这项研究得到了韩国卫生技术研发 & 发展研究所 (KHIDI) 的资助, 该项目由大韩民国卫生部 & 福利部提供资金 (赠款号: HI16C1559)。

该议定书是根据《赫尔辛基宣言》进行的, 《议定书》是在茶 Bundang 医疗中心的机构审查委员会批准的。
1. 退行性肌腱组织从患者中收获
<ol><li>诊断侧髁的病史和使用体检结果: 压痛在腱的起源接近侧髁;疼痛通过腕关节的伸展而诱发。</li>
	<li>使用以下纳入标准:18 岁以上;侧髁的历史至少六月;非手术治疗顽固;自注射皮质类固醇、富含血小板血浆或肉毒杆菌毒素以来, 至少有三月的时间过去了。</li>
	<li>排除下列情况: 孕妇;有颈椎病病史、肘关节关节炎、rheumatologic 病、肘关节手术及神经卡压综合征的患者;患者拒绝捐献组织。</li>
	<li>
外科技术 注: 见图 124。<ol>
<li>经异氟醚和气管插管全麻后, 将手臂放在桌子上, 应用止血带。然后在整个手臂上应用 betadine 剂, 用无菌手术片进行悬垂。</li>
		<li>将侧肘暴露在3–5厘米切口上, 用15号手术手术刀刀片内侧侧向髁和近端到关节的水平。</li>
		<li>视觉识别伸腕桡肌 (ECRL) 和伸腱界面, 并作出分裂切口2–3毫米深度之间 ECRL 和伸腱与15号手术刀刀片在确定的界面。</li>
		<li>提取 ECRL 前方, 使病理伸腕桡短肌 (ECRB) 的起源直接的看法。</li>
		<li>以暗灰色和通常水肿、易碎的组织为特征, 以视觉识别病理退行性组织。</li>
		<li>鉴定后, 用15号手术手术刀和迷你刀片仔细切除所有退行性组织。</li>
		<li>将切除的组织 (约1厘米3) 立即嵌入磷酸盐缓冲盐水 (pbs), 并尽快将肌腱组织以外的所有周围组织移除到大约10毫升的 pbs 中。</li>
		<li>在侧髁骨遗传性的情况下, 用钳将遗传性移除, 并抚平锉。在前外侧髁上制造多个钻孔 (直径1.6 毫米, 约6–8孔), 以增强血管供应。这是可选的。</li>
		<li>用不可吸收的缝合材料将伤口层层闭合。</li>
	</ol>
</li>
	<li>
健康控制 tenocyte
	<ol>
<li>在前交叉韧带重建过程中, 从自腿筋或自髌肌腱的剩余肌腱中获得健康肌腱细胞, 并以此为对照试验25。 注: 本研究中使用的样本数为每组三。</li>
	</ol>
</li>
</ol>2. 组织学
<ol><li>储存大约一半的收获组织在中性缓冲10% 福尔马林在化学油烟机或同等。</li>
	<li>将组织嵌入石蜡块 (第4-5 毫升), 并在第4-5 µm 节封闭。</li>
	<li>
染色部分从控制和退化肌腱与苏木精和伊红染色 (H & E) 和阿利新蓝染色的 pH 值为2.5。在自动染色机上进行 H & E 染色, 如下所示。
	<ol>
<li>Deparaffinize 使用二甲苯 (分别为3次, 5 分钟)。</li>
		<li>从分级醇到水的水合物 (100% 酒精, 4 分钟; 100% 酒精, 3 分钟; 95% 酒精, 2 分钟; 70% 酒精, 1 分钟; 水, 2 分钟)。</li>
		<li>在苏木精染色5分钟, 在水中洗好2分钟。</li>
		<li>区分在1% 酸酒精 (1% HCl 在70% 酒精) 为 3 s. 洗涤井在水中为3分钟。</li>
		<li>用0.5% 氨浸泡十年代, 然后用3分钟的水洗法中和 HCl。</li>
		<li>在 Eosion 染色5分钟。</li>
		<li>脱水通过酒精 (80% 酒精, 三十年代; 95% 酒精, 1 分钟; 100% 酒精1分钟, 100% 酒精2分钟)。</li>
		<li>在二甲苯中清除 (分别为3次、2分钟、2分钟和3分钟)。</li>
		<li>装上封面幻灯片。</li>
	</ol>
</li>
	<li>
immunohistochemitry (IHC) 使用粘结聚合物精制检测试剂盒, 根据制造商的指示, 稍加修改。
	<ol>
<li>deparaffinization 使用二甲苯后, 用键表位检索溶液进行抗原检索, 30 分钟100摄氏度。键表位检索解决方案含有柠檬酸盐基缓冲剂和表面活性剂 (1 升, pH 5.9–6.1)。</li>
		<li>用过氧化氢孵化组织内源性过氧化物5分钟。</li>
		<li>在室温下 (21-22 °c) 孵化出15分钟的切片, 主要抗体为血管内皮生长因子 (VEGF) (1:200), tenomodulin (1:200), 或莫霍克 (1:200)。</li>
		<li>用无生物素的高分子辣根过氧化物酶-链接器抗体共轭系统进行二次抗体标记。<ol>
<li>在 10% (v/v) 动物血清中使用二次兔抗小鼠 IgG, 在三缓冲盐水/0.09% 2-甲基 4-异噻唑啉 3-一溶液中定位小鼠抗体。</li>
			<li>在三缓冲盐水/0.09% 中使用含有 10% (v/v) 动物血清的高分子抗兔多酶 IgG (< 25 µg/毫升) 对兔抗体进行局部定位。</li>
			<li>使用 33 '-Diaminobenzidine tetrahydrochloride 水合物在稳定剂的解决方案, 以可视化的复杂通过褐色沉淀。</li>
			<li>Counterstain < 0.1% 苏木精 (蓝色), 以可视化细胞核。</li>
		</ol>
</li>
	</ol>
</li>
	<li>观察在光镜下用 H & E、阿利新蓝污渍和 IHC 为 VEGF、tenomodulin 和莫霍克染色的幻灯片, 并获得在一个高功率场 (400X) 下每张幻灯片的代表性显微图像。</li>
</ol>3. Tenocyte 文化
<ol><li>
组织准备
	<ol>
<li>用微剪刀和镊子彻底去除周围组织, 准备肌腱标本。</li>
		<li>用 PBS 和碎肉清洗成小块 (大约1毫米3或更少)。</li>
		<li>1小时的消化组织在30毫升的 Dulbecco 的改良鹰的培养基 (DMEM) 补充30毫克/毫升胶原酶 II 在37°c。</li>
		<li>通过100µm 细胞过滤器传递细胞。</li>
		<li>添加1毫升胎牛血清 (血清), 停用胶原酶 II。</li>
		<li>离心机在 196 x g 5 分钟。</li>
	</ol>
</li>
	<li>
细胞培养
	<ol>
<li>种子 3 x 105细胞成100毫米菜和文化与 DMEM 补充与10% 个血清和1% 个抗生素-防霉解答在37摄氏度在5% 个 CO2个孵化器为1星期。</li>
		<li>每三天更改一次培养基, 直到80% 汇合发生。培养健康的控制和退化细胞样本直到四, 并用于实验。</li>
	</ol>
</li>
</ol>4. 免疫细胞化学 (ICC)
<ol><li>将培养细胞的200µL (2 x 105细胞) 转移到腔室的八口井中, 并将细胞生长, 使其与新鲜培养基的添加融合在一起1天。</li>
	<li>用 PBS 冲洗细胞, 用500µL 4% 甲醛。</li>
	<li>在 PBS 中孵化0.5% 海卫 X-100, 通透膜的10分钟。</li>
	<li>以1% 牛血清白蛋白和 0.05% TWEEN-20 为莫霍克和 tenomodulin 的 PBS 块。</li>
	<li>在黑暗中孵育鼠抗莫霍克的单克隆抗体 (1:100 稀释) 和山羊抗 tenomodulin 抗体 (1:100 稀释) 在4°c 过夜。</li>
	<li>孵化二级抗体 (Alexa 555-抗山羊 (1:200) 为 tenomodulin;Alexa 488-反老鼠 (1:200) 为莫霍克) 在黑暗中为1小时的室温。</li>
	<li>安装0.5 µL 的荧光安装介质。</li>
	<li>使用荧光显微镜 (400X) 进行分析。</li>
</ol>5. 扩散试验
<ol><li>
用水溶性四氮唑 (WST)-1 测定细胞可行性。
	<ol>
<li>将健康和退化的细胞种子分为96个井板, 密度为 2 x 103细胞, 每井为24、72和 120 h。</li>
		<li>孵化后, 添加10µL 的 10% WST-1 解决方案, 每个井。</li>
		<li>孵化板材为3小时在37°c 在 5% CO2。</li>
		<li>使用微板块读取器测量 450 nm 的光学密度。在每个样品的时间点上至少取三次读数。</li>
	</ol>
</li>
	<li>
用 BrdU 法测定细胞 DNA 量。
	<ol>
<li>将健康和退化的细胞种子分为96井板, 密度为 2 x 103细胞, 每井为24、72和 120 h。</li>
		<li>孵化后, 添加10µL 10% BrdU 溶液的每一个井。</li>
		<li>孵化板材为3小时在37°c 在 5% CO2。</li>
		<li>添加100µL/变性溶液 (60–70% 乙醇, 0.1–0.5% 氢氧化钠), 并保持在室温下的板材30分钟。</li>
		<li>添加100µL/良好的1x 检测抗体溶液, 并保持在室温下的板材1小时。</li>
		<li>用1x 洗涤缓冲器去除溶液和洗涤板三次。</li>
		<li>添加100µL/精心制备的1x 酶共轭二次抗体溶液, 并保持在室温下的板30分钟。</li>
		<li>用1x 洗涤缓冲器去除溶液, 洗盘子三次。</li>
		<li>添加100µL 50% TMB 基板。</li>
		<li>室温下孵育30分钟。</li>
		<li>添加100µL 的停止解决方案。</li>
		<li>使用微板块读取器测量 450 nm 的光学密度。 注: 每样样品在所有时间点至少取三次读数。</li>
	</ol>
</li>
</ol>6. 统计分析
<ol><li>使用 SPSS 收集和分析数据。</li>
	<li>用平均标准误差描述数据。</li>
	<li>使用曼尼特. U 测试。</li>
	<li>当p值小于 0.05 (p < 0.05) 时, 请考虑统计学意义上的差异。</li>
</ol>

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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=Effect+of+dexamethasone+on+cultured+human+tenocytes+and+its+reversibility+by+platelet-derived+growth+factor.">Effect of dexamethasone on cultured human tenocytes and its reversibility by platelet-derived growth factor.</a> Journal of Bone and Joint Surgery American Volume. 85 (10), 1914-1920 (2003).</li><li>Tempfer, H., 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=Effects+of+crystalline+glucocorticoid+triamcinolone+acetonide+on+cultered+human+supraspinatus+tendon+cells.">Effects of crystalline glucocorticoid triamcinolone acetonide on cultered human supraspinatus tendon cells.</a> Acta Orthopaedics. 80 (3), 357-362 (2009).</li><li>Menon, 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=New+insights+in+extracellular+matrix+remodeling+and+collagen+turnover+related+pathways+in+cultured+human+tenocytes+after+ciprofloxacin+administration.">New insights in extracellular matrix remodeling and collagen turnover related pathways in cultured human tenocytes after ciprofloxacin administration.</a> Muscles Ligaments Tendons J. 3 (3), 122-131 (2013).</li><li>Backman, L. J., Fong, G., Andersson, G., Scott, A., Danielson, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Substance+P+is+a+mechanoresponsive,+autocrine+regulator+of+human+tenocyte+proliferation.">Substance P is a mechanoresponsive, autocrine regulator of human tenocyte proliferation.</a> PLoS One. 6 (11), 27209 (2011).</li><li>Backman, L. J., Eriksson, D. E., Danielson, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Substance+P+reduces+TNF-alpha-induced+apoptosis+in+human+tenocytes+through+NK-1+receptor+stimulation.">Substance P reduces TNF-alpha-induced apoptosis in human tenocytes through NK-1 receptor stimulation.</a> Britich Journal of Sports Medicine. 48 (19), 1414-1420 (2014).</li><li>Rolf, C. G., Fu, B. S., Pau, A., Wang, W., Chan, B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Increased+cell+proliferation+and+associated+expression+of+PDGFRbeta+causing+hypercellularity+in+patellar+tendinosis.">Increased cell proliferation and associated expression of PDGFRbeta causing hypercellularity in patellar tendinosis.</a> Rheumatology (Oxford). 40 (3), 256-261 (2001).</li><li>Hoppe, S., 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=Tenocytes+of+chronic+rotator+cuff+tendon+tears+can+be+stimulated+by+platelet-released+growth+factors.">Tenocytes of chronic rotator cuff tendon tears can be stimulated by platelet-released growth factors.</a> Journal of Shoulder and Elbow Surgery. 22 (3), 340-349 (2013).</li><li>Schipper, O. N., Dunn, J. H., Ochiai, D. H., Donovan, J. S., Nirschl, R. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Nirschl+surgical+technique+for+concomitant+lateral+and+medial+elbow+tendinosis:+a+retrospective+review+of+53+elbows+with+a+mean+follow-up+of+11.7+years.">Nirschl surgical technique for concomitant lateral and medial elbow tendinosis: a retrospective review of 53 elbows with a mean follow-up of 11.7 years.</a> American Journal of Sports Medicine. 39 (5), 972-976 (2011).</li><li>Cook, J. L., Feller, J. A., Bonar, S. F., Khan, K. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Abnormal+tenocyte+morphology+is+more+prevalent+than+collagen+disruption+in+asymptomatic+athletes'+patellar+tendons.">Abnormal tenocyte morphology is more prevalent than collagen disruption in asymptomatic athletes' patellar tendons.</a> Journal of Orthopedic Research. 22 (2), 334-338 (2004).</li><li>Yuan, 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=Creating+an+Animal+Model+of+Tendinopathy+by+Inducing+Chondrogenic+Differentiation+with+Kartogenin.">Creating an Animal Model of Tendinopathy by Inducing Chondrogenic Differentiation with Kartogenin.</a> PLoS One. 11 (2), 0148557 (2016).</li><li>Lui, P. P., Maffulli, N., Rolf, C., Smith, R. K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=What+are+the+validated+animal+models+for+tendinopathy.">What are the validated animal models for tendinopathy.</a> Scandinavian Journal of Medical Scientific Sports. 21 (1), 3-17 (2011).</li><li>Wilhelm, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Lateral+epicondylitis+review+and+current+concepts.">Lateral epicondylitis review and current concepts.</a> Journal of Hand Surgery American Volume. 34 (7), 1359-1360 (2009).</li></ol>

作者没有什么可透露的。

以前的一些研究报告了如何使用不同的程序, 如胶原酶或 kartogenin 注射, 跑步机跑步, 以及更多的26,27建立慢性 tendinopathic 动物模型。虽然许多研究显示有希望的治疗效果的基础上这些动物模型, 使用人类退行性 tenocyte 的实验将是至关重要的肌腱领域, 以重现疗效的治疗。在这篇文章中, 我们建立了一个成功地收获和文化的人类退化肌腱细胞一致的协议。?...

肌腱是一种慢性退行性肌肉骨骼的情况, 发展在身体的各个部分。最近, 由于越来越多地参与娱乐运动和提高预期寿命1、2, 发达世界的肌腱病例数量大大增加。肌腱的病因被认为是多因素的, 这些原因包括缺血、氧自由基损伤、收缩与血管扩张 innervations 的不平衡、内微泪和神经调节的变化3 ,4,5,6,7,8。大多数治疗肌腱只会缓解症状。此外, 没有组织再生的治疗需要很长的时间来恢复和获得有限的反应肌腱, 这给医生9的临床挑战。
目前治疗方案的无能以及缺乏退行性肌腱的自愈能力, 使研究人员对探索替代治疗策略感兴趣。最近, 新的研究报告了许多有希望的结果, 以提高肌腱肌腱的愈合效果, 使用生长因子, 干细胞为主的治疗, 和基因治疗10,11,12。
通过文献综述, 我们发现, 所涉及的研究可分为两类, 根据其分析材料: 动物模型, 如鼠, 一只老鼠, 或兔子;和人类模型。关于动物模型, 目前有两种常用的技术来产生肌腱: 化学诱导损伤或机械超载的模型。然而, 每个动物模型是有限的复制复杂的人类肌腱病理13,14。
大多数使用人体标本的论文都是在组织学上分析的, 或者是以健康的人类 tenocyte 为基础进行体外实验, 而不是变性 tenocyte15、16、17、18,19,20,21. 只有少数论文报告说, 他们使用了人类退化 tenocyte, 但他们没有详细描述用于从人类22、23获得变性 tenocyte 的协议。在这方面, 应该指出, 动物模型或健康组织/tenocyte 的成功结果不一定能预测人的疗效或有效剂量, 因为肌腱变性是一个复杂的过程, 而发病机制是仍未完全理解。
总的来说, 有必要描述从人体组织获得退行性 tenocyte 的标准协议, 而不会对捐献者造成不良影响。本文介绍了如何获得人类退行性 tenocyte 的协议。为验证该协议, 对所采集的组织进行组织学分析。然后, 采用免疫细胞化学 (ICC)、定量的实时聚合酶链反应 (qRT PCR) 和活性测定方法, 证实了 tenocyte 的退化性。

<table><tbody><tr><td>Scalpel</td><td>Kisanbio</td><td>KS-Q0306-15</td><td>No. 15</td></tr><tr><td>Mini-blade</td><td>Beaver</td><td>374769</td><td></td></tr><tr><td>Dulbecco&#039;s modified Eagle&#039;s medium (DMEM)</td><td>Gibco</td><td>11995065</td><td></td></tr><tr><td>PBS</td><td>Gibco</td><td>14190250</td><td></td></tr><tr><td>fetal bovine serum (FBS)</td><td>Gibco</td><td>16000044</td><td></td></tr><tr><td>50 mM ascorbic acid-2-phosphate</td><td>Sigma-Aldrich</td><td>A5960</td><td></td></tr><tr><td>Antibiotic-Antimycotic solution</td><td>Gibco</td><td>15240062</td><td></td></tr><tr><td>4% formaldehyde</td><td>Bio-solution</td><td>BP031</td><td></td></tr><tr><td>Triton X-100&amp;nbsp;</td><td>Sigma-Aldrich</td><td>X100-100ml</td><td></td></tr><tr><td>BSA</td><td>Rdtech</td><td>C0082</td><td></td></tr><tr><td>TWEEN 20</td><td>Sigma-Aldrich</td><td>P9416-100ml</td><td></td></tr><tr><td>MKX (C-5)</td><td>Santa cruz biotechnology</td><td>sc-515878</td><td></td></tr><tr><td>Tenomodulin (N-14)</td><td>Santa cruz biotechnology</td><td>sc-49325</td><td></td></tr><tr><td>Fluorescence Mounting Medium</td><td>DAKO</td><td>S3023</td><td></td></tr><tr><td>DAPI (4&#039;,6-Diamidino-2-Phenylindole, Dihydrochloride)</td><td>Thermo Fisher Scientific</td><td>D1306</td><td></td></tr><tr><td>WST-1</td><td>Dojindo Molecular Technologies</td><td>CK04</td><td></td></tr><tr><td>BrdU Cell Proliferation Assay Kit</td><td>Cell Signaling Technology</td><td>#6813</td><td></td></tr><tr><td>TRIzol Reagent</td><td>Invitrogen</td><td>15596018</td><td></td></tr><tr><td>iScript cDNA Synthesis Kit&amp;nbsp;</td><td>Bio-Rad</td><td>170-8891</td><td></td></tr><tr><td>TaqMan Gene Expression Master Mix</td><td>Applied Biosystems</td><td>4369016</td><td></td></tr><tr><td>GAPDH</td><td>Thermo Fisher Scientific</td><td>Hs02786624_g1</td><td></td></tr><tr><td>COL3A1</td><td>Thermo Fisher Scientific</td><td>Hs00943809_m1</td><td></td></tr><tr><td>ACTA2</td><td>Thermo Fisher Scientific</td><td>Hs00426835_g1</td><td></td></tr><tr><td>TAC1</td><td>Thermo Fisher Scientific</td><td>Hs00243225_m1</td><td></td></tr><tr><td>TACR1</td><td>Thermo Fisher Scientific</td><td>Hs00185530_m1</td><td></td></tr><tr><td>PTGS2</td><td>Thermo Fisher Scientific</td><td>Hs00153133_m1</td><td></td></tr><tr><td>ACTB</td><td>Thermo Fisher Scientific</td><td>Hs99999903_m1</td><td></td></tr><tr><td>Cell Strainers (100 &amp;micro;m)&amp;nbsp;</td><td>Corning</td><td>352360</td><td></td></tr><tr><td>100mm culture dish</td><td>Thermo Fisher Scientific</td><td>8188207</td><td></td></tr><tr><td>8-well Chamber Slide</td><td>Thermo Fisher Scientific</td><td>154534</td><td></td></tr><tr><td>96 Well Clear Flat Bottom Polystyrene TC-Treated Microplates</td><td>Corning</td><td>3596</td><td></td></tr><tr><td>Nikon Eclipse 50i Microscope&amp;nbsp;</td><td>Nikon</td><td></td><td></td></tr><tr><td>VERSA max microplate reader&amp;nbsp;</td><td>Molecular Devices</td><td></td><td></td></tr><tr><td>CFX96 Real-Time PCR Detection System</td><td>Bio-Rad</td><td></td><td></td></tr><tr><td>Formalin solution, neutral buffered, 10%</td><td>Sigma-Aldrich</td><td>HT501128</td><td></td></tr><tr><td>Paraffins</td><td>Leica Biosystems</td><td>3801340</td><td></td></tr><tr><td>Ethanol</td><td>JUNSEI CHEMICAL</td><td>90303-2185</td><td></td></tr><tr><td>Hematoxylin</td><td>DAKO</td><td>CS70030-2</td><td></td></tr><tr><td>Eosin</td><td>DAKO</td><td>CS70130-2</td><td></td></tr><tr><td>Alcian blue</td><td>DAKO</td><td>AR16011-2</td><td></td></tr><tr><td>Citric acid</td><td>Sigma-Aldrich</td><td>251275</td><td></td></tr><tr><td>Xylene</td><td>JUNSEI CHEMICAL</td><td>25165-0430</td><td></td></tr><tr><td>Endogenous peroxidases&amp;nbsp;</td><td>DAKO</td><td>S200380-2</td><td></td></tr><tr><td>Canada balsam</td><td>JUNSEI CHEMICAL</td><td>23255-1210</td><td></td></tr><tr><td>Microtome Blade</td><td>FEATHER</td><td>A35</td><td></td></tr><tr><td>Slide glass</td><td>SUPERIOR</td><td>1000612</td><td></td></tr><tr><td>Cover glass</td><td>Marienfeld-Superior</td><td>101050</td><td></td></tr><tr><td>VEGF</td><td>Santa cruz biotechnology</td><td>sc-7269</td><td></td></tr><tr><td>SPSS Software</td><td>IBM</td><td></td><td>Ver. 18.0</td></tr><tr><td>Multi-purpose Centrifuge</td><td>LABOGENE</td><td>1248R</td><td></td></tr></tbody></table>

a protocol to acquire the degenerative tenocyte from humans

肌腱, 一种因肌腱变性而发展起来的痛苦状态, 由于体力活动的增加和预期寿命的延长, 在发达世界中的上升。尽管患病率不断上升, 但潜在的发病机制仍不清楚, 治疗一般是有症状的。最近, 研究了多种治疗方法, 包括生长因子、干细胞和基因治疗, 以期提高退行性肌腱的愈合效力。然而, 这些研究中的大多数只对动物模型或健康的人类肌腱细胞进行。尽管一些研究使用病理学肌腱细胞, 我们最好的知识目前还没有描述如何获得人类退行性肌腱细胞的协议。本研究的目的是描述获取人类退行性肌腱细胞的标准协议。最初, 肌腱组织是在手术期间从侧髁的病人身上收获的。然后从腕桡肌肌腱中取出活检标本, 并与手术时观察到的结构变化有关。所有收获的肌腱看起来都是钝的、灰色的、易碎的和水肿的, 这使得它们在视觉上与健康的不同。肌腱细胞被培养并用于实验。同时, 在组织学上分析了一半的收获组织, 并表明它们具有相同的肌腱 (angiofibroblastic 发育不良或增生) 的主要特征。免疫细胞化学的二次分析证实, 培养的干细胞是肌腱细胞与大多数细胞有阳性染色的莫霍克和 tenomodulin 蛋白。用增殖试验或 qRT PCR 方法对细胞与健康对照进行比较, 确定肌腱细胞退行性的质量。退行性 tenocyte 显示出较高的增殖率和类似基因表达模式的肌腱, 匹配以前的报告。总的来说, 这一新的议定书可能为今后研究肌腱提供了有用的工具。

组织学分析表明, 从侧髁的收获组织具有 tendinopathic 肌腱的特征。肌腱退行性肌腱的 H & E 段揭示了一种无条理的胶原束, 极性和细直、强包装的平行纤维结构。在样品中常见的组织学征象如高细胞构成、大核无典型纺锤形等。此外, 退化肌腱的胶原束是由 H & E 染色的相对较弱的阿利新, 但呈阳性染色, 表明软骨和多糖物质增加。
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Watch this Scientific Journal Video about A Protocol to Acquire the Degenerative Tenocyte from Humans at JoVE.com

A Protocol to Acquire the Degenerative Tenocyte from Humans

体外应用退行性肌腱细胞是必要的, 当调查的效果, 新的治疗肌腱。然而, 大多数研究研究只使用动物模型或健康的 tenocyte。我们提出了以下的协议, 以隔离人类退行性肌腱细胞在手术期间。

从人类获得变性 Tenocyte 的一种协议

Tendinopathy, a painful condition that develops in response to tendon degeneration, is on the rise in the developed world due to increasing physical ...

a-protocol-to-acquire-the-degenerative-tenocyte-from-humans

Universidad de Cartagena UDEC

Research

JoVE Journal

Medicine

7.3K 次观看.  CHA University School of Medicine. 体外应用退行性肌腱细胞是必要的, 当调查的效果, 新的治疗肌腱。然而, 大多数研究研究只使用动物模型或健康的 tenocyte。我们提出了以下的协议, 以隔离人类退行性肌腱细胞在手术期间。

视频: A Protocol to Acquire the Degenerative Tenocyte from Humans

Direct Reprogramming of Human Fibroblasts into Myoblasts to Investigate Therapies for Neuromuscular Disorders

Investigations into both the pathophysiology and therapeutic targets in muscular dystrophies have been hampered by the limited proliferative capacity of human myoblasts. Several mouse models have been created but they either do not truly represent the human physiopathology of the disease or are not representative of the broad spectrum of mutations found in humans. The immortalization of human primary myoblasts is an alternative to this limitation; however, it is still dependent on muscle biopsies, which are invasive and not easily available. In contrast, skin biopsies are easier to obtain and less invasive to patients. Fibroblasts derived from skin biopsies can be immortalized and transdifferentiated into myoblasts, providing a source of cells with excellent myogenic potential. Here, we describe a fast and direct reprogramming method of fibroblast into a myogenic lineage. Fibroblasts are transduced with two lentiviruses: hTERT to immortalize the primary culture and a tet-inducible MYOD, which upon the addition of doxycycline, induces the conversion of fibroblasts into myoblasts and then mature myotubes, which express late differentiation markers. This quick transdifferentiation protocol represents a powerful tool to investigate pathological mechanisms and to investigate innovative gene-based or pharmacological biotherapies for neuromuscular disorders.

This protocol describes the conversion of skin fibroblasts into myoblasts and their differentiation into myotubes. The cell lines are derived from patients with neuromuscular disorders and can be used to investigate pathological mechanisms and to test therapeutic strategies.

将人类成纤维细胞直接重新编程到肌细胞中，以研究神经肌肉疾病的治疗方法

Investigations into both the pathophysiology and therapeutic targets in muscular dystrophies have been hampered by the limited proliferative capacity of ...

科研

遗传学

In Vitro Generation of Somite Derivatives from Human Induced Pluripotent Stem Cells

In response to signals such as WNTs, bone morphogenetic proteins (BMPs), and sonic hedgehog (SHH) secreted from surrounding tissues, somites (SMs) give rise to multiple cell types, including the myotome (MYO), sclerotome (SCL), dermatome (D), and syndetome (SYN), which in turn develop into skeletal muscle, axial skeleton, dorsal dermis, and axial tendon/ligament, respectively. Therefore, the generation of SMs and their derivatives from human induced pluripotent stem cells (iPSCs) is critical to obtain pluripotent stem cells (PSCs) for application in regenerative medicine and for disease research in the field of orthopedic surgery. Although the induction protocols for MYO and SCL from PSCs have been previously reported by several researchers, no study has yet demonstrated the induction of SYN and D from iPSCs. Therefore, efficient induction of fully competent SMs remains a major challenge. Here, we recapitulate human SM patterning with human iPSCs in vitro by mimicking the signaling environment during chick/mouse SM development, and report on methods of systematic induction of SM derivatives (MYO, SCL, D, and SYN) from human iPSCs under chemically defined conditions through the presomitic mesoderm (PSM) and SM states. Knowledge regarding chick/mouse&#160;SM development was successfully applied to the induction of SMs with human iPSCs. This method could be a novel tool for studying human somitogenesis and patterning without the use of embryos and for cell-based therapy and disease modeling.

We present here a protocol for the differentiation of human induced pluripotent stem cells into each somite derivative (myotome, sclerotome, dermatome, and syndetome) in chemically defined conditions, which has applications in future disease modeling and cell-based therapies in orthopedic surgery.

人诱导多能干细胞体外生成体细胞衍生物的研究

In response to signals such as WNTs, bone morphogenetic proteins (BMPs), and sonic hedgehog (SHH) secreted from surrounding tissues, somites (SMs) give ...

发育生物学

Isolation, Culture, Characterization, and Differentiation of Human Muscle Progenitor Cells from the Skeletal Muscle Biopsy Procedure

The use of primary human tissue and cells is ideal for the investigation of biological and physiological processes such as the skeletal muscle regenerative process. There are recognized challenges to working with human primary adult stem cells, particularly human muscle progenitor cells (hMPCs) derived from skeletal muscle biopsies, including low cell yield from collected tissue and a large degree of donor heterogeneity of growth and death parameters among cultures. While incorporating heterogeneity into experimental design requires a larger sample size to detect significant effects, it also allows us to identify mechanisms that underlie variability in hMPC&#160;expansion capacity, and thus allows us to better understand heterogeneity in skeletal muscle regeneration. Novel mechanisms that distinguish the expansion capacity of cultures have the potential to lead to the development of therapies to improve skeletal muscle regeneration.

We present techniques for isolating, culturing, characterizing, and differentiating human primary muscle progenitor cells (hMPCs) obtained from skeletal muscle biopsy tissue. hMPCs obtained and characterized through these methods can be used to subsequently address research questions related to human myogenesis and skeletal muscle regeneration.

从骨骼肌活检程序分离、培养、表征和分化人类肌肉祖细胞

The use of primary human tissue and cells is ideal for the investigation of biological and physiological processes such as the skeletal muscle ...

Isolation and Immortalization of Patient-derived Cell Lines from Muscle Biopsy for Disease Modeling

The generation of patient-specific cell lines represents an invaluable tool for diagnostic or translational research, and these cells can be collected from skin or muscle biopsy tissue available during the patient&#8217;s diagnostic workup. In this protocol, we describe a technique for live cell isolation from small amounts of muscle or skin tissue for primary cell culture. Additionally, we provide a technique for the immortalization of myogenic cell lines and fibroblast cell lines from primary cells. Once cell lines are immortalized, substantial expansion of patient-derived cells can be achieved. Immortalized cells are amenable to many downstream applications, including drug screening and in vitro correction of the genetic mutation. Altogether, these protocols provide a reliable tool to generate and preserve patient-derived cells for downstream applications.

This protocol describes techniques for live cell isolation and primary culture of myogenic and fibroblast cell lines from muscle or skin tissue. A technique for the immortalization of these cell lines is also described. Altogether, these protocols provide a reliable tool to generate and preserve patient-derived cells for downstream applications.

从肌肉活检患者来源的细胞系疾病建模隔离和永生化

The generation of patient-specific cell lines represents an invaluable tool for diagnostic or translational research, and these cells can be collected ...

医学

Anatomically Inspired Three-dimensional Micro-tissue Engineered Neural Networks for Nervous System Reconstruction, Modulation, and Modeling

Functional recovery rarely occurs&#160;following injury or disease-induced degeneration within the central nervous system (CNS)&#160;due to the inhibitory environment and the limited capacity for neurogenesis. We are developing&#160;a strategy to simultaneously address&#160;neuronal and axonal pathway loss within the damaged CNS. This manuscript presents the fabrication protocol for micro-tissue engineered neural networks (micro-TENNs), implantable constructs consisting of neurons and aligned axonal tracts spanning the extracellular matrix (ECM) lumen of a preformed hydrogel cylinder hundreds of microns in diameter that may extend centimeters in length. Neuronal aggregates are delimited to the extremes of the three-dimensional encasement and are spanned by axonal projections. Micro-TENNs are uniquely poised as a strategy for CNS reconstruction, emulating aspects of brain connectome cytoarchitecture and potentially providing means for network replacement. The neuronal aggregates may synapse with host tissue to form new functional relays to restore and/or modulate missing or damaged circuitry. These constructs may also act as pro-regenerative&#160;&#34;living scaffolds&#34; capable of exploiting developmental mechanisms for cell migration and axonal pathfinding, providing synergistic structural and soluble cues based on the state of regeneration. Micro-TENNs are fabricated by pouring liquid hydrogel into a cylindrical mold containing a longitudinally centered needle. Once the hydrogel has gelled, the needle is removed, leaving a hollow micro-column. An ECM solution is added to the lumen to provide an environment suitable for neuronal adhesion and axonal outgrowth. Dissociated neurons are mechanically aggregated for precise seeding within one or both ends of the micro-column. This methodology reliably produces self-contained miniature constructs with long-projecting axonal tracts that may recapitulate features of brain neuroanatomy. Synaptic immunolabeling and genetically encoded calcium indicators suggest that micro-TENNs possess extensive synaptic distribution and intrinsic electrical activity. Consequently, micro-TENNs represent a promising strategy for targeted neurosurgical reconstruction of brain pathways and may also be applied as biofidelic models to study neurobiological phenomena in vitro.

This manuscript details the fabrication of micro-tissue engineered neural networks: three-dimensional micron-sized constructs comprised of long aligned axonal tracts spanning aggregated neuronal population(s) encased in a tubular hydrogel. These living scaffolds can serve as functional relays to reconstruct or modulate neural circuitry or as biofidelic test-beds mimicking gray-white matter neuroanatomy.

解剖学启发的三维微组织工程神经网络用于神经系统重建，调制和建模

Functional recovery rarely occurs&#160;following injury or disease-induced degeneration within the central nervous system (CNS)&#160;due to the inhibitory ...

神经科学

利用 iPSC 来源的 iTenocytes 进行有效的基于细胞的治疗

Generation of Induced Pluripotent Stem Cell-Derived iTenocytes via Combined Scleraxis Overexpression and 2D Uniaxial Tension

Today&#39;s challenges in tendon and ligament repair necessitate the identification of a suitable and effective candidate for cell-based therapy to promote tendon regeneration. Mesenchymal stromal cells (MSCs) have been explored as a potential tissue engineering strategy for tendon repair. While they are multipotent and have regenerative potential in vivo, they are limited in their self-renewal capacity and exhibit phenotypic heterogeneity. Induced pluripotent stem cells (iPSCs) can circumvent these limitations due to their high self-renewal capacity and unparalleled developmental plasticity. In tenocyte development, Scleraxis (Scx) is a crucial direct molecular regulator of tendon differentiation. Additionally, mechanoregulation has been shown to be a central element guiding embryonic tendon development and healing. As such, we have developed a protocol to encapsulate the synergistic effect of biological and mechanical stimulation that may be essential for generating tenocytes. iPSCs were induced to become mesenchymal stromal cells (iMSCs) and were characterized with classic mesenchymal stromal cell markers via flow cytometry. Next, using a lentiviral vector, the iMSCs were transduced to stably overexpress SCX (iMSCSCX+). These iMSCSCX+ cells can be further matured into iTenocytes via uniaxial tensile loading using a 2D bioreactor. The resulting cells were characterized by observing the upregulation of early and late tendon markers, as well as collagen deposition. This method of generating iTenocytes can be used to assist researchers in developing a potentially unlimited off-the-shelf allogeneic cell source for tendon cell therapy applications.

作者聚焦:肌腱修复的细胞和组织工程进展 

This article describes a procedure to produce iTenocytes by generating iPSC-derived mesenchymal stromal cells with combined overexpression of Scleraxis using a lentiviral vector and uniaxial stretching via a 2D bioreactor.

通过联合巩膜过表达和 2D 单轴张力产生诱导多能干细胞衍生的 iTenocytes

Today&#39;s challenges in tendon and ligament repair necessitate the identification of a suitable and effective candidate for cell-based therapy to ...

生物工程

制造小鼠肌腱组合体以研究疾病中的细胞相互作用

Engineering Tendon Assembloids to Probe Cellular Crosstalk in Disease and Repair

Tendons enable locomotion by transferring muscle forces to bones. They rely on a tough tendon core comprising collagen fibers and stromal cell populations. This load-bearing core is encompassed, nourished, and repaired by a synovial-like tissue layer comprising the extrinsic tendon compartment. Despite this sophisticated design, tendon injuries are common, and clinical treatment still relies on physiotherapy and surgery. The limitations of available experimental model systems have slowed the development of novel disease-modifying treatments and relapse-preventing clinical regimes. 
In vivo human studies are limited to comparing healthy tendons to end-stage diseased or ruptured tissues sampled during repair surgery and do not allow the longitudinal study of the underlying tendon disease. In vivo animal models also present important limits regarding opaque physiological complexity, the ethical burden on the animals, and large economic costs associated with their use. Further, in vivo animal models are poorly suited to systematic probing of drugs and multicellular, multi-tissue interaction pathways. Simpler in vitro model systems have also fallen short. One major reason is a failure to adequately replicate the three-dimensional mechanical loading necessary to meaningfully study tendon cells and their function. 
The new 3D model system presented here alleviates some of these issues by exploiting murine tail tendon core explants. Importantly, these explants are easily accessible in large numbers from a single mouse, retain 3D in situ loading patterns at the cellular level, and feature an in vivo-like extracellular matrix. In this protocol, step-by-step instructions are given on how to augment tendon core explants with collagen hydrogels laden with muscle-derived endothelial cells, tendon-derived fibroblasts, and bone marrow-derived macrophages to substitute disease- and injury-activated cell populations within the extrinsic tendon compartment. It is demonstrated how the resulting tendon assembloids can be challenged mechanically or through defined microenvironmental stimuli to investigate emerging multicellular crosstalk during disease and injury.

作者聚焦：通过开发小鼠组合来了解细胞机制来推进肌腱研究

Here, we present an assembloid model system to mimic tendon cellular crosstalk between the load-bearing tendon core tissue and an extrinsic compartment containing cell populations activated by disease and injury. As an important use case, we demonstrate how the system can be deployed to probe disease-relevant activation of extrinsic endothelial cells.

从人类获得变性 Tenocyte 的一种协议

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将人类成纤维细胞直接重新编程到肌细胞中，以研究神经肌肉疾病的治疗方法

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从骨骼肌活检程序分离、培养、表征和分化人类肌肉祖细胞

从肌肉活检患者来源的细胞系疾病建模隔离和永生化

解剖学启发的三维微组织工程神经网络用于神经系统重建，调制和建模

通过联合巩膜过表达和 2D 单轴张力产生诱导多能干细胞衍生的 iTenocytes

通过联合巩膜过表达和 2D 单轴张力产生诱导多能干细胞衍生的 iTenocytes

通过联合巩膜过表达和 2D 单轴张力产生诱导多能干细胞衍生的 iTenocytes

工程肌腱组件以探测疾病和修复中的细胞串扰

工程肌腱组件以探测疾病和修复中的细胞串扰