Page 194 - IJB-10-5

International Journal of Bioprinting Structural design of D-surface scaffolds

2.5. Scaffold characterization well plate at 37°C with 5% CO and 95% humidity for 4
2
The surface microstructure of the scaffold structure h. Later, the medium culture was discarded to remove the
was observed by scanning electron microscope (SEM) non-adherent cells, and fresh medium was supplied. After
(QUANTA 450, FEI; Thermo Fisher Scientific, USA) 24, 48, and 96 h, the medium was removed from the 96-
after pretreatment with a thin gold layer. The internal well plate and washed with sterilized PBS. Thereafter, 100
microstructure was scanned using computed tomography μL of 10% Cell Counting Kit-8 (CCK8) working solution
(CT) (SIEMENS SOMATOM Drive; SIEMENS (XCM BIOTECH, China) was added and incubated for 2
Healthineers, Germany). The scanning parameters h. The optical density (OD) value of the reaction solution
were: a voltage of 70 kV, a current of 100 mA, and a slice was measured at 450 nm using a microplate reader.
thickness of 500 μm. The 3D models were reconstructed
using SIEMENS software analysis system, Syngo Via VB40 2.6.3. Calcein AM and nuclear propidium
(SIEMENS Healthineers, Germany). iodide staining
The scaffolds cultured after 24, 48, and 96 h were stained,
The compressive property of D-surface scaffolds was and the cell morphology was analyzed accordingly.
tested using a universal testing machine (TSE 105D; Calcein AM and PI dye (Beyotime, China) were added
Wance, China). According to ASTM D-695, the D-surface and incubated for 30 min. The scaffolds were then washed
samples were evaluated at a constant rate of 1 mm/min. with PBS and observed under an inverted phase contrast
The force and displacement curves were recorded, and the fluorescence microscope (LEICA, Germany).
compression process was pictured per 1 s by an advanced
contactless charge-coupled device. Each group of samples 2.6.4. Alkaline phosphatase assay
4
had three parallel samples, and the average value with A density of 4 × 10 cells/mL of MC3T3-E1 subclone 14 was
standard error bars was presented. seeded on cell plates, sterilized scaffolds, and PRP-loaded
scaffolds, and their alkaline phosphatase (ALP) activity
A micro-CT scanner (Xradia 610 Versa; Zeiss, Germany) was detected by using a kit (XCM BIOTECH, China)
was used to scan the internal structure of bone-implanted after 7 and 14 days of incubation. When the cells reached
scaffolds. The scanning power and voltage were 8.5 W and 70–80% confluence, the osteogenic media supplemented
70 kV, respectively. The sliced image was collected at every with 10 mM β-glycerophosphate （Macklin, China） and
30 μm and subsequently reconstructed into 3D models 0.05 mg/mL L-ascorbic acid （Xilong Scientific, China）
using Dragonfly software (Zeiss, Germany). was added to each cell-seeded well. On days 7 and 14, the
BCA protein concentration assay kit (XCM BIOTECH,
2.6. In vitro studies China) was used to detect the total protein concentration
2.6.1. Cell culture for each group. The relative ALP activity was measured
Cell culture was performed using mouse fibrosis L929 by normalizing the ALP activity (obtained at λ = 405 nm)
(Cell Bank of Typical Culture Preservation Committee with total protein concentration (obtained at λ =562 nm).
of Chinese Academy of Sciences, China) and mouse Each sample group was evaluated in triplicates.
osteoblast MC3T3-E1 subclone 14 (Shanghai Zhongqiao 2.7. In vivo studies
Xinzhou Biotech Co., Ltd., China). Roswell Park Memorial A total of six New Zealand white rabbits (2.5 ± 0.1 kg)
Institute (RPMI 1640; Gibco, USA) and Minimum Essential were purchased from Kangda Boai Biotechnology Co.,
Medium (α-MEM; Gibco, USA), supplemented with 10% Ltd., China. The rabbits were anesthetized using 1 mL/
fetal bovine serum (FBS) and penicillin/streptomycin, were kg of 3% pentobarbital sodium via auricular vein infusion
selected for cell culture. Trypsin-EDTA solution (0.25%; and 0.1 mL/kg for adequate anesthesia. The distal lateral
Gibco, USA) was used for subculture, and L929 cells were leg was then shaved and disinfected. The skin was incised
cultured at 95% humidity and 5% CO at 37°C. at the target surgical sites and disinfected using iodophor.
2
2.6.2. Proliferation on 3D-printed D-scaffolds A 1-cm-long incision was made in the distal lateral leg. A
Two scaffold groups were selected, i.e., bare scaffolds and low-speed electric drill was used to induce 4 × 8 mm bone
PRP-loaded D-scaffolds. Prior to cell culture, the scaffolds defects. The sterilized PRP-loaded graded scaffolds (8 mm
were washed three times with phosphate-buffered saline in height with a diameter of 4 mm) were then implanted
(PBS) (Hyclone, USA) and sterilized using 70% ethanol in the bone defect. The bare PBAT/PLA scaffold and the
solution accompanied by ultraviolet (UV) irradiation. L929 unfilled defect were set as controls.
cells with a density of 3 × 10 cells/mL were pre-seeded One week after surgery, the rabbits were examined by
3
into the graded D-surface scaffolds and incubated in a 96- CT. Then the rabbits were euthanized, and the implanted

Volume 10 Issue 5 (2024) 186 doi: 10.36922/ijb.3416