炎症响应型聚氨酯的构建及其对颈椎间盘置换后异位骨化抑制作用及机理研究

In order to overcome the limitation of preventing and restraining the heterotopic ossification (HO) after the cervical intervertebral disc replacement, it is proposed to prepare the inflammation response artificial cervical intervertebral disc for HO prevention. The nonsteroidal anti-inflammatory drugs (NSAIDs) ,which is commonly applied for preventing HO, will be fixed on the non-bone contact surface of the artificial cervical intervertebral disc by the amide bonds.The selective enzymatic hydrolysis effects on the amide bonds of the cholesterol esterase (CE) released by the inflammation macrophages will be utilized for the inflammation response release of the NSAIDs.Rupture of the amide bonds on surface will reduce the degradation rate in inflammatory conditions at the same time. The low temperature plasma modification, 1,1'-carbonyldiimidazole activation, and 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide hydrochloride/ N-hydroxysuccinimide activation and crosslinking methods will be used to bond the NSAIDs on the polycarbonate polyurethane, which is the non-bone contact surface material by amide bonds. The influences of the surface modification technological conditions on the drug loading and biocompatibility will be discussed. The concentration changes of CE after artificial cervical disc replacement will also be studied. The optimal drug loading quantity will be screened out. Linear regression equation of the CE concentration and release amount of NSAIDs will also be established. The molecular biology mechanism of the locally restraining HO by NSAIDs will be clarifyied. The inhibitory effect on HO will also be confirmed at tissue and moleculer level in this project. If the project complete succeefully , the serious adverse effects caused by commonly used oral NSAIDs in clinical will be overcome and the foundation of the design of controlable drug release materials by the inflammatory process will be laid.

针对预防抑制颈椎间盘置换后多发性并发症-异位骨化（HO）方法的局限性，提出将抑制HO的非甾体类抗炎药（NSAIDs）以酰胺键固定在人工颈椎间盘非骨接触面材料聚氨酯表面，利用炎症巨噬细胞释放的胆固醇酯酶（CE）对酰胺键的选择性酶解作用，构建炎症响应性释放NSAIDs的人工间盘非骨接触面外包膜用聚氨酯。同时利用膜表面大量酰胺键断裂降低材料本体在炎性环境中的降解率。采用低温等离子改性、羰基二咪唑及碳化二亚胺/羟基琥珀酰亚胺活化交联法在聚氨酯表面固定NSAIDs。探讨修饰工艺对载药量及生物相容性的影响，研究术后假体周围CE浓度变化规律，筛选最佳药物加载量，建立CE浓度与药物响应性释放间关系，阐明局部抑制HO的分子生物学效应及机理，从组织及分子水平验证术后HO的抑制效果。本申请项目如顺利实施将克服临床口服NSAIDs造成的严重副作用，实现局部高效可控释药，为设计炎症响应型可控药物释放材料奠定基础。

人工间盘置换后异位骨化（HO）具有较高的发病率，本项目针对临床预防抑制HO的局限性，提出构建炎症响应性人工颈椎间盘非骨接触面材料。基于人工颈椎间盘用 PCU膜及PCL膜，分别采用酰胺键及酯键将非甾体类抗炎药吲哚美辛（DM）以及甲硝唑接枝在PCU及PCL膜表面。探索构建炎症及感染响应表面的工艺条件，研究载药量、结构、理化性能及生物相容性; 体外研究模拟体内 CE酶浓度变化条件下药物释放规律，建立药物释放量与 CE浓度的相关性。炎症细胞模型中研究药物加载量对于炎症程度的抑制效果，确定不同炎症程度与CE酶及炎症因子浓度之间的关系。细胞模型中研究炎症对于成骨性能的影响。探索BMP-2诱导小鼠皮下及兔腿部肌袋内异位骨化模型的条件。重要结果及数据：（1）确定了在膜表面构建以酰胺键连接DM、以酯键连接甲硝唑的工艺、反应条件及载药量范围；（2）确定在PCU表面构建以酯键连接DM凝胶层的工艺及反应条件；（3）首次证明酯键对于CE酶的敏感性远高于酰胺键；（4）表面以酯键连接药物的材料在CE酶中呈现酶响应性释放规律，且在生理浓度0.05 u/mol和0.5u/mol下，药物持续释放时间可以达到50 h；（5）构建的三种表面改性材料均具有良好的细胞相容性；（6）成功构建出小鼠巨噬细胞炎症模型，确定脂多糖刺激浓度为0~1000ng/ml时，细胞分泌炎症因子呈上升趋势；（7）验证了炎症程度越高，巨噬细胞分泌CE酶越多；（8）炎症细胞模型中验证了DM的响应性释放及炎症抑制效果；（9）该炎症响应材料可通过抑制炎症从而降低前成骨细胞ALP的活力，抑制其分化；（10）BMP-2在小鼠皮下达到1.5µg/只以及在大鼠腿部肌肉中20µg/只能够刺激炎症反应，但未能诱导异位骨化。本项目的实施将克服临床口服NSAIDs造成的严重副作用，实现局部高效可控释药，为后续设计炎症响应型可控药物释放材料奠定基础。