Patent blue versus methylene blue and indigo carmine as a better dye for chromodiscography: in vitro staining efficacy and cytotoxicity study using bovine coccygeal intervertebral discs

Abstract

BACKGROUND CONTEXTChromodiscography is an integral part of full-endoscopic discectomy (FED), comprising ordinary discography with radiopacity produced by contrast medium and intradiscal stain for visualizing annular defects in the endoscopic field. Nevertheless, concerns remain about the cytotoxicity of the stains used. The study of their staining efficacy is also lacking. PURPOSETo evaluate the feasibility of methylene blue, patent blue, and indigo carmine for intradiscal injection, investigate the effectiveness of each dye, and define critical concentration with adequate staining efficacy and tolerable cytotoxicity for use in chromodiscography during FED. STUDY DESIGNAn experimental in vitro study. METHODSDye stock solutions were prepared from powder. The stock was diluted with culture medium or balanced saline and used for cytotoxicity or intervertebral disc staining assays, respectively. Bovine tails were obtained from the local slaughterhouse and functional spine units of intervertebral discs were acquired by transverse incision at the disc level. Each disc was punctured over the posterolateral aspect using a surgical knife to simulate an annular defect. The intradiscal injection was performed with each dye at different concentrations using a 22G needle from the contralateral aspect of the punctured site. Staining efficacy was quantified using ImageJ software. Primary cells of bovine tails were cultivated in each dye at different concentrations. Cytotoxicity was assessed 24 hours after stain exposure using the CCK-8 toxicity assay. RESULTSStaining efficacy and cytotoxicity were proportional to the concentration of tested dyes. Lower limits of concentration producing significant staining efficacy of indigo carmine, methylene blue, and patent blue were 0.25 mg/mL, 0.25 mg/mL, and 0.05 mg/mL, respectively. Compared with controls, concentrations showing significant toxicity for indigo carmine, methylene blue, and patient blue were 1 mg/mL, 0.5 mg/mL, and 2.5 mg/mL, respectively. CONCLUSIONSPatent blue can serve as a more suitable tissue stain than either indigo carmine or methylene blue due to the widest range of tradeoff concentration within 0.05 to 2.5 mg/mL. CLINICAL SIGNIFICANCEPatent blue with the characteristic of good staining efficacy and lower cytotoxicity may be a promising option for chromodiscography during FED.