Applications of nanoindentation in biology and medicine
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::: Application Report Advanced Mechanical Surface Testing Applications of nanoindentation in biology and medicine Anton Paar has for many years been active in the field of micromechanical testing of biological materials and biomaterials. After gaining significant experience in testing of hard biological materials such as dentin or bone in both dry and liquid environments, the next natural step was to evolve in the domain of soft tissues and materials. The ever increasing testing needs from the biology and medical community for characterization of soft biological and biocompatible materials initiated the development of a technique for localized testing of soft materials and a suitable testing device. The new bio-nanoindenter named Bioindenter was developed in collaboration with biology specialists from the Life Sciences Division of the CSEM Laboratory (Switzerland). This collaboration led to the adaptation and validation of nanoindentation protocols for the biological samples. The new Bioindenter is capable of applying loads from ~0.01 mN up to 20 mN with a total travel of the indenter larger than 100 mm. The Bioindenter is based on the successful Ultra Nanoindentation Tester (UNHT) from which it inherited superior thermal stability and high sensitivity for creep measurements. Special indenters with thin long shaft were developed in order to allow measurements of immersed samples and reduce capillary effects. The nature of the samples whose stiffness is very low (elastic modulus of ~10 MPa and less) dictated the requirements of the new instrument and of the corresponding nanoindentation technique. The new nanoindentation tester for biologists should therefore fulfill these specifications: • Large vertical displacement range, Ability to test immersed samples, • Automated procedure for testing irregularly shaped samples. Fig. 2 – Long shaft indenter for bioindentation applications. These long-shaft indenters are available in various geometries. The most commonly used geometries are spheres and flat punches. To facilitate the handling and testing of biological samples, the Bioindenter includes a specially designed Petri dish holder allowing easy sample transfer and switch. An optional heater is available for the Petri dish holder in order to control the samples at temperature up to 50°C. Motorized XY tables with repositioning accuracy of 1 mm provide precise lateral positioning; motorized Z-table allows automated indenter approach and retraction during the test. A microscope with long distance objectives and LED light source allows top view imaging of the sample and an optional microscope can be mounted under the sample to allow in-situ observation of the sample during the experiment. In addition, the new nanoindentation device should to offer visualization features, good thermal stability, sufficient lateral repositioning accuracy for localized testing and heating option to reproduce biological environments. The bioindentation technique The measurement protocol for nanoindentation of biological samples (sometimes referred as bioindentation) takes into account the irregularity of the surface of the samples by incorporating an automatic surface detection procedure in the measurement matrix. Surface detection by the Indentation software now Fig. 1: Full Bioindenter with control unit and heating cell

::: Application Report routinely uses the contact stiffness change to avoid false contact detection due to external forces (capillarity, etc.). The use of large spherical indenters facilitates contact detection on extremely soft samples (hydrogels, cartilage, scaffolds) by providing larger contact stiffness and averages surface and structural inhomogeneity. The penetrations seen in bioindentation are usually in the range of ten to several hundred micrometers, thus testing a large volume of tissue rather than single cells. Bioindentation yields averaged properties of the material and the...

::: Application Report and conclusions on the progress of the healing process would be difficult to draw. The measurements showed that the regenerating cartilage had much larger creep and more than ten times lower elastic modulus than the healthy cartilage. Obviously, the regeneration of the cartilage is at very early stage even after three months after the graft. Fig. 5: Different regions of the eye (a) and indentation depth versus time for the three regions of the eye: sclera, limbus and central cornea (indentation with 0.05 mN load, 180 seconds hold, 0.5 mm radius spherical indenter)...

::: Application Report Another great advantage of the Bioindenter is the ability to measure time dependent properties often linked to fluid flow in the tissues which is an important factor in understanding the life and behavior of many soft tissues. Authors Dr Jiri Nohava, Anton Paar TriTec SA b) Contact Anton Paar Fig. 6 –Bioindenter setup for indentation on hydrogels (a) and indentation load-depth curves for polyacrylamide hydrogel with various concentrations (0.05 mN maximum load, 100 seconds hold period, spherical ruby indenter with R = 0.5 mm) (b). info.tritec@anton-paar.com...