Studies On Preparation Of Water-soluble O-carboxymethyl Chitosan Hemostatic Sponge

Chitosan is the deacetylated derivative of chitin, which has the hemostasis effect by inducing erythrocytes aggregation, platelet activation, activating the blood clotting system and reducing fibrinolytic system. O-carboxymethyl Chitosan (O-CMC) with specific deacetylation degree was synthesized by using carboxymethylation reaction and deacetylation reaction. Then the sponge of main ingredients O-carboxymethyl chitosan, linker glutaral and calcium chloride to active blood clotting mechanism was prepared by using the vacuum freeze-drying technology. Through animal experiment, we expect to confirm that the self producing O-carboxymethyl chitosan hemostatic sponge has better hemostasis efficacy than other common hemostasis materials in clinical at present, it has the superordinary bioaffinity of chitosan. Moreover, the self producing sponge can promote the healing of skin wound with no scar or very few scar.The water-soluble O-carboxymethyl chitosan in the replacement of C6primary hydroxyl was synthesized by using carboxymethylation reaction and deacetylation reaction and ultrasound purification. The structure of the O-CMC was confirmed by IR, X-ray diffraction, and DSC-TG, the result shows that-CH2COOH substitution of product was only limited in the replacement of C6primary hydroxyl and the yield of product was78.14%. The product’s substitution degree and deacetylation degree was determined with the colloid titration method. Its viscosity-average molecular weight was acquired with one point method. The result shows that the substitution degree, deacetylation degree and viscosity-average molecular weight were0.64,44.53%and1.42×104, respectively. The product is soluble in neutral or alkaline water solution, the solubility was4.636g in water, the isoelectric point was4.50, and the pH value of0.5%aqueous solution was7.52.On the base of single-factor experiment, identified the three independent variables extent, with O-carboxymethyl chitosan, glutaral and Calcium chloride as indices factors (10:0.1:5by weight). Established the wounding model by ear vein and artery shearing of the Japanese LargeEar rabbit and femoral artery shearing of the rat, according to the bleeding time and bleeding volume as indexes. By central composite design and response surface methodology mechanism, Design-Expert (7.1.3) experimental design software was adopted to optimize prescription, then the prescription was validated. The results showed that the model of ear vein and artery shearing of the Japanese LargeEar rabbit predicts performance better, the optimum formulation of O-carboxymethyl chitosan, glutaral and calcium chloride were2.49%,1.08%,2.11%.The physics and chemistry nature of the self producing O-carboxymethyl chitosan hemostatic sponge was observed. Its outward appearance is the good toughness, loose texture, porous and white, the internal aperture probably is100～200μm, its porosity achieves above65.11%and water-absorptivity probably is38.29%, the density is0.045g·cm-3. The sponge was put in the physiological saline in8weeks, it does not damage and the shape integrity, so the self producing O-carboxymethyl chitosan hemostatic sponge has the good physics and chemistry nature.The hemostasis efficacy of O-carboxymethyl chitosan hemostatic sponge was observed. The efficacy was verified by two wounding models of ear vein and artery shearing of the Japanese LargeEar rabbit and femoral artery shearing of the rat. Positive control group is to use the commercial absorbable-gelatin sponge, negative control group is to use the medical gauze, and the bleeding time and bleeding volume as optimization indexes. The result shows that the self producing O-carboxymethyl chitosan hemostatic sponge and commercial absorbable-gelatin sponge have significant difference on the bleeding time (P<0.05), moreover the recurring hemorrhage was fewer. Accordingly, we consider that the self producing O-carboxymethyl chitosan hemostatic sponge has better hemostasis efficacy compare to commercial absorbable-gelatin sponge.We give further research to the the self producing O-carboxymethyl chitosan hemostatic sponge of skin wound healing. The Japanese LargeEar rabbit was used to make wound animal model by scalpel shearing. On either side of the wound was covered with the self-producing sponge and commercial absorbable-gelatin sponge respectively. Morphologic and histological sections are applied to study the different effects on wound healing. Results demonstrated that the self producing sponge can effective promote the healing of skin wound, the wounds recovered with clean,and surface exudation is minimal.