Page 144 - IJB-9-3

International Journal of Bioprinting Bioprinting tissue-engineered bone-periosteum biphasic complex.

evaluate the staining of cells. Live cells were shown in green, 2.6. Statistical analysis
and dead cells were in red. The cell viability was calculated The statistical results are expressed as mean ± standard
as the ratio of the number of live cells to total cells.
deviation. Student’s t-test and one-way analysis of variance
2.4.3. Laser confocal microscope (ANOVA) were used to analyze the differences of data. P <
RabBMSCs and rabPDSCs were labeled with two fluorescent 0.05 was considered statistically significant.
dyes (Invitrogen, USA), namely DiO (green) and DiI (red).
A laser scanning microscope (Zeiss, Germany) was used to 3. Results
analyze the distribution of fluorescent labeled cells in the 3.1. Co-culture of rabBMSCs and rabPDSCs
printed structure. promoted osteogenic differentiation in vitro

2.5. Calvarial bone reconstruction In order to obtain seed cells of the tissue-engineered bone-
2.5.1. Repair of critical-sized skull defect in rabbits periosteum biphasic complex, rabBMSCs and rabPDSCs
Fifteen male New Zealand White rabbits (2–2.5 kg) were were isolated. It was found that the cells were spindle-
divided into five groups (n = 6): (I) blank, (II) PLLA/HA, (III) shaped fibroblasts, which grew and proliferated rapidly
PLLA/HA+GelMA, (IV) PLLA/HA+GelMA+rabBMSCs, in vitro (Figure 2A and F). Multidirectional differentiation
and (V) PLLA/HA+GelMA+rabBMSCs+rabPDSCs. of the two sets of seed cells were induced in vitro. ALP
Rabbits were anesthetized and the calvarium were exposed. staining showed that the staining of cells in osteogenic
Two symmetrical 8 mm-diameter hole-shaped bone group was positive (Figure 2B and G). Alizarin red S
defects were established along the midline of the sutura staining showed calcium nodule deposition (Figure 2C
cranii with a circular drill. In group (I), the defect area and H), oil red O staining displayed lipid droplet formation
was retained without repair. In group (II), the defect area (Figure 2D and I), and Alcian blue staining resulted in the
was repaired with PLLA/HA scaffold alone. In group (III), formation of blue acid polysaccharide in the induction
PLLA/HA with GelMA to repair the defect area. In group group (Figure 2E and J). Besides, we detected the expression
(IV), bone-phase combined with scaffold was implanted of some osteogenesis-related genes in rabBMSCs and
in the defect area. In group (V), bone-periosteum biphasic rabPDSCs using qRCR after seven days of osteogenesis
complex was implanted in the defect area. All rabbits were induction. As shown in Figure S1, the expression of COL1,
sacrificed after 12 weeks, and the skulls were fixed with 4% OCN, OPN, and RUNX2 in rabBMSCs and rabPDSCs
paraformaldehyde. increased significantly after osteogenesis induction,
which was significantly different from the control group.
2.5.2. Micro-computed tomography (µCT) These above results illustrated that rabBMSCs and
All samples were imaged with high-resolution micro- rabPDSCs had multidirectional differentiation ability,
computed tomography (µCT) imaging system (Scanco which was in accordance with the characteristics of stem
μCT 100, Switzerland). The bone tissue was separated, and cells. RabBMSCs and rabPDSCs were then co-cultured
the defect area was reconstructed. Finally, the regenerated in transwell chambers. The results of ALP staining and
bone volume (BV), bone volume/total volume (BV/TV), Alizarin red S staining of the co-cultured cells and their
trabecular number (Tb. N), trabecular thickness (Tb. Th), osteogenic differentiation groups were significantly
and trabecular spacing (Tb. Sp) in the bone defect area different from those of the control group, indicating that
were measured and statistically analyzed. co-culturing of rabBMSCs and rabPDSCs could promote
osteogenic differentiation in vitro (Figure 2K and L).
2.5.3. Histological staining
The skull specimens were decalcified for 2 months. After 3.2. Characterization of PLLA/HA scaffolds
dehydration with graded alcohol, the skulls in the defect Bone scaffold should not only be able to bear certain
area were embedded in paraffin and cut into 7 μm-thick mechanical stress, but also have good biocompatibility
slices along the coronal axis. Hematoxylin and eosin in the process of in vivo repair. First, we applied synthetic
(H&E), Masson’s trichrome and immunohistochemical polymer–ceramic composites to construct 3D-printed
(IHC) staining of osteocalcin (OCN) were conducted to PLLA/HA tissue-engineered bone scaffolds by mixing
evaluate the newly formed bone within the defect area. PLLA of 3.2 W and 5.4 W with HA of 10%, 20%, and 30%
Image J software was applied to conduct quantitative mass fractions, respectively, and then explored the effect of
analysis of collagen volume fraction and mean density of different mixing ratios on the mechanical strength of the
OCN (%). The collagen volume fraction is equal to the scaffolds (Figure 3A). The results showed that when the
ratio of collagen area to total tissue area, and the mean mass fraction of HA was 20%, the maximal force was higher
density of OCN (%) represents the proportion of positive than that of 10% and 30% groups, and the maximal force of
expression of OCN in tissue. HA of 20% and 30% mass fraction groups were significantly

Volume 9 Issue 3 (2023) 136 https://doi.org/10.18063/ijb.698