GC and MAGO acknowledge all members of the CeMi. MSS acknowledges support via an EPSRC Programme Grant (EP/P001114/1).
GC: software (contribution to software development and algorithms), formal analysis (analysis of nanoindentation data), validation, Investigation (nanoindentation experiments on polyacrylamide gels), data curation, writing (original draft, review and editing), visualization (figures and graphs). MAGO: investigation (preparation of gels and cells samples, nanoindentation experiments on cells), writing (original draft, review and editing), visualization (figures and graphs). NA: validation, writing (review and editing). IL: software (contribution to software development and algorithms), validation, writing (review and editing); MV: conceptualization, software (design and development of original software and algorithms), validation, resources, writing (original draft, review and editing), supervision, project administration, funding acquisition MSS: resources, writing (review and editing), supervision, project administration, funding acquisition. All authors read and approved the final manuscript.&#160;&#160;

The authors have nothing to disclose.

The fundamental role of mechanics in biology is nowadays established1,2. From whole tissues to single cells, mechanical properties can inform about the pathophysiological state of the biomaterial under investigation3,4. For example, breast tissue affected by cancer is stiffer than healthy tissue, a concept that is the basis of the popular palpation test5. Notably, it has been recently shown that the coronavirus disease 2019 (COVID-19) caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is underlined by changes in the mechanical properties of blood cells, including decreased erythrocyte deformability and decreased lymphocyte and neutrophil stiffness as compared to blood cells from SARS-CoV-2-na&#239;ve individuals6.
In general, the mechanics of cells and tissues are inherently intertwined: each tissue has specific mechanical properties that simultaneously influence and depend on those of the constituent cells and extracellular matrix (ECM)5. Because of this, strategies to study mechanics in biology often involve engineering substrates with physiologically relevant mechanical stimuli to elucidate cell behavior in response to those stimuli. For example, the seminal work by Engler and colleagues demonstrated that mesenchymal stem cell linage commitment is controlled by matrix elasticity, as studied on soft and stiff two-dimensional polyacrylamide (PAAm) hydrogels7.
Many strategies to mechanically characterize the biomaterial under investigation exist, varying in spatial scale (i.e., local to bulk) and in the mode of deformation (e.g., axial vs shear), consequently yielding different information, which needs careful interpretation3,8,9,10. The mechanics of soft biomaterials is commonly expressed in terms of stiffness. However, stiffness depends on both material properties and geometry, whereas elastic moduli are fundamental properties of a material and are independent of the material&#39;s geometry11. As such, different elastic moduli are related to the stiffness of a given sample, and each elastic modulus encompasses the material&#39;s resistance to a specific mode of deformation (e.g., axial vs shear) under different boundary conditions (e.g., free expansion vs confinement)11,12. Nanoindentation experiments allow the quantification of mechanical properties through the E which is associated with uniaxial deformation (indentation) when the biomaterial is not laterally confined10,11,12.
The most popular method to quantify&#160;E of biological systems at the microscale is AFM13,14,15,16. AFM is an extremely powerful tool with force resolution down to the pN&#160;level and spatial resolution down to the sub-nm scale. Further, AFM offers extreme flexibility in terms of coupling with complementary optical and mechanical tools, extending its capabilities to extract a wealth of information from the biomaterial under investigation13. Those attractive features, however, come with a barrier-to-entry represented by the complexity of the experimental set-up. AFM requires extensive training before users can acquire robust data, and its use for everyday mechanical characterization of biological materials is often unjustified, especially when its unique force and spatial resolutions are not required.
Because of this, a new class of nanoindenters has recently gained popularity due to their ease of use, while still offering AFM-comparable data with sub-nN force resolution and &#181;m spatial resolution, reflecting forces exerted and perceived by cells over relevant length scales2. Particularly, ferrule-top nanoindentation devices based on optical fiber sensing technology17,18 have gained popularity among researchers active in the field of mechanobiology and beyond; and a wealth of works reporting the mechanical properties of biomaterials using these devices, including cells19,20, hydrogels8,21, and tissues22,23 have been published. Despite the capabilities of these systems to probe local dynamic mechanical properties (i.e., storage and loss modulus), quasi-static experiments yielding E remain the most popular choice8,19,20,21. In brief, quasi-static nanoindentation experiments&#160;consists of indenting the sample with a constant speed up to a set-point defined either by a maximum displacement, force, or indentation depth, and recording both the force and the vertical position of the cantilever in so-called force-distance (F-z) curves. F-z curves are then converted into force-indentation (F-&#948;) curves through the identification of the contact point (CP), and fitted with an appropriate contact mechanics model (usually the Hertz model13) to compute E.
While the operation of ferrule-top nanoindenters resembles AFM measurements, there are specificities worth considering. In this work, a step-by-step guide to robustly acquire F-z curves from cells and tissue-mimicking hydrogels using a commercially available ferrule-top nanoindenter is provided, in order to encourage standardization of experimental procedures between research groups using this and other similar devices. In addition, advice on how to best prepare hydrogel samples and cells to perform nanoindentation experiments is given, together with troubleshooting tips along the experimental pathway.
Furthermore, much of the variability in nanoindentation results (i.e., E and its distribution) depends on the specific procedure used to analyze data, which is non-trivial. To address this issue, instructions for the use of a newly developed open-source software programmed in Python and equipped with a user-friendly graphical user interface (GUI) for batch analysis of F-z curves are provided. The software allows for fast data screening, filtering of data, computation of the CP through different numerical procedures, the conventional computation of E, as well a more advanced analysis named the elasticity spectra24, allowing to estimate the cell&#39;s bulk Young&#39;s modulus, actin cortex&#39;s Young&#39;s modulus, and actin cortex&#39;s thickness. The software can be freely downloaded from GitHub and can be easily adapted to analyze data originating from other systems by adding an appropriate data parser. It is emphasized that this protocol can be used for other ferrule-top nanoindentation devices, and other nanoindentation devices in general, granted some steps are adapted according to the specific instrument&#39;s guidelines. The protocol is schematically summarized in Figure 1.

<table><tbody><tr><td>12 mm coverslips</td><td>VWR</td><td>631-1577P</td><td></td></tr><tr><td>35 mm cell treated culture dishes</td><td>Greiner CELLSTAR</td><td>627160</td><td></td></tr><tr><td>Acrylamide</td><td>Sigma-Aldrich</td><td>A4058</td><td></td></tr><tr><td>Acrylsilane</td><td>Alfa Aesar</td><td>L16400</td><td></td></tr><tr><td>Ammonium Persulfate</td><td>Merk</td><td>7727-54-0</td><td></td></tr><tr><td>Bisacrylamide</td><td>Merk</td><td>110-26-9</td><td></td></tr><tr><td>Chiaro nanoindenter</td><td>Optics 11 Life</td><td></td><td>&amp;nbsp;no catologue number</td></tr><tr><td>Ethanol</td><td></td><td></td><td>general</td></tr><tr><td>Fetal bovine serum</td><td>Gibco</td><td>16140071</td><td></td></tr><tr><td>High glucose DMEM</td><td>Gibco</td><td>11995065</td><td></td></tr><tr><td>Isopropanol</td><td></td><td></td><td>general</td></tr><tr><td>Kimwipe</td><td>Kimberly Clark</td><td>21905-026</td><td></td></tr><tr><td>Microscope glass slides</td><td>VWR</td><td>631-1550P</td><td></td></tr><tr><td>MilliQ system</td><td>Merk Millipore</td><td>ZR0Q008WW</td><td></td></tr><tr><td>OP1550 Interferometer</td><td>Optics11 Life</td><td></td><td>no catalogue number</td></tr><tr><td>Optics 11 Life probe (k = 0.02-0.005 N/m, R = 3-3.5 um)</td><td>Optics 11 Life</td><td></td><td>no catologue number</td></tr><tr><td>Optics 11 Life probe (k = 0.46-0.5 N/m, R = 50-55 um)</td><td>Optics 11 Life</td><td></td><td>no catologue number</td></tr><tr><td>Penicillin/Streptomycin</td><td>Gibco</td><td>15140122</td><td></td></tr><tr><td>RainX rain repellent</td><td>RainX</td><td>26012</td><td></td></tr><tr><td>Standard petri dishes (90 mm)</td><td>Thermo Scientific</td><td>101RTIRR</td><td></td></tr><tr><td>Tetramethylethylenediamine</td><td>Sigma-Aldrich</td><td>110-18-9</td><td></td></tr><tr><td>Vaccum dessicator</td><td>Thermo Scientific</td><td>531-0250</td><td></td></tr><tr><td>&lt;strong&gt;Software&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>Data acquisition software (v 3.4.1)</td><td>Optics 11 Life</td><td></td><td></td></tr><tr><td>GitHub Desktop (Optional)</td><td>Microsoft</td><td></td><td></td></tr><tr><td>Python 3</td><td>Python Software Foundation</td><td></td><td></td></tr><tr><td>Visual Studio Code (Optional)</td><td>Microsoft</td><td></td><td></td></tr></tbody></table>

experimental and data analysis workflow for soft matter nanoindentation

Nanoindentation refers to a class of experimental techniques where a micrometric force probe is used to quantify the local mechanical properties of soft biomaterials and cells. This approach has gained a central role in the fields of mechanobiology, biomaterials design&#160;and tissue engineering, to obtain a proper mechanical characterization of soft materials with a resolution comparable to the size of single cells (&#956;m). The most popular strategy to acquire such experimental data is to employ an atomic force microscope (AFM); while this instrument offers an unprecedented resolution in force (down to pN) and space (sub-nm), its usability is often limited by its complexity that prevents routine measurements of integral indicators of mechanical properties, such as Young&#39;s Modulus (E). A new generation of nanoindenters, such as those based on optical fiber sensing technology, has recently gained popularity for its ease of integration while allowing to apply sub-nN forces with &#181;m spatial resolution, therefore being suitable to probe local mechanical properties of hydrogels and cells.
In this protocol, a step-by-step guide detailing the experimental procedure to acquire nanoindentation data on hydrogels and cells using a commercially available ferrule-top optical fiber sensing nanoindenter is presented. Whereas some steps are specific to the instrument used herein, the proposed protocol can be taken as a guide for other nanoindentation devices, granted some steps are adapted according to the manufacturer&#39;s guidelines. Further, a new open-source Python software equipped with a user-friendly graphical user interface for the analysis of nanoindentation data is presented, which allows for screening of incorrectly acquired curves, data filtering, computation of the contact point through different numerical procedures, the conventional computation of E, as well as a more advanced analysis particularly suited for single-cell nanoindentation data.

Watch this Scientific Journal Video about Experimental and Data Analysis Workflow for Soft Matter Nanoindentation at JoVE.com

Experimental and Data Analysis Workflow for Soft Matter Nanoindentation

The protocol presents a complete workflow for soft material nanoindentation experiments, including hydrogels and cells. First, the experimental steps to acquire force spectroscopy data are detailed; then, the analysis of such data is detailed through a newly developed open-source Python software, which is free to download from GitHub.

Nanoindentation refers to a class of experimental techniques where a micrometric force probe is used to quantify the local mechanical properties of soft ...

experimental-data-analysis-workflow-for-soft-matter

Universidad San Sebastian

Research

JoVE Journal

Bioengineering

3.7K Views.  University of Glasgow. The protocol presents a complete workflow for soft material nanoindentation experiments, including hydrogels and cells. First, the experimental steps to acquire force spectroscopy data are detailed; then, the analysis of such data is detailed through a newly developed open-source Python software, which is free to download from GitHub.

Video: Experimental and Data Analysis Workflow for Soft Matter Nanoindentation

Soft Lithographic Functionalization and Patterning Oxide-free Silicon and Germanium

The development of hybrid electronic devices relies in large part on the integration of (bio)organic materials and inorganic semiconductors through a stable interface that permits efficient electron transport and protects underlying substrates from oxidative degradation. Group IV semiconductors can be effectively protected with highly-ordered self-assembled monolayers (SAMs) composed of simple alkyl chains that act as impervious barriers to both organic and aqueous solutions. Simple alkyl SAMs, however, are inert and not amenable to traditional patterning techniques. The motivation for immobilizing organic molecular systems on semiconductors is to impart new functionality to the surface that can provide optical, electronic, and mechanical function, as well as chemical and biological activity. 
Microcontact printing (&mu;CP) is a soft-lithographic technique for patterning SAMs on myriad surfaces.1-9 Despite its simplicity and versatility, the approach has been largely limited to noble metal surfaces and has not been well developed for pattern transfer to technologically important substrates such as oxide-free silicon and germanium. Furthermore, because this technique relies on the ink diffusion to transfer pattern from the elastomer to substrate, the resolution of such traditional printing is essentially limited to near 1 &mu;m.10-16
In contrast to traditional printing, inkless &mu;CP patterning relies on a specific reaction between a surface-immobilized substrate and a stamp-bound catalyst. Because the technique does not rely on diffusive SAM formation, it significantly expands the diversity of patternable surfaces. In addition, the inkless technique obviates the feature size limitations imposed by molecular diffusion, facilitating replication of very small (&lt;200 nm) features.17-23 However, up till now, inkless &mu;CP has been mainly used for patterning relatively disordered molecular systems, which do not protect underlying surfaces from degradation.
Here, we report a simple, reliable high-throughput method for patterning passivated silicon and germanium with reactive organic monolayers and demonstrate selective functionalization of the patterned substrates with both small molecules and proteins. The technique utilizes a preformed NHS-reactive bilayered system on oxide-free silicon and germanium. The NHS moiety is hydrolyzed in a pattern-specific manner with a sulfonic acid-modified acrylate stamp to produce chemically distinct patterns of NHS-activated and free carboxylic acids. A significant limitation to the resolution of many &mu;CP techniques is the use of PDMS material which lacks the mechanical rigidity necessary for high fidelity transfer. To alleviate this limitation we utilized a polyurethane acrylate polymer, a relatively rigid material that can be easily functionalized with different organic moieties. Our patterning approach completely protects both silicon and germanium from chemical oxidation, provides precise control over the shape and size of the patterned features, and gives ready access to chemically discriminated patterns that can be further functionalized with both organic and biological molecules. The approach is general and applicable to other technologically-relevant surfaces.

Here we describe a simple method for patterning oxide-free silicon and germanium with reactive organic monolayers and demonstrate functionalization of the patterned substrates with small molecules and proteins. The approach completely protects surfaces from chemical oxidation, provides precise control over feature morphology, and provides ready access to chemically discriminated patterns.

The development of hybrid electronic devices relies in large part on the integration of (bio)organic materials and inorganic semiconductors through a ...

Self-reporting Scaffolds for 3-Dimensional Cell Culture

Culturing cells in 3D on appropriate scaffolds is thought to better mimic the in vivo microenvironment and increase cell-cell interactions. The resulting 3D cellular construct can often be more relevant to studying the molecular events and cell-cell interactions than similar experiments studied in 2D. To create effective 3D cultures with high cell viability throughout the scaffold the culture conditions such as oxygen and pH need to be carefully controlled as gradients in analyte concentration can exist throughout the 3D construct. Here we describe the methods of preparing biocompatible pH responsive sol-gel nanosensors and their incorporation into poly(lactic-co-glycolic acid) (PLGA) electrospun scaffolds along with their subsequent preparation for the culture of mammalian cells. The pH responsive scaffolds can be used as tools to determine microenvironmental pH within a 3D cellular construct. Furthermore, we detail the delivery of pH responsive nanosensors to the intracellular environment of mammalian cells whose growth was supported by electrospun PLGA scaffolds. The cytoplasmic location of the pH responsive nanosensors can be utilized to monitor intracellular pH (pHi) during ongoing experimentation.

Biocompatible pH responsive sol-gel nanosensors can be incorporated into poly(lactic-co-glycolic acid) (PLGA) electrospun scaffolds. The produced self-reporting scaffolds can be used for in&#160;situ monitoring of microenvironmental conditions whilst culturing cells upon the scaffold. This is beneficial as the 3D cellular construct can be monitored in real-time without disturbing the experiment.

Culturing cells in 3D on appropriate scaffolds is thought to better mimic the in vivo microenvironment and increase cell-cell interactions. The resulting ...

Quantitative Hardness Measurement by Instrumented AFM-indentation

In this work, a combination of amplitude-modulated non-contact atomic force microscopy and atomic force spectroscopy is applied for instrumented hardness measurements on an Au(111) surface with atomistic resolution of single plasticity events. A careful experimental procedure is described that includes the force sensor selection, its calibration, the calibration of the cantilever deflection detection system, and the minimization of instrumental drift for accurate and reproducible force-distance measurements. Also, a method for the data analysis is presented that allows the extraction of force-penetration curves from recorded force-distance curves. A typical curve displays a clear elastic deformation regime up to the first plasticity event, or pop-in, with a length in the range of one to two Burger's vectors. Later plasticity events exhibit the same magnitude. The work of plasticity is further extracted from the measurements. Finally, the hardness is determined in combination with the indentation curve using non-contact atomic force microscopy images of the remaining indents.

This experimental protocol describes how atomic force microscopy can be used to measure hardness at the true nanometer scale and to detect single atomistic plasticity events.

In this work, a combination of amplitude-modulated non-contact atomic force microscopy and atomic force spectroscopy is applied for instrumented hardness ...

Engineering

Characterization Of Multi-layered Fish Scales (Atractosteus spatula) Using Nanoindentation, X-ray CT, FTIR, and SEM

The hierarchical architecture of protective biological materials such as mineralized fish scales, gastropod shells, ram&#8217;s horn, antlers, and turtle shells provides unique design principles with potentials for guiding the design of protective materials and systems in the future. Understanding the structure-property relationships for these material systems at the microscale and nanoscale where failure initiates is essential. Currently, experimental techniques such as nanoindentation, X-ray CT, and SEM provide researchers with a way to correlate the mechanical behavior with hierarchical microstructures of these material systems1-6. However, a well-defined standard procedure for specimen preparation of mineralized biomaterials is not currently available. In this study, the methods for probing spatially correlated chemical, structural, and mechanical properties of the multilayered scale of A. spatula using nanoindentation, FTIR, SEM, with energy-dispersive X-ray (EDX) microanalysis, and X-ray CT are presented.

Characterization Of Multi-layered Fish Scales Atractosteus spatula Using Nanoindentation, X-ray CT, FTIR, and SEM

This paper presents the methods used for probing spatially correlated chemical, structural, and mechanical properties of the multilayered scale of Atractosteus spatula (A. spatula) using nanoindentation, Fourier transform infrared (FTIR) spectroscopy, scanning electron microscopy (SEM), and X-ray computed tomography (X-ray CT). The experimental results have been used to investigate the design principles of protective biological materials.

The hierarchical architecture of protective biological materials such as mineralized fish scales, gastropod shells, ram&#8217;s horn, antlers, and turtle ...

Atomic Force Microscopy Cantilever-Based Nanoindentation: Mechanical Property Measurements at the Nanoscale in Air and Fluid

An atomic force microscope (AFM) fundamentally measures the interaction between a nanoscale AFM probe tip and the sample surface. If the force applied by the probe tip and its contact area with the sample can be quantified, it is possible to determine the nanoscale mechanical properties (e.g., elastic or Young&#39;s modulus) of the surface being probed. A detailed procedure for performing quantitative AFM cantilever-based nanoindentation experiments is provided here, with representative examples of how the technique can be applied to determine the elastic moduli of a wide variety of sample types, ranging from kPa to GPa. These include live mesenchymal stem cells (MSCs) and nuclei in physiological buffer, resin-embedded dehydrated loblolly pine cross-sections, and Bakken shales of varying composition.
Additionally, AFM cantilever-based nanoindentation is used to probe the rupture strength (i.e., breakthrough force) of phospholipid bilayers. Important practical considerations such as method choice and development, probe selection and calibration, region of interest identification, sample heterogeneity, feature size and aspect ratio, tip wear, surface roughness, and data analysis and measurement statistics are discussed to aid proper implementation of the technique. Finally, co-localization of AFM-derived nanomechanical maps with electron microscopy techniques that provide additional information regarding elemental composition is demonstrated.

Quantifying the contact area and force applied by an atomic force microscope (AFM) probe tip to a sample surface enables nanoscale mechanical property determination. Best practices to implement AFM cantilever-based nanoindentation in air or fluid on soft and hard samples to measure elastic modulus or other nanomechanical properties are discussed.

An atomic force microscope (AFM) fundamentally measures the interaction between a nanoscale AFM probe tip and the sample surface. If the force applied by ...

A Virtual Simulation Experiment of Mechanics: Material Deformation and Failure Based on Scanning Electron Microscopy

This work presents a set of comprehensive virtual experiments to detect material deformation and failure. The most commonly used pieces of equipment in mechanics and material disciplines, such as a metallographic cutting machine and a high-temperature universal creep testing machine, are integrated into a web-based system to provide different experimental services to users in an immersive and interactive learning environment. The protocol in this work is divided into five subsections, namely, the preparation of the materials, molding the specimen, specimen characterization, specimen loading, nanoindenter installation, and SEM in situ experiments, and this protocol aims to provide an opportunity for users regarding the recognition of different equipment and the corresponding operations, as well as the enhancement of laboratory awareness, etc., using a virtual simulation approach. To provide clear guidance for the experiment, the system highlights the equipment/specimen to be used in the next step and marks the pathway that leads to the equipment with a conspicuous arrow. To mimic the hands-on experiment as closely as possible, we designed and developed a three-dimensional laboratory room, equipment, operations, and experimental procedures. Moreover, the virtual system also considers interactive exercises and registration before using chemicals during the experiment. Incorrect operations are also allowed, resulting in a warning message informing the user. The system can provide interactive and visualized experiments to users at different levels.

This work presents a three-dimensional virtual simulation experiment for material deformation and failure that provides visualized experimental processes. Through a set of experiments, users can become familiar with the equipment and learn the operations in an immersive and interactive learning environment.

This work presents a set of comprehensive virtual experiments to detect material deformation and failure. The most commonly used pieces of equipment in ...

Characterizing Multiscale Mechanical Properties of Brain Tissue Using Atomic Force Microscopy, Impact Indentation, and Rheometry

To design and engineer materials inspired by the properties of the brain, whether for mechanical simulants or for tissue regeneration studies, the brain tissue itself must be well characterized at various length and time scales. Like many biological tissues, brain tissue exhibits a complex, hierarchical structure. However, in contrast to most other tissues, brain is of very low mechanical stiffness, with Young&#39;s elastic moduli E on the order of 100s of Pa. This low stiffness can present challenges to experimental characterization of key mechanical properties. Here, we demonstrate several mechanical characterization techniques that have been adapted to measure the elastic and viscoelastic properties of hydrated, compliant biological materials such as brain tissue, at different length scales and loading rates. At the microscale, we conduct creep-compliance and force relaxation experiments using atomic force microscope-enabled indentation. At the mesoscale, we perform impact indentation experiments using a pendulum-based instrumented indenter. At the macroscale, we conduct parallel plate rheometry to quantify the frequency dependent shear elastic moduli. We also discuss the challenges and limitations associated with each method. Together these techniques enable an in-depth mechanical characterization of brain tissue that can be used to better understand the structure of brain and to engineer bio-inspired materials.

We present a set of techniques to characterize the viscoelastic mechanical properties of brain at the micro-, meso-, and macro-scales.

To design and engineer materials inspired by the properties of the brain, whether for mechanical simulants or for tissue regeneration studies, the brain ...

Neuroscience

Viscoelastic Characterization of Soft Tissue-Mimicking Gelatin Phantoms using Indentation and Magnetic Resonance Elastography

Characterization of biomechanical properties of soft biological tissues is important to understand the tissue mechanics and explore the biomechanics-related mechanisms of disease, injury, and development. The mechanical testing method is the most straightforward way for tissue characterization and is considered as verification for in vivo measurement. Among the many ex vivo mechanical testing techniques, the indentation test provides a reliable way, especially for samples that are small, hard to fix, and viscoelastic such as brain tissue. Magnetic resonance elastography (MRE) is a clinically used method to measure the biomechanical properties of soft tissues. Based on shear wave propagation in soft tissues recorded using MRE, viscoelastic properties of soft tissues can be estimated in vivo based on wave equation. Here, the viscoelastic properties of gelatin phantoms with two different concentrations were measured by MRE and indentation. The protocols of phantom fabrication, testing, and modulus estimation have been presented.

This article presents a demonstration and summary of protocols of making gelatin phantoms that mimic soft tissues, and the corresponding viscoelastic characterization using indentation and magnetic resonance elastography.

Characterization of biomechanical properties of soft biological tissues is important to understand the tissue mechanics and explore the ...

Addressing Practical Issues in Atomic Force Microscopy-Based Micro-Indentation on Human Articular Cartilage Explants

Without a doubt, atomic force microscopy (AFM) is currently one of the most powerful and useful techniques to assess micro and even nano-cues in the biological field. However, as with any other microscopic approach, methodological challenges can arise. In particular, the characteristics of the sample, sample preparation, type of instrument, and indentation probe can lead to unwanted artifacts. In this protocol, we exemplify these emerging issues on healthy as well as osteoarthritic articular cartilage explants. To this end, we first show via a step-by-step approach how to generate, grade, and visually classify ex vivo articular cartilage discs according to different stages of degeneration by means of large 2D mosaic fluorescence imaging of the whole tissue explants. The major strength of the ex vivo model is that it comprises aged, native, human cartilage that allows the investigation of osteoarthritis-related changes from early onset to progression. In addition, common pitfalls in tissue preparation, as well as the actual AFM procedure together with the subsequent data analysis, are also presented. We show how basic but crucial steps such as sample preparation and processing, topographic sample characteristics caused by advanced degeneration, and sample-tip interaction can impact data acquisition. We also subject to scrutiny the most common problems in AFM and describe, where possible, how to overcome them. Knowledge of these limitations is of the utmost importance for correct data acquisition, interpretation, and, ultimately, the embedding of findings into a broad scientific context.

We present a step-by-step approach to identify and address the most common problems associated with atomic force microscopy micro-indentations. We exemplify the emerging problems on native human articular cartilage explants characterized by various degrees of osteoarthritis-driven degeneration.

Without a doubt, atomic force microscopy (AFM) is currently one of the most powerful and useful techniques to assess micro and even nano-cues in the ...

Multimodal Analysis of Bone Formation Using Explant Models

Comprehensive Characterization of Tissue Mineralization in an Ex Vivo Model

The extensive characterization of tissue mineralization in the context of bone regeneration represents a significant challenge, given the numerous modalities that are currently available for analysis. Here, we propose a workflow for a comprehensive evaluation of new bone formation using a relevant large animal osseous ex vivo explant. A bone defect (diameter = 3.75 mm; depth = 5.0 mm) is created in an explanted sheep femoral head and injected with a macroporous bone substitute loaded with a pro-osteogenic growth factor (bone morphogenetic protein 2 - BMP2). Subsequently, the explant is maintained in culture for a 28-day period, allowing cellular colonization and subsequent bone formation. To evaluate the quality and structure of newly mineralized tissue, the following successive methods are set up: (i) Characterization and high-resolution 3D images of the entire explant using micro-CT, followed by deep learning image analyses to enhance the discrimination of mineralized tissues; (ii) Nano-indentation to determine the mechanical properties of the newly formed tissue; (iii) Histological examinations, such as Hematoxylin/Eosin/Saffron (HES), Goldner's trichrome, and Movat's pentachrome to provide a qualitative assessment of mineralized tissue, particularly with regard to the visualization of the osteoid barrier and the presence of bone cells; (iv) Back-scattering scanning electron microscopy (SEM) mapping with internal reference to quantify the degree of mineralization and provide detailed insights into surface morphology, mineral composition, and bone-biomaterial interface; (v) Raman spectroscopy to characterize the molecular composition of the mineralized matrix and to provide insights into the persistence of BMP2 within the cement through the detection of peptide bonds. This multimodal analysis will provide an effective assessment of newly formed bone and comprehensive qualitative and quantitative insights into mineralized tissues. Through the standardization of these protocols, we aim to facilitate interstudy comparisons and improve the validity and reliability of research findings.

Author Spotlight: Advanced Techniques for Characterizing Tissue Mineralization in Bone Regeneration Research

The proposed protocol entails a global approach to assess bone formation in the context of bone regeneration using multimodal analyses. It aims to provide qualitative and quantitative information on new bone formation, enhancing the rigor and validity of basic and pre-clinical investigations.