Nanomechanical Mapping of Hard Tissues by Atomic Force Microscopy: An Application to Cortical Bone

Abstract

Force mapping of biological tissues via atomic force microscopy (AFM) probes the mechanical properties of samples within a given topography, revealing the interplay between tissue organization and nanometer-level composition. Despite considerable attention to soft biological samples, constructing elasticity maps on hard tissues is not routine for standard AFM equipment due to the difficulty of interpreting nanoindentation data in light of the available models of surface deformation. To tackle this issue, we proposed a protocol to construct elasticity maps of surfaces up to several GPa in moduli by AFM nanoindentation using standard experimental conditions (air operation, nanometrically sharp spherical tips, and cantilever stiffness below 30 N/m). We showed how to process both elastic and inelastic sample deformations simultaneously and independently and quantify the degree of elasticity of the sample to decide which regime is more suitable for moduli calculation. Afterwards, we used the frequency distributions of Young’s moduli to quantitatively assess differences between sample regions different for structure and composition, and to evaluate the presence of mechanical inhomogeneities. We tested our method on histological sections of sheep cortical bone, measuring the mechanical response of different osseous districts, and mapped the surface down to the single collagen fibril level.

Near-Surface Nanomechanics of Medical-Grade PEEK Measured by Atomic Force Microscopy

“…As a result, indentation becomes an irreversible process, or, in other words, residual indent is found on the sample, and elastic–plastic deformation regime should be assumed to fit the data [ 38 ]. In our work, the usage of nanometrically sharp tips for better spatial resolution implies h ~ R , therefore the modified Hertz model of Section 2.2 can be used to fit the data [ 41 , 42 , 43 ]. This allowed to numerically process nanometric indentations into PEEK, while the usage of low-stiffness cantilevers allows investigating the polymer mechanical response to small applied forces.…”

“…Nanoindentations were operated on a grid pattern within 5 × 5 µm 2 large topographies of the sample, with loading–unloading curves extracted at each point of the grid. Besides E , other useful quantities derived from the curves could be mapped, such as the maximum indentation depth h max and the elasticity index, 0 < η el < 1; the latter is defined as the ratio between the areas under the unloading and loading curves and indicates the degree of elasticity of the deformation at each point of indentation [ 35 , 42 ]. We show that these three quantities, which can be obtained simultaneously from a single indentation grid, provide comprehensive characterization of the nanomechanics of PEEK and promote AFM-based nanomechanical mapping as a mean to identify and quantify the spatial localization of the moduli originated by the contributions of different surface phases of the polymer, as well as to monitor changes of such distributions due to chemical and physical modification of the surface.…”

Trends in bioactivity: inducing and detecting mineralization of regenerative polymeric scaffolds

“…211 Atomic force microscopy (AFM), using contact or tapping modes, can be used to reveal nanoscale features (e.g., collagen fibrils and HAp crystals), while the nanoindentation mode is useful for nanomechanical modulus mapping. [229][230][231] Sub-resonance AFM (e.g., PeakForce Tapping mode) has also been developed for nanomechanical mapping 232 and was used by Zhou et al to evaluate bone tissue submerged in an aqueous environment. 233 It was also shown that micro-CT has also been coupled with mechanical testing to measure contact area and 3D full-field strain in bone/dental implant constructs; 234 such a method could likewise be highly informative to the assessment of bioactive scaffolds.…”

Determining Spatial Variability of Elastic Properties for Biological Samples Using AFM

“…As already mentioned, the equations presented in this section are valid for elastic half-spaces; however, they are extensively used when testing biological samples to create Young’s modulus maps [ 17 , 25 ]. A Young’s modulus map is created when multiple force–indentation curves are obtained on a selected region [ 26 , 27 , 28 ]. A typical illustration of the method is shown in Figure 2 c, where the selected area has been divided into 8 × 8 = 64 nanoregions.…”