Funding for this research was provided by:
Japan Society for the Promotion of Science (JP19K19088, JP21K17035)
Article History
Received: 1 November 2023
Accepted: 16 March 2024
First Online: 25 March 2024
Declarations
:
: All animal experimental protocols were approved by the Animal Care and Experimentation Committee of Niigata University (SA00959).
: No potential conflict of interest was reported by the authors.(A) Micro-CT analysis of mouse mandibular condyle at 5, 15, 26, 52, and 78 weeks. Bar, 0.5 mm. (B) Bone mineral density (BMD) of the mandibular condyle. (C) The mandibular condyle was stained with safranin O at five, 15, 26, 52, and 78 weeks. Bar, 50 μm. (D–G) Cartilage area, safranin O-positive area, cell density, and cell size distribution were quantified in the condylar cartilage with age. Data in (B), (D), (E), and (F) are represented as mean ± SD; n = 5 in each group. <sup>a</sup><i>P</i> < 0.05, vs. 5 w; <sup>A</sup><i>P</i> < 0.01, vs. 5 w; <sup>b</sup><i>P</i> < 0.05, vs. 15 w; <sup>B</sup><i>P</i> < 0.01, vs. 15 w; <sup>c</sup><i>P</i> < 0.05, vs. 26 w; <sup>C</sup><i>P</i> < 0.01, vs. 26 w; <sup>D</sup><i>P</i> < 0.01, vs. 52 w.(A) Immunohistochemistry of cis-Golgi marker GM130 in condylar cartilage. Bars, 10 μm. (B) The Golgi apparatus size of chondrocytes of all layers in the condylar cartilage was quantified. (C) Age-related changes in primary cilia were examined via immunohistochemistry of the condylar cartilage using anti-acetylated tubulin and anti-gamma-tubulin antibodies. Bars, 10 μm. (D) Primary cilia frequency in chondrocytes. (E) Immunohistochemistry analysis of pS6. Bars, 10 μm. (F) The ratio of pS6-positive chondrocytes. The white square indicates the region of interest in the high-magnification image shown in (A) (C) and (E). Data in (B) (D) and (F) are represented as mean ± SD; n = 5 in each group. <sup>a</sup><i>P</i> < 0.05, vs. 5 w; <sup>A</sup><i>P</i> < 0.01, vs. 5 w; <sup>b</sup><i>P</i> < 0.05, vs. 15 w; <sup>B</sup><i>P</i> < 0.01, vs. 15 w; <sup>c</sup><i>P</i> < 0.05, vs. 26 w.(A-D) ATDC5 cells, a chondroprogenitor cell line, were treated with DMSO as control (Cont) or MHY1485. (A) Immunocytochemistry analysis using anti-acetylated tubulin, anti-gamma-tubulin, and anti-pS6. Cells were counterstained with DAPI. Bars, 10 μm. (B) Cell number. (C) The ratio of pS6-positive cells. (D) Primary cilia frequency. (E–H) ATDC5 cells were treated with ethanol as control (Cont) or Rapamycin (RAP). (E) Immunocytochemistry analysis using anti-acetylated tubulin, anti-gamma-tubulin, and anti-pS6. Cells were counterstained with DAPI. Bars, 10 μm. (F) Cell number. (G) The ratio of pS6-positive cells. (H) Primary cilia frequency. The white square indicates the region of interest in the high-magnification image shown in (A) and (E). Arrowheads indicate the primary cilia. Data in (B), (C), (D), (F), (G), and (H) are represented as mean ± SD; n = 6 in each group. *: <i>P</i> < 0.05, as compared to vehicle.(A-C) Mice were injected with the vehicle as control (Cont) or rapamycin (RAP) for one week. (A) Immunohistochemistry analysis using anti-acetylated tubulin, anti-gamma-tubulin, and anti-pS6 antibodies in the condylar cartilage. Bars, 10 μm. (B) The ratio of pS6-positive cells. (C) Primary cilia frequency. (D-F) Mice were injected with a vehicle as control (Cont) or rapamycin (RAP) for 11 weeks. (D) Immunohistochemistry analysis in the condylar cartilage. Bars, 10 μm. (E) The ratio of pS6-positive cells. (F) Primary cilia frequency. The white square indicates the region of interest in the high-magnification image shown in (A) and (D). Arrowheads indicate the primary cilia. Data in (B), (C), (E), and (F) are represented as mean ± SD; n = 6 in each group. *: <i>P</i> < 0.05, as compared to control.Mice were injected with a vehicle as control (Cont) or rapamycin (RAP) for 11 weeks. (A) The mandibular condylar cartilage was stained with safranin O Bars, 100 μm. (B–E) Cartilage area, safranin O-positive area, cell density, and cell size distribution of the condylar cartilage in rapamycin/vehicle-injected mice. (F) cis-Golgi marker GM130 in condylar cartilage in rapamycin/vehicle-injected mice. Bars, 10 μm. (G) Golgi apparatus size. The white square indicates the region of interest in the high-magnification image shown in (F). Data in (B), (C), (D), and (G) are represented as mean ± SD; n = 6 in each group. *: <i>P</i> < 0.05, as compared to control.