Page 124 - IJB-9-5

International Journal of Bioprinting DLP-printed scaffold for bone regeneration

transwell assay. The scaffolds were placed in 24-well plates rabbits via micro-CT scans. The scanning parameters were
and then HUVECs (10 /well) were inoculated in the upper an effective pixel size of 17.34 μm, a current of 500 μA, a
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chamber with a pore size of 8 μm and cultured for 12 h voltage of 80 kV, and an exposure time of 1500 ms. The two-
and 24 h using low-serum medium (H-DMEM, 2% FBS, dimensional (2D) images were reconstructed into 3D images
1% penicillin–streptomycin), after which the cells were using Inveron Research Workplace (Siemens) to calculate
fixed and the upper layer was removed with cotton swabs, the bone regeneration parameters: bone mineral density
stained with 1% crystalline violet. After staining, the cells (BMD), bone volume/total volume (BV/TV), trabecular
were observed under a microscope, and cell detection was thickness (Tb.Th), and trabecular spacing (Tb.Sp).
performed using ImageJ.
2.9.3. Histology analysis
2.8.2. Scratch wound assay Samples were decalcified in 10% Ethylenediamine
HUVECs (10 ) were inoculated on 6-well plates and Tetraacetic Acid (EDTA), dehydrated in a gradient of
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cultured to 80%–90% confluence with complete medium ethanol, and cleared using xylene. The samples were then
(H-DMEM, 10% FBS, 1% penicillin–streptomycin). The embedded in paraffin and cut into 10-mm slices using a
cells were scratched with a 1000 µL sterile tip to form straight microtome for staining.
rows of scars, and then the suspended cells were washed
off, the medium was replaced with low-serum medium 2.10. Statistical analysis
(H-DMEM, 2% FBS, 1% penicillin–streptomycin), and the Statistical analysis was performed by one-way analysis of
culture was continued for 12 h, following which the extent variance (ANOVA) with post hoc tests using the GraphPad
of scratch healing was determined. Prism software (version 8, GraphPad, USA). The data are
expressed as the mean ± standard deviation (SD), and
2.8.3. Tube formation assay all experiments were performed at least three times. The
In order to explore the ability of different scaffolds to results were analyzed by one-way ANOVA with the Tukey–
promote angiogenesis, Matrigel (BD, growth factor- Kramer multiple comparison analysis. A value of p < 0.05
reduced, 356231) was covered on the surface of µ-Slide was regarded as statistically significant (*p < 0.05, **p
Angiogenesis (ibidi, 81506), and then 10 cells were < 0.01, and ***p < 0.001).
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inoculated on the surface of Matrigel. Different conditioned
culture medium was used to induce tubes for 6 h, and 3. Results
they were observed under a microscope. The extent of 3.1. Characterization and degradation of GelMA/
angiogenesis on the obtained images was determined PMAA scaffolds
using ImageJ software. The mechanical and biological properties of GelMA

2.9. Effect of scaffold in vivo hydrogels can be regulated by the degree of methacrylate (DS)
Eighteen of New Zealand white rabbits (2.5 kg ± 0.5 kg, substitution as well as the concentration and crosslinking
male) were randomly divided into three groups: blank, time of GelMA. First, we synthesized the macromonomer
GelMA, and GelMA/3%PMAA groups, and each sample via methacryloyl (MA) reacting with gelatin (Figure S1A
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was repeated three times. All animals used in this study in Supplementary File). The local high-resolution H NMR
were obtained from the Animal Experiment Center of spectra of gelatin (left) and GelMA (right) in Figure 2A
the PLA General Hospital and approved by the Ethics showed the appearance of new proton signals at 5.5 and 5.7
Committee (2022-x18-51). ppm (–CH=CH ) in the macromonomer, suggesting that the
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methyl acryloyl group was successfully grafted onto the side
2.9.1. Implantation in rabbit radio defects chain of gelatin. The substitution degree of methacrylate was
The rabbits were anesthetized, and the distal femur about 90%, and GelMA with interconnected pores could be
was shaved and disinfected. After cutting the skin and easily prepared by exposing it to UV for 10 s (Figure S1B
subcutaneous tissue, a cylindrical defect, which was 6 mm and S1C in Supplementary File).
in diameter, was created using a surgical drill, without
penetrating the contralateral cortex in the distal femur. The To better harmonize the mechanical strengths, we chose
sterile scaffolds were inserted into the defect site, and the PMAA concentrations of 3% and 6% as a way of exploring
subcutaneous tissue and skin were sutured layer by layer. more suitable concentrations for in vivo implantation.
The rabbits were sacrificed at week 4 and week 8 post- The introduction of PMMA increased the toughness and
operatively for the next step of treatment. strength of the scaffolds compared to GelMA (Figure 2D).
At the same time, the scaffolds’ modulus decreased from
2.9.2. Micro-CT analysis 66.72 ± 4.73 kPa to 28.42 ± 3.15 kPa with the increase
The Inveron MM System (Siemens, Munich, Germany) was of MAA concentration (Figure 2E). The structure of the
used to evaluate the amount of new bone in each group of scaffolds was observed by SEM, which had a macropore

Volume 9 Issue 5 (2023) 116 https://doi.org/10.18063/ijb.754