具有成骨诱导效应的可降解个体化仿生椎间融合器实验研究

The development of individual biodegradable bionic cage with osteogenic induction is the most desirable bone substitute of iliac bone for intervertebral fusion and further improvement of the fusion rate. In this experimental study, a brand-new type of biodegradable medical magnesium-zinc alloy with independent intellectual property rights was employed, and the individual simulation cage completely fitting for the size of goat cervical intervertebral space with porous inner environment suitable for seed cell growth and ideal toughness and stiffness was produced by 3D printing technology. The BMP-2 adenoviral vector was prepared with pAd/CMA/V5-Dest system, and then the BMP-2 expression in the mRNA level and protein level was determined by realtime PCR and immunofluorescence. The novel individual goat bionic cage with osteogenic induction was developed with simulation cage compound BMSC seed cells transfected by BMP-2 adenoviral vector, which enabled the sustained and stable release of BMP-2. After the implantation of this novel cage into the experimental goat cervical spine, the intervertebral fusion and cage biodegradation process will be closely observed in order to determine the osteogenic potential and degradation law of bionic cage. Molecular biological analyses will be employed to determine Runx2/Cbfa1, alpha 2 beta 1, alkaline phosphatase and type I collagen mRNA expression in osteoblasts, and observe the effects of degradation products on the adhesion, differentiation and proliferation of the osteoblasts. According to all the above researches, the laws of bone bearing, fusion and degradation of the novel cage can be explored in the molecular level, and the novel individual biodegradable bionic cage with osteogenic induction could be developed. This study could lay the solid foundation of the clinical use of novel bionic cage for intervertebral fusion.

研制具有成骨诱导效应的可降解个体化仿生椎间融合器是替代自体髂骨用于椎间融合及进一步提高植骨融合率的理想方法。本研究首先以自主研发的新型可生物降解医用镁锌合金为材料，运用3D打印技术制作既拥有促进种子细胞生长多孔环境、又完全适合山羊颈椎椎间隙形态大小的个体化仿真椎间融合器，其次构建含骨生长因子BMP2基因腺病毒载体，转染骨髓间充质干细胞BMSCs后，与仿真椎间融合器复合制备具有成骨诱导效应的新型可降解个体化仿生椎间融合器，最后植入山羊颈椎观察椎间骨融合效率及融合器降解过程，明确仿生椎间融合器的成骨能力及体内降解规律，通过测定成骨细胞Runx2/Cbfa1、α2β1、碱性磷酸酶和I型胶原mRNA表达方法，观察降解产物对成骨细胞黏附、分化和增殖的影响，从分子水平探明新型仿生融合器的骨承载、融合和降解规律，为新一代具有成骨诱导效应的个体化可降解仿生椎间融合器的临床实际应用打下坚实基础。

在颈椎前路椎间盘切除植骨融合手术中，融合器的植入对于恢复间隙高度及稳定性具有至关重要的作用。临床上使用以聚醚乙酮（PEEK）材料制作的颈椎融合器仍存在较大缺陷，如无诱导成骨效应，骨愈合所需时间长，椎间融合后材料不能降解吸收。而且，现有的颈椎融合器均为标准定制，而椎间隙的形态与大小存在个体差异。因此，研制具有诱导成骨效应且能随时间降解的完全适合颈椎椎间隙形态大小的个体化新型仿生椎间融合器以替代自体髂骨用于椎间植骨融合，已成为脊柱外科领域研发的新热点。.本研究旨在利用可降解的高纯镁材料，通过术前CT图像定制为个体化解剖型融合器，并用MgF进行表面修饰，制作成为在植入早期可以有效提供提供支撑，在融合后可以完全降解新型颈椎融合器。.本研究融合器材料的合作方为上海交通大学材料学院金属基国家重点实验室的张小农团队，合作方负责高纯镁融合器的加工制作及表面改性的加工，合作方共制作了三种融合器。融合器A为应用高纯镁仿照市售美敦力融合器进行制作，融合器B为应用高纯镁根据山羊颈椎CT进行三维重建并制作，融合器C为融合器A在氢氟酸中处理96小时表面改性后而成。.本研究应用了24只青山羊，分为4组，A组为融合器A+钛板，B组为融合器B+钛板，C组为融合器C+钛板，D组为PEEK+钛板。手术使用颈前路入路，术后即时拍颈椎正侧位X片，术后4、12、24周拍摄颈椎CT+三维重建，以明确椎体融合及融合器降解情况，并取血检测AST、ALT、CREA及镁离子，以明确镁降解产物对肝肾功能的影响及对血清镁离子浓度的影响。术后24周处死山羊，截取融合节段，行micro-CT检查，进一步明确椎体融合及融合器降解情况，随后进行硬组织切片，进行甲苯胺蓝染色，van Gieson染色，苏木精-伊红（HE）染色和Masson染色，以观察融合器与骨交界处结合情况，新生骨小梁的形态及生长情况。.研究结论，具有MgF涂层的高纯镁融合器早期稳定，融合效果佳，具有较广阔的临床应用前景。