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.

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.

The influence of carbon-encapsulated iron nanoparticles on elastic modulus of living human mesenchymal stem cells examined by atomic force microscopy.

Nanomaterials and nanoparticles are regarded as promising candidates for various biomedical applications due to their unique physicochemical properties. In this study, three types of carbon-encapsulated iron nanoparticles (CEINs) were synthesized and their impact on cellular changes was investigated by atomic force microscopy (AFM). The AFM experiment was additionally compared with conventional methods, such as colorimetric assay and other microscopic techniques. A significant difference of reduced Young's modulus of the cells was revealed, even at low concentration of nanoparticles in the culture medium. The AFM measurement proved to be a useful tool not only for visualization, but also for identification of local cellular changes at the nanoscale after exposure to carbon-encapsulated iron nanoparticles.

Discriminating the Independent Influence of Cell Adhesion and Spreading Area on Stem Cell Fate Determination Using Micropatterned Surfaces.

Adhesion and spreading are essential processes of anchorage dependent cells involved in regulation of cell functions. Cells interact with their extracellular matrix (ECM) resulting in different degree of adhesion and spreading. However, it is not clear whether cell adhesion or cell spreading is more important for cell functions. In this study, 10 types of isotropical micropatterns that were composed of 2 µm microdots were prepared to precisely control the adhesion area and spreading area of human mesenchymal stem cells (MSCs). The respective influence of adhesion and spreading areas on stem cell functions was investigated. Adhesion area showed more significant influences on the focal adhesion formation, binding of myosin to actin fibers, cytoskeletal organization, cellular Young's modulus, accumulation of YAP/TAZ in nuclei, osteogenic and adipogenic differentiation of MSCs than did the spreading area. The results indicated that adhesion area rather than spreading area played more important roles in regulating cell functions. This study should provide new insight of the influence of cell adhesion and spreading on cell functions and inspire the design of biomaterials to process in an effective manner for manipulation of cell functions.

Regulating the stemness of mesenchymal stem cells by tuning micropattern features.

There is increasing evidence that microstructures play an important role in the maintenance of the multipotency of stem cells. However, it is not clear how micropatterns affect the stemness of stem cells. We prepared micropatterns of different sizes, shapes and aspect ratios and used them for the culture of mesenchymal stem cells (MSCs) derived from human bone marrow to investigate their influence on the stemness of MSCs at the single cell level. The percentage of cells positively stained by stem cell markers decreased with increasing spreading area and aspect ratio. However, the cellular geometry controlled by the geometrical micropatterns had no significant influence on the expression of stem cell markers. The change in the stemness of stem cells was accompanied by changes in the nuclear activity and cytoskeleton. The nuclear activity increased with increasing spreading area and aspect ratio. The actin filament structure was significantly influenced by the spreading area and aspect ratio. The cells became stiffer when they had sufficient area to spread into or when they were elongated. These results suggest that controlling the cell morphology by micropatterns may be useful in varying the stemness of MSCs. This study will contribute to the design of materials for maintaining the multipotency of stem cells, enhancing their clinical application and helping to reveal the processes taking place under conditions of stem cell quiescence in vivo.

Cellular effects of magnetic nanoparticles explored by atomic force microscopy.

The investigation of subtle change of cells exposed to nanomaterials is extremely essential but also challenging for nanomaterial-based biological applications. In this study, atomic force microscopy (AFM) was employed to investigate the effects of iron-iron oxide core-shell magnetic nanoparticles on the mechanical properties of bovine articular chondrocytes (BACs). After being exposed to the nanoparticles even at a high nanoparticle-concentration (50 µg mL(-1)), no obvious difference was observed by using conventional methods, including the WST-1 assay and live/dead staining. However a significant difference of Young's modulus of the cells was detected by AFM even when the concentration of nanoparticles applied in the cell culture medium was low (10 µg mL(-1)). The difference of cellular Young's modulus increased with the increase of nanoparticle concentration. AFM was demonstrated to be a useful tool to identify the subtle change of cells when they were exposed to nanomaterials even at a low concentration.

Variation of mechanical property of single-walled carbon nanotubes-treated cells explored by atomic force microscopy.

With a range of biological properties, single-walled carbon nanotubes (SWCNTs) are a promising material for nanobiotechnology. Concerns about their potential effect on human health have led to the interest in understanding the interaction between SWCNTs and cells. There are many reports showing the potential cellular effects of SWCNTs but this issue is quite controversially discussed in the literature. In this study, we used conventional biological evaluation methods and atomic force microscopy (AFM) to compare the effects of SWCNTs on three different cell types: bovine articular chondrocytes, human bone marrow-derived mesenchymal stem cells and HeLa cells. No obvious effects of SWCNTs on cell morphology and viability were observed during 3 days in vitro culture. However, SWCNTs significantly increased the Young's modulus of all the three types of cells. The effect of SWCNTs on Young's modulus was in an increasing order of Hela cells < chondrocytes < mesenchymal stem cells. AFM was shown to be a useful tool for investigation of the effect of nanomaterials on mechanical property of cells.

Effect of single-wall carbon nanotubes on mechanical property of chondrocytes.

It is important to elucidate the effects of carbon nanotubes on cell functions for their biomedical applications. In this study, the effect of single-walled carbon nanotubes (SWCNTs) on the mechanical property of chondrocytes was investigated by atomic force microscopy. Chondrocytes were cultured in medium containing SWCNTs and showed an increase uptake of SWCNTs with culture time. The mechanical property of chondrocytes cultured with or without SWCNTs was measured at an indentation depth of 200 nm and 500 nm. The chondrocytes cultured with SWCNTs showed higher Young's modulus than that of cells cultured without SWCNTs at both indentation depths. The increase became significant after culture for more than 3 hours. Indentation at 500 nm depth magnified the change of Young's modulus compared to that monitored at 200 nm indentation depth. The results indicated uptake of SWCNTs increased the Young's modulus of chondrocytes.

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.

The combined influence of substrate elasticity and surface-grafted molecules on the ex vivo expansion of hematopoietic stem and progenitor cells.

Umbilical cord blood (UCB) is an attractive source of hematopoietic stem and progenitor cells (HSPCs) for transplantation. However, the low number of HSPCs from a single UCB donor limits the direct transplantation of UCB to patients. Because little is known about the effects of the physical microenvironment on HSPC expansion, we investigated the ex vivo expansion of HSPCs cultured on biomaterials with different elasticities and grafted with different nanosegments. Polyvinylalcohol-co-itaconic acid (PVA-IA)-coated dishes with different stiffnesses ranging from a 3.7 kPa to 30.4 kPa storage modulus were used. Fibronectin or an oligopeptide (CS1, EILDVPST) was grafted onto the PVA-IA substrates. High ex vivo fold expansion of HSPCs was observed in the PVA-IA dishes grafted with fibronectin or CS1, which displayed an intermediate stiffness ranging from 12.2 kPa to 30.4 kPa. The fold expansion was more than 1.4 times higher than that cultured in tissue culture polystyrene dishes (TCPS, 12 GPa). Furthermore, HSPCs cultured in fibronectin or CS1-grafted PVA-IA-coated dishes with a stiffness of 12.2-30.4 kPa generated more pluripotent colony-forming units (CFU-GM and CFU-GEMM) than those in TCPS dishes. This result indicates that both the physical and biological properties of biomaterials affect the ex vivo expansion of HSPCs.