Page 471 - IJB-10-1

International Journal of Bioprinting TPMS bone scaffold

osteogenic differentiation medium for 14 days. Following time 384 ms. Density measurements were calibrated to the
the incubation period, the cells were washed twice with manufacturer’s calcium hydroxyapatite (CaHA) phantom.
PBS and fixed in 4% paraformaldehyde for 30 min. Then, Analysis was performed using the manufacturer’s
the cells were washed again with PBS, stained with ALP evaluation software. Reconstruction was accomplished by
staining assay kit (Applygen Technologies, E1043, Beijing) NRecon (version 1.7.4.2). 3D images were obtained from
or Alizarin red staining kit (Oricell, RAXMX-90021, contoured two-dimensional (2D) images based on the
Guangzhou), and the staining was imaged under an distance transformation of the original grayscale images
inverted phase contrast microscope. (CTvox; version 3.3.0). 3D and 2D analyses were performed
using the software CT Analyzer (version 1.20.3.0). Analyses
2.8. Angiogenesis assessment of the bone microarchitecture were carried out in a region
Fifty microliter of Matrigel (10 mg/mL) was transferred to of interest (ROI), which was bone mineral density (BMD),
each well of a 96-well plate and placed in a 37°C incubator bone volume fraction (BV/TV), bone surface area density
for 1 h for fixation. Human umbilical vein endothelial (BS/TV), and trabecular number (Tb.N).
cells (HUVECs) were cultured in a vascular endothelial
medium. Cells were trypsinized and resuspended to a final 2.11. Histological analysis
concentration of 3 × 10 cells/mL. One hundred microliter A third of the tissue near the cephalic side was collected for
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of the cell suspension was inoculated in a 96-well plate subsequent histological analysis. The samples in the fixative
containing the Matrigel. After 2 h, the same volume of solution were dehydrated and embedded in paraffin wax.
culture medium and four groups of scaffold medium Then, samples were sectioned at 4 μm thickness using a
extracts were added, respectively, and cells were placed in paraffin microtome (RM2235, LEICA, Germany). The
the incubator for 12 h to observe the formation of blood histological structure was observed by H&E and Masson’s
vessels, which were then imaged under the inverted phase trichrome staining, ALP immunohistochemical staining,
contrast microscope. and OPG immunofluorescence staining, performed
following the standard protocols.
2.9. Preparation of rabbit mandibular critical bone
defect model 2.12. Statistical analysis
All animal surgical procedures were conducted under Quantitative data were expressed as mean ± standard
protocols approved by the Committee on the Use of Live deviation (SD). The data were compared using one-way
Animals in Teaching and Research, Guangdong Medical ANOVA or Student’s t test. P values < 0.05 are considered
University. Fifteen 6-month-old healthy male New Zealand statistically significant.
rabbits were anesthetized with a dose of 0.1 mL/kg of xylazine
hydrochloride injection diluted 4 times through the thigh 3. Results and discussion
muscle of the rabbits. The routine skin preparation in bilateral
submaxillary areas, disinfection, and laying of sterile sheets 3.1. Scaffold characteristics and release kinetics
were followed by making a transverse incision about 2–3 The TPMS structure has been introduced for its excellent
cm in length from the rabbit’s mandible to the front and rear structural properties and can be easily and rapidly fabricated
canthus of the rabbit, parallel to the corner of the mouth. The by 3D printing. The unique porous structure of TPMS
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incision cut through the rabbit’s epidermis, subcutaneous renders it a promising candidate for bone regeneration.
tissue, muscle, and periosteum layer by layer, followed by The current research shows that it has a good performance
blunt separation to the exposed bone surface. A circular bone in the study of bone regeneration. Researchers often take
drill was used to prepare an 8 × 4 mm full-thickness bone advantage of the fact that the TPMS structure is a smooth
defect at the middle and lower regions of the buccal side of the and connected shape inside. They inject biomaterials into
mandible, with paying attention to physiological saline cooling TPMS channels, which, upon curing, produce a powerful
during operation. According to the experimental group, internal framework to support the scaffolds. The results
different scaffold materials were implanted into the defect, show that the combination of 3D printing and biomaterials
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and then the wound was tightly sutured. Daily intramuscular can promote cell growth significantly. In recent years, both
injection of penicillin (80,000 units) was administered for 3 laboratory and clinical studies on I-PRF have demonstrated
days after operation to prevent infection. its positive impact on bone regeneration, bone induction,
and wound healing. Furthermore, combing I-PRF with
2.10. Micro-computed tomography (CT) analysis bone grafts has also shown superior performance in bone
Specimens were scanned using the Bruker Micro-CT regeneration. 46-48 However, I-PRF lacks rigidity due to its
Skyscan 1276 system (Kontich, Belgium). Scan settings fluid nature, so a mechanically robust scaffold is needed
were as follows: voxel size 9.921682 μm, medium to provide support for bone defects, a requirement that
resolution, 85 kV, 200 μA, 1 mm Al filter, and integration the TPMS scaffold fulfills. In terms of TPMS mechanical

Volume 10 Issue 1 (2024) 463 https://doi.org/10.36922/ijb.0153