Nano Scale Mechanical Analysis of Biomaterials Using Atomic Force Microscopy

Costal cartilage (CC) is one of the load-bearing tissues of the rib cage. Literature on material characterisation of the CC is limited. Atomic force microscopy (AFM) has been extremely successful in characterising the elastic properties of soft biomaterials such as articular cartilage and hydrogels, which are often the material of choice for cartilage models. But AFM data on CC are absent in the literature. In this study, AFM indentations using spherical beaded tips were performed on human CC to isolate the mechanical properties. A novel method was developed for modelling the relaxation indentation experiments based on Fung's quasi-linear viscoelasticity and a continuous relaxation spectrum. This particular model has been popular for uniaxial compression test data analysis. Using the model, the mean Young's modulus of CC was found to be about 2.17, 4.11 and 5.49 MPa for three specimens. A large variation of modulus was observed over the tissue. Also, the modulus values decreased with distance from the costochondral junction.

Studying local mechanical properties of metallic alloys and composites and their defect structures such as grain boundaries, cracks, and dislocations allows a better understanding and design of these structural materials. With a nanoindenting atomic force microscope (NI-AFM), which uses diamond tips, such measurements are possible on a nanometer scale. Measurements on the microstructure of superalloys are presented, where different precipitates with sizes in the range of 100 nm are characterized by their different hardness values. In addition, this new technique was used to characterize the nanohardness of the plastic zone and stress fields of cracks and grain boundaries. Therefore, crack tips of in-situ loaded bending specimens of intermetallic alloys (NiAl) were investigated. The measurements show a change in nanohardness and elasticity from the distance of interfaces. In material science the high-resolution capabilities of the atomic force microscopy (AFM) are used for studying metallographic sections, where different phases produce topographic contrasts in the image. A extremly small height difference in the microstructure is sufficient for a height contrast in the image, which allows a quantitative determination of particle size distributions and volume contents of precipitates [1, 2]. The plastic deformation behavior of metals is strongly influenced by the distribution and movement of defects as dislocations, grain boundaries and cracks. In-situ loading experiments in an AFM on specimens loaded in 4-point bending demonstrate the good imaging properties of an AFM for such defects [3]. Mechanical properties such as Young’s modulus of elasticity and hardness values were obtainable in the past by various indentation techniques, where a load displacement curve is recorded during the indentation process. These techniques were mainly applied for studying the mechanical properties of thin films. A combination of a nanoindenting instrument with an AFM now allows the local mechanical properties of different phases and particles in metallic alloys to be measured, since the indents can be positioned very exactly with the AFM. Such a combined nanoindenting AFM (NI-AFM) requires a special force measurement technique. Piezo hysteresis and creep of the used piezo scanner have to be eliminated. In addition, indenting should be performed by a tip which is centrally fixed and not at the end of a bending beam (cantilever) [4], because otherwise the location of the indent changes during loading. These properties are fulfilled by a capacitive transducer, which is used for load generation and measurement instead of the standard laser deflection unit. A diamond tip is used for the indentation experiments and topographic imaging in a conventional AFM mode. The resulting force indentation curves contain two pieces of information: the universal hardness H and the reduced modulus of elasticity Er. The possibilities of the NI-AFM for studying local mechanical properties are demonstrated on two different examples: first the hardness of the superalloy CMSX-6 was characterized; second, local changes of mechanical properties near a crack tip in a NiAl single crystal were examined. 1 Experimental setup and sample preparation The nanoindentation measurements shown in this paper were performed with an add-on-force transducer from Hysitron Inc. This transducer, mounted on a conventional AFM, controls the z movement of the tip (indentation, applied load, and feedback signal) while the piezos of the AFM control the lateral x-y movement. Further technical details on the transducer are described in [5]. Specimens of the superalloy CMSX-6 were examined with a Nanoscope II AFM from Digital Instruments. Since the x-y movement is achieved by scanning the whole specimen, only small specimens with a maximum diameter of 20 mm can be analyzed. Crack tips in NiAl single crystals were investigated with a bending apparatus specially constructed for the Explorer AFM from TopoMetrix. (This bending apparatus is described in [6].) Because the whole transducer assembly is mounted on the x-y scanner, the combined system is more sensitive to mechanical noise. However, large specimens can be examined with

<p>The composition, topography, adhesiveness and nanomechanical properties of biomaterials are all factors that affect biological processes, e.g. biofilm formation, cell differentiation, and morphogenesis [1-4]. Atomic Force Microscopy (AFM) is a highly versatile tool, ideal for the characterization of samples properties ranging from single molecules to complex biological systems, on the nm scale.</p> <p>JPK BioAFMs, like the NanoWizard<sup>®</sup> ULTRA Speed 2, enable fast imaging of challenging biological samples and the visualization of dynamic processes with high spatio-temporal resolution under near physiological conditions, e.g. the kinetics of collagen type I fibrillogenesis was imaged in situ revealing the formation of the 67 nm D-banding hallmark.</p> <p>Our distinctive Quantitative Imaging mode (QI™) measures various sample properties such as topography, nanomechanics and adhesion on the nanometer scale. Complex data like contact point, Young´s modulus or recognition images can also be extracted at the same resolution. To demonstrate the capability and flexibility of the QI™ mode, various biological samples like living cells have been investigated and their topographical and mechanical properties determined. QI™, based on fast force mapping, can also be used to determine the mechanical properties of different bacterial strains. We will discuss the application of HighSpeed AFM for the characterisation of dynamic biofilms with high spatiotemporal resolution, information which can then be directly correlated with advanced optical microscopy for immuno-characterisation of the sample.</p> <p>Investigating large, sticky and rough samples such as tissues and hydrogels using AFM has always been a challenge due to the limited z-axis of the AFM. The HybridStage™, equipped with an extended xyz scanner unit up to 300x300x300 µm<sup>3</sup>, an additional motorized unit for large sample movements in the mm range and optical tiling, is ideal for investigating such samples. This combination enables multi-region AFM probing over a large, uneven sample area and provides additional correlative optical data sets.</p> <p>Adhesion dynamics between cells and biomaterials play a crucial role in, e.g. the applicability of potential implant materials. The AFM based Single Cell Force Spectroscopy platform enables quantitative measurement of the interactions between individual cells and any substrate.</p> <p>A number of important research topics in the field of biomedicine, relate directly to the increased antimicrobial resistance of various biofilms to commonly prescribed drugs, and have identified adhesion, as a leading factor in biofilm formation, colony progression, and pathogenesis of microbial agents. JPK BioAFM has developed novel techniques for studying single-molecule forces and adhesion profiles at the cell/cell or cell/substrate interface. We will provide an overview of how to functionalize various surface substrates for the attachment of bacteria.</p> <p>We will also provide information on working with AFM under Biosafety Level L2/L3 conditions.</p> <p> </p> <p>[1]      Elter, P. et al., Eur Biophys J, <strong>2011</strong>, 40(3):317-27</p> <p>[2]      Engler AJ. Et al., Cell;<strong> 2006</strong>, 126(4):677-89</p> <p>[3]      Cisneros, DA. et al., Small,<strong> 2007</strong>, 3(6):956-63</p> <p>[4]         Koser DA. Et al., Nat. Neurosci.; <strong>2016</strong>, 19:1592-1598</p>

Costal cartilage is a promising donor source of chondrocytes to alleviate cell scarcity in articular cartilage tissue engineering. Limited knowledge exists, however, on costal cartilage characteristics. This study describes the characterization of costal cartilage and articular cartilage properties and compares neocartilage engineered with costal chondrocytes to native articular cartilage, all within a sheep model. Specifically, we (a) quantitatively characterized the properties of costal cartilage in comparison to patellofemoral articular cartilage, and (b) evaluated the quality of neocartilage derived from costal chondrocytes for potential use in articular cartilage regeneration. Ovine costal and articular cartilages from various topographical locations were characterized mechanically, biochemically, and histologically. Costal cartilage was stiffer in compression but softer and weaker in tension than articular cartilage. These differences were attributed to high amounts of glycosaminoglycans and mineralization and a low amount of collagen in costal cartilage. Compared to articular cartilage, costal cartilage was more densely populated with chondrocytes, rendering it an excellent chondrocyte source. In terms of tissue engineering, using the self-assembling process, costal chondrocytes formed articular cartilage-like neocartilage. Quantitatively compared via a functionality index, neocartilage achieved 55% of the medial condyle cartilage mechanical and biochemical properties. This characterization study highlighted the differences between costal and articular cartilages in native forms and demonstrated that costal cartilage is a valuable source of chondrocytes suitable for articular cartilage regeneration strategies.

Control of experiment is essential part of Atomic Force Microscopy (AFM). AFM is the technique in which is a minute probe is applied for high-resolution profiling of samples and studies of their mechanical and electric properties at the nanometer scale. The importance of this technique is increasing with miniaturization of functional industrial components and related need in comprehensive material characterization at small dimensions. In recent years control systems community expressed significant interest to AFM (several invited sessions at ACC 2008 and ACC 2009, publications in Control Systems Magazine). Further development of this method, which is related to transition to multi-frequency and broad band measurements and use increasing number of lock-in amplified, is inconceivable with proper advances in design and control of AFM instrumentation and applications. With increasing role of nanotechnology, AFM becomes an important tool in control education and research. However, few control experts have access to expensive AFM instrumentation. AFM Simulator allows control experts to demonstrate applicability of their theories and algorithms to AFM. This will increase the needed flow of ideas from control community to AFM instrument designers and practitioners. Simulator can be also introduced into control curriculums to expand them towards nanotechnology applications. All this shows the importance of this special session that will attract the control scientists and specialists to this valuable practical field.

Advances in cellular nanoscale force detection and manipulation

We used atomic force microscopy (AFM) technique to measure the viscoelastic response of cancer and normal thyroid cells on different stiffness polyacrylamide gels. After applying a step in contact we recorded the stress relaxation of cells in order to measure their viscous and elastic properties. With the help of an extended version of the Hertz model, we could quantify for the first time by AFM the elastic modulus and the dynamic viscosity of cells on substrates with different stiffnesses. We have cultured anaplastic carcinoma and normal thyroid cells on three different substrates: polyacrylamide gels with elastic modulus in a range of 3-5and 30-40kPa and "infinitely" stiff Petri dishes. Whereas normal thyroid cells adapted their mechanical properties to different stiffness substrates, cancer cells were less affected by the surrounding stiffness. Normal cells changed the elastic modulus from 1.2 to 1.6and to 2.6kPa with increasing substrate stiffness; the dynamic viscosity values varied from 230 to 515and to 470Pa·s, accordingly. By contrast, the values for cancer cells were rather constant regardless of substrate stiffness (in average the elastic modulus was 1.3kPa and the dynamic viscosity was 300Pa·s). This difference in sensing and reacting to the mechanical properties of the substrate shows that normal and cancer cells interact differently with the neighboring tissue, which may be related to the ability of cancer cells to form metastases.

The costal cartilage is a prominent feature of the anterior chest wall that is subject to developmental and acquired abnormalities. A fully automatic algorithm to reconstruct the human costal cartilage from multidetector computed tomography (MDCT) images was developed and tested. The reconstruction algorithm includes three steps: (1) estimation of length, curvature and end points for each costal cartilage centre-line, (2) costal cartilage cross-section area approximation, and (3) transformation of the estimated cross-section to the centre-line into a cylindrical coordinate system. Four different models were as follows: circle, vertical ellipse, horizontal ellipse and a non-geometric shape have been assumed for the cross-section. Shape estimates were based on each patient's dataset, so the algorithm is patient-specific and anatomically faithful. MDCT datasets from 15 patients were evaluated with the automated algorithm and the results compared with reference masks provided by an experienced radiologist. The costal cartilage reconstruction result and the reference mask were visually consistent. Based on evaluation results, the circular model cross-section with area of twice M (mean area of all rib cross-sections in the mid-coronal plane) had the highest Dice similarity coefficient (DSC = 77.5%) with only 2.12 mm registration distance. Costal cartilage 3D morphology can be extracted from MDCT scans with an automated method, using a circular cross-section with area equal to twice M.

In most communities, the risk of developing breast cancer is increasing. By affecting the cyclooxygenase 1 and 2 (COX-1 and COX-2) enzymes and actin filaments, acetylsalicylic acid (Aspirin) has been shown to reduce the risk of breast cancer and prevent cell migration in both laboratory and clinical studies. The purpose of this study is to determine the mechanical properties of normal and cancerous breast tissue cells, as well as the short-term effect of aspirin on cancer cells. To this end, the mechanical properties and deformation of three cell types were investigated: healthy MCF-10 breast cells, MCF-7 breast cancer cells, and MCF-7 breast cancer cells treated with a 5 μM aspirin solution. Atomic Force Microscopy (AFM) was used to determine the mechanical properties of the cells. Cell deformation was analyzed in all groups, and Young's modulus was calculated using the Hertz model. According to the obtained data, cancer cells deformed at a rate half that of healthy cells. Nonetheless, when aspirin was used, cancer cells deformed similarly to healthy cells. Additionally, healthy cells' Young's modulus was calculated to be approximately three times that of cancer cells, which was placed closer to that of healthy cells by adding aspirin to Young's modulus. Cell strength appears to have increased due to aspirin's intervention on actin filaments and cytoskeletons, and the mechanical properties of breast cancer cells have become more similar to those of normal cells. The likelihood of cell migration and metastasis decreases as cell strength increases.

Force Microscopy of Nonadherent Cells: A Comparison of Leukemia Cell Deformability

To investigate a new parameter for measuring the metastatic potential of cancer cells, we conducted nanomechanical analysis using atomic force microscopy (AFM). AFM, a probe microscope, has the advantage of measuring cell stiffness (elasticity) in physiological conditions without any pretreatment. Since cancer cells are softer than normal cells, the cell stiffness measured by AFM can be utilized as a new cancer diagnostic marker. Experimentally, we examined whether cell stiffness might reflect the metastatic potential of cancer cells using three subclones (B16-F10, B16-BL6, and B16-F1) of B16 melanoma cells. Migration potential was first determined by Transwell assay: B16-F10 and B16-BL6 cells showed 3- and 2- times more migrated cells into reverse sides of filter, at 5 μg/ml concentrations of fibronectin, than did B16-F1 cells. This indicates that B16-F10, B16-BL6, and B16-F1 cells have metastatic potential in the order of high, intermediate, and poor, respectively. AFM measurement clearly shows significant differences in cell stiffness of three B16 subclones, while the morphology and growth rate of these subclones are almost the same. Median values of Young’ modulus with B16-F10, B16-BL6, and B16-F1 cells were 395.12 Pa, 771.13 Pa, and 868.45 Pa, respectively. The results showed that highly metastatic B16-F10 cells are least stiff, suggesting that cell stiffness (Young's modulus) is directly associated with metastatic potential. Furthermore, we found that treatment with green tea catechin EGCG increased the cell stiffness of B16-F10 cells to greater than that of non-treated cells, and it also inhibited of cell migration in Transwell assay. Moreover, an inactive catechin, EC, did not have any effect on cell stiffness or cell migration. The results with EGCG strongly support our previous finding that EGCG in drinking water inhibits the metastasis of B16-F10 cells into the lungs of C57/BL6 mice. We have present for the first time evidence that the cell stiffness, as determined by AFM, is a useful biomarker for predicting metastatic potential and developing new preventive agents against metastasis. We discuss the significance of this mechanical and biochemical property (cell stiffness) in metastasis. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 1388. doi:10.1158/1538-7445.AM2011-1388

Cartilage is a complex avascular tissue composed of cells ("chondrocytes") embedded in an extracellular matrix (ECM) consisting of 70%-80% water. The primary components of the ECM are negatively charged aggrecans and collagen II fibrils, which possess a characteristic, ordered three-dimensional structure. The components interact to ensure that the cartilage is able to absorb shock and can function to protect the bone ends. Atomic force microscopy (AFM) can be used to examine structure-function relationships of cartilage at both micrometer and nanometer scales. When imaged at the micrometer scale with microspheres, only the ECM and chondrocytes can be distinguished. Correspondingly, mechanical testing of cartilage at the micrometer scale results in unimodal distribution of the stiffness because the bulk elastic property of the ECM is probed. In contrast, bare AFM tips are able to reveal the molecular components of the ECM at the nanometer scale. Mechanical testing at the nanometer scale reveals a bimodal distribution of the stiffness and reflects the distinct stiffness of the collagen network and the proteoglycan moiety. In this protocol, the corresponding AFM image and force map are used to reveal the distinct morphology of the collagen fibers and proteoglycan gel. Although, in principle, these experiments can be performed using any AFM, an AFM with tube scanners that have manual screws for tilting the sample is preferable because cartilage has macroscopically rough surface features. By manually tilting the probe over the sample, an optimal angle for tip approach can be achieved.

The measurement of small forces by atomic force microscopy (AFM) is of increasing importance in many applications. For example, in analytical applications where individual molecules are probed, or nanoindentation measurements as a source of information about materials properties on a nanometer scale. The fundamentals of AFM force measurement, and some of these applications, are briefly reviewed. In most cases absolute, not relative, measurements of forces are needed for valid comparisons with theory and other measurement techniques (such as optical tweezers). We review methods of AFM force calibration and the major uncertainties involved. The force range considered in this work is roughly from 10 pN to around 500 nN. We describe some issues of the repeatability of force measurements that can be important in common AFM instruments. In most cases the aspect that then limits the accuracy with which forces can be measured is the uncertainty in the stiffness (more specifically the normal force constant) of the atomic force microscope cantilever at the center of the instrument. It is known that commercially available microfabricated atomic force microscope cantilevers have a wide range of force constant, for cantilevers of nominally the same type and even the same production batch. Calibration is necessary, and many methods have been used over the years. We compare the accuracy that can be achieved and the ease of use of these different methods, including theoretical (dimensional), thermal, static and dynamic methods and their variants. A device developed at NPL should help overcome many of the problems of force constant calibration, at least for the most common AFM configurations. This is a microfabricated silicon device, which, because of its very small mass, is not susceptible to vibration as a larger device would be. A new calibration method based on electrical and Doppler measurements allows the calibration of the force constant of this device traceable to the SI newton. It can then be sent to AFM users for straightforward calibration of AFM force constants. We conclude with a brief discussion of the special problems of calibration of lateral forces, such as those obtained in frictional force measurements.

Quantifying mechanical, proliferation, and migrational properties of bulk ovarian cancer cells and ovarian cancer stem cells.

The influence of nivolumab on intercellular adhesion forces between T cells and cancer cells was evaluated quantitatively using atomic force microscopy (AFM). Two model T cells, one expressing high levels of programmed cell death protein 1 (PD-1) (PD-1high Jurkat) and the other with low PD-1 expression levels (PD-1low Jurkat), were analyzed. In addition, two model cancer cells, one expressing programmed death-ligand 1 (PD-L1) on the cell surface (PC-9, PD-L1+) and the other without PD-L1 (MCF-7, PD-L1−), were also used. A T cell was attached to the apex of the AFM cantilever using a cup-attached AFM chip, and the intercellular adhesion forces were measured. Although PD-1high T cells adhered strongly to PD-L1+ cancer cells, the adhesion force was smaller than that with PD-L1− cancer cells. After the treatment of PD-1high T cells with nivolumab, the adhesion force with PD-L1+ cancer cells increased to a similar level as with PD-L1− cancer cells. These results can be explained by nivolumab influencing the upregulation of the adhesion ability of PD-1high T cells with PD-L1+ cancer cells. These results were obtained by measuring intercellular adhesion forces quantitatively, indicating the usefulness of single-cell AFM analysis.