WO2018192152A1 - 一种检测血管支架材料体外内皮化的模型制备及应用 - Google Patents

一种检测血管支架材料体外内皮化的模型制备及应用 Download PDF

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WO2018192152A1
WO2018192152A1 PCT/CN2017/099397 CN2017099397W WO2018192152A1 WO 2018192152 A1 WO2018192152 A1 WO 2018192152A1 CN 2017099397 W CN2017099397 W CN 2017099397W WO 2018192152 A1 WO2018192152 A1 WO 2018192152A1
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implanted
model
collagen
endothelialization
cells
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French (fr)
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韩倩倩
莫志超
刘青
王春仁
杨昭鹏
孙雪
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中国食品药品检定研究院
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    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/5005—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells
    • G01N33/5008—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells for testing or evaluating the effect of chemical or biological compounds, e.g. drugs, cosmetics
    • G01N33/5044—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells for testing or evaluating the effect of chemical or biological compounds, e.g. drugs, cosmetics involving specific cell types
    • G01N33/5064—Endothelial cells
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/5005—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells
    • G01N33/5008—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells for testing or evaluating the effect of chemical or biological compounds, e.g. drugs, cosmetics
    • G01N33/5044—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells for testing or evaluating the effect of chemical or biological compounds, e.g. drugs, cosmetics involving specific cell types

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  • the invention relates to the field of biotechnology, in particular to a preparation method and application for detecting an endothelialization model of a blood vessel stent.
  • Vascular stents are implantable medical devices for the treatment of vascular diseases, coronary arteries, and peripheral vascular embolization.
  • Vascular stent products have undergone several generations of product technology development, from metal-based non-degradable vascular stents to polymer-based degradable vascular stents.
  • polymer-based degradable vascular stents are expected to solve a series of possible complications caused by permanent metal stents commonly used in clinical practice, such as advanced intimal hyperplasia, and in-stent Stenosis and thrombosis; the absorbability of the product prevents the patient from continuing to worry about the permanent retention of foreign bodies in the body, and also prevents the patient from taking anticoagulants for life.
  • Polylactic acid is one of the most synthetic materials used in the research and application of biomaterials. Its good biocompatibility and degradability make it suitable for use in the field of degradable vascular stents.
  • the invention provides a model comprising a porous plate, a collagen gel and a material to be implanted;
  • the collagen gel is placed in a hole of the porous plate, and the material to be implanted is placed on the collagen gel in a manner of being immersed and not immersed;
  • the material to be implanted is in the form of a grid.
  • the diameter of the grid-like material to be implanted is smaller than the diameter of the porous plate
  • the holes in the perforated plate are non-through holes (having a lower bottom surface).
  • the porous plate is a 48-well plate having a pore diameter of 1.2 cm;
  • the grid-like material to be implanted is in the form of a disk having a diameter of 1 cm.
  • Each grid is a rectangle 1 mm wide and 2 mm long.
  • the collagen gel is formed by reacting collagen in a system containing NaOH and acetic acid to form a collagen gel
  • the collagen gel is prepared as follows: firstly, the collagen is dissolved in an aqueous solution of acetic acid to prepare a collagen-acetic acid solution, and then the collagen-acetic acid solution is reacted with NaOH to form a collagen gel;
  • 1N NaOH (1M NaOH) was obtained by dissolving 4 g of NaOH (analytically pure AR) solid in 100 mL of deionized water and sterilizing by filtration.
  • reaction is carried out in the pores of the perforated plate.
  • reaction is carried out in the pores of the perforated plate.
  • the concentration of the collagen in the reaction system was 2 ug/ml.
  • the collagen is selected from commercially available collagens such as rat tail collagen, bovine tendon collagen, pig skin collagen or fish collagen.
  • the material to be implanted is PLLA.
  • the above model also includes cells; the cells are specifically endothelial cells and smooth muscle cells.
  • Another object of the present invention is to provide an in vitro endothelialization test kit for an implanted material.
  • the kit of the present invention comprises the porous plate, the collagen gel, the material to be implanted, and the cells in the above model.
  • a third object of the present invention is to provide a method for preparing an in vitro endothelialization detection model of a material to be implanted.
  • the method provided by the present invention comprises the steps of: preparing the collagen gel in a multi-well plate, and then placing the mesh-like material to be implanted into the pores of the porous plate in a manner of being immersed and not immersed. On the gel, the cell model to be accessed is obtained; finally, the cells are seeded on the cell model to be accessed in the well of the multi-well plate to obtain an in vitro endothelialization detection model of the material to be implanted;
  • the cells are specifically endothelial cells and smooth muscle cells.
  • the above model or the application of the above product in evaluating or detecting whether or not the implanted material is endothelialized is also the scope of protection of the present invention.
  • the above model or the use of the above product in evaluating or detecting the endothelialization effect of the material to be implanted is also within the scope of the present invention.
  • the evaluation or detection is an in vitro evaluation or an in vitro detection.
  • a third object of the present invention is to provide a method of detecting or assisting in detecting whether endothelialization occurs outside the material to be implanted.
  • the method provided by the invention comprises the following steps:
  • the collagen gel is prepared in the pores in the porous plate described above, and then the mesh-like material to be implanted is placed on the collagen gel in the pores of the porous plate in a manner of being immersed and not immersed, and is to be connected.
  • Into the cell model finally inoculation of the cells on the cell model to be accessed in the well of the multiwell plate; detecting whether the cells are adherent on the material to be implanted and proliferating, if adherent and proliferating If the material occurs or the candidate is endothelialized, if it is not adherent and proliferates, the material does not occur or the candidate does not undergo endothelialization;
  • the cells were endothelial cells and smooth muscle cells, and the inoculation concentration was 1 ⁇ 10 5 / species.
  • Figure 1 is a schematic view of a grid-like stent.
  • Figure 2 is a schematic diagram of a model prepared by inserting a grid-like stent into a rat tail collagen gel.
  • Figure 3 is a photomicrograph of HUVEC and HCASMC cells cultured in an endothelialization model for 4 days, wherein A and B are HCASMC; C and D are HUVEC.
  • Figure 4 is an immunofluorescence photograph of two kinds of cells cultured four days after climbing, in which A, B, and C are HCASMC, D, E, F are HUVEC.
  • Figure 5 shows the results of the exploration of the amount of cell inoculation.
  • the rat tail collagen sold company is Santa Cruz Biotechnology, catalog number SC-136157.
  • Figure 2 shows an in vitro endothelialization detection model of the material to be implanted, comprising a multiwell plate, a rat tail collagen gel, and a grid of PLLA 2 placed in the well 1 of the multiwell plate, the PLLA The collagen gel was placed in a immersed and non-immersed manner.
  • Rat tail collagen-acetic acid solution 100 mg of rat tail collagen was dissolved in 30 mL of 0.02 N aqueous acetic acid to obtain a rat tail collagen-acetic acid solution having a concentration of 3.33 mg/ml.
  • Synthetic whole grid-like PLLA scaffold material (PLLA scaffold provided by Beijing Amet Medical Devices Co., Ltd.), as shown in Figure 1, and each mesh of the grid-like PLLA scaffold material is 1mm wide and 2mm long. Rectangular.
  • the cell model to be accessed is irradiated with ultraviolet light (the ultraviolet irradiation intensity is ultra-clean operation table ultraviolet light illumination intensity ⁇ 300LX) for 1 hour and then used.
  • the ultraviolet irradiation intensity is ultra-clean operation table ultraviolet light illumination intensity ⁇ 300LX
  • the cells were seeded on the cell model to be accessed to obtain an in vitro endothelialization detection model of the PLLA scaffold.
  • Example 2 Application of in vitro endothelialization detection model of PLLA scaffold in detecting the endothelialization of PLLA materials
  • Coronary endothelial cells (HCAEC purchased from Shanghai Tongpai Biotechnology Co., Ltd.) and coronary smooth muscle cells (HCASMC, purchased from Shanghai Tongpai Biotechnology Co., Ltd.) in the logarithmic growth phase were all in accordance with the concentration of 1 ⁇ 105
  • the surface of the cell model to be accessed prepared in Example 1 prepared by co-inoculation in each well of the cell culture plate (adsorbed cells by means of a rat tail gel as an attachment on the scaffold) was cultured at 37 ° C in a 5% CO 2 incubator. Change the liquid once every 2 days and incubate for four days.
  • IF immunofluorescence
  • fluorescent secondary antibody name is Anti-Mouse IgG (H+L), F(ab') 2 Fragment, purchased from Cell Signaling Technology, catalog number 4408s.), incubated for 1 h at room temperature;
  • HCAEC coronary endothelial cells
  • P2B1 Anti-CD31 antibody [P2B1] (this antibody was purchased from Abcam, the number is ab24590), and the immunofluorescence primary antibody of coronary smooth muscle cells (HCASMC) was Anti-alpha Actinin.
  • Antibody this antibody was purchased from Cell Signaling Technology, Cat. No. 3528s.).
  • the PLLA material is endothelialized and can be used for in vivo implantation.
  • the method was basically the same as that of Example 1, except that the concentration of rat tail collagen in the well reaction system of the cell culture plate covered with the rat tail collagen gel was 1 ug/mL and 4 ug/mL, respectively.
  • the concentration of the rat tail collagen in the well reaction system of the cell culture plate covered with the rat tail collagen gel was 1 ug/mL, the shaped gel could not be obtained.
  • the concentration of the rat tail collagen in the well reaction system of the cell culture plate coated with the rat tail collagen gel was 4 ug/mL, the gel was too hard to be embedded in the grid-like PLLA scaffold material.
  • Example 2 the endothelial cell and coronary smooth muscle cells of the PLLA scaffold prepared in Example 1 were inoculated in vitro, and the concentration of both cells was 1 ⁇ 10 4 or 5 ⁇ 10 4 .
  • the invention is to prepare a grid scaffold structure and establish a method for cocultivating a grid scaffold and a cell line.
  • the grid scaffold structure simulates the lattice structure of the vascular scaffold, and the endothelial cells and smooth muscle cells are co-cultured with the mesh structure in vitro, and the growth state and the adherence state of the cells on the mesh structure are observed. And coverage status to judge whether this material is suitable as a preparation material for a blood vessel stent.
  • This invention is to establish a test method for screening As a material for vascular stents, the growth state of endothelial cells and smooth muscle cells on materials is a measure.
  • the invention establishes a method for evaluating materials in vitro, which helps to reduce the amount of animals, meets the requirements of animal welfare, and reduces the research and development costs of enterprises.

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Abstract

检测血管支架材料体外内皮化的模型制备及应用。一种待植入材料体外内皮化检测模型，其包括多孔板、胶原凝胶和待植入材料；所述胶原凝胶置于所述多孔板的孔中，所述待植入材料以浸入且不浸没的方式置于所述胶原凝胶上；所述待植入材料为网格状。建立了一种试验方法，用来筛选可以作为血管支架的材料，内皮细胞和平滑肌细胞在材料上的生长状态是衡量指标。

Description

一种检测血管支架材料体外内皮化的模型制备及应用 技术领域
本发明涉及生物技术领域，尤其涉及一种检测血管支架体外内皮化模型的制备方法及应用。
背景技术
血管支架是用于治疗血管疾病、冠脉、外周血管栓塞的植入类医疗器械。血管支架产品经历了几代产品技术的发展，从以金属材料为基础的不可降解血管支架到以高分子材料为基础的可降解血管支架。作为未来新一代的血管支架，以高分子材料为基础的可降解血管支架有望解决目前临床上普遍使用的永久性金属支架所带来的一系列可能并发症，如晚期内膜增生，支架内再狭窄及血栓形成等；产品的可吸收性让患者不必持续担心有异物永久地留在体内，也使患者避免终生服用抗凝药。
聚乳酸(PLLA)是目前生物材料研究和应用最多的合成材料之一，良好的生物相容性以及可降解性能使其在可降解血管支架领域得以应用。
良好的内皮化是植入的血管支架材料完成降解的前提，因此对植入的血管支架材料内皮化做出评价具有很大研究意义。但是在临床前安全性评价的体外试验中，没有针对血管支架材料内皮化效果的评价方法，导致只能通过大动物试验，即将材料通过介入手术植入大动物的血管以观察内皮化效果。这种操作费时费力且价格昂贵。现在缺乏在体外对血管支架材料的内皮化效果进行评判的模型装置和评价方法。
发明公开
本发明一个目的是提供一种待植入材料体外内皮化检测模型。
本发明提供的模型，其包括多孔板、胶原凝胶和待植入材料；
所述胶原凝胶置于所述多孔板的孔中，所述待植入材料以浸入且不浸没的方式置于所述胶原凝胶上；
所述待植入材料为网格状。
上述模型中，所述网格状待植入材料的直径小于所述多孔板的孔径；
或所述多孔板中的孔为非通孔(具有下底面)。
上述模型中，所述多孔板为48孔板，孔径为1.2cm；
或所述网格状待植入材料为圆片状，直径为1cm。
每个网格为宽1mm，长2mm的长方形。
上述模型中，所述胶原凝胶为将胶原在含有NaOH和乙酸的体系中进行反应形成胶原凝胶；
或所述胶原凝胶按照如下方法制备：先将胶原溶于乙酸水溶液制备胶原-乙酸溶液，再将所述胶原-乙酸溶液与NaOH反应，形成胶原凝胶；
具体为：
1)将100mg鼠尾胶原溶于30mL0.02N乙酸水溶液，得到浓度为3.33mg/ml鼠尾胶原-乙酸溶液；
2)取70ul1NNaOH于50ml洁净烧杯中，再加入500ulPBS缓冲液(品牌Hyclone，pH值为7.3)、1.5mlddH2O和3ml鼠尾胶原-乙酸溶液(最后加入)，共记5ml，迅速混匀，加入到48孔板(孔直径为1.2cm)中，每孔500ul，鼠尾胶原在每孔反应体系中的浓度为2ug/ml，37℃温箱放置30min凝固，得到铺有鼠尾胶原凝胶的细胞培养板。
1NNaOH(1M NaOH)为4g NaOH(分析纯AR)固体溶于100mL去离子水，过滤灭菌后得到。
上述中NaOH溶液(1N)：PBS缓冲液：ddH2O：鼠尾胶原-乙酸溶液的体积比为＝7:50:150:300。
或所述反应在所述多孔板的孔中进行。
或所述反应在所述多孔板的孔中进行。
上述模型中，所述胶原在所述反应的体系中的浓度为2ug/ml。
上述模型中，所述反应为37℃放置30-60min。
上述模型中，所述胶原选常见市售胶原，如鼠尾胶原、牛肌腱胶原蛋白、猪皮胶原或鱼胶原等。
上述模型中，所述待植入材料为PLLA。
上述模型还包括细胞；所述细胞具体为内皮细胞和平滑肌细胞。
本发明另一个目的是提供一种待植入材料体外内皮化检测套装产品。
本发明提供的套装产品，包括上述模型中的所述多孔板、所述胶原凝胶、所述待植入材料和所述细胞。
各自单独包装，但是使用时，按照如下方法配置。
本发明第三个目的是提供一种制备待植入材料体外内皮化检测模型的方法。
本发明提供的方法，包括如下步骤：先在多孔板中制备所述胶原凝胶，再将所述网格状待植入材料以浸入且不浸没的方式置于所述多孔板孔中的胶原凝胶上，得到待接入细胞模型；最后将细胞接种在所述多孔板孔中的所述待接入细胞模型上，得到待植入材料体外内皮化检测模型；
所述细胞具体为内皮细胞和平滑肌细胞。
上述的模型或上述产品在制备检测待植入材料体外是否发生内皮化的产品中的应用也是本发明保护的范围。
或，上述的模型或上述产品在评价或检测待植入材料是否发生内皮化中的应用也是本发明保护的范围；
或，上述的模型或上述产品在评价或检测待植入材料内皮化效果中的应用也是本发明保护的范围。
上述中，所述评价或检测为体外评价或体外检测。
本发明第三个目的是提供一种检测或辅助检测待植入材料体外是否发生内皮化的方法。
本发明提供的方法，包括如下步骤：
先在上述的多孔板中的孔中制备所述胶原凝胶，再上述网格状待植入材料以浸入且不浸没的方式置于所述多孔板孔中的胶原凝胶上，得到待接入细胞模型；最后将细胞接种在所述多孔板孔中的所述待接入细胞模型上；检测所述细胞是否在所述待植入材料上贴壁且增殖生长，若贴壁且增殖生长，则该材料发生或候选发生内皮化，若不贴壁且增殖生长，则该材料不发生或候选不发生内皮化；
所述细胞为内皮细胞和平滑肌细胞，接种浓度均为1×105个/种。
附图说明
图1为网格状支架示意图。
图2为网格状支架嵌入鼠尾胶原凝胶制备得到的模型示意图。
图3为HUVEC及HCASMC两种细胞于内皮化模型中培养4天后的显微照片，其中A、B为HCASMC；C、D为HUVEC。
图4为两种细胞培养四天后爬片的免疫荧光照片，其中A、B、C图为 HCASMC，D、E、F为HUVEC。
图5为细胞接种量的摸索的结果。
实施发明的最佳方式
下述实施例中所使用的实验方法如无特殊说明，均为常规方法。
下述实施例中所用的材料、试剂等，如无特殊说明，均可从商业途径得到。
所用鼠尾胶原出售公司为Santa Cruz Biotechnology，产品目录号为SC-136157。
实施例1、PLLA支架体外内皮化检测模型的制备
图2所示待植入材料体外内皮化检测模型，其包括多孔板、鼠尾胶原凝胶和网格状PLLA 2，所述胶原凝胶置于所述多孔板的孔1中，所述PLLA以浸入且不浸没的方式置于所述胶原凝胶上。
具体各组成及模型制备如下：
1、鼠尾胶原凝胶的制备
所有溶液均要4℃预冷。
鼠尾胶原-乙酸溶液：将100mg鼠尾胶原溶于30mL0.02N乙酸水溶液，得到浓度为3.33mg/ml鼠尾胶原-乙酸溶液。
1NNaOH(1M NaOH):4g NaOH(分析纯AR)固体溶于100mL去离子水，过滤灭菌后得到。
取70ul1NNaOH于50ml洁净烧杯中，再加入500ulPBS缓冲液(品牌Hyclone，pH值为7.3)、1.5mlddH2O和3ml鼠尾胶原-乙酸溶液(最后加入)，共记5ml，迅速混匀，加入到48孔板(孔直径为1.2cm)中，每孔500ul，鼠尾胶原在每孔反应体系中的浓度为2ug/ml，37℃温箱放置30min凝固，得到铺有鼠尾胶原凝胶的细胞培养板。
上述中NaOH溶液(1N)：PBS缓冲液：ddH2O：鼠尾胶原-乙酸溶液的体积比为＝7:50:150:300。
2、PLLA支架体外内皮化检测模型的制备
合成整片网格状PLLA支架材料(PLLA支架由北京阿迈特医疗器械有限公司提供)，如图1所示，且整片网格状PLLA支架材料的每个网格为宽1mm，长2mm的长方形。
将整片网格状PLLA支架材料剪裁为各个1cm直径圆形(该直径小于细胞培养板的孔直径即可)，然后将每个圆形用尖头镊子水平夹持放入铺有鼠尾胶原凝胶的细胞培养板孔中，并将其嵌入以浸入且不浸没的方式置于鼠尾胶原凝胶上，得到待接入细胞模型(如图2所示)；
将待接入细胞模型紫外照射(紫外照射强度为超净操作台紫外灯光照强度≥300LX)灭菌1h后备用。
将细胞接种在待接入细胞模型上，得到PLLA支架体外内皮化检测模型。
实施例2、PLLA支架体外内皮化检测模型在检测PLLA材料是否发生内皮化中的应用
1、利用细胞检测PLLA材料是否发生内皮化
取处于对数生长期的冠脉内皮细胞(HCAEC购自上海通派生物科技有限公司，)和冠脉平滑肌细胞(HCASMC，购自上海通派生物科技有限公司)均按照1×105个的浓度共同接种于细胞培养板孔各孔中的实施例1制备的待接入细胞模型表面(借助鼠尾凝胶作为支架上的附着物吸附细胞)，37℃，5％CO2温箱中培养，每2天换液一次，培养四天。
连续培养4天后进行荧光染色，采用免疫荧光(IF)检测细胞生长状态，具体如下：
1)培养第4天后(图3所示)，通过显微观察可见两种细胞在PLLA支架体外内皮化检测模型上均能正常生长。将已经爬好细胞的PLLA支架体外内皮化检测模型用冷PBS(市售gibico PBS缓冲液)浸洗2次，每次5min；
2)用4％(质量百分含量)多聚甲醛固定爬片15min，PBS浸洗材料3次，每次5min；
3)0.1％(质量百分含量)Triton X-100(PBS配制)室温通透20min；
4)PBS浸洗3次，每次5min，3％BSA室温封闭30min；
5)弃掉废弃液，不洗，每个孔滴加足量的稀释好的一抗，4℃孵育过夜(24h)；
6)一抗孵育完后，PBS浸洗2次，每次5min，0.1％Triton X-100(PBS配制)洗一次；
7)常温加荧光二抗(荧光二抗名称为Anti-Mouse IgG(H+L),F(ab')2Fragment，购自Cell Signaling Technology，货号为4408s。)，室温孵育1h；
8)孵育完成后PBS浸洗3次，每次5min，最后用ddH2O封片，荧光显微镜488nm观察荧光强度。
为防止荧光淬灭，自加二抗起，所有操作都尽量在暗处进行。
上述冠脉内皮细胞(HCAEC)免疫荧光检测的一抗为Anti-CD31抗体[P2B1](该抗体购自Abcam，货号为ab24590)，冠脉平滑肌细胞(HCASMC)免疫荧光一抗为Anti-alpha Actinin抗体(该抗体购自Cell Signaling Technology，货号为3528s。)。
结果如图4所示，两种细胞均能在凝胶基底的网格状支架表面粘附生长，且贴壁状态良好，证明这种PLLA材料有较好的的细胞相容性，PLLA材料发生内皮化，可用于体内植入。
上述结果表明，PLLA材料发生内皮化，可用于体内植入。
对比例1、鼠尾胶原浓度的摸索
方法与实施例1基本相同，不同的是，鼠尾胶原在铺有鼠尾胶原凝胶的细胞培养板的孔反应体系中的浓度分别为1ug/mL和4ug/mL。
结果鼠尾胶原在铺有鼠尾胶原凝胶的细胞培养板的孔反应体系中的浓度为1ug/mL时，无法得到成形凝胶。
鼠尾胶原在铺有鼠尾胶原凝胶的细胞培养板的孔反应体系中的浓度为4ug/mL时，得到凝胶太硬，无法嵌入网格状PLLA支架材料。
对比例2、细胞接种量的摸索
按照实施例2的方法，向实施例1制备的PLLA支架体外内皮化检测模型接种冠脉内皮细胞和冠脉平滑肌细胞，2种细胞的浓度均为1×104个或5×104个。
1×104个或5×104个结果均如图5，可以看出，在凝胶基底的网格状支架表面检测不到细胞，无法粘附生长。
工业应用
本发明是制备一种网格支架结构以及建立了一种网格支架与细胞系共培养的方法。这种网格支架结构模拟血管支架的网格状结构，在体外将内皮细胞和平滑肌细胞与这种网格状结构共培养，通过观察细胞在这种网格结构上的生长状态、贴壁状态和覆盖状态来评判这种材料是否适于作为血管支架的制备材料。这个发明就是建立了一种试验方法，用来筛选可以 作为血管支架的材料，内皮细胞和平滑肌细胞在材料上的生长状态是衡量指标。现在缺乏在体外对血管支架材料的内皮化效果进行评判的模型装置和评价方法，只能依靠动物实验来对材料进行评价。本发明建立一种在体外评价材料的方法，有助于减少动物用量，符合动物福利的要求，减少企业研发成本。

Claims (13)

  1. 一种待植入材料体外内皮化检测模型，其包括多孔板、胶原凝胶和待植入材料；
    所述胶原凝胶置于所述多孔板的孔中，所述待植入材料以浸入且不浸没的方式置于所述胶原凝胶上；
    所述待植入材料为网格状。
  2. 根据权利要求1所述的模型，其特征在于：
    所述网格状待植入材料的直径小于所述多孔板的孔径；
    或所述多孔板中的孔为非通孔。
  3. 根据权利要求1或2所述的模型，其特征在于：所述多孔板为48孔板；
    或所述网格状待植入材料为圆片状。
  4. 根据权利要求1-3中任一所述的模型，其特征在于：
    所述胶原凝胶为将胶原在含有NaOH和乙酸的体系中进行反应形成胶原凝胶；
    或所述胶原凝胶按照如下方法制备：先将胶原溶于乙酸水溶液制备胶原-乙酸溶液，再将所述胶原-乙酸溶液与NaOH反应，形成胶原凝胶；
    或所述反应在所述多孔板的孔中进行。
  5. 根据权利要求4所述的模型，其特征在于：所述胶原在所述反应的体系中的浓度为2ug/ml；
    或所述反应为37℃放置30-60min。
  6. 根据权利要求1-5中任一所述的模型，其特征在于：所述胶原为鼠尾胶原、牛肌腱胶原蛋白、猪皮胶原或鱼胶原。
  7. 根据权利要求1-6中任一所述的模型，其特征在于：所述待植入材料为用于制备血管支架的材料；
    或，所述待植入材料为用于制备血管支架的材料，所述用于制备血管支架的材料为PLLA。
  8. 根据权利要求1-7中任一所述的模型，其特征在于：所述模型还包括细胞；
    所述细胞具体为内皮细胞和平滑肌细胞。
  9. 一种待植入材料体外内皮化检测套装产品，包括权利要求1-8中任一所述模型中的所述多孔板、所述胶原凝胶、所述待植入材料和所述细胞。
  10. 一种制备待植入材料体外内皮化检测模型的方法，包括如下步骤：先按照权利要求1-8中任一所述多孔板中的孔中制备所述胶原凝胶，再将权利要求1-8任一中的网格状待植入材料以浸入且不浸没的方式置于所述多孔板孔中的胶原凝胶上，得到待接入细胞模型；最后将细胞接种在所述多孔板孔中的所述待接入细胞模型上，得到待植入材料体外内皮化检测模型；
    所述细胞具体为内皮细胞和平滑肌细胞。
  11. 权利要求1-8中任一所述的模型或权利要求9所述的产品在制备检测待植入材料体外是否发生内皮化的产品中的应用；
    或，权利要求1-8中任一所述的模型或权利要求9所述的产品在评价或检测待植入材料是否发生内皮化中的应用；
    或，权利要求1-8中任一所述的模型或权利要求9所述的产品在评价或检测待植入材料内皮化效果中的应用。
  12. 根据权利要求11所述的应用，其特征在于：所述评价或检测为体外评价或体外检测。
  13. 一种检测或辅助检测待植入材料体外是否发生内皮化的方法，包括如下步骤：
    先在权利要求1-8任一中的所述的多孔板中的孔中制备所述胶原凝胶，再将权利要求1-8任一中的网格状待植入材料以浸入且不浸没的方式置于所述多孔板孔中的胶原凝胶上，得到待接入细胞模型；最后将细胞接种在所述多孔板孔中的所述待接入细胞模型上；检测所述细胞是否在所述待植入材料上贴壁且增殖生长，若贴壁且增殖生长，则该材料发生或候选发生内皮化，若不贴壁且增殖生长，则该材料不发生或候选不发生内皮化；
    所述细胞为内皮细胞和平滑肌细胞。
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