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. ...

<ol>
	<li>Dunlop, D. 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=Risk+factors+for+functional+decline+in+older+adults+with+arthritis.">Risk factors for functional decline in older adults with arthritis.</a> Arthritis and rheumatism. 52 (4), 1274-1282 (2005).</li><li>Fitzpatrick, K., Tokish, J. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+military+perspective+to+articular+cartilage+defects.">A military perspective to articular cartilage defects.</a> The journal of knee surgery. 24 (3), 159-166 (2011).</li><li>Flanigan, D. C., Harris, J. D., Trinh, T. Q., Siston, R. A., Brophy, R. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Prevalence+of+chondral+defects+in+athletes'+knees:+a+systematic+review.">Prevalence of chondral defects in athletes' knees: a systematic review.</a> Medicine and science in sports and exercise. 42 (10), 1795-1801 (2010).</li><li>Martel-Pelletier, J., Boileau, C., Pelletier, J. P., Roughley, P. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cartilage+in+normal+and+osteoarthritis+conditions.">Cartilage in normal and osteoarthritis conditions.</a> Best practice &amp; research. Clinical rheumatology. 22 (2), 351-384 (2008).</li><li>Vinatier, 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=Cartilage+tissue+engineering:+towards+a+biomaterial-assisted+mesenchymal+stem+cell+therapy.">Cartilage tissue engineering: towards a biomaterial-assisted mesenchymal stem cell therapy.</a> Current stem cell research &amp; therapy. 4 (4), 318-329 (2009).</li><li>Taylor, D. A., Sampaio, L. C., Ferdous, Z., Gobin, A. S., Taite, L. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Decellularized+matrices+in+regenerative+medicine.">Decellularized matrices in regenerative medicine.</a> Acta biomaterialia. 74, 74-89 (2018).</li><li>Vashi, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Clinical+Outcomes+for+Breast+Cancer+Patients+Undergoing+Mastectomy+and+Reconstruction+with+Use+of+DermACELL,+a+Sterile,+Room+Temperature+Acellular+Dermal+Matrix.">Clinical Outcomes for Breast Cancer Patients Undergoing Mastectomy and Reconstruction with Use of DermACELL, a Sterile, Room Temperature Acellular Dermal Matrix.</a> Plastic Surgery International. 2014 (704323), 1-7 (2014).</li><li>Satterwhite, T. 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=Abdominal+wall+reconstruction+with+dual+layer+cross-linked+porcine+dermal+xenograft:+the+&amp;#34;Pork+Sandwich&amp;#34;+herniorraphy.">Abdominal wall reconstruction with dual layer cross-linked porcine dermal xenograft: the &amp;#34;Pork Sandwich&amp;#34; herniorraphy.</a> Journal of plastic, reconstructive &amp; aesthetic surgery : JPRAS. 65 (3), 333-341 (2012).</li><li>Martinello, 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=Successful+recellularization+of+human+tendon+scaffolds+using+adipose-derived+mesenchymal+stem+cells+and+collagen+gel.">Successful recellularization of human tendon scaffolds using adipose-derived mesenchymal stem cells and collagen gel.</a> Journal of tissue engineering and regenerative medicine. 8 (8), 612-619 (2014).</li><li>Benders, K. 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=Extracellular+matrix+scaffolds+for+cartilage+and+bone+regeneration.">Extracellular matrix scaffolds for cartilage and bone regeneration.</a> Trends in biotechnology. 31 (3), 169-176 (2013).</li><li>Benders, K. 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. F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Biologic+scaffolds+for+regenerative+medicine:+mechanisms+of+in+vivo+remodeling.">Biologic scaffolds for regenerative medicine: mechanisms of in vivo remodeling.</a> Annals of biomedical engineering. 43 (3), 577-592 (2015).</li><li>Gilbert, T. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Strategies+for+tissue+and+organ+decellularization.">Strategies for tissue and organ decellularization.</a> Journal of cellular biochemistry. 113 (7), 2217-2222 (2012).</li></ol>

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 ...

This method presents a way to create scaffolds derived from native tissue which can be used for cartilage regeneration. The main advantage of this technique is that components of native cartilage are preserved in the scaffolds that can promote cartilage formation and regeneration in vitro and in vivo. Although these method uses cartilage from equine stifle joints, cartilage from any other joints can be used as well.To begin, obtain an intact cadaveric joint for cartilage isolation and remove the skin excess fat and muscle tissue as described in the accompanying text protocol. Next, define the location where the joint articulates by performing extension inflection of the joint. Make a horizontal incision to reach the joint cavity.Next, make a vertical incision to open up the joint area. The joint cavity is filled with synovial fluid which will likely drip from the cavity when performing of correct incision. Continue to open up the joint completely by removing excessive fat, muscles and tendons, and by cutting through the tendons that keep the joint together.Carefully inspect the cartilage for any macroscopic damage. If the articular cartilage does not have a glossier smooth appearance or if evidence, blistering, clefts or defects are present, discard the cartilage and start again. Use a sterile scalpel to remove the cartilage from the bone.Cut all the way down to the subchondral bone to remove the deep zone cartilage. Collect the removed cartilage slices in 50 milliliter tubes containing previously prepared cartilage washing solution. While being processed, regularly drip cartilage washing solution on the cartilage to prevent the cartilage from drying out.After collecting the cartilage from all of the areas of interest, snap freeze the cartilage slices in liquid nitrogen for five minutes. Then transfer the cartilage slices into 50 milliliter tubes and immediately placed the frozen slices in a freeze dryer. Lyophilize the cartilage slices for 24 hours with the freeze dryer.When finished, store the freeze dried cartilage slices in a dry place at room temperature. For manual processing, pre chill a mortar and pestle with liquid nitrogen, then place the lyophilized cartilage slices in the mortar and submerge them and liquid nitrogen. Directly grind the samples by hand for approximately 45 minutes until they are sufficiently pulverized.Alternatively, to grind the samples automatically using a milling machine, begin by pre cooling the grinding compartment of the milling machine by adding liquid nitrogen. Then add the lyophilized cartilage slices and mill the sample at its preset speed for a few seconds up to a minute. Make sure that all particles are ground and that no particles stay in the bottom of the grinding compartment.Finally, follow along with the decellularization technique described in the accompanying text protocol prior to forming scaffolds. Transfer the cartilage particles with a small label to a cylindrical plastic mold. Press all of the air bubbles out to avoid cavities in the scaffold and fill the mold completely.To avoid cavities in the scaffoldS, ensure that air bubbles are pressed out while filling the mold with cartilage particles. After formation, freeze the mold for 10 minutes at minus 20 degrees Celsius. Then lyophilize the cartilage scaffolds within the mold for 24 hours in a freeze dryer.After lyophilization, carefully remove the scaffold from the mold. Place the scaffold under a 365 nanometer wavelength UV light and cross link it overnight. Form scaffold discs by cutting sterilized scaffolds into three millimeter thick slices.Then transfer the scaffolds into separate wellS of a six well plate. Add one milliliter of Chondrocyte Expansion Medium on top of the scaffolds and allow them to rehydrate for 30 minutes. While the scaffolds rehydrate, prepare the cells and pipette 50 microliter of the prepared cell suspension on top of the pre soaked scaffold.Then incubate the scaffold for one hour at 37 degrees Celsius. When using chondrocytes, make sure they have not been expanded past the first passage in order to minimize the number of the differentiated cells. After one hour, return the plate to the culture hood and carefully turn the scaffold over.Pipette the remaining 50 microliters of cell suspension on the scaffold and incubate for another hour at 37 degrees Celsius. After incubation, add three milliliters of medium to the wells with scaffolds. Avoid pipetting the medium directly on the scaffolds and handle the culture plate gently to avoid detachment of the cells.Return the plate to the incubator and culture cell ceded scaffolds at 37 degrees Celsius. A combination of historlogical staining and DNA quantification have been used to show that the methods presented in this article result in sufficient cartilage scaffold decellularization. The resulting scaffolds were found to contain no cells along with undetectable levels of DNA.They also lack glycosaminoglycans and are rich in collagen II.Here, neo matrix formation is observed on scaffold ceded with mesenchymal stem cells that were cultured for four weeks. The formation of glycosaminoglycans is abundant after four weeks. After attempting this procedure, it is important to examine whether decellularization has been successful, before using it in any application.Following this procedure, additional biochemical analysis like Western blot analyzer can be performed in order to answer specific questions about the presence of other components such as growth factors. This technique paved the way for researchers in the field of tissue engineering to explore strategies for cartilage regeneration using decellularized tissue from allogeneic and syngeneic sources. Precautions such as wearing a lab coat and gloves should always be taken.

Research

JoVE Journal

Bioengineering

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

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

Fabrication of Mechanically Tunable and Bioactive Metal Scaffolds for Biomedical Applications

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

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

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

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 ...

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 ...

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 ...

Decellularized Rat Spleen Matrix Preparation for Liver Tissue Engineering

Fabrication of Decellularized Spleen Matrix Derived from Rats

Author Spotlight: Utilization of Decellularized Spleen Matrix for Bioartificial Livers

Liver transplantation is the primary treatment for end-stage liver disease. However, the shortage and inadequate quality of donor organs necessitate the ...