时序释放白介素4和锌促进种植体早期骨结合的机理研究

Regulating cell responses at inflammatory phase and repair phase after implant insertion can affect early osseointegration. During the inflammatory phase, macrophages timely change to M2 polarization status timely can resolve inflammation effectively. Bone marrow mesenchymal stem cells (bMSCs) are the chief cells which play an important role in the repair phase. Aiming at exert the time sequential functions of macrophages and bMSCs, the present project observe the effects of time sequential release of IL-4 and zinc, in vivo and in vitro osteogenic effects and study the underlying osteogenesis-related mechanisms..(1) We first prepared TiO2 nanotube, then loaded zinc onto the nanostructure. After that, IL-4 was further loaded to the zinc-loaded TiO2 nanotube layer to form the combined IL-4/Zn/TiO2 nanotube composite, through combining the phase transited lysozyme (PTL) and a layer-by-layer (LbL) self-assembly technique. In this way, the time sequential release of IL-4 and zinc is achieved. .(2) We further employed in vitro experiment to do the following researches:.① We explore the mechanisms related to IL-4 inhibiting the histone de-SUMO enzyme SENP1 mediated IFN-γ-JAK-STAT1 pathway and activating the STAT3-SOCS3 pathway. We further elucidate the epigenetic mechanisms of macrophage polarization which improve early osseointegration of implant. .② We aim to study how zinc-loaded TiO2 nanotube inhibit NF-κB pathway and activate Wnt/β-catenin pathway, so as to induce bMSCs osteogenesis, and illuminate the mechanisms..(3) In addition, an in vivo rat model was used to prove the effect of time sequential release of IL-4 and zinc to improve the early osseointegration of implant. .This project is hopeful to found the theoretic basis and technical support for clinical purpose.

调控种植体植入后炎症期、修复期的细胞反应可影响早期骨结合。炎症期中巨噬细胞及时向M2型转化有效消退炎症；修复期中骨髓间充质干细胞是促进成骨的关键细胞。针对这两种细胞在骨结合中的作用和起效时间，本项目研究按先后次序释放白介素4和锌，观察体内、体外早期成骨效应，并探讨相关成骨机理。(1)在载锌TiO2纳米管上涂覆溶菌酶相转化产物和白介素4的组装层，实现白介素4和锌按时间次序释放。(2)通过体外细胞实验①研究白介素4通过抑制组蛋白去SUMO化酶SENP1介导的IFN-γ-JAK-STAT1通路，激活STAT3-SOCS3通路调控巨噬细胞M2极化的表观遗传学机制。②研究载锌TiO2纳米管通过抑制NF-κB通路、激活Wnt/β-catenin通路诱导bMSCs成骨分化的机制。(3)采用体内实验证实时序释放白介素4和锌促进骨结合的效果。本项目为临床促进种植体早期成骨提供理论依据和技术支持。

调控种植体植入后炎症期、修复期的细胞反应可影响早期骨结合。炎症期中巨噬细胞及时向M2型转化有效消退炎症，营造促成骨微环境，促进修复期成骨细胞分化，加速种植体骨结合。本项目研究按先后次序释放白介素4和锌，观察体内、体外早期成骨效应并进行机制研究。项目分两部分：第一部分为二氧化钛纳米管表面载白介素4光热控释涂层促进种植体早期骨结合的研究。将IL-4在第3天加入至用RANKL诱导的小鼠巨噬细胞中，可下调破骨相关标志物Rankl、Trap和Mmp9基因表达、上调促成骨基因Opg表达，促进成骨。据此构建二氧化钛纳米管表面载白介素4光热控释涂层，在近红外光照射下，实现IL-4第3天大量释放，大鼠体内植入实验显示，植入后7天，载白介素4光热涂层组可降低炎性细胞、抗酒石酸酸性磷酸酶（TRAP）阳性破骨细胞和诱导型一氧化氮合酶（iNOS）阳性M1型巨噬细胞数目，增加CD163阳性M2型巨噬细胞数目；植入后28天，载白介素4光热涂层组显著促进骨结合。在RANKL诱导的巨噬细胞中添加IL-4后的基因芯片和生物信息学分析显示，差异基因表达谱包含332个下调基因和443个上调基因。GOterm的生物学过程分析中，JNK级联的正调节，磷脂酰肌醇3-激酶活性的正调节，破骨细胞分化的正调节等与破骨功能密切相关的信号通路下调，提示IL-4通过抑制破骨，调控骨稳态向成骨方向倾斜、促进骨结合。第二部分为载锌二氧化钛纳米管涂层调控免疫微环境促进成骨的研究。适当管径的载锌纳米涂层（15VZn、25VZn组）中锌的缓释达21天以上且释放曲线平稳，该涂层可促进成骨细胞增殖、下调M1促炎因子（IL-6、TNF-α），上调M2促修复因子（TGF-β、HO-1）基因和蛋白表达。在巨噬细胞与成骨细胞共培养条件下，涂层可促进成骨细胞增殖、上调成骨相关基因（ALP、Opn、Col1a1、Runx-2）表达、增强ALP分泌、促进早期成骨分化和细胞外基质矿化。在体外模拟炎症条件下种植体植入和种植体植入后发生炎症两种情况下，载锌纳米涂层均有效抑制成骨细胞中P38和P65的磷酸化，阻断NF-κB信号通路的激活，减轻炎症反应，促进骨结合。本项目为促进种植体早期骨结合提供理论依据和技术支持。