The Mechanobiological Regulation of Autophagy in Mandibular Fibrochondrocytes

Background: One of the key initiating conditions of Temporomandibular Joint (TMJ) osteoarthritis (OA) is mechanical overloading. Neuron-glial antigen 2 (NG2/CSPG2) is a mechanically responsive transmembrane proteoglycan that modulates mTOR signaling, an upstream regulator of autophagy and cell stress. Objective: The objective of this study is to determine if biophysical stress on mandibular fibrochondrocytes regulates autophagy and cell stress in an NG2 and mTOR dependent manner. Methods: Primary mandibular fibrochondrocytes from WT and NG2 knockout (KO) mice were isolated and seeded in a 4% collagen-agarose scaffold at a density of 4 x 105 /mm3.Cell-collagen-agarose scaffolds were treated with and without the MHY1485 (mTOR agonist) and Rapamycin (mTOR antagonist) in low-serum media for 24 hours and loaded in compression at 2 N for 2 hour at 37° C and 5% CO2. For RT-PCR analysis, scaffolds were placed in 2 ml of lysis reagent (QIAzol, Qiagen) for 2 hours, processed using the RNeasy Plant Mini Kit (74903, Qiagen), and analyzed by RT-qPCR. Statistical significance was calculated by one-way ANOVA with post hoc Bonferroni test. (SPSS, Chicago IL) Results: Mechanical loading of WT cells increased expression of NG2 and TGFβ (p<0.05; n=4). Rapamycin and MHY1485 treatment increased MMP13, Col6a1, and TGFβ and decreased NG2 in a loading independent manner (p<0.05; n=4). Knockout of NG2 increased activation of mTOR at ser2481. Mechanical loading of NG2 KO cells increased the expression of MMP13, Col6a1, and TGFβ in a load