Page 64 - IJB-9-5

International Journal of Bioprinting 3D-printed PLA-BG composite induces angiogenesis

angiogenesis. First, in vitro analyses with human umbilical dithiocarbamate (ZDEC) and zinc dibutyl dithiocarbamate
vein endothelial cells (HUVECs) were performed to (ZDBC) (Food and Drug Safety Center, Hatano Research
characterize viability and gene expression of endothelial Institute, Hadano, Japan) were used as positive controls as
markers. Differentiation capacity of HUVECs in response to they induce a reproducible cytotoxic reaction.
the BG fraction in the test specimen was evaluated by means
of reverse transcription polymerase chain reaction (RT- 2.4. Cell culture and microscopy
PCR). The effect of the biomaterials on vascular formation HUVECs were purchased from Promocell (Heidelberg,
was analyzed by Matrigel assay and chicken chorioallantoic Germany) and cultured in complete EBM-2 medium
membrane (CAM) assay. The goal of this study was to detect (Promocell, Heidelberg, Germany) as recommended by
whether the 3D-printed PLA–BG composite scaffolds the supplier. For microscopic observations after seeding
are able to induce angiogenesis in addition to the already on PLA-BG samples, cells were labeled with Cell Tracker™
detected enhancement of osteogenesis. Green according to the manufacturer’s instructions
(Life Technologies, Carlsbad, CA, USA]. 2 × 10 /0.2 cm
2
5
2. Methods HUVECs were seeded onto the different PLA–BG disk
and cultivated overnight. The next day cell detection was
2.1. Filament fabrication performed with the EVOS® Digital Inverted Microscope
Composite filaments of PLA and BG were fabricated (Life Technologies, Carlsbad, CA, USA).
as described before . Shortly, PLA granules (PLA-
[17]
filament Kristall Natur, 3dk.berlin, Berlin, Germany) 2.5. Viability
with a grain size of 2–5 mm and bioglass Type S53P4 Cell viability was tested with the alamarBlue® assay. 2 ×
(bioglass composition: 53% SiO , 23% Na O, 20% CaO, 10 /0.2 cm HUVECs were seeded onto the different PLA–
2
5
2
2
4% P O , BonAlive Biomaterials Ltd., Turku, Finland) with BG disk and cultivated overnight. The alamarBlue® assay
2
5
a grain size of 25–42 µm were mixed manually to obtain (Gibco®Invitrogen™ Life Technologies, Carlsbad, CA, USA)
compositions with BG content of 0, 5, 10, and 20% (w/w). was performed 1 and 4 days after seeding. For this purpose,
For filament extrusion, a desktop filament extruder (NEXT cells were incubated with 320 µL of a 10% alamarBlue
1.0 Advanced, 3devo B.B., Utrecht, the Netherlands) was solution in medium for 4 h at 37°C. Subsequently, 100 µL
used. Screw speed was set to 4 U/min and fan speed to 65%. of the supernatant was transferred to a 96-well plate, and
The speed of the conveying mechanism was set to automatic the absorbance (presented as fluorescence intensity) of
to achieve the desired filament diameter of 1.75 mm. each was measured at 560/600 nm.
2.2. Sample fabrication 2.6. PCR
2
4
Two-dimensional round scaffolds of each PLA–BG 5 × 10 /0.2 cm HUVECs were seeded onto PLA–BG
composite were printed using fused filament fabrication disks and detached with accutase the next day. The cell
on a 3D printer (i3 MK3S, Prusa Research, Prague, Czech suspensions were centrifuged at 1,400 rpm for 5 min and
Republic) with a nozzle diameter of 0.4 mm. Filaments the cell pellet was stored at −80°C for future use. Isolation
were dried at 40°C for at least 12 h. Sample geometries of RNA was performed using PeqGold Total RNA Micro
were designed with computer-aided design software NX Kit (PeqLab) according to manufacturer’s instruction.
12 (Siemens NX 12, Siemens AG, Berlin and Munich, Total RNA (1 μg) was reverse-transcribed into cDNA
Germany) and preprocessed with Cura Ultimaker v.4.6. using dNTPs (4you4 dNTPs Mix (10 mM), BIORON
(Ultimaker, Utrecht, the Netherlands). A flat disk consisting GmbH, Ludwigshafen), Random Primers (Promega,
of two layers with a diameter of 0.5 cm was printed and Madison, WI, USA), and MuLV RT (M-MuLV Reverse
used for further experiments. Transcriptase, M0253S New England Biolabs, Ipswich,
MA, USA) according to the manufacturer’s instructions.
2.3. Biocompatibility assessment For gene expression analyses, cDNA template underwent
In vitro cytotoxicity was analyzed analogous to ISO PCR amplification (40 cycles) using the SYBR Green
10993-5 using the MTT ([3-(4,5-dimethylthiazol-2-yl)- (PowerUp™ SYBR® Green master mix, Applied Biosystems,
2,5-diphenyltetrazoliumbromid)]) assay. Mouse L929 Foster City, CA, USA) and sequence-specific primers
cells (20,000 cells/well) were seeded in a 96-well tissue (primer sequences listed in Table 1). GAPDH was used for
culture plate for 24 h. PLA–BG disks were incubated in normalization, and results were calculated using the well-
120 µL cell media for 48 h. About 100 µL of this extract established 2 −ΔΔCt method .
[32]
were given to L929 cells in the 96-well plate. After an
incubation time of 24 h, the MTT assay was performed and 2.7. Matrigel assay
the colorimetric readout was performed at a wavelength In this experiment, a series of conditioned media containing
of 570 nm (reference wavelength 650 nm). Zinc diethyl pure PLA and BG in different concentrations were prepared

Volume 9 Issue 5 (2023) 56 https://doi.org/10.18063/ijb.751