Page 74 - IJB-9-4

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

2.4.2. Cytoskeletal staining Table 1. TaqMan probes
The cells were first fixed with 4% formaldehyde solution Gene Catalog number of Catalog number
for 30 min and incubated in a cold cytoskeleton buffer TaqMan probe
(3 mM MgCl , 300 mM sucrose and 0.5% Triton X-100 in
2
PBS) for 5 min to permeabilize the cell membranes. The Collagen I (Col-I) Hs00164004_m1 4331182
permeabilized cells were incubated in blocking buffer Collagen II (Col-II) Hs00264051_m1 4331182
solution (5% FBS, 0.1% Tween-20, and 0.02% sodium Collagen X (Col-X) Hs00166657_m1 4331182
azide in PBS) for 30 min at 37°C. For F-actin, anti-mouse Aggrecan Hs00202971_m1 4331182
IgG (whole molecule)-FITC and rhodamine-phalloidin GAPDH Hs99999905_m1 4448490
(1:300) was added to the cells for 1 h. Further, the cells Sox9 Hs00165814_m1 4331182
were incubated in DAPI for 5 min to counterstain the
nucleus. These fluorescent dye-treated cells were observed
and imaged using a laser scanning confocal microscope n ≥ 3. Statistical analysis was performed using two-way
(Zeiss LSM 710 Inverted Confocal Microscope, Germany). analysis of variance (ANOVA), and values with p < 0.05

2.5. Chondrogenic differentiation were considered statistically significant.
Printed cell-laden constructs were placed in the CO
2
incubator for 3 days with complete growth media (α-MEM+ 3. Results and discussion
supplements). After 3 days, the constructs were washed
three times with 1× PBS for 5 min each, and chondrogenic 3.1. Ultrashort peptide gelation and
media were added for 30 days with media exchange characterization
every 3 days. The chondrogenic media is composed of Self-assembling peptide hydrogels have gained significant
DMEM High Glucose (Invitrogen, Thermo Fisher, USA) attention as cell-laden scaffolds due to their biocompatibility,
supplemented with 1% ITS+ premix (Corning, USA), 10 M mechanical tunability, and reproducibility. These non–
−7
dexamethasone (Sigma-Aldrich, Germany), 200 µM animal-derived materials can form hydrogels through
ascorbate 2-phosphate (Sigma-Aldrich, Germany), 10 ng/mL noncovalent interactions (e.g., hydrogen bonding,
TGF-β3 (R&D systems, USA), 40 µg/mL L-proline (Sigma- hydrophobic interaction, etc.). Compared to irreversible
Aldrich, Germany), and 1 mM sodium pyruvate and 1% covalently crosslinked hydrogels, peptide hydrogels
penicillin/streptomycin (Invitrogen, Thermo Fisher, USA). provide a dynamic mechanical microenvironment that can
promote better cell growth and spreading [32,33] . However,
2.6. Gene expression assessment (quantitative gene these physical interactions between peptide molecules
expression analysis by RT-PCR) generally contribute to the relatively poor mechanical
Total RNA was extracted from the 3D-bioprinted properties, limiting their further use in some emerging
constructs at each specific time point using the mixed biomedical applications, such as bioprinting [34-36] . Therefore,
isolation procedure using Trizol and Qiagen® RNeasy other mechanical integrity and gelation rate improvements
Mini kit (Qiagen, USA). Total RNA concentration while preserving the noncovalent interactions will be a
and purity were measured using the NanoDrop 2000 good strategy for developing a superior peptide bioink.
spectrophotometer system (Thermo Fisher Scientific,
USA). RNA was reverse-transcribed into cDNA using In our previous study, we developed amphiphilic
the SuperScript™ VILO™ cDNA synthesis according to the tetrapeptides that can self-assemble to form hydrogel at a
manufacturer’s instructions (Thermo Fisher Scientific, relatively low concentration (1 mg/mL) under physiological
[37]
USA). Quantitative polymerase chain reaction (PCR) was conditions (Figure 1A) . These ultrashort peptides were
performed using TaqMan gene expression assay (Table 1) mainly composed of a nonpolar hydrophobic tail and
and TaqMan™ Fast Advanced master mix (Thermo Fisher positively charged head group. The positively charged
Scientific, USA) using the Quantstudio 3 system (Thermo residue played an important role in solubility and cell
Fisher Scientific, USA). The thermal cycling parameters adhesion. Interestingly, two of these ultrashort peptides
were 50°C for 2 min, then 95°C for 2 min, followed by 40 that displayed faster aggregation kinetic and lower critical
cycles of 95°C for 1 s and 60°C for 20 s. GAPDH gene was gelation concentration contained a highly hydrophobic
used as an endogenous control. Results are displayed as cyclohexylalanine (Cha) in the middle of their sequences.
mean ± standard deviation. Through molecular dynamics simulations, the role of Cha
residue in the self-assembly of these ultrashort peptides
2.7. Statistical analysis was found to be related to the formation of Cha–Cha cross
All experimental approaches were executed in triplicates. interaction that can stabilize the β-sheet conformation of
[38]
Results are represented as mean ± standard deviation, the ultrashort peptides . Based on these results, we used
Volume 9 Issue 4 (2023) 66 https://doi.org/10.18063/ijb.719