The Effects Of Cytochalasin B And Uniaxial Tensile Stress On The Goat Temporomandibular Joint Disc Cells In Vitro

Objective:The purpose of this study was to investigate the change of cytoskeleton and cell function in goat temporomandibular joint(TMJ) disc cells by treated cytochalasin B(CB) and uniaxial tensile stress. To provide a theoretical basis for the study of tissue engineering of temporomandibular joint disc.Method:1. Isolation, culture and identification of goat TMJ disc cells. P1 cells were cryopreservation.2. To detect different concentrations of CB on the proliferation of TMJ disc cells and obtain an optimal concentration.3. To observe the uniaxial tensile stress on growth and morphology of TMJ disc cells and obtain an optimal parameters.4. Immunofluorescence staining was used to detect the change of TMJ disc cell cytoskeleton by treated CB and uniaxial tensile stress.5. Real-time PCR was used to detect actin, tubulin, vimentin, glycosaminoglycan and collagenⅠm RNA expression of TMJ disc cell by treated CB and uniaxial tensile stress.Result:1. TMJ disc cells culture and identificationThe primary cell isolated from goat TMJ disc had slowly proliferation ability in vitro.However, the cells accelerated growth with increased passage. Most of the primary TMJ disc cells presented long spindle-shape while the rest showed polygon-shape. Immunohistochemically staining of collagen Ⅰ and toluidine blue staining exhibited positive results.2. Optimal concentration of CBThe different concentration of CB could inhibit the TMJ disc cells proliferation. With prolonged culture, the proliferation ability in group of 2μmol/L recovered. The rest groups didn’t proliferate. So the optimum concentration of CB was 2μmol/L.3. Parameters of uniaxial tensile stressThe elongation of 2% could promote the proliferation and adjust growth direction of TMJ disc cells. Growth direction of cells was consistent and perpendicular to the direction of mechanical loading. While the elongation of 4% and 8% could inhibit the cells proliferation.4. Cell morphology and cytoskeletonThe group of 2μmol/L could make cells’ protrusions gradually disappear and cells’ morphology became smoother. Actin filaments obviously fractured and fluorescence intensity decreased.The group of stretch, which elongation is 2%, could increase actin and tubulin’s staining intensity and thickening. But it could decrease vimentin’ staining intensity, and make the filaments sparsely.5. The results of RT-PCRThe group of 2μmol/L could decrease m RNA expression of actin and collagen Ⅰ,respectively 57% and 41%. But GAG m RNA expression upregulation of 137%.In the group of stretch, which elongation is 2%, the m RNA expression of actin and tubulin and GAG respectively increased 90%, 71%, 83%. Collagen Ⅰ m RNA expression had no change.However, vimentin m RNA expression reduced 57%.Conclusion:1. Cytochalasin B can regulate morphology, phenotype and extracellular matrix synthesis of goat TMJ disc cells. Inhibiting actin expression may contribute to build self-assembly of TMJ disc.2. TMJ disc cells only adapt to low elongation of uniaxial tensile stress. But cell phenotype,growth direction and extracellular matrix synthesis can produce adaptable effects.