Methods

Alg + Ca was synthesized using the EDC(1-ethyl-3-(3′-dimethylaminopropyl)carbodiimide (EDC)/N-hydroxysuccinimide (NHS) coupling reaction, as described in a previous study [20]. Briefly, sodium alginate (1 g; ~ 5 mmol; Sigma-Aldrich, A2033-100G) was dissolved in 100 mL of 0.1 M MES buffer (pH 5.2). EDC (5 mmol) and NHS (5 mmol) were then added dropwise to the alginate solution. After 5 min of nitrogen bubbling, dopamine hydrochloride (5 mmol) (Alfa Aesar, Ward Hill, MA, USA, A11136-25G) dissolved in 3 mL MES (0.1 M, pH 5.2) was added dropwise to the alginate/EDC/NHS solution. The reaction was allowed to proceed for 1 h at room temperature. After the reaction, the mixture was dialyzed against (3.5 kDa MWCO dialysis membrane) 5 L of deionized water (pH 6) and 20 g of NaCl for 3 days. Subsequently, the mixture was dialyzed against pure deionized water for 4 h and lyophilized for 3 days (Fig. 1). The as-synthesized alginate-catechol was confirmed using ¹H NMR spectroscopy (AVANCE III 500, Bruker, Billerica, MA, USA). The degree of substitution (DOS) of catechol in alginate was determined by measuring the absorbance at 280 nm (A₂₈₀) using a UV–Vis spectrophotometer (UV-1800, Shimadzu, Japan). Various concentrations (0.02–0.1 mg/mL) of dopamine hydrochloride were used to plot a standard curve for DOS calculation.

Synthesis and chemical structure of alginate-catechol

A 3D printer (Invivo 4D2; ROKIT Healthcare, Seoul, Korea) was used to print the PLLA scaffold. The scaffold was printed with a thickness of 1 mm and diameter of 5 mm. PLLA plastic was extruded at a fixed temperature of 200 °C. For all supports, the speed control was set at 3 mm/s, ejection pressure was 200–300 kPa, and support density was 15%. The porosity (%) was calculated using the following equation.

The morphology of the 3D-printed PLLA scaffold was observed using SEM (Tescan MIRA3, Brno, Czech Republic) after coating with a thin film of gold (Quorum Q150T ES type, FEI, Hillsboro, OR, USA) at the Daegu Gyeongbuk Medical Innovation Foundation (DGMIF). The floor conditions were voltage, 10.0 kV; height, 9.1 mm; and magnification, 50×. The section conditions were voltage, 10.0 kV; height, 12.5 mm; magnification, 100×. The side-view conditions were voltage, 10.0 kV; height, 9.1 mm; magnification, 70×.

The PLLA scaffold was coated with an Alg + Ca solution (0.5 mg/mL) on a rocker (50 rpm) at room temperature for 24 h. Next, the Alg + Ca–coated PLLA scaffold was coated with rhBMP-2 by immersion in low concentration (L group; 1 µg/mL) and high concentration (H group; 2 µg/mL) at 4 °C for 24 h. After BMP-2 coating, the scaffolds were coated with collagen via immersion in collagen type 1 (3 mg/mL).

Sequential coating of Alg + Ca, rhBMP-2, and collagen onto the PLLA scaffold was performed twice to form multiple layers of coating. The coated samples were then freeze-dried overnight. The coating procedure is illustrated in Fig. 2.

Sequential coating of collagen and BMP-2 on the surface of the PLLA scaffold. Binding between PLLA, Alg + Ca, collagen, and BMP-2