Page 195 - IJB-9-3

International Journal of Bioprinting 3D-printed vascularized biofunctional scaffold

phosphate-buffered saline (PBS) in the ratio of 5:1 (w/w), Thereafter, they were removed at set time intervals, the
mixed thoroughly and then filtered with a 0.22-μm surface liquid was wiped off, and they were weighed until
membrane to prepare a 5% (w/v) GelMA and 1% (w/v) the weight remained constant, indicating the swelling was
AlgMA (GA) mixed hydrogel precursor solution, and then completed. The swelling ratio was calculated using the
PRP was added to construct the 20% PRP-GA (PRP-GA) following equation:
hydrogel. 1% (w/v) laponite was added to obtain the PRP- W − W
GA@Lap hydrogel. Swelling ratio% () = t 0 ×100 %
W 0
2.3. Printing of scaffolds where W and W refer to the weight of the hydrogel
0
t
The scaffolds used in this experiment were printed with scaffolds at the initial and set time points, respectively.
a 3D-Bioplotter (Bio-Architect; Regenovo, Hangzhou,
China). Briefly, for hydrogel scaffold printing, the mixed In the degradation test, the hydrogels were weighed,
bioink was loaded into the barrel with a needle diameter placed in PBS and incubated at 37°C. Samples were
of 250 µm, and by adjusting the temperature controller of removed at each set time point, lyophilized and weighed
the syringe barrel between 12°C and 20°C to achieve stable (dry weight, W ). W represents the initial dry weight, and
0
t
filament deposition. The temperature of the print platform the degradation rate (DR) for each sample was calculated
was set to approximately 4°C lower than the injection tube. by using the following equation:
The print speed was 10 mm/s, and the air compressor W − W t
0
pressure was 0.16 MPa. The 3D bioprinting process was DR % () = W ×100 %
performed under an ultraviolet (UV) light source (405 nm, 0
0.5 W cm ) for rapid curing. Finally, the scaffolds were 2.6. Growth factor release kinetics from the
−2
exposed to a 2% (w/v) CaCl solution for 10 min to obtain hydrogel scaffolds
2
a stable structure. The release of multiple growth factors from two groups of
hydrogel scaffolds containing PRP was tested by enzyme-
For the printing of bone repair scaffolds with PCL
(Perstorp, Sweden) combined with hydrogel ink, the linked immunosorbent assays (ELISAs). Briefly, groups
of hydrogels were added to 50-mL centrifuge tubes
fiber spacing was set to 1.0 mm, the PCL particles were containing PBS and placed at 37°C. The supernatant in
loaded into a nozzle with a 300-μm diameter barrel, the each group of test tubes was aspirated at each time point
temperature was kept at 60°C, and the printing speed was and stored in a −80°C refrigerator, and then an equal
10 mm/s. The PCL and each group of hydrogel inks were volume of fresh PBS solution was added to each test tube.
then printed layer-by-layer to construct scaffolds with a At last, the concentrations of VEGF, platelet-derived
diameter of 3 mm and a height of 4 mm.
growth factor (PDGF)-BB and transforming growth factor
2.4. Morphology and compressive modulus of each beta (TGF-β) were determined with ELISA kits according
group of hydrogels to the manufacturer’s protocol.
The samples of each group were sputtered with gold, 2.7. Cell proliferation assay
and the cross-sections of the lyophilized hydrogels were Briefly, BMSCs were grown in 96-well plates at a density of
observed by scanning electron microscopy (SEM, EVO 10,000 cells/well, and the proliferation assay was detected
MA10, Germany). C, Mg, and Si distribution in PRP- when the cells reached ~70% confluence. The Cell Counting
GA@Lap hydrogels was analyzed by energy dispersive Kit-8 (CCK-8) assay was performed according to the
spectroscopy (EDS). The pore size of the hydrogels was manufacturer’s instructions as previously described [28,29] .
analyzed and measured using ImageJ software.
Cylindrical hydrogel holders were prepared for uniaxial 2.8. Detection of osteogenic differentiation
compression tests. Stress–strain curves for the GA, PRP- Osteogenic differentiation of rat BMSCs was assessed by
GA, and PRP-GA@Lap hydrogels were obtained using alkaline phosphatase (ALP) and Alizarin red staining.
a universal mechanical testing machine (Instron 5969, Groups of hydrogel solutions were spread at the bottom
−2
USA) at a crosshead speed of 5 mm/min. The compression of 24-well plates, covering the bottom (~15 µL cm ),
2+
modulus of each group of hydrogels is represented by the and then treated with light-curing and Ca crosslinking.
slope of the 10%–20% compression phase. BMSCs were inoculated in the 24-well plates at a density
of 5 × 10 /well and cultured using α-MEM medium
4
2.5. Swelling and degradation of the hydrogel containing 10% fetal bovine serum. After the cell growth
scaffolds reached ~70% confluence, the BMSCs were cultured with
For the swelling test of the hydrogel scaffolds, preweighed osteogenic induction medium (supplemented with 0.1 μM
hydrogel scaffolds were immersed in PBS solution at 37°C. dexamethasone [Sigma], 10 μM sodium β-glycerophosphate

Volume 9 Issue 3 (2023) 187 https://doi.org/10.18063/ijb.702