Immortalization and Characterization of Mouse Temporomandibular Joint Disc Cells

Objective: Despite the importance of the TMJ disc in joint function and degeneration, studying the responses of its cells has been complicated by the lack of characterization of the cell subtypes comprising the disc. Our purpose was to immortalize and characterize TMJ disc cell clones, and to determine their responses to estrogen.  Methods: Disc cells from female mice were isolated, and immortalized by human telomerase reverse transcriptase. Four clones demonstrating successful immortalization were characterized for fibroblast and chondrocyte markers. Additionally, estrogen effects were determined by culturing the clones in the presence or absence of 0.05ng/ml estrogen, and analyzing for estrogen receptor ESR-1 and ESR-2, and matrix metalloproteinase MMP-9 and MMP-13.  Results: Two immortalized clones each were initially characterized as fibroblastic (clones 2 and 6) or chondrocytic (clones 1 and 11) on the basis of cell morphology and growth rate. Further characterization showed that clones 1 and 11 had higher mRNA expression levels of cartilage oligomeric matrix protein (COMP), collagen X, total collagen, collagen II, and collagen II/collagen I mRNA ratios relative to clones 2 and 6.  Clones 2 and 6 had high mRNA expression level of vimentin and fibroblastic specific protein-1 (FSP1). Immunofluorescence revealed cytoplasmic staining for FSP1 only in clones 2 and 6, while only clones 1 and 11 stained for COMP. Estrogen treatment of clones 1 and 11 resulted in enhanced mRNA expression levels of ESR-1 and ESR-2, and showed greater induction of MMP-9 in the chondrocytic vs the fibroblastic clones.  Conclusion: These studies are the first to immortalize TMJ disc cells and characterize chondrocytic and fibroblastic clones, which show different ESR expression profiles and MMP responses to estrogen. These cells offer a valuable resource for future studies to dissect the behavior of each of these cell types in health and disease. (Supported by NIH/NIDCR RO1 DE018455)