Name: synthesis of decellularized cartilage extracellular matrix hydrogels
Uploaded: 2023-07-21T14:10:00
Description: Watch this Scientific Journal Video about Synthesis of Decellularized Cartilage Extracellular Matrix Hydrogels at JoVE.com

本工作由浙江省医药卫生科技计划（2019KY050）、浙江省中医药科技计划（2019ZA026）、浙江省重点研发计划（批准号：2020C03043）、浙江省中医药科技计划（2021ZQ021）和浙江省自然科学基金（LQ22H060007）资助。

本研究经浙江省同德医院伦理委员会批准。
1. DC-ECM水凝胶的制备
注：在这项研究中，软骨是从12个月大的巴马微型猪的膝关节中获得的，避免了骨组织的收集。
<ol><li>取收集的软骨，用手术刀将其块状切成1-2毫米3 块。</li>
	<li>将 20 g 切碎的软骨放入 50 mL 离心管中，并加入 20 mL 低渗 Tris-HCl 缓冲液（10 mM Tris-HCl，pH 8.0）以完全浸没组?...

<ol>
	<li>Vega, S. L., Kwon, M. Y., Burdick, J. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Recent+advances+in+hydrogels+for+cartilage+tissue+engineering.">Recent advances in hydrogels for cartilage tissue engineering.</a> European Cells &amp; Materials. 33, 59-75 (2017).</li><li>Yang, J., Zhang, Y. S., Yue, K., Khademhosseini, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cell-laden+hydrogels+for+osteochondral+and+cartilage+tissue+engineering.">Cell-laden hydrogels for osteochondral and cartilage tissue engineering.</a> Acta Biomaterialia. 57, 1-25 (2017).</li><li>Bejleri, D., Davis, M. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Decellularized+extracellular+matrix+materials+for+cardiac+repair+and+regeneration.">Decellularized extracellular matrix materials for cardiac repair and regeneration.</a> Advanced Healthcare Materials. 8 (5), e1801217 (2019).</li><li>Brown, M., Li, J., Moraes, C., Tabrizian, M., Li-Jessen, N. Y. K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Decellularized+extracellular+matrix:+New+promising+and+challenging+biomaterials+for+regenerative+medicine.">Decellularized extracellular matrix: New promising and challenging biomaterials for regenerative medicine.</a> Biomaterials. 289, 121786 (2022).</li><li>Barbulescu, G. I., 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+extracellular+matrix+scaffolds+for+cardiovascular+tissue+engineering:+Current+techniques+and+challenges.">Decellularized extracellular matrix scaffolds for cardiovascular tissue engineering: Current techniques and challenges.</a> International Journal of Molecular Sciences. 23 (21), 13040 (2022).</li><li>Zhang, W., Du, A., Liu, S., Lv, M., Chen, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Research+progress+in+decellularized+extracellular+matrix-derived+hydrogels.">Research progress in decellularized extracellular matrix-derived hydrogels.</a> Regenerative Therapy. 18, 88-96 (2021).</li><li>Zhu, 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=Cell-derived+decellularized+extracellular+matrix+scaffolds+for+articular+cartilage+repair.">Cell-derived decellularized extracellular matrix scaffolds for articular cartilage repair.</a> International Journal of Artificial Organs. 44 (4), 269-281 (2021).</li><li>Li, T., Javed, R., Ao, Q. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Xenogeneic+decellularized+extracellular+matrix-based+biomaterials+for+peripheral+nerve+repair+and+regeneration.">Xenogeneic decellularized extracellular matrix-based biomaterials for peripheral nerve repair and regeneration.</a> Current Neuropharmacology. 19 (12), 2152-2163 (2021).</li><li>Xia, 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=Decellularized+cartilage+as+a+prospective+scaffold+for+cartilage+repair.">Decellularized cartilage as a prospective scaffold for cartilage repair.</a> Materials Science &amp; Engineering C-Materials for Biological Applications. 101, 588-595 (2019).</li><li>Chen, P., 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=Desktop-stereolithography+3D+printing+of+a+radially+oriented+extracellular+matrix/mesenchymal+stem+cell+exosome+bioink+for+osteochondral+defect+regeneration.">Desktop-stereolithography 3D printing of a radially oriented extracellular matrix/mesenchymal stem cell exosome bioink for osteochondral defect regeneration.</a> Theranostics. 9 (9), 2439-2459 (2019).</li><li>Saldin, L. T., Cramer, M. C., Velankar, S. S., White, L. 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=Extracellular+matrix+hydrogels+from+decellularized+tissues:+Structure+and+function.">Extracellular matrix hydrogels from decellularized tissues: Structure and function.</a> Acta Biomaterialia. 49, 1-15 (2017).</li><li>Yuan, X., 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=Stem+cell+delivery+in+tissue-specific+hydrogel+enabled+meniscal+repair+in+an+orthotopic+rat+model.">Stem cell delivery in tissue-specific hydrogel enabled meniscal repair in an orthotopic rat model.</a> Biomaterials. 132, 59-71 (2017).</li><li>Zheng, L., 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=Intensified+stiffness+and+photodynamic+provocation+in+a+collagen-based+composite+hydrogel+drive+chondrogenesis.">Intensified stiffness and photodynamic provocation in a collagen-based composite hydrogel drive chondrogenesis.</a> Advanced Science. 6 (16), 1900099 (2019).</li><li>Young, J. L., Holle, A. W., Spatz, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=P.Nanoscale+and+mechanical+properties+of+the+physiological+cell-ECM+microenvironment.">P.Nanoscale and mechanical properties of the physiological cell-ECM microenvironment.</a> Experimental Cell Research. 343 (1), 3-6 (2016).</li><li>Abdolghafoorian, 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=Effect+of+heart+valve+decellularization+on+xenograft+rejection.">Effect of heart valve decellularization on xenograft rejection.</a> Experimental and Clinical Transplantation. 15 (3), 329-336 (2017).</li></ol>

该方案为制备脱细胞软骨细胞外基质水凝胶提供了一种系统的方法，其紧密模拟软骨组织的天然ECM。该方案涉及物理、化学和酶促破坏的组合，以去除细胞物质，同时保留 ECM 的结构和组成。该协议的关键步骤包括调整脱细胞时间和方法，并确保完全脱细胞。
与其他现有的组织工程和再生医学方法相比，该协议具有几个优点。DC-ECM水凝胶具有优异的生物活性、空间结构和生物...

软骨组织工程是一个快速发展的领域，旨在再生受损或患病的软骨组织1。该领域的一个关键挑战是开发仿生支架，该支架可以支持软骨细胞的生长和分化，软骨细胞是负责产生软骨的细胞2。软骨组织的ECM在调节软骨细胞的行为中起着关键作用。DC-ECM 是组织工程应用的有效支架3.
已经开发了许多技术来从软骨组织生产 DC-ECM，包括化学、酶和物理方法。然而，这些方法通常会导致产生不充分仿生的ECM水凝胶，这限制了它们在组织工程应用中的潜力4,5。因此，需要一种更有效的方法来生产DC-ECM水凝胶。
这项技术的发展很重要，因为它可以通过提供一种新方法来创建可以支持组织再生和修复的仿生支架来推进组织工程领域。此外，该技术可以很容易地适应从其他组织生产ECM水凝胶，从而扩大其潜在应用。
在更广泛的文献中，人们对使用 DC-ECM 作为组织工程应用的支架越来越感兴趣6。大量研究表明，DC-ECM 水凝胶在促进各种组织（包括软骨）的细胞生长和分化方面是有效的 7,8。因此，开发一种用于生产与软骨组织天然ECM非常相似的DC-ECM水凝胶的方案是对该领域的重大贡献。
本文提出的方案通过提供一种生产DC-ECM水凝胶的新方法来解决这一需求，该水凝胶与软骨组织的天然ECM非常相似。该方案涉及使软骨组织脱细胞，分离所得的ECM，并通过将ECM与生物相容性聚合物交联来产生水凝胶。由此产生的水凝胶在支持软骨细胞的生长和分化方面显示出有希望的结果。

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>1 M Tris-HCl, pH7.6</td>
			<td>Beyotime</td>
			<td>ST776-100 mL</td>
			<td></td>
		</tr>
		<tr>
			<td>1 M Tris-HCl, pH8.0</td>
			<td>Beyotime</td>
			<td>ST780-500 mL</td>
			<td></td>
		</tr>
		<tr>
			<td>-80 &#176;C Freezer</td>
			<td>Eppendorf</td>
			<td>F440340034</td>
			<td></td>
		</tr>
		<tr>
			<td>Deoxyribonuclease</td>
			<td>Aladdin</td>
			<td>D128600-80KU</td>
			<td></td>
		</tr>
		<tr>
			<td>DNEasy Blood &#38;Tissue Kit</td>
			<td>Qiagen</td>
			<td>No. 69506</td>
			<td></td>
		</tr>
		<tr>
			<td>GAG colorimetric quantitative detection kit</td>
			<td>Shanghai Haling</td>
			<td>HL19236.2</td>
			<td></td>
		</tr>
		<tr>
			<td>HCP-2 dryer&#160;</td>
			<td>Hitachi</td>
			<td>N/A</td>
			<td></td>
		</tr>
		<tr>
			<td>Nanodrop8000</td>
			<td>Thermo Fisher</td>
			<td>N/A</td>
			<td>Spectrophotometer</td>
		</tr>
		<tr>
			<td>PBS (10x)</td>
			<td>Gibco</td>
			<td>70011044</td>
			<td></td>
		</tr>
		<tr>
			<td>Ribonuclease</td>
			<td>Aladdin</td>
			<td>R341325-100 mg</td>
			<td></td>
		</tr>
		<tr>
			<td>Sigma500</td>
			<td>ZIESS</td>
			<td>N/A</td>
			<td>Scanning electron microscope</td>
		</tr>
		<tr>
			<td>Spectra S</td>
			<td>Thermo Fisher</td>
			<td>N/A</td>
			<td>Transmission electron microscope</td>
		</tr>
		<tr>
			<td>Stainless steel sieve</td>
			<td>SHXB-Z-1</td>
			<td>Shanghai Xinbu</td>
			<td></td>
		</tr>
		<tr>
			<td>Triton X-100</td>
			<td>Beyotime</td>
			<td>P0096-500 mL</td>
			<td></td>
		</tr>
		<tr>
			<td>Trypsin&#160;</td>
			<td>Gibco</td>
			<td>15050065</td>
			<td></td>
		</tr>
		<tr>
			<td>Ultraviolet lamp</td>
			<td>Omnicure 2000</td>
			<td>N/A</td>
			<td></td>
		</tr>
		<tr>
			<td>Vitamin B2</td>
			<td>Gibco</td>
			<td>R4500-5G</td>
			<td></td>
		</tr>
		<tr>
			<td>Vortex mixer</td>
			<td>Shanghai Qiasen</td>
			<td>78HW-1&#160;</td>
			<td></td>
		</tr>
	</tbody>
</table>

synthesis of decellularized cartilage extracellular matrix hydrogels

脱细胞软骨细胞外基质（DC-ECM）水凝胶因其生物相容性和模仿天然组织特性的能力而成为组织工程和再生医学的有前途的生物材料。该协议旨在产生与软骨组织的天然ECM非常相似的DC-ECM水凝胶。该方案涉及物理和化学破坏以及酶消化的组合，以去除细胞物质，同时保留ECM的结构和组成。DC-ECM使用化学试剂交联，形成稳定且具有生物活性的水凝胶。DC-ECM水凝胶具有优异的生物活性、空间结构和生物诱导功能，以及低免疫原性。这些特性有利于促进细胞粘附、增殖、分化和迁移，并为细胞生长创造优越的微环境。该协议为组织工程领域的研究人员和临床医生提供了宝贵的资源。仿生水凝胶可以潜在地促进软骨修复和再生的有效组织工程策略的开发。

为了制备更好的DC-ECM软骨水凝胶，我们研究并回顾了以前的文献，并在脱细胞率、免疫原性和机械功能方面比较了各种脱细胞方案9。
在此基础上，我们制备了DC-ECM软骨水凝胶，并探讨了径向取向提取基质/间充质干细胞外泌体生物墨水治疗骨软骨缺损的效果。结果表明，DC-ECM软骨水凝胶具有较低的免疫原性，可以增强细胞迁移，促进软骨修复10<...

Watch this Scientific Journal Video about Synthesis of Decellularized Cartilage Extracellular Matrix Hydrogels at JoVE.com

Synthesis of Decellularized Cartilage Extracellular Matrix Hydrogels

本文介绍了一种合成脱细胞软骨细胞外基质（DC-ECM）水凝胶的新方法。DC-ECM水凝胶具有优异的生物相容性，为细胞生长提供了优越的微环境。因此，它们可以成为理想的细胞支架和生物递送系统。

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 ...

我们的方案意义重大，因为它能够在与软骨组织性质非常相似的水凝胶中产生疾病。该技术的主要优点是能够在水凝胶中产生疾病，在空间结构上具有明显的生物活性，免疫原性低，具有生物工业功能。首先，用手术刀将收集的软骨切成一到两立方毫米大小的碎片。在50毫米离心管中，将20克碎软骨浸入20毫升低渗Tris-盐酸盐缓冲液中。将管子在 80 摄氏度下放置三个小时，然后在 37 摄氏度的烤箱中放置三个小时。以 1000 RPM 涡旋离心管 30 秒。然后使用放置在50毫升离心管上的塑料筛过滤脱细胞软骨。在收集软骨之前，用无菌PBS洗涤软骨三次。向收集的软骨中加入10毫升胰蛋白酶溶液，在37摄氏度的振荡器上孵育24小时，每4小时更换一次胰蛋白酶。过滤悬浮液后，用高渗缓冲液洗涤胰蛋白酶消化软骨。保留软骨后，加入 10 毫升核酸酶溶液，并将其放在 37 摄氏度的摇床上 4 小时。用无菌PBS洗涤后，向软骨中加入高渗Tris-hydrochloride溶液，并将其放置在振荡器上，如图所示。用无菌PBS洗涤后，将组织浸入1%Triton X-100溶液中24小时。取出Triton X-100后，用蒸馏水冲洗脱细胞软骨三天，每12小时换一次水。三天后，在软骨中加入过氧乙酸溶液，浸泡四小时。用PBS洗涤后，如前所述，使用塑料筛收集软骨。使用液氮，制备脱细胞软骨粉。在两克软骨粉中加入80毫升0.5摩尔醋酸和20毫克胃蛋白酶，在37摄氏度下消化24小时。消化后，将悬浮液以 400 G 离心 10 分钟并收集上清液，现在称为 DC-ECM 溶液 为了储存 DC-ECM 溶液，向六孔板的每个孔中加入 1 毫升溶液，并将其放入冻干机中。溶液冷冻干燥后，将其转移到离心管中。要形成水凝胶，将 20 毫克冻干的 DC-ECM 溶液溶解在一毫升无菌蒸馏水中。涡旋后，向 DC-ECM 溶液中加入 1 毫克维生素 B2。孵育后，用紫外线照射三分钟。将缓冲液 GTL 和蛋白酶 K 加入 20 毫克 DC-ECM 软骨粉中，并使用涡旋振荡充分混合。为了完全溶解软骨，将混合物在 56 摄氏度下孵育 4 小时。涡旋后，加入 200 微升缓冲液 GTL，然后加入无水乙醇。涡旋后，将样品在4摄氏度下以6000G离心一分钟。然后按照手稿中的描述收集并定量DNA。为了分析胶原蛋白，将 5 毫克 DC-ECM 粉末在 100 摄氏度的盐酸中酸化 20 分钟。然后用五毫升六摩尔氢氧化钠溶液中和。使用570纳米处的吸光度与羟脯氨酸标准品计算中和样品的羟脯氨酸含量。混合后，将 500 微升试剂 A 加入 200 毫克 DC-ECM 粉末中。将样品在 4 摄氏度下孵育 16 小时。离心后，收集 50 微升样品溶液并加入 50 微升试剂 B，然后加入试剂 C.将样品孵育 10 分钟后，加入 750 微升试剂 D，在黑暗中孵育 30 分钟。离心后，向沉淀中加入一微升试剂E，并在离心前充分混合。然后在GAG含量测量之前解离样品。在制备的DC-ECM软骨中，DNA含量被显著消除。然而，与天然软骨相比，胶原蛋白和GAG含量被保留。SEM和TEM分析证明了所制备的DC-ECM的超超结构。倒置管中制备的水凝胶没有流到底部，从而证明凝胶化。此外，与仅 DC-ECM 溶液相比，由于 DC-ECM 水凝胶形成过程中诱导的交联，添加维生素 B2 缩短了凝胶时间。DC-ECM水凝胶黏度高于溶液黏度，剪切速率增加，溶液黏度降低。此外，DC-ECM溶液和水凝胶的高储能模量表明它们都具有凝胶而不是液体特性。SEM分析表明，交联和冷冻干燥后，DC-ECM溶液在水凝胶形态中的孔径显著减小。该协议涉及物理和化学破坏以及酶消化的组合，以去除细胞物质，同时保留ECM的结构和对话。

Research

JoVE Journal

Bioengineering

The Synthesis of RGD-functionalized Hydrogels as a Tool for Therapeutic Applications

RGD功能化水凝胶作为治疗应用的工具合成

The use of polymers as biomaterials has provided significant advantages in therapeutic applications. In particular, the possibility to modify and ...

科研

生物工程

Preparation of Extracellular Matrix Protein Fibers for Brillouin Spectroscopy

细胞外基质蛋白纤维的布里渊谱的制备

Brillouin spectroscopy is an emerging technique in the biomedical field. It probes the mechanical properties of a sample through the interaction of ...

Tunable Hydrogels from Pulmonary Extracellular Matrix for 3D Cell Culture

可调水凝胶由肺细胞外基质的三维细胞培养

Here we present a method for establishing multiple component cell culture hydrogels for in vitro lung cell culture. Beginning with healthy en bloc lung ...

Fabricating a Kidney Cortex Extracellular Matrix-Derived Hydrogel

肾皮质细胞外基质衍生水凝胶的制备

Extracellular matrix (ECM) provides important biophysical and biochemical cues to maintain tissue homeostasis. Current synthetic hydrogels offer robust ...

Fabrication of Decellularized Cartilage-derived Matrix Scaffolds

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 ...

Measuring Global Cellular Matrix Metalloproteinase and Metabolic Activity in 3D Hydrogels

三维水凝胶中全球细胞基质金属蛋白酶和代谢活性的测定

Three-dimensional (3D) cell culture systems often more closely recapitulate in vivo cellular responses and functions than traditional two-dimensional (2D) ...

Labeling of Extracellular Vesicles for Monitoring Migration and Uptake in Cartilage Explants

标记细胞外囊泡以监测软骨外植体的迁移和摄取

Extracellular vesicles (EVs) are used in different studies to prove their potential as a cell-free treatment due to their cargo derived from their ...

Fabrication and Characterization of Layer-By-Layer Janus Base Nano-Matrix to Promote Cartilage Regeneration

促进软骨再生的逐层Janus碱基纳米基质的制备和表征

Various biomaterial scaffolds have been developed to guide cell adhesion and proliferation in hopes to promote specific functions for in vitro and in vivo ...

牛肺 ECM 水凝胶脱细胞技术的比较研究

Development and Characterization of Decellularized Lung Extracellular Matrix Hydrogels

作者聚焦：优化器官型水凝胶的牛肺脱细胞

脱细胞肺细胞外基质水凝胶的开发与表征

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