重组人骨形态发生蛋白2/骨修复材料的制备与性能

Preparation and properties of recombinant human bone morphogenetic protein-2/bone repair material

背景:制备具有天然骨结构,能够结合生物因子的骨修复材料成为研究的热点。目的:制备重组人骨形态发生蛋白2/骨修复材料,评估其释放、活性及诱导异位成骨的能力。方法:首先制备含有胶原结合域的重组人骨形态发生蛋白2,然后将胶原结合域与骨修复材料中的胶原相结合,经过冷冻干燥制备重组人骨形态发生蛋白2/骨修复材料,采用ELISA法检测重组人骨形态发生蛋白2的体外释放情况。将C2C12细胞滴加到重组人骨形态发生蛋白2/骨修复材料上(重组人骨形态发生蛋白2剂量分别为0.25,0.5,1μg/块),72 h后检测细胞碱性磷酸酶活性。将含0,2,5,10μg重组人骨形态发生蛋白2的重组人骨形态发生蛋白2/骨修复材料分别埋置于SD大鼠肌肉内,埋置2,4周后,检测磷酸酶活性与新骨生长情况。将CY7标记的重组人骨形态发生蛋白2/骨修复材料埋置于SD大鼠后肢肌肉窝内,观察重组人骨形态发生蛋白2/骨修复材料的稳定性。结果与结论:重组人骨形态发生蛋白2/骨修复材料内的重组人骨形态发生蛋白2在45 d内基本没有释放;C2C12细胞的碱性磷酸酶活性随复合材料中重组人骨形态发生蛋白2剂量的升高而升高;重组人骨形态发生蛋白2/骨修复材料植入体内的稳定性较好,碱性磷酸酶活性、异位成骨随重组人骨形态发生蛋白2剂量的增加而升高。结果表明,重组人骨形态发生蛋白2/骨修复材料保证了重组人骨形态发生蛋白2的稳定性,使其不易释放,提高了其异位诱导成骨能力。

BACKGROUND： It has become a hotspot to prepare the bone repair material that exhibits natural bone structure and is used in combination with biological factors. OBJECTIVE： To prepare the recombinant human bone morphogenetic protein-2（rh BMP-2）/bone repair material, and to evaluate its capacities of release, activity and ectopic osteoinduction. METHODS： A collagen-binding domain was added to the N-terminal of native rh BMP-2 that allowed bind to collagens in the bone repair material. Then, rh BMP-2/bone repair material was obtained through freeze-dried method. The releasing ability of rh BMP-2 in vitro was assayed by ELISA. C2C12 cell lines were loaded to the composite material with 0.25, 0.5 and 1 μg rh BMP-2, respectively. Afterwards, alkaline phosphatase activity was detected at 72 hours. The composite materials with 0, 2, 5 and 10 μg rh BMP-2 were implanted into the quadriceps of Sprague-Dawley rats, respectively. Alkaline phosphatase activity and the newly formed bone were detected at 2 and 4 weeks after implantation. The CY-7-labeled composite material was implanted into the quadriceps of Sprague-Dawley rats to observe its stability. RESULTS AND CONCLUSION： Substantially no rhB MP-2 from the rh BMP-2/bone repair material was released within 45 days. The alkaline phosphatase activity of C2C12 was in a rise with the increased concentration of rhB MP-2. The stability of the composite material in vivo was better, the alkaline phosphatase activity and ectopic bone formation increased as the concentration of rh BMP-2 rose. To conclude, the rh BMP-2/bone repair material preserves the stability of rh BMP-2, and improves ectopic osteoinduction ability.