Effects of indigo carmine on human chondrocytes in vitro

Abstract

Joint infections following or accompanying superficious soft tissue infections are severe complication in orthopedic surgery. The use of intra-articular blue staining is a helpful method to visualize a fistula and to differentiate between superficial and intra-articular infections. Regarding this clinical implication data about the effects of indigo carmine, a frequently used blue staining substance, on cartilage is missing. The hypothesis of this study was that indigo carmine damages human chondrocytes in a time and concentration dependent manner. Human chondrocytes were isolated from donors with osteoarthritis who were treated with TKA. Cells were cultivated and treated with different concentrations of indigo carmine for 5 and 10 minutes. Morphologic damage was examined by light microscopy. Toxicity was quantified by counting vital cell number and lactate dehydrogenase (LDH) expression. Analysis by light microscopy showed defected cell structure and loss of cell number after treatment with 100% indigo carmine for 10 minutes. Treatment with 10% and 1% indigo carmine showed no significant cell defects and loss of cells. Counting vital cell number showed loss of vital cells after treatment with 100% and 10% indigo carmine for 10 minutes. LDH expression was significantly increased after treatment with 100% indigo carmine.Toxic effects were shown after treatment with indigo carmine. Therefore, it should be used in 1:100 dilution. This is both, sufficient for visualizing a fistula in a possible clinical application and could be protective for chondrocytes.

Keywords: Indigo carmine; human chondrocytes; toxicity..

Fig. (1)

Chondrocyte damage after exposure to IC. Chondrocytes were treated with IC and were analyzed by light microscopy. (A) Untreated control chondrocytes. (B) Chondrocytes treated with Triton X100, a known inducer of cell necrosis. (C) Chondrocytes treated with 1% IC. (D) Chondrocytes incubated with 10% IC. (E) Chondrocytes treated with 100% IC. One representative result is shown from at least three independently performed experiments.

Fig. (2)

Determination of vital cells after 5 and 10 minutes versus negative control. (A) Significant decrease of vital chondrocytes after treatment with 100 % IC for 5 minutes. (B) Significant decrease of living cells after treatment with 10 % and 100 % IC for 10 minutes. (A, B) n=6, Mean±SEM, A nonparametric Wilcoxon matched pairs test was used as indicated in the legends. A p value of < 0.05 was considered to be significant.

Fig. (3)

LDH release following treatment with IC. After the indicated time points supernatants were analyzed for LDH by ELISA. (A, B) Significant increase of LDH activity after treatment with 100 % IC for 5 and 10 minutes. n=6, Mean±SEM, A nonparametric Wilcoxon matched pairs test was used as indicated in the legends. A p value of < 0.05 was considered to be significant.