Tough And VEGF-releasing Scaffolds Composed Of Artificial Silk Fibroin Mats And Natural Acellular Matrix

Tissue engineering aims at developing functional substitutes for damaged or diseased tissuesand organs. Electrospinning has emerged to be a simple and effective technique to fabricate tissueengineering scaffolds, which were widely used in tissue engineering.Silk fibroin (SF) from Bombyx mori has been investigated widely by electrospinning tofabricate tissue engineering scaffolds. The mechanical properties of most electrospun SF scaffoldsare still poor, although people have tried many ways to reinforce the scaffolds, such as usingorganic solvents, adding some reinforcing agents, post-treated with different methods, or using thecylinder to collect alighed SF fibers instead of the random fibers. Bladder acellular matrix graft(BAMG), a tough natural material, was already used in urethral reconstruction, penilereconstruction, myocardial repair, esophageal repair and fascial tissue reconstruction. In this study,we used BAMG to toughen the electrospun SF scaffolds with random or aligned fibers.Considering the essential role of vascularization in the success of tissue regenerating, we fabricateSF scaffolds loaded VEGF (Vascular endothelial growth factor), which is essential for tissuevasculogenesis and angiogenesis, by blend and coaxial electrospinning.Compared with the conventional SF mats, the SF/BAMG composite scaffolds weresignificantly reinforced. The morphology, microstructure and biocompatibility of the preparedscaffolds were characterized by Scanning Electron Microscopy (SEM), Transmission ElectronMicroscopy (TEM), Fourier Transform Infrared Spectroscopy (FTIR), Wide angle X-raydiffraction (WAXD), Capillary Flow Porometer, Instron5969material testing machine and LaserScanning Confocal Microscope (LSCM). An Elisa kit was adopted to investigate the releasebehaviors of VEGF loaded in the scaffolds.In order to find the best parameters of coaxial electrospinning, the concentration of the SFaqueous solutions and the flow rates of the core and sheath spinning dopes were studied. Resultsshow that when the concentration of the SF aqueous solutions was39wt%and flow rates were0.3and1.2mL/h for core and sheath electrospinning dopes, we can gain SF fibers with stablecore-sheath structure. The voltage applied was20kV and the distance from the spinneret tocollector was10cm. The electrospinning surroundings were5±5oC and50±5%relativehumidity. For aligned fibers collected, the rotating speed of the cylinder was2000r/min whileother conditions were kept constant for SF mats.The best conditions of water vapor annealing were37oC,90%relative humidity, andpost-treated time of36h. The breaking energies of the composite scaffolds ranged from458to970J kg-1. The breaking strength and elongation at break of the blend and coaxially electrospunSF/BAMG composite scaffolds with random fibers reached14.4MPa and12.0%,12.9MPa and10.7%in dry state, respectively. In wet state, the breaking strength and elongation at break of theblend and coaxially composite scaffolds were3.9MPa and53.7%,3.3MPa and48.2%,respectively. These were much better than the bare SF scaffolds electrospun in the same conditions.The suture retention strength of the composite scaffolds was reinforced ranging from4.1N to4.3N in dry state and2.0N to2.3N in wet state, respectively. These satisfied the requirements for suturing in tissue engineering (2N).For the composite scaffolds with aligned SF fibers in multiple layers collected by the cylinder,the breaking strength and breaking elongation of the blend and coaxially electrospun compositescaffolds annealed in water vapor were22.7MPa and18.8%,21.0MPa and13.2%in dry state,respectively. In wet state, the breaking strength and breaking elongation of the blend and coaxiallyelectrospun composite scaffolds were5.9MPa and87.2%,5.5MPa and84.5%, respectively.These were higher than those of the composite scaffolds with random fibers. In addition, thesuture retention strength of the blend composite scaffolds was4.3N and1.9N in dry and wetstates, respectively. For the coaxially electrospun composite scaffolds, it is4.1N and1.8N in dryand wet states, respectively.The cumulative release profiles of VEGF from different scaffolds indicate that VEGF wassuccessfully loaded and released for more than2weeks. Comparing the two types ofpost-treatments, we found that water vapor annealing was a much better method than immersionin ethanol to keep VEGF alive and reinforce the SF/BAMG composite scaffolds. The proliferationof PIECs (porcine iliac endothelial cells) cultured on the scaffolds proved that both the randomand aligned-fiber scaffolds had a good biocompatibility. MTT assay indicates that VEGF-loadedscaffolds could promote PIECs to adhere, spread and proliferate better than their counterpartswithout VEGF. In addition, the well-aligned fibers are suitable to guide the cell growth withdesired anisotropy. Furthermore, PIECs could not only grow on the surface of the scaffolds, butalso infiltrate into the mats. In a word, these SF/BAMG electrospun composite scaffolds weresuccessful to deliver and release VEGF in a sustained manner, and could be considered asattractive candidates for tissue engineering applications.