Enzymatic Preparation Of Wool Keratin And Its Application In Composite Hemostatic Materials

The uncontrollable bleeding caused by trauma in wars,traffic accidents and surgery resulted in a high fatality rate.Traditional hemostatic materials,such as bandages and hemostatic accessories,have a good hemostatic effect on external wounds,however,they are not ideal for hemostasis of uncontrollable bleeding or bleeding from incompressible sites.Therefore,it is of great significance to develop new biomaterial with rapid hemostatic properties,excellent biocompatibility,and biodegradability.Wool keratin and chitosan are of nice coagulation properties,biodegradability,noncytoxicity and good biocompatibility.In this study,wool keratin and chitosan were served as the basic materials to prepare porous and spongy Ker/CS composite hemostatic material by freeze-drying methods.In addition,the preparation technology,physicochemical properties,coagulation effect in vitro and in vivo,coagulation mechanism,cytotoxicity and biocompatibility of this hemostatic material were evaluated.The main research contents of this paper are as follows:（1）Preparation of wool keratin/chitosan composite hemostatic material.The enzymatic preparation process of active wool keratin was established.The wool was treated with 300U m L^-1 of keratinase,and it was degraded under p H 10 and 40℃for 12 h.A large fragment of wool keratin between 25～35 k Da k Da was obtained.Subsequently,the keratin was used as the raw material,chitosan and auxiliary components were added to prepare the composite gel sponge,and the optimal preparation parameters with tannic acid and genipin as the cross-linking agent were obtained respectively.When 0.8%tannic acid was selected as the cross-linking agent,the optimized ratio is:keratin 4.0%,chitosan 4.0%,calcium chloride 20%and glycerol30%;when 0.8%genipin was selected the cross-linking agent,the optimized ratio is:keratin6.0%,chitosan 3.0%,calcium chloride 20%and glycerol 70%.Two kinds of composite sponges were prepared under the above conditions,and their in vitro coagulation index BCI was 30.83±3.34%（tannic acid cross-linking）,44.67±2.37%（genipin cross-linking）,so tannic acid was used as the cross-linking agent.（2）Properties of wool keratin/chitosan composite hemostatic material.The Ker/CS sponge cross-linked by tannic acid under the optimal preparation conditions has a uniform porous spongy structure with a porosity of 90.1±2.17%and a pore size ranging from 10μm to 100μm.The fourier transform infrared spectroscopy（FTIR）charaterization proved that the cross-linking of tannic acid resulted in the formation of a strong hydrogen bond force between keratin and chitosan,and the differential thermal gravity（DTG）curve indicated the thermal stability was improved.Ker/CS sponge could absorb whole blood of 34.23±1.85 times as its own weight,which showed that it has a strong ability to absorb liquid,and the number of adherent platelets and red blood cells is significantly higher than that of commercial gelatin sponge.Further research by the activated partial thromboplastin time（APTT）and prothrombin time（PT）tests showed that Ker/CS sponge exerted coagulation effect through the intrinsic coagulation pathway including the activation of FⅫ,followed by activation of FXⅠ,FⅠX and FⅧ,to the activation of FX.The hemolysis rate of Ker/CS sponge was less than 5%,indicating that the sponge owned good blood compatibility,and and the CCK-8 method proved that Ker/CS sponge had no potential cytotoxicity.（3）In vivo evaluation of wool keratin/chitosan composite hemostatic material.The hemostatic effect of Ker/CS sponge on rat tail-docking hemostasis and rat liver injury hemorrhage were better than or comparable to that of commercial gelatin sponge.Subcutaneous implantation experiments in the back of rats showed that the Ker/CS sponge could be completely degraded within 4 weeks.The results of enzyme linked immunosorbent assay（ELLSA method）showed that the pro-inflammatory cytokines interleukin-1β（IL-1β）,interleukin-6（IL-6）and tumor necrosis factor-α（TNF-α）of the Ker/CS sponge group were not expressed abnormally,which indicating that the implantation of Ker/CS sponges caused no significant inflammatory response.