基于"主动炎症调节"的骨组织工程技术的研究与机制探讨

Mesenchymal stem cell (MSC) based bone tissue engineering (BTE) strategy can fulfil the ever-increasing clinical need for the bone defect treatment in China. However, the major bottlenecking problem for the clinical translation of BTE is the poor cellular survival rate of tissue engineered bone graft (TEBG) after the in vivo transplantation, due to the strong inflammation reaction and lack of sufficient vascularization, which leads to the compromised defect healing efficacy. Based on our preliminary study showing that the local injection of cytokine IL-4 can help to modulate the inflammatory response, and resulted in the better defect healing. Thus we propose a novel “inflammation modulation” based bone tissue engineering strategy, where the first step aims to implant a bioactive scaffold with sustained release of IL-4 from the scaffold to modulate the inflammatory response and construct a pro-healing microenvironment for bone regeneration and the second step aims to develop a micro-sized bone tissue engineering graft（uTEBG）and deliver the uTEBG with pro-osteogenic treatment to the defect site after sufficient vascular network has been generated in order to supplement osteogenic cell pools for bone regeneration. In this study, we will develop and optimize this “inflammation modulation” BTE transplantation strategy and investigate its underlying mechanism: (1) to develop a bioactive scaffold incorporating IL-4 using 3D printing technique, and optimize the three dimensional structure of scaffold and amount of IL-4 within the scaffold; (2) to optimize the construction conditions of TEBG and investigate the optimal time point and dosage for uTEBG transplantation by injection; and (3) to investigate its underlying mechanism by comparing it with current BTE strategy at the tissue, cell and molecular levels. The development of “inflammation modulation” based new bone tissue engineering technique will help to boost up the healing efficacy of BTE technology, minimize the individual variation of BTE treatment and promote its fast clinical translation, on the other hand, it can supplement the current knowledge of BTE research and serve as a useful reference for developing tissue engineering strategies for other tissue defect treatment.

骨组织工程（BTE）技术有望满足我国骨科治疗巨大临床需求，但其临床转化瓶颈在于组织工程骨植入体内后由于剧烈炎症反应及血供不足，细胞存活率低、疗效有限。基于预实验发现适量抗炎因子IL-4注射可调节炎症反应、促进骨再生，我们首次提出基于“主动炎症调节”的BTE技术新理念：首先移植复合IL-4的功能型多孔支架材料，调节局部炎症反应，促进血管化，构建适宜再生的微环境；然后注射移植微型组织工程骨（uTEBG），促进骨再生。我们拟建立基于“主动炎症调节”BTE技术，并研究其作用机理：①应用3D打印技术制备复合IL-4的功能型支架材料，优化三维结构及IL-4载药量；②探索uTEBG最优构建条件与注射移植时间点及剂量；③与现有BTE技术对比，从组织、细胞、分子水平研究其作用机理。基于“主动炎症调节”BTE技术可提高疗效，减少个体差异，加速临床转化；是现有BTE理论的重要补充，对其他组织修复有重要借鉴意义。

证据表明骨替代材料在植入机体后的宿主的免疫反应决定了骨再生是否成功。而巨噬细胞是宿主炎症和免疫过程的核心细胞类型，因此巨噬细胞与材料的相互作用受到广泛关注。我们尝试研究基于 IL4 的精准递送主动精确调控炎症微环境，调控损伤部位巨噬细胞的极化表型，从而促进微环境中血管新生以及骨组织的修复。我们制备了四个负载梯度剂量的 IL-4 的 DBM 来探索主动、精确免疫调节策略对骨修复的影响。体内数据表明 IL-4 的正确递送可产生最优的巨噬细胞极化状态，形成促进组织愈合的微环境，从而增强下游的成骨和血管生成能力。该研究揭示了炎症调控因子与组织修复再生的量效关系，阐明了炎症调控因子稳态对炎症微环境、血管化微环境及组织再生的重要作用，为组织修复再生的局部炎症微环境的构建提供了新的数据支撑。. 此外，我们基于成骨微组织的骨组织工程修复策略可以直接利用体外构建的成骨微组织，复合支架材料作为支撑，直接移植入体内缺损部位进行节段性大段骨缺损修复。本研究中，我们设计实验比较了这种新策略与基于消化细胞的经典注射型组织工程骨修复策略。 复合成骨分化 BMSC 构建的微型组织工程骨结合 3D 打印可降解 PCL 支架，体内实验发现对比传统细胞注射技术，微型组织工程骨对骨修复再生的效果更优。. 另外，我们探究基于自体组织（血液、脂肪等）来源的高浓度复合细胞因子制剂的再生因子鸡尾酒治疗技术，阐明该制剂具有主动调控组织缺损的炎症微环境作用。其作用机制在于调节难愈性组织缺损微环境，将其由慢性炎症转化成急性炎症，从而重启组织修复。 . 通过“主动炎症调节”策略，我们实现骨组织工程中炎症微环境的研究，为领域提供新的理论基础。