Marine source collagen fiber hemostatic sponge and preparation method thereof

Abstract

The application discloses a marine-source collagen fiber hemostatic sponge and a preparation method thereof, and belongs to the field of biomedical materials. The preparation method is as follows: a true sea moss is cut open, a bag is removed, internal tissues are taken out, and digests, excrements and the like are removed after washing; the internal tissues are cut into pieces and then added into a NaCl solution, and are placed at 0-4 DEG C for 6-18 hours; urea solution is added after filtration and cleaning; a high-speed homogenizer is used for primary stirring to obtain a collagen-urea blend solution, and the solution is frozen and then thawed at 4 DEG C for 5-15 minutes; a high-speed homogenizer is used for secondary stirring; and finally, a vacuum filter press is used for solid-liquid separation of the solution to obtain collagen fibers; the collagen fibers are dispersed in water, and freeze-drying is carried out to obtain the collagen fiber hemostatic sponge. The application provides a porous hemostatic sponge with a capillary rapid liquid absorption capacity, which has the advantages of rapid preparation, low economic cost, ecological friendliness in a whole life cycle, strong liquid absorption, good hemostatic effect and low hemolysis rate.

Description

TECHNICAL FIELD

[0001] The present application belongs to the field of biomedical materials, and particularly relates to a marine-source collagen fiber hemostatic sponge and a preparation method thereof. BACKGROUND

[0002] In the case of war, accidental injury and surgery, excessive blood loss is the main cause of death. Therefore, how to achieve efficient hemostasis is particularly critical. In recent years, hemostatic sponge has gradually become a popular research object due to its advantages such as good liquid absorption and portability. Hemostatic sponge has a porous structure, which can trigger the aggregation and adhesion of platelets after contacting with blood, and promote the release of a large amount of coagulation factors, activate the coagulation system, and ultimately achieve the effect of hemostasis under the dual action of endogenous coagulation and exogenous coagulation of the human body itself.

[0003] At present, gelatin hemostatic sponge occupies the dominant position in the commercial hemostatic sponge market, and has the advantages of simple preparation and low cost. However, the water absorption of commercial gelatin hemostatic sponge is much lower than that of collagen hemostatic sponge, which cannot be in close contact with the wound, and the formed blood clots have poor adhesion and are easy to fall off, making it difficult to achieve ideal hemostatic effect in actual application. Commercial collagen hemostatic sponge prepared from bovine Achilles tendon is also a hemostatic material that has appeared in recent years, and plays an important role in hemostasis, absorption of tissue exudate, and promotion of wound healing. However, commercial bovine collagen hemostatic sponge has certain risks of zoonosis, such as mad cow disease, brucellosis and tuberculosis, and is also plagued by religious customs and other issues, resulting in more restrictions on its application. In addition, the preparation of commercial bovine collagen hemostatic sponge is relatively complex, and most of them use acid solution and enzyme solution as extraction medium, and use salt solution for separation and purification. In the whole preparation process, there are many types of experimental reagents, long preparation period, large energy consumption of experimental instruments, and high price, etc., which lead to high time and economic cost of product preparation; and if the experimental waste liquid is not properly treated, it may also cause environmental pollution and other problems.

[0004] Collagen is the main component of the extracellular matrix of the body and the main structural protein in skin tissue. Collagen has the biological function of accelerating the aggregation and adhesion of platelets, and therefore can help activate the coagulation system and accelerate the endogenous coagulation process. Collagen has strong water absorption and biocompatibility, and has good repair effect on the skin, so collagen is an ideal hemostatic sponge material.

[0005] The true sea squirt is a hermaphroditic organism, which can reproduce sexually and asexually by parthenogenesis. Due to the characteristics, the true sea squirt has a strong reproductive capacity and a fast growth rate, and can produce a large number of larvae in a short time. The true sea squirt is often attached to solid objects such as rocks, ships and fishing nets, and is a main member of the marine fouling organism community. The true sea squirt is composed of an outer pericyst and an internal tissue, and the internal tissue contains a large amount of collagen. Therefore, it is of great significance to develop the true sea squirt for use as a collagen hemostatic sponge. SUMMARY

[0006] The present application aims to provide a marine source collagen fiber hemostatic sponge and a preparation method thereof.

[0007] In order to achieve the above-mentioned purpose, the technical scheme adopted by the present application is as follows:

[0008] A preparation method of a marine source collagen fiber hemostatic sponge, comprising the following steps:

[0009] 1) Preparation of collagen fibers: fresh true sea squirt is cut open, the pericyst is removed, the internal tissue is taken out, and the digests, excrements and the like are removed by washing, and then the internal tissue is cut into pieces. 5-15 times volume (v / v) of 1-3 mol / L NaCl solution is added, and the mixture is placed at 0-4 DEG C for 6-18 h. After filtration, the mixture is repeatedly washed for 3-7 times. 5-15 times volume of urea solution with a concentration of 1-5 mg / mL is added, and then a high-speed homogenizer is used for high-speed stirring at a speed of 8000-15000 r / min for 1-5 times, each time for 1-5 min. The obtained collagen urea blend solution is frozen at-20 DEG C for 3-9 h, and then thawed at 4 DEG C for 5-15 min. The high-speed homogenizer is used for high-speed stirring at a speed of 8000-15000 r / min for 1-5 times, each time for 1-5 min. A vacuum filter is used for solid-liquid separation of the solution, and collagen fibers are obtained.

[0010] 2) Preparation of collagen fiber hemostatic sponge: the collagen fibers are dispersed in deionized water, and placed in a freeze dryer for freezing for 12-14 h to obtain collagen fiber hemostatic sponge.

[0011] The present application uses the marine fouling organism true sea squirt as a raw material for preparing collagen fiber hemostatic sponge. The preparation process mainly destroys the hydrogen bonds between the collagen fibers in the tissue by using a strong polar hydrogen bond breaker urea solution and physical and mechanical action, so as to obtain collagen fibers with micro-nano size. Finally, the collagen fibers are freeze-dried to prepare a porous sponge with a rapid liquid absorption capacity of capillary.

[0012] The present application has the following advantages:

[0013] 1、The preparation period of the present application is short, the preparation process only involves urea and sodium chloride reagents, and the urea solution and sodium chloride solution after use can be recycled by natural evaporation crystallization. At the same time, the sea squirt collagen fiber hemostatic sponge can be degraded by microorganisms in the soil within 15 days, which can effectively reduce environmental pollution and reduce waste disposal difficulty.

[0014] 2、Compared with commercial gelatin hemostatic sponge and commercial bovine collagen hemostatic sponge, the sea squirt collagen fiber hemostatic sponge has the advantages of short time, low economic cost and environmental friendliness, and also has the outstanding characteristics of strong liquid absorption, good hemostatic effect and low hemolysis rate. This is because the collagen fibers with micro-nano size in the sea squirt collagen fiber hemostatic sponge serve as a supporting structure, so that the sponge surface and the inside have a pore structure (pore size of 1-5 μm) that is mutually penetrating and connected. Blood can quickly soak into the inside of the sponge under the capillary action between the pores, thereby forming a blood clot and achieving good hemostatic effect. The sea squirt collagen fiber hemostatic sponge also has a certain flexibility and can adapt to different sizes and shapes of wounds, thereby better fitting the wound. In addition, the sea squirt collagen fiber hemostatic sponge does not contain other materials and does not contain any hemolytic components, so it will not cause hemolysis during application, and the hemolysis rate is relatively low.

[0015] 3、The sea squirt collagen fiber hemostatic sponge is prepared from marine fouling organisms, which can break through the application limitations of commercial bovine collagen hemostatic sponge. The sea squirt collagen fiber hemostatic sponge has the outstanding characteristics of short preparation time, few chemical reagents involved and recyclability, biodegradability and high-efficiency hemostasis. From the selection of raw materials to the processing and preparation process to the disposal of waste after product use, it meets the whole life cycle sustainable development concept. As a whole life cycle sustainable and eco-friendly material, the sea squirt collagen fiber hemostatic sponge not only fully develops and utilizes marine biomass resources to obtain high-performance hemostatic materials, but also fully meets the sustainable development concept of China, thereby helping to achieve the "double carbon" goal of China. BRIEF DESCRIPTION OF DRAWINGS

[0016] Figure 1 is the result of the rabbit ear artery incision hemostasis experiment. EMBODIMENT EMBODIMENT 1

[0017] A preparation method of a marine source collagen fiber hemostatic sponge, comprising the following steps:

[0018] (1) Fresh Styela coerulea is cut open, the tunic is removed, the internal tissue is taken out, and the internal tissue is washed to remove digests, excrements, and the like, and then is cut into pieces. A 5-fold volume (v / v) of a 1 mol / L NaCl solution is added, and the internal tissue is placed at 4°C for 6 h. After filtration, the internal tissue is repeatedly washed 5 times. A 5-fold volume of a urea solution is added, and the urea solution has a concentration of 1 mg / mL. A high-speed homogenizer is used for high-speed stirring at a speed of 8000 r / min. The high-speed stirring is performed 3 times, and each time lasts for 1 min. The obtained collagen-urea blend solution is frozen at -20°C for 3 h, is thawed at 4°C for 5 min, and is high-speed stirred again 3 times using the high-speed homogenizer, and each time lasts for 1 min. Finally, a vacuum filter pump is used to separate the solution into solid and liquid, and collagen fibers are obtained.

[0019] (2) The collagen fibers are dispersed in deionized water, are frozen for 12 h, and are placed in a freeze dryer. A collagen fiber hemostatic sponge is prepared by using a freeze-drying method. Example 2

[0020] A preparation method of a marine-source collagen fiber hemostatic sponge includes the following steps.

[0021] (1) Fresh Styela coerulea is cut open, the tunic is removed, the internal tissue is taken out, and the internal tissue is washed to remove digests, excrements, and the like, and then is cut into pieces. A 5-fold volume (v / v) of a 1 mol / L NaCl solution is added, and the internal tissue is placed at 4°C for 6 h. After filtration, the internal tissue is repeatedly washed 5 times. A 5-fold volume of a urea solution is added, and the urea solution has a concentration of 1 mg / mL. A high-speed homogenizer is used for high-speed stirring at a speed of 8000 r / min. The high-speed stirring is performed 3 times, and each time lasts for 1 min. The obtained collagen-urea blend solution is frozen at -20°C for 3 h, is thawed at 4°C for 5 min, and is high-speed stirred again 3 times using the high-speed homogenizer, and each time lasts for 1 min. Finally, a vacuum filter pump is used to separate the solution into solid and liquid, and collagen fibers are obtained.

[0022] (2) The collagen fibers are dispersed in deionized water, are frozen for 12 h, and are placed in a freeze dryer. A collagen fiber hemostatic sponge is prepared by using a freeze-drying method. Example 3

[0023] A preparation method of a marine-source collagen fiber hemostatic sponge includes the following steps.

[0024] (1) Fresh Styela coerulea was cut open, the tunic was removed, and the internal tissue was taken out, washed to remove digests, excrements, etc., and then chopped. A 15-fold volume (v / v) of 3 mol / L NaCl solution was added, and the mixture was placed at 4 °C for 18 h. After filtration, the solution was repeatedly washed 7 times. A 15-fold volume of urea solution was added, and the urea solution had a concentration of 5 mg / mL. Then, a high-speed homogenizer was used for high-speed stirring at a speed of 15000 r / min. The collagen-urea blend solution was frozen at -20 °C for 6 h, thawed at 4 °C for 15 min, and then high-speed stirring was performed 5 times at a speed of 15000 r / min for 3 min each time. Finally, a vacuum filter was used to separate the solution into solid and liquid, and collagen fibers were obtained.

[0025] (2) The collagen fibers were dispersed in deionized water, frozen for 12 h, and placed in a freeze dryer to obtain collagen fiber hemostatic sponges by a freeze-drying method.

[0026] The collagen fiber hemostatic sponge prepared in Example 1 was used for a rabbit ear artery incision hemostasis experiment.

[0027] Blank group: After the New Zealand white rabbits were anesthetized, the hair on the inner center of the rabbit ear was removed, and the surrounding skin was wiped with alcohol. The rabbit ear artery was exposed by gently patting, and the rabbit ear artery and vein were simultaneously incised horizontally (5 mm wide and 0.5 mm deep) using an 11# scalpel. After incision, the blood was allowed to flow freely until the blood coagulated, and the amount of blood loss was recorded.

[0028] Control group: After the New Zealand white rabbits were anesthetized, the hair on the inner center of the rabbit ear was removed, and the surrounding skin was wiped with alcohol. The rabbit ear artery was exposed by gently patting, and the rabbit ear artery and vein were simultaneously incised horizontally (5 mm wide and 0.5 mm deep) using an 11# scalpel. After incision, the wound was immediately covered with a commercial gelatin hemostatic sponge, and gauze and a 200 g weight were placed on the sponge to assist in compression hemostasis. After the wound stopped bleeding, if there was no secondary bleeding within 30 min, it was considered that hemostasis was successful, and the amount of blood loss was recorded.

[0029] Experimental group: After the New Zealand white rabbits were anesthetized, the hair on the inner center of the rabbit ear was removed, and the surrounding skin was wiped with alcohol. The rabbit ear artery was exposed by gently patting, and the rabbit ear artery and vein were simultaneously incised horizontally (5 mm wide and 0.5 mm deep) using an 11# scalpel. After incision, the wound was immediately covered with a Styela coerulea collagen fiber hemostatic sponge, and gauze and a 200 g weight were placed on the sponge to assist in compression hemostasis. After the wound stopped bleeding, if there was no secondary bleeding within 30 min, it was considered that hemostasis was successful, and the amount of blood loss was recorded.

[0030] The experimental results are as follows Figure 1As shown, in the rabbit ear artery laceration hemostasis experiment, compared with the untreated blank control group, the sea sponge collagen fiber hemostatic sponge can significantly reduce the amount of blood loss, and its blood loss is reduced by 73.80%, while the commercial gelatin hemostatic sponge only reduces the blood loss by 57.96%.

Claims

1. A method for preparing a hemostatic sponge of marine origin collagen fibers, characterized in that, The method comprises the following steps: 1) Preparation of collagen fibers: fresh sea squirts are cut open, the sacs are removed, the internal tissues are taken out, washed to remove digests and excrements, then chopped, 5-15 times volume of NaCl solution is added, and the mixture is placed at 0-4 ℃ for 6-18 h, then filtered and washed, 5-15 times volume of urea solution is added, a high-speed homogenizer is used for stirring once, a collagen-urea blend solution is obtained, and the solution is frozen, then thawed at 4 ℃ for 5-15 min, a high-speed homogenizer is used for stirring twice, and finally a vacuum filter is used for solid-liquid separation of the solution to obtain collagen fibers; 2) Preparation of collagen fiber hemostatic sponge: the collagen fibers are dispersed in water, and placed in a freeze dryer for freezing for 12-14 h to obtain a collagen fiber hemostatic sponge.

2. A process for the preparation of a hemostatic sponge of marine origin collagen fibers according to claim 1, characterized in that, The concentration of the NaCl solution is 1-3 mol / L.

3. The method for preparing a marine-derived collagen fiber hemostatic sponge according to claim 1, characterized in that, The concentration of the urea solution is 1-5 mg / mL.

4. The method for preparing a marine-derived collagen fiber hemostatic sponge according to claim 1, characterized in that, The stirring is high-speed stirring at a speed of 8000-15000 r / min by using a high-speed homogenizer.

5. A method for preparing a marine-derived collagen fiber hemostatic sponge according to claim 1, characterized in that, The number of times of the first stirring and the second stirring is 1-5 times, and each time is 1-5 min.

6. The method of claim 1, wherein the marine source collagen fiber hemostatic sponge is prepared by the steps of: The freezing is freezing at-20 ℃ for 3-9 h.

7. A collagen fiber hemostatic sponge prepared by the preparation method according to any one of claims 1-6.

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