Suitability of porcine chondrocyte micromasses to model osteoarthritis in vitro

Abstract

Purpose: In vitro models are useful tools for the development of new therapy strategies in cartilage repair. The limited availability of human primary tissue and high costs of animal models hamper preclinical tests of innovative substances and techniques. In this study we tested the potential of porcine chondrocyte micromasses to mimic essential aspects of osteoarthritis (OA) in vitro. Methods: Primary chondrocytes were enzymatically isolated from porcine femoral condyle and were maintained for 14 days in 96-multiwell format to establish cartilage-like extracellular matrix (ECM) in a high-throughput scale. Subsequently, 10ng/ml recombinant porcine tumor necrosis factor alpha (TNF-alpha) was added up to 14 days to induce OA changes. These changes were documented on histological (Alcian blue and Safranin O stainings histomorphometrically analyzed), biochemical (collagens: hydroxyproline assay, proteoglycans: DMMB-method) and gene expression level (Affymetrix porcine microarray, RT-PCR), and were compared with data from human OA cartilage. Further, the influence of 10 ng/ml transforming growth factor beta 1 (TGF-b1), 1 ng/ml interleukin 1 beta (IL-1b) and 5% platelet rich plasma (PRP) on TNF-alpha induced changes was analyzed to test the performance of the model. Results: After 14 days in micromass culture, porcine primary chondrocytes developed an ECM rich in proteoglycans and collagens. On gene expression level cartilage markers COL2A1, COMP, CSPG4 and Aggrecan were detected. After stimulation with 10 ng/ml TNF-alpha, proteoglycan content was found decreased by histomorphometry (score decline -64%) and biochemical analysis of GAG (-75%) and total collagen (-30%) content. Gene expression of catabolic enzymes (MMP13, MMP1, MMP2), and genes involved in inflammatory response (IL8, CCL2, CCL4 CXCL2) was significantly increased (>4-fold) and genes related to ECM formation (COL2A1, COMP, Aggrecan, COL8A1, COL11A1) were decreased (<-4-fold). Whereas histological and biochemical characteristics of the TNF-alpha induced model match with observations known from human OA cartilage, gene expression analysis revealed contrary gene expression pattern related to ECM formation (COL2A1, COL11A1, COMP) and to hypertrophic bone formation (COL10A1). The addition of TGF-b1 and PRP during TNF-alpha stimulation led to reduced GAG loss (both 40%) whereas IL1b enhanced the GAG loss by further 10% demonstrating responsiveness of the model to test substances. Conclusions: The use of porcine chondrocytes in micromasses and the addition of TNF-alpha resulted in the induction of some typical features of OA such as extensive ECM loss and induction of genes related to catabolic activity and inflammatory response. In contrast to human OA, ECM formation was inhibited, which might be a consequence of the exclusive use of TNF-alpha, which is only one of many impacting factors in OA. Although not all characteristics of human OA could be demonstrated, the presented test system was suitable to detect effects of test substances. TNF-alpha induced porcine micromasses can be a useful supplement to existing preclinical tests in OA and an alternative to the use of human tissue in vitro.