Fabrication And Optimization Of Biomimetic Composite Nanofibers Of Gelatin/PCL

As an emerging field in repair and reconstruction of tissues and organs, tissue engineering (TE) is believed to have far-reaching impact on the development of modern medicine. Among different efforts in TE, preparation of biomaterial scaffolds has always been one of the most important research areas. With the capability of producing nanofibrous scaffolds to mimic the natural extracellular matrix, electrospinning has become a pretty hot research topic in recent years. In order to impart balanced properties into electrospun nanofibers, preparation of biodegradable composite nanofibers made from natural and synthetic polymers, such as the representative gelatin /polycaprolactone (PCL) system has been widely practised by researchers. Since electrospinning of Gelatin/PCL composite nanofiber was reported for the first time in 2005, such composite nanofibers have been widely used for engineering tissues like skin, blood vessels, cartilage, bone, nerves and others. However, as the gelatin is highly hydrophilic and PCL is highly hydrophobic, admixture of the two will inevitably give rise to phase separation problem, which has received little attention so far. To address the noted problem, the aims of this study are first of all to study phase separation phenomena of Gelatin/PCL and its effects on the electrospinning process, and then to propose a possible solution to fix the problem, and finally to assess in vitro the biocompatibility of the composite nanofibers with improved phase homogenicity.Firstly, we dissolved a polymer hybrid of Gelatin/PCL (50:50) in trifluoroethanol (TFE) to examine the phase separation where a tiny amount of fluorescein isothiocyanate (FITC) conjugated gelatin was introduced to enhance observation contrast upon the two phases segregated. It was found that high loadings of gelatin in the solution observed more precipitate settled down. Additionally, dynamic light scattering experiments also confirmed the phase separation characteristic in this mixed solution. FTIR analysis of the sediment proved the identity of gelatin precipitate. Then, we probed the influence of phase separation on fiber morphology during electrospinning a prior homogeneously blended Gelatin/PCL (50:50) solution by collecting samples at predetermined time points followed by SEM observation. It was found that in the first 2 hours of electrospinning, the produced gelatin/PCL fibers are very smooth and homogeneous; however, as spinning time last longer, the fibrous structure became coarse with fiber diameters increased larger and contained many large splash defects. Further, FTIR quantitative analysis indicated that the ratio of gelatin to PCL in the electrospun gelatin/PCL nanofibers altered with collecting times. Nanofibers collected at 2.5 h contained the minimum gelatin (30%), whereas the amount of gelatin in the composite fibers increased steadily to about 65% after electrospinning for 5h.By addition of a tiny amount of acidic acid (HAc) to the mixed solution of gelatin/PCL was found to be a feasible approach to form a homogenous and transparent hybrid solution, which was confirmed by the previous examination methods. SEM results showed that the fiber diameter produced from the HAc mediated phase homogenity is smooth, uniform and finer with fiber diameter estimated to be 379±146 nm, a half of its original. The content of gelatin in the compounded nanofibers showed consistent at the level of 50%. Finally, we inspected the performance of the modified composite nanofibrous membrane of gelatin/PCL by measurement of its wettability and tensile properties before and after the modification. It was found that the modified composite nanofibrous membrane had much better hydrophilicity and the mechanical properties were greatly improved, with the average breaking strength around 2.96 MPa in comparison to the unmodified one of only 1.56 MPa.Lastly, using the GFP mouse fibroblast as a model cell type, we examined biocompatibility of the composite nanofibrous membrane of gelatin/PCL before and after modification. MTT assay revealed that there was no significant differentces in adhesion and proliferation of the fibroblast cells, but laser scanning confocal microscope (LSCM) examination showed better growth and more adhesion of fibroblasts on the modified gelatin/PCL membrane scaffolds. SEM observation of the cell-scaffold constructs indicated more cells with well spreaded morphology after one day of culture on the modified fibrous scaffolds. An even better cell-scaffold interaction could be observed after 7 days of culture, exhibiting much better cell-cell and cell-fiber integration. All these results suggested that the modified nanofibrous membranes of gelatin/PCL are of good biocompatibility compared to the non-modified counterparts.