Page 210 - IJB-9-1

International Journal of Bioprinting FeS /PCL scaffold for bone regeneration
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using trephine bur. The animals were sacrificed after 6 formaldehyde solution and were transparentized in 14%
and 12 weeks of implantation. Finally, the scaffolds were EDTA before scanning.
harvested and prepared for further examinations. For western blot analysis, the prepared samples

2.6. In vivo evaluation were minced and subjected to sodium dodecyl sulfate-
In order to visualize the whole calvaria, a micro-computed polyacrylamide gel electrophoresis at 100 V for 2 hours. The
tomography (CT) scanning was performed using the obtained proteins were then transferred to polyvinylidene
Quantum FX (PerkinElmer, USA) after 6 and 12 weeks. The difluoride membrane. The membrane was blocked with
samples were scanned with an X-ray tube voltage of 90 kVp, 5% skim milk in Tris-buffered saline/Tween20 (TBST) for
current intensity of 180 mA, and 40 mm voxel resolution. 30 minutes and incubated with the primary antibodies for
The field of view was 20 mm × 20 mm × 20 mm with a 12 hours at 4ºC. The membrane was washed three times
scan time of 4 minutes and 30 seconds. The reconstructed with TBST buffer and further incubated with secondary
3D images were acquired using Living Image® 4.4 software antibodies diluted 1:3,000 with 5% skim milk in TBST
(PerkinElmer, USA). for 1 hour at room temperature. The protein bands were
enhanced with chemiluminescent (Cat# 34577, Thermo
For histological assessment, the harvested scaffolds Scientific, Rochford, USA). The density of the bands was
were placed in 10% phosphate-buffered formalin solution quantified using ImageJ (National Institutes of Health,
and decalcified in ethylenediaminetetraacetic acid Bethesda, Maryland, USA).
(EDTA) solution. Subsequently, the samples were fixed
in 4% paraformaldehyde and paraffin, and then sectioned 2.7. Statistical analysis
to a thickness of 5 mm. Next, the sectioned samples Statistical analysis was performed with SPSS software 10.0
were stained with hematoxylin and eosin (H&E) (H&E (SPSS Inc., Chicago, Illinois, USA) using one-way analysis
Staining Kit, Abcam, United Kingdom [UK]) and Masson’s of variance. We considered the differences to be statistically
trichrome (MT) (Trichome Stain Kit, Sigma Aldrich, USA) significant at p < 0.05.
following a standard protocol. Immunohistochemical
(IHC) staining for bone morphogenetic protein (BMP)-2 3. Results and discussion
was performed on the animals. The prepared sections were
deparaffinized and blocked with 3% H O solution. Then, In this study, 3D scaffolds composed PCL and FeS were
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the sections were exposed to citrate buffer for antigen fabricated using a 3D bioprinting system to investigate the
retrieval and incubated with primary antibody (BMP-2, efficacy of FeS in bone regeneration. FeS was incorporated
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Bioss, USA) for 24 hours. Subsequently, the samples were in three different weight fractions (5, 10, and 20 wt%)
incubated with secondary antibody (Goat Anti-Rabbit and compared to pure PCL scaffold. First, the physical
IgG H+L [HRP], Abcam, UK) for 2 hours. The antibody characteristics (surface chemistry, surface roughness, and
complexes were then stained with diaminobenzidine mechanical properties) were analyzed depending on the
(DAB, Abcam, UK) solution. The stained images were FeS content. Then, biocompatibility test was performed
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acquired using an optical microscopy (Axio Scan.Z1, Carl on the hMSCs prior to conducting in vivo studies using a
Zeiss, Germany). rat calvarial defect model.
The harvested samples were perfused with MICROFIL® 3.1. Scaffold fabrication and morphology
MV-122 Yellow (Flow Tech, USA) to evaluate blood vessel The schematic of this study is illustrated in Figure 1A.
formation after 12 weeks. The anaesthetized rats were Before 3D bioprinting, FeS particles were incorporated in
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shaved and disinfected at the incision site. Then, 0.2 mL of the barrel attached to the heating chamber together with
heparin (5,000 IU/mL) was flushed through the tail vein. the melted PCL solution. The size of the FeS particles was

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The heart was then exposed by incision of the thorax and 100.8 ± 13.1 mm. The scaffold was 3D printed layer-by-
the rib cage after 10 minutes. Next, the left ventricle was layer to construct a stable structure with interconnected
exposed and penetrated using a feeding tube. The blood pores. The control scaffold was composed of pure PCL,
vessels were rinsed with heparin (1 IU/mL) containing whereas PF5, PF10, and PF20 were PCL-based scaffolds,
normal saline (200 mL). Thereafter, 20 mL of Microfil comprising 5, 10, and 20 wt% FeS , respectively. When
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(10.5 mL of Microfil compound, 13.2 mL of specific diluent, printing the PCL melt consisting of 30 wt% (or greater)
and 1.3 mL of specific curing agent) was perfused via the FeS , the enormous amount of FeS caused clogging at
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feeding tube at a pressure of 120 mmHg and perfusion the tip of the nozzle or resulted in discontinued struts.
rate of 2 mL/min. Following the perfusion, the animals Therefore, PF30 scaffold was left out from this study.
were stored at 4°C for 24 hours to allow Microfil’s primary The final scaffolds were 8 mm in diameter and 2 mm
setting. Finally, the animals were fixed in 10% buffered in height.

Volume 9 Issue 1 (2023)olume 9 Issue 1 (2023)
V 202 https://doi.org/10.18063/ijb.v9i1.636