Stephan Soeder

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-1&bgr; (IL-1&bgr;) 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-1&bgr; for forty-eight or ninety-six hours. Alternately, cartilage explants were cultured forty-eight hours with IL-1&bgr;, 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-1&bgr; 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-1&bgr; (recovery experiments) did not reverse its effect on OP-1 synthesis. A high dose of IL-1&bgr; 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-1&bgr;, 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.Clinical Relevance: A balance between anabolism and catabolism is perturbed and not fully synchronized in the degenerative processes seen in osteoarthritis. The aim of the current study was to investigate the function of a cartilage endogenous anabolic factor, i.e., OP-1, in a model of early degeneration induced by a catabolic mediator IL-1. The findings of the present study contribute to our understanding of the mechanisms involved in articular cartilage regeneration and repair.

Regulation of chondrocyte gene expression by osteogenic protein-1

IntroductionThe 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.MethodsChondrocytes 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.ResultsOP-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-β)/ 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.ConclusionsGene 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.

Knorpelschaden und -regeneration bei Osteoarthrose

ZusammenfassungDie Pathomorphologie der Osteoarthrose wird vom degenerativ veränderten Knorpel mit makroskopisch sichtbarem Matrixverlust sowie mikroskopischen Veränderungen von Zellen und Matrix bestimmt. Weiterhin kommt es zu teilweise dramatischen reaktiven Vorgängen angrenzender Gewebe, im Wesentlichen als Zeichen mechanischer Belastung im subchondralen Knochen und als Reaktion auf Degradationsprodukte in der Gelenkkapsel und der Synovialmembran. Zusätzlich finden sich reparative Knorpelneubildungsprozesse. Mesenchymale Vorläuferzellen aus umliegenden Geweben führen zur Bildung von sog. Brutkapseln und Randosteophyten. Ungeachtet der funktionellen Insuffizienz dieser regenerativen Prozesse stellt die Chondroneogenese in Osteophyten ein interessantes In-vivo-Modell der regenerativen Fähigkeiten und Differenzierungsmechanismen mesenchymaler pluripotenter Vorläuferzellen im erwachsenen Organismus dar. Die Analyse der hierbei relevanten zellulären Prozesse sowie der beteiligten Faktoren könnten nicht zuletzt auch zu neuen therapeutischen Ansätzen führen bzw. zu unserem Verständnis in diesem Kontext auftretender Prozesse beitragen.AbstractThe pathomorphology of osteoarthritis is mainly related to the destruction of the articular cartilage. Both macroscopically visible matrix loss as well as fissuring and matrix degradation on the microscopical level occurs. Additionally, other joint structures, namely, the subchondral bone and the synovial capsule and membrane, undergo significant, mostly reactive changes. Besides these destructive processes regenerative cartilage formation is also found: mesenchymal precursor cells differentiate to chondrocytic cells mainly during osteophyte formation. Though osteophytes are of little functional help during joint destruction, they represent an interesting in vivo model for therapeutically induced cartilage repair. Processes found in osteophyte formation and degeneration might point to new innovative therapeutic targets and can help to understand processes going on in joints during neocartilage formation.

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.