L929 mouse fibroblasts

The cytotoxicity analysis of TiO₂ nanoparticles was performed using L929 mouse fibroblasts (ATCC, Manassas, VA, USA) through MTT assay. These fibroblasts were maintained in standard culture conditions [41 ]. The temperature was maintained at 37 °C in 5% CO₂ at 95% humidity. Subsequently, trypsinization was performed, and the cell suspension was made in 10% DMEM containing 1 × 10⁴ cells [42 (link)]. Thereafter, 100 µL of cell suspension was obtained and seeded in each well of the standard 96-well plate for 24–48 h. The TiO₂ nanoparticles were added as 1 mg/mL stock solution concentration to test cytotoxicity. MTT dye (Sigma Aldrich, Saint Louis, MO, USA) was added to each well, and the plates were incubated at 37 °C for 2 h. The fluorescence of each well was measured at a wavelength of 490 nm with a fluorescence well plate reader (Thermo Fisher, Waltham, MA, USA) at day-1, day-3, day-5, day-7, day-21 and day-30 [43 (link)].

To measure the bioactive TNF-α released from the treated NR8383 cells, cell culture supernatants were centrifuged and analysed using the L929 cytolysis test described by Desch et al. [92 (link)]. Briefly, 50 μL supernatant was pipetted onto 80 % confluent L929 mouse fibroblasts (ATCC, USA) in the presence of actinomycin D (Sigma Aldrich, Germany). After 24 h, the L929 cells were washed with phosphate buffered saline (PBS; Biochrom GmbH, Germany), stained with 0.5 % crystal violet (Sigma Aldrich, Germany), washed extensively with PBS, and lysed in an acidic mixture of citrate-buffered 50 % ethanol (Carl Roth GmbH, Germany). OD was measured at 570 nm, and results were expressed as L929 fibroblast lysis relative to non-treated medium controls, which were set to 0 %. Additionally, the L929 cells’ responsiveness to TNF-α was controlled using a TNF-α standard (510-RT; Bio-Techne, Germany), and the uppermost value (1000 pg TNF-α/mL) was set to 100 %. As an additional PC, the TNF-α-forming capacity of NR8383 cells was confirmed by stimulation with lipopolysaccharide (LPS; 0.1 μg/mL, Sigma Aldrich, Germany). Finally, the direct effects of the test materials on L929 cells were assessed using 50 µL of the supernatants from cell-free NM-containing wells.

Pulp capping materials Dycal (Dentsply Caulk, Milford, DE, USA), Life (Kerr, Orange, CA, USA), MTA Gray (Dentsply Tulsa, Tulsa, OK, USA), and SB-C&B (Sun Medical Co., Shiga, Japan) were purchased and used without any modifications. Working times and setting times of these materials were summarized in Table 1. L929 mouse fibroblasts were obtained from American Type Culture Collection (ATCC, Manassas, VA, USA). Cells were grown in Dulbecco's Modified Eagle Medium (DMEM; Invitrogen, Carlsbad, CA, USA) supplemented with 10% fetal bovine serum (Hyclone Laboratories Inc., Logan, UT, USA) and 1% penicillin-streptomycin (Gibco BRL, Gaithersburg, MD, USA) under standard cell culture conditions (37°C and 5% CO₂).

L929 mouse fibroblasts were obtained from the American Type Culture Collection (Manassas, VA, USA). The cells were grown in complete Roswell Park Memorial Institute (RPMI) 1640 medium (with l-glutamine) supplemented with 10% fetal bovine serum (FBS), 100 IU/mL penicillin, and 100 µg/mL streptomycin.
MG-63 human osteoblast-like cells were purchased from the European Collection of Authenticated Cell Cultures (Salisbury, UK). The MG-63 cells were cultured in Eagle’s Minimum Essential medium (EMEM) supplemented with 10% FBS, 2 mM l-glutamine, 1 mM sodium pyruvate, nonessential amino acids, and antibiotics (100 U/mL penicillin, 100 μg/mL streptomycin).
Adipose-derived human mesenchymal stem cells (ADSCs) were purchased from PromoCell and cultured in Mesenchymal Stem Cell Growth Medium^® supplemented with 10% Supplement Mix^® (PromoCell GmbH, Germany), 100 U/mL penicillin, and 100 µg/mL streptomycin.
All cell lines were cultured at 37 °C in a humidified atmosphere with 5% CO₂ and passaged at 70–80% confluency using a 0.04% trypsin ethylenediaminetetraacetic acid (EDTA) solution (ADSC cells), 0.25% trypsin-EDTA solution (MG-63 cells), or cell scraper (L929 fibroblasts). All compounds used for cell culture were purchased from Sigma-Aldrich (Darmstadt, Germany).