To the moon and back: space dust and our skin

One of the many things discovered during the Apollo space missions was that exposure to lunar dust can cause skin irritation – but can space dust really tell us anything about keratinocytes? The skin serves as the main protective barrier shielding against harmful substances or pathogenic infection, and it protects the body from water loss. The outer-most layers of human skin are composed of keratinocytes, a highly regenerative cell type that undergoes rapid proliferation and terminal differentiation. Thus, keratinocytes build up the epidermal layers of human skin that are supported by dermis, a complex tissue containing fibroblasts as the main constituent. Environmental stressors are many fold but include dust particles as common contaminants of air or water. In a recent study, conducted by a research team at Jacobs University Bremen, skin cell cultures were used to evaluate the impact of dust exposure on mammalian skin1.

[caption id="attachment_25598" align="alignright" width="200" caption="Figure 1: HaCaT keratinocytes cultured on a 96-well E-plate and exposed to different concentrations of dust particles dispersed in culture medium; dark golden colour indicates exposure to dense dust particle-containing solutions"][/caption]

In this study, keratinocyte and fibroblast cell lines were chosen as representatives of cells in epidermal and dermal layers of the skin. The cells were cultured in specific micro-titre plates equipped with microelectronic sensors at the bottom of each well (Figure 1). Cultures were then exposed to different concentrations of particles ranging in size from 13µm to 1mm (Figure 2). Impedance was measured over a time period of four days after dust treatment. Simulants of lunar and Martian dusts were evaluated as motivated by previous observations of dermal irritations caused by lunar dust exposure of astronauts in spaceships during the Apollo missions. Elements present in lunar dust simulant are SiO2, TiO2, Al2O3, Fe2O3, FeO, MgO, CaO and Na2O; Martian dust simulant is comprised of a volcanic ash containing the same elements as the lunar dust simulant but in different concentrations.

Determinations of cell indices were conducted by impedance measurements. Steep increases of cell indices were indicative of rapid proliferation rates. Upon reaching confluence, as obvious from plateaus in cell indices, the keratinocyte cultures were exposed to several mg per cm2 of dust particles for up to four days (Figure 2). Impedance measurements of fibroblast cultures were characterised by rapid growth phases followed by increasingly longer plateau phases (Figure 3). Such characteristics reflected synchronised cell divisions with decreasing cell proliferation rates in long-term cultures reaching high cell densities.

[caption id="attachment_25601" align="alignleft" width="200" caption="Figure 2: HaCaT keratinocytes cultured on E-plates and after treatment with lunar (left) or Martian dust simulants (right) in culture medium"][/caption]

A rapid decrease in cell numbers was observed upon exposure of fibroblast cultures to lunar or Martian dust simulants while keratinocyte cultures resisted dust treatment without major changes in impedance values. This is an interesting finding because it implies that keratinocytes form stable and difficult to disturb cell layers when cultured on the so-called E-plates of the xCELLigence system that was used in these investigations. Fibroblast cultures, in stark contrast, were affected more severe by the exposure to dust particles since cell indices dropped rapidly upon treatment with similar concentrations of dust particle suspensions. Decreasing cell indices reflected cell death.

Proper controls are required for such measurements of cell viability and proliferation rates. In this regard, it is interesting to note that the addition of dust particles to wells without cells revealed only marginal impedance values that remained at hardly detectable levels over the entire time interval of up to several days.

[caption id="attachment_25605" align="alignright" width="300" caption="Figure 3: Determination of cell indices by impedance measurements of CHO-K1 fibroblast cultures (red and electric blue) and HaCaT keratinocytes (black and dark blue) before and after exposure to lunar or Martian dust simulants. Controls were performed with lunar (green) and Martian dust dispersions (chartreuse) in culture medium without cells"][/caption]

In addition to the impedance measurements, phalloidin staining of the actin cytoskeleton was employed to investigate structural integrity of individual cells. Inspection of the cytoskeleton demonstrated more severe impacts of dust simulants on fibroblast than on keratinocyte cultures. Shortly after dust exposure, fibroblasts lost their elongated, spindle-like shape and rounded up before most cells got lost due to cell death induced by the particulate stressors. Keratinocytes on the other hand maintained a cobble-stone appearance that was indicative of intact junctions between the well polarised cells for up to three day of dust treatment.

As an additional indicator of cellular integrity, the ability of dust-treated cells to incorporate propidium iodide was determined. Accumulation of propidium iodide within cells is only possible if the plasma membrane is leaky. Similar to the results previously achieved with impedance measurements and in line with the structural changes observed in the actin system, membranes of cells in dust-exposed fibroblast cultures became leaky within a few hours, whereas keratinocytes maintained their full integrity for up to two days.

Simulants of lunar and Martian dusts were evaluated as motivated by previous observations of dermal irritations caused by lunar dust exposure of astronauts in spaceships during the Apollo missions

Moreover, regeneration of keratinocyte cultures from scratch-wounding was analysed under conditions of lunar dust stimulant exposure which clearly decreased the cells’ abilities to perform proper wound healing. This conclusion was reached from the observation that the cellular ability to close a scratched area in the cultures was impaired.

[caption id="attachment_25609" align="alignleft" width="250" caption="Figure 4: Schematic representation of the differential impacts of dust particle exposure on keratinocyte (A) or fibroblast cultures (B)"][/caption]

Since both, cell migration and proliferation are important for proper wound closure the researchers further investigated the effects of lunar dust simulants on dividing cells in keratinocyte cultures at sub-confluent conditions. The proliferation rates of keratinocytes were analysed by staining for the proliferation marker Ki67. Total cell numbers were determined by staining of cell nuclei. Keratinocyte cultures exposed to lunar dust simulants were characterised by decreased proliferation rates in comparison to non-treated control cultures in such assays. These data clearly demonstrated a negative impact of lunar dust simulants on spontaneous proliferation of keratinocytes in submerse cultures.

The observed contrasting effects of dust particles on mammalian cell cultures can be explained by the notion of keratinocytes forming tight mono-layers in which the cells are well connected with each other. Thereby, they built up a sheet of cells that resists environmental stressors like dust particle exposure much better than the more readily exposed fibroblasts growing as single cells (Figure 4). In other words, intact human skin protects us better against environmental stressors than e.g. wounded skin in which dermal layers become exposed.

The study by the team of cell biologists at Jacobs University has demonstrated that impedance measurements on skin cell cultures grown on xCELLigence E-plates are suited for determination of cell proliferation rates and for constant monitoring of cytotoxic effects exerted by exposure of the cell cultures to dust particles. Further studies are intended to investigate the effects of particulate and other stressors in cell-based assays that allow assessing environmental impacts constantly in highly reproducible settings that are easily adaptable to variable experimental conditions or to other cell types.

The Authors Klaudia Brix and Maren Rehders School of Engineering and Science, Jacobs University Bremen,

Contact e: k.brix@jacobs-university.de e: m.rehders@jacobs-university.de

References

1. Rehders et al., Advances in Space Research, doi:10.1016/j.asr.2010.11.033