Bioengineering
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Application of Atomic Force Microscopy to Detect Early Osteoarthritis
Chapters
Summary May 24th, 2020
We present a method to investigate early osteoarthritic changes at the cellular level in articular cartilage by using atomic force microscopy (AFM).
Transcript
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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