Applications of nanoindentation in biology and medicine
Nanoindentation for biological materials
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 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:
- Low load range,
- Large vertical displacement range,
- Ability to test immersed samples,
- Automated procedure for testing irregularly shaped samples.
In addition, the new nanoindentation device should offer visualization features, good thermal stability, sufficient lateral repositioning accuracy for localized testing and heating option to reproduce biological environments.
Fig. 1: Full Bioindenter with control unit and heating cell
The Bioindenter instrument
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.
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.
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 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 technique therefore requires relatively low number of experiments.
The bioindentation can be used in the testing of cartilage, tendon, cornea, scaffold, tissue regeneration, plant, micro-tissue compression, hydrogel and elastomer (Ebenstein and Pruitt, 2006; Oyen, 2010). In addition to elastic modulus, the Bioindenter can also assess the creep and poroelastic properties of these materials (Hu et al., 2010; Kaufman et al., 2008; Menčík et al., 2009)
Applications of the Bioindenter
The applications of the Bioindenter are very wide. Many human tissues are subject to mechanical loading and their mechanical characterization can provide valuable information for disease evolutions, treatments and also developments of artificial replacements (implants, scaffolds). Bioindentation can be used in the diagnostic of disease (liver functions, arteries) and for fundamental research on treatment of these diseases (Hu et al., 2012; Levental et al., 2010; Oyen, 2010). This technique also finds its use in the growing area of biomimetic research where structure and mechanical properties of tissues must be carefully characterized to develop graft materials with properties as close as possible to the real tissues. Mechanical properties obtained with the Bioindenter are closely related to the local structure of the material and reveal important information about the biomechanical response of the tissue.
The fundamental advantage of the bioindentation is its ability to probe the properties of biological tissues at the scale of tissues in order to understand their mechanical behavior. This is often done to find potential replacement materials whose properties and behavior need to match closely the replaced tissues. The Bioindenter provides a unique tool to test both tissues and their potential replacement materials.
Osteoarthritis is one of the most common joint diseases that affect about fifty percent of the world’s population. Although some progress has been done in the treatment of this disease, the different mechanisms of disease initiation, propagation and the effects of medical treatments are not fully understood. Many laboratories are therefore conducting research that relies on the characterization of mechanical properties of cartilage in different stages of the disease along with the understanding of the effects of different treatments. These experiments are almost exclusively done on small laboratory animals, i.e. rats or mice yielding very small samples with small testing areas. The use of Bioindenter in this research offers suitable load and depth range together with high lateral resolution to test small cartilage samples. The results of the nanoindentation tests will be used in the development and evaluation of osteoarthritis treatment and the study of different effects on the evolution of osteoarthritis. One of the research topics is also mapping of the mechanical properties of cartilage in respect to the level of load applied on the respective zone. Detailed study was conducted on rat’s femur to determine the properties of three main zones on the femur: the anterior zone (1), the posterior zone (3) and the top zone (3). The anterior and posterior zones (1 and 3) are mildly loaded whereas the top zone (3) is heavily loaded during the life of a rat. The results of Bioindenter measurements with spherical indenter and the femur completely immersed in liquid showed that the highly loaded zone 2 has much higher elastic modulus and less creep than the anterior and posterior zones (1 and 3). This is an important finding as it points out the strong heterogeneity of the cartilage and the dependence of its properties on mechanical loading during the life cycle. All indentations were done with maximum depth of 15 mm (beginning of the hold period). The different stiffness and ability to fluid flow (creep) was reflected in various maximal forces needed to reach the depth of 15 mm. Clearly, the more loaded top zone 1 is much stiffer than the mildly loaded zones 1 and 3. Zones 1 and 3 also exhibit more creep (mainly due to fluid flow) than the stiffer zone 2. This example shows that also the creep properties are very important when characterizing biological materials.
Fig.3 – Image of Bioindenter during indentation on rat femur cartilage (a) and indentation response of cartilage in the loaded and less loaded region (b).
Tissue regeneration, scaffolds
Bioindenter can also be used for studying of degree of regeneration after lesion in cartilage. Research was performed on the evolution of cartilage regeneration after introducing a scaffold in the lesion of goat’s femur. The animal was sacrificed three months after the graft and both healthy and regenerating areas of the cartilage were tested. The Bioindenter measurements showed that the healthy and regenerating cartilage exhibited large differences both in elastic modulus and creep behavior. In this case the tested area was quite large (~few hundreds of micrometers) so that commonly present local variations of the stiffness of the healthy and regenerating cartilage were averaged. Highly localized measurements would lead to scattered experimental results 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. 4: Image of the Bioindenter when indenting goat’s femur (a) and comparison of nanoindentation on healthy and regenerating cartilage (goat’s femur, spherical indenter with 0.5 mm radius (b).
The cornea, corneoscleral rim and sclera represent regions of eye that play an important role in clear sight. Some cornea diseases or injuries can lead to partial or total blindness or chronic ocular surface pain. Treatments of such events can rely on regrowth of stem cells based in the limbal region. Survival and self-renewal of the limbal stem cells depends strongly on the biomechanical properties of the environment, i.e. cornea, corneoscleral rim and sclera. It is therefore of great interest to explore the elastic modulus and permeability of the corneoscleral rim to assess the biomechanical properties of this unique structure at the ocular surface. Some of the corneal treatments (such as UVA-crosslinking) can also affect the stiffness and the permeability of the cornea. Measurement of these changes is an important factor for indication of the efficiency of the treatment method. In our research the Bioindenter was used to indent the three distinct regions of the eye (cornea, limbus and sclera) in order to obtain elastic modulus and to compare creep properties (ability of fluid flow under mechanical loading) in these regions.
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) (b). Note different indentation depths and creep levels for each region.
Hydrogels are very soft materials suitable for tissue engineering that are used in various areas of biological and clinical research, e.g. osteoporosis or hemorrhage control. Many hydrogels are considered as potential candidates for replacement, regeneration, scaffolds or as growth substrates for soft tissues in the human body. Recently, it has been found that the elasticity of the substrate can significantly influence the homeostasis of tissues, which is important for tissue regeneration (Discher et al., 2005; Moers et al., 2013). Determination of elastic – and in general mechanical – properties of biological substrates is therefore of high interest. Elastic modulus and creep properties can easily be studied with the Bioindenter as the instrument is compatible with testing in liquids and can operate under various loading modes (Nohava et al., 2014). Time dependent response of hydrogels can then be easily studied.
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).
Adhesion – elastomers and gels
The bioindentation method applies not only to indentation of biological materials but also for measurements of various elastomers, gels and hydrogels that do not require immersion in liquid. Such materials can exhibit often very elastic behavior and strong adhesion (Kohn and Ebenstein, 2013). These phenomena can be studied using the Bioindenter and its ability to sense very small force on the indenter during the approach and retraction of the indenter from the surface of the material. As the indenter approaches the surface, pull-on adhesion generates a peak of normal force. This phenomenon is recorded in the load-displacement curve and can be used for calculation of adhesion force or surface energy. During retraction, when the indenter is removed from the surface, pull-off adhesion generates negative adhesive forces and can also be measured.
Fig. 7 – Adhesion on gel during spherical indentation using Bioindenter and equation based on the Johnson, Kendall, Roberts (JKR) theory for calculation surface energy W12 (R is the radius of the indenter and Fad is the pull-off adhesion force (Menčík, 2012).
The nanoindentation of biological tissues (bioindentation) is a growing field that has many specifics and requires new experimental and analytical approaches. Despite being a relatively new method, it has already found its place in many laboratories around the world and serves both the development of clinical treatments and fundamental biomechanical research. The initial lack of sensitive instrumentation has recently been addressed by Anton Paar with the Bioindenter. The Bioindenter offers unique capabilities for local in vitro characterization of biological materials to study their behavior and response to mechanical loading. 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.
Dr Jiri Nohava, Anton Paar TriTec SA
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