Standardized Evaluation of Chondrocyte Inflammation Model with TNF alpha

Abstract

This study aimed to develop a standardized chondrocyte inflammation model by evaluating the effects of TNFα concentration and cell passage, and to further refine it into a dynamic model that more accurately reflects pathological inflammatory conditions. To establish a standardized chondrocyte inflammation model, bovine chondrocytes at different passages (P1-P3) were cultured as pellets and stimulated with TNFα at different concentrations (0.1 ng/mL to 100 ng/mL) for 48 h. To refine this into a dynamic model, passage 3 chondrocyte pellets were initially stimulated with 20 ng/mL TNFα for 48 h, followed by exposure to different concentrations of TNFα (0.2 ng/mL to 20 ng/mL) for the next 12 days. Gene expression (collagen 1, collagen 2, aggrecan, COMP, PRG4, MMP3, MMP13, IL-6, IL-8, NFkb1, COX2, PTGES2, Caspase-3) was determined. IL-6, MCP-1, nitric oxide and GAG released into the medium were measured. Histology staining was performed on pellets. The statistical significance was defined as p<0.05. This study showed that TNFα supplementation caused a decrease of anabolic gene expression in 48h pellet culture and this detrimental effect had a concentration threshold. Cell passage also affected the gene expression. Certain genes (COMP, collagen 1, MMP13, and Caspase-3) showed significant changes at lower concentrations of TNFα on day 4, but increasing the TNFα concentration did not elicit a stronger response. In contrast, other genes (MMP3, IL6, and COX2) exhibited different patterns. The responses of gene expression to the TNFα stimulation could be markedly different between day 4 and day 14. Protein analysis and histology results were in line with PCR results. Overall, TNFα exhibited a detrimental effect on chondrocytes. A clear threshold for the induction of inflammation was identified at 10 ng/mL. Following inflammation induction, TNFα at 0.2 ng/mL could maintain the inflammation process. Cell passage had an effect on model construction.