Background

The topography of biomaterials can influence a broad range of cellular responses, from differentiation to growth to immunoregulation. Even subtle variations in the roughness and geometry of surface patterns, at both the micro- and nanoscale, can result in significantly different cellular responses. At the molecular level, subtle variations in the physical features of the surface may not be so subtle, affecting the way cell-surface adhesion proteins like integrins attach to the surface and activate downstream mechanotransduction pathways.

For instance, different surface patterns can promote osteogenic differentiation in mesenchymal stem cells (MSCs) and preosteoblasts in vitro. Understanding this link could enable advancements in the rational design of osteogenic patterns, an application with great clinical relevance for orthopedic implants. Yet how different feature sizes affect mechanotransduction to promote osteogenic differentiation is unclear, highlighting the need to better characterize this link.

The approach

The authors in this study perform an in-depth investigation of how two different 3D-printed patterns affect the adhesion behavior of mouse preosteoblasts (MC3T3-E1 cells). The two patterns, P1000 and P500, consist of pillars of the same geometry, diameter and interspace but different pillar heights, 1000nm and 500nm, respectively. In their previous publication the authors show that these selected patterns differ in osteogenic potential: P1000 shows osteogenic potential whereas P500 shows no such potential (Nouri-Goushki et al. ACS Appl Mater Interfaces 2021).

The study employs the FluidFM system to assess adhesion behavior by quantifying adhesion force and strength of the preosteoblasts. The authors also use Atomic Force Microscopy (AFM), Scanning Electron Microscopy (SEM) and optical microscopy to assess other parameters like cell morphology and cytoskeleton organization, settling behavior on the two patterns and mechanical properties of the pillars.

FluidFM experiments after 24h of MC3T3-E1 preosteoblasts cell culture on flat glass control surfaces (a-b), the P500 patterns (c-d) and P1000 patterns (e-f), showing representative force-distance curves (a,c,e) and optical images of experiments with FluidFM probe (b,d,f). Figure taken from Angeloni et al., Small 2022 (License: CC-BY-4.0) 

Thanks to the unique properties of the FluidFM, which allows for strong and controlled but reversible immobilization of cells, the authors can quantify the properties of live cells on two different patterns and at different times, 4h and 24h of culture. For each experiment, adhesion behavior was compared to cells grown on a control flat glass surface. 

Key findings

To characterize cell adhesion behavior on the two patterns, the authors first assessed mechanical properties of the patterns using Atomic Force Microscopy (AFM) and Scanning Electron Microscopy (SEM). The P1000 and P500 patterns differed in pillar stiffness, surface shear modulus and roughness of the surface: P1000 had a lower pillar stiffness, lower surface shear modulus and a higher average surface roughness compared to P500. 

Using the FluidFM system they next assessed adhesion behavior after 4h and 24h of cell culture. Patterns induced differences in adhesion behavior only after 24h, a finding consistent with increased cell-surface adhesion. The initial integrin-mediated interactions present at 4h of culture mature into focal adhesions that in turn increase adhesion strength at 24h. 

Interestingly, after 24h of culture cells cultured on the P500 or the flat control surface still exhibited similar adhesion strength (21.5±12.5 kPa and 22±7.5 kPa). On the taller P1000 pillars, cells were smaller and exhibited higher adhesion strength (32±7 kPa). The authors attribute this finding to partial attachment of cells on the sidewalls of the taller pillars (without touching the flat surface between pillars), thus requiring larger normal forces for detachment. 

The higher strength of adhesion on taller pillars also resulted in larger normal forces required for cells to detach. Indeed, the authors observed that upon retraction of the FluidFM probe, cells cultured on either glass or the P500 pattern completely detached, whereas cells on the P1000 pattern were not entirely detached. Although quantitative data cannot be retrieved using FluidFM given that in-plane forces cannot be measured, these qualitative observations indicate differences in resistance to shear forces. Cells on the taller pillars also had thicker fibers and higher elastic modulus at the leading edge, which suggests enhanced anchorage and thus higher resistance to shear forces for cells on the P1000 pattern.

Conclusions

Based on their findings, the authors conclude that the higher cell adhesion strength induced by P1000 patterns may be required for osteogenic differentiation. This is consistent with previous observations where high adhesion to the substrate was associated with signaling pathways involved in osteogenic differentiation of MSCs (Arnsdorf et al. J Cell Sci 2009), although further in-depth analysis is required to assess these pathways and the causal link between such topographies and osteogenic potential.

The authors highlight that this is the first study to provide measurements of adhesion strength of cells interacting with submicron patterns.

Related content

• Application note: Single-cell adhesion measurement
  

• Mechanobiology Uses Cases with FluidFM
  

References

Angeloni et al. "Fluidic Force Microscopy and Atomic Force Microscopy Unveil New Insights into the Interactions of Preosteoblasts with 3D‐Printed Submicron Patterns." Small (2022) DOI: 10.1002/smll.202204662

Nouri-Goushki et al. "3D-Printed Submicron Patterns Reveal the Interrelation between Cell Adhesion, Cell Mechanics, and Osteogenesis" ACS Appl Mater Interfaces (2021) DOI: 10.1021/acsami.1c03687

Arnsdorf et al. "Mechanically induced osteogenic differentiation – the role of RhoA, ROCKII and cytoskeletal dynamics" J Cell Sci (2009) DOI: 10.1242/jcs.036293