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. The enzymatic treatment steps must be performed in the described chronological order.
1. Harvesting of Articular Cartilage from Donor (Cadaveric) Joints
<ol>
	<li>Ahead of the harvesting step, prepare 1 L of cartilage washing solution, consisting of sterile phosphate-buffered saline (PBS), supplemented with 100 U/mL penicillin, 100 &#181;g/mL streptomycin, and 1% (v/v) Amphotericin B.</li>
	<li>Wear gloves and a lab coat during the entire harvesting procedure, since the donor can carry pathogens.</li>
	<li>Obtain an intact (cadaveric) joint for cartilage isolation. 
	NOTE: At this point, the skin around the joint is still intact. Equine stifle cartilage obtained from horse was used in this protocol, but other joints from other species can also be used.</li>
	<li>Remove the skin that is located around the joint area of interest by using a scalpel and a surgical tweezer. In addition, remove excessive fat and muscle tissue if necessary, without damaging the underlying joint. Optionally, transfer the prepared cadaver to a biological safety cabinet.</li>
	<li>To perform an arthrotomy, i.e., a separation of the joint, first define the location where the joint articulates by performing extension and flexion of the joint.</li>
	<li>Carefully remove more layers of fat, muscles, and tendons with a scalpel and surgical tweezer to open up the joint area.</li>
	<li>Make an incision to reach the joint cavity. 
	NOTE: The joint cavity is filled with synovial fluid, which can drip from the cavity when performing a correct incision.</li>
	<li>Continue to open up the joint completely by cutting through the tendons that keep the joint together.</li>
	<li>Inspect the cartilage for any macroscopic damage. 
	NOTE: If the articular cartilage does not have a glossy and smooth appearance or if evident blistering, clefts, or defects are present, discard the cartilage.</li>
	<li>Use a sterile scalpel to remove the cartilage from the subchondral bone. Cut all the way down to the subchondral bone to also remove the deep zone of the cartilage (Figure 1). To prevent the cartilage from drying out, regularly drip cartilage washing solution on the cartilage. 
	NOTE: At this time, the size of the cartilage slices does not matter.</li>
	<li>Collect the cartilage slices in 50 mL tubes containing previously prepared cartilage washing solution (Figure 2A).</li>
	<li>After collecting the cartilage from all the areas of interest, snap freeze the cartilage slices in liquid nitrogen for 5 min.</li>
	<li>Transfer the cartilage slices into 50 mL tubes and immediately lyophilize the cartilage slices for 24 h in a freeze dryer. Set the freeze dryer at approximately 0.090 mbar while the ice condenser is -51 &#176;C (Figure 2B).</li>
	<li>Store the cartilage slices in a dry place at room temperature until further use. 
	NOTE: Up to the point of scaffold formation, the solutions and cartilage do not have to be prepared and treated in a sterile way.</li>
</ol>
2. Creating Decellularized Cartilage Particles
<ol>
	<li>Submerse the lyophilized cartilage slices in liquid nitrogen.</li>
	<li>Directly grind the samples either manually or by a milling machine. When grinding the cartilage slices by hand, use a mortar and pestle and grind the slices for approximately 45 min until they are pulverized (Figure&#160;2C and 2D). When grinding automatically, use a milling machine at its pre-set speed for a few seconds up to a minute, until the snap-frozen cartilage slices are pulverized. 
	NOTE: Pre-cool the mortar or the grinding compartment of the milling machine by adding liquid nitrogen. When using a milling machine, make sure that all particles are ground, and that no particles stay in the bottom of the grinding compartment.</li>
	<li>Sieve the cartilage particles to get rid of larger parts by using a sieve with a 0.71 mm mesh size.</li>
	<li>Store the particles in a dry place at room temperature.</li>
</ol>
3. Enzymatic Decellularization with Trypsin 0.25%-EDTA
<ol>
	<li>Prepare the digestion solution, consisting of 0.25% trypsin with EDTA supplemented with 100 U/mL penicillin, 100 &#956;g/mL streptomycin, and 1% (v/v) Amphotericin B. Store the solution at 4 &#176;C. 
	NOTE: Trypin is a protease that degrades proteins residing in the cartilage tissue. This enzymatic step will open up the dense cartilage structure.</li>
	<li>Fill the 50 mL tubes with the pulverized cartilage particles up to a volume of approximately 7.5 mL per tube.</li>
	<li>Incubate the cartilage particles with the prepared digestion solution at 37 &#176;C for 24 h in total, while refreshing the digestion solution every 4 h.
	<ol>
		<li>First, add 30 mL of the digestion solution to each 50 mL tube that contains cartilage particles.</li>
		<li>Then, resuspend the particles by using a vortex or pipet so that all particles are exposed to the enzymatic solution.</li>
		<li>Incubate the samples for 4 h at 37 &#176;C on a roller.</li>
		<li>To refresh the digestion solution, centrifuge the tubes for 20 min at 3113 x g to cause sedimentation of the cartilage particles. Discard the supernatant. 
		NOTE: The supernatant will become clearer with every trypsin incubation period.</li>
		<li>Repeat step 3.3.1&#8211;3.3.4 until the particles have been incubated with the digestion solution for 6 cycles of 4 h.</li>
	</ol>
	</li>
	<li>After the final cycle, remove the supernatant after centrifuging and wash the particles in 30 mL of cartilage washing solution twice. Centrifuge the particles for 20 min at 3113 x g between each of the washes.</li>
</ol>
4. Enzymatic Decellularization with Nuclease Treatment
<ol>
	<li>Prepare a 10 mM Tris-HCl solution at pH 7.5 in deionized water.</li>
	<li>Add 50 U/mL deoxyribonuclease and 1 U/mL ribonuclease A to the Tris-HCl buffer, to obtain the nuclease solution. 
	NOTE: This step is performed to specifically degrade deoxyribonucleases and ribonucleases.</li>
	<li>To remove the cartilage washing solution from the cartilage particles (step 3.4), centrifuge for 20 min at 3,113 x g, and remove the supernatant.</li>
	<li>Add 30 mL of the nuclease solution to the cartilage particles and stir the particles through the solution, making sure that all particles are exposed to the nuclease solution.</li>
	<li>Incubate the samples on a roller for 4 h at 37 &#176;C.</li>
	<li>Remove the nuclease solution by centrifuging the cartilage particles for 20 min at 3,113 x g and discard the supernatant.</li>
	<li>Wash the samples by adding 30 mL of 10 mM Tris-HCl solution without the deoxyribonuclease and ribonuclease A to the cartilage particles. Resuspend the particles and leave the samples on a roller for 20 h at room temperature.</li>
</ol>
5. Detergent Decellularization
<ol>
	<li>Make the detergent solution by dissolving 1% (v/v) octoxynol-1 in PBS.</li>
	<li>Remove the Tris-HCl solution from the cartilage particles by centrifuging for 20 min at 3,113 x g and discarding the supernatant.</li>
	<li>Add 30 mL of the prepared 1% detergent solution to the cartilage particles. Resuspend the cartilage particles gently to avoid foaming of the solution. 
	NOTE: This step breaks down cellular membranes.</li>
	<li>Incubate the samples on a roller for 24 h at room temperature on a roller.</li>
	<li>Remove the detergent solution by centrifugation for 20 min at 3,113 x g and discard the supernatant.</li>
	<li>To remove all of the remnants of the decellularization solutions, wash the cartilage particles in 6 cycles of 8 h in 30 mL of cartilage washing solution. Perform washing on a roller at room temperature.
	<ol>
		<li>To change to the next wash, centrifuge the suspension for 20 min at 3,113 x g, discard the supernatant and add fresh cartilage washing solution.</li>
	</ol>
	</li>
	<li>Leave the last wash in the tubes and store the decellularized cartilage particles at -80 &#176;C.</li>
</ol>
6. Creating Scaffolds from the Decellularized Particles
<ol>
	<li>If the particles have been stored at -80 &#176;C, thaw the closed tubes that contain the cartilage particles in warm water before creating the scaffolds.</li>
	<li>Transfer the cartilage particles with a small ladle to a cylindrical mold, for example, a plastic vial with a diameter of 8 mm and a height of 2 cm.</li>
	<li>When placing the cartilage particles into the plastic mold, press all the air-bubbles out to avoid cavities in the scaffold, and fill the mold until the edge. 
	NOTE: It will be more difficult to take the scaffolds out of a metal mold after scaffold formation, which can lead to cracks in the scaffold.</li>
	<li>Freeze the molds with cartilage particles for 10 min at -20 &#176;C.</li>
	<li>Lyophilize the cartilage scaffolds within their molds for 24 h in a freeze dryer.</li>
	<li>After lyophilization, take the scaffold out of the mold (Figure 3) and cross-link them with ultraviolet (UV) light at 30 cm distance and 365 nm overnight.</li>
	<li>In order to use the scaffolds for in vitro cell culture or in vivo implantation, sterilize the scaffolds with, for example, ethylene oxide (EtO) gas. 
	NOTE: EtO sterilization is performed by an external party.</li>
</ol>
7. Characterization of the Decellularized Scaffolds with Histological Stainings
NOTE: To ensure complete decellularization and to visualize the remaining natural character of the cartilage, perform several histological stainings before using the scaffolds in any experiment, including hematoxylin and eosin (H&#38;E) staining to ensure decellularization, Safranin-O staining to visualize residual GAG presence, collagen type I immunohistochemistry to differentiate between collagen content, and collagen type II immunohistochemistry to differentiate between collagen content.
<ol>
	<li>Cut the scaffolds in thin slices of approximately 3 mm with a scalpel. 
	NOTE: If the scaffolds are cut in larger or smaller sizes, the durations of the dehydration cycles need to be adapted.</li>
	<li>Embed the scaffolds in a drop of 4% (w/v) alginate and induce cross-linking by adding a similar volume of 3.7% non-buffered formalin that contains 20 mM CaCl2. 
	NOTE: Alginate embedding makes the scaffold slices more rigid compared to the washing steps prior to paraffin embedding. In case the scaffolds have been cell-seeded or in vivo implanted, alginate embedding is not necessary, as the composition of the scaffolds will be resistant enough due to ECM incorporation by the cells.</li>
	<li>Dehydrate the samples by placing them in a graded ethanol series, starting with one-hour cycles of 70%, 96%, 96%, 100%, and 100% ethanol, followed by two&#160;1 h cycles of xylene, and ending with two 1 h cycles of paraffin at 60 &#176;C.</li>
	<li>After dehydration, paraffin-embed the samples in a mold and cool the samples down.</li>
	<li>Cut the samples with a microtome in slices of 5 &#956;m thick.</li>
	<li>Before staining, rehydrate the samples by placing them in a returned graded ethanol series. Start with two washes of xylene for 5 min, followed by 2 washes of 100%, 95%, and 70% ethanol for 3 min per wash. Finally, wash the samples 3 times in water for 2 min. 
	NOTE: In case of using alginate to process samples for paraffin embedding, make sure to wash off the alginate with 10 mM citric acid prior to rehydration of the paraffin sections.</li>
	<li>Stain the samples with hematoxylin and eosin, Safranin-O, collagen I, and collagen II as previous described11.</li>
</ol>
8. Characterization of the Decellularized Scaffolds with Quantitative Analyses
<ol>
	<li>Obtain papain digests of the scaffolds as described previously11.</li>
	<li>Perform an assay with a fluorescence-based DNA quantification kit to measure the double-strand DNA content of the scaffolds to ensure complete decellularization. Follow the protocol provided by the manufacturer. Express the amount of DNA per dry weight of the scaffold.</li>
	<li>Perform a dimethylmethylene blue assay to quantify the remainder of the GAGs within the scaffold, as described previously11. Express the amount in GAG per DNA.</li>
</ol>
9. Seeding of the Decellularized Scaffolds
<ol>
	<li>Cut sterilized scaffolds from step 6.7 into 3 mm thick slices.</li>
	<li>Transfer the scaffolds in separate wells of a 6-well plate.</li>
	<li>Rehydrate scaffolds with cell culture medium by pipetting 1 mL of medium on top of the scaffold and let it soak for 30 min. Use either chondrocyte or mesenchymal stem cell (MSC) expansion medium. 
	Note: Use the same medium for scaffold rehydration as for cell culture of the cells that will be seeded. Chondrocyte expansion medium consists of Dulbecco&#39;s modified media (DMEM) with 10% heat-inactivated fetal bovine serum (FBS), 100 U/mL penicillin, 100 &#956;g/mL streptomycin, and 10 ng/mL fibroblast growth factor-2 (FGF-2). MSC expansion medium consists of minimum essential medium alpha (a-MEM) with 10% heat-inactivated FBS, 0.2 mM L-ascorbic acid 2-phosphate, 100 U/mL penicillin, 100 &#956;g/mL streptomycin, and 10 ng/mL FGF-2.</li>
	<li>Prepare 3 x 106 cells in 100 &#956;L of medium, which is the required volume for each scaffold with a diameter of 8 mm and a height of 3 mm. 
	NOTE: Multiple cell types from different species can be used for seeding. When using chondrocytes or mesenchymal stromal cells, isolate the cells as previously described11. When using chondrocytes, make sure that they have not been expanded past the P1 passage in order to minimize the number of dedifferentiated chondrocytes. When using mesenchymal stromal cells, they must be tested on their ability for multi-lineage differentiation, as previously described14.</li>
	<li>Pipet 50 &#956;L of prepared cell suspension on top of the pre-soaked scaffold and incubate the scaffold for 1 h at 37 &#176;C.</li>
	<li>Carefully turn the scaffold upside down, pipet the remaining 50 &#956;L of cell suspension on this side of the scaffold and incubate for 1 h at 37 &#176;C.</li>
	<li>After incubation, add 3 mL of medium to the wells, and culture the cell-seeded scaffolds at 37 &#176;C. Handle the culture plate gently to avoid detachment of the cells.</li>
	<li>Culture the scaffolds for the period of time that is required for the experiment and change the medium 2&#8211;3 times a week. Pipet slowly and as far away from the scaffold as possible.</li>
	<li>After culturing of the cell-seeded scaffold, cut the scaffolds in half and process them for both histology and biochemical analyses.</li>
</ol>

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The authors declare that they have no competing financial interests.

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 ...

fabrication-of-decellularized-cartilage-derived-matrix-scaffolds

Research

JoVE Journal

Bioengineering

10.9K Views.  University Medical Center Utrecht, The Netherlands. 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.

Video: Fabrication of Decellularized Cartilage-derived Matrix Scaffolds

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 substrates for organ-scale tissue development. Sprague Dawley rat kidneys are cannulated by inserting a catheter into the renal artery and perfused with a series of low-concentration detergents (Triton X-100 and sodium dodecyl sulfate (SDS)) over 26 hr to derive intact, whole-kidney scaffolds with intact perfusable vasculature, glomeruli, and renal tubules. Following decellularization, the renal scaffold is placed inside a custom-designed perfusion bioreactor vessel, and the catheterized renal artery is connected to a perfusion circuit consisting of: a peristaltic pump; tubing; and optional probes for pH, dissolved oxygen, and pressure. After sterilizing the scaffold with peracetic acid and ethanol, and balancing the pH (7.4), the kidney scaffold is prepared for seeding via perfusion of culture medium within a large-capacity incubator maintained at 37 &#176;C and 5% CO2. Forty million renal cortical tubular epithelial (RCTE) cells are injected through the renal artery, and rapidly perfused through the scaffold under high flow (25 ml/min) and pressure (~230 mmHg) for 15 min before reducing the flow to a physiological rate (4 ml/min). RCTE cells primarily populate the tubular ECM niche within the renal cortex, proliferate, and form tubular epithelial structures over seven days of perfusion culture. A 44 &#181;M resazurin solution in culture medium is perfused through the kidney for 1 hr during medium exchanges to provide a fluorometric, redox-based metabolic assessment of cell viability and proliferation during tubulogenesis. The kidney perfusion bioreactor permits non-invasive sampling of medium for biochemical assessment, and multiple inlet ports allow alternative retrograde seeding through the renal vein or ureter. These protocols can be used to recellularize kidney scaffolds with a variety of cell types, including vascular endothelial, tubular epithelial, and stromal fibroblasts, for rapid evaluation within this system.

This protocol describes decellularization of Sprague Dawley rat kidneys by antegrade perfusion of detergents through the vasculature, producing acellular renal extracellular matrices that serve as templates for repopulation with human renal epithelial cells. Recellularization and use of the resazurin perfusion assay to monitor growth is performed within specially-designed perfusion bioreactors.

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 and low bioactivity of metals. In this study, we have developed a new method towards fabricating a new type of bioactive and mechanically reliable porous metal scaffolds-densified porous Ti scaffolds. The method consists of two fabrication processes, 1) the fabrication of porous Ti scaffolds by dynamic freeze casting, and 2) coating and densification of the porous scaffolds. The dynamic freeze casting method to fabricate porous Ti scaffolds allowed the densification of porous scaffolds by minimizing the chemical contamination and structural defects. The densification process is distinctive for three reasons. First, the densification process is simple, because it requires a control of only one parameter (degree of densification). Second, it is effective, as it achieves mechanical enhancement and sustainable release of biomolecules from porous scaffolds. Third, it has broad applications, as it is also applicable to the fabrication of functionally graded porous scaffolds by spatially varied strain during densification.

Bioactive and mechanically reliable metal scaffolds have been fabricated through a method which consists of two processes, dynamic freeze casting for the fabrication of porous Ti, and coating and densification of the Ti scaffolds. The densification process is simple, effective and applicable to the fabrication of functionally graded scaffolds.

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 cell-matrix interactions. Due to this complexity, recapitulation of lung development in vitro to promote differentiation of stem cells to lung epithelial cells has been challenging. In this protocol, decellularized lung scaffolds are used to mimic the 3-dimensional environment of the lung and generate stem cell-derived airway epithelial cells. Mouse embryonic stem cell are first differentiated to the endoderm lineage using an embryoid body (EB) culture method with activin A. Endoderm cells are then seeded onto decellularized scaffolds and cultured at air-liquid interface for up to 21 days. This technique promotes differentiation of seeded cells to functional airway epithelial cells (ciliated cells, club cells, and basal cells) without additional growth factor supplementation. This culture setup is defined, serum-free, inexpensive, and reproducible. Although there is limited contamination from non-lung endoderm lineages in culture, this protocol only generates airway epithelial populations and does not give rise to alveolar epithelial cells. Airway epithelia generated with this protocol can be used to study cell-matrix interactions during lung organogenesis and for disease modeling or drug-discovery platforms of airway-related pathologies such as cystic fibrosis.

This protocol efficiently directs mouse embryonic stem cell-derived definitive endoderm to mature airway epithelial cells. This differentiation technique uses 3-dimensional decellularized lung scaffolds to direct lung lineage specification, in a defined, serum-free culture setting.

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

Fabrication of Extracellular Matrix-derived Foams and Microcarriers as Tissue-specific Cell Culture and Delivery Platforms

Cell function is mediated by interactions with the extracellular matrix (ECM), which has complex tissue-specific composition and architecture. The focus of this article is on the methods for fabricating ECM-derived porous foams and microcarriers for use as biologically-relevant substrates in advanced 3D in vitro cell culture models or as pro-regenerative scaffolds and cell delivery systems for tissue engineering and regenerative medicine. Using decellularized tissues or purified insoluble collagen as a starting material, the techniques can be applied to synthesize a broad array of tissue-specific bioscaffolds with customizable geometries. The approach involves mechanical processing and mild enzymatic digestion to yield an ECM suspension that is used to fabricate the three-dimensional foams or microcarriers through controlled freezing and lyophilization procedures. These pure ECM-derived scaffolds are highly porous, yet stable without the need for chemical crosslinking agents or other additives that may negatively impact cell function. The scaffold properties can be tuned to some extent by varying factors such as the ECM suspension concentration, mechanical processing methods, or synthesis conditions. In general, the scaffolds are robust and easy to handle, and can be processed as tissues for most standard biological assays, providing a versatile and user-friendly 3D cell culture platform that mimics the native ECM composition. Overall, these straightforward methods for fabricating customized ECM-derived foams and microcarriers may be of interest to both biologists and biomedical engineers as tissue-specific cell-instructive platforms for in vitro and in vivo applications.

The tissue-specific extracellular matrix (ECM) is a key mediator of cell function. This article describes methods for synthesizing pure ECM-derived foams and microcarriers that are stable in culture without the need for chemical crosslinking for applications in advanced 3D in vitro cell culture models or as pro-regenerative bioscaffolds.

Cell function is mediated by interactions with the extracellular matrix (ECM), which has complex tissue-specific composition and architecture. The focus ...

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 prevent and treat sexually transmitted infections (STIs). Moreover, many EF technologies focus on encapsulating the active agent, relative to utilizing the surface to impart biofunctionality. Here we describe a method to fabricate and surface-modify poly(lactic-co-glycolic) acid (PLGA) electrospun fibers, with the potent antiviral lectin Griffithsin (GRFT). PLGA is an FDA-approved polymer that has been widely used in drug delivery due to its outstanding chemical and biocompatible properties. GRFT is a natural, potent, and safe lectin that possesses broad activity against numerous viruses including human immunodeficiency virus type 1 (HIV-1). When combined, GRFT-modified fibers have demonstrated potent inactivation of HIV-1 in vitro. This manuscript describes the methods to fabricate and characterize GRFT-modified EFs. First, PLGA is electrospun to create a fiber scaffold. Fibers are subsequently surface-modified with GRFT using 1-Ethyl-3-(3-dimethylaminopropyl)carbodiimide (EDC) and N-hydroxysuccinimide (NHS)chemistry. Scanning electron microscopy (SEM) was used to assess the size and morphology of surface-modified formulations. Additionally, a gp120 or hemagglutinin (HA)-based ELISA may be used to quantify the amount of GRFT conjugated to, as well as GRFT desorption from the fiber surface. This protocol can be more widely applied to fabricate fibers that are surface-modified with a variety of different proteins.

This manuscript describes the procedure to fabricate and characterize Griffithsin-modified poly(lactic-co-glycolic acid) electrospun fibers that demonstrate potent adhesive and antiviral activity against human immunodeficiency virus type 1 infection in vitro. Methods used to synthesize, surface-modify, and characterize the resulting morphology, conjugation, and desorption of Griffithsin from surface-modified fibers are described.

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 of a Biomimetic Nano-Matrix with Janus Base Nanotubes and Fibronectin for Stem Cell Adhesion

A biomimetic NM was developed to serve as a tissue-engineering biological scaffold, which can enhance stem cell anchorage. The biomimetic NM is formed from JBNTs and FN through self-assembly in an aqueous solution. JBNTs measure 200-300 &#181;m in length with inner hydrophobic hollow channels and outer hydrophilic surfaces. JBNTs are positively charged and FNs are negatively charged. Therefore, when injected into a neutral aqueous solution, they are bonded together via noncovalent bonding to form the NM bundles. The self-assembly process is completed within a few seconds without any chemical initiators, heat source, or UV light. When the pH of the NM solution is lower than the isoelectric point of FNs (pI 5.5-6.0), the NM bundles will self-release due to the presence of positively charged FN.
NM is known to mimic the extracellular matrix (ECM) morphologically and hence, can be used as an injectable scaffold, which provides an excellent platform to enhance hMSC adhesion. Cell density analysis and fluorescence imaging experiments indicated that the NMs significantly increased the anchorage of hMSCs compared to the negative control.

The goal of this protocol is to show the assembly of a biomimetic nanomatrix (NM) with Janus base nanotubes (JBNTs) and fibronectin (FN). When co-cultured with human mesenchymal stem cells (hMSCs), the NMs exhibit excellent bioactivity in encouraging hMSCs adhesion.

A biomimetic NM was developed to serve as a tissue-engineering biological scaffold, which can enhance stem cell anchorage. The biomimetic NM is formed ...

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

Various biomaterial scaffolds have been developed to guide cell adhesion and proliferation in hopes to promote specific functions for in vitro and in vivo uses. The addition of growth factors into these biomaterial scaffolds is generally done to provide an optimal cell culture environment, mediating cell differentiation and its subsequent functions. However, the growth factors in a conventional biomaterial scaffold are typically designed to be released upon implantation, which could result in unintended side effects on surrounding tissue or cells. Here, the DNA-inspired Janus base nano-matrix (JBNm) has successfully achieved a highly localized microenvironment with a layer-by-layer structure for self-sustainable cartilage tissue constructs. JBNms are self-assembled from Janus base nanotubes (JBNts), matrilin-3, and transforming growth factor beta-1 (TGF-&#946;1) via bioaffinity. The JBNm was assembled at a TGF-&#946;1:matrilin-3:JBNt ratio of 1:4:10, as this has been the determined ratio at which proper assembly into the layer-by-layer structure could occur. First, the TGF-&#946;1 solution was added to the matrilin-3 solution. Then, this mixture was pipetted several times to ensure sufficient homogeneity before the addition of the JBNt solution. This formed the layer-by-layer JBNm, after pipetting several times again. A variety of experiments were performed to characterize the layer-by-layer JBNm structure, JBNts alone, matrilin-3 alone, and TGF-&#946;1 alone. The formation of JBNm was studied with UV-Vis absorption spectra, and the structure of the JBNm was observed with transmission electron microscopy (TEM). As the innovative layer-by-layer JBNm scaffold is formed on a molecular scale, the fluorescent dye-labeled JBNm could be observed. The TGF-&#946;1 is confined within the inner layer of the injectable JBNm, which can prevent the release of growth factors to surrounding areas, promote localized chondrogenesis, and promote an anti-hypertrophic microenvironment.

This protocol describes the assembly of a layer-by-layer Janus base nano-matrix (JBNm) scaffold by adding Janus base nanotubes (JBNts), matrilin-3, and Transforming Growth Factor Beta-1 (TGF-&#946;1) sequentially. The JBNm was fabricated and characterized; additionally, it displayed excellent bioactivity, encouraging cell functions such as adhesion, proliferation, and differentiation.

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

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 viral gene transfer into T cells. The current gold standard of viral gene transfer involves spinoculation of retronectin-coated plates, which is expensive and time-consuming. There is a significant need for efficient and cost-effective methods to generate CAR-T cells. Described here is a method for fabricating inexpensive, dry macroporous alginate scaffolds, known as Drydux scaffolds, that efficiently promote viral transduction of activated T cells. The scaffolds are designed to be used in place of gold standard spinoculation of retronectin-coated plates seeded with virus and simplify the process for transducing cells. Alginate is cross-linked with calcium-D-gluconate and frozen overnight to create the scaffolds. The frozen scaffolds are freeze-dried in a lyophilizer for 72 h to complete the formation of the dry macroporous scaffolds. The scaffolds mediate viral gene transfer when virus and activated T cells are seeded together on top of the scaffold to produce genetically modified cells. The scaffolds produce &gt;85% primary T cell transduction, which is comparable to the transduction efficiency of spinoculation on retronectin-coated plates. These results demonstrate that dry macroporous alginate scaffolds serve as a cheaper and more convenient alternative to the conventional transduction method.

Herein is a protocol for creating dry macroporous alginate scaffolds that mediate efficient viral gene transfer for use in genetic engineering of T cells, including T cells for CAR-T cell therapy. The scaffolds were shown to transduce activated primary T cells with &gt;85% transduction.

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

The emergence of granular hydrogel scaffolds (GHS), fabricated via assembling hydrogel microparticles (HMPs), has enabled microporous scaffold formation in situ. Unlike conventional bulk hydrogels, interconnected microscale pores in GHS facilitate degradation-independent cell infiltration as well as oxygen, nutrient, and cellular byproduct transfer. Methacryloyl-modified gelatin (GelMA), a (photo)chemically crosslinkable, protein-based biopolymer containing cell adhesive and biodegradable moieties, has widely been used as a cell-responsive/instructive biomaterial. Converting bulk GelMA to GHS&#160;may open a plethora of opportunities for tissue engineering and regeneration. In this article, we demonstrate the procedures of high-throughput GelMA microgel fabrication, conversion to resuspendable dry microgels (micro-aerogels), GHS formation via the chemical assembly of microgels, and granular bioink fabrication for extrusion bioprinting. We show how a sequential physicochemical treatment via cooling and photocrosslinking enables the formation of mechanically robust GHS.&#160;When light is inaccessible (e.g., during deep tissue injection), individually crosslinked GelMA HMPs may be bioorthogonally assembled via enzymatic crosslinking using transglutaminases. Finally, three-dimensional (3D) bioprinting of microporous GHS&#160;at low HMP packing density is demonstrated via the interfacial self-assembly of heterogeneously charged nanoparticles.

This article describes protocols for high-throughput gelatin methacryloyl microgel fabrication using microfluidic devices, converting microgels to resuspendable powder (micro-aerogels), the chemical assembly of microgels to form granular hydrogel scaffolds, and developing granular hydrogel bioinks with preserved microporosity for 3D bioprinting.

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

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.

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 ...