The three critical steps of this protocol are i) developing the right composition and consistency of the cellulose hydrogel ink, ii) 3D printing of scaffolds into various pore structures with good shape fidelity and dimensions and iii) demonstration of the mechanical properties in simulated body conditions for cartilage regeneration.
This is the first report on 3D printed nanocellulose hydrogel scaffolds where we have a gradient pore structure and mechanical properties to mimic natural structures as cartilage. The two main advantages of using 3D printing techniques are customization and design freedom. That opens up an unlimited opportunity of fabricating new and unexplored geometric designs.
This protocol is user-friendly, and newcomers can easily reproduce the results. The choice of the slicing software and the nozzle movement have significant impact on the final product. To begin, prepare 40 milliliters of hydrogel ink by mixing 11%by weight CNC, 6%by weight sodium alginate, and 12%by weight gelatin in a container.
Heat the mixture to 40 degrees Celsius, and mix with a spatula until a smooth paste is obtained. Transfer the mixture into a 60-milliliter syringe. Next, with the help of a mechanical clamp, pass the mixture through a series of nozzles with different diameters into another 60-milliliter syringe.
Repeat the process until smoothly extruded filaments of hydrogel ink are obtained. Gently centrifuge the syringe filled with the hydrogel ink at 4, 000 times g to remove trapped air. From the SD card, select the saved files for uniform and gradient porosity scaffolds, and start printing.
If needed, adjust the speed and flow rate accordingly. To cross-link the scaffold after the 3D printing is complete, gently add drops of 3%by weight calcium chloride solution to the scaffold until it becomes completely wet. Wait for five minutes.
Very carefully transfer the scaffold from the printer bed to a 50-milliliter container filled with 3%by weight calcium chloride solution. Leave it overnight. Wash thoroughly with distilled water, and transfer the scaffold to a 50-milliliter container filled with 3%by weight glutaraldehyde solution.
Leave it overnight. Wash thoroughly, and store the 3D printed scaffold in distilled water. For compression testing, fill the container equipped with a submersible compression base plate with two liters of water, and start the heating system to reach 37 degrees Celsius.
Initialize Bluehill Universal Software, and set up the testing method. Select rectangular specimen geometry, and choose the option to enter dimensions before testing each sample. Set the strain rate to two millimeters per minute and end of result as 80%compressive strain together with 90-newton force.
In the Measurement section, select Force, Displacement, Compressive Stress, and Compressive Strain. Choose the option to export data as text files for future plotting. Set the zero extension point by using the jog controls to lower the crosshead plate as close as possible to the base plate.
Measure and record the dimensions of the samples to be tested. When the water temperature reaches 37 degrees Celsius, place the sample on the base plate. Secure the sample by moving the crosshead plate so that it starts to touch the sample.
Move the water bath up so that the plates with the sample in between them are immersed in water. Enter the sample name and dimensions, and start the test. After the test is complete, first move the water bath down, and then raise the crosshead plate.
Remove the sample and its pieces, if any, clean both the plates, and load a new sample. After all the samples are tested, export the raw data. Plot compressive stress versus compressive strain curves, and determine the compressive tangent modulus at strain values of one to 5%and 25 to 30%CNCs-based nanocomposite hydrogel ink shows a strong non-Newtonian shear thinning behavior with a five order of magnitude drop of the apparent viscosity.
The hydrogel ink exhibits a viscoelastic solid behavior, as the storage modulus is an order of magnitude greater than the loss modulus at low shear stress. At low strain rates of one to 5%the compressive modulus is similar for all types of porous scaffolds in comparison to the reference scaffold with no porosity, showing that the elastic nature of the hydrogel ink is preserved even in the presence of the macropores. However, at high strain rates of 25 to 30%the highest modulus is obtained for the reference scaffold with no porosity.
As soon as the pore size increases, the modulus decreases due to the decrease in density, indicating the expected relationship between porosity of the scaffolds and the corresponding mechanical properties. Furthermore, the compressive modulus of the 3D hydrogel scaffolds increases as the compression rate increases, exhibiting and mimicking the viscoelasticity of natural cartilage tissues. The homogeneous and the continuous flow of the ink during 3D printing are the most important things.
This method will be used by researchers to expand to other application areas by using nanocellulose hydrogel as a 3D printable platform. For example, we have already developed nanocellulose-based hybrids for controlled drug release following the same procedure.
