Hemostatic agents have been used in surgical operation for many years to control bleeding because human body cannot control the massive blood loss without any treatment. However, hemostatic agents used in operations are often ineffective, expensive or raise safety concerns from their shapes and mate...
Hemostatic agents have been used in surgical operation for many years to control bleeding because human body cannot control the massive blood loss without any treatment. However, hemostatic agents used in operations are often ineffective, expensive or raise safety concerns from their shapes and materials. In this study, enzyme-mediated injectable pullulan hydrogel containing blood coagulation agents such as thrombin and calcium chloride is fabricated to use as a hydrogel type hemostatic agent. The pullulan hydrogel hemostatic has many advantages such as biodegradability, flexibility, and blood coagulability. In this study, pullulan was oxidized through 2,2,6,6-tetramethyl-1-piperidinyloxy (free radical, TEMPO) in order to transform the primary alcohols into carboxyl groups. Then, carboxylated pullulan was modified to introduce tyramine moiety, which can be used as cross-linking site for the pullulan hydrogels, by 1-ethyl-3-(3-dimethylaminopropyl)-carbodiimide hydrochloride (EDC) - N-hydroxysulfosuccinimide (NHS) reaction. And then, the injectable in situ pullulan hydrogel was fabricated by enzymatic cross-linking method using horseradish peroxidase (HRP) and hydrogen peroxide (H2O2). The modified pullulan with tyramines was analyzed by ATR-FTIR, 13C NMR and 1H NMR to confirm the oxidation of primary alcohols and introduction of tyramines. Gelation time of hydrogels was determined as a function of concentration of HRP and pullulan solutions. Rheological properties were also examined according to the concentration of pullulan solutions. To optimize the concentration of thrombin and calcium chloride solution, blood coagulation time was investigated with platelet-poor-plasma (PPP). The in vitro red blood cell (RBC) hemolysis result showed that the absorbance of uncoagulated remained hemoglobin after contacting with hydrogel containing thrombin and calcium ions was lower than with control hydrogel without blood coagulation agents. The in vivo animal study using haemorrhaging liver rat model showed that the pullulan hydrogel containing thrombin and calcium ions significantly reduced hemorrhage quantity and bleeding time compared to the untreated model. As a result, the pullulan with tyramines was successfully synthesized and cross-linked by enzyme-mediated reaction. The modified pullulan hydrogel containing blood coagulation agents had suitable gelation time, biodegradability and hemostatic ability. From these results, the enzyme-mediated pullulan hydrogel containing calcium chloride and thrombin provided an effective and promising hemostatic agent to control massive blood loss.
Hemostatic agents have been used in surgical operation for many years to control bleeding because human body cannot control the massive blood loss without any treatment. However, hemostatic agents used in operations are often ineffective, expensive or raise safety concerns from their shapes and materials. In this study, enzyme-mediated injectable pullulan hydrogel containing blood coagulation agents such as thrombin and calcium chloride is fabricated to use as a hydrogel type hemostatic agent. The pullulan hydrogel hemostatic has many advantages such as biodegradability, flexibility, and blood coagulability. In this study, pullulan was oxidized through 2,2,6,6-tetramethyl-1-piperidinyloxy (free radical, TEMPO) in order to transform the primary alcohols into carboxyl groups. Then, carboxylated pullulan was modified to introduce tyramine moiety, which can be used as cross-linking site for the pullulan hydrogels, by 1-ethyl-3-(3-dimethylaminopropyl)-carbodiimide hydrochloride (EDC) - N-hydroxysulfosuccinimide (NHS) reaction. And then, the injectable in situ pullulan hydrogel was fabricated by enzymatic cross-linking method using horseradish peroxidase (HRP) and hydrogen peroxide (H2O2). The modified pullulan with tyramines was analyzed by ATR-FTIR, 13C NMR and 1H NMR to confirm the oxidation of primary alcohols and introduction of tyramines. Gelation time of hydrogels was determined as a function of concentration of HRP and pullulan solutions. Rheological properties were also examined according to the concentration of pullulan solutions. To optimize the concentration of thrombin and calcium chloride solution, blood coagulation time was investigated with platelet-poor-plasma (PPP). The in vitro red blood cell (RBC) hemolysis result showed that the absorbance of uncoagulated remained hemoglobin after contacting with hydrogel containing thrombin and calcium ions was lower than with control hydrogel without blood coagulation agents. The in vivo animal study using haemorrhaging liver rat model showed that the pullulan hydrogel containing thrombin and calcium ions significantly reduced hemorrhage quantity and bleeding time compared to the untreated model. As a result, the pullulan with tyramines was successfully synthesized and cross-linked by enzyme-mediated reaction. The modified pullulan hydrogel containing blood coagulation agents had suitable gelation time, biodegradability and hemostatic ability. From these results, the enzyme-mediated pullulan hydrogel containing calcium chloride and thrombin provided an effective and promising hemostatic agent to control massive blood loss.
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