Investigation of the hydromechanical threshold of high-mobility group box 1 release from synovial cells and chondrocytes

Abstract

Objective To investigate the effect of fluid flow shear stress (FFSS) on the fluid mechanic threshold of high-mobility group box 1 (HMGB1) release by synovial cells and chondrocytes. Moreover, the mechanism of chondrocyte and synovial cell damage induced by abnormal mechanical force was investigated to provide an experimental basis for exploring the pathogenesis and pathology of temporomandibular joint osteoarthritis. Methods With the approval of the Ethics Committee for Animal Experiments of the hospital, synovial tissue and cartilage tissue blocks were obtained from the knee joints of Sprague-Dawley (SD) rats, and synovial cells and chondrocytes were cultured and digested for subsequent experiments. Synovial cells and chondrocytes of 3-4 generations were acquired, and FFSS was applied to synovial and cartilage cells using a fluid shear mechanical device. The cells were divided according to the FFSS values of different sizes. Synovial cells were stimulated for 1 h with 1, 3, 5, or 10 dyn/cm2 of FFSS, and chondrocytes were stimulated for 1 h with 4, 8, 12, or 16 dyn/cm2 of FFSS. Resting cultures (0 dyn/cm2) were used as the control group. Changes in the morphology of the cells were observed. The expression and distribution of HMGB1 and interleukin-1β (IL-1β) were observed by immunohistochemistry. The expression of HMGB1 and IL-1β in the supernatant was analyzed by ELISA. The protein expression levels of intracellular HMGB1 and IL-1β were detected by Western blot. Results With increasing FFSS, the synovial cells and chondrocytes gradually swelled and ruptured, and the number of cells decreased. With increasing FFSS, the localizationof HMGB1 and IL-1β gradually shifted from the nucleus to the cytoplasm. In synovial cells, compared with those in the control group, the expression levels of HMGB1 and IL-1β were increased both in the supernatant and cells in the 1, 3, 5 and 10 dyn/cm2 intervention groups (P<0.01). In chondrocytes, compared with those in the control group, the expression levels of HMGB1 in the supernatant were increased in the 4, 12 and 16 dyn/cm2 intervention groups (P<0.05), and the protein expression levels of HMGB1 were significantly increased (P<0.01). The expression levels of HMGB1 in the supernatant were significantly increased in the 8 dyn/cm2 intervention groups (P<0.01); however, the protein expression levels of HMGB1 were significantly decreased. Compared with those in the control group, the expression levels of IL-1β in the supernatant gradually increased in the 4, 8, 12 and 16 dyn/cm2 intervention groups (P<0.01). With the exception of those in the 4 dyn/cm2 group, the protein expression levels of IL-1β gradually increased with increasing FFSS (P<0.05). Conclusion With increasing FFSS, synovial cells and chondrocytes gradually swelled and burst, and the hydromechanical thresholds of HMGB1 release were 1 dyn/cm2 and 8 dyn/cm2, respectively. Therefore, upon stimulation with a mechanical force, synovial damage was damaged before chondrocytes.

Keywords

• temporomandibular joint osteoarthritis|synovial cells|chondrocytes|fluid flow shear stress|threshold|high-mobility group box 1|interleukin-1β