基于超声控释技术研究威灵仙-多孔蚕丝蛋白-软骨细胞微球复合体修复软骨缺损的作用及机制

Traditional Chinese medicine (TCM) has highlighted its unique advantage in cartilage tissue engineering research as a result of its multi-factors and multi-targets characteristics. Considering of the advantages of three-dimensional space microenvironment, the present project aims at a new continuous and noninvasive method for cartilage defect by introducing the ultrasound controlled release technology into the intervention of TCM on the construction of cartilage tissue engineering and developing a porous silk fibroin microspheres under ultrasound irradiation as the drug delivery carrier of Weilingxian and seed cells. After Weilingxian and chondrocytes embedded in porous silk fibroin microspheres, chondrocytes will get amplification by suspension culture in vitro, and then the cells spatial distribution, phenotype maintainance and matrix expression will be observed under Weilingxian and ultrasonic irradiation. We will discuss the positive effects after implanted into the cartilage defect region in animal models of by ultrasound controlled release technology to help the release of Weilingxian effective components and slow down the rate of carrier degradation, after which the positive effect will be evaluated by the expression level of functional molecules (TGF-β1, MMP-13, IL-1β, and TIMP–1) in TGF-β/Smad signaling pathway.The combination of Chinese Medicine, biomedical ultrasound and biomaterials can open more possibilities for the utilization of Weilingxian and its effective components, which can also lead to new ideas of medical tissue engineering technology.

中药多因素、多靶点的作用特点彰显了其在软骨组织工程研究中的独特优势。基于三维空间微环境的积极意义，本项目将超声控释技术引入中医药干预组织工程软骨的构建，拟在超声辐照下以多孔蚕丝蛋白微球作为威灵仙药物缓释载体，并负载种子细胞建立微球复合体，摸索一种持续、无创修复软骨缺损的新方法。首先构建威灵仙-多孔蚕丝蛋白-软骨细胞微球复合体，经过体外悬浮培养扩增软骨细胞数量，然后观察威灵仙及超声辐照作用下软骨细胞空间分布、表型维持、基质表达情况。探讨其植入模型动物软骨缺损区域后通过超声控释技术帮助威灵仙有效成分的释放并缓冲载体降解速率的积极效应。最后通过软骨中 TGF-β/Smad信号通路，尤其是TGF-β1、MMP-13、IL-1β、TIMP-1等功能分子表达水平，评价其修复效果。本项目有机融合中医药与生物物理学和生物材料学技术，延伸威灵仙及其有效成分的应用和方法，开拓医学组织工程技术的新思路。

关节软骨损伤修复困难，在复杂力学环境下，由多条信号通路、各种细胞因子介导，软骨组织工程技术是软骨修复极具前景的一种治疗方式，如何获取表型良好的软骨种子细胞是其关键环节。我们的研究显示牵张拉伸力学环境能够促使软骨细胞退行性变，直接造成软骨细胞损伤；同时能够通过活化RhoA/ROCK-NF-κb信号促进RAW264.7巨噬细胞M1型极化诱导炎性微环境，间接造成软骨细胞损伤。中药多因素、多靶点的作用特点彰显了其在软骨组织工程研究中的独特优势。而威灵仙能够发挥类生长因子作用，通过TGF-β信号保护牵张拉伸力学环境下的软骨细胞；同时体外模拟微重力培养环境能够发挥协同作用，维持软骨细胞良好表型。项目组有机融合了中医药与细胞生物力学及生物材料学技术，创新性地制备了物理性状与缓释性质良好的威灵仙-多孔蚕丝蛋白微球，并在模拟微重力条件下构建威灵仙-多孔蚕丝蛋白-软骨细胞复合体进行体外三维培养，结合低频脉冲超声的声学及生物学效应对软骨细胞的表型及TGF-β/SMAD信号通路进行了深入探究。通过研究发现，超声能够促进威灵仙总皂苷释放，其干预下的威灵仙-多孔蚕丝蛋白三维培养可通过TGF-β/SMAD2，SMAD3信号维持软骨细胞表型，促进软骨基质合成代谢，抑制分解代谢。体内实验结果也验证了低强度脉冲超声介导的威灵仙-多孔蚕丝蛋白-软骨细胞复合体修复软骨缺损的效果，构建的组织工程软骨在12周后表面较光滑，修复组织以透明软骨为主，基质成分Ⅱ型胶原、蛋白聚糖表达丰富，同时在修复组织中检测到TGF-β/SMAD3的表达显著上调，这与细胞实验部分检测结果较一致。本项研究巧妙的将软骨组织工程三要素结合，成功构建了“威灵仙-多孔蚕丝蛋白-软骨细胞微球复合体”，延伸了威灵仙及其有效成分的应用和方法，开拓了医学组织工程技术的新思路。