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CTP Scaffolds Treated Bone Defects
to scan the radial defects in the rabbits. The regions of mean ± standard deviation. One-way analysis of variance
interest were selected and reconstructed. Then, 510 axial (ANOVA) was used for statistical analysis, and the data
images were reconstructed into 3D images. In the bone are indicated with * if the probability is <0.05 (P < 0.05).
tunnel, the residual scaffolds could be distinguished from
the newly formed bone in the tunnel by setting different 3. Results and discussion
gray levels. The gray threshold from 40 hu to 60 hu 3.1. Scaffold characterization, mechanical
represented the implanted composite scaffold, and the
gray threshold from 80 to 255 represented new bone. The properties and drug release behavior
changes in stent volume after implantation were analyzed. The morphology and structure of CTP scaffolds were
evaluated by SEM. As shown in Figure 2A-D, both
2.12. Microfil angiography and micro-CT scaffolds had a porous structure in both the horizontal
imaging and vertical (cross-sectional) directions, and the
After the New Zealand white rabbits had been interconnected gridded pores had a side length of 200 ±
anesthetized, their abdomen was fixed on the plate with 50 μm. Compared to TP scaffolds, CTP scaffolds had a
the abdomen facing upward. The skin and muscle layers rougher strut surface on which a number of micropores
were cut along the midline of the abdomen. The xiphoid and numerous TCP particles with a diameter around
process was lifted, the diaphragm was cut open, and the 200 nm can be seen (Figure 2E and F). The mechanical
heart was exposed. The heart was fixed with hemostatic properties of the TP and CTP scaffolds were measured
forceps, a 23 G needle was inserted into the left ventricle, through compression testing and the results are shown
and the right auricle was cut with ophthalmic scissors. in Table 2 and Figure 2G and 2H. The compressive
The perfusion pump was opened. The perfusion pump strengths of TP and CTP were 1.20 MPa and 1.34 MPa,
was irrigated with normal saline until no red liquid flowed respectively, which are in the low range of trabecular bone
[22]
out and was then changed to 4% paraformaldehyde. After (1.3 – 4.4 MPa) , and hence is suitable for treating bone
the muscle tissue was fixed, 40 – 50 ml of mixed microfil defects. The in vitro release behavior of CTP scaffold
liquid (solvent: solute = 4:5 and 1 – 2% coagulant) was was also studied. As shown in Figure 2I, 55% level of
infused. At the end of the perfusion, the small mesenteric released CFZ was observed on day 5, and the release
vessels turned yellow. The rabbit carcass was kept in a profile achieved a plateau after 10 days of incubation. The
4°C refrigerator overnight. After the contrast medium survival rate of C3H10T1/2 cultured on the TP and CTP
was fixed, the rabbits’ upper limbs were taken for micro- scaffolds for 3 days (as shown in Figure 2J) was more
CT scanning to observe the vascular development. than 90%, indicating that the TP and CTP scaffolds were
a biocompatible platform for C3H10T1/2 culture.
2.13. Histology analysis
At 12 weeks, the rabbits were sacrificed by injecting 3.2. CTP scaffolds promote osteogenic
differentiation of C3H10T1/2 cells
air into the ear edge vein. The skin and muscle tissues
were removed, and the radial specimens were obtained. To analyze the effect of CTP scaffold on osteogenesis,
The specimens were fixed in 4% PFA solution for we treated the mouse mesenchymal cells line C3H10T1/2
2 weeks and then decalcified with 10% EDTA solution with osteogenic media supplemented with extracts of the
for 4 – 6 weeks. The decalcification was continued until scaffold. Figure 3A shows the mRNA expression of the
the needle could be easily inserted into the bone tissue. osteogenic genes in C3H10T1/2 cells stimulated with
Gradient concentrations of ethanol solution were used control or CFZ for 7 days. It was seen that the expression
for dehydration, and a xylene soak for 30 min was used of osteogenic genes, including ALP, BMP2, Col1α1,
to clear the tissue. This was repeated twice. The treated OCN, Osterix, and Runx2, was all upregulated in CFZ
tissues were immersed in paraffin for 30 min, and the group compared with control group. With Western blot
paraffin was replaced 3 times. After paraffin solidification, analysis, we confirmed that the protein level of OCN,
the embedded specimens were taken out and cut into Col1α1, and Runx2 was increased, and the expression
5-μm slices. They were then put in distilled water, pasted of Runx2 and Col1α1 was more pronounced in the CFZ
to a slide, and baked at 60°C overnight. The prepared group (Figure 3B), which were accordant with the
paraffin sections were used for hematoxylin and eosin Table 2. Compression mechanical results of TP and CTP scaffolds
staining.
Scaffold Young Ultimate tensile Strain
2.14. Statistical analysis modulus (E) strength (UTS)

All quantitative data were obtained from four or five TP 31.52 1.20MPa 9.69%
independent experiments. The results are expressed as the CTP 33.13 1.34MPa 11.90%

104 International Journal of Bioprinting (2021)–Volume 7, Issue 4