Page 354 - IJB-9-6

International Journal of Bioprinting Surface modification of PCL scaffolds

2.4. Cell culture antibodies for an hour at room temperature. The nuclei were
Primary rat BMSCs were obtained from tibias and then counterstained using DAPI, and the resulting images
femurs of male Sprague-Dawley rats. BMSCs were were captured using confocal microscopy (Nikon, Japan).
culture in Modified Eagle Medium (MEM, Gibco, USA)
supplemented with 10% fetal bovine serum (Biological 2.8. Western blotting
industries, Australia). The cells were cultured at 37°C in a After being cultured on scaffolds for 14 days, the
5% CO incubator. BMSCs of passages 3–6 were used for BMSCs were lysed using RIPA. The total proteins were
2
further experiments. then separated by gel electrophoresis, transferred to
polyvinylidene fluoride membranes, and subsequently
2.5. Cell proliferation, toxicity, and morphology blocked with 5% milk. Following incubation with primary
For cell experiments, the scaffolds were sterilized with antibodies, the membranes were washed with TBST and
75% ethanol for 1 h and washed with phosphate-buffered treated with horseradish peroxidase-conjugated secondary
saline (PBS) to remove residual ethanol. After that, BMSCs antibodies. Finally, the membranes were photographed
(2 × 10 cells) were seeded on the scaffolds in the 24-well using Amersham Imager 600. Details of antibodies are
5
plate. After 1, 4, and 7 days of culture, the cell proliferation listed in Table S2 (Supplementary File).
was assessed with cell counting kit-8 (CCK-8, Beyotime,
China). Briefly, 100 µL of CCK-8 solution was added to the 2.9. Alizarin red S staining
cells and incubated for 30 min. The OD value at 540 nm After incubating for 14 and 21 days, the BMSCs on
was then detected using a microplate reader. Live/Dead the scaffolds were washed with PBS and fixed with 4%
staining kit was used for cytotoxicity analysis. In brief, the paraformaldehyde for 20 min. Subsequently, the cells were
working solution was prepared with 2 μM calcein AM and stained with Alizarin red S staining solution (Beyotime,
4.5 μM propidium iodide (PI) solution. After incubation China) for 30 min at room temperature. Finally, the gross
for various time intervals, the cells on the scaffold were and optical images were photographed. For quantification
stained with a working solution for 15 min. Subsequently, analysis, the scaffolds were loaded into 5% sodium dodecyl
the labeled cells were captured using immunofluorescence sulfate (SDS) in 0.5 N HCl for 1 h at room temperature.
microscopy (Zeiss, Germany). Cell viability was calculated The absorbance at 405 nm of this solution was measured
based on the ratio of the number of viable cells (green- using a microplate reader.
stained cells) to the total number of cells (both green- and
red-stained cells). For the quantification of results, three 2.10. Alkaline phosphatase (ALP) staining and
images per time point were analyzed using the ImageJ activity
software for two groups. For morphology analysis, the The BCIP/NBT Alkaline Phosphatase Color Development
BMSCs were fixed with glutaraldehyde and photographed Kit (Beyotime, China) was used for ALP staining. After
by SEM. being incubated for 7 and 14 days, the cells were fixed with
4% paraformaldehyde for 20 min and stained with working
2.6. Quantitative real-time PCR (qRT-PCR) solution for 30 min. After that, the stain was washed with
After being incubated at different time points, the BMSCs distilled water. In addition, Alkaline Phosphatase Assay
on the scaffold were digested by trypsin. Trizol reagent Kit (Beyotime, China) was used for ALP activity analysis
(Invitrogen, USA) was added into the 24-well plate to according to the manufacture’s protocol. The absorbance at
extract total RNA. The concentration of extracted RNA 405 nm was measured using a microplate reader.
was detected using a microplate reader. The cDNA was
synthesized by Reverse Transcription Kit (Takara, Dalian, 2.11. Transcriptome sequencing
China). The amplification process was conducted using the After being incubated for 14 days, the BMSCs on the
SYBR Taq Kit (Takara, Dalian, China). GAPDH was used scaffold were lysed using Trizol reagent (Invitrogen,
as an internal reference. The primers are listed in Table S1 USA) and the lysates were stored at -80°C for further
(Supplementary File). experiments. Illumina NovaSeq 6000 was used to perform
RNA sequencing. The criteria for screening differently
2.7. Immunofluorescence staining expressed genes were P < 0.05 and |log2FoldChange| >1.
After culturing on scaffolds for 7 and 14 days, the cells Free online platform of Novogene was used to conduct
were fixed with 4% paraformaldehyde, permeabilized using Kyoto Encyclopedia of Genes and Genomes (KEGG)
Triton-X, and blocked with goat serum. Following this, the pathway enrichment analyses.
cells were incubated with primary antibodies (anti-OPN,
1:200, Proteintech; anti-OCN, 1:200, Proteintech; anti- 2.12. Animal experiments
RUNX2, 1:300, Proteintech) overnight at 4°C. Following The animal study was approved by Ethics Committees of The
the PBS wash, the cells were incubated with secondary Fourth Military Medical University (IACUC-20220071). A

Volume 9 Issue 6 (2023) 346 https://doi.org/10.36922/ijb.1071