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.
