| Partâ… A novel compressing cultivation model of rabbit intervertebral disc cells in vitroObjective To establish and evaluate a novel gas compressing cultivation model of rabbit intervertebral disc cells, and observe the biological changes in the 12h-treated,24h-treated and 36-treated groups.Methods The rabbit nucleus pulposus and annulus fibrosus cells were divided randomly into control group,12h-treated group,24h-treated group and 36h-treated group separately with 1MPa pressure. The morphological changes, proliferation activity and the expression of matrix (COAâ… ,â…¡and AGG) of disc cells were detected.Results After the compressing culture treatment, the reduced cell size and reduced density were observed in the disc cells of treated groups. The inhibition of proliferation activity was detected in 24h-treated and 36h-treated groups in disc cells. The mRNA expression of AGG was decreased in >12h-treated group, and that of COAâ…¡was decreased in>24h-treated group in nucleus pulposus cells, while the mRNA expression of COAâ… ,â…¡was decreased in>24h-treated group in annulus fibrosus cells.Conclusion The gas compressing cultivation model can imitate the degeneration of rabbit intervertebral disc cells induced by mechanical pressure in short time, which provides a simple and safe laboratory scheme for pressurizing cells in vitro.Partâ…¡Mitochondrial Apoptotic Pathway involved in Rabbit Intervertebral Disc Cells Apoptosis Induced by Mechanical OverloadObjective To investigate the apoptotic signal pathways of rabbit nucleus pulposus and annulus fibrosus cells induced by mechanical overload in vitro.Methods The nucleus pulposus and annulus fibrosus cells were randomly divided into four groups separately, in which the cells were exposed to a overload pressure of 1MPa for different lengths of time (Oh,12h,24h and 36h). The cell apoptosis were detected by flow cytometry; the alterations in mitochondrial membrane potential were measured by fluorescence microscopy, fluorescence spectrophotometer and laser scanning confocal microscope; and the activities of caspase-8,9,3 were determined by spectrophotometry.Results The results showed that after the nucleus pulposus and annulus fibrosus cells were subjected to the pressure for 24h,36h (including 12h group, NP), the ratio of cells apoptosis were increased; the mitochondrial membrane potential were decreased; and the activity of caspase-9 was enhanced, no activity changes were observed in caspase-8 in annulus fibrosus cells groups, while in nucleus pulposus cells group, the caspase-8,9,3 activity were increased significantly.Conclusion The results suggested that 1MPa pressure treatment for more than 24 h can lead to the increase of apoptosis in disc cells, and the mitochondrial-dependent pathway is implicated in the pressure-induced annulus fibrosus cell apoptosis, while in that of nucleus pulposus cells the mitochondrial-dependent pathway and DISC pathway are both involved.Partâ…¢Effect of overload pressure on mitochondria function and ultrastructure of the inner and outer rabbit annulus fibrosus cells cultured in vitroObjective To investigate the effects of overload pressure on mitochondria function and ultrastructure of the inner and outer rabbit annulus fibrosus cells cultured in alginate beads in vitro.Methods The rabbit annulus fibrosus cells cultured in beads were divided into 4 groups respectively with overload pressure:OA1 group, the outer annulus cells, without pressure; OA2 group, the outer annulus cells, 1MPa pressure for 24h; IA1 group, the inner annulus cells, without pressure; IA2 group, the inner annulus cells, 1MPa pressure for 24h. After pressure treatment, ATP production was detected by fluorescence microplate reader, the mitochondrial membrane potential fluorescence and the reactive oxygen species were detected by microscope and fluorescence spectrophotometer, the respiratory chain enzyme complexes were detected by spectrophotometer and the mitochondria ultrastructure was observed by transmission electron microscopy.Results After the rabbit annulus fibrosus cells treated with 1MPa pressure for 24h, compared with the control group, both inner and outer annulus cells were significantly decreased in ATP production and mitochondrial membrane potential, the reactive oxygen species release was increased, and the mitochondrial ultrastructure presented pathological changes. The respiratory chain enzyme complexesâ… ,â…¢andâ…£activities were decreased obviously in outer annulus cells, while in inner annulus cells, only the respiratory chain enzyme complexesâ…¢andâ…£were decreased. Compared with the inner annulus fibrosus cells, the mitochondrial damage appeared more significantly.Conclusion Overload pressure influences mitochondrial energy metabolism and damage the mitochondria structure with obvious pathological changes, which may be an important mechanism in energy metabolization of disc degeneration.
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