摘要
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
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 staini
作者
王文博
徐敬之
吴亮
郗焜
辛天闻
唐锦程
顾勇
陈亮
Wang Wenbo;Xu Jingzhi;Wu Liang;Xi Kun;Xin Tianwen;Tang Jincheng;Gu Yong;Chen Liang(Department of Orthopedics,The First Affiliated Hospital of Soochow University,Suzhou 215000,Jiangsu Province,China)
出处
《中国组织工程研究》
CAS
北大核心
2023年第25期4028-4037,共10页
Chinese Journal of Tissue Engineering Research
基金
国家自然科学基金(81772312,81972078),项目负责人:陈亮。
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
black phosphorus
electrospinning
osteogenic differentiation
angiogenesis
bone defect
vascular endothelial growth factor
artificial periosteum
