Matrix composition of cartilaginous anlagen in achondrogenesis type II (Langer-Saldino).

Skeletal dysplasias represent in vivo models of genetic defects. Achondrogenesis type II (Langer-Saldino), caused by a genetic defect in the major cartilage matrix protein, collagen type II, is a rare and severe skeletal dysplasia. It comprises a severe derangement of the fetal growth plate cartilage with subsequent ossification defects. In this study, we analyzed the matrix composition and cell differentiation pattern in 3 relatives with achondrogenesis type II. Most strikingly we found a strongly reduced collagen type II and moderately reduced aggrecan proteoglycan content in the dysplastic cartilage matrix. Type II collagen is, at least to some extent, replaced by collagens type I III, and VI. Ultrastructural analysis of the dysplastic cartilage matrix demonstrated a distended rER (rough endoplasmic reticulum), which is typical for this condition and most likely related to improper processing and retention of genetically altered type II collagen. Immunostaining for type IIA and X collagens suggest a severe delay in chondrocyte maturation. Thus, the genetic defect in the present cases leads most likely to a severe retention of collagen type II in the rER and, therefore, a strongly reduced collagen deposition and replacement by other interstitial collagens. However, the latter are less efficient in binding aggrecan proteoglycans in the dysplastic cartilage matrix. Additionally, a delay in chondrocyte maturation appears to be important in achondrogenesis type II.

Modulation of endogenous osteogenic protein-1 (OP-1) by interleukin-1 in adult human articular cartilage.

BACKGROUND: Osteogenic protein-1 (OP-1, BMP-7) induces bone formation and cartilage growth. Since OP-1 is an anabolic factor expressed by human articular chondrocytes, we examined the response of endogenous OP-1 to interleukin-1beta (IL-1beta) in human articular cartilage. METHODS: Normal adult human articular cartilage explants were cultured for twenty-five days in the presence of medium only or were treated with a low dose (0.1 ng/mL) or high dose (1.0 ng/mL) of IL-1beta for forty-eight or ninety-six hours. Alternately, cartilage explants were cultured forty-eight hours with IL-1beta, followed by forty-eight hours in standard medium (recovery). Tissue was analyzed for OP-1 message (by means of the reverse transcriptase-polymerase chain reaction), protein (by means of enzyme-linked immunosorbent assay and Western blot analysis) and proteoglycan content. Medium was analyzed for released proteoglycans and OP-1. RESULTS: In the presence of medium, OP-1 maintained its steady state of mRNA and protein expression for as long as twenty-five days in culture. A low dose of IL-1beta led to some upregulation in message and a twofold (p < 0.02) increase in OP-1 protein characterized by enhanced processing and activation of OP-1. Removal of IL-1beta (recovery experiments) did not reverse its effect on OP-1 synthesis. A high dose of IL-1beta caused stronger upregulation of message and a twofold decrease in OP-1 protein content (p < 0.007) in the cartilage matrix. However, this decrease in the matrix was primarily due to a release of active OP-1 into the medium. After removal of the 1.0-ng/mL IL-1beta, the levels of OP-1 protein did not recover. CONCLUSION: The results of the present study indicate that human adult chondrocytes have an ability to respond anabolically to initial or early catabolic events through an upregulation of endogenous OP-1.

Analysis of protein distribution in cartilage using immunofluorescence and laser confocal scanning microscopy.

Protein localization in cartilage sections by antibodies that specifically bind to epitopes of a protein is one of the most powerful technologies in modern cartilage research. Studies using two or more primary antibodies that recognize different protein epitopes allow the colocalization of different gene products in one cartilage section. In addition, specific histochemical stains help to visualize nuclear DNA, mitochondria, and other subcellular compartments. By these immunohistological methods, the distribution of proteins can be analyzed throughout different zones of articular cartilage. In particular, with the use of laser scanning confocal microscopy, subcellular localization of proteins can also be determined (i.e, nuclear, cytoplasmic, membrane-associated, and extracellular). Overall, immunohistochemical methods are fairly simple to handle, and the reagents required are inexpensive, with the exception of basic technical equipment (fluorescence microscope or confocal microscope). However, as with many methodologies, technical knowledge and experience is important to avoid and/or interpret either false-positive or false-negative results.

Regulation of chondrocyte gene expression by osteogenic protein-1.

INTRODUCTION: The objective of this study was to investigate which genes are regulated by osteogenic protein-1 (OP-1) in human articular chondrocytes using Affimetrix gene array, in order to understand the role of OP-1 in cartilage homeostasis. METHODS: Chondrocytes enzymatically isolated from 12 normal ankle cartilage samples were cultured in high-density monolayers and either transfected with OP-1 antisense oligonucleotide in the presence of lipofectin or treated with recombinant OP-1 (100 ng/ml) for 48 hours followed by RNA isolation. Gene expression profiles were analyzed by HG-U133A gene chips from Affimetrix. A cut-off was chosen at 1.5-fold difference from controls. Selected gene array results were verified by real-time PCR and by in vitro measures of proteoglycan synthesis and signal transduction. RESULTS: OP-1 controls cartilage homeostasis on multiple levels including regulation of genes responsible for chondrocyte cytoskeleton (cyclin D, Talin1, and Cyclin M1), matrix production, and other anabolic pathways (transforming growth factor-beta (TGF-ß)/ bone morphogenetic protein (BMP), insulin-like growth factor (IGF), vascular endothelial growth factor (VEGF), genes responsible for bone formation, and so on) as well as regulation of cytokines, neuromediators, and various catabolic pathways responsible for matrix degradation and cell death. In many of these cases, OP-1 modulated the expression of not only the ligands, but also their receptors, mediators of downstream signaling, kinases responsible for an activation of the pathways, binding proteins responsible for the inhibition of the pathways, and transcription factors that induce transcriptional responses. CONCLUSIONS: Gene array data strongly suggest a critical role of OP-1 in human cartilage homeostasis. OP-1 regulates numerous metabolic pathways that are not only limited to its well-documented anabolic function, but also to its anti-catabolic activity. An understanding of OP-1 function in cartilage will provide strong justification for the application of OP-1 protein as a therapeutic treatment for cartilage regeneration and repair.

Identification of markers to characterize and sort human articular chondrocytes with enhanced in vitro chondrogenic capacity.

OBJECTIVE: To identify markers associated with the chondrogenic capacity of expanded human articular chondrocytes and to use these markers for sorting of more highly chondrogenic subpopulations. METHODS: The chondrogenic capacity of chondrocyte populations derived from different donors (n = 21) or different clonal strains from the same cartilage biopsy specimen (n = 21) was defined based on the glycosaminoglycan (GAG) content of tissues generated using a pellet culture model. Selected cell populations were analyzed by microarray and flow cytometry. In some experiments, cells were sorted using antibodies against molecules found to be associated with differential chondrogenic capacity and again assessed in pellet cultures. RESULTS: Significance Analysis of Microarrays indicated that chondrocytes with low chondrogenic capacity expressed higher levels of insulin-like growth factor 1 and of catabolic genes (e.g., matrix metalloproteinase 2, aggrecanase 2), while chondrocytes with high chondrogenic capacity expressed higher levels of genes involved in cell-cell or cell-matrix interactions (e.g., CD49c, CD49f). Flow cytometry analysis showed that CD44, CD151, and CD49c were expressed at significantly higher levels in chondrocytes with higher chondrogenic capacity. Flow cytometry analysis of clonal chondrocyte strains indicated that CD44 and CD151 could also identify more chondrogenic clones. Chondrocytes sorted for brighter CD49c or CD44 signal expression produced tissues with higher levels of GAG per DNA (up to 1.4-fold) and type II collagen messenger RNA (up to 3.4-fold) than did unsorted cells. CONCLUSION: We identified markers that allow characterization of the capacity of monolayer-expanded chondrocytes to form in vitro cartilaginous tissue and enable enrichment for subpopulations with higher chondrogenic capacity. These markers might be used as a means to predict and possibly improve the outcome of cell-based cartilage repair techniques.

Parosteal lipoma of the thigh with cartilaginous and osseous differentiation: an osteochondrolipoma.

Lipomas are very common benign soft tissue neoplasms. They are usually slow-growing and may occur anywhere in the body. Mature cartilage and bone arising in a lipoma is a rare event and is mostly associated with a parosteal localization of the neoplasm. We describe a new case of osteochondrolipoma showing not only major adipocytic differentiation but also areas of fibrocytic and cartilaginous cell differentiation and bone formation (both endochondral and membranous). The occurrence of at least 4 distinct directions of mesenchymal cell differentiation within a benign neoplasia underlines the concept of multilineage differentiation of pluripotent mesenchymal stem cells. Such a multidirectional potential was recently well established in vitro in stem cells present in adult adipocytic tissue.

Freshly isolated osteoarthritic chondrocytes are catabolically more active than normal chondrocytes, but less responsive to catabolic stimulation with interleukin-1beta.

OBJECTIVE: Interleukin-1beta (IL-1beta) is one potentially important cytokine during cartilage destruction. The aim of this study was to investigate whether there are different effects of low and high concentrations of IL-1beta on the expression level of anabolic genes (type II collagen, aggrecan), catabolic genes (matrix metalloproteinase 1 [MMP-1], MMP-2, MMP-3, MMP-13, and ADAMTS-4), and cytokines (IL-1beta, IL-6, and leukemia inhibitory factor [LIF]) by articular chondrocytes (normal and osteoarthritic). Determination of whether there was a difference in reactivity between normal and osteoarthritic chondrocytes was also a goal of this study. METHODS: Gene expression levels were detected by real-time polymerase chain reaction from isolated (nonpassaged) chondrocytes (normal [n = 6]; osteoarthritic [n = 7]) after stimulation with 0.01 ng, 0.1 ng, 1 ng, and 10 ng/ml IL-1beta. RESULTS: In normal adult articular chondrocytes the expression of both aggrecan and type II collagen genes was significantly down-regulated, whereas matrix-degrading proteases (except MMP-2), as well as the investigated cytokines, were induced by IL-1beta in a dose-dependent manner. The strongest regulation was found for IL-6 and LIF. Osteoarthritic chondrocytes showed strongly increased levels of catabolic enzymes and mediators, but were less responsive to further stimulation with IL-1beta. CONCLUSION: Our study confirms that IL-1beta activity is critically dependent on both the applied concentration and the reactivity of the cells stimulated. The responsiveness appears to be significantly reduced in late-stage osteoarthritic chondrocytes. However, these cells show high basic expression levels of catabolic enzymes and mediators. Thus, it remains open whether our data indicate that osteoarthritic chondrocytes are per se not responsive to IL-1beta or are already so strongly stimulated (e.g., by IL-1) during the disease process that they are refractory to further stimulation.

Hepatocyte growth factor/scatter factor is not a potent regulator of anabolic and catabolic gene expression in adult human articular chondrocytes.

Objective. Hepatocyte growth factor (HGF) has been reported to be present in articular cartilage and to be a potentially important inducing factor of anabolic and catabolic activity in chondrocytes. The aim of this study was to determine the expression levels of full length-functional-hgf and its receptor c-met in normal and osteoarthritic cartilage and the effect of HGF on anabolic and catabolic gene expression in adult human articular chondrocytes. Methods. Isolated adult human articular chondrocytes were stimulated for 48h with HGF (1, 10, and 100ng/ml). Synthesis of proteoglycans was determined by [(35)S]sulfate incorporation. mRNA levels for anabolic and catabolic genes as well as c-met and (functional) hgf were quantified using real-time PCR. Additionally, in situ mRNA expression levels of hgf and c-met were quantitatively measured from RNA directly isolated from normal and osteoarthritic adult human articular cartilage. Results. Proteoglycan synthesis in adult human articular chondrocytes was not stimulated by HGF nor was a selection of catabolic genes (collagenases and aggrecanases). Normal adult articular chondrocytes expressed only very low levels of hgf mRNA. Slightly higher levels of hgf were detected in chondrocytes isolated from osteoarthritic cartilage. Significant c-met expression was detected in both sample types. Conclusion. Despite the expression of its receptor c-met and its presence in articular cartilage, HGF does not appear to be a potent player in cartilage matrix turnover, at least not in terms of anabolic and catabolic gene expression in normal adult articular cartilage.