Preliminary study on the effect of inflamed TMJ synovial fluid on the intracellular calcium concentration and differential expression of iNOS and COX-2 in human immortalized chondrocyte C28/I2

ABSTRACT

OBJECTIVE: The objective of this study was to examine the hypothesis that inflammatory synovial fluid from TMJ internal derangement initiates a transient increase in intracellular calcium concentration ([Ca2+]i) in chondrocytes and the induced Ca2+ signaling affects iNOS/COX-2 gene expression patterns following exposure to inflamed synovial fluid. MATERIALS AND METHODS: Two female adult patients with symptoms of TMD who agreed to participate in the study were selected for this study. Immortalized human juvenile costal chondrocyte C-28/I2 was grown to 80% confluency and synovial fluids from two patients were added respectively to culture media for 24 hours at the concentration of 100ng/10ml. Confocal laser scanning microscope (CLSM) was used to examine changes of intracellular calcium concentration ([Ca2+]i). RT-PCR was performed to identify the expression profile of IL-1alpha, iNOS, COX-2. RESULTS: Increased [Ca2+]i was observed in chondrocytes subjected to inflamed synovial fluid compared to control cultures and in respective cultures exposed to inflamed synovial fluids from each patient, IL-1beta, COX-2 mRNA were detected. However, in neither case iNOS mRNA was expressed. IL-1alpha, COX-2, and iNOS mRNA were expressed in control culture. CONCLUSION: Our results show that immortalized chondrocytes cultured with inflamed synovial fluids from patients diagnosed as disc displacement without reduction and limitation in mouth opening showed increased calcium concentration and expression of COX-2 while inhibiting the production of iNOS, which in turn could adversely affect the chondrocytes in at least short term by hindering physiologic role of NO against inflammatory cascades. These findings suggest that inflamed synovial fluid may differentially regulate the transcriptomes of relevant inflammatory mediators, especially iNOS/COX-2 axis in chondrocytes through adjusting calcium transients.