氟化脱细胞兔主动脉制备人工小血管的可行性研究

doi:10.3969/j.issn.1674-8115.2023.07.003

上海交通大学学报（医学版） ›› 2023, Vol. 43 ›› Issue (7): 814-820.doi: 10.3969/j.issn.1674-8115.2023.07.003

• 生物材料与再生医学专题 • 上一篇

氟化脱细胞兔主动脉制备人工小血管的可行性研究

张如杰¹(), 周广东², 王建博¹, 翁光东¹, 雷东²(), 龚文辉¹()

^1.安徽医科大学第一附属医院心脏大血管外科，合肥 230088
^2.上海交通大学医学院附属第九人民医院整复外科，上海市组织工程研究重点实验室，上海 200125

收稿日期:2023-04-24 接受日期:2023-07-17 出版日期:2023-07-28 发布日期:2023-07-28
通讯作者: 雷东,龚文辉 E-mail:zhangrujie2021@163.com;370309803@qq.com;gongwenhui18@163.com
作者简介:张如杰（1995—），男，硕士生；电子信箱：zhangrujie2021@163.com。
基金资助:
安徽省自然科学基金(1908085MH241)

Feasibility study on the preparation of artificial small blood vessel by fluorinated decellularized rabbit aorta

ZHANG Rujie¹(), ZHOU Guangdong², WANG Jianbo¹, WENG Guangdong¹, LEI Dong²(), GONG Wenhui¹()

^1.Department of Cardio-Vascular Surgery, The First Affiliated Hospital of Anhui Medical University, Hefei 230088, China
^2.Department of Plastic and Reconstructive Surgery, Shanghai 9th People′s Hospital, Shanghai Jiao Tong University School of Medicine; Shanghai Key Lab of Tissue Engineering, Shanghai 200125, China

Received:2023-04-24 Accepted:2023-07-17 Online:2023-07-28 Published:2023-07-28
Contact: LEI Dong,GONG Wenhui E-mail:zhangrujie2021@163.com;370309803@qq.com;gongwenhui18@163.com
Supported by:
Natural Science Foundation of Anhui Province(1908085MH241)

摘要/Abstract

摘要：

目的·探讨氟化脱细胞兔主动脉作为组织工程人工小血管的可行性。方法·联合聚乙二醇辛基苯基醚（Triton X-100）、脱氧胆酸钠（sodium deoxycholate，SD）、十二烷基硫酸钠（sodium dodecyl sulfate，SDS）、DNA酶、RNA酶等对获取的兔主动脉进行脱细胞处理，分别对脱细胞组和未脱细胞组进行苏木精-伊红染色（hematoxylin-eosin staining，H-E染色）、Masson染色、Verhoff-von Gieson染色，用场发射扫描电镜鉴定脱细胞效果，观察脱细胞血管形态学变化。使用脱细胞后的兔主动脉作为动脉化人工小血管支架，利用液体全氟化碳涂层修饰脱细胞小血管内膜（氟化组）制备一种新型人工小血管，并对其特征基团进行定性且定量测定。观察血管内表面液体消散时间和液体在45°倾斜血管表面的流动情况来分析血管的疏水性。将脱细胞组和氟化组的血管置入富含血小板的血浆后孵育，在电镜下观察，评估体外抗血小板聚集性能。取未脱细胞组、脱细胞组以及氟化组的兔主动脉连接上球囊扩张压力泵进行爆破压力测试。结果·对血管进行组织学观察发现，联合脱细胞法可以有效去除细胞并保留胶原蛋白和弹性纤维，且在电镜下内膜并未出现破损。对3组进行压力爆破测试，3组间比较差异无统计学意义。在疏水性测试中，水滴在氟化组内膜上停留的时间超过5 min，且在45°斜板上未留下明显水迹。在血小板黏附性试验中，脱细胞组内膜聚集活化了血小板，而氟化组则得到抑制。结论·联合脱细胞法可使脱细胞血管具有良好力学性能和物理稳定性，氟化涂层可使血管具有抗凝血性及较好的生物相容性。`

关键词: 脱细胞, 组织工程, 兔, 小血管, 内膜, 涂层

Abstract:

Objective ·To explore the feasibility of fluorinated decellularized rabbit aorta as a small artificial blood vessel for tissue engineering. Methods ·The obtained rabbit aorta was decellularized in combination with Triton X-100, sodium deoxycholate (SD), sodium dodecyl sulfate (SDS), DNAse, and RNAse. Hematoxylin-eosin staining (H-E staining), Masson staining and Verhoff-von Gieson staining were performed in the decellularized group and undecellularized group, respectively. The effect of decellularization was identified by field emission scanning electron microscope, and the morphological changes of decellularized blood vessels were observed. The decellularized rabbit aorta was used as the arterialized artificial small vessel scaffold, and the decellularized small vessel intima was modified with liquid perfluorocarbons coating to prepare a new type of artificial small vessel. The characteristic groups of the artificial small vessel were qualitatively and quantitatively determined. The dissipation time of liquid on the inner surface of the vessel and the flow of liquid on the surface of the vessel tilted at 45° were observed to analyze the hydrophobicity of the vessel. The blood vessels in the decellularized group and the fluorinated group were implanted with platelet-rich plasma, incubated, and observed under an electron microscope to evaluate the antiplatelet aggregation in vitro. The balloon pressure pump was connected to the aorta of the undecellularized group, decellularized group and fluoride group for bursting pressure test. Results ·Histological observation of blood vessels showed that the combination could effectively remove cells while retaining collagen and elastic fibers, and there was no damage to the intima under the electron microscope. There was no significant difference in the pressure blasting test among the three groups. In the hydrophobicity experiment, the retention time of water droplets on the membrane of the fluorinated group was over 5 min, and no obvious water marks were left on the 45° inclined plate. In the platelet adhesion test, intimal aggregation activated platelets in the decellularized group, while they were inhibited in the fluorinated group. Conclusion ·The decellularized blood vessels have good mechanical properties and physical stability by combined decellularization, and the fluorinated coating makes the blood vessels have good anticoagulant and biocompatibility.

Key words: decellularization, tissue engineering, rabbit, small blood vessel, intima, coating

中图分类号:

R654.3

张如杰, 周广东, 王建博, 翁光东, 雷东, 龚文辉. 氟化脱细胞兔主动脉制备人工小血管的可行性研究[J]. 上海交通大学学报（医学版）, 2023, 43(7): 814-820.

ZHANG Rujie, ZHOU Guangdong, WANG Jianbo, WENG Guangdong, LEI Dong, GONG Wenhui. Feasibility study on the preparation of artificial small blood vessel by fluorinated decellularized rabbit aorta[J]. Journal of Shanghai Jiao Tong University (Medical Science), 2023, 43(7): 814-820.

图/表 9

图1 脱细胞兔主动脉

Fig 1 Acellular rabbit aorta

图2 血管压力爆破测试

Fig 2 Pressure burst testing of the vessels

图3 脱细胞效果组织学观察(×200)Note： A. H-E staining of the decellularized group. B. Masson staining of the decellularized group. C. Verhoff-von Gieson staining of the decellularized group. D. H-E staining of the undecellularized group. E. Masson staining of the undecellularized group. F. Verhoff-von Gieson staining of the undecellularized group

Fig 3 Histological observation of the decellularized group and non-decellularized group (×200)

图4 脱细胞组和未脱细胞组FSEM观察结果Note： A. The decellularized vessel group. B The undecellularized vessel.

Fig 4 FSEM scanning electron microscopy observation in the decellularized and undecellularized group

图5 血管内含氟涂层的表面形貌结构和元素分析

Fig 5 Surface morphology and elemental analysis of fluorine-containing coatings in blood vesselNote: A. SEM. B. EDS. cps—counts per second. eV—electron volt. 1 eV=1.602×10-19 J.

表1 含氟小血管内膜的元素分析

Tab 1 Elemental analysis of the intima of fluorinated small vessels

Element	Weight/%	Percentage of the atom/%
C	59.18	69.05
O	6.04	5.29
F	34.79	25.66
Total amount	100.00	100.00

图6 血管压力爆破测试结果Note： A. The decellularized group. B. The undecellularized group. C. The fluorinated group.

Fig 6 Results of the vascular pressure burst test

图7 血管内膜表面疏水性的结果Note： A. Water dropped in the decellularized blood vessel group. B. Water dropped in the fluorinated blood vessel group. C. Water dropped in the fluorinated vessel group with a 45° tilt. E. Water dropped in the the decellularized vessel group with a 45° tilt.

Fig 7 Results of hydrophobicity of the intimal surface of the vessel

图8 血管抗血小板黏附检测结果Note： A. The decellularized vessel group. B. The fluorinated vessel group.

Fig 8 Results of the vascular antiplatelet adhesion test

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氟化脱细胞兔主动脉制备人工小血管的可行性研究

Feasibility study on the preparation of artificial small blood vessel by fluorinated decellularized rabbit aorta

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