Study On Starch Gel Hemostatic Materials Fabricated By Regioselective Oxidation Coupled Hot Extrusion 3D Printing

Uncontrolled hemorrhage caused by traffic accident,natural disaster,surgical trauma and war has become a global concern of health problems.There is an urgent need to develop the fast,effective and safe hemostatic materials.Natural polymer-based hemostatic materials with excellent biocompatibility and biodegradability are popular and become the research frontier and hotspot in the field of modern hemostatic materials.Motivated by this,this thesis proposed the oxidized starch gel hemostatic materials constructed by regioselective oxidation coupled with hot extrusion 3D printing.Regioselective oxidation of Laccase-TEMPO system and its regulating mechanism of starch water absorption were established.The oxidized starch gels with high water absorption performance was constructed by hot extrusion 3D printing.After the relationship between hot extrusion 3D printing and material concentration-oxidized starch gel material network structure-water absorption performance was illuminated,the properties of oxidized starch gel hemostatic material were further improved by adding functional factor Ca²⁺.The hemostatic and wound healing functions of the material were evaluated by in vitro simulation and animal experiments.The specific results and conclusions are as follows.The effects on regioselective oxidation of starch by Laccase-TEMPO-system and its water absorption were investigated.The results showed that the Laccase-TEMPO system could selectively oxidize the primary alcohol hydroxyl group at the C6 position on the dehydration glucose unit of starch to prepare the oxidized starch with complete pyran ring structure.The suitable oxidation condition was as follows:laccase of 6%w/w,TEMPO of 15%w/w,reaction time of 12 h,reaction p H of 5 at room temperature.Regioselective oxidation of starch by Laccase-TEMPO system could lead to partial destruction of starch crystalline structure,nano-aggregates and short-range ordered structure,and degradation of molecular chains,but the destruction and reduction degree of molecular weight was much lower than that of sodium hypochlorite oxidized starch with similar carboxyl group content.The reaction process of regioselective oxidation of starch by Laccase-TEMPO system was as follows.Firstly,laccase oxidized TEMPO containing ammonium oxides（-N=O˙）groups.And then ammonium oxides reacted with the hydroxyl group of primary alcohol at C6 position on the starch dehydrated glucose unit,which was further oxidized to carboxyl group.Regioselective oxidation of Laccase-TEMPO system could effectively improve the water absorption performance of starch,which increased with the increase of oxidation degree.The enhanced water absorption mechanism is due to the introduction of hydrophilic carboxyl groups into starch molecules and the induction of the disordered transformation of starch multi-scale structure,which makes water molecules more easily migrate to the inside of starch molecules.The preparation of high-water absorption oxidized starch gel material by hot extrusion 3D printing technology was investigated.The influence of starch concentration on the rheological properties of oxidized starch were investigated,and the results showed the suitable concentration range for hot extrusion 3D printing was 11-19%.Hot extrusion 3D printing results in the complete destruction of crystalline lamellar structure of oxidized starch and the formation of a stable gel network structure.The Mt value of oxidized starch gel material were1213.71-724.05%within 10 second,and the Mt value at water absorption equilibrium state was increased by 1200.92-597.63%compared with that of oxidized starch.However,with the increasing concentration of oxidized starch,the inhomogeneity of the gel system increased,and the aggregation and winding between starch molecular chains increased,which made the mesh structure smaller and compact,thus reducing the space for binding water molecules,and decreasing the water absorption performance.After absorbing water,the oxidized starch gel materials still showed the elasticity and the intact network structure,indicating good water-holding properties.Oxidized starch gel materials with different concentrations had good biodegradability and could be degraded within 120 min.According to the correlation analysis between hot extrusion 3D printing and material concentration-oxidized starch gel material network structure-water absorption performance-printability,the best concentration for hot extrusion 3D printing of oxidized starch gel material was 13%.The fabrication of functional oxidized starch gel hemostatic material loaded with coagulation factor Ca²⁺was investigated.The results showed that Ca²⁺did not change the network structure of the oxidized starch gel material,but its volume increased.Ca²⁺could promote the formation of heterogeneous aggregates in the gel system,and its complexation with the carboxyl group in the oxidized starch molecule would lead to the increase of the network structure density and significantly improve the gel strength.The oxidized starch gel material loaded with Ca²⁺still had rapid and high-water absorption performance,but when the Ca²⁺concentration reached more than 10%,the water absorption performance was obviously reduced.In addition,these materials had excellent biodegradability and cell compatibility,and the cell survival rate is higher than 95%.Release test showed that Ca²⁺ions in oxidized starch gel material was slow released by different forms of free,chimeric and complex state.The coagulant effect of the oxidized starch gel material loaded with Ca²⁺was obviously better than that of the original oxidized starch gel material,and the 5%of Ca²⁺concentration was the best performance one.The hemostasis and wound healing effects of oxidized starch-based gel hemostasis materials（OMS）and Ca²⁺loaded starch-based gel hemostasis materials（Ca OMS）were evaluated by using the mouse back skin injury model.The experimental results showed that mice treated by the Ca OMS group showed no bleeding at the wound site.Its highest healing rate and the fastest weight recovery indicated that the presence of Ca²⁺benefited for wound healing and recovery caused by metabolic disorders in skin injury.The results of EGF,VEGF,TNF-αand FN factors showed that Ca OMS had a stronger ability to promote the growth of epidermis and blood vessels during wound healing,but had no obvious effect on inflammatory response and fibronectin production.The hemostatic mechanism of Ca OMS was mainly through the release of activated Ca²⁺,which could participate in cascade clotting,and combined with the rapid absorption of the fluid composition in the blood to improve the blood coagulation and promote the secretion of skin tissue growth factor（EGF and VEGF expression）.This paper has good innovation,academic value and practical significance.The regioselective oxidation of starch by Laccase-TEMPO system could provide a new way and green processing method for the preparation of novel oxidized starch.Besides,the research results on the construction of high-water absorbent oxidized starch gel materials by hot extrusion 3D printing technology could provide new ideas and new design methods for the preparation of high-water absorbent starch gel materials.The research results on the construction of oxidized starch gel hemostatic materials also could provide theoretical basis and technical support for the creation of safe and high-performance functional starch gel hemostatic materials.