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

The authors would like to acknowledge W. Boot for assistance in the production of the scaffolds. K.E.M. Benders is supported by the Alexandre Suerman Stipendium from the University Medical Center. R. Levato and J. Malda are supported by the Dutch Arthritis Foundation (grant agreements CO-14-1-001 and LLP-12, respectively).

For this protocol, equine stifle cartilage was obtained from horses that had died from other causes than osteoarthritis. Tissue was obtained with permission of the owners, in line with the institutional ethical regulations.
NOTE: This protocol describes the fabrication of scaffolds from decellularized equine cartilage, which can be used for applications such as in vitro tissue culture platforms or for in vivo implantation in regenerative medicine strategies. ...

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The ECM of articular cartilage is very dense and quite resilient to different enzymatic treatments. The multi-step decellularization protocol described in this article addresses such resistance and successfully generates decellularized matrices. To achieve that, the process spans over several days. Many decellularization processes have been proposed for different types of tissues18, and this article describes a protocol suitable for the decellularization of cartilage. In this protocol, it is, howe...

Articular cartilage defects in the knee caused by traumatic events can lead to discomfort, and above all can have a large impact on the lives of the young and active population1,2,3. Moreover, cartilage damage at a young age may lead to a more rapid onset of osteoarthritis later in life4. Currently, the only salvage therapy for generalized osteoarthritis of the knee is joint replacement surgery. As cartilage is a hypocellular, aneural, and avascular tissue, its regenerative capacity is severely limited. Therefore, regenerative medicine approaches are sought after to aid and stimulate the regenerative capacity of the native tissue. For this purpose, scaffolds are designed and used as either a cell-carrier or as an inductive material that incites differentiation and regeneration of tissue by the body&#39;s native cells5.
Decellularized scaffolds have been widely studied within regenerative medicine6. It has had some success, for example, in aiding the regeneration of skin7, abdominal structures8, and tendons9. The advantage of using decellularized scaffolds is their natural origin and their capacity to retain bioactive cues that both attract and induce cell differentiation into the appropriate lineage required for tissue repair6,10. Moreover, since extracellular matrix (ECM) is a natural biomaterial, and decellularization prevents a potential immune response by removing cellular or genetic content, issues regarding biocompatibility and biodegradability are overcome.
Cartilage-derived matrix (CDM) scaffolds have shown great chondrogenic potential in in vitro experiments when seeded with mesenchymal stromal cells11. In addition, these scaffolds have shown the potential to form bone tissue through endochondral ossification on ectopic locations in in vivo settings12. As CDM scaffolds guide the formation of both bone and cartilage tissue, these scaffolds may hold potential for osteochondral defect repair in addition to cartilage repair.
This article describes a protocol adapted from Yang et al. (2010)13 for the production of decellularized CDM scaffolds from equine stifle cartilage. These scaffolds are rich in collagen type II and devoid of cells, and do not contain any glycosaminoglycans (GAGs) after decellularization. Both in vitro and in vivo experiments on (osteo)chondral defect repair can be conducted using these scaffolds.

<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

Osteochondral defects lack sufficient intrinsic repair capacity to regenerate functionally sound bone and cartilage tissue. To this extent, cartilage research has focused on the development of regenerative scaffolds. This article describes the development of scaffolds that are completely derived from natural cartilage extracellular matrix, coming from an equine donor. Potential applications of the scaffolds include producing allografts for cartilage repair, serving as a scaffold for osteochondral tissue engineering, and providing in vitro models to study tissue formation. By decellularizing the tissue, the donor cells are removed, but many of the natural bioactive cues are thought to be retained. The main advantage of using such a natural scaffold in comparison to a synthetically produced scaffold is that no further functionalization of polymers is required to drive osteochondral tissue regeneration. The cartilage-derived matrix scaffolds can be used for bone and cartilage tissue regeneration in both in vivo and in vitro settings.

Decellularization of CDM scaffolds must always be confirmed using histological stainings as well as using DNA quantification to measure the amount of DNA remnants. Insufficient decellularization might lead to undesired immunological responses that influence the results in in vivo settings15,16,17. For this specific decellularization method, DNA was below the detection range, which started at 13.6...

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

Fabrication of Decellularized Cartilage-derived Matrix Scaffolds

Decellularized cartilage-derived scaffolds can be used as a scaffold to guide cartilage repair and as a means to regenerate osteochondral tissue. This paper describes the decellularization process in detail and provides suggestions to use these scaffolds in in vitro settings.

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

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