355 bm 050 cf

The following materials were obtained commercially and used as received unless otherwise noted: [2 (link),2 (link)]paracyclophane (Jiangsu Miaoqiao Synthesis Chemical Co., China, 98%), aluminum chloride (Alfa Aesar, 99%), dichloromethane (Macron Chemicals, USA), anhydrous magnesium sulfate (J.T. Baker, USA, 99.5%), trifluoroacetic anhydride (Sigma-Aldrich, 99%), Hydrochloric acid (Sigma-Aldrich, 37%), sodium hydroxide (Sigma-Aldrich, 99%), potassium hydroxide (Showa Kako Corp., 85.5%), Tetrahydrofuran (Sigma-Aldrich, 99.9%), N,N’-dicyclohexylcarbodiimide (Sigma-Aldrich, 99%), N-Hydroxysuccinimide (Alfa Aesar, 98%), recombinant human bone morphogenetic protein 2 (355-BM-050/CF, R&D systems, USA), and silicon wafers (Goldeninent Inc., Taiwan). Gold substrates were fabricated on a 4-inch silicon wafer by depositing a 300-Å layer of titanium followed by a 700-Å layer of gold with a thermal evaporator (Kao Duen Technology Co., Taiwan). All silicon substrates were cleaned using a piranha solution (3:1 v/v H₂SO₄:H₂O₂) before use.

To immobilize BMP-2 on the coating 5 modified surface, commercially obtained BMP-2 (355-BM-050/CF, molecular weight: 26 kDa, R&D systems, USA) was dissolved in deionized water. The concentration of BMP-2 solution used throughout this study was 50 μg ml^-1. The BMP-2 solution was dropped on the coating 5 modified surface directly after CVD polymerization, and kept at 4°C overnight. At the end of the reaction, the treated surfaces were washed by PBS solution at least three times.

Cells were osteoinduced at confluency in their appropriate media either supplemented with 50 μg/mL ascorbate (TCI A2521) and 2.5 mM β-glycerolphosphate (USB Corp Cleveland, OH) for 7 and 14 days, or 50 ng/mL BMP2 (R&D Systems 355-BM-050/CF) for 5 days. OB differentiation was assessed by alkaline phosphatase (Thermo NBT/BCIP 1-step 34042) and alizarin red staining. Crystal violet stain (Sigma C3886) was used at 0.5% to determine total cell count as previously described (Shares et al., 2018 (link)).