Nano Scale Mechanical Analysis of Biomaterials Using Atomic Force Microscopy (original) (raw)
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Atomic force microscopy characterization of collagen 'nanostraws' in human costal cartilage
Micron, 2013
Costal cartilage, a type of hyaline cartilage that bridges the bony ribs and sternum, is relatively understudied compared to the load bearing cartilages. Deformities of costal cartilage can result in deformation of the chest wall, , where the sternum is largely pushed towards or away from the spine, pectus excavatum and pectus carinatum respectively, with each condition having significant clinical impact. In the absence of extensive literature describing morphological features of costal cartilage, we characterized a sample from the costal margin immunohistologically and through atomic force microscopy. We had previously observed the presence of collagen 'nanostraws' running the length of costal cartilage. Hypothesizing that these structures may be responsible for fluid flow within this thick, avascular tissue, and prior to microfluidic analysis, we estimated the diameters and measured Young's modulus of elasticity of the collagen nanostraws. We found significant differences in results between treatment type and fixation. Significant differences in nanostraw strength and diameter obviously affect nano-fluidic transport calculations, and, therefore, we consider these results of importance to the scientific community relying upon measurements in the nanoscale.
Microscopy Research and Technique
Atomic force microscopy (AFM) is today an established tool in imaging and determination of mechanical properties of biomaterials. Due to their complex organization, those materials show intricate properties such as viscoelasticity. Therefore, one has to consider that the loading rate at which the sample is probed will lead to different mechanical response (properties). In this work, we studied the dependence of the mechanical properties of endothelial cells on the loading rate using AFM in force spectroscopy mode. We employed a sharp, four-sided pyramidal indenter and loading rates ranging from 0.5 to 20 μm/s. In addition, by variation of the load (applied forces from 100 to 10,000 pN), the dependence of the cell properties on indentation depth could be determined. We then showed that the mechanical response of endothelial cells depends nonlinearly on the loading rate and follows a weak power-law. In addition, regions of different viscous response at varying indentation depth could be determined. Based on the results we obtained, a general route map for AFM users for design of cell mechanics experiments was described.
Imaging articular cartilage tissue using atomic force microscopy (AFM)
Cold Spring Harbor protocols, 2010
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 na...
Atomic Force Microscopy in Bioengineering Applications
2013
The high lateral resolution imaging and its technical versatility in property evaluation, together with the relatively straightforward characterization of viable biological structures in liquid media, render the AFM an unrivaled instrument in the definition of novel structural-functional relationships in bioengineering domains. This chapter provides an overview of the AFM-based techniques employed in the analysis of biological structures and biomaterials. A brief introduction to the working principles of the AFM is followed by a description of application developments. Relevant findings on the structural and functional characterization of biomaterials and biological structures at submicrometric scales are highlighted. B. Bhushan (ed.
In this study, we present a new technique for the structural analysis of the collagen compound in historic tissues. We therefore used atomic force microscopy (AFM), a new high resolution technique which offers significant information on the fibrillar assembly and ultrastructure of collagen fibrils, which may provide insight into both the physiological and eventually pathogenic pattern of collagen fibrils, but also into possible diagenetic destructive changes of those fibrils. AFM figures three-dimensionally the surface of a sample with high resolution down to a nanometer scale. In our investigation we used the AFM to image paraffin embedded tissue sections from femoral bone tissue of a recent case and an age determined historic sample in ambient conditions. With this technique we were able to identify unambiguously collagen bundles and to determine their diameter. These results led us to differentiate the bundling pattern of collagen type I from that of collagen type II. In addition, we identified collagen type I in the historic sample, which provided a fibrillar pattern as that of recent bone. The results were compared to standard immunohistochemical staining techniques of the respective collagen types. In conclusion, our study presents circumstantial evidence that AFM analysis as a novel morphological technique can successfully be applied to historic tissue specimens.
Surface nanoscale imaging of collagen thin films by Atomic Force Microscopy
Materials Science and Engineering: C, 2013
Collagen, the most abundant protein in mammals, due to its unique properties is widely used as biomaterial, scaffold and culture substrate for cell and tissue regeneration studies. Since the majority of biological reactions occur on surfaces and structures at the nanoscale level it is of great importance to image the nanostructural surface of collagen based materials. The aim of this paper was to characterize, with Atomic Force Microscopy (AFM), collagen thin films formed on different substrates (glass, mica, polystyrene latex particle surfaces) and correlate their morphology with the used substrates, formation methodologies (spin coating, hydrodynamic flow) and original collagen solution. The results demonstrated that, by altering a number of parameters, it was possible to control the formation of collagen nanostructured films consisting of naturally occurring fibrils. The spin coating procedure enabled the formation of films with random oriented fibrils, while substrates influenced the fibril packing and surface roughness. The hydrodynamic flow was used for guiding fibril major orientation, while adsorption time, rinsing with buffer and solution concentration influenced the fibril orientation. The clarification of the contribution that different parameters had on thin film formation will enable the design and control of collagen nanobiomaterials with pre-determined characteristics.
Measurements of mechanical parameters of biological structures with atomic force microscope
Atomic force microscopy has been increasingly used for the measurement of mechanical parameters of biological materials in addition to imaging them. This article reviews recent contributions to the development of the methods used for such measurements and reliable interpretations of the data obtained. Kinds of mechanical properties that have attracted the attention of users of atomic force microscope include Young's modulus, binding force between single pairs of ligands and receptors, antigen and antibody binding, internal cohesive force of protein molecules, and the force of base pairing in double helical DNA. The mechanical manipulation of soft (or compliant to be exact) biological materials with atomic force microscope has also been attempted on chromosomes, cells, and DNA. Some of the recent work in chromosomal manipulation will be reviewed.
Observation of geometric structure of collagen molecules by atomic force microscopy
Applied Biochemistry and Biotechnology, 1998
Atomic force microscopy was used to study the geometric structure of collagen fibrils and molecules of rat calcanean tendon tissues. The authors found that the diameter of the fibrils ranged from 124 to 170 nm, and their geometric form suggested a helical winding with spectral period from 59.4 to 61.7 nm, close to the band dimensions reported by electron microscopy. At high magnification, the surface of these bands revealed images that probably correspond to the almost crystalline array of collagen molecules, with the triple helix structure almost visible. The typical helix width is 1.43 nm, with main periods of 1.15 and 8.03 nm, very close to the dimensions reported by X-ray diffraction.
Calibration of colloidal probes with atomic force microscopy for micromechanical assessment
Journal of the mechanical behavior of biomedical materials, 2018
Mechanical assessment of biological materials and tissue-engineered scaffolds is increasingly focusing at lower length scale levels. Amongst other techniques, atomic force microscopy (AFM) has gained popularity as an instrument to interrogate material properties, such as the indentation modulus, at the microscale via cantilever-based indentation tests equipped with colloidal probes. Current analysis approaches of the indentation modulus from such tests require the size and shape of the colloidal probe as well as the spring constant of the cantilever. To make this technique reproducible, there still exist the challenge of proper calibration and validation of such mechanical assessment. Here, we present a method to (a) fabricate and characterize cantilevers with colloidal probes and (b) provide a guide for estimating the spring constant and the sphere diameter that should be used for a given sample to achieve the highest possible measurement sensitivity. We validated our method by tes...