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Shi, et al.
submerged in phosphate-buffered saline (PBS) buffer MC3T3-E1 cells at a density of 5 × 10 cells/mL were
4
solutions at room temperature for 12 h to equilibrate. The inoculated on 3D printed scaffolds. After 2 h, 400 mL
strain rate was 10% s and the maximum compressive medium was added and cultured for 1, 3, and 5 days.
-1
strain was 70%. The preload force was set as 0.01 N. At the predetermined time point, 20 μL Counting Kit-8
The compressive modulus was calculated from the initial (CCK-8) reagent (Dojindo Kagaku, Japan) was added
strain range 1 – 3% and a linear fit. Five samples were to each group. After further incubation for 2 h, 100 μL
repeated for each group. mixed medium was transferred to 96-well plate and the
Tensile tests of printed monofilaments were absorbance of the solution was measured at 450 nm using
conducted on a Zwick mechanical testing machine a microplate reader (TECAN unlimited F50, Switzerland).
(Zwick Line 0.5, ZwickRoll, Germany). The extension
rate was 2 mm·min . The tensile modulus was calculated 2.14. Detection of Alkaline Phosphatase (ALP)
-1
from the initial strain range 1–3% and a linear fit. Fifteen activity
samples were repeated for each group. MC3T3-E1 cells at a density of 5 × 10 cells/mL were
3
2.11. Water retention test inoculated on 3D printed scaffolds which placed in a
48-well culture plate. After 2 h, 400 mL medium was
PBS solutions were used to measure the water retention added and cultured for 1, 4, 7, and 10 days. The complete
behavior of the scaffolds. After soaking the scaffolds in DMEM medium containing osteogenic induction fluid
PBS for 1–24 h, the excess water on the outer surface of (OIF) was added and replaced every other day (OIF:
the hydrated scaffold was absorbed carefully using filter 50 μg/mL ascorbic acid, 10 mM β-Glycerol phosphate,
paper and the mass was measured. The hydration degree and 10 μg/mL and 10 M dexamethasone). At the
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H of the scaffold was calculated by the Equation 3. predetermined time point, cells were lysed with 100 μL
H = ( M − M ) / M (3) RIPA lysis buffer, and the cell supernatant was collected
w d d
into 96-well plates. After the substrate was incubated
M is the weight of the scaffold before testing and with p-nitrophenol at 37°C for 30 min, ALP activity was
d
M is the weight of the hydrated scaffold. measured at 405 nm wavelength by ALP detection kit
w
In vitro degradation test, the degradation behavior (beyotime, China). Meanwhile, bicinchonic acid protein
of scaffolds in vitro was evaluated using enzyme Protease analysis kit (beyotime, China) was used to determine
XIV from Streptomyces griseus following a procedure the total protein content at 560 nm wavelength. Finally,
reported in ref . Protease XIV (3.5 U/mg) was dissolved the ALP level was standardized according to the protein
[42]
in PBS solution at 2 U/mL. Dry samples were weighed and content of each group, and the ratio of OD405/OD560
immersed in 1 mL protease XIV/PBS solution at 37°C. was calculated to determine the ALP level of each group.
The enzyme/PBS solution was changed every 3 days.
Samples were taken out after 7, 14, 21, and 28 days, 2.15. Quantitative real-time PCR (q-PCR)
rinsed with deionized water, freeze-dried, and weighed for Cells at a density of 10 cells/ml were seeded on the 3D
5
analysis. Three repeats were recorded for each time point. printed scaffold that was placed into the 6-well plates
The weight loss rate was calculated using Equation 4. for 2 h, and then ALP detection assay was performed.
L % () = ( M − M )/ M ×100% (4) After 10 days, total RNA in each group was extracted
1 2 1 with Trizol reagent (Invitrogen). Then, specific steps in
M is the initial mass of the scaffold and M is the the following were described according to our previous
1
2
remaining mass of the scaffold after varied degradation study . The Runx2, OPN, OCN, OSX, and Col1a mRNA
[43]
time. levels were normalized to that of GAPDH which was used
2.12. Cell culture as reference. The corresponding primers were synthesized
by Sangon Biotech Crop (Shanghai, China) and the
Mouse derived pre-osteoblasts (MC3T3-E1) were purchased corresponding primer sequences are listed in Table 1.
from American Type Culture Collection. Cell were cultured
by Dulbeccos Modified Eagle Medium (DMEM, GIBCO, 2.16. Statistical analysis
US) containing 10% fetal bovine serum (FBS, GIBCO, US) All experimental data were measured on n ≥ 3 samples.
and 1% penicillin streptomycin (PS, GIBCO, US) under an All data are presented as mean ± standard deviation
incubator containing 5% CO at 37°C.
2 (M ± SD). For n ≥ 5 measurements, one-way analysis
2.13. CCK-8 experiment of variance (ANOVA) was performed to determine the
statistical differences between groups. The statistical
3D printed scaffolds (SF scaffold 3 and SF scaffold significance was designated as * for P < 0.05, ** for
3-CaP10) were placed in 48-well culture plates. 0.01 < P < 0.05, and *** for 0.001 < P < 0.01.

International Journal of Bioprinting (2022)–Volume 8, Issue 4 5