超高压脱细胞方法制备组织工程血管支架

背景:构建组织工程血管支架的研究多集中于生物可降解支架和脱细胞同种或异种血管支架方面,但存在急需解决的若干问题:如生物可降解支架生物降解速度的控制以及植入脱细胞天然血管支架可能带来供体来源病毒细菌感染受体问题等.目的:采用超高压结合核酸酶洗涤方法(超高压脱细胞技术)处理同种异体血管,观察该方法的脱细胞效果以及猪内源性反转录病毒的去除情况.设计:对比观察实验.单位:日本国立心血管病中心研究所.材料:实验于2004-04/2005-04在日本国立心血管病中心研究所完成.选用健康雄性迷你猪幼猪,由日本九州鹿儿岛Japan Farm食用猪养殖场提供,体质量3～5 kg,猪龄1～3个月.实验过程中对动物的处置符合动物伦理学标准.实验涉及的主要试剂和仪器:Hoechst 33258为日本同仁化学研究所产品;超高压设备KOBELCO为神户制钢所产品:PCR为GENEAMP PCR SYSTEM 9700产品.方法:实验于2004-04/2005-04在日本国立心血管病中心研究所完成.无菌条件下取出猪降主动脉血管,用超高压设备以981 Mpa超高静水压(4 ℃)将供体来源细胞压碎,结合核酸酶的消化作用,PBS的搅拌洗涤,脱去细胞残片形成血管生物支架.主要观察指标: ①利用Hcechst 33258荧光探针,定量检测超高压脱细胞血管中DNA含量;用JEM100cx型透射电镜观察血管组织细胞成分去除和支架纤维保留情况;用JBM5200型扫描电镜观察支架的超微结构;100倍光镜下观察血管壁形态结构.即从组织学、分子生物学、免疫组组织化学水平评估超高压脱细胞方法的抗原去除效果. ②用PCR方法检测幼猪脱细胞血管中的猪内源性反转录病毒(PERV)前病毒DNA,评估超高压脱细胞方法对以猪内源性反转录病毒为代表的病原微生物的杀灭效果.结果: ①血管壁形态结构:血管壁纤维波浪状结构保存完好,组织? 内的细胞均被除去. ②细胞去除效果:透射电镜见细胞成分均已消失.但保留有胶原纤维和弹性纤维.扫描电镜可见细胞已被完全脱去,只剩脱细胞支架. ③病原微生物的杀灭效果:经过超高压处理,猪内源性反转录病毒被成功灭活,无法测出.而采用表面活性剂脱细胞组无法将猪内源性反转录病毒灭活. ④DNA含量:超高压脱细胞处理前,血管中DNA含量为(31.7±3.5) mg/L;超高压脱细胞处理后,DNA含量为(1.16±0.23)mg/L,DNA含量显著降低(P＜0.01).提示超高压脱细胞方法已将细胞核及内容物大部分除去.结论:实验证明采用超高压脱细胞方法可基本除去支架内细胞成分,可以将病源微生物杀灭(将猪内源性反转录病毒成功灭活).

Tissue-engineered vascular scaffolds prepared by ultrahigh pressure decellularization treatment

BACKGROUND: Studies on tissue-engineered vascular scaffold construction mostly focus on biodegradable scaffold and acellular allogenic or xenogenlc vascular scaffold. However, there are some problems to be urgently solved, such as control of degradable speed of biodegradable scaffold, and donor-sourced bacterial virus infecting recipients during the implantation of acellular natural vascular scaffold.OBJECTIVE: This study was designed to treat allogenic blood vessels by ultrahigh pressure in conjunction with nuclease washing (decellularization) to observe the decellularization effects and porcine endogenous retroviras (PERV) removal.DESIGN: A controlled observation.SETTING: National Cardiovascular Center, Japan.MATERIALS: This study was performed at the National Cardiovascular Center, Japan from April 2004 to April 2005.Young healthy male 1-3-month-old minipigs, weighing 3-5 kg, were provided by Japanese Farm. The protocol was performed in accordance with ethical guidelines for the use and care of animals. The main reagents and equipments used in the present study were as follows: Hoechst 33258 (Dojindo Laboratories, Kumamoto, Japan), ultrahigh pressure device (KOBELCO, Kobe Steel, Ltd, Japan), and PCR (GENEAMP PCR SYSTEM 9700).METHODS: Porcine descending aorta vessels were isolated under a sterile condition and treated by cold isostatic pressing (981 MPa, 4 ℃) for disruption of donor cells. The cell debris was digested by nuclease and washed out by phosphate buffered saline for vascular scaffold.MAIN OUTCOME MEASURES: After processing of decellularization by ultrahigh pressure treatment, vascular DNA levels were quantitatively determined by a fluorescent probe (Hoechst 33258); Removal of cell components from vascular tissue and retention of scaffold fibers were observed by a transmission electron microscope (JEM 100 cx); Scaffold ultrastructure was observed via a scanning electron microscope (JBM 5200); The morphological structure of vascular wall was observed via an optical microscope (100 augmentation) . All these were performed to evaluate the antigen-removal effects of decellularization by ultrahigh pressure treatment from histological, molecular biological, and immunohistochemical standpoints. Proviral DNA levels of acellular PERV were measured by PCR to evaluate the effects of decellularization by ultrahigh pressure treatment on killing PERV, a typical pathogenic microorganism.RESULTS: After decellularization by ultrahigh pressure treatment, the wavy structure of fibers was completely retained, and tissues were thoroughly cell free. Transmission electron microscope results demonstrated that collagen fibers and elastic fibers, but not cell components were detectable. Scanning electron microscope results demonstrated that only acellular scaffold was found. There was no PERV detected in the treated tissues. However, the PERV could not be inactivated in the tissues treated by surface active agent. Intravascular DNA levels significantly altered from (31.7±3.5 ) mg/L pre-decelhilarization by ultrahigh pressure treatment to (1.16±0.23) mg/L post- decellularization by ultrahigh pressure treatment(P＜0.01). Results demonstrated that decellularization by ultrahigh pressure treatment ridded of cellular nucleus and contents mostly.CONCLUSION: The study demonstrated that decellularization by ultrahigh pressure treatment could fundamentally rid cell components of scaffold, and concomitantly inactivate PERV successfully.