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

doi:10.12307/2023.163

Abstract

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

CLC Number:

Wang Wenbo, Xu Jingzhi, Wu Liang, Xi Kun, Xin Tianwen, Tang Jincheng, Gu Yong, Chen Liang. In vitro experiment of composite nanofibrous periosteum to promote vascularization and osteogenic mineralization[J]. Chinese Journal of Tissue Engineering Research, 2023, 27(25): 4028-4037.

Figures/Tables 11

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