Bioengineering
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Novel Process for 3D Printing Decellularized Matrices
Chapters
Summary January 7th, 2019
This protocol describes the production of polycaprolactone (PCL) filament with embedded polylactic acid (PLA) microspheres which contain decellularized matrices (DM) for 3D printing of structural tissue engineering constructs.
Transcript
This method allows co-printing of both structural synthetics and biologic components for tissue-engineered scaffolds. These scaffolds can replicate the native tissue environment more accurately, which can be beneficial for cultured cells. The main advantage of this technique is that it can print mechanically sound structures without damaging the biologic material encapsulated within the scaffolds in an all-in-one technique for tissue engineering.
Produce microspheres with the desired matrix encapsulated. These microspheres were made with decellularized cartilage from porcine hind limbs. They vary in size.
Work with a sieve machine to obtain microspheres of uniformed size. Ensure the three sieve trays have been thoroughly cleaned and dried prior to use. Assemble the sieve shaker with the larger mesh tray on top and the smaller mesh tray in the middle.
Put the sieve pan on the bottom. Place dry microspheres in the top-most tray. Then place the lid on the tray.
Coarse sieve for eight to ten minutes. Then fine sieve for eight to ten minutes. While waiting, arrange weigh paper for the sieved spheres.
When sieving is complete, carefully remove a sieve tray. Place the tray upside down on the weigh paper. Gently tap the sides to ensure most of the spheres fall out.
With all of the sieve plates emptied onto weigh paper, discard the oversized and undersized spheres. The remaining spheres have the correct size for use in subsequent steps. Place these spheres into a labeled centrifuge tube.
Store the tube in a 20 degree Celsius freezer until needed. Weigh the material necessary to make the filament material. Make a one-to-four weight ratio of microspheres to polycaprolactone powder with at least 25 grams of microspheres.
Transfer the powder mixture to a miniature rolling mixer. Mix the powder at 20 rotations per minute for five minutes. After five minutes, stop the rotation and flip the container.
Then mix at 20 rpm for an additional five minutes. To continue, take the mixture to an extruder and spooler setup. Setup the extruder so its outlet is about 60 centimeters from the inlet of a spooler.
Then at the extruder heating element, be sure there is no insulating jacket. Place a desktop fan about halfway between the extruder and spooler to cool the extrudate. Put another fan near the heating jacket to cool it with ambient air.
Next ensure the proper nozzle is attached to the extruder and set the heating element temperature. Start the cooling fans and allow the instrument to reach a steady state. After 20 to 30 minutes, get the microsphere PCL mixture and fill the extruder hopper.
Turn on the spooler and the extruder auger to initiate filament extrusion. Use forceps and manually pull the initial extruded filament. Feed the filament to the filament spooler.
As it is extruded, observe the filament to identify its composition. After some time, the filament composition will appear uniform which is desired. Wrap tape around the filament to mark the start of the uniform region.
Beyond the spooler rollers, monitor the diameter of the filament. Use calipers to test if the diameter is close to the desired 1.75 mm. Adjust the spooler and extruder speeds and temperatures as necessary.
To adjust adjust the filament diameter, first alter the spooler and extruder speeds. You may also change the extruder temperature, although this is usually not necessary. Continue refilling the hopper and extruding until all of powder is used.
When the hopper is almost empty, add PCL powder to the hopper to flush out the microsphere mixture. Monitor the extrudate while adding the PCL powder. At first, microspheres will still be visible in the extrudate.
Eventually when no more microspheres are visible, stop adding the PCL. Separate and label the filament with microspheres in the desired concentration. When there is minimal powder in the hopper, stop extruding and turn off the equipment.
The filament can be used in a standard fused deposition modeling printer. Load the filament into the printer that is fitted with nozzles of the desired diameter. With the model loaded, set the temperature and linear speed for the printing and begin depositing the filament.
The custom filament is deposited layer by layer. Pay special attention to the first layer and adjust settings as needed. These two 3D printed scaffolds are difficult to distinguish at this scale.
One has filament containing polycapralactone, PCL only. The other has PCL filament with embedded microspheres of polylactic acid and decelluarized matrices. Viewed using scanning electron microscopy, the PCL-only scaffold appears mostly smooth.
By contrast, for the other filament, microspheres embedded in the PCL are visible throughout the sample. While attempting this procedure, it is important to remember that over or undersized microspheres can impact the flow dynamics of the material in the extruder. For this reason, be sure to properly prepare your microspheres prior to use.
In order to minimize waste, it is advisable to create one large batch of filament required for all experiments rather than to create smaller batches more frequently. Expertise in filament production will come over time. Scaffolds produced following this procedure can be further assessed in vitro and in vivo to answer additional questions about enhanced induction and mechanical strength compared to other bio-printed scaffolds.
After its development, this technique paved the way for researchers in the field of tissue engineering to explore contragenic induction of decelluarized porcine matrix within the 3D scaffolds. Don't forget that working with small particulates can present respiratory hazards. Wearing a small particulate mask during this procedure is encouraged.
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