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Cultured Chondrocytes (cultured + chondrocyte)
Selected AbstractsThe Assembly and Remodeling of the Extracellular Matrix in the Growth Plate in Relationship to Mineral Deposition and Cellular Hypertrophy: An In Situ Study of Collagens II and IX and Proteoglycan,JOURNAL OF BONE AND MINERAL RESEARCH, Issue 2 2002Fackson Mwale Abstract The recent development of new specific immunoassays has provided an opportunity to study the assembly and resorption of type II and IX collagens of the extracellular matrix in relationship to endochondral calcification in situ. Here, we describe how in the bovine fetal physis prehypertrophic chondrocytes deposit an extensive extracellular matrix that, initially, is rich in both type II and type IX collagens and proteoglycan (PG; principally, aggrecan). The majority of the ,1(IX)-chains lack the NC4 domain consistent with our previous studies with cultured chondrocytes. During assembly, the molar ratio of type II/COL2 domain of the ,1(IX)-chain varied from 8:1 to 25:1. An increase in the content of Ca2+ and inorganic phosphate (Pi) was initiated in the prehypertrophic zone when the NC4 domain was removed selectively from the ,1(IX)-chain. This was followed by the progressive loss of the ,1(IX) COL2 domain and type II collagen. In the hypertrophic zone, the Ca2+/Pi molar ratio ranged from 1.56 to a maximum of 1.74, closely corresponding to that of mature hydroxyapatite (1.67). The prehypertrophic zone had an average ratio Ca2+/Pi ranging from 0.25 to 1, suggesting a phase transformation. At hypertrophy, when mineral content was maximal, type II collagen was reduced maximally in content coincident with a peak of cleavage of this molecule by collagenase when matrix metalloproteinase 13 (MMP-13) expression was maximal. In contrast, PG (principally aggrecan) was retained when hydroxyapatite was formed consistent with the view that this PG does not inhibit and might promote calcification in vivo. Taken together with earlier studies, these findings show that matrix remodeling after assembly is linked closely to initial changes in Ca2+ and Pi to subsequent cellular hypertrophy and mineralization. These changes involve a progressive and selective removal of types II and IX collagens with the retention of the PG aggrecan. [source] Effects of Alendronate on A Disintegrin and Metalloproteinase with Thrombospondin Motifs Expression in the Developing Epiphyseal Cartilage in RatsANATOMIA, HISTOLOGIA, EMBRYOLOGIA, Issue 2 2009M. S. Kim Summary A disintegrin and metalloproteinase with thrombospondin motifs (ADAMTS) have been reported to play a role in the degradation of aggrecan, a major component of cartilage. This study was performed to examine the effects of alendronate on the expression of ADAMTS in developing femoral epiphyseal cartilage. Primary cultured chondrocytes from this cartilage were treated with alendronate in vitro and postnatal day 1 rats were injected subcutaneously with alendronate (1 mg/kg) every second day in vivo. The number of cultured chondrocytes and their aggrecan mRNA levels were unaffected by the alendronate treatment at 10,6 to 10,4 m concentrations. The mRNA levels of ADAMTS-1, -2 and -9 in chondrocytes were also unaffected. However, the levels of ADAMTS-5 and -4 were reduced significantly by the same treatment. The thickness of the proliferating chondrocyte layers and the aggrecan mRNA levels in the epiphysis were unaffected by the alendronate treatment in vivo. However, the hypertrophied chondrocyte layers became significantly thicker, and the size of the secondary ossification centre was reduced significantly by the same treatment (P < 0.05). Both ADAMTS-4 and -5 mRNA expressions were also reduced significantly in vivo. The immunoreactivity against ADAMTS-4 was seen in hypertrophied chondrocytes and reduced significantly by the alendronate treatment. These results suggested that alendronate can inhibit the degradation of aggrecan in the articular cartilage by downregulating the expression of matrix enzymes such as ADAMTS-4 and -5. [source] Expression and modulation of ghrelin O -acyltransferase in cultured chondrocytesARTHRITIS & RHEUMATISM, Issue 6 2009Rodolfo Gómez Objective To use reverse transcription,polymerase chain reaction to detect ghrelin O -acyltransferase (GOAT) transcripts in both murine and human chondrocytes, to evaluate the effect of pharmacologic in vitro treatments with lipopolysaccharide (LPS), growth hormone, ghrelin, and dexamethasone on GOAT messenger RNA (mRNA) expression, and to study the GOAT mRNA profile during chondrocyte differentiation. Methods Murine and human GOAT and ghrelin mRNA levels were determined by the SYBR Green,based quantitative real-time polymerase chain reaction method. Results GOAT mRNA was expressed in murine cartilage explants as well as in the cultured murine chondrogenic ATDC-5 cell line. GOAT was also expressed in human immortalized chondrocyte cell lines and in human cultured primary chondrocytes. In addition, GOAT mRNA expression in differentiating ATDC-5 cells was lower at the early stage of differentiation (days 3,7), whereas GOAT mRNA levels increased progressively at the late stages. Finally, among the drugs and hormones tested, only LPS was able to strongly decrease GOAT mRNA expression. Conclusion These data indicate that chondrocytes are equipped with biochemical machinery for the synthesis of acylated ghrelin and suggest a novel role of the ghrelin axis in prehypertrophic and hypertrophic chondrocyte differentiation during endochondral ossification. [source] Differential expression of ,B-crystallin and evidence of its role as a mediator of matrix gene expression in osteoarthritisARTHRITIS & RHEUMATISM, Issue 1 2009Stijn Lambrecht Objective Alpha B,crystallin belongs to the family of small heat-shock proteins (HSPs). The role of this protein family in chondrocytes is not well understood. The present study was undertaken to investigate expression levels of ,B-crystallin in chondrocytes isolated from healthy subjects and patients with osteoarthritis (OA), and to explore the functional role of this potentially interesting protein in chondrocyte metabolism. Methods Western blot and real-time reverse transcriptase,polymerase chain reaction (RT-PCR) analyses were performed to determine expression levels of ,B-crystallin in healthy and OA chondrocytes cultured in alginate beads. RNA interference,mediated gene knockdown was used to explore the role of this small HSP in chondrocyte biology, by transfecting low concentrations of small interfering RNA (siRNA) in cultured chondrocytes. Results We initially identified ,B-crystallin as a small HSP that was differentially expressed between healthy and OA-affected chondrocytes. The decreased abundance of this protein in OA chondrocytes was confirmed by Western blotting. Moreover, real-time RT-PCR confirmed the differential expression between chondrocytes isolated from visibly intact and visibly damaged zones of OA cartilage. The proinflammatory cytokines interleukin-1, and tumor necrosis factor , both down-regulated ,B-crystallin expression. Transfection of low concentrations of siRNA in cultured chondrocytes resulted in efficient knockdown of ,B-crystallin gene expression. This was accompanied by altered expression of the chondrocyte-specific bone morphogenetic protein 2, aggrecan, and type II collagen genes. Conclusion The present findings identify the small HSP ,B-crystallin as a novel mediator of chondrocyte matrix gene expression that may contribute to altered chondrocyte metabolism during the development of OA. [source] Spontaneous oscillation and mechanically induced calcium waves in chondrocytesCELL BIOCHEMISTRY AND FUNCTION, Issue 2 2006Taisuke Kono Abstract The characteristics of spontaneous calcium (Ca2+) oscillation and mechanically induced Ca2+ waves in articular chondrocytes were studied. In some, but not all, chondrocytes in sliced cartilage and primary cultures, we observed spontaneous oscillation of intracellular Ca2+ that never spread to adjacent cells. In contrast, a mechanical stimulus to a single cell by touching with a glass rod induced an increase of intracellular Ca2+ that spread to neighboring cells in a wave-like manner, even though there was no physical contact between the cells. This indicated the release of some paracrine factor from the mechanically stimulated cells. Application of ultrasonic vibration also induced an oscillation of intracellular Ca2+. The application of a uridine 5,-triphosphate (UTP), UTP, induced a transient increase in intracellular Ca2+ and the release of adenosine 5,-triphosphate (ATP) in cultured chondrocytes. A P2 receptor antagonist (suramin) and blockers of Cl, channels, niflumic acid and 4,4,-diisothiocyanostilbene-2,2,-disulfonic acid (DIDS), reduced the UTP-induced ATP release. The results indicated that Cl, channels were involved in the extracellular release of ATP following mechanical or P2Y receptor stimulation. Thus, ATP stimulation of P2Y receptors elicits an increase in intracellular Ca2+, triggering further release of ATP from adjacent cells, thereby expanding the Ca2+ wave in chondrocytes. Copyright © 2005 John Wiley & Sons, Ltd. [source] | |||||||||||||