In Vitro investigation of the effects of bupivacaine and dexmedetomidine on wound healing and their antimicrobial properties

A wound is the disruption of the integrity of biological tissue in any part of the body due to trauma, cuts, burns, or other external factors. It leads to disruptions in anatomical, physiological, and functional functions. Wound healing is a complex biological process that not only affects the wound area but also impacts all systems of the body. This process involves cellular and physiological changes and is regulated by growth factors. Growth factors regulate cell proliferation and the production of blood cells and extracellular matrix, thus organizing cellular, physiological, and chemical changes during the healing process. Bupivacaine is a long-acting local anesthetic commonly used in various anesthesia techniques such as caudal, epidural, spinal anesthesia, and peripheral nerve blocks. It is an effective option for both anesthesia during surgical procedures and for acute and chronic pain management. The effects of bupivacaine on wound healing and its antimicrobial properties are controversial, and further research is needed on this topic. Dexmedetomidine, a potent agonist with high selectivity for alpha-2 adrenergic receptors, has sympatholytic, sedative, amnesic, and analgesic properties. Due to its lipophilic structure, it easily crosses the central nervous system and provides conscious sedation in patients. Recently, the use of dexmedetomidine for local infiltration in anesthesia has become more common. However, studies on its effects on wound healing, as well as its anti-inflammatory and antimicrobial effects, are quite limited. In our study, we planned to investigate the effects of bupivacaine, dexmedetomidine, and their combined use on wound healing and antimicrobial properties in vitro. In this study, human fibroblast cells from the stock of the Animal Cell and Tissue Culture Laboratory, Faculty of Pharmacy, Tokat Gaziosmanpaşa University, specifically foreskin cell lines (ATCC CRL2522; BJ Skin Fibroblast Human Lot: 70027151), were used. Initially, the effects of the relevant drugs on cell viability at different doses were determined using MTT, and the identified effective concentrations of bupivacaine and dexmedetomidine were used to perform cell migration and wound healing analysis. After seeding BJ cells onto 6-well culture plates, a 'scratch' was made on the cells with a sterile P200 pipette tip once they reached 80-90% confluence, and the cells were incubated with growth media containing bupivacaine and dexmedetomidine at the identified effective doses. The cells were cultured for 48 hours and photographed every 24 hours under an inverted microscope. Wound healing rates were calculated spatially using Image J software. The expression levels of Tnf-alpha, interleukin 6, and interleukin 10 in nucleic acids obtained from the cell culture pellet were analyzed by RT-PCR, and hydroxyproline levels were assessed using ELISA. Disk diffusion and microdilution tests were used to determine antimicrobial activity. To identify the antimicrobial activities of the drugs using the disk diffusion method, P. aeruginosa, K. pneumoniae, S. aureus, and E. faecalis pathogenic bacterial strains, as well as C. albicans and C. utilis pathogenic fungal strains, were initially used. After incubation, the zone diameter of each disk was measured, and antimicrobial activities were quantitatively assessed. The microdilution method was used to calculate the dose that inhibits 50% of bacterial and fungal cells, referred to as MIC50, using GraphPad Prism 9.0.0 (GraphPad Software, San Diego, California USA) software. In our study, it was found that bupivacaine reduced wound closure rates, while dexmedetomidine increased wound closure rates. When both drugs were used together, the wound closure rate was similar to the control. The MIC value for both bupivacaine and dexmedetomidine in each experimental group was determined to be below 50%. As a result, we found that when applied locally, bupivacaine delayed wound healing, while dexmedetomidine generally had a positive effect on wound healing. When both drugs were used together, we believe that dexmedetomidine may reduce the negative effects of bupivacaine. We determined that neither drug showed significant antimicrobial activity against the microorganisms tested at the applied doses. However, we believe that further studies are needed to explore their antimicrobial properties. Keywords: Bupivacaine, Dexmedetomidine, Wound healing, Inflammation, In vitro, Scratch test, Antimicrobial