| The body’s natural mechanisms can not resist severe bleeding caused by trauma or surgery,therefore the additional way to control bleeding was required. While in the current market effective hemostatic agents generally are expensive, low efficacy and lead to a variety of security issues. So, the development of rapid, cheap effective and non-toxic hemostatic materials has become an important resrearch topic in the field of medical materials.Silk fibroin, as a natural macromolecular material derived from silk, with the properties of readily available raw materials, non-toxic, non-irritating, biodegradable and so on, has been widely uesd on food additives, cosmetics, biosensor, drug controlled release materials, tissue engineering and other fields. In addition, silk fibroin can also be processed into a variety of shapes to meet all kinds of needs, such as membranous, spongy, gel, powder and so on. Now its applications in biological has been developed for so many years, while as a hemostatic material its effect is not perfect. So there is a potential market value to study the silk-based hemostatic material and improve its hemostatic effect.Mesoporous materials as a new material, have been received extensive attention due to its uniform mesoporous pore size, regular nanopore, enormous surface area and pore volum, good biocompatibility and strong adsorpotion etc. It is also a potential hemostatic material. However,as most of the shape of mesoporous material is powder, which cannot be well adhere to the wound, so it can be easily slide, meanwhile the materialcan release amout of heat, then the skin will burnt during the course of hemostasis as it can absorb lots of water from the tissue. So there are stil many problems to be resolved.To solve these problems, in this study, the freeze-drying method was used to prepare the cavernous silk fibroin membrane materials, the relationship between physical and chemical properties of silk fibroin and its hemostatic performance was studied, then silk fibroin and mesoporous bioactive glass composite material was prepared, and its hemostatic ability in vivo and in vitro and cytotoxicity experiments were conducted. The main contents and conclusions are as follows:1、The Alcalase was used to hydrolysis silk fibroin, the physicochemical properties of silkpeptide under different amount of enzyme was studied, and the functional group changes,aggregation structure and surface morphology were characterized by Fourier transform infrared(FTIR), X-ray diffraction(XRD) and scanning electron microscopy(SEM). The in vitro and in vivo hemostatic experiments were prepared by the activated partial thromboplastin time(APTT), prothrombin time(PT) and rat liver injury model. The results showed that: the aggregation structure was mainly α-hellix with a small amount of β-sheet. The surface morphology of silk prepared by freeze-drying method was mainly spongy structure with wrinkle porous, what’s more, with the increase amount of Alcalase, the silk peptide was changed from sheet to spherical then to sheet, and the porosity was also decreased. The hemostatic experiment results showed that silk peptide hemostatic material are mainly promoting the endogenous coagulation system and when compared with the gelatin sponge, the silk peptide material can singnificantly shorten the bleeding time, and the blood loss was also reduced, so we can say that silk peptide can be used as an effcient hemostasis material.2、The sol-gel merhod was used to synthesis the mesoporous bioactive glass, and freeze-drying method was adopted to prepare the silk fibroin/mesoporous bioactive glass composite material. The pore structure of the mesoporous bioactive, the functional group changes, surface morphology and porosity of the composite material were characterized by Transmission electron microscope(TEM), N2 adsorption- desorption analyzer, FTIR, SEM and porosity testing method. The TEM results showed that: the mateial has a uniform honeycomb tunels pore structure, and all of the tunels are highly ordered. The BET results showed that the spedific surface area was 400m2/g, and the average pore diameter was 7.3nm, which illustrate that the synthesis of bioactive glass have a pore structure of mesoporous scales and a large specific surface area, and belongs to the mesoporous category. SEM results showed that the mesoporous bioactive glass and silk fibroin was successfully compounded in physical, and the membrane material was presented “rime”-like structure. When the amount of mesopourous bioactive glass was increased, the prosity was decreased on the contrary.3、The in vitro and in vivo hemostatic experiments of silk peptide and the composite material were prepared by APTT, PT and rat liver injury model, the biocompatibility study was conducted by in vitro cytotoxicity experiment. The results showed that silk-based hemostatic material are mainly promoting the endogenous coagulation system. When compound withmesoporous bioactive glass, the hemostasis effect was significantly increased, the bleeding time and blood loss were 123±7.32 s, 0.296±0.02 g respectively, which was 10.87% shorted as for the bleeding time and 8.11% reduced for the blood loss compared to the silk peptide. In vitro cytotoxicity shows that the material is non-toxic side effect on cells, and the cell level is acceptable.4、The silver ions was loaded on the composite material by ion exchange method. The functional group, surface morphology and silver ion load capacity were characterized by SEM and Elemental analyze(EDS). The hemostatic experiment was conducted by in vivo animanl model, and the in vitro antibacterial effect was tested by inhibition rate method. The SEM results showed that: the composite material has a uniform pore structure, and a little siver particles adhered to the surface can be seen. The EDS results showed that the silver efficiency was more than 15% of all the materials. The liver injury experiment showed that when compared to the unload silver ion material(123±7.32 s, 0.296±0.02g), the hemostatic ability was significally improved when the silver was added(100.23±7.3s, 0.285±0.023g), with a 22.72% shorted in bleeding time and 3.86% reduced for blood loss. The antibacterial experiments show that when the silver ion concentration 0.15mol/L, the E coli and Staphylococcus aureus hardly grow, when the concentration was 0.2mol/L, the medium surface has no colonies at all. So we can say that the silver fibroin/mesoporous bioactive glass composite sponge has a good antibacterial ability.5、In summary, the silk fibroin material has a certain hemostatic effect, and the hemostatic get better when compound with the mesoporous bioactive glass. The antimicrobial performance became more obvious after the silver ion was loaded, and the in vitro cytotoxicity shows that the material is non-toxic side effect on cells, exhibit excellent biocompatibility, so it is expected to be developed as a new type of rapid hemostastic material. |
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