Compressive mechanics of hyaluronan-rich pericellular matrices - a study on a biomimetic model film, combining atomic force and reflection interference contrast microscopy

Resumo

Hyaluronan (HA) is a naturally occurring linear, negatively charged polysaccharide that plays a vital role in the mechanical integrity and function of pericellular matrices (PCM) surrounding many cell types and that is becoming increasingly popular in biomedical applications. Elucidating the mechanical properties of the highly hydrated HA-rich matrices would be valuable to understand how PCMs are organized and how they function. For a thorough investigation of the physical principles underlying the biological function of HA-rich pericellular matrices and provided the instrumental limitations in studying these highly hydrated systems in vivo, we have studied a model system that is based on films of HA that is end-grafted to a supported lipid bilayer. The major characterization techniques used are atomic force microscopy (AFM) and reflection interference contrast microscopy (RICM), as well as quartz crystal microbalance with dissipation monitoring (QCM-D) and spectroscopic ellipsometry. In this thesis, we combine AFM, a widely used analytical approach to determine the behavior of molecules or thin films under mechanical force, and colloidal probe RICM, an established microinterferometric technique to determine the thickness of soft hydrated films, into one instrument. The combination provides interaction forces as a function of the absolute distance between the two approaching surfaces, information that may not easily be obtained with either technique alone. We employ the combined setup to quantify the thickness of films of end-grafted HA, and their resistance to compression forces as a function of the external salt concentration. We find a swelling behavior that is consistent with expectations for a polyelectrolyte brush. Through comparison of the experimental data with polymer theory, molecular parameters that govern the swelling and force response are identified and quantified. Intercalation of aggrecan, a HA-binding proteoglycan prominent in the PCM of chondrocytes, drastically increased the thickness and resistance of HA films to compression. The combined AFM/RICM setup can serve as powerful tool to study the compression and frictional forces of surface-confined, hydrated films in general. The data on the mechanical properties of well-defined HA and HA/aggrecan composite films can be used as a reference point for future studies on the mechanics of PCMs.