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Exploring nanofibrous self-assembling peptide hydrogels using mouse myoblast cells for three-dimensional bioprinting and tissue engineering applications
3 mg/mL for CH-01 and CH-02, respectively, as shown changed to differentiated mode to study differentiation
in Figure 1. The final volume ratio of peptide solution and behavior for 8 days. The differentiation medium contained
10× PBS was 9:1. DMEM supplemented with 2% horse serum and 1% P/S.

2.2. Characterization of the Topography and 2.4. Biocompatibility Evaluation of Tetrameric
Morphology of Peptide Hydrogels Ultrashort Self-assembling Peptides in Two-
dimensional (2D) Culture
2.2.1. Evaluation of Fiber Structures by Field-emission
Scanning Electron Microscopy (SEM) 2.4.1. Cell Viability Assay (MTT Assay)
The peptide nanogels were dehydrated by gradually All biocompatibility studies were performed in a
increasing concentrations of 30%, 50%, 70%, 90%, 96-well plate. C2C12 (10,000 cells/well) were seeded
and 100% (v/v) ethanol solutions for 15 min in each in a complete medium. After 2 days, the medium was
solution. Further dehydration in 100% ethanol solution discarded, and the cells were incubated for 48 h with
was continued by changing the absolute ethanol solution different concentrations of peptide solution, at 37°C,
with a fresh one twice for 15 min each. The dehydrated 95% air, and 5% CO , Matrigel was used as a control.
2
samples were subsequently kept in 1:2 ratio of A colorimetric MTT assay was used to determine cell
hexamethyldisilazane (HMDS) and ethanol for 20 min, viability as advised in the manufacturer’s protocol.
followed by 20 min of incubation in a fresh solution of Briefly, the phenol-free fresh medium was mixed with
2:1 ratio of HMDS and ethanol and then in 100% HMDS, 10% MTT reagent. Each well was incubated for 4 h with
performed twice for 20 min. Finally, the samples were 100 μL MTT reagent including the positive control wells.
stored overnight in a fume hood to allow HMDS to Insoluble crystals of formazan were dissolved by adding
evaporate. Before imaging, the samples were mounted 100 μL of dimethyl sulfoxide to each well. Finally, the
onto SEM grids using conductive carbon tape, and then absorption of individual wells was recorded at 540 nm
sputter-coated with a 5 nm thick coating of iridium and a using a plate reader (PHERAstar FS, Germany).
3 nm thick coating of gold/palladium. Images were taken
of the coated samples with a field emission SEM system 2.5. 3D Bioprinting of Myoblast Cells
(FEI Nova Nano630 SEM, Oregon, USA).
2.5.1. 3D Bioprinting
2.3. Cell Culture and Growth Conditions
In two vials, 15 mg of CH-01 and CH-02 peptide powder
2.3.1. Mouse Myoblast Cells (C2C12) each were weighed out and placed under UV for 30 min
sterilization. The peptide powder was dissolved in 1mL of
Mouse myoblast cells (C2C12) were cultured either in a MilliQ water and the peptide solution was then vortexed and
T175 or T75 culture flask in complete DMEM media (10% sonicated to obtain a homogenous solution. The vials were
FBS and 1% P/S). The cells were placed in a humidified placed in an incubator at 37°C and 5% CO . The incubation
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incubator with 95% air and 5% CO at 37°C. Then they time for the CH-01 pre-gel bioink solution was 3.5 h, but
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were subcultured using trypsin at approximately 80% was 2 h when using the CH-02 pre-gel bioink solution.
confluence. Fresh culture media was added every 48 h. A custom-designed robotic 3D bioprinter [27,28] was set
up with commercial microfluidic pumps. A homemade
2.3.2. 3D Culture of Myoblast Cells in Peptide two-inlet nozzle was used for extrusion. A heatbed was
Hydrogels set to 37°C to create a suitable environment for the cells
In a 96-well plate, mouse myoblast cells were once extruded within the peptide bioink. Two commercial
encapsulated in peptide hydrogels, as previously microfluidic pumps were loaded for extrusion. A simple
described . Briefly, peptide solutions CH-01 (4 mg/ mL) gcode file was used to create a structure of 8 layers.
[1]
and CH-02 (3 mg/mL) were added at 40 μL/well. Mouse Pump 1 was loaded with the peptide pre-gel solution
myoblast cells (30,000 cells/well) that were re-suspended and set to a flow rate of 60 µL/min. Pump 2 was loaded
in 2xPBS were mixed gently with the peptide solutions. with myoblast cells containing serum-free DMEM culture
The gelation time was 3-5 min. Subsequently, the culture media. The same procedure was conducted for both
medium was added to the wells. peptides. 17-18 samples were printed for each peptide
with a height of 7-8 layers for each sample.
2.3.3. Differentiation of Myoblast Cells within 3D
Culture Construct 2.5.2. Live/Dead Assay
6 days of culturing myoblast cells inside the 3D environment A two-color fluorescence assay was used to assess the cell
in the growth medium, the culture conditions were then viability within the printed constructs. Calcein was used

76 International Journal of Bioprinting (2019)–Volume 5, Issue 2