犬股动脉置换壳聚糖-硫酸化丝素蛋白人工血管:内皮层及血管层的生成
|
摘要
背景:前期实验制备了壳聚糖-硫酸化丝素蛋白人工血管。目的:通过犬股动脉置换壳聚糖-硫酸化丝素蛋白人工血管,探讨组织工程血管的功效性能。方法:将8只比格犬随机分为2组,实验组于股动脉进行壳聚糖-硫酸化丝素蛋白人工血管置换;正常对照组不进行任何处理。置换后6个月,检测凝血四项后,采用苏木精-伊红染色观察血管结构,免疫组织化学观察血管结构形成,电镜观察血管内皮层及血管层生成情况。结果与结论:1苏木精-伊红染色:实验组血管形成了内皮细胞层、平滑肌层和成纤维细胞层3层主要血管组织,在组织形态上与正常对照组血管无显著差别;2免疫组织化学染色:实验组FactorⅧ染色可见组织边缘一深色带染色即为内皮层形成;á-SMA染色可见较多棕色颗粒沉淀,为血管平滑肌形成;Vimentin染色亦可见较多棕色颗粒沉淀,提示人工血管也形成了成纤维细胞层。该3层组织与正常对照组血管无明显差异;3电镜观察:实验组人工血管内表面及切面与正常对照组血管相似,切面也形成了3层组织,但排列不及正常血管整齐;4凝血四项:两组凝血酶原时间、活化部分凝血活酶时间、凝血酶时间、纤维蛋白原无差异;5结果表明:壳聚糖-硫酸化丝素蛋白人工血管具有较好的生物相容性及功效性能。
BACKGROUND: The chitosan-sulfated silk fibroin artificial blood vessel was obtained in our previous study. OBJECTIVE: To investigate the biocompatibility and function of the chitosan-sulfated silk fibroin artificial blood vessel replacement into the canine femoral artery.METHODS: Eight Beagles were randomly allotted to two groups, and chitosan-sulfated silk fibroin artificial blood vessel was implanted into the femoral artery(experimental group), but animalsin control group received no intervention. Six months after implantation, the coagulation function was detected, the vascular formation, including endothelial, smooth muscle and fibroblast layers were observed through hematoxylin-eosin staining under electron microscope. RESULTS AND CONCLUSION:(1) Hematoxylin-eosin staining showed that vascular endothelial cells, vascular smooth muscle cells and fibroblasts were found in the experimental group, which were similar with the normal vascular structures.(2) Immunohistochemistry: in the experimental group, there was a deeply stained band at the tissue edege through Factor VIII staining, suggesting the formation of the endothelium; there were abundant brown particles precipitated in á-SMA staining, suggesting the vascular smooth muscle cell formation; Vimentin staining showed various brown particle precipitations indicating the fibroblast formation, and all were close to the normal vascular structures.(3) Electron microscope observed that the inner surface and section of the artificial vessel were similar with the normal one, and the three-layer structures formed in the section arranged irregularly.(4) There were no differences in the prothrombin time, activated partial thromboplastin time, thrombin time and fibrinogen between groups. To conclude, these results suggest that the chitosan-sulfated silk fibroin artificial blood vessel holds good biocompatibility and performances.
BACKGROUND: The chitosan-sulfated silk fibroin artificial blood vessel was obtained in our previous study. OBJECTIVE: To investigate the biocompatibility and function of the chitosan-sulfated silk fibroin artificial blood vessel replacement into the canine femoral artery.METHODS: Eight Beagles were randomly allotted to two groups, and chitosan-sulfated silk fibroin artificial blood vessel was implanted into the femoral artery(experimental group), but animalsin control group received no intervention. Six months after implantation, the coagulation function was detected, the vascular formation, including endothelial, smooth muscle and fibroblast layers were observed through hematoxylin-eosin staining under electron microscope. RESULTS AND CONCLUSION:(1) Hematoxylin-eosin staining showed that vascular endothelial cells, vascular smooth muscle cells and fibroblasts were found in the experimental group, which were similar with the normal vascular structures.(2) Immunohistochemistry: in the experimental group, there was a deeply stained band at the tissue edege through Factor VIII staining, suggesting the formation of the endothelium; there were abundant brown particles precipitated in á-SMA staining, suggesting the vascular smooth muscle cell formation; Vimentin staining showed various brown particle precipitations indicating the fibroblast formation, and all were close to the normal vascular structures.(3) Electron microscope observed that the inner surface and section of the artificial vessel were similar with the normal one, and the three-layer structures formed in the section arranged irregularly.(4) There were no differences in the prothrombin time, activated partial thromboplastin time, thrombin time and fibrinogen between groups. To conclude, these results suggest that the chitosan-sulfated silk fibroin artificial blood vessel holds good biocompatibility and performances.
引文
[1]Batelaan NM,ten Have M,van Balkom AJ,et al.Anxiety disorders and onset of cardiovascular disease:the differential impact of panic,phobias and worry.J Anxiety Disord.2014;28(2):252-258.
[2]Charlton F,Tooher J,Rye K,et al.Cardiovascular Risk,Lipids and Pregnancy:Preeclampsia and the Risk of Later Life Cardiovascular Disease.Heart Lung Circ.2014;23(3):203-212.
[3]Futrega K,King M,Lott WB,et al.Treating the whole not the hole:necessary coupling of technologies for diabetic foot ulcer treatment.Trends Mol Med.2014;20(3):137-142.
[4]Zhang C,Ma L,Peng F,et al.Spontaneous gas gangrene of the scrotum in patient with severe diabetic ketoacidosis.Int J Diabetes Mellit.2010;2(3):196-198.
[5]Wang S,Zhao Y.Diabetes mellitus and the incidence of deep vein thrombosis after total knee arthroplasty:a retrospective study.J Arthroplasty.2013;28(4):595-597.
[6]薛光华.血管代用品[J].实用外科杂志,1987,7(3):119-120.
[7]Tara S,Kurobe H,Maxfield MW,et al.Evaluation of remodeling process in small-diameter cell-free tissue-engineered arterial graft.J Vasc Surg.2015;62(3):734-743.
[8]Campbell EM,Cahill PA,Lally C.Investigation of a small-diameter decellularised artery as a potential scaffold for vascular tissue engineering;biomechanical evaluation and preliminary cell seeding.J Mech Behav Biomed Mater.2012;14:130-142.
[9]Meiring M,Allers W,Le Roux E.Tissue factor:A potent stimulator of Von Willebrand factor synthesis by human umbilical vein endothelial cells.Int J Med Sci.2016;13(10):759-764.
[10]Wise ES,Brophy CM.The Case for Endothelial Preservation via Pressure-Regulated Distension in the Preparation of Autologous Saphenous Vein Conduits in Cardiac and Peripheral Bypass Operations.Front Surg.2016;3:54.
[11]Yin A,Bowlin GL,Luo R,et al.Electrospun silk fibroin/poly(L-lactide-?-caplacton)graft with platelet-rich growth factor for inducing smooth muscle cell growth and infiltration.Regen Biomater.2016;3(4):239-245.
[12]刘志芳,陈艰.自体血管在冠状动脉搭桥术中的应用[J].江西医药,2008,43(6):611-613.
[13]张凡,郭东阳,程悦,等.老年慢性肾功能衰竭合并糖尿病患者自体血管通路的建立及围手术期处理[J].实用医院临床杂志,2010,7(2):62-64.
[14]Wang Y,Shi H,Qiao J,et al.Electrospun tubular scaffold with circumferentially aligned nanofibers for regulating smooth muscle cell growth.ACS Appl Mater Interfaces.2014;6(4):2958-2962.
[15]Stollwerck PL,Kozlowski B,Sandmann W,et al.Long-term dilatation of polyester and expanded polytetrafluoroethylene tube grafts after open repair of infrarenal abdominal aortic aneurysms.J Vasc Surg.2011;53(6):1506-1513.
[16]Wang Y,Wu W,Allen R.Vitalize synthetic vascular grafts in vivo.Cardiovasc Pathol.2013;22(3):e51.
[17]Qi P,Maitz MF,Huang N.Surface modification of cardiovascular materials and implants.Surf Coat Technol.2013;233(11):80-90.
[18]吕静静,於学禅,沈秋霞,等.干细胞在工程化组织构建与再生中的应用[J].中国生物医学工程学报,2013,32(1):99-104.
[19]Laterreur V,Ruel J,Auger FA,et al.Comparison of the direct burst pressure and the ring tensile test methods for mechanical characterization of tissue-engineered vascular substitutes.J Mech Behav Biomed Mater.2014;34:253-263.
[20]Li C,Yang W,Zhou J,et al.Risk factors for predicting postoperative complications after open infrarenal abdominal aortic aneurysm repair:results from a single vascular center in China.J Clin Anesth.2013;25(5):371-378.
[21]张运芬.血管内支架置入术治疗布-加氏综合症护理体会[J].医学理论与实践,2003,16(6):700-701.
[22]Davoudi M,Tayebi P,Beheshtian A.Primary patency time of basilic vein transposition versus prosthetic brachioaxillary access grafts in hemodialysis patients.J Vasc Access.2013;14(2):111-115.
[23]彭辉,阳富春.同种异体血管移植的研究及临床应用[J].微创医学,2009,4(1):51-53.
[24]潘仕荣,杨世方,易武,等.小径微孔聚氨酯人工血管的制备条件对微观结构与性能的影响[J].中国修复重建外科杂志,2005,19(1):64-69.
[25]潘仕荣,陶军,郑欢玲,等.小径微孔聚氨酯人工血管的顺应性[J].生物医学工程学杂志,2006,23(3):517-520.
[26]Giudiceandrea A,Seifalian AM,Krijgsman B,et al.Effect of prolonged pulsatile shear stress in vitro on endothelial cell seeded PTFE and compliant polyurethane vascular grafts.Eur J Vasc Endovasc Surg.1998;15(2):147-154.
[27]冯亚凯,吴珍珍.可生物降解聚氨酯在医学中的应用[J].材料导报,2006,20(6):115-117.
[28]Saitow C,Kaplan DL,Castellot JJ Jr.Heparin stimulates elastogenesis:application to silk-based vascular grafts.Matrix Biol.2011;30(5-6):346-355.
[29]Sakiyama-Elbert SE.Incorporation of heparin into biomaterials.Acta Biomaterialia.2014;10(4):1581-1587.
[30]陈柄灿.聚醚氨酯结构修饰及其相关特性研究[D].重庆大学,2005.
[31]韩明川.石英晶体微天平研究蛋白质在壳聚糖及衍生物表面的吸附行为[D].天津大学,2004.
[32]Levenberg S,Khademhosseini A,Langer R.Chapter39–Embryonic Stem Cells in Tissue Engineering.Essentials of Stem Cell Biology(Third Edition),2009:581-592.
[33]Kuo ZK,Lai PL,Toh EK,et al.Osteogenic differentiation of preosteoblasts on a hemostatic gelatin sponge.Sci Rep.2016;6:32884.
[2]Charlton F,Tooher J,Rye K,et al.Cardiovascular Risk,Lipids and Pregnancy:Preeclampsia and the Risk of Later Life Cardiovascular Disease.Heart Lung Circ.2014;23(3):203-212.
[3]Futrega K,King M,Lott WB,et al.Treating the whole not the hole:necessary coupling of technologies for diabetic foot ulcer treatment.Trends Mol Med.2014;20(3):137-142.
[4]Zhang C,Ma L,Peng F,et al.Spontaneous gas gangrene of the scrotum in patient with severe diabetic ketoacidosis.Int J Diabetes Mellit.2010;2(3):196-198.
[5]Wang S,Zhao Y.Diabetes mellitus and the incidence of deep vein thrombosis after total knee arthroplasty:a retrospective study.J Arthroplasty.2013;28(4):595-597.
[6]薛光华.血管代用品[J].实用外科杂志,1987,7(3):119-120.
[7]Tara S,Kurobe H,Maxfield MW,et al.Evaluation of remodeling process in small-diameter cell-free tissue-engineered arterial graft.J Vasc Surg.2015;62(3):734-743.
[8]Campbell EM,Cahill PA,Lally C.Investigation of a small-diameter decellularised artery as a potential scaffold for vascular tissue engineering;biomechanical evaluation and preliminary cell seeding.J Mech Behav Biomed Mater.2012;14:130-142.
[9]Meiring M,Allers W,Le Roux E.Tissue factor:A potent stimulator of Von Willebrand factor synthesis by human umbilical vein endothelial cells.Int J Med Sci.2016;13(10):759-764.
[10]Wise ES,Brophy CM.The Case for Endothelial Preservation via Pressure-Regulated Distension in the Preparation of Autologous Saphenous Vein Conduits in Cardiac and Peripheral Bypass Operations.Front Surg.2016;3:54.
[11]Yin A,Bowlin GL,Luo R,et al.Electrospun silk fibroin/poly(L-lactide-?-caplacton)graft with platelet-rich growth factor for inducing smooth muscle cell growth and infiltration.Regen Biomater.2016;3(4):239-245.
[12]刘志芳,陈艰.自体血管在冠状动脉搭桥术中的应用[J].江西医药,2008,43(6):611-613.
[13]张凡,郭东阳,程悦,等.老年慢性肾功能衰竭合并糖尿病患者自体血管通路的建立及围手术期处理[J].实用医院临床杂志,2010,7(2):62-64.
[14]Wang Y,Shi H,Qiao J,et al.Electrospun tubular scaffold with circumferentially aligned nanofibers for regulating smooth muscle cell growth.ACS Appl Mater Interfaces.2014;6(4):2958-2962.
[15]Stollwerck PL,Kozlowski B,Sandmann W,et al.Long-term dilatation of polyester and expanded polytetrafluoroethylene tube grafts after open repair of infrarenal abdominal aortic aneurysms.J Vasc Surg.2011;53(6):1506-1513.
[16]Wang Y,Wu W,Allen R.Vitalize synthetic vascular grafts in vivo.Cardiovasc Pathol.2013;22(3):e51.
[17]Qi P,Maitz MF,Huang N.Surface modification of cardiovascular materials and implants.Surf Coat Technol.2013;233(11):80-90.
[18]吕静静,於学禅,沈秋霞,等.干细胞在工程化组织构建与再生中的应用[J].中国生物医学工程学报,2013,32(1):99-104.
[19]Laterreur V,Ruel J,Auger FA,et al.Comparison of the direct burst pressure and the ring tensile test methods for mechanical characterization of tissue-engineered vascular substitutes.J Mech Behav Biomed Mater.2014;34:253-263.
[20]Li C,Yang W,Zhou J,et al.Risk factors for predicting postoperative complications after open infrarenal abdominal aortic aneurysm repair:results from a single vascular center in China.J Clin Anesth.2013;25(5):371-378.
[21]张运芬.血管内支架置入术治疗布-加氏综合症护理体会[J].医学理论与实践,2003,16(6):700-701.
[22]Davoudi M,Tayebi P,Beheshtian A.Primary patency time of basilic vein transposition versus prosthetic brachioaxillary access grafts in hemodialysis patients.J Vasc Access.2013;14(2):111-115.
[23]彭辉,阳富春.同种异体血管移植的研究及临床应用[J].微创医学,2009,4(1):51-53.
[24]潘仕荣,杨世方,易武,等.小径微孔聚氨酯人工血管的制备条件对微观结构与性能的影响[J].中国修复重建外科杂志,2005,19(1):64-69.
[25]潘仕荣,陶军,郑欢玲,等.小径微孔聚氨酯人工血管的顺应性[J].生物医学工程学杂志,2006,23(3):517-520.
[26]Giudiceandrea A,Seifalian AM,Krijgsman B,et al.Effect of prolonged pulsatile shear stress in vitro on endothelial cell seeded PTFE and compliant polyurethane vascular grafts.Eur J Vasc Endovasc Surg.1998;15(2):147-154.
[27]冯亚凯,吴珍珍.可生物降解聚氨酯在医学中的应用[J].材料导报,2006,20(6):115-117.
[28]Saitow C,Kaplan DL,Castellot JJ Jr.Heparin stimulates elastogenesis:application to silk-based vascular grafts.Matrix Biol.2011;30(5-6):346-355.
[29]Sakiyama-Elbert SE.Incorporation of heparin into biomaterials.Acta Biomaterialia.2014;10(4):1581-1587.
[30]陈柄灿.聚醚氨酯结构修饰及其相关特性研究[D].重庆大学,2005.
[31]韩明川.石英晶体微天平研究蛋白质在壳聚糖及衍生物表面的吸附行为[D].天津大学,2004.
[32]Levenberg S,Khademhosseini A,Langer R.Chapter39–Embryonic Stem Cells in Tissue Engineering.Essentials of Stem Cell Biology(Third Edition),2009:581-592.
[33]Kuo ZK,Lai PL,Toh EK,et al.Osteogenic differentiation of preosteoblasts on a hemostatic gelatin sponge.Sci Rep.2016;6:32884.