We thank the orthopedic surgeons from the Department of Orthopaedic Surgery of the University Hospital of Tuebingen for providing the tissue samples.

Femoral condyles collected from patients undergoing total knee arthroplasty at the University Hospital of T&#252;bingen, Germany, were used. Only articular cartilage samples from patients with degenerative and posttraumatic joint pathologies were included in this study. Departmental, institutional, as well as local ethical committee approval were obtained before the commencement of the study (Project no.674/2016BO2). Written informed consent was received from all patients before participation.
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The authors have nothing to disclose.

As a progressive and multifactorial disease, OA triggers structural and functional changes in the articular cartilage.Throughout the course of OA, impairments in mechanical features are accompanied by structural and biochemical changes at the surface of the articular cartilage27,31. The earliest pathological events occurring in OA are proteoglycan depletion coupled with collagen network disruption32,33<su...

Due to the ever-shrinking size of electronic devices and systems, the rapid development of micro- and nano-based technology and equipment has gained momentum. One such device is atomic force microscopy (AFM), which can scan biological surfaces and retrieve topographic or biomechanical information at both nano- and micrometer scales1,2. Among its vast features, this tool can be operated as a micro- as well as a nano-indenter to obtain information about the mechanical properties of various biological systems3,4,5,6. The data are collected by physical contact with the surface through a mechanical probe, which can be as small as about 1 nm at its tip7. The resulting deformation of the sample is then displayed based on the indentation depth of the cantilever tip and the force applied on the sample8.
Osteoarthritis (OA) is a long-term degenerative chronic disease characterized by deterioration of the articular cartilage in the joints and surrounding tissues, which can lead to complete exposure of the bone surfaces. The burden of OA is substantial; currently, half of all women and one-third of all men aged 65 and over suffer from OA9. Traumas, obesity, and the resulting altered biomechanics of the joint10&#160;determine the articular cartilage degeneration, which is viewed as a common end result. The pioneering study of Ganz et al. posited that the early steps of the OA process may involve the biomechanical properties of cartilage11, and since then researchers have confirmed this hypothesis12. Likewise, it is generally accepted that the biomechanical properties of the tissue are functionally orchestrated by the ultrastructural organization as well as cell-cell and cell-matrix crosstalk. Any alterations can dramatically impact the overall tissue biomechanical functioning13. To date, OA diagnosis is clinical and is based on plain film radiography14. This approach is two-sided: firstly, the lack of a defined degenerative cut-off threshold to formulate the diagnosis of OA makes the condition difficult to quantify, and, secondly, imaging methods lack sensitivity and standardization and cannot detect localized cartilage damage15,16,17. To this end, the assessment of the mechanical properties of the cartilage has the decisive advantage that it describes a parameter that changes during the course of OA regardless of the etiology of the disease and has a direct influence on tissue functionality at a very early stage. Indentation instruments measure the force by which the tissue resists the indentation. This is, in fact, not a new concept; the earliest studies date back to the 1980s and 1990s. In this period, numerous studies suggested that indentation instruments designed for the arthroscopic measurements of articular cartilage could be well suited to detect degenerative changes in the cartilage. Even 30 years ago, some studies were able to demonstrate that indentation instruments were able to detect in vivo changes in the cartilage surface during tissue degeneration by conducting compressive stiffness measurements during arthroscopy18,19,20.
AFM indentation (AFM-IT) of the articular cartilage provides information about a pivotal mechanical property of the tissue, namely, stiffness. This is a mechanical parameter that describes the relation between an applied, nondestructive load and the resultant deformation of the indented tissue area21. AFM-IT has been shown to be capable of quantifying age-dependent modifications in stiffness in macroscopically unaffected collagen networks, thus, differentiating between the pathological changes associated with OA onset (grade 0 on the Outerbridge scale in articular cartilage)22. We have previously shown that AFM-ITs, on the basis of spatial chondrocyte organization as an image-based biomarker for early cartilage degeneration, allow for not only quantifying but also actually pinpointing the earliest degenerative mechanical changes. These findings have already been confirmed by others23,24. Hence, AFM-IT acts as an interesting tool to diagnose and identify early degenerative changes. These changes can be already measured at a cellular level, reshaping the understanding of the OA pathophysiological process.
In this protocol, we demonstrate a complete histological and biomechanical grading procedure of articular cartilage explants, from native cartilage explant preparation to AFM data acquisition and processing. Through a step-by-step approach, we show how to generate, grade, and visually classify articular cartilage tissue according to different stages of degeneration by means of 2D large mosaic imaging, followed by micro-AFM indentations.
Even though, currently, AFM-IT is one of the most sensitive tools to measure biomechanical changes in cartilage7, like any other instrumental technique, it has limitations and practical peculiarities25&#160;that can lead to erroneous data acquisition. To this end, we subject to scrutiny the most common problems that arise during AFM measurements of the cartilage explants and describe, where possible, how to minimize or overcome them. These include topographical aspects of the samples and the difficulties to stabilize them in an AFM-compatible environment, physical peculiarities of the tissue&#39;s surface, and the resulting difficulties in performing AFM measurements on such surfaces. Examples of erroneous force-distance curves are also presented, emphasizing the conditions that may cause them. Additional limitations inherent to the geometry of the cantilever tip and the use of the Hertz model for the data analysis are also discussed.

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>Amphotericin B</td>
			<td>Merck KGaA, Darmstadt, Germany</td>
			<td>1397-89-3</td>
			<td></td>
		</tr>
		<tr>
			<td>Atomic force microscop (AFM) head&#160;</td>
			<td>CellHesion 200, Bruker Nano GmbH, Berlin, Germany</td>
			<td>JPK00518</td>
			<td></td>
		</tr>
		<tr>
			<td>Biocompatible sample glue&#160;</td>
			<td>Bruker Nano GmbH, Berlin, Germany</td>
			<td>H000033</td>
			<td></td>
		</tr>
		<tr>
			<td>Calcein AM</td>
			<td>Cayman, Ann Arbor, Michigan, USA</td>
			<td>14948</td>
			<td>Cell membrane permeable stain, used for cartilage disc sorting- top view imaging</td>
		</tr>
		<tr>
			<td>Cantilever</td>
			<td>Bruker Nano GmbH, Berlin, Germany</td>
			<td>SAA-SPH-5UM</td>
			<td>Frequency Nom: 30KHz, k: 0.2N/m, lenght nom: 115&#956;m, width nom: 40&#956;m,&#160; geometry: rectangular, cylindrical tip with a 5&#956;m end radius</td>
		</tr>
		<tr>
			<td>Cartilage ctting device&#160;</td>
			<td>Self-made&#160;</td>
			<td>n/a</td>
			<td>Cutting plastic device containing predefined wholes of 4mmx1mm</td>
		</tr>
		<tr>
			<td>CDD camera integrated in the AFM</td>
			<td>The Imaging Source Europe GmbH, Bremen, Germany</td>
			<td>DFK 31BF03</td>
			<td></td>
		</tr>
		<tr>
			<td>CDD camera integrated in the fluorescence microscope</td>
			<td>Leica Biosystems, Wetzlar, Germany</td>
			<td>DFC3000G</td>
			<td></td>
		</tr>
		<tr>
			<td>Cryotome</td>
			<td>Leica Biosystems, Wetzlar, Germany</td>
			<td>CM3050S&#160;</td>
			<td></td>
		</tr>
		<tr>
			<td>Data Processing Software for the AFM</td>
			<td>Bruker Nano GmbH, Berlin, Germany</td>
			<td>n/a</td>
			<td>Version 5.0.86,&#160; can be downloaded for free from the following website https://customers.jpk.com</td>
		</tr>
		<tr>
			<td>Dulbecco&#39;s modified Eagle&#39;s medium (DMEM)&#160;</td>
			<td>Gibco, Life Technologies, Darmstadt, Germany</td>
			<td>41966052</td>
			<td></td>
		</tr>
		<tr>
			<td>Fluorescence Microscope (Leica DMi8)</td>
			<td>Leica Biosystems, Wetzlar, Germany</td>
			<td>11889113</td>
			<td></td>
		</tr>
		<tr>
			<td>Glass block cantiliver holder</td>
			<td>Bruker Nano GmbH, Berlin, Germany</td>
			<td>SP-90-05</td>
			<td>Extra long glass block with angled faces, designed especially for the use with the JPK PetriDishHeaterTM (Bruker).</td>
		</tr>
		<tr>
			<td>Inverted phase contrast microscope (integrated in the AFM)</td>
			<td>AxioObserver D1, Carl Zeiss Microscopy, Jena, Germany</td>
			<td>L201306_03</td>
			<td></td>
		</tr>
		<tr>
			<td>Leibovitz&#39;s L-15 medium without L-glutamine&#160;</td>
			<td>Merck KGaA, Darmstadt, Germany</td>
			<td>F1315</td>
			<td></td>
		</tr>
		<tr>
			<td>Microscope glass slides</td>
			<td>Sigma-Aldrich, St. Louis, Missouri, USA</td>
			<td>CLS294775X50</td>
			<td></td>
		</tr>
		<tr>
			<td>Mounting medium With DAPI</td>
			<td>ibidi GmbH, Gr&#228;felfing, Germany</td>
			<td>50011</td>
			<td>Mounting media with nuclear DAPI (4&#8242;,6-diamidino-2-phenylindole) counterstaining used for cartilage discs&#160; side view imaging</td>
		</tr>
		<tr>
			<td>Penicillin-Streptomycin</td>
			<td>Sigma-Aldrich, St. Louis, Missouri, USA</td>
			<td>P4333</td>
			<td></td>
		</tr>
		<tr>
			<td>Petri dish heater associated with AFM (Petri Dish Heater)</td>
			<td>Bruker Nano GmbH, Berlin, Germany</td>
			<td>T-05-0117</td>
			<td></td>
		</tr>
		<tr>
			<td>Scalpel</td>
			<td>Feather Medical Products, Osaka, Japan</td>
			<td>2023-01</td>
			<td></td>
		</tr>
		<tr>
			<td>Silicone Skirt</td>
			<td>Bruker Nano GmbH, Berlin, Germany</td>
			<td>n/a</td>
			<td>Protective silicone membrane (D55x0.25) which is placed on the basis of the base of the glas block to prevent&#160; medium condensation in the AFM head.</td>
		</tr>
		<tr>
			<td>Statistical program - SPSS</td>
			<td>IBM, Armonk, New York, USA</td>
			<td>SPSS Statistics 22</td>
			<td>Vesion 280.0.0.0 (190)</td>
		</tr>
		<tr>
			<td>Tissue culture dishes&#160;</td>
			<td>TPP Techno Plastic Products AG, Trasadingen, Switzerland</td>
			<td>TPP93040</td>
			<td></td>
		</tr>
		<tr>
			<td>Tissue-tek O.C.T. Compound</td>
			<td>Sakura Finetek, Alphen aan den Rijn, Netherlands</td>
			<td>SA6255012</td>
			<td>Water-soluble embedding medium&#160;</td>
		</tr>
	</tbody>
</table>

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.

Using a self-made cutting device, we were able to explant and generate small (4 mm x 1 mm) cartilage discs from fresh human condyles containing a single cellular spatial pattern30&#160;of single strings (SS, Figure 2A), double strings (DS), small clusters (SC), big clusters (BC; Figure 2A), and diffuse (Figure 2B). A representative cartilage explant is depicted in Figure 3A. The ...

Watch this Scientific Journal Video about Addressing Practical Issues in Atomic Force Microscopy-Based Micro-Indentation on Human Articular Cartilage Explants at JoVE.com

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

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 ...

The goal of this protocol was to create an in vitro model for studying articular cartilage mechanical properties during the onset and the progression of osteoarthritis. The main advantage of the present technique is that the cartilage explants are easy to generate and highly representative of native age cartilage. This model can be used to analyze and monitor various osteoarthritis, diagnostic, and therapeutic approaches at the biomechanical level.To begin, cut the cartilage away from the bone using a scalpel. Using a biopsy punch, generate discs of four millimeter diameter. Place the disc on a custom made cutting device.Using a spatula, fix and stabilize the cartilage disc. Cut the cartilage disc with a razor blade. Using a spatula, collect the disc and place it in a 1.5 milliliter tube.To stay in the cartilage samples, add 130 microliters of cell permeable fluorescence dye to each well of the 96 well-plate and distribute the disc on the plate as one disc per well. Place the 96 well-plate on the plate holder of the fluorescence microscope. Select the appropriate fluorescence filter and the objective.Position the objective underneath the preselected well containing individual cartilage. Focus on the disc to see the cellular pattern. Select the navigator function to get an overview of the entire well.Use the left mouse button and drag it to navigate to a different stage position. Select a square that encompasses the area of interest to be scanned. Select the software's focus map point option and then select each individual tile by left clicking in the center of it.Select the option focus map in the appeared window with all the previously selected tiles. Double click on a tile to display and bring it into proper focus. Next, click set Z to save the focal plan and proceed to the next tile.After adjusting the focal plan for each individual tile begin image acquisition by pressing start scan. Fix each preselected cartilage disc containing a cellular pattern in Petri dishes by adding sufficient biocompatible glue on the top, bottom, left, and right sides of the disc. Cover the discs with with 2.5 milliliters of Leibovitz medium without L-glutamine.Position the Petri dish in the AFM devices sample holder. To calibrate the AFM cantilever, place it on the surface of the glass block cantilever so that it rests on the polished optical plane in the center of the glass block. Lower the cantilever in 100 micrometers steps using the stepper motor function until it is completely submerged in the medium.To identify the desired cartilage measurement site, start a scanner approach with the cantilever on a clean sample free area of the Petri dish. Then retract the cantilever 1.5 millimeters away from the bottom of the plate. Switch from brightfield to fluorescence view and visually identify the top of the disc.Move the AFM sample holder exactly two millimeters toward the middle of the disc. Run a scanner approach to reach the surface of the cartilage disc and retract the cantilever by 100 micrometers. To generate a force distance curve, focus on the cells positioned in the desired measurement site.Click the run button to start the measurements and acquire five force distance curves on each measurement site. Save the curves once they are inspected. To estimate Young's moduli, using the open a batch of spectroscopy curves option, open the generated force distance curves to be analyzed.Select the Hertz-fit model followed by the elasticity-fit option. Then visualize and document Young's modulus. Adapt using the poisson ratio of 0.5 and the appropriate cantilever tip radius.Then visually check the force distance curve fit to ensure correctness. For indentation depth determination, open each of the generated force distance curves in the data analysis software and select the Hertz-fit model as the analysis process. Apply the subtract baseline offset option to zero the vertical deflection axis and select the offset plus tilt function.Use the find contact point function to automatically identify the contact point. Using the vertical tip position function, subtract the distance accounting solely for cantilever deflection from the raw piezo height during the indentation. Select the elasticity-fit option to display the processed force distance curve.Then select the area of the graph so that it lines up with the most negative value on the vertical tip position axis. In the parameter tab from the XMIN box, read and document the indentation. The discs containing single strings showed higher stiffness values with a median of 2.6 kilo pascals, which is representative of uncompromised, healthy cartilage areas.With osteoarthritis onset and progression, the AFM measurements showed a strong stepwise decrease in stiffness of 42%in double strings, 77%in small clusters, and ultimately 88%in advanced stages represented by big clusters. The discs containing a diffuse pattern displayed in elevated elasticity with an important variation of Young's modulus single values. For all the cartilage discs with assigned predominant cellular pattern organization, the indentation depth associated with the employed setpoint was found to be inversely proportional to stiffness.The artifacts seen in the generated force distance curves are indicative of either a suboptimal contact between the AFM cantilever and the cartilage surface or improper sample fixation to the Petri dish. Our cartilage explants can be further processed and analyzed using a variety of molecular biological techniques such as protein analysis via eliza, immuno labeling, western moding, or gen analysis via PCR. This method may be used to study the direct impact of new therapies on articular cartilege and may help researchers to gain important insights into the potoma mechanisms of osteoarthritis.

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Bioengineering

827 visualizações.  University Hospital Tübingen. O objetivo deste protocolo foi criar um modelo in vitro para o estudo das propriedades mecânicas da cartilagem articular durante o início e a progressão da osteoartrite. A principal vantagem da presente técnica é que os explantes cartilaginosos são de fácil geração e altamente representativos da cartilagem nativa da idade. Este modelo pode ser usado para analisar e monitorar várias abordagens osteoartrite, diagnósticas e terapêuticas no nível biomecânico.Para começar, co...