The cytotoxicity of local anesthetics to mesenchymal stem cells during early chondrogenic differentiation

Objective To identify the cytotoxicity of different local anesthetics (LAs) to the adipose-derived mesenchymal stem cells (ADMSCs) during early chondrogenic differentiation. Methods Adipose-derived stem cells (ADMSCs) were isolated from 4-month old New Zealand white rabbits, and cultured and induced to differentiate into cartilage. The third generation chondrogenic differentiating ADMSCs were isolated and incubated with 1% lidocaine, 2% methylpivacaine, 0.5% bupivacaine and 0.5% ropivacaine, respectively, for 30 min and 60 min, so as to screen out the LA of least toxicity. Control cells were incubated in phosphate buffer. The LAs were washed away and the chondrogenic differentiation medium was added to promote differentiation. Apoptosis and morphological changes during the chondrogenic differentiation were measured using flow cytometry and safranine-fast green double dyeing on the 3rd and 7th days of the culturing. The least toxic LA was diluted to 25%, 50%, 75% and 100% of the original concentration and incubated with ADMSCs for 30 and 60 minutes. On days 3 and 7 after the exposure, a quantitative real-time reverse-transcriptase polymerase chain reaction (qRT-PCR) and western blotting were employed to analyze the expression of chondrogenesis-related genes including the collagen I, collagen III and SOX9. Results Apoptosis increased significantly after the lidocaine, ropivacaine, bupivacaine and mepivacaine treatments compared to the PBS control. LAs are cytotoxic to rADMSCs during early chondrogenesis in a type- and time-dependent manner. Lidocaine was the least toxic LA tested. The chondrogenesis-related markers all decreased significantly by days 3 and 7 after the exposure to lidocaine at different concentrations compared with the control group. Conclusions LAs are cytotoxic to ADMSCs during early chondrogenesis in a dose- and time-dependent manner. 1% lidocaine was the least toxic LA tested. Key words: Mesenchymal stem cells; Local anesthetics; Cytotoxicity; Regenerative medicine