WO2020191906A1 - 一种海绵质止血或组织密封材料及其制备方法 - Google Patents

一种海绵质止血或组织密封材料及其制备方法 Download PDF

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WO2020191906A1
WO2020191906A1 PCT/CN2019/087551 CN2019087551W WO2020191906A1 WO 2020191906 A1 WO2020191906 A1 WO 2020191906A1 CN 2019087551 W CN2019087551 W CN 2019087551W WO 2020191906 A1 WO2020191906 A1 WO 2020191906A1
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spongy
hemostatic
sponge
sealing material
tissue sealing
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French (fr)
Inventor
陈军
陈经纬
王德祥
丁少雄
赵晶
欧徽龙
夏光远
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Xiamen University
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Xiamen University
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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61L—METHODS OR APPARATUS FOR STERILISING MATERIALS OR OBJECTS IN GENERAL; DISINFECTION, STERILISATION OR DEODORISATION OF AIR; CHEMICAL ASPECTS OF BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES; MATERIALS FOR BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES
    • A61L24/00—Surgical adhesives or cements; Adhesives for colostomy devices
    • A61L24/0005—Ingredients of undetermined constitution or reaction products thereof
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61L—METHODS OR APPARATUS FOR STERILISING MATERIALS OR OBJECTS IN GENERAL; DISINFECTION, STERILISATION OR DEODORISATION OF AIR; CHEMICAL ASPECTS OF BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES; MATERIALS FOR BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES
    • A61L24/00—Surgical adhesives or cements; Adhesives for colostomy devices
    • A61L24/001—Use of materials characterised by their function or physical properties
    • A61L24/0015—Medicaments; Biocides
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61L—METHODS OR APPARATUS FOR STERILISING MATERIALS OR OBJECTS IN GENERAL; DISINFECTION, STERILISATION OR DEODORISATION OF AIR; CHEMICAL ASPECTS OF BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES; MATERIALS FOR BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES
    • A61L24/00—Surgical adhesives or cements; Adhesives for colostomy devices
    • A61L24/001—Use of materials characterised by their function or physical properties
    • A61L24/0036—Porous materials, e.g. foams or sponges
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61L—METHODS OR APPARATUS FOR STERILISING MATERIALS OR OBJECTS IN GENERAL; DISINFECTION, STERILISATION OR DEODORISATION OF AIR; CHEMICAL ASPECTS OF BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES; MATERIALS FOR BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES
    • A61L2300/00—Biologically active materials used in bandages, wound dressings, absorbent pads or medical devices
    • A61L2300/20—Biologically active materials used in bandages, wound dressings, absorbent pads or medical devices containing or releasing organic materials
    • A61L2300/30—Compounds of undetermined constitution extracted from natural sources, e.g. Aloe Vera
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61L—METHODS OR APPARATUS FOR STERILISING MATERIALS OR OBJECTS IN GENERAL; DISINFECTION, STERILISATION OR DEODORISATION OF AIR; CHEMICAL ASPECTS OF BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES; MATERIALS FOR BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES
    • A61L2300/00—Biologically active materials used in bandages, wound dressings, absorbent pads or medical devices
    • A61L2300/40—Biologically active materials used in bandages, wound dressings, absorbent pads or medical devices characterised by a specific therapeutic activity or mode of action
    • A61L2300/418—Agents promoting blood coagulation, blood-clotting agents, embolising agents
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61L—METHODS OR APPARATUS FOR STERILISING MATERIALS OR OBJECTS IN GENERAL; DISINFECTION, STERILISATION OR DEODORISATION OF AIR; CHEMICAL ASPECTS OF BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES; MATERIALS FOR BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES
    • A61L2400/00—Materials characterised by their function or physical properties
    • A61L2400/04—Materials for stopping bleeding

Definitions

  • the invention relates to a hemostatic or tissue sealing material and a preparation method, in particular to a natural spongy material.
  • the body's own repair mechanism is through the joint action of blood vessels, platelets, fibrinolytic balance system and blood coagulation system.
  • the blood vessel wall shrinks to slow down the blood flow. Platelets adhere to the surface of the foreign body and deform, and release coagulation factors to activate the thromboplastin in the blood.
  • the generated thrombin acts on the dissolved fibrinogen in the blood and turns it into The solid fibrin connects into a network structure to adsorb red blood cells, and finally forms a thrombus on the surface of the wound under the combined action of the fibrin network and platelets to inhibit bleeding.
  • Coagulation factors such as the most common quick-acting hemostatic powder, are mainly composed of inorganic particles such as zeolite and kaolin. The principle is to slow the bleeding rate by absorbing large amounts of water in plasma from the wound through its van der Waals force, concentrate the clotting factors, and accelerate the formation of blood clots by exothermic .
  • This type of product is one of the earliest hemostatic drugs, which also shined in the Iraq War, but its shortcomings cannot be ignored. First of all, local high temperature will burn the tissue wound, which is not conducive to the subsequent wound repair.
  • the current mainstream hemostatic products on the market are collagen and chitosan polysaccharides. , Is an adhesive product. Collagen itself has very strong water absorption, and also has good affinity and adsorption to platelets. It can quickly concentrate and activate platelets at the wound and promote the formation of blood clots. However, it promotes the growth of bacteria, frequently contaminates wounds and causes infections, and its adhesion is poor and easy to fall off. Therefore, the development direction of collagen is mostly surgical suture products. Chitosan itself has excellent antibacterial properties and histocompatibility.
  • the modified chitosan can adsorb negatively charged red blood cells through the positive charge of the amino group, so that the red blood cells can aggregate and adhere.
  • the disadvantage of chitosan is that it has limited hemostatic effect and cannot deal with extensive bleeding wounds. However, it is still a very ideal hemostatic base material, and it is the focus of the current research on multi-material composite hemostatic agents.
  • hemostatic drugs There is also a special type of hemostatic drugs. It does not have any endogenous hemostatic function, but uses exogenous substances to quickly crosslink and polymerize at the wound to achieve the purpose of fast adhesion to the wound and sealing the injured tissue.
  • the main products are synthetic polymer materials such as ⁇ -cyanoacrylate and polyethylene glycol.
  • the above hemostatic materials also have a common shortcoming.
  • the above-mentioned drugs are often incapable of stopping bleeding from their wounds. Therefore, it is very necessary to develop hemostatic drugs that supplement coagulation factors.
  • This kind of procoagulant drugs carry high concentrations of fibrinogen and thrombin and other procoagulant factors to complete the third stage of coagulation, but its cost is often very expensive, and at the same time, it needs a normal coagulation stage so it needs a certain coagulation time.
  • the effect of promoting the coagulation rate is not obvious, and it is not suitable for large-area bleeding.
  • soluble procoagulant hemostatic drugs can interfere with the balance of the fibrinolytic system, easily cause thrombosis in the body, and bury unnecessary safety risks. All of the above limits the use of such drugs.
  • the purpose of the present invention is to provide a new type of hemostatic material, which can simultaneously cover the advantages of multiple types of hemostatic drugs, and can be used in special conditions such as platelet deficiency and hemophilia lacking clotting factors, and to help patients to stop bleeding through wounds to reduce Its drug cost.
  • the technical solution adopted by the present invention to solve the technical problem is: a hemostatic material or tissue sealing material, the main component or part of which is a sponge grown in the ocean, sea water or fresh water.
  • the sponge removes fleshy components.
  • spongy is a complex collagen-like macromolecular substance with a triple helix structure, and the spongy itself has a complex three-dimensional mesh skeleton structure. Can be used to anchor platelets to fix blood clots.
  • a method for preparing a spongy hemostatic material includes the following steps:
  • step (3) Soak the sponge skeleton of step (2) in a NaOH solution with a concentration of 0.1 to 0.2 mol/L for 2 to 3 days, and take out a clean water to soak for 3 to 4 days;
  • step (3) Add the sponge skeleton of step (3) to the Tris-HCl buffer solution, stir and pulverize into a homogenous suspension, the concentration of the Tris-HCl buffer solution is 0.1M, and the pH is 7.8 at 37°C; Add 10% trypsin to the suspension, shake and digest for 2 to 3 days;
  • step (4) Filtering and separating the product of step (4), soaking and rinsing the separated precipitate with clean water for 2 to 4 times, and drying to obtain spongy precipitate, which is insoluble large branched fiber sponging B;
  • step (5) spongy sediment to be broken into small particles with a freezing crusher, and sieve to obtain the spongy powder SFM; and/or use the hydrogen peroxide method to degrade the step (5) spongy sediment into soluble spongy SR .
  • the sponge is a mesh corner sponge.
  • any grown sponge rots to decompose the epidermis and fleshy components of the sponge, or puts it in sea water and vigorously scrubs to wash off the fleshy components on the surface of the net corner sponge.
  • the step (2) is immersed in the HCL solution with a concentration of 0.8 mol/L for 2 days.
  • the step (3) is immersed in a NaOH solution with a concentration of 0.1 mol/L.
  • the spongy powder SFM is obtained through a 200-mesh sieve in the step (6).
  • the spongy hemostatic material includes spongy powder SFM and soluble spongy SR, and has a scattered pore structure.
  • the hemostatic material has a scattered pore structure, so that the material itself has water absorption and air permeability comparable to medical cotton wool. Strong water absorption can help increase the concentration of coagulation factors near the wound, and excellent air permeability helps the normal metabolism of the tissues near the wound and facilitates subsequent repair.
  • the hemostatic material combines the advantages of collagen and chitosan at the same time, and shows excellent enrichment and adsorption capacity for red blood cells and platelets.
  • the hemostatic material has good biocompatibility. It is observed under the electron microscope that platelets can quickly activate and differentiate when contacted with it, and red blood cells actively undergo benign deformation after contacting with it, and stick out the artificial foot for attachment.
  • the hemostatic material has quite excellent hemostatic properties, and has special hemostatic capabilities that collagen and other common hemostatic drugs do not possess, and can effectively act on special situations where blood cannot be coagulated due to coagulation factor defects. Without the participation of fibrinogen and platelets, the blood can still be coagulated normally.
  • the hemostatic material is derived from marine organisms and is a pure natural biological material. Being alienated from humans, there is no risk of infectious diseases. It has the advantages of low sensitivity, safety and low cost.
  • Figure 1 shows the morphological changes of red blood cells in the detection of red blood cell adsorption capacity.
  • upper left original sheep red blood cells
  • upper right red blood cells on silicon wafers in the blank group
  • lower left red blood cells in the SFM group
  • lower right red blood cells in the SR coating group .
  • Figure 2 shows the adsorption of red blood cells to various materials in the detection of red blood cell adsorption capacity, where A is the blank group; B is the SR coating group; C is the original form of SFM; D is the SFM enriched with red blood cells.
  • Figure 3 shows the aggregation of platelets to various materials, where upper left: original platelet morphology; upper right: blank group; lower left: SR coating group; lower right: SFM group.
  • Figure 4 shows the characterization of platelet-poor blood coagulation.
  • Figure 5 shows the structure of platelet-poor blood coagulated clot under the microscopic view of SFM material.
  • Figure 6 shows the coagulation characterization of defiberized sheep blood.
  • Figure 7 shows the structure of the coagulated blood clot of defiberized blood under the microscopic view of SFM material.
  • Figure 8 shows the microscopic structure of defibrillated blood coagulated blood clot of SR material.
  • a method for preparing a spongy hemostatic material includes the following steps:
  • Fresh adult net-corner sponges are usually left to decay to decompose the epidermis and fleshy components of the sponge, or placed in seawater and scrubbed vigorously, and the fleshy components on the surface of the net-corner sponge are washed off by physical means, leaving its skeleton.
  • the obtained sponge skeleton is cut into small pieces to facilitate subsequent experiments.
  • the precipitate and supernatant are separated by filtration.
  • the supernatant is enzymatically soluble intercellular linear fiber spongin A and other enzymatically hydrolyzed impurities, and the precipitate is insoluble large branched fiber sponging B.
  • the sponge In the reticulated sponge, the sponge is mainly insoluble sponge B, which has a more complex three-dimensional structure.
  • Experimental method use glass test tube method to detect clotting time.
  • Table 1 shows that the main hemostatic mechanism of the above hemostatic material collagen is to increase the local coagulation factor concentration and absorb platelets through water absorption, so the effect is not obvious in the in vitro coagulation experiment.
  • the Panax notoginseng component of Yunnan Baiyao promotes hemostasis, but it is the same as SR, collagen and other materials dissolved in the blood. As the concentration increases, it will have the opposite effect. It is speculated that it is due to the high concentration of foreign substances. It breaks the internal balance system of blood and interferes with the function and vitality of blood clotting factors and thrombin.
  • the spongy powder SFM is insoluble in blood, and its clotting time gradually shortens with the increase of the amount used, which has a significant effect compared with the blank group.
  • the test method of water absorption of simulated body fluid (SBF) commonly used in porous hemostatic materials is adopted. Prepare the SBF solution and pour it into a dry petri dish. Bake the sample to a constant weight in an oven at 60°C, weigh about 0.5 g of the sample and record the initial weight W0, add it to the petri dish and immerse it for 30 minutes, take it out and weigh the final weight Wt. Each group of samples was repeated three times and the average value was taken. Considering that the soluble spongy SR material is soluble in water, the experimental group only uses SFM raw materials, and cotton wool is used as the control group. Calculate the water absorption rate according to the following formula:
  • Table 2 shows the water absorption characteristics of sponge materials.
  • the complete spongy skeleton itself has a rich microporous structure, and its water absorption performance is better than that of common materials on the market.
  • the large particles of spongy that have not been sieved are mixed into a flocculent structure, which has a water absorption performance of 2.5 times that of the same flocculent medical cotton.
  • some of the macroscopic microporous structure of the spongy ground into small particles is destroyed, which reduces its water absorption capacity, but it can still reach half of the water absorption performance of medical cotton. Therefore, the pore structure of the spongy powder is conducive to the penetration of water molecules. As a hemostatic powder, it can excellently absorb water from the wound, which is beneficial to increase the concentration of local coagulation factors and accelerate blood coagulation at the wound.
  • red blood cells change from a spherical shape to irregular granular deformation, which is a manifestation of red blood cell stress in a bad environment.
  • the red blood cells in the SR group and the SFM group basically maintained a healthy and complete red blood cell morphology.
  • Source SR coating and SFM powder surface can enhance the adsorption capacity and help stabilize the blood clot on the wound surface. Analysis shows that the spongy materials SR and SFM have good biocompatibility to red blood cells.
  • FIG. 2 Comparing the scattered and irregularly stressed red blood cells in the blank group silicon plate, the surface of the silicon plate with SR coating is enriched with a large number of red blood cells. Due to the benign induction of pseudopodia, a large number of red blood cells are still firm after repeated washing with PBS Firmly adsorb the surface of the silicon plate. C and D are the changes before and after SFM exposure to red blood cells. It is observed that a large area of red blood cells are adsorbed on the surface of SFM. At the same time, free red blood cells accumulate around the anchored red blood cells through the intercellular macromolecular bridging force. SFM is complicated The bifurcated three-dimensional structure also provides multi-angle and stable support for the red blood cell accumulation, which ultimately promotes the formation of a complete blood clot.
  • the material itself can not only adsorb red blood cells, induce red blood cells to differentiate into pseudopodia, and at the same time, the complex bifurcation structure of the insoluble SFM material itself can aggregate red blood cells.
  • the body provides solid support. It is speculated that it can replace fibrin in the wound in practical applications, skip the steps of thrombin, fibrinogen to generate fibrin, etc., directly and quickly adsorb red blood cells to aggregate into clumps, and has excellent hemostasis value.
  • Preparation of platelet PBS solution Take platelet-rich plasma, centrifuge at 3500r/15min, take the precipitate and wash with PBS, repeat three times to obtain platelet PBS solution.
  • Platelets are the first outpost to stop bleeding. After recognizing tissue trauma or vascular rupture, platelets will sequentially adhere to the collagen fibers exposed at the injury site, deform and activate, release coagulation factors, a large number of aggregates form loose platelet thrombi, and then shrink into compact thrombus. Subsequent formation of collagen fiber monomers under the action of thrombin will further form large thrombi around the platelet thrombus to intercept free red blood cells and block the further outflow of blood from the wound.
  • the SFM group can save a lot of time spent in the hemostasis process without relying on the formation of fibrin monomers to provide structural strength, quickly provide platelets to form highly differentiated thrombi, can accelerate the rate of hemostasis, and avoid Problems such as massive blood loss and large wounds and thrombosis are not easy to form.
  • Platelet anemia Take fresh plasma and centrifuge at 1000rpm/10min in a centrifuge to separate red blood cells and plasma. The red blood cells are cleaned by repeated centrifugation with PBS solution for 3 times, and the plasma is centrifuged again at 3500 rpm/15min to take the upper platelet-poor plasma, repeat 3 times, and mix the red blood cells and the platelet-poor plasma according to the original ratio to obtain platelet-poor blood.
  • Control group blank, collagen, Yunnan Baiyao
  • the coagulation time of whole blood was expressed by the glass test tube method, the degree of blood coagulation was observed by the method of lysing free red blood cells with pure water in the macroscopic view, and the structure of thrombus was observed in the microscopic view by scanning electron microscope.
  • Blood that lacks platelets to participate in coagulation is difficult to coagulate under normal conditions.
  • the coagulation factor released after platelet activation activates prothrombin to be converted into thrombin, so that fibrinogen in the blood surrounds the platelet thrombus to form fibrin monomer, and finally completes clot formation and coagulation.
  • the main difference is the size of the spongy particle size and the material gap distance. It is speculated that the complete spongy skeleton has a mesh structure visible to the naked eye. The large spacing of the skeletons cannot make the red blood cells adhere and adsorb firmly. Through grinding, the smaller the particle size of the spongy material, the larger the specific surface area of the sponge material. The shorter the interstitial distance, the higher the efficiency of intercepting and adsorbing red blood cells, the faster the blood clotting rate.
  • SR material dissolves in the blood and cannot have a stable solid structure like other insoluble spongy particles. Comparing the defibrillation experiment and the platelet-poor blood, we found that the SR group needed platelets to form thrombus to provide structural assistance during the coagulation process, and the blood could coagulate normally without the presence of fibrin components, so we speculated that spongy The material can well replace the fibrinogen in the blood.
  • the formation of a dense network structure can be observed under the microscope, and the fiber formed by cross-linking with the fibrin monomer
  • the protein network is similar, and it also has the properties of enriching red blood cells and agglomerating platelets, thus directly replacing the function of fibrin clot, and coagulating with red blood cells and other coagulation substances in the blood to form thrombi, reducing the process of generating fibrin monomers in the three stages of coagulation, greatly Shorten the time required for clotting.
  • spongy As a hemostatic material, spongy has good water absorption and biocompatibility. It not only has excellent performance in red blood cell adsorption and platelet activation, but also can effectively act on special blood with coagulation factor barriers. Low cost and high output, which can greatly alleviate the problems of hemophilia and other special populations such as expensive drugs and difficult to pay. It has a broad market prospect.
  • the invention discloses a spongy hemostatic material and a preparation method thereof.
  • the hemostatic material prepared by the hemostatic material can simultaneously cover the advantages of a variety of hemostatic drugs, and can be used in special conditions such as platelet deficiency and hemophilia lacking coagulation factors to help patients It performs wound hemostasis to reduce the cost of its medicine.
  • the hemostatic material itself has good water absorption and biocompatibility, not only has excellent performance in red blood cell adsorption and aggregation of platelet activation, but also can effectively act on special blood with coagulation factor disorder, and its low cost and high yield can greatly relieve Special populations such as hemophilia patients are expensive and difficult to pay. It has a broad market prospect and has good industrial applicability.

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Abstract

一种海绵质止血材料及其制备方法。海绵质止血材料的主要成份或部分成分为在海水或淡水中生长的海绵。该海绵质止血材料能同时涵盖多种类止血药物的优点，且能用于血小板贫乏、缺乏凝血因子的血友病等特殊情况，帮助患者其进行创口止血，以减少其药物成本。该止血材料本身具有良好的吸水性和生物相容性，不仅在红细胞吸附和聚集血小板活化上表现优异，同时能有效作用于凝血因子障碍的特殊血液。

Description

一种海绵质止血或组织密封材料及其制备方法 技术领域
本发明涉及一种止血或组织密封材料及制备方法，特别涉及天然海绵质材料。
背景技术
在紧急事故或大型灾害环境中，幸存的受害者往往因后续不可控的大量出血而丧生。据统计2011～2013年间，我国每年约有60万人死于交通事故，其中85％人员死亡是由于在创伤初期引起的大量出血，若在30min内出血能得到紧急处理和控制，受伤人员的存活率将能提高40％以上；在战争中，紧急有效的止血处理是减少士兵伤亡的最有效措施之一，止血药物是军需方重视开发的战略领域；在日常生活中，最普遍的意外事件导致小规模创口出血是人们频繁经历而又不可避免的问题之一，或是一场手术的成功与否的关键在于能否控制住病人的出血情况；同时关注血友病这一大类人群，面临凝血障碍的困境，需要大量药物辅予正常生活。可以说人在生与死的角逐中，我们的机体无时无刻都在在经历出血后如何快速修复的挑战。因此开发高效的止血药物一直是医药领域科研工作者关注的重点和需要不断突破的难题。
在创口产生造成出血的情况发生后，机体自身的修复机制是通过血管、血小板、纤溶平衡系统和凝血系统共同作用的。血管壁通过收缩来减缓血液流动速度，血小板黏附到异物表面变形，并释放出凝血因子来激活血液中的凝血活酶，生成的凝血酶作用到血液中溶解态的纤维蛋白原，使之转变为固态的纤维蛋白联结成网状结构吸附红细胞，最终在纤 维蛋白网和血小板的共同作用下在伤口表面形成血栓抑制出血情况。
对于以上不同阶段的凝血过程及机理，研发了各类凝血药物，基本可划分凝集因子类、黏附类和促凝类。凝集因子类如最常见的速效止血粉，主要成分为沸石高岭土等无机物颗粒，原理是通过其范德华力吸收伤口处血浆大量水分来减缓出血速度，浓缩凝血因子，同时放热加速血凝块形成。这类产品是最早的止血药物之一，在伊拉克战争中也曾大放异彩，但其缺陷也是不容忽视的。首先局部高温会灼烧组织创面，不利于之后伤口修复，同时小颗粒无机物容易进入到血液中，造成肺部血栓；市面上目前主流的止血产品为胶原蛋白和壳聚糖多糖这两大块，为黏附类产品。胶原本身具有非常强的吸水性，同时对血小板也有很好的亲和及吸附性，能在伤口处快速浓缩与激活血小板，促进血凝块形成。不过其本身会促进细菌生长，频繁发生污染伤口造成感染事件，并且其黏附性较差易脱落，故胶原开发方向多为手术缝合线产品。壳聚糖本身具有优秀的抗菌性及组织相容性，改性后的壳聚糖能通过氨基的正电来吸附带负电荷的红细胞，从而使红细胞聚集黏附。壳聚糖的不足在于其本身止血效果有限，无法处理广泛出血的创面，不过其仍是非常理想的止血基底材料，是目前多种材料复合止血剂研究的焦点；另外还有一类特殊止血药物，本身无任何内源性止血功能，而是利用外源物质在伤口处快速交联聚合来达到快速黏合伤口和封闭受伤组织目的。主要的产品为α－氰基丙烯酸酯和聚乙二醇等人工合成的高分子材料。虽然能快速封闭伤口，但在后期降解过程中会产生有害物质导致组织发炎坏死，所以运用情况较为特殊。
以上止血材料还有一大共通的缺憾，对于血液凝固障碍的患者，上述的各类药品对于其伤口止血往往无能为力，所以开发补充凝血因子的止血药物是非常有必要的。这类促凝类的药物携带高浓度的纤维蛋白原和凝血酶等促凝因子来完成凝血三阶段，但其成本往往十分昂贵，同时本身需要进行正常的凝血阶段所以需要一定的凝血时间，与上述止血材料相比促进凝血速率的效果不明显，不适用于大面积出血情况。尤其一点是可溶性的促凝止血药物会干扰到纤溶系统的平衡，容易在体内造成血栓，埋下不必要的安全隐患。以上种种限制了这类药物的使用。
发明内容
本发明的目的是提供一种新型的止血材料，能同时涵盖多种类止血药物的优点，且能用于血小板贫乏、缺乏凝血因子的血友病等特殊情况，帮助患者其进行创口止血，以减少其药物成本。
本发明解决其技术问题所采用的技术方案是：一种止血材料或组织密封材料，其主要成份或部分成分为在海洋或海水或淡水中生长的海绵。特别是所述的海绵去除肉质组份。
在海洋中最古老的生物之一海绵动物中，其独特的有机物质海绵质，是为一种含三螺旋结构的类胶原复杂大分子物质，且海绵质本身具有复杂的三维网孔骨架结构，可用于锚定血小板固定凝血块。
所提供了一种海绵质止血材料的制备方法，包括如下步骤：
(1)取新鲜的成株海绵，通过物理手段洗去海绵表面肉质组分，留下海绵骨架；
(2)将步骤(1)的海绵骨架浸泡于浓度为0.7～0.8mol/L的HCL 溶液中2～3d，取出以清水洗涤数次；
(3)将步骤(2)的海绵骨架浸泡于浓度为0.1～0.2mol/L的NaOH溶液中2～3d，取出一清水浸泡3～4d；
(4)将步骤(3)的海绵骨架加入Tris－HCl缓冲液中，搅拌粉碎为匀浆悬浊液，所述Tris－HCl缓冲液的浓度为0.1M，37℃下PH为7.8；接着在悬浊液中加入10％胰蛋白酶，震荡酶解2～3d；
(5)将步骤(4)的产物过滤分离，将分离得到的沉淀以清水浸泡冲洗2～4次，烘干得到海绵质沉淀，所述海绵质沉淀为难溶性大型分支纤维sponging B；
(6)取步骤(5)海绵质沉淀用冷冻破碎仪破碎成小颗粒，过筛得到海绵质粉末SFM；和/或用过氧化氢法将步骤(5)海绵质沉淀降解成可溶性海绵质SR。
在本发明一较佳实施例中，所述海绵为网角海绵。
在本发明一较佳实施例中，所述步骤(1)中任成株海绵腐烂分解掉海绵的表皮和肉质组分，或放入海水中大力搓洗，洗去网角海绵表面肉质组分。
在本发明一较佳实施例中，所述步骤(2)中浸泡于浓度为0.8mol/L的HCL溶液中2d。
在本发明一较佳实施例中，所述步骤(3)中浸泡于浓度为0.1mol/L的NaOH溶液。
在本发明一较佳实施例中，所述步骤(6)中过200目筛得到海绵质粉末SFM。
在本发明一较佳实施例中，所述海绵质止血材料包括海绵质粉末SFM和可溶性海绵质SR，具有错落孔隙结构。
本技术方案与背景技术相比，它具有如下优点：
1.该止血材料具有错落孔隙结构，使得该材料本身有能与医用棉絮比拟的吸水性和透气性。强吸水性能有助于提高伤口附近凝血因子浓度，优良透气性有助于伤口附近组织正常新陈代谢，便于后续修复。
2.该止血材料同时结合了胶原和壳聚糖的优点，对血红细胞、血小板表现出优秀的富集作用和吸附能力。
3.该止血材料具有良好的生物相容性，在电镜下观察到血小板与之接触能快速激活分化，血红细胞与之接触后主动进行良性变形，伸出拟足附着。
4.该止血材料具有相当优异的止血性能，且具有胶原和其它常见的止血药物所不具备的特殊止血能力，能有效作用于由于凝血因子缺陷而导致血液无法凝固的特殊情况。在无纤维蛋白原和血小板参与下仍能保证血液能够正常凝固。
5.该止血材料源于海洋生物，为纯天然的生物材料。与人类亲缘关系疏远，无传染疾病风险。具有低敏性、安全性和成本低廉等优点。
附图说明
图1为血红细胞吸附能力检测中血红细胞形态变化，其中，左上：原始羊血红细胞形态；右上：空白组硅片上血红细胞；左下：SFM组血红细胞；右下：SR涂层组血红细胞。
图2为血红细胞吸附能力检测中血红细胞对各材料吸附，其中，A为 空白组；B为SR涂层组；C为SFM原始形态；D为富集红细胞的SFM。
图3为血小板对各材料聚集性，其中，左上：血小板原始形态；右上：空白组；左下：SR涂层组；右下：SFM组。
图4为贫血小板血液凝固表征。
图5为SFM材料微观下贫血小板血液凝固血块结构。
图6为脱纤维羊血凝固表征。
图7为SFM材料微观下脱纤维血液凝固血块结构。
图8为SR材料微观下脱纤维血液凝固血块结构。
具体实施方式
实施例1
本实施例一种海绵质止血材料的制备方法，包括如下步骤：
(1)新鲜的成株网角海绵，通常是任其腐烂分解掉海绵的表皮和肉质组分，或放入海水中大力搓洗，通过物理手段洗去网角海绵表面肉质组分，留下其骨架。将所得海绵骨架裁剪成小块，方便后续实验。
(2)配置0.8mol/L浓度的HCL，将海绵骨架小块浸泡2d，目的是除去残余的表面杂质、含钙络合物和部分酸溶性蛋白。
(3)取不溶物海绵质骨架清水洗涤数次，后用0.1mol/L浓度的NaOH浸泡，清洗海绵质残留的部分碱溶性蛋白和碱溶性杂质。
(4)将不溶物海绵质骨架用清水浸泡3次，除去残余氢氧化钠溶液，加入Tris－Hcl缓冲液(0.1M，PH7.8，37℃)，搅拌粉碎为匀浆悬浊液。再加入10％胰蛋白酶，震荡酶解2d。
(5)过滤分离出沉淀和上清。上清中为酶溶性胞间线性纤维 spongin A和其余酶解杂质，沉淀为难溶性大型分支纤维sponging B。在网角海绵中海绵质主要为难溶性的spongin B，具有更复杂的三维结构。
(6)取沉淀用清水浸泡冲洗3次，烘干。
①用冷冻破碎仪破碎成小颗粒，过200目筛得到海绵质粉末SFM。
②用过氧化氢法将上述海绵质沉淀降解成可溶性海绵质SR。
一、止血性能检测(体外凝血时间、吸水率)
①体外凝血时间
实验方法：采用玻璃试管法检测凝血时间。
用枸橼酸钠采血管采取新鲜鸡血，取1mL血浆于玻璃管，在41℃水浴锅下预温，后加入2.8mg/mL的氯化钙溶液和各实验组材料，记录血液完全凝固时间，每组重复3次取均值。实验组材料分别为可溶性海绵质SR、不溶性海绵质粉末SFM，对照组为空白、云南白药(购于市面)、胶原蛋白。其实验结果如下表所示：
表1 各实验材料凝血时间
Figure PCTCN2019087551-appb-000001
表1可以看出，以上止血材料胶原的主要止血机理是通过吸水性增大局部凝血因子浓度及吸附血小板，因此在体外凝血实验中效果并不明显。云南白药的三七成分是起到促进止血的作用，但同SR、胶原这类溶解于血液中的材料相同，随着浓度的提升反而会起到相反效果，猜测是由于高浓度的外源物质打破了血液内在的平衡体系，干扰到凝血因子和凝血酶的作用及活力。而海绵质粉末SFM不溶于血液，随着使用量的增大其凝血时间逐渐缩短，与空白组相对照具有显著效果。
②吸水率
实验方法：采用多孔止血材料常用的模拟体液(SBF)吸水率测试方法。配置SBF溶液，倒入干燥培养皿中。将样品在60℃烘箱内烘至恒重，称取约0.5g样品记录初始重量W0，加入到培养皿浸没30min，取出称最终重量Wt。每组样品重复三次，取平均值。考虑到可溶海绵质SR材料溶于水，故实验组仅采用SFM原料，以棉絮为对照组。按照以下公式计算吸水率：
饱和吸水率＝(Wt－W0)/W0×100％
其实验结果如下表所示：
表2 止血材料吸水率
Figure PCTCN2019087551-appb-000002
表2为海绵质材料吸水率特性。完整海绵质骨架本身具有丰富微孔结构，其吸水性能优于市场上常见的各种材料。将海绵质骨架研磨至粉 末过筛后，未能过筛的大颗粒海绵质相互糅合成絮状结构，与同为絮状的医用棉花相比吸水性能是其2.5倍。但研磨成小颗粒的海绵质一些宏观的微孔结构被破坏，使得其吸水能力下降，但仍能达到医用棉花一半的吸水性能。故海绵质粉末自身的孔隙结构利于水分子的渗透，作为止血药物粉末能出色完成伤口处水分吸收，有利于提升局部凝血因子浓度，加速伤口处血液凝固。
二、血红细胞吸附能力、血小板聚集性检测
①血红细胞吸附能力
实验材料：新鲜羊血，海绵质粉末SFM，可溶海绵质SR
实验方法：
1、血红细胞获取：取新鲜羊血，离心1000r/15min，取沉淀用PBS冲洗，重复三次。从羊血中获得纯净的血红细胞PBS溶液。
2、材料涂片的制备：
①将可溶海绵质SR调配成溶液，滴在硅片上，烘干使其在表面形成薄膜
②将海绵质粉末SFM均匀撒在鸡蛋清液浸润过的硅片上，烘干使其固定。
③空白硅片用酒精清洗，烘干。
3、将实验组的硅片浸润在血红细胞PBS溶液中，于37℃环境下静置30min
4、取上述硅片用PBS轻轻冲洗3次，放入2.5％戊二醛固定过夜。
5、依次放入50％、60％、70％、80％、95％、100％乙醇梯度脱水，每次15min。
6、喷金，扫描电镜观察红细胞吸附情况。
实验结果：
图1，在30min的血红细胞和空白硅片的接触后，血红细胞由圆球形态转为不规则粒状形变，为不良环境下红细胞的应激表现。相较于空白组，SR组和SFM组中血红细胞基本都保持健康完整的血红细胞形态，同时观察到血红细胞在接触到外源物质时开始作出止血反馈，良性形变伸出伪足主动吸附在外源SR涂层、SFM粉末表面，，能增强吸附能力有助于伤口表面血凝块的稳固。分析可知海绵质材料SR和SFM对血红细胞有良好的生物相容性。
图2，对比空白组硅板中零散不规则的应激形态血红细胞，具有SR涂层的硅板表面富集了大量血红细胞，由于良性诱导伪足产生，在PBS反复冲洗后大量红细胞仍牢牢吸附硅板表面。C、D为SFM接触血红细胞前后变化图，观察发现大面积血红细胞吸附在SFM表面，同时通过细胞间大分子桥联力作用使得游离的血红细胞聚积在已锚定血红细胞周围，而SFM复杂分叉的三维结构也给血红细胞聚积团提供多角度稳定的支撑力，最终促使形成完整的血凝块。
分析可知海绵质材料对血红细胞有良好生物相容性，材料本身不仅能对血红细胞有吸附作用，诱导红细胞分化出伪足吸附，同时不溶性的SFM材料本身复杂的分叉结构能给血红细胞聚集体提供牢靠的支撑力，推测其在实际应用中能在创口处取代纤维蛋白的作用，跳过凝血酶、纤 维蛋白原生成纤维蛋白等步骤，直接快速吸附血红细胞凝聚成块，具有优秀的止血价值。
②血小板聚集性
实验材料：富血小板血浆，海绵质粉末SFM，可溶海绵质SR
实验方法：
1、血小板PBS溶液制备：取富血小板血浆，离心3500r/15min，取沉淀用PBS冲洗，重复三次，获得血小板PBS溶液。
2、材料涂片的制备：
①将可溶海绵质SR调配成溶液，滴在硅片上，烘干使其在表面形成薄膜
②将海绵质粉末SFM均匀撒在鸡蛋清液浸润过的硅片上，烘干使其固定。
③空白硅片用酒精清洗，烘干。
3、将实验组的硅片浸没在血小板PBS溶液中，于37℃环境下静置30min
4、取上述硅片用PBS轻轻冲洗3次，放入2.5％戊二醛固定过夜。
5、依次放入50％、60％、70％、80％、95％、100％乙醇梯度脱水，每次15min。
6、喷金，扫描电镜观察血小板聚集情况。
实验结果：
血小板是止血的第一前哨。在识别到组织创伤或血管破裂后，血小板会依次在损伤处暴露的胶原纤维上进行黏附、变形活化、释放凝血因 子、大量聚集形成松软血小板血栓、再收缩成紧实血栓。后续凝血酶作用下形成胶原纤维单体也会围绕血小板血栓进一步形成大血栓拦截游离的红细胞，堵截伤口血液进一步外流。
在图3中，对比空白组硅板上分散稀释的未分化变形的血小板，具有SR涂层的硅板上有黏附了大量血小板，同时已活化变形，释放出凝血因子使得血小板开始聚集反应，在硅板平面上拉伸成树枝状结构，形成初步的网格脉络。而SFM组中我们可以观察到，血小板不止大量黏附在海绵质材料表面，同时高度分化变形，聚集形成血小板血栓，且已进行收缩因子的释放，最终在SFM表面形成紧实的血栓层。在实际应用中，SFM组能在不依赖纤维蛋白单体的形成来提供结构力的前提下，能节约大量止血过程所耗费的时间，快速提供结合血小板形成高度分化血栓，能加速止血速率，避免血液大量流失及创口过大血栓不易形成等问题。
三、特殊的止血性能
海绵质材料最为特殊的止血性能表现在对于有凝血因子缺陷的血液与之接触作用后能表现正常凝血功能。我们实验中试验了贫血小板血液和脱纤维蛋白原血液进行检测。
实验原料如下：
无菌脱纤维羊血：采购于南京茂捷微生物有限公司
贫血小板血：取新鲜血浆于离心机离心1000rpm/10min，分离红细胞和血浆。将红细胞用PBS溶液反复离心清洗3次，血浆再离心3500rpm/15min，取上层贫血小板血浆，重复3次，将红细胞与贫血小板血浆按原比例混合得到贫血小板血。
实验组：小颗粒海绵质粉末SFM、可溶性降解海绵质SR、大颗粒絮状海绵质SX、完整海绵质骨架SP
对照组：空白、胶原蛋白、云南白药
实验方法：采用玻璃试管法表达全血凝固时间，宏观上采用纯水裂解游离红细胞法观测血液凝固程度，微观上采用扫描电镜观测形成血栓的结构。
实验结果：
缺乏血小板参与凝血的血液在正常条件下是难以凝固的。血小板活化后释放的凝血因子激活凝血酶原转化为凝血酶，使得血液中纤维蛋白原围绕血小板血栓形成纤维蛋白单体，最终完成血块生成与凝固。
表3 贫血小板血液对各材料凝血时间(单位：min)
Figure PCTCN2019087551-appb-000003
结合表3、图4，可以看出对照组各材料最终无法促使存在血小板缺陷的血液完成凝固。而海绵质材料对于贫血小板血液有显著的止血效果，同时其止血效果与海绵质本身的形态结构有关。
对比SFM和SX、SP的结构，其主要差别是海绵质粒径大小和材料间隙距离。推测是完整的海绵质骨架为肉眼可见的网孔结构，骨架间距大无法使得血红细胞牢靠附着和吸附，而通过研磨，海绵质材料的粒径越小，海绵质材料的比表面积越大，海绵质间距缩短，其拦截吸附血红细胞的效率越高，血液的凝固速率愈快。
对比图5、图6可以看出，贫血小板血液形成的血栓表面相对光滑，缺少了血小板形成血栓支撑物。而SR材料是溶解于血液，本身亦无提供支撑力的稳定结构，所以在实验中观测到SR组中血液未能完全凝固，但在重力沉降和缺少支撑力的相互作用下，血液在底部形成局部血块。
表4 脱纤维羊血对各材料凝血时间(单位：min)
Figure PCTCN2019087551-appb-000004
表4、图6、图7为脱纤维血液的凝固实验结果。我们所知血栓形成的过程中各凝血机制最终目的是将血液中的纤维蛋白原转化为固态的纤维蛋白单体网络，配合血小板血栓去固定游离红细胞和血浆成分。而脱纤维血液实验我们排除了纤维蛋白原这一成分，实验结果表明仅有SR 材料和SFM能使得脱纤维羊血能够正常凝固。结合上述实验，分析认为：
1、SR材料溶解在血液中，无法像其他不溶态海绵质颗粒物有稳定的固态结构。对比脱纤维血实验和贫血小板血液，我们发现SR组在凝血过程中需要血小板形成血栓提供结构力上的协助，而在无纤维蛋白组分的存在下能使得血液正常凝固，因此我们推测海绵质材料能够良好的替代血液中纤维蛋白原的成分。
2、对比SFM、SX、SP的实验结果，发现不同于其它止血药物过高浓度会干扰血液凝结反应的现象，海绵质在血液中含量越高，材料本身粒度越小，其凝血性能越好。推测是海绵质不仅是单纯的替代纤维蛋白原，并且不溶态的海绵质粉末个体分枝间相互交叉重叠在微观下能观察到形成致密的网状结构，与纤维蛋白单体交联形成的纤维蛋白网相似，本身也具有富集红细胞和聚集血小板特性，从而直接替代纤维蛋白凝块功能，与血液中红细胞和其他凝血物质凝结成血栓，缩减了凝血三阶段生成纤维蛋白单体的过程，大大缩短了凝血所需的时间。
综上实验所述，海绵质作为止血材料，本身具有良好的吸水性和生物相容性，不仅在红细胞吸附和聚集血小板活化上表现优异，同时能有效作用于凝血因子障碍的特殊血液，且其成本低廉产量高，能大大缓解血友病患者等特殊人群用药昂贵难以支付等问题，具有广泛的市场前景。
以上所述，仅为本发明较佳实施例而已，故不能依此限定本发明实施的范围，即依本发明专利范围及说明书内容所作的等效变化与修饰，皆应仍属本发明涵盖的范围内。
工业实用性
本发明公开了一种海绵质止血材料及其制备方法，其制备的止血材料能同时涵盖多种类止血药物的优点，且能用于血小板贫乏、缺乏凝血因子的血友病等特殊情况，帮助患者其进行创口止血，以减少其药物成本。且该止血材料本身具有良好的吸水性和生物相容性，不仅在红细胞吸附和聚集血小板活化上表现优异，同时能有效作用于凝血因子障碍的特殊血液，且其成本低廉产量高，能大大缓解血友病患者等特殊人群用药昂贵难以支付等问题，具有广泛的市场前景，具有良好的工业实用性。

Claims (11)

  1. 一种海绵质止血或组织密封材料的制备方法，其特征在于，包括如下步骤：
    (1)取新鲜的成株海绵，通过物理手段洗去海绵表面肉质组分，留下海绵骨架；
    (2)将步骤(1)的海绵骨架浸泡于浓度为0.7～0.8mol/L的HCL溶液中2～3d，取出以清水洗涤数次；
    (3)将步骤(2)的海绵骨架浸泡于浓度为0.1～0.2mol/L的NaOH溶液中2～3d，取出一清水浸泡3～4d；
    (4)将步骤(3)的海绵骨架加入Tris－HCl缓冲液中，搅拌粉碎为匀浆悬浊液，所述Tris－HCl缓冲液的浓度为0.1M，37℃下PH为7.8；接着在悬浊液中加入10％胰蛋白酶，震荡酶解2～3d；
    (5)将步骤(4)的产物过滤分离，将分离得到的沉淀以清水浸泡冲洗2～4次，烘干得到海绵质沉淀，所述海绵质沉淀为难溶性大型分支纤维sponging B；
    (6)取步骤(5)海绵质沉淀用冷冻破碎仪破碎成小颗粒，过筛得到海绵质粉末SFM；和/或用过氧化氢法将步骤(5)海绵质沉淀降解成可溶性海绵质SR。
  2. 根据权利要求1所述的一种海绵质止血或组织密封材料的制备方法，其特征在于：所述海绵为网角海绵。
  3. 根据权利要求1所述的一种海绵质止血或组织密封材料的制备方法，其特征在于：所述步骤(1)中任成株海绵腐烂分解掉海绵的表皮 和肉质组分，或放入海水中大力搓洗，洗去网角海绵表面肉质组分。
  4. 根据权利要求1所述的一种海绵质止血或组织密封材料的制备方法，其特征在于：所述步骤(2)中浸泡于浓度为0.8mol/L的HCL溶液中2d。
  5. 根据权利要求1所述的一种海绵质止血或组织密封材料的制备方法，其特征在于：所述步骤(3)中浸泡于浓度为0.1mol/L的NaOH溶液。
  6. 根据权利要求1所述的一种海绵质止血或组织密封材料的制备方法，其特征在于：所述步骤(6)中过200目筛得到海绵质粉末SFM。
  7. 根据权利要求1所述的一种海绵质止血材料的制备方法，其特征在于：所述海绵质止血材料具有错落孔隙结构。
  8. 一种止血材料或组织密封材料，其特征在于其主要成份或部分成分为在海水或淡水中生长的海绵。
  9. 根据权利要求8的止血材料或组织密封材料，其特征在于所述的海绵去除表面肉质组份。
  10. 根据权利要求8或9的止血材料或组织密封材料，其特征在于所述的海绵经沉淀用冷冻破碎，得到海绵质粉末SFM。
  11. 根据权利要求8或9的止血材料或组织密封材料，其特征在于所述的海绵质材料经沉淀降解成可溶性海绵质SR。
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