大动物长段气管缺损长期功能重建的关键科学问题

Tissue engineering technique, which can regenerate cartilage with specific shape, provides a new promising strategy for solving the problem of reconstructing segmental tracheal defect. However, no breakthrough has been achieved so far for the treatment of the segmental tracheal defect in a large animal model, which apparently impeded the clinical translation of the tissue engineering tracheal (TET). According to our previous studies, the failure of regenerating tubular cartilage was mainly related to the acute inflammatory reactions mainly caused by the degradation byproduct from biodegradable scaffolds and the silicon tube used for moulding tubular cartilage. In addition, the other leading reasons that resulted in the failure of segmental tracheal defect reconstruction were mainly related to inferior blood supply, weak mechanical strength, and the inappropriate surgical strategy used in the tracheal reconstruction. To address the above-mentioned problems and the possible reasons, this project proposed a series of improved strategies from the following aspects: 1) Optimizing the strategies of in vitro cartilage regeneration to alleviate inflammation caused by the scaffolds and to realize stable regeneration of cartilage in large animals；2) Modifying in vivo pre-shaped model by using a fascia tissue to segregate the tubular TE cartilage and silicon tube together with the intramuscular implantation to promote the regeneration of vascularized tubular cartilage；3) Using polycarpolaction as the core of biodegradable scaffolds to increase the mechanical strength of TET；4) Improving reconstructive operation model and strengthening nursing care post-operation. Through the improvements of these strategies, in this project, it is expected to establish a large animal model of long-term functional reconstruction for segmental tracheal defect, which will provide a feasible strategy for the clinical translation of TET.

组织工程技术可以再生特定形态软骨，有望解决长段气管修复的临床难题，但大动物长段气管缺损的长期功能重建一直未取得突破，已成为阻碍这一技术临床转化的主要瓶颈。申请人前期研究发现，支架材料炎症反应及预塑形支撑管的异物反应是干扰大动物体内管状软骨再生的关键因素，而预构建管状软骨缺乏血供、强度不足、以及手术修复模式不当是影响重建术后长期存活的重要原因。针对上述原因，本项目拟采取以下几项改进方案：1）优化体外构建技术，避免材料炎症反应，实现大动物体内软骨再生；2）改进体内预塑形模式，通过筋膜隔离支撑管结合肌内埋植，避免异物反应并形成带稳定血供的管状软骨；3）优化支架设计，添加聚已内酯内核支架提高预构建管状软骨的力学强度；4）全面优化修复方案，加强术后护理。通过上述多种方案的有机整合，最终建立长段气管缺损长期功能重建的大动物模型，为组织工程技术修复长段气管缺损的临床转化奠定技术基础和理论依据。

组织工程技术再生特定形态软骨有望解决长段气管修复的临床难题，但大动物模型的长期功能重建一直未取得突破，成为阻碍临床转化的主要瓶颈。我们前期研究发现支架材料炎症反应是干扰大动物体内软骨再生质量的关键因素，而预构建管状软骨缺乏充分的血供是影响气道重建术后长期存活的重要原因。针对这两个主要问题，我们实施了以下改进：1）优化体外构建技术：一是延长细胞-材料复合物体外预培养时间，待支架材料已基本降解再植入体内，减少材料造成的局部组织炎症，再生出均质成熟的软骨组织。二是利用软骨细胞膜片技术，构建不含支架材料的组织，也可以有效地避免材料在体内引起的炎症反应，在大动物皮下再生出大面积优质成熟的片状软骨组织。同时软骨细胞膜片可以模拟软骨微环境促进BMSCs异位软骨稳定诱导再生，扩大了种子细胞来源。2）我们利用肌肉瓣直接包裹软骨细胞膜片方式在大动物体内分层有序预制带血供管状肌肉-软骨复合组织，可以一步就预制出带血供管状软骨，避免了先皮下构建再肌肉内构建的两步法，减少了手术次数，节省了构建时间，为后期气管缺损修复手术奠定基础。3）我们采用内核概念，在PGA/PLA复合支架中增加一层具有较大的孔隙率，一定弹性和较大的力学强度强度，降解缓慢的PCL内核支架。不仅增加了组织工程软骨的力学强度，还具有精确塑形，可以长期维持形状的优点，通过实验我们确立了含内核复合物体外培养及体内再生软骨的关键技术参数。4）我们通过术后及时吸痰，加强营养，抗感染等系列治疗措施提高了移植组织的存活与功能恢复；通过纤维支气管镜检查，MRI，通气功能检测综合评估移植组织的存活及功能重建情况；建立长期随访气管功能远期修复效果的检测体系。通过上述多种方案的有机整合，我们取得了大动物术后长期存活的突破，取材发现再生软骨存活良好，内壁已为气道上皮完全覆盖，成功建立了长段气管缺损长期功能重建的大动物模型，为组织工程技术修复长段气管缺损的下一步临床转化奠定扎实的基础。