具有调控炎性反应能力的钛基复合植入材料及其在组织再生过程的作用机理

It is accepted that angiogenesis is critical for osteogenesis after implanting materials. In fact, natural angiogenesis and revascularization in wound healing depend on pro-inflammatory phenotype by macrophage M1 and then anti- inflammatory phenotype by macrophage M2. According to the natural sequential events, this project develops an idea that biomaterials stimulate vascularization and tissue regeneration by modulating inflammatory events. The novel titanium implants will be prepared by surface modification based on this idea. Layer-by-layer self assembly and sol-gel routine will be used to preparing multilayers and gel layers on titanium surfaces. The activating factor to M2 distributed in the middle-down-layer will release after inflammatory response and stimulate M2 to secrete cytokines and chemotactic factors. For samples used in macrophage culture, the activating factor to M1 will be located at the up-layer besides the M2 factor in the middle-down-layer. At the first stage, the materials iwill initiate inflammatory and then induce M1 phenotype which secrete inflammatory factors and some pro-angiogenesis factors. At the second stage, the activating factor to M2 will release. The release kinetics and dose will be controlled by the molecular weights, cross ratios of the components and the distribution of loaded factors in the layers. The in vivo experiment combining with macrophage culture will be used to study the behavior and mechanism of inflammatory response modulated by the materials, and subsequently revascularization and tissue regeneration. The novel titanium implants will be obtained. This work will also take a new direction for development of biomaterials.

材料植入体内后骨组织再生过程中新血管生长的重要性已广为接受。而创伤愈合过程自然发生的血管化则依赖于炎性刺激下的巨噬细胞M1促炎-巨噬细胞M2抑炎序列事件。本项目参照此自然过程，提出材料调控炎性历程促血管化及组织再生的概念，由此制备新型表面改性医用钛。以层层自组装和溶胶凝胶法制备钛表面多层膜和凝胶层，膜层中下区分布激活M2的生物因子，在抑炎阶段释放，引发M2分泌血管化所需细胞因子和趋化因子。为机理研究，对体外细胞培养试样，膜层上部携载激活M1的生物因子，中下区载促M2激活的因子。该材料在细胞培养初始引发炎性反应，激活M1，分泌炎性因子和部分促血管化因子；第二时段分泌激活M2的因子。通过膜层组分的分子量、交联度、因子的空间分布实现因子阶段性释放。巨噬细胞共培养结合体内植入试验，研究材料通过调控炎性反应促进血管化及组织再生的规律和理论基础。由此研制相关钛基骨植入材料。为生物材料研发开辟一新方向。

自然的创伤愈合过程必然经历促炎-抑炎两个阶段。在促炎阶段M1型巨噬细胞等免疫细胞会产生诱导新生血管的生物因子，但若此阶段过长则产生慢性炎症，因此必须适时转化为抑炎型或称愈合型巨噬细胞M2，这也是组织修复的必要条件。材料植入人体后组织再生同样历经创伤-愈合过程。本项目由此提出材料调控炎性历程促血管化及组织再生的概念，设计具有时序性调控巨噬细胞表型转变的新型表面改性医用钛。制备了多种携载炎性因子的溶胶凝胶涂层和多层组装层。基于涂层组分及其分子量和交联度等，调节其降解速率。并结合生物因子在涂层中的空间分布，调控M1或M2的激活因子适时释放。考察了改性层的理化性能，在细胞、蛋白质和基因水平评价了体内外生物学性能。结果表明所制备的钛表面改性层具有设计所预期性能。在材料释放炎性因子或含炎性因子培养基所产生的炎性微环境，初期(3-5天) 主要形成促炎型巨噬细胞M1，释放血管新生所需生物因子。其后明显释放的抑炎因子诱导M1转变为M2抑炎表型，进入抑炎-愈合阶段。由此，这类表面改性材料能通过调控炎性响应促进组织再生与修复。结合多种表面改性扩展了骨免疫研究。通过材料与巨噬细胞、间充质干细胞、内皮细胞共培养及体内植入实验，发现携载炎性因子再结合粘附性多肽、钛亲和肽和抗菌化改性的钛表面，不仅能时序性调控M1-M2表型转化，同时促进了干细胞成骨分化和内皮细胞的成血管化行为，通过材料的免疫调节能力和诱导功能协同增强了组织修复能力。其中所制备的载银钛表面具有极低的银释放量，表现出良好的生物相容性和抗菌性。也开展了经皮器械的炎性响应与生物密封行为。发现纳米化改性钛表面经皮植入时初期的炎性反应有利于血管化因子分泌，并能适时进入炎性消退期，组织愈合形成良好的生物密封。