Suitability of Porcine Chondrocyte Micromass Culture To Model Osteoarthritis in Vitro

In
vitro tissue models are useful tools for the development of novel
therapy strategies in cartilage repair and care. 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 micromass cultures to mimic human
articular cartilage and essential aspects of osteoarthritis (OA) in
vitro. Primary chondrocytes were enzymatically isolated from porcine
femoral condyles and were maintained in 96-multiwell format to establish
micromass cultures in a high-throughput scale. Recombinant porcine
tumor necrosis factor alpha (TNF-α) was used to induce OA-like
changes documented on histological (Safranin O, collagen type II staining),
biochemical (hydroxyproline assay, dimethylmethylene blue method),
and gene expression level (Affymetrix porcine microarray, real time
PCR) and were compared with published data from human articular cartilage
and human micromass cultures. After 14 days in micromass culture,
porcine primary chondrocytes produced ECM rich in proteoglycans and
collagens. On gene expression level, significant correlations of detected
genes with porcine cartilage (r = 0.90), human cartilage
(r = 0.71), and human micromass culture (r = 0.75) were observed including 34 cartilage markers such
as COL2A1, COMP, and aggrecan. TNF-α stimulation led to significant proteoglycan (−75%)
and collagen depletion (−50%). Comparative expression pattern
analysis revealed the involvement of catabolic enzymes (MMP1, -2, -13, ADAM10), chemokines (IL8, CCL2, CXCL2, CXCL12, CCXL14), and genes associated with cell death (TNFSF10, PMAIPI, AHR) and skeletal development
(GPNMB, FRZB) including transcription
factors (WIF1, DLX5, TWIST1) and growth factors (IGFBP1, -3, TGFB1) consistent with published data from human
OA cartilage. Expression of genes related to cartilage ECM formation
(COL2A1, COL9A1, COMP, aggrecan) as well as hypertrophic bone formation
(COL1A1, COL10A1) was predominantly
found decreased. These findings indicating significant parallels between
human articular cartilage and the presented porcine micromass model
and vice versa confirm the applicability of known cartilage marker
and their characteristics in the porcine micromass model. TNF-α
treatment enabled the initiation of typical OA reaction patterns in
terms of extensive ECM loss, cell death, formation of an inflammatory
environment through the induction of genes coding for chemokines and
enzymes, and the modulation of genes involved in skeletal development such as growth factors, transcription factors, and cartilage ECM-forming genes. In conclusion, the porcine micromass model represents an alternative tissue platform for the evaluation of innovative substances and techniques for the treatment of OA.