CN103230309B - A tissue engineered blood vessels and their preparation and use - Google Patents

A tissue engineered blood vessels and their preparation and use Download PDF

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CN103230309B
CN103230309B CN201310163477.7A CN201310163477A CN103230309B CN 103230309 B CN103230309 B CN 103230309B CN 201310163477 A CN201310163477 A CN 201310163477A CN 103230309 B CN103230309 B CN 103230309B
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gelatin
electrospun
cells
blood vessel
tissue engineered
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CN103230309A (en
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付炜
冯蓓
殷猛
王伟
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上海交通大学医学院附属上海儿童医学中心
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Abstract

本发明涉及一种组织工程血管及其制备方法和用途,所述的组织功能血管是以管状明胶/聚己内酯电纺膜为支架,平滑肌细胞均匀分布于支架内部。 The present invention relates to engineered blood vessels and tissue preparation method and use thereof, is a tubular vascular tissue function gelatin / PCL film electrospun scaffold, smooth muscle cells uniformly distributed inside the stent. 制备方法为:a)将消毒好的明胶/聚己内酯材料平铺于培养皿中;b)收集脐动脉平滑肌细胞均匀的接种于明胶/聚己内酯电纺膜表面后培养过夜,形成细胞材料复合电纺膜;c)将细胞材料复合电纺膜缠绕在PVC管上培养,最终形成管状的组织工程血管。 Method for the preparation of: a) disinfection of the gelatin / PCL material plated in petri dishes; b) collecting a uniform umbilical artery smooth muscle cells were seeded in gelatin / poly after overnight culture hexyl lactone electrically spinning membrane surface, formed cells composite electrospun membranes; c) cells composite electrospun film is wound on PVC tube culture, eventually forming a tubular vascular tissue engineering. 该方法制备得到的组织工程血管细胞分布更加均匀,同时又具备弹性好、硬度适中、内壁光滑、组织结构致密、胶原纤维丰富、与自身组织分界清晰等优良特性,其特征更加接近于活体血管。 The preparation obtained in tissue engineering of vascular cell more uniform, but also have good elasticity, moderate hardness, smooth wall, organizational structure is dense, rich in collagen fibers with their clear organizational boundaries and other excellent properties, characterized in closer to the in vivo blood vessel.

Description

一种组织工程血管及其制备方法和用途 A tissue engineered blood vessels and their preparation and use

技术领域 FIELD

[0001] 本发明涉及组织工程技术领域,具体地说,涉及一种组织工程血管及其制备方法和用途。 [0001] The present invention relates to the field of tissue engineering, particularly, to a tissue engineered blood vessel and its preparation and use.

背景技术 Background technique

[0002] 血管疾病是我国目前死亡率最高的疾病之一,血管移植是其主要的外科治疗手段。 [0002] Vascular disease is one of China's current highest mortality, vascular graft surgery is the main treatment. 血管移植手术中需要利用组织工程血管来替代病患血管。 Vascular graft surgery requires the use of tissue engineered blood vessels to replace the patient. 组织工程血管是利用组织工程学方法,用血管种子细胞(内皮细胞、平滑肌细胞)与相关细胞外基质(支架材料)复合,以构建从形态到功能都接近活体血管的组织工程化血管。 Tissue engineered blood vessel using tissue engineering techniques, seeds with vascular cells (endothelial cells, smooth muscle cells) associated with the extracellular matrix (scaffold material) is compounded to form the construct functions to close blood vessels in the living tissue engineered blood vessels. 性能优良的组织工程血管应当具备与自身组织分界清晰、具有一定的弹性和硬度、组织结构致密、细胞分布均匀等优点,以发挥近似于活体血管的功能。 Excellent performance tissue engineered blood vessel tissue itself should have clear boundaries, it has a certain elasticity and hardness, dense structure and uniform cell distribution, etc., to exert similar biological function of blood vessels.

[0003]目前,将明胶和聚己内酯混合在一起电纺成纳米纤维膜已被广泛应用于组织工程化不同的组织,如皮肤、神经、骨、血管等。 [0003] Currently, polycaprolactone and gelatin were mixed together electrically spun nanofiber films have been widely used in different tissue engineered tissues, such as skin, nerve, bone, blood vessels and the like. Chong等人[Chong EJ, et al.ActaB1materialia 2007,3(3):321-330]将明胶/聚己内酯混纺纳米纤维膜用于皮肤伤口愈合的研宄,结果显示所制备的明胶/聚己内酯纤维膜明显有利于成纤细胞的粘附、增值和分化。 Chong et al [Chong EJ, et al.ActaB1materialia 2007,3 (3): 321-330] The gelatin / PCL blend nanofiber membrane for wound healing study based on the results show that the prepared gelatin / poly caprolactone fiber membrane clearly advantageous as adhesion, proliferation and differentiation of cells of the fiber. Laleh Ghasem1-Mobarakeh等人[Laleh Ghasem1-Mobarakeh, et al.B1materials2008, 29(34):4532-4539]将明胶/聚己内酯电纺成平行度很高的纳米纤维膜用于神经组织的修复,神经干细胞的培养结果显示,明胶/聚己内酯纳米纤维膜是一种很好的组织工程支架材料,所纺的平行纳米纤维有利于神经细胞的增值和分化,并有利于神经突触的伸长° Sepideh Heydarkhan-HagvalI 等人[Sepideh Heydarkhan-Hagvall, et al.B1materials 2008, 29(19):2907-2914]将明胶和聚己内酯电纺成三维组织工程支架并成功用于心血管组织工程,结果显示混纺的纳米纤维膜展现出合成材料所拥有的优越的力学性能及天然材料所独有的细胞亲和性。 Laleh Ghasem1-Mobarakeh et al [Laleh Ghasem1-Mobarakeh, et al.B1materials2008, 29 (34): 4532-4539] Gelatin / polycaprolactone high parallelism electrically spun nanofiber membranes for repairing nerve tissue , neural stem cell culture results show that the gelatin / PCL nanofiber membrane is a good tissue engineering scaffolds, nano-spun fibers parallel facilitate proliferation and differentiation of nerve cells, and facilitates synaptic elongation ° Sepideh Heydarkhan-HagvalI et al [Sepideh Heydarkhan-Hagvall, et al.B1materials 2008, 29 (19): 2907-2914] and gelatin polycaprolactone electrically spun into a three-dimensional tissue engineering scaffold for use in cardiovascular and successfully tissue engineering, nano-fiber membrane results show that blends of synthetic materials exhibit have superior mechanical properties of natural materials and unique cell affinity. 其结果均表明明胶/聚己内酯是一种良好的支架材料。 The results show that the gelatin / PCL is a good scaffold.

[0004] 但是本领域技术人员均知,组织工程构建的思路通常是先构建与特定组织形状近似的支架载体,然后种植细胞进行培养。 [0004] those skilled in the art are aware, tissue engineered idea is generally similar to scaffolding vector specific shape of the tissue, cultured cells were then seeded. 例如目前利用明胶/聚己内酯作为支架材料构建组织工程血管时,通常都是明胶和聚己内酯混合在一起电纺成近似血管的管状结构,然后将细胞直接种植在支架的外表面,并进一步培养。 Currently, for example, when constructing a vascular tissue engineering scaffolds using a gelatin / PCL, usually gelatin polycaprolactone are mixed together and electrically spun vascular approximately tubular structure, and the cells were planted directly on the outer surface of the stent, and further training. 为模拟活体血管,直接电纺成管状的支架材料的壁厚与活体血管厚度基本一致,总体较厚,不适于细胞迀移和增殖,又因为细胞是直接种植在支架材料外表面,因而造成构建的组织工程血管内、外细胞分布不均匀,性能不佳。 To simulate in vivo blood vessel, is directly electrically spun into a blood vessel of the living body wall thickness of the stent material thickness of the tubular consistent, generally thicker, Gan suitable cell migration and proliferation, and because the cells are grown directly on the outer surface of the scaffold, resulting in construct in vascular tissue engineering, extracellular uneven distribution, poor performance. 如何构建细胞分布均匀、弹性和硬度适宜、与自身组织分界清晰、组织结构致密等各项性能均优异的组织工程血管十分重要。 How to build a uniform distribution of cells, a suitable elasticity and hardness, with a clear demarcation self-organization, the performance of dense tissue structure excellent in vascular tissue engineering is important.

发明内容 SUMMARY

[0005] 本发明的目的是针对现有技术中的不足,提供一种组织工程血管。 Objective [0005] The present invention is directed to the prior art is insufficient, there is provided a tissue engineered blood vessel.

[0006] 本发明的再一的目的是,提供上述组织工程血管的制备方法。 Then one [0006] The object of the present invention is to provide a method for preparing the above-described tissue engineered blood vessel.

[0007] 本发明的另一的目的是,提供上述组织工程血管的用途。 Another object of the [0007] present invention, there is provided the use of the above-described tissue engineered blood vessels.

[0008] 为实现上述目的,本发明采取的技术方案是: [0008] To achieve the above object, the present invention takes the following technical solution:

[0009] 一种组织工程血管,以管状明胶/聚己内酯电纺膜为支架,平滑肌细胞均匀分布于所述的支架的内部。 [0009] A tissue engineered blood vessels, tubular gelatin / PCL electrospun film, inner support smooth muscle cells uniformly distributed in the scaffold.

[0010] 所述的组织工程血管是按照以下方法制备的: [0010] The tissue engineered blood vessels prepared according to the following method:

[0011] a)将消毒好的明胶/聚己内酯材料平铺于器皿中; [0011] a) disinfecting the gelatin / PCL plated material in the vessel;

[0012] b)收集脐动脉平滑肌细胞均匀的接种于明胶/聚己内酯电纺膜表面后培养过夜,形成细胞材料复合电纺膜; [0012] b) collecting a uniform umbilical artery smooth muscle cells were seeded in gelatin / poly after overnight culture hexyl lactone electrically spinning membrane surface to form a cell composite electrospun membranes;

[0013] c)将细胞材料复合电纺膜缠绕在PVC管上培养,最终形成管状的组织工程血管。 [0013] c) Cells composite electrospun membranes wound on PVC tube culture, eventually forming a tubular vascular tissue engineering.

[0014] 优选的,步骤b)中脐动脉平滑肌细胞与明胶/聚己内酯电纺膜的接种比例是10 X 16个细胞/(2cmX 12cm)电纺膜;所述的细胞材料复合电纺膜缠绕在PVC管上的圈数为6-7圈,然后培养I周以形成供移植的组织工程血管,最好在该培养过程总隔2天半量换液一次。 [0014] Preferably, step b) and umbilical artery smooth muscle cells gelatin / polycaprolactone ratio seeded electrospun film is 10 X 16 cells / (2cmX 12cm) of electrospun membranes; Cell said composite electrospun the number of turns wound on the film 6-7 ring PVC pipe, and then cultured to form a peripheral I for transplantation tissue engineered blood vessel, preferably during the total culture medium was changed every 2 days and a half time.

[0015] 为实现上述第二个目的,本发明采取的技术方案是: [0015] To achieve the above second object, the present invention takes the following technical solution:

[0016] 如上所述的组织工程血管的制备方法,包括以下步骤: [0016] The method of preparing the tissue engineered blood vessel as described above, comprising the steps of:

[0017] a)将消毒好的明胶/聚己内酯材料平铺于器皿中; [0017] a) disinfecting the gelatin / PCL plated material in the vessel;

[0018] b)收集脐动脉平滑肌细胞均匀的接种于明胶/聚己内酯电纺膜表面后培养过夜,形成细胞材料复合电纺膜; [0018] b) collecting a uniform umbilical artery smooth muscle cells were seeded in gelatin / poly after overnight culture hexyl lactone electrically spinning membrane surface to form a cell composite electrospun membranes;

[0019] c)将细胞材料复合电纺膜缠绕在PVC管上培养,最终形成管状的组织工程血管。 [0019] c) Cells composite electrospun membranes wound on PVC tube culture, eventually forming a tubular vascular tissue engineering.

[0020] 优选的,步骤b)中脐动脉平滑肌细胞与明胶/聚己内酯电纺膜的接种比例是10 X 16个细胞/(2cmX 12cm)电纺膜;所述的细胞材料复合电纺膜缠绕在PVC管上的圈数为6-7圈,然后培养I周以形成供移植的组织工程血管,最好在该培养过程中隔2天半量换液一次。 [0020] Preferably, step b) and umbilical artery smooth muscle cells gelatin / polycaprolactone ratio seeded electrospun film is 10 X 16 cells / (2cmX 12cm) of electrospun membranes; Cell said composite electrospun the number of turns wound on the film 6-7 ring PVC pipe, and then cultured to form a peripheral I for transplantation tissue engineered blood vessel, preferably during the culture medium was changed every 2 days and a half time.

[0021] 为实现上述第三个目的,本发明采取的技术方案是:如上所述的组织工程血管在制备人工血管中的用途。 [0021] To achieve the above third object, the present invention takes the following technical solution: tissue engineered blood vessel as described above in the manufacture of an artificial blood vessel.

[0022] 本发明优点在于: [0022] The advantages of the present invention comprising:

[0023] 1、本发明采用“蛋卷”法,将接种了平滑肌细胞的明胶/聚己内酯电纺膜缠绕于PVC管上培养获得可供移植的组织工程血管,扭转了组织工程血管构建中的惯性思维,相比于现有技术中先制备管状支架再接种细胞的做法,可保证制备得到的组织工程血管细胞分布更加均匀,另外,经过一系列测定,表明该方法制备的组织工程血管弹性好、硬度适中、内壁光滑,组织结构致密,胶原纤维丰富,与自身组织分界清晰,即具备组织工程血管的各种优良特性,其特征更加近似于活体血管; [0023] 1, the present invention is a "cone" method, the smooth muscle cells seeded gelatin / PCL electrospun film is wound on the tube culture obtained PVC available for transplant tissue engineered blood vessel, a tissue engineered blood vessel torsion the inertia of thinking, as compared to prior art practices is first prepared the tubular stent reseeded cells can be prepared to ensure the tissue engineering of vascular cells distributed more evenly, additionally, through a series of measurements, indicating that a blood vessel tissue engineering produced by this method good elasticity, moderate hardness, smooth wall, the organizational structure of dense, rich in collagen fibers, with a clear demarcation own tissues, i.e., have the excellent characteristics of the tissue engineered blood vessel, wherein the blood vessel is more similar to the living body;

[0024] 2、利用本发明的方法构建组织工程血管,可先统一制作细胞材料复合电纺膜,然后选取不同管径的PVC管缠绕,即可获得不同直径且细胞分布均匀的组织工程血管,满足个性化的需求,尤其对于构建直径较小的组织工程血管尤为适用; [0024] 2. Construction of tissue engineered blood vessel using the method of the present invention, cells can be made uniform first composite electrospun membranes, PVC and select different diameters of winding tubes, to obtain a uniform distribution of the different diameters of vascular tissue and cell engineering, to meet individual needs, especially for the construction of small diameter vascular tissue engineering is particularly applicable;

[0025] 3、本发明的方法提供了合适的细胞与明胶/聚己内酯电纺膜的接种比例、细胞材料复合电纺膜缠绕在PVC管上的圈数、缠绕在PVC管上的细胞材料复合电纺膜培养成可供移植的组织工程血管的时间,在该条件下,可保证获得的组织工程血管具备优良特性。 [0025] 3. The method of the present invention provides a suitable cell gelatin / poly-caprolactone inoculation ratio of electrospun membranes, the cell number of turns composite electrospun membranes wound on PVC pipe, PVC pipe is wound on a cell composite electrospun membrane into the culture time for tissue engineered vascular graft, in this condition, the device can be made with excellent properties of vascular tissue engineering.

附图说明 BRIEF DESCRIPTION

[0026] 附图1是“蛋卷”法构建组织工程血管示意图。 [0026] Figure 1 is a schematic view of vascular tissue engineered constructs "cone" method.

[0027] 附图2是“蛋卷”法构建组织工程血管实际操作图。 [0027] Figure 2 is a "cone" Method Construction of vascular tissue engineering practice FIG. A:明胶/聚己内酯电纺膜;B:明胶/聚己内酯电纺膜接种平滑肌细胞后;C:接种好平滑肌细胞的明胶/聚己内酯电纺膜沿PVC导尿管卷曲成管;D:形成组织工程血管。 A: gelatin / PCL electrospun membranes; B: gelatin / polycaprolactone after inoculation electrospun membranes of smooth muscle cells; C: Good Gelatin smooth muscle cells were seeded / polycaprolactone electrospun membranes curl in PVC catheter into the tube; D: forming tissue engineered blood vessels.

[0028] 附图3是明胶/聚己内酯材料鉴定结果。 [0028] Figure 3 is a gelatin / caprolactone polycaprolactone material identification results. A:明胶/聚己内酯电纺膜大体观;B:人脐动脉平滑肌细胞;C:单纯明胶/聚己内酯扫描电镜;D:接种平滑肌细胞后的明胶/聚己内醋扫描电镜。 A: gelatin / PCL electrospun membranes gross appearance; B: human umbilical artery smooth muscle cells; C: pure gelatin / PCL SEM; D: gelatin after inoculation smooth muscle cells / poly caprolactone SEM.

[0029] 附图4是组织工程血管体内成熟与鉴定结果。 [0029] Figure 4 is a result of maturation in vivo and identification of vascular tissue engineering. A:组织工程血管裸鼠体内8周的大体观(带PVC导尿管);B、C:组织工程血管裸鼠体内8周的大体观(取掉PVC导尿管);D:HE染色整体观;E:HE染色;F:Masson染色。 A: blood vessel tissue engineering in nude mice 8 weeks gross appearance (with PVC catheter); B, C: vascular tissue engineering in nude mice 8 weeks gross appearance (PVC catheter profusely); D: HE staining whole view; E: HE staining; F: Masson staining.

具体实施方式 Detailed ways

[0030] 下面结合附图对本发明提供的具体实施方式作详细说明。 [0030] DETAILED DESCRIPTION OF THE DRAWINGS Embodiment of the present invention provides detailed description.

[0031] 实施例1本发明组织工程血管的制备(一) [0031] Example 1 Preparation invention tissue engineered blood vessel (a)

[0032] I材料和方法 [0032] I Materials and Methods

[0033] 1.1明胶/聚己内酯电纺膜的制备 Preparation of [0033] 1.1 gelatin / PCL film electrospun

[0034] 用电子分析天平称取0.5g明胶和0.5g聚己内醋溶于1ml的三氟乙醇中,搅拌24h至完全溶解,得到浓度为10%(克/毫升)的明胶/聚己内酯纺丝液,向溶液中加入10μ1乙酸,搅拌10-20min至溶液由浑浊变澄清。 [0034] trifluoroethanol, 0.5g of gelatin and 0.5g polycaprolactone vinegar, said electronic balance was dissolved in 1ml, 24h stirring until completely dissolved, to give a concentration of 10% (g / ml) in gelatin / polycaprolactone ester spinning solution, acetic acid was added to the solution 10μ1, a cloudy solution was stirred for 10-20min to become clear. 选用1ml的注射器,1.2mm内径的针头,抽取明胶/聚己内酯纺丝液,固定在静电纺丝装置上,调整静电压为10kv,接受距离为12cm,注射速率为2ml/h,进行电纺,采用铝箔为接受装置,纺丝3h,得到明胶/聚己内酯复合纳米纤维膜。 Selection 1ml syringe, needle inner diameter 1.2mm, extracting gelatin / PCL dope, is fixed to the electrospinning apparatus, adjust the static voltage 10kV, accepted distance is 12cm, the injection rate was 2ml / h, electrically spinning, means to accept an aluminum foil, spinning 3h, to give gelatin / PCL nanofiber composite membrane. 构建血管时,剪裁成宽2厘米、长12厘米的长方形条块,对其进行真空冷冻干燥消毒处理后备用。 When building vessels, cut into 2 cm wide, 12 cm long rectangular bar, subjected to vacuum freeze-drying process disinfection standby.

[0035] 1.2人血管平滑肌细胞培养 [0035] 1.2 human vascular smooth muscle cells

[0036] 取人脐动脉,于超净工作台上清除结缔组织,剥除动脉外膜,纵向剖开血管,钝性刮除内膜面,去除内皮细胞,剩余血管中膜组织。 [0036] Take human umbilical artery, clean table to remove connective tissue, stripping the adventitia, opened longitudinally vessel, blunt scraping intima, endothelial cells is removed, the remaining vascular membrane tissue. 含青、链霉素的生理盐水反复冲洗后,用眼科弯剪反复剪切成Imm X Imm大小的组织块,并将剪切好的组织小块均匀摆置于瓶底,组织块间距0.5cm。 Containing cyan, streptomycin saline repeatedly washed with ophthalmic scissors to cut tissue pieces is repeatedly bent Imm X Imm size, and good cutting tissue placed uniformly disposed small bottom, tissue blocks spacing 0.5cm . 盖好瓶盖,轻轻翻转培养瓶,让瓶底朝上并向瓶内注入适量低糖DMEM(Hyclone,美国)含10%胎牛血清(FBS) (Hyclone,美国)的培养液,置于含5% CO2的37°C孵箱内放置2-4h使组织块干涸并与瓶壁贴附后,将培养瓶慢慢翻转平放,使组织块完全浸入培养液中,继续静置培养3-5d。 Cover caps, flasks by gentle inversion, so that the bottle bottom and up into the amount of sugar DMEM (Hyclone, USA) containing 10% fetal bovine serum (FBS) (Hyclone, USA) broth, placed in containing of 5% CO2 37 ° C incubator for 2-4h placed dry tissue mass and the rear wall of the bottle is attached, the flask was slowly inverted flat, fully immersed in the tissue culture medium block, static culture continue 3- 5d. 待有细胞从组织块周围游出后换液,待90%融合后传代。 After medium change cells have to be run from the tissue mass, to be 90% after passage fusion.

[0037] 1.3扫描电镜观察 [0037] SEM 1.3

[0038] 消化收集平滑肌细胞,并种植在一片明胶/聚己内酯电纺膜上,体外培养Id后取出复合物,经2.5%戊二醛固定,然后乙醇梯度脱水干燥,离子喷射仪喷金,进行扫描电镜观察并拍照,以单纯明胶/聚己内酯电纺膜材料组作为对照组。 [0038] smooth muscle cells were harvested, planted and removing the composite in a gelatin / polycaprolactone after electrospinning film, Id vitro, by 2.5% glutaraldehyde, dehydrated in graded ethanol and then dried, ion spray spraying device , scanning electron microscope and photographed, to pure gelatin / PCL film material is electrospun as a control group.

[0039] 1.4 “蛋卷”法构建组织工程血管 Construction of tissue engineered blood vessels [0039] 1.4 "egg roll" method

[0040] 将消毒好的宽2厘米、长12厘米的长方形明胶/聚己内酯电纺膜平铺于培养皿上备用,用0.25%胰蛋白酶消化收集培养的平滑肌细胞,将其重新悬浮于10% FBS的低糖DMEM培养液中,细胞浓度为10X106/ml,接种Iml细胞悬液于明胶/聚己内酯电纺膜上,使细胞悬液铺满整个电纺膜,放置于含5% 0)2的37°C培养箱培养,每隔30min后添加少许培养液于材料表面,直至2h后添加1ml培养液到培养皿后过夜。 [0040] A 2 cm wide good disinfection, length 12 cm rectangular gelatin / PCL electrospun film is plated on the spare dish, cultured smooth muscle cells were harvested with 0.25% trypsin, resuspended in low glucose DMEM medium containing 10% FBS, the cells at a concentration of 10X106 / ml, the cell suspension was seeded Iml gelatin / PCL electrospun film, covered the entire cell suspension electrospun membranes, placed in 5% 37 ° C incubator 0) 2, a little after adding broth every 30min on the surface of the material, until the broth is added after 2h 1ml petri dish overnight. 第二天将细胞材料复合电纺膜沿直径为6_的PVC导尿管“蛋卷”法卷曲成管状(圈数约为:12cm/ (6 X 3.14) =6.4圈),并静置半个小时后放入50ml离心管中,加低糖DMEM,10% FBS的培养液覆盖复合物,置于含5%0)2的37°0培养箱培养。 The next day the cells were electrospun composite film in a tubular crimp diameter (about the number of turns: 12cm / (6 X 3.14) = 6.4 turns) of PVC catheter 6_ of "cone" method, and the left half after hours into 50ml centrifuge tube, add sugar DMEM, 10% FBS culture medium covering the composite, is placed 37 ° 0 incubator containing 5% 0) 2. 隔2天半量换液,培养I周后裸鼠皮下移植,8周后取材。 Medium was changed every two and a half days, weeks cultured xenografted I, drawn after 8 weeks. 上述组织工程血管的构建过程可参见图1和图2。 Vascular tissue engineering construct the above process can be found in Figures 1 and 2.

[0041] 1.5构建的组织工程血管的鉴定 Construction of tissue engineered blood vessel identification [0041] 1.5

[0042] 1.5.1组织学检测 [0042] 1.5.1 histology

[0043] HE染色:将构建的组织工程血管切片脱蜡水化后苏木素染色5min,冲洗后盐酸酒精分化数秒,自来水洗返蓝30min,伊红染色Imin后乙醇脱水,二甲苯透明后中性树脂封片。 [0043] HE staining: after the tissue engineered blood vessel sections were deparaffinized hydration hematoxylin 5min, rinsed several seconds after differentiation hydrochloric acid alcohol, water wash back blue 30min, after eosin staining Imin ethanol dehydration, xylene neutral resin mounted.

[0044] Masson染色:将构建的组织工程血管切片脱錯水化后Weiger氏铁苏木素5_10min水洗后1%盐酸酒精分化后再次水洗,丽春红酸性品红液染5-10min,水洗后1%磷钼酸水溶液处理5min,绿液复染5min,1%冰醋酸处理lmin,95%酒精脱水多次,无水酒精脱水,二甲苯透明,中性树胶封片。 [0044] Masson staining: tissue engineered blood vessel sections were de-hydration washing again after error Weiger 1% hydrochloric acid alcohol's iron hematoxylin 5_10min washed after differentiation, stained with Ponceau 5-10min acid fuchsin solution, washed with water, 1% aqueous solution of phosphomolybdic acid 5min, stained green liquor 5min, 1% glacial acetic acid lmin, 95% dehydrated alcohol several times, ethanol dehydration, xylene, mounted with neutral gum.

[0045] 1.5.2生物力学检测 [0045] 1.5.2 biomechanical test

[0046] 将构建的组织工程血管剪成3_宽的环状后剪断,用千分尺测量其厚度后通过万能拉力检测仪(Instron-3343)检测其力学性能,计算弹性模量。 After [0046] The cut tissue engineered blood vessel 3_ wide annular cut which detects the thickness of the mechanical properties by Tensilon tester (Instron-3343), calculated modulus of elasticity measured with a micrometer. 取内径同样为6mm的小猪主动脉作为对照组。 Taking an inner diameter of 6mm likewise pig aorta as the control group.

[0047] 2 结果 [0047] 2 Results

[0048] 2.1明胶/聚己内酯电纺膜的鉴定 [0048] 2.1 Identification of gelatin / PCL film electrospun

[0049] 静电纺方法制备出来的明胶/聚己内酯电纺膜材料具有一定的力学强度(断裂强度2.74±0.20MPa,弹性模量33.43±3.07 Mpa,断裂时的应变1.77±0.15mm/mm),可操作性比较强(图3A)。 Gelatin prepared out of [0049] the electrostatic spinning process / polycaprolactone films electrospun material having a certain mechanical strength (breaking strength of 2.74 ± 0.20MPa, the elastic modulus of 33.43 ± 3.07 Mpa, strain at break 1.77 ± 0.15mm / mm ), strong operability (FIG. 3A). 单纯明胶/聚己内酯电纺膜材料的扫描电镜结果显示,纳米纤维均匀、光滑、连续,较好的仿生了天然细胞外基质胶原的纤维细度(图3C)。 Pure gelatin / PCL electrically SEM results show the film material spun nanofibers uniform, smooth, continuous, preferred fiber fineness biomimetic natural extracellular collagen matrix (FIG. 3C). 与人平滑肌细胞复合后体外培养1山扫描电镜可见平滑肌细胞粘附于该材料表面,细胞铺展良好(图3D)。 In vitro cultured human smooth muscle cells with a complex scanning electron microscopy showed Hill smooth muscle cells adhered to the surface of the material, good cell spreading (Figure 3D).

[0050] 2.2 “蛋卷”法体外构建组织工程血管 [0050] 2.2 "cone" in vitro tissue engineered blood vessels in Establishing

[0051] “蛋卷”法体外构建组织工程血管可参见图1和图2。 [0051] "cone" in vitro tissue engineered blood vessels may be constructed Method Referring to Figures 1 and 2. 先将消毒好的明胶/聚己内酯材料平铺于大的培养皿中(图2A),收集人脐动脉平滑肌细胞均匀的接种于明胶/聚己内酯电纺膜表面后过夜(图2B),第二天将接种了细胞的明胶/聚己内酯电纺膜缠绕在PVC导尿管上,最终形成管状的组织工程血管(图2C,2D),体外培养I周后然后移植到裸鼠皮下,使其进一步成熟。 After sterilization first the gelatin / PCL material plated on a large dish (FIG. 2A), collected uniformly inoculated human umbilical artery smooth muscle cells in gelatin / poly-caprolactone electrically spun overnight film surface (FIG. 2B ), the next day, cells were seeded gelatin / PCL electrospun PVC film wrapped around the catheter, forming a tubular tissue engineered blood vessels (FIG. 2C, 2D), cultured in vitro and then transplanted into nude peripheral I mouse skin, making it more mature.

[0052] 2.3构建的组织工程血管的检测 [0052] 2.3 Construction of a tissue engineered blood vessel detection

[0053] 裸鼠皮下移植8周后取材,大体看:构建的组织工程血管与裸鼠自身组织分界清晰,弹性好,硬度适中,内壁光滑,平放可独立支撑成管状(图4A,4B, 4C)。 [0053] 8 weeks after subcutaneous transplantation drawn, see generally: tissue engineered blood vessel and the tissue itself nude clear demarcation, good elasticity, moderate hardness, smooth wall, can be independently flat tubular support (FIG. 4A, 4B, 4C). HE切片显示组织结构致密,细胞分布均匀(图4D, 4E),Masson染色显示构建的组织工程血管中胶原纤维丰富(图4F)。 HE sections showed dense structure, a uniform distribution of cells (FIG. 4D, 4E), Masson staining tissue engineered vascular rich collagen fibers (FIG. 4F).

[0054] 实施例2本发明组织工程血管的制备(二) Vascular tissue engineering invention prepared in Example 2 (b) [0054] Embodiment

[0055] 制备方法同实施例1,不同之处仅在于:制备的长方形明胶/聚己内酯电纺膜宽2厘米、长13厘米;步骤1.4中,细胞材料复合电纺膜沿直径为6mm的PVC导尿管“蛋卷”法卷曲成管状,则卷曲的圈数为:13cm/(6X3.14)=6.9圈。 [0055] prepared in Example 1, except that only: Preparation of Gelatin rectangular / polycaprolactone electrospun membranes 2 cm wide, 13 cm long; in step 1.4, the cell membrane composite electrospun 6mm in diameter PVC catheter curly "omelet" tubular method, the number of turns of the curl: 13cm / (6X3.14) = 6.9 turns. 结果:制备的组织工程血管和实施例I 一样,与裸鼠自身组织分界清晰,弹性好,硬度适中,内壁光滑,平放可独立支撑成管状;HE切片显示组织结构致密,细胞分布均匀,Masson染色显示构建的组织工程血管中胶原纤维丰富。 Results: The tissue engineered blood vessels prepared as in Example I, with a clear demarcation nude self-organization, good elasticity, moderate hardness, smooth wall, can be independently supported to lie flat tube; HE sections showed dense tissue structures, uniform cell distribution, Masson staining tissue engineered blood vessels collagen fiber rich.

[0056] 对比例I [0056] Comparative Example I

[0057] 制备方法同实施例1,不同之处仅在于:制备的长方形明胶/聚己内酯电纺膜宽2厘米、长11厘米;步骤1.4中,细胞材料复合电纺膜沿直径为6mm的PVC导尿管“蛋卷”法卷曲成管状,则卷曲的圈数为:11cm/(6X3.14)=5.8圈。 [0057] prepared in Example 1, except that only: Preparation of Gelatin rectangular / polycaprolactone electrospun membranes 2 cm wide, 11 cm long; in step 1.4, the cell membrane composite electrospun 6mm in diameter PVC catheter curly "omelet" tubular method, the number of turns of the curl: 11cm / (6X3.14) = 5.8 turns. 结果:制备的组织工程血管力学性能不足。 Results: insufficient mechanical properties of vascular tissue engineering prepared.

[0058] 对比例2 [0058] Comparative Example 2

[0059] 制备方法同实施例1,不同之处仅在于:制备的长方形明胶/聚己内酯电纺膜宽2厘米、长14厘米;步骤1.4中,细胞材料复合电纺膜沿直径为6mm的PVC导尿管“蛋卷”法卷曲成管状,则卷曲的圈数为:14cm/(6X3.14)=7.4圈。 [0059] prepared in Example 1, except that only: Preparation of Gelatin rectangular / polycaprolactone electrospun membranes 2 cm wide, 14 cm long; in step 1.4, the cell membrane composite electrospun 6mm in diameter the number of turns of coiled PVC catheter "omelet" tubular method, the curling of: 14cm / (6X3.14) = 7.4 turns. 结果:制备的组织工程血管内部的细胞存在死亡现象。 Result: there is the phenomenon of cell death inside the tissue engineered blood vessels prepared.

[0060] 对比例3 [0060] Comparative Example 3

[0061] 制备方法同实施例1,不同之处仅在于:细胞浓度为8 X 107ml。 Preparation of [0061] the same way as Example 1, except that only: a cell concentration of 8 X 107ml. 结果:制备的组织工程血管未能形成良好的组织。 Results: The tissue engineered blood vessels prepared failed to form a good organization.

[0062] 对比例4 [0062] Comparative Example 4

[0063] 制备方法同实施例1,不同之处仅在于:组织工程血管体外培养的时间为2天半。 Preparation of [0063] the same way as Example 1, except that only: Time vascular tissue engineering in vitro culture for 2 ½ days. 结果:制备的组织工程血管强度不足,易松散。 Results: insufficient strength tissue engineered blood vessels prepared, easy to loose.

[0064] 对比例5 [0064] Comparative Example 5

[0065] 制备方法同实施例1,不同之处仅在于:组织工程血管体外培养的时间为7天半。 Preparation of [0065] the same way as Example 1, except that only: Time vascular tissue engineering in vitro culture for 7 and a half days. 结果:制备的组织工程血管内部的细胞存在死亡现象。 Result: there is the phenomenon of cell death inside the tissue engineered blood vessels prepared.

[0066] 以上所述仅是本发明的优选实施方式,应当指出,对于本技术领域的普通技术人员,在不脱离本发明方法的前提下,还可以做出若干改进和补充,这些改进和补充也应视为本发明的保护范围。 [0066] The above are only preferred embodiments of the present invention, it should be noted that for those of ordinary skill in the art, without departing from the method of the present invention, can make various improvements and additions, modifications and additions of these also it is considered the scope of the present invention.

Claims (3)

1.一种组织工程血管,以管状明胶/聚己内酯电纺膜为支架,其特征在于,平滑肌细胞均匀分布于所述的支架的内部;它是按照以下方法制备的: a)将消毒好的明胶/聚己内酯材料平铺于器皿中; b)收集脐动脉平滑肌细胞均匀的接种于明胶/聚己内酯电纺膜表面后培养过夜,形成细胞材料复合电纺膜; c)将细胞材料复合电纺膜缠绕在PVC管上培养,最终形成管状的组织工程血管, 步骤b)中脐动脉平滑肌细胞与明胶/聚己内酯电纺膜的接种比例是10 X 16个细胞/(2cm X 12cm)电纺膜;所述的细胞材料复合电纺膜缠绕在PVC管上的圈数为6_7圈,然后培养I周以形成可供移植的组织工程血管;所述的培养I周的过程中隔2天半量换液一次;所述的明胶/聚己内酯电纺膜是按照以下方法制备的:用电子分析天平称取0.5g明胶和0.5g聚己内酯溶于1ml的三氟乙醇中,搅拌24h至完全溶解,得到 A tissue engineered blood vessel, a tubular gelatin / PCL electrospun film holder, characterized in that the smooth muscle cells uniformly distributed inside the stent; which is prepared in the following manner: a) a disinfecting the gelatin / PCL plated material in the vessel; b) collecting a uniform umbilical artery smooth muscle cells were seeded in gelatin / poly after overnight culture hexyl lactone electrically spinning membrane surface to form a cell composite electrospun membranes; c) cells composite electrospun film is wound on PVC tube culture, eventually forming a tubular tissue engineered blood vessel, in step b) and umbilical artery smooth muscle cells gelatin / polycaprolactone ratio seeded electrospun film is 10 X 16 cells / culturing said peripheral I; (2cm X 12cm) of electrospun membranes; the number of turns electrospun composite membrane is wound on the cell PVC pipe 6_7 ring is then cultured to form a peripheral I for the transplanted tissue engineered blood vessel during half days every 2 medium was changed once; the gelatin / PCL electrospun membranes were prepared in the following ways: electronic analytical balance weighing 0.5g and 0.5g of gelatin polycaprolactone dissolved in 1ml of trifluoroethanol, stirred for 24h until complete dissolution, to give 浓度为10%的明胶/聚己内酯纺丝液,向溶液中加入10μ1乙酸,搅拌10-20min至溶液由浑浊变澄清,选用1ml的注射器,1.2mm内径的针头,抽取明胶/聚己内酯纺丝液,固定在静电纺丝装置上,调整静电压为10kv,接受距离为12cm,注射速率为2ml/h,进行电纺,采用铝箔为接受装置,纺丝3h,得到明胶/聚己内酯电纺膜。 Concentration of 10% gelatin / PCL spinning solution, acetic acid was added to the solution 10μ1, the turbid solution was stirred for 10-20min until became clear, the choice of 1ml syringe, needle inner diameter 1.2mm, gelatin extraction / polycaprolactone ester spinning solution, fixed to the electrospinning apparatus, adjust the static voltage 10kV, the distance 12cm is accepted, the injection rate was 2ml / h, electrospinning, an aluminum foil as a reception apparatus, the spinning 3h, to give gelatin / polyhexamethylene lactone electrospun membranes.
2.权利要求1所述的组织工程血管的制备方法,其特征在于,包括以下步骤: a)将消毒好的明胶/聚己内酯材料平铺于器皿中; b)收集脐动脉平滑肌细胞均匀的接种于明胶/聚己内酯电纺膜表面后培养过夜,形成细胞材料复合电纺膜; c)将细胞材料复合电纺膜缠绕在PVC管上培养,最终形成管状的组织工程血管, 步骤b)中脐动脉平滑肌细胞与明胶/聚己内酯电纺膜的接种比例是10 X 16个细胞/(2cm X 12cm)电纺膜;所述的细胞材料复合电纺膜缠绕在PVC管上的圈数为6_7圈,然后培养I周以形成供移植的组织工程血管;所述的培养I周的过程中隔2天半量换液一次。 The method of preparing the tissue engineered blood vessel 1 according to claim 1, characterized in that it comprises the steps of: a) disinfection of the gelatin / PCL plated material in the vessel; b) collecting a uniform umbilical artery smooth muscle cells seeded in gelatin / film surface after spinning polyhexamethylene lactone electrically cultured overnight, the cells formed composite electrospun membranes; c) cells composite electrospun film is wound on PVC tube culture, eventually forming a tubular tissue engineered blood vessel, a step b) in the umbilical artery smooth muscle cells and the gelatin / polycaprolactone ratio seeded electrospun film is 10 X 16 cells / (2cm X 12cm) electrospun membranes; the cells composite electrospun membranes wound on PVC pipe 6_7 number of turns of coil, I then cultured to form a tissue engineered blood vessel circumference for transplantation; I periphery of the culture process in every two and a half days medium was changed once.
3.权利要求1所述的组织工程血管在制备人工血管中的用途。 Tissue engineered blood vessel of claim 1 for the preparation of the artificial blood vessel of claim 1.
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