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>CaCl&lt;sub&gt;2&lt;/sub&gt;</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 &amp;amp; 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>Superlite 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...

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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

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Bioengineering

11.0K Views.  University Medical Center Utrecht, The Netherlands. Deze methode biedt een manier om steigers afgeleid van inheemse weefsel die kunnen worden gebruikt voor kraakbeen regeneratie te creëren. Het belangrijkste voordeel van deze techniek is dat componenten van inheemse kraakbeen worden bewaard in de steigers die kraakbeenvorming en regeneratie in vitro en in vivo kunnen bevorderen. Hoewel deze methode maakt gebruik van kraakbeen van paarden verstikken gewrichten, kraakbeen uit andere gewrichten kan ook worden gebruikt.Om te beginnen, het ...

Video: Fabrication of Decellularized Cartilage-derived Matrix Scaffolds

Tissue Engineering: Construction of a Multicellular 3D Scaffold for the Delivery of Layered Cell Sheets

Many tissues, such as the adult human hearts, are unable to adequately regenerate after damage.2,3 Strategies in tissue engineering propose innovations to assist the body in recovery and repair. For example, TE approaches may be able to attenuate heart remodeling after myocardial infarction (MI) and possibly increase total heart function to a near normal pre-MI level.4 As with any functional tissue, successful regeneration of cardiac tissue involves the proper delivery of multiple cell types with environmental cues favoring integration and survival of the implanted cell/tissue graft. Engineered tissues should address multiple parameters including: soluble signals, cell-to-cell interactions, and matrix materials evaluated as delivery vehicles, their effects on cell survival, material strength, and facilitation of cell-to-tissue organization. Studies employing the direct injection of graft cells only ignore these essential elements.2,5,6 A tissue design combining these ingredients has yet to be developed. Here, we present an example of integrated designs using layering of patterned cell sheets with two distinct types of biological-derived materials containing the target organ cell type and endothelial cells for enhancing new vessels formation in the &#8220;tissue&#8221;. Although these studies focus on the generation of heart-like tissue, this tissue design can be applied to many organs other than heart with minimal design and material changes, and is meant to be an off-the-shelf product for regenerative therapies. The protocol contains five detailed steps. A temperature sensitive Poly(N-isopropylacrylamide) (pNIPAAM) is used to coat tissue culture dishes. Then, tissue specific cells are cultured on the surface of the coated plates/micropattern surfaces to form cell sheets with strong lateral adhesions. Thirdly, a base matrix is created for the tissue by combining porous matrix with neovascular permissive hydrogels and endothelial cells. Finally, the cell sheets are lifted from the pNIPAAM coated dishes and transferred to the base element, making the complete construct.

For creation of highly organized structures of complex tissue, one must assemble multiple material and cell types into an integrated composite. This combinatorial design incorporates organ-specific layered cell sheets with two distinct biologically-derived materials containing a strong fibrous matrix base, and endothelial cells for enhancing new vessels formation.

Many tissues, such as the adult human hearts, are unable to adequately regenerate after damage.2,3 Strategies in tissue engineering propose innovations to ...

3D Hydrogel Scaffolds for Articular Chondrocyte Culture and Cartilage Generation

Human articular cartilage is highly susceptible to damage and has limited self-repair and regeneration potential. Cell-based strategies to engineer cartilage tissue offer a promising solution to repair articular cartilage. To select the optimal cell source for tissue repair, it is important to develop an appropriate culture platform to systematically examine the biological and biomechanical differences in the tissue-engineered cartilage by different cell sources. Here we applied a three-dimensional (3D) biomimetic hydrogel culture platform to systematically examine cartilage regeneration potential of juvenile, adult, and osteoarthritic (OA) chondrocytes. The 3D biomimetic hydrogel consisted of synthetic component poly(ethylene glycol) and bioactive component chondroitin sulfate, which provides a physiologically relevant microenvironment for in vitro culture of chondrocytes. In addition, the scaffold may be potentially used for cell delivery for cartilage repair in vivo. Cartilage tissue engineered in the scaffold can be evaluated using quantitative gene expression, immunofluorescence staining, biochemical assays, and mechanical testing. Utilizing these outcomes, we were able to characterize the differential regenerative potential of chondrocytes of varying age, both at the gene expression level and in the biochemical and biomechanical properties of the engineered cartilage tissue. The 3D culture model could be applied to investigate the molecular and functional differences among chondrocytes and progenitor cells from different stages of normal or aberrant development.

Cartilage repair represents an unmet medical challenge and cell-based approaches to engineer human articular cartilage are a promising solution. Here, we describe three-dimensional (3D) biomimetic hydrogels as an ideal tool for the expansion and maturation of human articular chondrocytes.

Human articular cartilage is highly susceptible to damage and has limited self-repair and regeneration potential. Cell-based strategies to engineer ...

3D Magnetic Stem Cell Aggregation and Bioreactor Maturation for Cartilage Regeneration

Cartilage engineering remains a challenge due to the difficulties in creating an in vitro functional implant similar to the native tissue. An approach recently explored for the development of autologous replacements involves the differentiation of stem cells into chondrocytes. To initiate this chondrogenesis, a degree of compaction of the stem cells is required; hence, we demonstrated the feasibility of magnetically condensing cells, both within thick scaffolds and scaffold-free, using miniaturized magnetic field sources as cell attractors. This magnetic approach was also used to guide aggregate fusion and to build scaffold-free, organized, three-dimensional (3D) tissues several millimeters in size. In addition to having an enhanced size, the tissue formed by magnetic-driven fusion presented a significant increase in the expression of collagen II, and a similar trend was observed for aggrecan expression. As the native cartilage was subjected to forces that influenced its 3D structure, dynamic maturation was also performed. A bioreactor that provides mechanical stimuli was used to culture the magnetically seeded scaffolds over a 21-day period. Bioreactor maturation largely improved chondrogenesis into the cellularized scaffolds; the extracellular matrix obtained under these conditions was rich in collagen II and aggrecan. This work outlines the innovative potential of magnetic condensation of labeled stem cells and dynamic maturation in a bioreactor for improved chondrogenic differentiation, both scaffold-free and within polysaccharide scaffolds.

Chondrogenesis from stem cells requires fine tuning the culture conditions. Here, we present a magnetic approach for condensing cells, an essential step to initiate chondrogenesis. In addition, we show that dynamic maturation in a bioreactor applies mechanical stimulation to the cellular constructs and enhances cartilaginous extracellular matrix production.

Cartilage engineering remains a challenge due to the difficulties in creating an in vitro functional implant similar to the native tissue. An approach ...

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 ...

Two Methods for Decellularization of Plant Tissues for Tissue Engineering Applications

The autologous, synthetic, and animal-derived grafts currently used as scaffolds for tissue replacement have limitations due to low availability, poor biocompatibility, and cost. Plant tissues have favorable characteristics that make them uniquely suited for use as scaffolds, such as high surface area, excellent water transport and retention, interconnected porosity, preexisting vascular networks, and a wide range of mechanical properties. Two successful methods of plant decellularization for tissue engineering applications are described here. The first method is based on detergent baths to remove cellular matter, which is similar to previously established methods used to clear mammalian tissues. The second is a detergent-free method adapted from a protocol that isolates leaf vasculature and involves the use of a heated bleach and salt bath to clear the leaves and stems. Both methods yield scaffolds with comparable mechanical properties and low cellular metabolic impact, thus allowing the user to select the protocol which better suits their intended application.

Here we present, and contrast two protocols used to decellularize plant tissues: a detergent-based approach and a detergent-free approach. Both methods leave behind the extracellular matrix of the plant tissues used, which can then be utilized as scaffolds for tissue engineering applications.

The autologous, synthetic, and animal-derived grafts currently used as scaffolds for tissue replacement have limitations due to low availability, poor ...

Novel Process for 3D Printing Decellularized Matrices

3D bioprinting aims to create custom scaffolds that are biologically active and accommodate the desired size and geometry. A thermoplastic backbone can provide mechanical stability similar to native tissue while biologic agents offer compositional cues to progenitor cells, leading to their migration, proliferation, and differentiation to reconstitute the original tissues/organs1,2. Unfortunately, many 3D printing compatible, bioresorbable polymers (such as polylactic acid, PLA) are printed at temperatures of 210 &#176;C or higher - temperatures that are detrimental to biologics. On the other hand, polycaprolactone (PCL), a different type of polyester, is a bioresorbable, 3D printable material that has a gentler printing temperature of 65 &#176;C. Therefore, it was hypothesized that decellularized extracellular matrix (DM) contained within a thermally protective PLA barrier could be printed within PCL filament and remain in its functional conformation. In this work, osteochondral repair was the application for which the hypothesis was tested. As such, porcine cartilage was decellularized and encapsulated in polylactic acid (PLA) microspheres which were then extruded with polycaprolactone (PCL) into filament to produce 3D constructs via fused deposition modeling. The constructs with or without the microspheres (PLA-DM/PCL and PCL(-), respectively) were evaluated for differences in surface features.

This protocol describes the production of polycaprolactone (PCL) filament with embedded polylactic acid (PLA) microspheres which contain decellularized matrices (DM) for 3D printing of structural tissue engineering constructs.

3D bioprinting aims to create custom scaffolds that are biologically active and accommodate the desired size and geometry. A thermoplastic backbone can ...

Production of Extracellular Matrix Fibers via Sacrificial Hollow Fiber Membrane Cell Culture

Engineered scaffolds derived from extracellular matrix (ECM) have driven significant interest in medicine for their potential in expediting wound closure and healing. Extraction of extracellular matrix from fibrogenic cell cultures in vitro has potential for generation of ECM from human- and potentially patient-specific cell lines, minimizing the presence of xenogeneic epitopes which has hindered the clinical success of some existing ECM products. A significant challenge in in vitro production of ECM suitable for implantation is that ECM production by cell culture is typically of relatively low yield. In this work, protocols are described for the production of ECM by cells cultured within sacrificial hollow fiber membrane scaffolds. Hollow fiber membranes are cultured with fibroblast cell lines in a conventional cell medium and dissolved after cell culture to yield continuous threads of ECM. The resulting ECM fibers produced by this method can be decellularized and lyophilized, rendering it suitable for storage and implantation.

The goal of this protocol is production of whole extracellular matrix fibers targeted for wound repair which are suitable for preclinical evaluation as part of a regenerative scaffold implant. These fibers are produced by the culture of fibroblasts on hollow fiber membranes and extracted by dissolution of the membranes.

Engineered scaffolds derived from extracellular matrix (ECM) have driven significant interest in medicine for their potential in expediting wound closure ...

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 ...

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 ...

Decellularized Rat Spleen Matrix Preparation for Liver Tissue Engineering

Fabrication of Decellularized Spleen Matrix Derived from Rats

Liver transplantation is the primary treatment for end-stage liver disease. However, the shortage and inadequate quality of donor organs necessitate the development of alternative therapies. Bioartificial livers (BALs) utilizing decellularized liver matrix (DLM) have emerged as promising solutions. However, sourcing suitable DLMs remains challenging. The use of a decellularized spleen matrix (DSM) has been explored as a foundation for BALs, offering a readily available alternative. In this study, rat spleens were harvested and decellularized using a combination of freeze-thaw cycles and perfusion with decellularization reagents. The protocol preserved the microstructures and components of the extracellular matrix (ECM) within the DSM. The complete decellularization process took approximately 11 h, resulting in an intact ECM within the DSM. Histological analysis confirmed the removal of cellular components while retaining the ECM's structure and composition. The presented protocol provides a comprehensive method for obtaining DSM, offering potential applications in liver tissue engineering and cell therapy. These findings contribute to the development of alternative approaches for the treatment of end-stage liver disease.

Author Spotlight: Utilization of Decellularized Spleen Matrix for Bioartificial Livers

The decellularized spleen matrix (DSM) holds promising applications in the field of liver tissue engineering. This protocol outlines the procedure for preparing rat DSM, which includes harvesting rat spleens, decellularizing them through perfusion, and evaluating the resulting DSM to confirm its characteristics.

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

Fabrication of Decellularized Cartilage-derived Matrix Scaffolds

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