含重组人骨形态发生蛋白2复合材料的制备及体内外成骨实验研究

Preparation of the Composites Loaded with Recombinant Human Bone Morphogenetic Protein 2 and Evaluation of the Osteogenic Activity in vitro and in vivo

将重组人骨形态发生蛋白2(Recombinant human bone morphogenetic protein-2,rh BMP-2)负载于所制备的明胶/羟基磷灰石/卵磷脂复合材料中,评价其体内外诱导成骨的能力。利用冷冻干燥技术,制备出负载rh BMP-2的明胶/羟基磷灰石/卵磷脂复合支架材料,通过体外细胞实验,评价复合材料的毒性及体外诱导成骨活性;建立小鼠肌袋植入模型,24只动物分4组处理,空白组做假手术,其余3组分别植入空白复合材料、负载rh BMP-2的复合材料或直接注射rhBMP-2溶液,术后4 w评价复合材料体内的异位成骨能力。结果显示,复合材料在电镜下呈多孔海绵状,孔隙直径平均为2050μm,孔间互相连通。L929细胞与复合材料培养1,3,5 d后细胞相对增殖率均在90%以上,说明材料无细胞毒作用;体外成骨诱导3 d后,C2C12细胞中碱性磷酸酶活性显著高于对照组(P<0.05),且随rh BMP-2浓度的增加,表现出剂量依赖性升高。术后4 w X-射线及大体观察发现,rh BMP-2溶液组、负载rh BMP-2复合材料组有骨样物质形成,且负载rh BMP-2复合材料组血液中碱性磷酸酶活力显著高于其余3组(P<0.05);组织学观察发现,负载rh BMP-2复合材料组的骨小梁数量、厚度以及胶原纤维生成量明显优于rh BMP-2溶液组,展现出良好的异位成骨能力。研究结果表明制备的负载rh BMP-2的明胶/羟基磷灰石/卵磷脂复合材料具有良好的生物相容性及骨诱导性,可望作为骨组织工程支架用于修复骨损伤、促进骨愈合。

Recombinant human bone morphogenetic protein 2(rh BMP-2)was loaded into the prepared composite consisting of gelatin/hydroxyapatite/lecithin,and its ability to induce bone formation was evaluated in vivo and in vitro.The freeze-drying technique was used to prepare the gelatin/hydroxyapatite/lecithin composite loaded with rh BMP-2,and the toxicity and osteogenic activity of the composites were evaluated by cell experiment in vitro;the muscle bag implantation model was established,and 24 animals were divided into four groups.The blank group was treated by sham surgery,and the other three groups were implanted with blank composite,composite loaded with rh BMP-2,or directly injected with rh BMP-2 solution,respectly.The heterotopic osteogenesis ability of the composite material in vivo was evaluated 4 weeks after surgery.The results showed that the composite was spongy with an average diameter of 20~50μm under the electron microscope.The RGR of L929 cells cultured with the composite for 1,3 and 5 days was above 90%,indicating that the composite had no cytotoxic effect.After 3 days induction of osteogenesis,alkaline phosphatase activity in C2 C12 cells was significantly higher than that in the control group(P<0.05),and showed dose dependence with the increase of rh BMP-2 concentration.The mice were dissected after 4 weeks,and X-ray and gross observation showed that bone like tissue was formed in the rh BMP-2 solution group and the composite loaded with rh BMP-2 group,and the activity of alkaline phosphatase in blood of the composite loaded with rh BMP-2 group was significantly higher than that of the other three groups(P<0.05).Histological observation showed that the number and thickness of bone trabeculae and the production of collagen fibers in the composite loaded with rh BMP-2 group were significantly better than those in the rh BMP-2 solution group,showing good ectopic osteogenesis ability.The results showed that the prepared gelatin/hydroxyapatite/lecithin composite loaded with rh BMP-2 had good biocompatibility and bone inductivity,and it is expected to repair bone injury and promote bone healing as a kind of bone scaffold material.