Our protocol is significant as it enables the creation of disease in hydrogels that closely resemble the nature of cartilage tissue. The main advantage of this technique is ability to produce disease in hydrogels with evident biological activity in spatial structure, low immunogenicity, and biological industrial function. To begin, chop the collected cartilage using a scalpel into pieces of one to two cubic millimeters in size.
In a 50-millimeter centrifuge tube, submerge 20 grams of the minced cartilage in 20 milliliters of hypotonic Tris-hydrochloride buffer. Place the tube at 80 degrees Celsius for three hours, and then in the oven at 37 degrees Celsius for three hours. Vortex the centrifuge tube for 30 seconds at 1000 RPM.
Then filter the decellularized cartilage using a plastic sieve placed on a 50-milliliter centrifuge tube. Wash the cartilage thrice with sterile PBS before collecting it. Add 10 milliliters of trypsin solution to the collected cartilage and incubate it on a shaker at 37 degrees Celsius for 24 hours, replacing the trypsin every four hours.
After filtering the suspension, wash the trypsinized cartilage with hypertonic buffer. Once the cartilage is preserved, add 10 milliliters of nuclease solution and place it on a shaker at 37 degrees Celsius for four hours. After washing with sterile PBS, add hypertonic Tris-hydrochloride solution to the cartilage and place it on the shaker as demonstrated.
Once washed with sterile PBS, submerge the tissue in 1%Triton X-100 solution for 24 hours. After removing the Triton X-100, rinse the decellularized cartilage with distilled water for three days, changing the water every 12 hours. After three days, add peracetic acid solution to the cartilage and soak it for four hours.
Once washed with PBS, collect the cartilage using a plastic sieve as previously demonstrated. Using liquid nitrogen, prepare decellularized cartilage powder. Add 80 milliliters of 0.5 molar acetic acid and 20 milligrams of pepsin to two grams of cartilage powder, and digest the mixture for 24 hours at 37 degrees Celsius.
After digestion, centrifuge the suspension at 400 G for 10 minutes and collect the supernatant, which is now called DC-ECM solution For storing the DC-ECM solution, add one milliliter of the solution to each well of a six-well plate and place it in a lyophilizer. Once the solution is freeze dried, transfer it to a centrifuge tube. To form a hydrogel, dissolve 20 milligrams of freeze dried DC-ECM solution in one milliliter of sterile distilled water.
Once vortexed, add one milligram of vitamin B2 to the DC-ECM solution. After incubation, irradiate it with UV light for three minutes. Add buffer GTL and proteinase K to 20 milligrams of DC-ECM cartilage powder, and mix thoroughly using vortex oscillation.
For complete cartilage dissolution, incubate the mixture at 56 degrees Celsius for four hours. Once vortexed, add 200 microliters of buffer GTL followed by anhydrous ethanol. After vortexing, centrifuge the sample at 6000 G for one minute at four degrees Celsius.
Then collect and quantify the DNA as described in the manuscript. For analyzing collagen, acidify five milligrams of DC-ECM powder in hydrochloric acid at 100 degrees Celsius for 20 minutes. Then neutralize it with five milliliters of six molar sodium hydroxide solution.
Calculate the hydroxyproline content of the neutralized sample using absorbance at 570 nanometers against the hydroxyproline standard. Add 500 microliters of reagent A to 200 milligrams of DC-ECM powder after mixing. Incubate the sample at four degrees Celsius for 16 hours.
Once centrifuged, collect 50 microliters of sample solution and add 50 microliters of reagent B, followed by reagent C.After incubating the sample for 10 minutes, add 750 microliters of reagent D and incubate for 30 minutes in the dark. After centrifugation, add one microliter of reagent E to the pellet and mix well before centrifuging. Then dissociate the sample before GAG content measurement.
In the prepared DC-ECM cartilage, the DNA content was significantly eliminated. However, the collagen and GAG content were retained compared to the native cartilage. The SEM and TEM analysis demonstrated the ultra structure of the prepared DC-ECM.
The prepared hydrogel in the inverted tube did not flow to the bottom, thus demonstrating gelation. Further, compared to the DC-ECM only solution, the addition of vitamin B2 reduced the gelation time due to the induced crosslinking during the DC-ECM hydrogel formation. The DC-ECM hydrogel viscosity was higher than the solution viscosity and increased shear rates decreased the solution viscosity.
Moreover, the high storage modulus of the DC-ECM solution and hydrogel indicated that they both had gel rather than liquid properties. The SEM analysis demonstrated that the pore size of the DC-ECM solution was significantly decreased in the hydrogel form after crosslinking and freeze drying. The protocol involves a combination of physical and chemical disruption and enzymatic digestion to remove cellular material while preserving the structure and the conversation of the ECM.
