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Oct 2019

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Evaluating Whole Blood Clotting in vitro on Biomaterial Surfaces
生物材料表面进行全血凝固的体外评估   

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Abstract

Biomaterial-associated thrombosis is still a major concern for blood-contacting implants. After the medical device is implanted and comes in contact with blood, several complex reactions occur, which may lead to thrombus formation and failure of the device. Therefore, it is essential to evaluate the biomaterial interaction with the whole blood. Several studies have been reported in the literature that evaluate different steps in the coagulation cascade, such as protein adsorption, plasma activation, and platelet adhesion in vitro, however, evaluation of whole blood clotting on biomaterial surfaces is not widely reported. Here, a protocol to evaluate whole blood clotting in vitro on 2D biomaterials surfaces via a simple and fast hemolysis assay is presented. Whole human blood is placed onto the biomaterial surfaces and is allowed to clot for different time periods. After the specific time intervals, the surfaces are transferred into deionized (DI) water to release the free hemoglobin and the absorbance of this solution is measured. The absorbance value is proportional to the free hemoglobin concentration in the DI water due to lysis of red blood cells and gives an indirect correlation to the extent of blood clotting on the biomaterial surfaces. This protocol provides a fast, facile and effective method to measure the anti-thrombogenic properties of biomaterials.

Keywords: Blood clotting (全血凝固), Biomaterials (生物材料), Blood-contacting implants (血液接触植入物), Thrombosis (血栓形成), Hemocompatibility test (血液相容性实验), Thrombogenicity (血栓形成)

Background

Investigating the blood clotting on medical devices is essential for successful development of biomaterials for implantable medical devices. Until now, no biomaterial surface has been truly able to prevent blood clotting (Sabino et al., 2019). After contact with blood, the implant surface gets an adsorbed layer of blood protein, which can further activate the coagulation cascade, leading to platelet adhesion and activation, and finally to the development of the fibrin mesh (Gorbet and Sefton, 2004). Many published methods to investigate the anti-thrombogenic properties of biomaterials focus on studying the early stages of blood clotting, such as protein adsorption, and platelet adhesion and activation. Although they are important to understand the interaction between blood and the implant surface, these studies do not provide significant information about the overall coagulation process (Damodaran et al., 2013; Simon‐Walker et al., 2018; Obstals et al., 2018).

Preventing whole blood clotting on surfaces is crucial for long term success of blood-contacting implants. The formation of a fibrin clot is one of the latest stages of thrombosis, and this fibrin mesh traps the red blood cells (Leszczak et al., 2013). In this protocol, human blood is allowed to clot on biomaterial surface for up to 45 min. When the surface is transferred to DI water, only the red blood cells that are not trapped in the fibrin mesh are dissolved in water and get lysed due to pressure change. Hemolysis is the rupture of the red blood cells, followed by the release of their components, such as hemoglobin. Thus, a higher amount of hemoglobin released indicates less blood clotting on the surface. Positive control with just blood in DI water is prepared and is considered the maximum hemoglobin release. The absorbance measured at a wavelength of 540 nm is directly proportional to the concentration of free hemoglobin in water (Sabino et al., 2019). Therefore, a higher absorbance value indicates higher hemoglobin concentration, which means less clotting on the biomaterial surface.

Materials and Reagents

  1. 3.0 ml vacuum blood tubes with no anticoagulant (BD Vacutainer, catalog number: 366703)
  2. 24-well plate (Greiner Bio-One CELLSTAR, catalog number: 662-160)
  3. 96-well plate (Greiner Bio-One CELLSTAR, catalog number: 687-100)
  4. Standard pipette tips with a volume capacity of 20 μl and 1,000 μl (Eppendorf, catalog numbers: 22491911 and 22491351)
  5. Deionized (DI) water
  6. Human blood
  7. Biomaterial surface (average top area ~25 mm2)
  8. Vacuum line
  9. Marker (from any commercial source)
  10. 70% ethanol
  11. PBS (see Recipes)

Equipment

  1. Microplate reader (BMG LABTECH, FLUOstar Omega)
  2. Horizontal shaker (VWR, catalog number: 97109-890)
  3. Measurement pipettes (Eppendorf, catalog numbers: ES-20F and ES-1000F)
  4. Timer (from any commercial source)
  5. Tweezers (from any commercial source)
  6. Bench

Software

  1. Software Omega and MARS Data Analysis (BMG LABTECH, https://www.bmglabtech.com/reader-control-software/)
  2. Software Microsoft Excel or Origin (OriginLab, https://www.originlab.com/)

Procedure

Note: All procedures are performed twice (using blood from two different donors) for three samples of each surface in the hood.

  1. Biomaterial sterilization
    1. Place biomaterial surfaces in each well of a 24-well plate. Prepare one plate for each time point (15, 30 and 45 min) and label them on the lid with the corresponding time point.
    2. Add 500 μl of ethanol for 5 min at room temperature.
    3. Aspirate the ethanol and rinse the biomaterial surfaces three times with 500 μl of PBS.
    4. Aspirate the PBS and rinse the biomaterial surfaces once with 500 μl of DI water.
    5. Aspirate the DI water and let the biomaterial surfaces dry for at least 30 min.

  2. Adding blood to the biomaterial surface
    1. Draw human whole blood in a vacuum tube with no anticoagulant.
    2. Immediately after the blood draw, carefully open the vacuum tube and pipette 7 μl of blood out of the tube and put onto each biomaterial surface (Figure 1).
    3. Close the lid and start counting the timer for each plate (15, 30 and 45 min).


      Figure 1. Whole blood on biomaterial surfaces. 7 μl of blood are placed onto each surface and allowed to clot for 15, 30 and 45 min. Group A and B are different biomaterial surfaces being studied.

  3. Preparing the positive control (maximum hemoglobin release)
    1. Prepare a new 24-well plate with 500 μl of DI water in 3 wells.
    2. Add 7 μl of blood to each well with DI water (Figure 2A).


      Figure 2. Positive Control. A. the maximum hemoglobin release is obtained adding 7 μl of blood in 500 μl of DI water. B. 200 μl of each solution is transferred to a 96-well plate to read the absorbance.

    3. Place the plate on a horizontal shaker for 30 s (100 rpm).
    4. Move the plate to a bench and wait for 5 min to release the free hemoglobin.
    5. After 5 min, pipette 200 μl of the solution to a 96-well plate (Figure 2B).
    6. Read the absorbance using a microplate reader at a wavelength of 540 nm.

  4. Transferring samples to release hemoglobin
    1. Prepare a new 24-well plate with 500 μl of DI water in each well.
    2. After 15 min, gently transfer the biomaterial surfaces from the first plate to a new well with DI water. Be careful while moving the biomaterial surfaces to not disturb the blood on the surface. The samples should under water after this step. If your samples are thicker, use more water in order to cover the whole with the blood.
    3. Place the plate on a horizontal shaker for 30 s (100 rpm).
    4. Move the plate to a bench and wait for 5 min to release the free hemoglobin (Figure 3).


      Figure 3. Different biomaterial surfaces, A and B, in DI water to release the free hemoglobin. The lysis of red blood cells occurs due to pressure change and hemoglobin is released in DI water.

  5. Reading the absorbance
    1. After 5 min, pipette 200 μl of the “water + hemoglobin released” solution to a 96-well plate. Change the pipette tip for each solution (Figure 4).
    2. Read the absorbance using a microplate reader at a wavelength of 540 nm.
    3. Repeat Procedures C and D after 30 and 45 min for the second and third plates, respectively.


      Figure 4. Solutions from different biomaterial surfaces, A and B (water + hemoglobin released). 200 μl of each solution in Figure 3 is transferred to a 96-well plate to read the absorbance.

Data analysis

  1. Absorbance values at 540 nm are read using a microplate reader. Export the data to an excel format file.
  2. Any protocols involving blood are recommended to be performed at least twice using two different blood donors. However, in order to avoid donor-to-donor variability, only present the results obtained from one donor (Damodaran et al. 2013; Sabino et al., 2019).
  3. Using Origin/Excel (or similar software), plot the mean/standard deviation of absorbance values from each group (Figure 5). Insert a line corresponding to the average absorbance measured for the positive control.
  4. Conduct analysis of variance (ANOVA) for the experimental data using software Origin (or similar) at a 5% significance level (P < 0.05).


    Figure 5. Free hemoglobin concentration values measured in terms of absorbance for different biomaterial surfaces after 15, 30 and 45 min. The results were significantly different for all time points. Group B shows significant higher hemoglobin concentration, which indicates that these surfaces significantly delay whole blood clotting in comparison with group A.

Notes

This protocol can be applied to evaluate the whole blood clotting in vitro of any biomaterial surface that is stable underwater. If the sample floats in water, use double-sided tape to attach the sample to the well bottom. Sterilization process may vary depending on the biomaterial surface.

Recipes

  1. PBS (10x)
    Dissolve 100 ml of 10x PBS in 900 ml of DI water and autoclave or filter sterilize

Acknowledgments

This work was supported by National Heart, Lung and Blood Institute of the National Institutes of Health under award number R01HL135505 and R21HL139208. This protocol was adapted from previous publications from our group (Leszczak et al., 2013; Sabino et al., 2019).

Competing interests

The authors declare no conflict of interest.

Ethics

All experiments were conducted in agreement with the National Institutes of Health's “Guiding Principles for Ethical Research”. Colorado State University Institutional Review Board approved the protocol (17-7195H, valid from 04/01/2017 to 03/31/2020) for blood isolation from healthy participants. Whole human blood was acquired through venipuncture from healthy individuals, and formal consents were obtained from the donors.

References

  1. Damodaran, V. B., Leszczak, V., Wold, K. A., Lantvit, S. M., Popat, K. C. and Reynolds, M. M. (2013). Anti-thrombogenic properties of a nitric oxide-releasing dextran derivative: evaluation of platelet activation and whole blood clotting kinetics. RSC Adv 3(46).
  2. Gorbet, M. B. and Sefton, M. V. (2004). Biomaterial-associated thrombosis: roles of coagulation factors, complement, platelets and leukocytes. Biomaterials 25(26): 5681-5703.
  3. Leszczak, V., Smith, B. S. and Popat, K. C. (2013). Hemocompatibility of polymeric nanostructured surfaces. J Biomater Sci Polym Ed 24(13): 1529-1548.
  4. Obstals, F., Vorobii, M., Riedel, T., de Los Santos Pereira, A., Bruns, M., Singh, S. and Rodriguez-Emmenegger, C. (2018). Improving hemocompatibility of membranes for extracorporeal membrane oxygenators by grafting nonthrombogenic polymer brushes. Macromol Biosci 18(3).
  5. Sabino, R. M., Kauk, K., Movafaghi, S., Kota, A. and Popat, K. C. (2019). Interaction of blood plasma proteins with superhemophobic titania nanotube surfaces. Nanomedicine 21: 102046.
  6. Simon-Walker, R., Cavicchia, J., Prawel, D. A., Dasi, L. P., James, S. P. and Popat, K. C. (2018). Hemocompatibility of hyaluronan enhanced linear low density polyethylene for blood contacting applications. J Biomed Mater Res B Appl Biomater 106(5): 1964-1975.

简介

[摘要 ] 生物材料相关的血栓形成仍然是血液接触植入物的主要问题。医疗设备植入并与血液接触后,会发生一些复杂的反应,这可能导致血栓形成和设备故障。因此,评估生物材料与全血的相互作用至关重要。文献中已有几项研究评估了凝血级联反应的不同步骤,例如体外蛋白质吸附,血浆活化和血小板粘附,但是,对生物材料表面全血凝块的评估尚未广泛报道。这是评估体外全血凝块的方案 提出了一种通过简单快速的溶血分析在二维生物材料表面上的应用。将全人类血液放置在生物材料表面上,并允许其凝结不同的时间段。在特定的时间间隔后,将表面转移到去离子(DI)水中以释放出游离的血红蛋白,并测量该溶液的吸光度。由于红细胞的溶解,吸光度值与去离子水中游离血红蛋白的浓度成正比,并且与生物材料表面上的血凝程度间接相关。该协议提供了一种快速,简便而有效的方法来测量生物材料的抗血栓形成特性。

[背景 ] Investigatin 克血液凝固上的医疗设备为生物材料用于可植入医疗器械的成功发展至关重要。迄今为止,还没有生物材料表面能够真正阻止血液凝结(Sabino 等人,2019)。与血液接触后,植入物表面会吸附一层血液蛋白,该蛋白可进一步激活凝血级联反应,从而导致血小板粘附和激活,最终导致纤维蛋白网的发育(Gorbet 和Sefton ,2004)。研究生物材料抗血栓形成特性的许多公开方法都集中于研究血液凝结的早期阶段,例如蛋白质吸附,血小板粘附和活化。虽然他们是重要的是了解血液和植入物表面之间的相互作用,这些研究没有提供关于整体凝血过程显著信息(达莫达伦等人。2013年,西蒙- 沃克等人。2018 ; Obstals 等人。2018 )。

防止表面上的全血凝结对于血液接触型植入物的长期成功至关重要。血纤蛋白凝块的形成是血栓形成的最新阶段之一,而血纤蛋白网捕获了红血球(Leszczak 等,2013)。在此方案中,允许人类血液在生物材料表面凝结长达45分钟。当表面转移到去离子水中时,只有未捕获在纤维蛋白网中的红细胞才会溶解在水中,并由于压力变化而被溶解。溶血是红细胞破裂,然后释放其成分(例如血红蛋白)。因此,释放出更多的血红蛋白表明较少的血液凝结在表面上。准备在去离子水中仅含血液的阳性对照,被认为是最大的血红蛋白释放。在540 nm波长处测得的吸光度与水中游离血红蛋白的浓度成正比(Sabino et al 。,2019)。因此,较高的吸光度值表示较高的血红蛋白浓度,这意味着在生物材料表面上的团块较少。

关键字:全血凝固, 生物材料, 血液接触植入物, 血栓形成, 血液相容性实验, 血栓形成

材料和试剂


 


3.0 ml无抗凝剂的真空血管(BD Vacutainer,目录号:366703)
24孔板(Greiner Bio-One CELLSTAR,目录号:662-160)
96孔板(Greiner Bio-One CELLSTAR,目录号:687-100)
标准移液管尖端与20的体积容量微升和1000 微升(的Eppendorf ,目录号:22491911和22491351)
去离子水
人血
生物材料表面(平均顶部面积〜25 mm 2 )
真空线
标记(来自任何商业来源)
70%乙醇
PBS(请参阅食谱)
 


设备


 


酶标仪(BMG LABTECH,FLUOstar Omega)
卧式振动筛(VWR,目录号:97109-890)
测量移液器(Eppendorf,目录号:ES-20F和ES-1000F)
计时器(来自任何商业来源)
镊子(任何商业来源)
板凳
 


软件


 


软件Omega 和MARS数据分析(BMG LABTECH ,https: //www.bmglabtech.com/reader-control-software/ )
Microsoft Excel或Origin软件(OriginLab ,https : //www.originlab.com/ )
 


程序


 


注意:对于抽油烟机每个表面的三个样本,所有程序都要进行两次(使用来自两个不同供体的血液)。


 


Biomater IAL 消毒
将生物材料表面放在24孔板的每个孔中。在每个时间点(15、30和45分钟)准备一块板,并在相应的时间点在盖上贴上标签。
加入500 微升的乙醇,在室温下5分钟。
吸出乙醇并用500冲洗生物材料表面三次微升PBS中。
吸出PBS并冲洗所述生物材料的表面一旦用500 微升去离子水中。
吸入去离子水,并使生物材料表面干燥至少30分钟。
 


向生物材料表面添加血液
在没有抗凝剂的真空管中抽取人全血。
抽血后,立即小心地打开真空管和吸管7 微升的血液从管的,并把在每个生物材料表面(图1)。
合上盖子,开始为每个板计数计时器(1、5、30和45分钟)。
 


 


图1 。生物材料表面上的全血。将7μl 血液置于每个表面上,使其凝结15、30和45分钟。A组和B组是正在研究的不同生物材料表面。


 


准备阳性对照(最大血红蛋白释放)
准备一个新的24孔板用500 微升在3个孔DI水。
添加7 微升的血液用DI水(图2A)每个孔中。
 


 


 


图2 。积极控制。A. 最大血红蛋白释放获得添加7 微升的血液在500 微升去离子水中。B.将200μl 每种溶液转移到96孔板中以读取吸光度。


 


将板放在水平振荡器上30 s(100 rpm)。
将板移到工作台上,等待5分钟以释放游离的血红蛋白。
5分钟后,吸移管200 微升的溶液至96孔板(图2B)的。
使用酶标仪在540 nm波长处读取吸光度。                                                       
 


转移样品以缓解血红蛋白
P repare新的24孔板用500 微升在每个去离子水的井。
15分钟后,用去离子水将生物材料表面从第一块板轻轻转移到新孔中。移动生物材料表面时要小心,以免干扰表面的血液。在此步骤之后,样品应在水下。如果您的样本较厚,请使用更多的水以使整个血液覆盖。
将板放在水平振荡器上30 s(100 rpm)。
将板移到工作台上,等待5分钟以释放游离的血红蛋白(图3)。
 


 


图3 。去离子水中不同的生物材料表面A和B 释放出游离的血红蛋白。红细胞的溶解是由于压力变化而发生的,血红蛋白在去离子水中释放。


 


              读取吸光度
5 分钟后,将200μl “水+释放血红蛋白”溶液移液至96孔板。更改每种溶液的移液器吸头(图4)。
使用酶标仪在540 nm波长处读取吸光度。                                                       
30和45分钟后分别对第二块板和第三块板重复步骤C和D。
 


 


图4.来自不同生物材料表面的溶液,A和B(水+血红蛋白释放)。将200μl 图3中的每种溶液转移到96孔板中以读取吸光度。


 


数据分析


 


使用酶标仪读取540 nm处的吸光度值。将数据导出到excel格式文件。
建议任何涉及血液的方案至少要使用两个不同的献血者进行两次。如何以往,以避免供体到供体变性,仅呈现从一个供体获得的结果(达莫达兰等人,2013; 萨比诺等人。,2019)。
使用Origin / Excel(或类似软件),绘制每个组的吸光度值的平均值/标准差(图5)。插入与阳性对照测得的平均吸光度对应的线。
使用Origin软件(或类似软件)以5%的显着性水平对实验数据进行方差分析(ANOVA)(P < 0.05)。
 


 


 


 


图5 。游离血红蛋白浓度值的吸光度为不同来测量生物材料ス后15,30和45 rfaces 分钟。WER结果Ë显著LY 所有时间点不同。B组显示明显较高的血红蛋白浓度,这表明与A组相比,这些表面明显延迟了全血凝结。


 


笔记


 


该协议可用于评估水下稳定的任何生物材料表面的全血体外凝结。如果样品漂浮在水中,请使用双面胶带将样品粘贴到井底。灭菌过程可能会因生物材料表面而异。


 


菜谱


 


PBS(10x)
将100 ml的10x PBS溶于900 ml的去离子水中,高压灭菌或过滤灭菌


 


致谢


 


这项工作得到了美国国立卫生研究院国家心,肺和血液研究所的支持,授予的编号为R01 HL135505和R21HL139208。该协议改编自以前的出版物从我们的组(Leszczak 等人,2。013 ; 小号abino 等人,2019)。


 


利益争夺


 


作者宣称没有利益冲突。


 


伦理


 


所有实验均按照美国国立卫生研究院的“伦理研究指导原则”进行。科罗拉多州立大学机构审查委员会批准了从健康受试者中分离血液的方案(17-7195H ,有效期从04/01/2017至03/31/2020 )。通过静脉穿刺从健康个体获得全血,并从供体获得正式同意。


 


参考文献


 


Damodaran,VB,Leszczak ,V.,Wold ,KA,Lantvit ,SM,Popat ,KC和Reynolds,MM (2013)。一氧化氮释放葡聚糖衍生物的抗血栓形成特性:评估血小板活化和全血凝固动力学。RSC Adv 3(46)。
Gorbet ,MB和Sefton,MV(2004)。生物材料相关的血栓形成:凝血因子,补体,血小板和白细胞的作用。生物材料25(26):5681-5703。
Leszczak ,V.,Smith,BS和Popat ,KC(2013)。聚合物纳米结构表面的血液相容性。J Biomater Sci Polym Ed 24(13):1529-1548。
Obstals,F.,Vorobii,M.,Riedel,T.,de Los Santos Pereira,A.,Bruns,M.,Singh,S.和Rodriguez-Emmenegger,C.(2018)。通过嫁接非血栓形成性聚合物刷改善体外膜氧合器的膜的血液相容性。Macromol Biosci 18(3)。
萨比诺,RM,Kauk ,K.,Movafaghi ,S.,哥打,A。和Popat ,KC(2019)。血浆蛋白与超疏水二氧化钛纳米管表面的相互作用。纳米医学21:102046。
西蒙·沃尔克(R.Simon -Walker,R .),卡维奇亚(Cavicchia ,J.),普拉维尔(Prawel),DA,达西(Dasi),LP,詹姆斯(James),SP和波帕特(Popat),KC(2018)。透明质酸增强的线性低密度聚乙烯在血液接触应用中的血液相容性。生物医学研究杂志B应用生物学杂志106(5):1964-1975。
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引用:Sabino, R. M. and Popat, K. C. (2020). Evaluating Whole Blood Clotting in vitro on Biomaterial Surfaces. Bio-protocol 10(3): e3505. DOI: 10.21769/BioProtoc.3505.
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