Microindentation of cartilage before and after articular loading in a bioreactor: assessment of length-scale dependency using two analysis methods.

Background: Microindentation is a technique with high sensitivity and spatial resolution, allowing for measurements at small-scale indentation depths. Various methods of indentation analysis to determine output properties exist. Objective: Here, the Oliver-Pharr Method and Hertzian Method were compared for stiffness analyses of articular cartilage at varying length-scales before and after bioreactor loading. Methods: Using three different conospherical tips with varying radii (20, 100, 793.75 µm), a bioreactor-indenter workflow was performed on cartilage explants to assess changes in stiffness due to articular loading. For all data, both the Oliver-Pharr Method and Hertzian Method were applied for indentation analysis. Results: The reduced moduli calculated by the Hertzian Method were found to be similar to those of the Oliver-Pharr Method when the 20 µm tip size was used. The reduced moduli calculated using the Hertzian Method were found to be consistent across the varying length-scales, whereas for the Oliver-Pharr Method, adhesion/suction led to the largest tip exhibiting an increased average reduced modulus compared to the two smaller tips. Loading induced stiffening of articular cartilage was observed consistently, regardless of tip size or indentation analysis applied. Conclusions: Overall, geometric linearity is preserved across all tip sizes for the Hertzian Method and may be assumed for the two smaller tip sizes using the Oliver-Pharr Method. These findings further validate the previously described stiffening response of the superficial zone of cartilage after articular loading and demonstrate that the finding is length-scale independent.

Elastic modulus and hydraulic permeability of MDCK monolayers.

The critical role of cell mechanics in tissue health has led to the development of many in vitro methods that measure the elasticity of the cytoskeleton and whole cells, yet the connection between these local cell properties and bulk measurements of tissue mechanics remains unclear. To help bridge this gap, we have developed a monolayer indentation technique for measuring multi-cellular mechanics in vitro. Here, we measure the elasticity of cell monolayers and uncover the role of fluid permeability in these multi-cellular systems, finding that the resistance of fluid transport through cells controls their force-response at long times.

Mechanical properties derived from phase separation in co-polymer hydrogels.

Hydrogels can be synthesized with most of the properties needed for biomaterials applications. Soft, wettable, and highly permeable gels with a practically unlimited breadth of chemical functionalities are routinely made in the laboratory. However, the ability to make highly elastic and durable hydrogels remains limited. Here we describe an approach to generate stretchy, durable hydrogels, employing a high polymer-to-crosslink ratio for extensibility, combined with an aggregating copolymer phase to provide stability against swelling. We find that the addition of aggregating co-polymer can produce a highly extensible gel that fails at 1000% strain, recovers from large strains within a few minutes, maintains its elasticity over repeated cycles of large amplitude strain, and exhibits significantly reduced swelling. We find that the gel׳s enhanced mechanical performance comes from a kinetically arrested structure that arises from a competition between the disparate polymerization rates of the two components and the aggregation rate of the unstable phase. These results represent an alternative strategy to generating the type of stretchy elastomer-like hydrogels needed for biomedical technologies.

Surface indentation and fluid intake generated by the polymer matrix of Bacillus subtilis biofilms.

Bacterial biofilms are highly structured, surface associated bacteria colonies held together by a cell-generated polymer network known as EPS (extracellular polymeric substance). This polymer network assists in adhesion to surfaces and generates spreading forces as colonies grow over time. In the laboratory and in nature, biofilms often grow at the interface between air and an elastic, semi-permeable nutrient source. As this type of biofilm increases in volume, an accommodating compression of its substrate may arise, potentially driven by the osmotic pressure exerted by the EPS against the substrate surface. Here we study Bacillus subtilis biofilm force generation by measuring the magnitude and rate of deformation imposed by colonies against the agar-nutrient slabs on which they grow. We find that the elastic stress stored in deformed agar is orders of magnitude larger than the drag stress associated with pulling fluid through the agar matrix. The stress exerted by the biofilm is nearly the same as the osmotic pressure generated by the EPS, and mutant colonies incapable of producing EPS exert much lower levels of stress. The fluid flow rate into B. subtilis biofilms suggest that EPS generated pressure provides some metabolic benefit as colonies expand in volume. These results reveal that long-term biofouling and colony expansion may be tied to the hydraulic permeability and elasticity of the surfaces that biofilms colonize.

Polymer fluctuation lubrication in hydrogel gemini interfaces.

Interfacial sliding speed and contact pressure between the sub-units of particulate soft matter assemblies can vary dramatically across systems and with dynamic conditions. By extension, frictional interactions between particles may play a key role in their assembly, global configuration, collective motion, and bulk material properties. For example, in tightly packed assemblies of microgels - colloidal microspheres made of hydrogel - particle stiffness controls the fragility of the glassy state formed by the particles. The interplay between particle stiffness and shear stress is likely mediated by particle-particle normal forces, highlighting the potential role of hydrogel-hydrogel friction. Here we study friction at a twinned "Gemini" interface between hydrogels. We construct a lubrication curve that spans four orders of magnitude in sliding speed, and find qualitatively different behaviour from traditional lubrication of engineering material surfaces; fundamentally different types of lubrication occur at the hydrogel Gemini interface. We also explore the role played by polymer solubility and hydrogel-hydrogel adhesion in hydrogel friction. We find that polymer network elasticity, mesh size, and single-chain relaxation times can describe friction at the gel-gel interface, including a transition between lubrication regimes with varying sliding speed.

Cell friction.

Cells sense and respond to their environment. Mechanotransduction is the process by which mechanical forces, stress, and strains are converted into biochemical signals that control cell behavior. In recent decades it has been shown that appropriate mechanical signals are essential to tissue health, but the role of friction and direct contact shearing across cell surfaces has been essentially unexplored. This, despite the obvious existence of numerous biological tissues whose express function depends on sliding contacts. In our studies on frictional interactions of corneal cells we find that the friction coefficients are on the order of mu = 0.03-0.06 for in vitro and in vivo experiments. Additionally, we observe cell death after single cycles of sliding at contact pressures estimated to be approximately 12 kPa. These experimental results suggest that frictional contact forces produce mechanical stresses and strains that are in the cellular mechanosensing ranges.

Direct observation of counterion organization in F-actin polyelectrolyte bundles.

Attractions between like-charged polyelectrolytes have been observed in a variety of systems (W.M. Gelbart, R.F. Bruinsma, P.A. Pincus, V.A. Parsegian, Phys. Today 53, September issue, 38 (2000)). Recent biological examples include DNA, filamentous viruses, and F-actin. Theoretical investigations on idealized systems indicate that counterion correlations play a central role, but no experiments that specifically probe such correlations have been performed. Using synchrotron X-ray diffraction, we have directly observed the organization of multivalent ions on cytoskeletal filamentous actin (a well-defined biological polyelectrolyte) and found an unanticipated symmetry-breaking collective counterion mechanism for generating attractions. Surprisingly, the counterions do not form a lattice that simply follows actin's helical symmetry; rather, the counterions organize into "frozen" ripples parallel to the actin filaments and form structures reminiscent of charge density waves. Moreover, these 1D counterion charge density waves form a coupled mode with twist deformations of the oppositely charged actin filaments. This counterion organization is not sensitive to thermal fluctuations in temperature range accessible to protein-based polyelectrolyte systems. Moreover, the counterion density waves are "pinned" to the spatial periodicity of charges on the actin filament even if the global filament charge density is varied, indicating the importance of charge periodicity on the polyelectrolyte substrate.