In vivo hemostatic capability of a novel Tetra-PEG hydrogel

TetraStat is a novel synthetic sealant created with a tetra-armed polyethylene glycol (PEG) hydrogel. It has no risk of infection from biological pathogens and has a hemostatic mechanism independent of the blood coagulation pathway and controllable gelation. We evaluated the hemostatic effect of TetraStat in ex vivo and in vivo experiments for future clinical application. In ex vivo experiments using a circulatory system filled with phosphate-buffered saline under high pressure, needle punctures were astricted with TetraStat and two commercially available hemostatic agents (SURGICEL and TachoSil). For in vivo experiments, rat vena cavae were punctured with 14, 18, and 20 gauge needles, and hemorrhage occurred for several seconds. A porous PEG sponge soaked with TetraStat was applied as a hemostatic system for the massive hemorrhage. In the ex vivo experiment, punctures were sealed completely after 1 min astriction with TetraStat gel; in contrast, SURGICEL and TachoSil failed to seal the hole. In vivo experiments demonstrated that TetraStat successfully caused hemostasis in the punctured vena cava within 1 min of application in a dose-dependent manner. For SURGICEL and TachoSil, successful hemostasis occurred after 5 min astriction but was less frequent after 1 min astriction. Ex vivo and in vivo experiments revealed TetraStat’s high hemostatic ability under high pressure and in rat vena cava injuries under massive hemorrhage. A porous PEG sponge soaked with TetraStat is a promising advancement in hemostatic systems.

There are a wide variety of hemostatic agents with various types of materials and processes.

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The basic mechanism of most commercially available agents is to accelerate blood 44 coagulation via fibrin formation and platelet activity. However, the hemostatic effects can be 45 negatively affected by abnormal coagulation conditions. Synthetic sealants using 46 polymerization have been developed under strong demand for reliable intraoperative 47 hemostatic agents independent of blood conditions, especially in the field of cardiovascular 48 surgery (Fig 1).  50 We designed a next-generation synthetic sealant using a polyethylene glycol (PEG) 51 hydrogel (TetraStat) that solidified instantly in response to pH changes. Formed by the 52 reaction of two types of tetra-armed PEGS, TetraStat responds slowly in acidic conditions 53 and extremely fast in neutral conditions [1,2]. When TetraStat comes in contact with a neutral 54 body fluid such as blood, apparent solidification occurs. This principle, which is independent 55 of the biological blood coagulation reaction, allows for the selective chemical sealing of 56 bleeding sites. Therefore, this non-biomaterial sealant could be used in situations with 57 uncontrolled hemorrhaging and should be theoretically effective under anti-coagulant and 58 anti-platelet drug administration and abnormal coagulation states.

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In this study, we evaluated the ex vivo and in vivo effectiveness of TetraStat. First, 60 we applied TetraStat to various sized holes in a prosthetic graft connected to a circulatory   The rats were anesthetized using isoflurane. Laparotomy was performed at the midline, and 97 the infrarenal vena cava was exposed. Each vena cava was punctured with a different size 98 needle (14, 18, or 20 gauge) 1 cm above the junction of the left and right common iliac veins.

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After withdrawing the needle, bleeding occurred for 5-7 s, and the punctures were then 100 astricted with the three hemostatic agents for 1 min. To adhere to the commercial instructions 101 for use, SURGICEL and TachoSil were also applied for 5 min. In vivo evaluation of hemostasis 104 We removed any pooled blood and confirmed hemostasis macroscopically. Then, the 105 abdominal incision was closed, and the subject was recovered from anesthesia. Seven days 106 after the operation, the rats were euthanized with an isoflurane overdose. A 2-cm section of 107 the treated vena cava was excised and fixed in a buffered 4% formalin solution for 24 h. After 108 formalin fixation, the excised vessels were embedded in paraffin. The embedded tissue was 109 cut into 5-mm thick slices and stained with hematoxylin and eosin (HE) stain. The

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Ex vivo experiments 116 The punctures were successfully sealed in most TetraStat cases (S1 Video). In one case with 117 50 g/L TetraStat, the sealing was unsuccessful. However, with concentrations ranging from 118 60 to 100 g/L, we increased the intraluminal pressure up to 600 mmHg after sealing was 119 confirmed without leakage (  with a 14-gauge needle in one case with 120 g/L TetraStat (Table 1).  The use of the porous PEG sponge was the key to this hemostatic system, which was 174 effective even under massive hemorrhage. Initially, we attempted to only use the solidifying 175 liquid. However, we found that the liquid became diluted, and a large amount of TetraStat thereby preventing the liquid from mixing with or becoming diluted by the blood.