Articular cartilage vesicles contain RNA

Small membrane-bound extracellular organelles known as articular cartilage matrix vesicles (ACVs) participate in pathologic mineralization in osteoarthritic articular cartilage. ACVs are also present in normal cartilage, although they have no known functions other than mineralization. Recently, RNA was identified in extracellular vesicles derived from mast cells, suggesting that such vesicles might carry coding information from cell to cell. We found that ACVs from normal porcine and human articular cartilage and primary chondrocyte conditioned media contained 1 μg RNA/80 μg ACV protein. No DNA could be detected. RT-PCR of ACV RNA demonstrated the presence of full length mRNAs for factor XIIIA, type II transglutaminase, collagen II, aggrecan, ANKH and GAPDH. RNA in intact ACVs was resistant to RNase, despite the fact that ACV preparations contained measurable levels of active RNases. Significantly, radiolabeled RNA in ACVs could be transferred to unlabeled chondrocytes by co-incubation and produced changes in levels of chondrocyte enzymes and proteins. The demonstration that ACVs contain mRNAs suggests that they may function to shuttle genetic information between articular cells and indicate novel functions for these structures in articular cartilage.     

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.     

Tenascin-C and the development of articular cartilage

In comparison to the vast literature on articular cartilage structure and function, relatively little is known about how articular cartilage forms during embryo-genesis and is endowed with unique phenotypic properties, most notably the ability to persist and function throughout postnatal life. In this minireview, we summarize recent studies from our laboratory suggesting that the extracellular matrix protein tenascin-C is involved in the genesis and function of articular chondrocytes. These and other data have led us to propose that tenascin-C may be part of in vivo mechanisms whereby articular chondrocytes develop at the epiphysis of long bone models, remain functional throughout postnatal life, and avoid the endochondral ossification process undertaken by the bulk of chondrocytes located in the metaphysis and diaphysis of skeletal models.     

Sclerostin Immunoreactivity Increases in Cortical Bone Osteocytes and Decreases in Articular Cartilage Chondrocytes in Aging Mice

Sclerostin is a 24-kDa secreted glycoprotein that has been identified as a negative modulator of new bone formation and may play a major role in age-related decline in skeletal function. Although serum levels of sclerostin markedly increase with age, relatively little is known about whether cells in the skeleton change their expression of sclerostin with aging. Using immunohistochemistry and confocal microscopy, we explored sclerostin immunoreactivity (sclerostin-IR) in the femurs of 4-, 9-, and 24-month-old adult C3H/HeJ male mice. In the femur, the only two cell types that expressed detectable levels of sclerostin-IR were bone osteocytes and articular cartilage chondrocytes. At three different sites along the diaphysis of the femur, only a subset of osteocytes expressed sclerostin-IR and the percentage of osteocytes that expressed sclerostin-IR increased from approximately 36% to 48% in 4- vs. 24-month-old mice. In marked contrast, in the same femurs, there were ~40% fewer hypertrophic chondrocytes of articular cartilage that expressed sclerostin-IR when comparing 24- vs. 4-month-old mice. Understanding the mechanism(s) that drive these divergent changes in sclerostin-IR may provide insight into understanding and treating the age-related decline of the skeleton.     

Chondromalacia induced by patellar subluxation: morphological and morphometrical aspects in rabbits

The purpose of this study was to describe morphologically and quantify the changes of the articular cartilage in chondromalacia, concerning both the chondrocytes and extracellular matrix. Eight rabbits were submitted daily to patellar subluxation, causing chondromalacia after two weeks. The knee fragments obtained were processed by the standard methods. These experimental conditions caused degenerative alterations of the articular cartilage, varying from a slight decrease of proteoglycans, to fibrillations, clefts, and horizontal splitting. The results showed a significantly increase number of chondrocytes (p < 0,000139), although smaller in size (p < 0,000109). The immobilization for 2 weeks and the intermittent passive daily motion afterwards for a period of 2 weeks, was effective to cause patellar chrondomalacia in rabbits.     

Mesenchyme-specific Knockout of ESET Histone Methyltransferase Causes Ectopic Hypertrophy and Terminal Differentiation of Articular Chondrocytes

The exact molecular mechanisms governing articular chondrocytes remain unknown in skeletal biology. In this study, we have found that ESET (an ERG-associated protein with a SET domain, also called SETDB1) histone methyltransferase is expressed in articular cartilage. To test whether ESET regulates articular chondrocytes, we carried out mesenchyme-specific deletion of the ESET gene in mice. ESET knock-out did not affect generation of articular chondrocytes during embryonic development. Two weeks after birth, there was minimal qualitative difference at the knee joints between wild-type and ESET knock-out animals. At 1 month, ectopic hypertrophy, proliferation, and apoptosis of articular chondrocytes were seen in the articular cartilage of ESET-null animals. At 3 months, additional signs of terminal differentiation such as increased alkaline phosphatase activity and an elevated level of matrix metalloproteinase (MMP)-13 were found in ESET-null cartilage. Staining for type II collagen and proteoglycan revealed that cartilage degeneration became progressively worse from 2 weeks to 12 months at the knee joints of ESET knock-out mutants. Analysis of over 14 pairs of age- and sex-matched wild-type and knock-out mice indicated that the articular chondrocyte phenotype in ESET-null mutants is 100% penetrant. Our results demonstrate that expression of ESET plays an essential role in the maintenance of articular cartilage by preventing articular chondrocytes from terminal differentiation and may have implications in joint diseases such as osteoarthritis.     

Articular cartilage and changes in Arthritis: Collagen of articular cartilage

The extracellular framework and two-thirds of the dry mass of adult articular cartilage are polymeric collagen. Type II collagen is the principal molecular component in mammals, but collagens III, VI, IX, X, XI, XII and XIV all contribute to the mature matrix. In developing cartilage, the core fibrillar network is a cross-linked copolymer of collagens II, IX and XI. The functions of collagens IX and XI in this heteropolymer are not yet fully defined but, evidently, they are critically important since mutations in COLIX and COLXI genes result in chondrodysplasia phenotypes that feature precocious osteoarthritis. Collagens XII and XIV are thought also to be bound to fibril surfaces but not covalently attached. Collagen VI polymerizes into its own type of filamentous network that has multiple adhesion domains for cells and other matrix components. Collagen X is normally restricted to the thin layer of calcified cartilage that interfaces articular cartilage with bone.     

人关节软骨细胞的体外分离、培养与鉴定

目的:研究人关节软骨细胞的体外分离、培养及鉴定方法,观察各代人关节软骨细胞的形态学特性。方法:取人创伤性截肢的无菌膝关节软骨,采用两步酶消化法分离培养人关节软骨细胞,并进行传代培养。通过倒置相差显微镜下观察细胞形态,绘制生长曲线,甲苯胺蓝染色及Ⅱ型胶原免疫组织化学染色对细胞进行鉴定。结果:两步酶消化法消化出的软骨细胞呈圆形,培养2-3天,细胞贴壁、变形,呈三角形或多角形,2周左右细胞融合成层,传代5次后出现去分化。软骨细胞增殖和生长缓慢。形态学、免疫组织化学染色显示细胞培养5代以内可以保持表型的稳定。结论:本研究采用胰蛋白酶及Ⅱ型胶原酶联合消化法获得大量高纯度、高活性的人关节软骨细胞。5代以内细胞生长良好,生物学特性明显,适合于实验研究,5代以后出现去分化现象。     

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 .     

Long noncoding RNA MALAT-1 inhibits apoptosis and matrix metabolism disorder in interleukin-1β-induced inflammation in articular chondrocytes via the JNK signaling pathway

Proinflammatory cytokine such as interleukin (IL)-1β causes inflammation of articular cartilage. In this current study, we explored the chondroprotective effects of long noncoding RNA (lncRNA) MALAT-1 on cell proliferation, apoptosis, and matrix metabolism in IL-1β-induced inflammation in articular chondrocytes. Articular chondrocytes from knee joints of normal rats were isolated and cultured, followed by identification through observation of toluidine blue and COL II immunocytochemical stainings. The proliferation of chondrocytes at passage 2 was detected by the 3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2H-tetrazolium bromide (MTT) assay. The inflammatory chondrocytes induced by 10 ng/mL IL-1β were observed and identified by toluidine blue and COL II immunocytochemical stainings. pcDNA 3.1 and pcDNA-MALAT-1 were transfected in the chondrocytes. Ultrastructure of chondrocytes was observed by using a transmission electron microscope. The MTT assay was carried out to evaluate chondrocyte viability. Hoechst 33258 staining and flow cytometry were adopted to assess chondrocyte apoptosis. The chondrocytes at passage 2 with the biological characteristics of chondrocytes were used for subsequent experiments. In IL-1β-treated chondrocytes, the growth rate of chondrocytes slowed down, the cells became narrow and long, the vacuoles were seen in the cells, and the morphology of the chondrocytes was irregular. The toluidine blue staining and the immunohistochemical staining of COL II became weaker. In response to IL-1β induction, articular chondrocytes showed reduced MALAT-1 expression; moreover, obvious cartilage injury was observed with decreased chondrocyte viability and Col II expression and elevated chondrocyte apoptosis, MMP-13 expression, and p-JNK expression. With the treatment of pcDNA-MALAT-1, the cartilage injury was alleviated with increased chondrocyte viability and type II collagen (Col II) expression and reduced chondrocyte apoptosis, MMP-13 expression and p-JNK expression. Taken together these results, lncRNA MALAT-1 blocked the activation of the JNK signaling pathway; thereby, IL-1β-induced inflammation in articular chondrocytes was reduced with enhanced chondrocyte proliferation and suppressed chondrocyte apoptosis and extracellular matrix degradation.     

Partial purification of a factor from human synovium that possesses interleukin 1, chondrocyte stimulating and catabolin-like activities

Human synovial explants in culture release material that stimulates the production of prostaglandin E2 (PGE2) and several extracellular enzymes by human chondrocytes. Fractionation of conditioned medium by gel filtration revealed a protein of approx. 15 kDa, which in addition to stimulating production of PGE2 and plasminogen activator by human articular chondrocytes, possessed interleukin 1 activity and induced cartilage degradation. Further purification using iso-electric focussing again showed co-elution of these activities with a major pI of 6.9 and a minor pI of 5.1-5.3. This study indicated that human synovium releases a factor that is closely related to or identical with interleukin 1 and suggests that this protein may participate in cellular interactions that occur within the rheumatoid joint.     

Identification of Fibroblast Growth Factor-18 as a Molecule to Protect Adult Articular Cartilage by Gene Expression Profiling

To identify genes that maintain the homeostasis of adult articular cartilage and regenerate its lesions, we initially compared four types of chondrocytes: articular (AA) versus growth plate (AG) cartilage chondrocytes in adult rats, and superficial layer (IS) versus deep layer (ID) chondrocytes of epiphyseal cartilage in infant rats. Microarray analyses revealed that 40 and 186 genes had ≥10-fold higher expression ratios of AA/AG and IS/ID, respectively, and 16 genes showed ≥10-fold of both AA/AG and IS/ID ratios. The results were validated by real-time RT-PCR analysis. Among them, Hoxd1, Fgf18, and Esm1 were expressed more strongly in AA than in IS. Fgf18 was the extracellular and secreted factor that decreased glycosaminoglycan release and depletion from the cartilage, and enhanced proliferation of articular chondrocytes. Fgf18 was strongly expressed in the articular cartilage chondrocytes of adult rats. In a surgical rat osteoarthritis model, a once-weekly injection of recombinant human FGF18 (rhFGF18) given 3 weeks after surgery prevented cartilage degeneration in a dose-dependent manner at 6 and 9 weeks after surgery, with significant effect at 10 μg/week of rhFGF18. As the underlying mechanism, rhFGF18 strongly up-regulated Timp1 expression in the cell and organ cultures, and inhibition of aggrecan release by rhFGF18 was restored by addition of an antibody to Timp1. In conclusion, we have identified Fgf18 as a molecule that protects articular cartilage by gene expression profiling, and the anticatabolic effects may at least partially be mediated by the Timp1 expression.     

Characterization of subpopulated articular chondrocytes separated by Percoll density gradient

The aim of this study was to develop a method for fractionation of articular chondrocytes from the entire thickness of the tissue. Isolated chondrocytes from rabbit articular cartilage fractionated by centrifugation in a discontinuous Percoll gradient resulted in four cell fractions with two differing properties. The lowest-density fraction consisted mainly of large cells with small nuclei proliferated actively, maintained the chondrocytic phenotype, and secreted larger amounts of proteoglycan. In contrast, the highest-density fraction consisted of small cells with large nuclei proliferated slowly, did not express the chondrocytic phenotype, and produced larger amounts of interleukin 1-induced nitric oxide. Comparing our results with other previous reports, we find that fraction 1 cells are likely originated from the deep layer of the articular cartilage, whereas fraction 4 cells are tentatively categorized as chondrocytes from the superficial layer of cartilage. Centrifugal fractionation of articular chondrocytes via Percoll density gradient permits clear separation of these heterogeneous cells into different phenotypic populations and allows distinguishing of cells from the different layers of articular cartilage. This simple novel method will provide ready separation of articular chondrocytes for the investigation of the pathogenesis of 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.     

Osteopontin promotes pathologic mineralization in articular cartilage.

Calcium pyrophosphate dihydrate (CPPD) crystals are commonly found in osteoarthritic joint tissues, where they predict severe disease. Unlike other types of calcium phosphate crystals, CPPD crystals form almost exclusively in the pericellular matrix of damaged articular cartilage, suggesting a key role for the extracellular matrix milieu in their development. Osteopontin is a matricellular protein found in increased quantities in the pericellular matrix of osteoarthritic cartilage. Osteopontin modulates the formation of calcium-containing crystals in many settings. We show here that osteopontin stimulates ATP-induced CPPD crystal formation by chondrocytes in vitro. This effect is augmented by osteopontin's incorporation into extracellular matrix by transglutaminase enzymes, is only modestly affected by its phosphorylation state, and is inhibited by integrin blockers. Surprisingly, osteopontin stimulates transglutaminase activity in cultured chondrocytes in a dose-responsive manner. As elevated levels of transglutaminase activity promote extracellular matrix changes that permit CPPD crystal formation, this is one possible mechanism of action. We demonstrate the presence of osteopontin in the pericellular matrix of chondrocytes adjacent to CPPD deposits and near active transglutaminases. Thus, osteopontin may play an important role in facilitating CPPD crystal formation in articular cartilage.     

Preservation of the chondrocyte's pericellular matrix improves cell‐induced cartilage formation

The extracellular matrix surrounding chondrocytes within a chondron is likely to affect the metabolic activity of these cells. In this study we investigated this by analyzing protein synthesis by intact chondrons obtained from different types of cartilage and compared this with chondrocytes. Chondrons and chondrocytes from goats from different cartilage sources (articular cartilage, nucleus pulposus, and annulus fibrosus) were cultured for 0, 7, 18, and 25 days in alginate beads. Real‐time polymerase chain reaction analyses indicated that the gene expression of Col2a1 was consistently higher by the chondrons compared with the chondrocytes and the Col1a1 gene expression was consistently lower. Western blotting revealed that Type II collagen extracted from the chondrons was cross‐linked. No Type I collagen could be extracted. The amount of proteoglycans was higher for the chondrons from articular cartilage and nucleus pulposus compared with the chondrocytes, but no differences were found between chondrons and chondrocytes from annulus fibrosus. The expression of both Mmp2 and Mmp9 was higher by the chondrocytes from articular cartilage and nucleus pulposus compared with the chondrons, whereas no differences were found with the annulus fibrosus cells. Gene expression of Mmp13 increased strongly by the chondrocytes (>50‐fold), but not by the chondrons. Taken together, our data suggest that preserving the pericellular matrix has a positive effect on cell‐induced cartilage production. J. Cell. Biochem. 110: 260–271, 2010. © 2010 Wiley‐Liss, Inc.     

The Frictional Coefficient of Bovine Knee Articular Cartilage

1 Introduction Cartilage has excellent biomechanical and tri- bological properties with low friction and minimum wear in diarthrodial joints throughout the lifetime of most people, and the lifetime of articular cartilage can be 40 years or longer. This has inspired material and bionic scientists to study the mechanism of such excellent tri- bological characteristics in order to develop artificial joints. Various mechanisms have been proposed to ex- plain the remarkable low friction behavior of…     

Human articular chondrocytes express three facilitative glucose transporter isoforms: GLUT1, GLUT3 and GLUT9

Glucose is an important metabolite and a structural precursor for articular cartilage and its transport has significant consequences for cartilage development and functional integrity. In this study the expression of facilitative glucose transporters (GLUTs) in human chondrocytes was investigated. Results showed that at least three GLUT isoforms (GLUT1, GLUT3 and GLUT9) are expressed by normal chondrocytes. Given the central role of glucose in chondrocyte physiology and metabolism, its regular provision via GLUTs will influence the metabolic activity and survival of chondrocytes in cartilage matrices.