Glucosamine HCl alters production of inflammatory mediators by rat intervertebral disc cells in vitro.

BACKGROUND CONTEXT: Studies on cartilage have shown anti-inflammatory effects of glucosamine related to inhibition of inflammatory mediators. Intradiscal injection of glucosamine has been proposed as a treatment for chronic discogenic low back pain. However, there have been no studies of the direct effects of glucosamine on disc cells. PURPOSE: To determine the effects of glucosamine HCl on pro-inflammatory mediator production by intervertebral disc cells. STUDY DESIGN: An in vitro, experimental study of interleukin-1 (IL-1) stimulated rat intervertebral disc cells treated with and without glucosamine HCl. METHODS: Rat annulus and nucleus cells were cultured in alginate beads and exposed to IL-1a (10 ng/mL)+glucosamine HCl (4.5 mg/mL), IL-1 alone, or neither for 4 and 7 days. Cell viability and IL-6, tumor necrosis factor alpha (TNF-alpha), prostaglandin E(2) (PGE(2)), and NO levels in the medium were quantified and compared across treatments. RESULTS: Annulus cells, 7 days: Glucosamine completely inhibited IL-6 and TNF-alpha, increased NO (by 75%), and reduced viability (by 89%) compared with IL-1 alone. Nucleus cells, 7 days: Glucosamine reduced IL-6 (by 89%), PGE(2) (91%), and NO (90%) with no effect to viability. CONCLUSIONS: Glucosamine inhibits inflammatory mediator production by IL-1 stimulated disc cells, but also adversely affects the viability of rat annulus cells. The response is cell-type dependent, illustrated by differences for annulus and nucleus cells.

The short-term effects of electrosurgical ablation on proinflammatory mediator production by intervertebral disc cells in tissue culture.

BACKGROUND CONTEXT: Percutaneous discectomy can be performed by a variety of methods. One method, electrosurgical ablation, has been shown in a chronic animal model to alter the expression of inflammatory cytokines in degenerated discs. PURPOSE: To determine whether electrosurgical ablation has an acute direct effect on proinflammatory mediator production by disc cells. STUDY DESIGN: A short-term in vitro study using normal and interleukin (IL)-1alpha stimulated porcine disc cells cultured in alginate gel to evaluate the biochemical effects of electrosurgical ablation. METHODS: Porcine annulus and nucleus cells were embedded into alginate gels and cultured using control culture media or IL-1alpha-treated media for 6 days before ablation treatment. Treated gels were ablated by using a radiofrequency-based electrosurgical device for 5 seconds and cultured an additional 3 or 6 days. IL-1beta, IL-6, IL-8, tumor necrosis factor alpha (TNF-alpha), prostaglandin E2 (PGE2), nitric oxide (NO), and heat shock protein-70 (Hsp70) levels in culture medium were measured. Levels were normalized to DNA and compared between ablated and shams. RESULTS: For normal annulus cells, there were no significant changes in cytokine levels between ablation and sham groups. For normal nucleus cells, ablation produced significantly greater levels of IL-8 at 3 days and 6 days, Hsp70 at 3 days but not 6 days, and NO at 6 days. PGE2 was also increased at 3 days and 6 days but not significantly. For IL-1-stimulated annulus cells, IL-6 and NO in the ablation group were decreased at 3 days relative to the control group. However, IL-6, IL-8, PGE2, and Hsp70 were significantly increased in the 6-day ablation group. For degenerated nucleus cells, IL-6, IL-8, and TNF-alpha were significantly decreased in the ablation group at both 3 days and 6 days. Ablation resulted in reduced PGE2 at 3 days but not 6 and reduced Hsp70 and NO at 6 days. CONCLUSIONS: The results show that electrosurgical ablation has an acute direct effect on proinflammatory mediator production by disc cells. The effect produced depends on disc cell phenotype, the mediator, and time. These direct biologic effects may be a mechanism of pain relief after percutaneous discectomy using electrosurgical ablation. However, the measured responses are limited to the short-term (1 week), and the existence of a prolonged effect remains to be determined.

Intervertebral disc cell therapy for regeneration: mesenchymal stem cell implantation in rat intervertebral discs.

This study explores the use of mesenchymal stem cells (MSCs) for intervertebral disc regeneration. We used an in vivo model to investigate the feasibility of exogenous cell delivery, retention, and survival in the pressurized disc space. MSC injection into rat coccygeal discs was performed using 15% hyaluronan gel as a carrier. Injections of gel with or without MSCs were performed. Immediately after injection, fluorescently labeled stem cells were visible on sections of cell-injected discs. Seven and 14 days after injection, stem cells were still present within the disc, but their numbers were significantly decreased. At 28 days, a return to the initial number of injected cells was observed, and viability was 100%. A trend of increased disc height compared to blank gel suggests an increase in matrix synthesis. The results indicate that MSCs can maintain viability and proliferate within the rat intervertebral disc.

Biological response of the intervertebral disc to dynamic loading.

Disc degeneration is a chronic remodeling process that results in alterations of matrix composition and decreased cellularity. This study tested the hypothesis that dynamic mechanical forces are important regulators in vivo of disc cellularity and matrix synthesis. A murine model of dynamic loading was developed that used an external loading device to cyclically compress a single disc in the tail. Loads alternated at a 50% duty cycle between 0MPa and one of two peak stresses (0.9 or 1.3MPa) at one of two frequencies (0.1 or 0.01Hz) for 6h per day for 7 days. An additional group received static compression at 1.3MPa for 3h/day for 7 days. A control group wore the device with no loading. Sections of treated discs were analyzed for morphology, proteoglycan content, apoptosis, cell areal density, and aggrecan and collagen II gene expression. Dynamic loading induced differential effects that depended on frequency and stress. No significant changes to morphology, proteoglycan content or cell death were found after loading at 0.9MPa, 0.1Hz. Loading at lower frequency and/or higher stress increased proteoglycan content, matrix gene expression and cell death. The results have implications in the prevention of intervertebral disc degeneration, suggesting that loading conditions may be optimized to promote maintenance of normal structure and function.

In vivo growth factor treatment of degenerated intervertebral discs.

STUDY DESIGN: An in vivo model was used to investigate the response of degenerated discs to various exogenous growth factors. OBJECTIVES: To study growth factor-induced alterations of the spatial and temporal patterns of disc cellularity and matrix gene expression. SUMMARY OF BACKGROUND DATA: Cell proliferation and proteoglycan synthesis have been stimulated by growth factors in normal disc cells, suggesting that growth factors may play a therapeutic role for degeneration. However, the response in situ in degenerated discs has not been characterized. METHODS: Degeneration was induced in murine caudal discs by static compression. Degenerated discs were given single or multiple injections of growth and differentiation factor-5, transforming growth factor-beta, insulin-like growth factor-1, basic fibroblast growth factor, or saline as control. Comparisons of disc morphology, anular cell density, proliferating cells, disc height, and aggrecan and type II collagen gene expression were made either 1 week or 4 weeks after treatment. RESULTS: In some growth and differentiation factor-5 and transforming growth factor-beta treated discs, expansion of inner anular fibrochondrocyte populations into the nucleus was observed. The cells actively expressed aggrecan and type II collagen mRNA. A lesser effect was observed for insulin-like growth factor-1 and little or no effect for basic fibroblast growth factor. Differences in cell density and proliferating cells were not significant between treatments but suggested a trend of increased cellularity and proliferation following growth factor treatment. A statistically significant increase in disc height 4 weeks after growth and differentiation factor-5 treatment was measured. CONCLUSIONS: Anular fibrochondrocytes in degenerated discs are responsive to some growth factors in vivo. The results have implications in the early intervention of disc degeneration to arrest or slow the degenerative process.