Page 285 - IJB-9-1

International Journal of Bioprinting 3D-Printed scaffolds

2.5. In vitro biocompatibility assay and further characterized. Chemical characterization of

3D-printed PTMC, PTMC/TCP, PCL/TCP, and PTMC/ PTMC, PCL, TCP, PTMC/TCP, PCL/TCP, and PTMC/
PCL/TCP scaffolds with 25% TCP content (diameter PCL/TCP scaffolds was analyzed using Fourier-transform
[mm] × height [mm]: 6 × 2) were sterilized and placed into infrared spectroscopy. The spectra of PTMC/TCP
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the 24-well plate. MC3T3-E1 cells (6 × 10 , 200 µL culture displayed characteristic 1740 cm peaks of ester and
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medium) were seeded onto the scaffolds and cultured for 2900 cm peaks of CH, and accordingly, peak intensities
1 day. Subsequently, paraformaldehyde (4%, 1 mL) was decreased with increasing of TCP content from 0% to
added for 10 min and washed. The scaffolds were then 25% (Figure 1A). Moreover, PTMC/TCP indicated 1040,
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stained using fluorescein isothiocyanate (FITC)-labeled 960, 603, and 564 cm peaks of P-O and P=O. PCL/TCP
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phalloidin (500 nmol/L, Invitrogen Co., USA) for 30 min also displayed typical 1740 cm peaks of ester, 2900 cm
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and washed. Cells were stained again with 4’,6-diamidino- peaks of CH, and 1040, 960, 603, and 564 cm peaks of
2-phenylindole (100 nmol/L, Invitrogen Co., USA) for spectral features of P-O and P=O (Figure 1B). Meanwhile,
10 min and washed, and the scaffolds were stored or the spectra of PTMC/PCL/TCP also demonstrated 1948
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photographed at 4°C. The adhesion and growth of the cells cm peaks of typical ester, 2960 cm peaks of CH, and
in scaffolds were observed using a fluorescence inverted accordingly, peak intensities decreased with increasing
microscope (IX-70, Olympus Co., Ltd., Japan). of TCP content (Figure 1C). Moreover, PTMC/PCL/TCP
scaffolds showed P-O and P=O peaks at 1181, 960, 603,
2.6. Osteogenic gene expression and 564 cm .
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rBMSCs (initial density: 8 × 10 cells/mL) were cultured Figure 2 demonstrates that the water contact angles
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in 3D-printed scaffolds for 7 and 14 days. At the indicated of all scaffolds increased with TCP content. Moreover,
time point, RNA was extracted from the cells, and 1 mg PTMC/PCL/TCP scaffolds showed higher water contact
of RNA was reverse transcribed into complementary DNA angles than PTMC/TCP scaffolds and lower water contact
(cDNA). siRNA Transfection Reagent (QIAGEN, Hilden, angles than PCL/TCP scaffolds. This result indicated that
Germany) was utilized following the manufacture’s protocol. PTMC/PCL/TCP scaffolds possessed lower hydrophilicity
The expression of actin, osteocalcin (OCN), alkaline than PTMC/TCP and higher hydrophilicity than PCL/
phosphatase activity (ALP), runt-related transcription TCP scaffolds. Therefore, the modification of PCL with
factor 2 (Runx), and type I collagen (Col-I) were quantified PTMC can enhance hydrophilicity of composites.
and analyzed in triplicate. The relative expression level of
each gene was measured and the expression level of each 3.2. Scanning electron microscopy of PCL/TCP,
target gene was normalized to the reference housekeeping PTMC/TCP, and PTMC/PCL/TCP scaffolds
gene (GAPDH). Micrographs of all scaffolds were measured by a scanning
2.7. In vivo implantation of PCL/TCP, PTMC/TCP, and electron microscope. Figure 3A-C indicates that TCP
PTMC/PCL/TCP scaffolds scattered uniformly and presented a few aggregations in
the scaffolds. The intensity of TCP appeared to increase
Twenty-four Sprague Dawley (SD) rats (weight: 200 ± 20 g) on the basis of the enlargement of TCP content. Therefore,
bearing a circular thighbone defect (diameter [mm]: 4) TCP displayed good compatibility to PTMC and PCL. The
were divided into four groups. 3D-printed PCL/TCP, micrographs of PTMC/PCL/TCP scaffolds indicate that
PTMC/TCP, and PTMC/PCL/TCP scaffolds with 25% TCP can enhance the compatibility of PCL and PTMC,
TCP content (diameter [mm] × height [mm]: 4 × 6) in a way similar to the thermals properties above. The
were implanted in the thighbone defect of the rats. New phase composition was measured by X-ray diffraction
bone formation was measured using micro-computed diffractometer (XRD, German Bruker Co., Germany).
tomography (Skyscan1276 X-Ray Microtomograph, PCL/TCP, PTMC/TCP, and PTMC/PCL/TCP scaffolds
Bruker, Belgium). New bone area and percentage were displayed the same typical XRD data as PTMC, PCL, and
calculated, and 3D reconstruction of interest was also TCP (Figure 4A-C).
determined.
Compressive modulus of scaffolds was determined
3. Results and discussion using MTS universal electronic testing machine according
to the standard GB/T 1041-2008; the results are presented
3.1. Production of PTMC/TCP, PCL/TCP, and PTMC/ in Figure 5A-C and Table 1. All scaffolds showed an
PCL/TCP scaffolds increase in compressive modulus with the increase of
Biodegradable PCL/TCP, PTMC/TCP, and PTMC/PCL/ TCP content. PTMC/PCL/TCP scaffolds displayed higher
TCP scaffolds were produced using 3D printing technique compressive modulus than PTMC/TCP and lower values

Volume 9 Issue 1 (2023) 277 https://doi.org/10.18063/ijb.v9i1.641