载Defactinib的Gt-PCL电纺纤维支架维持BMSCs构建软骨皮下表型稳定性的作用机制研究

The currently BMSCs-engineered cartilage is unsatisfactory for clinical application due to unstable phenotype in the subcutaneous environment, this phenomenon mainly results from hypertrophic differentiation of chondrocytes induced from BMSCs cultured in vitro and vascularization subcutaneously. Our previous studies indicate that FAK play an important role in hypertrophy and vascularization of BMSCs-engineered cartilage. Based on this, a scientific hypothesis is presented out: an electrsopun gelatin-PCL (Gt-PCL) nanofibrous scaffold loaded with a novel FAK inhibitor-defactinib to construct a niche to inhibit hypertrophy and vascularization of BMSCs-engineered cartilage, finally preventing phenotype lose. For this purpose, we will first prepare and characterize a stable defactinib-loaded nanofibrous scaffold that has controlled release of drug via electrospinning. Then the anti-hypertrophy and anti-vascularization capability of the scaffold will be investigated.At last，we will discuss the molecular mechanism how the drug-loaded scaffold functions in hypertrophy and vascularization of BMSCs-engineered cartilage under subcutaneous condition. This study is expected to provide a novel theoretical basis and construction strategy for cartilage tissue engineering.

BMSCs构建的软骨在皮下环境中表型易于丧失阻碍了其临床应用，究其原因主要与构建过程中BMSCs软骨的肥大化及皮下移植后的血管化有关。我们前期研究证实FAK在BMSCs软骨肥大化及血管化中发挥了重要作用。基于此，本项目提出以下科学假说：将一种新型的FAK抑制剂Defactinib载入电纺明胶-聚己内酯(Gt-PCL)纳米纤维中，从而形成一个抑制肥大化和血管化的构建微环境，最终维持皮下BMSCs软骨的表型稳定。为此，本项目将开展以下研究内容：1.Defactinib-Gt-PCL载药材料的制备与鉴定；2.载药材料对BMSCs软骨体外肥大化的影响；3.载药材料对BMSCs软骨皮下血管化的影响；4.载药材料在维持BMSCs软骨表型稳定中的作用机制研究。该项目的成功将为基于BMSCs的软骨组织工程提供了新的理论基础和构建策略。

BMSCs构建的软骨在皮下环境中表型易于丧失阻碍了其临床应用，究其原因主要与构建过程中BMSCs软骨的肥大化及皮下移植后的血管化有关，我们希望将一种新型的FAK抑制剂Defactinib载入电纺明胶-聚己内酯(Gt-PCL)纳米纤维中，从而形成一个抑制肥大化和血管化的构建微环境，最终维持皮下BMSCs软骨的表型稳定。我们的研究使用了modified RNA技术对ADSCs进行了改造，构建了一个血管化的微环境，证明了其可以明显提高移植后脂肪存活率，而Defactinib的Gt-PCL电纺纤维复合干细胞则希望构建乏血管的微环境，两者相互补充。另外，我们对Gt-PCL电纺纤维支架材料的构建体系进行了优化，针对实验中出现电纺纤维材料粘连不足的问题，为了增加材料的层间粘附，我们制作了软骨脱细胞基质凝胶作为层间填充物，并对该凝胶进行了一系列的表征，流动学，以及生物学的鉴定，证明了软骨脱细胞凝胶具有良好的生物相容性，并且在体内具有促进软骨形成的能力，该凝胶有望在软骨组织工程和骨关节炎的治疗中进行临床转化。

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