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Jiao, et al.
system (Bruker SkyScan 1176; Germany) was used to A B
analyze the samples, which were scanned with a spot size
of 10 μm, current of 250 μA, and maximum voltage of
40 kV. Then, the 3D images were reconstructed, and the
bone volume fraction (BV/TV) and bone mineral density
(BMD) were calculated using auxiliary software.

(3) Hematoxylin and eosin (H&E) staining and
Masson staining C D
The skull samples were fixed with 4% paraformaldehyde
and decalcified in 10% ethylenediaminetetraacetic
acid. Then, the samples were embedded in paraffin
and sectioned at a thickness of 5 μm. H&E staining
and Masson’s trichrome staining were performed as
previously described .
[26]
(4) Immunohistochemistry
Figure 1. Characterization and biocompatibility of β-TCP extract.
The sample sections were incubated with primary (A) Ion concentrations of Ca and P of α-MEM medium (Ctrl),
antibodies at 4°C overnight. The slides were then α-MEM with 1/32 dilution of β-TCP extract (1/32), α-MEM with
incubated with horseradish peroxidase (HRP)-conjugated 1/64 dilution of β-TCP extract (1/64), and α-MEM with 1/128
secondary antibodies. Images were captured using a dilution of β-TCP extract (1/128). (B) pH values of Ctrl, 1/32, 1/64,
microscope and the numbers of positive cells were and 1/128 dilution groups. (C) OD values of BMSCs in Ctrl, 1/32,
1/64, and 1/128 dilution groups on day 3 by CCK-8. (D) OD values
counted.
of BMSCs in Ctrl, 1/32, 1/64, and 1/128 groups on day 7 by CCK-
2.12. Statistical analysis 8. *P < 0.05; **P < 0.01.

All results are expressed as mean ± standard deviation The pH values were 7.353 ± 0.012, 7.343 ± 0.012, 7.427
(SD). Error bars represent the SD. A two-tailed Student’s ± 0.009, and 7.427 ± 0.021, respectively (Figure 1B).
t-test and one-way analysis of variance were used to The pH of the Ctrl and 1/32 dilution was significantly
analyze the difference between two groups and more than different from that of 1/64 and 1/128 dilutions (P < 0.01).
2 groups, respectively; subsequently, Tukey’s post hoc The cytotoxicity of the β-TCP extract was examined
analysis was used to compare the differences between using the CCK-8 assay. It was found that BMSCs
three or more groups. Statistical significance was set at proliferated, and the cell numbers increased over time
*P < 0.05 and **P < 0.01. throughout the assay period. No significant difference
was observed between the Ctrl and β-TCP extracts on day
3. Results 3 after treatment, revealing no cytotoxicity of the β-TCP
extract on day 3 (Figure 1C). On day 7 after treatment,
3.1. Characterization and biocompatibility of however, the absorbance of 1/32 and 1/64 dilutions of
β-TCP extract β-TCP extract obviously decreased, showing a lower cell

We prepared extract of β-TCP made of β-TCP powder. viability than the Ctrl and 1/128 dilution of β-TCP extract
Size distribution analysis revealed a particle size on day 7 (Figure 1D).
smaller than 10,000 nm and mainly between 300 and 3.2. β-TCP promoted the osteogenic
1000 nm (Figure S1). The zeta potential of β-TCP
extract was −7.04 ± 0.69 mV (Table S1). Next, we differentiation of BMSCs
diluted β-TCP extract into the ratios of 1/32, 1/64, and We then tested the osteogenic differentiation of the BMSCs
1/128. The concentrations of Ca and P ions (Ctrl, 1/32, induced by β-TCP. As shown in Figure 2A and B, after
1/64, and 1/128) were detected by ICP-AES, as shown in induction for 7 days, all the groups treated with β-TCP
Figure 1A. The concentrations of Ca were 66.20 ± 1.53, extract showed approximately twice the ALP expression
65.37 ± 0.81, 64.26 ± 0.97, and 65.79 ± 1.26 mg/L in than those of the Ctrl. However, the expression among
Ctrl, 1/32, 1/64, and 1/128 dilutions, respectively, while the three β-TCP groups showed no significant difference,
the concentrations of P were 32.05 ± 1.64, 29.96 ± 0.65, suggesting that the β-TCP extract could induce
29.80 ± 0.96, and 30.99 ± 0.19 mg/L, respectively, which osteogenic differentiation of BMSCs. We also examined
were consistent with a previous study and showed no the expression of bone formation-related genes.
significant difference in the concentrations of Ca and P . Figures 2C-F show that the levels of collagen type I
[27]
International Journal of Bioprinting (2022)–Volume 8, Issue 2 35