Construction of vascularized tissue-engineered bone with polylysine-modified coral hydroxyapatite and a double cell-sheet complex to repair a large radius bone defect in rabbits
Construction of vascularized tissue-engineered bone with polylysine-modified coral hydroxyapatite and a double cell-sheet complex to repair a large radius bone defect in rabbits
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用聚赖氨酸修饰的珊瑚羟基磷灰石和双细胞片复合物构建血管化组织工程骨来修复兔大半径骨缺损
DOI：
10.1016/j.actbio.2019.04.024
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发表时间：
2019
影响因子：
9.7
通讯作者：
He Yalan
中科院分区：
文献类型：
--
作者：
Zhang Hualin;Zhou Yueli;Yu Na;Ma Hairong;Wang Kairong;Liu Jinsong;Zhang Wen;Cai Zhuoyan;He Yalan
本研究探讨了双细胞复合体与聚赖氨酸(PLL)修饰珊瑚羟基磷灰石(CHA)复合构建的血管化组织工程化骨修复兔大段骨缺损的能力。首先，对兔脂肪间充质干细胞(ADSC)进行诱导培养，获得复合体。其次，采用浸渍和真空冷冻干燥法制备了不同浓度的PLL-CHA复合支架，并用X射线衍射仪、傅立叶变换红外光谱、压缩性能测试和细胞相容性评价等方法对支架进行了表征。第三，体外构建DCs-PLL-CHA血管化组织工程骨，并将其移植到兔大段骨缺损模型中。最后，通过大体观察、激光散斑成像、扫描电子显微镜、组织学染色、放射学观察和RT-PCR等方法评价了DCSPLL-CHA血管化组织工程化骨修复大块骨缺损的能力。体外实验结果表明，该复合体提供了非常大的细胞储备，携带了大量的体外诱导的成骨细胞和血管内皮细胞。将复合后的复合体与PLL-CHA支架在体外结合，PLL对细胞的黏附、增殖和分化的影响与体内的趋化作用相似，使复合复合体比单纯的复合体更有利于移植物的细胞化。活体实验显示，各组骨痂表面均有血供，缺损区表面的血流量各组间基本相同。12 时，DCSPLL-CHA组表面已完全被骨组织和类骨质包裹，皮质骨显像基本连续，髓腔以穿孔为主。形成大量排列整齐的板层状骨，少量未降解的CHA呈线状排列，孔洞内可见大量骨填充。12 时，各组BGLAP、SPP1和VEGF3种蛋白的表达水平相似，但DCSPLL-CHA组PECAM1的表达高于自体骨组和CHA组。结果表明，PLL能有效促进ADSCs的黏附、增殖和分化，DCSPLL-CHA血管构建的组织工程骨具有骨再生和骨重建的潜能，可用于修复较大的骨缺损。采用浸泡和真空冷冻干燥两种方法制备了不同浓度PLL的PLL-CHA复合支架。采用双细胞复合片复合PLL-CHA支架构建血管化组织工程化骨。DCs-PLL-CHA血管化组织工程化骨具有骨再生和骨重建的潜能，可用于修复较大的骨缺损。
In this study, the potential of vascularized tissue-engineered bone constructed by a double cell-sheet (DCS) complex and polylysine (PLL)-modified coralline hydroxyapatite (CHA) to repair large radius bone defects was investigated in rabbits. Firstly, the DCS complex was obtained after rabbit adipose-derived mesenchymal stem cell (ADSC) culture was induced. Secondly, PLL-CHA composite scaffolds with different concentrations of PLL were prepared by the soaking and vacuum freeze-drying methods, and then the scaffolds were characterized by X-ray diffraction (XRD), Fourier transform infrared (FTIR) spectroscopy, compression performance testing and cytocompatibility evaluation. Thirdly, DCS-PLL-CHA vascularized tissue-engineered bone was constructedin vitroand transplanted into a large radius bone defect model in rabbits. Finally, the potential of the DCS-PLL-CHA vascularized tissue-engineered bone to repair the large bone defect was evaluated through general observations, laser speckle imaging, scanning electron microscopy (SEM), histological staining, radiography observations and RT-PCR. Thein vitroexperimental results showed that the DCS complex provided a very large cell reserve, which carried a large number of osteoblasts and vascular endothelial cells that were inducedin vitro. When the DCS complex was combined with the PLL-CHA scaffoldin vitro, the effects of PLL on cell adhesion, proliferation and differentiation led to a situation similar to the chemotaxis of the body, making the combined complex more conducive to graft cellularization than the DCS complex alone. Thein vivoexperiments showed blood supply on the surface of the callus in each group, and the amount of blood perfusion on the surface of the defect area was almost equal among the groups. At 12 weeks, the surface of the DCS-PLL-CHA group was completely wrapped by bone tissue and osteoids, the cortical bone image was basically continuous, and the medullary cavity was mainly perforated. A large amount of well-arranged lamellar bone was formed, a small amount of undegraded CHA exhibited a linear pattern, and a large amount of bone filling could be seen in the pores. At 12 weeks, the expression levels of BGLAP, SPP1 and VEGF were similar in each group, but PECAM1 expression was higher in the DCS-PLL-CHA group than in the autogenous bone group and CHA group. The results showed that PLL could effectively promote the adhesion, proliferation and differentiation of ADSCs and that DCS-PLL-CHA vascularized tissue-engineered bone has potential for bone regeneration and bone reconstruction and can be used to repair large bone defects.Statement of Significance1. PLL-CHA composite scaffolds with different concentrations of PLL were prepared by the soaking and vacuum freeze-drying methods.2. The vascularized tissue-engineered bone was constructed by the double cell sheet (DCS) complex combined with PLL-CHA scaffolds.3. The DCS-PLL-CHA vascularized tissue-engineered bone has potential for bone regeneration and bone reconstruction and can be used to repair large bone defects.