Determining the effectiveness of vitamin C in skin care by atomic force microscope.

This article presents the results of experiments, which examine cell mechanisms with the goal of confirming the effective action of the active ingredients used in anti-aging cosmetics. Skin stiffness measurements with the use of an atomic force microscope on two forms of vitamin C (ascorbyl tetraisopalmitate and l-ascorbic acid) have been presented. The estimated Young's modulus for three types of cells (a control as well as cells treated with two forms of vitamin C) has shown significant differences in the stiffness of the tested cells which was confirmed in the histological staining experiment. The presented results indicate the dependence between collagen synthesis and the stiffness of cells treated with two forms of vitamin C.

New lupeol esters as active substances in the treatment of skin damage.

The purpose of the research was to obtain new derivatives of natural triterpene lupeol and to evaluate their potential as active substances in the treatment of skin damage. Four new lupeol esters (propionate, succinate, isonicotinate and acetylsalicylate) and lupeol acetate were obtained using an eco-friendly synthesis method. In the esterification process, the commonly used hazardous reagents in this type of synthesis were replaced by safe ones. This unconventional, eco-friendly, method is particularly important because the compounds obtained are potentially active substances in skin care formulations. Even trace amounts of hazardous reagents can have a toxic effect on damaged or irritated tissues. The molecular structure of the esters were confirmed by 1H NMR, 13C NMR and IR spectroscopy methods. Their crystal structures were determined using XRD method. To complete the analysis of their characteristics, physicochemical properties (melting point, lipophilicity, water solubility) and biological activity of the lupeol derivatives were studied. Results of an irritant potential test, carried out on Reconstructed Human Epidermis (RHE), confirmed that the synthesized lupeol derivatives are not cytotoxic and they stimulate a process of human cell proliferation. The safety of use for tested compounds was determined in a cell viability test (cytotoxicity detection kit based on the measurement of lactate dehydrogenase activity) for keratinocytes and fibroblasts. The results obtained showed that the modification of lupeol structure improve its bioavailability and activity. All of the esters penetrate the stratum corneum and the upper layers of the dermis better than the maternal lupeol. Lupeol isonicotinate, acetate and propionate were the most effective compounds in a stimulation of the human skin cell proliferation process. This combination resulted in an increase in the concentration of cells of more than 30% in comparison to control samples. The results indicate that the chemical modification of lupeol allows to obtain promising active substances for treatment of skin damage, including thermal, chemical and radiation burns.

The Effect of Anti-aging Peptides on Mechanical and Biological Properties of HaCaT Keratinocytes.

Atomic force microscopy (AFM) and fluorescence microscopy was applied to determine the influence of the anti-aging peptides on the morphology and the mechanical properties of keratinocytes. Immortalized human keratinocytes (HaCaT) were treated with two anti-aging bioactive peptides: Acetyl Tetrapeptide-2 and Acetyl Hexapeptide-50 (Lipotec). The AFM measurement of the keratinocyte stiffness were carried after 48 h exposure at an indentation depth of 200 nm. AFM analysis showed increase of the cell stiffness for cells treated with Acetyl Tetrapeptide-2 (P1) in concentration range. Acetyl Hexapeptide-50 (P2) at concentration of 0.05 µg/ml also increased the stiffness of HaCaT cells but at higher concentrations 0.5 and 5 µg/ml cell stiffness was lower as compared to untreated control. Fluorescence microscopy revealed remodeling of actin filaments dependent on the concentration of P2 peptide. The mechanical response of HaCaT cells treated with P2 peptide corresponds to change of transcription level of ACTN1 and SOD2 which activity was expected to be modulated by P2 treatment.

A study of the activity and effectiveness of recombinant fibroblast growth factor (Q40P/S47I/H93G rFGF-1) in anti-aging treatment.

INTRODUCTION: Fibroblast growth factor 1 (FGF-1) is a powerful mitogen involved in the stimulation of DNA synthesis and the proliferation of a wide variety of cell types. Fibroblast growth factor 1 was genetically modified to improve its thermal stability and resistance to protease degradation without losing its biological activity. AIM: To study the impact of Q40P/S47I/H93G rFGF-1 on skin cells, its penetration through the skin and the evaluation of the rFGF-1-cosmetic product properties. MATERIAL AND METHODS: In vitro studies included the examination of primary fibroblast and keratinocyte viability after the incubation with rFGF-1. The penetration abilities of rFGF-1 in various formulations and carrier systems were examined ex vivo by the Raman spectroscopy. In vivo studies - HF Ultrasound and 3D Imaging System - were used to evaluate the anti-aging properties of creams containing rFGF-1. RESULTS: In vitro studies demonstrated that rFGF-1 strongly enhanced the viability of the treated cells. The Raman Spectroscopy analysis indicated that rFGF-1 encapsulated in lipid spheres penetrate through the stratum corneum to the depth of 60 µm, and added to the o/w formulation - could penetrate to a depth of 90 µm. The results obtained from Primos revealed the reduction of the volume and the depth of the wrinkles. Changes in the skin structure in the analyzed areas were evaluated by HF Ultrasonography. CONCLUSIONS: Recombinant FGF-1 strongly stimulated fibroblast and keratinocyte proliferation. However, the transition of this protein through the SC required an appropriate carrier system - lipid spheres. All tests - in vitro, ex vivo and in vivo - have proved that rFGF-1 is a substance with a potentially wide spectrum of use.

Age-Related Changes in the Mechanical Properties of Human Fibroblasts and Its Prospective Reversal After Anti-Wrinkle Tripeptide Treatment.

One of an essential characteristic of human skin are time dependent mechanical properties. Here, we demonstrate that stiffness of human dermal fibroblast correlates with age and it can be restored after anti-wrinkle tripeptide treatment. The stiffness of human fibroblasts isolated from donors of 30-, 40- and 60 years old were examined. Additionally the effect of anti- wrinkle tripeptide of latter cells was investigated. The atomic force microscopy measurements were performed on untreated fibroblast as well as on treated with the peptide. The Young's modulus for two indentation depths 200 and 600 nm of each cell type was determined. The Young's modulus increases with age of the cells. The highest values of Young's modulus were obtained for fibroblasts collected from 60 years old donors, for indentation depth of ~200 nm. For larger indentation depth of 600 nm there are no significant differences in stiffness between cells. Fibroblasts treated with the anti-wrinkle tripeptide exhibit lower Young's modulus. The cells derived from 40- and 60-years old donors restored stiffness characteristic to the level of 30 years old subjects. The results show correlation between stiffness and age of the human fibroblast as well as impact of anti-wrinkle tripeptide on the mechanical properties of skin cells.

Imaging of the skin and subcutaneous tissue using classical and high-frequency ultrasonographies in anti-cellulite therapy.

BACKGROUND: The development of ultrasonography allowed for skin imaging used in dermatology and esthetic medicine. By means of classic and high-frequency ultrasonographies, changes within the dermis and subcutaneous tissue can be presented. OBJECTIVE: The aim of this study was to show the possibilities of applying classic and high-frequency ultrasonographies in esthetic dermatology based on monitoring various types of anti-cellulite therapies. METHODS: Sixty-one women with cellulite were assigned to two smaller groups. One group was using anti-cellulite cream and the second group was a placebo group. The ultrasound examination was carried out before the initiation and after the completion of the treatment and evaluated epidermal echoes, the thickness of the subcutaneous tissue and the dermis, dermis echogenicity, the length and surface area of the subcutaneous tissue fascicles growing into the dermis, and the presence or absence of edemas. RESULTS: After the completion of the treatment, a statistically significant difference was observed. The most useful parameters were as follows: the thickness of the subcutaneous tissue, echogenicity, the surface area and length of the subcutaneous tissue, as well as the presence of edemas. The discussed changes were not observed in the placebo group. CONCLUSION: Classic and high-frequency ultrasonographies are useful methods for monitoring anti-cellulite therapies.