Spermidine-Induced Attraction of Like-Charged Surfaces Is Correlated with the pH-Dependent Spermidine Charge: Force Spectroscopy Characterization.

The ubiquitous molecule spermidine is known for its pivotal roles in the contact mediation, fusion, and reorganization of biological membranes and DNA. In our model system, borosilicate beads were attached to atomic force microscopy cantilevers and used to probe mica surfaces to study the details of the spermidine-induced attractions. The negative surface charges of both materials were largely constant over the measured pH range of pH 7.8 to 12. The repulsion observed between the surfaces turned into attraction after the addition of spermidine. The attractive force was correlated with the degree of spermidine protonation, which changed from +3 to +1 over the measured pH range. The force was maximal at pH 7.8. To explain the observed pH and spermidine concentration dependence, two different theoretical approaches were used: a chemical model of the charge equilibrium of spermidine and Monte-Carlo simulations of the orientation of the rodlike spermidine molecules in the gap between the borosilicate and mica surfaces. Monte-Carlo simulations of the orientational ordering of the rodlike spermidine molecules suggested the induction of attractive interactions between the surfaces if the gap was bridged by the molecules. For larger gaps, the orientational distribution function of the spermidine molecules predicted a considerable degree of parallel attachment of the molecules to the surfaces, resulting in reduced effective surface charge densities of both surfaces, which reduced their electrostatic repulsion.

A comparative analysis of detachment forces and energies in initial and mature cell-material interaction.

Single cell force spectroscopy (SCFS) enables data on interaction forces to be acquired during the very early adhesion phase. However, SCFS detachment forces and energies have not been compared so far with the forces and energies after maturation of the cell-material contact on a single cell level and with comparable time resolution. We used FluidFM® to physically attach single cells to the cantilever by aspiration through a microfluidic channel, in order to achieve the higher forces required for detaching maturely adhering cells. Combining these two approaches allowed us to compare cell adhesion in the initial and maturation phases of adhesion for two exemplary cell-substrate combinations - L929 fibroblasts on fibronectin and MC3T3 osteoblasts on collagen type I. Uncoated glass substrates were used as a reference. For both cell lines, SCFS measurements after contact times of 5, 15 and 30 s revealed significantly higher maximum detachment forces (MDFs) and energies on glass compared to the protein-coated surfaces in the 0.5-4 nN (1-40 fJ) range. FluidFM® measurements after 1, 2 and 3 days of culture revealed a significant absolute increase in the MDFs and detachment energies for both cell lines on protein-coated substrates to values of about 600 nN and 10 pJ. On glass, the MDFs were similar for MC3T3 cells, while they were significantly lower for L929 cells. For both cell types, the differences in detachment energy were significant. These differences underline the importance of investigating early and mature adhesion states to obtain a holistic assessment of the cell-material interactions.

Surface Coatings Modulate the Differences in the Adhesion Forces of Eukaryotic and Prokaryotic Cells as Detected by Single Cell Force Microscopy.

Single cell force microscopy was used to investigate the maximum detachment force (MDF) of primary neuronal mouse cells (PNCs), osteoblastic cells (MC3T3), and prokaryotic cells (Staphylococcus capitis subsp. capitis) from different surfaces after contact times of 1 to 5 seconds. Positively charged silicon nitride surfaces were coated with positively charged polyethyleneimine (PEI) or poly-D-lysine. Laminin was used as the second coating. PEI induced MDFs of the order of 5 to 20 nN, slightly higher than silicon nitride did. Lower MDFs (1 to 5 nN) were detected on PEI/laminin with the lowest on PDL/laminin. To abstract from the individual cell properties, such as size, and to obtain cell type-specific MDFs, the MDFs of each cell on the different coatings were normalized to the silicon nitride reference for the longest contact time. The differences in MDF between prokaryotic and eukaryotic cells were generally of similar dimensions, except on PDL/laminin, which discriminated against the prokaryotic cells. We explain the lower MDFs on laminin by the spatial prevention of the electrostatic cell adhesion to the underlying polymers. However, PEI can form long flexible loops protruding from the surface-bound layer that may span the laminin layer and easily bind to cellular surfaces and the small prokaryotic cells. This was reflected in increased MDFs after two-second contact times on silicon nitride, whereas the two-second values were already observed after one second on PEI or PEI/laminin. We assume that the electrostatic charge interaction with the PEI loops is more important for the initial adhesion of the smaller prokaryotic cells than for eukaryotic cells.