Page 106 - IJB-7-1

HA15-loaded Bone Tissue Scaffold
HSPA5 gene is a reliable indicator of ER stress in human 1.2 was used to perform aperture measurement on the
diseases . Furthermore, HSPA5 promotes cell survival SEM micrograph of the bracket. In this regard, 30
[33]
and drug resistance under ER stress conditions . Hence, measurement holes were randomly selected to calculate
[34]
it seems that β-TCP/PLGA-loaded HA15 material the mean and standard deviations (SD) to make a
targeting HSPA5 which is a master regulator of the preliminary assessment of the aperture range.
anti-apoptotic UPR signaling network can be a good
[35]
therapeutic option for bone defect problems. 2.3. Mechanical properties of scaffolds
In this study, we constructed a 3D-printed β-TCP/ An electronic universal testing machine was used to test
PLGA-loaded HA15 targeting HSPA5 bone tissue the mechanical response of the bone tissue scaffolds
scaffold according to a rabbit model of radial bone defect with a dimension of 15 mm × 3 mm × 2 mm according
and performed the implantation. The effect of 3D-printed to the GB/T1041-1992 Chinese standard protocol. The
β-TCP/PLGA-loaded HA15 bone tissue scaffold on bone prepared sample was inserted in the testing area and the
defect treatment and the healing condition is thoroughly compression loading was conducted with a speed of 0.5
discussed in this article. This study also provides a mm/min until the complete deformation, then the strength
theoretical and experimental guideline for the treatment
of bone defect with drug-loaded biomaterials that may be and modulus of the scaffold were measured.
a promising treatment of bone lesions. 2.4. In vitro experiments

2. Materials and methods (1) Cell culture

2.1. Scaffold fabrication Murine mesenchymal stem cell line C3H10 was incubated
under the standard condition in DMEM medium
The 3D-printed bone tissue scaffold formation procedure supplemented with 10% fetal bovine serum, 100 U/ml
along with in vitro and in vivo experiments is briefly penicillin G, and 100 mg/ml streptomycin. To seed the
shown in Figure 1. First, 3 g of PLGA (Shandong C3H10 cells onto the 3D-printed scaffolds, the scaffolds
Medical Device Company, P.R. China) with an inherent were sterilized with ultraviolet light and 70% ethanol
viscosity of 0.6–0.8 dL/g was dissolved in 10 mL of and were placed in 24-well tissue culture plates. For cell
dichloromethane (DCM). Then, 3 g of β-TCP was added seeding, samples were pre-soaked in DMEM complete
to prepare PLGA/DCM solution, which was subject culture medium for 24 h. Subsequently, 1 mL complete
to a 20-min ultra-sonication. After that, 200 μg of culture medium (3 × 10 C3H10 cells) was poured on
4
HA15 was added to 1.5 ml of deionized water to form the top surface of the scaffolds. Then, the samples were
an aqueous solution. The HA15 aqueous solution was incubated for 2 h to permit the cells to attach the scaffolds.
mixed with β-TCP/PLGA/DCM composite solution The cells were stimulated with two concentrations of
with the assistance of ultra-sonication to form uniform
HA15/water/TCP/PLGA/DCM composite emulsions leaching solution from β-TCP/PLGA/HA15 scaffolds;
as printing inks. The inks were subsequently added into the scaffold was infiltrated with 5 ml (HA15-1) or 10 ml
(HA15-2) culture medium for 48 h.
a 20 mL syringe connected to a V-shape nozzle (inner
diameter: 0.4 mm). A pre-designed STL file shown in (2) siRNA knockdown
Figure 2D and E with dimensions of sample and multi-
section views was imported in a cryogenic 3D printer We transiently transfected C3H10T1/2 cells with HSPA5
(Shenzhen Creality 3D Technology Co., Limited, P.R. siRNA using Lipofectamine RNAiMAX (Invitrogen,
China) and cylindrical scaffolds with 3D grid patterns Carlsbad, CA, USA) in Opti-MEM medium (Invitrogen),
were printed. The printing procedure was carried out according to the manufacturer’s instructions. The
according to the pre-set parameters; the feed speed was sequences of HSPA5 siRNA are as follows: Forward,
0.005 mL/s and the printing speed was 8 mm/s. Finally, a 5’-AAGGUUACCCAUGCAGUUGTT-3’ and reverse,
cylindrical scaffold with a diameter of 4 mm and a height 5’-AGAUUCAGCAACUGGUUAAAGTT-3’. Universal
of 15 mm were obtained (Figure 2). The printed scaffolds Non-targeting Control siRNA was considered and
were freeze-dried for 24 h to remove all DCM. used as the control for non-sequence-specific effects.
The capability of siRNA knockdown was evaluated by
2.2. Detection using scanning electron microscope Western blotting. Each experiment was performed in
Scanning electron microscopy (SEM) (TESCAN-Vega triplicates.
3 system) was used to observe the pore structure and (3) Western blot analysis
micro-morphology of the prepared 3D-printed scaffold as
well as the scaffold aperture size, pore connectivity, and Cells were lysed in 2% sodium dodecyl sulfate
surface morphology of pore walls. The Nano Measurer (SDS) with 2 M urea, 10% glycerol, 10 mM Tris–

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