Anthos 2020

Plasma IL-6 was measured using human quantitative IL-6 enzyme-linked immunosorbent assay (ELISA) kit, as per the manufacturer's instructions (R&D, Sigma-Aldrich). The absorbance was measured at 450 nm using a microplate reader (Biochrom Anthos 2020, UK).

The hemocompatibility test was carried out on human fresh anticoagulated blood collected form volunteers. The study was approved by the Ethics Committee in Kermanshah University of Medical Sciences (Ethics board approval number: IR.KUMS.REC.1397.545). The methods in the study were in accordance with the guidelines of the Declaration of Helsinki. Written informed consent was obtained from all subjects before blood donation. The Each scaffold was incubated with 0.2 ml of the blood diluted with normal saline for 60 min at 37 °C. After passing the incubation time, the incubated blood was centrifuged at 1,500 rpm for 10 min and the resulted supernatant transferred to a 96-well plate, and the absorbance was measured at 545 nm by utilizing the Anthos 2020 (Biochrom, Berlin, Germany) microplate reader. The absorbance was attributed to the hemoglobin leaked from red blood cells (RBC) and hemolysis degree was calculated using Eq. (3)^{43 (link)}: $$Hemolysis\%=\frac{Dt-Dnc}{Dpc-Dnc}\times 100$$ where D_t, D_nc, and D_pc are the absorbance of the sample, the absorbance of the negative control, and the absorbance of the positive control. The negative and positive controls were 0.2 ml of blood diluted with normal saline and deionized water, respectively.

Cell culture studies were performed using MG-63 cell lines and the MTT assay kit was used to quantitatively measure the cell proliferation rate. The prepared scaffolds were cut spherically and put in the bottom of the 96-well plate under sterile conditions. MG-63 cells were cultured at the density of 7 × 10³ cells on the scaffolds in DMEM/F12 culture media supplemented with 10% (v/v) FBS, 100 unit/ml penicillin, and 100 µg/mL of streptomycin incubated in a humidified incubator at 37 °C with 5% CO₂. At each time point (1 and 3 days after cells seeding), the culture medium was removed from the 96-well plate and 0.2 mL of MTT (0.5 mg/1 mL DMEM) was added to each well, and the cells were incubated at 37 °C for 4 h in a dark place. Then, the supernatant was discarded and 150 mL DMSO was added to each well to dissolve the formed formazan crystals. After 10 min, 100 μL from each well was transferred to a new 96-well plate and the optical density was read at 570 nm using Anthos 2020 microplate reader (Biochrom, Berlin, Germany).

The presence of nitric oxide-derived species (NO) was determined by a test based on the Griess reagent (Sigma-Aldrich, USA)^{80 (link)}. Briefly, 100 µL of supernatant collected from untreated or treated cells were incubated with 100 µL of the Griess reagent for 15 min at room temperature. 1 µM lipopolysaccharide Escherichia coli O111:B4 (Sigma-Aldrich, USA) was used as a positive control. Samples were read in a microplate reader (Biochrom Anthos 2020, UK) at 562/630 nm. The results were extrapolated to a nitrite standard curve, which was performed using NaNO₂ (Sigma-Aldrich, USA).