Characterization And Preparation Of Regenerated Silk Fibroin/Bam Mats And Hydrogel

As a natural polymer material,regenerated silk fibroin(RSF)is well known owing to the tunable mechanical properties,excellent biocompatibility,biodegradability,and very limited immune response.Electrospun RSF scaffolds retain the good properties of RSF and have unique characteristics,such as large specific surface area,high porosity,and narrow fiber diameter distribution.Therefore,RSF scaffolds have wide applications in biological tissue engineering,which mimicked the structure of extracellular matrix.To improve the bioactivity of RSF scaffolds and promote the proliferation and adhesion of cells,exogenous growth factors had been added into the scaffolds.Compared to exogenous growth factors,natural growth factors are more effective for cell growth.Bladder acellular matrix(BAM)is a natural biomaterial with good biocompatibility and biodegradability,which composed of collagen,laminin,fibronectin,glycoprotein and growth factors.In this thesis,RSF/BAM aqueous solutions were applied as the electrospinning dope to prepare RSF/BAM scaffolds with bioactivity,which combined the growth factors in BAM.Moreover,viscous hyaluronic acid(HA)with good biocompatibility and biodegradability was added into the RSF/BAM spinning dope to further improve the spinnability.The morphology,structure and property of the RSF/BAM/HA scaffolds were characterized by Scanning electron microscope(SEM),Wide-angle X-ray diffraction(WAXD),Fourier infrared spectrometer(FTIR),and Differential scanning calorimeter(DSC).Determination and release curves of the growth factors in the RSF/BAM/HA scaffolds were performed by ELISA kit.The biocompatibility of RSF/BAM/HA scaffolds were investigated by cultivating porcine iliac endothelial cells(PIECs)in vitro.The influence of BAM concentration,total solute concentration of the spinning dopes on the rheological behaviors,conformation,structures and properties were also studied.For the RSF/BAM blended solution,the viscosity and surface tension of the solution reduced,while the conductivity increased with the increasing of BAM content.Overall,the conductivity decreased obviously,whereas the viscosity and surface tension had limited change.With the increasing of BAM content,the fiber diameter decreased and the fiber with wide diameter distribution was easy to be fractured.The RSF/BAM/HA scaffolds showed smoother fiber surface than the RSF/BAM scaffolds.The RSF/BAM/HA scaffolds became compact,while fiber diameter decreased when the total solute concentration ranged from 22.0 wt% to 24.5 wt%.Further increase of the total solute concentration of the spinning dope had limited influence on the fiber diameter and diameter distribution.No evident change of the conformation,crystallinity and thermal stability of the RSF/BAM/HA scaffolds were found,when the total solute concentration varied.Hydrogels have great biomedical application because of variable geometry,tunable strength and porous structure.The cross-linked three-dimensional polymer networks lead the hydrogels promising for drug delivery and tissue engineering.It has been shown that the biological scaffolds composed of BAM can be partially digested with pepsin,solubilized and polymerized to form hydrogel.BAM hydrogel has attracted attention due to its injectability and the ability to fill an irregularly shaped space.The rheological behavior,morphology,and cell response in vitro and in vivo of BAM hydrogel have been characterized by rotational rheometer,micro-rheometer,SEM,FTIR,DSC.The influence of BAM concentration on the properties of the hydrogel was discussed.The precursor solution of 4 mg/mL and 6 mg/mL(BAM 4-P,BAM 6-P)showed gel times of 3.95±0.07 min and 23.77±0.05 min,respectively.However,the precursor solution of 8 mg/mL could not form a gel within the detection time.The new absorption peaks at 985 cm-1 and 870 cm-1 in FTIR spectra indicated that chemical reaction or interaction occurred in the gel process.Compared to the gel precursor solution,the BAM hydrogel showed higher denaturation temperature and thermal stability.The lower BAM content in the hydrogel resulted in a higher denaturation temperature.The three dominant growth factors,VEGF,PDGF-BB,KGF were confirmed to store in the both RSF/BAM/HA scaffolds and BAM hydrogels by ELISA kit.In the RSF/BAM/HA scaffolds,the three growth factors can be released for 16 days.VEGF had the maximum release amount with the most rapid release rate among the three growth factors.In the BAM hydrogel,the three growth factors have the same phenomenon of burst release in the release process.The release amounts increased evidently in the first two days,and declined gradually with a consequent stable release in the following days.BAM 4-G had better release effect than BAM 6-G with more release amounts of the three growth factors.The existence of growth factors in the scaffolds promoted the cell proliferation and adhesion.PIECs had better cytoskeletal structure in the RSF/BAM/HA scaffolds and the BAM hydrogel than control groups.Moreover,PIECs could not only adhere on the surface of scaffolds but also grew inside the scaffolds,which might accelerate the tissue regeneration.