Page 287 - IJB-10-5

International Journal of Bioprinting 3D printing of collagen II-scaffolds

marrow, of SD mice. Before cell seeding, all scaffolds were (i) RNA extraction: Cells were digested using a
immersed in 75 wt% ethanol for 10 min, washed once TRK Lysis Buffer (Omega Bio-tek, USA), and
with phosphate-buffered saline (PBS; Beyotime, China), impurities were removed by centrifugation in an
exposed to ultraviolet (UV) radiation for 30 min, and RNA homogenizer spin column (Omega Bio-tek,
immersed in Minimum Essential Medium α (α-MEM; USA). The filtrate was collected and transferred to a
Vivacell, China), supplemented with 10 wt% fetal HiBind RNA mini-column (XXX, YYY). Nuclease-
bovine serum (Gibco, Australia), 10 kU/mL penicillin free water was then used to collect RNAs attached
(Beyotime, China), and 10 mg/mL streptomycin to the column. The total RNA concentration was
(Beyotime, China) (henceforth denoted as complete measured using a NanoDrop spectrophotometer
medium) for 24 h. MSCs passage 2 (P2) were seeded onto (Thermo Fisher Scientific, USA) and the 260/280 nm
scaffolds at a density of 10 cells/mL for CCK-8 testing absorbance ratio was recorded.
5
and 10 cells/mL for all other tests. Cell culture was (ii) Reverse transcription: Following the kit’s
6
conducted in an incubator (Thermo Fisher Scientific, instructions, gDNA Purge and 2× NovoScript Plus
®
USA) at 37°C and 5% CO . After 24 h with observable 1 Strand cDNA Synthesis SuperMix were added
st
2
cell attachment, the complete medium was replaced to the extracted RNA for reverse transcription into
by a chondrogenic differentiation medium to induce cDNA. The mixture was incubated at 50°C for 15 min
the chondrogenic differentiation. The chondrogenic and subsequently at 75°C for 5 min. The resultant
differentiation medium is composed of the as-prepared cDNA was then directly used for qPCR.
α-MEM culture medium, supplemented with 10 μg/mL
transforming growth factor-β3 (Abbkine, China), 10 (iii) qPCR: According to the manufacturer’s instructions,
®
mM β-glycerol phosphate (Sigma-Aldrich, USA), and 2× NovoStart SYBR qPCR SuperMix Plus, forward
100 nM dexamethasone (Sigma-Aldrich, USA). and reverse primers, template, and RNase-free water
were sequentially added to the cDNA sample with
2.5.2. CCK-8 test a final volume of 20 μL. qPCR amplification was
CCK-8 test was conducted on days 1, 3, and 5 of culture. performed and the reaction program was set to 95°C
Before the test, the existing culture medium was replaced for 1 min for initiation, followed by 35–45 cycles of
with α-MEM medium, supplemented with 10 wt% CCK- incubation at 95°C for 20 s and 60°C for 1 min.
8 cell-counting agent (Beyotime, China), and incubated
for 1 h. During the test, 100 μL of cell supernatant was The results were subsequently analyzed using the
29
extracted and its optical density (OD) value was measured ΔΔCt method. The cycle threshold (Ct) value is the
at a wavelength of 450 nm using a spectrophotometer cycle number at which the fluorescence signal exceeds
(Multiskan GO; Thermo Fisher Scientific, USA). Each the background noise. The Ct values of the reference and
sample type was assessed in triplicates, and data are target genes of the experimental and control groups were
presented as mean ± standard deviation. One-way analysis measured using qPCR. The ΔCt value was then calculated
of variance (ANOVA) was performed for comparison for each sample as the difference between the Ct values of
between sample types using GraphPad Prism 7. Statistical the experimental and control groups:
significance is considered for p < 0.05.
ΔΔCt = ΔCt (experimental group) − ΔCt (control group) (I)
2.5.3. Reverse transcription-quantitative polymerase
chain reaction
®
On day 7 of culture, the E.Z.N.A. Total RNA Kit Next, the relative expression of the target gene in the
(Solarbio, China) was used to extract total RNA from experimental group compared to the control group was
®
bone MSCs (BMSCs). NovoStart SYBR qPCR SuperMix determined as follows:
plus (Novoprotein, China) was used to measure the
gene expression levels of collagen I (COL1), collagen Relative expression = 2 −ΔΔCt (II)
II (COL2), hypoxia-inducible factor-1α (HIF1A), SRY-
box transcription factor 9 (SOX9), runt-related transcription 2.5.4. Cell morphology staining
factor-2 (RUNX2), Aggrecan core protein (ACAN), focal Cell morphology staining was performed using Actin-
adhesion kinase (FAK), and N-cadherin (NCAD). Relative Tracker Red (Beyotime, China) and 4´,6-diamidino-2-
gene expression was normalized to the reference gene of phenylindole (DAPI; Beyotime, China) staining to visualize
glyceraldehyde-3-phosphate dehydrogenase (GAPDH). the cell cytoskeleton and nucleus, respectively. The stained
Quantitative reverse transcription polymerase chain cells were fixed with a mounting medium (Biosharp,
reaction (RT-qPCR) was conducted as follows: China) and examined under an upright microscope (Leica,

Volume 10 Issue 5 (2024) 279 doi: 10.36922/ijb.3371