Chitosan-based nanofibrous scaffold for cell growth

Abstract

Chitosan-based electrospun nanofibrous scaffolds are selected as tissue scaffolds because of their extracellular matrix nature and biocompatible properties. However, two major issues are involved with regard to this nanofibrous scaffold. The first is that crosslinking of the scaffold is necessary to avoid lysozyme degradation in an aqueous environment. The second is that topological cues are necessary to guide fibroblast migration. These two issues affect the cell behavior and cell fate of fibroblasts including adhesion, proliferation, and migration/infiltration. Autoclaving (physical) and genipin crosslinking (chemical) methods are employed to stabilize chitosan-based scaffolds individually. However, the differences in scaffold microstructure and physiochemical properties induced by the individual or combined crosslinking methods have yet to be investigated systematically. For the first research objective, we investigate the microstructure-properties relationship in the electrospun chitosan/PEO nanofibrous scaffolds with these crosslinking methods. Major scaffold properties including nanofibrous morphology, chemical crosslinks, crystallinity, tensile strength, and hydrophilicity are analyzed by scanning electron microscopy (SEM), Fourier transform infrared spectroscopy (FTIR), x-ray Diffraction (XRD), tensile testing, and water contact angle measurement, respectively. It is found that autoclaving crosslinking improves mainly the structural properties (stiffness and crystallinity), but it also expands the chitosan and PEO network, which significantly enlarges the fiber diameter and causes chitosan chain degradation. Meanwhile, genipin crosslinking improves the physiochemical properties, primarily hydrophilicity, with less improvement on the structural properties. The combined crosslinking significantly improves both structural and physiochemical properties through the unique reorganization of the polymeric network. The intact nanofibrous structure as well as the genipin crosslinks result in confinement for maximal crystallization of chitosan and amorphization of PEO chains. Unfortunately, the combined physical-chemical crosslinking demonstrates the lowest antibacterial activity because of the consumption of amino groups in the crosslinking process. Nevertheless, the scaffold achieves the best resistance to lysozyme degradation and therefore combined crosslinking is preferred over autoclaving or genipin crosslinking alone. For the second research objective, the individual or combined crosslinked thin-film scaffolds are cultured with L929 fibroblasts for 7 days to investigate the effect of crosslinks on cell behavior and cell fate. Single cell behavior (cell and nuclear shape, filamentous actin (F-actin) expression and chromatin condensation) is quantified by confocal fluorescence microscopy. The global cell fate including cell adhesion, proliferation, and infiltration are quantified by SEM imaging, and cell count from the fluorescence imaging, respectively. We categorize the heterogeneous fibroblasts first by location within nanofibers then by cell aspect ratio. We identify subgroups of round, elongated, and infiltrative morphology with increasing cell (nuclear) area. Compared with fibroblasts on uncrosslinked scaffolds, fibroblasts on the combined crosslinked scaffolds have synergistic (greater than the sum of individual methods) cell (nuclear) spreading, F-actin expression, and chromatin decondensation. Fibroblasts on the autoclaving crosslinked scaffolds exhibit a medium increase in the aforementioned parameters. By contrast, fibroblasts on genipin crosslinked scaffolds exhibit longer protrusions, slightly increased cell (nuclear) spreading, yet depressed F-actin expression and chromatin decondensation. By scatter-plotting, we find exponential correlations of the cell and nuclear behavior maximally increased, especially in the infiltrative population, on combined crosslinked scaffolds. This coincides with the reported mechanosensitive coupling of the cytoplasmic and nuclear compartments via cell spreading, F-actin expression, and chromatin decondensation. On the other hand, the correlations decrease in the genipin crosslinked scaffolds despite slight cell spreading (an effect of genipin crosslinks), suggesting that mechanosensing is alleviated by uncrosslinked genipin. It is found that crosslink-induced cell behavior is also associated with cell fate. On the combined crosslinked scaffold, in light of the strongest F-actin expression, maximum cell adhesion and proliferation are detected. However, infiltration is minimized in light of maximized scaffold crosslinks and previously mentioned lowest lysozyme degradation. By contrast, on the genipin crosslinked scaffolds, cell adhesion and proliferation are alleviated in light of depressed F-actin expression. Infiltration is maximized in light of less scaffold crosslinks and higher lysozyme degradation. For the third research objective, we investigate the effect of both structural cue (microrod) and physiochemical cue (genipin) on L929 fibroblast migration. A gradient scaffold with microrod-nanofibrous density is fabricated by electrospinning and post crosslinked with genipin. The radial gradient starts from the middle and increases with microrod-nanofibrous density towards the edge. In 5-day static culture, the microrod-nanofibrous density gradient demonstrates a significant increase in fibroblast proliferation and F-actin expression. By using time-lapse brightfield microscopy scanning the long range gradient (radius of 0.5cm) for 2 hours, we demonstrate that major fibroblasts migrate up the microrod-nanofibrous density gradient with significantly the fastest speed in the edge region. In the middle region, fibroblasts are sensitive to the underlying glass stiffness when the scaffold is thinner and subsequently migrates down the density gradient at the slowest speed. In the region between the edge and the middle, major fibroblasts migrate along the gradient contour at medium speed. Collectively, the microrod-nanofibrous density gradient scaffold exhibits zonal differences in fibroblast proliferation, migration speed, and migration direction. In conclusion, we demonstrate that crosslinking methods produce significantly different microstructures that correlate with significantly different cell behavior and cell fate. In particular, genipin crosslinked nanofibrous scaffold favors more fibroblast infiltration, while combined crosslinked nanofibrous scaffold favors more fibroblast proliferation and adhesion. In addition, microrod-nanofibrous density gradient scaffold is a new topological cue for both fibroblast proliferation and directed migration at the fastest speed. Therefore, both crosslink-induced microstructure and microrod-nanofibrous topology are important considerations in designing tissue scaffold.