Suitability of Porcine Chondrocyte Micromass Culture To Model Osteoarthritis in Vitro

Abstract

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.

Scaffoldless tissue‐engineered cartilage for studying transforming growth factor beta‐mediated cartilage formation

“…9,10 Tissue-engineered cartilage models that are focused on mechanical trauma and cytokines are very different from the model described here with alterations in TGF-β signaling. 10 Multiple genes, including matrix metalloproteinase 13 (MMP-13), were regulated differently in the TNFα-related model when compared to the DNIIR mouse model with reduced TGF-β signaling. 30 The DMM mice did not exhibit altered expression of Tgfbr2, parathyroid hormone-like hormone (Pthlh), and two genes encoding for matrix-modifying enzymes, Papss2 and procollagen lysine 2-oxoglutarate 5-dioxygenase 2 (Plod2).…”

Postnatal changes to the mechanical properties of articular cartilage are driven by the evolution of its collagen network

“…Osteochondral cores (Ø 6 mm) were harvested from the femoropatellar groove of healthy porcine knee joints within 3 h of sacrifice. The porcine model was chosen, as biochemical levels are reported of a similar order of magnitude to human articular cartilage (Malda et al, 2013;Schlichting et al, 2014). Specimens collected were divided into groups, representing different stages of postnatal development: 4 week and 8 week old, immature articular cartilage; 1 year old, approaching skeletal maturity; and 3 years old, representing fully mature articular cartilage.…”

“…The brightness and contrast of all images were optimised; no other post processing procedures were performed. Fibril diameters were measured using ImageJ (Schneider et al, 2012).…”

Chemokine CCL25 Induces Migration and Extracellular Matrix Production of Anulus Fibrosus-Derived Cells

“…Photographs of the staining were taken and analyzed histomorphometrically. For the determination of red values for safranin O staining and the red stained areas of immunohistochemical collagen types I and II staining a histomorphometric analysis was performed as described before [36]. …”