研究目的
Investigating the differences in adhesion forces between eukaryotic and prokaryotic cells on various surface coatings using single cell force microscopy to understand initial adhesion mechanisms and potential for discriminating against bacterial adhesion.
研究成果
The research concludes that initial cell adhesion is dominated by electrostatic interactions, with surface coatings modulating adhesion forces. PEI coatings induce higher MDFs due to flexible loops that can span laminin layers, particularly benefiting prokaryotic cells. Normalization methods (relative and specific MDFs) effectively reduce variability and highlight coating effects. PDL/laminin discriminates against prokaryotic cells, suggesting potential for designing surfaces that favor eukaryotic over prokaryotic adhesion. The findings support the use of specific MDFs for comparing cell adhesion in SCFM studies and indicate that silicon nitride and PEI have similar adhesion-enhancing properties, with laminin acting as a geometric spacer that reduces electrostatic interactions.
研究不足
The study is limited to initial adhesion forces (up to 5 seconds), so longer-term adhesion mechanisms involving specific integrin interactions are not captured. SCFM measurements have high variability due to biological differences between individual cells, requiring large sample sizes. The method may not fully predict proliferation outcomes, as other mechanisms become effective after the initial phase. Additionally, the use of poly-dopamine for cell attachment, while improving comparability, may introduce unknown effects on adhesion.
1：Experimental Design and Method Selection:
The study used single cell force microscopy (SCFM) to measure maximum detachment forces (MDFs) of cells on different surface coatings. The experimental design involved comparing adhesion forces of primary neuronal mouse cells (PNCs), osteoblast-like MC3T3 cells, and Staphylococcus capitis subsp. capitis (Scc) cells on silicon nitride, PEI, PEI/laminin, and PDL/laminin coatings. The rationale was to understand how surface coatings affect initial cell adhesion, with a focus on electrostatic interactions. Methods included SCFM force-distance curve measurements with controlled contact times (1, 2, 5 seconds), normalization of MDFs to a reference surface to reduce variability, and statistical analysis using two-way ANOVA.
2：Sample Selection and Data Sources:
Cell samples included PNCs from mouse embryos, MC3T3 cells from DSMZ, and Scc bacteria from ATCC. Selection criteria involved using cells relevant to implant integration (eukaryotic) and biofilm formation (prokaryotic). Data were sourced from SCFM measurements on coated surfaces, with each cell probed on multiple surfaces to minimize inter-cell variability.
3：List of Experimental Equipment and Materials:
Equipment included SCFM setup (CellHesion/Nanowizard II AFM, JPK Instruments), microscope (Axio Observer A1, Zeiss), sputter coater (PPS-90UV, Von Ardenne), plasma cleaner (Zepto LF, Diener electronic), and Petri dish heater (PetriDishHeater, JPK). Materials included glass cover slips, silicon nitride, PEI, PDL, laminin, poly-dopamine, cell culture media, and buffers. Specific models and brands are detailed in the products section.
4：Experimental Procedures and Operational Workflow:
Surface preparation involved sputter-coating glass cover slips with silicon nitride, then applying polymer coatings (PEI or PDL) followed by laminin where specified. Cantilevers were functionalized with poly-dopamine for cell attachment. SCFM measurements involved calibrating cantilevers, attaching single cells, and recording force-distance curves on different surfaces with varying contact times in a randomized grid pattern to avoid surface damage. Data were collected for multiple cells per type.
5：Data Analysis Methods:
MDFs were normalized to reference surfaces (silicon nitride with 5-second contact time) to derive relative and specific MDFs, reducing effects of individual cell properties. Statistical analysis used two-way ANOVA to assess significance of differences between coatings and cell types, with p-values indicating significance levels.
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