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Materials Science in Additive Manufacturing 3D-printed nozzle for 3D bioprinting

single outlet, as shown in Figure 1. A range of constructs pumps connected to the DNC at a range of flow rates for
were then 3D-bioprinted through a microfluidic syringe peptide and PBS, to determine optimal gelation parameters.
pump-based extrusion system with peptide-based and A g-code file for a continuous 5-segment line was used to
live cells. To determine the feasibility of the proposed trace any clumps or clogs and gelation time was recorded.
connectors, an evaluation of the 3D-bioprinted construct To assess printability, a six-layer semi-filled cube
was conducted in terms of gelation continuity, printability, was 3D-printed with the DNC. Based on the gelation
biocompatibility, and shape fidelity. The methods applied continuity test, the optimized flow rates were set
for designing, fabricating, assembling, and evaluating to 55 µL/min, 20 µL/min, and 20 µL/min for peptide, PBS,
DNCs for cellular 3D bioprinting with peptide bioinks are and the cells inlet, respectively. Constructs were evaluated
detailed in the following.
for print shape, consistent formation of bioink thread, and
2.1. Designing DNC continuous layer deposition.
To ensure uniformity, the connectors were designed 2.4. Creating acellular 3D-printed scaffolds
using the NX CAD software with millimeter precision. Finally, to evaluate the shape fidelity of bioprinted constructs,
Considering the desired needle tip diameter and angle, acellular samples were printed and observed for print
the connector was designed to fit into a Luer lock needle resolution and mechanical stiffness. The DNC was mounted
tip. By design, the two inlets of the connector merge into on the robotic 3D bioprinter to print hollow cylinders of 10
one channel considering the volume of the two solutions × 10 × 13 mm and grid structures of 20 mm . To enhance
3
2
flowing inside the connector. This was done to reduce any flow for longer periods of time, automated time-dependent
material clogging before extrusion. The mixing region
length was taken from a previous study to complement pumping was exploited by programming the microfluidic
pumps with alternating square wave flow profiles. Based on
the characteristic requirements of our peptide hydrogels the optimized parameters reported previously, the square
for 3D bioprinting [25-27] . This can be modified based on the wave flow profile for the peptide hydrogel solution was set
characteristics of the desired printing materials. The DNC to a range of 50 – 55 µL/min with a 75% duty cycle and a
was designed with an additional holder for the cells inlet
to enable extruding cells at the tip of the nozzle. An ideal period of 115 s. For the PBS, the square wave flow profile
design was narrowed down based on ease of flow through was set to a range of 15 – 20 µL/min with a 25% duty cycle
[28]
the mixing region while maintaining an inlet angle closest and a period of 115 s .
to 90° angle. 2.5. 3D cell culture
2.2. 3D printing connectors using vat The biocompatibility of peptide hydrogel biomaterials of
polymerization IVZK peptide was tested with human neonatal dermal
Connectors were 3D-printed using FormLabs 3B 3D fibroblasts (HDFn). HDFn was cultured in a 3D-bioprinted
printer in the recommended settings for the white polymer construct with self-assembling ultra-short IVZK peptide-
resin. Before 3D printing, the design files were converted to based hydrogels. An optimal gelation concentration of
the STL format and then processed with PreForm software the IVZK peptide (13 mg/mL) was used. Purified and
to prepare for slicing. The materials were chosen, and lyophilized peptide powder was sterilized using a UV light
the model configuration took place during this process. for 30 min before each experiment.
Following the printing process, the 3D-printed model was HDFn was obtained from Thermo Fisher Scientific,
washed with isopropanol for 30 min and then cured at a and cell suspensions were used after seven passages for
temperature of 40°C for 60 min using the Form Washer every experiment. First, 1 mL of Dulbecco’s modified
and Form Cure post-processing devices. eagle medium (DMEM, ×1) was supplemented with
4.5 g/L glucose, 1-glucamine, sodium pyruvate, 10%
2.3. Parameter optimization for 3D bioprinting fetal bovine serum (FBS), and 1% penicillin/streptavidin
For seamless material extrusion with the DNC, optimization (10,000 units/mL). After adding cells to the growth
experiments were run with an in-house developed medium, the mixture was centrifuged for 5 min (250 ×g),
robotic 3D bioprinter to evaluate gelation, printability, at room temperature, to remove the DMSO storage buffer
and shape fidelity. For all experiments, 13 mg/mL IVZK from the stock solution. Then, cells were cultured in 75 mL
(Ac-Ile-Val-Cha-Lys-NH2) peptide and ×7 phosphate- of growth medium within cell-treated flasks and incubated
buffered saline (PBS) were used. The cells inlet was pumped for 2 days at 37°C. After incubation, the cells were viewed
with ×1 PBS for acellular simulation tests. A gelation under a microscope to determine their confluency. Then,
continuity test was conducted by running the microfluidic the growth medium was removed and approximately 5 mL

Volume 2 Issue 1 (2023) 3 https://doi.org/10.36922/msam.52