartilage and chondrosarcoma tissue. These structures, termed chondrons, are thought to anchor the chondrocytes in the matrix and protect them from the compressive forces experienced when articular cartilage is under load. The capsule of these chondrons contains both type II and type IX collagens and is composed of fine fibrillar material, unlike the large banded fibres of type II collagen found in the rest of the matrix. This suggests a rote for type IX collagen in regulating the diameter of type II fibres to produce the fine fibrillar structure of the chondron capsules.     

Characteristics of cartilage engineered from human pediatric auricular cartilage

In the repair of cartilage defects, autologous tissue offers the advantage of lasting biocompatibility. The ability of bovine chondrocytes isolated from hyaline cartilage to generate tissue-engineered cartilage in a predetermined shape, such as a human ear, has been demonstrated; however, the potential of chondrocytes isolated from human elastic cartilage remains unknown. In this study, the authors examined the multiplication characteristics of human auricular chondrocytes and the ability of these cells to generate new elastic cartilage as a function of the length of time they are maintained in vitro. Human auricular cartilage, harvested from patients 5 to 17 years of age, was digested in collagenase, and the chondrocytes were isolated and cultured in vitro for up to 12 weeks. Cells were trypsinized, counted, and passaged every 2 weeks. Chondrocyte-polymer (polyglycolic acid) constructs were created at each passage and then implanted into athymic mice for 8 weeks. The ability of the cells to multiply in vitro and their ability to generate new cartilage as a function of the time they had been maintained in vitro were studied. A total of 31 experimental constructs from 12 patients were implanted and compared with a control group of constructs without chondrocytes. In parallel, a representative sample of cells was evaluated to determine the presence of collagen. The doubling rate of human auricular chondrocytes in vitro remained constant within the population studied. New tissue developed in 22 of 31 experimental implants. This tissue demonstrated the physical characteristics of auricular cartilage on gross inspection. Histologically, specimens exhibited dense cellularity and lacunae-containing cells embedded in a basophilic matrix. The specimens resembled immature cartilage and were partially devoid of the synthetic material of which the construct had been composed. Analyses for collagen, proteoglycans, and elastin were consistent with elastic cartilage. No cartilage was detected in the control implants. Human auricular chondrocytes multiply well in vitro and possess the ability to form new cartilage when seeded onto a three-dimensional scaffold. These growth characteristics might some day enable chondrocytes isolated from a small auricular biopsy to be expanded in vitro to generate a large, custom-shaped, autologous graft for clinical reconstruction of a cartilage defect, such as for congenital microtia.     

Chondrocyte transplantation for osteochondral defects with the use of suspension culture

Cartilage graft is considered to be useful in repairing chondral or osteochondral defects. One method of the cartilage graft is achieved by autologous chondrocyte transplantation following cell culture. However, chondrocytes change their phenotype during culture. We used costal chondrocytes cultured over agarose (suspension culture) as a source of graft materials. The suspension-cultured chondrocytes formed aggregate in culture. We first examined the expressions of cartilage-specific matrices of cultured chondrocytes after two weeks in culture. The chondrocytes cultured over agarose expressed more type II collagen mRNA than those cultured on plastic dishes did after two weeks in culture. Safranin O staining showed the presence of glycosaminoglycans in the chondrocyte culture over agarose, while glycosaminoglycans were not observed in the culture on plastic dishes. We then examined the changes of rat articular osteochondral defects after transplantation of suspension-cultured chondrocytes. The aggregate of suspension-cultured chondrocytes was easily picked up with forceps and transplanted in the osteochondral defects. The defects were filled with safranin O-stained hyaline cartilage tissue two weeks after chondrocyte transplantation. On the contrary, the fibrous materials, which were not stained with safranin O, were observed in the control defects. These results suggest that the suspension-cultured chondrocytes are useful for autologous cartilage grafts by preserving chondrocyte phenotype.     

Application of mesenchymal stem cell therapy for the treatment of osteoarthritis of the knee: A concise review

Osteoarthritis(OA) refers to a chronic joint disease characterized by degenerative changes of articular cartilage and secondary bone hyperplasia. Since articular cartilage has a special structure, namely the absence of blood vessels as well as the low conversion rate of chondrocytes in the cartilage matrix, the treatment faces numerous clinical challenges. Traditional OA treatment(e.g., arthroscopic debridement, microfracture, autologous or allogeneic cartilage transplantation,chondrocyte transplantation) is primarily symptomatic treatment and pain management, which cannot contribute to regenerating degenerated cartilage or reducing joint inflammation. Also, the generated mixed fibrous cartilage tissue is not the same as natural hyaline cartilage. Mesenchymal stem cells(MSCs) have turned into the most extensively explored new therapeutic drugs in cell-based OA treatment as a result of their ability to differentiate into chondrocytes and their immunomodulatory properties. In this study, the preliminary results of preclinical(OA animal model)/clinical trials regarding the effects of MSCs on cartilage repair of knee joints are briefly summarized, which lay a solid application basis for more and deeper clinical studies on cell-based OA treatment.     

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.     

The hyaline layer is a collagen-containing extracellular matrix in sea urchin embryos and reaggregating cells

Evidence is given to support the classification of the hyaline layer of sea urchin embryos and reaggregating cells as a collagen-containing extracellular matrix. Ruthenium red staining shows the presence of striated fibril-like structures, dense spheroids, crystalline lattice structures and filamentous material. Collagenase digestion causes disappearance of the fibril-like structures; hyaluronidase treatment causes a diminution of other matrix components, but does not affect the fibrillar structures. The hyaline layer maintains the integrity of the embryo and is also involved in morphogenesis, which are among the functions of extracellular matrices.     

Effects of matrix macromolecules on chondrocyte gene expression: synthesis of a low molecular weight collagen species by cells cultured within collagen gels

Chick-embryo sternal chondrocytes have been cultured within three- dimensional collagen gels as part of a study concerned with the effects of extracellular matrix macromolecules on chondrocyte gene expression. Data are presented indicating that chondrocytes cultured within such a collagenous environment synthesize significantly more of an hitherto unidentified, low molecular weight collagen species than do cells grown on plastic tissue-culture dishes in the conventional manner. This low molecular weight collagen species contains noncollagenous domains (as indicated by its decreased molecular size after mild pepsin digestion), is distinct from the known collagen types (as judged by CNBr peptide analysis), and forms part of the insoluble collagenous matrix produced by the chondrocytes. Cells growing within the gel tend to form colonies consisting of a linear array of cells reminiscent of the cellular organization in growth cartilage.