Calcium liquicolor assay

PCL‐TCP scaffolds were placed into separate wells in a 24‐well plate (n = 3 per time point) and treated for 30 min with 70% ethanol to mimic the sterilization process used in later cell culture experiments. Ethanol treatment also increased the hydrophilicity of the scaffold for the Ca²⁺ release experiments. The scaffolds were then fully submerged in 0.5 ml of PBS and maintained in an incubator (37°C, 5% CO₂) for the duration of the study. PBS was collected at time points of 1, 2, 7, 14, and 28 days and stored at −25°C until assayed. A Calcium LiquiColor® Assay (StanBio, Boerne, TX) was used to quantify calcium content of each sample. Using the measured weight of the scaffolds along with the molar mass of calcium ions (Ca²⁺ 40.08 g•mol⁻¹), orthophosphates (PO₄³⁻94.97 g•mol⁻¹), and TCP [Ca₃(PO₄)₂ 310.18 g•mol⁻¹] the weight of Ca²⁺ doped within each scaffold was calculated to be 11.9 μg.

Cells were seeded onto a 12-well plate, with 1 × 10⁴ MC3T3-E1 cells per well. OGFs (100 nM dexamethasone, 0.05 mM ascorbic acid 2-phosphate, and 10 mM b-glycerophosphate; Sigma-Aldrich, St. Louis, MO, USA) were added to trigger osteoblast differentiation under different treatments. After the cells had been treated, they were fixed in 60% citrate-buffered acetone and then incubated with a mixture of Fast Violet B Salt and naphthol AS-MX phosphate alkaline solutions (Sigma-Aldrich, St. Louis, MO, USA) for the detection of ALP expression. ALP activity and the total number of cells were measured using the TRACP & ALP assay kit (TakaRa Bio, Kusatasu, Shiga, Japan) and water-soluble tetrazolium salt (WST-1) reagent (Roche, Basel, Switzerland), respectively, in accordance with the manufacturers’ instructions. Cells were fixed with 10% paraformaldehyde after treatment, and then a 2% Alizarin Red S solution (ScienCell, Carlsbad, CA, USA) was used for detecting calcification. Calcium LiquiColor Assay (Stanbio Laboratory, Boeme, TX, USA) and WST-1 reagent (Roche, Basel, Switzerland) were respectively used in accordance with the manufacturers’ instructions to determine the calcium content and number of cells after treatment.