Page 304 - IJB-10-5

International Journal of Bioprinting Proteins-loaded 3D-printed PEEK cage

2.5. Mobility assessment of mesenchymal stem cells samples for 20 min for qualitative observation. Acetic acid
The migration capacity of MSCs was assessed using the solution (10% [v/v]) (Southwest, China) and ammonia
transwell assay. A 24-well transwell chamber (pore size: solution (10% [v/v]) (Southwest, China) were then used
8 mm; Corning, China) was used to evaluate the effect of to treat the stained samples. Finally, the mineralization
different samples on the migration of MSCs. Approximately of MSCs was measured at an OD value of 405 nm using
1 × 10 cells were seeded in the upper chamber, and samples a spectrophotometric microplate reader (Thermo
4
with different coatings were placed in the bottle chamber Fisher, USA).
and immersed in 600 mL of medium supplemented with
1% fetal bovine serum (FBS; ExCell, China). After 12 2.10. Osteogenesis-related gene expression
and 24 h of incubation, non-migrated MSCs in the upper The osteogenesis-related gene expression of MSCs
chamber were gently removed with a cotton swab. The was evaluated by quantitative reverse-transcription
remaining MSCs were treated with 4% paraformaldehyde polymerase chain reaction (qRT-PCR). MSCs were seeded
(Sigma, USA) and stained with 0.1% crystal violet solution on the surface of different samples and after 3 days of
(Glpbio, USA). Each group (n = 4) was imaged using an incubation, qRT-PCR experiments were performed. The
Olympus inverted microscope (Olympus, Japan), and cell cells were lysed with TRK lysate and then centrifuged
migration capacity is determined from the mean number for RNA extraction. The mRNA concentration of each
of stained cells. sample was then determined using a NanoDrop 2000
Spectrophotometer (Thermo Fisher, USA). The reagent
2.6. Cell viability assay was then removed from the frozen Takara PrimeScript RT
MSCs were seeded on the surface of each sample and Reagent Kit with gDNA Eraser (Takara, Japan) and thawed
processed for fluorescein diacetate/propyl iodide (FDA/ on the ice box. The RNA obtained was reverse transcribed
PI) staining and CCK-8 experiments after 4 and 7 days of into cDNA using the kit. The expression of genes (ALP,
incubation. The media of different samples were washed COLI, OCN, OPN, OPG, and RUNX2) was quantified using
with PBS, and the working solution was added to the a qPCR instrument (Bio-Rad, USA), and the primers of
different samples. When the FDA/PI solution was used, the osteogenesis-related genes are listed in Table 1.
samples had to be incubated for 10 min and subsequently
photographed using a fluorescence microscope (Olympus, 2.11. In vivo animal evaluation
Japan). For the CCK-8 experiment, we had to determine New Zealand white rabbits (6 months old; n = 16) were
the absorbance value (optical density [OD]) at 450 nm randomly divided into four groups, and the nucleus
using a spectrophotometric microplate reader (Thermo pulposus in the L4/5 intervertebral disc was removed from
Fisher, USA). The OD value is directly proportional to the the rabbits in all groups. Subsequently, different PEEK
cell activity. cages were implanted into the L4/5 intervertebral disc
for 2 months. All animal experiments were approved by
2.7. Osteogenic response of mesenchymal stem cells the Experimental Animal Ethics Committee of Southwest
In this study, MSCs were cultured by conditional culture. Medical University (approval number: 20230228-013).
The chromogenic and activity detection of ALP, collagen After 2 months, the L4 and L5 vertebral bodies were
secretion detection, and mineralization detection were sectioned to assess the interbody fusion effect using micro-
performed. MSCs were co-cultured for 4 and 7 days, computed tomography (CT) (PerkinElmer, USA) and the
respectively. The BCIP/NBT ALP kit was used for push-out test using an electronic static material testing
qualitative observation. The MicroBCA Protein Assay Kit system (Instron, UK).
(Abcam, UK) and the p-nitrophenyl phosphate assay kit
(Millipore, USA) were employed for quantitative analysis. 2.12. Statistical analysis
Data are presented as mean ± standard deviation. One-way
2.8. Collagen secretion assay analysis of variance (ANOVA) and student’s t-test were
MSCs were co-cultured for 14 days. Sirius Red was used performed to determine the statistical significance of the
to stain the various samples for qualitative observation of data, i.e., *p < 0.05; **p < 0.01; and ***p < 0.001.
collagen secretion. Thereafter, 1 mM NaOH was used to treat
stained samples for quantitative analysis. Finally, collagen 3. Results
secretion was measured at an OD value of 540 nm using a
spectrophotometric microplate reader (Thermo Fisher, USA). 3.1. Sample characterization
Substance P (SP) is a peptide composed of a chain of 11
2.9. Mineralization assay amino acid residues in its sequence. To graft SP onto Gel/
MSCs were co-cultured for 14 days. Alizarin Red (Glpbio, Chi multilayer films and maintain its bioactivity, a Schiff
USA) working solution (pH 4.1) was applied to stain the base reaction utilizing chitosan with an aldehyde group

Volume 10 Issue 5 (2024) 296 doi: 10.36922/ijb.3574