周细胞与外膜细胞复合PHBHHx/P3HB4HB在软骨组织缺损修复中的研究

The damaged cartilage cannot fully regenerate because of its limited intrinsic healing capacity. Cartilage tissue engineering has emerged as a promising strategy for cartilage regeneration and repair. Two populations of perivascular stem cells (pericytes (CD31negCD45negCD146+CD34neg) and adventitial cells (CD31negCD45negCD146negCD34+)) represent many features of MSCs including morphology, growth, surface markers, clonogenicity and multilineage differentiation potentials. Polyhydroxyhexanoate (PHBHHx) and poly(3-hydroxybutyrate-co-4-hydroxybutyrate) (P3HB4HB) as the member of bacterial biopolyesterpolyhydroxyalkanoate (PHA) family, is becoming a desirable candidate for tissue engineering. In this project, the scaffold of PHBHHx/P3HB4HB will be prepared and the morphology characterization, surface roughness, hydrophilicity and mechanical properties of the scaffolds will be measured firstly. Then, adipose-derived stem cells and chondrocytes will be acquired from Rabbits. Pericytes and adventitial cells from digested fat tissue will be sorted by fluorescence activated cell sorting. The cells will be seeded with scaffold materials and assay the cell morphology, adhesion and proliferation in vitro. After making the full thickness cartilage defects at the knee joints in rabbits, the implantation will be carried out by filling the scaffolds with cells into the defect holes. Rabbits will be sacrificed after 10 and 20 weeks to assess the repair process of cartilage effects by ICRS scores, histological staining, immunohistochemistry and GAG assay respectively. The implantation results based on pericytes and adventitial cells with PHBHHx/P3HB4HB scaffold may shed light on their potential of in cartilage tissue engineering.

软骨损伤后自我修复能力有限，组织工程技术为大面积软骨缺损的修复提供了新的方法。周细胞和外膜细胞这两种血管外周细胞亚群能够表达间充质干细胞的很多特征，PHBHHx和P3HB4HB是优良的组织工程生物材料。本研究首先构建PHBHHx/P3HB4HB三维多孔和静电纺丝软骨仿生材料，并对材料各项特性进行相关检测。然后分离、培养兔软骨/脂肪细胞，并从脂肪细胞中流式分选出周细胞和外膜细胞两种血管周干细胞亚群，细胞经过荧光标记后三维共培养于仿生材料上，检测不同支架材料对细胞形态、增殖及功能的影响，最后建立兔膝关节软骨缺损修复模型，观察细胞/支架材料植入缺损部位后软骨再生及关节运动功能的影响。通过对比周细胞与外膜细胞在体内外成软骨能力的差异以及不同处理方式及生长因子对软骨再生的调控作用，为软骨再生筛选最佳的种子细胞和分化调控条件，并合成软骨再生理想仿生材料，构建组织工程化软骨，成功修复大动物极量软骨缺损。

软骨损伤后自我修复能力有限，组织工程技术为大面积软骨缺损的修复提供了新的方法。尽管脂肪源性间充质干细胞（ASCs）对于软骨再生修复有着众多的优点，但是临床应用还是存在很多困难。作为ASCs的替代，脂肪源性基质血管组分（SVF）因其不需要经过体外扩增培养，被越来越多的使用。SVF由多种细胞群体组成，并且能够表达间充质干细胞的很多特征。因此本研究利用组织工程手段，借助支架材料，与软骨细胞共培养的方法，构建软骨缺损动物模型，比较ASCs和SVF在动物体内软骨生成的差异。本研究选择PHA生物材料家族的PHB、PHBHHx生物材料构建支架，并对支架材料的各项特性进行相关检测。然后分离、培养兔软骨细胞和脂肪细胞，共培养于支架材料上，检测细胞的形态、增殖及功能。最后建立兔膝关节软骨缺损修复模型，观察细胞/支架材料植入缺损部位后软骨再生及关节运动功能的影响。研究结果显示细胞体外培养于PHB/PHBHHx支架材料上，表现出良好的生物相容性。将SVF和ASCs分别与软骨细胞等比例混合接种于支架材料上，移植入兔膝关节软骨缺损处，SVF比ASCs更能促进软骨修复。研究表明，与软骨细胞混合培养，脂肪源性SVF比ASCs更能促进软骨组织的生成，SVF多样化的细胞亚群在软骨形成中表达更高水平的营养效应。PHB/PHBHHx共混体系是一种优秀的软骨组织工程材料。这些结果支持了临床上将来源于脂肪组织的SVF与来源于非负重关节面的软骨细胞同时获取混合后重新移植入患处的单步手术治疗软骨损伤的可能。

内容获取失败，请点击重试