鱼鳔作为新型心血管外科手术生物材料的试验研究

发布时间：2018-07-25 17:22

【摘要】：生物材料（Biomaterials）已在心血管外科领域得到广泛使用，其中又以生物心脏瓣膜和人工血管使用较多。生物瓣膜因血流动力学出色、血栓栓塞发生率低、术后生活质量高等优点，尤其在发达国家已有较高的认可度，但由于牛心包等异种材料本身原因，早期即可能出现机械性毁损和钙化衰败，大大限制了生物瓣膜的适用范围。大口径人工血管已成功应用于心血管手术且术后效果好，而小口径人工血管（口径＜6mm）因血液相容性差导致的术后通畅率低等原因，仍未获得临床认可。因此，目前人们正在努力通过对现有材料加工改性及寻找新材料的角度出发以期解决以上问题。由此目的出发，本文分别从物理性能、体外生物相容性及动物实验三个不同层面深入研究鱼鳔性质，旨在寻求一种优于目前临床常用材料的新型心血管外科生物材料。第一部分鱼鳔的物理性能试验研究目的：研究鱼鳔各项生物力学性能和热稳定性，以及交联处理（0.625%戊二醛溶液）对其性能的影响。方法：1、通过单轴拉伸试验（Uniaxial Tensile Test）获得被测材料拉伸至毁损时的极限阿尔曼西应变（FailureAlmansi strain，f）、极限柯西应力（Failure Cauchy stress，f）以及极限弹性模量（Peak Elastic Modulus，MP）,比较新鲜、戊二醛处理后的鱼鳔在不同方向上和牛心包的力学差异。结合预试验中维多利亚蓝-苦味酸酸性复红染色（Victoria blue-van Gieson, VG）染色、透射电镜观察以及本试验结果，对纤维排列做出描述。2、通过差示量热扫描技术（Differential Scanning Calorimetry，DSC）检测新鲜和戊二醛处理后鱼鳔的热变性温度并分析交联处理对其热稳定性的影响。结果：1、单轴拉伸试验：（1）无论新鲜或是戊二醛交联后鱼鳔在周向的三个力学结果均高于纵向。（2）戊二醛处理后鱼鳔周向的f、MP均高于同方向的新鲜鱼鳔，两者f无显著统计学差异。（3）戊二醛交联后鱼鳔纵向f大于同方向新鲜鱼鳔，两者f、MP无显著统计学差异。（4）戊二醛处理后牛心包f、MP均高于新鲜牛心包，两者f无统计学差异。（5）两材料经戊二醛处理后，鱼鳔周向及纵向f、MP均低于牛心包，f显著高于牛心包。2、差示量热扫描：新鲜鱼鳔热变性温度高于正常人体温范围，且戊二醛处理后鱼鳔热稳定性优于新鲜时。结论：1、鱼鳔纤维排列较牛心包规则，胶原纤维主要沿周向排列，弹力纤维在周向及纵向均有分布。2、戊二醛处理后鱼鳔僵硬度（Stiffness）低于牛心包，延展性优于牛心包，更接近人正常主动脉瓣膜力学范围。3、鱼鳔，尤其经过戊二醛处理后，热稳定性可达到体温要求。第二部分鱼鳔生物相容性体外试验评价目的：根据国际ISO10993医疗器械生物学评价指南研究鱼鳔体外细胞毒性及血液相容性。方法：1、体外细胞毒性试验：用含10%胎牛血清的MEM培养液浸提新鲜鱼鳔，采用MTT法检测浸提液对小鼠成纤维细胞L929的细胞毒性作用。2、体外血小板激活试验：用人全血浸提鱼鳔，通过CD62p检测法计算活化血小板数量。3、体外溶血试验：用无菌生理盐水浸提鱼鳔，采用游离血红蛋白直接测定法检测浸提液与人血混合后的游离血红蛋白含量并计算溶血率。以上试验均以牛心包材料作为试验对照组。结果：1、100%新鲜鱼鳔和100%新鲜牛心包浸提液接触后的L929细胞活力分别为85.7%和94.2%，可认为两材料对L929细胞无毒性影响。2、戊二醛处理后鱼鳔和牛心包组CD62P百分率分别为0.940.27%和1.730.35%，可认为两材料均对体外人血血栓形成没有影响。3、戊二醛处理后鱼鳔和牛心包组溶血率分别为0.2%和0.6%，可认为两材料对体外溶血没有影响。结论：鱼鳔材料在体外细胞毒性检测和体外血液相容性检测中表现出良好的生物相容性并为进一步动物试验提供理论基础。第三部分鱼鳔体内钙化研究目的：采用大鼠背部皮下埋植模型，定性及定量评估鱼鳔不同时间点钙化程度，并与牛心包进行对比。方法：1、采用已在牛心包中验证有效的DSC(Denaturant-surfactant-crosslinking)抗钙化方法（美国Edwards公司专利，Patent No.:US6214054B1）预处理鱼鳔及牛心包材料，行幼年大鼠背部皮下埋植，于术后7天、21天、56天取出样本，定性及定量检测钙化情况并明确与免疫反应的关系。2、钙定性检测：采用X线摄影、HE染色、VG染色及Von Kossa染色从样本形态学和组织学水平评价各时间点钙盐沉积情况。3、钙定量测定：采用电感耦合等离子体发射光谱仪（Indutive Coupled Plasma EmissionSpectrimeter,ICP）对比分析样本钙含量。4、免疫组织化学：CD68＋巨噬细胞及CD8＋T细胞特异性免疫组化染色研究材料内部炎性细胞浸润情况，并与钙化程度关联。结果：1、鱼鳔各期钙化程度均低于牛心包，鱼鳔纤维完整排列有序，仅在后期局部钙化处发生纤维断裂，牛心包早期出现钙盐沉积，纤维破坏严重。定量测定表明牛心包各期钙含量高于鱼鳔组，以21天时差异最显著。2、免疫组化染色显示各时期鱼鳔内部未见炎性细胞浸润，而牛心包各期均有CD68＋巨噬细胞及CD8＋T细胞浸入。结论：大鼠皮下埋植钙化模型显示经戊二醛-DSC抗钙化方法处理后鱼鳔各期钙化程度显著低于牛心包，鱼鳔在体内具有更出色的抗钙化性能。第四部分新型材料小口径人造血管的试验研究目的：采用大鼠腹主动脉置换模型评估鱼鳔作为小口径人造血管材料的效果，并与牛心包相比较，进一步评估两者的血液相容性。方法：根据ISO10993指南,使用鱼鳔和牛心包材料制作小口径人造血管并行大鼠腹主动脉置换，于30天及60天取出并观察如下指标。1、通畅率及血管瘤观察：采用320排CT扫描并三维成像观察通畅率及血管瘤形成。2、内皮化及新生内膜观察：采用扫面电镜直观观察内皮细胞黏附，采用HE、VG染色观察通畅血管管壁纤维排列及弹力纤维增生，采用第八因子相关抗原及α-SMA免疫组化染色观察人造血管各期纤维排列、内皮化及平滑肌细胞增生程度。3、钙化研究：采用X线体外摄影及离体样本Micro CT扫描初步评估血管壁钙盐沉积，采用组织切片Von Kossa染色镜下进一步观察钙盐沉积情况。结果：1、各期鱼鳔人造血管通畅率均为100%，牛心包人造血管均为16.67%。通畅血管均未见血管瘤形成。2、通畅血管各期纤维排列有序，仅60天牛心包血管可见纤维破坏断裂，两者通畅血管均可见弹力纤维覆盖，鱼鳔血管中弹力纤维更丰富；鱼鳔内皮化与牛心包比较速度更快程度更完整；两材料吻合口处均可见平滑肌细胞增生，30天及60天鱼鳔内膜厚度无明显差异，30天时牛心包内膜增生较鱼鳔显著，60天时牛心包内膜几乎堵塞管腔。3、X线体外摄影及离体样本Micro CT扫描均未发现血管中明显钙化点，经组织切片Von kossa染色显示，仅60天时牛心包血管材料管壁可见钙盐点状沉积并致纤维断裂，其余时期两者通畅血管中均未见钙盐沉积。结论：鱼鳔材料血液相容性优于牛心包，可以作为小口径人造血管材料。全文总结：本课题研究表明，鲤鱼鳔最大断裂强度低于牛心包，延展性及柔韧性优于牛心包，热稳定性能达到人体内使用要求。鱼鳔生物相容性优良，无细胞毒性、致血栓形成及致溶血的性质。大鼠皮下埋植模型结果表明，经抗钙化处理后，钙化率显著低于牛心包。鱼鳔小口径人造血管行大鼠腹主动脉置换结果表明，鱼鳔材料作为人造血管，血液相容性满意，30及60天通畅率高于牛心包组，内皮覆盖迅速完全，，且无动脉瘤发生。
[Abstract]:Biomaterials (Biomaterials) have been widely used in the field of cardiovascular surgery, including biological heart valves and artificial blood vessels. Biological valves have the advantages of excellent hemodynamics, low incidence of thromboembolism and high quality of life after operation, especially in the developed countries, but because of the heterogenous materials such as the cow heart bag The cause of material itself is that mechanical damage and calcification may occur in the early stage, which greatly limits the scope of application of biological valves. Large caliber artificial blood vessels have been successfully applied to cardiovascular surgery and have good postoperative effect, while small caliber artificial blood vessels (caliber < 6mm) have not been clinically obtained because of low postoperative patency rate caused by poor blood compatibility. Therefore, at present, people are trying to solve the above problems through the modification of the existing materials and the search for new materials. This purpose is to study the nature of the fish swim bladder from three different levels, physical performance, biocompatibility and animal experiments, in order to seek a better than current clinical use. Material for new cardiovascular surgical biomaterials.
Experimental study on the physical properties of the first part of the swim bladder
Objective: To study the biomechanical properties and thermal stability of swimming bladder, and the effect of cross-linking treatment (0.625% glutaraldehyde solution) on its performance.
Methods: 1, through the uniaxial tensile test (Uniaxial Tensile Test), the limit Arman strain (FailureAlmansi strain, f), the ultimate Cauchy stress (Failure Cauchy stress, f) and the ultimate modulus of elasticity (Peak Elastic) were obtained by the uniaxial tensile test (Test), and the swim bladder in the treatment of glutaraldehyde was in different directions. The mechanical differences with the bovine pericardium. Combined with the pre test of Vitoria blue bitter acid acid red red staining (Victoria blue-van Gieson, VG), transmission electron microscopy and the results of this test, the fiber arrangement was described by.2, and the treatment of fresh and glutaraldehyde was detected by differential thermal scanning (Differential Scanning Calorimetry, DSC). The thermal denaturation temperature of fish bladder was analyzed and the effect of cross-linking treatment on its thermal stability was analyzed.
Results: 1, uniaxial tensile test: (1) the three mechanical results of the swim bladder in the swim bladder of both fresh or glutaraldehyde were higher than that in the longitudinal direction. (2) the F of the swim bladder in the swim bladder was higher than that of the fresh swim bladder in the same direction after the treatment of glutaraldehyde. There was no significant difference in F between them. (3) the longitudinal F of the swim bladder was larger than the same direction fresh swim bladder, and both F, There was no significant statistical difference in MP. (4) after glutaraldehyde treatment, F and MP were higher than those of fresh bovine pericardium, and there was no significant difference in F. (5) after treatment with glutaraldehyde, two materials were treated with glutaraldehyde, and F, MP were lower than those of bovine heart bag, f was significantly higher than that of bovine heart pack.2, and the thermal denaturation temperature of fresh swim bladder was higher than that of normal human body, and the temperature of fresh swim bladder was higher than that of normal human body. After two aldehyde treatment, the thermal stability of the swim bladder is better than that of fresh water.
Conclusions: 1, the fibers of the swim bladder are arranged in the pericardial rule. The collagen fibers are arranged mainly along the circumferential direction. The elastic fibers are distributed.2 in both the circumference and the longitudinal direction. The stiffness of the swim bladder (Stiffness) is lower than that of the bovine pericardium after glutaraldehyde treatment. The ductility of the fish is better than that of the bovine pericardium. It is closer to the mechanical range of the normal aortic valve,.3, and the swim bladder, especially after glutaraldehyde treatment. Thermal stability can meet the requirements of body temperature.
The second part is the evaluation of the biocompatibility of swim bladder in vitro.
Objective: To study in vitro cytotoxicity and blood compatibility of swim bladder in accordance with international ISO10993 guidelines for biological evaluation of medical devices.
Methods: 1, in vitro cytotoxicity test: using MEM culture containing 10% fetal bovine serum to extract fresh fish swim bladder, MTT method was used to detect the cytotoxic effect of the extract on the cytotoxicity of L929 in mouse fibroblasts. The platelet activation test in vitro was tested in vitro. The number of activated platelets was calculated by CD62p detection, and the number of activated platelets was calculated by CD62p assay, and the in vitro hemolysis test was carried out. The free hemoglobin was directly measured and the free hemoglobin content was measured and the hemolysis rate was calculated by direct determination of free hemoglobin. The results were as follows: 1100% fresh fish swim bladder and 100% fresh bovine pericardium extract were exposed to L929 cell viability, respectively. For 85.7% and 94.2%, it is considered that two materials have no toxic effect on L929 cells.2, the percentage of CD62P in the swim bladder and heart pack group after glutaraldehyde treatment is 0.940.27% and 1.730.35% respectively. It is considered that two materials have no effect on the formation of human blood thrombus in vitro, and the hemolysis rate of the swim bladder and bovine pericardium group after glutaraldehyde treatment is 0.2% and 0.6%, respectively, and two material can be considered. The material has no effect on the hemolysis in vitro.
Conclusion: the biocompatibility of bladder material in cytotoxicity detection and in vitro blood compatibility detection in vitro provides a theoretical basis for further animal test.
The third part of the study on calcification of the swim bladder in the swim bladder: the model of the rat's back subcutaneous implantation was used to determine the calcification degree of the fish swim bladder at different time points qualitatively and quantitatively, and compared with the bovine pericardium.
Methods: 1, the DSC (Denaturant-surfactant-crosslinking) anti calcification method (Edwards patent of the United States, Patent No.: US6214054B1) was used to pretreat the fish swim bladder and bovine pericardium material in the bovine pericardium. The subcutaneous implantation of the young rat's back was performed on the back of the young rat. The samples were taken at 7 days, 21 days and 56 days after the operation, and the calcification was determined qualitatively and quantitatively. The relationship between the immune response and.2, calcium qualitative detection: using X-ray photography, HE staining, VG staining and Von Kossa staining, the calcium salt deposition in each time point was evaluated from the sample morphology and histology level.3, calcium quantitative determination: The inductively coupled plasma emission spectrometer (Indutive Coupled Plasma EmissionSpectrimeter, ICP) was compared. Analysis of samples of calcium content.4, immunohistochemistry: CD68 + macrophage and CD8 + T cell specific immunohistochemical staining to study the infiltration of inflammatory cells in the material, and related to the degree of calcification.
The results were as follows: 1, the degree of calcification in all stages of the swim bladder was lower than that of the bovine pericardium, and the fiber of the swim bladder was arranged in a complete order. The fibrous fracture occurred in the local calcification at the later stage. The calcium salt was deposited early in the bovine pericardium and the damage of the fiber was serious. The quantitative determination showed that the calcium content in the bovine pericardium was higher than the swim bladder group, and the difference was most significant at the time of 21 days, and the immuno histochemical staining showed that each group was.2. There was no inflammatory cell infiltration in the internal swim bladder, and CDC + macrophage and CD8 + T cells were immersed in all stages of the bovine pericardium. Conclusion: the calcification model of subcutaneous implantation in rats showed that the calcification of the swim bladder was significantly lower than that of the bovine pericardium after treatment with glutaraldehyde -DSC anti calcification, and the swim bladder had a better anti calcification performance in the body.
The fourth part is the experimental study of new material small diameter artificial blood vessel.
Objective: To evaluate the effect of the swim bladder as a small caliber artificial vascular material by rat abdominal aortic replacement model, and to compare the blood compatibility with the bovine pericardium to further evaluate the blood compatibility of the two.
Methods: according to the ISO10993 guide, small caliber artificial blood vessels and abdominal aortic replacement were made in parallel with the swim bladder and bovine pericardium. The following indexes,.1, patency and angioma observation were observed on 30 days and 60 days. The patency rate of 320 rows of CT scans and three-dimensional imaging and the formation of.2, endothelialization and neointima of angioma were observed. The adherence of endothelial cells was observed by scanning electron microscopy. HE, VG staining was used to observe the arrangement of vascular wall fibers and elastic fiber proliferation. Eighth factor associated antigen and alpha -SMA immunohistochemical staining were used to observe the arrangement of fibers in various stages of artificial blood vessels, endothelialization and smooth muscle cell proliferation.3, calcification study: X ray photography in vitro And the Micro CT scanning of the isolated samples was used to evaluate the calcium deposition of the vessel wall. The calcium deposition was further observed under the microscope of tissue Von Kossa staining.
The results were as follows: 1, the patency rate of the artificial blood vessel of the swim bladder of the fish was 100%. All the artificial blood vessels of the bovine pericardium were 16.67%. unobstructed vessels and no angioma formed.2. The fibers in all phases of the patency vessels were arranged in order. Only 60 days of the pericardium vessels of the cattle were found to be damaged by fibrous destruction. Both of the smooth vessels were covered with elastic fiber, and the elastic fibers in the blood vessel of the swim bladder were more abundant. The speed of endothelialization of the swim bladder was more complete than that of bovine heart bag; smooth muscle cell proliferation was seen at the anastomosis of two materials, and there was no significant difference between the thickness of the intima of the swim bladder in 30 days and 60 days. At the time of 30 days, the intima hyperplasia of the bovine heart was more significant than that of the swim bladder. At 60 days, the intima of the bovine pericardium almost blocked the.3 of the lumen, and the X-ray photogrammetry and the Micro CT scanning of the isolated samples were all scanned. No obvious calcification in the blood vessels was found. The tissue section Von Kossa staining showed that calcium salt punctate deposition and fibrous fracture were found in the wall of the bovine pericardial vascular material only at 60 days, and no calcium salt was found in the other patency vessels at the rest of the period.
Conclusion: the blood compatibility of fish bladder material is superior to that of bovine pericardium and can be used as a small caliber vascular prosthesis material.
The full text summary:
The study shows that the maximum breaking strength of the swim bladder of the carp is lower than that of the bull's heart bag, the ductility and flexibility are superior to the cow's heart bag, and the thermal stability has reached the requirements of the human body. The biocompatibility of the swim bladder is excellent, no cytotoxicity, the formation of thrombus and the nature of hemolysis. The result of the rat subcutaneous implant model shows that the calcification rate is obvious after the treatment of anti calcification. The results of abdominal aorta replacement of the small caliber artificial blood vessel of the swim bladder showed that the bladder material was used as artificial blood vessel, the blood compatibility was satisfactory, the patency rate of the 30 and 60 days was higher than that of the bovine pericardium group, the endothelium was covered rapidly and completely, and no aneurysm occurred.
【学位授予单位】：第二军医大学
【学位级别】：博士
【学位授予年份】：2013
【分类号】：R318.08

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