The Novel Hydrogel Membranes Of Silk Fibroin And Pupa Chitosan Derived From Silkworm Bombyx Mori And Their Characteristics In Vivo And In Vitro

In this paper, we used the silkworm cocoons(Bombyx mori) as experimental material. Silk fibroin derived from the cocoons was treated with various degumming and dissolving methods to process various kinds of test material, degummed fiber, liquid fibroin and regenerated silk fibroin membrane. The effect of the silk degumming and fiber dissolution system on the molecular structure and properties of silk fibroin were studied in detail. On this basis, we developed a novel method for the preparation of the silk fibroin hydrogel membrane(FHM). The effect of the regenerated liquid silk fibroin and electrophoretic deposition conditions on the formation, mechanical and structural properties of the FHM were studied in detail. Besides, in vitro and in vivo experiments were tested to evaluate its biological safety. In addition, we extended this improved electrophoretic deposition technology to pupa chitosan and prepared the chitosan hydrogel membrane(CHM) which was smooth, soft and transparent. Similarly, mechanical and structural properties and biological safety of the CHM were also tested.The main results are listed as follows:(1) Silk fibroin derived from cocoons of the female silkworm(Bombyx mori) was made into three kinds of test material, degummed fiber, liquid fibroin and regenerated silk fibroin membrane. The effect of the silk degumming and fiber dissolution system on the molecular structure and properties of silk fibroin were studied in detail. Degumming with Na2CO3 solution had the greatest impact on silk fibroin fiber, resulting in significant reduction in the thermal stability and mechanical properties. The thermal decomposition temperature of the degummed fiber was 7?C lower than that of the fiber degummed with urea buffer. The breaking stress, breaking energy and elongation decreased 37%, 62% and 45% than those of the fiber degummed with 8 M urea separately. The moderate degumming solution was urea buffer followed by strongly alkaline electrolyzed water. SDS-PAGE showed that the silk fibroin degummed with 8.0 M urea at 80?C and dissolved in 9.3 M LiBr at 25?C was similar to the natural silk fibroin in vivo, but boiling in Na2CO3 solution leaded to serious breakage of the silk fibroin peptide chain. The degree of breakage of the peptide chain was positively correlated with the storage time of liquid silk fibroin. DSC showed the more nearly intact the silk fibroin peptide chain, the higher the thermal decomposition temperature. Besides, treatment with methanol increased the glass transition temperature and the thermal decomposition temperature of the regenerated silk fibroin membranes. XRD indicated that dipping in methanol could induce the characteristic diffraction peaks of β-sheet crystalline structure and improve the silk fibroin membranes’ crystallinity significantly. The results showed that the silk fibroin degumming method had more important impact than the dissolving method on the regenerated silk fibroin molecular structure and its properties.(2) Regenerated silk fibroin solution means the silk fibroin solution prepared after degumming and dissolving procedure which can be made into different kinds of biomaterials. Regenerated silk fibroin is metastable and can turns into hydrogels in response to acid, ion and other additives. We used the silk fibroin degummed with 8.0 M urea at 80?C and dissolved in 9.3 M LiBr at 25?C as material and prepared the FHM by a novel method. The hydrogel membrane was formed on a nanoporous film as a barrier on the principle of electrophoretic deposition by our home-made device. The experimental setup consisted of two electrodes made of platinum wire. The negative electrode(cathode) in the inner chamber and the positive electrode(anode) in the outer chamber were separated by the nano-membrane and its support. When the regenerated silk fibroin solution was added into the reservoir with negative charge and the silk molecules migrated toward the positive charge at a higher DC voltage, the FHM formed on the barrier film. The results showed that a smooth, semi-transparent and soft membrane could be prepared under 80 VDC and Tris buffer(pH 6.55-7.55). The barrier film with a MWCO of 10 kDa was favorable to the formation of the FHM and the recovery of the FHM was as high as 97.4%. The FHM with the swelling ratio of 1056.4% was predominantly a mixture of β-sheets and α-helix crystalline structures. SEM observation showed that the FHM characterizes 3D mesh structure woven by a chain of silk fibroin nanoparticles with size of about 30 nanometers as a pearl necklace. In vitro tests indicated that the FHM was degradable and could satisfy the cell adhesion and growth requirements. Although these scaffolds demonstrated a slight initial pro-inflammatory response, the amount of inflammatory cells decreased at the forth week and returned to baseline control values at the sixth week. The expressions of TNF-α and IL-6 had no significant difference between the experimental and control groups. Therefore, the FHM is a promising candidate for loading bioactive protein and appropriate cells, artificial skin or using for transplantation.(3) Silkworm pupa shells contain 33%~44% of chitin. The chitin which is extracted from silkworm pupa shells can prepare chitosan by degrease, decolorization, deproteinization and deacetylation. Chitosan can be made into gel system by using different methods. Here, we report a novel pupa chitosan hydrogel membrane which was processed by improved electrophoretic deposition technology. The inner chamber was changed into the anode chamber and the outer chamber was changed into the cathode chamber accordingly. When chitosan dissolved in mildly acidic aqueous solution was added into the anode chamber and the chitosan molecules migrated toward the negative charge at 60 VDC, the CHM formed on the barrier film. The maximal recovery of 81.8% could be achieved after electrophoretic deposition for 1 h. The transparent CHM with the elongation of 42.46% was a mixture of type I and type II crystal structures. SEM studies revealed that the CHM had an irregular net structure. In vitro studies indicated that the CHM was sufficient for L-929 mouse fibroblast cells adhesion and growth. To demonstrate immuno-compatibility with host tissues, H&E and TGF-β1 were observed and the expressions of TNF-α and NF-κB were measured up to 3 weeks post-implantation. Although these scaffolds demonstrated an initial pro-inflammatory response, the amount of inflammatory cells and the expressions of TGF-β1 returned to baseline control values at 3 weeks. The expressions of TNF-α and NF-κB had no significant difference between the experimental and control groups. In vivo animal experiments showed that the CHM didn’t incite serious inflammatory reactions. Thus, the CHM is a promising medical biomaterial candidate for loading appropriate cells, as artificial skin or for use in transplantation.In conclusion, this paper studied the effect of the silk degumming and fiber dissolution system on the molecular structure and properties of silk fibroin in detail. In addition, we developed a novel method for the preparation of the FHM and the CHM. Besides, in vitro and in vivo experiments were also tested to evaluate its biological safety. The results of this paper help us gain a deeper understanding of the effect of silk degumming and fiber dissolution on the molecular structure and properties of silk fibroin and provide important experimental data for screening and using silk protein and pupa as advanced functional biomaterials. Besides, the improved electrophoretic deposition method we develop may establish the certain foundation for tissue engineering medical materials.