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

This work was sponsored by the Medicine and Health Technology Plan of Zhejiang Province (2019KY050), the Traditional Chinese Medicine Science and Technology Plan of Zhejiang Province (2019ZA026), the Key Research and Development Plan in Zhejiang Province (Grant No.2020C03043), the Traditional Chinese Medicine Science and Technology Plan of Zhejiang Province (2021ZQ021), and the Zhejiang Provincial Natural Science Foundation of China (LQ22H060007).

This study was approved by the Ethics Committee of Tongde Hospital of Zhejiang Province.
1. Preparation of the DC-ECM hydrogel
NOTE: In this study, the cartilage was obtained from the knee joints of 12 month old Bama miniature pigs, avoiding the collection of bone tissue.
<ol>
	<li>Take the collected cartilage, and block and chop it into 1-2 mm3 pieces with a scalpel.</li>
	<li>Place 20 g of the minced cartilage in a 50 mL centrifuge tu...

<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>

This protocol provides a systematic approach for the preparation of decellularized cartilage extracellular matrix hydrogels that closely mimic the native ECM of cartilage tissue. The protocol involves a combination of physical, chemical, and enzymatic disruption to remove cellular material while preserving the structure and composition of the ECM. The protocol's critical steps include adjusting the decellularization time and methods and ensuring complete decellularization.
Compared to other ex...

Cartilage tissue engineering is a rapidly developing field that seeks to regenerate damaged or diseased cartilage tissue1. One key challenge in this field is the development of biomimetic scaffolds that can support the growth and differentiation of chondrocytes, the cells responsible for producing cartilage2. The ECM of cartilage tissue plays a critical role in regulating the behavior of chondrocytes. DC-ECM is an effective scaffold for tissue engineering applications3.
A number of techniques have been developed to produce DC-ECM from cartilage tissue, including chemical, enzymatic, and physical methods. However, these methods often result in the generation of ECM hydrogels that are insufficiently biomimetic, which limits their potential for use in tissue engineering applications4,5. Thus, there is a need for a more effective method for producing DC-ECM hydrogels.
The development of this technique is important because it can advance the field of tissue engineering by providing a new approach for creating biomimetic scaffolds that can support tissue regeneration and repair. Furthermore, this technique could be easily adapted to produce ECM hydrogels from other tissues, thereby expanding its potential applications.
In the broader body of literature, there has been growing interest in using DC-ECM as a scaffold for tissue engineering applications6. Numerous studies have demonstrated the effectiveness of DC-ECM hydrogels in promoting cell growth and differentiation in various tissues, including cartilage7,8. Therefore, the development of a protocol for producing DC-ECM hydrogels that closely mimic the natural ECM of cartilage tissue is a significant contribution to the field.
The protocol presented in this paper addresses this need by providing a novel method for producing DC-ECM hydrogels that closely mimic the natural ECM of cartilage tissue. The protocol involves decellularizing cartilage tissue, isolating the resulting ECM, and creating a hydrogel by cross-linking the ECM with a biocompatible polymer. The resulting hydrogel has shown promising results in supporting the growth and differentiation of chondrocytes.

<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

Decellularized cartilage extracellular matrix (DC-ECM) hydrogels are promising biomaterials for tissue engineering and regenerative medicine due to their biocompatibility and ability to mimic natural tissue properties. This protocol aims to produce DC-ECM hydrogels that closely mimic the native ECM of cartilage tissue. The protocol involves a combination of physical and chemical disruption and enzymatic digestion to remove the cellular material while preserving the structure and composition of the ECM. The DC-ECM is cross-linked using a chemical agent to form a stable and biologically active hydrogel. The DC-ECM hydrogel has excellent biological activity, spatial structure, and biological induction function, as well as low immunogenicity. These characteristics are beneficial in promoting cell adhesion, proliferation, differentiation, and migration and for creating a superior microenvironment for cell growth. This protocol provides a valuable resource for researchers and clinicians in the field of tissue engineering. Biomimetic hydrogels can potentially enhance the development of effective tissue engineering strategies for cartilage repair and regeneration.

To prepare a better DC-ECM cartilage hydrogel, we studied and reviewed the previous literature and compared the various decellularization protocols in terms of the decellularization ratio, immunogenicity, and mechanical functionality9.
On this basis, we prepared the DC-ECM cartilage hydrogel and explored the effect of a radially oriented extractive matrix/mesenchymal stem cell exosome bio-ink in treating osteochondral defects. The results showed that the DC-ECM cartilag...

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

Synthesis of Decellularized Cartilage Extracellular Matrix Hydrogels

This paper introduces a new method for the synthesis of decellularized cartilage extracellular matrix (DC-ECM) hydrogels. DC-ECM hydrogels have excellent biocompatibility and provide a superior microenvironment for cell growth. Therefore, they can be ideal cell scaffolds and biological delivery systems.

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

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