Name: fabrication of decellularized cartilage-derived matrix scaffolds
Uploaded: 2019-01-07T14:00:00
Description: Watch this Scientific Journal Video about Fabrication of Decellularized Cartilage-derived Matrix Scaffolds at JoVE.com

作者希望感谢 w. boot 在制作脚手架方面的帮助。benders 由大学医学中心的亚历山大·苏尔曼基金会提供支持。r. levato 和 j. malda 得到荷兰关节炎基金会的支持 (分别为 co-14-001 和 llp-12 赠款协议)。

在这个协议中, 马窒息软骨是从死于骨关节炎以外的其他原因的马身上获得的。根据机构道德条例, 组织是经业主许可获得的。
请注意:该协议描述了脱细胞马软骨支架的制作, 可用于体外组织培养平台或体内植入再生医学策略等应用。酶处理步骤必须按所述的时间顺序进行。
1. 从供体 (尸体) 关节中收集关节软骨
<ol><li>在收获步...

<ol>
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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=Multipotent+Stromal+Cells+Outperform+Chondrocytes+on+Cartilage-Derived+Matrix+Scaffolds.">Multipotent Stromal Cells Outperform Chondrocytes on Cartilage-Derived Matrix Scaffolds.</a> Cartilage. 5 (4), 221-230 (2014).</li><li>Gawlitta, 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=Decellularized+cartilage-derived+matrix+as+substrate+for+endochondral+bone+regeneration.">Decellularized cartilage-derived matrix as substrate for endochondral bone regeneration.</a> Tissue engineering. Part A. 21 (3-4), 694-703 (2015).</li><li>Yang, Z., 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=Fabrication+and+repair+of+cartilage+defects+with+a+novel+acellular+cartilage+matrix+scaffold.">Fabrication and repair of cartilage defects with a novel acellular cartilage matrix scaffold.</a> Tissue engineering. Part C, Methods. 16 (5), 865-876 (2010).</li><li>Pittenger, M. 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=Multilineage+potential+of+adult+human+mesenchymal+stem+cells.">Multilineage potential of adult human mesenchymal stem cells.</a> Science. 284 (5411), 143-147 (1999).</li><li>Meyer, S. R., 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=Decellularization+reduces+the+immune+response+to+aortic+valve+allografts+in+the+rat.">Decellularization reduces the immune response to aortic valve allografts in the rat.</a> The Journal of thoracic and cardiovascular surgery. 130 (2), 469-476 (2005).</li><li>Brown, B. N., Valentin, J. E., Stewart-Akers, A. M., McCabe, G. P., Badylak, S. F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Macrophage+phenotype+and+remodeling+outcomes+in+response+to+biologic+scaffolds+with+and+without+a+cellular+component.">Macrophage phenotype and remodeling outcomes in response to biologic scaffolds with and without a cellular component.</a> Biomaterials. 30 (8), 1482-1491 (2009).</li><li>Keane, T. J., Londono, R., Turner, N. J., Badylak, S. F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Consequences+of+ineffective+decellularization+of+biologic+scaffolds+on+the+host+response.">Consequences of ineffective decellularization of biologic scaffolds on the host response.</a> Biomaterials. 33 (6), 1771-1781 (2012).</li><li>Crapo, P. M., Gilbert, T. W., Badylak, S. F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=An+overview+of+tissue+and+whole+organ+decellularization.">An overview of tissue and whole organ decellularization.</a> Biomaterials. 32 (12), 3233-3243 (2011).</li><li>Malda, 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=Of+mice,+men+and+elephants:+the+relation+between+articular+cartilage+thickness+and+body+mass.">Of mice, men and elephants: the relation between articular cartilage thickness and body mass.</a> PloS One. 8 (2), e57683 (2013).</li><li>Malda, 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=Comparative+study+of+depth-dependent+characteristics+of+equine+and+human+osteochondral+tissue+from+the+medial+and+lateral+femoral+condyles.">Comparative study of depth-dependent characteristics of equine and human osteochondral tissue from the medial and lateral femoral condyles.</a> Osteoarthritis and Cartilage. 20 (10), 1147-1151 (2012).</li><li>Londono, R., Badylak, S. 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关节软骨的 ecm 是非常密集的, 对不同的酶处理有相当的弹性。本文描述的多步骤去电磁化协议解决了这种电阻问题, 并成功地生成了去电磁化矩阵。为了实现这一目标, 这一过程持续了几天。针对不同类型的组织18提出了许多脱髓化过程,本文介绍了一种适用于软骨去细胞化的方案。然而, 在这个协议中, 有必要遵循酶处理与洗涤剂步骤, 以消除所有细胞。dna 的数量在涉及胰蛋白酶?...

创伤事件引起的膝关节关节软骨缺损会导致不适, 最重要的是会对年轻和活跃人群的生活产生很大的影响 1,2,3。此外, 年轻时的软骨损伤可能会导致以后更迅速地开始骨关节炎 4.目前, 对全身性膝关节骨关节炎的唯一抢救疗法是关节置换术。由于软骨是一种下细胞、听觉和血管组织, 其再生能力受到严重限制。因此, 我们寻求再生医学的方法来帮助和刺激原生组织的再生能力。为此, 支架被设计并用作细胞载体或诱导材料, 通过人体的原生细胞促进组织的分化和再生5。
脱光支架在再生医学中得到了广泛的研究。例如, 它在帮助皮肤7、腹部结构8和肌腱9的再生方面取得了一些成功。使用脱细胞支架的优点是它们的自然来源和保留生物活性线索的能力, 这些线索既能吸引和诱导细胞分化, 也能诱导细胞分化为组织修复所需的适当谱系 6,10.此外, 由于细胞外基质 (ecm) 是一种天然的生物材料, 去细胞化通过去除细胞或遗传含量来防止潜在的免疫反应, 从而克服了生物相容性和生物降解性方面的问题。
软骨衍生基质 (cdm) 支架在体外实验中显示出巨大的软骨生质潜力, 当使用间充质基质细胞 11播种时。此外, 这些支架还显示了在体内环境中通过软骨内骨化在异位形成骨组织的潜力.由于 cdm 支架引导骨和软骨组织的形成, 这些支架除了软骨修复外, 还可能具有骨软骨缺损修复的潜力。
本文介绍了杨等人 (2010年)13所采用的一种协议, 用于生产从马窒息软骨中脱病毒的 cdm 支架。这些支架富含 ii 型胶原蛋白, 没有细胞, 脱髓化后不含任何糖胺多糖 (gag)。在 (骨) 软骨缺损修复的体外和体内实验都可以使用这些支架进行。

<table>
	<tbody>
		<tr>
			<td>Cadaveric joint</td>
			<td></td>
			<td></td>
			<td>This can be obtained as rest material from the local butcher or veterinary center.</td>
		</tr>
		<tr>
			<td>Sterile phosphate-buffered saline (PBS)</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Penicillin-Streptomycin</td>
			<td>Gibco</td>
			<td>15140</td>
			<td></td>
		</tr>
		<tr>
			<td>Amphotericin B</td>
			<td>Thermo Fischer Scientific</td>
			<td>15290026</td>
			<td></td>
		</tr>
		<tr>
			<td>Liquid nitrogen</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Trypsin-EDTA (0.25%), phenol red</td>
			<td>Thermo Fischer Scientific</td>
			<td>25200072</td>
			<td></td>
		</tr>
		<tr>
			<td>Tris-HCl pH 7.5</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Deoxyribonuclease I from bovine pancreas</td>
			<td>Sigma-Aldrich</td>
			<td>DN25</td>
			<td></td>
		</tr>
		<tr>
			<td>Ribonuclease A from bovine pancreas</td>
			<td>Sigma-Aldrich</td>
			<td>R6513</td>
			<td></td>
		</tr>
		<tr>
			<td>Triton X-100 (octoxynol-1)</td>
			<td>Sigma-Aldrich</td>
			<td>X100</td>
			<td></td>
		</tr>
		<tr>
			<td>Papain</td>
			<td>Sigma-Aldrich</td>
			<td>P3125</td>
			<td></td>
		</tr>
		<tr>
			<td>Trisodium citrate dihydrate</td>
			<td>Sigma-Aldrich</td>
			<td>S4641</td>
			<td></td>
		</tr>
		<tr>
			<td>Alginate</td>
			<td>Sigma-Aldrich</td>
			<td>180947</td>
			<td></td>
		</tr>
		<tr>
			<td>Formalin</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>CaCl2</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Ethanol</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Xylene</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Paraffin</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Ethylene oxide sterilization</td>
			<td>Synergy Health, Venlo, the Netherlands</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Multipotent Stromal cells/chondrocytes from equine donors</td>
			<td></td>
			<td></td>
			<td>MSCs and chondrocytes can be isolated from donor joints that are rest material, coming from the local butcher or veterinary center.</td>
		</tr>
		<tr>
			<td>MEM alpha</td>
			<td>Thermo Fischer Scientific</td>
			<td>22561</td>
			<td></td>
		</tr>
		<tr>
			<td>L-ascorbic acid 2-phosphate</td>
			<td>Sigma-Aldrich</td>
			<td>A8960</td>
			<td></td>
		</tr>
		<tr>
			<td>DMEM</td>
			<td>Thermo Fischer Scientific</td>
			<td>41965</td>
			<td></td>
		</tr>
		<tr>
			<td>Heat inactivated bovine serum albumin</td>
			<td>Sigma-Aldrich</td>
			<td>10735086001</td>
			<td></td>
		</tr>
		<tr>
			<td>Fibroblast growth factor-2 (FGF-2)</td>
			<td>R &#38; D Systems</td>
			<td>233-FB</td>
			<td></td>
		</tr>
		<tr>
			<td>DNA quantification kit (Quant-iT PicoGreen dsDNA Reagent)</td>
			<td>Thermo Fischer Scientific</td>
			<td>P7581</td>
			<td></td>
		</tr>
		<tr>
			<td>1,9-Dimethyl-Methylene Blue zinc chloride double salt</td>
			<td>Sigma-Aldrich</td>
			<td>341088</td>
			<td></td>
		</tr>
		<tr>
			<td>Freeze-dryer</td>
			<td>SALMENKIPP</td>
			<td>ALPHA 1-2 LD plus</td>
			<td></td>
		</tr>
		<tr>
			<td>Analytical mill</td>
			<td>IKA</td>
			<td>A 11 basic</td>
			<td></td>
		</tr>
		<tr>
			<td>mortar/pestle</td>
			<td>Haldenwanger 55/0A</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Roller plate</td>
			<td>CAT</td>
			<td>RM5</td>
			<td></td>
		</tr>
		<tr>
			<td>Centrifuge (for 50 mL tubes)</td>
			<td>Eppendorf</td>
			<td>5810R</td>
			<td></td>
		</tr>
		<tr>
			<td>Capsule (cylindric mold)</td>
			<td>TAAB</td>
			<td>8 mm flat</td>
			<td></td>
		</tr>
		<tr>
			<td>Superlight S UV</td>
			<td>Lumatec</td>
			<td>2001AV</td>
			<td></td>
		</tr>
		<tr>
			<td>Incubator</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Microtome</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Sieve (mesh size 0.71 mm)</td>
			<td>VWR</td>
			<td>34111229</td>
			<td></td>
		</tr>
		<tr>
			<td>Scalpel</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Scalpel holder</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Small laddle</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
	</tbody>
</table>

fabrication of decellularized cartilage-derived matrix scaffolds

骨软骨缺损缺乏足够的内在修复能力来再生功能健全的骨骼和软骨组织。在这方面, 软骨研究的重点是再生支架的开发。本文介绍了支架的发展, 这是完全来自天然软骨细胞外基质, 来自一个马供体。支架的潜在应用包括生产用于软骨修复的同种异体移植, 作为骨软骨组织工程的支架, 以及提供研究组织形成的体外模型。通过去细胞化组织, 供体细胞被切除, 但许多天然的生物活性线索被认为是被保留的。与合成支架相比, 使用这种天然支架的主要优点是, 不需要聚合物的进一步功能化来推动骨软骨组织再生。软骨衍生基质支架可用于体内和体外组织的骨和软骨组织再生。

cdm 支架的去序列化必须始终使用组织学标记以及 dna 定量来测量 dna 残留量来确认。去细胞化不足可能会导致不想要的免疫反应, 影响在体内的结果15,16,17。对于这种特定的去细胞化方法, dna 低于检测范围, 从 13.6±2.3 ng/mg dna/干重 (n = 3) 开始。使用此协议的完全去电化将导致一个支架的生产, 该支架富含 i...

Watch this Scientific Journal Video about Fabrication of Decellularized Cartilage-derived Matrix Scaffolds at JoVE.com

Fabrication of Decellularized Cartilage-derived Matrix Scaffolds

脱形软骨衍生支架可用作指导软骨修复的支架, 也可用作骨软骨组织再生的手段。本文详细介绍了脱髓化过程, 并对在体外环境中使用这些支架提出了建议。

脱碳软骨衍生基质支架的制备

Osteochondral defects lack sufficient intrinsic repair capacity to regenerate functionally sound bone and cartilage tissue. To this extent, cartilage ...

此方法提供了一种从原生组织中提取的脚手架，可用于软骨再生的方法。该技术的主要优点是，原生软骨的成分保留在脚手架中，可以促进软骨形成和体外和体内再生。虽然这些方法使用软骨从马窒息关节，软骨从任何其他关节也可以使用。首先，获得一个完整的尸体关节软骨分离，并删除皮肤多余的脂肪和肌肉组织，如随附的文本协议所述。接下来，定义关节通过执行关节的延伸拐点来表达的位置。做一个水平切口，以达到关节腔。接下来，做一个垂直切口，以打开关节区域。关节腔充满突触液，在执行正确的切口时，可能会从腔中滴落。继续完全打开关节，去除过多的脂肪，肌肉和肌腱，并通过切开肌腱，保持关节在一起。仔细检查软骨是否有任何宏观损伤。如果关节软骨没有光泽光滑的外观，或者如果存在证据、起泡、裂口或缺陷，请丢弃软骨并重新开始。使用无菌手术刀从骨骼上取下软骨。切到亚骨，去除深区软骨。将去除的软骨片收集到含有先前准备的软骨洗涤溶液的 50 毫升管中。在加工过程中，定期在软骨上滴软骨洗涤溶液，以防止软骨干燥。从所有感兴趣区域收集软骨后，将软骨片在液氮中捕捉冻结五分钟。然后将软骨片转移到50毫升管中，并立即将冷冻切片放入冷冻干燥器中。使用冷冻干燥机将软骨片冻干 24 小时。完成后，将冷冻干燥的软骨片存放在干燥的地方，在室温下。对于手动加工，用液氮预冷却砂浆和害虫，然后将冻干软骨片放在砂浆中，然后将它们和液氮淹没。用手直接研磨样品约45分钟，直到样品充分粉碎。或者，要使用铣床自动研磨样品，请首先通过添加液氮来预冷却铣削机的磨削室。然后添加冻干软骨切片，以预设速度碾磨样品几秒钟，长达一分钟。确保所有颗粒都是接地的，并且砂削室底部没有颗粒。最后，在形成基架之前，请遵循随附的文本协议中描述的去细胞化技术。将带小标签的软骨颗粒转移到圆柱形塑料模具上。将所有气泡压出，以避免脚手架中出现空腔，并完全填充模具。为避免脚手架出现空腔，确保在用软骨颗粒填充模具时将气泡压出。形成后，在零下20摄氏度下冷冻模具10分钟。然后在冷冻干燥机中将软骨脚手架在模具中冻干 24 小时。干冻后，小心地从模具中取出脚手架。将脚手架放在365纳米波长的紫外光下，并通宵交叉连接。通过将消毒的脚手架切割成三毫米厚的切片，形成脚手架盘。然后将脚手架转移到六井板的单独井中。在脚手架上加入一毫升的胆囊细胞膨胀介质，让它们补充水分30分钟。当脚手架补充水分时，在预浸泡脚手架顶部准备电池和移液器 50 微升的预制电池悬架。然后在37摄氏度下孵育脚手架一小时。使用软体细胞时，请确保它们没有通过第一通道展开，以尽量减少分化细胞的数量。一小时后，将板返回到文化罩，并小心地将脚手架翻过来。将剩余的50微升细胞悬浮液移出脚手架，并在37摄氏度下再孵育一小时。孵育后，用脚手架在井中加入三毫升的介质。避免将介质直接移液在脚手架上，并轻轻处理培养板，以避免细胞分离。将盘子返回到孵化器，培养细胞在摄氏37度时将脚手架放在一起。利用组织染色和DNA定量相结合的方法表明，本文提出的方法导致足够的软骨支架去细胞化。结果发现，脚手架不含细胞以及检测不到的DNA水平。它们还缺乏糖糖，富含胶原蛋白II. 在这里，在与培养了四周的中层干细胞一起生长的脚手架上观察到新基质的形成。糖糖在四周后形成丰富的糖糖。尝试此过程后，在尝试此过程之前，在任何应用程序中使用它之前，必须检查去细胞化是否成功。按照此过程，可以执行额外的生化分析，如西印迹分析仪，以回答有关其他成分（如生长因子）存在的具体问题。这项技术为组织工程领域的研究人员探索软骨再生策略铺平了道路，这些策略来自异源和合成源的去细胞化组织。应始终采取佩戴实验室外套和手套等预防措施。

Research

JoVE Journal

Bioengineering

Epithelial Cell Repopulation and Preparation of Rodent Extracellular Matrix Scaffolds for Renal Tissue Development

上皮细胞复育和肾组织的发展准备啮齿动物细胞外基质的支架

This protocol details the generation of acellular, yet biofunctional, renal extracellular matrix (ECM) scaffolds that are useful as small-scale model ...

科研

生物工程

Fabrication of Mechanically Tunable and Bioactive Metal Scaffolds for Biomedical Applications

加工机械可调和生物活性金属支架的生物医学应用

Biometal systems have been widely used for biomedical applications, in particular, as load-bearing materials. However, major challenges are high stiffness ...

Generation of ESC-derived Mouse Airway Epithelial Cells Using Decellularized Lung Scaffolds

用脱细胞支架龙ESC衍生鼠气道上皮细胞的生成

Lung lineage differentiation requires integration of complex environmental cues that include growth factor signaling, cell-cell interactions and ...

Fabrication and Characterization of Griffithsin-modified Fiber Scaffolds for Prevention of Sexually Transmitted Infections

Griffithsin 修饰纤维支架预防性传播感染的制备与表征

Electrospun fibers (EFs) have been widely used in a variety of therapeutic applications; however, they have only recently been applied as a technology to ...

Fabrication and Use of Dry Macroporous Alginate Scaffolds for Viral Transduction of T Cells

干式大孔海藻酸盐支架的制备和使用用于T细胞的病毒转导

Genetic engineering of T cells for CAR-T cell therapy has come to the forefront of cancer treatment over the last few years. CAR-T cells are produced by ...

Gelatin Methacryloyl Granular Hydrogel Scaffolds: High-throughput Microgel Fabrication, Lyophilization, Chemical Assembly, and 3D Bioprinting

明胶甲基丙烯酰基颗粒水凝胶支架：高通量微凝胶制造、冻干、化学组装和 3D 生物打印

The emergence of granular hydrogel scaffolds (GHS), fabricated via assembling hydrogel microparticles (HMPs), has enabled microporous scaffold formation ...

Synthesis of Decellularized Cartilage Extracellular Matrix Hydrogels

脱细胞软骨细胞外基质水凝胶的合成

Decellularized cartilage extracellular matrix (DC-ECM) hydrogels are promising biomaterials for tissue engineering and regenerative medicine due to their ...

Comparative Study of Decellularization Techniques for Bovine Lung ECM Hydrogels

Development and Characterization of Decellularized Lung Extracellular Matrix Hydrogels

Author Spotlight: Optimizing Bovine Lung Decellularization for Organotypic Hydrogels

The use of extracellular matrix (ECM)-derived hydrogels in tissue engineering has become increasingly popular, as they can mimic cells&#39; natural ...