Page 294 - IJB-10-6

International Journal of Bioprinting 3D-Printed Zn/MgHA-PCL for angio/osteogenesis

Figure 1A. Briefly, the prepared ink was extruded through for real-time extraction. Fresh complete culture media
a conical plastic nozzle (23 G, 0.33 mm) at a speed of 5 were replenished every other day to generate real-time
mm/s and pressure of 0.2 MPa to obtain a porous 3D extracts of the composite scaffolds.
scaffold. Finally, the 3D-printed HA-PCLs, 10Zn@HA-
PCLs, 5Mg10Zn@HA-PCLs, 10Mg10Zn@HA-PCLs, 2.3.3. Cell proliferation and cytotoxicity
and 15Mg10Zn@HA-PCLs were freeze-dried for 24 h to Cell proliferation of BMSCs and HUVECs was evaluated
remove the organic solvent. using the Cell Counting Kit-8 (CCK-8; Bimake, China).
BMSCs and HUVECs were seeded at a density of 1 × 10
3
2.2.4. Characterization of composite scaffolds cells/well in a 96-well plate and incubated with α-MEM and
The morphology and elements of the scaffolds were DMEM complete culture media until cell attachment. After
examined using SEM (VEGA3; TESCAN, Czech Republic) discarding the original culture media, cells were washed
equipped with EDS, respectively. A microcomputer- with PBS and then treated with the extracts prepared in
controlled electronic universal testing machine (WDW-5; Section 2.3.2, with fresh extracts replaced every 48 h.
Bairoe, China) was used for compression testing, and the Cell proliferation was assessed on days 1, 3, and 7 and 12,
stress and strain curves were monitored to calculate the 24, and 48 h using a microplate reader (BioTek, USA) at
compression modulus. A scaffold with a size (H × Φ) of a wavelength of 450 nm according to the manufacturer’s
5 × 6 mm was compressed to 60% strain at a speed of 60 instructions. The experiments were repeated four times
mm/min. The compression modulus was determined by and statistically analyzed.
drawing a compressive stress–strain curve for each scaffold Cell cytotoxicity of the composite scaffolds was
(n = 4).
assessed using the Live/Dead Cell Staining Kit (YEASEN,
2.2.5. In vitro ion release of HA-PCLs, Zn@HA-PCLs, China). After co-culturing the scaffolds with BMSCs and
and Zn/Mg@HA-PCLs HUVECs for 72 h, the cells were stained with 600 mL of
A total of 40 mg of the scaffolds were dispersed into 40 mL Live/Dead staining working solution for 30 min, followed
phosphate-buffered saline (PBS; Servicebio®, China) at 10 by observation of live and dead cells under a fluorescence
mg/mL. The solutions were kept in a constant temperature microscope and image capture.
oscillation chamber (SHZ-82A; Aohua Instrument, China) 2.3.4. In vitro angiogenic capacity assessment
at 37°C and 90 rpm for 1, 2, 3, 7, and 14 days. At each time For the wound healing assay, HUVECs were cultured in a
point, 10 mL of supernatant was collected and replaced six-well plate using DMEM complete culture media until
with 10 mL of fresh PBS before continuing the shaking reaching 90% confluence. A scratch was made directly
process until the end of the release process.
with a 1 mL pipette tip, and suspended cells were washed
2.3. In vitro cell experiments of composite scaffolds with PBS. The culture media were then replaced with
the extracts of the composite scaffolds, and images of
2.3.1. Cell isolation, source, and cultivation migrated cells were captured after 12 and 24 h using an
The RBMSCs were extracted from the femur and cranial optical microscope (Olympus, Japan) and subjected to
bone of male Sprague-Dawley rats (4 weeks old; Shengchang statistical analysis.
Biotech, China). HUVECs were provided by the American
Type Culture Collection (ATCC, United States of America Quantitative reverse transcription polymerase chain
2+
[USA]). They were all used to study the effects of Zn reaction (RT-qPCR) was performed to evaluate the
and Mg on cell proliferation, viability, osteogenesis, and expression of angiogenesis-related genes, including VEGF,
2+
angiogenesis. The RBMSCs and HUVECs were cultured in FLT1 (VEGFR1), KDR (VEGFR2), bFGF, and GAPDH.
cell culture dishes with Alpha Minimum Essential Medium Briefly, after co-culturing sterile composite scaffolds
(α-MEM; Gibco, USA) and Dulbecco’s Modified Eagle with HUVECs for 24 h, the total RNA was extracted
Medium (DMEM; Gibco, USA) supplemented with 10% from the cells using TRIzol (Sangon Biotech, China).
fetal bovine serum (FBS; Gibco, USA) and 1 % penicillin- Complementary DNA (cDNA) synthesis was performed
streptomycin (PS; Biosharp, China) at 37°C in 5% CO . using the PrimeScript RT kit (Takara, DaLian, China),
2 followed by amplification using the SYBR Green RT-PCR
2.3.2. Preparation of composite scaffold extracts kit (Bimake, China) and real-time PCR equipment (CFX
The composite scaffolds were immersed in 75% ethanol Connect™ Optics Module; Bio-Rad, USA). Each step
solution for 30 min, followed by washing with sterile PBS was performed in accordance with the manufacturer’s
three times, each for 10 min. Subsequently, they were added instructions. The sequences of the primers are summarized
to α-MEM and DMEM culture media at a concentration of in Table S1, Supporting Information. The experiments
10 mg/mL and incubated at 37°C in a sterile environment were repeated four times.

Volume 10 Issue 6 (2024) 286 doi: 10.36922/ijb.4243