| Background:It is clear that mechanical forces are involved in initiating disc degeneration but also have the potential to exert beneficial effects. On the one hand, excessive mechanical stress promote accelerated decomposition of disc tissue cells and matrix, on the other hand,appropriate pressure may promote anabolic to repair disc. However, the signaling pathways initiated by mechanical stress and thresholds to these responses have not been elucidated. It was cleared that vertebral endplate cartilage is the important channel that disc nutrition is transmitted and metabolites is excreted. The cartilage cells are the material basis for the normal function of the endplate and its physiological function depends on normal cells. When the cartilage endplate became degenerate with sclerosis and calcify, the channel function of nutrition and metabolites was disturbed. Currently, the research of endplate cartilage cells were cultured using a static pressure, that can not accurately reflect the impact of physiological stress load on cartilage endplate. We had developed a metabolically active compression system which has the ability to test cells in vitro like in vivo. The cells in this system were exposured to environmental factors and intervention characteristics, such as pressure, time and medicine. We hypothesized that vertebral endplate cartilage cells would respond to compressive stress and TMP with different thresholds for alterations in catabolic and anabolic gene expression.Objective:The purpose of the study was to establish the utility of a compression chamber and examine the effects of various magnitudes (0.1MPa,0.7MPa,2MPa,4MPa), durations (4and24hours) and TMP intervention of compression on vertebral endplate chondrocytes in rabbits. The RT-PCR test was used to detect the gene expression of endplate cartilage cells about inflammatory, catabolic, and anabolic. We explored the mechanism of hydrostatic pressure and drug intervention for vertebral endplate cartilage degeneration. We try to find new way of treating degenerative disc disease.Methods:1. The endplate cartilage were isolated, cultured and passaged, with testing endplate cartilage cell counts,0D values by MTT methods. Then the cells were identified by the methods of morphology and immunohistochemistry.2. By using of the compression chamber, the cells were exposure to various magnitudes (0.1MPa,0.7MPa,2MPa,4MPa), durations (4hours,24hours) and TMP intervention. In turn with RNA extration, reverse transcription reaction quantitative PCR reactions, the gene expression was analysised, which included inflammatory markers inducible nitric oxide synthase (Inducible nitric oxide synthase iNOS) and cyclooxygenase-2(Cyclooxygenase COX-2),matrix catabolic markers:matrix metalloproteinase-3(Matrix metalloproteinases-3MMP-3), anti-catabolic markers:matrix metalloproteinase inhibitor1(Matrix metalloproteinase inhibitor TIMP-1) and reference gene (GAPDH).3. TMP, cxtracting from Chuanxiong, was used to intervent the compression test of rabbit vertebral endplate chondrocytes. The pressure reached a certain threshold (0.1MPa,0.7MPa,2MPa,4MPa) and effect time (4hours,24hours) in vitro hydrostatic compression system. It was completed by fluorescence quantitative PCR to detect the differences in gene expression, including inflammatory markers iNOS, COX-2, matrix catabolism markers MMP-3, anti-catabolic markers TIMP-1and reference gene GAPDH.Results:1. The rabbit vertebral endplate chondrocytes had good growth state in vitro monolayer culture conditions. The first four generations of cell morphology had normal state with strong proliferation and phenotypic expression. With the increased passage numbers, it appeared "de-differentiation" phenomenon, which the expression of collagen II reduced with decreasing of cell proliferation and cell aging. It was not available for vertebral endplate chondrocytes research.2. TMP with different density of0.01g/L,0.02g/L,0.04g/L,0.08g/L,0.16g/L can promote variedly degrees of endplate cartilage cell proliferation and differentiation. Compared with other groups,0.04g/L was the best density of endplate chondrocyte, which had better proliferation and the largest number of colony.3. With the hydrostatic pressure effting4hours, when the pressure was rising, anabolic and inflammatory markers firstly incresesed and lately decreased. However, catabolic and anti-catabol ic genes gradually increased, but the increase of catabolic genes is greater than it catabolic genes. The overall performance of catabolism is greater than the anabolism; After the intervention of TMP, with the pressure gradually increased, anabolic genes was increased and the genes of inflammatory markers, catabolic and anti-catabolic increased early and decreased lately. It reached crest stage in2MPa followed by decline, but anti-catabolic gene expression greater than the catabolic gene. It exhibited that TMP was good at anabolic gene expression;4. With the hydrostatic pressure effecting24hours, when the pressure was rising, anabolic and inflammatory factors firstly incresesed and then decreased. The catabolic and anti-catabolic genes incresesed firstly and decreased lately. However the increase volume of catabolic is greater than anti-catabolic gene. The overall performance of catabolism is greater than the anabolism. After the intervention of TMP, with the pressure gradually increasing, the anabolic, inflammatory markers and anti-catabolic genes increased early and decreased lately. At0.7MPa, the inflammatory markers and catabolic genes reached the highest value. It exhibited that TMP was good at anabolic gene expression;5. Under atmospheric (0.1MPa), with the time prolonged to24hours, the anabolism gene promoted after the early rejection. The genes of inflammatory markers and anti-catabolic exhibits were promoted; Under low pressure(0.7MPa), with the time prolonged to24hours, the gene of without TMP group’s anabolism and catabolism were combined into a depression, the gene of TMP group’s anabolism and catabolism were promoted excepting for TIMP1; Under moderate pressure pressure(2MPa), with the time prolonged to24hours, the anabolism gene of TMP group and without TMP group were weaker than catabolism; Under high pressure (4MPa), with the time prolonged to24hours, anabolism and catablism of blank group was inhibited. The anabolism gene of TMP group was inhibited and the genes of catabolic and anti-catabolic were promoted.Conclusion:With a short time and low pressure, the gene expression in each group showed more anti-decomposition and synthesis tendencies. Then increasing the load and time, it was showed more catabolic tendencies. If applied TMP intervention, the gene expression occurs classes some cytoprotective effect. The study shows that moderate pressure in favor of the matrix homeostasis, and sustained pressure is good on the promotion of catabolic genes and TMP favor cell repair. We concluded that vertebral endplate chondrocytes have a dependency of "load-time-drugs". It provided the basis for further exploration restoration endplate cartilage cells and which is a new ideas for the effective prevention of intervertebral disc degeneration. |
PDF Full Text Request