Fibroblasts play a crucial role in the proliferative phase. They migrate from normal tissue into the wound area from its margins, where they grow and form a new, provisional extracellular matrix by excreting collagen and fibronectin. Due to the crucial role of fibroblasts in the wound healing process, we investigated the effects of different concentrations of local anaesthetics on viability and proliferation of fibroblasts. Based on previous results in an inflammatory model of acute lung injury [13], we hypothesized that local anaesthetics do not have

an adverse effect on fibroblasts. In this study, human osteosarcoma cells (LGC Standard GmbH, Wesel, AZD6244 cell line Germany), osteoblast-like cell types with the morphology of human fibroblasts, were used. According to a study from Jukkola et al. in 1993, these cells have the characteristics of proliferative wound fibroblasts [14]. Cells were cultured in α-modified Eagle’s medium (MEM; LGC Standard GmbH) with 10% fetal bovine serum

(FBS; LGC Standard GmbH) and 10 000 U/l penicillin/streptomycin (LGC Standard GmbH) at 37°C and 5% CO2. Lidocaine (Lidocain CO2 2% Sintetica®) was purchased from Sintetica AG, Mendrisio, Switzerland, bupivacaine (Bucain®) from DeltaSelect GmbH, Munich, Germany and ropivacaine (Naropin®) from AstraZeneca, Wedel, Germany. Serial dilutions were chosen with lidocaine, bupivacaine and ropivacaine resulting in concentrations Opaganib manufacturer of 0·3 mg/ml and 0·6 mg/ml, representing comparable tissue concentrations measured in clinical practice [15]. In group 1, cells were exposed to the LA for 2 days followed by another incubation time of 1, 4 or 7 days with normal medium without LA. In group 2, cells were exposed permanently to local anaesthetics for 3, 6 or 9 days. The LA-containing medium was changed every second day to provide stable and constant drug concentrations. Control cells were incubated with medium only for the STK38 according period of time. All changes

of medium performed in the treated group were performed similarly in control cells. On days 3, 6 and 9, living cells were counted manually in the Neubauer chamber, using trypan blue [16,17]. The tetrazolium bromide (MTT) assay is a well-known and recognized method to measure cell viability in vitro[18]. The method is based on the reduction of yellow tetrazoliumsalt 3-[4, 5-dimethylthiazol-2-yl]-2, 5-diphenyl tetrazolium bromide into purple formazan crystals by mitochondrial dehydrogenases. Dehydrogenases are active only in living cells. Conversion of MTT is therefore related directly to cell viability. Proliferation tests were performed with the help of the colorimetric bromodeoxyuridine (BrdU) assay (Roche, Basel, Switzerland). The test analyses the proliferation of cells by utilizing BrdU as an analogue of the DNA nucleotide thymidine, which is incorporated into the synthesized DNA of actively dividing cells.