技术领域technical field
本发明涉及一种新型多孔止血材料及其制备方法，属于医药新剂型技术领域。The invention relates to a novel porous hemostatic material and a preparation method thereof, belonging to the technical field of new pharmaceutical dosage forms.
背景技术Background technique
我国传统医学对于创伤的认识起源于在三千年前的周代，从那时起，外科学就已经成为一门独立的学科，据《周礼·家宰》，早在大约公元前11世纪中医就分为四科，分别是疡医、食医、疾医和兽医，并明确提出：“疡医，掌肿疡、溃疡、疡、折疡之祝药，刮杀之齐”。这里的“祝药”就是指外敷药，由此来看，外伤及刀剑伤归为“疡”的范畴，古代的疡医，即指现代的外科医生，专治一切创伤和疮疡。而“金疡”类似于今天外科学中所称的物理性切割伤，就是指由刀、釜、剑、矢等利物导致的损伤。“疡”伴随着整个人类生产实践的过程同时也见证着人类文明的发展。中医学整体观认为，人体是由脏腑、经络、皮肉、筋骨、气血、精与津液等共同组成的整体，外因作用所带来的损害，可导致脏腑及经络、气血功能失调。《杂病源流犀烛》说:“损伤之患，……而经络脏腑并与俱伤”。故中医学认为金疡的造成的结果为皮肉受害、筋骨病损、筋络阻塞、以致气滞血癖，邪毒与气血相博，不通、不荣则痛。The understanding of trauma in traditional Chinese medicine originated in the Zhou Dynasty 3,000 years ago. Since then, surgery has become an independent discipline. It is divided into four subjects, which are ulcer medicine, food medicine, disease medicine and veterinary medicine, and it is clearly stated: "Ulcer medicine, palm swollen ulcers, ulcers, ulcers, ulcers, and sores, scraping and killing." The "Zhuyao" here refers to the medicine for external application. From this point of view, trauma and sword wounds are classified into the category of "sores". Ancient ulcer doctors refer to modern surgeons who specialize in treating all wounds and sores. The "golden ulcer" is similar to the physical cutting injury called in today's surgery, which refers to the injury caused by sharp objects such as knives, cauldrons, swords, and arrows. "Ulcer" accompanies the entire process of human production practice and also witnesses the development of human civilization. The holistic view of traditional Chinese medicine believes that the human body is composed of viscera, meridians, skin, muscles, bones, qi and blood, essence and body fluid. "Origin of Miscellaneous Diseases Rhinoceros Candle" said: "The suffering of injury... and the viscera of the meridians and collaterals are all injured." Therefore, traditional Chinese medicine believes that the results of gold sores are skin damage, muscle and bone damage, and blockage of tendons and collaterals, resulting in qi stagnation and blood addiction.
传统医学根据整体观和辩证论治两大原则，建立起了一套完整的关于创伤修复的理论体系。在中医药治疗外科疾病的手段中，外治法最为关键，是从体外进行治疗，即施于体表的方法，相比于内治法，它直接作用于体表病灶，在这一点上，要优于内治法。《素问·至真要大论》就明确主张“外者外治”。在中医理论指导下，历代医家多从“收敛、益气、祛腐、补益、清热、养血、解毒”角度来治疗外科疾病。徐灵胎《医学源流论》云：“外科之法，最重外治”。我国现存最早的外科专著《刘涓子鬼遗方》，共载处方140个，对外伤的治疗措施有对止血、止痛、收敛、切开、排脓、引流、解毒等诸法，并用黄连、水银、雄黄等解毒、去腐药物制成药膏以治疗外科疾病，如皮肤溃疡。其阐述的许多治法至今依然颇为实用。晋代葛洪所著《肘后备急方》中就已经针对化脓性伤口提出了“清洗疮口”的办法，这种提法的学术价值很高。隋代巢元方《诸病源候论》一书中有“夫金疮伤之时，若碎骨不去，脓血不止”一说，就已经认识到对于复杂伤口进行彻底清创和早期缝合是极其必要和有效的，这一观点较西方学者于1363年提出的清创引流早了约700多年。Based on the two principles of holistic view and dialectical treatment, traditional medicine has established a complete theoretical system on trauma repair. Among the means of treating surgical diseases with traditional Chinese medicine, external treatment is the most critical method, which is to treat from outside the body, that is, the method applied on the body surface. Compared with the internal treatment method, it directly acts on the body surface lesions. In this regard, It is better than internal treatment. "Suwen Zhizhenyaodalun" clearly advocates "the outsider governs outside". Under the guidance of TCM theory, physicians of past dynasties have treated surgical diseases from the perspective of "converging, replenishing qi, removing rot, nourishing, clearing away heat, nourishing blood, and detoxifying". Xu Lingtai's "On the Origin and Development of Medicine" says: "The method of surgery is the most important external treatment." The earliest surviving surgical monograph in my country, "Liu Juanzi Guiyi Fang", contains a total of 140 prescriptions. The treatment measures for external injuries include hemostasis, pain relief, convergence, incision, pus drainage, drainage, and detoxification. Coptidis, mercury, Realgar and other detoxification and antiseptic drugs are made into ointments to treat surgical diseases, such as skin ulcers. Many of the treatments he described are still quite practical today. In "Elbow Reserve Emergency Prescriptions" written by Ge Hong in the Jin Dynasty, the method of "cleaning the sores" has been proposed for purulent wounds. This formulation has high academic value. Chao Yuanfang of the Sui Dynasty wrote in his book "On the Causes and Symptoms of Various Diseases" that "when the husband's sore is injured, if the broken bone does not go away, the pus and blood will not stop." It has been recognized that thorough debridement and early suturing of complex wounds Extremely necessary and effective, this view is about 700 years earlier than the debridement and drainage proposed by Western scholars in 1363.
与常规的促愈合手段相比较，中医学理论指导下的具有止血、消肿生肌作用的中药疗效确切且独特，中国古代医家常将具有止血、消肿生肌的中药制成粉剂、膏剂外用，具有悠久的历史，中药外治的优势在于：药物的化学成分可以直达病灶，使病灶局部表面的有效化学成分浓度明显高于体内血药浓度，以此来发挥迅捷的作用，但此类外用制剂使用不便，易受污染，所以局限性很大。基于此，本发明运用材料工程学和制药工程学中的真空冷冻干燥技术，结合了中药现代化创新成果-微纳米中药技术，旨在制成一种我国传统中药有效止血并促进创面肉芽组织生长、毛细血管再生的多孔海绵止血材料。Compared with conventional means of promoting healing, under the guidance of the theory of traditional Chinese medicine, traditional Chinese medicines with the functions of hemostasis, detumescence and muscle regeneration have exact and unique curative effects. Ancient Chinese doctors often made traditional Chinese medicines with hemostasis, detumescence and muscle regeneration into powders and ointments for external use. , has a long history. The advantage of external treatment of traditional Chinese medicine is that the chemical components of the drug can directly reach the focus of the lesion, so that the concentration of the effective chemical component on the local surface of the lesion is significantly higher than the blood concentration in the body, so as to exert a rapid effect. The preparation is inconvenient to use and is easily contaminated, so it has great limitations. Based on this, the present invention uses the vacuum freeze-drying technology in materials engineering and pharmaceutical engineering, and combines the innovative achievements of traditional Chinese medicine modernization-micro-nano Chinese medicine technology, aiming to make a traditional Chinese medicine that can effectively stop bleeding and promote the growth of wound granulation tissue. Porous sponge hemostatic material for capillary regeneration.
发明内容Contents of the invention
本发明的目的之一在于提供一种新型多孔止血材料的组成配比；One of the objectives of the present invention is to provide a composition ratio of a novel porous hemostatic material;
本发明的目的之二在于提供该止血材料的制备方法。The second object of the present invention is to provide a preparation method of the hemostatic material.
本发明提供的新型多孔止血材料，是由如下质量配比的材料组成：三七粉0.5-3份，白芨胶0.5-3份，明胶2-10份，水80-120份；The novel porous hemostatic material provided by the present invention is composed of materials with the following mass proportions: 0.5-3 parts of Panax notoginseng powder, 0.5-3 parts of Bletilla striata glue, 2-10 parts of gelatin, and 80-120 parts of water;
其中，对于上述本发明止血材料进一步优选地方案，所述的材料质量配比为：Wherein, for the further preferred solution of the above-mentioned hemostatic material of the present invention, the mass ratio of the materials is:
三七粉1份，白芨胶1份，明胶6份，水100份；1 part of notoginseng powder, 1 part of bletilla striata gum, 6 parts of gelatin, 100 parts of water;
进一步，本发明止血材料进一步优选地方案，所述的材料质量配比为：Further, in a further preferred solution of the hemostatic material of the present invention, the mass ratio of the materials is:
三七粉2份，白芨胶2份，明胶8份，水110份；2 parts of notoginseng powder, 2 parts of bletilla striata gum, 8 parts of gelatin, 110 parts of water;
又进一步，本发明止血材料进一步优选地方案，所述的材料质量配比为：Still further, in a further preferred solution of the hemostatic material of the present invention, the mass ratio of the materials is:
三七粉3份，白芨胶3份，明胶10份，水120份。3 parts of notoginseng powder, 3 parts of bletilla striata gum, 10 parts of gelatin, and 120 parts of water.
上述本发明所有组成配比的止血材料，进一步来说，所述的三七粉为微米粉，其粒径小于75μm；所述的三七微米粉是利用球磨法制得，具体方法是将三七粉碎成粗粉后放入球磨罐中，在真空状态下，粉碎参数设定为球料比为6:1，转速为300转每分钟，时间20-40分钟。The above-mentioned hemostatic material with all the composition ratios of the present invention, further, the said notoginseng powder is a micron powder, and its particle size is less than 75 μm; the described notoginseng micron powder is made by ball milling, and the specific method is to grind the After pulverizing into coarse powder, put it into a ball mill jar. Under vacuum, the pulverization parameters are set to a ball-to-material ratio of 6:1, a rotating speed of 300 rpm, and a time of 20-40 minutes.
上述本发明所有组成配比的止血材料，进一步来说，所述的白芨胶是由如下方法制备而成：取白芨药材，切制成片，第一次浸泡提取，加入与药材质量比为7～9倍量的水，浸泡24小时，滤过,滤液备用；第二、三次浸泡提取，滤过的药渣中加入与药材质量比为3倍量的水，浸泡8-12小时，滤过,合并滤液后，加入乙醇使含醇量达70％-90％，静置2-4h，将沉淀物干燥，即得。The above-mentioned hemostatic material with all the composition ratios of the present invention, further, the bletilla striata glue is prepared by the following method: take the bletilla striata medicinal material, cut into slices, soak and extract for the first time, and add to the medicinal material with a mass ratio of 7 ~9 times the amount of water, soaked for 24 hours, filtered, and the filtrate was used for later use; for the second and third times of soaking and extraction, add water with a mass ratio of 3 times the medicinal material to the filtered medicinal residue, soaked for 8-12 hours, filtered , after combining the filtrates, add ethanol to make the alcohol content reach 70%-90%, let it stand for 2-4h, and dry the precipitate to obtain.
上述本发明所有组成配比的止血材料，进一步来说，所述止血材料的剂型为海绵剂。The above-mentioned hemostatic material with all compositional ratios in the present invention, further, the dosage form of the hemostatic material is a sponge.
更进一步，所述止血海绵剂的制备方法如下：按质量比取三七粉、白芨胶、明胶、水，混匀，置入50℃-60℃水浴中搅拌溶解后，置于冻干设备中进行预冷冻，持续时间36h～48h后，再进行冻干处理，其温度设置为-55℃，压力为0.400mbar，冷冻时间至少48h以上，取出，切块，包装。Further, the preparation method of the hemostatic sponge is as follows: take notoginseng powder, bletilla striata gum, gelatin, and water according to the mass ratio, mix them evenly, put them in a water bath at 50°C-60°C, stir and dissolve, and place them in a freeze-drying device Pre-freeze for 36-48 hours, and then freeze-dry. The temperature is set at -55°C, the pressure is 0.400mbar, and the freezing time is at least 48 hours. Take it out, cut it into pieces, and pack it.
有益效果Beneficial effect
一、多孔止血材料的性能研究1. Performance research of porous hemostatic materials
1.试验用材料及仪器1. Test materials and instruments
1.1主要实验材料:1.1 Main experimental materials:
白芨、三七药材，由陕西兴盛德药业有限公司生产提供；明胶是由上海试一化学公司生产制造；本发明多孔止血材料(按照下述实施例1中所述的方法制得)。Bletilla striata and Panax notoginseng are produced and provided by Shaanxi Xingshengde Pharmaceutical Co., Ltd.; gelatin is produced by Shanghai Shiyi Chemical Company; porous hemostatic material of the present invention (prepared according to the method described in Example 1 below).
1.2主要实验仪器：1.2 Main experimental instruments:
ACS-30型电子称：浙江杰力衡器有限公司生产制造；VaCo5型冷冻干燥仪：德国ZIRBUS公司生产制造；SHB3型真空泵：郑州长城科工贸公司生产制造；QM-3SP2型球磨机：南京大学仪器厂生产制造；HHS4型恒温水浴锅：北京科伟永兴仪器公司生产制造；Mastersizer 3000型激光粒度仪：英国Malvern公司生产制造；101型鼓风干燥箱：北京科伟永兴仪器公司生产制造；CT-6021型酸度计：深圳柯迪达电子生产制造。ACS-30 electronic scale: manufactured by Zhejiang Jieli Weighing Apparatus Co., Ltd.; VaCo5 type freeze dryer: manufactured by German ZIRBUS company; SHB3 type vacuum pump: manufactured by Zhengzhou Great Wall Technology Industry and Trade Company; QM-3SP2 ball mill: Nanjing University Instrument Factory manufacturing; HHS4 constant temperature water bath: Beijing Kewei Yongxing Instrument Co., Ltd.; Mastersizer 3000 laser particle size analyzer: British Malvern company; 101 blast drying oven: Beijing Kewei Yongxing Instrument Co., Ltd.; CT-6021 acidity meter: manufactured by Shenzhen Kodida Electronics.
2.参数筛选及测定：2. Parameter screening and determination:
2.1外观观察：2.1 Appearance observation:
结果显示：本发明多孔止血材料，经肉眼观察，具有弹性，呈多孔似海绵，呈暗黄色。The results show that the porous hemostatic material of the present invention has elasticity, is porous like a sponge, and is dark yellow when observed by naked eyes.
2.2理化测定结果：2.2 Physical and chemical test results:
⑴密度测定：取本发明多孔止血材料5片(规格为:长2cm×宽2cm×高1cm)，得到体积V；用精密天平称取质量(m)，以公式：ρ＝m/v计算其单位体积内的质量(ρ)。(1) Density measurement: get 5 pieces of porous hemostatic material of the present invention (specification is: length 2cm * width 2cm * height 1cm), obtain volume V; Take quality (m) with precision balance, calculate its with formula: ρ=m/v Mass per unit volume (ρ).
⑵材料溶液PH值测定：取本发明多孔止血材料5片(规格同上密度测定)，分别浸泡于20mL蒸馏水中30min，使其溶解，以酸度计测算。(2) Measurement of the pH value of the material solution: Take 5 pieces of the porous hemostatic material of the present invention (the specification is the same as the density measurement above), soak them in 20 mL of distilled water for 30 minutes, dissolve them, and measure them with a pH meter.
⑶数据分析方法：密度、PH值及测定后数据用Microsoft Excel 2010中Average函数和STDEV.S函数进行分析，具体结果见表1。(3) Data analysis method: Density, PH value and measured data were analyzed with the Average function and STDEV.S function in Microsoft Excel 2010, and the specific results are shown in Table 1.
表1 多孔止血材料理化性质测定结果Table 1 Measurement results of physical and chemical properties of porous hemostatic materials
由表1的测定结果可知：It can be seen from the measurement results in Table 1 that:
3.溶血实验：3. Hemolysis test:
取本发明止血材料，平均放入3支无菌试管中，再取6个无菌试管，分为2个对照组，阴性对照组中每支试管加入10mL生理盐水；阳性对照组中每支试管加入10mL蒸馏水。将所有试管密封，一同置于37摄氏度恒温水浴箱中，温浴30min。取稀释兔血(心脏取血，草酸钾抗凝，生理盐水稀释)，每支试管中缓慢混合人0.2mL，再将所有试管密封放入37℃恒温水浴箱中，温浴1h。取出所有试管离心吸取上清液，紫外分光光度计540nm波长下测定各管吸光度。每组取均值，按此公式计算溶血百分率：溶血百分率＝[(浸提液吸光度值-阴性对照吸光度值)/(阳性对照吸光度值-阴性对照吸光度值)]×100％。Get the hemostatic material of the present invention, put into 3 sterile test tubes on average, get 6 sterile test tubes again, be divided into 2 control groups, add 10mL normal saline to each test tube in the negative control group; Add 10 mL of distilled water. Seal all test tubes and place them together in a constant temperature water bath at 37°C for 30 minutes. Take diluted rabbit blood (take blood from the heart, anticoagulate with potassium oxalate, dilute with normal saline), slowly mix 0.2 mL of human in each test tube, then seal all the test tubes and put them in a 37°C constant temperature water bath for 1 hour. Take out all the test tubes and centrifuge to absorb the supernatant, and measure the absorbance of each tube at a wavelength of 540 nm by an ultraviolet spectrophotometer. Take the average value for each group, and calculate the percentage of hemolysis according to this formula: percentage of hemolysis=[(absorbance value of extract solution-absorbance value of negative control)/(absorbance value of positive control-absorbance value of negative control)]×100%.
研究结果显示：本发明止血材料，其RBC溶血百分率平均计算值为4.18％，小于国家药品应用标准5％，充分证实该新型止血材料安全有效。The research results show that: the hemostatic material of the present invention has an average calculated value of RBC hemolysis percentage of 4.18%, which is less than 5% of the national drug application standard, which fully proves that the new hemostatic material is safe and effective.
二、作用效果实验2. Effect experiment
1实验材料及分析方法：1 Experimental materials and analysis methods:
1.1实验动物：选用SPF级健康SD雄性大鼠60只，体重为250～280g，购自西安交通大学医学部实验动物中心(许可证号：SCXK(陕)2012-003)；1.1 Experimental animals: 60 SPF-grade healthy SD male rats, weighing 250-280 g, were purchased from the Experimental Animal Center of the Medical Department of Xi'an Jiaotong University (permit number: SCXK (Shaanxi) 2012-003);
1.2实验药物：4％多聚甲醛溶液，SABC试剂盒EP，苏木素、伊红染液，兔抗鼠VEGF，抗体DAB显色剂，本发明多孔止血材料(按照下述实施例1中所述的方法制得)。1.2 Experimental drugs: 4% paraformaldehyde solution, SABC kit EP, hematoxylin, eosin staining solution, rabbit anti-mouse VEGF, antibody DAB chromogen, porous hemostatic material of the present invention (according to the method described in the following example 1) method made).
1.3实验环境：本实验中动物实验部分在陕西省中药产业化协同创新中心SPF级动物实验室内完成。环境温度22℃～26℃，湿度40％～70％左右；1.3 Experimental environment: The animal experiment part of this experiment was completed in the SPF level animal laboratory of the Collaborative Innovation Center of Traditional Chinese Medicine Industrialization in Shaanxi Province. Ambient temperature 22℃～26℃, humidity 40%～70%;
1.4实验结果的统计方法：实验数据以均值±标准差表示，运用统计软件对各组进行组间比较。1.4 Statistical method of experimental results: experimental data is expressed as mean ± standard deviation Indicates that statistical software was used to compare between groups.
2造模方法2 modeling method
2.1动物分组：60只SD大鼠按照数字表法随机分为4组，每组15只。A组：本发明多孔材料组；B组：明胶海绵组；C组(空白对照组)：无菌纱布(用注射用水浸湿)；D组(材料基质对照组)：明胶/白芨胶多孔材料。2.1 Animal grouping: 60 SD rats were randomly divided into 4 groups according to the digital table method, 15 in each group. Group A: porous material group of the present invention; Group B: gelatin sponge group; Group C (blank control group): sterile gauze (soaked with water for injection); Group D (material matrix control group): gelatin/Bletilla striata gum porous material .
2.2大鼠创伤模型制作2.2 Establishment of Rat Trauma Model
2.2.1实验前准备：将笼具消毒，适应性的喂养7天，大鼠投以饲料，自由饮水，通风及光照条件良好；实验前12h禁食，不禁水，观察健康状况良好后用于实验。2.2.1 Preparations before the experiment: Disinfect the cages, feed them adaptively for 7 days, feed the rats, drink water freely, and have good ventilation and light conditions; fast for 12 hours before the experiment, without water, and observe the health status before use experiment.
2.2.2麻醉：术者及助手戴防护手套及口罩；每只大鼠称重，60只均以10％水合氯醛腹腔注射麻醉(0.3ml/100g)。腹腔注射10％水合氯醛时，左手抓取、固定好动物，将腹部朝向上；助手先以碘伏给予动物腹部消毒，然后手持注射器从右下腹位置以几乎平行于皮肤的角度刺入皮下，进针深约3mm-4mm，再使针头与皮肤呈45°角推注射器向前，当针尖穿过腹肌进入腹腔时，助手可自觉有落空感，然后固定保持针尖不动，回抽观察确定无回血、肠液及尿液后，便可缓缓将麻醉药物注入腹腔。注射过程完成后，将大鼠放回各自笼具，约1分钟后用手捏住大鼠尾部将其提起，观察见大鼠无反抗、呼吸无明显异常即可视为麻醉已生效。2.2.2 Anesthesia: The operator and assistants wear protective gloves and masks; each rat is weighed, and 60 rats are anesthetized by intraperitoneal injection of 10% chloral hydrate (0.3ml/100g). When injecting 10% chloral hydrate intraperitoneally, grasp and fix the animal with the left hand, and turn the abdomen upward; the assistant first sterilizes the abdomen of the animal with povidone iodine, and then inserts the syringe into the subcutaneous tissue from the right lower abdomen at an angle almost parallel to the skin. Insert the needle to a depth of about 3mm-4mm, and then push the syringe forward with the needle tip and the skin at an angle of 45°. When the needle tip passes through the abdominal muscle and enters the abdominal cavity, the assistant will feel a sense of falling, and then fix the needle tip and keep it still. Pull back and observe to confirm After there is no return of blood, intestinal fluid and urine, anesthetic drugs can be slowly injected into the abdominal cavity. After the injection process is completed, the rats are put back into their respective cages, and after about 1 minute, the tails of the rats are pinched by hand to lift them up. It is considered that the anesthesia has taken effect if the rats do not resist and there is no obvious abnormality in breathing.
2.2.3背部创面制作：麻醉生效后，用宠物剃毛机剔毛。将大鼠固定在手术板上，取俯卧位，常规消毒、铺巾，准备手术。创面制作：麻醉成功后碘伏消毒，用自制圆形印章(直径为1.8cm，内部注有亚甲蓝溶液)在大鼠脊柱两侧标明界限，以手术剪及手术刀沿亚甲蓝所显示界限制作机械性全层皮肤圆形创面2处。创面制作在严格的无菌条件下进行，用生理盐水冲洗伤口，用无菌纱布进行压迫止血。2.2.3 Preparation of the back wound: After the anesthesia takes effect, use a pet shaver to remove the hair. The rats were fixed on the operating board, placed in a prone position, routinely disinfected and draped, and prepared for surgery. Wound preparation: Iodophor disinfection after successful anesthesia, use a self-made circular stamp (1.8 cm in diameter, with methylene blue solution inside) to mark the boundaries on both sides of the rat spine, and use surgical scissors and a scalpel along the methylene blue to show Two mechanical full-thickness circular wounds were made on the boundary. The wound surface was made under strict aseptic conditions, the wound was rinsed with normal saline, and sterile gauze was used to stop bleeding.
2.2.4创面覆盖：A、B、C、D四组每只鼠2个创面分别用各组材料覆盖创面后，材料上再覆盖一块无菌纱块后最外层用绷带包扎。每天换敷料一次，至术后第14d。2.2.4 Wound coverage: 2 wounds of each mouse in groups A, B, C, and D were covered with the materials of each group, and then covered with a piece of sterile gauze, and the outermost layer was wrapped with a bandage. The dressing was changed once a day until the 14th day after operation.
2.2.5术后处理：待动物麻醉苏醒后，投入单笼分开饲养以避免群养时互相舔舐伤口而影响实验观察；不限制饮水进食。2.2.5 Postoperative treatment: After the animals wake up from anesthesia, they are put into a single cage and raised separately to avoid licking each other's wounds and affecting the experimental observation during group breeding; drinking and eating are not restricted.
2.2.6观察样本的取材：分别于造模成功后第3天、第7天、第14天共3个时间点分三批经10％水合氯醛腹腔注射麻醉后(方法同造模麻醉方法)，在创面切取肉芽组织，放入装有4％多聚甲醛溶液的规格为5ml的EP管中完全浸泡、固定，4℃冰箱过夜后制作HE切片及免疫组化切片。2.2.6 Observation samples were collected: three batches were anesthetized by intraperitoneal injection of 10% chloral hydrate on the 3rd day, 7th day, and 14th day after successful modeling (the method was the same as that of modeling anesthesia) ), cut the granulation tissue from the wound, put it into a 5ml EP tube filled with 4% paraformaldehyde solution, soak it completely, fix it, and make HE slices and immunohistochemical slices after overnight in the refrigerator at 4°C.
3石蜡切片制作及HE染色步骤3 Paraffin section preparation and HE staining steps
3.1固定与石蜡切片3.1 Fixation and paraffin section
取材后立即投入4％多聚甲醛中固定24h，经梯度酒精(70％乙醇→80％乙醇→90％乙醇→95％乙醇×2→100％乙醇×2)脱水，经二甲苯Ⅰ液、Ⅱ液透明，浸蜡3次，每次2h，石蜡包埋后切片。Immediately put the samples into 4% paraformaldehyde for 24 hours, dehydrate with graded alcohol (70% ethanol → 80% ethanol → 90% ethanol → 95% ethanol × 2 → 100% ethanol × 2), and pass through xylene solution I, II The liquid was transparent, dipped in wax three times, 2 hours each time, and sectioned after paraffin embedding.
3.2HE染色及免疫组化步骤3.2 HE staining and immunohistochemical steps
(1)二甲苯脱蜡Ⅰ液、Ⅱ液各15min；(1) xylene dewaxing liquid Ⅰ and liquid Ⅱ each for 15 minutes;
(2)100％酒精Ⅰ液、Ⅱ液各5min；(2) 100% alcohol solution Ⅰ and Ⅱ for 5 minutes each;
(3)90％酒精5min；(3) 90% alcohol for 5 minutes;
(4)80％酒精5min；(4) 80% alcohol for 5 minutes;
(5)70％酒精5min；(5) 70% alcohol for 5 minutes;
(6)无水乙醇5min；(6) Absolute ethanol 5min;
(7)浸入双蒸水ddH2O 5min；(7) Immerse in double distilled water ddH2 O for 5 minutes;
(8)苏木精染色5分钟；(8) Hematoxylin staining for 5 minutes;
(9)流水洗去苏木精染液；(9) running water washes away the hematoxylin dye solution;
(10)伊红染色5分钟；(10) Eosin staining for 5 minutes;
(11)流水洗去伊红染液；(11) running water washes away the eosin dye solution;
(12)70％乙醇30秒；(12) 70% ethanol for 30 seconds;
(13)80％乙醇30秒；(13) 80% ethanol for 30 seconds;
(14)90％乙醇30秒；(14) 90% ethanol for 30 seconds;
(15)无水乙醇30秒；(15) absolute ethanol for 30 seconds;
(16)100％乙醇Ⅱ液2min(16) 100% ethanol II solution for 2 minutes
(17)100％乙醇Ⅰ液2min；(17) 100% ethanol solution I for 2 minutes;
(18)无水乙醇2min；(18) dehydrated alcohol 2min;
(19)二甲苯Ⅰ液5min；(19) xylene solution I for 5 minutes;
(20)二甲苯Ⅱ液5min；(20) Xylene II solution for 5 minutes;
(21)中性树胶封片。(21) Seal the slide with neutral gum.
3.3免疫组织化学染色(SABC法)的步骤3.3 Steps of immunohistochemical staining (SABC method)
(1)石蜡切片，常规脱蜡水化；(1) Paraffin sections, conventional dewaxing and hydration;
(2)3％H2O2-甲醇溶液作用60min以封闭内源性过氧化物酶活性；(2) 3% H2 O2 -methanol solution for 60 minutes to block endogenous peroxidase activity;
(3)PBS缓冲液冲洗，5min×1次；(3) Rinse with PBS buffer, 5min×1 time;
(4)微波炉辐射抗原修复；(4) Microwave oven radiation antigen restoration;
(5)PBS缓冲液冲洗，5min×1次；(5) Rinse with PBS buffer, 5min×1 time;
(6)滴加山羊血清封闭液封闭60min，倾去多余液体；(6) Add goat serum blocking solution dropwise to seal for 60 minutes, and pour off excess liquid;
(7)滴加适当比例稀释的一抗(抗兔抗鼠VEGF(浓度为1:200)，4℃过2夜；(7) Add the primary antibody (anti-rabbit anti-mouse VEGF (concentration: 1:200)) diluted in an appropriate ratio dropwise, and spend 2 nights at 4°C;
(8)复温30min后，PBS冲洗，2min×3次；(8) After rewarming for 30 minutes, wash with PBS, 2 minutes × 3 times;
(9)滴加二抗(生物素化山羊抗兔或山羊抗小鼠IgG抗体)工作液，37℃孵育60min；(9) Add secondary antibody (biotinylated goat anti-rabbit or goat anti-mouse IgG antibody) working solution dropwise, and incubate at 37°C for 60 minutes;
(10)PBS冲洗，2min×3次；(10) Rinse with PBS, 2min×3 times;
(11)加SABC，30min；(11) Add SABC, 30min;
(12)PBS冲洗，5min×4次；(12) Rinse with PBS, 5min×4 times;
(13)DAB显色，镜下观察显示程度；(13) DAB color development, the degree of display under the microscope;
(14)ddH2O洗3min×2次；(14) Wash with ddH2 O for 3 min×2 times;
(15)用苏木素复染2mi；(15) Counterstain 2mi with hematoxylin;
(16)1％盐酸酒精分化；(16) 1% hydrochloric acid alcohol differentiation;
(17)ddH2O洗5秒→70％乙醇5秒→80％乙醇5秒→90％乙醇5秒→100％乙醇Ⅱ液1min→100％乙醇Ⅰ液5min→二甲苯Ⅱ液5min→二甲苯Ⅰ液5min；(17) Wash with ddH2 O for 5 seconds→70% ethanol for 5 seconds→80% ethanol for 5 seconds→90% ethanol for 5 seconds→100% ethanol II solution for 1 min→100% ethanol solution I for 5 min→xylene II solution for 5 min→xylene Ⅰ liquid 5min;
(18)中性树胶封片。(18) Mount the slides with neutral gum.
3.4光镜观察3.4 Light microscope observation
将制作好的H.E染色切片置于光镜下观察细胞渗出、肉芽组织及新生毛细血管生长情况。The prepared H.E stained sections were placed under a light microscope to observe cell exudation, granulation tissue and new capillary growth.
3.5创面愈合速率计算3.5 Calculation of wound healing rate
分别于术后3、7、14天观察记录各组大鼠的创面愈合情况，用消毒的透明膜贴于创面，沿创缘划线将动物创面大小描画在透明膜上，扫描仪扫描后储存为JPG图片格式，以Adobephotoshop cs6软件得出描记区域像素值，来计算各时期创面愈合率，以创面愈合率超过95％为愈合标准。计算公式如下：Observe and record the wound healing of the rats in each group at 3, 7, and 14 days after the operation, stick a sterile transparent film on the wound, draw the animal wound size on the transparent film along the wound edge, and store it after scanning with a scanner It is in JPG image format, and the pixel value of the tracing area is obtained by Adobe Photoshop cs6 software to calculate the wound healing rate in each period, and the wound healing rate exceeds 95% as the healing standard. Calculated as follows:
3.6创面肉芽组织中毛细血管计数统计3.6 Statistics of capillary count in wound granulation tissue
将制作好的各组H.E切片于光镜下(10×)观察，每张切片随机选取5个视野，观察毛细血管数量增长情况，计数均数。The prepared H.E slices of each group were observed under a light microscope (10×), and 5 fields of view were randomly selected for each slice to observe the increase in the number of capillaries and count the average number.
3.7VEGF阳性细胞计数统计3.7 VEGF positive cell count statistics
采用免疫组化SABC法染色后，检测每张切片的VEGF蛋白的阳性表达，在血管内皮细胞中表达最明显，胞浆呈黄褐色、棕黄色或者深棕色颗粒状物。采用MiE图像处理系统摄片，用BI-2000图像分析系统进行分析，每张切片随机选取5个视野，测定棕黄色阳性表达颗粒的平均吸光度值。判断标准如下：无特异性着色者为阴性(-)；轻度特异性着色，呈黄褐色弱阳性(+)；中度特异性着色明显，呈棕黄色(++)；呈深棕色者为强阳性(+++)。After immunohistochemical SABC staining, the positive expression of VEGF protein in each section was detected, and the expression was most obvious in vascular endothelial cells, and the cytoplasm was yellowish brown, brownish yellow or dark brown granular. The MiE image processing system was used to take pictures, and the BI-2000 image analysis system was used for analysis. Five fields of view were randomly selected for each slice, and the average absorbance value of brown-yellow positive expression particles was measured. Judgment criteria are as follows: no specific staining is negative (-); mild specific staining is yellow-brown weak positive (+); moderate specific staining is obvious and brownish-yellow (++); dark brown is Strongly positive (+++).
3.8统计学分析方法3.8 Statistical Analysis Methods
图像分析获取到的数据采用IBM公司SPSS 19.0软件作统计分析处理，分别对各组数据进行方差分析及两两组之间t检验，以P<0.05为差异显著，表示有统计学意义。The data obtained by image analysis was processed by IBM SPSS 19.0 software for statistical analysis. The variance analysis and t-test between the two groups were performed on the data of each group, and P<0.05 was considered significant, indicating statistical significance.
4.实验结果4. Experimental results
4.1光镜组织学观察结果4.1 Light microscope histological observation results
伤后第3天，可观察到各组间无明显差异，亦无明显感染现象，但各组均有较多的炎性细胞，A、B、D三组新生的毛细血管计数比C组多；伤后第7天，四组创面均可看到增生的肉芽组织，新生的毛细血管以A组为最多；伤后第14天，A、B、D三组与皮肤结构基本相同，C组、B组两组新生毛细血管较A、D组少。On the 3rd day after injury, there was no significant difference among the groups and no obvious infection, but there were more inflammatory cells in each group, and the number of new capillaries in groups A, B, and D was more than that in group C On the 7th day after injury, hyperplastic granulation tissue could be seen on the wounds of the four groups, and group A had the most new capillaries; on the 14th day after injury, the three groups A, B, and D had basically the same skin structure, and group C New capillaries in group B and group A were less than those in group A and D.
4.2创面愈合速率4.2 Wound healing rate
造模后第3天，A组与B、C、D三组有显著的差异(P<0.05)，A、D组与C组有显著的差异(P<0.05)；伤后第7天，A、D组与C有显著的差异(P<0.05)，A、D组与B组有显著的差异(P<0.05)；A组与D组比较有显著差异(P<0.05)；伤后第14天，A、B、C、D四组间比较无差异。说明A组、D组均有促进创面愈合的作用，且本发明多孔材料的促愈合作用要优于明胶海绵或明胶/白芨胶多孔材料，结果见表2。On the 3rd day after modeling, there were significant differences between group A and groups B, C, and D (P<0.05), and there were significant differences between groups A, D and group C (P<0.05); on the 7th day after injury, There was a significant difference between groups A and D and C (P<0.05), and there was a significant difference between groups A and D and B (P<0.05); there was a significant difference between group A and group D (P<0.05); On the 14th day, there was no difference among groups A, B, C, and D. Explain that group A and group D all have the effect of promoting wound healing, and the promoting effect of porous material of the present invention is better than gelatin sponge or gelatin/Bletilla striata glue porous material, the results are shown in Table 2.
表2 各组创面愈合速率比较Table 2 Comparison of wound healing rates in each group
注：△：与明胶海绵组对照组比较，P<0.05；★：与材料基质组比较，P＜0.05Note: △: Compared with the gelatin sponge group, P<0.05; ★: Compared with the material matrix group, P<0.05
4.3毛细血管数量的统计4.3 Statistics of the number of capillaries
造模后第3天，D组与B组比较差异有统计学意义(P＜0.05)。造模后7天、第14天各组新生毛细血管计数均明显增多，A组、D组与C组比较具有明显差异(P<0.05)，说明本发明多孔材料组(A组)与明胶/白芨胶多孔材料组(D组)毛细血管数量明显高于空白对照组。以上研究结果可以充分证明本发明多孔材料对大鼠创面毛细血管的生成具有一定的促进作用。结果见表3。On the 3rd day after modeling, there was a statistically significant difference between group D and group B (P<0.05). On the 7th day and the 14th day after modeling, the counts of new capillaries in each group were significantly increased, and there was a significant difference between the A group, the D group and the C group (P<0.05), indicating that the porous material group of the present invention (Group A) and gelatin/ The number of capillaries in the bletilla striata glue porous material group (group D) was significantly higher than that in the blank control group. The above research results can fully prove that the porous material of the present invention has a certain promoting effect on the generation of rat wound capillaries. The results are shown in Table 3.
表3 各组各时间点毛细血管数量变化Table 3 Changes in the number of capillaries in each group at each time point
4.4VEGF的阳性表达结果4.4 Positive expression results of VEGF
造模后第3天，VEGF在四组创面的肉芽组织中均有阳性表达，A组、B组、D组表达为弱阳性(+)，C组表达为阴性到弱阳性(－～+)。具体结果见表4。On the 3rd day after modeling, VEGF was positively expressed in the granulation tissue of the wounds of the four groups, the expression in groups A, B, and D was weakly positive (+), and the expression in group C was negative to weakly positive (-～+) . The specific results are shown in Table 4.
表4 各组各时间点大鼠创面VEGF阳性表达的平均吸光度值(A)Table 4 The average absorbance value of the positive expression of VEGF on rat wounds in each group at each time point (A)
结果显示：造模后第7天，VEGF的阳性表达在四组创面肉芽组织中均上升，为中度阳性(++)；而C组表达为弱阳性(+)。A组中VEGF阳性表达含量高于B组和D组。造模后第14天，A组VEGF在四组创面肉芽组织中的表达最为明显，为强阳性(+++)。而C则表达为中度阳性(++)，C组中VEGF阳性表达开始下降。The results showed that: on the 7th day after modeling, the positive expression of VEGF increased in the wound granulation tissue of the four groups, which was moderately positive (++); while the expression in group C was weakly positive (+). The positive expression level of VEGF in group A was higher than that in group B and group D. On the 14th day after modeling, the expression of VEGF in group A was the most obvious in the wound granulation tissue of the four groups, which was strongly positive (+++). The expression of C was moderately positive (++), and the positive expression of VEGF in group C began to decline.
具体实施方式detailed description
此处，应当理解本领域的普通技术人员基于此处内容公开的启示下，但凡在本发明的精神和实质范围内，所作的任何改变、等同替换和改进。此外，应当理解，此处提供的具体实施例仅仅是示意性的、指导性的，而不应当解释为对本发明方案的限制。Here, it should be understood that those skilled in the art can make any changes, equivalent replacements and improvements within the spirit and essential scope of the present invention based on the enlightenment of the content disclosed herein. In addition, it should be understood that the specific embodiments provided here are only illustrative and instructive, and should not be construed as limiting the solutions of the present invention.
以下通过具体实施例来阐述本发明多孔止血材料的制备成型过程，进一步让本领域的技术人员能够全面理解、实施本发明的技术内容。The preparation and molding process of the porous hemostatic material of the present invention will be described below through specific examples, further enabling those skilled in the art to fully understand and implement the technical content of the present invention.
实施例1Example 1
⑴三七微米粉的制备：取三七药材，粉碎成粗粉后放入球磨罐中，在真空状态下，设定球料比为6:1，转速为300转每分钟，时间20-40分钟，过9号筛，取粒径小于75μm粉末，留用；⑴Preparation of notoginseng micron powder: Take notoginseng medicinal material, crush it into coarse powder and put it into a ball mill jar. Minutes, pass through a No. 9 sieve, take the powder with a particle size of less than 75 μm, and keep it for use;
⑵白芨胶的制备：取白芨药材，切制成片，第一次浸泡提取，加入与药材质量比为7～9倍量的水，浸泡24小时，滤过,滤液备用；第二、三次浸泡提取，滤过的药渣中加入与药材质量比为3倍量的水，浸泡8-12小时，滤过,合并滤液后，加入乙醇使含醇量达80％，静置2-4h，将沉淀物干燥，粉碎，留用；⑵Preparation of Bletilla striata glue: take Bletilla striata medicinal material, cut into slices, soak and extract for the first time, add water with a mass ratio of 7 to 9 times that of the medicinal material, soak for 24 hours, filter, and the filtrate is used for later use; soak for the second and third times Extraction, adding water with a mass ratio of 3 times that of the medicinal material to the filtered medicinal residue, soaking for 8-12 hours, filtering, combining the filtrate, adding ethanol to make the alcohol content reach 80%, standing for 2-4h, and The precipitate is dried, pulverized, and retained;
⑶多孔海绵剂的成型：分别取上述步骤⑴中留用的三七微米粉1质量份，步骤⑵中留用的白芨胶1质量份，6％质量浓度的明胶6质量份，蒸馏水100质量份，混匀，置入50℃-60℃水浴中搅拌溶解后，置于冻干设备中进行预冷冻，持续时间36h～48h后，再进行冻干处理，其温度设置为-55℃，压力为0.400mbar，冷冻时间至少48h以上，取出，切块，包装。(3) Forming of the porous sponge: take respectively 1 mass part of the notoginseng micron powder retained in the above step (1), 1 mass part of the bletilla striata gum retained in the step (2), 6 mass parts of gelatin of 6% mass concentration, 100 mass parts of distilled water, and mix Stir and dissolve in a water bath at 50°C-60°C, put it in a freeze-drying equipment for pre-freezing, and then freeze-dry after 36h-48h. The temperature is set at -55°C and the pressure is 0.400mbar. , Freeze for at least 48 hours, take it out, cut into pieces, and pack.
实施例2Example 2
⑴三七微米粉的制备：取三七药材，粉碎成粗粉后放入球磨罐中，在真空状态下，设定球料比为5:1，转速为350转每分钟，时间20分钟，过9号筛，取粒径小于75μm粉末，留用；(1) Preparation of notoginseng micron powder: Take notoginseng medicinal material, grind it into coarse powder and put it into a ball mill jar. Pass through a No. 9 sieve, take the powder with a particle size of less than 75 μm, and keep it for use;
⑵白芨胶的制备：取白芨药材，切制成片，第一次浸泡提取，加入与药材质量比为7～9倍量的水，浸泡24小时，滤过,滤液备用；第二、三次浸泡提取，滤过的药渣中加入与药材质量比为3倍量的水，浸泡10小时，滤过,合并滤液后，加入乙醇使含醇量达70％，静置2-4h，将沉淀物干燥，粉碎，留用；⑵Preparation of Bletilla striata glue: take Bletilla striata medicinal material, cut into slices, soak and extract for the first time, add water with a mass ratio of 7 to 9 times that of the medicinal material, soak for 24 hours, filter, and the filtrate is used for later use; soak for the second and third times Extraction, add water with a mass ratio of 3 times that of the medicinal material to the filtered medicinal residue, soak for 10 hours, filter, combine the filtrate, add ethanol to make the alcohol content reach 70%, let stand for 2-4h, and remove the precipitate dried, pulverized, retained;
⑶多孔海绵剂的成型：分别取上述步骤⑴中留用的三七微米粉2质量份，步骤⑵中留用的白芨胶2质量份，6％质量浓度的明胶8质量份，蒸馏水110质量份，混匀，置入50℃-60℃水浴中搅拌溶解后，置于冻干设备中进行预冷冻，持续时间36h～48h后，再进行冻干处理，其温度设置为-55℃，压力为0.400mbar，冷冻时间至少48h以上，取出，切块，包装。(3) Forming of the porous sponge: take respectively 2 mass parts of notoginseng micron powder retained in the above step (1), 2 mass parts of bletilla striata gum retained in the step (2), 8 mass parts of gelatin of 6% mass concentration, 110 mass parts of distilled water, and mix Stir and dissolve in a water bath at 50°C-60°C, put it in a freeze-drying equipment for pre-freezing, and then freeze-dry after 36h-48h. The temperature is set at -55°C and the pressure is 0.400mbar. , Freeze for at least 48 hours, take it out, cut into pieces, and pack.
实施例3Example 3
⑴三七微米粉的制备：取三七药材，粉碎成粗粉后放入球磨罐中，在真空状态下，设定球料比为7:1，转速为300转每分钟，时间40分钟，过9号筛，取粒径小于75μm粉末，留用；(1) Preparation of notoginseng micron powder: Take notoginseng medicinal material, grind it into coarse powder and put it into a ball mill jar. Pass through a No. 9 sieve, take the powder with a particle size of less than 75 μm, and keep it for use;
⑵白芨胶的制备：取白芨药材，切制成片，第一次浸泡提取，加入与药材质量比为7～9倍量的水，浸泡24小时，滤过,滤液备用；第二、三次浸泡提取，滤过的药渣中加入与药材质量比为3倍量的水，浸泡8-12小时，滤过,合并滤液后，加入乙醇使含醇量达90％，静置2-4h，将沉淀物干燥，粉碎，留用；⑵Preparation of Bletilla striata glue: take Bletilla striata medicinal material, cut into slices, soak and extract for the first time, add water with a mass ratio of 7 to 9 times that of the medicinal material, soak for 24 hours, filter, and the filtrate is used for later use; soak for the second and third times Extraction, add water with a mass ratio of 3 times that of the medicinal material to the filtered medicinal residue, soak for 8-12 hours, filter, combine the filtrate, add ethanol to make the alcohol content reach 90%, let it stand for 2-4h, and The precipitate is dried, pulverized, and retained;
⑶多孔海绵剂的成型：分别取上述步骤⑴中留用的三七微米粉3质量份，步骤⑵中留用的白芨胶3质量份，6％质量浓度的明胶10量份，蒸馏水120质量份，混匀，置入50℃-60℃水浴中搅拌溶解后，置于冻干设备中进行预冷冻，持续时间36h～48h后，再进行冻干处理，其温度设置为-55℃，压力为0.400mbar，冷冻时间至少48h以上，取出，切块，包装。(3) Forming of the porous sponge: take respectively 3 mass parts of notoginseng micron powder retained in the above step (1), 3 mass parts of bletilla striata gum retained in the step (2), 10 mass parts of gelatin of 6% mass concentration, 120 mass parts of distilled water, and mix Stir and dissolve in a water bath at 50°C-60°C, put it in a freeze-drying equipment for pre-freezing, and then freeze-dry after 36h-48h. The temperature is set at -55°C and the pressure is 0.400mbar. , Freeze for at least 48 hours, take it out, cut into pieces, and pack.
实施例4Example 4
⑴三七微米粉的制备：取三七药材，粉碎成粗粉后放入球磨罐中，在真空状态下，设定球料比为6:1，转速为300转每分钟，时间30分钟，过9号筛，取粒径小于75μm粉末，留用；(1) Preparation of notoginseng micron powder: Take notoginseng medicinal material, grind it into coarse powder and put it into a ball mill jar. Pass through a No. 9 sieve, take the powder with a particle size of less than 75 μm, and keep it for use;
⑵白芨胶的制备：取白芨药材，切制成片，第一次浸泡提取，加入与药材质量比为7～9倍量的水，浸泡24小时，滤过,滤液备用；第二、三次浸泡提取，滤过的药渣中加入与药材质量比为3倍量的水，浸泡8-12小时，滤过,合并滤液后，加入乙醇使含醇量达80％，静置2-4h，将沉淀物干燥，粉碎，留用；⑵Preparation of Bletilla striata glue: take Bletilla striata medicinal material, cut into slices, soak and extract for the first time, add water with a mass ratio of 7 to 9 times that of the medicinal material, soak for 24 hours, filter, and the filtrate is used for later use; soak for the second and third times Extraction, adding water with a mass ratio of 3 times that of the medicinal material to the filtered medicinal residue, soaking for 8-12 hours, filtering, combining the filtrate, adding ethanol to make the alcohol content reach 80%, standing for 2-4h, and The precipitate is dried, pulverized, and retained;
⑶多孔海绵剂的成型：分别取上述步骤⑴中留用的三七微米粉0.5质量份，步骤⑵中留用的白芨胶0.5质量份，6％质量浓度的明胶2质量份，蒸馏水80质量份，混匀，置入50℃-60℃水浴中搅拌溶解后，置于冻干设备中进行预冷冻，持续时间36h～48h后，再进行冻干处理，其温度设置为-55℃，压力为0.400mbar，冷冻时间至少48h以上，取出，切块，包装。(3) Forming of the porous sponge: take respectively 0.5 mass parts of the notoginseng micron powder retained in the above step (1), 0.5 mass part of the bletilla striata gum retained in the step (2), 2 mass parts of gelatin of 6% mass concentration, 80 mass parts of distilled water, and mix Stir and dissolve in a water bath at 50°C-60°C, put it in a freeze-drying equipment for pre-freezing, and then freeze-dry after 36h-48h. The temperature is set at -55°C and the pressure is 0.400mbar. , Freeze for at least 48 hours, take it out, cut into pieces, and pack.
| Application Number | Priority Date | Filing Date | Title |
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| CN201610250387.5ACN105709265A (en) | 2016-04-21 | 2016-04-21 | Novel porous hemostatic material and preparation method thereof |
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| CN201610250387.5ACN105709265A (en) | 2016-04-21 | 2016-04-21 | Novel porous hemostatic material and preparation method thereof |
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| CN105709265Atrue CN105709265A (en) | 2016-06-29 |
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| CN201610250387.5APendingCN105709265A (en) | 2016-04-21 | 2016-04-21 | Novel porous hemostatic material and preparation method thereof |
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