Page 138 - IJB-9-5

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

2.2.2. Water contact angle CO for 30 min, and then, a microplate reader (BIO-
2
The water contact angle of the untreated, sulfuric acid- RAD 680, USA) was used to test the absorbance value
treated and nitric acid-treated scaffold was measured (OD ) of the culture solution at 450 nm. The adhesion
450
by using a Drop Shape Analysis System DSA100 (Kruss, and distribution of macrophages on the surface of PEEK
Germany) at room temperature. scaffolds were observed by fluorescence microscope. After
macrophage seeding for 7 days, the cells were fixed in 4%
2.2.3. Parameters of scaffold pore paraformaldehyde for 30 min. All samples were rinsed
According to the SEM image of the scaffold, the distance several times with PBS and permeabilized with 0.5% Triton
(pore size) between the printed filaments in the SEM image X-100. After that, cells were stained with 70 nM FITC-
of the scaffold was counted using ImageJ software, and the labeled rhodamine phalloidin (Solarbio, China), washed
average value of 20 pore sizes was counted and calculated. with PBS after incubation for 30 min. The fluorescence
According to the pore size measured by SEM and the width images of the constructs were observed with a confocal
of the printing wire, the average porosity of five scaffolds microscope (LSM880, Zeiss, Germany).
was calculated. The connectivity between the pores of the
scaffold was observed, and the pore connectivity of the 20 2.3.3. Cytokine measurements
scaffolds was counted and calculated. M1 macrophages were seeded on the surface of PEEK0,
PEEK200, and PEEK400 scaffolds at a density of 2 × 10
4
2.3. Characteristics of macrophages incubated in cells/well, and the supernatant of each experimental group
3D-printed PEEK scaffolds was collected after 3 days of incubation. The concentrations
2.3.1. Macrophage seeding of TNF-α, IL-6, IL-10, BMP-2, VEGF, and PDGF-bb were
RAW264.7 (murine monocyte/macrophage cell line) quantified by ELISA kit (4A Biotech).
were obtained from American Type Culture Collection
(ATCC) and incubated in basal culture media composed 2.3.4. Expression of macrophage polarization-related
of Dulbecco’s modified eagle medium (DMEM; Gibco) genes
supplemented with 10% fetal bovine serum (FBS; Gibco) To simulate the state of acute inflammatory response
and 1% penicillin and streptomycin (HyClone) in 25 cm produced by biomaterials in the early stage of implantation,
2
culture flask. The induction of M1 macrophages followed the M1 macrophages were seeded on the surface of 3D-printed
steps below after RAW264.7 cells were adherently cultured PEEK scaffolds with different pore sizes at a density of 1 ×
5
in culture flasks overnight, and the medium was changed 10 cells/well. After 3 days of co-culture, the cells were
to induction medium containing 20 ng/mL interferon collected and washed with PBS for three times. Total RNA
gamma (IFN-γ) and 100 ng/mL lipopolysaccharide (LPS). was isolated using the Trizol Reagent (Life Technologies,
After induction culture for 12 h, the induction medium USA) method as described by the manufacturer (BIO-
was replaced with complete medium, and M1 macrophages RAD 680, USA). cDNA was synthesized from the isolated
were obtained after culturing for 24 h. RNA using PrimeScript™ cDNA synthesis kit (TaKaRa
Bio, Japan). The sequences of forward and reverse primers
For cell seeding, 3D-printed PEEK scaffolds (10 mm of housekeeping gene and target gene (C-C chemokine
in diameter, 3 mm in height) were sterilized and pre- receptor type 7 [CCR7], TNF-α, inducible nitric oxide
incubated with DMEM for 12 h. Subsequently, the M1 synthase [iNOS], VEGF, CD206, TGF-β, BMP-2, and
macrophages were washed three times with phosphate- PDGF-bb) are listed in Table S1 (Supplementary File).
buffered saline (PBS), and then the concentrated RT-PCR samples were prepared by mixing the cDNA,
suspensions of macrophage were injected evenly on the SYBR Green qPCR Master Mix (Life Technologies), and
surface of the PEEK scaffold. The scaffolds were cultured the primers, and the RT-qPCR was performed utilizing the
in a humidified atmosphere of 5% CO , 37 °C for 1 h, and 7500HT Fast Real-Time PCR (Applied Biosystems, USA).
2
basal culture media were added for further incubation; the The relative gene expression was calculated using the 2 −ΔΔCt
medium was changed every 2 days. method with GAPDH as the reference gene.

2.3.2. Macrophage proliferation and adhesion on 2.4. Paracrine effect of macrophage on BMSCs
scaffolds 2.4.1. Preparation of macrophage-conditioned media
M1 macrophages were seeded on the surface of PEEK0, Conditioned medium (CM) is the supernatant of the
PEEK200, and PEEK400 scaffolds at a density of 2 × medium produced after co-culture of macrophages and
4
10 cells/well and then cultured for a further 1, 4, and 7 scaffolds . After the scaffolds were co-cultured with
[27]
days. Then, the original medium was removed, CCK-8 macrophages for 3 days, the supernatant was collected,
working solution (a mixture of 90% complete medium centrifuged at 300×g for 1 h to remove any residual, and
and 10% CCK-8) was added and incubated at 37°C, 5% then filter-sterilized using a 0.22-μm sterile microporous

Volume 9 Issue 5 (2023) 130 https://doi.org/10.18063/ijb.755