高强度镁合金/纳米银复合膜在引导骨再生中成骨效应和抗菌性能的基础研究

Current available guided bone regeneration membranes (GBRMs) are not yet ideal. Absorbable magnesium (Mg) alloys have high strength and good osteogenic properties. Silver nanoparticles are good in antibacterial properties and are not easy to induce bacterial resistance. Combined application of magnesium alloy and silver nanoparticles can produce synergistic effects in enhancing osteogenesis and antibacterial effects. Silver nanoparticles may also promote the osteogenic activity of magnesium based biomaterial through osteoimmunomodulatory response. Biodegradable polymer coating can be loaded or grafted with nanomaterials, and can also slow down the degradation of magnesium alloys. Based on all the above, a Mg alloy/ nano silver composite membrane, which is high in strength, easy to shape, absorbable, and antibacterial, will be fabricated. Porous Mg alloys matrix will be prepared, and filled with biodegradable polymer, inside the pores and on the one side. Then, a second nano silver coating will be prepared on this polymer coating by loading silver nanoparticles into biopolymers and dip coating. Finally, a nano silver layer will be prepared on the nano silver coating by layer-by-layer assembly. The Mg side is designed to face bone defect and promote bone regeneration, while the polymer/ nano silver side is designed to barrier soft tissue and antibacterial. The effects of Mg alloy pore size, the content of polymer and silver nanoparticles on the properties of composite membrane will be evaluated. In vitro cell assay, in vivo animal test and antibacterial tests will be performed to evaluate the effects of composite membrane on osteogenesis and antibacterial. Related signaling pathway which involves in the osteogenic properties of Mg alloy and synergistic effects of Mg alloy and silver nanoparticles will also be explored. The results will provide new sights in designing GBRM and will also be helpful for further investigation of the underneath mechanisms of Mg alloy with silver nanoparticles in osteogenesis.

现有引导骨再生膜尚不够理想。镁合金可吸收、强度高且有成骨活性。纳米银可抗菌且不易产生细菌耐药。联合应用镁合金和纳米银还可产生协同作用增强成骨和抗菌效能。纳米银也可能通过对成骨免疫的调节，进而促进镁基生物材料的成骨活性。可降解高分子聚合物可用于制备涂层负载或接枝纳米银，还可以减缓镁合金的降解。基于此，本项目拟制备一种高强度、易塑形、可吸收且可抗菌的镁合金/纳米银复合膜。以多孔镁合金为基体，聚合物充填其内部孔隙并作为涂层包裹一侧，且作为纳米银的载体在聚合物涂层上做纳米银层并再接枝纳米银。以镁侧接触骨缺损促进成骨，聚合物/纳米银侧接触软组织发挥屏障和抗菌功能。研究孔隙尺寸、聚合物及纳米银含量对膜的影响，以细胞实验、动物实验和抑菌实验评价膜在体内外的成骨、抑菌效能以及镁/纳米银的协同作用和相关机制通路。研究结果将为引导骨再生膜的设计提供新思路，并为镁合金/纳米银在骨再生中的联合应用提供重要基础。

由于抗菌性差、成骨诱导活性和骨免疫调节作用弱，现有可吸收引导骨再生膜尚不理想。本研究使用镁合金、纳米银（AgNPs）和可吸收高分子聚合物，制备具有成骨诱导活性和成骨免疫调节作用的抗菌可吸收膜，以实现更理想的骨缺损修复。首先在镁合金（Mg3Gd）薄膜表面制备壳聚糖涂层（CS-Mg），发现涂层能降低镁合金的降解速率、改善生物相容性和成骨诱导活性并在体内促进骨修复。然而，CS-Mg机械强度过高，直接裁剪难以匹配复杂的骨缺损形态，产生的锐利边缘易再次损伤周围组织，因此，需对膜进行改良。通过3D打印技术，制备一侧致密另一侧多孔的聚己内酯膜（PCL）；添加镁合金粉末可打印含Mg的复合膜，细胞毒性实验筛选最佳添加比例（3%体积分数),记为PCL-3Mg；通过层层自组装法（LBL)在PCL上接枝AgNPs，细胞毒性实验和抗菌实验筛选最佳接枝层数(3层)，记为PCL-3Ag；在PCL-3Mg上接枝3层AgNPs，记为PCL-Mg-Ag。我们发现以上4种膜均易于裁剪和塑形。与PCL相比，PCL-Mg-Ag杨氏模量增加，能促进大鼠骨髓间充质干细胞（rBMSCs）的增殖。与PCL和PCL-3Mg相比，PCL-3Ag和PCL-Mg-Ag对金黄色葡萄球菌和大肠杆菌有良好抗菌性，但二者间差异不大，提示在本研究中未观察到Mg对膜的抗菌性产生协同效应。与PCL和PCL-3Ag相比，PCL-3Mg和PCL-Mg-Ag能同时上调rBMSCs成骨相关基因（ALP、Col-1、RUNX2）以及Mg2+通道激活相关基因（MagT1）的表达，说明PCL-3Mg和PCL-Mg-Ag可通过释放Mg2+激活下游信号通路以促进rBMSCs的成骨向分化。但上述基因的表达在PCL-Mg和PCL-Mg-Ag间无明显差异，提示在本研究中Ag对膜的成骨可能无直接影响。此外，与PCL-3Mg相比，PCL-Mg-Ag能促进巨噬细胞M2极化，与巨噬细胞共培养后，能促进rBMSCs的成骨向分化，说明复合膜中的Ag可通过免疫调节促进成骨。最后，动物实验结果显示，在第4周，PCL-Mg-Ag具有最佳的骨修复效果。综上，本研究应用3D打印技术结合层层自组装法制备抗菌PCL-Mg-Ag膜，通过激活Mg2+相关信号通路并调控巨噬细胞M2极化，从而直接和间接促进干细胞成骨向分化，最终实现更理想的骨缺损，为引导骨再生膜的设计提供新思路。