Purpose: This study was to investigate the cytotoxic potency of local anesthetics on human mesenchymal stem cells during chondrogenesis.
Methods: Aggregates were created from density-gradient centrifugation-separated bone marrow-derived mesenchymal stem cells. After 7, 14, and 21 days, aggregates were analyzed histologically and immunohistochemically and exposed to equipotent concentrations of bupivacaine, ropivacaine, and mepivacaine for 1 h. Cell viability, apoptosis, and necrosis were determined using live-dead and caspase staining. Additionally, following a 1-h exposure on day 7, aggregates were cultured under chondrogenic conditions until day 21 to assess the effects of local anesthetics on differentiation potency of mesenchymal stem cells.
Results: In the course of chondrogenesis, mesenchymal stem cells were embedded in varying amount and structure of cartilage-specific extracellular matrix. Contents of sulfated glycosaminoglycan, type I and II collagen increased from day 7 to day 21. Compared to control, death rates of mesenchymal stem cells were significantly elevated 1 day after treatment at 7 and 14 days. Four days after exposure, death rates were 13-15 % at 7 and 11-17 % at 14 days. Mesenchymal stem cell viability in aggregates at 21 days was unchanged to controls. The width of the superficial aggregate zone containing stem cell necrosis decreased with elongated differentiation time. Apoptosis rates were elevated in the edge regions of aggregates, reaching maximum values 4 days after treatment. Local anesthetic exposure on day 7 reduced Collagen II, but not DNA contents in aggregates at 21 days. Bupivacaine, ropivacaine, and mepivacaine did not differ in mesenchymal stem cell cytotoxicity in aggregates.
Conclusion: Local anesthetic exposure results in cytotoxicity of mesenchymal stem cells undergoing chondrogenesis, especially in superficial layers. Therefore, induced cell damage should be avoided during chondrogenesis of mesenchymal stem cells, particularly early after cartilage repair.