[摘要]目的:探讨利用生物酶连续消化法制备犬颈总动脉脱细胞血管基质时对材料生物力学的影响。方法:利用胰酶和核酸酶连续消化法制备犬颈总动脉脱细胞血管基质材料,通过组织学、扫描电镜观察和DNA含量的检测,判断细胞的脱除情况;检测脱细胞材料的拉伸强度、爆裂强度及顺应性,判断脱细胞过程对材料生物力学性质的影响。结果:组织学染色及扫描电镜的结果表明血管细胞成分被完全去除,血管细胞外基质成分保留完整;与新鲜犬颈总动脉相比,脱细胞材料的拉伸强度和爆裂强度没有明显的改变,而材料的顺应性有所降低。结论:生物酶连续消化法制备的犬颈总动脉脱细胞基质材料保持了与新鲜血管大部分生物力学性质,但材料的顺应性有所降低。
[关键词]血管;组织工程;细胞外基质;生物力学
[中图分类号]R318[文献标识码]A[文章编号]1008-6455(2010)07-0996-04
Effects of decellularization using biotic enzymes on the mechanical properties of the canine carotid artery
LIU Guo-feng1,HE Zhi-juan2,YANG Da-ping1,XU Xue-wu1,LIU Ying1,REN Li-hong1,LI Qing-chun1
(1.Department of Plastic Surgery,Second Affiliated Hospital of Harbin Medical University,Harbin 150086,Heilongjiang,China; 2.Department of Obstetrics and Gynecology,First Affiliated Hospital of Harbin Medical University,Harbin 15001 0,Heilongjiang,China)
Abstract:ObjectiveThe objective of this study is to investigate the effects of decellularization using biotic enzymes on the mechanical and structural properties of the canine carotid artery.MethodsIntact canine carotid artery were decellularized by using Trypsin/EDTA,ribonuclease and desoxyribonuclease. Residual cellular and extracellular matrix composition was evaluated with hematoxylin and eosin (H&E) staining,quantitative DNA analysis and scanning electron microscopy. Tensile strength, burst strength and compliance were measured in vitro to determine the effects of decellularizedprocess on the biomechanical properties of the canine carotid artery.ResultsHistology and scanning electron microscopy examination demonstrate that scaffolds were completely decellularized and scaffolds revealed a well-preserved extracellular matrix. Compared with fresh canine carotid artery, decellularized artery had similar burst and breaking strength and had lower compliance.Conclusion This study demonstrates that the decellularized artery using biotic enzymes had similar burst and breaking strength and had lower compliance compared with fresh canine carotid artery.
Key words: vascular grafts; tissue engineering; extracellular matrix; biomechanical
血管组织工程学研究为临床上小口径血管移植物的制备提供了光明的前景,口径小于6mm的小口径组织工程血管研究与大口径血管移植物的研究有很大的差别[1]。因为血管移植物与受区血管生物力学性质的匹配程度对不同口径的组织工程血管移植物通畅率的影响差别较大,血管移植物的口径越小受到的影响越大,小口径血管移植物在受体内更容易发生内膜增生、中膜增厚,最后导致移植物的官腔狭窄甚至闭塞[2]。血管组织工程研究中支架材料的力学性质对血管移植物的生物力学性质起着决定性的作用,所以在小口径组织工程工程血管的研究中制备与受区血管生物力学性质完全匹配的支架材料是最关键的科学问题,这将决定小口径组织工程血管移植的远期通畅率[3]。本实验将对小口径组织工程血管脱细胞基质生物支架材料的生物力学性质进行研究,明确生物酶联合消化法对犬颈总动脉脱细胞基质材料生物力学的影响。
1材料和方法
1.1 实验用动物:普通杂种家犬,体重25～30kg,6个月龄,雌雄不限。
1.2 实验材料及主要仪器:胰蛋白酶(Trypsin)、核糖核酸酶(Ribonuclease,RNase)、脱氧核糖核酸酶(Desoxyribonu clease,DNase):美国Sigma公司;EDTA(Ethylenediamine tetraacetic acid)、磷酸缓冲盐溶液(Phosphate buffer solution,PBS):北京中杉生物公司;普通光学显微镜:日本Nikon公司;S-3400N型扫描电子显微镜:日本Hitachi公司;电子万能实验材料机:德国Zwick-Roell公司。
1.3 犬颈总动脉脱细胞基质材料制备: 无菌条件下切取犬颈总动脉,大量无菌PBS冲洗,去除血液成分及外层附属软组织,选择切取长度约为7cm,内径约为3mm的动脉,利用胰蛋白酶和核酸酶连续消化法去除动脉细胞及其碎片成分[4]。先用0.1% 胰蛋白酶加0.02% EDTA 溶液消化20h,中间更换1次消化液,大量PBS冲洗后用20μg/ml RNase +200μg/ml DNase溶液消化2h,大量无菌PBS 溶液冲洗。以上步骤均在5%CO2、37℃,80次/ min持续震荡条件下进行。监测制备的脱细胞血管基质,使材料的细胞DNA残留量少于0.1%。同时选取新鲜犬颈总动脉作为对照组(每组6例),进行以下检测。
1.3 组织学染色 将标本置于10%中性甲醛溶液中固定24h,常规石蜡包埋、切片,进行HE染色。在普通光学显微镜下对标本的结构进行组织学评价照相。
1.4 扫描电镜观察 将标本在1%戊二醛溶液中固定24h以上,用1%饿酸作后固定2h,50%、70%、90%、100%丙酮梯度脱水,50%、70%、90%、100%醋酸异戊酷置换,应用临界点干燥仪、液体C02等进行干燥;干燥的标本固定在铝质标本台上,用溅射镀膜机镀金后,用S-3400N型扫描电子显微镜系统观察并照相。
1.5 生物力学检测:利用Zwick/Roell Z010型电子万能力学实验机测定标本的拉伸强度、爆裂强度及轴向顺应性。拉伸强度及轴向顺应性检测:将管状标本(长度为5cm)两端固定于微型夹具上,以0.5mm/s的速度拉伸,直至标本断裂,系统自动记录标本的应力-应变曲线及极限拉伸强度,计算出标本的轴向顺应性。爆裂强度检测:把管状标本一端用丝线结扎,另一端固定于装满生理盐水的5ml医用注射器上,注射器筒壁固定于250ml玻璃葡萄糖瓶中,整个装置平置在力学实验机上,以40kPa/s的力向标本内注射生理盐水,直至标本爆裂或漏液,系统自动记录标本的爆裂强度。
1.6统计学分析: 所有定量实验结果均用x±s表示,利用SPSS11.0统计学软行独立样本t检验,P0.05)(图4)。极限拉伸强度检测结果如下:脱细胞犬颈总动脉基质组为4 122.91±118.05 KPa,新鲜犬颈总动脉组为4 212.99±103.80 KPa,两组标本极限拉伸强度相似,差别无统计学意义(P>0.05)。轴向顺应性计算结果如下,脱细胞犬颈总动脉基质组为0.945±0.158 mm/mm,新鲜犬颈总动脉组为1.195±0.22 mm/mm,脱细胞犬颈总动脉基质组轴向顺应性低于新鲜犬颈总动脉组,差别有统计学意义(P [3]Tiwari A,Cheng KS,Salacinski H,et al. Improving the patency of vascular bypass grafts: the role of suture materials and surgical techniques on reducing anastomotic compliance mismatch[J]. Eur J Vasc Endovasc Surg,2003,25(4):287-295.
[4]刘国锋,杨大平,郭铁芳,等. 小口径组织工程血管脱细胞生物支架材料的研究[J]. 中国康复理论与实践,2008,14(3): 234-236,301.
[5]Stegemann JP,Kaszuba SN,Rowe SL. Review: advances in vascular tissue engineering using protein-based biomaterials[J]. Tissue Eng,2007,13(11):2601-2613.
[6]Meredith JE Jr,Fazeli B,Schwartz MA. The extracellular matrix as a cell survival factor[J]. Mol Biol Cell,1993,4(9): 953-961.
[7]Schmidt CE,Baier JM. Acellular vascular tissues: natural biomaterials for tissue repair and tissue engineering[J]. Biomaterials,2000,21(22):2215-2231.
[8]MacNeill BD,Pomerantseva I,Lowe HC,et al. Toward a new blood vessel[J]. Vasc Med,2002,7(3):241-246.
[9]Amiel GE, Komura M,Shapira O,et al. Engineering of blood vessels from acellular collagen matrices coated with human endothelial cells[J]. Tissue Eng,2006,12(8):2355-2365.
[10]Courtman DW, Pereira CA, Kashef V. Development of a pericardial acellular matrix biomaterial: biochemical and mechanical effects of cell extraction[J]. J Biomed Mater Res,1994,28:655-666.
[11]Ballyk PD, Walsh C, Butany J,et al. Compliance mismatch may promote graft-artery intimal hyperplasia by altering suture-line stresses[J]. J Biomech, 1998,31(3):229-237.
[12]Abbott WM, Megerman J,Hasson JE,et al. Effect of compliance mismatch on vascular graft patency[J]. J Vasc Surg,1987,5(2):376-382.
[13]Howard AB,Alexander RW,Nerem RM,et al. Cyclic strain induces an oxidative stress in endothelial cells[J]. Am J Physiol,1997,272(2 Pt 1):C421-427.
[14]Trubel W,Moritz A,Schima H,et al. Compliance and formation of distal anastomotic intimal hyperplasia in Dacron mesh tube constricted veins used as arterial bypass grafts[J]. Asaio J,1994,40(3):M273-278.
[收稿日期]2010-05-25[修回日期]2010-07-10
编辑/张惠娟