2.1. Human bone marrow and adipose MSCs
The human BMMSCs and AMSCs were obtained by Lonza (CH) and cultivated according manufacturer’s instruction.
2.2. Preparation of MPs
The MP particles derived from uncolored PET water bottles. The polymer type of the bottles was verified by FTIR spectroscopy (FTIR, Thermo-Scientific, MS, USA). In brief, a bottle sample (10 grams) was flash-frozen with liquid nitrogen and then grounded in a swing mill to obtain irregular particles having heterogeneous form and size. The obtained samples were filtered with a 30 µm sieve and then diluted in 100 ml of MQ water. The obtained plastic dust was successively filtered through small pores of cellulose nitrate filters (Ø = 47 mm, 2.76 µm, and 1 µm) (Healthcare Life Science, UK) to obtain two solutions with two size ranges of MPs (2.67–1 µm and under 1µm).We performed a Scanning Electron Microscopy qualitative analysis of the particle surface structure (Cambridge Instruments, UK - Mod. Stereoscan 360). The microscopy analysis was associated with SEM-EDX (X Energy Dispersion Detector) using the Inca software. The SEM-EDX was also used to count the number of particles per ml for each size range and further transformed to µg/L based on PET density [12].
2.3. MSC treatment with PET
For an evaluation of PET effects on in-vitro MSC functions, the cells were incubated for 48 hours in aMEM containing FBS (10%), FGF2(3ng/ml) and PETs (10μg/ml). We used two different sizes—<1μm (PET1) and <2.6μm (PET2.6). After 48 hours, the cultures were used for the programmed experiments.
2.4. Proliferating assay
Cell proliferation was determined by CCK-8(Colorimetric Cell Counting)assay by Dojindo (MD, USA) according manufacturer’s instruction.
2.5. Immunocytochemistry and Senescence-Associated Beta-Galactosidase
After PETs exposure, the cells grown in 24 multi-wells were fixed in a 2% formaldehyde solution for 10 minutes. Cells were then stained for detecting Senescence-Associated Beta-Galactosidase as already reported [13].
Following senescence staining, cells were permeabilized with 0.3% Triton-X100 (Roche, CH) and further incubated at room temperature in a blocking solution maid of 0.1% Triton-X100and5% FBS. The samples were then incubated with the antibodies against pRPS6 (Cell Signaling, MA, USA, code 4858) and Ki67 (Santa Cruz Biotechnology, CA, USA, code sc7846) at 4 °C overnight. We used a goat anti-rabbit antibody (FITC-conjugated) secondary antibody(Gtx-Rb-003D488) and a goat anti-mouse antibody(TRITC-conjugated) secondary antibody (Gtx-Mu-003D594) that were from Immuno Reagents (NC, USA). DAPI staining was employed to recognize nuclei and images were obtained using a DM2000Leica fluorescence microscope., For each analyzed marker, the percentage of positive cells was calculated as reported by Alessio and co-workers(2021) [14].
2.6. Immunocytochemistry (ICC) for detection of Ataxia-telangiectasia mutated kinase (ATM) and gamma-H2AX
The ICC procedure was performed according our previous published [13].
2.7. DCF-DA assay
Reactive oxygen species (ROS)were evaluated by intracellular conversion of fluorescent DCFH-DA (2,7-dichlorodihydrofluorescein). Cells were incubated with 0.1% Pluronic F-127 and DCFH-DA (2 μM) for 30 minutes at 37°C, then were PBS washed analyzed with easyCyte™ flow cytometer (Millipore, MS,USA) with easyCyte™ software.
2.8. Annexin-V assay
Apoptotic cells were identified with a fluorescein-conjugated Annexin V kit (Millipore, MS, USA) on a Guava easyCyte cytometer following the manufacturer's instructions. We grouped together early and late apoptotic cells.
2.9. Colony Forming Units assay (CFU)
Following treatments with MPs, 1,000 cells were seeded in 100 mm plate and incubated in a growth medium for 14 days according our previous published procedure [13].
2.10. Spontaneous differentiation
MSC cultures were trypsinized after being treated with MPs and 1.5x104 cells were seeded in six-well plates pre-coated with gelatin solution (Sigma Aldrich, MO, USA). Cells were incubated in aMEM medium supplemented with 10% FBS for 21 days.
2.11. In-vitro differentiation and staining
After being treatment with MPs, 1.5 x 104/cm2 cells were seeded and cultivated either in osteogenic or chondrogenic or adipogenic medium at 37°C for 21 days as already reported [15]. Cell staining for detecting osteocytes, adipocytes and chondrocytes was performed as previously described [15].
2.12 Western blotting
Cells were lysed in buffer containing 0.1% Triton-X100 (Roche, CH) and analyzed for western blot as already reported [16]. The following primary antibodies were used: RB2/P130 from BD Biosciences (CA, USA); P27KIP1 and RB1 from Cell Signaling Technology (MS, USA), P107 (code sc-318), P53 (code DOI-1), and P21CIP1 (code C-19) from Santa Cruz Biotechnology (CA, USA); P16INK4A from ABCAM (UK).
2.13 Soft agar
After being treated with MPs, 2,500 cells were incubated in 0.5 mL DMEM containing agarose plus FBS in 35 mm Petri dishes and incubated for 21 days as already reported [13].
2.14 Statistical analysis
Statistical significance was processed using the one-way test ANOVA followed by post hoc test (Tukey test). All data were analyzed using the statistical software package GraphPad Prism version 5.01 (GraphPad, CA, USA).