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International Journal of Bioprinting PCL/Fe3O4@ZIF-8 for infected bone repair

by using a magnetometer (MPMS-XL7 SQUID, Quantum Committee of the Laboratory Animal Center of the Fourth
Design, USA). The concentration of zinc ions released from Military Medical University. SD rats were euthanized to
the scaffolds was measured by inductively coupled plasma obtain bone marrow from the femurs and tibias, and the
atomic emission spectrometry (ICP-AES; IRIS Advantage, whole bone marrow adhesion method was used to culture
Thermo Scientific). The PCL/Fe O @ZIF-8 scaffolds were bone marrow mesenchymal stem cells (BMSCs), according
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soaked in simulated body fluid composed of 2 mL PBS and to procedures described in a previous study. BMSCs
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2.6 U/mL hyaluronidase at 37°C, for 3 h, 6 h, 12 h, 1 day, 2 were cultured in α-minimum essential medium (α-MEM;
days, 4 days, and 7 days. At each time point, the leachates Hyclone, USA) containing 10% fetal bovine serum (FBS;
were collected and detected. The thermal stability of the Shaanxi Zhonghui Hecai Bio-pharmaceutical Technology
scaffolds was also measured. Co., Ltd., China) and 1% penicillin–streptomycin
(Hyclone, USA) at 37°C in a 5% CO incubator, and the
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2.5. Evaluation of antibacterial activity in vitro culture medium was refreshed every 3 days. BMSCs were
S. aureus (ATCC 25923) and E. coli (ATCC 25922) were sub-cultured when they reached 80% confluence, and
used to evaluate the antibacterial activity of the PCL/ cells between passages 3 and 5 were used in subsequent
Fe O @ZIF-8 scaffolds. Pure PCL, PCL/5%Fe O @ZIF-8, experiments. The surface makers, including CD45, CD31,
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PCL/10%Fe O @ZIF-8, and PCL/15%Fe O @ZIF-8 were CD90, and CD29, were detected by flow cytometric
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sterilized with ethylene oxide and then placed in a 24- analysis to identify BMSCs.
well plate with three wells for each group. Then, 500 µL of
bacterial suspension containing S. aureus and E. coli [1 × 2.7. BMSCs proliferation and attachment on the PCL/
10 colony forming unit (CFU)/mL] was added into each Fe O @ZIF-8 scaffolds
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well containing scaffolds and incubated at 37°C for 4 h. The Pure PCL, PCL/5%Fe O @ZIF-8, PCL/10%Fe O @ZIF-
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non-attached bacteria on the surfaces of the scaffolds were 8, and PCL/15%Fe O @ZIF-8 scaffolds were sterilized by
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gently removed by rinsing with aseptic phosphate-buffered ethylene oxide and co-cultured with 2 mL of the BMSCs
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saline (PBS) and discarded. Next, the adherent bacteria on suspension at a density of 2 × 10 cells/mL in 24-well plates.
the scaffolds were detached by ultrasonication (50 Hz for The co-cultured BMSCs was incubated at 37°C in 5%
10 min) and collected. After being diluted 100 times, the CO atmosphere, and the culture medium was refreshed
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collected bacterial suspensions were plated onto Luria– every 2 days. After 1, 4, and 7 days of co-culture, BMSCs
Bertani (LB) solid medium in triplicate and then incubated proliferation was evaluated by cell counting kit-8 (CCK-8)
at 37°C for 24 h. The bacteria CFU were imaged and counted assay. On the third day of co-culture, BMSCs and scaffolds
by using a fully automatic colony counter (Interscience were fixed in 4% paraformaldehyde solution for 15 min
Scan 1200, France). The counts of the colonies on the LB at room temperature. Then, cytoskeleton of BMSCs was
indirectly reflected the number of CFUs adhered on the stained with phalloidin-rhodamine (C2207S, Beyotime,
surface of scaffolds. The anti-bacterial adhesion efficiency China) for 30 min, and the nuclei was stained with
of the PCL/Fe O @ZIF-8 scaffolds was calculated using 4′,6-diamidino-2-phenylindole (DAPI; D9542, Sigma,
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the formula: Anti-bacterial adhesion efficiency (%) = (A USA) for 15 min. After washing with PBS, attachment of
- B)/A × 100%, where A is the average CFU of pure PCL BMSCs on the scaffolds was observed and imaged by laser
scaffold (control group), and B is the average CFU of PCL/ confocal scanning microscopy.
Fe O @ZIF-8 scaffolds (experimental groups). 32 2.8. Evaluation of osteogenic differentiation in vitro
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Furthermore, the adherent bacteria on the surfaces of After sterilization, pure PCL, PCL/5%Fe O @ZIF-8,
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the scaffolds were detected by SEM. Briefly, suspensions of S. PCL/10%Fe O @ZIF-8, and PCL/15%Fe O @ZIF-8 were
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aureus and E. coli were incubated with the scaffolds at 37°C respectively co-cultured with BMSCs at a density of 2
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for 24 h and then fixed overnight with 2.5% glutaraldehyde × 10 cells/mL in six-well plates, and incubated at 37℃
at 4°C. The specimens were dehydrated with graded in 5% CO atmosphere. When the confluence of co-
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ethanol series (50%, 75%, 90%, and 95% for 15 min and cultured BMSCs reached 70%, the culture medium was
100% for 30 min), dried overnight in a vacuum desiccator, replaced with osteogenic induction medium containing
and observed under SEM, and ImageJ Vol.6.0 software was 100 nM dexamethasone, 50 mM vitamin C, and 10 mM
used to quantify the adherent bacteria number. glycerophosphate (Beyotime, China). Alkaline phosphatase
(ALP) staining and ALP activity were performed after 7
2.6. Isolation and culture of bone marrow and 14 days of induction, and Alizarin red staining was
mesenchymal stem cells performed after 21 days of induction. After 7 and 14 days
All Sprague–Dawley (SD) rats in this study were obtained of induction, the expression of osteogenic-related genes
from the Laboratory Animal Center of the Fourth Military and proteins, including ALP, type I collagen (Col-1), runt-
Medical University and approved by the Welfare and Ethics related transcription factor 2 (RUNX2), and osteocalcin
Volume 10 Issue 4 (2024) 300 doi: 10.36922/ijb.2271