Name: characterization of leukocyte-platelet rich fibrin, a novel biomaterial
Uploaded: 2015-09-29T15:00:00.000+00:00
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Description: Watch this Scientific Journal Video about Characterization of Leukocyte-platelet Rich Fibrin, A Novel Biomaterial at JoVE.com

The project was supported by CTSA (UL1TR000058) from the National Center for Advancing Translational Sciences) and the CCTR Endowment Fund of Virginia Commonwealth University. The contents are solely the responsibility of the authors and do not necessarily represent official views of the National Center for Advancing Translational Sciences or the National Institutes of Health.

所有血液采集程序应当由授权和认证的专业人士来完成。使用人类受试者的研究涉及的机构审查委员会或其他有关机关批准。有关知情同意，保护参与者的身份特殊的预防措施，需要遵循。在这个协议中列出的所有实验涉及处理的人血和/或血液制品和适当的个人防护设备需要在任何时候都被磨损。废物应被视为生物危害和设置按规定的。 1.静脉穿刺<ol><li>确定病人/参与者和确认与现有的记录。解释研究的细节，并得到了知情同意书。 </li><li>有一个托盘设置为个别病人有标注在平整稳定的表面管。 </li><li>解释程序，使患者座椅舒适与支撑臂。告知病人，他或SHË会觉得小捏，应保持不动整个过程。 </li><li>附加针到适配器。 </li><li>洗手和戴手套。 </li><li>通过用70％的异丙醇从中心清洗同心圆向外制备肘窝静脉穿刺。允许站点风干30秒。 </li><li>通过触诊确定适当的静脉。 </li><li>在穿刺部位确保它是不是太紧张（同时仍感觉桡动脉）上述应用止血带3-4。 </li><li>通过将针15-30度的斜角，以在皮肤上平滑的动作进行静脉穿刺。通过针推血液收集管，并收集9毫升全血。 </li><li>取出止血带。从针移除管。 </li><li>撤回针头并应用纱布平方在之前针移除穿刺部位。处置针以适当的生物危害的容器。 </li><li>询问病人保持PRessure在穿刺部位。 </li><li>标签管</li><li>检查穿刺部位，以确保出血已停止。 </li><li>应用粘性绷带或磁带上的纱布方，询问患者的感觉好吗（无疼痛，肿胀或轻头晕） </li><li>感谢在出院前病人/参与者。 </li></ol> 2，L-PRF准备<ol><li>紧接着静脉采血的红色平顶玻璃干管，将其放置在离心机。 </li><li>将适当的反平衡后离心400 XG在室温为12分钟。 </li><li>取出管在周期的末端。注意三层:贫血小板血浆（PPP），富血小板纤维蛋白（L-PRF）和RBC基（ 图1）。 </li><li>用吸管吸出PPP。使用镊子轻轻一拉L-PRF出来，并将其放置在一个无菌，冲孔网。 </li><li>使用手术刀，刮去大部分RBC层精心留下的buffŸ上衣完好。 </li><li>轻轻压缩的L PRF凝块（使用无菌金属板，近似的重量225克）30秒。贫血小板血浆将被挤掉。 </li><li>取出板，轻轻抬起L-PRF膜。在L-PRF膜是准备在实验中使用的12。 </li></ol> 3.单轴拉伸试验<ol><li>在滤纸上以易于处理代替L- PRF膜（6）和冲压成"狗骨头"使用定做的金属模具（2.75毫米宽在其最窄处为7.5毫米的测量长度）。 </li><li>测量每个样品的厚度在三个点并取平均值。 </li><li>仔细地接合的L- PRF膜在单轴测试系统的颚夹具的中心。 </li><li>小心撕文件管理器文件支持，以揭露L-PRF膜。 </li><li>程序的仪器，以使可动头工作在一个恒定的速率（10.0毫米/分钟）和实验开始时为L-PRF仍然是湿的。 </li><li>记录弹性模量，断裂能，和应变从伴随单轴试验系统中的软件中断。这些值被自动计算并没有用户定义输入是必需的。请参见图3。 </li></ol> 4.缝合保留实力<ol><li>在滤纸上以易于处理代替L- PRF膜（N = 3）并切成使用外科手术刀矩形样品进行测定（10毫米×25毫米）。 </li><li>测定各样品（平均3）的厚度。 </li><li>使用不锈钢正畸绷带导线（直径为220微米），在样品的中心的针孔。 </li><li>通过针孔传递结扎线，以形成一个环，并将其固定在拉力试验机。将L-PRF膜的边缘下颚把手13。 </li><li>编程仪器，以使可动头工作在一个恒定的速率（10毫米/分钟）和起动实验。 </li><li>记录弹性模量，断裂能，和应变从伴随单轴试验系统中的软件中断。这些值被自动计算并没有用户定义输入是必需的。请参见图3。 </li></ol> 5.形态学考察<ol><li>使用10 mm的皮肤活检穿孔准备L-PRF样本进行扫描电镜检查，并放置在一个24孔板。 </li><li>洗用PBS样品，用2.5％戊二醛（于PBS中）20分钟固定。 </li><li>脱水试样通过浸渍在连续增加的乙醇浓度（50％，70％，80％，90％和100％），每次5分钟。 </li><li>治疗与0.5毫升100％的HMDS（六甲基二）5分钟。充气O / N，除去过量的HMDS 14。 </li><li>使用双面胶带上存根安装样品，溅射涂层铂为70秒，并检查在扫描型电子显微镜在一个加速操作20千伏关合作电压（或相应的设置）。 </li></ol> L-PRF，胰蛋白酶敏感性和茚三酮测定6.京尼平交联<ol><li>为了制备京尼平交联的L- PRF，冲洗膜用PBS和48小时浸泡在4ml 1％京尼平溶液（在70％的乙醇）。用PBS实验前漂洗以除去过量的京尼平15,16。 </li><li>由它的胰蛋白酶降解的抗性评估L-PRF的京尼平交联的稳定性。代替L- PRF膜（N = 3）和京尼平交联的L-PRF在500μl0.01％胰蛋白酶和胰蛋白酶的每日变化在37℃下进行3天。 <ol><li>称取样品在1天前酶在开始曝光，并在3天的差异，并最终权重代表酶降解17。 </li></ol></li><li>通过茚三酮测定法量化京尼平处理的L- PRF（G-PRF）的交联的量。首先准备的标准CURVe。使用甘氨酸（1毫-0.031毫米）的曲线来建立游离氨基酸浓度（FAA）​​和吸光度之间的​​关系。 <ol><li>热PRF样品与1ml 2％（重量/体积）茚三酮为15分钟，在100℃。 </li><li>让溶液冷却至室温，并添加1.5 ml的50％的乙醇。 </li><li>分析使用适宜分光光度计570nm处的吸光度。 </li><li>确定的交联百分比使用低于 15式</li></ol></li></ol><img alt="式（1）" src="/files/ftp_upload/53221/53221eq1.jpg" /> 7. MTS细胞增殖试验<ol><li>在T-75烧瓶生长MC3T3（小鼠颅盖前成骨细胞）的极限必需培养基-alpha修饰（αMEM），直到获得80％的汇合单层。 </li><li>准备好新鲜，无菌L-PRF膜（打开L-PRF管细胞培养罩内）和膜转移到一个新的细胞培养皿。 </li><li>从烧瓶中吸出介质并冲洗用PBS单层，加入5 ml的0.05％胰蛋白酶，并放置在37℃培养箱烧瓶5分钟，吸取烧瓶的内容到一个离心管中并离心，在400×g离心5分钟。 </li><li>滗析出上清液，轻轻拍打管打破细胞沉淀和重新悬浮用4ml新鲜αMEM，分配细胞悬浮液（50微升）和台盼蓝（50微升）的混合物放入血球和计数的数细胞</li><li>种子内放置在L-PRF膜保留细胞的细胞膜中的前10毫米的玻璃克隆环4×10 5个细胞 （环能24小时后删除）。 </li><li>在第4天，冲洗构建体用PBS三次10分钟。 </li><li>加入1毫升不含血清的培养基，并加入200μl的MTS试剂至各孔，孵育2小时，在37℃。 </li><li>测量从200微升等份490nm处的吸光度。 </li></ol>

<ol>
	<li>Marx, R. E., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Platelet-rich+plasma+Growth+factor+enhancement+for+bone+grafts.">Platelet-rich plasma Growth factor enhancement for bone grafts.</a> Oral Surg Oral Med Oral Pathol Oral Radiol Endod. 85 (6), 638-646 (1998).</li><li>Rodriguez, I. A., Growney Kalaf, E. A., Bowlin, G. L., Sell, S. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Platelet-rich+plasma+in+bone+regeneration:+Engineering+the+delivery+for+improved+clinical+efficacy.">Platelet-rich plasma in bone regeneration: Engineering the delivery for improved clinical efficacy.</a> BioMed Res In. 2014, (2014).</li><li>Del Corso, M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Current+Knowledge+and+Perspectives+for+the+Use+of+Platelet-Rich+Plasma+(PRP)+and+Platelet-Rich+Fibrin+(PRF).">Current Knowledge and Perspectives for the Use of Platelet-Rich Plasma (PRP) and Platelet-Rich Fibrin (PRF).</a> in Oral and Maxillofacial Surgery Part 1: Periodontal and Dentoalveolar Surgery. Curr Pharm Biotechno. 13 (7), 1207-1230 (2012).</li><li>Mazzucco, L., Balbo, V., Cattana, E., Guaschino, R., Borzini, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Not+every+PRP-gel+is+born+equal+Evaluation+of+growth+factor+availability+for+tissues+through+four+PRP-gel+preparations:+Fibrinet,+RegenPRP-Kit,+Plateltex+and+one+manual+procedure.">Not every PRP-gel is born equal Evaluation of growth factor availability for tissues through four PRP-gel preparations: Fibrinet, RegenPRP-Kit, Plateltex and one manual procedure.</a> Vox San. 97 (2), 110-118 (2009).</li><li>Fern&#225;ndez-Barbero, J. E., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Flow+cytometric+and+morphological+characterization+of+platelet-rich+plasma+gel.">Flow cytometric and morphological characterization of platelet-rich plasma gel.</a> Clin Oral Implants Re. 17 (6), 687-693 (2006).</li><li>Li, Z., Guan, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Hydrogels+for+cardiac+tissue+engineering.">Hydrogels for cardiac tissue engineering.</a> Polymer. 3 (2), 740-761 (2011).</li><li>Zhu, J., Cai, B., Ma, Q., Chen, F., Wu, W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cell+bricks-enriched+platelet-rich+plasma+gel+for+injectable+cartilage+engineering+&#8211;+an+in+vivo+experiment+in+nude+mice.">Cell bricks-enriched platelet-rich plasma gel for injectable cartilage engineering &#8211; an in vivo experiment in nude mice.</a> J Tissue Eng Regen Med. 7 (10), 819-830 (2013).</li><li>Dohan, D. M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Platelet-rich+fibrin+(PRF):+a+second-generation+platelet+concentrate.+Part+I:+technological+concepts+and+evolution.">Platelet-rich fibrin (PRF): a second-generation platelet concentrate. Part I: technological concepts and evolution.</a> Oral Surg Oral Med Oral Pathol Oral Radiol Endod. 101 (3), e37-e44 (2006).</li><li>Dohan, D. M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Platelet-rich+fibrin+(PRF):+a+second-generation+platelet+concentrate.+Part+II:+platelet-related+biologic+features.">Platelet-rich fibrin (PRF): a second-generation platelet concentrate. Part II: platelet-related biologic features.</a> Oral Surg Oral Med Oral Pathol Oral Radiol Endod. 101 (3), e45-e50 (2006).</li><li>Choukroun, J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Platelet-rich+fibrin+(PRF):+a+second-generation+platelet+concentrate.+Part+V:+histologic+evaluations+of+PRF+effects+on+bone+allograft+maturation+in+sinus+lift.">Platelet-rich fibrin (PRF): a second-generation platelet concentrate. Part V: histologic evaluations of PRF effects on bone allograft maturation in sinus lift.</a> Oral Surg Oral Med Oral Pathol Oral Radiol Endod. 101 (3), 299-303 (2006).</li><li>Dohan Ehrenfest, D. M., Bielecki, T., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Do+the+Fibrin+Architecture+and+Leukocyte+Content+Influence+the+Growth+Factor+Release+of+Platelet+Concentrates?+An+Evidence-based+Answer+Comparing+a+Pure+Platelet-Rich+Plasma+(P-PRP)+Gel+and+a+Leukocyte-+and+Platelet-Rich+Fibrin+(L-PRF).">Do the Fibrin Architecture and Leukocyte Content Influence the Growth Factor Release of Platelet Concentrates? An Evidence-based Answer Comparing a Pure Platelet-Rich Plasma (P-PRP) Gel and a Leukocyte- and Platelet-Rich Fibrin (L-PRF).</a> Curr Pharma Biotechno. 13 (7), 1145-1152 (2012).</li><li>Dohan Ehrenfest, D. M., Rasmusson, L., Albrektsson, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Classification+of+platelet+concentrates:+from+pure+platelet-rich+plasma+(P-PRP)+to+leucocyte-+and+platelet-rich+fibrin+(L-PRF).">Classification of platelet concentrates: from pure platelet-rich plasma (P-PRP) to leucocyte- and platelet-rich fibrin (L-PRF).</a> Trends Biotechno. 27 (3), 158-167 (2009).</li><li>Mine, Y., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Suture+Retention+Strength+of+Expanded+Polytetrafluoroethylene+(ePTFE)+Graft.">Suture Retention Strength of Expanded Polytetrafluoroethylene (ePTFE) Graft.</a> Acta Med Okayam. 64 (2), 121-128 (2010).</li><li>Braet, F., De Zanger, R., Wisse, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Drying+cells+for+SEM+,+AFM+and+TEM+by+hexamethyldisilazane:+a+study+on+hepatic+endothelial+cells.">Drying cells for SEM , AFM and TEM by hexamethyldisilazane: a study on hepatic endothelial cells.</a> J Micros. 186 (1), 84-87 (1997).</li><li>Yuan, Y., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+effect+of+cross-linking+of+chitosan+microspheres+with+genipin+on+protein+release.">The effect of cross-linking of chitosan microspheres with genipin on protein release.</a> Carbohydr Poly. 68 (3), 561-567 (2007).</li><li>Sell, S. A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cross-linking+methods+of+electrospun+fibrinogen+scaffolds+for+tissue+engineering+applications.">Cross-linking methods of electrospun fibrinogen scaffolds for tissue engineering applications.</a> Biomed Mater. 3 (4), (2008).</li><li>Gorczyca, G., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Preparation+and+characterization+of+genipin+cross-linked+porous+chitosan-collagen-gelatin+scaffolds+using+chitosan-CO2+solution.">Preparation and characterization of genipin cross-linked porous chitosan-collagen-gelatin scaffolds using chitosan-CO2 solution.</a> Carbohydr Poly. 102, 901-911 (2014).</li><li>Rozman, P., Semenic, D., Wislet-Gendebien, S. a. b. i. n. e. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Chapter+15.+The+Role+of+Platelet+Gel+in+Regenerative+Medicine.">Chapter 15. The Role of Platelet Gel in Regenerative Medicine.</a> Advances In Regenerative Medicine. , 319-349 (2011).</li><li>Dohan Ehrenfest, ., Lemo, D. M., Jimbo, N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Selecting+a+relevant+animal+model+for+testing+the+in+vivo+effects+of+Choukroun&#8217;s+platelet-rich+fibrin+(PRF):+Rabbit+tricks+and+traps.">Selecting a relevant animal model for testing the in vivo effects of Choukroun&#8217;s platelet-rich fibrin (PRF): Rabbit tricks and traps.</a> Oral Surg Oral Med Oral Pathol Oral Radiol Endod. 110 (4), 413-416 (2010).</li><li>Guilak, F., Baaijens, F. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Functional+tissue+engineering:+Ten+more+years+of+progress.">Functional tissue engineering: Ten more years of progress.</a> J Biomec. 47 (9), 1931-1932 (2014).</li><li>Guilak, F., Butler, D. L., Goldstein, S. A., Baaijens, F. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Biomechanics+and+mechanobiology+in+functional+tissue+engineering.">Biomechanics and mechanobiology in functional tissue engineering.</a> J Biomec. 47 (9), 1933-1940 (2014).</li><li>Collet, J. P., Shuman, H., Ledger, R. E., Lee, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+elasticity+of+an+individual+fibrin+fiber+in+a+clot.">The elasticity of an individual fibrin fiber in a clot.</a> Proc Natl Acad Sci. 102 (26), 9133-9137 (2005).</li><li>Liu, W., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Fibrin+fibers+have+extraordinary+extensibility+and+elasticity.">Fibrin fibers have extraordinary extensibility and elasticity.</a> Science. 313 (5787), 634 (2006).</li><li>American National Standard. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Chapter+8:+Test+methods+for+vascular+prostheses.">Chapter 8: Test methods for vascular prostheses.</a> Association for the Advancement of Medica lnstrumentation Guidance document: Cardiovascular Implants - Tubular vascular prostheses. , 33-34 (2001).</li></ol>

作者什么都没有透露，并确认不存在任何已知的利益冲突与该发布关联。

自体血小板浓缩物是有希望的，因为生长因子的丰度在再生医学18的领域。但是，这些准备工作往往缺乏一个明确的结构，使手术操作非常困难。许多次，悬浮液和凝胶不能有效地在递送位点保留，导致不可预测的结果。 L-PRF是一个巨大的进步在血小板的演变集中在它本质上是一个牢固的血纤维蛋白膜截留的血小板。这些固体膜具有优异的操控性能，并且可以在开放式手术安全地缝合在解...

The use of blood and blood-derived products to seal wounds and improve healing in different clinical situations started with fibrin glues, which are mainly fibrinogen concentrates. Addition of platelets to fibrin glue not only improved their strength but also promoted neoangiogenesis and regeneration. These benefits are attributed to the release of a variety of peptide growth factors from the alpha-granules of platelets upon activation1. Platelet concentrates (PC) were seen as a practical way to deliver growth factors2 and its use was driven by commercial interests rather than research characterization3. In fact, PCs are difficult to characterize unlike homogenous and defined pharmacological preparations, they are a potpourri of signaling molecules and blood cells (platelet and leukocytes) entrapped within a fibrin matrix. Different commercial and proprietary preparations yield a variety of PC that are different in cellular composition, growth factor recovery and kinetics of release4. 
It is important to realize that in most oral surgeries, platelet-rich plasma (PRP) preparations are used as a gel in open surgical wounds and not as platelet suspensions. In these situations, the gelation is induced by the addition of thrombin, calcium chloride, batroxobin or other agents and directly placed in the site of injury5. Due to rapid activation, fibrinogen polymerization is often incomplete and results in friable fibrin gels with very little mechanical strength. In addition, injectable PRP gels undergo rapid fibrinolysis6,7.
In contrast, the processes of blood coagulation (fibrinogen polymerization), platelet enrichment and activation occur simultaneously in the preparation of L-PRF8. The coagulation cascade is triggered when whole blood contacts the walls of a dry glass tube and continues throughout the centrifugation process. This results in the formation of a mechanically-strong blood clot (L-PRF) that can be surgically handled and used. 
Even though L-PRF has been investigated in terms of optimal methods of preparation, growth factor release and cell distribution9-11, detailed mechanical characterization of these membranes are lacking. This is significant gap in knowledge, given the popularity of these membranes in clinical practice as well as its potential to be used as a biomaterial. Current study focusses on the protocol for deriving L-PRF as well as methods that can be employed to study its mechanical properties. This data is intended to serve as baseline for ongoing studies investigating the viscoelastic properties of this interesting natural biomaterial.

<table><tbody><tr><td>Needle 19G</td><td>BD</td><td>305186</td><td></td></tr><tr><td>Needle Disposal Container</td><td>Fisherbrand</td><td>14-827-122</td><td></td></tr><tr><td>Red-Topped Glass Collection Tube</td><td>BD</td><td>8020129</td><td></td></tr><tr><td>Gauze Pads</td><td>Tyco</td><td>5750</td><td></td></tr><tr><td>Bandage</td><td>Johnson &amp;amp; Johnson</td><td>5005989</td><td></td></tr><tr><td>Surshield</td><td>Terumo</td><td>SV*S19BL</td><td>Safety winged infusion set</td></tr><tr><td>Blood Collection Assembly</td><td>BD</td><td>303380</td><td></td></tr><tr><td>Tourniquets</td><td>BD</td><td>367203</td><td></td></tr><tr><td>Brand Luer Adapter</td><td>Vacutainer</td><td>L42179</td><td></td></tr><tr><td>Intra-Spin System&amp;nbsp;</td><td>Intra-Lock International</td><td>ISS110</td><td>Centrifuge and Xpression L-PRF FabricationKit&amp;nbsp;</td></tr><tr><td>Pipettes (Serological &amp;amp; Micro)</td><td>Corning</td><td></td><td></td></tr><tr><td>Scalpel</td><td>Exelint</td><td>29552</td><td></td></tr><tr><td>MTS Bionix 200</td><td>MTS Systems Corporation</td><td></td><td>Material testing systems</td></tr><tr><td>MTS Test Works 4</td><td>MTS Systems Corporation</td><td></td><td></td></tr><tr><td>Whatman Filter Paper</td><td>Whatman</td><td>1004 070</td><td></td></tr><tr><td>SS Orthodontic ligature wire</td><td>Patterson Dental</td><td>628-4228</td><td></td></tr><tr><td>200 Proof Ethanol</td><td>Koptec</td><td>V1001</td><td></td></tr><tr><td>Hexamethyldisilazane (HMDS)</td><td>Aldrich</td><td>440191</td><td></td></tr><tr><td>Aluminium Mounting Stubs</td><td>Ted Pella</td><td>16324</td><td></td></tr><tr><td>Double Sided Carbon Tape</td><td>PELCO Tabs</td><td>16084-1</td><td></td></tr><tr><td>Scanning Electron Microscope</td><td>JEOL</td><td>LV 5610</td><td></td></tr><tr><td>Trypsin</td><td>HyClone</td><td>SH30042.01</td><td></td></tr><tr><td>Cell Culture Incubator</td><td>Thermo Fisher Scientific Inc</td><td>51026282</td><td></td></tr><tr><td>Antibiotic-Antimicotic</td><td>Gibco</td><td>15240-062</td><td></td></tr><tr><td>Genipin</td><td>Wako</td><td>078-03021</td><td></td></tr><tr><td>Cell Culture Media</td><td>Gibco</td><td>12000-022</td><td>Minimum Essential Medium-Alpha</td></tr><tr><td>MTS Reagent</td><td>Promega</td><td>G1118</td><td></td></tr><tr><td>PMS Reagent</td><td>Sigma</td><td>P9625</td><td></td></tr><tr><td>Spectrophotometer</td><td>BioTek</td><td></td><td>Epoch Spectrophotometer</td></tr><tr><td>10mm Glass Cloning Rings</td><td>Corning</td><td>3166-10</td><td></td></tr><tr><td>T-75 Flask</td><td>Corning</td><td>430641</td><td></td></tr><tr><td>DPBS</td><td>Corning</td><td>55-031-PB</td><td></td></tr><tr><td>Ninhydrin 98%</td><td>Aldrich</td><td>454044</td><td></td></tr><tr><td>24 Well Plate</td><td>Corning</td><td>3987</td><td></td></tr><tr><td>Biopsy Punch</td><td>Acu Punch</td><td>P1025</td><td></td></tr><tr><td>Digital Micrometer</td><td>Pittsburgh</td><td>68305</td><td></td></tr><tr><td>Glutaraldehyde</td><td>Sigma</td><td>G6257</td><td></td></tr><tr><td>12 Well Plate</td><td>Corning</td><td>3336</td><td></td></tr><tr><td>96 Well Plate</td><td>Corning</td><td>3596</td><td></td></tr></tbody></table>

characterization of leukocyte-platelet rich fibrin, a novel biomaterial

Autologous platelet concentrates represent promising innovative tools in the field of regenerative medicine and have been extensively used in oral surgery. Unlike platelet rich plasma (PRP) that is a gel or a suspension, Leukocyte-Platelet Rich Fibrin (L-PRF) is a solid 3D fibrin membrane generated chair-side from whole blood containing no anti-coagulant. The membrane has a dense three dimensional fibrin matrix with enriched platelets and abundant growth factors. L-PRF is a popular adjunct in surgeries because of its superior handling characteristics as well as its suturability to the wound bed. The goal of the study is to demonstrate generation as well as provide detailed characterization of relevant properties of L-PRF that underlie its clinical success.

L形的PRF凝块在不同的部分（顶部，中部和底部）层表示在图2的扫描电子显微镜的图像。如可以看到的，顶部主要是纤维蛋白网状的组成无细胞。中间层富含血小板与他们的活化和脱颗粒的证据。下层具有白细胞和血纤蛋白基质中包埋的红血细胞的混合物。 单轴拉伸测试和缝合保留强度试验:机械性能是在两种模式进行评价。结果证明L-PRF的粘弹性行?...

Watch this Scientific Journal Video about Characterization of Leukocyte-platelet Rich Fibrin, A Novel Biomaterial at JoVE.com

Characterization of Leukocyte-platelet Rich Fibrin, A Novel Biomaterial

Leucocyte-Platelet Rich Fibrin (L-PRF) represents an FDA cleared preparation of autologous platelet concentrates that possesses unique fibrin architecture, enriched platelets and abundant growth factors. Here, we present a protocol for chair-side generation of L-PRF as well as evaluate its mechanical properties including uniaxial testing and suture retention strength testing.

白细胞 - 富血小板纤维蛋白，一种新型生物材料的表征

Autologous platelet concentrates represent promising innovative tools in the field of regenerative medicine and have been extensively used in oral ...

characterization-of-leukocyte-platelet-rich-fibrin-a-novel-biomaterial

Research

JoVE Journal

Bioengineering

12.9K 次观看.  School of Dentistry, Virginia Commonwealth University. Leucocyte-Platelet Rich Fibrin (L-PRF) represents an FDA cleared preparation of autologous platelet concentrates that possesses unique fibrin architecture, enriched platelets and abundant growth factors. Here, we present a protocol for chair-side generation of L-PRF as well as evaluate its mechanical properties including uniaxial testing and suture retention strength testing. 

视频: Characterization of Leukocyte-platelet Rich Fibrin, A Novel Biomaterial

Engineering a Bilayered Hydrogel to Control ASC Differentiation

Natural polymers over the years have gained more importance because of their host biocompatibility and ability to interact with cells in vitro and in vivo. An area of research that holds promise in regenerative medicine is the combinatorial use of novel biomaterials and stem cells. A fundamental strategy in the field of tissue engineering is the use of three-dimensional scaffold (e.g., decellularized extracellular matrix, hydrogels, micro/nano particles) for directing cell function. This technology has evolved from the discovery that cells need a substrate upon which they can adhere, proliferate, and express their differentiated cellular phenotype and function 2-3. More recently, it has also been determined that cells not only use these substrates for adherence, but also interact and take cues from the matrix substrate (e.g., extracellular matrix, ECM)4. Therefore, the cells and scaffolds have a reciprocal connection that serves to control tissue development, organization, and ultimate function. Adipose-derived stem cells (ASCs) are mesenchymal, non-hematopoetic stem cells present in adipose tissue that can exhibit multi-lineage differentiation and serve as a readily available source of cells (i.e. pre-vascular endothelia and pericytes). Our hypothesis is that adipose-derived stem cells can be directed toward differing phenotypes simultaneously by simply co-culturing them in bilayered matrices1. Our laboratory is focused on dermal wound healing. To this end, we created a single composite matrix from the natural biomaterials, fibrin, collagen, and chitosan that can mimic the characteristics and functions of a dermal-specific wound healing ECM environment.

This protocol focuses on utilizing the inherent ability of stem cells to take cue from their surrounding extracellular matrix and be induced to differentiate into multiple phenotypes. This methods manuscript extends our description and characterization of a model utilizing a bilayered hydrogel, composed of PEG-fibrin and collagen, to simultaneously co-differentiate adipose-derived stem cells1.

工程双层凝胶控制升序分化

Natural polymers over the years have gained more importance because of their host biocompatibility and ability to interact with cells in vitro and in ...

科研

生物工程

PRP as a New Approach to Prevent Infection: Preparation and In vitro Antimicrobial Properties of PRP

Implant-associated infection is becoming more and more challenging to the healthcare industry worldwide due to increasing antibiotic resistance, transmission of antibiotic resistant bacteria between animals and humans, and the high cost of treating infections.
In this study, we disclose a new strategy that may be effective in preventing implant-associated infection based on the potential antimicrobial properties of platelet-rich plasma (PRP). Due to its well-studied properties for promoting healing, PRP (a biological product) has been increasingly used for clinical applications including orthopaedic surgeries, periodontal and oral surgeries, maxillofacial surgeries, plastic surgeries, sports medicine, etc.
PRP could be an advanced alternative to conventional antibiotic treatments in preventing implant-associated infections. The use of PRP may be advantageous compared to conventional antibiotic treatments since PRP is less likely to induce antibiotic resistance and PRP's antimicrobial and healing-promoting properties may have a synergistic effect on infection prevention. It is well known that pathogens and human cells are racing for implant surfaces, and PRP's properties of promoting healing could improve human cell attachment thereby reducing the odds for infection. In addition, PRP is inherently biocompatible, and safe and free from the risk of transmissible diseases.
For our study, we have selected several clinical bacterial strains that are commonly found in orthopaedic infections and examined whether PRP has in vitro antimicrobial properties against these bacteria. We have prepared PRP using a twice centrifugation approach which allows the same platelet concentration to be obtained for all samples. We have achieved consistent antimicrobial findings and found that PRP has strong in vitro antimicrobial properties against bacteria like methicillin-sensitive and methicillin-resistant Staphylococcus aureus, Group A Streptococcus, and Neisseria gonorrhoeae. Therefore, the use of PRP may have the potential to prevent infection and to reduce the need for costly post-operative treatment of implant-associated infections.

PRP as a New Approach to Prevent Infection: Preparation and In vitro Antimicrobial Properties of PRP

Implant-associated infection is a significant clinical complication. This study describes an approach using platelet-rich plasma (PRP) to prevent implant-associated infections, presents the protocol for preparing PRP with constant platelet concentration, and reports the newly identified antimicrobial properties of PRP and related protocols for examining such antimicrobial properties in vitro.

PRP作为一种新的方法，以防止感染的制备及<em在体外</em&gt;抗菌性能的PRP

Implant-associated infection is becoming more and more challenging to the healthcare industry worldwide due to increasing antibiotic resistance, ...

免疫与感染

Microfluidic Flow Chambers Using Reconstituted Blood to Model Hemostasis and Platelet Transfusion In Vitro

Blood platelets prepared for transfusion gradually lose hemostatic function during storage. Platelet function can be investigated using a variety of (indirect) in vitro experiments, but none of these is as comprehensive as microfluidic flow chambers. In this protocol, the reconstitution of thrombocytopenic fresh blood with stored blood bank platelets is used to simulate platelet transfusion. Next, the reconstituted sample is perfused in microfluidic flow chambers which mimic hemostasis on exposed subendothelial matrix proteins. Effects of blood donation, transport, component separation, storage and pathogen inactivation can be measured in paired experimental designs. This allows reliable comparison of the impact every manipulation in blood component preparation has on hemostasis. Our results demonstrate the impact of temperature cycling, shear rates, platelet concentration and storage duration on platelet function. In conclusion, this protocol analyzes the function of blood bank platelets and this ultimately aids in optimization of the processing chain including phlebotomy, transport, component preparation, storage and transfusion.

Microfluidic Flow Chambers Using Reconstituted Blood to Model Hemostasis and Platelet Transfusion In Vitro

Platelet transfusion and hemostasis was modeled using blood reconstitution and microfluidic flow chambers to investigate the function of blood banking platelets. The data demonstrate the consequences of platelet storage lesion on hemostasis, in vitro.

微流体流钱伯斯再造使用血止血建模和血小板输注<em&gt;体外</em

Blood platelets prepared for transfusion gradually lose hemostatic function during storage. Platelet function can be investigated using a variety of ...

Wet Chemistry and Peptide Immobilization on Polytetrafluoroethylene for Improved Cell-adhesion

Endowing materials surface with cell-adhesive properties is a common strategy in biomaterial research and tissue engineering. This is particularly interesting for already approved polymers that have a long standing use in medicine because these materials are well characterized and legal issues associated with the introduction of newly synthesized polymers may be avoided. Polytetrafluoroethylene (PTFE) is one of the most frequently employed materials for the manufacturing of vascular grafts but the polymer lacks cell adhesion promoting features. Endothelialization, i.e., complete coverage of the grafts inner surface with a confluent layer of endothelial cells is regarded key to optimal performance, mainly by reducing thrombogenicity of the artificial interface.
This study investigates the growth of endothelial cells on peptide-modified PTFE and compares these results to those obtained on unmodified substrate. Coupling with the endothelial cell adhesive peptide Arg-Glu-Asp-Val (REDV) is performed via activation of the fluorin-containing polymer using the reagent sodium naphthalenide, followed by subsequent conjugation steps. Cell culture is accomplished using Human Umbilical Vein Endothelial Cells (HUVECs) and excellent cellular growth on peptide-immobilized material is demonstrated over a two-week period.

Cell-adhesiveness is key to many approaches in biomaterial research and tissue engineering. A step-by-step technique is presented using wet-chemistry for the surface modification of the important polymer PTFE with peptides.

湿化学和肽固定在聚四氟乙烯为改善细胞粘附

Endowing materials surface with cell-adhesive properties is a common strategy in biomaterial research and tissue engineering. This is particularly ...

A Microfluidic Flow Chamber Model for Platelet Transfusion and Hemostasis Measures Platelet Deposition and Fibrin Formation in Real-time

Microfluidic models of hemostasis assess platelet function under conditions of hydrodynamic shear, but in the presence of anticoagulants, this analysis is restricted to platelet deposition only. The intricate relationship between Ca2+-dependent coagulation and platelet function requires careful and controlled recalcification of blood prior to analysis. Our setup uses a Y-shaped mixing channel, which supplies concentrated Ca2+/Mg2+ buffer to flowing blood just prior to perfusion, enabling rapid recalcification without sample stasis. A ten-fold difference in flow velocity between both reservoirs minimizes dilution. The recalcified blood is then perfused in a collagen-coated analysis chamber, and differential labeling permits real-time imaging of both platelet and fibrin deposition using fluorescence video microscopy. The system uses only commercially available tools, increasing the chances of standardization. Reconstitution of thrombocytopenic blood with platelets from banked concentrates furthermore models platelet transfusion, proving its use in this research domain. Exemplary data demonstrated that coagulation onset and fibrin deposition were linearly dependent on the platelet concentration, confirming the relationship between primary and secondary hemostasis in our model. In a timeframe of 16 perfusion min, contact activation did not take place, despite recalcification to normal Ca2+ and Mg2+ levels. When coagulation factor XIIa was inhibited by corn trypsin inhibitor, this time frame was even longer, indicating a considerable dynamic range in which the changes in the procoagulant nature of the platelets can be assessed. Co-immobilization of tissue factor with collagen significantly reduced the time to onset of coagulation, but not its rate. The option to study the tissue factor and/or the contact pathway increases the versatility and utility of the assay.

This paper describes an experimental model of hemostasis that simultaneously measures platelet function and coagulation. Platelet and fibrin fluorescence is measured in real-time, and platelet adhesion rate, coagulation rate, and onset of coagulation are determined. The model is used to determine platelet procoagulant properties under flow in concentrates for transfusion.

微流体流室模型血小板输注与止血措施，实时血小板沉积和纤维蛋白形成

Microfluidic models of hemostasis assess platelet function under conditions of hydrodynamic shear, but in the presence of anticoagulants, this analysis is ...

Laminar Flow-based Assays to Investigate Leukocyte Recruitment on Cultured Vascular Cells and Adherent Platelets

The recruitment of leukocytes upon injury or inflammation to sites of injury or tissue damage has been investigated during recent decades and has resulted in the concept of the leukocyte adhesion cascade. However, the exact molecular mechanisms involved in leukocyte recruitment have not yet been fully identified. Since leukocyte recruitment remains an important subject in the field of infection, inflammation, and (auto-) immune research, we present a straightforward laminar flow-based assay to study underlying mechanisms of the adhesion, de-adhesion, and transmigration of leukocytes under venous and arterial flow regimes. The in vitro assay can be used to study the molecular mechanisms that underlie the interactions between leukocytes and their cellular partners in different models of vascular inflammation. This protocol describes a laminar flow-based assay using a parallel-flow chamber and an inverted phase contrast microscope connected to a camera to study the interactions of leukocytes and endothelial cells or platelets, which can be visualized and recorded then analyzed offline. Endothelial cells, platelets, or leukocytes can be pretreated with inhibitors or antibodies to determine the role of specific molecules during this process. Shear conditions, i.e. arterial or venous shear stress, can be easily adapted by the viscosity and flow rate of the perfused fluids and the height of the channel.

Leukocytes avidly interact with vascular cells and platelets after vessel wall injury or during inflammation. Here, we describe a straightforward laminar flow-based assay to characterize the molecular mechanisms that underlie the interactions between leukocytes and their cellular partners.

以层流为基础的检测培养血管细胞和粘附血小板白细胞的研究

The recruitment of leukocytes upon injury or inflammation to sites of injury or tissue damage has been investigated during recent decades and has resulted ...

Ferric Chloride-induced Murine Thrombosis Models

Arterial thrombosis (blood clot) is a common complication of many systemic diseases associated with chronic inflammation, including atherosclerosis, diabetes, obesity, cancer and chronic autoimmune rheumatologic disorders. Thrombi are the cause of most heart attacks, strokes and extremity loss, making thrombosis an extremely important public health problem. Since these thrombi stem from inappropriate platelet activation and subsequent coagulation, targeting these systems therapeutically has important clinical significance for developing safer treatments. Due to the complexities of the hemostatic system, in vitro experiments cannot replicate the blood-to-vessel wall interactions; therefore, in vivo studies are critical to understand pathological mechanisms of thrombus formation. To this end, various thrombosis models have been developed in mice. Among them, ferric chloride (FeCl3) induced vascular injury is a widely used model of occlusive thrombosis that reports platelet activation and aggregation in the context of an aseptic closed vascular system. This model is based on redox-induced endothelial cell injury, which is simple and sensitive to both anticoagulant and anti-platelets drugs. The time required for the development of a thrombus that occludes blood flow gives a quantitative measure of vascular injury, platelet activation and aggregation that is relevant to thrombotic diseases. We have significantly refined this FeCl3-induced vascular thrombosis model, which makes the data highly reproducible with minimal variation. Here we describe the model and present representative data from several experimental set-ups that demonstrate the utility of this model in thrombosis research.

We report a refined procedure of the ferric chloride (FeCl3)-induced thrombosis models on carotid and mesenteric artery as well as vein, characterized efficiently using intravital microscopy to monitor time to occlusive thrombi formation.

三氯化铁诱导小鼠模型的血栓形成

Arterial thrombosis (blood clot) is a common complication of many systemic diseases associated with chronic inflammation, including atherosclerosis, ...

医学

Live-cell Imaging of Platelet Degranulation and Secretion Under Flow

Blood platelets are essential players in hemostasis, the formation of thrombi to seal vascular breaches. They are also involved in thrombosis, the formation of thrombi that occlude the vasculature and injure organs, with life-threatening consequences. This motivates scientific research on platelet function and the development of methods to track cell-biological processes as they occur under flow conditions.
A variety of flow models are available for the study of platelet adhesion and aggregation, two key phenomena in platelet biology. This work describes a method to study real-time platelet degranulation under flow during activation. The method makes use of a flow chamber coupled to a syringe-pump setup that is placed under a wide-field, inverted, LED-based fluorescence microscope. The setup described here allows for the simultaneous excitation of multiple fluorophores that are delivered by fluorescently labeled antibodies or fluorescent dyes. After live-cell imaging experiments, the cover glasses can be further processed and analyzed using static microscopy (i.e., confocal microscopy or scanning electron microscopy).

This work describes a fluorescence microscopy-based method for the study of platelet adhesion, spreading, and secretion under flow. This versatile platform enables the investigation of platelet function for mechanistic research on thrombosis and hemostasis.

活细胞成像血小板脱水和分泌流量

Blood platelets are essential players in hemostasis, the formation of thrombi to seal vascular breaches. They are also involved in thrombosis, the ...

生物学

<meta charset="utf8"></head><body>用于再生医学的纤维蛋白水凝胶中的机械基质血管组分

The Combination of Mechanically Isolated Stromal Vascular Fraction and Fibrin Hydrogel: A Processing Protocol

The regenerative potential of adipose-derived stromal cells (ASCs) has gained significant attention in regenerative and translational research. In the past, the extraction of these cells from adipose tissue required a multistep enzyme-based process, resulting in a heterogenous cell mix consisting of ACSs and other cells, which are jointly termed the stromal vascular fraction (SVF). More recently introduced mechanical SVF (mSVF) isolation protocols are less time-consuming and bypass regulatory concerns. We recently proposed a protocol that generates mSVF rich in stromal cells based on a combination of emulsification and centrifugation. One current issue in mSVF application for wound therapy application is the lack of a scaffold providing protection from mechanical manipulation and desiccation. Fibrin hydrogels have been shown to be a useful adjunct in cell transfer for wound healing purposes in the past. In the work herein, we delineate the preparation steps of an mSVF-fibrin hydrogel construct as a novel approach for translational research and clinical application.

<meta charset="utf8"></head><body>作者聚焦：探索脂肪衍生基质血管成分对伤口愈合的潜力

Mechanically isolated stromal vascular fraction (SVF) in combination with a fibrin hydrogel offers an easy and efficient carrier for viable adipose-derived stromal cells for various indications, including tissue engineering and or wound healing purposes. Here, we present the preparation of a mechanical SVF (mSVF)-fibrin hydrogel construct for translational research and clinical application.

机械分离的基质血管组分和纤维蛋白水凝胶的组合：一种加工方案

The regenerative potential of adipose-derived stromal cells (ASCs) has gained significant attention in regenerative and translational research. In the ...

白细胞 - 富血小板纤维蛋白，一种新型生物材料的表征

摘要

探索更多视频

此视频中的章节

工程双层凝胶控制升序分化

PRP作为一种新的方法，以防止感染的制备及

微流体流钱伯斯再造使用血止血建模和血小板输注

湿化学和肽固定在聚四氟乙烯为改善细胞粘附

微流体流室模型血小板输注与止血措施，实时血小板沉积和纤维蛋白形成

以层流为基础的检测培养血管细胞和粘附血小板白细胞的研究

三氯化铁诱导小鼠模型的血栓形成

活细胞成像血小板脱水和分泌流量

机械分离的基质血管组分和纤维蛋白水凝胶的组合：一种加工方案

机械分离的基质血管组分和纤维蛋白水凝胶的组合：一种加工方案