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Materials Science in Additive Manufacturing From 3D printed molds to bioprinted scaffolds

To demonstrate this approach, an open-source CAD wave profile for the peptide hydrogel solution was set to
model of a human ear was obtained and modified to create a range of 55 – 60 µl/min with a 75% duty cycle and a
a negative mold using NX SIEMENS software, as described period of 80 s. For the phosphate-buffered soline (PBS),
in the process above. A scale of 1:1 ratio was maintained to the square wave profile had a range of 15 – 20 µl/min with
keep the model as realistic as possible. After implementing a 25% duty cycle and a period of 80 s. The microfluidic ink
a cut into the extruded block and adding feature delivery system was loaded with peptide hydrogel and PBS
modifications, the mold was 3D printed with a Formlabs ® to facilitate bioink formation.
3B+ 3D printer using biocompatible elastic resin.
2.3. Creating the 3D bioprinted scaffolds
Finite element analysis (FEA) was performed to
test the design’s flexibility further and ease of releasing To evaluate shape fidelity and biocompatibility of 3D
the desired structure after 3D printing. Using the NX bioprinted structures with mold support, a human ear
SIEMENS software, the material was set to the selected model was 3D bioprinted with peptide bioink. The robotic
material elasticity of the 3D printed mold. The mold was 3D bioprinter was mounted with a homemade dual coaxial
nozzle consisting of three inlets as described previously .
[17]
3D meshed and constrained from the bottom side and was One inlet is for the peptide solution, another one for the
set to experience a force against the upper side with F=15 PBS buffer and a third inlet for the cells. Initially, the molds
N (Figure 3B).
were tested for shape fidelity by printing an acellular 3D
2.2. Parameter optimization for 3D bioprinter and human ear construct. In this experiment, IVZK peptide was
G-code dissolved in water at an initial concentration of 13 mg/ml.
Furthermore, a 5× PBS buffer was used to induce gelation
For seamless material extrusion into the mold, the g-code and solidify the hydrogel before extrusion. All solutions
file was optimized to conform to the mold profile. First, were loaded into the microfluidics pumps and extruded
the CAD model for the human ear model was sliced through the nozzle using the automated pumping program
using Cura slicer software and adjusted in terms of print described earlier. The constructs were left to solidify inside
speed, layer height, and orientation to be suitable for 3D the mold for 30 min after printing. They were then removed
bioprinting. Then, the g-code was modified to ensure free from the mold and shape fidelity was subjectively assessed
movement by removing any features that would cause in comparison to the 3D model design.
collision and adding layers where necessary to maintain the
desired shape. Bottom layers of the g-code were removed 2.4. Cell bioprinting and bioimaging
to allow the mold to serve its purpose. Noteworthy, for Experiments were done to assess the suitability of using
non-symmetrical shapes like the human ear, the positive the mold support method with cellular constructs.
CAD model has to be mirrored along the horizontal Initially, IIZK peptide was dissolved in sterile water at
X-axis to ensure that the inner features align with those of an initial concentration of 13 mg/ml and loaded into the
the negative mold. For orientation, it is essential that the microfluidics pumps along with a sterile 5×PBS buffer.
g-code path aligns with the position of the mold placed The mold was washed with 70% ethanol and sterilized for
on the printbed. This was done by creating a user-defined 30 min under UV light before bioprinting. MSCs were
home position for the robotic arm. The mold was then cultured in T175 flasks until they reached 95% confluency
fixed to the printbed such that its start point in the g-code at passage eight. The cells were suspended in a 1×PBS
aligned with the user-defined home position. Likewise, in buffer supplemented with 5% FBS. Cells were loaded
the Repetier printing software, the printbed dimensions into the microfluidics pumps and extruded at a constant
were entered accurately, and the g-code file was loaded to flow rate of 15 µl/min. After printing, the constructs were
be at the center of the user-defined home position. left in the mold and incubated overnight at 37℃ with
Our in-house developed robotic 3D bioprinting Dulbecco’s Modified Eagle Medium supplemented with 5%
system [30,31] also required optimization to print with the L-glutamine. The constructs were removed from the mold
mold. The robotic 3D bioprinter was prepared for printing the next day. At day 1, cell viability was assessed using live-
with peptide-based hydrogels to assess suitability of the dead imaging staining with confocal microscopy.
molds for soft bioink materials. For optimal material
extrusion into the mold, the microfluidic pumps were 3. Results
programmed with alternating square wave flow profiles Fabrication of the optimal mold for 3D bioprinting was
to enable automated time-dependent pumping of the found to be an experimental process that required several
solutions. The optimization process for this parameter was iterations to achieve quality results. The first iteration was
developed in a previously reported study . The square fabricated with standard resin, which is a stiff and rigid
[32]

Volume 1 Issue 1 (2022) 5 https://doi.org/10.18063/msam.v1i1.7