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HA15-loaded Bone Tissue Scaffold
(4) Gene expression and real-time polymerase chain rabbits were assigned to either left or right radial defect
reaction (PCR) analysis model group using the random numbering method.
The rabbits were anesthetized with 3% pentobarbital
The expression of osteogenic genes, including OCN, sodium at 20 mg/kg through ear intravenous injection
HSPA5, bone morphogenetic protein 2 (BMP2), alkaline (Figure 3A). The rabbits were fixed in the supine position
phosphatase (ALP), collagen type I (col1a1), transcription and the forearm hairs were shaved to reveal the surgery
factor Sp7 (Osterix), and Runx2, in C3H10 cells cultured site, which was disinfected with 2% iodine tincture 3
on various specimens was analyzed by real-time PCR times. About 75% alcohol was used for deiodination.
assay. After culturing for 7 days, total RNA was extracted The corneal reflex of animals was observed to check if
using Trizol reagent from cells. The concentration of they were ready for surgery. The surgery began with a
RNA was measured by a NanoDrop spectrophotometer 2.0 cm long incision parallel to the radius of the forearm
(Thermo Fisher Scientific, USA). The primers used are in the middle and upper segment of the forearm. Then,
shown in Table 1. the subcutaneous tissue and muscle space were separated

(5) Alizarin red S staining to expose the radius, after that the periosteum of the
radius was cut (Figure 3B). An orthopedic mini-drill was
Following osteogenic induction for 14 days, the cells used to cut a radius of about 1.5 cm in the upper-middle
were placed in 48-well plates (3-wells for each group) and segment of the radius, and then the surgical site was rinsed
fixed with 4% paraformaldehyde (PFA) for 20 min, and with 0.9% sodium chloride solution. The samples were
then rinsed twice with phosphate buffered saline (PBS). implanted at this site (indicated by a circle in Figure 3C).
After that, the cells were stained using 40 mM alizarin The subcutaneous tissue was sewed with 4-0 absorbable
red working solution for 10 min at room temperature. The risk (Figure 3D). The incision was sutured with No. 0
cells were then rinsed twice with PBS and visualized under suture and the wound was then disinfected and bandaged.
a light microscope. Next, 100 mmol/l cetylpyridinium The rabbits were kept in separate cages and each was
chloride was poured into each well and semi-quantitative administered an intramuscular penicillin injection at a
analyses were done by optical density measurement at dose of 1.6 × 10 units/day for 3 days post-surgery. All
6
560 nm. the animals were sacrificed 12 weeks after surgery by
air embolization at the ear’s marginal vein. The original
(6) Immunofluorescence analysis surgical site was incised to observe the healing condition
For immunofluorescence analysis, after incubation with of the local bone defects. The surgical process is shown
primary antibodies, followed by Alexa Fluor 594 donkey in Figure 3.
anti-mouse IgG1 (Life Technologies, Carlsbad, CA, (2) Micro-computed tomography (micro-CT) scan
USA) and Alexa Fluor 488 goat anti-mouse IgG2b (Life
Technologies) secondary antibodies, cells were washed All experimental animals were subjected to 40-row micro-
3 times in PBS, after which nuclei were counterstained CT scanning through SIEMENS Inveon MMCT micro-
with 4',6-diamidino-2-phenylindole (Life Technologies). CT instrument with working parameters of voltage and
Images were obtained on a confocal laser-scanning power of 55 kV and 80 W, respectively. Furthermore, the
microscope (Olympus, Tokyo, Japan). 3D reconstruction examination of the surgical sites was
performed 12 weeks after surgery to thoroughly clarify
2.5. In vivo experiments the specific and intuitive bone callus growth and healing
(1) Radial defect treatment of rabbit condition at the bone defect. Moreover, the quantitative
analysis of bone volume/total volume (BV/TV), bone
All in vivo animal experiments were conducted under mineral density, trabecular thickness, and the structural
the “Regulations on the Administration of Laboratory model index was done according to the Hedberg’s
Animals” approved by the State Council, issued by the standard for evaluating the condition of new bone area.
National Science and Technology Commission and
approved by the Experimental Animal Ethics Committee (3) Microfil angiography and micro-CT imaging
of Youjiang Nationalities Medical College. Six New The anesthetizing procedure was carried out according to
Zealand white rabbits, with an average weight of 2.5 ± a previously described method. The abdomen was fixed
0.5kg, that were used in this experiment were provided by upward on the plate and the skin and muscle layer was cut
the Science Experiment Center of Youjiang Nationalities along the midline of the abdomen. Then, the diaphragm
Medical College. Among them, the male rabbits and was cut and the heart was exposed afterward. The heart
female rabbits were divided into β-TCP/groups using the was fixed with hemostatic forceps, and inserted with a 23G
random number method; these groups were PLGA/HA15 needle. The right atrial appendage in the left ventricle was
group, β-TCP/PLGA group, and bone defect group. The cut with ophthalmic scissors. Subsequently, the infusion

104 International Journal of Bioprinting (2021)–Volume 7, Issue 1