Page 210 - IJB-10-5

International Journal of Bioprinting Biomimetic osteochondral scaffold

to a confocal laser scanning microscopy for fluorescence and incubation in 1% BSA, samples were treated with
staining image observation. monoclonal antibodies for RUNX2, COL I, or OCN
(Boster Biological Technology, China) diluted at 1:500
2.10. Cell viability and proliferation on and incubated at 4°C overnight. After rinsing with DPBS,
osteochondral scaffolds samples were incubated in a solution of Alexa Fluor
The rBMSCs suspension (0.2 mL, 5 × 10 cells/mL) was 488-conjugated secondary antibodies diluted at 1:800 for
5
dropped onto the subchondral and interface layers, and 1 h at 37°C. The samples were then treated with a solution
rBMSC microspheres/GelMA solution (0.2 mL, 10 of Alexa Fluor 594 phalloidin and DAPI for F-actin and
3
microspheres/mL) was dropped onto the cartilage and nucleus staining. The fluorescence of each sample was
interface layers. Cell viability was detected by Live/Dead observed using a confocal laser scanning microscope. To
staining. Briefly, after 1 and 3 days of cultivation, cell-laden investigate ALP production, after fixing with 4% PFA for
scaffolds were rinsed with DPBS and incubated in ethidium
homodimer-1 (4 μM) and calcein AM (2 μM) for 20 min at 20 min, samples were treated with ALP staining (described
37°C, where living cells and dead cells were presented with in Section 2.6). Furthermore, for real-time quantitative
green and red colors, respectively. ImageJ software was used polymerase chain reaction (RT-qPCR) analysis, Trizol
to analyze cell viability. The cell proliferation properties (Thermo Fisher Scientific, USA) was used to extract total
were examined using Cell Counting Kit-8 (CCK-8, GlpBio, RNA. Thereafter, reverse transcription was performed
USA) after 1, 4, and 7 days of cultivation. Briefly, samples using a reverse transcription reagent kit (ABM, Canada).
were incubated in CCK-8 solution at 37°C for 4 h, and MagicSYBR mixture, ROX, cDNA, and mixed primers
the absorbance was measured using a microplate reader were loaded onto a 96-well plate, and RT-qPCR was
(Synergy HT, Bio-Tek Instruments, USA) at 450 nm. performed in an Applied Biosystems QuantStudio™ 3 Real-
Time PCR Instrument (Singapore). The primer sequences
2.11. Osteogenic differentiation of rat bone marrow are displayed in Table 1.
mesenchymal stem cells in osteochondral scaffolds
Rat bone marrow mesenchymal stem cells (rBMSCs) 2.12. Chondrogenic differentiation of
seeded on tissue culture plates and cultured with ordinary osteochondral scaffolds
culture medium (DMEM with 10% FBS and antibiotics) After 21 days of cultivation, osteochondral scaffolds
were set as the negative control group. After 14 days of were subjected to staining of the nucleus, F-actin, and
cultivation, osteochondral scaffolds were subjected to chondrogenic markers (sry-related HMG box protein-9
staining of the nucleus, F-actin, and osteogenic markers [SOX9], COL II, and aggrecan [ACAN]). Briefly, after
(Runt-related transcription factor-2 [RUNX2], collagen fixation with 4% PFA, permeabilization in 0.2% Triton
I [COL I], or osteocalcin [OCN]). Briefly, after fixation X-100, and incubation in 1% BSA, samples were treated
with 4% PFA, permeabilization in 0.2% Triton X-100, with SOX9, COL II, and ACAN monoclonal antibodies

Table 1. Primers used in real-time quantitative polymerase chain reaction (RT-qPCR) analysis

Gene Primer (5’ to 3’) Sequence (5’ to 3’)
GAPDH Forward ACCACAGTCCATGCCATCAC
Reverse TCCACCACCCTGTTGCTGTA
RUNX2 Forward GAACCAAGAAGGCACAGAC
Reverse AATGCGCCCTAAATCACTG
COL I Forward CAAGAAGACATCCCTGAAGTC
Reverse GCATACATCAGGTTTCCACG
OCN Forward CTTTGTGTCCAAGCAGGAG
Reverse CTCCCAGCCATTGATACAG
SOX9 Forward CTCTGGAGACTTCTGAACGA
Reverse ACTTGTAATCCGGGTGGTC
COL II Forward AGGAGACAGAGGAGAAGCT
Reverse CTTGAGGACCCTGGATTCC
ACAN Forward CTGACCAGACTGTCAGATACC
Reverse TCCTCACACCAGGAAACTC

Volume 10 Issue 5 (2024) 202 doi: 10.36922/ijb.3229