摘要
目的:观察聚乙二醇法在组织工程瓣膜准备中的应用价值,比较聚乙二醇去细胞前后组织工程瓣膜的物理特性。方法:实验于2005-10/2006-03在华中科技大学同济医学院基础医学院生物化学系实验室完成。①实验分组:取猪10只,由于猪主动脉瓣为三叶瓣结构,共取得瓣叶组织30个,麻醉后宰杀取其心脏动脉瓣膜,分为去细胞组和对照组,每组各15个。②实验方法:去细胞组用聚乙二醇和DNase I处理;瓣叶组织放入1kg/L聚乙二醇,室温下浸泡30～45min,振荡器加以振荡;含抗生素磷酸盐缓冲液浸泡24h,反复3次洗脱;以5×104U/L DNase I液浸泡处理1h;对照组仅以含抗生素磷酸盐缓冲液浸泡24h,反复3次洗脱。③实验评估:苏木精-伊红染色、扫描电镜观察去细胞情况,吸光度(A)值,计算去细胞率(%)=(对照组A值-去细胞组A值)/对照组A值×100%。猪去细胞瓣膜条置于力学测试仪测定最大负荷、最大应力、最大应变和弹性模量。结果:纳入猪10只,均进入结果分析。①去细胞组织形态学观察:去细胞组猪瓣膜组织中看不到细胞成分,且细胞外基质结构保存完整,胶原纤维排列整齐,无明显断裂,仍呈波浪状平行排列,结构紧凑,弹性纤维结构清晰,组织无明显水肿。②DNA含量分析:聚乙二醇处理后去细胞百分率为95.32%。③生物力学检测:与对照组比较,去细胞组瓣膜组织最大负荷[(12.586±1.693),(10.242±1.435)N,P>0.05]、最大应力[(2.346±0.342),(1.877±0.572)N/mm,P>0.05]、弹性模量(15.152±1.579,14.549±0.678,P>0.05)、最大应变[(31.685±7.533),(28.118±6.045)mm/N,>0.05]等均无显著差异。P结论:聚乙二醇法去除细胞完全,细胞外基质保存完整,对组织机械性能无明显影响,适于构建组织工程瓣膜。
AIM： To study a new method of preparing porcine tissue engineering valves by decellularization with poly ethylene glycol （PEG） and observe the change of mechanical properties. METHODS： The experiment was carded out in the laboratory of Department of Biochemistry, Tongji Medical College, Huazhong University of Science and Technology from October 2005 to March 2006. ①Thirty porcine aortic valves were taken from 10 hybrid porcine and randomly divided into PEG group and control group, each contained 15 valves②PEG group： The valves were dipped in a decellularizing solution containing 1 kg/L PEG in distilled water and placed for 30-45 minutes at room temperature with gentle vibrating, then dipped in phosphate-buffered saline （PBS） containing antibiotics for 24 hours, washed three times, and incubated for 1 hour in PBS containing 5×10^4 U/L DNAae I. Control group： The valves were only dipped in PBS containing antibiotics for 24 hours and washed three times.③Histochemical studies were performed on sequential valve sections with hematoxylin-eosin stain and scanning electron microscope. The decellularization percentage was assessed with such equation： Decellularization percentage=（A value of control group-A value of PEG group）/A value of control groupx×100%（A： absorbance）. The mechanical properties were measured, including peak load, maximum stress, maximum strain, and modulus of elasticity. RESULTS： Ten porcine all entered the result analysis.①Histomorphological detection： No cellular component was found in porcine valve of PEG group, and the structure of extra cellular matrix （ECM） was well reserved. Collagen fiber arrangement was parallel and orderly on waving without obvious fracture. Elastic fiber was clear and no marked edema was observed in structure. ②DNA content： The decelluladzation percentage was 95.32%. ③The mechanical properties had no significant difference between the two groups [peak load： （12.586＋1.693）, （10.242＋1.435） N, P 〉 0.05; maximum stress： （2.346±0.342）, （1.877±0,572） N/mm, P 〉 0.05; maximum strain： （31.685±7.533）, （28.118±6.045） mm/N, P 〉 0.05; modulus of elasticity： 15.152±1.579, 14.549±0.678, P〉 0.05]. CONCLUSION： The method of decelluladzation with PEG can remove cellular components effectively and not disrupt the histological structure of ECM and the mechanical properties. It is a suitable valve for tissue engineering construction.
出处
《中国组织工程研究与临床康复》
CAS
CSCD
北大核心
2007年第26期5094-5096,共3页
Journal of Clinical Rehabilitative Tissue Engineering Research
