Effect of adenovirus-mediated overexpression of bovine ADAMTS-4 and human ADAMTS-5 in primary bovine articular chondrocyte pellet culture system.

Articular cartilage matrix synthesis and degradation are dynamic processes that must be balanced for proper maintenance of the tissue. In osteoarthritis (OA), this balance is skewed toward degradation and ultimate loss of matrix. The transcriptional and/or activity levels of hundreds of genes are dysregulated in chondrocytes from osteoarthritic cartilage, and a subset of these genes may represent pivotal factors that could be modulated if their specific role in the disease process could be identified. To investigate the role of ADAMTS-4 and ADAMTS-5 in cartilage matrix degradation by developing a chondrocyte pellet culture assay in combination with adenoviral gene expression, and to demonstrate the utility of this assay by assessing the specific functional contribution of these genes to cartilage matrix metabolism. A full-length cDNA for bovine ADAMTS-4 (bADAMTS-4) was isolated, and used to evaluate the expression, regulation, and activity of this gene in bovine cartilage. Adenoviruses expressing bADAMTS-4, human ADAMTS-5 (hADAMTS-5) or human bone morphogenetic protein 2 (BMP-2) were used to infect primary chondrocytes, and their effect on extracellular matrix metabolism was assessed by monitoring the accumulation and release of glycosaminoglycans (GAG) in three-dimensional chondrocyte pellet cultures. Analysis of bADAMTS-4 transcriptional regulation in chondrocytes revealed that interleukin-1alpha (IL-1alpha) was the most potent inducer of bADAMTS-4 mRNA and subsequent aggrecan degradation in cartilage explant cultures of those cytokines tested. bADAMTS-4 mRNA induction by IL-1alpha was greater in nasal cartilage than in articular cartilage. Chondrocytes infected with adenovirus expressing either bADAMTS-4 or hADAMTS-5 genes showed increased aggrecan degradation in newly synthesized matrix by pellet cultures while chondrocytes overexpressing BMP-2 showed increased aggrecan synthesis. Adenoviral delivery of genes to primary bovine chondrocytes, followed by culture in three-dimensional pellet format and evaluation of extracellular matrix protein metabolism, is a useful functional assay for assessing the role of genes on cartilage matrix synthesis and degradation.