Fabrication of three-dimensional nanofibrous gelatin scaffolds using one-step crosslink technique

Electrospun nanofibers have been considered to be an ideal scaffold for tissue engineering, because of the extracellular-matrix-like structure and the well-controlled fabrication. Here, a new method was used to fabricate electrospun three-dimensional macroporous nanofibrous gelatin scaffolds in ethanol bath by one-step crosslink with glutaraldehyde. The mean diameter of the one-step crosslinked fibers was significantly smaller than that of the traditional two-step crosslinked fibers (p < 0.05), and scaffolds prepared by one-step crosslink were fluffy and porous. No significant difference was found in the degradation rates for both fibers within 14 days. After immersion in PBS for 14 days, numerous two-step crosslinked fibers merged together. By contrast, the morphology and macroporous structure of one-step crosslinked fibers showed no evident change and were generally maintained. Approximate crosslinking degrees of the two-step and one-step crosslinked gelatin fibers were 40% and 54%, respectively (p < 0.05). Results from fluorescence microscopy and hematoxylin-eosin staining showed that MC3T3-E1 subclone four cells were distributed more evenly and diversely in the one-step crosslinked fiber scaffolds. The one-step crosslinked fibers enhanced the proliferation and differentiation potential of MC3T3-E1 cells. Furthermore, one-step crosslinked fibers were beneficial in repairing defects in the skulls of rats. Thus, one-step crosslink by glutaraldehyde in ethanol bath is a cost-effective and simple method to fabricate three-dimensional macroporous nanofiberous scaffolds. This technique retains the morphology and structure of the gelatin fibers, and enhances the biological performance of scaffolds in vitro and in vivo.