MTT assay

MTT (Sigma, St. Louis, MO, USA) assay [28] was used to evaluate cellular energy metabolism and indicate cell proliferation indirectly. According to the Chinese national standard (GB-16886.5T-2003) for biological evaluation, the in vitro cytotoxicity experiment was performed at 37 °C with at least 24 h of scaffold extraction. Scaffold leaching solutions with different concentrations of 25, 50, and 100% were prepared and divided into four groups (n = 6), namely A, B, C, and D (single control). According to the previously reported protocol [29], rabbit bone marrow stem cells (rBMSCs) (Cyagen, USA) were applied to investigate the cytotoxicity of 3D porous NP ECM-derived scaffolds. The cells were cultured in the 25 cm² tissue culture flasks at 37 °C in a humidified atmosphere of 5% CO₂ in air. The applied cell culture medium was Dulbecco’s Modified Eagle Medium, i.e., nutrient mixture F12 (DMEM/F12, Gibco, USA) culture medium, which contains 8% fetal calf serum (FBS, Gibco, USA). The passage 3 (P3) generation of rBMSCs (Cyagen, USA) with a density of about 5 × 105 cells/cm² was suspended in the culture medium, and then dispersed over the four groups in a 96 well plate by MTT assays. The assays were performed at the second, fourth, and sixth day, respectively.