Toxicity of methylene blue to human nucleus pulposus cells detected by cell counting-kit 8 assay

He Sheng-hua; Feng Hua-long; Sun Zhi-tao; Lai Ju-yi; Wang Ye-guang; Wang Jian; Huang Fei-qiang

doi:10.3969/j.issn.2095-4344.0801

Abstract：BACKGROUND: Methylene blue is used as a developer to identify intervertebral disc degeneration in the transforaminal endoscopic surgery. However, many scholars have indicated that methylene blue can accelerate the degeneration process, whilst foreign researches have reported that it may play therapeutic effect on degenerative intervertebral discs under acidic conditions due to its acidophily. Therefore, whether methylene blue holds toxic effect on the disc remains controversial. OBJECTIVE: To determine whether methylene blue exerts toxic effect on nucleus pulposus cells by cell counting-kit 8 (CCK-8) assay. METHODS: The discarded nucleus pulposus from two patients with intervertebral disc herniation were selected. After digestion, nucleus pulposus cells were extracted and cultured until proliferated to 80% of the medium. Then, the cells were digested to make cell suspensions, divided into six groups and inoculated into the 96-well plates: blank control (only the medium, CCK-8 solution), control group (only medium, cells and CCK-8 solution), and the other groups were cultured with 1%, 2%, 3%, and 4% methylene blue, respectively. The absorbance values were measured by CCK-8 assay at 24, 48, 72 and 96 hours after incubation to calculate the cell viability, and the color change was observed. RESULTS AND CONCLUSION: The color in the control group was the deepest, and the color became lighter with the concentration of methylene blue increasing. The cell viability was the highest in the control group, and it was decreased with the concentration of methylene blue increasing. Thus, methylene blue may exert toxic effect on human nucleus pulposus cells.