Page 27 - IJB-9-1

International Journal of Bioprinting Biocompatible materials and Multi Jet Fusion

cells and incubated for 30 min at room temperature and DNA content was quantified using Quant-iT™ PicoGreen
examined under fluorescence microscope (n = 3 per dsDNA Reagent and Kits (Molecular Probes Inc, USA) by
group). The strength of the fluorescence signal correlates measuring the fluorescence using 480 nm excitation and
with GSH level and consequently the health of cells. 520 nm emission. ALP activity was normalized to the total
DNA content. The results were expressed in nanomoles of
2.3.7. Proliferation state characterization by p-nitrophenol produced per microgram of DNA.
immunostaining of Ki67 and p53
To characterize the proliferative state of cells grown on 2.3.9. Long-term cell culture
3D-printed PA-12, the expression of Ki67 (proliferation In practice, cells are often subcultured for multiple rounds.
marker) and p53 (anti-proliferation marker) at protein Accordingly, the effect of growing/subculturing cells on the
level were determined by immunostaining. Cells, when 3D-printed PA-12 cell culture chambers on the viability and
fixed and permeabilized, were blocked with 1% BSA in proliferative ability was determined. On reaching confluence
PBS-Tween (0.1%) for 1 h at room temperature and washed (at day 4), cells were subcultured from 3D-printed PA-12
before staining. Ki67 was detected with monoclonal into new PA-12 cell culture chambers and conventional cell
anti-Ki67 antibody (#AB16667, Abcam, USA), while p53 culture plates, as indicated in Figure S1. As a control, cells
was detected with anti-p53 antibody (#AB26, Abcam, were also subcultured from conventional cell culture plates
USA) according to manufacturer’s instruction. Cells were to plates. Two rounds of subculture were performed on day
incubated with both antibodies overnight at 4°C. Cells were 4 and day 8, for which the cell viability and proliferation
then washed before incubation with FITC-conjugated goat were tested on day 8 and day 12, respectively, by MTT assay
anti-Mouse IgG (H+L) secondary antibody (Thermofisher, and fluorescence microscopy, according to the protocol
#F2761) and TRITC-conjugated goat anti-Rabbit IgG mentioned in section 2.3.2 (n = 4 per group).
(H+L) secondary antibody (Thermofisher, #T2769) for
1 h at room temperature and counterstained with Hoechst 2.3.10. Bacterial adhesion and viability
33342 (Sigma-Aldrich, #B2261) as described earlier. Cells A. Strains and growth conditions
were then dried at room temperature before viewing under Escherichia coli strain 0114 (ATCC 25922) was used in
the microscope (n = 3 per group). this study. Overnight cultures from frozen stock were
2.3.8. Alkaline phosphatase activity recovered in 5 mL of brain–heart infusion (BHI) broth and
cultured aerobically at 37°C in a shaking incubator. From
Before differentiation, osteoblasts were seeded at a density the overnight culture, an inoculum was prepared in fresh
of 4000 cells/cm and left in culture to attain confluence. To BHI. 300 µL (equivalent to 10 bacteria/chamber) of each
2
7
regulate osteoblast differentiation, osteogenesis induction bacterial suspension was freshly seeded in the 3D-printed
medium 1 and 2 were used. The osteogenic medium 1 PA-12 and positive control (polystyrene plate) cell culture
was composed of complete αMEM supplemented with 0.2 chambers. Both O plasma-treated or untreated PA12 cell
2
mM of ascorbic acid and 10 mM of glycerol 2-phosphate. culture chambers were tested.
The osteogenic medium 2 was composed of osteogenic
medium 1 with the addition of 50 nM melatonin. When B. MTT assay
cells were confluent, the normal medium was removed, The viability of E. coli cultured on the 3D-printed PA-12
and osteogenic medium 1 was added. This medium change cell culture chamber was measured using MTT assay
corresponded to differentiation day 0, and the medium was (CellTiter 96 AQueous One Solution Cell Proliferation
®
changed twice a week. On differentiation day 6, osteogenic Assay, Promega, USA) to obtain a quantitative value for
medium 2 -was added, and the medium was replenished the absorbance of bacterial units after being cultured for
every 3 days. 24 days after differentiation, the alkaline 24 h at 37°C (n = 3 per group). CellTiter 96® AQueous
phosphatase (ALP) activity (Alkaline Phosphatase Assay One tetrazolium reagent was added to each well (20 µL/
Kit, Abcam, USA) was tested colorimetrically (n = 3 per every 100 µL of media) and incubated for 24 h at 37°C.
group). Cells were harvested from cell culture chambers, Absorbance was read at 490 nm according to the
washed with ×1 PBS, and resuspended in 50 µL of manufacturer’s instructions.
assay buffer. Cells were frozen at −20°C and thawed at
37°C for 5 min to test the ALP activity according to the C. Live/dead imaging
manufacturer’s instructions. Enzyme activity was measured The bacterial viability on 3D-printed PA-12 cell culture
by recording the absorbance at OD . To determine the chambers was observed by staining live bacteria with
405
number of cells, deoxyribonucleic acid (DNA) was extracted SYTO9 green and dead bacteria with impermeant
using DNeasy Blood and Tissue Kit (Qiagen, USA), and the propidium iodide (PI) (LIVE/DEAD Bac-Light Bacterial

Volume 9 Issue 1 (2023) 19 https://doi.org/10.18063/ijb.v9i1.623