PMC:4137170 / 6762-10443 JSONTXT

{"project":"2_test","denotations":[{"id":"24928668-19993-61779598","span":{"begin":1169,"end":1171},"obj":"19993"},{"id":"24928668-2547836-61779599","span":{"begin":3677,"end":3679},"obj":"2547836"}],"text":"Injectable rhBMP-2 carrier fabrication and an rhBMP-2 release test\n\nPorous β-tricalcium phosphate microsphere\nThe porous β-TCP (Cerectron Co., Kimpo, South Korea) microspheres were prepared by the spray-dry method. The resulting spherical particles were subsequently sintered at 1,250 °C for 2 h, resulting in diameters ranging from 45 to 75 μm (Fig. 1). The β-TCP that was sintered at high temperature had a porosity of 59.3 %, as measured with a mercury porosimeter.\nFig. 1 SEM images of β-TCP microspheres generated by the spray-dry method after sintering at 1,250 °C\n\nHyaluronic acid (HA)-based powder gel\nThe powder gel was prepared to facilitate the loading of rhBMP-2 and to improve the fixation force of the carrier at the site of a bone defect by absorbing the blood and body fluids generated at the defect site. The powder gel was a cross-linked hyaluronic acid-based hydrogel consisting of HA (Bioland Co., Ochang, South Korea) with a high molecular weight of 3 million Daltons. This HA-based hydrogel was prepared through cross-linking by adding butanediol diglycidyl ether (Sigma-Aldrich, St. Louis, USA), a cross-linking agent, to 2.7 wt% of HA solution [17]. The remaining reagents were removed without resuspending by dialysis against 1X PBS (Sigma-Aldrich, USA) for 5 days. The resulting HA-based hydrogel was lyophilized for 4 days and then ground and sieved to yield a gel powder with particles less than 100 µm in diameter.\n\nCarrier preparation\nConformational change of the rhBMP-2 due to sterilization was avoided by mixing the rhBMP-2 solution with a powder gel composite to a final concentration of 1 mg/ml rhBMP-2. The in situ mixing process is a method used to generate an rhBMP-2 loaded injectable carrier by mixing the powder gel and the porous spherical particles for loading rhBMP-2.\nIn other words, one syringe contained porous β-TCP microspheres and the other syringe held the HA-based powder-gel containing 1 mg/ml rhBMP-2, and the relative weight ratio of the materials was 1:9. These two syringes were interconnected through a 2-way connector and 30 cycles of piston movement of each syringe were performed to ensure that the mixture was homogeneous. Collectively, β-TCP microspheres and HA were used as an rhBMP-2 carrier in this study.\n\nAnalysis of the physical properties of the HA-based powder gel\nThe reticular microstructure of the HA-based powder gel was induced by cross-linking and analyzed using a scanning electron microscope (SEM, S-4700, Hitachi, Tokyo, Japan). The swelling properties of the HA-based powder gel were measured by incubating it in PBS at room temperature. The swelling ratio was measured by comparing the change in the wet weight of the hydrogel before and after 3 days of incubation. The percentage of water absorbed (Wa) was calculated by the following formula: Swelling ratio (%) = ((Ww-Wi)/Wi) X 100 % (Ww: wet weight of hydrogel; Wi: initial weight of hydrogel).\n\nrhBMP-2 release rate study\nThe release rate for rhBMP-2 was evaluated by impregnating rhBMP-2 into the hyaluronic acid-based powder gel composite. The experiment was carried out by impregnating rhBMP-2 into the composite and then analyzing the rhBMP-2 release, as detected by an antigen-antibody reaction and ELISA analysis. A collagen type I sponge (Bioland, Ochang, South Korea), which is currently used as a periodontal tissue regeneration-inducing agent in dentistry, was impregnated with 100 µg rhBMP-2 and used for comparison. The release patterns from the sponge and the composite were analyzed following treatment with hyaluronidase (100 units/ml) and collagenase (20 CDU/ml) (Sigma, USA) for 7 days, and incubation at 37 °C in PBS [18, 19]."}