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Study Design: This in vitro study clarifies the role of nitric oxide (NO) in human lumbar intervertebral disc metabolism., Objective: To investigate the effects of NO on proteoglycan synthesis in human lumbar discs and to test the hypothesis that NO is a mediator of the changes in proteoglycan synthesis in response to hydrostatic pressure., Summary of Background Data: The authors have clarified that hydrostatic pressure has an apparent effect on proteoglycan synthesis as well as matrix metalloproteinase production in the intervertebral disc. The cellular mechanisms underlying the response of disc cells to hydrostatic pressure remain to be clarified. Herniated lumbar discs produce NO in response to interleukin (IL)-1 beta. In articular cartilage, NO mediates the change of proteoglycan synthesis by IL-1 or shear stress., Methods: Fifty-eight lumbar intervertebral disc specimens were obtained from patients who had undergone posterior discectomy. The specimens were chopped into 1-2-mm cubes and were incubated in a plastic syringe with 1 mL Dulbecco's modified Eagle's medium (DMEM). The syringes were placed in a water-filled pressure vessel kept at 37 C. Hydrostatic pressures of 1 (control), 3, and 30 atmospheres (atm) were applied. Proteoglycan synthesis was determined from (35)S-sulfate incorporation rates. Nitrite (the stable oxidation product of NO) concentration in DMEM was determined by a spectrophotometric method based on the Griess reaction. As a competitive inhibitor of NO synthases, N(G)-methyl-l-arginine (l-NMA, 10-1000 micromol) and as an organic donor of NO, S-nitroso-N-acetylpenicillamine (SNAP, 1-200 micromol) were used., Results: Addition of l-NMA suppressed NO production and increased proteoglycan synthesis rates in the intervertebral disc specimens in a dose-dependent fashion. Addition of SNAP increased exogenous NO content in the medium significantly and suppressed proteoglycan synthesis rates in a dose-dependent fashion. Three-atmosphere hydrostatic pressure stimulated the proteoglycan synthesis rates. Rates were approximately 1.3-fold greater than at 1 atm, whereas 30-atm pressure inhibited proteoglycan synthesis rates. However, the hydrostaticpressure had inverse effect on NO production. At 3 atm, NO production decreased slightly relative to 1 atm, whereas at a pressure of 30 atm, NO production was increased and was approximately 1.32-fold greater than at 1 atm. L-NMA enhanced the 3-atm pressure-induced increase in proteoglycan synthesis and also relieved the suppression of proteoglycan synthesis at a pressure of 30 atm., Conclusion: The current study confirmed the previous finding that human herniated lumbar disc cultures spontaneously produce NO. Endogenously generated and exogenously supplied NO inhibited proteoglycan synthesis in the intervertebral disc. Hydrostatic pressure influenced NO production by disc cells, and NO is one of the mediators that changes proteoglycan synthesis in response to hydrostatic pressure. These results may show that autocrine and paracrine mechanisms of NO play an important role in the regulation of disc cell metabolism under mechanical stress and in the pathophysiology of intervertebral disc degeneration.

Study Design: A case-control study using magnetic resonance imaging findings and a polymerase chain reaction assay to investigate the association between aggrecan gene polymorphism and lumbar disc degeneration., Objective: To analyze whether the aggrecan gene polymorphism is related to lumbar disc disease in young women., Summary of Background Data: It has been suggested that a genetic factor or familial predisposition contributes to the development of lumbar disc herniation. However, the precise genetic component related to disc disease remains unclear. Recently, a polymorphism has been identified in the region of the human aggrecan gene. The expressed variable numbers of tandem repeat polymorphism occur in the highly conserved repeat region., Methods: The participants were 64 young women with or without low back problems. Magnetic resonance imaging was used to evaluate the degeneration and herniation of the intervertebral disc. Genomic deoxyribonucleic acid was extracted from all participants. A polymerase chain reaction assay was carried out to detect the alleles of the aggrecan gene. The association of intervertebral disc degeneration and herniation with the distribution of the aggrecan gene alleles was analyzed., Results: Findings showed an overrepresentation of alleles with small numbers of repeats in subjects with multilevel disc degeneration, thus indicating a significant distribution difference. There also was a significant difference between the distribution of alleles and the severity of disc degeneration. No significant association was found between any of the alleles either in number or type of disc herniation., Conclusions: The current study showed that multilevel and severe disc degeneration was present in the participants with shorter variable numbers of tandem repeat length of the aggrecan gene. This suggests that subjects with shorter variable numbers of tandem repeat length of the aggrecan gene have a risk of having multilevel disc degeneration develop at an early age.

Study Design: This study is a unique in vitro study on the effects of hydrostatic pressure on human intervertebral disc metabolism., Objective: To investigate the effects of hydrostatic pressure on matrix synthesis and matrix metalloproteinase production in the human lumbar intervertebral disc., Summary of Background Data: Mechanical stress and hydrostatic pressures influence proteoglycan and protein synthesis rates in bovine articular cartilage and coccygeal discs. However, the mechanism of matrix synthesis regulation of the intervertebral disc under mechanical stress has not been elucidated., Methods: Twenty-eight human lumbar intervertebral discs obtained from surgery and from cadavers at autopsy were used. Each tissue fraction was charged with medium in a plastic syringe and placed in a water-filled hydrostatic pressure-control vessel. The hydrostatic pressures applied were 1 (control), 3, and 30 atm (atm = atmospheres) for 2 hours. The proteoglycan and protein synthesis rates were determined by radioisotope incorporation. The production of matrix metalloproteinase-3 and tissue inhibitor of metalloproteinases-1 were measured by a one-step enzyme immunoassay method using monoclonal antibodies., Results: Three atm pressure stimulated proteoglycan synthesis rates in the nucleus pulposus and inner anulus (n = 14 in each tissue). Compared with the control group, 30 atm pressure significantly inhibited proteoglycan synthesis in the inner anulus (P = 0.011). In the nucleus pulposus, matrix metalloproteinase-3 production was stimulated at a pressure of 30 atm relative to 3 atm (P = 0.014, n = 16 in each tissue). The highest tissue inhibitor of metalloproteinases-1 production showed highest values at 3 atm pressure in the inner anulus (n = 16 in each tissue)., Conclusion: The results suggest that hydrostatic pressure influences intervertebral disc cell metabolism. A physiologic level of hydrostatic pressure (3 atm) may act as an anabolic factor for stimulation of proteoglycan synthesis and tissue inhibitor of metalloproteinases-1 production. This may be essential for maintaining the matrix of the disc. If the pressure was 30 atm or more or 1 atm or less, a catabolic effect will be predominant, with reduction of proteoglycan synthesis rate and increase of matrix metalloproteinase-3 production. Abnormal hydrostatic pressure, therefore, may accelerate disc degeneration.

The present study was undertaken to investigate the effect of insulin-like growth factor-1 on proteoglycan synthesis and the autocrine/paracrine mechanism involving insulin-like growth factor-1 in the bovine coccygeal intervertebral disc. Insulin-like growth factor-1 stimulated proteoglycan synthesis in cultured cells of the nucleus pulposus of bovine intervertebral discs in a dose-dependent manner, and the effect was inhibited by an anti-insulin-like growth factor-1 monoclonal antibody. In situ hybridization histochemistry revealed the expression of insulin-like growth factor-1 mRNA in the cultured cells, and its production in these cells was demonstrated by radioimmunoassay. Insulin-like growth factor-1 receptor in the cultured cells was also demonstrated immunohistochemically. Scatchard analysis using an [125I]insulin-like growth factor-1 binding assay showed that the cells cultured in monolayer had a single type of insulin-like growth factor-1 receptor, whose affinity and number were estimated to be 7.38 x 10(8)/M and 9.27 x 10(4)/cell, respectively. These results suggest that insulin-like growth factor-1 stimulates proteoglycan synthesis in cells of the nucleus pulposus and that these cells in culture have an insulin-like growth factor-1 autocrine/paracrine mechanism. The expressions of insulin-like growth factor-1 mRNA and insulin-like growth factor-1 receptor in disc tissue were greater in cells of the nucleus pulposus of fetal bovine intervertebral discs than in those of the adult discs. These findings suggest that the action of autocrine/paracrine insulin-like growth factor-1 is more active in cells of the young nucleus pulposus than in cells of mature subjects.