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Ma, et al.
– 10 s ), and the rheological properties of GelMA and released from the 3D-printed scaffolds were calculated by
-1
30DE-GelMA inks were measured through varying the adding the differential concentrations at each time point
temperature from 40°C to 4°C. together.
2.6. Cell culture 2.9. Gene expression of the HUVECs in the
Two types of cells, human umbilical vein endothelial cell scaffolds
(HUVEC) and human dermal fibroblast (HDF), were used The expression of genes related to angiogenesis in
in this study. HDFs were cultured in Dulbecco’s Modified HUVECs seeding on the 3D-printed scaffolds was detected
Eagle Medium (DMEM, Gibco, USA) with accessory by real-time quantitative polymerase chain reaction
fetal bovine serum (FBS, 10% v/v) and penicillin/ (RT-qPCR). After culturing for 5 days, Trizol reagent
streptomycin (P/S, 1% v/v). HUVECs were cultured in (Invitrogen, USA) was used to extract the ribonucleic
the endothelial cell culture medium (ECM, Sciencell, acid (RNA) of HUVEC cells on scaffolds, then, the
USA) containing 5% (v/v) FBS, 1% (v/v) endothelial cell obtained RNA was transcribed into complementary DNA
growth factor/heparin kit (ECGS/H), and 1% (v/v) P/S. (cDNA) by applying cDNA synthesis kit (TOYOBO,
Cells were all cultured in an incubator with a temperature Japan). Next, RT-qPCR was conducted by applying
of 37°C and atmosphere of 5% CO . the SYBR Green QPCR Master Mix (Takara, Japan),
2
2.7. Proliferation assay and cell morphology and the angiogenic genes such as vascular endothelial
growth factor (VEGF), hypoxia-inducible factor-1α
The proliferation of HDFs and HUVECs seeded on the (HIF-1α), vascular endothelial cadherin (VE-cad), and
3D-printed scaffolds was determined by cell counting kinase domain receptor (KDR) were studied. Meanwhile,
kit-8 (CCK-8) (Beyotime, China). ECM/DMEM medium GAPDH served as the housekeeping gene for endogenous
containing 10% CCK-8 solution was used to culture the control. The procedure was performed using StepOnePlus
cell-laden 3D-printed scaffolds at 37°C for 3 h, and then, Real-Time PCR Systems (Applied Biosystems, Thermo
the supernatant was transferred to the wells in a 96-well Fisher, USA). First, the pre-denaturation was conducted
plate. Next, the microplate reader (Tecan, Germany) was at 95°C for 30 s followed by 5 s and 30 s of the PCR
applied to measure the absorbance of the supernatant of reactions at 95°C and 60°C in turn. The process was
CCK8-containing medium at 450 nm. looped 40 times in total. After that, the relative expression
For the observation of cell morphology, scaffolds levels of these angiogenic genes were normalized by the
were put in a 48-well plate, and then, cells were seeded on 2 -ΔΔCt method. For RT-qPCR, the primer sequences were
the 3D-printed scaffolds with a density of 2 × 10 per well. listed in Table 1.
4
After cultured for 1 day and 5 days, the scaffolds were
fixed in 4% paraformaldehyde solution for 12 h and rinsed 2.10. In vivo burn wound-healing study
with PBS solution. The DAPI (Sigma-Aldrich, USA) and The wound-healing animal experiment was proceeded
Alex Fluor 647-conjugated phalloidin (Molecular Probes, according to the guidelines sanctified by the Institutional
USA) were applied to stain the nuclei and cytoskeleton Animal Care and Utilization Committee of Nanjing
of cells on the scaffolds separately. After that, confocal First Hospital, Nanjing Medical University. BALB/c
laser scanning microscopy (CLSM, TCS SP8, Leica, mice (8 weeks old, male, SPF grade) which purchased
Germany) was employed to take the fluorescence images from the Charles River Laboratories Research Model
of cell distribution. The cell number and spreading area Technology Co. Ltd. (Beijing, China) were used in this
of HUVECs on the scaffolds on day 1 were determined in study. In this study, the animal models with second-
four different areas of CLSM images using ImageJ (NIH, degree burn skin wounds were established. First,
USA), an image processing software. mice were anesthetized by intraperitoneal injection
and the hair on the murine back was removed. After
2.8. Ionic release behavior of the scaffolds disinfection, a metal rod (10 mm in diameter of section)
With the aim to determine the cumulative Si ions immersed in boiling water at 100°C was pressed on the
released from the 3D-printed scaffolds with different back of the mice for 5 s to create a deep burn wound
concentrations of DE during the cell-cultured process, with circular shape (10 mm in diameter). After that, the
the supernatant medium was collected after the cell- mice were split into three groups randomly: Blank, Gel,
laden scaffolds culturing for 1, 2, 3, and 5 days. After and 5DE-Gel. The 3D-printed scaffolds (5 mm of radius
filtration, the concentrations of Si element in ECM and 1 mm of thickness) were transplanted onto the burn
were determined by inductively coupled plasma atomic wound site on murine back and then medical dressings
emission spectrometry (ICP-AES) with model number of (3M, USA) were used to fix the scaffolds. Next, the
715-ES (Varian, USA). The concentrations of the Si ion wounds were recorded by phone camera on days 0, 2,

International Journal of Bioprinting (2022)–Volume 8, Issue 3 165