人残耳软骨细胞体外构建人耳形态软骨及其体内转归

Due to lack of appropriate seed cells, tissue engineered human-ear-shaped cartilage has made no breakthrough so far. Our previous research found that chondrocytes from the patient’s microtia ear (microtia chondrocytes, MCs) could proliferate robustly but accompany with rapid loss of chondrogenic phenotype. Fortunately, the proliferated MCs could re-differentiate to form cartilage-like tissue under chondrogenic induction, indicating that MCs might be the ideal cell source for regeneration of human-ear-shaped cartilage in terms of cell quantity and function. However, it is still unknown whether it would be feasible to use the extensively expanded MCs to regenerate real-sized human-ear-shaped cartilage, whether the engineered cartilage would have sufficient mechanical strength to sustain the ear-shape after in vivo implantation, and what would be the long-term in vivo fate of the engineered human-ear-shaped cartilage in immuno-competent animal model. To address the above issues, based on the previous studies and technologies established in human-ear-shaped cartilage regeneration in vitro, the current project will establish the in vitro human-ear-shaped cartilage regeneration technologies based on expanded MCs through optimizing chondrogenic re-differentiation parameters and in vitro tissue regeneration systems. Meanwhile, slow degraded supporting core in scaffold combining with bioreactor will be applied to enhance the mechanical properties and maturity of the in vitro engineered cartilaginous tissue, so as to solve the issues of shape maintenance and host inflammation response caused by residual scaffold in immuno-competent animals. Finally, animal models for simulating clinical external ear reconstruction will be established to further investigate the long-term fate of the engineered human-ear-shaped cartilaginous tissue. The current project would provide practical feasible strategies in terms of seed cell, in vitro reconstruction technology, and animal model, and thus promote clinical translation of the tissue engineered human-ear-shaped cartilage.

由于缺乏理想种子细胞，组织工程耳廓软骨的临床应用一直未获突破。本课题组前期研究发现：小耳症患者的残耳软骨细胞（MC）体外增殖能力极强，但扩增后迅速丧失软骨表型，而在特定条件下又能重分化形成软骨。因此，MC在数量上和功能上均有望成为耳廓软骨再生的理想种子细胞。然而，MC体外构建正常大小人耳形态软骨是否可行、体内植入后其力学强度能否维持人耳形态、长期转归如何等系列问题目前仍不清楚。针对上述问题，本项目将基于前期人耳形态软骨构建相关研究基础，通过优化体外软骨再生技术参数建立MC体外构建人耳形态软骨技术平台；同时，利用慢降解内核支架结合生物反应器提高体外构建软骨力学性能及成熟度，解决体内形态维持及材料引发的炎症反应问题；最后，建立模拟临床耳再造的动物模型，探讨耳廓形态软骨的体内长期转归。本项目有望从种子细胞、体外构建技术、动物模型三方面为组织工程人耳软骨的应用提供切实可行的方案，推动其临床转化。

由于缺乏理想的种子细胞，组织工程耳廓形态软骨的大规模临床推广应用一直未获突破。前期研究发现，小耳症患者的残耳软骨细胞（MC）体外增殖能力极强，但扩增后迅速丧失软骨表型，而在特定条件下又能重分化形成软骨。如果能调控MC的软骨表型分化，将有望成为耳廓软骨再生的理想种子细胞。然而，MC复合支架材料体外构建正常大小人耳形态软骨是否可行、体内植入后其力学强度能否维持人耳形态、长期转归如何等系列问题目前仍不清楚。针对上述问题，本项目基于前期人耳形态软骨构建相关研究基础，优化了体外软骨再生技术参数，建立了MC体外构建人耳形态软骨技术平台；同时，利用慢降解内核支架结合生物反应器提高了体外构建软骨力学性能及成熟度，解决了体内形态维持及材料引发的炎症反应问题，并进一步探讨了天然生物材料支架构建人耳形态软骨的可行性；最后，建立了模拟临床耳再造的动物模型，探讨了耳廓形态软骨的体内长期转归。