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Mechanical Stress-Induced Apoptosis Of Endplate Chondrocytes In Organ-Cultured Mouse Intervertebral Discs: An Ex Vivo Study

K. Ariga, K. Yonenobu, T. Nakase, N. Hosono, S. Okuda, W. Meng, Y. Tamura, H. Yoshikawa
Published 2003 · Biology, Medicine

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Study Design. Various amounts of static mechanical load were applied to mouse intervertebral discs in organ cultures. The apoptosis then was examined using nick end labeling. Two mitogen-activated protein kinase (MAPK) inhibitors were added to the medium. Objectives. To establish an experimental model for detecting factors regulating chondrocyte apoptosis induced by mechanical stress, and to determine the role of MAPK and p38 in the stress-induced apoptotic pathway of endplate chondrocytes. Summary of Background Data. The cause of degenerative change in the cartilaginous endplate (CEP) remains unclear. The authors’ previous findings using a mouse model suggested that apoptosis in the cartilaginous endplate may play a role in intervertebral disc degeneration, and that mechanical stress may induce apoptosis. If apoptosis of endplate chondrocytes is involved in the cascade of intervertebral disc degeneration, then how apoptosis is induced by mechanical stress should be important in preventing disc degeneration. However, the mechanism of apoptosis induced by mechanical stress remains unclear. Methods. Mouse coccygeal discs were harvested and organ cultured. Various static compression loads (0, 0.2, 0.4, 0.8, and 1.0 MPa) were applied on intervertebral discs placed in culture bottles for 24 hours. Paraffin-embedded sections of the harvested discs were stained using Safranin-O and the nick end labeling procedure. The apoptotic cells were counted in the cartilaginous endplate and junctional anulus fibrosus of each intervertebral disc. In addition, U0126 (MAPK inhibitor) and SB202190 (p38 inhibitor) were added to the culture medium to determine their regulatory roles in the apoptosis of endplate chondrocytes induced by mechanical load. Results. Histologically, loaded discs became bulged, and the disc space became narrow. Apoptosis was absent in discs without load, but was particularly noticeable in loaded discs (load weight, 1.0 MPa). The number of apoptotic cells increased depending on the weight of the load. The two MAPK inhibitors significantly increased the number of apoptotic cells. Conclusions. Chondrocyte apoptosis was induced using a static mechanical load especially in the cartilaginous endplate in an organ culture. Apoptosis occurred similarly to previous findings using an in vivo model. This culture system thus reflected the apoptosis demonstrated in vivo. Because biologically active reagents such as MAPK inhibitors can be simply added to culture media, this system may be a useful method for detecting factors that influence apoptosis induced by mechanical stress. Both MAPK inhibitors increased the occurrence of apoptosis. This suggests that these two MAPKs can counteract the apoptotic pathway induced by mechanical stress.
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