The work presented here was carried out with the collaboration of all the authors. Authors GB and BT defined the research theme and designed the methods and experiments, analyzed the data, interpreted the results and wrote the paper. Author PA was involved in the writing process of the manuscript, author RV co-designed the experiments, discussed the analyses, interpretation, and presentation of data. All authors have contributed to, seen and approved the manuscript.

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An interesting effect of gene induction by administration of NAC and amino acids in vitro was demonstrated. Upregulation of elastin-, hyaluronic acid-, and GDF6-encoding genes supports the evidence of clinical improvement induced by NAC biostimulation in the prevention and correction of skin aging.

IntroductionAesthetic medicine uses many injective techniques; biostimulation (BS) and biorevitalization (BR) are among these. The term BS indicates stimulation of the anabolic functions of dermal fibroblasts such as replication, protein synthesis and production of extracellular matrix components (ECM). BR instead uses the same injective technique but different medical devices. It is a direct supplementation of hyaluronic acid (HA) alone or added to other molecules (i.e. vitamins). This study discusses BS and BR and their effects on human skin fibroblasts. Materials and methodsIn order to verify the different metabolic effects of BS and BR fibroblast cell cultures, RNA extraction, cDNA synthesis and PCR were performed. Results BS and BR produce different metabolic effects in fibroblast cell cultures, thus showing that they are different therapies. For example, neutrophil elastine is activated by BS and to a lesser extent by BR, whereas hyaluronan synthase 1 is activated to a higher extent by BR using the medical device with the lowest HA content. Neutrophil elastase, responsible for the degradation of one of the fibrillar components of ECM, is activated to a lesser extent by BS. ConclusionFurther experiments using more time points (i.e. not only 24 h of cell cultures but also 12, 48 and 72 h) are necessary to give additional insights on fibroblast behaviour after BS and BR. A better comprehension of fibroblast biology will result in a proper clinical application of BS and BR.

Reactive oxygen species (ROS) are chemically reactive molecules with impaired electrons that make them unstable and able to react easily with a great variety of molecules. The main targets of ROS are DNA, proteins, and membrane phospholipids. In the skin, ROS are able to affect the production of collagen and elastin, the main components of the extracellular matrix (ECM). This action contributes to the skin's aging. N-Acetylcysteine (NAC) is an acetylated cysteine residue with excellent anti-oxidant activity that boosts glutathione (GSH) levels. This study evaluates the effect of a solution of NAC and amino acids, which is used in aesthetic medicine as an intra-dermal injective treatment, on fibroblast behavior. To this aim, the expression levels of some ECM-related genes (HAS1, HYAL1 ELN, ELANE, MMP2, MMP3, MMP13, COL1A1, COL3A1) were analyzed on cultured dermal fibroblasts using real-time reverse transcription polymerase chain reaction (RT-PCR). All but two collagen genes were up-regulated after 24 hr of treatment.