Background: Sterile surgical marker pens are commonly used in cartilage repair surgery to aid in the placement of periosteal patches or collagen membranes in autologous chondrocyte implantation.
Purpose: To investigate the effects that methylene blue and crystal violet marker pen ink have on human chondrocytes when cultured on collagen membranes in vitro.
Study Design: Controlled laboratory study.
Methods: Human chondrocytes were applied to Chondro-Gide collagen membranes at a volume of 12 million cells. In the first experiment, 2 sterile marker pens, one containing methylene blue and the other crystal violet inks, were used to mark membranes immediately before the addition of cells. In the second experiment, the same marker pens marked the membranes after 7 days of cell culture. In each experiment, 3 groups of membrane were tested for each pen. Group A consisted of no ink mark, group B had only the uppermost “smooth” layer marked, and group C had the lower “porous” layer marked. All membranes were then cultured in standard growth media for 24 hours. Cell viability was assessed at 24 hours on all membranes using a live/dead-cell viability assay. Cell viability was quantified with florescent microscopy with mean percentage of live cells in each marker pen group compared with control membranes using the Student t test (P < .05).
Results: Control membranes (group A) with no ink showed cell viability approaching 100%. A statistically significant reduction in cell viability with both methylene blue (23.1%; P < .0001) and crystal violet (18.9%; P < .0001) was found adjacent to the ink mark on the smooth side (group B) and on the porous side remote from the ink (group C) in both experiments (<30%; P < .0001). Areduction in cell viability was noted on the smooth side remote from the ink mark but did not reach statistical significance. Marked cell death was seen with both dyes (<15%; P < .0001) adjacent to the ink on the porous side.
Conclusion: Chondrocyte viability is significantly reduced when cells are cultured in vitro on collagen membranes marked with methylene blue and crystal violet pen ink.
Clinical Relevance: Surgeons should be aware of the potential negative effect of marker pens in cell-based therapies.
BartlettWSkinnerJAGoodingCR. Autologous chondrocyte implantation versus matrix-induced autologous chondrocyte implantation for osteochondral defects of the knee: a prospective, randomised study. J Bone Joint Surg Br. 2005;87(5):640-645.
2.
BrittbergMLindahlANilssonAOhlssonCIsakssonOPetersonL. Treatment of deep cartilage defects in the knee with autologous chondrocyte transplantation. N Engl J Med. 1994;331(14):889-895.
3.
BrouzasDDroutsasDCharakidasA. Severe toxic effect of methylene blue 1% on iris epithelium and corneal endothelium. Cornea. 2006;25(4):470-471.
ChangYSTsengSYTsengSHChenYTHsiaoJH. Comparison of dyes for cataract surgery: Part 1. Cytotoxicity to corneal endothelial cells in a rabbit model. J Cataract Refract Surg. 2005;31(4):792-798.
GoodingCRBartlettWBentleyGSkinnerJACarringtonRFlanaganA. A prospective, randomised study comparing two techniques of autologous chondrocyte implantation for osteochondral defects in the knee: periosteum covered versus type I/III collagen covered. Knee. 2006;13(3):203-210.
8.
JakobMDemarteauOSchaferDStummMHebererMMartinI. Enzymatic digestion of adult human articular cartilage yields a small fraction of the total available cells. Connect Tissue Res. 2003;44(3-4):173-180.
9.
LeeYSWursterRD. Methylene blue induces cytotoxicity in human brain tumor cells. Cancer Lett. 1995;88(2):141-145.
