中国组织工程研究

中国组织工程研究 ›› 2023, Vol. 27 ›› Issue (25): 4028-4037.doi: 10.12307/2023.163

• 组织工程骨材料 tissue-engineered bone • 上一篇    下一篇

复合纳米纤维骨膜促进血管化和成骨矿化的体外实验

王文博，徐敬之，吴  亮，郗  焜，辛天闻，唐锦程，顾  勇，陈  亮   

  1. 苏州大学附属第一医院骨科，江苏省苏州市  215000
  • 收稿日期:2022-02-16 接受日期:2022-05-09 出版日期:2023-09-08 发布日期:2023-01-17
  • 通讯作者: 陈亮，主任医师，教授，博士生导师, 苏州大学附属第一医院骨科，江苏省苏州市 215000 顾勇，副主任医师，副教授，硕士生导师，苏州大学附属第一医院骨科，江苏省苏州市 215000
  • 作者简介:王文博，男，1991年生，江苏省苏州市人，汉族，苏州大学附属第一医院在读硕士，主要从事脊柱外科及骨组织工程研究。 徐敬之，男，1995年生，江苏省无锡市人，汉族，苏州大学附属第一医院在读硕士，主要从事脊柱外科及骨组织工程研究。
  • 基金资助:
    国家自然科学基金(81772312，81972078)，项目负责人：陈亮

In vitro experiment of composite nanofibrous periosteum to promote vascularization and osteogenic mineralization

Wang Wenbo, Xu Jingzhi, Wu Liang, Xi Kun, Xin Tianwen, Tang Jincheng, Gu Yong, Chen Liang   

  1. Department of Orthopedics, The First Affiliated Hospital of Soochow University, Suzhou 215000, Jiangsu Province, China
  • Received:2022-02-16 Accepted:2022-05-09 Online:2023-09-08 Published:2023-01-17
  • Contact: Chen Liang, Chief physician, Professor, Doctoral supervisor, Department of Orthopedics, The First Affiliated Hospital of Soochow University, Suzhou 215000, Jiangsu Province, China Gu Yong, Associate chief physician, Associate professor, Master’s supervisor, Department of Orthopedics, The First Affiliated Hospital of Soochow University, Suzhou 215000, Jiangsu Province, China
  • About author:Wang Wenbo, Master candidate, Department of Orthopedics, The First Affiliated Hospital of Soochow University, Suzhou 215000, Jiangsu Province, China Xu Jingzhi, Master candidate, Department of Orthopedics, The First Affiliated Hospital of Soochow University, Suzhou 215000, Jiangsu Province, China
  • Supported by:
    the National Natural Science Foundation of China, No. 81772312, 81972078 (to CL)

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摘要：

文题释义：

黑磷：磷中最稳定、最不活泼的同素异形体，其本质的特征是由单一的磷元素组成。黑磷水解氧化以后形成磷酸盐、亚磷酸酯等产物，与人体天然骨的无机成分具有高度的同源性，因此在治疗骨缺损方面具有充分的优势。
血管内皮生长因子：是一种可以有效促血管生成的细胞生长因子，主要效应是促使内皮细胞分化增殖、增加血管通透性及促进血管发生。骨膜血管的有效生长在骨组织修复中起着重要作用。

背景：骨膜对骨骼的发育、重塑具有重要作用，因此，从结构与功能的角度设计一种高生物相容性、生物降解性能及成骨能力强的仿生骨膜，对骨缺损的研究发展具有重要意义。
目的：利用复合纳米纤维骨膜负载黑磷纳米片和血管内皮生长因子，探讨纤维骨膜的理化特征，检测其生物相容性及体外促血管化和骨分化能力。 
方法：将黑磷通过液相剥离术剥离成黑磷纳米片，将血管内皮生长因子及黑磷纳米片包裹于透明质酸钠水溶液形成微溶胶颗粒，加入含左旋聚乳酸的二氯甲烷溶液中混合均匀，再加入N，N-二甲基甲酰胺形成混合溶液，通过静电纺丝技术得到纳米复合人工纤维骨膜(记为PLLA-BP@VEGF)；同理制备单纯的左旋聚乳酸纤维骨膜(记为PLLA)、左旋聚乳酸-黑磷纤维骨膜(记为PLLA-BP)、左旋聚乳酸-血管内皮生长因子纤维骨膜(记为PLLA-VEGF)。将骨髓间充质干细胞分别与上述4组纤维骨膜共培养，通过活死荧光染色、CCK-8、细胞形态黏附实验检测纤维骨膜的细胞生物相容性，通过碱性磷酸酶染色、茜素红染色与骨钙素免疫荧光染色和成骨相关基因PCR检测纤维骨膜的成骨性能。以人脐静脉内皮细胞为实验对象，通过成血管实验检测4组纤维骨膜的成血管能力。

结果与结论：①活死荧光染色、CCK-8实验显示，骨髓间充质干细胞可在4组纤维骨膜上良好增殖并存活，其中PLLA-BP@VEGF组活细胞数量最多；细胞形态黏附实验显示，各纤维骨膜上的骨髓间充质干细胞黏附铺展形态良好；②碱性磷酸酶染色、茜素红染色、骨钙素免疫荧光染色及成骨相关基因检测显示，PLLA-BP@VEGF组、PLLA-BP组纤维骨膜可促进骨髓间充质干细胞的成骨分化；成血管实验显示，相较于PLLA组和PLLA-BP组，PLLA-VEGF组、PLLA-BP@VEGF组血管长度更长，血管样染色结构呈网状形态，交叉节点更多；③结果表明，负载黑磷纳米片和血管内皮生长因子的复合纳米纤维骨膜有良好的生物相容性，体外可促进骨髓间充质干细胞的成骨分化能力与血管内皮细胞的成血管能力。

https://orcid.org/0000-0002-1033-830X(王文博)

中国组织工程研究杂志出版内容重点：生物材料；骨生物材料；口腔生物材料；纳米材料；缓释材料；材料相容性；组织工程

关键词: 黑磷, 静电纺丝, 成骨分化, 成血管, 骨缺损, 血管内皮生长因子, 人工骨膜

Abstract: BACKGROUND: Periosteum is essential for the development, remodelling and integrity of bone. Therefore, designing a biomimetic periosteum with high biocompatibility, biodegradability and strong osteogenic ability from the perspective of structure and function is of great significance to the research and development of bone defects.
OBJECTIVE: To investigate the physicochemical characteristics, biocompatibility and in vitro pro-vascularisation and bone differentiation capabilities of fibrous bone membranes using composite nanofibrous bone membranes loaded with black phosphorus nanosheets and vascular endothelial growth factor (VEGF).
METHODS: Black phosphorus (BP) was stripped into black phosphorus nanosheets by liquid phase exfoliation. VEGF and black phosphorus nanosheets were encapsulated in aqueous solution of sodium hyaluronate to form BP@VEGF to form microsoluble particles, which were added to dichloromethane solution containing L-polylactic acid and mixed well. N,N-dimethylformamide was added to form a mixed solution to obtain nanocomposite artificial fibrous bone membrane by electrostatic spinning technique (denoted as PLLA-BP@VEGF). In the same way, simple L-polylactic acid fibrous periosteum (denoted as PLLA), L-polylactic acid-black phosphorus fibrous periosteum (denoted as PLLA-BP), and L-polylactic acid-VEGF fibrous periosteum (denoted as PLLA-VEGF) were prepared. Bone marrow mesenchymal stem cells were co-cultured with the above four groups of fibrous periosteum. The cell biocompatibility of fibrous periosteum was detected by live-dead fluorescent staining, CCK-8 assay and cell morphological adhesion experiments. The osteogenic properties of fibrous periosteum were detected by alkaline phosphatase staining, alizarin red staining, osteocalcin immunofluorescence staining and osteogenesis-related gene PCR. Taking human umbilical vein endothelial cells as the experimental object, the angiogenic ability of the fibrous periosteum of the four groups was detected by angiogenic experiment.
RESULTS AND CONCLUSION: (1) Live/dead fluorescent staining and CCK-8 assay showed that bone marrow mesenchymal stem cells could proliferate and survive well on the fibrous periosteum of the four groups, among which the PLLA-BP@VEGF group had the largest number of living cells. The cell morphological adhesion experiment showed that the bone marrow mesenchymal stem cells on each fibrous periosteum adhered well and spread well. (2) Alkaline phosphatase staining, alizarin red staining, osteocalcin immunofluorescence staining and osteogenesis-related gene detection showed that the fibrous periosteum of PLLA-BP@VEGF group and PLLA-BP group could promote the osteogenic differentiation of bone marrow mesenchymal stem cells. Angiogenesis experiments showed that compared with the PLLA group and the PLLA-BP group, the blood vessels in the PLLA-VEGF group and the PLLA-BP@VEGF group were longer in length; the vessel-like staining structure was network-like; and there were more crossing nodes. (3) The results show that the composite nanofibrous periosteum loaded with black phosphorus nanosheets and VEGF has good biocompatibility, and can promote the osteogenic differentiation ability of bone marrow mesenchymal stem cells and the angiogenic ability of vascular endothelial cells in vitro. 

Key words: black phosphorus, electrospinning, osteogenic differentiation, angiogenesis, bone defect, vascular endothelial growth factor, artificial periosteum

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