魚鰾作為新型心血管外科手術(shù)生物材料的試驗(yàn)研究
發(fā)布時(shí)間：2018-07-25 17:22
【摘要】：生物材料（Biomaterials）已在心血管外科領(lǐng)域得到廣泛使用，其中又以生物心臟瓣膜和人工血管使用較多。生物瓣膜因血流動(dòng)力學(xué)出色、血栓栓塞發(fā)生率低、術(shù)后生活質(zhì)量高等優(yōu)點(diǎn)，尤其在發(fā)達(dá)國(guó)家已有較高的認(rèn)可度，但由于牛心包等異種材料本身原因，早期即可能出現(xiàn)機(jī)械性毀損和鈣化衰敗，大大限制了生物瓣膜的適用范圍。大口徑人工血管已成功應(yīng)用于心血管手術(shù)且術(shù)后效果好，而小口徑人工血管（口徑＜6mm）因血液相容性差導(dǎo)致的術(shù)后通暢率低等原因，仍未獲得臨床認(rèn)可。因此，目前人們正在努力通過對(duì)現(xiàn)有材料加工改性及尋找新材料的角度出發(fā)以期解決以上問題。由此目的出發(fā)，本文分別從物理性能、體外生物相容性及動(dòng)物實(shí)驗(yàn)三個(gè)不同層面深入研究魚鰾性質(zhì)，旨在尋求一種優(yōu)于目前臨床常用材料的新型心血管外科生物材料。 第一部分魚鰾的物理性能試驗(yàn)研究 目的：研究魚鰾各項(xiàng)生物力學(xué)性能和熱穩(wěn)定性，以及交聯(lián)處理（0.625%戊二醛溶液）對(duì)其性能的影響。 方法：1、通過單軸拉伸試驗(yàn)（Uniaxial Tensile Test）獲得被測(cè)材料拉伸至毀損時(shí)的極限阿爾曼西應(yīng)變（FailureAlmansi strain，f）、極限柯西應(yīng)力（Failure Cauchy stress，f）以及極限彈性模量（Peak Elastic Modulus，MP）,比較新鮮、戊二醛處理后的魚鰾在不同方向上和牛心包的力學(xué)差異。結(jié)合預(yù)試驗(yàn)中維多利亞藍(lán)-苦味酸酸性復(fù)紅染色（Victoria blue-van Gieson, VG）染色、透射電鏡觀察以及本試驗(yàn)結(jié)果，對(duì)纖維排列做出描述。2、通過差示量熱掃描技術(shù)（Differential Scanning Calorimetry，DSC）檢測(cè)新鮮和戊二醛處理后魚鰾的熱變性溫度并分析交聯(lián)處理對(duì)其熱穩(wěn)定性的影響。 結(jié)果：1、單軸拉伸試驗(yàn)：（1）無(wú)論新鮮或是戊二醛交聯(lián)后魚鰾在周向的三個(gè)力學(xué)結(jié)果均高于縱向。（2）戊二醛處理后魚鰾周向的f、MP均高于同方向的新鮮魚鰾，兩者f無(wú)顯著統(tǒng)計(jì)學(xué)差異。（3）戊二醛交聯(lián)后魚鰾縱向f大于同方向新鮮魚鰾，兩者f、MP無(wú)顯著統(tǒng)計(jì)學(xué)差異。（4）戊二醛處理后牛心包f、MP均高于新鮮牛心包，兩者f無(wú)統(tǒng)計(jì)學(xué)差異。（5）兩材料經(jīng)戊二醛處理后，魚鰾周向及縱向f、MP均低于牛心包，f顯著高于牛心包。2、差示量熱掃描：新鮮魚鰾熱變性溫度高于正常人體溫范圍，且戊二醛處理后魚鰾熱穩(wěn)定性優(yōu)于新鮮時(shí)。 結(jié)論：1、魚鰾纖維排列較牛心包規(guī)則，膠原纖維主要沿周向排列，彈力纖維在周向及縱向均有分布。2、戊二醛處理后魚鰾僵硬度（Stiffness）低于牛心包，延展性優(yōu)于牛心包，更接近人正常主動(dòng)脈瓣膜力學(xué)范圍。3、魚鰾，尤其經(jīng)過戊二醛處理后，熱穩(wěn)定性可達(dá)到體溫要求。 第二部分魚鰾生物相容性體外試驗(yàn)評(píng)價(jià) 目的：根據(jù)國(guó)際ISO10993醫(yī)療器械生物學(xué)評(píng)價(jià)指南研究魚鰾體外細(xì)胞毒性及血液相容性。 方法：1、體外細(xì)胞毒性試驗(yàn)：用含10%胎牛血清的MEM培養(yǎng)液浸提新鮮魚鰾，采用MTT法檢測(cè)浸提液對(duì)小鼠成纖維細(xì)胞L929的細(xì)胞毒性作用。2、體外血小板激活試驗(yàn)：用人全血浸提魚鰾，通過CD62p檢測(cè)法計(jì)算活化血小板數(shù)量。3、體外溶血試驗(yàn)：用無(wú)菌生理鹽水浸提魚鰾，采用游離血紅蛋白直接測(cè)定法檢測(cè)浸提液與人血混合后的游離血紅蛋白含量并計(jì)算溶血率。以上試驗(yàn)均以牛心包材料作為試驗(yàn)對(duì)照組。結(jié)果：1、100%新鮮魚鰾和100%新鮮牛心包浸提液接觸后的L929細(xì)胞活力分別為85.7%和94.2%，可認(rèn)為兩材料對(duì)L929細(xì)胞無(wú)毒性影響。2、戊二醛處理后魚鰾和牛心包組CD62P百分率分別為0.940.27%和1.730.35%，可認(rèn)為兩材料均對(duì)體外人血血栓形成沒有影響。3、戊二醛處理后魚鰾和牛心包組溶血率分別為0.2%和0.6%，可認(rèn)為兩材料對(duì)體外溶血沒有影響。 結(jié)論：魚鰾材料在體外細(xì)胞毒性檢測(cè)和體外血液相容性檢測(cè)中表現(xiàn)出良好的生物相容性并為進(jìn)一步動(dòng)物試驗(yàn)提供理論基礎(chǔ)。 第三部分魚鰾體內(nèi)鈣化研究目的：采用大鼠背部皮下埋植模型，定性及定量評(píng)估魚鰾不同時(shí)間點(diǎn)鈣化程度，并與牛心包進(jìn)行對(duì)比。 方法：1、采用已在牛心包中驗(yàn)證有效的DSC(Denaturant-surfactant-crosslinking)抗鈣化方法（美國(guó)Edwards公司專利，Patent No.:US6214054B1）預(yù)處理魚鰾及牛心包材料，行幼年大鼠背部皮下埋植，于術(shù)后7天、21天、56天取出樣本，定性及定量檢測(cè)鈣化情況并明確與免疫反應(yīng)的關(guān)系。2、鈣定性檢測(cè)：采用X線攝影、HE染色、VG染色及Von Kossa染色從樣本形態(tài)學(xué)和組織學(xué)水平評(píng)價(jià)各時(shí)間點(diǎn)鈣鹽沉積情況。3、鈣定量測(cè)定：采用電感耦合等離子體發(fā)射光譜儀（Indutive Coupled Plasma EmissionSpectrimeter,ICP）對(duì)比分析樣本鈣含量。4、免疫組織化學(xué)：CD68＋巨噬細(xì)胞及CD8＋T細(xì)胞特異性免疫組化染色研究材料內(nèi)部炎性細(xì)胞浸潤(rùn)情況，并與鈣化程度關(guān)聯(lián)。 結(jié)果：1、魚鰾各期鈣化程度均低于牛心包，魚鰾纖維完整排列有序，僅在后期局部鈣化處發(fā)生纖維斷裂，牛心包早期出現(xiàn)鈣鹽沉積，纖維破壞嚴(yán)重。定量測(cè)定表明牛心包各期鈣含量高于魚鰾組，以21天時(shí)差異最顯著。2、免疫組化染色顯示各時(shí)期魚鰾內(nèi)部未見炎性細(xì)胞浸潤(rùn)，而牛心包各期均有CD68＋巨噬細(xì)胞及CD8＋T細(xì)胞浸入。結(jié)論：大鼠皮下埋植鈣化模型顯示經(jīng)戊二醛-DSC抗鈣化方法處理后魚鰾各期鈣化程度顯著低于牛心包，魚鰾在體內(nèi)具有更出色的抗鈣化性能。 第四部分新型材料小口徑人造血管的試驗(yàn)研究 目的：采用大鼠腹主動(dòng)脈置換模型評(píng)估魚鰾作為小口徑人造血管材料的效果，并與牛心包相比較，進(jìn)一步評(píng)估兩者的血液相容性。 方法：根據(jù)ISO10993指南,使用魚鰾和牛心包材料制作小口徑人造血管并行大鼠腹主動(dòng)脈置換，于30天及60天取出并觀察如下指標(biāo)。1、通暢率及血管瘤觀察：采用320排CT掃描并三維成像觀察通暢率及血管瘤形成。2、內(nèi)皮化及新生內(nèi)膜觀察：采用掃面電鏡直觀觀察內(nèi)皮細(xì)胞黏附，采用HE、VG染色觀察通暢血管管壁纖維排列及彈力纖維增生，采用第八因子相關(guān)抗原及α-SMA免疫組化染色觀察人造血管各期纖維排列、內(nèi)皮化及平滑肌細(xì)胞增生程度。3、鈣化研究：采用X線體外攝影及離體樣本Micro CT掃描初步評(píng)估血管壁鈣鹽沉積，采用組織切片Von Kossa染色鏡下進(jìn)一步觀察鈣鹽沉積情況。 結(jié)果：1、各期魚鰾人造血管通暢率均為100%，牛心包人造血管均為16.67%。通暢血管均未見血管瘤形成。2、通暢血管各期纖維排列有序，僅60天牛心包血管可見纖維破壞斷裂，兩者通暢血管均可見彈力纖維覆蓋，魚鰾血管中彈力纖維更豐富；魚鰾內(nèi)皮化與牛心包比較速度更快程度更完整；兩材料吻合口處均可見平滑肌細(xì)胞增生，30天及60天魚鰾內(nèi)膜厚度無(wú)明顯差異，30天時(shí)牛心包內(nèi)膜增生較魚鰾顯著，60天時(shí)牛心包內(nèi)膜幾乎堵塞管腔。3、X線體外攝影及離體樣本Micro CT掃描均未發(fā)現(xiàn)血管中明顯鈣化點(diǎn)，經(jīng)組織切片Von kossa染色顯示，僅60天時(shí)牛心包血管材料管壁可見鈣鹽點(diǎn)狀沉積并致纖維斷裂，其余時(shí)期兩者通暢血管中均未見鈣鹽沉積。 結(jié)論：魚鰾材料血液相容性優(yōu)于牛心包，可以作為小口徑人造血管材料。 全文總結(jié)： 本課題研究表明，鯉魚鰾最大斷裂強(qiáng)度低于牛心包，延展性及柔韌性優(yōu)于牛心包，熱穩(wěn)定性能達(dá)到人體內(nèi)使用要求。魚鰾生物相容性優(yōu)良，無(wú)細(xì)胞毒性、致血栓形成及致溶血的性質(zhì)。大鼠皮下埋植模型結(jié)果表明，經(jīng)抗鈣化處理后，鈣化率顯著低于牛心包。魚鰾小口徑人造血管行大鼠腹主動(dòng)脈置換結(jié)果表明，魚鰾材料作為人造血管，血液相容性滿意，30及60天通暢率高于牛心包組，內(nèi)皮覆蓋迅速完全，，且無(wú)動(dòng)脈瘤發(fā)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.
【學(xué)位授予單位】：第二軍醫(yī)大學(xué)
【學(xué)位級(jí)別】：博士
【學(xué)位授予年份】：2013
【分類號(hào)】：R318.08
本文編號(hào)：2144510
[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.
【學(xué)位授予單位】：第二軍醫(yī)大學(xué)
【學(xué)位級(jí)別】：博士
【學(xué)位授予年份】：2013
【分類號(hào)】：R318.08
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本文編號(hào)：2144510
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