Page 26 - IJB-9-1

Biocompatible materials and Multi Jet Fusion

fibroblasts and osteoblasts under conditions pertaining at 490 nm in a microplate reader, according with the
to short-term (day 2 and day 4) and long-term (day 28) manufacturer’s instructions. MTT assay was performed
culture. Moreover, long-term studies were also conducted at the aforementioned timepoints to determine the
to investigate the effect of two rounds of subculture on the time-dependent change in cell proliferation and cell
viability of cells on 3D-printed cell culture chambers. The number (n = 4 per group).
24-well plates with a similar surface area as the printed
PA-12 chambers were used as the comparison basis for 2.3.3. Surface coating
assessing the printed chamber’s biocompatibility in all The surface of 3D-printed PA-12 cell culture chambers
assays throughout the study. and polystyrene 24-well plates was coated with PDL and
collagen (CLG) (50 µg/mL) through physical absorption
2.3.1. Cell culture conditions
to enhance the attachment of cells on the surface. The
Murine L929 fibroblasts were cultured in DMEM PDL- and CLG-coated cell culture chambers were
supplemented with 10% fetal bovine serum (FBS) and 1% incubated for ~1 h at room temperature following which
penicillin/streptomycin. MC3T3e1 mouse osteoblasts were the chambers were washed thrice with distilled water and
propagated in α-MEM supplemented with 10% FBS and PBS, respectively. The above-mentioned coatings were
1% penicillin/streptomycin. All cell culture reagents were included for all cell culture experiments.
obtained from Gibco, USA. Both cell lines were maintained
at 37°C in a humidified incubator supplied with 5% CO . 2.3.4. Cell morphology and proliferation
2
For all experiments, fibroblasts and osteoblasts were The adherence and growth of cells on 3D-printed PA-12
seeded at a density of 5000 and 4000 cells/cm at day 0, cell culture chambers were visualized by fluorescence
2
respectively, and allowed to grow to confluence for 4 days. microscopy and SEM by staining the cells with fluorescent
dyes (n = 3 per group). For fluorescence microscopy,
2.3.2. Effect of PA-12 leachate
cells cultured on 3D-printed PA-12 cell culture chambers
The effect of substance leachate from 3D-printed PA-12 on were fixed, stained, and viewed according to the protocol
cell adherence and viability was determined by culturing mentioned in section 2.3.2. The viability of cells cultured on
cells in the leachate medium. The leachate medium was 3D-printed PA-12 cell culture chambers was performed at
prepared by soaking fourteen 3D-printed PA-12 cell the aforementioned time-points and assessed by MTT assay
culture chambers (weight = 1.344 g ± 0.02 per chamber; according to the protocol mentioned in section 2.3.2. For
total surface area of the cup: 452.388 mm ) in respective SEM, cell culture chambers with cells were fixed with 2.5%
2
culture medium (~30 mL) for 5 days at 37°C, filtered and glutaraldehyde for 6 h, washed with PBS, and subjected to
used for cell culture. Here, cells were grown in normal sequential ethanol series dehydration (50%, 70%, 85%, and
and leachate medium on 24-well plates, tested for cell 100%) before critical point drying (BALTEC, CPD 030,
adherence and viability by performing MTT assay and Critical Point Dryer, Scotia, NY, USA). Samples were then
fluorescence microscopy (n = 4 per group). sputter coated and imaged using the protocol mentioned

For fluorescence microscopy, Alexa Fluor 546 in 2.2.1.
®
phalloidin (#A22283, Thermofisher, USA) was used to 2.3.5. Toxicity analysis
stain actins, while Hoechst 33342 (Sigma-Aldrich, USA)
was used as a counterstain to visualize the nuclei. Briefly, Toxicity analysis was performed by lactate dehydrogenase
cells were fixed with 4% paraformaldehyde, washed (LDH) assay at predetermined timepoints using
with ×1 PBS, and permeabilized with 0.1% Triton-X for CyQUANT™ LDH Cytotoxicity Assay Kit (Invitrogen,
15 min. Cells were then stained with Alexa Fluor 546 USA) according to manufacturer’s instructions. LDH,
®
phalloidin for 1 h and counterstained with Hoechst 33342 which was secreted into the supernatant, was determined
for 10–15 min, according to manufacturer’s instructions. colorimetrically at OD 490 using a microplate reader (n = 3
Cells were washed after staining and air-dried before per group).
being viewed under the microscope using the TRITC
and DAPI filters. The proliferation and viability of cells 2.3.6. Intracellular redox status
cultured on 3D-printed PA-12 in normal and leachate Cellular glutathione (GSH) level was detected by live-
medium were assessed by MTT assay. Briefly, 20 µL of the staining cells with a GSH-labeling probe, to detect any
CellTiter 96 AQueous One Solution Reagent (Promega, oxidative stress induced on the cells by the cell culture
®
USA) was added per 100 µL of the culture medium to chambers sand surface coatings. About 40 µM of
each sample and incubated for 4 h at 37°C in a humidified monochlorobimane (mBCI) (#M1381MP, Thermofisher,
5% CO incubator before the absorbance was measured USA) diluted in the culture medium was added to the
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Volume 9 Issue 1 (2023) 18 https://doi.org/10.18063/ijb.v9i1.623