关键词: 硬脊膜修补, 人工硬脊膜, 静电纺丝, 胶原蛋白, 骨髓间充质干细胞, 聚乳酸

Abstract: BACKGROUND: Currently, the dura mater is clinically repaired using autologous tissue or materials such as gelatin sponge, but all of them have their inherent defects. Therefore, there is an urgent need for a biomaterial that can promote dural repair.
OBJECTIVE: The two-sided anisotropic electrospun membrane was constructed by using directional electrospinning technology and collagen self-assembly technology, and was used as a carrier for bone marrow mesenchymal stem cells to investigate various physicochemical properties and biological characteristics of the artificial dura mater.
METHODS: Ordered polylactic acid electrospun fibers with double-sided (collagen protein on one side and polylactic acid on the other side) anisotropic electrospun membranes (collagen group), disordered polylactic acid electrospun membranes (disordered fiber group), and ordered oriented polylactic acid electrospun membranes (ordered fiber group) were prepared by electrospinning technique as well as collagen self-assembly technique. Scanning electron microscopy, mechanical stretching, water contact angle testing, and degradation experiments were used to characterize the physicochemical properties of the electrospun membranes. Electrospun membranes in the collagen group (bone marrow mesenchymal stem cells were inoculated on the collagen surface to obtain the stem cell-engineered electrospun membranes), disordered fiber group and ordered fiber group were cocultured with bone marrow mesenchymal stem cells. The biocompatibility of electrospun membranes was evaluated using CCK-8 assay and live/dead staining. Integrin β1 immunofluorescence staining was used to evaluate the adhesion characteristics of electrospun membranes. The stem cell-engineered electrospun membrane and the electrospun membrane in the collagen group were cocultured with bone marrow macrophages respectively. Immunomodulatory properties were assessed by detecting the expression of inflammation-related genes using inducible nitric oxide synthase (M1 type), CD206 (M2 type) immunofluorescence staining, and qRT-PCR.
RESULTS AND CONCLUSION: (1) The oriented electrospun fiber membrane could mimic the structure of the longitudinally aligned natural dura mater, and the addition of collagen increased the hydrophilicity of the fiber membrane by about 2-fold and the mechanical properties by 1.2-fold. (2) When cocultured with bone marrow mesenchymal stem cells, CCK-8 assay and live/dead staining suggested that the cellular bioactivity in the collagen group was significantly higher than that in the disordered fiber group and ordered fiber group. Immunofluorescence staining revealed that the expression of integrin β1 in the collagen group was about 2.6 times higher than that of the disordered and ordered fiber groups, and the cell spreading morphology was good. (3) When cocultured with bone marrow macrophages, immunofluorescence staining exhibited that the fluorescence intensity of M1 type macrophages in the stem cell-engineered electrospun membrane group was lower than that in the collagen group (P < 0.01), and the fluorescence intensity of M2 type macrophages was higher than that in the collagen group (P < 0.01). qRT-PCR demonstrated that proinflammatory gene tumor necrosis factor α and interleukin-1β mRNA expression in the stem cell-engineered electrospun membrane group was lower than that of the collagen group (P < 0.001); anti-inflammatory genes such as interleukin-10 and transforming growth factor β mRNA expressions were higher than those in the collagen group (P < 0.001). (4) The above results suggest that the stem cell-engineered amphipathic artificial dura mimics the directional structure of normal dura, with the inner surface facilitating cell growth and adhesion and the outer edge avoiding tissue adhesion, while the polarization of macrophages to the M2 subtype is promoted and the local inflammatory microenvironment is regulated through the mesenchymal stem cell paracrine component.

Key words: dural repair, artificial dura, electrospinning, collagen, bone marrow mesenchymal stem cell, polylactic acid