Alp kit

The preconditioned PDLSCs were cultured in 96-well plate at a density of 2 × 10³ cells/well with maintenance medium containing 10% FBS. When the cells adhered up to 80%, medium was changed into osteogenic inductive medium (10⁻⁸ M dexamethasone (Sigma-Aldrich, USA), 10 mM β-glycerophosphate (Sigma-Aldrich, USA) and 50 ng/ml ascorbic acid (Sigma-Aldrich, USA) in DMEM-F12 containing 10% FBS). Seven days later, 1% TritonX-100 (Solarbio) was added for cell lysis and intracellular proteins were collected via supersonic splitting and centrifuging. Then, a BCA Protein assay kit (Nanjing Jiancheng Bioengineering Institute, Nanjing, China) was used to detect the total protein concentration and ALP kits (Nanjing Jiancheng Bioengineering Institute, Nanjing, China) were used to assay alkaline phosphatase (ALP) activity according to the manufacturer’s instructions. At last, the absorbance was measured at 520 nm wavelength.

Recombinant porcine IFN-α was obtained as a gift sample from Harbin Veterinary Institute of Chinese Academy of Sciences. Caco-2 cell line (Human colon carcinoma) was purchased from Stem Cell Bank, Chinese Academy of Sciences. High Glucose DMEM and Hank's balanced salt solution (HBSS) were obtained from Gibco, USA. HOOK^TM-Dye Labeling Kit was purchased from Biosciences, USA. ALP Kit was purchased from Jiancheng Bioengineering institute, Nanjing, China. Trypsin-EDTA, penicillin and streptomycin solution, glutamine and non-essential amino acids all were obtained from Hyclone, USA. Fetal bovine serum (FBS) was a product of PAN Biotech, Germany. 12-well Transwell inserts (0.4 μm pore size, 1.13 cm² surface area), 6-, 12-, and 96-well plates all were purchased from Costar, Corning, USA. MTT assay dye and DMSO were purchased from Amresco, USA. Hoechst 33,258 was purchased from Solarbio, China. Chlorpromazine, β-cyclodextrin, Wortmannin and Amiloride were all purchased from Sigma-Aldrich, USA.

DPCs were grown in osteogenic medium containing 10 μM E3330. At days 3 and 5, ALP activity of E3330 treated and untreated DPCs was determined according to the manufacturer’s recommendations with an ALP kit (Jiancheng, Nanjing, China) and normalized on the basis of equivalent protein concentrations. The absorbance of each well was determined using a microplate reader at 520 nm. Calcium deposition in the extracellular matrix was determined by Alizarin Red S staining after 14 days of osteogenic differentiation. Cells were fixed in 4% polyoxymethylene (Sigma-Aldrich, USA) and then incubated in 0.1% Alizarin Red S solution (pH = 4.3; Sigma-Aldrich, USA). Calcium deposition was observed and visualized under a light microscope, and the picture of 6-wells plate was captured by camera.

ALP assay was performed to assess the mineralization activity of hJBMMSCs cultured on scaffolds (n = 3, for each group). hJBMMSCs were seeded on scaffolds and incubated in standard medium and osteogenic differentiation medium, respectively, at 37°C for 1, 3, 7, and 14 days. Culture medium was refreshed at 3-day intervals. After culture, cell-seeded scaffolds were rinsed with PBS and cell lysate was obtained by incubating the scaffolds with 0.2% Triton X-100 overnight. ALP activity was evaluated using an ALP kit (Jiancheng Bioengineering Institute, China) according to manufacturer’s instructions. The OD was measured at 520 nm with an automatic microplate reader (Bio-Tek, VT, USA).

MC3T3-LP, MC3T3-BM, and MC3T3-KO cells with a density of 2.0 × 10⁴ cells/well were inoculated in 24-well plates and cultured in differentiation media. The cells were eventually harvested after 3, 5, 7, 9, 14, 18, and 21 days. Alkaline phosphatase (ALP) activities were determined using an ALP kit (Nanjing Jiancheng, China) in accordance with the manufacturer’s instructions. Protein concentrations were measured using a BCA protein assay kit (KeyGEN BioTECH, China). Enzyme activity was quantified via absorbance measurements at 520 nm using a 96-well microplate reader (TECAN, China) and calculated according to protein concentrations. Total protein content was used to normalize ALP activity, and all assays were repeated at least three times.