Study On Structural Regulation And Hemostatic Mechanism Of Hydrolyzed Silk Fibrion

In the war and daily life,it is hard to avoid sudden traumatic bleeding,and rapid hemostasis is important during the emergency medical treatment.The previous researches indicated that the common hemostatic materials like zeolite,gelatin sponge,chitosan,oxidized cellulose,etc,have some disadvantages such as low biocompatibility,susceptible to infection,complicated preparation,high price,and slow hemostasis,etc.Silk,a kind of natural polymeric protein fiber,possesses some advantages like non-toxicity,low inflammatory reaction,biodegradability and easy processing,which has been widely used in medical products such as biosensors,drug release carrier,tissue engineering.Nevertheless,there are only a few reports on the preparation of silk fibroin hemostasis products.It is thought that the application of silk fibroin on the hemostatic materials may not only improve the hemostatic properties and biocompatibility,but also provide new methods and theoretical basis for the preparation of the silk fibroin hemostatic materials and the related hemostatic mechanism.In our study,with the silkworm cocoon as the raw material,we applied different methods to hydrolyze silk fibroin macromolecules under various conditions,focusing on the correlation and synergistic effects among structure,molecular weight,solubility,surface electrical potential and hemostatic properties of the hydrolyzed silk fibroin materials to reveal the related hemostatic mechanism.The detailed researches include the following three aspects:（1）Preparing the hydrolyzed silk fibroin materials by CaCl₂-ethanol-H₂O and enzymes including sing enzymes,two-step enzymolysis and mesoporous bioactive glass composite method,and studying the effects of the different hydrolysis systems and conditions on the surface morphology,molecular weight,solubility and surface potential of the hydrolyzed silk fibroin materials;（2）Applying the in vitro coagulation performance test system and animal liver hemorrhage model to investigate the influences of the structure,molecular weight,solubility and surface potential of the hydrolyzed silk fibroin materials on the hemostatic performances,and reveal the main differences of the hemostatic properties and the possible reasons among different hydrolysis systems and conditions;（3）Analyzing the relationship between the structure and physical&chemical propertiesc of the hydrolyzed silk fibroin materials and the related activated partial thrombin time（APTT）,prothrombin time（PT）,platelet adhesion,platelet factor 4（PF4）content and blood viscosity,illuminating the pathway by which the hydrolyzed silk fibroin materials promote blood coagulation,and revealing the hemostatic mechanism.The results show that:（1）The silk fibroin materials hydrolyzed by CaCl₂-ethanol-H₂O and enzymes have coarse and porous structure,and are sticky,and soluble in water with negative charge in a simulated blood environment.However,the surface morphology,molecular weight,dissolution rate and surface potential of the silk fibroin materials hydrolyzed by different hydrolysis methods are significantly different.Compared to CaCl₂-ethanol-H₂O hydrolysis system,the silk fibroin materials hydrolyzed by the enzymes had clearer honeycomb or pore-like structures,lower molecular weights,faster dissolution rates,and higher electronegativity.Two-step enzymolysis has stronger enzyme hydrolysis ability than one-step method,in which the low molecular weight components and electronegativity increase significantly;（2）The molecular weight,solubility and surface potential of the silk fibroin materials hydrolyzed by CaCl₂-ethanol-H₂O and enzymes are closely related to their hemostatic performances.The lower the molecular weight of the hydrolyzed silk fibroin materials,the faster the dissolution rates,the higher the electronegativity in the simulated blood environment and the better the hemostatic performance have.The enzyme hydrolysis method is preferable to prepare silk fibroin materials of low molecular weight,having lower zeta potential and good procoagulant properties in vitro and hemostatic effect in vivo,in which the silk fibroin materials hydrolyzed by alkaline protease,two-step method and 15%M58S/SF composite materials have better hemostatic effect than Yunnan Baiyao;（3）Coagulation factors,blood platelets,blood viscosity and other indicators are strongly linked with the structure,the physical&chemical properties of the hydrolyzed silk fibroin materials.With the increase of the electronegativity of the hydrolyzed silk fibroin materials,the APTT time decreases,which activates the coagulation factor Ⅻ in blood plasma and promotes the intrinsic coagulation.The more the adhesion of the blood platelets on the surface of the hydrolyzed silk fibroin materias,the more release of PF4 and the stronger hemostatic performance have.The more apparent the honeycomb or pore-like structure of the hydrolyzed silk fibroin materials,the higher of the blood viscosity has,which promotes the formation of the blood coagulation and is beneficial for hemostasis.In particular,the enzyme hydrolyzed silk fibroin materials with lower molecular weight and stronger electronegativity have better adhesion and activation to blood platelets;（4）When the hydrolyzed silk fibroin materials contact with blood,muti-factors promote the hemostasis including activating blood coagulation factor Ⅻ by liquid phase,accelerating the initiation of endogenous coagulation,inducing the generation of thrombin,promoting the adhension and activation of blood platelet by electronegative surface to accelerate the formation of platelet thrombus,and absorbing the liquid substances in the blood to thicken the blood to promote coagulation with the coarse and porous physical structure.