IntroductionSignals from the epidermal growth factor receptor (EGFR) have typically been considered to provide catabolic activities in articular cartilage, and accordingly have been suggested to have a causal role in osteoarthritis progression. The aim of this study was to determine in vivo roles for endogenous EGFR signal activation in articular cartilage.MethodsTransgenic mice with conditional, limb-targeted deletion of the endogenous intracellular EGFR inhibitor Mig-6 were generated using CreLoxP (Mig-6-flox; Prx1Cre) recombination. Histology, histochemical staining and immunohistochemistry were used to confirm activation of EGFR signaling in the articular cartilage and joints, and to analyze phenotypic consequences of Mig-6 loss on articular cartilage morphology, proliferation, expression of progenitor cell markers, presence of chondrocyte hypertrophy and degradation of articular cartilage matrix.ResultsThe articular cartilage of Mig-6-conditional knockout (Mig-6-cko) mice was dramatically and significantly thicker than normal articular cartilage at 6 and 12 weeks of age. Mig-6-cko articular cartilage contained a population of chondrocytes in which EGFR signaling was activated, and which were three to four times more proliferative than normal Mig-6-flox articular chondrocytes. These cells expressed high levels of the master chondrogenic regulatory factor Sox9, as well as high levels of putative progenitor cell markers including superficial zone protein (SZP), growth and differentiation factor-5 (GDF-5) and Notch1. Expression levels were also high for activated β-catenin and the transforming growth factor beta (TGF-β) mediators phospho-Smad2/3 (pSmad2/3). Anabolic effects of EGFR activation in articular cartilage were followed by catabolic events, including matrix degradation, as determined by accumulation of aggrecan cleavage fragments, and onset of hypertrophy as determined by type × collagen expression. By 16 weeks of age, the articular cartilage of Mig-6-cko knees was no longer thickened and was degenerating.ConclusionsThese results demonstrate unexpected anabolic effects of EGFR signal activation in articular cartilage, and suggest the hypothesis that these effects may promote the expansion and/or activity of an endogenous EGFR-responsive cell population within the articular cartilage.
The extracellular matrix (ECM) plays a critical role in governing cell behavior and phenotype during limb skeletogenesis. Chondroitin sulfate proteoglycans (Cspgs) are highly expressed in the ECM of precartilage mesenchymal condensations and are important to limb chondrogenesis and cartilage structure, but little is known regarding their involvement in formation of synovial joints in the embryonic limb. Matrix versican Cspg expression has previously been reported in the epiphysis of developing long bones and presumptive joint; however, detailed analysis has not yet been conducted. In the present study we immunolocalized versican and aggrecan Cspgs during chick elbow joint morphogenesis between HH st25-41 of development. In this study we show that versican and aggrecan expression initially overlapped in the incipient cartilage model of long bones in the wing, but versican was also highly expressed in the perichondrium and presumptive joint interzone during early stages of morphogenesis (HH st25-34). By HH st36-41 versican localization was restricted to the future articular surfaces of the developing joint and surrounding joint capsule while aggrecan localized in an immediately adjacent and predominately non-overlapping region of chondrogenic cells at the epiphyses. These results suggest a potential role for versican proteoglycan in development and maintenance of the synovial joint interzone.
Mesenchymal cell aggregation is critical for cartilage formation in the vertebrate limb. The extracellular matrix (ECM) plays a critical role in governing cell behavior and cell phenotype in this tissue, and the hyalectin versican is highly expressed in the ECM of precartilage mesenchymal cells and developing synovial joints. Although several in vitro studies have been conducted in an attempt to address versican's role during limb mesenchymal condensation, factors such as differences in cell density in culture, variations between chondrogenic cell lines, and the inability to prolong the viability of limb explants have led to conflicting data, mandating an in vivo analysis. By using a morpholino directed strategy in ovo, we performed knock-down of versican expression in the presumptive ulnar region of the developing chick wing at time points critical to skeletogenesis. These data indicate that in ovo misexpression of versican compromised mesenchymal condensation with resulting ulnar cartilages reduced in length distally by an average of 53% relative to contralateral control limbs. In select versican morphants the olecranon process was also reduced in size proximally and failed to cup the humerus, likely impairing joint morphogenesis. This study represents the first report assessing the role of versican in the developing chick limb in ovo, further demonstrating the importance of versican proteoglycan expression during chondrogenesis and extending previous findings to suggest a role for versican during synovial joint development.
Osteoarthritis is a debilitating joint disease present in epidemic proportions worldwide. Osteoarthritis results from degeneration of the articular cartilage of the joint surfaces due to acute trauma, or chronic wear and tear. Due to limited ability of cartilage to repair itself, and lack of available treatments, there is an urgent need for development of approaches to repair articular cartilage damage due to injury or osteoarthritic disease. Cell-based repair strategies are among the most promising of these approaches. Various adult cell sources for cartilage repair are proposed including autologous adult chondrocytes as well as adult Mesenchymal Stem Cells (MSC). Disadvantages such as destructive harvest protocols; poor proliferation, and particularly for MSC, considerable cellular heterogeneity, have limited success of these cell types for cartilage repair. Chondrogenic cells derived from human embryonic stem cells (hESC) offer a highly proliferative cell source, which when directed into the chondrogenic lineage, could provide an ideal source of cells for cartilage repair. Chondrogenic cells derived from human induced pluripotent stem cells (iPSC) offer additional advantages for patient-specific therapy. Recently protocols have been established for directed differentiation of hESC into the chondrogenic lineage. Harnessing the potential of hESC-derived chondrogenic cells will require comprehensive testing of their efficacy for in vivo cartilage repair, as well as considerations of safety and immunogenicity of the cells. Use of pro-chondrogenic factors and/or bioactive scaffolds may assist in optimizing cartilage repair by chondrogenic cells. Repair of cartilage damage in osteoarthritis is a special challenge because of the widespread damage and presence of signals and stressors which disrupt normal joint homeostasis. Particular promise in cell-based repair of osteoarthritis may be provided by chondrogenic progenitor cells which may mimic endogenous repair responses. This review discusses the current status of cell-based cartilage repair strategies and in particular the potential role of hESC-derived chondrocytes or chondroprogenitor cells for treatment of articular cartilage damage due to injury and osteoarthritis.
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