Abstract: BACKGROUND: Schwann cells play a pivotal role in the process of peripheral nerve repair. By migrating to form cell bridges and secreting neurotrophic factors, Schwann cells provide essential nutrients and topographical cues for axon extension. The high specific surface area of nanofibers is conducive to the release of drugs and bioactive substances, and provides sufficient space for cell adhesion, spreading and proliferation, which is of great significance for the rapid repair of nerve tissue.
OBJECTIVE: To explore the regulation of Schwann cell behavior by polycaprolactone aligned nanofibers deposited endothelial cell derivative microparticles.
METHODS: Functionalized endothelial cell derivatives were extracted from the conditioned medium of human umbilical vein endothelial cells by low-temperature freeze-drying technology. Polycaprolactone aligned nanofibers were prepared by electrospinning technology. Endothelial cell derivatives were deposited on the surface of polycaprolactone aligned nanofibers in the form of microparticles by electrospraying technology, and the deposition time was 10, 20, and 30 minutes, respectively. Human umbilical vein endothelial cells (or Schwann cells) were inoculated on polycaprolactone oriented nanofibers deposited with endothelial cell derivatives, and the optimal deposition time of endothelial cell derivatives was determined by cell proliferation and growth for subsequent experiments. During the electrostatic spraying process, the glass slide was divided into five areas of equal size and a shield was covered on the glass slide. By changing the area covered by the shield (i.e., gradually decreasing from 4 areas to 0 areas), eposition time of microparticles in different regions was regulated to obtain polycaprolactone aligned nanofibers with unidirectional linear gradient deposition of endothelial cell derivative microparticles. Schwann cells were inoculated on polycaprolactone aligned nanofibers (polycaprolactone group), polycaprolactone aligned nanofibers with uniform deposition of endothelial cell derivative microparticles (uniform deposition group), and polycaprolactone aligned nanofibers with unidirectional linear gradient deposition of endothelial cell derivative microparticles (gradient deposition group), and the cell activity, morphology, and migration ability were detected.
RESULTS AND CONCLUSION: (1) The optimal deposition time of endothelial cell derivatives was determined to be 10 minutes based on cell proliferation and growth. Scanning electron microscopy showed that endothelial cell derivatives were uniformly deposited on the polycaprolactone aligned nanofiber membrane and had superhydrophilicity. (2) CCK-8 assay results showed that the Schwann cell viability in the uniform deposition group and gradient deposition group was higher than that in the polycaprolactone group. The results of fluorescence microscopy showed that Schwann cells in the polycaprolactone group, uniform deposition group, and gradient deposition group grew along the direction of nanofibers and were in good growth state. The results of immunofluorescence staining showed that compared with the polycaprolactone group, Schwann cells in the uniform deposition group and gradient deposition group showed stronger migration ability, and the migration distance of Schwann cells in the gradient deposition group was the largest. These results indicate that unidirectional linear gradient deposition of endothelial cell derivative microparticles polycaprolactone aligned nanofibers can promote the proliferation and directional migration of Schwann cells.

Key words: electrospinning, electrospray, endothelial cell derivative, Schwann cell, cell migration, peripheral nerve tissue, nanofiber membrane