Polydimethylsiloxane (PDMS) is a bioinert synthetic polymer with tunable elastic properties that is commonly used as a cell culture substrate. Although plasma treatments are widely used to biofunctionalize otherwise hydrophobic PDMS surfaces, plasma altered surface mechanical properties and implications for cell-substrate mechanical interactions are poorly understood. We performed a multi-scale mechanical characterization of PDMS following plasma treatment: spherical indentation tests were performed with a universal testing machine (indenter diameter, d ¼ 4.75 mm) and atomic force microscopy (AFM) with round tips of 2 different diameters (d ¼ 2 mm, d ¼ 20 mm). Results indicated substantial surface stiffening at indentation depths up to 1 micron, with exponentially decreasing effects to depths of 1 mm. AFM indentation results were analyzed using a finite element (FE) based optimization to determine the substrate material properties, and thus separate the confounding influence of the underlying substrate on surface indentation experiments. We found that a two-layer material model composed of a thin, stiff plasma-oxidized layer (296 nm and 3.66 MPa, respectively) superimposed on a thick layer of bulk polymer (elastic modulus of 10.5 kPa) was able to robustly fit the experimental data. We then investigated the repercussions of the biopolymer surface modifications on cell mechanics, using an inverse finite element model to interpret cell-matrix force exchange. Estimates of cell elastic modulus neglecting the mechanical effects of plasma treatment were more than an order of magnitude lower than estimates accounting for the surface layer (9.6 AE 4.2 kPa vs. 124 AE 55 kPa, respectively). This study thus highlights the need to accurately consider biomaterial surface modifications and how they may influence cell-biomaterial interaction. It further provides a novel approach to characterizing cell-relevant mechanical properties of a polymer substrate. These advances may lead to an improved quantitative assessment of actin cytoskeleton function, with potential relevance to biomaterial based therapies.

Osteosarcoma is the most frequent primary tumor of bone and is characterized by its high tendency to metastasize in lungs. Although treatment in cases of early diagnosis results in a 5-yr survival rate of nearly 60%, the prognosis for patients with secondary lesions at diagnosis is poor, and their 5-yr survival rate remains below 30%. In the present work, we have used a number of analytical methods to investigate the impact of increased metastatic potential on the biophysical properties and force generation of osteosarcoma cells. With that aim, we used two paired osteosarcoma cell lines, with each one comprising a parental line with low metastatic potential and its experimentally selected, highly metastatic form. Mechanical characterization was performed by means of atomic force microscopy, tensile biaxial deformation, and real-time deformability, and cell traction was measured using two-dimensional and micropost-based traction force microscopy. Our results reveal that the low metastatic osteosarcoma cells display larger spreading sizes and generate higher forces than the experimentally selected, highly malignant variants. In turn, the outcome of cell stiffness measurements strongly depends on the method used and the state of the probed cell, indicating that only a set of phenotyping methods provides the full picture of cell mechanics.