Abstract
BACKGROUND:Studies on tissue-engineered vascular scaffold construction mostly focus on biodegradable scaffold and acellular allogenic or xenogenic 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 retrovirus (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-decellularization 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.





INTRODUCTION

Tissue-engineered vessels have predominant advantages in pathogenicity and growth over the vessels used currently in the clinical practice. Studies on tissue-engineered vessel reconstruction primarily focus on biodegradable scaffold and acellular allogenic or xenogenic 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 [1]. In the present study, decellularization by ultrahigh pressure treatment, was used to treat allogenic young porcine descending aorta to construct an ideal acellular natural vascular scaffold.

MATERIALS AND METHODS

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: Triton-X 100 (AMRESCO, USA), RNase I, DNase I, D-Hank solution, phosphate buffered saline (PBS), and evidence-based medicine (EBM) solutions (Sigma, USA), MiliQ water and second boiling high pure water distiller (Hitachi Ltd, Japan), ethylenediamine tetraacetic acid (EDTA, Amresco. Inc, USA), Hoechst 33258 (Dojindo Laboratories, Kumamoto, Japan), ultrahigh pressure device (Kobelco, Kobe Steel, Ltd, Japan), optical microscope (Nikon Eclipss TE200, Japan), optical digital microscope (Nikon Digital Camera DXM 1200), PCR (GeneAmp PCR System 9700), antibiotics (Japan), bench (Japan), breathing machine (900-C, Siemens, Germany), centrifugal pump (SP101, Japan), thermostatic waterbath cabin
(Thermo Minider SM-05), electro-scalpel (Mera MS-BM2), shaker (Vortex-2Genie), high-speed centrifuge (SorvaccMC), supercentrifuge(Microlite), and thermostatic incubator (BNA-111, Espec, Osaka, Japan).

Methods
Vascular decellularization treatment
After anesthesia, young porcine descending aorta vessels were isolated under sterile condition and washed by D-Hank's solution and MilliO water, and then divided into 4 groups: control group, surface active agent Ⅰ-treated group (Triton 24 hours), surface active agent Ⅱ-treated group (Triton 24 hours and washing), and ultrahigh pressure-treated group (decellularization by ultrahigh pressure treatment and washing). In the control group, porcine descending aorta vessels were given no treatments, and they were only stored in a well-closed container filled with PBS (antibiotics included) at 4 ℃ for future use. In the surface active agent Ⅰand Ⅱ- treated groups, porcine descending aorta vessels were soaked and suspended in 1% Tritox X-100 solution, which was supplemented with 20 mg/L RNase A, 200 mg/L DNase I, and 0.2 g/L EDTA, for 24 hours at 37 ℃ in a CO2 (0.05 volume fraction)-air environment. Immediately or 2 weeks after PBS washing, the aorta vessels were stored in a well-closed container filled with PBS (antibiotics included) for future use. In the ultrahigh pressure-treated group, porcine descending aorta vessels were placed in a plastic bag made specially, which was filled with MilliQ water. After the plastic bag was pressurized by an ultrahigh pressure machine for 10 minutes (981 MPa, 4 ℃), the porcine descending aorta vessels were taken out from the plastic bag made specially under a sterile condition, and soaked and suspended in EBM solution, which was supplemented with 20 mg/L RNase A, 200 mg/L DNase I and 0.02% EDTA2Na for 1 week of washing at 37 ℃ in a CO2 (volume fraction 0.05)-air environment. Next, the vessels were soaked in ethanol (volume fraction 0.8) for 3 days. The vessels were then rinsed using PBS and stored in well-closed container filled with PBS ( antibiotics included) at 4 ℃ for future use.

Histological observation
The vessel tissues were fixed in 40 g/L neutral formalin for 48 hours. Subsequently, the vessel tissues were dehydrated by ethanol, cleared by dimethyl benzene, routinely embedded by paraffin, sliced in 5μm-thickness sections, and stained by hematoxylin-eosin. Finally, they were observed via an optical microscope (100 augmentation). Results demonstrated that collagen fibers were slightly red-stained, and cellular nuclei were blue-stained.

Ultrastructural observation
Vessel tissues were fixed by 40 g/L glutaraldehyde and 10 g/L osmic acid. Prior to and after fixation, PBS washing was performed. Subsequently, the vessel tissues were gradually dehydrated by alcohol acetone, soaked and embedded by Epon 812 epoxide resin, sliced using LKB ultramicrotome, stained by uranium for 20 minutes and by lead for 10 minutes. Finally, removal of cell components from vascular tissue and retention of scaffold fibers were observed via a transmission electron microscope (JEM 100 cx). Vessel tissues were fixed by 30 g/L glutaraldehyde and 10 g/L osmic acid. Prior to and after fixation, PBS washing was performed again. The vessel tissues were gradually dehydrated by alcohol acetone, replaced isoamyl acetate, critical point dried, and platinum-palladium plated. Finally, the ultrastructure of scaffold was observed via a scanning electron microscope.

Porcine endogeneous retrovirus (PERV) detection
PERV gag gene (Department of Regenerative Medicine and Tissue Engineering, National Cardiovascular Center, Osaka, Japan) was taken as target sequence. After selecting a specific primer, proviral DNA of PERV could be detectable from young porcine acellular vessels by PCR method.

Determination of DNA levels
Using a Hoechst 33258 fluorescent probe, DNA levels were quantitatively determined in the decellularized vessels by ultrahigh pressure treatment.

Statistical analysis
Statistical analysis was performed by the first author using SPSS 8.0 software. All data were expressed as Mean±SD. Intergroup or paired t test was used for the comparison of means between two samples. A level of P < 0.05 was considered significant.

RESULTS

Morphological structure of vascular wall
Wavy structure of fibers was intact, and tissues were thoroughly cell free (Figure 1).

Removal of cells
Transmission electron microscope results demonstrated that collagen fibers and elastic fibers, but not cell components were detectable, and collagen fibers exhibited a good arrangement and clear transverse striation without dissolution and fragmentation. Scanning electron microscope results demonstrated that only acellular scaffold was found, and the structure of collagen fiber bundles, which were on the scaffold surface, was well retained and not covered by cells (Figure 2).

All PERV killed
After ultrahigh pressure treatment, there was no PERV detected in the treated tissues. However, the PERV could not be inactivated in the tissues treated by surface active agent (Figure 3).

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Detection of DNA levels
Intravascular DNA levels significantly altered from （31.9±3.7）mg/L pre-decellularization by ultrahigh pressure treatment to (1.2±0.1) 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.

DISCUSSION

Surgical treatment of heart disease acquires considerable vessel substitutes. Previously, people used artificial vessels for transplant operation, which can alleviate clinical symptoms, but concomitantly, which can also easily lead to thrombus, vascular restenosis, poor compliance, and not growing [2-4]. Since the concept of tissue engineering, a novel tissue engineering technique, was purposed in 1987, study and development of human tissue organs are always the hotspot research objective [5]. To overcome the limitations of artificial vessels, the substitutes of impaired vessels should be prepared by tissue engineering technique. The tissue-engineered vessels should have viable tissues that can give response to blood flow motivation and chemical stimulations, and concomitantly secrete substances. These vessels can also self-heal, and self reconstitute according to the surrounding environment, even grow in children's bodies, and are characterized by good compliance, not easy to cause thrombus, and with resistance to infection [6].
There are three fundamental elements: structural scaffold, seeded cells, and nutrient-providing environment in preparing tissue-engineered vessels [7-15]. Before cell produces extracellular matrix, scaffold provides the skeleton for tissue growth. Based on collagen matrix and biodegradable polymer, most vessel scaffolds are often implanted in vivo. A tubular axis is used as a sustentaculum and removed prior to implantation [16]. Autologous smooth muscle cells, fibroblasts and endothelial cells can be inoculated onto the scaffold and differentiate into vessels with ideal mechanical characteristics. A bioreactor is used to simulate the intravascular environment with pulsatile flow [14,17-18]. Some other vascular scaffolds are acellular natural vascular scaffolds. The current research of acellular porcine vascular scaffold focuses on how to use decellularization properly, i.e. to decellularize the vessels thoroughly and maintain the integrity of structure and function of extracellular matrix, to reduce the use of interfacial active agent to lower the toxicity to human body, and to prevent the infection to animal-derived pathogenic microorganism.
At present, the methods used commonly acquire to be improved. The cell components in the biovalve scaffold are mostly removed by in conjunction of surface active agent, pancreatic enzyme, and nuclease. To implant autologous valved vessles into human bodies, porcine bacterial virus was ensured not to bring to humans (such as PERV). Among the methods to kill such pathogenic microorganisms, ultrahigh pressure treatment should be more effective and safer than surface active agent, which was commonly used. We hoped to remove allogenic or xenogenic antigens, and concomitantly kill vascular pathogenic microorganisms by ultrahigh pressure treatment.
In the present study, ultrahigh pressure (981 MPa), which can inactivate gemma, was used in conjunction with low-concentration nuclease digestion as well as buffered solution washing to safely and thoroughly decellularize the vessels and kill the pathogenic microorganisms. The present study proved that such a method could remove donor-sourced cells easily, effectively, and safely.
Transplantation of animal-soured tissues and organs has the risk of animal-sourced bacterium and virus (such as PERV) infection [19]. Whether the virus and bacterium from the porcine valved vessels can be thoroughly removed is the precondition of a safe clinical application. Ultrahigh pressure sterilization uses the characteristic that ultrahigh pressure can lead to the alteration of noncovalent bond (such as hydrogen bond, hydrophobic bond, and ionic bond) in the high molecular stereochemical structure to inactive and kill pathogenic microorganisms, because the alteration of noncovalent bond can denaturalize protein, inactive enzyme, disrupt cell membrane, leak bacterial components, and arrest vital movement. Ultrahigh pressure has a strong destructive effect on viral envelope. Ultrahigh pressure can cause the leakage of strain protein, which leads to reverse transcriptase inactivation and infectivity loss. The present study confirmed that decellularization by ultrahigh pressure treatment, but not decellularization by use of surface active agent, could successfully inactive PERV.
To be concluded, the present study demonstrated that decellularization by ultrahigh pressure treatment could fundamentally rid cell components of scaffold, and concomitantly successfully inactivate PERV. Decellularization by ultrahigh pressure treatment provides a new thought for tissue engineering.

REFERENCES

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超高压脱细胞方法制备组织工程
血管支架**☆○

殷 猛1，刘锦纷1，藤里俊哉○2，郑 海1，凑谷谦司○2，中谷武嗣○2
1上海交通大学医学院上海儿童医学中心胸外科,上海市 200127; 2日本国立循环器病中心研究所再生医学部，脏器移植部，日本大阪府吹田市

殷 猛☆，男，1972年生，黑龙江省哈尔滨市人，汉族，2006年上海交通大学医学院毕业，博士，主治医师，主要从事组织工程方法治疗胸外科疾病的研究。
中国卫生部笹川医学奖学金*; 日本国厚生劳动省科学研究费*
摘要
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 Hoechst 33258 荧光探针，定量检测超高压脱细胞血管中DNA含量；用JEMl00cx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。④ DNA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）。
关键词：支架；脱细胞；猪内源性反转录病毒；超高压
中图分类号: R318.08 文献标识码: A 文章编号: 1673-8225(2008)10-01969-04
殷猛，刘锦纷，藤里俊哉，郑海，凑谷谦司，中谷武嗣.超高压脱细胞方法制备组织工程血管支架[J].中国组织工程研究与临床康复，2008，12(10):1969-1972
[www.zglckf.com/zglckf/ejournal/upfiles/12-10/10k-1969(ps).pdf]
(Edited by Xing WH/Song LP/Wang L)