| Objective: 1. To fabricate the oriented PCL/SF hybrid micro-fiber membranes by wet spinning method.2. To evaluate the biological safety and biocompatibility of PCL/SF hybrid micro-fiber membranes.3. To construct the ±30°angle-ply multi-lamellar tissue engineered annulus fibrosus scaffold with the complexes of rabbit AF cells and the oriented PCL/SF hybrid micro-fiber membranes.Methods: 1. PCL/SF-HFIP co blended spinning solution was established with HFIP as solvent, then it was fabricated into oriented PCL/SF hybrid micro-fiber membranes using wet spinning method. The surface morphology, fiber diameter and arrangement direction of fiber membranes were investigated by general observation, stereomicroscope and scanning electron microscopy(SEM), and the chemical composition of the mixture was detected by FTIR analysis. The tensile modulus of fiber membranes inwhich the arrangement of fibers were cross or parallel were evaluated.2. The rabbit AF cells were isolated and cultured to P2 generation, then they were used in CCK-8 assay to examine cytotoxicity of the extraction of hybrid materials. P2 AF cells were seeded onto the fiber membranes, after a 7 days culture in vitro, the cells- fiber membrane complexes were investigated by LIVE/DEAD staining and SEM images, and compatibility of the hybrid material was evaluated.3. After a 3 days culture in vitro, the cells- fiber membrane complexes were constructed to ±30°angle-ply multi-lamellar tissue engineered annulus fibrosus scaffold, and the histological and immunohistochemical staining were used to evaluate the biological behavior of cell-scaffold composite and its feasibility for annulus fibrosus tissue engineering.Results: 1. Stereomicroscope and SEM images showed that the orientation of fibers is quite consistent, the diameter of one fiber was uniform, the fiber surface was rough and irregular. The average diameter of hybrid micro fibers was 18.05 ± 3.51μm. The tensile modulus of fiber membranes inwhich the arrangement of fibers were cross or parallel were 9.91 ± 0.63 MPa and 8.25 ± 1.09 MPa, respectively, and the difference was statistically significant(P<0.05). FTIR analysis showed that the chemical composition of mixture had the characteristics of PCL and SF.2. CCK-8 proliferation assay showed that OD value increased significantly with the time. There are significant differences between the OD values of extraction groups at different time points(P<0.05). There was a significant difference between extraction group and control group at the tenth day during culture in vitro(P<0.05). LIVE/DEAD staining confocal images showed that after a 7 days culture, the growth activity of AF cells seeded on fiber membranes was good, a large number of cells were observed and the cells could grow along the direction of fiber orientation. SEM images showed that AF cells could adhere to the surface of the material and secrete a large quantity of extracellular matrix.3. The fixation of the angle-ply multi-lamellar tissue engineered annulus fibrosus was firm, and the cross section shows that there is no delamination between each layer, the layers contacted well. HE staining showed that the spindle shaped AF cells could adhere to the surface of the fiber, and the cross orientation structure of hybrid fiber could be distinguished in the images. Toluidine blue and safranin O staining results were positive, the positive region mainly concentrated in outer layers and the gaps between adjacent layers. Collagen I immunohistochemical staining results were positive, with light brown, evenly distributed between the fibers.Conclusion: 1. In this experiment, the oriented PCL/SF hybrid micro-fiber membranes were fabricated by wet spinning method, the fibers were aligned in the same direction practically and uniform in diameter. It had a certain degree of mechanical strength when it was arranged in a ±30°angle-ply overlapping structure.2. AF cells could be led to adhere onto the surface of the material and secrete a large quantity of extracellular matrix by oriented hybrid fibers. PCL/SF hybrid micro-fiber membrane has good biological safety and biocompatibility, and it could be an ideal scaffold to construct tissue engineered annulus fibrosus.3. In this study, we used cell- fiber membrane complexes to construct a biomimetic annulus fibrosus scaffold which mimicked the ±30° angle-ply multi-lamellar structure of outer annulus fibrosus. It provided structural support for the survival of AF cells and the secretion of ECM in the scaffold, and it could also be used to construct tissue engineered annulus fibrosus scaffolds. |
PDF Full Text Request