Page 221 - IJB-10-2

International Journal of Bioprinting Property of scaffolds with different lattices

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S (5 × 10 /ml) were cultured in 24-well plates, and the culture
(IX) medium (α-MEM medium, 10% fetal bovine serum, 1%
V s streptomycin) was changed every 2 days. After 3 days of
culture, phalloidin/DAPI staining (Yeasen, Shanghai,
uL Q
K A p (VI) China) and scanning electron microscopy (SEM) were
employed to further observe the cell adhesion status.

where δ represents the specific surface area; S denotes the 2.5.3. Cell proliferation
internal surface area and refers to the interface between Similar to the cell adhesion experiment, mouse MC3T3-E1
solid and pore in the porous structure; V shows the cells at a concentration of 2 × 10 /ml were seeded onto
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S
volume of the porous structure; L represents the thickness 24-well plates containing Ti6Al4V scaffolds. After 5 days
of the scaffold; u denotes the dynamic viscosity of the fluid; of incubation, a CCK8 assay was performed to measure
A represents the cross-sectional area of the scaffold; Δp cell growth.
shows the pressure gradient when the fluid passes through
the scaffold at the flow rate Q; and K is the permeability. 2.5.4. Cell differentiation
Ti6Al4V scaffolds seeded with mouse MC3T3-E1 cells (5
2.5. In vitro cell experiments × 10 /ml) were grown in 24-well plates containing α-MEM
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2.5.1. Scaffold processing medium supplemented with 10% fetal bovine serum and 1%
Ti6Al4V porous scaffolds (Φ10 mm × 3 mm) with various penicillium–streptomycin. The following day, the medium
unit cells were pickled (2% HF, ultrasonic pickling for 5 was changed to an induction medium (α-MEM medium,
min) to eliminate powder residue, followed by sonication 10% fetal bovine serum, 1% streptomycin, 10 nmol/l
(KQ5200DE, Kunshan Ultrasonic Instrument Co., LTD., dexamethasone, 10 mmol/l β-glycerophosphoid sodium,
China) with deionized water, acetone, 95% ethanol, and 50 µg/ml vitamin C), and the medium was replaced after
deionized water for 30 min in each liquid or solution. The every 3 days. Each experiment required three copies of the
scaffolds were then wrapped with sterile gauze and then porous Ti6Al4V scaffold for each type of unit cell. Alkaline
subjected to sterilization at high temperature (121°C, 90 phosphatase (ALP) activity was determined after 7 and
min). Finally, the sterilized scaffolds were dried in air 14 days of culture using an ALP kit, and the expression
and subsequently placed in sterile tubes for further cell of osteogenesis-related genes (ALP, OCN, Runx2) was
experiments. determined using reverse-transcription polymerase chain
reaction (RT-PCR). Primer sequences are listed in Table 3.
2.5.2. Cell adhesion After 30 days of culture, 10% cetylpyridinium chloride
In the logarithmic phase of growth, mouse pre-osteoblasts was employed to dissolve the calcium salt deposited, and
MC3T3-E1 cells (5 × 10 /ml) were seeded into 24-well plates the optical density (OD) value was measured using a
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containing Ti6Al4V scaffolds at 1 ml per well, and scaffolds microplate reader at the wavelength of 590 nm.
of each unit cell were set up in triplicate. The cells were
grown for 3 h under preset conditions (37°, 95% relative 2.6. In vivo animal experiments
humidity, 5% CO ). The number of adherent cells was 2.6.1. Surgical procedure
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quantitatively and qualitatively determined using CCK8 kit Twenty healthy adult male New Zealand rabbits (2 kg)
(Beyotime, Shanghai, China) and acridine orange staining were divided into four groups at random. Via the auricular
(Source Leaf Biology, Shanghai, China), respectively. vein, pentobarbital sodium (30 mg/kg) was injected to
Similarly, Ti6Al4V scaffolds seeded with MC3T3-E1 cells induce anesthesia. After preparing the skin, the surgical
region was cleansed three times with iodophor, and
Table 3. Sequences of upper and lower primers for RT-PCR then the skin and fascia were sliced to expose the lateral
epicondyle of the femur. With a 3.0 Kirschner wire and a
Gene Forward primers Reverse primers manual grinding drill, a circular hole with a diameter of
ALP 5’-GGCACCTGCCT- 5’-GTTGTGGTGTAGCTGG- 4 mm and a depth of 10 mm was created perpendicular
TACCAACTCT-3’ CCCTTA-3’ to the lateral epicondyle of the femur. After washing and
OCN 5’-ACAG- 5’-AAGAACTCAAACATAC- sterilizing, Ti6Al4V scaffolds of four different unit cell
GCTTCCTAGAACAAG- GGGCAA-3’ types (Φ4 mm × 10 mm) were implanted into the femurs of
GGC-3’ four groups of rabbits, one scaffold per femur (Figure 3E).
Runx2 5’-ATGACACTGC- 5’AGGGATGAAATGCTTGG- After the wound was treated with iodophor and saline, 2-0
CACCTCTGACTTCT-3’ GAACT-3’ silk thread was used to close the wound layer by layer. After
GAP- 5’-CCTCGTCCCGTAG- 5’-TGAGGTCAATGAAGGG- surgery, all animals received injectable penicillin (10,000
DH ACAAAATG-3’ GTCGT-3’ U) for 3 days to prevent infection.

Volume 10 Issue 2 (2024) 213 doi: 10.36922/ijb.1698