Preparation And Property Of A Silkworm Pupa-derived Chitosan Compound Haemostatic Sponge

In the first aid of war wound and daily surgical treatment, excessive blood loss is the main cause of death. At present, the domestic hemostatic material all have its own shortcomings, and the foreign hemostatic material are expensive. Chitosan is an ideal material to make dressings because of its excellent hemostasis property, antibiotic property and biocompatibility. Chinese silk resources are. abundantly. A lot of chitosan resources are contained in the by-products of silkworm reeling, and silkworm pupa-derived chitosan also has some better performance than that derived of shrimp and crab. Therefore, a composite sponge-like haemostatic material was prepared using silkworm pupa-derived chitosan as the major ingredient which was mixed with gelatin, calcium chloride, glutaraldehyde, and glycerol by means of freeze vacuum drying. The single factor experiment and the orthogonal experiments are used to test its Blood Clotting Index (BCI), its hemostatic performance at rabbits, its promote wound healing properties, antibacterial properties and toxicology biological safety performance. The main results are reported as follows:1. Chitin and chitosan was extracted from silkworm pupa, and the silkworm pupa-derived chitosan with different degree of deacetylation was extracted by controlling the reaction time. The highest degree of deacetylation can reach93.13%. A composite sponge-like haemostatic material was prepared using this silkworm pupa-derived chitosan as the major ingredient which was mixed with auxiliary agents by means of freeze vacuum drying. The sample has flexibility texture with rich and uniform pore in the surface and the internal.2. The single factor experiment was studied by the blood clotting test. Experiment results showed that the best proportion of dosage was:Pre frozen1h at-40℃. The results of orthogonal experiment showed that the silkworm pupa-derived chitosan compound sponge, which is made by 2.5%chitosan,3.0%gelatin,0.005%glutaraldehyde.0.1%calcium chloride.0.05%glycerol, has the best haemostatic property. The sequence affecting the BCI is that:chitosan> calcium chloride> glutaraldehyde> gelatin> glycerol.3. The key of leading the silkworm pupa-derived chitosan compound sponge’s haemostatic property is the concentration of chitosan. The sample made by silkworm pupa-derived chitosan has better haemostatic property than made by chitosan derived of shrimp and crab. The excellent haemostatic property of silkworm pupa-derived chitosan compound sponge was proved by the Blood Clotting Index and the model of rabbit ears arterial hemorrhage. Its haemostatic property was better than hospital gauze with the BCI reduced by62.71%, the blood loss reduced by92.08%, the haemostatic time reduced by80.55%. Its haemostatic property was better than hospital gelatin sponge with the BCI reduced by35.61%, the blood loss reduced by88.35%, the haemostatic time reduced by57.01%.4. The healing function of silkworm pupa-derived chitosan compound sponge was studied by the experiment of rabbit wound model. The results showed that the sample had obvious wound contraction. The wound areas are smaller than the blank group and the healing rates are taller than the blank group at the3,7,14,21d. The results of tissue section examination showed that the silkworm pupa-derived chitosan compound sponge had better wound contraction than the blank group with active growth of tissues, less inflammatory cell infiltration and no ulcer.5. The antibacterial properties of silkworm pupa-derived chitosan compound sponge tested by inhibitory rate and bacterial inhibition diameters with Escherichia coli and Staphylococcus aureus. The results showed that the hospital gelatin sponge had no antibacterial properties. The inhibitory rate of silkworm pupa-derived chitosan compound sponge was100%within72h to Escherichia coli and Staphylococcus aureus. Its bacterial inhibition diameters reached1.8-2.1cm to Escherichia coli and1.6-1.8cm to Staphylococcus aureus.6. According to the national standard, the toxicology test was used to study the silkworm pupa-derived chitosan compound sponge, including hemolysis test, primary skin stimulation test, intradermal injection test, heat source test and short-term systemic toxicity test. The results showed that the silkworm pupa-derived chitosan compound sponge is an excellent hemostatic material with good biocompatibility. The result provides basic feasibility theory for further research on the silkworm pupa-derived chitosan compound sponge.