Name: effects of allogeneic platelet-rich plasma (prp) on the healing process of sectioned achilles tendons of rats: a methodological description
Uploaded: 2018-03-19T12:30:00.000+00:00
Duration: 7 min 51 s
Description: Watch this Scientific Journal Video about Effects of Allogeneic Platelet-Rich Plasma PRP on the Healing Process of Sectioned Achilles Tendons of Rats: A Methodological Description at JoVE.com

这项研究得到了里昂 Frédéricq 基金标准 de 列日和勒琼-Lechien 赠款的支持。

对动物的护理和处理是按照国家科学院编写并由国家卫生研究院 (美国) 出版的《护理和使用实验动物指南》进行的。欧洲和国家立法得到了仔细的遵循。
1. 动物制剂
<ol><li>使用 132 2 月大的雄性大大鼠重320-450 克 (120 只老鼠进行实验, 12 只老鼠进行血液取样,图 1)。基于 Dell13, 每个组的15只老鼠足够的功率为 0.8 (如果指定的效果存在, 则发现统计意义的概率 80%)。</li>
	<li>在传统的设施中, 所有的老鼠都在隔离的条件下, 在一个装有变站的常规设备里。在静脉 (单独通风的笼) 架上进行繁殖, 用 SPF (特定病原体免费) 大鼠被专门购买。</li>
	<li>使用以下住房条件: 温度范围从19-24 °c;相对湿度: 40-60%;通风: 空气更新频率: 每小时 10-15;光/暗循环:12 h-12 h。</li>
	<li>消毒所有锁定设备, 使用辐照床上用品。通过在笼子内放置无菌纸板托盘和毛巾系统地实施网箱浓缩。</li>
	<li>允许进入受限条件下的区域: 新的实验室大衣, 面具, 手套, 鞋类, 和头发帽在入口处。</li>
	<li>根据 Felasa14关于常规设施的建议, 每年对存放在肮脏床上用品上的定点动物进行菌落健康监测。保持四到五动物每笼, 与适当的辐照饮食和提供酸化水ad 随意。每日监控。</li>
	<li>转移大鼠选择的手术干预, 过滤顶笼到手术室2小时前手术。</li>
</ol>2. 手术方法
<ol><li>准备手术器械: 细剪刀, 钳, 两个止血钳, 和一个针架。在所有的程序, 戴手套, 面具, 罩, 和实验室大衣。</li>
	<li>将大鼠随机分为两组 (PRP 和盐水溶液)。</li>
	<li>把老鼠从笼子里拿出来称重。使用编号的耳朵标记来识别老鼠。为了避免眼部干燥, 请将 waterdrops 放在角膜上。</li>
	<li>麻醉鼠腹腔与甲苯噻嗪 (10 毫克/千克体重) 和氯胺酮 (80 毫克/公斤的体重)。要确认麻醉, 请将动物置于心肺监测之下, 检查是否有眼部反射。</li>
	<li>在颈部区域注射50µL 的丁丙诺啡 (0.01-0, 05 毫克/千克, 每8-12 小时) 作为止痛药。</li>
	<li>用电动剃须刀剃掉左后肢, 用 3 betadine/酒精 (稀释 1:10) 溶液的交替磨擦消毒, 然后将老鼠放在一个温暖的垫子上 (20 °c) 下解剖显微镜。将大鼠放在侧褥疮上, 用腿在上级位置进行手术。用手术钳握住爪子。</li>
	<li>使用剪刀, 在左跟腱周围的皮肤上做一个小的侧向切口 (20-25 毫米), 用细剪刀解剖筋膜以暴露跟腱复合体 (图 2a)。</li>
	<li>用剪刀把跖肌腱取下 (图 2b)。</li>
	<li>剪断跟腱横向5毫米到其跟骨插入, 并删除一个5毫米长的部分使用剪刀。不要缝合肌腱 (图 2c, 2d)。</li>
	<li>用可吸收纱缝合筋膜, 然后用 overjet 缝合 (连续缝合)。也可以使用单丝缝合, 防止皮肤进入手术切口。</li>
	<li>把老鼠放在加热灯下, 直到醒来, 然后把老鼠放回笼子里 (58 x 38 厘米) (不给出): 不进行固定化.</li>
</ol>3. PRP 准备15
<ol><li>麻醉甲苯噻嗪 (10 毫克/千克体重) 和氯胺酮 (80 毫克/千克体重) 的供体大鼠腹腔注射。PRP 注射液术后2小时进行, 无再麻醉。</li>
	<li>注射50µL 的丁丙诺啡作为止痛药。</li>
	<li>收集全血 (每只老鼠20毫升, 最终出血) 的心脏穿刺到含有3.2% 缓冲柠檬酸钠 (0.109 米, 抗凝血) 的不育管。然后用心内注射弄死大鼠。</li>
	<li>为了获得 PRP, 将血液离心10分钟, 在 150 x g 和室温。这一初始的低速离心步骤产生两个不同的阶段: 一个较低的阶段, 由红细胞 (红细胞) 占总体积的约 80-90%, 和一个上阶段由富含血小板的血浆 (PRP) (通常为10-20% 的血液示例)。</li>
	<li>使用塑料转移吸管轻轻地将 PRP (上部相) 收集在二次塑料管上。由于 PRP 与红细胞之间的界面非常松散, 不能使吸管太靠近界面。如果处理得当, 在 PRP 中污染红细胞应低于 0.05 x 103单元格/µL. 丢弃余下的下部和原血收集管。</li>
	<li>确定收集的 PRP 的体积和测量血液分析仪的血小板计数。在接下来的步骤中, 需要使用这些值来调整血小板浓度。血液细胞计数也是有用的评估潜在的污染与红细胞和 WBCs. 在 PRP 的血小板浓度在这个阶段通常是 1-1.5 x106/µL。</li>
	<li>在 1000 x g 和室温下进行10分钟的 PRP 第二次离心。这种高速离心步骤产生松散的血小板颗粒和上清, 由自体血小板贫乏血浆 (PPP) 组成。使用步骤3.6 中确定的值, 计算要丢弃的上清液的体积, 以浓缩 PRP 并达到 2.5 x106/µL 的最终浓度。</li>
	<li>使用塑料转移吸管轻轻地在二级塑料管中收集上清 (PPP), 在步骤3.7 中计算最终体积的大约两个。由于颗粒松散, 当 PPP 被丢弃时, 大量的血小板丢失, 因此有可能减少最终所需的浓度。</li>
	<li>用温和重复的吹打仔细并用重悬血小板颗粒与剩余清液。测量这个浓缩的 PRP 的血小板计数。如果需要, 添加适当体积的自体 PPP 达到最终目标浓度。 注: 使用 PRP 在3小时内准备。</li>
	<li>添加50µL 的 CaCl2 (11 mEq 10 毫升) 每毫升 PRP 激活血小板。</li>
	<li>将50µL 的新鲜 PRP 或盐水溶液与21G 针直接插入缝合手术部位, 约1小时后进行准备。在这段时间, 保持 PRP 在室温下。</li>
	<li>在手术后和跟腱切除前密切监测大鼠的功能恢复。</li>
</ol>4. 切除跟腱和生物力学测试16
<ol><li>5、15或30天后5, 对老鼠进行称量。然后, 麻醉与甲苯噻嗪 (10 毫克/千克体重) 和氯胺酮 (80 毫克/千克体重) 腹腔注射。</li>
	<li>注射50µL 的丁丙诺啡作为止痛药。在牺牲动物之前去掉肌腱, 尽可能长时间保持生理条件。</li>
	<li>剃掉左后肢, 把老鼠放在解剖显微镜下。将大鼠放在侧褥疮上, 用腿在上级位置进行手术。用手指握住爪子。</li>
	<li>在前一个手术部位做一个小切口 (10 毫米), 并延伸至三头肌 suralis 暴露。</li>
	<li>为了去除跟腱的愈合, 将跟骨横向5-10 毫米的跟腱连接。然后解剖一个部分的三头肌 suralis, 这是附加到跟腱 (图 3b)。肌肉部分必须足够大, 以适应低温颚 (图 3c-d)。使用镊子将样品立即放入冷冻颚 (图 3e)。</li>
	<li>切除肌肌腱-骨复合物后, 弄死大鼠心内注射戊巴比妥 (200 毫克/千克)。通过评估心脏节律和呼吸缺乏15分钟来确认死亡。</li>
	<li>将肌肉单元放入下颚 (图 3e), 关闭它并垂直放置到通用测试机 (106.2 kN,图 3a)。然后在机器的下部夹具之间修复骨骼 (图 3f)。</li>
	<li>在上颌骨的两个盆地中加入液氮, 以冻结肌肉, 使其在拉伸试验期间不变形, 比肌腱坚硬。</li>
	<li>当冻结区到达金属夹边时, 开始拉伸试验, 这样肌腱就能保持其结构。将机器的位移率设定在1毫米/秒的恒定速度直到破裂。记录在计算机上牛顿 (N) 给出的极限抗拉强度 (众信)。</li>
	<li>要计算横截面积, 请将两个相机垂直放置, 形成椭圆形形状, 拍照。</li>
	<li>为了解释愈合肌腱横截面积的差异, 将众信正常化为单位面积 (N 每平方毫米), 代表组织所经历的机械应力。</li>
</ol>5. 组织学分析
注: 每组15肌腱进行组织学分析。
<ol><li>切除后, 立即将跟腱浸入4% 多聚甲醛以保持其结构 (1 毫米的组织每小时固定, 所以固定时间因肌腱大小而异)。</li>
	<li>用70% 乙醇代替多聚甲醛, 在这个溶液中至少留出一晚。</li>
	<li>将肌腱放在一个带有孔的小塑料容器中, 然后将该容器放入新鲜的70% 乙醇溶液中1小时。</li>
	<li>将容器放置在95% 乙醇中, 重复1小时。</li>
	<li>将容器放置在100% 乙醇中, 重复1小时。</li>
	<li>将容器放置在100% 二甲苯中, 重复1小时。</li>
	<li>现在安置塑料容器, 包含肌腱, 在液体石蜡 (56 °c) 和离开它隔夜。</li>
	<li>使用嵌入站 (配有熔融蜡、热板和冷板), 选择最适合组织样品的金属模具, 然后用少许液体石蜡填充它, 以使底座覆盖。 注: 定位样品以获得切片的垂直剖面。</li>
	<li>当样品正确定位时, 用液体石蜡将模具填满, 放到冷冻机上。让它冷却至少15分钟。</li>
	<li>从石蜡块中取出, 放在冰上至少30分钟。</li>
	<li>准备几张干净的玻璃滑梯, 把一些去离子水滴在他们身上。</li>
	<li>在切片, 削减5µm 部分的区块 (只使用部分从肌腱的中部), 并将该节放在幻灯片上。</li>
	<li>将幻灯片放在65摄氏度的加热烤箱中, 使石蜡开始融化, 将组织粘到玻璃上。</li>
	<li>将含有石蜡切片的幻灯片放在切片架中。</li>
	<li>现在 deparaffinize 和水化组织通过放置在以下连续的浴池: 8分钟在二甲苯 (浴 1) 4分钟在二甲苯 (浴 2) 2分钟100% 乙醇 2分钟95% 乙醇 2分钟70% 乙醇 去离子水2分</li>
	<li>对于苏木精-伊红染色, 将组织置于下列溶液中。<ol>
<li>将组织浸泡在苏木精溶液中8分钟 (1 克苏木精一水合物, 0.2 g 克钦体3, 50 克 AIK (所以4)2. 12H2O; 200 毫升甘油; 用蒸馏水调整至1升)。</li>
		<li>在自来水下浸泡8分钟的组织。</li>
		<li>将组织浸泡在三十年代的伊红溶液中 (0.25% 在100毫升蒸馏水中含有200µL 的未稀释醋酸)。</li>
		<li>在自来水下浸泡2分钟的组织。</li>
	</ol>
</li>
	<li>对于马尾松的三色染色, 请按照下列步骤操作。<ol>
<li>在补液后, 将各部分放置在带有铁明矾的容器中 (0.5 克 (NH4) Fe (因此4)2. 12H2o 在10毫升 H2o) 并关闭容器。用微波炉加热3分钟, 280 瓦特。</li>
		<li>让它冷却一分钟, 然后用去离子水冲洗。</li>
		<li>将节放在一个封闭的容器中, Regaud 的苏木精溶液 (0.5 克苏木精溶解在50毫升 H2O 在50°c, 然后添加5毫升95% 乙醇和5毫升甘油), 加热它九十年代在 280 W。</li>
		<li>用去离子水冲洗。</li>
		<li>将剖面放置在苦味酸酸溶液中 (在甲醇饱和度) 为3分钟, 在运行自来水下冲洗5分钟, 然后将其放在去离子水中几秒钟。</li>
		<li>现在放置在酸性品红 (0.5 克50毫升 H2O 包含0.25 毫升未稀释的乙酸) 为 5 s 和冲洗在自来水下, 直到染料消失。</li>
		<li>在钼酸溶液中孵育切片 (0.5 克, 50 毫升 H2O) 为5分钟, 将其放在去离子水中几秒钟。</li>
		<li>将各节置于浅绿色 (0.5 克50毫升 H2O, 含有0.25 毫升未稀释醋酸) 溶液, 另5分钟, 并将其置于自来水中, 直到染料消失。</li>
	</ol>
</li>
	<li>现在再次脱水组织。<ol>
<li>将切片浸入70% 乙醇中1分钟。</li>
		<li>将切片浸入95% 乙醇中1分钟。</li>
		<li>将切片浸入100% 乙醇中1分钟。</li>
		<li>将该剖面浸入二甲苯 (浴 1) 中1分钟。</li>
		<li>将该剖面浸入二甲苯 (浴 2) 中1分钟。</li>
		<li>拆卸部分, 而不去除过多的二甲苯, 并在样品上放一滴安装介质。</li>
		<li>在上面放一个盖玻片 (不引发任何气泡), 让它硬化至少2小时。</li>
		<li>水解 6% HCl 中每组5个组织学切片 (每个不同的肌腱), 并测定胶原蛋白的浓度, 方法是在5µm 无瑕切片中测定羟脯氨酸作为指数。</li>
	</ol>
</li>
	<li>使用制造商的软件对分区区域的结果进行规范化。</li>
	<li>通过计算机辅助分析, 对部分带有浅绿色染色的部分进行染色, 以可视化和量化纤维胶原蛋白。</li>
	<li>扫描各部分, 并使用 IrfanView 软件转换为灰色细微差别的图片。使用软件进行半量化 (例如, 数量一)。</li>
</ol>6. 分子评价
<ol><li>肌腱破裂后, 在机械测试发生, 采取的样品, 捕捉冻结他们在液氮, 并储存在-80 °c (PRP 和盐水溶液)17。</li>
	<li>使用商业总 rna 套件隔离总 rna17。</li>
	<li>通过 rt-pcr17,18, 测量胶原蛋白 (i. 和 III.)、基质金属蛋白酶 (MMP-2、MMP-3 和 MMP-9) 和 tenomodulin (TNMD) 的表达。</li>
	<li>将 mRNA 的表达式级别规范化为 28S19的级别。</li>
</ol>

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C., 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=Growth+Factor-Rich+Plasma+Increases+Tendon+Cell+Proliferation+and+Matrix+Synthesis+on+a+Synthetic+Scaffold:+An+In+Vitro+Study.">Growth Factor-Rich Plasma Increases Tendon Cell Proliferation and Matrix Synthesis on a Synthetic Scaffold: An In Vitro Study.</a> Tissue Eng. Part A. 16, 1021-1029 (2010).</li><li>Zhang, J., Wang, J. H. -. C. <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+Releasate+Promotes+Differentiation+of+Tendon+Stem+Cells+Into+Active+Tenocytes.">Platelet-Rich Plasma Releasate Promotes Differentiation of Tendon Stem Cells Into Active Tenocytes.</a> Am. J. Sports Med. 38, 2477-2486 (2010).</li><li>Kajikawa, 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=Platelet-rich+plasma+enhances+the+initial+mobilization+of+circulation-derived+cells+for+tendon+healing.">Platelet-rich plasma enhances the initial mobilization of circulation-derived cells for tendon healing.</a> J. Cell. Physiol. 215, 837-845 (2008).</li><li>Pasternak, B., Aspenberg, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Metalloproteinases+and+their+inhibitors-diagnostic+and+therapeutic+opportunities+in+orthopedics.">Metalloproteinases and their inhibitors-diagnostic and therapeutic opportunities in orthopedics.</a> Acta Orthop. 80, 693-703 (2009).</li><li>Dell, R. B., Holleran, S., Ramakrishnan, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Sample+size+determination.">Sample size determination.</a> ILAR J. 43, 207-213 (2002).</li><li>M&#228;hler Convenor, 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=FELASA+recommendations+for+the+health+monitoring+of+mouse,+rat,+hamster,+guinea+pig+and+rabbit+colonies+in+breeding+and+experimental+units.">FELASA recommendations for the health monitoring of mouse, rat, hamster, guinea pig and rabbit colonies in breeding and experimental units.</a> Lab. Anim. 48, 178-192 (2014).</li><li>Kaux, J. -. F., 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=&#201;tude+comparative+de+cinq+techniques+de+pr&#233;paration+plaquettaire+(platelet-rich+plasma).">&#201;tude comparative de cinq techniques de pr&#233;paration plaquettaire (platelet-rich plasma).</a> Pathol. Biol. 59, 157-160 (2011).</li><li>Wieloch, P., Buchmann, G., Roth, W., Rickert, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+cryo-jaw+designed+for+in+vitro+tensile+testing+of+the+healing+Achilles+tendons+in+rats.">A cryo-jaw designed for in vitro tensile testing of the healing Achilles tendons in rats.</a> J. Biomech. 37, 1719-1722 (2004).</li><li>Kaux, J. -. F., 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=Vascular+Endothelial+Growth+Factor-111+(VEGF-111)+and+tendon+healing:+preliminary+results+in+a+rat+model+of+tendon+injury.">Vascular Endothelial Growth Factor-111 (VEGF-111) and tendon healing: preliminary results in a rat model of tendon injury.</a> Muscles. Ligaments Tendons J. 4, 24-28 (2014).</li><li>Docheva, D., Hunziker, E. B., F&#228;ssler, R., Brandau, O. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Tenomodulin+is+necessary+for+tenocyte+proliferation+and+tendon+maturation.">Tenomodulin is necessary for tenocyte proliferation and tendon maturation.</a> Mol. Cell. Biol. 25, 699-705 (2005).</li><li>Lambert, C. A., Colige, A. C., Munaut, C., Lapi&#232;re, C. M., Nusgens, B. V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Distinct+pathways+in+the+over-expression+of+matrix+metalloproteinases+in+human+fibroblasts+by+relaxation+of+mechanical+tension.">Distinct pathways in the over-expression of matrix metalloproteinases in human fibroblasts by relaxation of mechanical tension.</a> Matrix Biol. 20, 397-408 (2001).</li><li>Nurden, A. T., Nurden, P., Sanchez, M., Andia, I., Anitua, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Platelets+and+wound+healing.">Platelets and wound healing.</a> Front. Biosci. 13, 3532-3548 (2008).</li><li>Woodall, J., Tucci, M., Mishra, A., Benghuzzi, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cellular+effects+of+platelet+rich+plasma:+a+study+on+HL-60+macrophage-like+cells.">Cellular effects of platelet rich plasma: a study on HL-60 macrophage-like cells.</a> Biomed. Sci. Instrum. 43, 266-271 (2007).</li><li>Taylor, D. W., Petrera, M., Hendry, M., Theodoropoulos, J. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+systematic+review+of+the+use+of+platelet-rich+plasma+in+sports+medicine+as+a+new+treatment+for+tendon+and+ligament+injuries.">A systematic review of the use of platelet-rich plasma in sports medicine as a new treatment for tendon and ligament injuries.</a> Clin. J. Sport Med. 21, 344-352 (2011).</li><li>Mazzocca, A. D., 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+positive+effects+of+different+platelet-rich+plasma+methods+on+human+muscle,+bone,+and+tendon+cells.">The positive effects of different platelet-rich plasma methods on human muscle, bone, and tendon cells.</a> Am. J. Sports Med. 40, 1742-1749 (2012).</li><li>McCarrel, T. M., Minas, T., Fortier, L. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Optimization+of+leukocyte+concentration+in+platelet-rich+plasma+for+the+treatment+of+tendinopathy.">Optimization of leukocyte concentration in platelet-rich plasma for the treatment of tendinopathy.</a> J. Bone Joint Surg. Am. 94 (1-8), e143 (2012).</li><li>Boswell, S. 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=Increasing+platelet+concentrations+in+leukocyte-reduced+platelet-rich+plasma+decrease+collagen+gene+synthesis+in+tendons.">Increasing platelet concentrations in leukocyte-reduced platelet-rich plasma decrease collagen gene synthesis in tendons.</a> Am. J. Sports Med. 42, 42-49 (2014).</li><li>Virchenko, O., Aspenberg, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=How+can+one+platelet+injection+after+tendon+injury+lead+to+a+stronger+tendon+after+4+weeks?:+Interplay+between+early+regeneration+and+mechanical+stimulation.">How can one platelet injection after tendon injury lead to a stronger tendon after 4 weeks?: Interplay between early regeneration and mechanical stimulation.</a> Acta Orthop. 77, 806-812 (2006).</li></ol>

没有宣布利益冲突。

血小板对肌腱愈合过程的早期炎症期至关重要。当这些血小板暴露于结合组织或诱发凝血的因素时, 它们将释放在α颗粒中储存的生长因子。由于这种相互作用, 胞外基质大分子综合升华, 间充质细胞增殖。血小板也有一个趋化活性的祖细胞在血液循环, 增强血管生成和刺激细胞分化6,20。
生物力学测试是使用专门为体外拉伸试验的大?...

凝血, 炎症过程和血小板的免疫调节作用是众所周知的1。最近, 已经证明它们还具有恢复性属性2,3。事实上, 在脱粒期间血小板释放各种细胞因子和生长因子 (VEGF、血小板因子、TGF、生长因子 I 和 HGF)。这些生长因子促进血管生成, 组织重塑和伤口愈合 (骨骼, 皮肤, 肌肉, 肌腱)2。离心自体血液产生血小板丰富的血浆 (PRP), 其中含有高血小板浓度取决于隔离方法 (3 和10倍的血液基线浓度)。事实上, 各种 PRP 制备技术不能提供相同的最终产品。到目前为止, 在这一问题上尚未达成国际普遍协议。总的来说, PRP 可能是治疗慢性肌肉骨骼疾病的一种有吸引力的治疗方案, 如肌腱、足底筋膜炎、骨关节炎和不愈合的4。它第一次用于口腔外科和种植4 , 以改善和加速骨愈合后放置一个牙科植入。在本研究中, 我们描述了一种可重现的方法, 允许获取 PRP 的动物实验4。
由于肌腱的损伤在运动员和体力工作者中经常被观察到, 加强愈合过程, 从而减少恢复的时间是非常感兴趣的5。新的治疗方法往往涉及使用生长因子, 而 PRP 的管理是一种简单和微创的方式, 以提供混合的内生生长因子4。
一些体外或动物研究表明, 通过释放生物介质来管理含有高水平血小板的血浆, 可以通过释放生物介质来刺激肌腱和韧带的修复6 ,7,8,9。此外, 其他研究表明, PRP 可刺激肌腱细胞的 i 型和 III. 类胶原合成9,10,11。也有人建议, PRP 可以减少基质金属蛋白酶的活化, 从而减少基体的降解。参与炎症过程的细胞可以产生 MMP-9, 它在炎症12引起的组织重塑 (生理学和病理) 中起着作用。
根据这一信息, 我们假设单个 PRP 注射到大鼠的跟腱, 可以改善修复组织的恢复过程和机械强度。这是通过测量愈合肌腱在恢复过程中的生物力学性质, 并通过进行组织学和分子分析, 以评估新形成的组织胶原重塑的测试。本研究的目的是观察单注射同种异体 PRP 是否会影响切片跟腱的愈合。

<table><tbody><tr><td>Xylazine (Xyl-M)</td><td>VMD</td><td>none</td><td>anesthetic</td></tr><tr><td>Ketamin (J&amp;eacute;tamine 1000 CEVA)</td><td>CEVA Sant&amp;eacute; Animale</td><td>none</td><td>anesthetic</td></tr><tr><td>Buprenorphin (Veterg&amp;eacute;sic Multidosis)</td><td>ALSTOE</td><td>none</td><td>Painkiller</td></tr><tr><td>iso-Betadine</td><td>MEDA-Pharma</td><td>none</td><td>Desinfectant</td></tr><tr><td>resorbable yarn Vicryl 6/0</td><td>Johnson &amp;amp; Johnson</td><td></td><td></td></tr><tr><td>Nembutal</td><td>CEVA Sant&amp;eacute; Animale</td><td>none</td><td>Anesthetic</td></tr><tr><td>Paraformaldehyde</td><td>Sigma-Aldrich</td><td>P6148</td><td>Preserves structure of the tissue</td></tr><tr><td>Isopropanol 100%</td><td>VWR</td><td>20,922,364</td><td></td></tr><tr><td>Ethanol 95%</td><td>VWR</td><td>20,823,362</td><td></td></tr><tr><td>Xylene</td><td>VWR</td><td>28973.363</td><td></td></tr><tr><td>Paraffin</td><td>VWR</td><td>LEIC3950.1006</td><td></td></tr><tr><td>Hematoxylin</td><td>Millipore</td><td>1.15938.0025</td><td>Colorant</td></tr><tr><td>Eosin</td><td>Millipore</td><td>1.15935.0100</td><td>Colorant</td></tr><tr><td>Eukitt</td><td>Sigma-Aldrich</td><td>3989</td><td>Mounting Medium</td></tr><tr><td>CaCl2</td><td></td><td></td><td></td></tr></tbody></table>

effects of allogeneic platelet-rich plasma (prp) on the healing process of sectioned achilles tendons of rats: a methodological description

本文介绍了用于观察 PRP 是否能对肌腱愈合有积极影响的实验程序。有4个主要步骤要遵循: 诱发跟腱损伤;准备 PRP 并注入 (或盐水溶液);取下肌腱;并进行生物力学、分子和组织学评价。在每个步骤中, 都详细描述了所有的过程和方法, 因此可以很容易地进行复制。
跟腱已经手术切片 (切除5毫米长的部分)。然后, 注射 prp 或生理盐水来研究 prp 是否对肌腱愈合有积极作用。三组40只动物 (共120只大鼠在本研究中使用) 分为2组: PRP 注射液组和生理盐水注射控制小组。大鼠在增加的时间点牺牲 (A 组: 5 天;B 组:15 天;C 组:30 天) 和肌腱被删除。90肌腱在进行 transcriptomic 分析前进行生物力学测试, 其余30肌腱提交组织学分析。

结果以平均标准偏差表示, 并与方差分析进行了比较。使用了双向方差分析和后端测试 de Scheffé (即参数测试)。
引起大鼠非损伤性跟腱断裂的最终抗拉强度为 42.0, 5.7 n (n = 10)。5天后, 两组抗拉强度显著增加 (p < 0.0001)。对照组比较, 在任何时间测量, 特别是在15和30天, PRP 组的信众均较高。通过测量在进行生物力学评估前被...

Watch this Scientific Journal Video about Effects of Allogeneic Platelet-Rich Plasma PRP on the Healing Process of Sectioned Achilles Tendons of Rats: A Methodological Description at JoVE.com

Effects of Allogeneic Platelet-Rich Plasma PRP on the Healing Process of Sectioned Achilles Tendons of Rats: A Methodological Description

本协议描述了大鼠经同种异体血小板丰富血浆 (PRP) 或盐水溶液取出部分跟腱后愈合肌腱的评价过程。采用不同类型的分析方法对肌腱愈合的进展进行评价。

同种异体富血小板血浆 (PRP) 对大鼠分段腱愈合过程的影响: 方法学描述

This article describes the experimental procedures used to observe if PRP can positively affect tendon healing. There are 4 main steps to follow: induce a ...

effects-allogeneic-platelet-rich-plasma-prp-on-healing-process

Research

JoVE Journal

Medicine

Effects of Allogeneic Platelet-Rich Plasma (PRP) on the Healing Process of Sectioned Achilles Tendons of Rats: A Methodological Description

9.2K 次观看.  University of Liège. 本协议描述了大鼠经同种异体血小板丰富血浆 (PRP) 或盐水溶液取出部分跟腱后愈合肌腱的评价过程。采用不同类型的分析方法对肌腱愈合的进展进行评价。

视频: Effects of Allogeneic Platelet-Rich Plasma PRP on the Healing Process of Sectioned Achilles Tendons of Rats: A Methodological Description

Use of Human Perivascular Stem Cells for Bone Regeneration

Human perivascular stem cells (PSCs) can be isolated in sufficient numbers from multiple tissues for purposes of skeletal tissue engineering1-3. PSCs are a FACS-sorted population of 'pericytes' (CD146+CD34-CD45-) and 'adventitial cells' (CD146-CD34+CD45-), each of which we have previously reported to have properties of mesenchymal stem cells. PSCs, like MSCs, are able to undergo osteogenic differentiation, as well as secrete pro-osteogenic cytokines1,2. In the present protocol, we demonstrate the osteogenicity of PSCs in several animal models including a muscle pouch implantation in SCID (severe combined immunodeficient) mice, a SCID mouse calvarial defect and a femoral segmental defect (FSD) in athymic rats. The thigh muscle pouch model is used to assess ectopic bone formation. Calvarial defects are centered on the parietal bone and are standardly 4 mm in diameter (critically sized)8. FSDs are bicortical and are stabilized with a polyethylene bar and K-wires4. The FSD described is also a critical size defect, which does not significantly heal on its own4. In contrast, if stem cells or growth factors are added to the defect site, significant bone regeneration can be appreciated. The overall goal of PSC xenografting is to demonstrate the osteogenic capability of this cell type in both ectopic and orthotopic bone regeneration models.

Human perivascular stem cells (PSCs) are a novel stem cell class for skeletal tissue regeneration similar to mesenchymal stem cells (MSCs). PSCs can be isolated by FACS (fluorescence activated cell sorting) from adipose tissue procured during standard liposuction procedures, then combined with an osteoinductive scaffold to achieve bone formation in vivo.

人类血管周围干细胞用于骨再生

Human perivascular stem cells (PSCs) can be isolated in sufficient numbers from multiple tissues for purposes of skeletal tissue engineering1-3. PSCs are ...

科研

生物工程

Athymic Rat Model for Evaluation of Engineered Anterior Cruciate Ligament Grafts

Anterior cruciate ligament (ACL) rupture is a common ligamentous injury that often requires surgery because the ACL does not heal well without intervention. Current treatment strategies include ligament reconstruction with either autograft or allograft, which each have their associated limitations. Thus, there is interest in designing a tissue-engineered graft for use in ACL reconstruction. We describe the fabrication of an electrospun polymer graft for use in ACL tissue engineering. This polycaprolactone graft is biocompatible, biodegradable, porous, and is comprised of aligned fibers. Because an animal model is necessary to evaluate such a graft, this paper describes an intra-articular athymic rat model of ACL reconstruction that can be used to evaluate engineered grafts, including those seeded with xenogeneic cells. Representative histology and biomechanical testing results at 16 weeks postoperatively are presented, with grafts tested immediately post-implantation and contralateral native ACLs serving as controls. The present study provides a reproducible animal model with which to evaluate tissue engineered ACL grafts, and demonstrates the potential of a regenerative medicine approach to treatment of ACL rupture.

Animal models are important tools for the evaluation of tissue-engineered grafts. This paper presents the protocol for preparing an electrospun biodegradable polymer graft for use in anterior cruciate ligament tissue engineering, as well as a surgical protocol for implantation in a rat model.

无胸​​腺大鼠模型工程前交叉韧带移植评估

Anterior cruciate ligament (ACL) rupture is a common ligamentous injury that often requires surgery because the ACL does not heal well without ...

Preparing Activated Platelet-Rich Plasma for Culturing Human Adipose-Derived Stem Cells

Activated platelet-rich plasma (PRP) prepared from whole blood via centrifugation demonstrated a proliferation-stimulating effect in several kinds of cultured cells, implying a possible use in regenerative medicine. Here, a double-spin method was used to prepare PRP from whole blood. PRP was further activated by autologous thrombin. The platelet count was measured in the activated PRP and the proliferation-stimulating effect in human adipose-derived stem cells (hASCs) was examined. The resulting platelet count was 11.5-times higher in PRP than in whole blood plasma. The proliferation of hASCs was markedly enhanced by incubation with 1% PRP. The described method can be used to reproducibly prepare PRP with a high concentration of platelets. PRP prepared by this method markedly promotes proliferation of hASCs.

In this report, we describe a double-spin method for preparing activated platelet-rich plasma (PRP). Using autologous thrombin, human adipose-derived stem cells (hASCs) were cultured. Activated PRP was shown to promote proliferation of hASCs.

Activated platelet-rich plasma (PRP) prepared from whole blood via centrifugation demonstrated a proliferation-stimulating effect in several kinds of ...

发育生物学

A Rat Tibial Growth Plate Injury Model to Characterize Repair Mechanisms and Evaluate Growth Plate Regeneration Strategies

A third of all pediatric fractures involve the growth plate and can result in impaired bone growth. The growth plate (or physis) is cartilage tissue found at the end of all long bones in children that is responsible for longitudinal bone growth. Once damaged, cartilage tissue within the growth plate can undergo premature ossification and lead to unwanted bony repair tissue, which forms a "bony bar." In some cases, this bony bar can result in bone growth deformities, such as angular deformities, or it can completely halt longitudinal bone growth. There is currently no clinical treatment that can fully repair an injured growth plate. Using an animal model of growth plate injury to better understand the mechanisms underlying bony bar formation and to identify ways to inhibit it is a great opportunity to develop better treatments for growth plate injuries. This protocol describes how to disrupt the rat proximal tibial growth plate using a drill-hole defect. This small animal model reliably produces a bony bar and can result in growth deformities similar to those seen in children. This model allows for investigation into the molecular mechanisms of bony bar formation and serves as a means to test potential treatment options for growth plate injuries.

The growth plate is a cartilaginous region in children's long bones where longitudinal growth occurs. When injured, bony tissue can form and impair growth. We describe a rat model of growth plate injury that leads to bony repair tissue, allowing the study of repair mechanisms and growth plate regeneration strategies.

大鼠胫骨生长板损伤模型，用于表征修复机制并评估生长板再生策略

A third of all pediatric fractures involve the growth plate and can result in impaired bone growth. The growth plate (or physis) is cartilage tissue found ...

医学

Evaluation of Stem Cell Therapies in a Bilateral Patellar Tendon Injury Model in Rats

Regenerative medicine provides novel alternatives to conditions that challenge traditional treatments. The prevalence and morbidity of tendinopathy across species, combined with the limited healing properties of this tissue, have prompted the search for cellular therapies and propelled the development of experimental models to study their efficacy. Umbilical cord matrix-derived mesenchymal stem cells (UCM-MSC) are appealing candidates because they are abundant, easy to collect, circumvent the ethical concerns and risk of teratoma formation, yet resemble primitive embryonic stem cells more closely than adult tissue-derived MSCs. Significant interest has focused on chitosan as a strategy to enhance the properties of MSCs through spheroid formation. This paper details techniques to isolate UCM-MSCs, prepare spheroids on chitosan film, and analyze the effect of spheroid formation on surface marker expression. Consequently, creation of a bilateral patellar tendon injury model in rats is described for in vivo implantation of UCM-MSC spheroids formed on chitosan film. No complication was observed in the study with respect to morbidity, stress rising effects, or tissue infection. The total functional score of the operated rats at 7 days was lower than that of normal rats, but returned to normal within 28 days after surgery. Histological scores of tissue-healing confirmed the presence of a clot in treated defects evaluated at 7 days, absence of foreign body reaction, and progressing healing at 28 days. This bilateral patella tendon defect model controls inter-individual variation via creation of an internal control in each rat, was associated with acceptable morbidity, and allowed detection of differences between untreated tendons and treatments.

This paper describes the preparation and evaluation of umbilical cord matrix-derived mesenchymal stem cells spheroids with a bilateral patellar tendon defect model in a rat. This model was associated with an acceptable morbidity and was found to detect differences between untreated and treated tendons, and between the two treatments tested.

大鼠双侧髌腱损伤模型中干细胞治疗的评价

Regenerative medicine provides novel alternatives to conditions that challenge traditional treatments. The prevalence and morbidity of tendinopathy across ...

Preparation, Procedures and Evaluation of Platelet-Rich Plasma Injection in the Treatment of Knee Osteoarthritis

Knee osteoarthritis (KOA) is one of the most frequently encountered diseases in the orthopedic department. Existing non-surgical treatments have a limited effect on the repair of cartilage and on bone regeneration. Platelet-rich plasma (PRP) is an autologous bioactive substance that can repair cartilage injury and accelerate bone regeneration effectively. However, reporting of PRP preparation protocols in clinical studies is highly inconsistent, with the majority of studies providing insufficient information to allow the protocol to be reproduced. We describe a repeatable method of preparing PRP visually, the treatment of KOA using PRP intra-articular injection, and methods of evaluating the outcome. PRP was prepared using manual double centrifugation. The PRP layer was extracted from peripheral blood and used for knee joint cavity injection. Evaluations included assessments of blood platelet concentrations and clinical outcomes. Preparation of PRP by manual centrifugation requires less apparatus and is less costly than plasma filtration or centrifugation using equipment. The centrifugation time of our double centrifugation method was 6 and 5 minutes for the respective centrifugations at forces of 800 and 1400 x g, respectively, to allow for the consistent preparation of standardized PRP. However, a manual method is susceptible to operator error, and PRP batch preparation is not available. Intra-articular injection of PRP proved to be an effective treatment for knee osteoarthritis. The entire treatment procedure took less than 30 minutes, the blood platelet concentration of PRP could be standardized, and treatment was proven to be effective when evaluated by follow-up.

Knee osteoarthritis is frequently seen in the orthopedic department. We introduce in detail the entire knee osteoarthritis treatment process with platelet-rich plasma injection, including preparation, procedures, and evaluation.

富血小板血浆注射液治疗膝关节骨关节炎的制备、程序及评价

Knee osteoarthritis (KOA) is one of the most frequently encountered diseases in the orthopedic department. Existing non-surgical treatments have a limited ...

Clinical Protocol of Producing Adipose Tissue-Derived Stromal Vascular Fraction for Potential Cartilage Regeneration

Osteoarthritis (OA) is one of the most common debilitating disorders.&#160;Recently, numerous attempts have been made to improve the functions of the knees by using different forms of mesenchymal stem cells (MSCs). In Korea, bone marrow concentrates and cord blood-derived stem cells have been approved by the Korean Food and Drug Administration (KFDA) for cartilage regeneration. In addition, an adipose tissue-derived stromal vascular fraction (SVF) has been allowed by the KFDA for joint injections in human patients. Autologous adipose tissue-derived SVF contains extracellular matrix (ECM) in addition to mesenchymal stem cells. ECM excretes various cytokines that, along with hyaluronic acid (HA) and platelet-rich plasma (PRP) activated by calcium chloride, may help MSCs to regenerate cartilage and improve knee functions. In this article, we presented a protocol to improve knee functions by regenerating cartilage-like tissue in human patients with OA. The result of the protocol was first reported in 2011 followed by a few additional publications. The protocol involves liposuction to obtain autologous lipoaspirates that are mixed with collagenase. This lipoaspirates-collagenase mixture is then cut and homogenized to remove large fibrous tissue that may clog up the needle during the injection. Afterwards, the mixture is incubated to obtain adipose tissue-derived SVF. The resulting adipose tissue-derived SVF, containing both adipose tissue-derived MSCs and remnants of ECM, is injected into knees of patients, combined with HA and calcium chloride activated PRP. Included are three cases of patients who were treated with our protocol resulting in improvement of knee pain, swelling, and range of motion along with MRI evidence of hyaline cartilage-like tissue.

Here, we present a protocol to produce an adipose tissue-derived stromal vascular fraction and its application to improve knee functions by regenerating cartilage-like tissue in human patients with osteoarthritis.

制备脂肪组织基质血管部分用于潜在软骨再生的临床协议

Osteoarthritis (OA) is one of the most common debilitating disorders.&#160;Recently, numerous attempts have been made to improve the functions of the ...

了解肌腱撞击 – 用于研究机械生物学的小鼠外植体模型

Murine Hind Limb Explant Model for Studying the Mechanobiology of Achilles Tendon Impingement

Tendon impingement upon bone generates a multiaxial mechanical strain environment with markedly elevated transverse compressive strain, which elicits a localized fibrocartilage phenotype characterized by accumulation of glycosaminoglycan (GAG)-rich matrix and remodeling of the collagen network. While fibrocartilage is a normal feature in impinged regions of healthy tendons, excess GAG deposition and disorganization of the collagen network are hallmark features of tendinopathy. Accordingly, impingement is clinically recognized as an important extrinsic factor in the initiation and progression of tendinopathy. Nevertheless, the mechanobiology underlying tendon impingement remains understudied. Prior efforts to elucidate the cellular response to tendon impingement have applied uniaxial compression to cells and excised tendon explants in vitro. However, isolated cells lack a three-dimensional extracellular environment crucial to mechanoresponse, and both in vitro and excised explant studies fail to recapitulate the multiaxial strain environment generated by tendon impingement in vivo, which depends on anatomical features of the impinged region. Moreover, in vivo models of tendon impingement lack control over the mechanical strain environment. To overcome these limitations, we present a novel murine hind limb explant model suitable for studying the mechanobiology of Achilles tendon impingement. This model maintains the Achilles tendon in situ to preserve local anatomy and reproduces the multiaxial strain environment generated by impingement of the Achilles tendon insertion upon the calcaneus during passively applied ankle dorsiflexion while retaining cells within their native environment. We describe a tissue culture protocol integral to this model and present data establishing sustained explant viability over 7 days. The representative results demonstrate enhanced histological GAG staining and decreased collagen fiber alignment secondary to impingement, suggesting elevated fibrocartilage formation. This model can easily be adapted to investigate different mechanical loading regimens and allows for the manipulation of molecular pathways of interest to identify mechanisms mediating phenotypic change in the Achilles tendon in response to impingement.

作者聚焦：揭开肌腱撞击的力学生物学 – 多轴鼠后肢外植体模型 

We present a custom experimental platform and tissue culture protocol that recreates fibrocartilaginous change driven by impingement of the Achilles tendon insertion in murine hind limb explants with sustained cell viability, providing a model suitable for exploring the mechanobiology of tendon impingement.

小鼠后肢外植体研究跟腱撞击力学生物学的模型

Tendon impingement upon bone generates a multiaxial mechanical strain environment with markedly elevated transverse compressive strain, which elicits a ...

同种异体富血小板血浆 (PRP) 对大鼠分段腱愈合过程的影响: 方法学描述

摘要

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此视频中的章节

人类血管周围干细胞用于骨再生

无胸腺大鼠模型工程前交叉韧带移植评估

Preparing Activated Platelet-Rich Plasma for Culturing Human Adipose-Derived Stem Cells

大鼠胫骨生长板损伤模型，用于表征修复机制并评估生长板再生策略

大鼠双侧髌腱损伤模型中干细胞治疗的评价

富血小板血浆注射液治疗膝关节骨关节炎的制备、程序及评价

制备脂肪组织基质血管部分用于潜在软骨再生的临床协议

小鼠后肢外植体研究跟腱撞击力学生物学的模型

小鼠后肢外植体研究跟腱撞击力学生物学的模型

小鼠后肢外植体研究跟腱撞击力学生物学的模型

同种异体富血小板血浆 (PRP) 对大鼠分段腱愈合过程的影响: 方法学描述

摘要

探索更多视频

此视频中的章节

人类血管周围干细胞用于骨再生

无胸​​腺大鼠模型工程前交叉韧带移植评估

Preparing Activated Platelet-Rich Plasma for Culturing Human Adipose-Derived Stem Cells

大鼠胫骨生长板损伤模型，用于表征修复机制并评估生长板再生策略

大鼠双侧髌腱损伤模型中干细胞治疗的评价

富血小板血浆注射液治疗膝关节骨关节炎的制备、程序及评价

制备脂肪组织基质血管部分用于潜在软骨再生的临床协议

小鼠后肢外植体研究跟腱撞击力学生物学的模型

小鼠后肢外植体研究跟腱撞击力学生物学的模型

小鼠后肢外植体研究跟腱撞击力学生物学的模型

无胸腺大鼠模型工程前交叉韧带移植评估