(293c) Single Cell Behavior On Single Electrosprayed Nanofiber with Controllable Pore Size

Electrospinning has recently emerged as a technique for tissue regeneration; however, the tiny pore size of electrosprayed fibers has inhibited mammalian cells from infiltrating into the layers below the surface. Hence, cell growth is restricted to the surface only. To overcome this barrier, we have recently developed a thin layer of electroprayed fibers with large pore sizes using our innovative collector plate. Because of the aluminum frame of the fibers, it is also easy to handle single nanofiber without any mechanical damages. Furthermore, in this study, we address the fabrication of innovative electrosprayed fibers made of polycarprolactone and gelatin (PCL/gelatin) with controllable pore size, gelatin distribution on single PCL/gelatin fiber while its degradation, and single cell behavior on the single electrosprayed fiber.

PCL/gelatin in hexafluoro-2-propanol was used to fabricate electrosprayed fibers. The electrospraying setup consists of a syringe pump, high voltage power supply, earth grounding, syringe, syringe tip and the novel collector or a conventional one. 6 different fibers were fabricated by increasing the deposit volume of the polymer solution along with the novel or conventional collector. Using SEM, CCD camera, and Sigma Scan Pro software, the physical properties (diameter, pore size, shape factor) of fibers were confirmed. Degradation study of fibers in Krebs Henseleit buffer solution (pH 7.4) was also carried out in CO2 incubator at 37°C for 2 weeks. Carboxyfluorescein diacetate-succinimidyl ester (CFDA-SE) was used to confirm the gelatin distribution on the single fiber. After human foreskin fibroblasts (HFF-1s) were pre-stained by CFDA-SE, HFF-1s were seeded onto the fibers, and the samples were cultured in serum free media. The cell morphology was confirmed by inverted fluorescent microscope, confocal microscope and SEM. The shape and size of HFF-1s on different single fibers were compared to those on 2D film made of the same PCL/gelatin solution.

Novel (Fiber A, B, and C) and conventional (Fiber D, E, and F) fibers were fabricated under the same conditions except for exchanging novel (A, B, C) and conventional collectors (D, E, and F) and for increasing the deposit volume of PCL/gelatin solution from 0.3 microliter (A and D) via 0.6 microliter (B and E) to 0.9 microliter (C and F). Physical properties of the fibers were very similar except pore sizes. The pore sizes were 339, 129, and 51 micrometers for A, B, and C, and about 5 micrometers for D, E, and F. However, the diameters of fibers were about 800 nanometers for all the fibers (A to F). Also, the fluorescent images using CFDA-SE show that gelatin was well distributed on the entire single PCL/gelatin fiber at day 0. Furthermore, the single fiber was stable under the physiological condition for 2 weeks, similar to the previous results of the conventional electrosprayed fibers from other research groups. After cell culture using Fiber C, images of HFF-1s on single fibers were collected. The images showed that cells on the fibers were attaching, reshaping, and merging with adjacent cells. The shapes and sizes of HFF-1s on the single fibers were various, compared to those on the 2D film. Thus, we strongly believe that the single cell behavior study on the innovative electrosprayed fiber will expand its implications to other cells and other fibers made of various synthetic and natural biomaterials.