The Influence of Donor Age and Cholesterol Inhibition on Fibroblast Mechanical Properties and Susceptibility to Bacterial Infection

Cell mechanics depend upon cell membrane mechanics (which, in part, depends upon cholesterol content) and the rigidity and organization of the cytoskeleton. The latter was shown to depend on the properties of the substrate upon which cells are cultured, where tensegrity models were used to explain reorientation of the scaffold. With aging, measurements of the viscoelastic properties of cells were shown to stiffen, but these measurements were performed on individual cells without mechanical cues from surrounding tissue or the substrate. We report on experiments performed on dermal fibroblasts, which were obtained from female donors: “young” (ages 28 and 29 years) and “old” (ages 69 and 71 years), where we applied the technique of scanning force modulation microscopy (SFMM) which allows for measurements of cell modulus that account for the underlying substrate mechanics. The results represent an average of n=9 measurements.<br/><br/>Cells were cultured on polybutadiene thin films whose moduli were varied (ratio of 3:1) by adjusting the film thickness without additional chemical additives. On both substrates the moduli of the cells became significantly harder with age, consistently 40±1.6%, in agreement with previous work. For both young and old cells the cell modulus responded to changes in the substrate, where the moduli between cells plated on the hard and soft substrates differed significantly, 50±2% and 60±2.4% for the old and young cells, respectively. These results indicate that even though cells become harder as they age their ability to adjust to mechanical changes in their environment is not diminished. We were also able to image the actin filaments and we found that the moduli of the filaments in the older cells were consistently 20±0.8% higher than those of the younger cells independent of substrate.<br/><br/>Cholesterol is the most dominant sterol found in mammalian tissue and is a major structural component of the cell membrane. We investigated the impact of cholesterol on the moduli of dermal fibroblasts. To prevent uptake of cholesterol, we cultured cells in lipid-free media. We then added AY9944, a known disruptor of cholesterol synthesis, which has been shown to cause a shift to 7-dehydrocholesterol (7DHC), a precursor to cholesterol, as the major sterol. In the lipid-free media, we observed that while the proliferation of the cells was reduced, the moduli of the cells increased significantly by 100±4% and 66±2.64% for the young and old cells, respectively, relative to the cultures in standard media. Introduction of AY9944 at a concentration of 50nM decreased the modulus in the lipid-free media by 25±1% for both ages, which represented an overall increase relative to the control media of only 20±0.8% for young and 60±2.4% for old. This confirmed prior observation of increased membrane ductility.<br/><br/>Finally, it has been shown that cholesterol increases the susceptibility of cells to infection by Staphylococcus aureus bacteria. Therefore, we exposed cells to S. aureus at an MOI of 1000 for 90 minutes. The infection rate of old cells appeared to be approximately ⅓ that of younger cells, and the infection rate overall was nearly zero when AY9944 was added. These results illustrate that changes in the sterol composition of the membrane can have a significant impact on dermal fibroblasts. Hence, further studies are in progress to quantify the cholesterol to 7DHC quantities, as well as the impact on fibroblast functions, which are affected by aging such as collagen contraction, deposition, and turnover.