Page 73 - IJB-9-4

International Journal of Bioprinting 3D bioprinting of ultrashort peptides for chondrogenesis

gradual increase of strain from 0.01% to 100% at 1 rad/s to hold shape with taller structures without sagging due
angular frequency. to excess water. Imperfect quality constructs had signs of
sagging, clumpy deposits of gel, and low-resolution shapes,
2.3. 3D bioprinting and could not define structure details.
2.3.1. 3D bioprinter setup and printing parameters
optimization 2.3.2. Bioprinting of cell-laden constructs
An in-house developed robotic 3D bioprinter was used for The study was approved by the Institutional Biosafety and
the 3D bioprinting experiments . The printer components Ethics Committee (IBEC) at King Abdullah University of
[30]
included a five-degree-of-freedom robotic arm, a custom- Science and Technology (KAUST). Human bone marrow
designed coaxial nozzle, a set of microfluidic pumps, and a mesenchymal stem cells (hBM-MSCs) were expanded in
heated bed. The robotic arm was interfaced with Repetier- 2D culture, as described before . Briefly, the cells were
[31]
Host to slice files into gcode for 3D printing, and printing cultured at a seeding density of 4 × 10 cells/cm in T175
3
2
files were designed in SolidWorks®. The coaxial nozzle was tissue culture flasks. When cultures reached 70%–80%
fabricated to house three inlets and a single outlet, with a confluence, the cells were subcultured using 0.25% trypsin.
final inner diameter of 0.5 mm. The three inlets included a The cells were cultured and maintained in complete growth
channel for the peptide, another one for the cells, and the media, consisting of α-modified minimum essential
third inlet for PBS concentration >1× to fasten the gelation medium (α-MEM) supplemented with 10% mesenchymal
process of the peptide. The commercial microfluidic pumps stem cell-qualified fetal bovine serum (FBS), 2 mM
were controlled simultaneously during printing through a L-glutamine, and 1% penicillin/streptomycin (GIBCO,
Labview-based graphical user interface. Thermo Fisher, USA). Cells at passages 4–8 were used in
The printing parameters used were as described printing experiments. For bioprinting, hBM-MSCs were
6
before ; the peptide concentration was set to 13 mg/mL mixed with PBS at a final concentration of 8 × 10 cells/
[29]
for the two ultrashort peptides, a concentration of 7× PBS mL and loaded into the microfluidic pumps of the robotic
was used for the gelation of both ultrashort peptides, and arm bioprinter. In the printing process, the flow rates were
the heatbed was set to 37°C. The pump flow rates were 10 µL/min, 55 µL/min, and 8 µL/min for cells, peptide
optimized at a range of 55–60 µL/min for the peptide, 15– solution, and 5× PBS, respectively. Different cell-laden
20 µL/min for PBS, and 10 µL/min for cells. structures were printed, including cuboids with 10-mm
edges and 2.6-mm height and cylinders with 10-mm
The two ultrashort peptides, IIZK and IZZK, were diameter and 10-mm height. After printing, the printed
compared for printability and the ability to support the cell-laden constructs were placed in the CO incubator
2
chondrogenic differentiation of hBM-MSCs. For 3D for 5 min before the addition of complete growth media.
bioprinting, three solutions were prepared—peptide The printed cell-laden constructs were placed in standard
solution (13 mg/mL), 7× PBS, and cells in 1× PBS. conditions (37°C, 5% CO , and 95% relative humidity),
2
Each solution was dispensed into an individual inlet and the media were changed every 3 days.
of the coaxial nozzle through the microfluidic pumps.
Immediately before printing, the selected peptide was 2.4. Assessment of cell-laden constructs
dissolved in MilliQ water and loaded in Pump 1. A solution 2.4.1. Cell viability
of 7× PBS was loaded in Pump 2. A solution of 1× PBS was The viability of 3D-bioprinted cells was assessed using
loaded in Pump 3. Flow rates of the microfluidic pumps the LIVE/DEAD Viability/Cytotoxicity Kit (Thermo
were optimized at a range of 55–60 µL/min for Pump 1, Fisher, USA), in which calcein acetoxymethyl ester
15–20 µL/min for Pump 2, and 10 µL/min for Pump 3. (Calcein-AM) is used to detect viable cells, and ethidium
The flow rates were adjusted within the optimized range, homodimer-I (EthD-I) is used to detect dead cells. Cell-
depending on the viscosity of the peptide being used. The laden 3D-bioprinted constructs were washed twice with
printed structures were designed in SolidWorks®, converted Dulbecco’s phosphate-buffered saline (D-PBS). Then,
into gcode, and bioprinted. The structures included a filled a staining solution of 2 μM of Calcein-AM and 4 μM
cube (10 × 10 × 1.5 mm), a hollow cylinder (10 × 10 × of EthD-1 was added to the 3D cell-laden bioprinted
10 mm). Multiple samples were printed for each shape to constructs and incubated for 45 min in the CO incubator.
2
assess shape fidelity. Print resolution, refinement of details, After incubation, the staining solution was removed, and
and heights of the samples were compared. A rubric for the 3D-bioprinted constructs were washed with 1× D-PBS.
fidelity assessment was developed to examine printed Stained printed cell-laden constructs were imaged using an
constructs. The best quality constructs were expected inverted laser scanning confocal microscope (Zeiss LSM
to have excellent resolution, visibly refined details, a 880 Inverted Confocal Microscope, Germany). Viability
consistent thread of gel without any gaps within layers, and percentage was calculated using ImageJ software.

Volume 9 Issue 4 (2023) 65 https://doi.org/10.18063/ijb.719