Name: application of atomic force microscopy to detect early osteoarthritis
Uploaded: 2020-05-24T14:00:00
Description: Watch this Scientific Journal Video about Application of Atomic Force Microscopy to Detect Early Osteoarthritis at JoVE.com

We thank our co-authors from the original publication for their help and support.

The human cartilage samples were obtained from patients undergoing total knee arthroplasty in the Department of Orthopaedic Surgery of the University Hospital of Tuebingen, Germany, and the Winghofer-hospital, Rottenburg a.N., Germany, for end-stage OA of the knee. Full departmental, institutional, and local ethical committee approval were obtained before commencement of the study (project number 674/2016BO2). Written informed consent was received from all patients before participation. The methods were carried out in ac...

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A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Horizontally+oriented+clusters+of+multiple+chondrons+in+the+superficial+zone+of+ankle,+but+not+knee+articular+cartilage.">Horizontally oriented clusters of multiple chondrons in the superficial zone of ankle, but not knee articular cartilage.</a> The Anatomical Record. 266 (4), 241-248 (2002).</li><li>Rolauffs, B., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Onset+of+preclinical+osteoarthritis:+the+angular+spatial+organization+permits+early+diagnosis.">Onset of preclinical osteoarthritis: the angular spatial organization permits early diagnosis.</a> Arthritis Rheumatology. 63 (6), 1637-1647 (2011).</li><li>Maver, U., Velnar, T., Gaber&#353;&#269;ek, M., Planin&#353;ek, O., Fin&#353;gar, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Recent+progressive+use+of+atomic+force+microscopy+in+biomedical+applications.">Recent progressive use of atomic force microscopy in biomedical applications.</a> TrAC Trends in Analytical Chemistry. 80, 96-111 (2016).</li><li>Polini, A., Yang, F., Ramalingam, M., Ramakrishna, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Physicochemical+characterization+of+nanofiber+composites.">Physicochemical characterization of nanofiber composites.</a> Nanofiber Composites for Biomedical Applications. , 97-115 (2017).</li><li>Danalache, M., Jacobi, L. 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Using AFM as a novel and powerful technique to measure the biomechanical properties of biological materials at a nanoscale level, we measured the elastic properties of the ECM and PCM in human osteoarthritic articular cartilage. Cartilage samples were selected according to their predominant spatial pattern of chondrocyte organization as an image-based biomarker for local tissue degeneration. As expected, a strong decline in the values of elasticity of both ECM and PCM was observed along spatial chondrocyte reorganization...

Articular cartilage is an avascular, aneural tissue. Sparsely scattered chondrocytes produce, organize, and maintain an expansive extracellular matrix (ECM) into which they are embedded. As a distinct and specialized part of the ECM, chondrocytes are surrounded by a thin layer of specialized matrix known as the pericellular matrix (PCM). The PCM acts as a mechanosensitive cell-matrix interface1 that protects the chondrocytes2 and modulates their biosynthetic response3. As previously described4, in healthy cartilage, chondrocytes are arranged in specific, distinct spatial patterns that are specific for each tissue layer and joint4,5 and depend on joint-specific mechanical loading mechanisms6. These patterns change from pairs and strings in healthy cartilage to double strings with the onset of osteoarthritis (OA). With further progression of the disease the chondrocytes form small clusters, increasing gradually in size to big clusters in advanced OA. A complete loss of any organizational structure and induction of apoptosis is observed in end stage OA. Thus, chondrocyte cellular arrangement can be used as an image-based biomarker for OA progression4.
Biomechanical properties of cells and tissues not only regulate their shape and function but are also crucial for maintaining their vitality. Changes in elasticity can propagate or trigger the onset of major diseases like cancer or OA. Atomic force microscopy (AFM) has emerged as a powerful tool to qualitatively and quantitatively characterize the biomechanical properties of specific biological target structures on a microscopic scale, measuring a wide range of force, from piconewton to the micronewton. The major application of AFM is to measure the surface topography and mechanical properties of samples at subnanometer resolution7. The measurement device consists of three main components: 1) An AFM probe, which is a sharp tip mounted on a cantilever and is used for the direct interaction with the surface of the sample. When force is applied to the cantilever, deformation of the latter occurs according to the measured tissue&#39;s properties. 2) An optical system that projects a laser beam onto the cantilever, which is then reflected to a detector unit. 3) A photodiode detector that catches the light deflected from the cantilever. It converts the received information regarding the laser deflection by the cantilever into a force curve that can be analyzed.
Thus, the main principle of AFM is the detection of the force acting between the AFM probe and the target structure of the sample. The force curves obtained describe the mechanical properties of the target structures on the sample surface like elasticity, charge distribution, magnetization, yield stress, and elastic plastic deformation dynamics8. An important advantage of AFM over other imaging techniques is that AFM can be used to measure the mechanical properties of live cells in medium or tissues in a native state without damaging the tissue. AFM can operate both in liquid or dry conditions. There is no requirement for sample preparation. AFM provides the possibility to image a specimen and measure its mechanical properties simultaneously in specimens that are near physiological conditions. In the present study we describe a novel approach to assess OA progression by measuring the elasticity of the PCM and ECM in native articular cartilage. The correlation of spatial organization of chondrocytes with the degree of local tissue degeneration provides a completely new perspective for early detection of OA. The functional relevance of these patterns has not been evaluated so far, however. Because the major function of articular cartilage is load bearing at low friction, the tissue must possess elastic properties. AFM allows measuring not only the elasticity of the ECM but also of the spatial cellular patterns embedded into their PCM. The observed correlation of elasticity with spatial pattern change of the chondrocytes is so strong that measuring elasticity alone may allow stratification of local tissue degeneration.
Elastic moduli of the PCM and ECM were assessed in 35 &#181;m-thin sections using an AFM system integrated into an inverted phase contrast microscope that allowed simultaneous visualization of the cartilage sample. This protocol is based on a study already published from our laboratory9 and specifically describes how to characterize the spatial arrangement of the chondrocytes and how to measure the elasticity of their associated PCM and ECM. With each pattern change of the chondrocytes, significant changes in elasticity can also be observed for both the PCM and ECM, allowing this technique to be used to directly measure the stage of degeneration of the cartilage.
This validated approach opens up a new way to evaluate OA progression and therapeutic effects at early stages before macroscopic tissue degradation actually starts to appear. Performing AFM measurements consistently is an arduous process. In the following protocol we describe how to prepare the sample to be measured by AFM, how to perform the actual AFM measurements starting with preparation of the cantilever, how to calibrate the AFM, and then how to perform the measurements. Step-by-step instructions give a clear and concise approach to obtain reliable data and provide basic strategies for processing and interpreting it. The discussion section also describes the most common pitfalls of this rigorous method and provides helpful troubleshooting tips.

<table>
	<tbody>
		<tr>
			<td>Amphotericin B</td>
			<td>Merck</td>
			<td>A2942</td>
			<td></td>
		</tr>
		<tr>
			<td>Atomic Force Microscope (AFM)</td>
			<td>CellHesion 200, JPK Instruments, Berlin, Germany</td>
			<td>JPK00518</td>
			<td></td>
		</tr>
		<tr>
			<td>AFM head</td>
			<td>(CellHesion 200) JPK</td>
			<td>JPK00518</td>
			<td></td>
		</tr>
		<tr>
			<td>Biocompatible sample glue</td>
			<td>JPK Instruments AG, Berlin, Germany</td>
			<td>H000033</td>
			<td></td>
		</tr>
		<tr>
			<td>Cantilever</td>
			<td>tip C, k &#188; 7.4 N/m, All-In-One-AleTl, Budget Sensors, Sofia, Bulgaria</td>
			<td>AIO-TL-10</td>
			<td></td>
		</tr>
		<tr>
			<td>Dulbecco&#39;s modified Eagle&#39;s medium (DMEM)</td>
			<td>Gibco, Life Technologies, Darmstadt, Germany</td>
			<td>41966052</td>
			<td></td>
		</tr>
		<tr>
			<td>Inverted phase contrast microscope (Integrated with 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</td>
			<td>(Merck KGaA, Darmstadt, Germany)</td>
			<td>F1315</td>
			<td></td>
		</tr>
		<tr>
			<td>Microspheres</td>
			<td>Polysciences</td>
			<td>07313-5</td>
			<td></td>
		</tr>
		<tr>
			<td>Penicillin-Streptomycin</td>
			<td>Sigma</td>
			<td>P4333</td>
			<td></td>
		</tr>
		<tr>
			<td>Petri dish heater associated with AFM</td>
			<td>JPK Instruments AG, Berlin, Germany</td>
			<td>T-05-0117</td>
			<td></td>
		</tr>
		<tr>
			<td>Scalpel</td>
			<td>Feather</td>
			<td>2023-01</td>
			<td></td>
		</tr>
		<tr>
			<td>Tissue culture dishes</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></td>
		</tr>
	</tbody>
</table>

application of atomic force microscopy to detect early osteoarthritis

Biomechanical properties of cells and tissues not only regulate their shape and function but are also crucial for maintaining their vitality. Changes in elasticity can propagate or trigger the onset of major diseases like cancer or osteoarthritis (OA). Atomic force microscopy (AFM) has emerged as a strong tool to qualitatively and quantitatively characterize the biomechanical properties of specific biological target structures on a microscopic scale, measuring forces in a range from as small as the piconewton to the micronewton. Biomechanical properties are of special importance in musculoskeletal tissues, which are subjected to high levels of strain. OA as a degenerative disease of the cartilage results in the disruption of the pericellular matrix (PCM) and the spatial rearrangement of the chondrocytes embedded in their extracellular matrix (ECM). Disruption in PCM and ECM has been associated with changes in the biomechanical properties of cartilage. In the present study we used AFM to quantify these changes in relation to the specific spatial pattern changes of the chondrocytes. With each pattern change, significant changes in elasticity were observed for both the PCM and ECM. Measuring the local elasticity thus allows for drawing direct conclusions about the degree of local tissue degeneration in OA.

Along the physiopathological model from strings to double strings, to small and finally to big clusters, both ECM (Figure 3A) and PCM (Figure 3B) elastic moduli decreased significantly between each pattern change. The only exception was the difference in ECM between strings and double strings (p = 0.072). The results show that the ECM/PCM ratio (Figure 4B) did not change significantly, whereas a marked decrease in the absolute diffe...

Watch this Scientific Journal Video about Application of Atomic Force Microscopy to Detect Early Osteoarthritis at JoVE.com

Application of Atomic Force Microscopy to Detect Early Osteoarthritis

We present a method to investigate early osteoarthritic changes at the cellular level in articular cartilage by using atomic force microscopy (AFM).

Biomechanical properties of cells and tissues not only regulate their shape and function but are also crucial for maintaining their vitality. Changes in ...

The biomechanical properties of cells and tissues regulate their function and vitality. AFM allows the early quantification of osteoarthritis at the cellular level based on elasticity measurements. AFM allows the acquisitions of measurements at a cellular level without damaging the sample and with a high resolution, reliability, and sensitivity.Biomechanical property changes occur early in osteoarthritis. Measuring the local elasticity of articular cartilage allows valid conclusions to be drawn about the degree of local tissue degeneration. To evaluate degenerative changes within the human knee joint, first use a scalpel to cut the articular cartilage as a whole from the subchondral bone and embed the cartilage sample in water-soluble embedding medium on the Cryotome knob that freezes under low temperature in the Cryotome device.When the sample has frozen, use a standard Cryotome to acquire a 35 micrometer thick sections of tissue from the topmost layer of the articular cartilage, collecting each section onto individual glass slides. When all of the sections have been obtained, rinse the samples three times in fresh PBS for five minutes per wash to remove the water-soluble embedding medium. Remove the first slide from the wash.After this, add two to three drops of biocompatible sample glue per section into individual AFM-compatible Petri dishes. Remove any excess solution from the sample and gently press the edges of the dried section onto the glue spots. After two minutes, carefully cover the bonded tissues with Leibovitz's L-15 medium without L-glutamine and place the dishes in a cell culture incubator until the samples are to be analyzed.To prepare the AFM device, adjust a gas block for measuring in a liquid environment on the AFM holder to that the upper surface is parallel to the holder, and use tweezers to carefully mount the selected cantilever onto the surface of the glass block so that the AFM tip with the microsphere protrudes over the polished optical plane. To stabilize the cantilever on the glass block, slide the metallic spring into the groove of the block, and use tweezers to clamp the top of the cantilever with the spring. Carefully place the glass block with the cantilever on the AFM head and secure the block with the integrated locking mechanism.Then mount the AFM head with the cantilever onto the AFM device with the spring facing to the left. To mount the sample onto the AFM device, place the sample Petri dish onto the AFM sample holder and set the Petri dish heater to 37 degrees Celsius. After about 20 minutes, open the device software and laser alignment and approach parameter setting windows.Next, turn on the stepper motor, the laser light, and the CCD camera and use the stepper motor function to lower the cantilever in 100 micrometer steps until the cantilever is fully submerged in the medium. Use the adjustment screws to direct the laser on top of the cantilever and use the AFM device screws to adjust the laser beam so that the reflected beam falls onto the center of the photodetector. Once the laser cantilever has been adjusted, adjust the photodetector as necessary to set the sum signal to one volt or above and the lateral and vertical deflections to close to zero.To obtain the calibration force curve, run a scanner approach with the approach parameters as indicated in the table. Once the bottom of the tissue culture dish is reached, retract the cantilever by 100 micrometers. Set up the run parameters as indicated in the table.And click run to start a measurement and to obtain a calibration force distance curve. The force curve is obtained as illustrated in the figure. On the calibration force distance curve, select the region for a linear fit of the retracted curve.Once the linear fit is in place, the values will be saved by the software. Then as the measurements are performed in medium at 37 degrees Celsius temperature, set the temperature variable in the software to 37 degrees to mimic the physiological conditions as closely as possible. To identify the conversite patterns in the cartilage sample sections, locate and identify the specific cellular patterns of articular cartilage from the osteoarthritic knee on the phase contrast microscope, which may include single strings indicative of healthy tissue areas, double strings indicative of the beginning of tissue degeneration, small clusters, which are signs of advanced tissue degeneration, and big clusters, indicative of end-stage tissue destruction.Once a specific desired pattern has been identified, for pericellular matrix measurements, position the cantilever in close proximity to the cells and measure two sites per chosen pattern per matrix type, nine times per measurement site including a sample size large enough to account for possible inaccuracies. Focus on the cellular matrix of the pattern to be measured and fix the computer mouse at that point. Next, focus on the probe and move the tip of the probe to the point previously fixed by the computer arrow.To conduct measurements of the extracellular matrix, select a region without any cells and perform an approach followed by a retraction so that the cantilever is positioned 100 micrometers above the tissue. Then click run to start the measurements, using the setpoint parameter obtained by calibration of the cantilever. To process the obtained data, open data processing software compatible with the data obtained from the AFM device and select the Hearst model in the software for processing the force distance curves.Set the Poisson ratio to 0.5, the tip shape as spherical, and the tip radius to 12.5 micrometers. Once all of the parameters have been adjusted, the results will be fitted and the Young's Modulus will be calculated by the software. Along the physiopathological model from strings to double strings and from small to big clusters, both extracellular and pericellular matrix elastic moduli decrease significantly between each pattern change except between strings and double strings.In addition, the extracellular-pericellular matrix ratio does not change significantly, whereas a marked decrease in the absolute differences in elasticity between the extracellular and pericellular matrices is observed. Elasticity values depend on various conditions such as the indentation depth or the cantilever tip properties. AFM is thus best suited to comparing different conditions within the same experimental setup.As the AFM measures the local elasticity of the sample, the sections need to be fixed in place in order to allow for precise measurements and to avoid cantilever damage. To further analyze the processes responsible for tissue elasticity changes, biochemical quantification of the structure of proteins of interest can be performed via western blots or ELISAs.

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