Chondrocyte cluster formation in agarose cultures as a functional assay to identify genes expressed in osteoarthritis

Abstract

Understanding altered gene expression in osteoarthritic cartilage can lead to new targets for drug intervention. We established a functional assay based on chondrocyte cluster formation, a phenotype associated with osteoarthritis (OA), to screen an OA cartilage gene library. Previous reports have demonstrated that normal chondrocytes grown in suspension culture maintain their chondrocytic phenotype, however, certain growth factors such as basic fibroblast growth factor (bFGF) will induce the cells to proliferate in tight clusters similar to those seen in osteoarthritic cartilage. In this study we validate that overexpression of bFGF by retrovirally transduced normal chondrocytes would similarly induce the proliferation of tight cell clusters. We then used this approach as a basis to set up a functional screen where an entire OA cartilage cDNA library was tranduced into normal chondrocytes to search for other genes that would also induce cluster formation. Seven potential genes were isolated from the OA gene library, including BPOZ, IL-17 receptor C, NADH ubiquinone oxidoreductase, COMP, Soluble carrier 16 (MCT 3), C1r, and bFGF itself. None of the identified genes were upregulated by bFGF, however, all of them upregulated the expression of bFGF suggesting a common pathway. Although cluster formation is not considered to be destructive in OA cartilage, it is consistent with the disease and could yield answers to the altered phenotype. Further studies are needed to elucidate how these genes are linked to the disease state.

Biological impact of the fibroblast growth factor family on articular cartilage and intervertebral disc homeostasis

“…Surprisingly, this antagonistic effect of bFGF does not inhibit cellular proliferation mediated by IGF-1 and/or BMP7 as was seen with PG production; rather, bFGF further promoted cellular proliferation when combined with other anabolic growth factors, leading to decreased PG production per cell and clustering of cells. Similarly, others have found bFGF to be a potent stimulator of cell cluster formation after retroviral transduction of a cDNA library in functional genomic screens for the novel mediators of OA Quintavalla et al, 2005).…”

Correlation between plasma and synovial fluid basic fibroblast growth factor with radiographic severity in primary knee osteoarthritis

“…In recent years, bFGF expression in osteoarthritic articular cartilage has been explored and implicated in OA development [25,27]. Previous studies also demonstrated upregulated bFGF expression in chondrocytes in human OA cartilage, which suggests that human chondrocytes can synthesise bFGF [22]. These findings prompted us to speculate that bFGF may be responsible for OA pathogenesis.…”

ADAM‐12 (meltrin α) is involved in chondrocyte proliferation via cleavage of insulin‐like growth factor binding protein 5 in osteoarthritic cartilage

“…OA chondrocytes at passage 1 were subjected to agarose gel culture according to the methods described by Quintavalla et al (4). Briefly, bottom agarose gel consisting of 1,300 l DMEM-Ham's F-12 containing 10% FBS and 0.8% low melt agarose (Takara Shuzo, Shiga, Japan) was cast into 6-well culture plates and then overlaid with 1,300 l of a chondrocyte suspension (5 ϫ 10 3 cells) in the same medium except for 0.4% low melt agarose at 37°C.…”

“…an effort by chondrocytes to repopulate the cartilage tissue and enhance matrix synthesis, or it could signify reversion to an earlier developmental stage of cartilage (3,4). However, little is known about the mechanisms of this phenotypic change in OA.…”