缓释型重组人骨形态发生蛋白2/壳聚糖生物骨修复材料诱导骨形成

背景：重组人骨形态发生蛋白2在体内半衰期短、易降解代谢，达不到理想的骨再生效果。 目的：制备缓释型重组人骨形态发生蛋白2/壳聚糖生物骨修复材料，并观察其缓释性能、骨诱导活性。 方法：将重组人骨形态发生蛋白2与壳聚糖混合制备壳聚糖膜，涂覆于生物骨修复材料表面，ELISA方法检测其体外释药性能。茜素红染色检测缓释型人骨形态发生蛋白2/壳聚糖生物骨材料、重组人骨形态发生蛋白2生物骨材料、单纯骨填充材料诱导C2C12细胞骨钙蛋白的形成，观察其诱导成骨细胞能力。同时将3种骨修复材料植入清洁级KM小鼠股部肌袋内，2周后检测新生骨Ca2+离子含量，评价其异位骨诱导能力。 结果与结论：材料表面的壳聚糖膜分布均匀，负载的重组人骨形态发生蛋白2呈团簇状。重组人骨形态发生蛋白2/壳聚糖生物骨修复材料体外释药存在突释，前4 d释放量达总药量的50%，持续至12 d，释药量达到90%，第18天时释放完全。与单纯骨填充材料、重组人骨形态发生蛋白2生物骨材料相比，缓释型人骨形态发生蛋白2/壳聚糖生物骨修复材料诱导C2C12细胞向成骨晚期分化能力与异位骨形成能力显著增强(P < 0.05)。结果提示缓释型人骨形态发生蛋白2/壳聚糖生物骨修复材料缓释性能好，促进骨形成能力强。

Enhancement of bone formation by recombinant human bone morphogenetic protein-2/chitosan bone biomaterials

BACKGROUND:The recombinant human bone morphogenetic protein-2 (rhBMP-2) administered in solution often loses its bioactivity in a short time and does not always exhibit efficacy required in bone regeneration in vivo. It would be ideal to develop a system for the sustained delivery of biologically active rhBMP-2 over an extended period of time. OBJECTIVE:To evaluate rhBMP-2 sustained delivery and osteoinductive effects of bone biomaterials which were combined with chitosan films containing rhBMP-2. METHODS:①The rhBMP-2 was added into a chitosan solution to prepare chitosan films which were spread on the bone materials. The effect of slow release was analyzed by ELISA assays. ②In vitro, the osteoinductive to differentiation marker (osteocalcin) of C2C12 cells after induction of rhBMP-2/chitosan (CS) bone biomaterials was assayed by Alizarin red staining.  ③Intramuscular implantation test was made in vivo, rhBMP-2/CS carriers were implanted in muscles of the hind leg of rat for two weeks. The amount of calcium deposited in the implants was measured to assay bioactivity. RESULTS AND CONCLUSION: ①A scanning electron micrograph of the rhBMP-2/CS bone biomaterials showed uniform distribution of chitosan films on the material surface with rhBMP-2 scattering like cluster. ②An abrupt rhBMP-2 release from rhBMP-2/CS carriers was observed. 50% of the loaded rhBMP-2 was released in 4 days. Thereafter, the release rate was nearly constant until day 12 when 90% of rhBMP-2 was released. All of it was released by day 18 at last. ③In vitro rhBMP-2 released from the rhBMP-2/CS carriers stimulated an increase in osteocalcin of C2C12 cells after 4 weeks. ④In vivo bone formation studies showed the rhBMP-2/CS biomaterials induced bone formation to a much greater extent than control groups by Ca²⁺ test assay. These results demonstrate that the rhBMP-2/CS bone biomaterials delivery system is capable of potentiating the osteogenic efficacy of rhBMP-2 and underscore its importance as a possible bone regeneration strategy.