2.3.3. In Vitro rhPDGF-BB and rhBMP-2 Release

To determine the releases of rhPDGF-BB and rhBMP-2 from PDGF/Hepa/Ti, BMP/Hepa/Ti, and PDGF/BMP/Hepa/Ti, a prepared disc was placed in a 15 mL conical tube (Falcon, North Haledon, NJ, USA) containing PBS buffer (pH 7.4) at 37 ◦C with 100 rpm. At predetermined times of 1 h, 3, 6, and 10 h, and 1, 3, 5, 7, 10, 14, 21, and 28 days, supernatants were collected and buffer was replenished with an equal volume of fresh PBS. Amounts of rhPDGF-BB and rhBMP-2 released were determined using an enzyme-linked immunosorbent assay kit (ELISA), according to the manufacturer's instruction using a Varioskan Flash Multimode Reader (Thermo Fisher Scientific, Waltham, MA, USA) at 450 nm.
