体外三维软骨再生体系调控去分化软骨细胞功能及机制

Limited cell source, rapid dedifferentiation and loss of chondrogenic phenotype of the chondrocytes are still the major obstacles for the clinical translation of tissue engineered cartilage. Our previous research found that the in vitro three-dimensional (3D) cartilage regeneration system can regulate the dedifferentiated chondrocytes’ function and induce the dedifferentiated cells to redifferentiated chondrocytes. More importantly, these redifferentiated chondrocytes not only achieve a similar cell function to native chondrocytes but also can repeat the dedifferentiated process during in vitro successive passages. These results indicated that it is possible to repeatedly regulate the dedifferentiated chondrocytes using this in vitro 3D cartilage regeneration, which might help to obtain sufficient amounts of fully functional chondrocytes for clinical application with this regulation model. Nevertheless, several important issues still need to be elucidated, for example: how many rounds the circular regulation system would last, what would be the function and biosecurity of chondrocytes after repeated regulation, and what is the underlying mechanisms of this regulate system. To address the above issues, based on the previous studies and technologies established in chondrocyte regulation, the current project will establish a circulatory regulation model (chondrocytes will undergo several circulatory regulation with the in vitro 3D cartilage regeneration system) to investigate the feasibility of repeating reverse the function of the dedifferentiated chondrocytes. Meanwhile, after undergo several circular regulations, chondrocytes in each regulated cycle will be collected and used for the function examination and biosecurity evaluation to predict the clinical efficacy and safety of these cells. Finally, the role of the 3D culture system, the extracellular matrix microenvironment and the TGF-β growth factor will be further investigate to elucidate the underlying mechanisms how the regulation system reverse the function of the dedifferentiated chondrocytes. The current project would provide practical feasible strategies in terms of limited cell source, rapid dedifferentiation and loss of chondrogenic phenotype of chondrocytes, and thus promote clinical translation of the tissue engineered cartilage.

软骨细胞来源有限、扩增后去分化、功能丧失等问题已成为阻碍软骨组织工程临床转化的重要瓶颈。本项目组前期研究发现：体外三维软骨再生体系可调控逆转去分化软骨细胞功能，且调控后的细胞与天然软骨细胞功能相似、去分化规律相同。因此，应用该体系多轮循环调控去分化软骨细胞有望获得功能完全且数量充足的细胞。然而，多轮循环调控方案是否可行、多次调控后的细胞功能及生物安全性如何、其调控机制是什么等问题目前仍不清楚。针对上述问题，本项目将基于前期的相关研究基础，通过探讨体外三维软骨再生体系多轮循环调控去分化软骨细胞功能实现指数级细胞扩增的可行性；评估多次调控后细胞的功能、生物安全性能变化情况，明确其临床应用可行性；最后，进一步探讨该体系的调控机制及其作用原理，深化软骨细胞调控理论。本项目有望从细胞数量、功能、生物安全性、调控方案等方面为解决软骨细胞去分化、功能丧失问题提供切实可行的解决方案，推动其临床转化。

软骨细胞体外培养的去分化、功能丧失等问题仍是软骨组织工程临床应用转化的重要难题。有研究表明三维培养及细胞因子可促进去分化软骨发生重分化。本项目在前期研究基础上建立了聚乙醇酸/聚乳酸支架联合多种细胞因子的体外三维软骨再生体系，并应用这一体系对去分化软骨细胞进行多轮循环调控，以期获得功能完全且数量充足的细胞。项目研究发现，去分化残耳软骨细胞经过第一轮调控后其细胞（第一轮重分化细胞，RMC-C1）行为特性及功能几乎完全恢复到天然软骨细胞水平；但RMC-C1通过扩增去分化后进行二次调控（第二轮重分化细胞，RMC-C2）时，细胞的表型及功能出现快速丧失。通过对天然软骨细胞、去分化软骨细胞、RMC-C1及RMC-C2进行转录组测序发现，与天然软骨细胞相比，去分化软骨细胞、RMC-C1及RMC-C2有诸多上调及下调基因。这些基因的表达变化可能是二次重分化软骨细胞不能完全恢复到天然软骨细胞水平的关键因素。上述研究为软骨细胞去分化和重分化的机制研究提供了参考，同时也为后续优化三维软骨培养体系提供了新的方向。