Expression of Sox and fibrillar collagen genes in lamprey larval chondrogenesis with implications for the evolution of vertebrate cartilage

Lampreys possess unique types of cartilage in which elastin-like proteins are the dominant matrix component, whereas gnathostome cartilage is mainly composed of fibrillar collagen. Despite the differences in protein composition, the Sox-col2a1 genetic cascade was suggested to be conserved between lamprey pharyngeal cartilage and gnathostome cartilage. We examined whether the cascade is conserved in another type of lamprey cartilage, the trabecular cartilage. We found that SoxD and SoxE are expressed in both trabecular and pharyngeal cartilages. However, trabecular cartilage shows no clade A fibrillar collagen gene expression, including genes expressed in pharyngeal cartilage of this animal. On the basis of these observations, we propose that lampreys possess an ancestral type of cartilage that is similar to amphioxus gill cartilage, and in this respect, gnathostome cartilage can be regarded as derived for the loss of elastin-like protein as a cartilage component and recruitment of fibrillar collagen, which is included as a minor component in the ancestral cartilage, as the main component.     

Articular cartilage regeneration with microfracture and hyaluronic acid

Microfracture used to treat articular cartilage injuries can facilitate access to stem cells in the bone marrow and stimulate cartilage regeneration. However, the regenerated cartilage is fibrocartilage as opposed to hyaline articular cartilage and is thinner than native cartilage. Following microfracture in rabbit knee cartilage defects, application of hyaluronic acid gel resulted in regeneration of a thicker, more hyaline-like cartilage. The addition of transforming growth factor-β3, an inducer of chondrogenic differentiation in mesenchymal stem cells, to the treatment with microfracture and hyaluronic acid did not significantly benefit cartilage regeneration.     

ADAM-10 protein is present in human articular cartilage primarily in the membrane-bound form and is upregulated in osteoarthritis and in response to IL-1alpha in bovine nasal cartilage.

The objective of our study was to determine the tissue distribution and localization of ADAM-10 protein in human and bovine cartilage and the changes it undergoes with cartilage degeneration seen in osteoarthritis (OA) and under the influence of interleukin-1 (IL-1). Human normal and OA articular cartilage and bovine nasal cartilage cultured in the presence of IL-1alpha were processed for histology and immunohistochemistry. ADAM-10 protein was extracted from human cartilage and analyzed by Western blotting using anti-ADAM-10 antibodies. Fluor S Image analyzer and Quantity One software program were applied to quantify the total amount of ADAM-10. ADAM-10 protein was detected in both human and bovine cartilage. The strongest immunostaining was found in the cytoplasm and/or cell membranes of the superficial and upper middle layer of normal adult human cartilage, in the clusters and fibrillated areas of OA cartilage, and in IL-1alpha-stimulated bovine nasal cartilage. The distribution of ADAM-10 protein in bovine nasal cartilage was dependent on the length of exposure to IL-1alpha and corresponded to the areas of proteoglycan depletion. By Western blotting analysis of human cartilage, ADAM-10 was primarily detected in the membrane-enriched fraction and its levels were increased in degenerated and OA cartilage compared to normal cartilage. The results of this study suggest that ADAM-10 might be an important factor associated with cartilage degenerative processes. (J Histochem Cytochem 49:1165-1176, 2001)     

Expression profile of carbonic anhydrases in articular cartilage

Carbonic anhydrases (CAs), which catalyze the reversible reaction of carbonate hydration, are important for cartilage homeostasis. The full spectrum of CA activity of all 13 isoenzymes in articular cartilage is unknown. This study quantified the mRNA profile of CAs in rat articular cartilage, using quantitative polymerase chain reactions. Among the 13 functional CAs, CAs II, III, Vb, IX, XII and XIII were significantly expressed at mRNA level by the chondrocytes in articular cartilage. To verify these significantly expressed CAs in articular cartilage at protein level, immunohistochemistry was performed. While CAs III, Vb and XII distributed in the full-thickness of cartilage, including the calcified zone of cartilage, CA II was mainly localized in the proliferative zone of cartilage. CA IX was limited in the superficial zone of cartilage and CA XIII expressed in the superficial and partially mid zone. These results provide a framework for understanding individual CAs as well as the integrated CA family in cartilage biology, including matrix mineralization.     

Os penis of the rat. IV. The proximal growth cartilage

Histomorphological and histochemical aspects of the proximal cartilage of os penis and its surrounding perichondrium in 60 rats aged between 1 and 100 days are described. Comparisons at 11-14 days with the mandibular condylar cartilage reveal a slight difference in their general morphological composition. The developmental changes which take place in the os penis cartilage reveal histomorphologic events, some of which may be brought into agreement with previous observations of patterns of transformations of the bone. Observations on an age-dependent morphological appearance of the area adjacent to the proximal surface of the cartilage suggest certain agreements between the mandibular angular cartilage and the os penis cartilage. The study of phosphomonoesterases in the os penis cartilage and its perichondrium reveals significant, unexplained differences in the distribution of alkaline phosphatase between this cartilage and the mandibular condylar cartilage.     

Development and aging of the articular cartilage of the rabbit knee joint: Distribution of biglycan, decorin, and matrilin-1.

We determined the distributions of the small proteoglycans biglycan and decorin and the glycoprotein matrilin-1 (cartilage matrix protein) during development and aging of articular cartilage in the rabbit knee joint. Before cavitation, the matrices of the interzone and the adjacent epiphyseal cartilage do not contain biglycan or decorin, but some chondrocytes express their mRNAs. Matrilin-1 is found only in the deeper epiphyseal cartilage. After cavitation, biglycan and decorin are detected in the presumptive articular cartilage, but there is no matrilin-1. All are present in the underlying epiphyseal cartilage. In the neonate, the epiphyseal cartilage is ossified and the articular cartilage becomes a discrete layer. Biglycan and decorin accumulate in the articular cartilage, but matrilin-1 remains confined to the residual epiphyseal cartilage. In the adult, the distributions of biglycan and decorin are highly variable. Decorin tends to be confined to the central region; matrilin-1 is absent. The findings indicate that the articular and epiphyseal cartilages are different from the earliest developmental stages. The epiphyseal cartilage can be identified by its possession of matrilin-1. Epiphyseal cartilage is removed during development to leave the articular cartilage. The relationships between the distributions of decorin and matrilin-1 and the fibrillar collagens are discussed. (J Histochem Cytochem 47:1603-1615, 1999)     

Immunochemical analysis of cartilage proteoglycans. Cross-reactivity of molecules isolated from different species.

Antibodies directed against whole bovine nasal-cartilage proteoglycan and against the hyaluronic acid-binding region and chondroitin sulphate peptides from the same molecule were used in immunodiffusion and immunoelectromigration experiments. Proteoglycans from bovine nasal and tracheal cartilage showed immunological identity, with all three antisera. Proteoglycans from pig hip articular cartilage, dog hip articular cartilage, human tarsal articular cartilage and rat chondrosarcoma reacted with all the antisera and showed immunological identity with the corresponding structures isolated from bovine nasal-cartilage proteoglycans. In contrast, proteoglycans from rabbit articular cartilage, rabbit nasal cartilage and cultured chick limb buds did not react with the antibodies directed against the hyaluronic acid-binding region, though reacting with antibodies raised against whole proteoglycan monomer and against chondroitin sulphate peptides. All the proteoglycans gave two precipitation lines with the anti-(chondroitin sulphate peptide) antibodies. Similarly, the proteoglycans reacting with the anti-(hyaluronic acid-binding region) antibodies gave two precipitation lines. The results indicate the presence of at least two populations of aggregating proteoglycan monomers in cartilage. The relative affinity of the antibodies for cartilage proteoglycans and proteoglycan substructures from various species was determined by radioimmunoassay. The affinity of the anti-(hyaluronic acid-binding region) antibodies for the proteoglycans decreased in the order bovine, dog, human and pig cartilage. Rat sternal-cartilage and rabbit articular-cartilage proteoglycans reacted weakly, whereas chick limb-bud and chick sternal-cartilage proteoglycans did not react. In contrast, the affinity of antibodies to chondroitin sulphate peptides for proteoglycans increased in the order bovine cartilage, chick limb bud and chick sternal cartilage, dog cartilage, rat chondrosarcoma, human cartilage, pig cartilage, rat sternal cartilage and rabbit cartilage.     

Thionin Staining of Paraffin and Plastic Embedded Sections of Cartilage

The usefulness of thionin for staining cartilage sections embedded in glycol meth-acrylate (GMA) and the effect of decalcification on cartilage sections embedded in paraffin and GMA were assessed. Short decalcification periods using 5% formic acid or 10% EDTA did not influence the staining properties or the morphology of cartilage matrix and chondrocytes. The standard stain safranin O-fast green for differential staining of cartilage was used as control in these experiments. Prolonged exposure of safranin P stained sections to fast green resulted in disappearance of the safranin O stained matrix, thereby hampering the quantitative measurement of negatively charged glycosaminoglycans (GAG). Thionin stained evenly throughout all cartilage layers, independent of the staining times. In contrast to safranin 0, thionin did not show meta-chromasia in nondehydrated cartilage sections, which made it more suitable for assessing cartilage quality in GMA embedded cartilage. To evaluate the selectivity of thionin staining in cartilage, chondroitinase ABC and trypsin digestions were carried out. Thionin staining was prevented by these enzymes in the territorial matrix, representing the interlacunar network and the chondrocyte capsule. Staining with thionin of the interterritorial matrix was only slightly reduced, possibly representing keratan sulfate and hyaluronic acid in cartilage of elderly patients. Comparison of thionin stained GMA embedded cartilage with safranin O stained paraffin embedded sections showed significant similarity in optical densitometry, indicative of the specificity of thionin bound to negatively charged GAG in cartilage. In GMA embedded cartilage morphology was relatively intact compared to paraffin embedded sections due to less shrinkage of chondrocytes and the interlacunar network.     

Biochemical analysis of normal and osteoarthritic human cartilage

Non-collagenous proteins from the articular cartilage of normal subjects and patients with degenerative joint disease were extracted sequentially. Proteoglycans and the other glycoproteins were more extractable from the osteoarthritic cartilage at lower ionic strength than those from the normal cartilage. A 50-kD protein which seems specific to osteoarthritic cartilage was identified. Three different populations of proteoglycans were purified from normal and only two from osteoarthritic cartilage. Moreover, greater amounts of albumin and fibronectin were found in the pathological cartilage. No differences were observed between link proteins from normal and osteoarthritic cartilage, nor in their molecular weight or the amounts extracted.     

Maturation-dependent spontaneous healing of partial thickness cartilage defects in infantile rats

Partial-thickness articular cartilage defects (PTCDs) do not heal spontaneously and are thought to be a predisposing factor for the development of osteoarthritis. Younger and smaller animals have a better healing capacity for many types of injuries including those to articular cartilage. Our aim was to examine the longitudinal histological changes of immature murine articular cartilage after the creation of small PTCDs and to compare them to PTCDs in mature cartilage. Single linear PTCDs were created in 3-week-old and 16-week-old rats in the direction of joint motion. At 6 and 12?weeks after PTCD creation, histological changes were examined in the defect sites and surrounding cartilage. Immature cartilage showed a higher repair capability than mature cartilage. Although repaired immature cartilage had fibrocartilage, it exhibited better quality than any PTCD model, except for a fetus model and comparable quality to full-thickness cartilage defects (FTCD) after bone marrow stimulation. Elucidation of the underlining mechanisms that immature cartilage possesses for repairing PTCDs is necessary in order to aid the prevention or develop treatment for osteoarthritis.     

A simple in vitro culture system for tracheal cartilage development

Semi-circular tracheal cartilage is a critical determinant of maintaining architectural integrity of the respiratory airway. The current effort to understand the morphogenesis of tracheal cartilage is challenged by the lack of appropriate model systems. Here we report an in vitro tracheal cartilage system using embryonic tracheal–lung explants to recapitulate in vivo tracheal cartilage developmental processes. With modifications of a current lung culture protocol, we report a consistent in vitro technique of culturing tracheal cartilage from primitive mouse embryonic foregut for the first time. This tracheal culture system not only induces the formation of tracheal cartilage from the mouse embryonic foregut but also allows for the proper patterning of the developed tracheal cartilage. Furthermore, we show that this culture technique can be applied to culturing other types of cartilage in vertebrae, limbs, and ribs. We believe that this novel application of our in vitro culture system will facilitate the manipulation of cartilage development under various conditions and thus enabling us to advance our current limited knowledge on cartilage biology and development.     

Pathways of load-induced cartilage damage causing cartilage degeneration in the knee after meniscectomy

Results of both clinical and animal studies show that meniscectomy often leads to osteoarthritic degenerative changes in articular cartilage. It is generally assumed that this process of cartilage degeneration is due to changes in mechanical loading after meniscectomy. It is, however, not known why and where this cartilage degeneration starts. Load induced cartilage damage is characterized as either type (1)--damage without disruption of the underlying bone or calcified cartilage layer--or type (2), subchondral fracture with or without damage to the overlying cartilage. We asked the question whether cartilage degeneration after meniscectomy is likely to be initiated by type (1) and/or type (2) cartilage damage. To investigate that we applied an axisymmetric biphasic finite element analysis model of the knee joint. In this model the articular cartilage layers of the tibial and the femoral condyles, the meniscus and the bone underlying the articular cartilage of the tibia plateau were included. The model was validated with data from clinical studies, in which the effects of meniscectomy on contact areas and pressures were measured. It was found that both the maximal values and the distributions of the shear stress in the articular cartilage changed after meniscectomy, and that these changes could lead to both type (1) and type (2) cartilage damage. Hence it likely that the cartilage degeneration seen after meniscectomy is initiated by both type (1) and type (2) cartilage damage.     

In Vivo Evaluation of a Novel Oriented Scaffold-BMSC Construct for Enhancing Full-Thickness Articular Cartilage Repair in a Rabbit Model

Tissue engineering (TE) has been proven usefulness in cartilage defect repair. For effective cartilage repair, the structural orientation of the cartilage scaffold should mimic that of native articular cartilage, as this orientation is closely linked to cartilage mechanical functions. Using thermal-induced phase separation (TIPS) technology, we have fabricated an oriented cartilage extracellular matrix (ECM)-derived scaffold with a Young''s modulus value 3 times higher than that of a random scaffold. In this study, we test the effectiveness of bone mesenchymal stem cell (BMSC)-scaffold constructs (cell-oriented and random) in repairing full-thickness articular cartilage defects in rabbits. While histological and immunohistochemical analyses revealed efficient cartilage regeneration and cartilaginous matrix secretion at 6 and 12 weeks after transplantation in both groups, the biochemical properties (levels of DNA, GAG, and collagen) and biomechanical values in the oriented scaffold group were higher than that in random group at early time points after implantation. While these differences were not evident at 24 weeks, the biochemical and biomechanical properties of the regenerated cartilage in the oriented scaffold-BMSC construct group were similar to that of native cartilage. These results demonstrate that an oriented scaffold, in combination with differentiated BMSCs can successfully repair full-thickness articular cartilage defects in rabbits, and produce cartilage enhanced biomechanical properties.     

Correction of abnormal matrix formed by cmd/cmd chondrocytes in culture by exogenously added cartilage proteoglycan

The cartilage matrix deficiency (cmd/cmd) mouse fails to synthesize the core protein of cartilage-characteristic proteoglycan (cartilage PG). Chondrocytes from the cmd/cmd cartilage cultured in vitro produced nodules with greatly reduced extracellular matrix. Immunofluorescence staining revealed that the nodules of mutant cells differed from the normal in lacking cartilage PG and in uneven and reduced deposition of type II collagen. Exogenously added cartilage PG prepared from either normal mouse cartilage or Swarm rat chondrosarcoma to the culture medium was incorporated exclusively into the extracellular matrices of the nodules, with a concurrent correction of the abnormal distribution pattern of type II collagen. The incorporation of cartilage PG into the matrix was disturbed by hyaluronic acid or decasaccharide derived therefrom, suggesting that the incorporation process involves the interaction of added proteoglycan with hyaluronic acid. Both the hyaluronic acid-binding region and the protein-enriched core molecule prepared from rat chondrosarcoma cartilage PG could also be incorporated but, unlike the intact cartilage PG, they were distributed equally in the surrounding zones where fibroblast-like cells predominate. The results indicate that the intact form of cartilage PG is required for specific incorporation into the chondrocyte nodules, and further suggest that cartilage PG plays a regulatory role in the assembly of the matrix macromolecules.     

Articular cartilage destruction in experimental inflammatory arthritis: insulin-like growth factor-1 regulation of proteoglycan metabolism in chrondrocytes

Summary Rheumatoid arthritis, a disease of unknown aetiology, is characterized by joint inflammation and, in its later stages, cartilage destruction. Inflammatory mediators may exert not only suppression of matrix synthesis but also cartilage degradation, which eventually leads to severe cartilage depletion. Systemically and locally produced growth factors and hormones regulate cartilage metabolism. Alterations in levels of these factors or in their activity can influence the pathogenesis of articular cartilage destruction in arthritic joints. The main topic of the present review is the role of the anabolic factor insulin-like growth factor-1 in the regulation of chondrocyte metabolic functions in normal and in diseased cartilage. This is the most important growth factor that balances chondrocyte proteoglycan synthesis and catabolism to maintain a functional cartilage matrix. A brief overview of how chondrocytes keep the cartilage matrix intact, and how catabolic and anabolic vactors are thought to be involved in pathological cartilage destruction precedes the review of the role of this growth factor in proteoglycan metabolism in cartilage.     

Characteristics of mineral particles in the human bone/cartilage interface

Bone and cartilage consist of different organic matrices, which can both be mineralized by the deposition of nano-sized calcium phosphate particles. We have studied these mineral particles in the mineralized cartilage layer between bone and different types of cartilage (bone/articular cartilage, bone/intervertebral disk, and bone/growth cartilage) of individuals aged 54 years, 12 years, and 6 months. Quantitative backscattered electron imaging and scanning small-angle X-ray scattering at a synchrotron radiation source were combined with light microscopy to determine calcium content, mineral particle size and alignment, and collagen orientation, respectively. Mineralized cartilage revealed a higher calcium content than the adjacent bone (p<0.05 for all samples), whereas the highest values were found in growth cartilage. Surprisingly, we found the mineral platelet width similar for bone and mineralized cartilage, with the exception of the growth cartilage sample. The most striking result, however, was the abrupt change of mineral particle orientation at the interface between the two tissues. While the particles were aligned perpendicular to the interface in cartilage, they were oriented parallel to it in bone, reflecting the morphology of the underlying organic matrices. The tight bonding of mineralized cartilage to bone suggests a mechanical role for the interface of the two elastically different tissues, bone and cartilage.     

Thionin Staining of Paraffin and Plastic Embedded Sections of Cartilage

The usefulness of thionin for staining cartilage sections embedded in glycol meth-acrylate (GMA) and the effect of decalcification on cartilage sections embedded in paraffin and GMA were assessed. Short decalcification periods using 5% formic acid or 10% EDTA did not influence the staining properties or the morphology of cartilage matrix and chondrocytes. The standard stain safranin O-fast green for differential staining of cartilage was used as control in these experiments. Prolonged exposure of safranin P stained sections to fast green resulted in disappearance of the safranin O stained matrix, thereby hampering the quantitative measurement of negatively charged glycosaminoglycans (GAG). Thionin stained evenly throughout all cartilage layers, independent of the staining times. In contrast to safranin 0, thionin did not show meta-chromasia in nondehydrated cartilage sections, which made it more suitable for assessing cartilage quality in GMA embedded cartilage. To evaluate the selectivity of thionin staining in cartilage, chondroitinase ABC and trypsin digestions were carried out. Thionin staining was prevented by these enzymes in the territorial matrix, representing the interlacunar network and the chondrocyte capsule. Staining with thionin of the interterritorial matrix was only slightly reduced, possibly representing keratan sulfate and hyaluronic acid in cartilage of elderly patients. Comparison of thionin stained GMA embedded cartilage with safranin O stained paraffin embedded sections showed significant similarity in optical densitometry, indicative of the specificity of thionin bound to negatively charged GAG in cartilage. In GMA embedded cartilage morphology was relatively intact compared to paraffin embedded sections due to less shrinkage of chondrocytes and the interlacunar network.     

Scaffoldless tissue-engineered cartilage for studying transforming growth factor beta-mediated cartilage formation

Reduced transforming growth factor beta (TGF-β) signaling is associated with osteoarthritis (OA). TGF-β is thought to act as a chondroprotective agent and provide anabolic cues to cartilage, thus acting as an OA suppressor in young, healthy cartilage. A potential approach for treating OA is to identify the factors that act downstream of TGF-β's anabolic pathway and target those factors to promote cartilage regeneration or repair. The aims of the present study were to (a) develop a scaffoldless tissue-engineered cartilage model with reduced TGF-β signaling and disrupted cartilage formation and (b) validate the system for identifying the downstream effectors of TGF-β that promote cartilage formation. Sox9 was used to validate the model because Sox9 is known to promote cartilage formation and TGF-β regulates Sox9 activity. Primary bovine articular chondrocytes were grown in Transwell supports to form cartilage tissues. An Alk5/TGF-β type I receptor inhibitor, SB431542, was used to attenuate TGF-β signaling, and an adenovirus encoding FLAG-Sox9 was used to drive the expression of Sox9 in the in vitro-generated cartilage. SB431542-treated tissues exhibited reduced cartilage formation including reduced thicknesses and reduced proteoglycan staining compared with control tissue. Expression of FLAG-Sox9 in SB431542-treated cartilage allowed the formation of cartilage despite antagonism of the TGF-β receptor. In summary, we developed a three-dimensional in vitro cartilage model with attenuated TGF-β signaling. Sox9 was used to validate the model for identification of anabolic agents that counteract loss of TGF-β signaling. This model has the potential to identify additional anabolic factors that could be used to repair or regenerate damaged cartilage.     

MR水激励序列显示膝关节软骨的临床应用研究

目的：探讨三维快速梯度回波水激励膝关节软骨成像序列（3D-FFE-WATS）相对于三维快速梯度回波预饱和反转恢复法脂肪抑制序列（3D-FFE-SPIR）在显示膝关节软骨方面的优势,选择显示膝关节软骨的最佳序列。方法：应用3D-FFE-WATS及3D-FFE-SPIR序列组合对20名志愿者及30例疑诊关节软骨损伤的单膝关节进行检查,获得膝关节各软骨的3D图像,并利用3D最大密度投影法（MIP）进行横断面和冠状面3D重建。分析上述2种序列对软骨病变的显示及检出能力,计算其对关节软骨的信噪比（SNR）和对比噪声比（CNR）,并进行统计学分析。结果：两序列在显示膝关节软骨SNR方面,无统计学意义（P〉0.05）;两种序列在显示软骨与关节液的CNR、软骨与骨皮质的CNR、软骨与骨髓的CNR、软骨与肌肉的CNR差异方面t值分别为（-30.619;2.348;-2.408;2.216）,有统计学意义（P〈0.05）。结论：3D-FFE-WATS序列可作为膝关节软骨成像的首选序列。