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International Journal of Bioprinting 3D printed PEEK scaffold mediates macrophages to affect osseointegration

were allowed to recover. On dates of scheduled explant scaffold is relatively regular, which can also be proven by the
retrieval, rats were sacrificed by CO asphyxiation. The SEM results. The SEM results also show that the diameter
2
repaired femur, with the PEEK plate fixator intact, was of the printing wire of PEEK scaffold is relatively uniform,
carefully separated from the adjacent hip and knee joints and no broken wire and defects are found. To improve
for analysis. the hydrophilicity of the 3D-printed PEEK scaffolds, the
PEEK scaffolds were treated with concentrated sulfuric
2.6.2. In vitro micro-CT analysis acid and concentrated nitric acid in turn, and the contact
After 4, 8, and 12 weeks of implantation, the rabbits were angle and microstructure before and after the treatment
killed by excessive injection of chloral hydrate anesthesia, were tested, as shown in Figure 1B. The contact angle of
and the specimens were obtained and fixed in 10% PEEK scaffolds treated with concentrated sulfuric acid and
formaldehyde solution. The specimens were scanned at concentrated nitric acid decreased from 90° to 65°, and
a pixel size of 30 μm using a micro-CT (SkyScan 1176, the hydrophilicity was significantly improved. In addition,
Bruker, Germany), and the 3D structure was reconstructed SEM results showed that the surface of the acid-treated
from the acquired two-dimensional (2D) continuous scaffolds formed a uniformly distributed layered porous
tomographic images. The region of interest (ROI) was structure with a pore size of about 1–2 μm.
accurately located according to the 3D image to ensure
that the bone defect, scaffold, and new bone can be fully In addition, based on the SEM results of the scaffolds,
covered. After the new bone and residual scaffold in the pore size, porosity, and pore connectivity of the
the ROI region was distinguished, the bone volume/ 3D-printed PEEK scaffolds were analyzed, as shown in
total volume ratio (BV/TV), bone surface/bone volume Table 1. The actual pore size obtained by measurement and
(BS/BV), trabecular thickness (Tb.Th), and trabecular statistics is basically consistent with the preset pore size,
Number (Tb.N) at different implantation time points were and the porosity of the scaffold gradually increases with
systematically analyzed. the increase of the pore size. However, the connectivity
rates of scaffolds with different pore sizes are all around
2.6.3. Histological staining 100%, which indicates that scaffolds with different pore
After micro-CT scanning, the specimens were dehydrated sizes all have good 3D connectivity structures. The
and embedded in polymethyl methacrylate. Then, the results of the study of mechanical properties showed
specimens were serially sectioned at a thickness of 5 μm that the compressive strength of the scaffold gradually
and placed on the glass slide. The sections were stained decreases with the increase of the pore size (Figure S1 in
with hematoxylin & eosin (H&E), Masson’s trichrome, and Supplementary File). In addition, the results of proliferation
von Kossa to assess the bone surrounding the scaffold, and and fluorescent staining of L929 cells on the 3D-printed
then observed under a light microscope (ZEISS, Germany). scaffold group showed that the 3D-printed PEEK scaffolds
with different pore sizes had good biocompatibility
2.6.4. Biomechanical analysis (Figure S2 in Supplementary File).
A biomechanical testing machine (Electro Force 3510,
USA) was used to test the bonding strength between the 3.2. In vitro macrophage polarization
PEEK scaffold and the host bone. The compression rate The proliferation results (Figure 2A) of macrophages co-
was 2 mm/min, and the maximum load force and stiffness cultured with the scaffold for 1, 4, and 7 days showed that
were calculated. there was no significant difference in the number of cells
in the scaffold groups with different pore sizes, which
2.7. Statistical analysis indicated that the pore size of the scaffold had no effect on
All statistical analyses were carried out by using SPSS cell proliferation. Fluorescence staining results (Figure 2B)
version 10.1 software (SPSS Inc., USA). Statistically after 7 days of cell culture showed that macrophages grew
significant differences (p < 0.05) between the various well on the scaffold surface, and there was no significant
groups were adjusted using the Tukey–Kramer post- difference between different groups, which was consistent
hoc test. All the data are expressed as means ± standard with the cell proliferation results.
deviation (SD).
The expression of polarization-related genes in
3. Results macrophages was detected by RT-PCR, as shown in
Figure 3A. Compared with the PEEK group, the expression
3.1. Characteristics of 3D-printed PEEK scaffolds levels of the M1 macrophage-related genes iNOS and
The macro- and micro-morphologies of 3D-printed PEEK nuclear factor kappa B (NF-κB) in the PEEK400 group
scaffolds with different pore sizes are shown in Figure 1A. were relatively lower, while the expression levels of the
It can be seen that the structure of the prepared PEEK M2 macrophage-related genes CD206, TGF-β, and IL-

Volume 9 Issue 5 (2023) 132 https://doi.org/10.18063/ijb.755