The overall goal of this procedure is to apply dynamic mechanical compressive loading to cell seeded hydrogels. To do this, primary cells are obtained through dissection and enzymatic digestion of cartilage slices. The isolated cells are encapsulated in a 2%agros, hydrogel, and cell seeded constructs are created using a biopsy punch.
The samples are then placed into the compressive loading rig and loaded into a mach one micro mechanical stimulator. Dynamic compressive loading is then applied to cell seeded constructs to assess collagen and prot glycan synthesis. The constructs are radio labeled following incubation incorporated.
Isotopes are quantified by scintillation counting the resulting data, which is normalized to DNA. Content shows changes in extracellular matrix synthesis following mechanical stimulation. Though this method can provide insight into counter site response and mechanical stimulation.
It can also be applied to other systems such as stem cells, other connective tissues, or mechano receptive cells. The metacarpal phal joints from Skeletally mature cows used here were obtained from a local abattoir working in a sterile dissection area. In a laminar flow hood place the cartilage slices into a 100 millimeter diameter Petri dish containing 20 milliliters of hams, F 12 containing 0.5%protease and incubate for two hours at 37 degrees Celsius.
Following the incubation vacuum, aspirate the protease solution and rinse the slices by adding hams. F 12 culture medium gently swirling, then aspirating. Repeat this, wash two more times after the third wash, vacuum aspirate the culture medium.
Add 20 milliliters of hams F 12 containing 0.15%collagenase A and incubate the dishes overnight at 37 degrees Celsius. The next day, pipette the cell suspension through a 200 mesh screen Filter into a clean Petri dish to wash the filter, pipette an additional five milliliters of hams F 12, collecting the flow through in the same dish. Transfer the cell suspension to a 50 milliliter conical tube.
Wash the Petri dish with 10 milliliters of hams F 12. Then add the wash solution to the conical tube. Centrifuge the tube at 600 to 800 RCF for six to eight minutes at room temperature.
Once the spin has completed, aspirate the supernatant taking care not to disturb the cell pellet. Resuspend the pellet in 40 milliliters of culture medium. Centrifuge the tube at 600 to 800 RCF for six to eight minutes.
Repeat the aspiration, resus suspension and centrifugation twice more for a total of three times before the third spin.Resus. Suspend the palate by agitation and take a 500 microliter aliquot for cell counting. During the third spin, use a hemo cytometer and an inverted light microscope to count the viable cells in the aliquot by trian blue exclusion.
Then once the cell number has been determined, and the third spin is complete, aspirate the snat and resus. Suspend the pellet in a volume of culture medium at double the desired seating density. Here the chondrocytes are resuspended at 20 times 10 to the sixth cells per milliliter.
The next step of the procedure is to encapsulate the isolated primary articular chondrocytes in an aros hydrogel in a beaker suspend 0.8 grams of aros. In 20 milliliters of sterile PBS. Place the beaker on a hot plate set to 120 degrees Celsius as soon as the aros dissolves.
Lower the heat to 60 degrees Celsius in a 50 milliliter conical tube. Combine equal volumes of the cell suspension with double the desired concentration of the heated aro solution. Here five milliliters of cell suspension is added to five milliliters of 4%agro suspension for a final 2%Agros hydrogel mix thoroughly by pipetting while avoiding the creation of large bubbles which may affect cell viability.
Next carefully Eloqua 10 milliliters of the chondrocyte agros mixture into a 60 millimeter diameter Petri dish. Again, avoiding the creation of large bubbles. Cover the dish with the lid.
Then let the mixture stand for 30 minutes at room temperature. Once the mixture has solidified, use a four millimeter biopsy punch to obtain as many chondrocyte agros hydrogel samples as possible. Typically, this amounts to 20 to 30.
Place the samples in a clean Petri dish using a micrometer measure and record the diameter and height of each sample. Ideally, construct should be approximately five millimeters in height, discard any samples obtained from a region where the sample height is greater than 2%Transfer the samples to a fresh 60 millimeter Petri dish containing 10 millimeters of complete medium. In this part of the protocol, the cell seated constructs will be mechanically stimulated by dynamic compression.
This will be accomplished using a custom designed compression rig that mechanically couples the construct loading PLAs to a mock one micro mechanical system, which controls the amount of compression applied to the constructs. The most difficult aspect of the procedure is loading samples into the compression rig. It's important to properly adjust the plates in the rig to ensure the appropriate application of dynamic compressive loading.
Prior to use, sterilize the metallic components of the compression rig in an autoclave. Sterilize the plastic components with 70%ethanol overnight and UV light for at least 15 minutes. To keep the chondrocyte aros hydrogel constructs from falling over.
Use forceps to place 12 sterile plastic retaining rings into the wells of a 24 well culture plate. The inner diameters of the retaining rings should be larger than the construct diameters. Carefully using forceps.
Transfer the chondro aros hydrogels from the Petri dish to the 24 well plate securing each one in a retaining ring. Transfer 400 microliters of complete medium to each sample. Well next assemble the sterilized compression rig and using set screws, secure the PLAs.
Attach the compression rig to the 24 well culture plate to establish hydrogel contact release and resecure. The set screws construct deformation will be applied to this unloaded reference state or zero strain state. Load the assembled compression rig into a Mach one micro mechanical testing system with the locating pin.
Secure the rig to the vertical stage and then using a set screw, lock it into place. Remove the spacing jig. Use the Mach one software interface to set the frequency to one hertz, the amplitude to 350 microns and the number of cycles to 1, 200.
This applies dynamic compressive loading to 10%strain amplitude based on the original height of the samples. To begin the mechanical stimulation, press the start button, the mach one actuator. The loading device that attaches to the culture plate has six indent that move up and down, stimulating six samples simultaneously.
The unstimulated control samples are also in the same culture plate, but are not placed into the wells that have the indents. When the run is complete, the mach one will beep and the actuator will move to the unloaded reference state. Upon the completion of mechanical stimulation, replace the spacing jig.
Loosen the locating pin set screw, and remove the compression rig and culture plate from the testing system next to radio label, the cell hydrogel constructs pipette both five micro curies of Tritium Prolene protein label for the chondrocyte specific collagen synthesis and S 35 sulfur for proteoglycan synthesis into each, well incubate the radio labeled constructs for 24 hours at 37 degrees Celsius, 5%carbon dioxide to harvest the radio labeled hydrogel constructs. Use forceps to rinse each hydrogel construct three times by immersion in PBS. To remove any unincorporated isotope, place the rinsed construct into a 1.5 milliliter tube.
Next, add 500 microliters of pape solution to the 1.5 milliliter tubes. Place them on a heating block and allow them to digest at 65 degrees Celsius for 72 hours. Once the digest is completed, the sample should look relatively clear.
Immediately following pape digestion, take aliquots of 100 microliters from each sample to determine the DNA content using the pico green D assay. Then take a 100 microliter aliquot of each sample to determine incorporated isotope activity by beta liquid scintillation counting. The resulting isotope activity should then be normalized to DNA content for analysis.
Bovine articular chondrocyte aros hydrogel constructs composed of 2%aros with 10 times 10 to the sixth. Cells per milliliter of encapsulated cells were mechanically stimulated at an amplitude of 10%compressive strain at a frequency of one hertz for 20 to 60 minutes, or 1, 200 to 3, 600 cycles. They were then assay for DNA content and extracellular matrix synthesis by radioisotope incorporation.
As can be seen here, DNA and extracellular matrix synthesis was affected in a dose dependent manner. DNA content exhibited a significant 35%decrease as a result of 20 or 30 minutes of stimulation with a p value of less than 0.01 60 minutes of stimulation exhibited no effect. Cartilage specific collagen and proteoglycan synthesis was determined by tritium, proline and S 35 sulfur incorporation respectively.
These exhibited significant increases of approximately 60%in response to 20 or 30 minutes of stimulation with a p value of less than 0.01 60 minutes of stimulation did not exhibit an observable effect. After watching this video, you should have a good understanding of how to mechanically stimulate hydrogel cultures using dynamic impression and adapt this protocol for use in similar applications.
