Effects of epinephrine, lidocaine, and prilocaine on viability and differentiation capacity of human adipose stem cells.

INTRODUCTION: Local anesthetics (LAs) are routinely administered in plastic and reconstructive surgery, e.g., as tumescent anesthesia adjunct in liposuction. Historically, these substances were assumed to act cytotoxically. Thus, the application of LA was avoided when handling adipose stem cells (ASCs). We recently determined that most LAs are not cytotoxic when ASCs are exposed to concentrations used for tumescent liposuction. However, there is limited information when combining LA with epinephrine and about the effects of prilocaine on ASCs. METHODS: We analyzed the effects of prilocaine or lidocaine in co-exposure with epinephrine on the viability of primary human ASCs, i.e., proliferation, metabolic activity, and cytotoxicity, using crystal violet-staining, PrestoBlue®-, and WST-1 assay. We quantified the impact of short-term incubation of lidocaine and epinephrine on the differentiation of ASCs into the adipogenic, chondrogenic, and osteogenic lineage. RESULTS: After 2 h, prilocaine (10 mM) significantly reduced metabolic activity and cell numbers, whereas lidocaine only inhibited metabolic activity. After 6 h, prilocaine (10 mM) and lidocaine significantly decreased metabolic activity as well as cell numbers. The application of high concentrations of epinephrine did not affect cell numbers but diminished metabolic activity. Combining lidocaine with epinephrine had no additional cytotoxic effect. Differentiation into the chondrogenic lineage was significantly inhibited by epinephrine. CONCLUSIONS: Deducing from our data, neither lidocaine combined with epinephrine nor prilocaine has a cytotoxic impact on ASCs in vitro at concentrations equivalent to those in tumescent anesthesia and has no long-lasting effect on the differentiation capacity of ASCs into the osteogenic and adipogenic lineage.

Comprehensive Analysis of Local Anesthetics Affecting Adipose Stem Cells In Vitro.

BACKGROUND: Adipose stem cells (ASCs) hold a great regenerative capacity because of their differentiation capability and their secretory activity. Thus, ASC survival is of great significance during perioperative harvesting. Various local anesthetics are commonly applied during fat grafting procedures. These substances are known to impair cellular viability, which would affect graft survival and final outcomes, but the exact extent of their impact on ASC biology is unknown. METHODS: The authors analyzed the short- and long-term effects of lidocaine, mepivacaine, ropivacaine, and bupivacaine at increasing concentrations (0.1 to 10 mM) on primary human ASC proliferation and metabolic activity. Trilinear differentiation was assessed by oil red O stain (adipogenesis), safranin O (chondrogenesis), and cresolphthalein (osteogenesis) labeling. In supernatants, cytokine [interleukin (IL)-6/IL-8, vascular endothelial growth factor, hepatocyte growth factor] secretion was analyzed by enzyme-linked immunosorbent assay. RESULTS: Bupivacaine at greater than 100 µM demonstrated the strongest anti proliferative effects, whereas lidocaine and ropivacaine did not affect cell numbers. Mepivacaine evoked reciprocal results regarding cell count at greater than 1 mM. Each compound impaired trilinear differentiation. Secretion of hepatocyte growth factor and IL-8 was reduced significantly by local anesthetic exposure; levels were restored after substances were washed out. CONCLUSIONS: In vitro data show that lidocaine, mepivacaine, and ropivacaine could be applied at concentrations of 1 to 10 mM without affecting ASC survival. In contrast, bupivacaine at concentrations greater than 100 µM should be administered with great caution. The differentiation of ASCs and the ASC's secretome might already be decreased by each local anesthetic at 1 mM. CLINICAL RELEVANCE STATEMENT: The authors' experimental data can be of great significance to the clinical practice, as local anesthetics are routinely administered during liposuction as a tumescent anesthesia adjunct. CLINICAL QUESTION/LEVEL OF EVIDENCE: Therapeutic, V.

Genetic deletion of the cannabinoid receptors CB1 and CB2 enhances inflammation with diverging effects on skin wound healing in mice.

AIMS: Inflammation during wound healing is both essential and critical for restoring tissue integrity. Participating cells secrete soluble factors to regulate the inflammatory phase and to induce the adjacent regenerative processes. If pro-inflammatory signals are overexpressed, the wound stagnates in the inflammatory phase, which decelerates regular wound healing. The endocannabinoid system is ascribed great significance in maintenance of tissue homeostasis. It mediates several effects through the cannabinoid receptors CB1 and CB2. MAIN METHODS: In order to clarify the role of these receptors in wound healing, excisional wounds were created on wildtype and CB1 and CB2 knockout mice. The wound closure was analyzed over a period of 14 days, and cytokine concentrations of tissue homogenisates were measured by ELISA. MSCs were isolated from the animals' subcutaneous adipose tissue and analyzed for viability and differentiation capacity, in vitro. KEY FINDINGS: Deletion of CB2 increased Interleukin (IL)-6 and tumor necrosis factor (TNF)-α but did not affect tissue regeneration. In CB1-deficient animals, wound closure was delayed during early phases of healing, which was accompanied by increased concentrations of monocyte chemoattractant protein (MCP)-1 and TNF-α. CB1 and CB2 knockout MSCs presented altered viability and differentiation capacity compared to wildtype MSCs. The CB1-deficient MSCs released high levels of MCP-1 upon stimulation with TNF-α and IL-1ß. SIGNIFICANCE: The data indicate that both cannabinoid receptors regulate inflammation, and this study emphasizes the important role of CB1 in wound repair. Furthermore, our findings suggest that the secretome of CB1-deficient MSCs may contribute to the wound healing delay, in vivo.