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TetraStat is a tetra-armed polyethylene glycol (PEG) hydrogel. It is a synthetic sealant that solidifies instantly in response to pH changes. This study aimed to evaluate the hemostatic effect of TetraStat through experiments evaluating future clinical applications.
We used TetraStat, oxidized regenerated cellulose (SURGICEL®), and fibrinogen and thrombin sealant patch (TachoSil®) using in vitro and in vivo experiments. For the in vitro experiment, a closed circulatory system filled with phosphate-buffered saline under high pressure was used. Needle punctures were created and closed using the various sealants. For the in vivo experiment, rat venae cavae were punctured with 18- and 20-gauge (G) needles, and hemorrhage was allowed to occur for several seconds. A porous PEG sponge soaked with TetraStat was applied as a hemostatic system. Hemostasis outcomes were compared among the various concentrations (40–100 g/L) of TetraStat, SURGICEL, and TachoSil.
The punctured holes in the prosthetic graft were successfully sealed with TetraStat in 1 min. The success rate of hemostasis with TetraStat for the punctured holes in the rat vena cava was dose-dependent. TetraStat was effective in sealing the holes created with a 20 G needle at all concentrations; however, the holes created with an 18 G needle could be sealed only when the concentration ≥60 g/L. Hemostasis using SURGICEL or TachoSil was less successful and sometimes required up to 5 min.
TetraStat has a high hemostatic ability. A porous PEG sponge soaked with TetraStat is a useful choice for effective hemostasis during massive hemorrhage.
Hemorrhage control is crucial for the success of surgical procedures. Even after attempting to control hemorrhage through suturing the bleeding sites, surgeons sometimes encounter situations where achieving hemostasis is difficult. These include disseminated intravascular coagulation owing to cancer, pregnancy, infection, massive tissue injury, and uncontrolled oozing from needle holes under heparinization in cardiovascular surgeries. A wide variety of hemostatic agents with various types of materials and processes are available. The basic mechanism underlying most commercially available agents is the acceleration of blood coagulation via fibrin formation and platelet activity. However, the hemostatic effects is negatively affected under abnormal coagulation conditions. In consideration of the limited biological resources and the cost, synthetic sealants using polymerization were developed to meet the needs for reliable intraoperative hemostatic agents, which are independent of blood conditions, especially in the field of cardiovascular surgery.
We designed a next-generation synthetic sealant using a polyethylene glycol (PEG) hydrogel (TetraStat) that solidifies instantly in response to pH changes. TetraStat was formed by the reaction of two types of tetra-armed PEGs; it responds slowly under acidic conditions but rapidly under neutral conditions.
When TetraStat comes in contact with a neutral body fluid, such as blood, solidification occurs. This principle is independent of the biological blood coagulation reaction; it allows for the selective chemical sealing of bleeding sites. Therefore, this nonbiomaterial sealant could be used in situations with uncontrolled hemorrhaging. It is theoretically effective under anticoagulant and antiplatelet drug administration and abnormal coagulation states.
In this study, we aimed to evaluate the hemostatic effects of TetraStat and to optimize the dosage for clinical use through in vitro and in vivo experiments.
Materials and Methods
Preparation of TetraStat
We used mutually reactive tetra-armed PEGs with sulfhydryl groups (Tetra-PEG-SH; SUNBRIGHT PTE-100SH, NOF CORPORATION, Tokyo, Japan) and maleimide groups at the termini (Tetra-PEG-MA; SUNBRIGHT® PTE-100 MA, NOF CORPORATION, Tokyo, Japan). Aqueous solutions of tetra-PEG-SH and tetra-PEG-MA were prepared by dissolving the desired amount of PEG in 1 mM citrate phosphate buffer (pH 3.0). Equal volumes of tetra-PEG-SH and tetra-PEG-MA solution were combined to form the TetraStat solution.
For the in vivo experiment, a porous PEG sponge (Gellycle Co., Ltd., Tokyo, Japan) was soaked in an excess amount of TetraStat (Fig. 1).
In Vitro Experiment
A circulatory system mimic was created using a silicone tube connected to a 6-mm diameter expanded polytetrafluoroethylene prosthetic graft (Propaten, Gore Medical, Flagstaff, AZ, USA). This closed circuit was filled with phosphate-buffered saline (PBS) under human systemic pressure (100–150 mm Hg); the pressure was controlled and measured using a liquid fluid pump and pressure gauge (Fig. 2).
The graft was punctured using a 27-gauge (G) needle (outer diameter: 0.41 ± 0.02 mm). A porous PEG sponge soaked with TetraStat was applied to the punctures and compressed for 1 min. The PEG sponge was removed with nontoothed forceps. We initially tried a larger needle size; however, bigger punctures resulted in excess water spillage, which rendered the circuit pressure unstable. Therefore, we only used a 27-G needle and assumed that this size was sufficient to highlight the effect of TetraStat.
In Vivo Experiment with Rats
To compare the hemostatic capability of TetraStat with that of existing products, we chose two products widely used in Japan: oxidized regenerated cellulose (SURGICEL®, Johnson & Johnson Medical Ltd., Tokyo, Japan) and a fibrinogen and thrombin sealant patch (TachoSil®, Takeda Nederland b.v. Takeda, Zurich, Switzerland).
All animal experiments were performed according to the Guideline for the Care and Use of Laboratory Animals, established by the University of Tokyo (Permit No. KA20-7, approved on August 11, 2020). As the values of arterial blood gas and electrolytes of anesthetized rats were very similar to those in humans, we used Male Sprague-Dawley rats (320–380 g of body weight) for the experiments.
They were fed a normal diet and were kept in air conditioning (21 ± 1°C) with a 12-hr light-dark cycle.
The rats were anesthetized using isoflurane. A midline laparotomy was performed, and the infrarenal vena cava was exposed. Each vena cava was punctured with the respective-sized needle (18-G and 20-G) 1 cm above the junction of the left and right common iliac veins. After withdrawing the needle, bleeding was allowed for 5–7 s. The punctures were sealed with one of the three hemostatic agents for 1 min. SURGICEL and TachoSil were applied for 5 min to adhere to the manufacturer’s instructions for usage.
In Vivo Evaluation of Hemostasis
We removed any pooled blood and confirmed complete hemostasis macroscopically. The abdominal incision was closed, and the animal was allowed to recover from anesthesia. Seven days after the experiment, the rats were euthanized using an isoflurane overdose. A 2-cm section of the treated vena cava was excised and fixed in a buffered 4% formalin solution for 24 hr; then, the excised vessels were embedded in paraffin. The embedded tissue was cut into 5-mm thick sections and stained with hematoxylin and eosin (HE). The inflammatory reaction was evaluated using a grading criterion from 0 to 3 (0, no inflammation; 1, mild inflammation; 2, moderate inflammation; and 3, extensive inflammation).
The punctures were successfully sealed in most rats treated with TetraStat (Video, Supplemental Digital Content 1). In one of the cases, which used 50 g/L of TetraStat, sealing was unsuccessful. However, with TetraStat concentrations ranging from 60 to 100 g/L, sealing was effective without any leakage, up to an intraluminal pressure of 150 mm Hg (Table I).
Table IResults of sealing the punctured sites (“hemostasis”)
Most of the in vivo punctures were successfully sealed after 1-min astriction with TetraStat, despite needle rotation and allowing bleeding for several seconds, followed by massive bleeding (Video, Supplemental Digital Content 2) (Fig. 3). TetraStat was effective at all applied concentrations in sealing punctures made using the 20-G needle. Successful hemostasis was observed in some cases in which SURGICEL and TachoSil were used. In case of punctures using the 18-G needle, which has an outer diameter approximately equal to that of the vena cava, hemostasis was less effective with 40 and 50 g/L TetraStat, indicating a dose-dependent effect. Hemostasis was successful in one case with a puncture using a 14-G needle and in 1 of 2 cases with 120 g/L TetraStat. In the SURGICEL and TachoSil groups, pressing the sponge against the vena cava for 1 min was insufficient for hemostasis; subsequently, we pressed the sponge for 5 min and found a successful hemostatic effect after puncture with an 18-G needle (Table I).
We initially set the “control” group in which punctures were created with 27-G needles, and bleeding was stopped by manual or PEG sponge compression only. However, we failed to stop fatal bleeding in the preliminary trial. Therefore, we terminated the additional trial with the “control” group from an animal ethics standpoint.
TetraStat is acidic; therefore, intraperitoneal inflammation after the surgery was expected. Severe adhesions were found in 2 out of 8 and in 1 out of 4 cases with 100 g/L and 80 g/L TetraStat, respectively. However, there were no macroscopic adhesions in the group that used below 80 g/L of TetraStat.
HE-stained sections of the regions around the puncture site in TetraStat, SURGICEL, and TachoSil (n = 4 for each group) groups were investigated and compared. The amount of residual agent in the puncture site was significantly lower in the TetraStat group, when compared to that in the other two groups (Fig. 4A). Inflammatory reactions such as inflammatory cell infiltration, granulation, and fibrosis were observed in all groups; grade 1 for the TetraStata and grade 3 for the SURGICEL and TachoSil groups.
Remarkable venous wall thickening with severe inflammatory cell infiltration was observed in the SURGICEL group (Fig. 4B).
A thrombus in the vena cava was observed in the SURGICEL group (1 case) and the TachoSil group (3 cases), but not in the TetraStat group (Fig. 4C).
We demonstrated the high hemostatic ability of TetraStat in both in vitro and in vivo experiments. Material details cannot be provided at this time because a patent application is currently pending; therefore, our main purpose was to evaluate the ability and optimize the dosage. TetraStat, being a synthetic sealant, has less risk of infections, such as viral infections, compared to that with biological sealants. Its hemostatic mechanism is independent of the blood coagulation process and enables controlled gelation. TetraStat’s sealing ability was high enough to seal the hole where PBS was spouting out in the in vitro experiment.
We compared the ability of TetraStat with that of two commercially available hemostatic agents widely used in Japan. TachoSil is a reliable biological sealant with high hemostatic ability; however, it is expensive owing to its limited availability. SURGICEL is popular because of its low cost and nonbiological nature; however, it is usually used only supportively after suture hemostasis because of its relatively low hemostatic ability. TetraStat has the advantages of both rapid hemostasis (within 1 min) and high hemostatic ability, which is critical for managing massive uncontrollable hemorrhages during surgery. TetraStat has limitations; intraperitoneal administration of high concentrations causes inflammation, most likely because of its acidity. However, SURGICEL is also acidic.
Considering its high hemostatic performance, inflammation with TetraStat could be an acceptable clinical adverse event, which would, however, need further studies.
The use of a porous PEG sponge was the key to this hemostatic system. It was effective even during massive hemorrhage. We evaluated using only the solidifying liquid; however, the liquid was quickly diluted in case of massive hemorrhage that occurred under blood pressure; a large amount of TetraStat was needed. This limitation was overcome with the use of the porous PEG sponge as a temporary liquid reservoir for the TetraStat. This prevented the liquid from mixing with or becoming diluted by blood. The blood-TetraStat composite was removable after confirmation of hemostasis (Fig. 1). The PEG sponge is not overly sticky and is easy to handle, which is a novel and strong advantage of our hemostatic system.
Hydrofit (Sanyo Medical Industry, Ltd., Kyoto, Japan) is a novel synthetic sealant developed in Japan. It is formed by a reaction between a copolymer of PEG and fluorinated hexamethylene diisocyanate (FHD). Hydrofit has high hemostatic ability and is safe and effective in thoracic aortic surgery.
We assumed that this sealant could be strongly competitive with TetraStat as it has similar PEG components and hemostatic procedures. However, Hydrofit is a liquid agent, which can be difficult to use, requiring skill and experience.
In addition, the long-term safety of residual FHD following hemostasis is not established. The TetraStat hemostatic system using a PEG sponge could be used in massive hemorrhage and then removed after hemostasis. After removal of the gel, a thin PEG component was observed at the puncture site; theoretically, the PEG could be modified to be biodegradable.
The hydrogel liquid was directly applied to the injured liver, and gelation occurred after a certain period. This is potentially useful for very small amounts of bleeding or for postoperative bleeding. However, this hydrogel material lacks the blood-responsive gelation observed with the TetraStat and PEG sponge system. The ongoing bleeding could dilute the hydrogel liquid, rendering it ineffective. In addition, a porous sponge is clinically necessary for pinpoint hemostasis in massive bleeding lesions with high blood pressures. Our design ensures that the hemostatic component is not easily diluted with blood during massive bleeding. These factors could contribute to the cost-effectiveness.
In the in vivo experiments, we injured the vena cava and not the aorta. The aorta would have been an appropriate puncture site owing to its high blood pressure; however, the difference between the aortic hemostatic effects of the different agents was not remarkable because of the thickness of the aortic wall. We initially performed aortic hemostasis and found that all punctures made by 18-G and 20-G needles were successfully sealed by all sealants used in this study and the effect of TetraStat could not be distinguished from other sealants. Additionally, the demand for sealants for uncontrollable venous hemorrhage is high because venous injury repair is technically difficult and requires experience owing to the thin, fragile venous wall structure.
In contrast to that in clinical cardiovascular situations, we did not perform heparinization in the in vivo experiment. However, we demonstrated the effect of TetraStat compared to that of commonly used hemostatic agents. Future experiments would focus on evaluating the effects in larger animals to confirm the safety and effectiveness and to optimize the dose in models more closely mimicking humans. Additionally, we are exploring the possibility of applying TetraStat on vascular closure or endoscopic hemostatic devices.
The leakage of PBS, which lacks coagulation ability, was stopped with a 1-min press with TetraStat in an in vitro circulation mimic. TetraStat exhibited a high hemostatic ability in sealing large holes in the rat’ vena cava, during massive hemorrhage, without major adverse events such as intraluminal thrombus. A porous PEG sponge soaked with TetraStat could be applied at the bleeding point in cases of massive hemorrhage to ensure effective hemostasis.
Conflict of Interest Statement
The authors have no conflicts of interest.
Declarations of Interest
This study was funded by Japan Agency for Medical Research and Development .
Study design: Katsuyuki Hoshina, Takamasa Sakai, Writing: Shinya Okata, Katsuyuki Hoshina, Animal Surgery: Shinya Okata, Ayano Fujisawa, Data collection: Hiroyuki Kamata, Yuki Yoshikawa, Critical review and revision: all authors, Final approval of the article: all authors, Accountability for all aspects of the work: all authors.
Ethical Approval for Research
All animal experiments were performed according to the Guideline for the Care and Use of Laboratory Animals established by the University of Tokyo (Permit No. KA20-7, approved on August 11, 2020).
The porous PEG sponge used in the experiments was a generous gift from Gellycle Co.
Dr. Hoshina and his colleagues have developed a new hemostatic sealant (TetraStat) based off a polyethylene glycol (PEG) hydrogel PEG that solidifies upon contact with body fluids to achieve hemostasis. This hemostasis is independent of the blood coagulation factors. They tested their product using 27 G needle injuries in water filled pressurized PTFE grafts, and then on an inferior vena cava injury in vivo rat model with acceptable results.