Page 543 - IJB-10-6

International Journal of Bioprinting Design and property of PLPG/PDLA scaffold

Testing Machine Co., LTD, China) at a compression speed washed with phosphate-buffered saline (PBS) and analyzed
of 1 mm/min. Each experiment had three parallel samples. using a fluorescence microscope (LEICA DMi8; LEICA,
Germany). Each experiment had three parallel samples.
2.6. Degradation behavior of the
PLPG/PDLA scaffolds 2.7.3. Immunofluorescent staining
The enzymatic degradation behavior of the PLPG/PDLA MC3T3-E1 cells (3 × 10 cells per mL) co-cultured on the
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scaffolds was assessed in a proteinase K solution over 15 PLPG/PDLA scaffolds for 12 and 72 h were fixed with
days. Initially, the PLPG/PDLA scaffolds were immersed in paraformaldehyde for 0.5 h and then permeabilized with
3 mL of proteinase K solution containing tris buffer (0.05 Triton X-100 solution. The cells were co-cultured with an
M; pH 8.5). To maintain enzyme activity, the proteinase anti-vinculin antibody for 12 h at 4°C. Cell actin stress
K-tris buffer was replaced every two days. The scaffolds fibers were stained using a rhodamine-phalloidin solution
were removed at specified time points, washed with plenty for 0.5 h, and cell nuclei were stained with 10 µg/mL DAPI.
of water, and then placed in a vacuum drying oven (37°C) The adhesion morphology of the MC3T3-E1 cells cultured
until their mass stabilized. Mass loss was calculated using on the scaffolds was analyzed using a confocal laser
Equation II: scanning microscope (LEICA DMi8 S; LEICA, Germany).
Each experiment had three parallel samples.
2.7.4. Alkaline phosphatase staining
Mass loss (%) 100 (II)
To assess alkaline phosphatase (ALP) activity, MC3T3-E1
cells (6 × 10 cells per mL) were co-cultured with the PLPG/
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where M and M are the initial dry mass and final PDLA scaffolds for 7 and 14 days at 37°C. After fixing
d
i
dry mass, respectively. Each experiment had three the cells with paraformaldehyde and rinsing with PBS,
parallel samples. a certain amount of BCIP/NBT solution was added, and
the samples were incubated in the dark for 24 h. Finally,
2.7. Biocompatibility of the PLPG/PDLA scaffolds the staining was captured using an inverted microscope
MC3T3-E1 cells were cultured in an incubator (5% CO ) (LEICA DMi8 S; LEICA, Germany). Each experiment had
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at 37°C for 5 h. The PLPG/PDLA scaffolds were sterilized three parallel samples.
under ultraviolet light for two days. MC3T3-E1 cells For the assessment of ALP, MC3T3-E1 cells (6 × 10
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were then detached from the culture plates using trypsin cells per mL) were co-cultured with the PLPG/PDLA
and subsequently centrifuged. The cell concentration scaffolds for 14 days. Post-incubation, RIPA lysis buffer was
was adjusted to 6 × 10 cells/mL. A 30 µL cell suspension added to each well. The resulting supernatants with 150 μL
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was inoculated onto the PLPG/PDLA scaffolds. After were mixed with 150 μL pNPP and incubated in the dark
incubation for 6 h, a fresh medium was added to each well, for 1 h. The supernatants of 20 μL were then introduced
fully immersing the scaffolds. into a 200 μL BCA detection solution and incubated for

2.7.1. Cell proliferation another hour in the dark. Finally, the OD of the solution
A cell counting kit-8 (CCK-8) assay was used to was measured at 405 and 562 nm using a multifunctional
quantitatively assess the proliferation of MC3T3-E1 cells enzyme-labeled instrument (TECAN Infinite M200 PRO;
at 6 × 10 cells per mL in the PLPG/PDLA scaffolds. Tecan, Switzerland), and the relative activity of ALP (R ALP )
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MC3T3-E1 cells and the scaffolds were co-cultured for 1, 3, was calculated using Equation III:
and 5 days in a 48-well plate. 10 μL of CCK-8 reagent (100
μL, 10%), diluted 10 times, was then added to the wells and R ALP =OD /OD ×100% (III)
405
562
incubated in the cell incubator for 2 h. The optical density
(OD) was measured at 450 nm using a multifunctional
microplate reader (Infinite M200PRO; Tecan, Switzerland). 3. Results and discussion
Each experiment had three parallel samples.
3.1. Characterization of the PLPG/PDLA scaffolds
2.7.2. Live/dead viability assay The morphology of the PLPG/PDLA scaffolds was
The live/dead staining assay was performed to evaluate observed via SEM. As displayed in Figure 1a–d, the PLPG/
the viability of MC3T3-E1 cells cultured with the PLPG/ PDLA scaffolds, with pore sizes of 200–300 μm, exhibited
PDLA scaffolds. The cells and scaffolds were co-cultured a regular network structure and uniform struts, indicating
for 1, 2, and 3 days in the incubator. Calcein-AM and that the PLPG/PDLA composites are suitable for 3D
propidium iodide solutions were then added, and the printing to fabricate complex scaffolds. The top and side
samples were cultured for 0.5 h. Finally, the cells were views of the pore structure inside the scaffold demonstrated

Volume 10 Issue 6 (2024) 535 doi: 10.36922/ijb.4645